Laboratory Manual Invertebrate Zoology

BIO 353

University of Maine Seth Tyler

2016

Table of Contents Introduction to the Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Comparative-morphology checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Diversity checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Laboratory Notebook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Microsopes — use and care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Annelida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Polychaeta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Polychaete diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Oligochaeta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Hirudinea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Arthropoda / Onychophora / Tardigrada Panarthropoda: Onychophora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Panarthropoda: Tardigrada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Arthropoda I: Crustacea I (Decapoda, Anostraca, Phyllopoda) . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Arthropoda II: Crustacea II (Peracarida, Maxillopoda) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Arthropoda III: Myriapoda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Arthropoda IV: Chelicerata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36 Mollusca System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Mollusca I (Polyplacophora, Gastropoda) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Mollusca II (Bivalvia, Scaphopoda, Cephalopoda) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Porifera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Cnidaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Ctenophora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Lophophorata, Tunicata, Hemichordata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Echinodermata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Meiofauna Extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Platyhelminthes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Gnathifera (Gnathostomulida, Rotifera) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Gastrotricha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Nematoda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Priapulida, Kinorhyncha, Cycliophora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Invertebrate Zoology — laboratory

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Laboratory, Invertebrate Zoology Study Nature, not books. theme of the Anderson School of Natural History, founded by Louis Agassiz, 1873

Saper vedere (knowing how to see) Leonardo da Vinci’s guiding principle

Study of live animals is the focus of this course. In each laboratory period, you will have access to animals representing the groups of invertebrates we are covering at the time in lecture. You will examine these animals in depth, learning about their anatomy and behavior and making notes and sketches in your notebook of the things you find. You will focus on certain organ systems in each animal you study and prepare hypotheses of how those organs work. You will gain some understanding of the diversity of invertebrate animals by making comparisons among the animals you study and by having opportunity to see other representatives of the groups we cover, to gain some understanding of the diversity of invertebrates, learning as you go how to identify certain animals and to know their place in the taxonomic system, Studying animals. Use your textbook, the laboratory manual, and other resources available online or as texts provided in lab to guide your study. For each animal, you should identify its major organ systems and try to determine how those systems adapt the animal for the particular habitat in which it lives. The accompanying checklist should help, and you should use it to track your progress and to note where you have kept sketches and notes of your observations. Some species may not be specifically mentioned in the written material, and you will have use your knowledge of the group to which a species belongs to discover how it, in particular, accomplishes what it has to to survive. In many cases, a good approach for studying the animal is to simply watch it, looking for features from the checklist and applying things you learn in class and from the textbooks. Often, simply trusting your own powers of observation and listening to the thoughts and questions that arise in your mind as you watch will be productive; you may discover new things about the animals no one else has seen before. In a way, you let the animal speak to you. Remember that the animal is always right; don’t necessarily trust textbooks. To study an animal in depth, it is often necessary to anesthetize it so that it remains still under the microscope and so it can be humanely sacrificed to expose internal organs. Small animals can often be studied by squeezing them on a slide, flattening them under the pressure of a coverslip so that the arrangement of internal organs can be seen. Larger animals must be dissected—that is, the body wall must be cut open and the organs gently teased apart to expose their relationships with one another. Dissection actually involves very little cutting, contrary to popular belief; most of it should be done with a blunt probe and forceps. You will need to make a cut through the body wall to expose the organs, and you will sometimes need to cut the whole organ system or pieces of it out to see what tissues and cells compose it and to see its internal parts. Dissections are best done on fully anaesthetized live animals, because the parts of the body are much easier to distinguish in the living condition; fixatives tend to remove colors and homogenize texture. Paramount in dealing with such living specimens is humane treatment: the animals must be anaesthetized so that they do not suffer. For the most part, animals for dissection will be provided to you already anaesthetized. If you work on a live animal that has not been anaesthetized, you will need to administer an anaesthetic yourself, typically, for marine animals at least, a solution of magnesium chloride that is isotonic with sea water; other anaesthetics provided in lab are ethanol and phenoxy propanol. Dissections should be carried out under liquid rather than dry. Only when liquid completely covers the specimen is the tissue sufficiently supported to reveal internal features. Having the specimen submerged also prevents glare from reflective surfaces. Use a microscope to examine all animals, even the big ones. The beauty of an animal is often in the finer detail, and to appreciate the texture of the integument or the morphology of a sensory bristle or eye, for example, you must see it magnified. Use at least a dissecting microscope on all organisms, a compound microscope if the animals fits on a slide; and examine small parts of the animal with the compound microscope. Dissections should also be observed with a microscope; in fact, as the name specifies, dissecting microscopes should be used to do the dissections, at least to see what has been exposed with dissecting if that is done free-hand to the side of the microscope. Notebooks. Observations of the animals should be recorded with sketches and notes in a laboratory notebook. While skill at drawing is useful in making this notebook, it is certainly not required—anyone should be able to make the kind of sketches useful for recording observations of these animals. The emphasis in recording observations should be on the sketches: your notes should be used to explain the illustrations,

2 not drawings to illustrate your notes. (See also separate section on how to make the notebook.) Examples of notes and sketches an invertebrate zoologist made in studying an animal are on the next two pages. These were made by Donald Abbott and are taken from a book of his sketches his students published after his death (D. P. Abbott [1978] Observing marine invertebrates. Stanford Univ. Press, Stanford, CA. 382 pp.) Copies of this book are available in the lab, and you are encouraged to peruse it. It is a very useful source of information about invertebrates. Some of this information would have gone into publications by Abbott and his students on specific animals; all would have been useful to him as he dealt with invertebrates he encountered in the field and lab. You will keep your notebook in the lab in the filing cabinet by the door; pick it up there when you come to lab and turn it in to the TA when you leave. You will get feedback on the notebook periodically during the semester, and it will be graded at the end of the course. Teamwork. To study most animals, particularly animals that you work on in depth, you will work with a lab partner or two. If there are enough specimens of a species you want to study, you should take your own specimen and then collaborate with your partners as you study them, showing each other what you find. For some species, you may need to work together on the same specimen. When specimens must be sacrificed for dissection, we will conserve animals and minimize the sacrifice by working together on a single specimen in teams of two or three. You should still keep your own notes and sketches of the study in your notebook, but you should cooperate with your partner or other team members to share in the dissections and share your thoughts about what you are seeing in a specimen. Everyone in the team should have some records of a dissection, but you can share these for making your reports. Also, share your observations with others in the lab outside your team, particularly when you find an animal doing something interesting, or find its heart beating, or something like that that may not last long. Not everyone will be concentrating on the same animals or organ systems, so we can all benefit from the work others in the lab are doing to learn about them. When you present your findings to the rest of the class, you can do this as a team, reporting your shared observations. Checklists. The following two pages are checklists you should use to keep track of what you accomplish: • Functional-morphology checklist. To summarize your studies of each animal and to provide a guide to where you can find information about them in the textbook, lab manual, and your notebook, use the checklist on functional morphology. Even if you don’t find all organ systems listed here in any particular animal you look at, you should think about what those organ systems should be in that animal, so starting this checklist before lab is important. For each lab period, fill in what you expect to find and turn in the sheet as you enter lab. You’ll get the sheet back so that you can consult it during lab; then, once you have recorded where in your notebook you have made sketches and notes on the animals you see in lab, turn in the sheet with your notebook as you leave lab. • Diversity checklist. To understand what a taxonomic group of invertebrates is, it helps tremendously to compare its representatives for their similarities and differences. We will have a variety of animals representing the groups of invertebrates we study, and the diversity checklist will help you track which representatives you have seen and how they compare. You can keep these checklists in your notebook. Copies of these checklists are also available online as separate pdf’s in the Handouts section.

Invertebrate Zoology — laboratory

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Name:

Date: Functional-morphology checklist Pre-lab expectations & Lab findings

For each of the systems below, list and describe two features that you expect to see in an animal representing the group we are studying this week in lab and indicate the page numbers in the textbook (RFB — Ruppert, Fox, & Barnes) and lab manual (Manual) where information can be found about this system. Sketch the general form of the body of this animal representing this group here Ú Submit this sheet when you come into lab (it’s your ticket for entry). As you work in lab, check off the features you actually do find (and add any you did not list beforehand) and fill in the last column with the page number in your notebook where you sketch them. Class:

Phylum Structure Body wall: epidermis protective coverings musculature Digestive system: digestive tract glands

Circulatory system: vascular elements coelomic

Respiratory system

Excretory organs

Nervous system: brain nerve cords sensory organs

Reproductive system

Special features: food-capture mechanism locomotory mechanisms or . . .

features to find

pp. RFB

Manual

Notebook

4 Name:

Diversity Checklist Keep track of the diversity of animals you study by listing them on copies of this worksheet, one copy for each of the major groups we study. First check the box (one box only) for which this sheet is representative (the one encompassing all the animals you list here). Then name in the first column the animals you have studied that belong to this group. In the second column, list a few characters for each of the animals that make it a member of the group and a few that distinguish it from the others you name. In the third column, note the pages in your notebook that have notes and sketches of the animal. Check the box by the group to which this particular worksheet applies (check only one): 2 Annelida “Polychaeta” 2 Arthropoda Myriapoda 2 Cnidaria 2 Annelida Clitellata 2 Mollusca Gastropoda 2 Bryozoa 2 Arthropoda “Crustacea” 2 Mollusca Bivalvia 2 Urochordata (Tunicata) 2 Arthropoda Chelicerata 2 Porifera 2 Echinodermata Animal (genus)

Characters you saw that are in common and distinctive Characters placing this species within the group:

Characters that are distinctive of this species:

Characters placing this species within the group:

Characters that are distinctive of this species:

Characters placing this species within the group:

Characters that are distinctive of this species:

Characters placing this species within the group:

Characters that are distinctive of this species:

Notebook pages

Invertebrate Zoology — notebook

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Laboratory Notebook How to keep records of laboratory study Your laboratory notebook should be a log of what you see and do in laboratory—your drawings together with notes that explain those drawings and notes of thoughts and questions that come to mind as you observe animals. What you can see, of course, depends on knowing how to see—saper vedere (and how to observe invertebrates in particular)—which is a skill you must develop. Forcing yourself to draw what sits in front of you, to look at all its details as well as general form, is the best way to hone that skill. Comparative morphology is the emphasis in this lab, so look always for similarities and differences among the animals we study; think about what is similar about species in the same genus, family, or higher taxonomic rankings and about critical differences that distinguish them. You should be able to use your notebook to remind yourself of what you saw and thought and to provide the primary material by which you document your lab reports. It is not expected to be a letter-perfect, publishable work of art, but a log. The notebook as a whole should be loose-leaf—that is, pages assembled in a binder, such as a three-ringed binder, so that you can organize its pages into appropriate sections. The pages on which you make your sketches and notes should be plain drawing paper, without rule lines. (Plain white paper is available in lab, and this is sufficient for our purposes; if you want high-quality drawing paper, you will need to provide your own.) Your notebook should have these sections: 1. Table of contents listing all the animals you have sketches of and citing the page numbers where those sketches and notes can be found. Group the list by major taxa (e.g., phylum and class as a heading for a group of species). 2. Sketches and notes—your log of what you do in lab. These pages should be left in the order in which you made them and all together in one section. 3. Functional-morphology checklists (of the animals you studied in detail for their organ systems). 4. Diversity checklists (of the animals you briefly compare to those studied in detail). Keep the following points in mind as you work on your sketches and notes: Honest and realistic reporting Notes and sketches should record what you yourself actually see of the animals. Do not copy the textbook’s or other published figures; rather, draw what you see (and not what you think ought to be seen) and use the book or other guides to determine what it is you are seeing. Try to relate what you can actually find to what the lab guide is pointing out as being significant features. Record copiously Record all that you do in lab and what you think as you do it. Text in the notebook, therefore, should be not only annotations of drawings but questions and other thoughts that occur to you as you work. Don’t just idly oggle the animals, but actively think and write as you watch them. Drawings should form the bulk of the material recorded, but always write about what you draw— label, annotate, comment. Record anything that strikes you as interesting, not just what you think you should see, and don’t hesitate to make any notes, no matter how silly or unscientific they may seem to you, even notes about your own confusion. Records of such things as behavior and interactions with other organisms may require an almost narrative style of note-taking. Drawings Start with a simple sketch of the whole animal. As you start to work on a species, first sketch what the whole animal looks like in the upper right quarter of a blank page. Show its general shape—something that you can use to recognize the animal again—and label that sketch as accurately as possible with the animal’s scientific name (species, if possible—or genus, family, or at least class). Most animals provided for you will have a label by the dish from which you get it; use that name or ask others what it is. Note the size of the animal (for example, report its length) or draw a scale bar next to it, and note whether the animal is live, fixed, or dried; this will make a difference when it comes to recognizing the animal again. This should be a simple sketch, so don’t spend much time on it. More important are sketches you make of details of selected parts of the animal on the rest of this page and following pages. Set this whole-animal sketch off from the rest of the page with lines. (Box it off.)

6 Large-format drawings. The primary drawings for each animal should be of selected parts of it and should be no smaller than a quarter of a page, even for the smallest and simplest of the animals. It is actually easier to draw a large-format drawing than a tiny one, and then you have room to put in whatever details are significant. You may also want to include smaller, cartoon-style drawings to indicate movements, habitus, actual size—i.e., not all drawings have to be large. Keep drawings simple. Your drawings should have clean, simple lines. Clear outlines of whole animals and of selected internal features are all that are necessary. Keep shading or stipling to a minimum; if you want to indicate texture, do so on limited portions of the drawing or in a small sketch to one side. Don’t color your drawings, but note coloration with text. Be accurate in drawing; don’t simply render an impression of an animal or its parts, but specifically show exactly what can be discerned with some scrutiny. Examine animals at a range of magnifications. Use lower magnification to gauge the general appearance of the animal and then use higher magnification to sketch and take notes on details in structure, especially of features the lab guide is pointing out as important, distinguishing features. If it’s not obvious from your whole-animal sketch what the relative size is of a part you sketch, note its size (length, e.g.) or place a scale bar next to it. Don’t crowd the page / Write on only one side. Notes and sketches should have plenty of room around them and not be crowded together. Good separation will make it easier to refind information. Avoid confusion from bleed-through by using only one side of each page. Mechanics. Use a soft pencil (#2 or, with mechanical pencils, F, HB, or 2B lead), not a pen. Draw with a light touch, with a relaxed hand, and use mostly long, flowing lines. Feel free to edit the drawings as necessary—erasing, revising, and retouching; don’t, however, spend a lot of time trying to get something exactly right; this is, after all, a working log. Never recopy a drawing unless you actually have the animal available to make sure you are not introducing error in the copy. For those rare cases in which you need to shade or stipple, make the drawing appear as if light falls on the object from the upper left (the convention). Stippling is not random dotting, by the way; we’ll show you how to do it properly if you want. Notes Label and explain. Label your drawings fully and write notes to explain them. Use the textbook and lab manual to be sure you know what you are sketching and that you have it identified correctly. Write explanations that will help you remember what something looks like, what its texture is, or how it moves. Questions and other thoughts. Posing good questions is as important a skill as observing. This is the essence of meaningful research. Write those questions down! Record any questions that come to mind, even questions expressing your confusion over material you see. Do not include notes from reading or other illustrations. Notes on the reading material and copies of figures from textbooks or other published material should not be part of this laboratory log. Keep such things in your class notebook for the lecture portion of the class. Cooperate Sharing your work with others is the best way to improve it, and that benefits us all in our efforts to learn about the animals. Show your animals and drawings to your neighbors and look carefully at their material, including their dissections or whole animals and drawings. Discuss what you see. Help others if you can.

Invertebrate Zoology — notebook

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8

Use of microscopes

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Use and Care of Microscopes Two microscopes, a dissecting microscope and a compound microscope, are issued for each student in each laboratory section. These microscopes are of high quality and must be treated with the care and respect that such precision instruments require. To get the most out of the microscopes, follow these precautions and instructions for use. Precautions Avoid getting sea water on the microscopes. Wipe any dishes and slides that are to be placed on the microscopes with a towel before they contact the microscopes. While it is virtually impossible to keep sea water from the microscopes entirely, exposure should be minimized, and any spills on the microscopes should be wiped off as soon as possible. Before putting a microscope away, wipe up any water on it and then wipe it down with a second towel dampened with fresh water and dry it. Don’t touch the lenses. Occasional cleaning to remove oil or grease may be necessary; this should be done only with special lens tissue. Please consult the instructor before attempting cleaning. Please do not loosen any set screws, such as the screw holding the head of the microscope in its mounting ring. (Please do not turn the head to show lab partners views under the scope; keep the microscope fully stationary and make them move to look in it.) Dissecting microscope The oculars (eye pieces) adjust in focus independently and should be adjusted when you first start work on the microscope. The spacing between the oculars (interpupilary spacing) is also adjustable and should be set so that both eyes see the specimen simultaneously. It is best to focus on a specimen with the large focus knob of the microscope while looking first through the right ocular (only) with your right eye; then look through the left ocular (only) with your left eye and turn the knurled ring on it until the same object you saw with your right eye in the other ocular is in focus. Illumination. Lights for reflected and transmitted illumination are controlled separately, each with its own knob. In most cases you would use either transmitted or reflected light, not both at once. Before turning on the rocker switch (on the right side of the base), make sure the two knobs for these lamps (labeled TRANSMITTED and REFLECTED) are all the way down to off; then turn on the rocker switch and turn up either of the two lamp knobs to an appropriate brightness. (Similarly, in turning the illuminatoion off, turn the two lamp knobs down to off before turning off the rocker switch.) Turn the lamp knobs gradually up or down; don’t just crank them up to full power routinely. This gradual on-and-off greatly lengthens the life of the bulbs. When turning the lights off, turn the switches counterclockwise until you hear a click and no farther (these switches can break). Even opaque animals can sometimes be studied with transmitted light—to see structures along the edges, for instance. Note that color of an animal may look different with transmitted than it does with reflected light. Magnification is changed by rotating the knurled knob on the right side of the head. To obtain parfocal zoom in magnification (i.e., so that the image stays in focus at all magnifications), first focus on a specimen at high magnification, then zoom to low magnification and adjust focus with the oculars only. Compound microscope Turning the illumination on and off should be done gradually by using the rheostat slider switch appropriately. Before turning on the rocker switch, make sure the slider is all the way to 0; then turn it on and slide the rheostat slowly up until the appropriate brightness is attained. Similarly, in turning the illuminator off, turn the rheostat down to 0 before turning off the rocker switch. This gradual on-and-off greatly lengthens the life of the bulb. Do not run the illuminator at full throttle (10) except briefly when making observations at high magnification; this setting is higher than the voltage rating of the bulbs. Bright-field K¨ ohler illumination. Optimum resolution is obtained only with appropriate setting of the lenses and apertures, specifically a condition called K¨ohler illumination. Because these

10 microscopes do not have a field aperture, strict K¨ohler cannot be set, but we can approximate it by setting the microscope this way: 1. Focus roughly on a specimen (starting with a low-magnification objective—i.e., the 4×); if the two oculars are not set so that both eyes are focused on the specimen, adjust them by turning the knurled knobs on them. 2. Place a piece of paper on the substage illuminator so that a corner of it is centered and then use the knob on the condenser lens to focus on that corner. Remove the paper. 3. Adjust the condenser diaphragm: while looking at a specimen, open the diaphragm fully, then close it down slowly until the brightness of the image just starts to dim. 4. As you view specimens, adjust brightness with the rheostat on the illuminator, not with the condenser aperture. 5. Contrast can be improved on transparent specimens by placing a narrow strip of paper across the substage illuminator (to produce oblique illumination). Contrast will also go up with a narrowed condenser aperture, but this severely reduces resolution. The binocular head should be firmly clamped in its support ring. (Please do not loosen the set screw on this ring and turn the head to show lab partners views under the scope.) Use the dissecting microscope to find and mount specimens. Specimens on slides must be covered with a coverslip (never use the microscope to examine a specimen without a cover slip.) Specimens also usually need to be compressed with the cover slip but not squashed by it. To achieve the correct compression, place small wax or clay feet on the corners of the coverslip by picking each corner into a piece of wax or clay and getting just a very small fleck on one side at the corners. Then lower the coverslip onto the specimen by resting first one edge on the slide and then lowering the other gradually with a dissecting needle, thus minimizing trapping of air bubbles on the specimen. Compression can be adjusted by pressing gently on the wax/clay feet of the coverslip and by wicking out fluid from under the coverslip with bibulous paper or filter paper (or other absorbent paper).

Putting microscopes away Each microscope is numbered (number scratched into base at back) and should be returned to the correspondingly numbered slot in the cabinets. Remove all specimens from a microscope before placing it in the cabinets. Ensure that no sea water or other fluids are on the microscope. Wipe off sea water with a towel dampened in fresh water and then dry the spot. Replace the plastic dust cover on the microscope before placing it in the cabinet.

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Annelida ANNELIDA Polychaeta Hediste – live, preserved, slides Neanthes – live Alitta – preserved, slides Glycera – live Clymenella – live, perserved Spio – live Aphrodita – preserved demo Arenicola – preserved Nephtys – live, preserved Pectinaria – preserved Amphitrite – preserved others live as available: Phyllodoce, Sabella, Spirorbis, Amphitrite, Lepidonotus, Harmothoe Oligochaeta Enchytraeus – live Lumbricus – live, preserved, slides miscellaneous freshwater oligochaetes – live Hirudinoidea Hirudinea Macrobdella – live, preserved, slides Haemopis – preserved Glossiphonia? – slides (Ozobranchus – demo) Branchiobdellida Cambarincola – slide demo

Annelida: Polychaete Nereis and other nereids – family Nereidae (Figs. 13-10, 13-13, and 13-6). Note: the species the book names in the genus Nereis are actually now classified in the genera Neanthes, Alitta, and Hediste; the distinctions are rather subtle, and the Figures apply to all three genera. The live species available in lab are Hediste diversicolor, which lives in marine sand flats, and the quite similar, but smaller Neanthes succinea, which is common in brackish water–i.e., in estuaries. The preserved specimens are Alitta virens, a marine species more common south of Maine. Species of nereids are often taken as quintessential polychaetes since they have a long series of similarappearing segments, well-developed parapodia, and well-developed sensory structures on the head, including cirri, palps, and eyes. The cuticle may show richly opalescent and iridescent colors–green, coppery-brown, or red. These animals are commonly known as clamworms, sandworms, or ragworms. They prey on many kinds of invertebrates and also feed on algae (i.e., they are omnivores); and they are, in turn, favorite prey of fishes and crabs. Neanthes succinea is common in coastal areas where freshwater runoff from streams and rivers dilutes the sea water (i.e., brackish habitats). It makes mucus-lined muddy tubes in the sediment, under rocks, or in the fouling communities attached to pilings and floats. Hediste diversicolor is commonly found in mud or muddy sand, forming tubes in which the sediment of the walls is glued together with mucus. Both of these species remain in their tubes most of the time, occasionally protruding the anterior end of the body in search of food. The paddle-like parapodia make these worms good swimmers, and they use this skill particularly in breeding swarms appearing during the dark of the moon in June through September. To study Hediste or Neanthes and get a general idea of what the body parts of polychaetes look like and how they work, first, watch a live specimen in a dish of seawater under a dissecting microscope. You will see a fairly well-defined head followed by a long series of segments. Note that most of the segments have appendages, the parapodia, that are somewhat paddle-shaped. Next, study the behavior of the animal.

12 Watch how it crawls on the bottom of the dish or swims. Are the parapodia used differently in crawling and swimming? Try placing the animal on sand (small aquaria with sand are available) and watch it burrow. What parts of the body are used to start the burrow and how does the activity change as the worm enters the burrow? (Compare with Fig. 13-13.) To get a more detailed look at the morphology of the worm, you may need to anesthetize it (if it moves too much) by placing it in magnesium chloride that is isotonic to sea water. (This solution works as an anesthetic because the magnesium ions interfere with calcium binding and so prevent neuromuscular synapses from working; it may take 10-15 minutes to fully take effect). You may also work with one of the larger preserved specimens to study morphology; just be sure not to contaminate the live animal or its dish with fluids from the preserved animal. As is true for polychaetes in general, the body is composed of an anterior prostomium followed by a variable number of segments and, at the posterior tip, the pygidium. Only the segments between the prostomium and pygidium are considered true segments. Larger specimens of Hediste may have 200 or more segments. Growth and addition of segments takes place by production of new ones just anterior to the pygidium. With the exception of the first segment, the peristomium (so named because it bears the mouth), these segments possess lateral appendages, the parapodia. Nereids have an eversible pharynx which, in most preserved specimens, is protruding through the mouth because of contraction of body-wall muscles when the animal reacts to fixative; this same mechanism is used in life to evert the pharynx and grab prey. Such an eversible pharynx is often called a proboscis. If the pharynx is protruded in your preserved specimen, you will be able to see the large black jaws and other denticles at its end (if it is not everted, look at a neighbor’s specimen that does have it everted). Find the anus in the pygidium which also bears two long ventral cirri. In front of the peristomium and overhanging the proboscis is the prostomium (Figs. 13-10, 13-14). The prostomium bears a pair of stubby palps on its anterolateral edge, and between these, at the anterior margin, are a pair of small, slender tentacles. Behind the palps and lateral to them are four pairs of tentacular cirri (actually arising from the peristomium), and in the posterodorsal part of the prostomium are two pairs of eyes. Look closely at the parapodia, and see how they differ along the length of the worm. (Compare Figs. 13-6, 13-10.) Try to see, especially, how the ventral and dorsal projections of the parapodia are arranged. It is easiest to see such things on a body segment that has been cut from the body, and such cut-out segments are available on the prepared slides. Study one of these slides under a dissecting scope and verify that the live and the preserved worms also have the same structure. Note that a parapodium is biramous, having one dorsal and one ventral ramus, the notopodium and neuropodium, respectively. Each of these is supported by a large tapering modified chaeta called an aciculum which is embedded in the parapodium. At the distal end of each aciculum is a bundle of smaller projecting chaetae, which are fine bristles composed of a long blade joined to a sturdier shaft; each chaeta arises from a sac in the parapodium secreted by a single cell at the base of the sac. Use Figures 13-6, 13-30 and 13-21, and the Figure next to the demonstration slides to find parts of the parapodia. Find internal features in the section, including epidermis, body-wall muscles (circular, longitudinal, and oblique), coelom, blood vessels, nerve cord, nephridia, and gut (Fig. 13-10). The live worms are small and transparent enough that you may be able to see internal organs in them without having to dissect them. Find structures identified in Figure 13-10. Blood vessels should stand out by the red blood they contain. Find metanephridia, each visible as a whitish mass closely applied to the body wall on either side of the gut (cf. Figs. 13-10, 13-20). On the ventral side of the find the ventral nerve cord, its ganglia, and lateral nerves arising from it. Try to find the circumesophageal nerve ring and the dorsal ganglia on it which constitute the bilobed brain (lying dorsal in the prostomium); from the brain are numerous nerves extending to the sensory organs of the head (Figs. 13-14, 13-15). See if these specimens are in reproductive condition, with gonads developed as proliferations of the coelomic lining of many segments. See if the posterior segments are better differentiated for swimming (see Fig. 13-30)—i.e., whether the animal is differentiating as an epitoke. Glycera – family Glyceridae (Figs. 13-4A, 13-32, 13-39) Take care in handling these animals, avoiding giving them the soft skin between the fingers to nip. Holding them in your flattened palm should be okay; just be careful when you see the pharynx being protruded, which can be a surprisingly rapid process. They have four fangs on the end of this protrusible pharynx and can inject a toxin through those fangs. Normally, this toxin is used to quell prey, but given the opportunity, the animals could conceivably inject it through human skin, producing a sting some have compared to a bee sting.

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These animals are called bloodworms because the red coloemic fluid can easily be seen through the translucent body wall of the animal, sloshing back and forth as the animal moves. The fluid is red because cells in it (coelomocytes) contain hemoglobin, and it sloshes so freely because the septa between segments are reduced to facilitate protrusion and retraction of the rather large pharynx, which is used in prey capture and burrowing. Another red structure in these animals is the major nerve cord, visible as a red line running down the ventral side of the animal; again it is hemoglobin that makes the red color, this time in glial cells that surround the cord. Morphology If large specimens of Glycera are available, it is best to study their morphology after they have been anesthetized in magnesium chloride and placed at one edge of a dissecting tray so that they can be viewed under a dissecting microscope. Support the tray tilted a bit under the microscope so that a small puddle of magnesium chloride pools around the worm and submerges it. Compare the head of Glycera with that of Hediste. The head is sharply pointed in Glycera, largely because the prostomium has the shape of an elongate cone, and it has only four tiny antennae (two pairs of prostomial tentacles). These antennae are highly sensitive to vibration and are normally used to detect movements of potential prey for which Glycera lies in ambush at the entrance to its burrow. Other sensory organs visible are ciliated pits called nuchal organs, a pair of which opens just behind the point at which the body broadens (the peristomium), in a position you might expect to see eyes. Observe the parapodia, noting the shape of noto- and neuropodia, shape and number of chaetae, and the presence of two red-colored finger-like gills (branchiae) on each parapodium, one above and one below the bristly lobe of the parapodium. Look at the gills under higher magnification to see the red coelomocytes circulating in them. What drives this circulation? Look down the rest of the body of the worm to see how the parapodia differ along its length, and find the red nerve cord and the pygidium with its caudal filaments. Behavior Watch a live worm in a dish of seawater or an aquarium with sand to see how it swims and burrows. What differences in behavior can you see between Glycera and Hediste? Does it use different parts of the body to start the burrow? How does the activity change as the worm enters the burrow? What differences in the shape of the parapodia can account for the differing abilities of Hediste and Glycera to swim and crawl?

Dissection of a polychaete We will use whatever large and expendable polychaetes are available. An animal for dissection should be fully anesthetized in magnesium chloride before proceeding, and it should be opened in a tray that has magnesium chloride to a level that completely covers it. Orient an anesthetized worm dorsal side up and prepare it for dissection by pinning the prostomium to the tray with a pin through its right side (make sure that you can still view the worm with your microscope). Make a preliminary small dorsal incision about a third of the way back from the anterior end and collect a drop of coelomic fluid. Examine the drop on a slide to see coelomocytes and other cells here; if the fluid appears colored, determine whether the color is in the fluid itself or cells (coelomocytes) floating in it. Try to find amoebocytes, amoeboid and transparent cells with pseudopodia projecting from them; as the preparation sits some of these amoebocytes will disintegrate and form fibers that clump all the cells together in clots. Oocytes of various stages or sperm packets may also be present. Open the worm by cutting along the dorsal midline, keeping the inner point of your scissors pulled up against the dorsal body wall to avoid damaging internal organs. Cut anteriorly to just behind the prostomium, then cut laterally so that you can spread the body wall out; pin the body wall back with pins. Make sure the worm is still submerged in magnesium-chloride/sea-water and visible under your dissecting microscope. You may need to flush the opened body cavity with magnesium chloride in a pipet so that the coelomocytes don’t obscure your view. The septa between the segments will appear like flaps in the opened worm; some species (Glycera, Aberinicola) may have incomplete septa, forming just ladd erlike flaps under the gut, so that the coelom is continuous through the length of the body; this presumably accommodates movement of the gut as the large pharynx is extended and retracted. Find teeth or fangs on the pharynx. (The black fangs on the pharynx of Glycera and the poison sacs that connect to them, lying in the muscular bulb of the pharynx are good examples; Hediste will have jaws and denticles on the pharynx.) The pharynx typically attaches to the body wall with a fan of retractor muscles–silvery ribbons that reach to either side of the body. The gut itself looks yellowish green.

14 Protruding into the coelom and looking like small teepees on either side of the body are the muscles of the parapodia, especially the protractor muscles that attach to the inner ends of the acicula, the skeletal supports of the parapodia. On the dorsal side of the gut you may see the dorsal blood vessel, pumping blood toward the head; if the blood vascular system is reduced (as in Glycera), then the large coelom serves in circulation and the gills may open directly to the coelomic space. Find nephridia as pairs of whitish tuft-like protrusions on either side of the gut. In those without a blood-vascular system there will be protonephridia instead for excretion and osmoregulation. These protonephridia sit on white, green-capped lollipop-shaped structures projecting between the setal muscles. To see the individual nephridia, mount one on a microscope slide for viewing under the compound scope. Protonephridia will look like tiny, thick-stemmed lollipops themselves, studding the surface of the protonephridial organ. Look for ciliary activity inside the protonephridia as well as in the sac-like appendage into which the protonophridia project. Cut through the esophagus fairly far forward and turn the gut out to expose the ventral midline. Note the segmental ganglia and nerves and trace the cord forward to the circumesophageal connectives which converge dorsally in the prostomium. The minute brain is embedded in the prostomium. See if you can expose it. Nephthys (Fig. 13-40) superficially resembles Hediste. Its common names are shimmy worm (because it swims with a rapid shimmying motion) and red-lined worm (for the red ventral nerve cord visible through the body wall). The two jaws and fringe of sensory papillae on the pharynx are visible when it is everted. Lepidonotus and Harmothoe – scale worms; family Polynoidae (Fig. 13-41C) Scale worms have dorsal cirri modified to cover the dorsal surface of the body as scale-like projections (called elytrae). Not only do these scales serve a protective function, they also create a respiratory space between the dorsal body wall and the underside of the scales: cilia on the epidermis pump water through this space to ventilate it. Scale worms live in a wide range of habitats, especially in crevices and among other fouling and sedentary animals; some live as commensals with other invertebrates. Species of Lepidonotus have 12–13 pairs of overlapping scales; Harmothoe species have 15 pairs. Aphrodita (Fig. 13-41 A-B), the sea mouse, is a close relative of these scale worms; its scales are covered with a mat of hair-like chaetae that give the worm a mouse-like appeaence. Preserved specimens are on demonstration. Clymenella – family Maldanidae (Fig. 13-36 B-D) Species in the family Maldanidae have segments that are longer than they are wide, giving the animal a jointed appearance like bamboo, hence the common name “bamboo worms.” There are relatively few segments and the parapodia are quite reduced. The worms live head-down in tubes constructed in muddy sand by gluing sediment grains together with mucus, and they feed by consuming sediment in a head gallery at the bottom of the tube (something like Arenicola). The pygidium is funnel- or crown-shaped and fits the tube like a reamer, keeping it clear of sediment and fecal castings. The parapodia are reduced, looking like low ridges, with the neuropodium bearing short hook-like chaetae called uncini. The bamboo worm Clymenella torquata has commensals living with it, i.e., animals that live in its tube in a more or less obligate relationship, feeding on material brought into the tube by the worm’s movements; they apparently cause no harm to their host (are not parasitic) but whether the worm benefits from their presence is not known. The commensals of C. torquata are a small clam, which lives attached to the head end of the tube, and an amphipod, which lives inside the upper end of the tube. Gently remove a bamboo worm from its tube and determine which is the anterior and which the posterior end. (Watch for commensals as you open the tube.) Observe movements of the worm, watching in particular to see eversion of the pharynx, a rather bulbous structure that essentially mops up sediment. Try placing the worm back in sediment in a dish and see if it can reburrow. Is its burrowing activity like that of the other polychaetes? Arenicola – the lug worm (Figs. 13-34, 13-35, 13-20) is also on demonstration. In the preserved specimens, the pharynx is protruded and appears like a warty ball at the anterior end of the animal (Fig. 13-21). The body has distinct regions (trunk and abdomen), and along the dorsal side of the trunk are paired tufts of gills that are red in life. Note how the parapodia are reduced in this sedentary, burrow-dwelling polychaete; each neuropodium appears zipper-like, consisting of a row of short hook-like chaetae called uncini. Amphitrite – the spaghetti worm (Figs. 13-20, 13-24B). We will eventually see a spaghetti worm alive, on

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the field trip or with other collections, if it is not available in today’s lab. Like other terebellids, it lives in a mud tube, usually under rocks, and extends tentacles over the sediment to pick up individual particle for food (selective deposit feeding). Behind the tentacles is a cluster of gills, usually bright red from the blood flowing through them. Preserved specimens are on demonstration. Pectinaria – The ice-cream cone worm (Figs. 13-49C-D) feeds in a similar fashion to Amphitrite, but headdown in the sediment. From larger particles it collects, it builds a conical tube. The large opening of the tube can be closed off by opposing sets of bristles visible in the specimen that has been removed from its tube. Sabella, Hydroides, Spirorbis (Fig. 13-51) – The fanworms are sedentary polychaetes, spending their entire juvenile and adult lives in tubes fixed to one spot. The prostomium is transformed into a crown of simple or branched, pinnate tentacles called radioles. These are heavily ciliated and produce a feeding current and capture particles from it—hence, these worms are active suspension feeders. The peristomium is collarlike, folded over the rim of the tube which it molds from material it secretes. Sabellids build a mucus tube with the peristomium, and in most sabellids it is tough and leathery and has sand grains, which the worm collects as by-products of the particle-gathering activities of its crown of tentacles, incorporated into it. Serpulids and spirorbids have a calcareous tube which is secreted by the peristomium, and one of the radioles in the crown is modified into an operculum, a stopper-like plug that seals off the tube when the worm is retracted. The body of fanworms is divided into a thoracic and abdominal region and there is a conspicuous fecal groove running along its length by which fecal material is dumped out the tube opening.

Polychaete Diversity The relationships among the many species of polychaete annelids are difficult to decipher, but these animals do fall into fairly easily recognizable family groupings. For reference, the following text describes features of key families that we might come across in looking at marine samples. Macrophagous polychaetes (omnivores and carnivores) Family Nereidae — These polychaetes have a well-developed prostomium in comparison with other families. The eversible pharynx of Nereis bears a bear of jaws and small denticles used in burrowing and feeding. Most nereids eat algae, and the local species Nereis vexillosa often “farms” the green alga Ulva. They can be carnivorous, however. Nereids have an errant lifestyle—that is, they move around all the time, never having a permanent home tube or burrow. (Figs. 13-6, -10, -30A.) Family Glyceridae — Glycerids are active worms that live beneath the surface sediment. The long proboscis is effective in prey capture and in burrowing. By rapid eversion of this structure the worm hammers the sand, causing it to liquefy, and expansion of the tip of the extended proboscis and subsequent retraction pulls the worm rapidly through the sand. Glycerids have four small, sharp jaws on the proboscis. At the base of each jaw is a poison gland. Glycerids are called blood worms because their coelomic fluid contains hemoglobin in blood cells (unlike most other polychaetes, where the hemoglobin is dissolved in the blood plasma); a blood vascular system is lacking. Note the four small antennae at the tip of the prostomium. (Fig. 13-39, -32.) Family Nephtyidae — These resemble nereids but behave more like glycerids. Their large but short proboscis lacks jaws but may have papillae or denticles (little teeth). Nephtyids swim actively with their strong longitudinal muscles, and they can swim into the sand substrate (with a motion that earns them their common name “shimmy worms”). They are reputed to be carnivorous, but very little is actually known of their diet. Place them on the surface of some mud to watch them burrow. (Fig. 13-40.) Family Polynoidae — These are also known as scaleworms, because they have paired dorsal scales. The scale worms are commonly encountered with collections that have rubble or material with crevices in which the worms may hide. Many species live associated with other invertebrates. (For example, species of Arctonoe live as ectosymbionts of keyhole limpets, sea stars, and a few

16 other marine invertebartes.) The body is flattened and covered dorsally by a series of paired, imbricated scales (“elytra”). Alternating segments bear scales and dorsal cirri. Respiratory flow of seawater under the scale is driven by the ciliated epidermis. Both prostomium and peristomium usually bear specialized appendages. The eversible pharynx bears two dorsal and two ventral jaws. (Figs. 13-41C.) Family Aphroditidae — aphroditids, or “sea mice”, are actually closely related to scaleworms and have dorsal scales. However, those scales are hidden under a huge expanse of tangled hairs that are actually long, flexible notosetae. These animals move wholly by parapodial stepping and can hold the body off the substrate; body wall musculature stabilizes the body wall. These too are carnivores, feeding on other polychaetes. (Fig. 13-41A,B.) Family Phyllodocidae — Rather unmodified segmentation and leaf-like dorsal parts of the parapodia characterize this family, Most phyllodocids are long, actively crawling worms, and most prey on other polychaetes. The proboscis is similar to that of Nereis but lacks jaws. (Figs. 13-11B, -37C.) Family Tomopteridae — These worms are modified for planktonic existence. The parapodia are enlarged with spacious coelomic extensions—gonads may even be included—but they lack acicula and setae. The head is specialized with highly developed tentacles and palps. (Fig. 13-38.) Family Syllidae — Syllids are active worms often found among sedentary animals (such as sponges) and algae. While most are quite small, they are often extremely abundant. Syllids can often be recognized by the characteristic beaded appearance of the tentacles and or the parapodial cirri. The family is also known for elaborate reproductive modifications including asexual budding, transverse fission, and specializations for swimming associated with sexual maturity and breeding. Ripe females may swim and carry conspicuous, colorful masses of developing eggs. (Fig. 13-31.) Family Onuphidae — Tubiculous worms with five long tentacular processes and two smaller frontal antennae. They possess a complex set a paired replaceable jaws. (Figs. 13-9, -43A,B, p. 413.) Family Lumbrineridae — Superficially resemble earthworms. Lumbrinerids have a prostomium with no appendages whatsoever. They possess a complex set of paired, replaceable jaws. Suspension feeders Family Cirratulidae — These worms have feeding tentacles arranged along the body, not just on the head. Long respiratory cirri or gills also emerge dorso-laterally from the segments. One noteworthy example of this family is Dodecaceria which is a clonal worm that builds calcareous tubes. Each individual breaks up and regenerates multiple individuals. Because asexual reproduction is faster than tube-building, lots of tubes end up with more than one worm living in them. This worm has numerous gills coming off the head, as well as a single pair of grooved, ciliated palps (which look quite similar to the gills. The animal sticks out the cluster of gills and palps into flow. Particles adhere to the sticky gills, and the palps grab them and move them to the mouth via their ciliated grooves. Remove a couple of worms from the tubes and examine their parapodia, setae, Family Serpulidae — Members of this family are sedentary polychaetes, spending their entire juvenile and adult lives in a tube fixed to one spot on the sea floor. The prostomium is transformed into a crown of simple or branched, pinnate tentacles called radioles. These are heavily ciliated and produce a feeding current and capture particles from it—hence, these are active suspension feeders. Another appendage of the head is an operculum, a trapdoor that seals off the tube when the worms are retracted. The calcium carbonate tube is secreted by the peristomium. They are very shy, and pop back into their tubes at the slightest provocation. This response is mediated by a giant nerve axons. (Fig. 13-51.) Family Sabellidae — Members of this family are sedentary, spending their entire juvenile and adult lives in a tube fixed to one spot on the sea floor. They have no proboscis or other complexity of the pharynx, and the prostomium is transformed into a crown of simple or branched, pinnate tentacles called radioles. The mucus tube is secreted by the peristomium.

Annelida – Polychaeta In most sabellids such as Sabella it is tough and leathery, but in Myxicola it is slimy and watery. The body is conspicuously divided into a thoracic and abdominal region. Along the dorsal side of both regions is a conspicuous fecal groove that transports feces to the tube opening. Sabellid eyes are unusual among annelids in being compound, that is each is made up of about 10-60 units, the ommatidia, each with its own lens. Compound eyes do not make single clear images, but they can readily detect very slight motion. (Figs. 13-51, -52.) Family Sabellariidae — Sabellaria builds firm tubes of sand cemented with mucus. The body of the worm has a complicated serial arrangemnt. Two anterior segments form an operculum capped with a crown of setae, the whole serving to stopper the tube. The mass of slender feeding tentacles arises from the ventral side of the base of the stalk of the opercular segments. Each tentacle has a ciliated groove which moves particles to the mouth. (Fig. 13-50.)

Mucus-Bag Feeding Polychaetes Family Chaetopteridae — Chaetopterids are some of the most highly modified of all polychaetes. They show profound differentiation of segments in different body regions, and in some the parapodia are variously strongly modified to make a mucus-bag filter, form fans to pump water through a U-shaped tube, and make a ciliated cup that collects mucus and food from the bag into little balls that are transported forward to the mouth. Other members of the family make long vertical tubes that protrude from the sand, and they collect most of their food with two long palps rather than pumping water through the tube. (Fig. 13-47.)

Deposit feeders Family Arenicolidae — These worms are subsurface deposit feeders. They have parapodial gills on just certain segments and reduced parapodia and setae. (Figs. 13-35, -34, -16, -21.) Family Terebellidae — These are surface deposit feeders. Terebellids (a.k.a. spaghetti worms) hide their bodies in cracks or burrows and extend their long tentacles over the substratum (how?). They use these to pull nutritious particles of muck to their mouths. Note the gills behind the cluster of feeding tentacles. Can you see blood flowing in and out of them? Particles that they catch but don’t want to eat often end up as part of their mucus/mud tubes. Note that some terebellids are quite large; they occur in areas where much organic detritus falls to the bottom. Accordingly, the tentacles are fanned out to cover as much feeding surface as possible. Large terebellids may have feeding areas as large as 1 m2 . (Figs. 13-48, -24, -20.) Family Sternaspidae — Sternaspis, the only representative of this family, is a short, robust worm of only 13 segments. It lives head down in mud, swallowing the sediment from which it digests the organic matter. Its filamentous gills extend up into the water column, and a pair of heavy brown cuticular plates protect its rear end. This worm lacks most septation, and the arrangement of internal organs is unique. Family Maldanidae — Commonly called bamboo worms. The long, cylindrical maldanid body consists of relatively few segments. Clymenella builds a heavy permanent tube of sand bound with mucus. (Figs. 13-36B,C.) Family Owenidae — The tube of Owenia is thinner and more flexible than that of maldanids. The tubes are short and the worms are often capable of moving around with the tube. (Fig. 13-36A.) Family Spionidae — These worms are often very abundant in colonies locally in soft sediment in the intertidal. They build tubes and from these protrude the characteristic two tentacles, each with a ciliated feeding groove. The tentacles lash about in the water or over the surface of the substrate. (Fig. 13-45.) Family Chrysopetalidae — Bodies usually strongly flattened; notosetae are arranged in transverse rows, held erect over the dorsum or covering the back as tiles on a roof.

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18 Annelida: Oligochaeta I. Earthworm Lumbricus terrestris, the common earthworm or crawler, is a cosmopolitan species. Some believe it was introduced to North America from Europe, but recent fossil evidence shows at least some of its relatives in the family Lumbricidae were here 10,000 years ago. Like other lumbricids, L. terrestris lives in soils that don’t dry out and that contain organic matter on which it feeds. Earthworms burrow partly by pushing the earth aside and partly by swallowing it; and the burrows, which are lined with a cement of defecated material, may extend deeper than 6 feet. Locomotion Use a live earthworm to study locomotion, testing how it crawls on a wet paper towel and on a wet glass surface. Determine, for example, how peristaltic waves pass the length of the body (do they pass anteriorly or posteriorly?), how independently the coelomic compartments of each segment function, and how the chaetae are used to gain traction. (Compare with Fig. 13-60, 13-68.) External anatomy Study external anatomy of a worm anesthetized in ethanol (or of a preserved worm) and find the landmarks depicted in Figure 13-59. The clitellum is the obvious non-annulated region, and the anterior end of the worm is that with the fewer number of segments on one side of this band. Note that the mouth is subterminal; in front of it is the prostomium; the first segment, the peristomium, surounds the mouth. At the other end of the body, find the anus. Run your finger over the body to feel the chaetae. In which direction (anteriorly or posteriorly) are they directed? How many setae are there per segment and what is their position relative to the ventral surface of the worm? The openings through which the setae project can be readily observed by peeling off a section of the cuticle covering the worm and mounting it in water on a slide. Remove this cuticle from some place in the caudal half of the worm. Species of earthworms and other oligochaetes are distinguished according to which segments are occupied by which internal organs, particularly reproductive organs. It is necessary, therefore, to keep track of segment numbering, which starts with the peristomium as number 1 and successively designates segments behind in order. Which segments does the clitellum occupy? In Lumbricus terrestris, the vasa deferentia appear on somewhat swollen bumps on the ventral side of the worm on segment 15. The smaller openings of the oviducts are just in front of them on segment 14, and the minute openings of the two pairs of spermathecae lie in the grooves between segments 9 and 10 and 10 and 11 (use the dissecting microscope to find these). The relatively prominent grooves leading back from the vasa deferentia to the clitellum are seminal grooves through which sperm pass to the spermathecae of a partner during copulation. Try to find nephrostomes, openings of the metanephridia, just dorsal to and in front of the chaetae on most segments (excepting the first three segments and the last). Along the mid-dorsal line in the creases between the segments are the minute unpaired dorsal coelomic pores, which permit discharge of coelomic fluid to moisten or lubricate the skin. Dissection (Fig. 13-61, 13-62, 13-67) Place the anesthetized earthworm ventral-side-down on a dissecting tray and attach it to the tray with a pin through the prostomium. Then open its dorsal body wall by snipping across its back and inserting one point of the scissors into the snipped hole to cut forward along the dorsal midline as far as the peristomium; keep the scissors horizontal to avoid cutting deep into internal tissues. Using forceps and teasing with a dissecting needle, free the body wall from the septa and open the cut, pinning the worm to the bottom of the tray by putting pins on each side in the cut edges. If you place the pins at segments 10, 20, 30, etc., it will be easier to keep track of the segment numbers. Slant all pins outward to provide room for dissection. Place the dissecting tray under a dissecting microscope so that you can see the worm and then add tap water to the tray to float the internal organs. Internal anatomy Anterior digestive tract, brain, circulatory system Observe the septa separating the segments internally. Occupying most of the first five segments is the pharynx, with many fine muscle fibers radiating to the body wall. Above the front end of the pharynx is a bilobed brain or dorsal ganglion, with a pair of circum-esophageal connectives passing around either side of the pharynx and uniting to form the ventral nerve cord (which will be exposed later). (See Fig. 13-61.)

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Behind the pharynx the digestive tract narrows into a smaller esophagus, extending through segments 6 to 14. It is largely obscured by the large whitish seminal vesicles and by the five pairs of hearts. With care, two or three pairs of small outpouchings, the calciferous glands, can be seen along the sides of the esophagus. They are believed to play a role in controlling the level of calcium ions in the blood. The esophagus leads into an enlarged, thin-walled crop, where the food is stored and then into a thickwalled gizzard for grinding the food into small particles. From the gizzard extends a uniform intestine, in which most of the digestion occurs. In the cut end of the intestine a fold of tissue, the typhlosole, can be seen projecting into the cavity of the intestine from the top. What is its function? The yellowish tissue surrounding the intestine is the chlorogogue tissue, a tissue functioning a manner analogous to the vertebrate liver. Along the dorsal surface of the intestine lies the dorsal blood vessel. This is continuous anteriorly with the five pairs of pseudohearts in segemnt 7 through 11. You will probably have to cut away portions of the septa to see these clearly. Trace these around the esophagus and see that they join a ventral blood vessel running just below the intestine. Carefully remove one inch of the intestine at the caudal end of your dissection to expose this blood vessel and other ventral structures. Can you detect any lateral branches from the ventral blood vessel or dorsal blood vessel? The latter are the parietal vessels running among the chlorogogue cells. What is the path of circulation in the earthworm? Nerve cord. You have already observed the brain and circum-esophageal connectives. Now in the caudal part of your dissection where the intestine has been removed, observe with a dissecting microscope or hand lens the ventral nerve cord with its segmental ganglia and lateral nerves. What is the arrangement of these structures in each segment? The nervous system in the annelids was originally double in structure, but because of centralization it now appears to be a single cord. If the nerve cord and its branches are not very distinct, remove a portion of the cord from the worm and mount it separately on a slide. Metanephridia. The pair of white fluffy, transverse structures toward the ventral midline of each segment are the metanephridia. Each consists of a ciliated funnel or nephrostome near the midline of the anterior surface of a septum. The tubule from the nephrostome passes through the septum into the following segment and makes three loops, opening to the outside by means of a nephridiopore near the ventral pair of chaetae. Pluck one out of the body, mount it on a slide under a coverslip, and study it under a compound scope. A demonstration of a stained whole-mounted nephridium is on display. (See also Fig. 13-65.) Reproductive organs (Fig. 13-61, 13-67) The earthworms (and oligochaetes in general) are hermaphroditic. In segments 10 and 11 are two small, hand-shaped testes attached to the posterior side of the septa. These are not readily visible because they are masked by the three pairs of seminal vesicles, which are diverticula of segments 10 and 11. Often by carefully removing the top portion of the seminal vesicles and washing out the contents, the testes can be seen. They do not give rise to sperm directly, but rather to cells that form sperm balls and eventually sperm during their period of storage in the seminal vesicles. Make a water smear of the contents of a seminal vesicle and examine under high power. Note sperm balls and the boat-shaped cells of the sporozoan. Monocystis, which is parasitic in sperm-forming cells. The sperm get to the outside of the body by a system of ducts which are not easily found. Associated with each testis is a sperm funnel leading into a vas efferens. The two vasa efferentia on each side unite to form a vas deferens, which extends backward as a thin tube to its opening on the ventral side of segment 15. The ovaries are located in segment 13, attached to the posterior face of the septum in the same manner that the testes are. Two ovarian funnels, also located in segment 13, pass through the septum and open to the outside in segment 14. The two pairs of seminal receptacles located in segments 9 and 10 contain the spermatozoa from another worm transferred during sexual union. How do these sperm reach an egg to fertilize it? What role does the clitellum play and how are fertilized eggs laid? Draw a dorsal view of your dissection of the earthworm, showing the various parts of the digestive, circulatory, nervous, and reproductive systems observed. Label fully. Number the segments on the left side of the drawing. Histological sections Study the longitudinal sections of Lumbricus with 10× and 40× objectives in the compound microscope to see histological structure of the brain and the anterior end of the digestive tract (pharynx, crop, gizzard).

20 Study the cross-sections of the earthworm to see the body wall with cuticle, epidermis, layer of circular muscle fibers, longitudinal muscle fibers (see Fig. 13-61); below the body wall is a flattened layer of peritoneum surrounding the coelom. In the wall of the intestine find the large chlorogogue cells, making up the splanchnic peritoneum; a submucosa, consisting of longitudinal muscle fibers (appearing as dark dots at the base of the chlorogogue layer) and circular muscle fibers; and a muscosa, consisting of a columnar intestinal epithelium with a thick investing layer of cilia. The dark line near the surface of the epithelium is aligned basal bodies of cilia. Find the dorsal blood vessel and the ventral blood vessel, the latter attached to the intestine by a mesentery. The typhlosole can be readily distinguished. The space between the body wall and intestinal wall is the coelom. In the section of the nerve cord, make out the nerve cells, nerve fibers, and the three giant fibers near the top of the cord. The entire nerve cord is enclosed in a connective tissue sheath, in which are located the subneural blood vessel and two lateral neural blood vessels. Between the body wall and the intestinal wall may be present portions of nephridia or septa, but the structure of these cannot be made out from just one slide. Some slides show portions of the setigerous sacs or of the chaetae themselves (Fig. 13-59 B). Diagram the entire cross section of the earthworm, showing the extent and relative thickness of the various layers and structures. Make this drawing at least four inches in diameter. Draw in detail at a greater magnification (1) the ventral nerve cord and its surrounding sheath, and a small section of (2) the body wall, and (3) the intestinal wall. II. Other oligochaetes (Enchytraeus, Dero, etc.) Examine some of the other live oligochaetes available and compare their behavior and anatomy to that of Lumbricus. The small worms, such as Enchytraeus, and various aquatic species such asDero and Chaetogaster , should be mounted on a slide to study them; most are so transparent that it is possible to see internal anatomy on the whole worm, without necessity of dissection. (Mount the worm in a drop of water on the slide and cover it with a coverslip that has wax feet at each of its corners to support it above the worm). If the worm is very active, it can be quelled by applying pressure with the coverslip or by introducing spring water containing the anaesthetic phenoxy propanol; blot away water from under the coverslip a little at a time with a piece of filter paper as you watch the worm and until it appears trapped. The freshwater oligochaete Dero has gills surrounding the anal region (see Figure 13-64). The gills in the species we have look almost like a hand at the posterior tip of the body; the fingerlike gills originate from the margin of a shallow cup-like posterior end. Like other gill-bearing species of oligochaetes, Dero sits with its posterior end protruding from the sediment, its anterior end buried as it feeds. Look for differences and similarities in segmentation, septa, presence of chaetae, position of the pharynx and intestine, etc. Watch for peristaltic motion in the intestine and movement of coelomocytes in the coelomic compartments as the intestine moves. Is the coelom divided completely by the septa? Find metanephridia and blood vessels. How does the blood move in the vessels? (Compare Fig. 13-61.) Try to find parts of the reproductive system, using your knowledge of that of Lumbricus to distinguish parts of both male and female systems. Ovaries lie in segment 12 and spermathecae (to receive sperm from a partner) in segment 5. There are no ovarian funnels or oviducts; the eggs are released to the outside through simple rupture of the body wall. Testes lie in segments 10 and 11 and sperm funnels may be conspicuous; aquatic forms have only a single pair of testes (one segment). The nerve cord can be seen usually only when the worm turns onto its side or back.

Annelida: Hirudinea Live leeches, collected from local rivers and ponds, are available for study of locomotion. Watch the animals for their manner of locomotion, including the characteristic leech-like looping movement (Fig. 13-74), involving anterior and posterior suckers, and swimming movements, which involve dorso-ventral flexure of the body. The coelom is reduced in leeches (cf. Fig. 13-72), invaded by mesenchymal tissue called botryoidal tissue, and constituting a system of sinuses called the hemocoelomic system because it is a circluatory system derived from coelom, not blood vessels (Fig. 13-73). Leeches, therefore, cannot rely on segmentally arranged hydraulic compartments, as do oligochaetes, but use the entire internal body tissue as a single compartment. External anatomy (Fig. 13-70) may also be studied in the preserved specimens that are available. Note the absence of chaetae and the annulation of each segment in both live and preserved animals. In preserved

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animals, the single opening of the vas deferens midventral in segment 10 may have the penis protruding. The vagina opens in the segment behind this. The clitellum is on segments 9–11. For studying internal anatomy, more transparent living specimens may be used (mounted under a coverslip with wax feet) or preparations of cleared whole mounts on slides that you can take to your stations and on demonstrations of stained, whole-mounted specimens. Consult Figures 13-77 and 13-78, and use them to find parts of the digestive system, nervous system, and reproductive organs.

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Arthropoda IV: Trilobita / Onychophora, Tardigrada Onychophora, Tardigrada, Trilobita ARTHROPODA Trilobita (trilobites) fossil trilobite on demonstration Panarthropoda ONYCHOPHORA Peripatus – preserved, slides TARDIGRADA Hypsibius – live Milnesium – live, slides

In comparison with the modern-day representatives of the Arthropoda we have covered, the trilobites have yet another arrangement of head appendages and limb structure, but they clearly belong in the phylum Arthropoda. They were strictly marine and have long gone extinct. Onychophorans and tardigrades have a more flexible cuticle than the other arthropodan groups we have covered, so the whole body as well as the appendages can bend. Both Onychophorans and tardigrades are ancient groups with origins in the marine environment but with most living representatives now specialized for terrestrial life. (There are marine tardigrades, specialized for living between sand grains, for example.)

Arthropoda: Trilobite A fossil trilobite is on demonstration. Examine it for evidence, in particular, of its segmentation, compound eyes, antennae, and appendages (Fig. 17-1, p. 544).

Onychophora Peripatus sp. (Figs. 15-1, -2, -3, pp. 505-507). Onychophorans live in humid tropical and subtropical habitats and are commonly referred to as velvet worms. They crawl through spaces beneath logs, stones, or leaf litter where they feed on small invertebrates which they capture by shooting an entangling adhesive secretion from modified appendages that flank the mouth. While onychophorans have many characters in common with the arthropods, they also have many characters that are quite annelid-like. The cuticle, while being chitinous, like that of arthropods, is relatively soft, allowing the flexibility the animals use to crawl through tight places. (Its lack of a waxy epicuticular layer, as is found in other arthropods, limits these animals to humid habitats.) The cuticle is ridged and covered with rows of sensory spines on tubercles. Study the ventral side of the animal and find the anterior-most, annulated antennae, behind which are the paired oral papillae that are used to secrete the prey-entangling adhesive. The mouth, lying in a buccal depression between the papillae, is surronded by lip-like peribuccal lobes, and is flanked by claw-like mandibles. Down the length of the trunk are non-jointed, lobe-form paired appendages bearing terminal claws. (How do these appendages differ from parapodia of annelids and legs of arthropods?) Opening at the base of each appendage is a nephridiopore. At the very posterior tip of the body is the anus, and, subterminal just in front of it, the gonopore. Sexes are separate with paired gonads, and males produce spermatophores. Internal anatomy, can be seen from histological sections on slides. In these cross sections, look first at the body wall, with its relatively thin cuticle and well-developed muscle layer of circular and longitudinal muscles, more like the body wall of an annelid than of arthropods. Look for the papillae and sensory complexes in the epidermis (Fig. 15-2). In the section through the middle of the body (the middle of the three sections on the slides), find the spacious intestine in the center, the dorsal blood vessel (heart, bearing ostia not necessarily visible in all sections), and pair of nerve cords which sit ventro-laterally (these have a

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central neuropile composed of nerve axons and a rind of darker staining nuclei of the cell bodies). Dorsal and slightly lateral to the intestine are several profiles of branches of the uterus (eggs may be visible in some profiles) and, more laterally, branches of the slime gland (salivary gland). Also ventro-lateral, lateral to the nerve cords, are profiles through the nephridia. See also if you can find any trachea. The spacious cavity in which these internal organs sit is the hemocoel.

Tardigrada Live tardigrades (cf. Figs. 15-4, -5, -6, -7, -8, pp. 510-515) from moss have been brought from their cryptobiotic state by wetting locally collected mosses with fresh spring water. Preserved tardigrades on whole-mount slides are also available. Examine the living animals with a dissecting microscope as they crawl over bits of leaves and debris from the moss and mount one on a slide with a wax-foot-supported coverslip to study its anatomy with the compound microscope. These animals are notoriously cute, looking something like animated teddy bears. In the crawling locomotion, notice how the first three pairs of stubby legs are used to move forward and the last pair is used for retreat or to simply hang on to surfaces. Under the compound microscope, the most prominent internal organs will probably be parts of the digestive tract, including the muscular pharynx and the stylet that is used to puncture moss cells, the midgut, Malpighian tubules, and hindgut. Eggs in the ovary will be obvious in females. What is the fluid-filled space in which these organs slosh? The nervous system will be evident as a circumesophagal ring plus a ganglionated chain extending posteriorly on the ventral side of the body. Is the cuticle ornamented? Can you see evidence of molting? Are the claws separate segments of the appendage (as in euarthropods) or cuticular elaborations? Our textbook lumps the tardigrades with the pseudocoelomate phyla, but probably the similarities between tardigrades and nematodes are convergent, due to their occupying similar habitats. What nematode-like and arthropod-like characters can you discern?

24 Arthropoda I: Crustacea I (anostracans, phyllopods, decapods) ARTHROPODA Crustacea Anostraca (fairy shrimps) Artemia – live Streptocephalus – slides Phyllopoda Cladocera (water fleas, clam shrimps) Daphnia – live, slides Malacostraca Phyllocarida (leptostracans) Hoplocarida (mantis shrimps) Squilla – preserved on demonstration Eumalacostraca Syncarida (anaspidaceans and bathynellaceans) Eucarida Euphausiacea (krill) Euphausia – preserved on demonstration Decapoda (shrimp, crabs, crayfish, lobster) Crangon (sand shrimp) – live Pandalus and Lebbeus (shrimp) – demonstration Homarus (lobster) – live, demonstration Cambarus (crayfish) – preserved for dissection Panulirus (spiny lobster) – demonstration Calinectes (blue crab) – demonstration Carcinus (green crab) – live, preserved Pagurus (hermit crab) – live, demonstration Libinia (spider crab) – live, demonstration Paralithodes (king crab) – demonstration Cancer (rock crab) – preserved and live as available Peracarida Cumacea – demo Mysidacea (mysids or opposum shrimps) Praunus – live, preserved Heteromysis – preserved Isopoda (isopods, pill bugs, gribble) Idotea – live (marine) Caecidotea (=Asellus) – live (freshwater) Amphipoda (amphipods, beach hoppers, scuds) Gammarus – live (freshwater and marine), slides Cirrepedia (barnacles) Semibalanus – live, dry Balanus – dry Lepas – preserved Copepoda (copepods) Diaptomus – live (marine), slides Cyclops – live (freshwater), slides Ostracoda (ostracods or seed shrimps) ostracods – live, slides

larval stages of crustaceans in plankton and on demonstration (nauplius, zoea, megalops, lobster larvae)

Arthropoda: Crustacea

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Arthropods and their close relatives the onychophorans and tardigrades are segmented animals, and so structures such as appendages and certain internal organs are repeated in a serial arrangement along the body. Their body wall is strengthened by a cuticle whose main fibrous component is chitin, and it is tanned (cross-linked) to provide protection to the underlying epidermis and internal organs. In onychophorans and tardigrades, this chitinous cuticle remains flexible, so the whole body can bend; the appendages are also soft and flexible—relatively simple stub-like outgrowths of the body wall. In the more familiar arthropods, the tanning is to the degree that it becomes stiff and rigid, and so it can provide mechanical support to the body. Not only does the stiffer cuticle provide protection, it also makes the appendages stronger, so that they can be used to better mechanical advantage in swimming and walking; they can be strong enough, in fact, to support the animal’s body out of water, as you know from familiar examples among insects and spiders. Arthropoda, Onychophora, and Tardigrada share a common ancestor, and so these three phyla can be grouped into a superphylum called the Panarthropoda; some systematists even consider these groups all as a single phylum, Arthropoda, with the traditional arthropods separated in a group Euarthropoda (“true” arthropods). Arthropoda: Crustacea Crustaceans are the quintessential aquatic arthropods and a very large and successful group. The anastracans (fairy and brine shrimps) appear to have a form much like what would be expected of an ancestral crustacean; the decapods include the more familiar crustaceans and so we use them as models of the body form of crustaceans against which we can compare other crustacean groups. Anostraca and Phyllopoda have leaf-like thoracic appendages (phyllopods) that are used in a rowing motion to propel the animals as well as to capture unicellular algae and other particulates from the water for food (Fig. 1). The name phyllopoda (“leaf foot”) derives from the broad shape of the thoracopods, including the presence of a relatively large exite on these appendages. The prime examples we have available for study are the brine shrimp Artemia (Anostraca) and the water flea Daphnia (Phyllopoda), and we’ll use them as representatives of more primitive non-malacostracan crustacean groups that have a long series of homonomous (similar-appearing) appendages.

Figure 1: Phyllopod of Artemia (its sixth left trunk appendage, anterior-side view). From R. Fox Invertebrate Zoology Online Brine shrimp (Artemia) and Fairy shrimp (Streptocephalus); cf. Fig. 19-11, p. 618. Anostracans are found in inland relictual waters such as vernal pools and saline and alkaline lakes where they are free from fish and other predators; the brine shrimp, in particular, lives in saline water, notably the Great Salt Lake. Artemia is a good subject for in-depth study and as a model of how the arthropod body plan is structured. It is almost transparent, showing the workings of the gut and circulatory system and other internal organs, and, because it is so easily cultured, its developmental stages can be studied to see how crustaceans grow from the hatching nauplius through the adult.

26 Adult male and female specimens of the fairy shrimp Streptocephalus are available for study on slide preparations. Start with the nauplius and metanaupliar stages of Artemia available in separate cultures. Study both actively swimming specimens and some that have been relaxed fully with magnesium chloride.

Figure 2: Nauplius in dorsal and ventral views. From R. Fox Invertebrate Zoology Online The body of the freshly hatched nauplius (Fig. 2) consists of just three of the head segments (identified by their appendages, the two pairs of antennae and the mandibles) and a short unsegmented trunk (really just the telson). In front of the mandibles is an upper-lip-like overhang, the labrum. The single ocellus, called a naupliar eye, is prominent, and you may be able to make out the brain around the eye. Abundant yolk in the nauplius gives it an orange color and may obscure much of the internal anatomy. Look at the three pairs of appendages in a subdued larva and try to discern how their structure is appropriate for their functions. How does the larva sense where it is going? How does it swim? How does it feed? Try adding a bit of algae or yeast suspension to a dish with free-swimming larvae and then check them to see if you can see how the larva catches and ingests the cells as food. In older stages of Artemia (Fig. 3) or Streptocephalus distinguish the body regions in the specimens, namely, the head, the thorax (limb-bearing region), and the abdomen (region devoid of limbs). No carapace is present. On the head find a sessile, median eye and a pair of stalked lateral compound eyes; first antennae, which are small and filiform; second antennae, which arise from a fleshy base and are short and rudimentary in the female but strongly developed in the male; the labrum, a shelf-like projection overhanging the mouth anteriorly; and a pair of mandibles, which bare heavily sclerotized (tanned) flattened plates at the sides of the mouth. On the thorax find eleven pairs of leaf-like appendages, each having a setose gnathobase for passing food to the mouth. The abdomen consists of nine segments and terminates with a telson bearing the anus and caudal furcae.

Figure 3: Adult female Artemia in lateral view. From R. Fox Invertebrate Zoology Online A male can be distinguished from a female especially by its more strongly developed second antennae with which it clasps the female in mating; it also has paired frontal organs, which are leaf-like expansions arising from the anterior border of the head, and a complex copulatory organ, which is built around fused extremities of the vasa deferentia, on the abdomen. The female has on her abdomen a brood pouch, formed by the union of the two oviducts, in which eggs are carried until they hatch. The vascular system of Artemia can easily be seen in action. Look at the limbs of an animal held under a coverslip and watch for the motion of cells in the blood that is flowing through them. Can you see a pattern

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to the flow and tell from it where the channels are in the hemocoel? Orient the animal so that you can see it in lateral view and look for the heart beating. Find in it the ostia. How does this heart exemplify the arthropod condition? What is the relation of the heart to the hemocoel? For studying Artemia in depth, refer to the relevant pages in Rick Fox’s online lab manual (http://webs.lander.edu/rsfox/invertebrates/artemia.html). Cladocerans (water fleas). (Daphnia or Bosmina spp. Fig. 19-15, p. 621.) Living cladocerans from pond water are available; slurp some with a pipet from the bucket into a finger bowl. (Also in these samples are copepods, which we will study in the next lab; you are welcome to study them now, too, of course.) Whole-mounted cladocerans on slides are also available. The body is laterally compressed, and the trunk enclosed in the carapace while the head is free. The first antennae are small, and project from the ventral surface of the head. The second antennae, on the other hand, are quite large, and are biramous; they project anteriorly from the head. There is a conspicuous median eye, which is formed by the fusion of two lateral, compound eyes. In Daphnia there are five pairs of thoracic limbs. The abdomen is bent forward under the thorax, and is without appendages. Find the heart in the dorsal midline and see if its ostia are visible. Identify the intestine, and, if possible, the gonads. If your specimen is a female (as most specimens will be) it will have a conspicuous space just posterior to the heart; this is the brood sac. Here eggs are carried until development is completed; the young emerge as miniature adults. Some of the whole-mounted specimens bear ephippia or resting eggs, thickshelled eggs that withstand periods of freezing and desiccation. (Preserved whole mounts of cladocerans, some with parthenogenetic eggs, some with ephippia, are also available, and can be studied optionally.) Decapod crustaceans Lobster (or Crayfish) (Homarus americanus, Cambarus sp.). Living and preserved specimens are available for studying behavior and anatomy. (Figures on pages 607-609 and 639 of the textbook, are useful.) Like other malacostracans, the lobster has a trunk composed of 14 segments, namely 8 thoracic plus 6 abdominal segments; and the head comprises 5 adult segments (plus the acron, an embryonic segment, in front of them). These segments are best characterized by the specific appendages they carry. As each body region is studied using the following instructions, remove the appendages from the left side of the body and line them up in order so that you can study and draw them. The head is fused with the thorax to form a cephalothorax, and it is enclosed in a cephalothoracic shield, the carapace. The head is still distinguishable as the region anterior to a fold of the carapace, the cephalothoracic groove. The first two sets of appendages on the head are the first and second antennae (or antennules and antennae). Behind these are the first mouth parts, the mandibles, and, closely associated with them (and almost hidden by appendages behind them), two pairs of accessory feeding appendages, the first and second maxillae (or maxillules and maxillae). Three pairs of maxillipeds (first, second, third) follow the maxillae. These are actually appendages of the thorax that have moved forward to serve as mouthparts. The remaining thoracic appendages are the chelipeds and the four pairs of walking legs or pereopods: the five pairs give the Decapoda its name (meaning “10-footed”). The chelipeds and first two pairs of walking legs are chelate, having the distal joints modified to form chelae for gripping and tearing food. The two chelipeds can be distinguished in the lobster: the so-called crusher claw is massive and is used to crush or open shells of its prey; the cutter claw is narrower. Lobsters feed mostly as scavengers. Crustacean appendages are fundamentally biramous, composed of an endopod medially and an expopod laterally. In the Decapoda, the first antennae look biramous, but the branches are actually so-called flagella and not homologous to the branches of the typical biramous appendage. The second antenna is biramous and has the exopod appearing as a flattened scale, while the chelipeds and walking legs have lost the exopod and so appear uniramous, the endopod developed to serve in walking or prehension. Are the abdomenal appendages biramous? Dissection (of lobster). Remove the mouthparts and one walking leg from one side of the body and arrange them in order on a slide to compare their structure. Do the mouthparts in the lobster have the typical biramous structure? The gills are exites (= epipodites), processes from the coxa of the appendage, and are developed in the lobster as filamentous branches on the second and third maxilliped and on each of the walking legs. They are protected in a gill chamber formed from an overhang of the carapace, the branchiostegite. Expose the gills by cutting with scissors from the left posterodorsal edge of the carapace forward until the branchiostegite can be lifted off. Move the appendages to see how their motion affects the gills. Carefully remove all the thoracic appendages from the left side of the body.

28 Look at the appendages to see how the muscles move them. How do the muscles attach to the exoskeleton and how do they reach across the joints to bend them? Look at the stained histological section of cuticle and apodeme on display and see how cuticle, epidermis, and muscle are linked. (This is the same section in which the compound eye is visible.) Can you tell from this if the muscle attaches directly to the cuticle? (Knowing what you do about secretion of the cuticle would this be possible?) The particularly strong muscles of the crusher and cutter claws have particularly large apodemes. Open a claw and see how the apodeme links muscle and digit. Why is the apodeme so broad and flat, and how do you think it relates to the epidermis of the claw? Turn the animal ventral-side-up and find the genital openings which are on the base of the fifth thoracic pereopods (the eighth thoracic segment) if the animal is a male or on the third pair of pereopods (the sixth thoracic segment) if it is a female. The abdominal appendages are five pairs of pleopods (swimmerets) and, forming the tail fan, the uropods and telson. The first and second pairs of pleopods are modified in males to serve as copulatory organs. Watch a live lobster to see how the pleopods and tail fan are used in swimming as well as how the chelipeds, legs, and mouthparts are used in walking and feeding. How does a disturbed animal execute the rapid escape response? Open the lobster by cutting the dorsal wall of the carapace off: start cutting on the side of the lobster at the posterior free margin of the carapace, cut forward to just behind the eyestalk on the same side, then cut over the dorsal surface to just behind the other eyestalk and then back posteriorly past the free edge of the carapace on that side down the sides of the abdomen. Then extend the cut on the other side down the side of the abdomen and peel back the dorsal exoskeleton of both the thoracic and abdominal regions. Find the heart and principal blood vessels and the gut as well as other organs depicted in Figure 19-2. The two large greenish masses surrounding the midgut are the digestive glands (hepatopancreas), which are diverticula of the midgut. These secrete digestive enzymes and process small particulates in the food, absorbing digested components; they also store reserve materials (and hence are sites of accumulation of heavy metals and other pollutants, for example, in lobster as well as lobsters, crabs, and shrimp). Large brown mandibular muscles lie on either side of the anterior end of the gut. Ovaries (pink, yellow, or greenish) or testis (whitish) may obscure parts of the digestive tract; they can be removed. Remove also the heart and place it in tap water to study the valves of the three pairs of ostia. On the gut, find the short vertical esophagus, the stomach, which consists of ananterior cardiac chamber (a grinding region) and a smaller posterior pyloric chamber (a sorting region), and the midgut leading from the stomach to the hindgut and having a caecum projecting from it over the pyloric stomach. Remove the stomach and open it under water to see the chitinous teeth of the gastric mill and the rows of setae in the pyloric chamber. Excretory organs are the paired maxillary and antennary glands (green glands) which open at the bases of the appendages for which they are named. The green glands are the easiest to find. Remove the gut so that you can see the muscles, and flex the animal’s body and legs to see how these muscles might work. Open a cheliped to see how its muscles can cause opening and closing. Cut away the cuticle of the head to expose the supraesophageal ganglion (brain), circumesophageal connectives, and ventral nerve cord with segmental ganglia. In the abdomen, separate the muscles, cutting close to their origins on the sterna, to see the abdominal part of the nerve cord and nerves emanating from it. Remove a first antenna (if you have not already done so) and cut open its basal segment to show the statocyst. Remove a compound eye and bisect it longitudinally for study under a dissecting scope. A stained histological section of a compound eye is on display on the demonstration table. Study it to find ommatidia and their components (compare with Fig. 16-13, pp. 536–537). Study a piece of the cuticle under a dissecting microscope to see pores and setae. Which of these are likely to be sensory organs? Exuvia (molted exoskeletons) of lobster and crab are on display. Crabs: Carcinus maenus (the green crab), Carcinus irroratus (the rock crab), or C. borealis (the Jonah crab). (See Figures on pages 633, 636–637, 640.) Crabs have a specialized short body form, not typical of decapods in general, but it can be viewed as simply having the abdomen tucked under the thorax (in fact, this is how the abdomen develops from the larva of crabs; see the demonstration of a megalopa larva showing its fully extended abdomen). These are all crabs with the carapace expanded laterally and dorsoventrally flattened, as is characteristic of the Brachyura. Lateral extensions of the carapace produce the branchiostegites which form a protected space within which the gills lie. Slits above each of the stout pereopods admit water into the gill chamber, and the water exits via a large opening in front of the mouth. This opening can be nearly completely closed by the heavy third maxillipeds, which also enclose the mouth. If you are dealing with a live crab, you will see bubbles being blown from this region when the crab is out of water. If you are studying a preserved or heat-killed specimen, pull them down in order to find this exhalant opening and the

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mouthparts. At the front of the cephalothorax note the two depressions, the orbits, into which the eyes can be withdrawn. If the eyes are withdrawn, as is likely in preserved specimens, use a needle to force them out so that you can see them. Another pair of depressions closer to the center line are used to tuck the first antennae away; locate them and, if you are dealing with a preserved specimen, extend these first antennae with your needle. Where are the second antennae? The live animal flicks its first antennae jerkily almost constantly, and the currents they test for odors are generated by the beating of the exopods of the maxillipeds. Note the chelipeds, plus the four, non-chelate posterior pairs of periopods. The abdomen is bent under the thorax and lacks uropods (Fig. 19-31). In the male it is narrow, and by pulling it outward you may see that the first two pairs of pleopods are modified as sperm-transferring organs. These are the only abdominal appendages in the male. In the female, the abdomen is quite broad, and only the second through fifth abdominal segments bear pleopods. In mature females, these develop elongate setae to which the eggs are glued. Find the rather large genital openings of the female on the sternum of the sixth thoracic segment; in the male, the genital openings are on flexible papillae on the coxae of the last legs. Dissection (of crab). With a heat-killed or preserved crab, remove the mouthparts from one side of the body and arrange them in order on a slide to compare their structure. Do the mouthparts have the typical biramous structure? To open the body, cut around just inside the edge of the carapace so that you can pry up almost the entire dorsal surface, scraping the underlying epidermis from the cuticle. You will have to scrape off the attachments of muscles, especially large ones attaching to two calcified projections on either side of the midline. Once you have the cuticle removed, look at the epidermis under a dissecting scope to see the chromatophores (red, black, white). With the epidermis removed, the internal organs will be visible. Refer to Figure 19-35 to find the heart, digestive gland, gonad, and stomach. The heart, lying in the pericardial sinus, has three pairs of ostia. The pyloric portion of the stomach will be visible dorsally; lift it to see the more muscular cardiac stomach and the esophagus. The intestine is quite short, only about 1 cm. The digestive gland is divided into two lateral parts and is highly lobed. Lifting the stomach will also expose the pale green antennal glands, the excretory organs, on the ventral surface of the head; their bladders are remarkably large and thin-walled. Remove the thin roof of the gill chamber. The gills are exites of the last two maxillipeds and first three pereopods. Over the gills, one of the gill cleaners will be visible, the long exite (epipodite) of the first maxilliped. The gill bailer (scaphognathite) that drives the water through the gill chamber is a projection of the second maxilla. Move the appendages to see how their motion affects the gills. Look at the appendages to see how the muscles move them. How do the muscles attach to the exoskeleton and how do they reach across the joints to bend them? Look at the stained histological section of cuticle and apodeme on display and see how cuticle, epidermis, and muscle are linked. (This is the same section in which the compound eye is visible.) Can you tell from this if the muscle attaches directly to the cuticle? (Knowing what you do about secretion of the cuticle would this be possible?) The particularly strong muscles of the claws have particularly large apodemes. Open a claw and see how the apodeme links muscle and digit. Why is the apodeme so broad and flat, and how do you think it relates to the epidermis of the claw? Try to see the central nervous system by exploring around the base of the esophagus. The supraesophageal ganglion lies immediately behind the rostrum; the subesophageal ganglion includes fusion of many of the segmental ganglia of the thorax. Remove a first antenna and cut open its basal segment to show the statocyst. Remove a compound eye and bisect it longitudinally for study under a dissecting scope. A stained histological section of a compound eye is on display on the demonstration table. Study a piece of the cuticle under a dissecting microscope to see pores and setae. Which of these are likely to be sensory organs? Exuvia (molted exoskeletons) of crab and lobster are on display. Other decapods (shrimp, crayfish) Shrimps (Pandalus, Crangon, Lebbeus; see Figure 19-25, p. 630). Specimens of the boreal red shrimp, Pandalus, are on the demonstration table; live specimens of Crangon and Lebbeus may also be available. Pandalus is the commercial shrimp common on the Maine coast. In general, the morphology of shrimps is like that of the lobster, but a few differences deserve mention. First, note that in contrast to the lobster, which is primarily a bottom-dweller, the pereopods in the swimming shrimps are more delicate, and the pleopods, by means of which they do most of their swimming, are relatively large. The first three pairs of pereopods are chelate, but in these species the first pair are not enlarged into chelipeds, as in the lobster.

30 Crayfish. (Cambarus sp., Astacus sp., etc.) Preserved and living specimens are on display. Much of what you learned of anatomy of the lobster applies to crayfish. Spider crabs (Libinia; compare Figure 19-32). Spider crabs typically have long spindly legs, proportionally like spiders, and an elongate rostrum, a narrow anterior projection of the carapace. They also actively attach and encourage fouling organisms to colonize their exoskeleton, presumably as an effective camouflage. If you examine one under a dissecting microscope, you can see the hook-shaped setae by which they initially attach such organisms. Hermit crabs (Pagurus sp., Coenobita; see Figure 19-28, p. 631.) These crabs are classified in the Anomura, characterized by having the fifth legs reduced and turned upward. Hermit crabs have a soft, assymetrical abdomen with reduced pleopods on the right side and its shape is adapted to protection inside abandoned snail shells. Specimens of Pagurus in shells and removed from their shells are on demonstration. Note how the last walking leg is small and tucked under the thorax, adapted to holding onto the snail shell. King crab (Paralithodes). This crab is also an anomuran crab, possibly derived from a hermit-crab-like ancestor. Note how the fifth legs are reduced so that it appears at first glance that the crab has only four pairs of legs.

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Arthropoda II: Crustacea II other malacostracans (incl. mysids, isopods, amphipods) / barnacles / copepods / ostracods Euphausia (Figure 19-51, p. 651.) Animals in the Euphausiacea are commonly known as krill. They live in the water column of the open ocean, capturing planktonic organisms for food with a forward-directed basket-like arrangement of the setose thoracic appendages, and they are a vital part of the food chain, being prey in turn for baleen whales as well as many other marine predators. The most obvious distinguishing feature of euphausiaceans is the bush of gills along the side of the thorax; rather than being protected in a fold of the carapace as the decapods have them, the gills are exposed. They arise from the bases of the thoracopods. Note also on the animals on demonstration how the thoracopods are adapted for filter-feeding, with long bristles and forming a basket. Also distinctive is that none of the thoracopods are modified as maxillipeds in euphausiaceans (cf. the decapods). Mantis shrimp (Squilla sp.; Fig. 19-22, -23, pp. 627–628.) Specimens of the mantis shrimp, Squilla, are on demonstration. Like the insect praying mantises, Squilla, stands out for a large pair of subchelate appendages used to capture prey; mantis shrimps deliver stunning blows or spear prey with them. On the head are prominent eyes borne on a pseudosegment. Behind this identify a free head segment which bears the first antennae, and behind that the segment bearing the biramous second antennae, the exopod of which is a large, posteriorly directed antennal scale. The carapace covers the last four head segments, plus thoracic segments 1-4, with which the carapace is fused. You may be able to see the massive mandibles. Thoracic segments 5-8 are free; there is a pair of subchelate maxillipeds; the pair of large, raptorial subchellae; three pairs of subchelate appendages which aid in handling food; and three pairs of biramous walking legs. The abdomen bears broad, biramous pleopods, on 1-5 of which there are delicate gills. In the male the first two pairs of pleopods are modified into copulatory organs. Terminally there is the broad telson, which forms a fan with the sixth pair of pleopods. Peracarid malacostracans This is the largest of the crustacean groups, and it is characterized by possession of a marsupium—a brood chamber on the ventral side of females, formed of ventral plates, the o¨ostegites, extending medially from the thoracic appendages. Mysidacea. (Praunus or Heteromysis sp.; Fig. 19-53, p. 653.) The carapace covers only the posterior head segments plus the anterior ones of the thorax in mysids. While mysids have the general shape of shrimp, they are distinct by possession of the marsupium; and an especially distinctive feature of mysids is presence of statocysts in the endopods of the uropods. The first two thoracic appendages are maxillipeds, and the others are biramous, swimming and filter-feeding appendages, having highly setose exopods. The abdominal appendages are biramous pleopods; in the female these are frequently reduced or absent. Isopoda. (Idotea [marine] or Caecidotea (=Asellus) [freshwater] sp. or Haplophthalmus [small terrestrial isopod from leaf litter] or Oniscus sp., etc. [terrestrial “pill bug”] Figs. 19-61–63, p. 661–663.) Watch live isopods for the way they swim, walk, and respire (keep them cool and avoid overexposing them to the bright light of your microscope); it may be easier to scrutinize fixed specimens for details of anatomy; the small terrestiral isopods can be gently stuck to a slide upside-down with a spot of vaseline jelly or relaxed in 10% ethanol. A distinctive feature of isopods is that the body is dorso-ventrally flattened. Like amphipods (below), isopods lack a carapace. There is a short, compact cephalothorax which is composed of the head and the first thoracic segment. It bears a pair of sessile, compound eyes; long second antennae; and short, rudimentary first antennae, located just medial to the bases of the second antennae. There are seven free thoracic segments, each of which bears a pair of pereopods. The appendages of thoracic segment 1 are maxillipeds. If the animal is a reproductive female, pereopods 1–5 will bear o¨ ostegites, leaf-like flaps that project medially to form a brood pouch. If it is a male, pleopods 1 and 2 are modified into copulatory organs. The pleopods on the abdominal segments are flat and biramous; note in the living aquatic species how they beat as respiratory structures. On the terrestrial isopod, you may be able (through low power on the compound microscope to see tubular structures within the pleopods; these are pseudotrachea for breathing air. Does the cuticle look different in the terrestrial form as if adapted to

32 drier conditions? On an animal mounted on a slide, you should be able to see the heart beating through the dorsal body wall. The head appendages associated with the mouth are typically crustacean (mandibles, first and second maxillae). On demonstration are other isopods, including a giant deep-sea form and parasitic species. The parasitic species are globose and have a large marsupium. Amphipoda. (Gammarus sp. (Fig. 19-54, -55 pp. 654–655.) Both live and fixed specimens are available; as with the isopods, it may be easier to scrutinze fixed specimens for morphology. Amphipods are easily distinguished from isopods by the lateral flattening of the body (they are laterally compressed). Like the isopods, there is no carapace, and the cephalothorax is composed of the fused head appendages plus the first thoracic segment. Its appendages are similar to those of the isopods. Thoracic segments 2–8 are free and bear pereopods. Appendages on thoracic segments 2 and 3 are subchelate and aid in feeding; those on seqments 4 and 5 are not subchelate, but they are directed forward and aid in feeding, also. Finally, pereopods on the last three thoracic segments serve in walking. Now examine the abdomen and its appendages. Those on the first three abdominal segments are biramous, swimming appendages. The appendages on segments 4–6, however, are stout and are used for kicking. If your specimen is a female, o¨ ostegites are present on thoracic legs 2–5. On demonstration are other amphipods, including skeleton shrimp (caprellids—members of the family Caprellidae—which we have seen live on samples of hydroids we have studied; live ones may also be available in this lab) and an ectocommensal of larger marine vertebrates (possibly Cyamus, the whale louse). Cirripedia Members of the class Cirripedia include the familiar barnacles, which live attached to substrates of various sorts, as well as a large number of parasitic forms, many of which are highly modified. We will study morphology of free-living barnacles. Look at least briefly at external anatomy of both Semibalanus (the common rock barnacle) and Lepas (the gooseneck barnacle). Then choose either barnacle to study internal anatomy. See the description below (“Internal anatomy...”) for identifying internal parts of either barnacle species. Rock barnacle (Semibalanus balanoides, the common rock barnacle (and species of Balanus on demonstration; Figs. 19-78, -82, pp. 679, 684). These barnacles are sessile, without a prominent stalk or peduncle attaching them to the substrate (compared to the gooseneck barnacles). The two pairs of plates that open to permit the protrusion of the thoracic appendages when the animal is feeding are the scuta and terga, the scuta are the larger of the two pairs. Around these opercular plates several fused plates form a solid protective parapet. The carina is in the midline behind the terga; opposite it, and behind the scuta, is a large plate, the rostrum; laterally two lateral plates are fused on either side. Watch the living specimens of Semibalanus to see how the thoracic appendages are protruded and swept through the water to capture food. Gooseneck barnacle (Lepas sp. Fig. 19-80, pp. 680.) Preserved specimens of this barnacle should be studied submerged in water; the dissecting scope will be needed for most of your observations. Note the peduncle, by means of which the animal was attached to the substrate. At the end of the peduncle there are vestigial first antennae, but these are not readily seen. Within the fleshy mass of the peduncle is a cement gland and an ovary. The upper part of the animal is enclosed by calcareous plates which are secreted by a mantle that encloses the rest of the body. There are five calcareous plates. On the dorsal side, opposite the openining, is the single plate called the carina. Ventral and anterior (toward the peduncle) are a pair of scuta (one scutum on each side), and posterior and dorsal a pair of smaller terga (one per side). From the aperture on the central surface some of the thoracic appendages probably protrude. For studying internal anatomy as described in the next paragraph, use an animal that has had the plates removed from one side. Internal anatomy of barnacles. Specimens of Semibalanus chipped off their attachments and anaesthetized in magnesium chloride are available on the side counter; preserved specimens of Lepas, including opened specimens, are available on the demonstration table. For Semibalanus, look first at the underside (basal attachment disc) of the freed specimens. Visible through the thin basal membrane is the ovary, a yellow, lobulated and branched structure. The anterior end of the animal is also visible in the center, between the lobes of the ovary. Once these parts have been

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found, separate the peanut-shaped body of the animal from the tissue attaching it to the calcareous plates. This can be done by gently scooping the animal out of its shell or by breaking the carina and rostrum and drawing them apart to free the body. In an opened barnacle, either Semibalanus or Lepas, identify the six pairs of biramous thoracic appendages, the oral cone, and the finely lobate testis visible through the body wall (and which extends up into the bases of the appendages). Possibly a plate of eggs will be present on either side of the mantle cavity (in Lepas) or in its anterior end, attached to the anterior end of the body (in Semibalanus); the mantle cavity acts as a brood chamber during the reproductive season. On the base (coxa) of the first thoracic appendage are two to four fleshy epipodites, which are respiratory. Locate the mouth at the apex of the oral cone; there may be food material in the mouth, which might have to be removed in order for you to clearly see the feeding appendages. The anus is located between two small flaps, the caudal rami, on the dorsal surface between the bases of the last pair of thoracic appendages. Just ventral to the anus there arises the large penis. Bear in mind that barnacles are hermaphroditic and need the long penis to inseminate neighbors, which may be some distance away. Copepoda (Fig. 19-68–19-74, pp. 670–675.) Living copepods should be available from the live plankton sample. Preserved plankton samples are also available and can be used in the absence of or in addition to live samples. As you look at these plankton samples, keep an eye out for other crustaceans and crustacean larvae. Various shrimps, amphipods, and larvae (nauplii and zoea, in particular) will likely be found, as well as a host of other invertebrates. Plankton-identification guides are available on the front table for identification of mystery animals. If you are not sure how to recognize copepods, you might first examine the slides of whole-mounted specimens of the calanoid Diaptomus and the cyclopoid Cyclops (Fig. 19-68) to form a search-image for copepods in your mind and to get a general idea of anatomy. Then find examples of copepods in the plankton samples and study several animals in a finger bowl or watch glass, using intermediate and higher powers of your dissecting scope. Representatives of two major groups of copepods are present in these plankton samples, a cyclopoid and a calanoid. Watch the live animals for their manner of swimming. The jerky swimming movements are produced by the biramous thoracic appendages (numbers 2–5). The long first antennae serve for flotation, like parachutes, after each forward spurt; and the second antennae beat in what looks almost like a rotary motion to produce currents that drive the animal forward in a steadier forward movement. These copepods are likely to be herbivorous, capturing suspended particulates with the maxillae from the current created by the second antennae. Other copepods are predacious, cruising continuously in search of prey and grabbing it with mouthparts that are more clawlike than the paddle-like maxillae of herbivorous species. Many other copepods are parasitic, some highly modified, and lead a sedentary life, simply absorbing nutrients from a root-like anchor in a host’s tissue; mouthparts in these, then, as well as the other appendages, may be completely reduced. (See the demonstration of parasitic copepods.) Mount some specimens on a microslide and cover them with a wax-supported coverslip. Note that the thorax has two segments fused with the head (the two segments cannot be distinguished), behind which are several distinct thoracic segments. There is a narrow abdomen, composed of several segments, which terminates in paired rami that bear setae or spines. Cyclopoid females carry eggs in ovisacs (one or a pair) which are suspended beneath the abdomen; the calanoid females in our samples probably do not have ovisacs, instead releasing the eggs singly, but some calanoids have a single medial ovisac. On the head, look for a median eye (naupliar eye). Identify, also, the long first antennae and the shorter second antennae. Under the thorax you will be able to see the thoracic appendages, which in the living specimens execute quick backward thrusts. Harpacticoid copepods are more elongate than the calanoids and cyclopoids, almost worm-like in apparent adaptation to living in sediments and crawling between the sediment grains. Many of the sediment-dwelling species scrape the sediment grains for food, capturing the epigrowth of diatoms and bacteria there. Look at specimens available and see what other adaptations you can see for sediment dwelling. (For example, are their antennae as long as those of the planktonic copepods?) Parasitic copepods are on demonstration to show simply how extremely modified some members of this class can be. Some appear to be little more than sacks of gonad. How do you think we know these really are copepods if they don’t even look like crustaceans? (See also Figs. 19-73, -74, pp. 674–675.)

Ostracoda

34 The carapace of ostracods (seed shrimp; see Fig. 19-85) is bivalved, hinged at the dorsal midline and so large that it can be closed over the body. The shell is typically fairly opaque (often because of impregnation with calcium carbonate), but the living specimens can be studied at least to watch behavior and to see some of the external anatomy. Slides of preserved, cleared whole mounts of ostracods are available, and in them you can see some internal anatomy, such as the eyespot, the gut (brownish and convoluted in these specimens), and the adductor muscle that closes the carapace (in the middle of the shell in lateral view). Appendages may also be discerned; these are, in order from anterior to posterior, first and second antennae, mandible, first and second maxillae, and first and second thoracic appendages. Only the first and second antennae may be protruding from the opening of the carapace; you may also see the tip of the caudal ramus protruding from the posterior edge. Crustacean larvae (Figs. 19-8, 19-48, 19-47, 19-72, 19-79 [pp. 614, 648–649, 673, 680]). Search for crustacean larvae in the samples of living plankton. Plentiful in them are nauplii of barnacles (identifiable as nauplii by having only three pairs of appendages; identifiable as barnacles in having horns). Also present are cyprid larvae of barnacles, looking something like ostracods (their name, in fact, is derived from their resemblance to Cypris, an ostracod). Try to find other crustacean larvae in the plankton samples, including zoea. On demonstration are nauplii (of the barnacle, Lepas) and two larval stages in the life cycle of a brachyuran crab, a zoea and a megalops. In the nauplius, note that there are only three pairs of appendages, the first and second antennae and the mandibles. In the zoea, note the long rostral and dorsal spines. The megalops has an elongate abdomen, which, upon metamorphosis, would be tucked under the cephalothorax, producing the typical crab form. Also on demonstration are larval stages of the lobster. These are zoea.

Arthropoda: Myriapoda

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Arthropoda III: Myriapoda ARTHROPODA (cont.) Myriapoda Chilopoda Scolopendra — preserved Otocryptops(?) or Lithobius(?) and geophilomorphs — live, as available Diplopoda Spirobolus — preserved Julus(?) and polydesmid millipedes — live as available [Pauropoda] [Symphyla]

The “multi-legged arthropods” or myriapods (centipedes, millipedes, and the two groups of smaller litterdwelling relatives, the pauropods and symphylans) comprise a somewhat controversial group. Some arthropod systematists consider them a true monophyletic taxon and apply the official name Myriapoda to it. Some others believe this taxon is not, strictly speaking, monophyletic, that the elongate, multilegged body form was arrived at by several independent evolutionary lines. In some phylogenies it is said to be paraphyletic because some of its members may be more closely related to the insects, the Hexapoda (that is, insects might have descended from an ancestor that would be classified as a myriapod). Other more recent evidence, including characters from molecular sequences and development of the nervous system, points to a closer relationship between crustaceans and insects, leaving the Myriapoda as a monophyletic taxon (but still leaving the insects as a paraphyletic taxon). Centipede. (Scolopendra sp.) The head is relatively flat, with the antennae on the front margin. The most prominent of the mouthparts is the forcipule, which is a maxilliped with poison glands opening at its tip. Its large coxae and the associated sternum cover the ventral side of the head. The mandible, which bears teeth and a fringe of setae, and two pairs of maxillae lie in front of it, partly covered by it. Behind the first trunk segment (with the forcipules) lie 15 or more leg-bearing segments. The last pair of legs is sensory and not locomotory; the last two segments lack legs. Examine living centipedes from leaf litter in covered dishes (they are fast and escape easily) to see how they behave and how their morphology compares to that of Scolependra. The shorter-bodied, dark brown ones, may be Lithobius or Otocryptops. The more elongate, narrow-bodied and pale forms are geophiliomorph centipedes; they are not as fast but are well adapted to snaking their way through the interstices of forest litter and rotting logs. Millipede. (Spirobolus marginatus) Millipedes are mostly herbivorous, feeding on decaying vegetation. The body of Spirobolus, like that of other so-called juliform millipedes, is rounded in cross section, apparently adapted to bulldozing through leaf litter and similar habitats. The head itself is rounded dorsally and flattened ventrally and bears a pair of antennae and two clusters of ocelli constituting the eyes. Prominent on its sides are the bases of the large mandibles. Behind the mandible is the gnathochilarium, a relatively large plate formed from the fused pair of maxillae. (There are no second maxillae.) The first trunk segment, called the collum, is legless and forms a collar behind the head. The three segments behind it each have one pair of legs, but the remaining segments are double, such that each bears two pairs of legs. the third trunk segment bears the genital pore. Watch living millipedes from leaf litter and compost and note how differently they use their legs and antennae and how they adopt a defensive posture by curling up. Try smelling some to see if you can detect any repugnatorial secretions (hydrogen cyanide, for example, smells something like almonds; quinones and aldehydes will be pungent).

36

Arthropoda IV: Chelicerata Arthropoda: Chelicerata ARTHROPODA Chelicerata Merostomata (horseshoe crabs; extinct eurypterids) Limulus — live, preserved, slide (demo) Arachnida Scorpiones (scorpions) Centruroides — preserved Uropygi (whiptail scorpions) Thelyphonus — preserved Araneae (spiders) Miranda and Argiope — preserved (use either) live spiders as available Pseudoscorpiones Chelifer — live, preserved (demo) Opiliones Liobunum — preserved (demo) live as available “Acari” orders (mites and ticks) mites from leaf litter — live Dermanyssus — slide Dermacentor — slide Pycnogonida (sea spiders) Anoplodactylus — preserved Pycnogonum — preserved demo live as available

Chelicerates are ancient groups with origins in the marine environment. The most successful group of chelicerates is the Arachnida, containing scorpions, spiders, and mites among others, and all are specialized for terrestrial life. The body plan of chelicerates is well represented by the marine group Merostomata, containing the horseshoe crabs, so it serves as a good starting point. Horseshoe crab: Limulus polyphemus The Xiphosura is an ancient group of marine arthropods that now has very few living representatives, among them Limulus, often referred to as a living fossil (animals virtually identical to Limulus lived 230 million years ago, and very similar species trace back to 400-million-year-old fossil deposits). Watch living animals to see how the appendages are moved in locomotion, including crawling and swimming. The animals are hardy and can stand being removed from the seawater briefly, so (with clean hands) you should hold an animal on its back to see how the appendages move. Watch also the bases of the appendages, which are spiny and known as gnathobases, to see how they can grind together to tear food as the animal moves. Limulus places worms, clams, and other small invertebrates in among the gnathobases with the chelicerae, and as the appendages move in walking, the grinding motion of the gnathobases prepares the food for ingestion. (We will demonstrate feeding with bits of mussel or clam; you may try dropping pieces of such food on the gnathobases yourself.) Obtain a preserved specimen for study of external anatomy. The body is covered dorsally by the depressed and laterally expanded exoskeleton. It is divided into two major body regions (tagmata): the cephalothorax (or prosoma), which bears appendages surrounding the mouth, and, behind this, the abdomen (or opisthosoma), bearing the gill flaps an operculum covering them. The long tail-like telson is terminal. Note that the body can be flexed at the joint between abdomen and cephalothorax and the joint between abdomen and telson. On the lateral margins of the abdomen are six movable spines; these mark the six anterior-most somites of the abdomen. On the dorsal surface of the cephalothorax, there are a pair of lateral compound eyes and a pair of median simple eyes. Not immediately apparent are other photoreceptors, namely on the ventral surface in front of the mouth and near the base of the telson. These smaller photoreceptors appear to be instrumental particularly in setting the internal clock of Limulus, so that the compound eyes

Arthropoda Chelicerata

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adapt to conditions of night and day on a regular schedule (even in complete 24-h darkness in experimental conditions). Examine the ventral surface. The first of the appendages are the chelicerae, a pair of small, threesegmented, chelate appendages—that is, the last two segments articulate together in a pincer-like fashion. The remaining five pairs of appendages, the walking legs, are more or less similar to one another and composed of six segments. The basal segment on each (the segment called the coxa) is a spiny gnathobase, so-called; the spines on the gnathobases grind together to macerate food and push it toward the mouth. Find the mouth, located between the bases of the first pair of walking legs; the anus is also ventral, where the telson joins the abdomen. The second pair of appendages are sometimes called pedipalps, though they are not nearly as distinctive (from the walking legs) as pedipalps in the other chelicerates we will study. In Limulus, these appendages (the first walking legs), as well as the following four pairs of walking legs, are chelate like the chelicerae. The sixth pair of appendages (the fifth walking legs) are modified as ski-pole-like appendages, having large, flattened spines which arise on the penultimate (next-to-last) segment, and a slender spine on the terminal segment; the spines spread against the substrate, allowing the animal to pole its way along. Arising on the lateral side of the basal segment of this fifth leg is a spatulate process used for cleaning the gills (called a flabellum). The last appendages, a small seventh pair lying almost between the sixth pair of appendages, are called chilaria. They are one-segmented movable processes bearing a row of spines on their medial surface and presumably function in preventing food from escaping posteriorly from between the gnathobases. The first walking legs become subchelate in males—that is they have a thickened penultimate segment and a hook-like distal segment that folds against it. Males use these appendages to clasp the abdomen of a female during pairing. In the breeding season, pairs of horseshoe crabs linked in tandem can be seen migrating onto sandy beaches where the female buries the eggs. The male releases sperm as the eggs are being buried, and the young develop in the sand into a “trilobite larva,” so-called because of its lack of a long tail-spine and consequent superficial resemblance to trilobites; technically speaking, it is not a true larva because it is morphologically quite like the adult. Examine the “trilobite larva” on display. Look at the ventral surface of a preserved specimen for the abdomenal appendages. Each of the six pairs of appendages is fused in the midline to form a single plate. The first pair acts as an operculum, covering and protecting the other five, and it bears the genital openings on a pair of papillae on its posterior surface near the midline. Each of the five remaining appendages bears a large number of gill lamellae on its lateral margins; each group of lamellae is called a book-gill. The gills are aerated by the beat of the abdomenal flaps and they can be used to swim by beating faster. Scorpion: Centruroides sp. Examine the dorsal surface of a preserved specimen of this scorpion and note that the body is divisible into a relatively short cephalothorax of six fused segments, a mesosoma of seven segments, and a slender metasoma of six segments. The terminal segment bears a poison-wielding stinging apparatus and usually a spine ventral to it. Dorsally on the so-called carapace of the cephalothorax you will see a pair of median eyes, and anterolaterally, a row of three lateral eyes on each side. Examining the ventral surface, you will find the anteriormost of the appendages to be a pair of small chelicerae. Next are a pair of large, strongly chelate pedipalps, which are composed of six segments. The walking legs are in four pairs, and each has seven segments and a terminal pair of claws. Note that the basal segments of the pedipalps and of the first two pairs of legs are modified as gnathobases. On the first mesosomal segment is a small plate that is cleft in the midline and that serves to protect the genital opening; this is the genital operculum. On the next posterior segment is a pair of comb-like sensory appendages, the pectines. Segments 3-6 each bear a pair of slit-shaped spiracles which are openings into book-lungs. The genital operculum, pectines, and book-lungs presumably represent modified appendages. Superficially, at least, the book lungs resemble the book-gills of Limulus. Spiders (Araneae): Argiope aurantia or Miranda sp. Preserved specimens of either of these two species can be studied as representatives of the spiders. (A. aurantia is the common yellow garden spider; Miranda is another orb-weaver.) Large individuals are females. Males, which are distinguishable particularly by their swollen pedipalps, are smaller, have a cylindrical instead of a bulbous abdomen, and are less often seen. Note that the body is divided into a cephalothorax and an abdomen, neither of which shows distinct segmentation in the adult. On the anterior, dorsal part of the cephalothorax you will find four pairs of simple eyes; their positions enable the animal to command a view forward, ventrally, dorsally, and laterally,

38 all simultaneously. The anterior-most appendages of the cephalothorax constitute most of the animal’s “face” when viewed from the anterior end. These are the chelicerae, each of which has a wide, stout basal segment, and a dark, conical fang. The fangs can be extended so as to stand at right angles to the basal segment, but at rest they are reflected against the basal segment. Behind the chelicerae is a labrum, a sort of upper-lip. The second pair of appendages are pedipalps. These are six-segmented and have an enlarged coxa (basal segment) which bears bristles that aid in feeding. The slender, distal segments are generally carried in front of the chelicerae and serve as sensory organs. A fleshy protuberance, the labium, between the coxae of the pedipalps forms a posterior wall (lower lip) to the mouth cavity. Examine the walking legs. There are four pairs, each of which is similar to the others. They have seven segments, which are, beginning proximally, the coxa, trochanter, femur, patella, tibia, metatarsus, and tarsus. The tarsus bears one straight and two curved claws. A slender waist connects the cephalothorax to the abdomen. The latter bears, at its anterior end on the ventral surface, two lateral brownish spots which are triangular in shape; these mark the positions of the book lungs. Posterior to each is a long, transverse slit, the spiracle. Spread the spiracle to see that it connects to respiratory lamellae of the book lung. The free edges of the leaves are supported by a hardened rib. Between the spiracles is a triangular process, the epigynum, which covers the female genital openings. Posteriorly on the ventral surface of the abdomen are three pairs of spinnerets and, behind these, the anal papilla. The anterior and posterior spinnerets are large, two-segmented, and movable. The middle pair is small and immovable. Anterior to the group of spinnerets look for a transverse slit; this is the tracheal spiracle (it is typically difficult to see) which opens to the tracheal system, a system of internal tubes that conduct respiratory gases directly between tissues and environment. The book lungs and spinnerets all represent modified appendages. Some specimens of the spiders have been bisected to show internal organs. Examine one of these to see if you can find the heart, gut, ovary, book lungs, and silk glands. Once you are familiar with the parts of the spider, watch a living spider and see if you can determine how the various appendages are used, how the spider uses its sensory organs, and other features of spider behavior. Mites and ticks Living water mites (hydracarina) are available for study of swimming motion. Most mites and ticks, of course, are terrestrial. Living mites extracted from leaf litter with Berlese funnels are available for study. Watch them under your dissecting scope to see how they walk, then mount one on a slide in a drop of water or 10% ethanol, apply a coverslip, and study its morphology using Figures in the textbook to see what you can of external and internal anatomy. Note the head-like capitulum (not a true head since it does not bear the brain) on which the mouth parts sit, the chelicerae and pedipalps. The four pairs of walking legs are located on a distinct division of the cephalothorax. The abdomen lacks appendages. In its ventral midline lie the anus and the genital pore, the anus behind the genital pore. If the cuticle is sufficiently translucent, you may be able to see some internal structures, such as the brain, the branched gut, and reproductive organs. Two parasitic acarines, Dermacentor variabilis and Dermanyssus, a common dog tick and the chicken mite are available as whole mounts on slides. Because these are cleared and flattened, it may be easier for you to see parts of the anatomy mentioned in the preceding paragraph. Note the dorso-ventrally flattened body (these specimens are not engorged on blood meals). Examining the specimen under the scope, you can see how the outer edges of the chelicerae are serrated so they are effective for cutting into skin. Beneath the chelicerae is the hypostome, an anterior projection of the sternum of the capitulum, with numerous spines on its sides; this is also a cutting organ, and together with the chelicerae, forms a sucking channel. The pedipalps lie lateral to the chelicerae; they are sensory. A scutum covers the dorsal side of the part of the cephalothorax that bears the walking legs. The abdomen in engorged specimens is greatly swollen. Pseudoscorpion (Pseudoscorpiones): Chelifer As their name suggests, pseudoscorpions resemble true scorpions, particularly because they have proportionally large chelate pedipalps; but they are quite small and the abdomen is simple (not divided into two parts) and does not bear a sting. A preserved specimen of Chelifer is on demonstration; live specimens may be available from the leaf-litter extraction or from student-captures in houses or the library. Pseudoscorpions prey on other small arthropods which they kill with poison glands in the pedipalps; the chelicerae are then used to tear open the prey so that its fluids can be sucked in. On the specimen available, find these appendages and the walking legs. The demonstration specimen is a male and on its ventral side two ram’shorn-like scent organs can be seen protruding from the genital aperture; the male uses these in a courtship

Arthropoda Chelicerata

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dance to bring the female to a spermatophore he deposits. Daddy longlegs (Opiliones): Liobunum Specimens of daddy longlegs, or harvestmen—most likely Liobunum sp., which is common locally in the late summer and early fall—will be available preserved on demonstration and possibly live. Note that the cephalothorax is covered by a single shield, or carapace. In the dorsal midline is a pair of eyes. The cephalothorax is united along its entire posterior margin to the segmented abdomen. The cephalothorax bears chelicerae, pedipalps, and four very long walking legs. Opiliones are non-poisonous predators. Adults arise from eggs in the course of a summer, and the eggs they produce are the overwintering stage. The adults generally do not survive the winter.

Pycnogonida: Anaplodactylus sp. or other pycnogonids Preserved specimens of sea spiders should be studied to see external anatomy. Males and females can be distinguished especially when the males are carrying egg masses in a pair of curled legs on the first segment; these are the ovigerous legs and appear as an extra pair of appendages. (Ovigerous legs are reduced or absent often in female pycnogonids; the males regularly carry the eggs). The head or cephalon bears a tubercle with four simple eyes and the anteriorly directed proboscis flanked by the chelicerae and pedipalps (but pedipalps are absent from some pycnogonids, including Anoplodactylus). The trunk usually comprises four segments, each with walking legs; the first segment is fused with the cephalon so it appears as if the first walking legs arise from the sides of the head. At the posterior end of the last trunk segment is a vestigial abdomen. The legs attach to lateral processes of the segments. You may be able to see eggs in the legs of female specimens; this is a peculiarity of pycnogonids—that is, having the gonads as well as branches of the gut extend into the legs.

40 MOLLUSCA Aplacophora (chaetodermomorphs and neomeniomorphans) Meiomenia - preserved (demonstration) Polyplacophora: chitons Ischnochiton - live, preserved Katharina, Cryptochiton - preserved Conchifera Monoplacophora (e.g., Neopilina) Ganglioneura Gastropoda (snails nudibranchs, slugs, etc.) (prosobranchs): Vetigastropoda Haliotis, Diodora – shells Patellogastropoda Testudinalia (= Acmaea) – live, shell Caenogastropoda (mesogastropods) Littorina – live, shell Crepidula – live, shell, slides (development) Neptunea, Strombus, Cassis – shells Neogastropoda Buccinum – live, shell Busycon – preserved (demonstration) Nucella (= Thais) – live, shell Conus – shell Euthyneura (opisthobranchs): Nudibranchia Dendronotus, Onchidoris, Aeolidia – live (pulmonates): Basommatophora Lymnaea, Bulimnea, Physa – live Planorbella – live (as available) Laevapex – live (as available) Stylommatophora Helix – preserved (demo) Limax, Arion – live, preserved Cephalopoda (squids, octopuses, nautilus) Nautilus — preserved, shell (demonstration) Doryteuthis (Loligo) paeleii (squid) — frozen, preserved, demonstration of eggs, eye Octopus — preserved (demonstration) Bivalvia (= Pelecypoda, Lamellibranchia) Protobranchia Nucula, Nuculana – preserved Metabranchia Mytilus (blue mussel) — live, shell Modiolus (horse mussel) — live, shell Mya (soft-shelled or steamer clam) — live, shell Mercenaria (quahog) — preserved, shell Placopecten, Aequipecten (scallop) — live, preserved, shell Crassostrea (oyster) — live, preserved, shell Aequipecten (bay scallop) — shell Ensis (razor clam) — shell Tridacna (giant clam) — shell Anodonta, etc. (freshwater mussels) — shells Teredo (shipworm; wood-boring clam) — preserved and shell Dreissena (zebra mussel) —shell Scaphopoda (tusk shell) Dentalium — preserved and shells on demonstration

Mollusca I

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Mollusca I – Aplacophora, Polyplacophora, Gastropoda aplacophorans (solenogasters, caudofoveates), chitons, gastropods Polyplacophora All chitons are marine, and most feed by grazing on attached organisms, usually algae but also sessile animals. The shell of chitons is composed of eight plates, and the mantle which secretes the plates and anchors them extends as a girdle encircling the edge of the animal. The mantle also covers the visceral mass. Most of the ventral surface is occupied by the broad, flat, muscular foot. Between the mantle edge and the foot is a groove in which the ctenidia (the technical term for the gills characteristic of the Mollusca) lie. Ischnochiton spp. (Figs. 12-6 – 12-9, pp. 293–296.) Living specimens of Ischnochiton ruber, which is the most common chiton on the New England coast, are available for study of behavior and external anatomy. Some of these animals have been placed on slides so that you can view them in extended position from the underside. Look also at living animals in the aquarium; if any are attached to the sides of the aquarium, you may be able to see grazing activity through the glass. Chitons are slow-moving, so watching for behavior may not be particularly exciting; watch at least for creeping motion of the foot on the substrate, testing of the substrate with the mouth and radula, and water currents generated by the ciliary activity of the ctenidia (gills) in the pallial groove flanking the foot (see below). We will also use preserved specimens of another species of Ischnochiton, probably a West Coast species, for studying external anatomy. It will be easier to poke and turn preserved specimens; be careful not to contaminate living animals with preservative from the fixed specimens. Some of these fixed specimens have had shell plates removed so that you can see mantle and muscles below and even outlines of some internal organs. In an intact animal, find the shell’s eight calcareous plates. These are partially embedded in the mantle of the dorsal surface and interlock by flanges, each plate overlapping the one posterior to it. Having such a jointed shell allows the animal to roll up when dislodged, almost like a pill bug. The mantle completely covers the dorsal surface of the animal and forms a so-called girdle around the margin of the body. The girdle bears small scale-like spicules on its dorsal surface. View the chiton under a dissecting scope to see the spicules and other details of the shell and mantle. In the animals with plates removed, note how the mantle folds around the space originally occupied by the plates and how muscles of the mantle insert on them. Turn the animal over and identify the broad, flat foot. Around the margins of the foot, between it and the mantle edge, is a deep groove, the mantle cavity or pallial groove. In the mantle cavity on each side lie serially arranged ctenidia. It may be necessary to pull the foot and mantle edge apart in order to see these gills. Note that each has a central axis which supports numerous gill-plates. By gently moving some of the posterior gills to the side, you may be able to see the renal openings (nephridiopores) and, anterior to those, the genital openings. Identify the head at the anterior end. The mouth is ventral. With a living specimen, study the manner in which currents are made to flow through the pallial groove by watching an animal attached to glass; add a drop of carmine suspension near the head of the animal and trace the motion of the particles. The current enters the pallial groove via temporary passages the animal makes by lifting the girdle at any point along its length. On demonstration are specimens of Ischnochiton with the shell plates partially and wholly removed. Examine these to see the musculature, and, in the dissected animals, internal organs. At the anterior end, find the buccal mass with its radula and the radula sac extending back toward mid-body. Flanking the bucal mass are two large glands, the so-called sugar glands used to digest carbohydrate in the algal diet. Exposed behind the radula sac are the digesive gland (mop-like in its branchiness), the coiled intestine, and the unpaired gonad lying dorsal in the mid-line (over the digestive gland for the most part), and, at the posterior end of the body, the pericardium with central ventricle and two lateral atria. Slides of the radula of a chiton are available. Study one to see the arrangement of teeth. Chitons harden the teeth by incorporating magnetite, an iron compound that is magnetic. On demonstration on the side table are two additional chitons for you to examine, Katharina and Cryptochiton. Both are West Coast species, and both have an expanded mantle that nearly (in Katharina) or completely (in Cryptochiton) covers the shell plates. One of the Katharina specimens has been bisected to show internal anatomy. On it, see if you can find the buccal mass with its radula at the anterior end of the animal, the unpaired gonad lying dorsal in the mid-line, and, beneath the gonads, the coiled intestine and the digestive gland. The space in which the viscera lie is a haemocoel. A pair of salivary glands, the so-called sugar glands, open into the pharynx. A short esophagus leads into the capacious stomach.

42 Internal anatomy of chitons1 . If you are interested in delving further into chiton anatomy, you may elect to dissect one of the preserved specimens. There are two approaches to this dissection, dorsal or ventral, to the internal anatomy of a chiton. Both require a sharp scalpel. Most students seem to find the dorsal approach easier. Dorsal approach. Starting at the anterior end of a fresh specimen that has been thoroughly relaxed or parboiled, cut away the girdle by holding the scalpel nearly parallel to the body surface and slicing through the thick, muscular girdle, removing the tissue a little at a time. Take care not to cut down into the mantle cavity or to let the scalpel slip under or between the valves. When the girdle has been mostly cut away, leaving only a thin portion next to the ctenidia, work around the shell plates, carefully cutting away all the muscular and connective tissue. When the shells are relatively clean, note how much of each valve was covered by the girdle. Then, beginning at the anterior end, and using only the tip of the blade, carefully free the first valve from the next posterior one. When the connections have been cut, place the tip of a dissecting needle or other small instrument under the center of the valve, at its posterior border, and pry up and forward. If the muscular tissue has been carefully cut, the valve will be easily removed. Keep the valves in order and examine them. Note that the most anterior valve varies in shape from the remaining ones. On the anterior valve the number of slits in the portion originally covered by the girdle is one of the characters used to classify chitons. Examine the dorsal surface of the chiton’s body and note how thin the mantle is directly under each valve and how it is expanded into muscular partitions between the shells. There is also a pair of longitudinal muscles near the midline of the dorsal surface. Note also that each valve is held laterally by a set of diagonal muscles. Working carefully, remove the mantle only from the anterior half of the body, exposing the viscera in this region. Ventral approach, while easier to visualize, may not give as good a result. For this method, cut away the foot of a specimen that has been parboiled. To do this, start at the posterior end and slice through the body wall, toward the midline, just above the sole of the foot and medial to the gill chamber. Take care not to cut too deeply into the visceral mass. Continue the cut nearly to the anterior edge of the foot, then make a similar cut on the other side. Carefully cut all the muscular and connective tissue away, so that the entire foot can be folded forward. Before attempting to expose the various organ systems in detail, you should recognize some of the main viscera. Note the long, coiled intestine, dark green in color, and the many-lobed greenish-yellow digestive gland, which follows the coils of the intestine. The single gonad (really a fusion of two in a pair) is located in the mid-dorsal region and extends from a point usually less than half way back from the anterior end to the pericardium in the posterior part of the body. The gonad is large and lobed. Two big arteries proceed from the pericardium directly forward just above the center of the gonad. Wash the specimen gently and then place it in the dissecting pan and cover it with water. Carry on the remainder of the dissection under water. Many of the structures in small organisms are so delicate that they cannot be seen unless “floated up” in water. Reproductive System. Carefully lift up the anterior border of the gonad and cut it away progressively, moving carefully, to expose the genital ducts. In the female, the ducts are large, somewhat expanded, and pale yellow-white in color. The ducts double back under the gonad, so that it is necessary to remove the reproductive gland to get a clear view of the ducts. Locate them and determine that the ducts bend laterally and ventrally to enter the mantle cavity through the genital pore already located. Circulatory System. The dorsal posterior one-fourth of the body is separated from the more anterior larger portion by a thin partition and forms the pericardium, containing the heart. Note the large, median ventricle, a roughly triangular structure located in the most posterior part of the pericardial cavity. Leading forward from the ventricle, two conspicuous arteries continue anteriorly dorsal to the gonad. To the sides and slightly anterior to the ventricle are the two auricles in which blood accumulates before being pumped to the ventricle. Of the vessels and channels composing the hemocoel, only the large vessels may be seen; the circulatory system is open, meaning the blood is not confined to cylindrical vessels but occupies sinuses and channels through most of the body. The blood contains a blue pigment, hemocyanin, which functions in oxygen transport. Excretory System. The kidneys can be seen as thin, long, flattened, many-lobed, light yellow structures lying along the ventrolateral portions of the body cavity in Katharina, or, in Mopalia, embedded in the 1 Adapted

from laboratory handout of Alan Kohn

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ventrolateral muscular body wall. They extend from near the anterior end to the pericardial cavity and then expand into a pair of thin tubular ducts lying beside and below the ventricle and leading to the outside through the excretory pore you already found in the mantle cavity. If you used the ventral approach, now examine the dorsal surface of the chiton. Beginning at the anterior end and using only the tip of the blade, carefully free the first valve from the next posterior one. When the connections have been cut, place the tip of a dissecting needle or other small instrument under the center of the valve, at its posterior border, and pry up and forward. If the muscular tissue has been carefully cut, the valve will be easily removed. Place the first valve to one side, and repeat the process with the next succeeding valve. Note that the most anterior valve differs in shape from the remaining ones. On the anterior valve, the number of slits in the portion originally covered by the girdle is one of the species-specific characters used to classify chitons. Digestive System. Note the heavy bands of muscles surrounding the anterior part of the digestive system. On each side of the esophagus, just lateral to these muscles, is located a lobed structure, hard and light green in preserved material, the salivary glands. The small paired salivary ducts enter the posterior and ventral part of the mouth; they are not easily demonstrated in preserved specimens. Beginning at the anterior tip, cut into the dorsal part of the mouth cavity; continue the cut a very short distance posteriorly (do not cut through the heavy muscle bands) and extend it laterally to expose the anterior end of the radula. Note the position of the radula and the form and structure of its teeth, and be able to describe its functioning. The heavy reddish muscle bands in this area may now be recognized as those that operate the buccal mass or odontophore. Cut through them, continuing the cut in the mid-dorsal line, but working carefully and looking for the nerve ring which surrounds the esophagus. Continue the cut into the dorsal surface of the esophagus, revealing the full extent of the radula. Observe that the posterior part of the radula is enclosed within the radular sac. Remove the radula with its sac and examine its teeth under the microscope. With the radula removed, examine the floor of the mouth and anterior end of the digestive system. Note the cartilaginous material over which the radula slides. The term odontophore is usually applied to the entire complex of radula, the muscles which operate it, and the cartilage, which is then called a bolster. Some authors use the term odontophore for just the cartilage. Tracing backward from the esophagus, note the long, coiled intestine, dark greenish in color, and the yellow, many-lobed digestive gland that follows the coils of the intestine. If you have not yet studied the gonad, remove only its anterior part which may conceal other structures in this region. Locate the straight posterior part of the intestine lying in the ventral part of the body cavity. Cut through the intestine anterior to the genital pores and posterior to the odontophore and remove the entire tract. Nervous System. Dissection of the nervous system is difficult. The anteriormost portion of the nervous system is a half-ring, lying in a canal-like space in connective tissue in front of the mouth and more or less in the horizontal plane of the animal. Take small slices from the top and front of the head and work down gradually to try to expose the nerve ring. Proceeding posteriorly on either side of the body from the nerve ring are two longitudinal cords that extend back into the body of the chiton. The more lateral of these is the pleurovisceral cord (also called pallial cord), lying above the gills. The more medial and ventral cord is the pedal cord. At the juncture of the anterior half-ring with the two major longitudinal cords, three additional smaller nerves arise and proceed medially. The middle one is actually the buccal commissure, and it completes the circumbuccal ring. Fore and aft of the commissure, paired nerves arise as the remainder of the trio on each side. The anterior pair innervate the buccal mass, and in it there is a pair of buccal ganglia, connected to the nerve cords. Posterior to the buccal commissure the subradular nerve cords run to a pair of ganglia that lie on a subradular organ, which they innervate. The buccal ring proper gives off a very large number of fine cerebral nerves to the buccal aperture and to the head. When the dissection of the general features of the nervous system has been done (or attempted), the remaining emptied husk of the chiton body, mainly foot and lateral body walls, should be sectioned cleanly in the transverse plane at about the middle. Inspection of the cut surfaces should show the passages—and perhaps the contained cords—of the pedal and pallial nerves.

44 Aplacaphora (Fig. 12-5, p. 292). Representatives of this class of small vermiform (worm-like) molluscs live in sand and mud or on soft corals in deeper marine environments. Rather than having a shell, these molluscs have a spiny cuticle, and the foot is reduced. In the specimen of Meiomenia on demonstration, note the brightly refractile spicules in the cuticle and the absence of a pronounced foot, which here is in the form of a ciliated groove. Gastropoda This class is by far the largest and most diverse in the phylum. It includes snails, limpets, slugs, sea slugs, and many more. The major unifying feature of the class is the ontogenic phenomenon of torsion, where the visceral mass is twisted 180o relative to the head and foot, bringing the gills to the front, just behind the head (“proso” means front, “branch” refers to the gills, and so the name “prosobranch” refers to the position of the gills in front—specifically in front of the heart). Three degrees of torsion and its loss are evident among gastropods. Prosobranchs are the marine gastropods with complete torsion—that is, the mantle cavity, with its ctenidia and the anus and pores of the excretory and reproductive organs, is positioned over the head—and they typically have large external shells. In specimens of Littorina, Buccinum, Busycon, Nucella, or other rather typical prosobranchs, note the conispirally coiled shell, the foot with its operculum or door that can close off the shell aperture, the inhalant siphon (a fold of the mantle), and the head. Peak under the shell opening to appreciate the position of the mantle cavity and to see if you can find any gills. Within this mantle cavity will also be the anus and nephridiopore, etc., facing forward. Also observe prosobranchs with highly modified shells, such as the limpets (Testudinalia) (=Acmaea)) or “slipper shells” (Crepidula, Fig. 12-49). Opisthobranchs, almost all of which are marine, have the openings of the reproductive and excretory systems (pores associated with the mantle cavity) on the right side of the body, while the anus is often posterior. (They are said to be “detorted” because the arrangement of internal organs appears to reflect reversal of torsion rather than partial torsion.) The shell of opisthobranchs tends to be thin, small, or absent, and the body is often not coiled but flattened and quite bilaterally symmetrical. Associated with reduction of the shell is loss, in some species, of the ctenidia, so respiratory exchange occurs through secondary respiratory extensions or generally through the skin. Some typical living opisthobranchs we have available are the nudibranchs which lack a shell (others have a shell though often reduced). Pulmonates are terrestrial. Among them are both snails and shell-less slugs. Many live in fresh water but must come to the surface to breathe air; these typically have a large but thin shell. Like opisthobranchs, pulmonates have undergone considerable detorsion. In a living slug, are the head and foot demarcated from each other? Note the two pairs of tentacles. What sensory modalities do you think they represent? Note the mantle and the opening to its cavity, called the pneumostome. Do you see evidence of air-breathing? The mantle cavity in pulmonates is, as this name indicates, modified as a lung. Prosobranch gastropods The prosobranch gastropods are so-called because the mantle cavity, with its ctenidia, is in an anterior position. Different groups of prosobranchs have adapted differently to the problems with sanitation that moving the anus and excretory pores over the head near the gills produce. Vetigastropoda (“archaeogastropods,” in part) Haliotis sp. (Fig. 12-21). Shells of Haliotis, the abalone, are on demonstration at the side table. Haliotis has a pair of ctenidia which are more or less displaced to the left, under the row of respiratory perforations in the shell. The anus opens under the perforations, also. Abalone is a choice Westcoast seafood, and a favorite prey for skindivers. After proper tenderization (pounding) by the chef, the muscular foot is served as abalone steak. Because of overcollecting, abalone species are endangered, and attempts are underway to culture abalones for marketing. Important primitive features include the respiratory perforations of shell and mantle, the spiralled shell, presence of two ctenidia and two auricles, discharge of the right gonad via the right kidney, and separate sexes, with no sexual dimorphism such that the gonads and gametes provide the only evidence of the sex of the individual. Diodora sp. (also on demonstration; Fig. 12-22), a keyhole limpet, has a single hole at the top of the shell for the exhalant water current. Patellogastropoda (“archaeogastropods,” in part)

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Tectura (= Acmaea) , the tortoiseshell limpet. (Fig. 12-24, p. 309). Patellogastropods have limpet-like shells without an apical opening, a single (left) ctenidium or none, and a markedly different radula from that of the vetigastropods. Like most other prosobranchs, Tectura has only one ctenidium, the left one; and you can see it extending from the left over the back of the head. The ctenidium shows the more primitive condition of ctenidia in being bipectinate, having filaments on both sides of its axis. Find an animal that has attached to glass and watch it from its underside and watch for motion of the foot, tentacles, and radula. Using a pipette, add a drop of carmine suspension near the head and record how the currents flow through the mantle cavity. The intestine is visible from the ventral side as a dark line circling around the visceral mass to open via the anus on the right of the ctenidium. The patellogastropod limpets represent an advance in structure over that of vetigastropods, with a number of basic features that are rather typical of the coiled gastropods. If time and interest permit, look more closely at anatomy of the limpet by removing the body from the shell. The head bears somewhat reduced tentacles. The mantle cavity is shallow. Cut the mantle to expose the mantle cavity and look at the single ctenidium on the left side. On the right side, the anus terminates in the tubular rectum. Osphradial tissue may be visible as colored patches in the “neck” region. In a carefully dissected specimen, the auricle may be seen at the base of the ctenidium; the ventricle is ventral to it. Nephridiopores are found to either side of the anus in favorable specimens. The kidneys are paired but the right kidney is considerably larger than the other, its duct serving also as gonoduct. The visceral mass is elaborately coiled and includes a great deal of spongy digestive gland. Entwined with this are coils of the radula; it reaches amazing lengths in some species. The shell, mantle cavity, and shell muscle are quite symmetrical, but asymmetry is seen in the loss of the ctenidium and auricle on the right (downstream) side, in the reduction of the kidney and gonad on the left (upstream) side (gonad lost, kidney smaller), and in the deflection of the anus to the right. Caenogastropoda constitute the remainder (and most) of the prosobranch gastropods. These gastropods have a monopectinate gill, which may have facilitated expansion into habitats with sediments. Some species, informally grouped into what are known as mesogastropods, are mostly herbivorous grazers; others, informally grouped as neogastropods, are largely carnivores. These groups are represented here, respectively, by Littorina littorea and Nucella. Littorina littorea is the common periwinkle on the Maine coast. It is an imported species, not native to North America but probably brought with ballast in ships from western Europe; it was introduced first in Nova Scotia and has spread south as far as Maryland and at densities greater than in its native Europe where it is limited by competitors. Spreading has come through its planktonic egg capsules which the females release during the warmer months of the year; each capsule is a delicate transparent case in the shape of a flying saucer; and each encloses one, sometimes two, eggs. Adults feed on algae growing on rocks in the intertidal zone. Find an animal that has attached to glass and watch it move by waves of muscular contraction in the foot. Note the division of the foot along a median ventral line. Pull gently on the shell to see how the snail is held in place by suction action of the mucus-lubricated foot. The snails to be dissected have been relaxed in magnesium chloride and are in a separate finger bowl. Observe the state of contraction and note the position of the operculum, which serves as a cover over the shell aperture when the snail is withdrawn. Crack the shell with the special red snail-cracking pliers our use a gentle blow with a hammer while the shel sits aperture-down on a metal plate. Once the white columellar muscle that attaches the shell to the body is exposed, scrape it from the shell remnant (don’t cut it off the animal) and place the shell-free body in a finger bowl with magnesium chloride. Perform the dissection under a dissecting microscope. Figures of Littorina removed from its shell are on the page at the end of this section; also compare the appearance of your animal with the textbook Figs. 12-14 and 12-45, pp. 301 and 325. Note how the visceral mass is coiled. Observe the snail from all sides in order to understand the spatial relationships among its various parts. Find head, with snout, tentacles and eyes; mantle, with its free outer fold extending down over the head; foot, located ventrally, with operculum attached to its posterior dorsal surface; dorsal coiled visceral mass; columellar muscle, the whitish mass extending back from the foot. Before dissecting, try to identify all the structures visible through the body wall, again noting the relationships between parts. Keep in mind that the head is anterior and the foot ventral. Try to visualize

46 the orientation of the visceral mass within the shell of an undissected individual. Note that “right” always refers to the snail’s right. The dark area behind the mantle edge on the snail’s left side marks the place of attachment of the single pectinibranch gill. To the left of the gill is a thin tan line that extends back from the mantle edge along the left side of the animal; this is the osphradium. Just behind the osphradium and dorsal to it is the pericardium which looks like a delicate sac and contains the two-chambered heart. Above the pericardial cavity, and extending posteriorly from it, is a conspicuous, light-colored, mottled organ, the kidney. To the right of the gill lie the terminal portions of the digestive and reproductive systems. The major portions of the digestive and reproductive systems make up most of the rest of the visceral mass, which extends upward and posteriorly in a coil which narrows to a tip. This coil consists mainly of the brownish digestive gland. Blood vessels are scattered over the surface of the visceral mass; they appear as delicate silvery canals. The gonad is diffuse; it lies over the digestive gland and generally follows the pattern of the blood vessels. When a snail is in a non-reproductive condition, the gonad is reduced and consists of small amounts of dark material associated with the blood vessels. The stomach can be identified easily; through the body wall it appears as a long, sac-like structure extending backward in the visceral mass and terminating approximately one full turn before the tip. Along its surface runs a conspicuous, highly branched blood vessel. With fine scissors cut through the mantle along the right side of the gill, beginning at the mantle’s free edge and continuing to the anterior end of the kidney. Pin back the mantle on both sides. (It is possible to pin the cut flaps simply by anchoring the pins in the foot; you may prefer to use a dissecting tray for the pinning. Compare the appearance of the opened animal with Figs. 12-14 and 12-45). If the animal is a male, there will be a penis behind the base of the right tentacle. On the left fold are the delicate gill filaments. The osphradium appears as a channel on the gill’s left side along the base of attachment. The rectum extends along the cut edge of the right mantle flap. Its terminal part hangs free, directed toward the opening of the mantle; it terminates with the anus. Lying between the right edge of the gill and the rectum, where you have cut, is a modified portion of the epithelium forming a glandular mass called the hypobranchial gland. The genital system parallels the rectum and lies lateral to it in the undissected animal, medially to it with the right mantle flap pinned out. If your specimen is a female, find the opening to the female system; it is on a projection somewhat behind the anus. The opening of the kidney into the mantle cavity lies at the extreme posterior portion of the cavity in both sexes. With fine scissors cut open the kidney; note that it is a sac-like structure, the walls of which are variously modified. Look for ciliary currents along these walls. Put the tips of fine scissors into the mouth and cut posteriorly a short distance along the snout in the mid-dorval line, opening the buccal cavity. Continue this cut posteriorly, being careful to cut only through the superficial skin layers so as to avoid damage to underlying structures. Note within the opened buccal cavity the functional portion of the radula, extending across a mass of reddish muscles. The radular sac lies behind the buccal cavity and appears as a conspicuous coiled tube. The esophagus extends back from the buccal cavity and can be traced easily some distance behind the head. At the anterior end of the esophagus are two conspicuous lateral outpockets, the esophageal pouches. Just behind these will be found an anterior concentration of nerves. The ganglia can be recognized by their yellowish color. Carefully dissect away nonnervous tissue, including the radular sac and the salivary glands, which appear as whitish masses on either side of the esophagus. This will reveal the nerve ring (Fig. 12-53). There are two dorsal cerebral ganglia, connected by a commissure. Lying behind each cerebral ganglion is a pleural ganglion, and below each cerebral ganglion is a large pedal ganglion. The pleural and pedal ganglia are attached to their respective cerebral ganglia by cerebro-pleural and cerebro-pedal connectives. On either side of the esophagus, in front of the esophageal pouches, are conspicuous buccal ganglia; each is connected to its respective cerebral ganglion. There is a statocyst associated with each pedal ganglion; these are not easy to see, however. From the right pleural ganglion a conspicuous nerve passes posteriorly and crosses over the esophagus; it leads back to a ganglion on the left side, the supra-esophageal ganglion (the pretorsion right parietal ganglion). A corresponding nerve passes from the left pleural ganglion, running below the esophagus to a ganglion on the right side, the sub-esophageal ganglion (the pretorsion left parietal ganglion). From each parietal ganglion connectives pass posteriorly to a visceral ganglion. The tight visceral ganglion is the pretorsion left one, and vice-versa. An appreciation of the complications of torsion can be achieved by trying to visualize the twisting required to convert a bilaterally symmetrical nervous system to the one seen here. Students feeling sufficiently interested can dissect out the entire digestive system by opening the esophagus and tracing it into the stomach, and then cutting along the length of the intestine and rectum to the anus. An alternative dissection could also be done by cutting into the stomach and finding the openings of the esophagus and intestine. Note the relationship of these openings to each other. On the wall of the stomach

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is the gastric shield and various folds and ridges. The appearance of both male and female reproductive systems varies considerably depending on the reproductive state (see Fig. 12-56). As noted before, the gonads are quite diffuse and appear in developed individuals as highly branched structures lying over the surface of much of the digestive gland. In females there is, on the right side, a conspicuous spiral-patterned oval structure of grey or cream color. From this structure a duct parallels the rectum on the right site of the snail, and leads to the female opening. In males, the lower extremity of the sperm duct is highly coiled and enlarged with sperm during the reproductive season. This duct opens into the mantle cavity by a pore situated deep within the mantle cavity, to the right of the rectum (medial to it in the dissected animal). This pore discharges to a ciliated groove which runs forward along the floor of the mantle cavity. Along most of the length of the rectum, this groove is covered over by a rich glandular field, the prostate gland. The groove passes forward until it reaches a position posterior and ventral to the right tentacle, where it runs onto the large penis. The penis is highly muscular. It is divided into a stout basal portion and a narrower terminal part. It serves to transfer sperm to the female during the reproductive season. Nucella lapillus (Dogwinkle, dogwhelk). This is a drill, preying on mussels and barnacles by drilling holes in their shells and consuming the soft parts by protruding the proboscis through the hole. It was once proposed that color variations arise from diet: those feeding on mussels supposedly having blue in the shell, those feeding on barnacles whiter or yellower shells; this proposition has been questioned recently. Shells of other prosobranchs on demonstration Topshells, primitive vetigastropods. Buccinum undatum, wavy whelk. (Fig. 12-14, p. 301). This is common on the Maine coast; juveniles are found in the intertidal zone, adults in deeper water (often in lobster traps and in Downeast grocery stores). Busycon canaliculata, common east coast whelk. Shell, preserved animal, and egg case on demontrastion; the preserved animal can be compared (briefly) with the dissected Littorina. Cassis, the helmet shell, and Strombus (Fig. 12-32B), the queen conch. Conus, a cone shell (Fig. 12-47). Cone shells harpoon their prey with a single radula tooth that is modified as a dart and connected with venom glands. Many species are potent enough to be fatal to human beings. ˙ Opisthobranch gastropods (Figs12-34 – 12-36, pp. 317-318). The mantle cavity is displaced to the right or the posterior side in opisthobranch gastropods–that is, they have gone through detorsion–and they show trends to reduction and loss of the shell. Opisthobranchs probably evolved from prosobranchs, and because of the detorsion, the nervous system is no longer crossed (X-shaped). More primitive members retain the single ctenidium of the prosobranch ancestor; in others it is lost and commonly replaced by secondary respiratory structures. The opisthobranch reproductive system is hermaphroditic; even in the most symmetrical species, the openings of the reproductive system are on the right side. As in the mesogastropods, only the left kidney is present and the gonad and its duct are entirely derived from the post-torsional right side. Morphologically the Opisthobranchia is a very diverse subclass–including bubble shells, sea hares, sea butterflies, nudibranchs, and minute interstitial forms–and there are many distinctive orders; physiologically the Opisthobranchia is quite conservative as almost all members live in subtidal marine habitats. We will concentrate on the Nudibranchia, more commonly represented on the Maine coast. Nudibranchia, the sea slugs. In all members of this order, there is no shell, ctenidium, or mantle cavity. Instead of the ctenidium, most have conspicuous secondary gills, either as branching plumes surrounding the anus or as projections called cerata (singular: ceras) on the dorsal surface. Some nudibranchs feeding on cnidarians are able to use the nematocysts (stinging organelles of the cnidarians) they ingest to defend themselves by moving those nematocysts in an undischarged state out to the cerata where they are positioned to fire. Branches of the gut extend into the cerata, and these branches deliver the nematocysts to the cerata. Aeolidia (Fig. 12-34G). The many cerata (up to 400 on each side) and golden color of this animal give it its common name, maned nudibranch. When it is contracted, it looks something like a sea anemeone (as in the case of the preserved sample we have available). Dendronotus (Fig. 12-34I). The most conspicuous morphological feature is the cerata which cover the dorsal surface and which in this genus are branched (hence the common name, bushy-backed sea slug); these function as respiratory organs. On the head are two pairs of tentacles; the anterior pair are called oral tentacles, the posterior pair are called rhinophores. Near the base of the rhinophores are eyes. On the ventral side of the head find the mouth. On the right side of the head there is a conspicuous genital aperture, and behind it the small anus. Pulmonate gastropods. Like the opisthobranchs, the pulmonates are usually regarded as a highly spe-

48 cialized offshoot of the prosobranchs. The mantle cavity functions as a lung; there are no ctenidia. The nervous system has lost its cross, but by shortening of the connectives rather than by untwisting as in the opisthobranchs (Fig. 12-54B). The effects of torsion remain: e.g., the auricle is anterior to the ventricle. The right kidney is absent. Elaborate accessory sex organs are present. Like opisthobranchs, pulmonates are simultaneous hermaphrodites. Reproductive, anal, and excretory openings are typically just outside the mantle cavity. The shell is typically spiralled, although there are some limpet-like fresh water and marine species. Also, (most) pulmonates lack an operculum. Basommatophora. The eyes sit at the bases of the tentacles in these pulmonates. The freshwater snail Lymnaea stagnalis is available live for observation as a representative of this group. (Similar in shape are Bulimnea, which has a thicker, less pointed shell, and Physa, distinguishable by having fingerlike projections of the mantle on either side of the shell aperture.) The shell of Lymnaea and relatives has transparent parts through which you may be able to see the heart beat and some of the feeding motions. Watch also for breathing motions. Specimens crawling on the water surface or on glass can be watched to see how the underside of the foot has waves of contraction that propel the snail along. Specimens of other freshwater pulmonates we have available, Laevapex (which has a limpet like shell) and Planorbella (which has a planospiral shell), may be even more transparent so that you can see virtually all internal organs through the shell. Watch one under a dissecting scope and note the beating heart and the motions of the radula. What other internal organs are visible? Stylommatophora. The eyes are at the tips of the tentacles in this group (Fig. 12-28, 12-43, pp. 312, 323). Helix pomatia, the common land snail, is a much-cited example and the stuff of escargot in French cuisine; some preserved specimens are available. Slugs are common representatives seen locally, including Limax maximus and Arion. We will dissect either of these with the following directions. (With such a lengthy description and complex organism it would be best to carry out this work in pairs, with one person reading the description aloud as the other follows it and identifies the structures.) Limax maximus and Arion spp.. The regions of the body visible externally are the head, mantle, elongate visceral mass or trunk, and foot. The shell is rudimentary in these slugs and is covered by the mantle, which takes the form of a fold of tissue on the anterodorsal surface. The muscular foot forms the entire ventral surface. It has three longitudinal muscular zones in Limax, and the medial one is particularly apparent on a live slug crawling on a glass plate; Arion shows no subdivided zones in its foot. The head bears two pairs of elongate tentacles. The upper, longer pair (the superior tentacles) taper anteriorly, and when fully extended terminate in distal bulbous enlargements that bear the eyes. The shorter, more ventrally placed inferior tentacles are also tapered and terminate dorsally in a slight enlargement. They seem to function as olfactory and tactile organs. The mouth, bounded by fleshy lips, is anterior and ventral, just ventral to the inferior tentacles. When the slug eats, it pushes the lips apart, and protrudes the active edge of the radula and the single, dorsal horny jaw. Just below the mouth is the opening of the pedal mucus gland, a horizontal slit. On the right side of the head, a little behind the superior tentacle, is the genital aperture. It cannot always be seen with the naked eye. When it is apparent, it appears as either a brownish depression or sometimes as a white disc. In preserved slugs, part of the genital atrium complex protrudes from the center of the disc, and in some, the penis protrudes as well. Just behind the head is the oblong fleshy mantle, under which the head is pulled when the slug is inactive or irritated. On the right side of the mantle is the pneumostome, the opening to the mantle cavity or “lung”; it opens and closes at times to allow air to enter or leave. A little posterior and lateral to the pneumostome and under the flap of the mantle is the anal opening. The vestigial shell is housed on the inner surface of the posterior end of the mantle.

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Optional dissections Dissection of a neogastropod (Buccinum)2 . If Buccinum is available, and you are inclined to study its internal anatomy further, follow these directions for dissection. Most organs are similarly positioned in both, but Buccinum has unusually large salivary glands. The specimens have been parboiled to facilitate dissection. Sometimes the animal can be twisted intact out of the shell. More likely, however, you will have to crack the relatively thin shell with a vise or hammer. If done carefully, this will not damage the underlying tissues. You should work in pairs. To study the internal anatomy, open the body wall that forms the floor of the mantle cavity. If you are working on Buccinum, the two salivary glands exposed here will be quite large, filling the body space. To the right of the salivary glands, find the gut closely applied to them and partially below them. These glands can be seen to be made of two parts, with the anterior smaller portion differing externally in color and texture. It is firmer, yellow, and closely applied to the posterior, softer, more granular, light yellow portion. These two salivary gland complexes are actually paired structures, but when Buccinum is opened, the larger right one is seen to be displaced so as to lie posteriorly to the smaller left one. Together the two form a smooth, cone-shaped mass. If the two glands are carefully displaced to the left side, their small, white ducts will be seen running from the ventral surface of the anterior portion, along the esophagus and through the nerve ring to the buccal mass. To observe the esophagus and buccal mass, slit the proboscis musculature carefully dorsally along its length. Here, the two salivary ducts are stout, firm cords that enter the buccal mass near the entry of the esophagus. The salivary secretions of Buccinum have not been studied but undoubtedly are toxic substances used in overcoming prey. In related species they produce an acidic saliva which may help overcome prey. From the buccal mass at the tip of the proboscis, the esophagus runs down the proboscis and through the nerve ring. It then receives a pair of closely applied esophageal glands. The anterior one is whitish and thick-walled, the posterior one thinner walled and brown. The esophagus then passes to the stomach, which receives the digestive gland ducts. The large digestive gland surrounds the stomach and extends posteriorly to the end of the coiled visceral mass. The intestine leaves the stomach posteriorly and proceeds anteriorly through the kidney and finally along the roof of the mantle cavity where the anus opens to the right of the hypobranchial gland. Feces are flushed out of the mantle cavity with the exhalant water flow. Note that the intestine does not pass through the heart as in Diodora or bivalves. The kidney is clearly visible posterior to the hypobranchial gland. The nephridiopore is slit-like and opens anterior to the kidney into the mantle cavity. A large blood vessel runs from the kidney to the thin-walled auricle of the heart. Another vessel enters the auricle from the ctenidium, bringing oxygenated blood. Blood from the auricle enters the muscular ventricle and is pumped into an aorta that divides into two branches a short distance from the heart. One branch proceeds anteriorly as the cephalic artery and the other posteriorly as the visceral artery. The former can be seen passing to the right of and parallel to the esophagus; this vessel transports blood to the head and foot. The testis of the male is a pink mass in living specimens, found in the coil of the visceral mass next to the digestive gland. A highly convoluted sperm duct (vas deferens) is visible between where the intestine leaves the stomach and then passes through the kidney. From here, the sperm duct can be traced along the floor of the mantle cavity to the base of the large penis, where the duct opens into a ciliated groove. The ovary of the female is a brownish mass located in the same position as the testis. The oviduct runs anteriorly to the capsule gland on the roof of the mantle cavity, passing first through a small, round structure that may be an albumen gland. Anteriorly, the ducts opens into a wide area that is likely a copulatory bursa. Eggs contained in a membrane-bound capsule are passed from the opening of the duct down a temporary groove in the foot. The capsule passes into a glandular area on the sole of the foot, where its outer covering is toughened. It is then cemented to the substratum. Make sure you see both male and female specimens. The circumesophageal nerve ring is located anterior to the large salivary glands. Carefully remove the thin membrane of connective tissue that covers it dorsally to expose the nerve ring. Cut the ring and remove the esophagus. Laterally, the dark red cerebral and pleural ganglia are fused. Ventral to these structures are the pedal ganglia. The buccal ganglia can be found at the anterior end of the esophagus by lifting the esophagus up to reveal the ventral area just posterior to the buccal mass. The visceral ganglia are difficult to locate. 2 Adapted

from laboratory handout of Alan Kohn

50 When you have completed your observations of the nervous system, remove the radula, make a water mount of it and observe the arrangement of the teeth. How many teeth are in each row?

Optional dissections Dissection of sea slug (Onchidoris)3 . Onchidoris. The dorsum is covered with small bumps or tubercles and black-brown spots found both on and between the tubercles. The pair of sensory rhinophores is located anterodorsally. Each consists of a stalk with numerous pairs of folds. The rhinophores can be retracted into the body wall. Posterodorsally the anus opens medially, surrounded by the circle of branchial plumes; these also are retractable into the body wall. Seven plumes, each branching 3-4 times, would be the normal complement. The large, broad foot is ventral, as is the mouth, at the anterior tip of the body. The genital opening is on the right anterior tip of the body. The genital opening is on the right anterior side between the foot and the dorsal wall. To view internal structures, make a longitudinal incision the length of the dorsal midline, using very sharp scissors. End the incision at the branchial plumes. The dorsal wall is very thick (3 to 4 mm) so a deep cut must be made but care must be taken not to damage internal organs. Lateral incisions made just posterior to the rhinophores and at the anterior edge of the branchial plumes make it possible to pin out the two dorsal flaps, exposing internal structures. Note first that all internal organs are enclosed in a thin, transparent coat, the outer perivisceral membrane. There is also an inner perivisceral membrane, difficult to distinguish from the outer. See the diagram of internal structures for orientation with regard to location of heart, buccal mass, dorsal ganglia, digestive gland, intestine and the anterior genital mass, then proceed to explore in more detail. Heart. The heart is mid-dorsal, just slightly posterior to the middle of the body. it is enclosed in a large transparent sac, the pericardium, formed by a delicate membrane which is easily broken and may not have survived the original incision. The ventricle is usually yellowish. If the animal is anesthetized the ventricle will be seen to contract regularly to pump blood through the single anterior aorta, which must be made out with the aid of a dissecting microscope. The delicate auricle stretches from the ventricle to the base of the branchial plumes. Varying degrees of relaxation of the animal will vary the position in which the heart will be found. It may be as described, or may be pulled very close to the branchial plumes by contraction of the body wall. Kidney. The renopericardial canal is found within the pericardium, just to the right of the auricle. It is a round orange mass with a dorsally located distinct opening, the renal syrinx. This conducts to the kidney, a branching system of white canals, located on the surface of the hermaphrodite gland, which in turn surrounds the large digestive gland. The kidney duct is very difficult to trace. Nervous system. Nervous structures should be traced next; this necessitates removing the perivisceral membranes. [You do not need to spend a lot of time on the nervous system; the details are provided here for completeness. But remember that the opisthobranch brain has proved to be exceedingly valuable as a biological model in studies of how nerve cells interact to control complex behaviors. Why is this so?] Note the bright orange ganglia positioned dorsally over the junction between the buccal mass and the esophagus (in contracted specimens, they may appear to lie over the buccal mass). Use 2 pairs of forceps to tear the thin membrane from the center of the animal toward the sides, keeping an eye on the ganglia and nerves to avoid damage. Remove the membrane from over the buccal mass, digestive gland and anterior genital mass. Now return to the ganglia and note still additional covering, a thin semi-transparent connective tissue and a large orange hematic gland, both associated with the vascular system. Remove these structures with great care, rendering the ganglia more sharply visible. Refer to the diagram and identify the following: the cerebral ganglia are most anterior, with black eyespots located at their lateral edges. Nerves can be traced from these ganglia which innervate the buccal mass, the mouth and related structures. Just posterior to the cerebral ganglia, somewhat more ventral, are the bright orange, round pedal ganglia, which innervate the foot. The pleural ganglia are most posterior in position, somewhat dorsal to the pedals, on about the same level as the cerebrals, best identifiable by their nerves which can be traced to the mantle. Individual neurones can be observed in the pleural ganglia with the aid of high power of the dissecting microscope. They look like dark orange spots on the surface of the ganglia. 3 Adapted

from laboratory handout of Alan Kohn

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The cerebro-buccal connective can be seen circling the esophagus at the point where it connects to the buccal mass. By gently pulling the esophagus with forceps up and to the left, the connective can be followed to the ventral orange subesophageal ganglia. These lie at the posterior end of the buccal mass, ventral to where the esophagus joins it and just dorsal to the radular sac. The largest, most ventral of these subesophageal ganglia are the buccal ganglia, giving rise to nerves to the buccal mass and radular sac. Just dorsal are the smaller gastroesophageal ganglia with nerves to the ventral side of the esophagus. Parallel to the cerebro-buccal connective can be seen the much larger circumesophageal commissure, circling the esophagus to connect the pedal ganglia. Digestive system. Proceed to study the digestive system beginning with the buccal mass. Locate it as the anteriormost structure and with a sharp scalpel sever the mass from the inner body wall as far anterior as possible - at the mouth. Carefully work around, severing all the nerves and muscles holding it in place but carefully avoiding damage to the anterior genital mass. Gently pull the buccal mass to the left and turn it on its side to locate ventrally the radular sac, a slight protrusion ventrally at the most posterior end. Just dorsal to the radular sac are the buccal and gastroesophageal ganglia examined earlier. Slightly dorsal to the ganglia are two slender, yellow salivary glands. Locate the esophagus and follow it from the buccal mass to where it enters the stomach ventrally. The stomach is a very thin-walled organ embedded in the large, roughly heart-shaped digestive gland. By gently working between the stomach and digestive gland, severing thin connectives holding them together the large opening of the stomach into the digestive gland may be located. It is actually very close to where the esophagus enters the stomach. Just ventral to this junction of esophagus and stomach, and slightly to the left, is a small diverticulum, the caecum. Note that the stomach wall becomes quite thick and muscular anteriorly as it approaches the intestine, which exists from the dorsal anterior part of the stomach, doubling back over the surface of the stomach and the right lobe of the digestive gland to exit at the anus located within the circle of branchial plumes. Finally return to the buccal mass and carefully cut it open dorsally to reveal the radula and the cartilaginous odontophore. By slitting the entire stomach through the caecum and into the digestive gland, the contents (if any) may be observed and once these are removed, the openings from the stomach to the digestive gland will be apparent. Reproductive system. Study of the reproductive system should start by locating the hermaphrodite gland (ovotestis), a light yellow layer of tissue completely enclosing the large, centrally located digestive gland (in ripe individuals the gland may be a rusty color streaked with white). The hermaphrodite duct originates on the anterior tip of the right lobe of the hermaphrodite gland and runs anteriorly to the anterior genital mass. The duct widens into the white, thin-walled ampulla, quite convoluted, and attached by connective tissue to the left, anterior tip of the large, orange mucus-albumen gland. The ampulla stores sperm, so constitutes a seminal vesicle. Examination will show the mucus-albumen gland is made up of a soft, bright orange mucus gland externally and a harder, more dull orange albumen gland internally. As the convoluted ampulla enters the mucus gland, just beneath the surface of the latter, it bifurcates, giving rise to a flesh-colored vas deferens and an oviduct. The vas deferens is a convoluted, hard-walled tube which leaves the mucus gland very close to where the ampulla enters. The vas deferens can be carefully untwisted with a pair of forceps by cutting the minute connectives which hold it in close proximity to the mucus gland. The vas deferens proceeds laterally to enter the large, hard-walled penis, the distal portion of which enters the genital pore. Within the mucus gland at the point where the ampulla bifurcates there is a valve, the hermaphrodite valve, which serves to prevent eggs from entering the vas deferens. Just past the valve the oviduct bifurcates again, giving rise to the androgynous duct and another branch which appears to become confluent with the albumen gland. The albumen gland passes through the mucus gland to open into the genital pore. The androgynous duct passes anteriorly, leaves the mucus gland and enters the spermatocyst. From the spermatocyst, the androgynous duct passes to the spermatheca which gives rise to another duct which terminates in the genital pore. The spermatheca is purple to black colored, a bag-like structure located laterally to the vas deferens. In some specimens the light-colored, pear-shaped spermatocyst will be seen lying very close to it; in others the spermatocyst will be hidden from view underneath. By use of a fine forceps under a dissecting microscope, the fine, transparent tissue holding the spermatocyst and the spermatheca tightly to the mucus gland can be removed. By gently flipping the spermatheca anteriorly the androgynous duct and the spermatocyst should be revealed. The duct can now be traced from where it leaves the mucus gland and enters the spermatocyst. If the base of the spermatocyst lies close to the spermatheca the connection cannot be traced. A terminal duct passes from the spermatheca to open into the genital pore. In the functioning of this complex system the sperm from the donor passes in a non-motile state into the spermatheca of the recipient. The sperm then passes to the spermatocyst where it matures and becomes

52 motile. Fertilization occurs in the oviduct.

Optional dissection Dissection of land slug (Ariolimax )4 . The following directions for dissection are based on Ariolimax , but should work as well for Arion. With such a lengthy description and complex organism it would be best to carry out this work in pairs, with one person reading the description aloud as the other follows it and identifies the structures. The body is thickly vermiform; the secondary body axis is of minimal importance in slugs. The regions of the body are thus the head, mantle, elongate visceral mass or trunk, and foot. The head bears two pairs of elongate tentacles. The upper, longer pair (the superior tentacles) taper anteriorly, and when fully extended terminate in distal bulbous enlargements that bear the eyes. The shorter, more ventrally placed inferior tentacles are also tapered and terminate dorsally in a slight enlargement. They seem to function as olfactory and tactile organs. The mouth, bounded by fleshy lips, is on the antero-ventral portion of the head, just ventro-medial to the inferior tentacles. In eating, the lips are pushed apart and through the opening a part of the radula and the single, dorsal horny jaw may appear. It may be visible in extended, anesthetized slugs. Just below the mouth is the opening of the pedal mucus gland, a horizontal slit measuring 1 cm across in adult slugs. On the right side of the head region, about 2-3 mm postero-ventral to the superior tentacle, is the genital aperture. It cannot always be seen with the naked eye. When it is apparent, it appears as either a brownish depression or sometimes as a white disc. In preserved slugs, part of the genital atrium complex protrudes from the center of the disc. Just behind the head is the oblong fleshy mantle, under which the head is pulled when the slug is inactive or irritated. Viewed dorsally on the extended slug, the mantle is oval with the small end anterior. On the right side of the mantle is an opening, the pneumostome, which opens and closes at times to allow air to enter or leave the single “lung” or mantle cavity, to be noted in more detail later. A little posterior and lateral to the pneumostome and under the flap of the mantle is the anal opening. The vestigial shell is housed on the inner surface of the mantle in a posterior position. To extract the shell, carefully cut open the mantle by a longitudinal slit and find the shell cavity; do not cut so deeply as to penetrale below the mantle cavity. The trunk is somewhat keeled middorsally, especially at the posterior end, and the keel is particularly evident when the slug is contracted. Just dorsal to the foot at the posterior end of the trunk is a pore, usually blocked with a plug of mucus. The muscular foot forms the entire ventral surface. There is often a coloration pattern on the foot and there are three longitudinal muscular zones. The medial one is particularly apparent when a live slug is observed in locomotion on a glass plate. The mantle cavity, which lies ventral to the mantle just below its shell cavity, can be opened by partly detaching the mantle by a cut along its right edge beneath the pneumostome, continued across the front and then along the left edge. The posterior margin is thus left as a hinge, permitting the mantle to be pulled back. It is now possible to identify the heart, on the left, in the pericardium. It is surrounded by the large kidney just to the right. The openings of the kidney duct and the rectum are on the extreme right, near the pneumostome. All these structures lie in the turned back flap of the mantle. The kidney, thus seen posterior to the heart, must be remembered as a dorsal, anterolateral structure occupying much of the roof of the mantle cavity. A muscular diaphragm forming the floor of the mantle cavity separates it from the visceral cavity. The duct from the kidney, not often readily seen, courses beside the rectum. Having made these observations, cut across the back of the mantle flap, leaving only the ventricle in place, and remove the flap with the attached organs. Now open the body proper by a careful, longitudinal, mid-dorsal incision from the tip of the head to the tip of the tail; pin out the flaps. The visceral cavity contains the digestive and reproductive systems, enclosed as a compact, tapered mass within a tough, thin transparent membrane which is attached to the body wall by connective tissue. Careful separation of the connective tissue attachments permits deflection of the visceral complex completely to one side to expose the ventral midline where the pedal slime gland is obvious as a long band. Digestive system. The mouth opens into the buccal mass which contains the single jaw, located dorsoanteriorly just internal to the dorsal lip, and the radula, located ventrally on the odonotophore cartilage. The salivary glands are creamish-white masses of lobules surrounding the anterior part of the crop (see 4 Adapted

from laboratory handout of Alan Kohn

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below), one on each side, the right being the larger. The glands are joined by connective tissue on the dorsal side of the crop and joined ventrally by arteries and connective tissue. The tissue also holds the salivary ducts to the crop and esophagus. The ducts run along the medial surface of the glands, with small auxiliary ducts joining them at intervals along the glands. The ducts enter the buccal mass dorsally, one on each side of the esophagus. As these structures are noted, you can also see the cerebral ganglia, lying over the esophagus more or less at the point where it leaves the buccal mass. These ganglia are a part of the circumesophageal nerve complex; take care to preserve this and the nerves radiating from the vatious components as you make the following observations. The fairly short and narrow esophagus leads into the crop, which swells to a width about twice that of the esophagus. From dorsal view the crop angles slightly to the left and extends posteriorly about 3/4 the body length. A constriction marks the posterior end of the crop, where it leads into the left anterior portion of the sac-like stomach. The three branches of the digestive gland arise from this region. The intestine emerges from the right ventro-anterior portion of the stomach and is about 1/4 the width of the stomach. The intestine passes anteriorly on the right of the crop for about half its length, then passes to the left under the crop and rectum. At its anterior limit, this anterior intestine loops to the left through the loop of the aorta and the anterior artery, then runs posteriorly about 3/4 the length of the body as the posterior intestine, passing back to the right beneath the rectum, the crop, and the anterior intestine. It reaches its posterior limit somewhat posterior, ventral, and slightly to the left of the stomach. It then loops to the right and forms the rectum, which is very thin-walled, passes anteriorly, crossing obliquely to the left ventral to the posterior intestine, anterior intestine and crop, then turns obliquely to the right, passing dorsal to the posterior intestine, anterior intestine and crop. Sometimes it passes up the midline before proceeding to the right of the body where it passes through the diaphragm, entering the mantle cavity wall to the right and exits at the anus. The length of the rectum is about 2/3 the body length. The two anterior branches of the digestive gland arise laterally opposite each other at the point of union of the crop and stomach. The posterior branch arises just behind these and extends to the posterior end of the animal. There may be variations in the entrance of these glands into the stomach. Each branches and rebranches, forming lobules. The main posterior branch extends just posterior to the stomach and sends out two branches the lobules of which weave over and under each other and the posterior loop of the intestine, almost hiding it from view. This branch extends still posterior to the posterior loop of the intestine, thus making up the posterior third of the visceral complex of the animal. Various major muscle strands are seen in the body cavity as the viscera are examined. The major anterior strands are the large retractor muscles of the superior tentacles and the buccal mass. These originate in the body wall on the right postero-lateral rim of the diaphragm. They pass anteriorly and obliquely to the left just ventral to the entrance of the rectum into the mantle cavity. Among the strands sharing this general region of insertion is a muscle proceeding dorsally to the genitalia and inserting near the genital atrium. Another strand bifurcates and branches, passing through the nerve ring to furnish muscles to the buccal mass. The superior tentacular muscles give rise to smaller branches which are the retractors of the inferior tentacles, which also receive some other minor muscle strands which traverse the visceral cavity. The reproductive system is especially prominent seasonally, but its hypertrophy may persist for most of the year. Another feature of this species is “aphallation,” the absence of the penis. This occurs after copulation, because the complexities of the sex act are so great that the animals can only separate after sperm deposition by the mutual act of chewing off the partner’s penis at the genital aperture. Just internal to the genital aperture is a considerable collection of structures, the atrium, a vagina, duct of the spermatheca, part of the free oviduct and part of the spermatheca, all extending interiorly from the genital aperture on the right side of the body. The major portion of the spermatheca and free oviduct cross obliquely to the left, dorsal to the salivary glands and crop. In atrophy the ovotestis, or hermaphrodite gland, is located on part of the anterior end of the left anterior digestive gland and against the left side of the crop or, sometimes, ventral to the crop. The hermaphrodite duct, albumen gland and ovisperm duct or common duct are all located in this general area. The hermaphrodite duct is the part extending from the ovotestis to the albumen gland, and the ovisperm or common duct is the part extending from the albumen gland to the free oviduct where the vas deferens or sperm duct separates from it. In hypertrophy the ovisperm duct is so enlarged that it pushes the ovotestis and albumen gland more posteriorly. Further complicating the elaborate pattern of ducts is the fact that a prominent tubule is seen among them, actually the genital artery, running for a considerable distance along the ovisperm duct, as shown in the accompanying diagram. The ovotestis, which produces eggs and sperm, varies in shape. It is an aggregation of pockets, arranged in larger lobes, these in turn held together by a closely adherent membrane, which also surrounds and holds the loops of the hermaphrodite duct together. This duct arises in a groove on the ventral surface of the

54 ovotestis. It is a tightly coiled tube, and functions to carry eggs and sperm to the common duct. The albumen gland varies greatly in size with the degree of hypertrophy. In atrophy it may be less than 1 cm long; in hypertrophy it may be over 7 cm long. It consists of large lobes that fill a portion of the posterior part of the body and partly cover the digestive tract dorsally. It is broadest anteriorly, gradually diminishing posteriorly. In hypertrophy the common duct takes the form of a greatly sacculated, convoluted structure with its greatest width of almost 1 cm near its attachment to the albumen gland. The common duct tapers anteriorly; in hypertrophy a flattened, lobed structure can be seen to follow and closely adhere to the common duct. The latter, at its anterior end, gives rise to the large, loosely coiled oviduct and the slender, straight sperm duct. In hypertrophy, the oviduct is more convoluted and the sperm duct longer and wider. The vagina is the basal part of the female tract between the spermatheca and atrium; normally it receives the penis in copulation. The free oviduct is that portion between the spermatheca and the point of separation of the sperm duct from the ovisperm duct. The spermatheca is stalked and attached by connective tissue to the side of the free oviduct. The genital atrium is a small sac-like region. In aphallates the ejaculatory duct, the terminal portion of the sperm duct, either is joined to the atrium by connective tissue or is open and free in the body cavity. In phallates the substantial penis is usually partially evaginated; internally its loop is attached to a retractor muscle. Nervous system. The supraesophageal ganglia lying dorsal to the esophagus just posterior to the buccal mass were noted earlier. To work out further details of the nervous system cut through the esophagus and the salivary duct immediately behind the ganglia and turn back the digestive tract. Pull the stumps of esophagus and salivary ducts forward through the nerve ring. Remove head retractor muscles and salivary glands, and other tissues obscuring the nervous structures. Carefully dissect away the connective tissue sheath of the ganglia and remove the epithelium which covers the larger nerves passing back in the body. The largest nerve leaving the cerebral ganglion is the superior tentacular nerve. It gives rise on its dorsal surface to the smaller optic nerve, and proceeds to the tip of the tentacle. A smaller nerve arising lateral to the superior tentacular nerve proceeds anteriorly and supplies the right dorsal lip and adjacent body wall. A third nerve arises near these; it runs forward about 4 mm and branches to give off to the right the inferior tentacular nerve and also branches supplying the lip and body wall. Arising from about the mid-lateral section of the ganglionic mass are three nerves about equal in size. The anteeriormost proceeds with the tentacular nerves but supplies the ventral lip and body wall. The middle of the three is the commissural cord connecting the supresophageal ganglia with the buccal ganglia. The latter ganglionic masses lie on each side of the esophagus as it leaves the buccal mass, but are hidden by the salivary ducts running over them. The posterior nerve of the trio supplies the buccal retractor muscles. Posterior to these nerves and arising from the posterolateral section of the ganglionic mass the large, two-stranded connective cords that join the cerebral ganglia to the subesophageal mass emerge. The subesophageal ganglionic mass is a much fused complex forming a sort of ring around the cephalic artery. The very great number of nerves radiating from the mass include tracts to the viscera, the body wall and mantle, the foot, and still other structures in the body. This distribution indicates the mass is a fused complex of pedal, pleural, visceral, and perhaps still other ganglionic bodies.

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Mollusca II — Bivalvia, Scaphopoda, Cephalopoda bivalves, tusk shells, squid and octopus Bivalvia The clams and their relatives are generally sedentary molluscs with large, external, bivalved shells. The head is reduced, while the visceral mass, mantle, and mantle cavity, and, usually, the foot are large. Except in the protobranch bivalves, which probably represent a more primitive condition, the ctenidia are greatly expanded within the mantle cavity and serve as filter-feeding structures in most bivalves. The shell valves are lateral—that is left and right—and joined by a hinge, with specialized interlocking teeth, on the dorsal side of the animal. In clams, the mantle is often elaborated into siphons at its posterior edge. Protobranch bivalves. Nut shells (Nucula and Nuculana spp. Figs. 12-94, p. 373). While most modern bivalves are suspension feeders, the protobranchs adopt what is probably a more primitive mode of feeding for bivalves, namely selective deposit feeding. This is accomplished with the labial palps, which appear as a pair of flaps on each side of the mouth. From each set (pair) of palps an elongate tentacle extends, and these probe the substrate to pick up particles of food which are then sorted by the labial palps themselves. The gills in protobranchs serve only a respiratory function. Look at the preserved specimens of nut shells on demonstration. Find the foot, ventral-most in the mantle cavity, with its frilled edge. The ctenidium is situated at the posterior end of the body; its axis, and the gill-plates arranged along the axis, can clearly be seen. Anterior to the ctenidium is a prominent fold of tissue, the labial palp; in Nucula the palp is larger than the ctenidium. At the posterior edge of the labial paip, between the palp and the ctenidium, the proboscis may be visible. Flanking the visceral mass on the dorsal side are the posterior and anterior adductor muscles, looking like brown ovals. Identify the mantle, lining the shell, and, on the shell itself, note how the hinge has many teeth in a row, a characteristic of this primitive group of bivalves. (How do the hinge teeth compare in the other bivalve shells on demonstration?) Anatomy of the bivalves We will take a close look at bivalve anatomy using Mytilus (the blue mussel) or other bivalves that may be available, including Modiolus (the horse mussel), Mya (the soft-shelled clam), oyster, quahog, and surf clam. You may choose any one of these for your own dissection; then compare what you find with what your neighbors find working on other (and the same) species. Figures of Mytilus opened to reveal internal anatomy are on the page preceding this section, and they can apply to other bivalves as well. Mytilus. Blue mussel (Mytilus edulis. Figs. 12-110 A, B; 12-98 A, B; pp. 388, 378; see also figures on page at the end of the preceding section.) Like most bivalves Mytilus is a filter feeder, using the expanded surface of its gills to sort suspended particles from the water the gills pump into the mantle chamber. Mytilus occurs in immense beds in rocky intertidal regions on both coasts of the United States. The animals attach to rocks, shells, and each other by byssal threads which are collagenous threads secreted by glands at the base of the foot. Watch live animals in the aquarium to see how the shell gapes to allow water to flow through the mantle folds at the rounded posterior end of the shell. Two openings are evident, something akin to siphons but not called such; the one with the fimbriated margin is the inhalant opening; the other one, dorsal to it, is the exhalant. Note how byssus threads in attached animals fan out from a point on the shell where the foot can be protruded. The animal can break its attachment and move to a new site as the need arises. Young animals, smaller than about 1 cm long, are more mobile and may be seen in the aquarium or in one of the specimen dishes gliding around, or hitching themselves along, with the foot. Modiolus. Horse mussel. Like Mytilus, this mussel attaches to hard substrates with byssus threads. It is easily distinguished by its thick, brown periostracum which usually even extends as beard-like threads from the shell. The beaks of the umbo are placed to one side, while those of Mytilus are at the apex. (For anatomy, treat as Mytilus.) Spisula. Surf clam. This is the largest bivalve on the north Atlantic Coast, a heavy-shelled clam, found mostly subtidally. It is commercially important (good to eat) in some areas. (For anatomy, compare to Mercenaria, Figs. 12-89, 12-92; p. 368, 370) Mercenaria. Quahog (a native-American name, pronounced ”ko-hog”). The shell is thick and solid, and rounded, as if the animal inflates it. A commercially important (good-to-eat) clam. Crassostrea. (American oyster. Fig. 12-113.) The shell is rough and heavy and generally flattened. The uper valve is smaller and flatter than the lower shell which is cemented to the substrate. Contrary to popular

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belief, valuable pearls are not likely to be found in this bivalve; the nacreous layer of the shell is rather dull, as would any pearls produced by the animal. Examine one of the whole MgCl2 -relaxed animals at your work station. We will be removing the left shell; to identify it, remember that the anterior end of the shell of most species is that toward which the umbo points; the siphons are at the posterior end. Lay the animal on its right side and, while propping both hands against the shell, insert a scalpel blade at the postero-ventral end, slipping it between the mantle and the left (upper) shell. (Use caution! Keep your hands propped against the shell, and don’t let the blade slip and cut you.) Carefully, while gently lifting the left shell, scrape the mantle from right shell, working toward the hinge until you cut through the posterior adductor muscle. Then scrape further forward to cut the pedal-retractor and anterior adductor muscles from their attachments on the shell. Gently pry the shell up and away from the mantle and rest of the animal. (Don’t just rip the shell upward, or you will tear the flesh and make it unrecognizable.) It will be easier to identify some structures with the animal submerged in liquid, as usual, so use a dish with magnesium chloride or sea water. Identify the white muscle masses: the anterior and posterior adductor muscles, the six or so pedal retractor muscles, and the anteriorly positioned pedal protractors. Note their scars on the shell. Note also the pallial line on the shell, a line showing the site of attachment of the edge of the mantle. Find the inhalant and exhalant openings again. While the animal is still fresh, it may be easier to find the heart as it beats inside the rectangular pericardium on the dorsal side of the animal above the attachment of the gills. Gently slit open the pericardium to expose the heart and note how the rectum goes through the ventricle! The auricles are thin sacs opening into the ventricle at its midpoint (and covered by brown pericardial glands). Peel up the edge of the mantle and cut through it, then view the cut edge-on to see the inner and outer mantle lobes and the middle fold and periostracal groove. Raise the mantle flap and cut it off near its attachment to the rest of the body. This will expose the two gills, each of which appears as an inner and outer demibranch on either side of the visceral mass. Each demibranch can be seen to be a double sheet, a descending and an ascending lamella of filaments. In the mussel, these filaments are only loosely joined together by ciliary tufts (filibranch condition); in the oyster they have more tissue junctions keeping the filaments together (pseudolamellibranch); and in the other bivalves, they are in more continuous, more solid sheets (eulamellibranch). Gently separate a few filaments to test the strength of these junctions. The easy fraying in the mussel is typical of the filibranch gill. The axis of each gill is fused to the dorsal wall of the mantle cavity, and by the folding of the gill into lamellae it separates the dorsal part of the mantle cavity from the ventral part as discrete chambers. Water enters the ventral chamber, passes through the gill lamellae, and leaves the mantle cavity via the upper chamber and exhalant siphon. Find the two labial palps at the anterior edge of the gills; a pair on either side encloses the narrow ends of the two demibranchs. Between the palp pairs lies the mouth. Place a drop of carmine suspension in the mantle cavity and watch how the cilia of the gill and labial palps move the carmine particles. The cilia can be seen even better by cutting a small square of gill out and examining it mounted on a slide and coverslip under a compound microscope. The foot is a muscular projection directed anteriad. In Mercenaria and Spisula, it is substantial and used for burrowing; in Mya it somewhat smaller. In Mytilus the foot is rather small, brown, and finger-like and serves not only for creeping but to lay down the byssal threads, molding them in a groove in its ventral surface from secretions released by the byssal glands at its base. In Crassostrea, the foot is reduced. Cut away the gills on the upper side of the animal. Find the kidney (renal organ) extending from the labial palps to the posterior adductor. The kidneys are reddish brown, and lie dorsal to the bases of the gill. The renal pore, halfway along the length of the renal organ sits on a papilla with the genital opening. While most bivalves have the gonads in the connective tissue of the foot, Mytilus (with such a small foot) has its gonads in the mantle, appearing here as ramifying gonadal tubules, white in the male and orange in the female. The tubules can be traced in younger animals to their convergence at the genital opening. The nervous system can be traced through the translucent body wall. The visceral ganglia and their commissure lie on the posterior adductor muscle. The visceral connectives run forward superficially in the wall of the kidney. The cerebral ganglia and their commissure lie in front of the esophagus, deep to the mouth. Pedal commissures run back from the esophagus with the visceral trunks to diverge into the foot. You might open the base of the foot to expose the pedal ganglia. The digestive gland is the greenish compact mass in front of the pericardium. Cutting into it will expose the stomach and the proximal limb of the intestine within which is the glass-clear crystalline style (which

58 will probably pop out once you cut into this area). Where the larger end of the crystalline style rests in the stomach is the gastric shield and the ridged ciliary sorting areas. Other bivalves on demonstration table and live animals in aquarium Various living bivalves are in the aquarium, including oysters (Crassostrea), scallops (Placopecten, cf. Fig. 12-115), and horse mussels (Modiolus). You are welcome to take any to your seats in a clean, living-animal finger bowl. Examine the shells of Mytilus and other bivalves on the demonstration table. Identify the anterodorsally located umbo and the dorsal hinge. Growth lines on the shells are evident. Find the dark, horny periostracum; and, where the periostracum is worn away, the calcareous prismatic layer. A section through a shell of a bivalve is on demonstration. Use it to refresh your knowledge of the structure of the molluscan shell and its three layers (cf Fig. 12-91): the periostracum (outermost, dark organic layer), the ostracum or prismatic layer (calcareous, underlying the periostracum), and the hypostracum or nacreous layer (also calcareous, lying next to the mantle). Which of the whole shells shows a nacreous layer? Filibranch and eulamellibranch gills (Fig. 12-98). The histology of these two types of gill is shown in microscopes on demonstration. One demonstration shows cross sections of the filibranch gills of Mytilus, and on it you can see the epithelial cells of the filaments and their ciliation. Note how the filaments are separate, linked only by ciliary tufts. In the eulamellibranch condition, however, as in the other microscope showing sections of gills of a freshwater mussel, the filaments are completely fused. In the whole opened quahog (Mercenaria mercenaria) beside this microscope, you can see how this eulamellibranch condition makes the gills appear as solid sheets, the outer and inner lamella, therefore, perforated by ostia. Between the lamellae are the water tubes. On the microscopes, try to identify frontal, laterofrontal, and lateral cilia. You may be able to see the chitinous supporting rods on the inner walls of the filament of the eulamellibranch gill. Look at the other demonstrations of preserved bivalves and note gill structure (for example, the intermediate nature of gill fusion in the pseudolamellibranch gills of the oyster) and differences in size of the gills, foot, digestive gland, and other internal organs. Shipworm. (Teredo sp. Fig. 12-117). The specimens on demonstration of the shipworm, Teredo, show how highly modified this eulamellibranch is, as it is adapted to boring into and feeding on wood. Most of what you see is mantle. At the anterior end the reduced right and left valves form an efficient boring mechanism. Between them is the foot. At the posterior end, two shell remnants, called pallets, protect the dorsal exhalant and the ventral inhalant siphons. Scaphopoda Dentalium. (Figs. 12-123, -124.) Preserved whole specimens and shells of the tusk shell Dentalium are on demonstration. Though rarely seen, this animal is actually quite common in deeper water of the Gulf of Maine. The mantle and shell are elongate and fused ventrally to form a tapered tube open at both ends. The larger opening is anterior, and through it the foot can be protruded. On the specimens here, note how the margin of the mantle and foot are visible through the larger opening, and note that the foot consists of two lateral lobes and a median process; it is very effective in burrowing motions. To feed, tusk shells extend tentacle-like captacula from this anterior opening; an adhesive knob at the end of each captaculum captures small organisms from the sand such as foraminiferan protozoans. Cephalopoda This class includes the squids, octopus, and their relatives. Many have streamlined bodies and prominent, well developed eyes on the sides of the head and are swiftly moving carnivores. In most living cephalopods the shell is poorly developed or absent. Some fossil cephalopods had gigantic external shells; octopus has none. The eight arms are derived from the foot but surround the mouth. The siphon or funnel, also derived from the foot, marks what has become the topographically ventral side of the animal (even though the foot as a whole indicates the true morphological ventral side, as we know in comparison with other molluscs). Nautilus. (Figs. 12-65, -66.) Nautilus shows what may be the primitive condition of the shell; specimen shells are on demonstration. One shell is bisected so that the partitions and chambers are revealed. Look also at the preserved specimen of Nautilus that has been removed from its shell and note the short tentacles, the eyes (in which the pupils are simple slits), and the siphon which is used to propel the animal by a jet-propulsion-like mechanism.

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Squid. Doryteuthis (Loligo) paeleii (Fig. 12-79). Frozen specimens of the squid Doryteuthis are available for dissection. (Also available are preserved specimens, and these are larger and injected with latex to reveal the circulatory system; they are not as pleasant to work with, but can be informative for comparison, if desired.) Dissection of the frozen specimens is worthwhile, and for the appropriate detail we will use Rick Fox’s online manual, specifically his chapter on dissection of Lolliguncula brevis, a southern species but quite similar to Doryteuthis: http://webs.lander.edu/rsfox/invertebrates/lolliguncula.html. In general (and if the online manual is not accessible), you can use the following brief instructions for studying the squid. Note, also, its references to the appropriate Figures in the textbook. Examine the head and foot, noting the modification of the foot into five pairs of appendages that surround the mouth. Notice that one pair is different from the other four: this pair consists of retractile tentacles, and the remaining four are considered arms (cf Fig. 12-74). The terminal portion of the tentacle is widened into a club. Examine the lower left arm; if your specimen is a male this will be modified into a hectocotylous arm, bearing small suckers on the terminal portion. Study the suckers on one of the arms. Note that each is composed of a cup that is attached to the arm by a pedicle. Around the rim of the cup there is a supporting, toothed chitinous ring. The central basin of the cup is formed by a piston, the action of which creates a partial vacuum when the cup attaches. Spread the arms aside in order to find the mouth. Around the mouth is a peristomial membrane, and inside its opening are the chitinous beaks (cf Figs. 12-77, -78). If your specimen is a female, the horseshoe organ can be seen in the midline, just below the mouth; this is the sperm receptacle. Study the pair of eyes on the head. Identify the cornea, iris, pupil, and lens (cf Fig. 12-82). The remainder of the body is called the visceral hump; it is completely enveloped by the mantle. The anterior margin of the mantle, or collar, is divided into three scallops. The projection in the dorsal midline marks the position of the internal rudimentary shell, called the pen. The two ventral marginal projections mark the position of the pallial cartilages. Note the lateral fins which project from the posterior part of the mantle. By undulating these fins, the squid can swim forward. It can propel itself backwards quickly in rapid-escape jet propulsion by contracting the mantle to force water out the funnel which lies on the mid-ventral line (cf Figs. 12-64, -75). One specimen has its mantle cavity cut open so that the gills and some other organs are visible. Compare this specimen to the Figures beside the demonstration and in the textbook and find the gills and their branchial hearts, kidney, and parts of the digestive tract (rectum, caecum) and reproductive organs (gonad, penis or oviduct). Octopus (Octopus vulgaris). Examine the demonstration specimens of Octopus. Note that there are eight arms and no tentacles. Observe the suckers on the arms. Note the head, with the eyes. On either side of the head the incurrent siphons of the mantle cavity can be seen. The single, excurrent siphon is hidden under the visceral mass and mantle, which are folded back behind the head in this relaxed specimen. This position of the visceral mass is more or less characteristic in a living specimen that is relaxed.

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Porifera PORIFERA Demospongiae Haliclona — live, dry (demo) Isodictya — live, dry Halichondria — live, dry Microciona — live, dry Cliona — live, wet (demo) Spongilla — live, dry Calcarea Leucosolenia — live, preserved, dry, slides Sycon (=Scypha) — preserved, dry, slides Hexactinellida (Symplasma) Euplectella — dry (demo) Rossella — dry (demo) Sponges are an important part of fouling communities, growing over almost any substrate that provides solid surface for attachment, and they provide habitat for many other invertebrates. As you look at the living sponges in this lab, watch also for other invertebrate members of this fouling community—for example, shelled amoebas and ciliate protozoans, skeleton shrimp (amphipods), tube-forming amphipods, isopods, pycnogonids, polychaetes, flatworms, hydroids, and bryozoans. Most sponges have their choanoderm—the layer of flagellated cells called choanocytes—divided into millions of small flagellated chambers scattered in a substantial mesohyl and interconnected by pinacodermlined canals. This kind of arrangement is known as the leuconoid type. In contrast, some smaller calcareous sponges have a reduced canal system and more extended choanoderm; these are known as asconoid if the choanoderm lines the whole inside of a sack-like, thin-walled body, and syconoid if the choanoderm lines many small sack-like outfoldings in a relatively thickened wall. Because it is easier to see choanoderm in such calcereous sponges, we will start with them. Leucosolenia. (Figs. 5-2 A, 5-3 A, 5-5). This small calcareous sponge has the asconoid type of body structure: individuals are sack-like, with a single osculum at the free end and with the entire inside of the sack lined with choanoderm. If living specimens are available, we will concentrate on those. Take a specimen in a dish of sea water to your microscope. Whole-mount slides of Leucosolenia colonies and wet-preserved specimens in dishes of alcohol (please keep these dishes covered when you are not looking at them; do not let them dry) are also available for studying external morphology. Leucosolenia grows in colonies of interconnected asconoid individuals; each individual is an elongate, almost tubular sack attached to the rest of the colony by its base. At the distal tip of each individual is an osculum. The whole colony has an irregular branching structure. Note budding young individuals. On the living specimen, first see if you can find evidence of water currents generated by the sponge. Watch the osculum in particular to see if particles in its vicinity are swept away from the sponge. You may want to add a drop of the suspension of carmine particles in sea water if other particles are not visible. Cut an individual lengthwise and mount both halves on a slide, one half with its inner surface facing up, the other half with its inner surface facing down. Cover with a coverslip and add sea water, if necessary, to cover the sponge. Porocytes should be visible on both halves as clear cells, each with a canal running through its center (sometimes rather crooked). Focus especially on the surface levels of the pieces. Activity of the flagella of choanocytes should be visible on the inner surface; the cells themselves are small and closely packed. Pinacocytes form a pavement-like surface on the outer surface. Try cutting a very thin cross section from either half using a razor blade; then study the section under the compound microscope to see if you can find choanocytes in profile. Also, simply try macerating a small piece of the sponge by slicing scissor-like against it with two dissecting needles, then apply a coverslip and examine the preparation for cell types. Choanocytes will stand out for their flagellar activity; amoebocytes will eventually produce elongate pseudopodia and crawl over the slide; try also to distinguish pinacocytes. Use the prepared slides of stained sections of Leucosolenia to see the cell arrangement. At low and intermediate magnification, find the spacious central spongocoel. At high power, identify individual cell types. (You may want to use the oil-immersion lens for this.) Find choanocytes lining the spongocoel and discern their flagella and collars; identify also pinacocytes on the outside, porocytes forming the pores (ostia), and amoeboid cells in the mesohyl.

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Sycon (=Scypha) (Figs. 5-2 C, 5-3 C). Sycon is a syconoid calcareous sponge, having the choanoderm divided into radial canals facing the spongocoel. Examine living or preserved and dried specimens and note how these are clearly separate individuals except where budding is going on. Find the prominent osculum surrounded by long monaxon spicules. The surface of the sponge appears bristly because of protruding spicules. Use the sliced dry or wet-preserved specimens to study the arrangement of the radial canals. Each opens into the spongocoel by a conspicuous pore, the apopyle, through which water is discharged. Water enters the radial canals through prosopyles that connect to incurrent canals from the outside. Ostia on the surface of the sponge are openings to the incurrent canals. Living specimens, if available, can be studied in a dish of sea water in the same way as Leucosolenia, if you want, but you will need to cut thin cross sections to see flagellar activity and cell types. Study the prepared slides of stained sections of Sycon to see the relationship of radial and incurrent canals and to find, at high magnification, the cell types (choanocytes, pinacocytes, amoeboid cells) and developmental stages lying in the mesohyl (eggs, amphiblastula larvae; Fig. 5-16 B). Look also at the whole-mount slides of spicules isolated from Sycon (labeled “Grantia”). How many different types (e.g., distintuished as monaxons, triaxons, etc.) can you find? How many axes of symmetry and rays do these spicules have? What is the composition of these spicules? (Some slides labeled “spicules” show sections of the sponge with the spicules in place in the mesohyl.) Demosponges: Halichondria, Isodictya, Cliona (Fig. 5-14), Microciona, Spongilla (Fig. 5-6), and others. Most sponges (including many calcareous sponges and all the demosponges), are of the leuconoid type (Fig. 5-2 D). Such sponges can reach a far greater size than asconoid or syconoid sponges, not just because of the organization of the choanoderm into many flagellated chambers, but because these sponges are able to form a more massive scaffolding of their spicules by gluing them together with spongin; calcareous sponges are incapable of such interspicule scaffolding. Study any of the demosponges under a dissecting microscope. (The following text applies especially to Spongilla, a freshwater sponge that is green from algal symbionts (Chlorella) living in the mesohyl, but other than color, this description should fit other live demosponges available.) Note how the entire colony seems to be covered with a thin transparent skin, the outer pinacoderm, supported over the greener inner mass by tent-pole like spicule aggregates. The space between the two is a so-called subdermal cavity, also bounded by pinacoderm, essentially the first of the incurrent canals; the remaining incurrent canals entering the mass of the sponge are visible as rather large openings from the subdermal cavity. Look for particles being drawn into the sponge through the numerous inconspicuous ostia in the skin and then being transported quickly through the subdermal cavity to any of the incurrent canals. If no such naturally occuring particles are visible, add a small drop of carmine suspended in water near the point where your microscope is focused and watch their motion. Oscula, through which excurrent water is released are on raised chimneys and have a larger diameter, usually 0.5-2.0 mm. Examine the preparations of pieces of the live sponge mounted on a slide (with a coverslip) as closely as you can to distinguish cells and spicules. Pinacocytes can be made out in the covering pinacoderm; you may be able to see flagellated chambers and amoebocytes wandering the mesohyl in thinner portions of the colony near its growing edges or near a torn edge. If you can, look for the activity of the flagella of the choanocytes and the slow amoeboid motion of the cells in the mesohyl. On demonstration is a slide of Spongilla sectioned and stained to show the flagellated chambers. Water enters each chamber through several prosopyles connected to incurrent canals and exits through an apopyle to excurrent canals (see the diagram accompanying the demonstration and Fig. 5-6 in the textbook). If whole specimens of Spongilla on sticks and bark are available, look at the base of these encrusting colonies for asexually produced overwintering bodies known as gemmules (Fig. 5-15) which consist of archaeocytes protected by a wall of amphiaster spicules (short spicules having star like spikes at either end). After being subjected to freezing or at least prolonged cooling (“vernalization”), these gemmules will “hatch,” releasing the archaeocytes which differentiate into whole new colonies. (If you do not find gemmules in your specimen, check the demonstrations for dried specimens of Spongilla showing them.) Look at the various marine demosponges available for study and note differences in growth form, color, and texture. Try smelling the sponges also; many use chemical defenses that give the sponge a distinctive odor. Use a dissecting microscope to study the surface of one or several of the living marine demosponges if you have not already done so. Differentiate between ostia and oscula scattered over this surface. Try adding a small drop of carmine suspension to see if water currents can be traced into or out of these pores. Break pieces of the sponge off and look at the inner surface to find canals and sites of flagellated chambers; note how the external surface is set off as a dense cortex. Use a razor blade to slice into the sponge, and see whether you can find the tiny flagellated chambers. Try also to cut thin slices that can be mounted on a slide with a coverslip, and see if you can detect flagellar activity; or simply macerate a small piece of sponge on

62 a slide, and a coverslip, and look for flagellated and amoeboid cells. Note on these preparations the spongin fibers and spicules. Use the fragments of demosponges available in the small dishes beside the demonstrations to make your own preparations of spicules. Tear off a very small chunk of sponge (1-2 mm3 ), place it on a slide, and add a small amount of bleach or hydrogen peroxide to dissolve its organic constituents. Once bubbling of the preparation has ceased cover the drop with a coverslip and examine it under a compound microscope to find spicules. How many different types of spicules can you find? What are these spicules made of? How differrent ar the spicules from the different specimens? Morphology of spicules provides key taxonomic features for distinguishing species of sponges (see Fig. 5-9). Halichondria spp. are among the commonest local members of the Demospongiae and are known as crumb-of-bread sponges. H. panicea forms large encrusting yellow to green masses on rocks in the intertidal and on submerged timbers; H. bowerbanki is typically found on floating docks. Cliona celata (Fig. 5-14) bores into mollusc and barnacle shells and other calcareous objects and forms and occupies channels within them; it is visible on the surface only as small yellow buttons protruding from the openings of the channels. C. celata may severely damage the shells of commercial oysters and clams. Other species of Cliona form non-boring upright masses. Take a quick look at the piece of commercial sponge (Spongia? Hippospongia?) available on the demonstration table to see how the skeleton of such spicule-less keratose sponges is composed of very large reticulating spongin fibers (cf Fig. 5-11 A-B). Glass sponges (Hexactinellida): Euplectella (Figs. 5-4 A, 5-7), Rossella; dried specimens on demonstration. These are deep-water sponges. Like demosponges, hexactinellids have a leuconoid body form and siliceous spicules. They are distinguished from demosponges, however, by the fact that their spicules are six-rayed (hexactine) triaxons. They also differ significantly in the nature of the pinaco- and choanoderm: these are continuous sheets of syncytial tissue rather than cellular. Look at the dried specimens on demonstration and see if you can find the distinguishing character of the spicules. The rays of some spicules are very long, and so very like glass wool. (Be careful in handling the specimens to avoid getting impaled!) Look at the spicule debris in the dish under the microscope beside the whole hexactinellids. Note that the spicules are hexactines—that is, they have six rays. In the long spicules, one of these rays is very long, giving the glass-wool like look to the spicule, while the others are short, standing at right angles to the long axis like the spikes of a child’s jack. In some spicules the short rays are arrow-shaped or barbed, perhaps functioning in anchoring the spicule in the sediment. Euplectella has a beautiful skeleton, with lattice-like interweaving of long-rayed spicules and the large osculum at the distal tip has a seive-like protective closure. A certain species of shrimp inhabits Euplectella in male-female pairs that enter the spongocoel as juveniles. They use currents through the spongocoel for their own feeding; as the oscular seive grows shut they become forced—but presumably willing—permanent residents of the sponge, protected from large predators there by its cage-like growth form.

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Cnidaria Alcyonarians, stony corals, anemones, jellyfish, hydroids CNIDARIA Anthozoa Alcyonaria (Octocorallia) Stolonifera Tubipora – dry Gorgonacea Gorgonia – dry Leptogorgia – preserved (demo) Pennatulacea Pennatula – preserved (demo) Renilla – preserved (demo) Zoantharia (Hexacorallia) Ceriantharia Cerianthus – preserved (demo) Zooanthidea Zoanthus? – preserved (demo) Actiniaria Nematostella – live Metridium – live, preserved, slides Aiptasia – live Anthopleura – preserved (demo) Scleractinia Acropora – dry Diploria – dry Fungia – dry Medusozoa Scyphozoa Cubomedusae Stauromedusae Haliclystus – preserved (demo) Semaeostomeae Aurelia – preserved, slides (demo of life cycle) Rhizostomeae Hydrozoa Anthoathecatae (Anthomedusae, Athecata) Ectopleura (=Tubularia) – live Halocordyle (Pennaria) – slides Hydra – slides, live Hydractinia – live Siphonophora Physalia – preserved (demo); video Leptothecatae (Leptomedusae, Thecata) Obelia – live, slides Plumularia – slides Limnomedusae Gonionemus – preserved CTENOPHORA Pleurobrachia — live, preserved, video

64 Anthozoans Alcyonarians (Octocorals) (Figs. 7-33 – 7-40, pp. 141-145) The sea-whip, Leptogorgia (=Plexaura), is on demonstration under a dissecting scope, preserved (cf. Figs. 7-33, -39). As in other alcyonarians, the polyp of Leptogorgia has eight pinnate tentacles. Locate an adequately expanded polyp and identify the tentacles to see their hollow nature and the manner in which they arise from the margin of the flattened oral disc. Can you see the stomodaeum and the septal filaments through the wall of the polyp? Note the horny axis (a central rod composed of the protein gorgonin) and the yellow coenenchyme (composed of anastomosing tubules of the gastrovascular system within the mesoglea, which is stiffened by numerous calcareous spicules). The sea pansy Renilla reniformis, which is also on demonstration under a dissecting microscope, preserved, demonstrates how different forms of polyp co¨operate to make a colony (Fig. 7-40B). The primary polyp is broad and leaflike, with a stem-like peduncle that anchors the colony in sand; and on its upper surface the other zooids project, including the tentacle-bearing autozooids (the feeding polyps) and clusters of low, wart-like siphonozooids (zooids responsible for pumping water through the gastrovascular cavity). A strip extending from the peduncle in the middle of the upper surface is free of zooids, and at the end of this tract is a single, large, exhalant siphonozooid. The under surface of the colony bears no polyps. Various other alcyonarians are on demonstration to give you an idea of diversity of form: Pennatula (Fig. 7-40), the sea pen, also with a large primary polyp on whose feather-like surface the other zooids are arranged; Tubipora (Fig. 7-35), the organ-pipe coral, which has long polyps encased in parallel in calcareous tubes of fused spicules; Gorgonia the sea fan, in which the branches anastomose to form a lattice; and Pterogorgia and Eusimilia, large branching colonies. Stony corals (Figs. 7-23 – 7-26) form massive skeletons of calcium carbonate and are the major builders of coral reefs. Most live as colonies of thousands of polyps connected by a common sheet of tissue; the skeleton is secreted by the basal epidermis and so is an exoskeleton. A variety of growth forms in corals can be seen in the skeletons (coralla) on demonstration: Acropora (2 spp.), with well separated, small corallites, are important reef-building corals; Diploria, a brain coral, in which the corallites are confluent, forming sinuous rows sharing elongate mouths (a broken piece of Diploria shows how growth of the corallum occurs); Agaricia, the lettuce coral, which is flat and leaflike, feeding not by stinging animal prey but by mucociliary capture of small planktonic organisms. The form of the coral relates to how the polyps bud (cf. Fig. 7-27). Sea anemones (Nematostella vectensis, Metridium senile, Aiptasia pallida Figs. 7-16 – 7-18). The body of anemones is polypoid, consisting of an elongate column bearing an oral disc at the free end and a basal end that typically is flattened into a pedal disc that attaches to solid substrates. Some anemones are adapted to burrowing in sediment and so have a more pointed basal end. Numerous hollow tentacles arise from the margin of the oral disc, and at the center the disc is the mouth, a flattened slit that leads to the pharynx. The column is supported internally by longitudinal septa that act as struts for the body wall and that divide the gastrovascular cavity into a radial array of chambers. Nematostella is a burrowing anemone adapted to the muddy sediments in low-salinity habitats such as salt marshes and estuaries. Rather than attaching to solid substrates, as other more typical anemones do, it sits within the sediment, with its bulbous base burrowed into the sediment and its tentacles and mouth sitting at the surface in wait for passing prey. Because it is easy to keep alive in the laboratory, it is used as a model organism by scientists studying development and phylogenetic relationships of lower metazoans (Darling et al., 2005, BioEssays 27:211-221), and its full genome has been sequenced. The column of Nematostella appears divided into somewhat distinct regions: the capitulum (“head-like” and where the pharynx sits), the scapus (the main length of the column), and the physa (a bulbous region that anchors the animal in the sediment). The pharynx is discernible within the capitulum as an elongate, pearly, opaque mass, and it is suspended in the gastrovascular cavity by eight septa (also called mesenteries) which are visible arising alongside the pharynx and continuing through the length of the scapus. Toward the basal end of the scapus, the septa are distinctly convoluted—these ruffled, innermost edges of the septa being the septal filaments that are richly glandular and loaded with nematocysts. Just next to (under) the filament in each septum is a thickening constituting a retractor ’muscle’ (actually the enlarged myofilamentcontaining bases of the gastrodermal cells here). Another thickened band of longitudinal ’muscle’ runs along the length of the septum where it attaches to the body wall. The septum may also be thickened toward its free edge with gonads. What happens to the septa as they reach the physa? The oral disk is circular and bears two whorls of tentacles, an inner whorl of smaller tentacles and an

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outer whorl of larger ones, usually together totalling 16 but ranging in number from 10 to 20. Note how gastrovasuclar fluid is circulated through the tentacles. (What drives this circulation?) Look closely at the mouth and find the siphonoglyph, a ciliated groove that runs down the length of the pharynx. See if you can see movement of particles in the water near the siphonoglyph to track the cilia-driven currents. The pharynx opens into the gastrovascular cavity, and the siphonoglyph serves to pump water into the cavity to charge its hydrostatic skeleton—that is, to expand the gastrovascular cavity and allow the anemone to elongate and to spread its tentacles and body. Because the pharynx is not radially symmetrical (being flattened and having the siphonoglyph), it imposes a biradial symmetry on the animal (as opposed to the true radial symmetry of the Hydrozoa and Scyphozoa). Watch how the tentacles are used, first in their reaction to a clean probe that you brush by them and then in reaction to passing prey. What makes them appear so sticky? Watch how the animals capture and consume Artemia nauplii. Try retrieving a captured nauplius and looking at it under the compound microscope for the nematocysts that killed it. What happens to the nauplii that are shoved into the pharynx? Can you see how the septal filaments play a role in killing (and digesting) the prey? Metridium and Aiptasia (or other anemones that may be available, such as Anthopleura or Tealia) have a more typical anemone shape—that is with an adhesive pedal disk by which they attach to solid substrates such as rocks and pilings. We have both live and preserved specimens of Metridium and Aiptasia, and it would be best to study a live one first for external anatomy and behavior. Aiptasia is fairly transparent, so internal anatomy can be seen with whole animals, while Metridium, especially larger specimens, are opaque, so you will need to use the preserved specimens to see internal anatomy. Aiptaisia offers the advantage, too, that it has zooxanthellae, so you can use it to study relationships of these symbionts to the host anemone’s tissues by pulling off a tentacle and observing it in a squeeze-prep. Live Metridium and Aiptasia typically respond defensively to the handling necessary to get them into a dish by ejecting acontia—white thread-like, nematocyst-laden extensions of the internal septa. These acontia are shot out through pores in the wall of the column called cinclides. Find some acontia and cinclides. Place a small piece of acontium on a slide, squash it gently with a coverslip, and examine it under the compound microscope to find nematocysts and ciliated cells. Live Metridium in the aquarium will probably be more expanded and responsive than those in dishes. Find such an expanded animal and gently touch an inert object to the tentacles, noting how they react, direction of bending, etc. As this stimulus continues, what is the order in which other parts of the body are brought into the reaction? Excess stimulation will cause the localized raising of the edge of the oral disc, and this will close around the disc by contraction of the sphincter ’muscle’ (contractile fibers of epitheliomuscular cells here). What do these probing experiments show about the nervous system of anemones? Small live specimens of Metridium or Aiptasia are often transparent enough to see food inside the gastrovascular cavity. Offer the anemone a few live brine shrimp by squirting them into the dish or use clean forceps to touch a small piece of fresh mussel to the tentacles of an expanded anenome and watch the feeding reaction. Are nematocysts discharged? Is the food stuck to the tentacle? Retrieve the food and examine it under a compound microscope to see if nematocysts are attached. Watch to see how the anemones move food into the gastrovascular cavity. How do the acontia at the base of the pharynx behave? Does the food get into the tentacles? Try offering a piece of paper or a stick to an anemone and see if it is rejected. What does this tell you about sensory responses? From a larger anemone, pull off a tentacle and place it on a slide so that you can study it under the compound microscope. Leave a small drop of sea water around the tentacle and cover it with a coverslip. First try to find the two tissue layers, the epidermis and gastrodermis; then look for cnidocytes with their bristle-like cnidocils and nematocysts. Nematocysts will stand out as refringent rods. Some will have discharged, shooting the thread out; look for spines on the thread. You may be able to make unexploded nematocysts discharge by applying pressure with the coverslip or by adding a bit of methylene blue. Where are the zooxanthellae in a tentacle of Aiptasia? Are they localized to certain cells? How can you tell these are algal cells? Look at the mouth in the center of the oral disk and find the siphonoglyph (some specimens may have more than one siphonoglyph). See if you can see evidence for its cilia driving water currents into the pharynx. Especially with a contracted anemone left undisturbed and in the process of re-expanding, the siphonoglyph should be quite active in pumping water into the gastrovascular cavity to lengthen the body and spread the tentacles. Using a preserved specimen, identify the major parts mentioned above; they may be easier to find in these pre-relaxed specimens. Look also at the cross-sectioned and longitudinally-sectioned specimens and at the prepared slides of histological sections of Metridium. In a cross-sectioned portion identify the siphonoglyph

66 in the pharynx. The gastrovascular cavity is divided by septa (also called mesenteries) running parallel with the axis of the column; these are folds of the gastrodermis and its supporting mesoglea and arise from the column wall, extending toward the center of the column. Those septa joining the pharynx are the primary septa; there are six pairs of primary septa, including two pairs of directives, the septa that anchor the ends of the pharynx. Between the primary septa are secondary and tertiary pairs of septa, septa that are incomplete, not reaching the pharynx; secondary, tertiary, etc., are differentiated by relative length. Often irregularities are present in the septa of Metridium, and so it may be difficult to distinguish between secondary septa. At the inner margins of the secondary septa are septal filaments which are continuous with the coiled acontia lying in the gastrovascular cavity. They bear ciliated cells responsible for moving fluid through the cavity as well as cnidocytes and gland cells secreting digestive enzymes and mucus. Examine the slides (histological sections) to find these cell types on the septal filaments. Retractor fibers lie back from the edge of the primary and secondary septa; in the septa of the preserved animals they will appear as cords and in the histological sections as red-staining feather-like masses. These contractile fibers are mainly derived from the gastrodermis, so note that they are in the epitheliomuscular cells and simply more developed than other contractile bases of the epithelia.. The retractor fibers on the directives face away from each other, away from the space between the members of each pair (the so-called exocoelic surface); those on each of the other primary septa face toward each other, to the inside of the space between the members of the pair (the endocoel). Parietal contractile fibers run in the septa close to the column wall. Circular contractile fibers are also present in the column, pedal disc, and tentacles and will be visible in the histological sections. Though anemones look rather sedentary, they are actually moving all the time; if you keep track of the position of an anemone in the aquarium over a few days, you can detect its lateral gliding movement. Pieces of the pedal disc that are left behind can regenerate into new anemones. Both Metridium and Aiptasia will be producing small daughter anemones by this process. Examine the longitudinally cut specimens to find, at the oral end of the primary and secondary septa, the ostia through which the chambers of the coelenteron communicate. Also find in these specimens the gonads in the septa. Other actiniarians. Anthopleura, a West Coast anemone, is available as preserved specimens on demonstration. Compare the arrangement of tentacles in Anthopleura and Metridium. Zoanthidean (Fig. 7-28, -29). Like other zoantharians, Zoanthus (preserved on demonstration) is a colonial anemone-like organism in which the rather small polyps arise from a common spreading mat. Scyphozoans – the jellyfish Saemeostome scyphozoans Aurelia sp. (Fig. 7-47 – 7-49.) Preserved specimens of this semaeostome jellyfish are available and should be studied submerge in water. Identify the marginal tentacles, eight rhopalia, and the oral arms. Push the oral arms to one side and identify the gastric pouches with the gonads in their walls, and with a fringe of gastric tentacles. On the subumbrellar surface find the subgenital pit. The gastrovascular cavity of Aurelia is divided into a complex system of canals which are named by the body axis in which they sit. Perradial canals are those in line with the four arms of the manubrium, interradial canals lie between these, and adradials in the axes between these, etc. Fluid is pumped through the canals in a defined order: outward through adradials, inward through inter- and perradials. In drawing an adult Aurelia, show in one quadrangle details of tentacles, canals, gastric pouch, and the oral arms. A rhopalium, whole-mounted and stained, is on demonstration. Stages in the life cycle of Aurelia are on demonstration as prepared slides (cf. Fig. 7-49). Look first at the slide on demonstration of a planula of Aurelia; this is a simple larva with an epidermis outside and a parenchyma-like filling inside. The other stages are available on slides for you to take to your microscope. Next in line is the scyphistoma, which is the polypoid generation. Find the tentacles, stolon, and the base of attachment. Features that distinguish the scyphozoan polyp from polyps of other cnidarians are tetramerous symmetry–with four septa separating four gastric pouches–and septal funnels, which each appear as an indentation of the epidermis into each septum. The number of tentacles also reflects this tetrammery, but tentacle number is not definite, ranging from 8-20 in scyphopolyps in general; in this species, the number is probably 32. Note how the gastrodermis in the tentacles forms a solid core and is not epithelial. Some of the scyphistomae can be seen to be budding to produce new scyphistomae. Next is the strobila, which is budding developing ephyrae like stacks of saucers. The released ephyra of Aurelia looks like a scalloped disk. The marginal lappets are the most conspicuous feature; the primary

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tentacles develop between these. Find the mouth; its corners point in the direction of the perradii. Arms lie along the perradii and interradii, and canals develop inside (perradial, interradial, and adradial canals). Look for gastric filaments in the stomach (the interradial portion). Stauromedusan scyphozoans Haliclystus sp. (Fig. 7-55), a stalked jellyfish, is on demonstration preserved. Stauromedusans have the body form of a polyp whose oral end is partially differentiated into a medusa, including development of gonads in the gastral pockets. They are sessile, attaching to such substrates as rocks and seaweeds by an adhesive pedal disk. What look superficially like rhopalia between the clusters of tentacles are adhesive knobs that the stauromedusan can use to loop along, summersault-like, to new attachment sites.

Hydrozoans – the hydroids Hydrozoans typically have colonial polypoid forms alternating in the life cycle with medusoid forms. The medusa is the generation bearing the gonads and the larvae that it produces through sexual reproduction settle and grow as colonies of hydroids from which special zooids bud free-swimming medusae or sessile medusoids (reduced forms that do not leave the parent colony). In the Athecata and Thecata, the polypoid generation is most conspicuous and typically forms relatively large colonies; the two are easily distinguished by differences in the extent of the cuticular covering, the perisarc: it extends as a cuplike sheath, the hydrotheca, around the hydranth in Thecata, whereas members of the Athecata have naked hydranths. In the Limnohydrina, the polypoid generation is small and inconspicuous, and the most commonly encountered form is the medusa. The Siphonophora comprises planktonic animals that are colonies of both polyps and medusoids. Athecate hydroids Ectopleura (=Tubularia) crocea. (Figs. 7-58, 7-64B, pp 160, 165.) This pink hydroid is commonly seen on pilings and floats. The flower-like hydranths arise on long unbranched stalks from a stolon called a hydrorhiza and have two whorls of filiform tentacles, one of longer tentacles at the base of the hydranth and one of shorter tentacles around the manubrium. Between the whorls, gonophores develop in grape-like clusters borne on blastostyles. The gonophores enclose attached medusoids which produce the gametes; males have purple gonophores, females pinker ones. Larvae, including a planula and an actinula larva, develop inside the female gonophores and leave the gonophore in the actinula stage which looks like a small polyp with only a dozen or fewer tentacles. Try to find both larval stages in the gonophores and actinula in the process of escaping the gonophores or settled on or near the parent colony. Other tubulariids have free-swimming medusae. Try feeding copepods or other small crustaceans to the hydranths. Pull off a tentacle or two from a hydranth and use a pipet to place it on a slide with a drop of sea water; cover the tentacle with a coverslip. First try to find the two tissue layers, the epidermis and gastrodermis, and look for cnidocytes with their bristle-like cnidocils and nematocysts and note how they compare to those of the anemones you studied. Nematocysts will stand out as oval, refringent bodies. Some will have discharged, shooting the thread out; you may be able to make others discharge by applying pressure with the coverslip. To see details of nematocyst structure, you will need to use the oil-immersion lens. Halocordyle (Pennaria) (Figs. 7-58, -62, pp. 160, 164), also an athecate species, is available on slides demonstrating both polypoid and medusoid generations. The gonophores form on the hydranths of Pennaria, sometimes more than one on a single hydranth. Each gonophore will become a single medusa which eventually buds off the polyp. The medusae are free-swimming for a brief period and have relatively degenerate tentacles, appearing as only bumps in these preparations. Hydractinia sp. (Fig. 7-61, pp. 163), may show up on hermit crabs available in the lab. It typically grows on such crab-inhabited snail shells (and so gets the name “snail fur”) and also on other substrates. The colony is polymorphic—that is, differentiated into several types of hydroids: gastrozooids for feeding, gonozooids for reproduction, and dactylozooids for defense. Thecate hydroids Obelia sp. (Figs. 7-63, 7-64A, p. 165), is available both as living colonies and as whole mounts on slides; start with the living material but use the whole mounts to find structures not easily seen on the living colonies, particularly if you cannot find gonophores on the living material. Note how the hydranth is protected by a hydrotheca (a structure absent from the Athecata). Identify also the perisarc with its annuli; the coenosarc; the tentacles (occurring in a single basal whorl); the manubrium and stomach region of the hydranth; a

68 gonangium, composed of blastostyle, medusa buds, and the enclosing gonotheca. Try feeding copepods or other small crustaceans to the hydranths. Study the histological sections of the polypoid generation of Obelia and find the cell types mentioned by the textbook for cnidarians (Figs. 7-2, 7-7 p. 113, 118; epitheliomuscular cells, cnidocytes, gland cells, interstitial cells, sensory cells). The gastrodermal cells are large and watery (clear and almost rectangular in these sections with cytoplasm appearing as web-like strands); covering them are the more densely staining epidermal cells and cnidocytes, the latter recognizable by the nematocysts they bear. Between the two epithelial layers is the mesoglea, so thin in hydrozoans that it is referred to as a mesolamella. Find also nerve cells (lying within the plain of the epidermis at its base) and mucous and other gland cells. Study the cells and nematocysts under the oil-immersion lens. Study prepared slides of Obelia medusae, looking both at slides of young specimens and of mature medusae. In the mature medusa identify subumbrellar and exumbrellar surfaces, the manubrium, circular canal, radial canals with the four gonads suspended from them, tentacles, and statocysts. Plumularia sp. (cf. Fig. 7-60, p. 162) forms featherlike colonies and has hydrotheca like stemless cups attached along one edge to the stalk or branches. Special defensive polyps called nematophores are interspersed among the gastrozooids; these are a kind of heterozooid, distinguished from the autozooids or feeding polyps. Limnohydrozoans Gonionemus sp. (Fig. 7-59, p. 161) is a common medusa which arises from a reduced, one-tentacled polyp stage. Study preserved Gonionemus medusa. First note the velum, a feature which distinguishes hydrozoan medusae from scyphozoan medusae. Identify the manubrium, mouth, and the oral lobes. The gonads take the form of a curtain of tissue hanging in the subumbrellar space, beneath each radial canal. The tentacles possess welts of nematocysts along their length, and about two-thirds along their length from the bell there is a conspicuous adhesive pad. Statocysts and tentacular bulbs may be identified around the margin of the bell. Siphonophoran hydrozoan. Physalia pelagica (Fig. 7-70), the Portugese Man-of-War, is a siphonophoran colony; specimens are on demonstration. This is a tropical oceanic species regularly washed into inshore waters from the Gulf Stream. The balloonlike float is brilliantly blue, pink, and purple in life, with a deflatable sail-like ridge above. It can be as long as 1 foot; the tentacles trailing below it can be up to 40-50 feet. Try to locate the different polypoid forms below the float: gastrozooid, dactylozooid, and gonozooids.

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While ctenophores resemble jellyfish in their gelatinous, transparent appearance and use of tentacles, they differ distinctly in the way they move—i.e., by comb rows of giant cilia—and the way they capture prey—by specialized adhesive cells. Sea gooseberry: Pleurobrachia pileus (Figs. 8-1, 8-8, 8-9, pp. 182–189). Study one of the live specimens of Pleurobrachia under the demonstration dissecting scope. If living animals are not available, something of ctenophore morphology can be seen in a preserved specimen. Turn these specimens carefully; these animals are quite fragile. Pleurobrachia has an ovoid shape. The ciliated comb-rows, bearing fused rows of cilia, called ctenes, are clearly visible (Fig. 8-2). These cilia are among the longest cilia in the animal kingdom, being measured in millimeters; they give the living animal a vibrant iridescence. The ctenes can be seen under high magnification of the dissecting scope. At the oral pole find the mouth. Through the transparent body, observe that the mouth opens into a laterally compressed pharynx, which leads to the stomach, in the center of the animal. At the aboral pole, find the statocyst with its statolith suspended on balancer cilia (Fig. 8-4, 8-7). Ciliated tracts from the statocyst leading to each of the comb rows conduct signals that coordinate their beating. On opposite sides of the specimen, toward the aboral pole, find the two conspicuous openings to the tentacular sheath within which the contracted tentacle can be seen. The tentacular sheath runs inward and downward, to end at the tentacle base, an elongated, opaque structure lying parallel to the oral-aboral axis. The tentacles are branched and retractable. Pleurobrachia captures prey with these tentacles by setting them out like a driftnet. It relies on chance encounters of the prey with the colloblast-laden tentilla (Fig. 8-5). When food has been captured, the animal spins its body to wrap the tentacles around it and so bring the base of the food-laden tentacle within reach of the mouth (Fig. 8-8); the mouth then closes around the tentacle, and longitudinal muscles of the tentacle contract to pull it through the lips and to wipe the adhering food off. Try feeding Artemia nauplii to a live specimen and watch how they stick to the tentilla but keep kicking. (Compare this to the capture by cnidarians, in which the prey rather quickly becomes immobilized.) The live ctenophore may try to execute the spin-capture, but if there is not enough room in the dish, it may not be able to wipe the tentacles off on the mouth so well. The gastrovascular system of Pleurobrachia is relatively complex in comparison with that of the cnidarians, consisting of canals radiating from the central stomach and paralleling the comb rows. You may be able to make out some of these through comparison with Figures 8-4 and 8-9.

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Lophophorata, Tunicata, Hemichordata bryozoans, brachiopods, sea squirts, acorn worms Lophophorata PHORONIDA BRYOZOA Phylactolaemata Pectinatella - slides, demonstration Gymnolaemata Bugula – live, slides Membranipora – live Schizoporella – dry other bryozoans in marine fouling communities BRACHIOPODA Inarticulata Lingula – preserved demonstration Articulata Terebratella – preserved demonstration CHORDATA Tunicata (Urochordata) Ascidiacea (tunicates, sea squirts) Ciona — live Ecteinascidia — slides Boltenia — live, preserved Botryllus — live or preserved Botrylloides — live Didemnum or Lissoclinum — live tadpole larvae — slides Thaliacea (salps) Pyrosoma — preserved demonstration Salpa — preserved demo. Larvacea (larvaceans, appendicularians) Cephalochordata Vertebrata . . . HEMICHORDATA Enteropneusta Saccoglossus — live or preserved demo Pterobranchia Rhabdopleura — demo

Brachiopoda Brachiopods bear a bivalved shell, one valve ventral, the other dorsal (contrast this with the lateral valves of bivalve molluscs). The brachiopods we have for study are all on demonstration: Lingula pyramidata (Fig. 25-9A, 25-11, pp. 823, 825), Terebratulina and Terebratella (Figs. 25-8, 25-10, pp. 822, 824). In inarticulate brachiopods like Lingula, the valves are not hinged together. There is a long peduncle, by means of which the animal anchors itself in the muddy bottom; by contracting the peduncle, it can withdraw its body from the surface of the mud. Projecting from between valves you will see numerous setae; these arise from the mantle. In the specimens that have been opened, find the major organs using the accompanying diagram as a guide. The coiled lophophore fills about half of the shell; inside the body you can see digestive gland, gonad, and muscles to the valves. In articulate brachiopods like Terebratella and Terebratulina, the valves are hinged at the posterior edges, and the peduncle attaches the animal to a hard substrate. The ventral valve has a posterior umbo which is pierced by a formamen through which the peduncle passes. The tentacle-bearing lophophore consists of

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two horseshoe-shaped lateral arms and a median process that coils anteriorly and dorsally (Fig. 25-13). A calcareous loop supports the lophophore. Look at the opened specimens of Lingula and Terebratulina to see the general layout of the lophophore and internal organs.

Bryozoans General observation of bryozoans (Figs. 25-15 – 25-33, pp. 829–844). As you look at any of the living bryozoans available for study, note colony form and shape of the zooecia housing the individuals of the colony. Encrusting bryozoans, such as Membranipora and Schizoporella, have box-like zooecia arranged like bricks in a patio; in upright branching forms such as Bugula, Crisia, and Bowerbankia, overlapping zooecia compose branches a few individuals wide. More than likely, we will have living specimens of Membranipora (or Electra) spp. and of Bugula. Take a specimen in a fingerbowl of sea water. Also take prepared slides of Bugula and Pectinatella to see the internal morphology of individuals. In studying the living specimens under the dissecting microscope, avoid placing the light too close to the specimens so that the animals are not overheated. From the apertures of some of the zooecia in the living colonies you should be able to find expanded polypides with their tentacular apparatus, the lophophore, extended in feeding (Figs. 25-24, 25-25). The ciliated tentacles generate a feeding current and conduct food to the mouth, which is located in the center of the lophophore, by ciliary tracts and by flicking movements of individual tentacles. The gut leading from the mouth is U-shaped. Leading downward from the mouth is a bulbous pharynx, and under that an esophagus; the junction of esophagus and stomach forms the caecum and represents the bottom of the U, the stomach being the large chamber on the other arm of the U. Above the stomach there is a short, bulb-like intestine, which opens via an anus. Note that the anus is situated outside the lophophore. From the caecum a double strand, the funiculus, runs to the wall of the zooecium. Retractor muscles run from the lophophore to the wall of the zooecium. Look for brown bodies present in some of the zooids; these are accumulations of waste material alongside the gut and are expelled by degeneration of the polypide which then regenerates from tissues remaining in the cystid. Polymorphic colonies have defensive zooids (Fig. 25-21) such as avicularia (so-called because they look like birds’ heads; typically perched alongside the apertures of the feeding zooids in Bugula, for example) and vibracula (whip-like projections), and hemispherical ovicells overhanging the distal ends of some of the zooecia (Fig. 25-27). Look for these zooids in the colonies you examine. Membranipora membranacea is an invasive species that has become very common; it grows as encrusting lacy patches on the brown alga Laminaria. (It was probably established from cyphonautes larvae dumped with ship ballast water, and is outcompeting other bryozoans that are native.) The zooids are typically very active and their internal parts can be viewed easily through the transparent frontal membrane. Are any predators of Membranipora present on the colonies you see? Is there evidence of intercolony competition? Examine also preparations of a living strip cut with a razor blade as a one-zooid-wide radius from a colony; this can be manipulated under a coverslip for viewing from the side. Look for the rather flattened eggs in the coelom and the very thin, refringent spermatozoeugmata in the tentacles. With patient observation you may see release of gametes. The Bugula species we have available has spirally arranged branches and well-developed avicularia alongside the apertures of its tubular feeding zooids. What other types of zooids are present? If living specimens are not available, use the preserved ones to get an idea of the three-dimensional arrangement of these zooids; you may also get a sufficient image of these zooids from the prepared slides. A large colony of the freshwater bryozoan Pectinatella magnifica (cf. Fig. 25-18, p. 832) is on demonstration. The bulk of the colony consists of a gelatinous mass which was secreted by the zooids. The whitish substance over the surface are zooids, and the numerous black objects are statoblasts. Slides of extracted zooids with statoblasts (asexually produced overwintering bodies) are available for study (cf. Fig. 25-33, p. 844). Tunicata (Urochordata) Ascidians (Figs. 29-12 – 29-25, pp. 941–950). Solitary (simple) and colonial ascidians: Ciona intestinalis, Molgula manhattensis, and Ecteinascidia turbinata. (cf Fig. 29-15, p. 942). Watch a living specimen of Ciona or Molgula in a finger bowl of sea water to study the form and behavior of a solitary (simple) ascidian. Also obtain a wholemount slide of Ecteinascidia to study internal anatomy and to compare with Molgula or Ciona.

72 Allow the living sea squirt to relax while it is completely covered with sea water. As the siphons extend, you will see that they are of unequal length. The longer inhalant or buccal siphon appears to lie at the apex of the animal; it opens into the mouth and is anterior. The exhalant or atrial siphon opens into the side of the atrium and marks the dorsal side of the animal. Both siphons have lobes or papillae with sensory structures to monitor water flow and, between them in Ciona, red ocelli. At the other end of the body or even along the sides where the body contacts substrate are stolons, finger-like projections by which the animal attaches to the substrate (and to the remainder of the colony in Ecteinascidia). Verify the identity of the siphons by watching flow of particles in or out (with a drop of carmine suspension if necessary, or see what the animals will do with the yeast suspension). Try poking the animal, including both the exterior and interior of the two siphons, with a fine probe and see which parts of the body are most sensitive and how the body-wall longitudinal muscles (which appear as clear bands running the length of the body unde the tunic) contract. The animal is covered with a thick tunic, a cuticle, and in Molgula this characteristically incorporates much foreign material as a sort of camouflage. You may be able to see through the tunic (especially of Ciona) and identify some of the internal organs, or you can compare your specimen with the wholemount slide of Ecteinascidia in which the tunic has been cleared and internal organs have been stained. On either animal you will see that the body consists of two regions, the thorax and the abdomen. The thorax is mostly the branchial or pharyngeal portion of the gut. The abdomen contains the viscera (gonads, gut, and heart). Dissection. To clearly see internal organs in Ciona or Molgula, it will be necessary to dissect off the tunic. Insert scissors into the buccal siphon and cut downward slightly to the right of the median line. Continue this cut to one side around the base of the body and then spread the halves apart. Note the large, lattice-like gill basket (cf., also, Fig. 29-23). Its cilia create the current which passes into the buccal siphon, through the slits in the gill basket, into a space called the atrium which lies between the pharyngeal basket and the body wall, and then out via the atrial siphon. On the side of the pharyngeal basket opposite the atrial siphon (the ventral side) there is a conspicuous band running the length of the basket. This is the endostyle, which secretes mucus in a sheet that spreads around the pharynx and serves in filtering food from the inhalant water current. The ciliated fold opposite the endostyle is the dorsal lamina; this wraps up the mucus and transports it to the mouth. Identify the esophagus, which leads into a sac-like stomach, which in turn leads to the intestine that courses upward into the atrium to open via the anus not far below the atrial siphonal aperture. The intestine typically contains fecal material. In the bend of the intestine lies a circular mass of gonads. The ovary contains eggs, and the testis appears to comprise numerous small nodules. A sperm duct accompanies the intestine up into the atrium. This appears as a thin cord. On the side opposite the gonads, eggs and larvae in various stages of development may be found. If so, examine these, noting especially any tadpole larvae. Between the buccal and atrial siphons and near the surface lies a small dense structure, the nerve ganglion. The heart of ascidians is peculiar in that it reverses beat periodically. Try to see heartbeat through the tunic and to see blood cells as they are pumped through blood vessels (cf. Fig. 29-21, p. 947). After finding the internal organs, try mounting a piece of the pharyngeal basket on a slide with coverslip and observing the shape of stigmata (modified gill slits) in it and motion of its cilia (Fig. 29-26). A slide showing a stained piece of the pharyngeal basket of Molgula is on demonstration. Compound ascidians: Botryllus schlosseri, Botrylloides sp., Didemnum sp. or Lissoclinum sp. (Figs. 29-12, 29-16, pp. 941, 943). Botryllus shows best how compound ascidians are arranged—that is, how individuals are grouped into a colony. If live colonies of Botryllus are available, start with those; otherwise, study the preserved specimen of Botryllus on demonstration. The rosettes scattered over the surface of the tunic are groups of individuals sharing a common tunic. Each individual has its own buccal siphon; these siphons lie to the outer edge of the group. In the group’s center is a common atrial chamber serving the entire group. The other compound tunicates (such as Botrylloides and Lissoclinum) are similarly constructed but not in such neat star-shaped clusters of zooids, and they have so many spicules and pigment that discerning internal structures is a little more difficult. Give it a try, anyway, and also look at any of these compound ascidians, Botryllus included, for blood vessels and spicules in the tunic. Individual zooids as well as larvae they brood can be isolated from the colonies of Botrylloides simply by squeezing or teasing apart the edge of a colony. Do this over a bowl of sea water and then check the bottom of the bowl first for swimming tadpole larvae. Mount some larvae as well as some freed zooids on a slide. In the zooids, you should be able to see the terminal oral siphon (and dorsal atrial siphon) and the pharyngeal basket; use the higher powers of the microscope to see the gill slits and the cilia in the pharynx. You may be able to distinguish the three regions of the body: the thorax, abdomen, and postabdomen. The gonads

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and heart lie in the postabdomen; other internal organs are as described above. Tadpole larva. If you cannot get larvae out of Botrylloides, study the preserved whole-mount preparations (slides) of the larva of an unidentified tunicate. On either, try to distinguish the various structures highlighted in the textbook’s figure (Fig. 29-24, p. 950). The larva is called a tadpole larva because of its resemblance to amphibian tadpoles. It consists of a body and tail, both enclosed in tunic. Prominent at the anterior end are adhesive papillae and in the brain the eyespot and statolith of the cerebral vesicle. The pharynx is well-developed even in the larva though it does not open to the exterior; ventral to it are the stomach, intestine, and heart. The tail is supported by the notochord which stretches about two-thirds of its length and ends next to the pharynx. Above the notochord is the nerve cord. Pyrosoma and Salpa on demonstration (Fig. 29-27 – 29-29, pp. 953–954) These urochordates are pelagic; because the buccal and atrial siphons lie at opposite ends of the body, the current through the pharynx can be used for jet propulsion, propelling the colony through the water. In Pyrosoma (so-named [“fire body”] because of its bioluminescence) the colony is thimble-shaped; individuals of the colony have their buccal siphons directed toward the outside and their atrial siphons directed toward the inside so that water processed by the individuals must exit through the open end of the thimble and so propel the colony through the water column. In Salpa, zooids are are linked together in chains, and circularly arranged muscle bands in individual zooids are responsible for driving water through the pharynx (replacing the cilia-driven currents of other urochordates). The stage on demonstration is such a chain produced by asexual reproduction from a stolon on the so-called oozoid stage; each individual here reproduces sexually and bears an egg that will develop into a new oozoid. Hemichordata Acorn worm: Saccoglossus bromophenolicum (Figs. 27-1, 27-4, 27-5, pp. 858–862). The name of this species derives from its iodine-like smell (actually a bromophenol compound). The body is divided into an anterior proboscis (cream-colored in live animals), a collar (orange), and, posteriorly, an elongate trunk. The proboscis attaches to the collar region by means of a thin proboscis stalk. The mouth is ventral at the base of the proboscis stalk and the ventral side of the collar. Posteriorly, the free edge of the collar of Saccoglossus overlaps the anterior part of the trunk. Find the pharyngeal slits (“gill slits”) on the front dorsolateral part of the trunk (the animals curl ventrally, so the dorsal side is to the outside), starting immediately behind the collar and extending posteriorly in a row on either side of the mid-line of the trunk. Behind this so-called branchial region lies a region without gill slits, the beginning of which is marked arbitrarily by the posterior pair of gill slits. This is the genital region and contains the gonads. Gonads open to the outside by genital pores which may be too small to distinguish. The abdominal region, usually tightly coiled, contains the intestine and lateral pouches of the hepatic caeca; it appears greenish in living animals. At its tip is the anus (out of which you may see sandy fecal material being extruded). If live animals are available, look for the effect of cilia over the proboscis and rest of the body by watching particles. Try gently putting a little sand on the proboscis, or use carmine suspended in sea water, or see if the worms will consume suspended yeast particles (all available at the side counter). Notice how the worm can reject particles it does not want entering the mouth by bringing the edge of the collar up close to the proboscis; otherwise the gap between proboscis and collar is larger. Pterobranch: Rhabdopleura. (Figs. 27-9, 27-10, pp. 865–866). Pterobranchs are best known from deepwater samples collected in the southern hemisphere, but they have recently been discovered in shallow water in Bermuda. A portion of a colony and an individual zooid of the pterobranch Rhabdopleura collected from Bermuda are on demonstration. Each zooid bears two antler-shaped arms with tentacles by whose cilia it collects suspended material for food. Its body is rather pear shaped, and from the ventral side of its base a stolon arises, connecting the zooid with others of the colony. At the base of the arms is a cephalic shield, homologous with the proboscis of the enteropneusts and by which the zooid can creep within the branching tubes of the colony and out to extend its tentacles at its tube’s opening. The mouth sits just behind the cephalic shield, and the gut is U-shaped so that the anus opens dorsally behind the arms (i.e., closer to the tube opening than if the gut were straight).

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Echinodermata ECHINODERMATA Crinoidea (sea lilies, feather stars) Antedon — whole-mount demonstration Promachrocrinus — preserved demonstration Asteroidea (sea stars, starfish) Asterias — live, preserved, dry, slides Henricia — live Ctenodiscus, Solaster, Crossaster, Oreaster — dry Ophiuroidea (brittle stars, serpent stars) Ophiopholis — live, dry Ophiura — dry Gorgonocephalus (basket star) — dry Echinoidea (sea urchins, sand dollars) Strongylocentrotus droebachiensis – live, dry Echinarachnius – live, dry Mellita – dry Brisaster - dry Holothuroidea (sea cucumbers) Cucumaria – live, preserved Leptosynapta – preserved, slides

Crinoids (on demonstration): Promachrocrinus sp. and Antedon sp. (Figs. 28-54 – 28-59, pp. 918–923). Crinoids have long feathery arms with branching pinnules bearing the tube feet, and the arms and oral surface are directed upward. Most crinoids have a long aboral stalk by which they are anchored to the substrate, and this flower-like morphology is the basis for calling them sea lilies. Promachrocrinus on demonstration comes from Antarctica. Note the stalk’s finger-like basal part, which anchors in the sediment, and the soft feathery arms. More modern crinoids like the feather-star Antedon have the stalk only as juveniles and break away from the stalk when adult. On the plastic-embedded specimen of Antedon on demonstration (Please handle these specimens with care!) note how there are curled cirri on the aboral surface of the disk; these are used for temporary attachment and for climbing up to good feeding sites on prominences. The animal has five pairs of arms, each pair representing a division, during development, of one ray. Pinnules on the arms appear as alternating lateral branches. The arms can be used for swimming by alternate up and down strokes; every other arm makes a downward stroke as the alternate arms are swung in upward recovery; then the former recover as the latter make their downward stroke. Because the arms of these specimens are closed over the body, it is difficult to see the mouth, which lies in the center of the disk, and the anus, which lies also on the oral surface on a prominent, eccentric anal papilla. Five ciliated food grooves converge on the mouth from the arms; they branch out onto the arms and onto the pinnules. Along the sides of the grooves are rows of yellowish sacculi, or dermal vesicles. Tube feet, or podia, covered with mucous glands, project inward over the grooves, originating in single rows on either side of the ambulacral grooves. Food is caught in the mucus on the podia and passed to the mouth along the ciliary currents in the food grooves. Use the microscope to find the podia and food grooves. The leathery skin or tegmen is embedded with minute calcareous plates. There are no spines or pedicellariae. Neither is there a madreporite, but the tegmen is perforated by numerous pores that open into the visceral coelom. Gonads lie in the pinnules that are nearer the disc (but are not so prominent in this specimen); gametes are released simply by rupture of the gonads. Asteroids. The arms are more or less set off from a central disk in the asteroids (Fig. 28-5, p. 877). Asterias and Henricia are local intertidal representative with the typical 5-rayed body; Ctenodiscus is a deeper-water, mud-living form; Crossaster and Solaster are found from the intertidal to subtidal hard-bottom habitats and have many arms; Labidiaster is a many-armed predator from Antarctica, notorious for using its large monster-jaw-like pedicellariae to capture prey, including small fish. Note the spines on the aboral surfaces of the sea stars (cf. Figs. 28-6–11, pp. 878–880) and the madreporite, a stonelike plate at one edge of the central disc. The mouth lies in the center of the central disc, and running down the center of each ray or arm is the open ambulacral groove with its many tube feet arranged in four rows.

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Live sea stars (Asterias sp.) are available for observing behavior. Watch how the animal moves on a glass surface such as the sides of the aquarium, a glass dish, or on a slide introduced into the animal’s dish. Think about how the individual tube feet move, what muscles are involved in extending a tube foot or retracting and bending it, and how the sucker works. Turn the specimen so that its oral surface faces you and find the mouth in the center of the central disc. Running down the center of each ray or arm is the open ambulacral groove with its many tube feet arranged in four rows. At the tip of each ray is a special modified tube foot (or tentacle) which lacks a sucker and has a light-sensitive pigmented eyspot at its base. A thick white radial nerve cord runs down the center of each ray and is visible if you spread the tube-feet rows apart. Movable spines bordering the ambulacral groove can be positioned to protect it; those spines surrounding the mouth are larger and can be used to push food into the mouth. Conspicuous on the aboral surface is the madreporite. The microscopic anus lies in the next interradius in a clockwise direction from the madreporite. The aboral surface is covered with calcareous tubercles, warts, and spines; with pincer-like pedicellariae, which are modified spines; and small fleshy papulae which are protrusions of the coelom that emerge between the plates of the endoskeleton and serve in respiratory-gas exchange. Dissection of sea star Dissection can be done on either an anesthetized or a preserved specimen. Carefully remove the aboral surface of the central disc and of the three arms opposite the madreporite to produce a view similar to that depicted in Figure 28-16 (p. 884). Start cutting by inserting the point of a pair of heavy scissors through the body wall at the tip of one arm and extend the cut down both sides of the arm toward the central disc, being careful not to disturb the underlying organs. Carefully separate the madreporite from the body wall so that it remains connected to the rest of the vater vascular system. Lift the cut aboral body wall completely free from the body, pin the animal to a waxed dissecting tray, and flood it with water (seawater if the animal is alive). Loosen the internal organs from the mesenteries by which they are suspended. Find the digestive tract. It consists of a short esophagus leading from the mouth to the stomach which is divided into a globular evertible cardiac portion and an aboral pyloric portion. A pair of hepatic (pyloric) caecae extend from the pyloric stomach into each of the arms; these function in digestion and food storage. The rectum runs from the pyloric stomach to the aboral body wall. Also prominent in each arm are a pair of gonads which lie mostly under the pyloric caecae. Ovaries are usually red; testes white or yellow. Trace the stone canal from the madreporite to the ring canal on the oral side of the body. (You may want to remove the digestive tract to make finding the more oral parts of the water vascular system.) Try to find the nine Tiedemann’s bodies on the ring canal. Radial canals run into the arms; trace one radial canal by lifting the pyloric caecae in that arm and find the connections to the tube feet and their ampullae. Under the radial canal, lies the radial nerve, which may be visible with the arm held up to shine light through it. Slides of cross sections through the arm of a sea star are also available. Compare these to Figures 28-12 and 28-15 (pp. 881, 883) to find those structures identified in the dissection. Brittle stars and other ophiuroids (Figs. 28-21–27, pp. 891–896). The arms of brittle stars are more distinctly set off from the disk than are arms of the sea stars, and they do not contain parts of the gut or gonads. Also unlike sea stars, brittle stars use their long, sinuous arms in rowing motions for locomotion, not their tube feet which instead are used only in feeding; most brittle stars feed on small zooplankton and other suspended material; they sometimes feed on deposit material and sometimes act as scavengers. Ophiura and Ophioderma on display are deeper-water species from the Gulf of Maine; Ophiopholis, the daisy star, is common in intertidal and shallow subtidal habitats; Gorgonocephalus, the basket star is found in the shallow subtidal, using its branched, curling arms to capture sizeable zooplankters. In the live specimens that are available of the daisy brittle star, Ophiopholis aculeata, look at the oral surface and find the mouth and its triangular jaws (cf. Fig. 28-22, p. 892); the madreporite is also on the oral surface, on one of the oral shields. The tube feet on the arms project between the skeletal plates or shields of the oral surface of the arms. The ambulacra, therefore, are covered. Note that the tube feet lack suckers. Bending in the arms takes place through articulation of central ossicles, the so-called vertebrae. At the base of each arm is a pair of genital bursae. (Some brittle stars, notably the dwarf brittle star Axiognathus squamata, brood their young in the bursae.) On the aboral surface note the covering of spines and the lack of an anus. Echinoderm development. Slides of echinoid (sand dollar and urchin) developmental stages are on demonstration to show some of the features mentioned in class as characteristic of all echinoderms. The

76 slide labeled “Segmentation to Larva” shows cleavage stages (note that cleavage is total, equal, and radial), blastulae, gastrulae, and early pluteus larvae. The other demonstration slide shows more developed plutei.

Sea urchin: Stongylocentrotus droebachiensis (cf. Figs. 28-29, 28-30, 28-31, 28-34, 28-36, pp. 898–902). Use a dissecting microscope to study a living sea urchin in a bowl of seawater. Over the surface of the urchin are spines, tube feet, and pedicellariae. The tube feet (Figs. 28-34, 28-37, pp. 901, 903) are different from those of Asterias in having a calcalcareous plate supporting the sucker, and the pedicellariae are different in having three jaws. The tube feet are restricted to five radia, the ambulacra; and unlike the ambulacra of sea stars, these ambulacra are closed–i.e., they are covered by plates so that the tube feet emerge through holes in the plates. Five interambulacral regions with no tube feet separate the ambulacra from each other. All of the epidermis is ciliated, including that covering the spines. In the center of the aboral surface (cf. Fig. 28-31, p. 899) is the periproct which is a membranous area bearing many small ossicles; the anus sits in this in an excentric position. Around the periproct are five larger plates, the genital plates, each with a genital pore; the largest of these plates bears the madreporite. At the outer edge of each genital plate is an ocular plate bearing a single light-sensitive tube foot. On the opposite pole the mouth is centrally located; its opening is surrounded by a lip, within which is a sphincter to close the mouth. The tips of five protrusible teeth should be visible through the mouth; these are part of the jaw apparatus known as Aristotle’s lantern (Fig. 28-30; cf. 28-38). A soft membrane known as the peristome surrounds the mouth; it is covered with cilia and is perforated by five pairs of large buccal tube feet which are probably sensory. The ambulacra radiate from the peristome toward the aboral side as five double rows of tube feet. At the edge of the peristome are five pairs of gills, so-called peristomial gills, that are branching, papillate evaginations of the body wall open internally to the coelom. Also on the peristome are numerous large pedicellariae. These pedicellariae comprise three broad blades supported by a peduncle which is stiffened by an internal calcareous rod. Remove one for closer study. Examine the spines. Note that those nearest the equator are longer than the ones located toward the poles. The spines bear longitudinal flutings. Near the peristome there are spines which are somewhat spatulate in shape. The spine sits on a tubercle of the ossicle beneath it; it is attached to the tubercle by rings of muscles plus the outer epidermal covering. The muscles are capable of holding the spine in a fixed position, or they may slowly move it in any direction. Examine one of the dried tests of Strongylocentrotus. Radiating from the genital plates downward are the five ambulacra. They are bounded by a double row of pores on either side, through which the tube feet protrude. Each ambulacral area has two rows of alternating ambulacral plates lying between the podial pores. The interambulacral areas similarly consist of two rows of alternating plates, but these are larger than plates of the ambulacra. Note the tubercles on the plates, to which the spines were attached and on which they move; the tubercles form the ball of a ball-and-socket joint with the spine. In the dried skeleton, the five large genital plates around the periproct are relatively conspicuous, and the genital pore in each stands out. Under magnification the madreporic plate can be seen to be perforated over its entire surface. Inside the peristomial edge of the test are five pairs of projections at the ends of the ambulacra. These are points of attachment for muscles that operate the jaw apparatus and are called auricles. Dissection. Open the test of the live relaxed specimen by cutting around its equator with your heavy scissors. Gently lift the top half a short distance and carefully separate the mesenteries that hold the intestine and loops of the stomach to the body wall, then invert the top next to the bottom half. As you fold back the top half, look for the stone canal stretching from the madreporic plate toward the oral region. The five gonads, delectable to sushi afficionados, may be prominent (cf. Fig. 28-36). Ovaries are yellow-orange, testes gray. Determine the sex of your specimen. In the center of the oral region and surrounding the end of the esophagus is the complex jaw mechanism, Aristotle’s lantern (Fig. 28-38, p. 904). The esophagus, after leaving the lantern, enters the stomach which makes an almost complete clockwise turn around the test; from the stomach the smaller-diameter intestine forms a counterclockwise loop and joins an aboral bend leading into the rectum and anus. Paralleling the stomach, running along its inner edge is the siphon, which is a tube that probably serves to shunt excess water past the stomach so that gut contents are not diluted. Cut the esophagus just above the point where it leaves the lantern and carefully remove the intestine. Examine the top of the lantern under the low power of your dissecting scope. The ring canal may be

77 identified as a thin-walled, clear channel lying on the margin of the aperture of the lantern through which the esophagus passes. Polian vesicles are visible as five grape-like structures on the ring canal. The five radial canals pass down through the lantern and emerge on the oral body wall between the auricles. Right at this point lateral canals to the first tube feet can be seen branching from the radial canal. Note the ampullae of the tube feet along the ambulacra. These connect to each tube foot by a pair of canals, thus accounting for the two rows of pores seen on each side of the ambulacra in the cleaned test. Under the radial canals there are hyponeural canals and haemal channels, as in asteroids, but these cannot be identified readily in dissection. The axial gland can be identified, however, as a mass of spongy tissue attached along the stone canal. The Aristotle’s lantern is amazingly complex, consisting of as many as 40 distinct ossicles (see demonstration) and an arrangement of muscles that move them. Motion of the lantern serves not only in feeding but in pumping coelomic fluid through the peristomial gills as well. Contraction of the circular compass elevator muscles of the lantern elevates the compasses and lifts the lantern membrane, a membrane that covers the whole lantern and separates the perivisceral coelom from the lantern coelom. This elevation enlarges the cavity of the lantern coelom and causes retraction of the peristomial gills, the cavity of which is continuous with the lantern coelom. The compass depressors lower the compasses, thus decreasing the size of the lantern coelom and expanding the gills. Protractor muscles serve to extend the mouth parts, while retractor muscles open the jaws. The rotulae, with their muscles of attachment (running from rotula to alveolus), bind the lantern together. With your specimen under the dissecting scope, remove the radial canal in one or more ambulacra and find the radial nerve cord of the oral nervous system. It is broad and whitish. By cutting away the alveoli of the lantern it should be possible to demonstrate the union of the radial nerve with the peripharyngeal nerve ring, a pentagon-shaped circle of nervous tissue encircling the pharynx. Other echinoids. Other urchins, as well as dissections of the Aristotle’s lantern of urchins are on demonstration. Also here are irregular echinoids (Fig. 28-40–42, pp. 905-907) which have secondarily adopted a bilateral symmetry so that they move through the substrate with one edge leading and with the anus shifted to the trailing edge. The sand dollar Echinarachnius is like this. Find the anus on these animals. Note that the velvety appearance of the animal is a function of the short spines. The ambulacra are arranged in petaloid shapes. Each ambulacrum has a double row of tube feet running to the edge of the disk. If living specimens are available, look at the surface of the body with a dissecting microscope and find spines, pedicellariae, and tube feet. Both spines and tube feet play a role in moving particles toward the mouth for feeding (Fig. 28-43). Tube feet completely ring the edge of the body because the ambulacral grooves that radiate from the mouth branch repeatedly toward the edge. Other irregular echinoids on demonstration include a southern sand dollar, Mellita, with marked lunules, and Brisaster , a heart urchin (Fig. 28-40). Sea cucumbers: Cucumaria frondosa (Fig. 28-45). Holothurians are worm-like, with the body elongated along the oral-aboral axis. The mouth at the oral end is surrounded by tentacles which are modified tube feet, the buccal podia. Of the ten tentacles, the two ventral ones are small, and between the two dorsal tentacles is a genital papilla. The tentacles are typically wiped one-by-one through the mouth in feeding, and the entire tentacular complex can be withdrawn into the body because it is situated on a retractable introvert. At the opposite end of the body is the cloacal aperture; it has the respiratory trees as well as the rectum opening into it. The five ambulacra with their tube feet run along the length of the body in Cucumaria. Three of these ambulacra are kept in contact with the substrate and have well-developed suckers; they mark what is called the trivium, the functional ventral surface of the animal. The bivium is functionally dorsal and has less-developed tube feet, reduced to wart-like sensory structures. Having a ventral and dorsal side makes it possible to define an anterior-posterior axis in holothurians. The endoskeleton is reduced to little more than scattered microscopic spicules and plates, making the body wall leathery to the touch. The madreporite is not visible on the body surface; rather it is internal, opening into the coelom. On demonstration is Leptosynapta, a burrowing holothurian that lacks tube feet along the body; the only remaining tube feet are those modified as modified buccal podia, the tentacles. Ossicles in the body wall can be seen in these relatively translucent specimens, and noteworthy among them are anchor-shaped ones (Fig. 28-47). A mounted piece of body wall showing these and shield-shaped ossicles is on demonstration.

78 The Extraction and Observation of Interstitital Fauna Animals inhabiting the interstices of sand, mud, and gravel are rather cryptic in their native habitat, and they are more easily studied after they have been extracted from the sediments in which they live. Because many of these animals cling tenaciously to sediment grains, getting them out of sediments requires some trickery. A brute-force extraction technique is to fix entire sediment samples so that the animals are killed and then can be washed out with water washs; this approach works well with hard-bodied animals such as nematodes and arthropods, but it renders most soft-bodied members of the meiofauna into unrecognizable wisps. Techniques to force live animals out use anesthetization and sieving, sedimentation in high-density solutions, or gradual degradation of the sediment so that the animals are induced to migrate out of the sediment on their own volition (various techniques are summarized by Westheide & Purschke, 1988, in Introduction to the Study of Meiofauna, Smithsonian Instuitution Press, pp. 16–160). The two simplest and most practical techniques are the magnesium-chloride anaesthetization technique (in which the narcotizing effect of magnesium chloride facilitates the animals’ release from substrates and prevents their reattachment) and the sea-water-ice technique (in which changes in temperature and salinity forced on a block of sediment cause animals in it to migrate downward) as follows. (See also attached figure.) Magnesium-cloride anesthetization and sieving (Sterrer 1969, Ark. Zool. 221–125) The sediment to be extracted is placed in a one-liter Erlenmeyer flask to a depth of up to 1 inch, and the flask is filled with a solution of MgCl2 made isotonic to sea water (32–35 ppt, or roughly 7.2% in nonchlorinated fresh water). The flask is then inverted once gently to ensure that all the sediment is exposed to MgCl2 and allowed to sit for 5–10 minutes. Ten-minutes’ exposure to the MgCl2 is sufficient to relax most meiofaunal animals; some, such as acoels, other small turbellarians, or gastrotrichs, relax almost immediately. After an appropriate wait, the flask is shaken by inverting it a few times to completely suspend the sediment and then set upright briefly so that the sediment settles to the bottom of the flask. As soon as the major part of the sediment has settled (usually no more than a few seconds) the supernatant—which should contain most of the animals because they are lighter than the sediment—is poured through a plankton-netting sieve (with mesh size of 42 µm or 64 µm) into a second container. The MgCl2 should be returned then to the flask and the processes of shaking and decanting through the sieve repeated once or twice, with perhaps more vigorous shaking to dislodge more tenacious animals. Once the sieving process is complete, the sieve should be rinsed briefly but thoroughly with a little sea water and its contents then washed into a Petri dish using a wash-bottle with sea water. The animals can be examined immediately in the dish using a dissecting microscope, preferably with transmitted illumination. Sea-water-ice technique (Uhlig, 1965, Verh. Dtsch. Zool. Ges. Jena, 1965:151–157) Some animals can be induced to migrate downward in sand by applying melting sea-water ice to the sediment’s surface. In a method that takes advantage of this phenomenon, sediment is placed in a tube that has a 64-µm or 125-µm mesh netting covering its bottom, and the tube is positioned a few millimeters above the bottom of a Petri dish that has just enough sea water in it to contact the bottom of the tube. The upper surface of the sand is covered with a wad of glass wool, and crushed sea-water ice is placed on top of the wool. As the ice melts, the tube’s height should be adjusted upward so that its bottom is just at the surface of the water in the dish. Animals will migrate into the dish, first appearing there within about 15 min. The sample will continue to produce animals over a period of several hours. If the extraction period is carried out for long periods, it will be necessary to replace the Petri dish with fresh empty dishes to avoid overflow of sea water. Microscopic examination The tiny animals of the meiofauna are best studied alive under a compound microscope. They can be picked from the exraction dish with fine-bore pipets (conventional Pasteur pipets are usually too coarse; they can be pulled to narrower bores in a gas flame). Animals conventionally are mounted individually in so-called squeeze preparations in which they can be differentially flattened under a cover slip. To prepare this, place the animal on a slide in a small drop of sea water and add a drop of anesthetizing MgCl2 solution (isotonic to sea water); then apply a coverslip that is supported at its corners with minute feet of soft wax such as beeswax. (Make the feet by picking the corners of the coverslip one at a time into wax, to pick just a tiny crumb of wax onto each corner, before positioning the coverslip over the animal on the slide.) The wax feet prevent crushing of the animal but allow compression so that the animal can be gently squeezed to hold it in place and to press it flat to reveal internal anatomy. Pressure can be differentially applied by wicking out small amounts of water from under the coverslip with a piece of filter paper (or it can be released by

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adding more water under the coverslip). If wax feet are at first too resistant, they can be gently pressed down with the tip of a pencil.

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Platyhelminthes, Nematoda, Gnathostomulida, Rotifera, Gastrotricha, Cycliophora turbellarians and other lower worms of the meiofauna; cycliophorans PLATYHELMINTHES Cycloneuralia Acoelomorpha GASTROTRICHA Nemertodermatida Macrodasyida * Acoela * Turbanella Catenulida Tetranchyroderma Rhabditophora Chaetonotida Macrostomorpha Chaetonotus Haplopharyngida Macrostomida * NEMATODA *** Trepaxonemata “Adenophorea” Polycladida * ... Neoophora Enoplida * Lecithoepitheliata Secernentea (parasitic) Prolecithophora * NEMATOMORPHA (parasitic) Seriata Proseriata * PRIAPULIDA Tricladida Priapulus Rhabdocoela LORICIFERA “Typhloplanoida” * “Dalyellioida” * KINORHYNCHA Kalyptorhynchia * Echinoderes Neodermata (the major parasitic flatworms) Trematoda Aspidobothrea Digenea Cercomeromorphae Monogenea Cestoda Gnathifera . GNATHOSTOMULIDA Filospermoidea Halognathia Bursovaginoidea Gnathostomula * “ROTIFERA” * Seisonida Bdelloida Philodina Monogononta Brachionus Proales? ACANTHOCEPHALA (parasitic) CYCLIOPHORA Symbion pandora

* groups likely to be encountered in sand samples in laboratory

Platyhelminthes, Nematoda, etc.

81 Platyhelminthes

The phylum Platyhelminthes encompasses non-segmented, acoelomate worms. It has three clearly distinguishable monophyletic groups (whose relationships are indeterminate), the Acoelomorpha, the Catenulida, and the Rhabditophora; and within the Rhabditophora are the major parasitic flatworms (flukes, tapeworms, etc., which are now grouped in the monophyletic taxon Neodermata and which constitute most of the described species of flatworms). In the phylogenetic classification above, those taxa above the Neodermata have mostly free-living members and were, in more traditional classification schemes, collectively placed in a class Turbellaria. Because the turbellarians constitute a paraphyletic group, the name for them is no longer a valid taxon and now has to be placed in quotation marks (“Turbellaria”) to indicate that. We will study marine representatives of these free-living worms. Most turbellarians are benthic, living on and in sediment and on algae, typically in the interstices of these habitats. Most large turbellarians, namely polyclads and triclads, are typically epibenthic, found crawling over substrates. It is easiest to see internal anatomy of the smaller turbellarians, and we will concentrate on these. We will extract them from sand, mud, and algal mats using magnesium-chloride anesthetization and sediment-deterioration methods. A separate handout describes the anesthetization extraction which is best used on clean sands; the deterioration method simply relies on the animals’ tendency to rise to the surface of stagnating containers and is useful particularly on soft muddy sediments. Pick individual animals from the extraction dishes with a micropipet and mount them, one at a time, on a slide in a drop of sea water with an added drop of MgCl2 . Place a coverslip fitted with small wax feet (just prick each corner of the coverslip into the wax to get a small fleck) over the animal and draw out excess fluid until the animal begins to flatten. It is easiest to see things in these mounts when the animals are compressed quite flat, but start out with only a fairly light squeezing; then, as you need to see more detail, wick out water from the edge of the coverslip with a piece of filter paper. If the water edge retracts from the edge of the coverslip as you try this, you will need to push down gently on the wax feet. Acoels will be found in sediment samples and on algae. Acoels are so-called because they have no digestive cavity; the digestive tissue is a syncytium rather than an epithelium-lined gut. Acoels are typically ovoid in shape and have a statocyst at the anterior end. Watch some swimming free in a dish for a while to see ciliary swimming and to see how the body (of many) folds ventrally to produce a longitudinal groove. In an animal mounted on a slide, watch the pattern of ciliary beating over the epidermis and look for diatoms in the syncytium (many acoels are herbivorous, others carnivorous). Find the statocyst and, at the anterior tip, the slight indentation that marks the opening of the frontal organ; many glands opening alongside the frontal organ reach the tip from positions behind the statocyst and brain. Mature animals will have relatively large oocytes in one or two strings (central or paired on either side), with the more mature ones at the posterior end of the string(s). Usually anterior to these are bundles of developing sperm, the testes. Notice that the gonads are indistinct, not forming a compact body but arising scattered in the parenchyma. Toward the posterior end of the animal will be copulatory structures (e.g., penis or stylet connected to accumulations of sperm, and a copulatory bursa with sclerotized bursa-mouthpiece); these also may be rather indistinct. The mouth in the center of the ventral side is often difficult to see and will show up best when the animal is quite flattened. Macrostomids. These have a simple sack-shaped gut with an anteriorly positioned muscular simple pharynx. Many species have eyes and tracts of glands with rhabdiform secretions terminating at the anterior tip of the body. At the posterior of the body is sometimes a tail plate which bears adhesive papillae. The copulatory organ is a sclerotized stylet, usually hook-shaped, near the posterior end of the body, with a bulbous seminal vesicle attached. Distinct vasa deferentia conduct sperm to it from two testes on either side just behind the pharynx. The ovary sits in front of the copulatory stylet. Some macrostomids reproduce asexally by paratomy, forming chains of individuals that eventually separate from one another; one such paratomizing animal we are likely to encounter it the sediment samples is Microstomum. Polyclads. These large animals (Notoplana) live under boulders and eat barnacles or small molluscs. A specimen should be mounted on a slide with a few added drops of MgCl2 . If the animal is not too large and thick, use a coverslip with small wax feet to compress it; larger ones might require, instead, a second slide to compress them. Find eyespots along the anterior and anterio-lateral margins of the body or over the brain. The large ruffled pharynx sits centrally and the gut radiates from it with many branches (hence the name of the group). Copulatory structures will be behind the pharynx (compare with figures.)

82 Proseriates are common in sand where waves are breaking, among them the very fast-swimming otoplanids (which have prominent sensory bristles on the head as well as the statocyst and many adhesive organs on the side as well as on a posterior tail plate) and nematoplanids (which are more threadlike and slower gliders). In a specimen mounted on a slide, look for the statocyst just in front of the distinct brain capsule, the plicate pharynx typically in the posterior third of the body, and lateral diverticulae of the gut that stretches anteriorly and posteriorly from the pharynx. Copulatory stylets can be seen toward the posterior end of the body; prominently filling the lateral sides of the body and mostly lying between gut diverticulae are the vitellaria which produce yolk for the eggs. The ovaries (i.e., germaria which produce oocytes) are small and sit just in front of the pharynx; the paired testes lie further anterior. Found crawling over algae or rocks or in the sediment is often a species of Monocelis or Minona, an elontage worm with an eyespot over its statocyst and a fairly pronounced lanceolate tailplate with adhesive pads. Feeding can often be observed by breaking open small amphipods that co-occur with the flatworms. Note how the plicate pharynx toward the posterior end of the body is inserted into the prey and undergoes peristaltic contractions to suck tissue out.

Triclads are more commonly found in freshwater (though there are marine and terrestrial species) and are often used as model flatworms in biology classes. They are big and thick compared to the other flatworms available in this laboratory, so it is less easy to discern their internal organs. Specimens collected from under stones in the Penobscot River may be available for study, and on them you should at least be able to see eyespots and the underlying brain, the large pharynx in the middle of the body, and the three-branched gut, the branches of which are in turn lobulated. Some specimens may be transparent enough to show a pair of ovaries, fairly close behind the brain, and, behind those, a row of testes on either side; the copulatory organ sits behind the pharynx. Rhabdocoels. These animals have a bulbous pharynx, somewhat barrel-shaped, linked to a simple sackshaped gut. Because the basement membrane around internal organs is so well developed in rhabdocoels, it is often easy to discern parts of the reproductive system in them. Compare animals you find with the attached figures to find these parts. Kalyptorhynch rhabdocoels have a prominent proboscis at the anterior tip of the body and use it to capture prey. It is not connected to the gut; the animal bends to place captured food in the pharynx which is oriented perpendicular to the body axis. Kalyptorhynchs will show up on algae as well as in sand. Dalyellioids, which will more likely be encountered on algae, have a more elongate barrel-shaped pharynx oriented parallel with the body axis at the anterior end of the body. Typhloplanoids, common in both sand and algae, have a pharynx more like that described for the kalyptorhynchs, and, instead of a proboscis, well-developed tracts of secretory granules converging on the anterior tip of the body. Prolecithophorans are more likely to show up in extractions of algae. Some have a prominent large pharynx at the anterior tip of the body; others have a less-distinct pharynx at the posterior end and with the reproductive organs opening with it. Some have multiple eyes, typically four over the brain. Gnathiferan phyla Gnathostomulids will be probably be present in some of the muddy, finer-grained sediments we have, especially those smelling of hydrogen sulfide with a black subsurface layer; they will be found mostly at the interface between the black and the lighter-colored surface layer. If specimens appear, study one mounted on a slide in a drop of MgCl2 , comparing its form to figures in your textbook. Rotifers. The most plentiful samples of rotifers are in fresh-water settings. Philodina, for example, is very common in hay infusions (dried or fresh grass steeped in fresh water) and in waste-water treatment facilities. Rotifers can also be found commonly in samples of marine fouling communities we have, and can be extracted from growths of algae, hydroids, and bryozoans, as well as from some sediment samples. In these samples, we see monogonont rotifers. The rotifer Brachionus is cultured in enormous numbers for feeding larval fish in aquaculture facilities, and we have specimens on hand from the aquaculture facility on campus (courtesy Linda Kling). Pick one of any of whatever representatives are available (Philodina, Brachionus, Proales), and mount it on a slide so that you can study its anatomy. Compare what you see in the animal with figures in your textbook. Females rule in the world of rotifers, so you will probably see only females. The bdelloid rotifers reproduce strictly by parthenogenesis; and while monogononts do seasonally have males (which are dwarf and short-lived), the dominant form is female reproducing through parthenogenesis. In your specimens, look

Platyhelminthes, Nematoda, etc.

83

especially for the corona, the mastax, adhesive toes, and protonephridia. The corona appears in these forms as two circlets of cilia around the anterior end; in the bdelloids like Philodina, the anterior-most circlet is divided into two arcing trochal discs which look like rotating wheels as the cilia beat. The mastax (the pharynx) bears cuticular jaw-like pieces, the trophi, and should be moving in these animals. See if you can tell what type of trophi your specimen bears–whether specialized for grabbing prey or for grinding bacteria and phytoplankton, for example. The adhesive glands going out into the toes should be evident, and if you squeeze the animal, protonephridia should be easily visible in the pseudocoel, looking like tiny flickering flames. Cycloneuralian phyla Gastrotrichs of marine environments inhabit the interstitial spaces of sandy sediments. The pioneer in study of this environment, Adolf Remane, discovered the first-known representatives of the Macrodasyida, strap-shaped primitive gastrotrichs. The more derived chaetonotid gastrotrichs, which are shaped like a bowling-pin, have been much longer known from common representatives in fresh water; chaetonotids are also found in marine sediments, especially coarse-grained ones. Turbanella and Tetranchyroderma may appear in the extraction dishes from cleaner sediments. If you find either, watch how it glides over the dish bottom. In an animal mounted in a drop of MgCl2 on a slide, note how the ciliation responsible for this movement is restricted to the ventral surface. The cuticle is elaborate with four-pronged spines in Tetranchyroderma; Turbanella has a smooth cuticle. Adhesive organs, in the form of tubular cuticular extensions enwrapping gland necks are prominent along the sides of the body and in “toes” of foot-like tail plates. Note the large tubular pharynx with two pores at its posterior end and a pronounced buccal capsule at the tip where the mouth is. This animal probably feeds on bacteria it vacuums off sand grains. The brain overlies the pharynx. The gut is straight, ending at the anus near the posterior tip of the body. Eggs in the ovary may be prominent; the testes sit in front of the largest egg; behind the ovary and anus is a small copulatory organ. Copulation is a complicated process involving transfer of sperm into the copulatory organ before they can be used in gamete exchange. Chaetonotid gastrotrichs are very small and easily overlooked in extraction dishes. More plentiful are freshwater species from the slime on various algae. We may have some of these in lab if time permits. Reproduction in such animals is by parthenogenesis. Though sperm have been found rarely, their use is unknown. Nematodes are extremely common as a group; every habitat we look at will have nematodes, and many marine sediments are completely dominated by nematodes. We know pitifully little about the local nematode fauna. Despite the tremendous diversity of habitats nematodes occupy, they are quite uniform in morphology: most are uniformly cylindrical in form, generally thread-like, with a narrow tapering posterior end and a blunter anterior end. The most distinctive shapes are seen in epsilonematids, named for the epsilon shape (more like a number 3) and desmoscolecids which look like ringed elongated footballs; both can be found in coarse-grained shallow subtidal sediments and may turn up in samples we examine. By far more common are thread-like forms that move in a nematode-characteristic sinuous, almost snake-like mode. In the extraction dishes we have of nematodes, compare the abilities (or inabilities) of nematodes to move on the flat bare bottom of the dish to their movement among sediment particles or algal filaments. Do any of the sedimentdwelling nematodes have the ability to adhere to substrata? In the sand samples, see if you can find any nematodes that hop on their tail tips. Study internal anatomy by mounting a large mature worm on a slide (it may be expedient to mount several at once). Females can be recognized by the eggs visible through the body wall. In such a mount, nematodes invariably land on their sides because their bending for locomotion is by dorso-ventral flexure, so note that you are seeing a lateral view. Find the anterior end with the prominent tubular pharynx and sensory bristles arranged around the mouth and sides of the head (there are usually 6 small labial setae, 6 + 4 cephalic setae); facing up will be one of the two amphids, which is a sensory organ appearing like a pocket in the cuticle, in the form of a spiral in some nematodes. The pharynx may have tooth-like or spear-like thickenings in the cuticle of the buccal capsule. The gut extends straight back from the pharynx, ending short of the tail at the ventral anus. The ovaries of the females are seen anterior and posterior to a ventral opening, the vulva. The testes of males are similarly placed, one extending anterior toward the pharynx, the other extending toward the hindgut; their common duct ends at the hindgut (hence a cloaca) near two cuticular copulatory stylets. Note any pattern of ornamentation in the cuticle (especially with higher magnification), the brain around the pharynx and its connection to longitudinal nerve cords, and evidence of a fluid-containing body cavity

84 (do the internal organs slosh freely in an internal space?). Priapulida. Only one species, Priapulus caudatus, is known to occur in this area. It is macroscopic and burrows in fine subtidal sediments. It is probably carnivorous. The introvert has a complex array of fine teeth and the central part of the complete gut has the form of a broad sac, seemingly just right for accomodating macroscopic prey. If specimens are available, examine an intact specimen for external body morphology and its use of the introvert during locomotion. When placed in an “ant-farm”-type aquarium, specimens can sometimes be observed burrowing beneath the surface and sometimes lurking with the oral end near the surface of the burrow opening, possibly waiting for prey. Kinorhynchs are unmistakable members of the marine meiofauna. Their bodies appear almost arthropod-like in showing distinct superficial segments yet they have no appendages and move, very characteristically, by the protrusion and retraction of a spiny head-like introvert (“kinorhynch” means “moving by a proboscis”). They are very small (0.2–0.8 mm) and benign creatures, feeding on small particulates or, in a few species, on diatoms. The body is divided into 13 zonites, segment-like divisions that involve only the body wall, not any internal organs, and their cuticle is divided into plate-like parts. In an animal mounted in a drop of MgCl2 on a slide, study the form of the head with its spine-like scalids, other sensory bristles on the body, and whatever of internal anatomy may be visible through the body wall, such as pharynx, gut and gonads.

Cycliophora Cycliophora is the newest of phyla, described in only 1995 even though specimens had been collected decades earlier and filed away in museum drawers (Funch & Kristensen, 1995, Nature 378:711-714). Countless billions of them have been cast away with lobster dinners, too. The only described species so far, Symbion pandora, is a tiny sessile filter feeder living attached to the mouthparts of lobsters from cold, deep waters. The specimen on demonstration is a new species from Maine, living on Homarus, the American lobster. Cycliophorans have a complex life cycle involving both sexual and asexual production of motile stages. The feeding sessile stage, on demonstration, has a buccal funnel with a ring of cilia at its margin, a trunk, and a stalked attachment disk by which it is fixed to bristles of the host’s mouthparts. The attachment disk is an elaboration of the cuticle covering the body.

Platyhelminthes, Nematoda, etc.

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87

Index Acanthocephala, 80 Acari, 36 Acmaea, 40, 44 Acmaea testudinalis, 45 Acoela, 80, 81 Acoelomorpha, 80, 81 acorn worm, 73 Acropora, 63 Adenophorea, 80 Aeolidia, 40, 47 Aequipecten, 40 Aiptasia, 63–65 Alcyonaria, 63 Amphipoda, 24, 32 amphipods, 24 Amphitrite, 11, 14 Anaplodactylus, 39 Annelida, 11 Anodonta, 40 Anoplodactylus, 36 Anostraca, 24, 25 Antedon, 74 Anthoathecatae, 63 Anthopleura, 63, 66 Anthozoa, 63 Aphrodita, 11 Aphroditidae, 16 Aplacaphora, 44 Aplacophora, 40 Arachnida, 36 Araneae, 36, 37 Arenicola, 11, 14 Arenicolidae, 17 Argiope, 36, 37 Ariolimax, 52 Arion, 40, 48, 52 Arthropoda, 24, 36 Ascidiacea, 70 Ascidians, 71 Asellus, 24, 31 Aspidobothrea, 80 Asterias, 74, 75 Asteroidea, 74 Aurelia, 63, 66 Axiognathus, 75 Balanus, 24, 32 barnacle, 32 barnacles, 24 basket star, 74 Bdelloida, 80 beach hoppers, 24 Bivalvia, 40, 56 blue crab, 24 Boltenia, 70

Bosmina, 27 Botrylloides, 70, 72 Botryllus, 70, 72 Bowerbankia, 71 Brachionus, 80, 82 Brachiopoda, 70 Branchiobdellida, 11 Brisaster, 74, 77 brittle star, 75 brittle stars, 74 Bryozoa, 70 Bryozoans, 71 Buccinum, 40, 44, 47, 49 Bugula, 70, 71 Bulimnea, 40, 48 Bursovaginoidea, 80 Busycon, 40, 47 Caecidotea, 24, 31 Calcarea, 60 Calinectes, 24 Cambarincola, 11 Cambarus, 24, 27 Cancer, 24 Carcinus, 24, 28 Cassis, 40, 47 Catenulida, 80, 81 Centruroides, 36, 37 Cephalochordata, 70 Cephalopoda, 40, 58 Cercomeromorphae, 80 Ceriantharia, 63 Cerianthus, 63 Cestoda, 80 Chaetogaster, 20 Chaetonotida, 80, 83 Chaetonotus, 80 Chaetopteridae, 17 Chelicerata, 36 Chelifer, 36, 38 Chilopoda, 35 chitons, 40, 41 Chordata, 70 Chrysopetalidae, 17 Ciliophora, 86 Ciona, 70, 71 Cirratulidae, 16 Cirrepedia, 24 Cirripedia, 32 Cladocera, 24 Cladocerans, 27 clam shrimps, 24 clams, 56 Cliona, 60–62 Clymenella, 11, 14 88

89 Cnidaria, 63 Coenobita, 30 Conchifera, 40 cone shell, 47 Conus, 40, 47 Copepoda, 24, 33, 87 copepods, 24 corals, 64 crabs, 28 Crangon, 24, 29 Crassostrea, 40, 56, 58 crayfish, 24, 27, 30 Crepidula, 40, 44 Crinoidea, 74 Crisia, 71 Crossaster, 74 Crustacea, 24, 25 Cryptochiton, 40, 41 Ctenodiscus, 74 Ctenophora, 63 Cubomedusae, 63 Cucumaria, 74, 77 Cumacea, 24 Cyamus, 32 Cycliophora, 80, 84 Cycloneuralia, 80 Cyclops, 24, 33 daddy longlegs, 39 Dalyellioida, 80 dalyellioids, 82 Daphnia, 24, 25, 27 Decapoda, 24 Demospongiae, 60 Dendronotus, 40, 47 Dentalium, 40, 58 Dermacentor, 36, 38 Dermanyssus, 36, 38 Dero, 20 development, Echinoderm, 75 Diaptomus, 24, 33 Didemnum, 70, 72 Digenea, 80 Diodora, 40, 44 Diplopoda, 35 Diploria, 63 dogwhelk, 47 Doryteuthis, 40, 59 Dreissena, 40 Earthworm, 18 Echinarachnius, 74 Echinoderes, 80 Echinodermata, 74 Echinoidea, 74 echinoids, 77 Ecteinascidia, 70, 71 Ectopleura, 63, 67

Electra, 71 Enchytraeus, 11, 20 Enoplida, 80 Ensis, 40 Enteropneusta, 70 Eucarida, 24 Eumalacostraca, 24 Euphausia, 24, 31 Euphausiacea, 24 Euplectella, 60, 62 fanworms, 15 feather stars, 74 Filospermoidea, 80 Fungia, 63 Gammarus, 24, 32 Gastropoda, 40, 44 Gastrotricha, 80, 83, 87 geophilomorphs, 35 glass sponges, 62 Glossiphonia, 11 Glycera, 11, 12 Glyceridae, 12, 15 Gnathifera, 80, 82 Gnathostomula, 80 Gnathostomulida, 80, 82 Gonionemus, 63, 68 Gorgonia, 63 Gorgonocephalus, 74, 75 green crab, 24 gribble, 24 Gymnolaemata, 70 Haemopis, 11 Halichondria, 60–62 Haliclona, 60 Haliclystus, 63, 67 Haliotis, 40, 44 Halocordyle, 63, 67 Halognathia, 80 Haplopharyngida, 80 Haplophthalmus, 31 Harmothoe, 11, 14 Hediste, 11 Helix, 40, 48 Hemichordata, 70, 73 Henricia, 74 hermit crab, 24 hermit crabs, 30 Heteromysis, 24, 31 Hexacorallia, 63 Hexactinellida, 60, 62 Hippospongia, 62 Hirudinea, 11, 20 Hirudinoidea, 11 Holothurians, 77 Holothuroidea, 74

90 Homarus, 24, 27 Hoplocarida, 24 horse mussel, 56 horseshoe crab, 36 Hydra, 63 hydracarina, 38 Hydractinia, 63, 67 Hydroides, 15 hydroids, 67 Hydrozoa, 63 Hydrozoans, 67 Idotea, 24, 31 Ischnochiton, 40, 41 Isodictya, 60, 61 Isopoda, 24 isopods, 24 jellyfish, 66 Julus, 35 Kalyptorhynchia, 80, 82 Katharina, 40, 41 king crab, 24, 30 Kinorhyncha, 80, 84 krill, 24, 31 Laevapex, 40, 48 larva, tadpole, 73 Larvacea, 70 larvae, 76 larvae, Crustacea, 34 Lebbeus, 24, 29 Lecithoepitheliata, 80 leeches, 20 Lepas, 24, 32 Lepidonotus, 11, 14 Leptogorgia, 63 Leptosynapta, 74, 77 Leptothecatae, 63 Leucosolenia, 60 Libinia, 24, 30 Limax, 40, 48 Limnomedusae, 63 Limulus, 36 Lingula, 70 Liobunum, 36, 39 Lissoclinum, 70, 72 Lithobius, 35 Littorina, 40, 44, 45, 55 lobster, 24, 27 Loligo, 40, 59 Lophophorata, 70 Loricifera, 80 lug worm, 14 Lumbricus, 11, 18 Lumbrineridae, 16 Lymnaea, 40, 48

Macrobdella, 11 Macrodasyida, 80 Macrostomida, 80, 81 Macrostomorpha, 80 Malacostraca, 24 Maldanidae, 14, 17 mantis shrimp, 31 mantis shrimps, 24 Medusozoa, 63 Meiomenia, 40, 44 Mellita, 74, 77 Membranipora, 70, 71 Mercenaria, 40, 56 Merostomata, 36 Metridium, 63, 64 Microciona, 60, 61 Microstomum, 81 millipedes, 35 Minona, 82 Miranda, 36, 37 mites, 36 Modiolus, 40, 56, 58 Molgula, 71 Mollusca, 40, 41 Monocelis, 82 Monogenea, 80 Monogononta, 80 Monoplacophora, 40 mussel, 40 Mya, 40 Myriapoda, 35 Mysidacea, 24 mysids, 24 Mytilus, 40, 55, 56, 58 Nautilus, 40, 58 nautilus, 40 Neanthes, 11 Nematoda, 80, 83, 87 Nematomorpha, 80 Nematostella, 63, 64 Nemertodermatida, 80 Neodermata, 80 Neoophora, 80 Neopilina, 40 Nephthys, 14 Nephtyidae, 15 Nephtys, 11 Neptunea, 40 Nereidae, 15 Notoplana, 81 Nucella, 40, 44, 45, 47 Nucula, 40, 56 Nuculana, 40, 56 Nudibranchia, 40, 47 nudibranchs, 40 Obelia, 63, 67

91 Octocorallia, 63 Octopus, 40, 59 octopuses, 40 Oligochaeta, 11, 18, 86 Onchidoris, 40, 50 Oniscus, 31 Onuphidae, 16 Onychophora, 22 Ophioderma, 75 Ophiopholis, 74, 75 Ophiura, 74, 75 Ophiuroidea, 74 ophiuroids, 75 Opiliones, 36, 39 Opisthobranch, 47 Opisthobranchs, 44 opisthobranchs, 40 opposum shrimps, 24 Oreaster, 74 Ostracoda, 24, 33 ostracods, 24, 34 Otocryptops, 35 Owenidae, 17 oyster, 40, 56 Ozobranchus, 11

Polycladida, 80 Polynoidae, 14, 15 Polyplacophora, 40, 41 Porifera, 60 Praunus, 24, 31 Priapulida, 80, 84 Priapulus, 80, 84 Proales, 80, 82 Prolecithophora, 80, 82 Promachrocrinus, 74 Proseriata, 80 Proseriatea, 82 Prosobranchs, 44 Pseudoscorpiones, 36 pseudoscorpions, 38 Pterobranch, 73 Pterobranchia, 70 Pulmonate, 47 Pulmonates, 44 pulmonates, 40 Pycnogonida, 36, 39 pycnogonids, 39 Pycnogonum, 36 Pyrosoma, 70, 73 quahog, 56

Pagurus, 24, 30 Pandalus, 24, 29 Panulirus, 24 Paralithodes, 24, 30 Patellogastropoda, 44 Pauropoda, 35 Pectinaria, 11, 15 Pectinatella, 70, 71 Pennaria, 63 Pennatula, 63 Peracarida, 24 peracarids, 31 Peripatus, 22 periwinkle, 45 Philodina, 80, 82 Phoronida, 70 Phylactolaemata, 70 Phyllocarida, 24 Phyllodoce, 11 Phyllodocidae, 16 Phyllopoda, 24, 25 Physa, 40, 48 Physalia, 63, 68 pill bugs, 24 Placopecten, 40, 58 Planorbella, 40, 48 Platyhelminthes, 80, 81 Pleurobrachia, 63 Plumularia, 63, 68 pluteus, 76 Polychaeta, 11 Polyclada, 81

razor clam, 40 Renilla, 63 Rhabditophora, 80, 81 Rhabdocoela, 80, 82 Rhabdopleura, 70, 73 rock crab, 24 Rossella, 60, 62 Rotifera, 80, 82, 87 Sabella, 11, 15 Sabellariidae, 17 Sabellidae, 16 Saccoglossus, 70, 73 Salpa, 70, 73 sand dollars, 74 sand shrimp, 24 scale worms, 14 scallop, 40 Scaphopoda, 40, 58 Schizoporella, 70, 71 Scolopendra, 35 scorpion, 37 Scorpiones, 36 scorpions, 36 scuds, 24 Scypha, 60, 61 Scyphozoa, 63 Scyphozoans, 66 sea anemones, 64 sea cucumbers, 74, 77 sea gooseberry, 69

92 sea lilies, 74 sea spiders, 36, 39 sea squirts, 70 sea stars, 74 sea urchin, 76 sea urchins, 74 Secernentea, 80 Seisonida, 80 Semaeostomeae, 63 Semibalanus, 24, 32 Seriata, 80 serpent stars, 74 Serpulidae, 16 shipworm, 40, 58 shrimp, 24 shrimps, 29 Siphonophora, 68 slugs, 40 snails, 40 Solaster, 74 spaghetti worm, 14 spider crab, 24 spider crabs, 30 spiders, 36 spiny lobster, 24 Spio, 11 Spionidae, 17 Spirobolus, 35 Spirorbis, 11, 15 Spisula, 56 Spongia, 62 Spongilla, 60, 61 squids, 40, 58 Squilla, 24, 31 starfish, 74 Stauromedusae, 63 Sternaspidae, 17 Stongylocentrotus, 76 Streptocephalus, 24 Strombus, 40 Strongylocentrotus droebachiensis, 74 surf clam, 56 Sycon, 60, 61 Syllidae, 16 Symbion, 84 Symbion pandora, 80 Symphyla, 35 Symplasma, 60 Syncarida, 24 Tardigrada, 22 Tectura, 45 Terebellidae, 15, 17 Terebratella, 70 Terebratulina, 70 Teredo, 40, 58 Testudinalia, 40, 44 Tetranchyroderma, 80, 83

Thais, 40 Thaliacea, 70 Thelyphonus, 36 ticks, 36 Tomopteridae, 16 Trematoda, 80 Trepaxonemata, 80 Triclada, 82 Tricladida, 80 Tridacna, 40 Trilobita, 22 trilobite, 22 Tubipora, 63 Tubularia, 63 Tunicata, 70, 71 tunicates, 70 Turbanella, 80, 83 Turbellaria, 81, 86 tusk shell, 40 Typhloplanoida, 80 Urochordata, 70 Uropygi, 36 Vertebrata, 70 water fleas, 24, 27 water mites, 38 whelk, 47 whiptail scorpions, 36 zebra mussel, 40 Zoantharia, 63 Zoanthidean, 66 Zoanthus, 63, 66 Zooanthidea, 63