Insect Physiology (Ento 306) Insect Trehalose Synthesis

HORMONE REGULATION OF TREHALOSE SYNTHESIS BY THE FAT BODY Introduction: The primary blood sugar of vertebrate animals is glucose, but in insects the primary hemolymph sugar is trehalose (a disaccharide formed from two glucose units; shown to the left). Trehalose levels in the hemolymph are regulated by a neurohormone called hypertrehalosemic hormone (HTH). This hormone is a peptide consisting of 10 amino acids, and it is synthesized from neurosecretory cells of corpora cardiaca, a pair of neurohemal organs located on the walls of the aorta just behind the brain (indicated by the red arrows in the picture below). HTH acts by stimulating fat body to degrade glycogen (stored sugar) to produce the glucose precursors that are then synthesized to trehalose by the fat body. Hence the net effect of HTH is to elevate the synthesis of the hemolymph sugar. The corpora allata (blue arrows), which is located just behind the corpora cardiaca, are neurosecretory cells that secrete juvenile hormone (JH), which maintains insects in their juvenile forms.

Corpora cardiaca

Corpora allata

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Insect Physiology (Ento 306) Insect Trehalose Synthesis

Figure 1. The head of a cockroach. The dotted line is the plane upon which razorblade should cut.

Objectives: HTH activity can be observed in vitro (in a test tube) by exposing harvested fat body to corpora cardiaca extracts. In this lab we will explore the effect of HTH by comparing trehalose content in a sample containing HTH-treated fat body with a sample containing fat body that has not been exposed to HTH. This laboratory has 3 key objectives: 1) Demonstrate the action of HTH on the fat body to increase the synthesis of trehalose. 2) Illustrate a hormone bioassay. 3) Illustrate how physiologists can work with insect tissue in vitro. Materials and Methods: Saline solution

Cockroaches

Dissecting kit

Microscopes

Shaker

Petri dish

Spectrophotometer

Water bath

Anthrone reagent

Incubation vials

Dissecting tray

Balance

Test tubes

Vortex

STEP 1: Extraction of HTH from corpora cardiaca 1)

Decapitate a cockroach (Blaberus discoidalis) and use the head for dissecting the brain to isolate corpora cardiaca. Keep the abdomen aside for dissecting the fat bodies later. 2

Insect Physiology (Ento 306) Insect Trehalose Synthesis 2)

Place the head with the frons (face) upwards on the paraffin wax surface of dissecting dish.

3)

At the base of antenna align the razor blade perpendicular to the frons (see figure 1).

4)

With a quick push of razor blade cut through the head and in to paraffin. Keep your fingers out of the way!

5)

Discard the top portion of the head and grasp the lower part of the head by the mouth parts, and using your thumb nail squeeze the head upwards as if squeezing tooth paste (use enough pressure). This will push the cranial viscera out of the cut.

6)

Using fine tip forceps remove the viscera that you’ve just squeezed out.

7)

Place the viscera on a drop of saline solution (a.k.a., physiological saline) and examine it under a microscope.

8)

Carefully remove the fat bodies, muscle and trachea surrounding the opalescent-colored brain. Try to maintain the orientation of the brain in an anterior-posterior position.

9)

The corpora cardiaca and corpora allata are located on the posterior side of the brain, and are attached to the brain by two nerves. The corpora cardiaca are the elongate structures closest to the brain and may have a pale blue appearance.

10) Expose these glands by working under the microscope with the help of saline solution and make sure they don’t dry up any time. You can ask your TA to help you locate them. 11) If your dissection is appropriate then the T.A will collect the brain with the attached corpora cardiaca in a pre-cooled centrifuge tube containing 500 ul saline and extract the HTH using the sonicator (Power 6, Pulse 4-5 times with 2 seconds each time). Corpora cardiaca obtained from 3-4 groups will be pooled and sonicated to get enough HTH. 12) Mark two incubation vials as Control (C) and HTH with a marker. Pipette 1 ml of saline solution in to each vial and cover with foil. 13) Weigh covered incubation vials containing saline solution using pan balance. Record the weights. STEP 2: Fat body isolation 1)

Excise the abdomen from the cockroach that you have used for dissecting the corpora cardiaca.

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Insect Physiology (Ento 306) Insect Trehalose Synthesis 2)

Cut off the posterior segment of the abdomen and slit the lateral intersegmental membrane on each side from back to front.

3)

Pin down the ventral half and remove the dorsal sclerites. Gently pull out the gut so that it doesn’t rupture and contaminate the fat body.

4)

The fat body consists of two large lobes, one on each side. Slip the forceps underneath the fat body on one side and lift the tissue as one large piece. Do not grasp the fat body with the forceps. Use the forceps like a fork and lift the tissue on the forcep blades without crushing it. Leave any trachea attached.

5)

Isolate two pieces of fat bodies about this size -

- by teasing them away from the rest of

the tissue (do not grab and pull). 6)

Place one piece in to the preweighed Control vial and the other piece in to preweighed HTH vial containing 1 ml saline solution. STEP 3: HTH bioassay

1)

Reweigh both the vials containing the fat body and determine the wet weight of the fat body samples in each vial. Make sure that aluminum foil cover is in place during both weighings. Now we calculate the weight of the fat body by subtracting the initial weight from the final weight as follows: (Weight of vial+ Saline + Fat body) - (Weight of vial + Saline) = Fat Body

2)

Add 50 µl of saline to the control vial.

3)

Add 100 µl of Corpora cardiaca extract to the HTH vial.

4)

Place both the vials on the shaker immediately and begin timing.

5)

Incubate vials at room temperature (approx. 25°C) with gentle shaking for 30 minutes. STEP 4: Trehalose assay

1)

Obtain 6 tubes and label them 1 to 6 on the top part of the tubes.

2)

Aliquot 50 µl saline in to test tube 1 for the reagent blank.

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Insect Physiology (Ento 306) Insect Trehalose Synthesis 3)

Aliquot trehalose standards (20, 40, and 100 µg of pure trehalose) in to test tubes 2 to 4.

4)

Tubes 5 and 6 will be aliquots from Control and HTH tubes, respectively.

5)

Pour the incubation medium into an eppendorf tube by tilting the tubes slowly to leave the fat body in the tube. Try to drain the liquid as much as possible into the eppendorf tube and centrifuge for 10 minutes to sediment any remaining fat body.

6)

Aliquot 50 µl of supernatant from the control incubation medium and place in tube 5; aliquot 50 µl of supernatant from the HTH treatments and place in tube 6.

7)

Now, there are six test tubes: i) 50 µl aliquot of the saline (blank) ii) 50 µl aliquot of trehalose (20 µg bottle) iii) 50 µl aliquot of trehalose (40 µg bottle) iv) 50 µl aliquot of trehalose (100 µg bottle) v) 50 µl aliquot of HTH untreated fat body (control) vi) 50 µl aliquot of HTH treated fat body

8)

Perform the carbohydrate (trehalose) analysis by the Anthrone method as described next. CAUTION: Anthrone reagent is made of concentrated sulfuric acid, hence corrosive and dangerous. Avoid spillage and contact. Calorimetric carbohydrate analysis and anthrone assay

1)

To each test tube add 2.9 ml Anthrone reagent. Immediately after adding the Anthrone, cover each test tube with foil and vortex for 30 s. Label the foil cover so the tubes can be identified even after the label on the tubes was washed off in the hot water. CAUTION: Be careful not to let the contents splash out of the tubes when vortexing.

3)

Quickly put the six test tubes into a boiling water bath. Keep the temperature at 100°C (make sure there are always boiling bubbles in the water bath).

4)

Boil for 15 minutes.

5)

Remove the tubes and place them in the rack for 30 s, then place tubes in cool water.

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Insect Physiology (Ento 306) Insect Trehalose Synthesis 6)

When cool read tubes at 620 nm on a Spectrophotometer.

7)

Set the Spectrophotometer to zero Absorbance with reagent blank (tube#1), then read the next 5 tubes against the blank.

TUBE #

SAMPLE

CONTENT

ANTHRONE

1

Saline (blank)

50 µl saline

2.9 ml

2

Standard

(50 µl) 20 µg Trehalose

2.9 ml

3

Standard

(50 µl) 40 µg Trehalose

2.9 ml

4

Standard

(50 µl) 100 µg Trehalose

2.9 ml

5

HTH-untreated

50 µl Incubation medium

2.9 ml

6

HTH-treated

50 µl Incubation medium

2.9 ml

ABS.

Data Analysis: 1) Here we will use simple linear regression to estimate the amount of trehalose in the HTHuntreated (control) and HTH-treated samples, based on their absorbance. 2) Based on the regression output, we can calculate the total amount of trehalose produced by the two treatments (control and HTH-treated). 3) We can standardize trehalose production using the amount of fat body collected. We can do this by dividing the amount of trehalose by the mass of the fat body (unit: ug / mg). 4) Next, we can use a t-test to compare the standardized amount of trehalose between the untreated fat body (control) and the HTH-treated fat body. 5) Your TA will conduct the statistical analysis, and then post it on-line as a PDF on the laboratory web page. You will need to specifically address the following questions based upon your observations:

•

What is reason for generating a standard curve?

•

What is the effect of adding HTH to insect fat body?

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