Achtheres - Morone Relationships in Reservoirs Submitted to Virginia Department of Game and Inland Fisheries

Prepared by: Thomas Shahady Ph.D., Associate Professor, Environmental Science Program Kevin Peterson Ph.D., Associate Professor, Math Program George Schuppin Ph.D., Assistant Professor, Biology Program

Lynchburg College 1501 Lakeside Drive Lynchburg VA, 24501 434-544-8545

August 30, 2007

Achtheres- Morone Relationships in Reservoirs

Table of Contents List of Tables……………………………………………………………………..

3

List of Figures…………………………………………………………………….

4

Acknowledgements……………………………………………………………….

5

Summary and Explanation……………………………………………………….

6

Section 1: Studies of Parasite Life Cycle and Its Impact on Striped Bass Health………………………………………………………………………………

7

1.1 Background on the Parasite (Achtheres)……………………………. 1.2 Life Cycle of Parasite Attached to Striped Bass…………………….. 1.3 Impact of Parasite on Fish Respiration……………………………… 1.4 Effect of Parasite on Blood of Striped Bass…………………………

7 14 16 22

Section 2: Bioenergetics Model…………………………………………………..

26

2.1 Background……………………………………………………………. 2.2 The Facts and Hypothesis…………………………………………… 2.3 Study Site……………………………………………………………. 2.4 Introduction to the Model…………………………………………… 2.5 Slope Fields………………………………………………………….. 2.6 The Model…………………………………………………………… 2.7 Sensitivity…………………………………………………………… 2.8 Stability……………………………………………………………… 2.9 Results and Discussion………………………………………………. 2.10 Conclusion…………………………………………………………...

26 26 28 28 29 31 34 38 39 40

Section 3: Work in Progress and Future Direction…………………………….

45

3.1 Histology Studies……………………………………………………. 3.2 Parasite/Striped Bass Aquaculture Model………………………........ 3.3 Validation of Blood Parameters for Striped Bass in Aquaculture…… 3.4 Effects of Achtheres Exposure on Striped Bass in Aquaculture…….. 3.5 Field Studies of Fish Infected with the Parasite in the Wild………….

45 45 45 46 47

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Achtheres- Morone Relationships in Reservoirs

List of Tables

1.3 1.4 1.5 1.6 1.7

Section 1 Mean Comparisons of Fish Size and Parasite Counts for All Fish Examined…………………………………………………………………… Physical Locations of the Parasites in the Oral Cavities of Fish Examined... Respiration Rates of Experiment Fish……………………………………… Summary of Respiration Experiments……………………………………... Individual Oxygen Consumption for Each Tested Fish……………………. Effect of Parasite on %PCV of Wild Striped Bass………………………… Effect of Parasite on %WBC of Wild Striped Bass………………………...

2.1

Section 2 Specie Specific Parameters Used for Striped Bass in Model……………….

3.1 3.2

Section 3 Effect of Aquaculture on %PCV…………………………………………… Effect of Aquaculture on %WBC…………………………………………...

1.1 1.2

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Achtheres- Morone Relationships in Reservoirs

List of Figures Section 1 1.1 Infected Striped Bass Showing Parasites Aligned on Gill Racers…………. 1.2 View of Infected Striped Bass Showing Attached Parasite on Tongue Area………………………………………………………………………… 1.3 Life Cycle of Achtheres……………………………………………………. 1.4 Fish Size vs. Numbers of Parasites………………………………………… 1.5 Picture of Development Stage on Gill Arch………………………………... 1.6 Picture of Chalimus Stage Removed from Gill Arch………………………. 1.7 Picture of Developing Young on Gill Arch………………………………... 1.8 Extrapolated Respiration Rates…………………………………………….. 1.9 Culture Facilities at the Claytor Nature Center…………………………….. 1.10 Typical Blood Smear from Striped Bass……………………………………

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17

Section 2 Number of Citations of Striped Bass, Largemouth Bass, and Crappies in Smith Mountain Lake………………………………………………………. Slope Field (name of graph) (name of graph) (name of graph) Change in Respiration Dependent on Weight and Temperature…………… (name of graph) (name of graph) (name of graph) Slope Field of Bioenergetics Model of Striped Bass Growth (name of graph) (name of graph) (name of graph) Positive Growth Weight After Eight Years at 1920g………………………. Positive Growth Weight After Eight Years at 1860g………………………. Positive Growth Weight After Eight Years at 1815.2g……………………. Growth Rate of 1814g Fish with Nine Times the Normal Respiration……...

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Achtheres- Morone Relationships in Reservoirs

Acknowledgements Department of Game and Inland Fisheries of Virginia: This research was funded by Department of Game and Inland Fisheries of Virginia. Dan Wilson, Fisheries Biologist, was integral to the success and our ability to conduct this research. This project would not be possible without his continued support and help. He is the driving force behind our inquiry. With continued partnership, we will understand the impact of this parasite on striped bass populations.

Lynchburg College: Dr. Thomas Shahady, Associate Professor of Environmental Science Dr. Kevin Peterson, Associate Professor of Mathematics Dr. George Schuppin, Assistant Professor of Biology

Lynchburg College Students: Doug Thomasey, Math Program Ali Manns, Math Program Joseph Ashwell, Environmental Science Program David Ford, Environmental Science Program Travis Wray from the Environmental Science Program Brian Cocchiola, Biology/BioMedical Science program

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Achtheres- Morone Relationships in Reservoirs

Summary and Explanation: Achtheres is a parasitic copepod in the family Lemaeopodidae. As early as 2000 in Tennessee reservoirs it was found associated with Striped Bass (Morone). Concern developed among fisheries biologists as this parasite spread throughout additional reservoirs in striped bass populations. The concerns were twofold. First, oral cavities of striped bass were covered very densely with parasites to the degree that the fishing public became concerned. Secondly, fish kills of striped bass occurred in several reservoirs coincidentally at the time Achtheres developed in striped bass populations. In response to this concern we began to study fish infected by Achtheres. Both fish extracted from reservoirs and cultured fish were studied to develop some understanding of the ecological relationship between Acthheres and Morone. This is only an ecological study and not intended to present parasitological or other related findings. Our findings are presented in three logical sections: Section 1: Includes a background on the Achtheres parasite, further characterization of the Achtheres life cycle stages associated with Striped Bass, and the negative impact of the parasite on the health of Striped Bass, Section II: Bioenergetics model used to predict the impact of the parasite on Striped Bass growth. Section III: Describes work in progress and planned/proposed future direction.

6

Achtheres- Morone Relationships in Reservoirs

Section 1: Studies of Parasite Life Cycle and Its Impact on Striped Bass Health 1.1

Background on the Parasite (Achtheres):

Achtheres (gill maggot) is a copepod parasite known to infect the gills of freshwater fish. Earliest descriptions of Achtheres infecting Morone date back to 1915 (Wilson). Wilson’s work described infections of A. lacae in Morone saxatilis in the Potomac River. In 1957, Causey (Causey, 1957) identified two species of Achtheres from Louisiana freshwater fish. He described two parasite species: (1) A. micropteri with maxillae (arms extending from the bulla) shorter than the body length and (2) A. lacae with maxillae more slender but as long as the body. In his study, Achtheres infected large and small mouth bass (Micropterus spp.), bullhead (Ictalurus) and other sport species. More recent identification (Hoffman 1999) suggests two species of Achtheres may exist in the southeastern US. Hoffman (1999) describes A. micropteri / A. pimelodi / A. ambloplitis as synonyms for the same species typically identified as A. pimelodi with maxillae shorter than bulla. A second species A. lacae with maxillae longer than bulla may exist as well (Causey 1957). Hoffman (1999) describes the life cycle of this parasite. Both male and female are attached to the host. At maturity the male releases it hold from the fish and crawls to the female. After copulation an egg sac is produced. These egg sacs are very visible on infected fish (Figures 1.1 and 1.2). The remainder of the life cycle is described as shown in Figure 3. Eggs hatch as first copepodids and molt into second copepodids 1 –2 days later. This is the only free living stage of the parasite. They copepodids must attach to fish or die within 24 hours. Remaining time to maturity is spent attached to host. There is not a nauplier stage in this organism typical of copepods. Males do not develop past the second copepodite stage. Only females mature into adult copepods. The impact of infestations of this parasite on fish populations is poorly understood. It is generally believed that infestations are so low that damage does not occur to the host fish (Valtonen 1993). The highest prevalence of infection documented to date occurred in Finnish lakes. Achtheres had infected 30% with 0-7 parasites per fish in oligotrophic lakes and 6% with 0-1 parasites per fish in these polluted eutrophic lakes. Higher rates of infestation and prevalence for Achtheres have not been documented in literature.

7

Achtheres- Morone Relationships in Reservoirs

. Figure 1.1. Infected striped bass showing parasites aligned on gill rakers. Egg sacs of the copepod are the most visible portion. Fish collected from Smith Mountain Lake courtesy of Dan Wilson DGIF.

Figure 1.2. View of infected striped bass showing attached parasite on tongue area. White area is the body of the parasite. Egg sacs appear in brown. Fish courtesy of Dan Wilson DGIF.

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Achtheres- Morone Relationships in Reservoirs

Figure 1.3 – Life cycle of Achtheres. Figure is from Hoffman (1999). In our previous study, we found that sampled fish were very highly infected (Table 1.1). Striped bass (Morone saxatilis) from Smith Mountain Lake (VA) caught during the winter (January and February of 2007) showed the greatest infections rates in our study (Table 1.1). Conversely, Lake Norris (TN) fish were the least infected. Prevalence of collected fish was 100%. Gravid adult females dominated all parasite populations (Table 1). Percentages of gravid females from Lake Norman (NC) were significantly lower than the other lakes studied. Numbers of parasites per fish declined into the summer months on Smith Mountain Lake.

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Achtheres- Morone Relationships in Reservoirs

Table1.1. Mean comparisons of fish size and parasites counts for all fish examined (N=48). Statistical comparisons using ANOVA to examine differences in data set for a given parameter. Significance levels of * = 0.1 and ** 0.05. SML = Smith mountain Lake in winter (February 2004) and summer (June 2004). Lake Norris Tennessee (June 2004 and Lake Norman North Carolina (July 2004).

Parameter

SML Winter N = 21

SML Summer N = 13

Lake Norris Summer N=9

Lake Norman Summer N=5

Mean Fish Size (inches)

**20.3

23.6

25.6

25.6

Mean Number of Parasites

**142.3

87.8

*61.3

89.8

Percent Gravid Parasites

89.5

84.6

83.3

**65.8

We found a weak but positive linear relationship between the numbers of parasites on the fish and the size of the fish (Figure 4). This relationship was weakly significant (r2 = 0.08 and p = .04). Without the three fish infected with over 300 parasites the relationship would be much stronger. It would appear from our initial work that size of fish allows more room for the parasite to attach to the fish. This is not absolute as even bigger fish did not have increasing numbers of parasite. We concluded other mechanism must be involved pertaining to predictive numbers of parasites on individual fish.

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Achtheres- Morone Relationships in Reservoirs

500

r2 = 0.08 p = 0.04

Total Number of Parasites

400

300

200

100

0

10

15

20

25

30

35

40

Fish Size (in)

Figure 1.4. Fish Size vs. Numbers of Parasites. Size of fish is in inches and total numbers of parasites is the number of parasites on each fish dissected. Infections varied in intensity depending upon location in the oral cavity of the fish (Table 1.2). The gill arches were predominately the most heavily infested areas followed by top of the mouth, then tongue, and finally lips. Two patterns emerged. With lower rates of infection, the parasites were more evenly distributed throughout the oral cavity (Lake Norris). With increasing rates of infection, the gill arches become more heavily infected (Smith Mountain Lake and Lake Norman). Species Identification We are certain the current outbreak of fish parasites observed in our study is Achtheres. We are less certain of species identification. The specimens of Achtheres are quite mixed relative to length maxillae in relation to the bulla. Zooplankton keys (Edmondson 1959) give indicators of general body shape and size but no specific indicators allowing identification of the species. While it is most probable this parasite is Achtheres pimelodi, we could not rule out the potential presence of Achtheres lacae. Our species identification is based upon descriptions in Causey (1957) and Hoffman (1999).

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Achtheres- Morone Relationships in Reservoirs

Table 1.2. Physical locations of the parasites in the oral cavities of fish examined. Oral cavity locations as described in text. Numbers represent means + standard deviations. SML Winter (N = 21), SML Summer (N=13), Lake Norris Summer (N=9), Lake Norman Summer (N=5). Mouth locations significantly different from each other at 0.05 (ANOVA) in all lakes except Lake Norman Summer. Bottom Lip

Tongue

Top of mouth

Gill Arches

Total

4.5 + 4

20.3 + 14.4

34.2 + 28

47.5 + 42

SML Winter SML Summer Lake Norris Summer Lake Norman Summer

6 + 3.9

23.4 + 18.7

47.1 + 36.2

65.3 + 54.1

3.8 + 2.6

21.4 + 10.1

26.9 + 11.7

35.6 + 14.7

2 + 3.1

13.6 + 9.8

21.4 + 10.1

24.2 + 14

5+7

16.8 + 5.5

22 + 15.6

46 + 42.5

The infection rate discovered in our study was extremely high for Achtheres (Valtonen 1993) and parasitic copepods (Brickle et. al. 2003). Our prevalence rate was 100% and infections ranged from 16 to 424 parasites per fish. Some of the higher infection rates such as Brickle et. al. (2003) found prevalence of 49% and infections ranging from 0 to 24, with only 2% of the sample having more than 5 of the parasites for Sphyrion laevigatum on Genypterus blacodes. Valtonen (1993) found 30% prevalence with 0-7 parasites per fish for Achtheres. Our findings are incomparable to these other incidences of infection. This type of prevalence and intensity has not been reported in prior literature for any type of copepod parasite / bass species relationship. While parasites such as Achtheres have been found and documented, this outbreak is much higher than any other documented in the literature Two factors contributed to the difficulty in finding a relationship between fish size and infection rates (Figure 1.1): (1) very high variability between all fish and (2) some very infected fish (300-400 parasites). We logged removed the five greatest outliers and still only developed a regression coefficient of 0.21 with p < 0.05. There is indication that infection rate is correlated with size (ex. Hudson et. al. 1995) but this relationship is less certain as infection rates increase. All sizes may be equally vulnerable however the largest fish have the highest infections. Predictable Patterns: Attachment of the parasite in the oral cavity of the fish showed some predictable patterns. Locations on the tongue and gill rakers seem to be preferable for the adult parasite. The parasite was located most frequently on fleshy tissue and these sites were often completely covered with parasites. The roof of mouth does not contain the same amount

12

Achtheres- Morone Relationships in Reservoirs

of fleshy tissue although areas where the parasite could attach were often heavily infested. All parasites we observed during this initial investigation on all fish were adult or late copepodite stages. Almost all copepods were females (a few males were discovered attached to females) with a majority fecund. Seasonal Cycles: Achtheres has been observed to have seasonal cycles of abundance (Boxrucker 1979) with highest abundances in summer. We found the reverse to be true in Smith Mountain Lake where the highest incidences were in the winter months. This may correlate with behavior of the host. Striped bass can often experience stress from higher temperatures and lower oxygen levels in reservoirs over summer periods (Schaffler and Isely 2002). Winter provides greater range of movement. Abundance of parasite may reflect the overall condition of the host. Impact on Fish Mortality: The impact of this parasite on direct and indirect mortality of the host is difficult to predict. Little is known of this organism impacting striped bass in reservoirs. Some reports in the literature (Hoffman 1977, Stepanova and Vjuskova 1985) suggest direct injury to the host due to hyperplasia in the gills. We have no made such observations. We do believe many fish are infected to levels that may impact respiration. Muzzall et. al. (1995) observed an increase in respiration rates of gobys infected with parasites. Fish kills of striped bass have been reported with initial onset of the parasite in a reservoir (VA Game and Inland Fisheries personal communication.). The lower mean size of fish studied in winter from Smith Mountain Lake (Table 1.1) reflected the loss of many larger striped bass to fish kill. It is possible this loss was an indirect impact from the parasite increasing respiration leading to fish kill under summer reservoir oxygen and temperature regime. It is therefore possible the parasite is increasing respiration rates in striped bass thus contributing to summer fish kills during periods of high temperatures and low oxygen in southeastern reservoirs. Outbreak / Spread: Spread of this parasite is also difficult to predict both among fish in a reservoir and among reservoirs. Outbreaks of introduced copepod parasites occur (Cakic et. al. 2004) and can disperse over distances very quickly (Hudson and Bowen 2002). This parasite infection on Striped Bass was first reported in Tennessee in 2002, later spread to Virginia (2003) and now is reported in North Carolina (2004). A possible mechanism is angler transfer through live wells or introductions of infected species. Once the parasite enters a reservoir behavior of the fish may facilitate the spread. Schooling of striped bass may lead directly to rapid spread of the parasite among other fish. Fecundity is very high among the parasites possibly leading to high rates of spread and infection. Autoinfection is likely with such high prevalence and rates of infection on fish. This parasite is currently a threat to stocked reservoir striped bass populations throughout the southeastern US. While infection rates may decrease through time (Lake Norris TN and Smith Mountain Lake VA) this parasite does not lack availability of hosts and

13

Achtheres- Morone Relationships in Reservoirs

continues to proliferate. Annual stocking programs will continue to provide new hosts. While infections observed in Lake Norris do suggest a decrease when compared to Smith Mountain Lake, infection rates are much higher than ever observed in the literature elsewhere. The potential direct and indirect impact on respiration and metabolic costs will likely decrease survivability of stocked fishes. 1.2

Life Cycle of Parasite Attached to Striped Bass.

Initially based on preliminary findings we hypothesize the following description of the life cycle of this parasite and how it affects its fish host: •Attachment of Chalmus (first and second copepodids) to gill filaments – this provides maximum blood flow and easy attachment for this stage of parasite. It is also very small and can live among the filaments. •The copepod completes life cycle growing to adult male or female attached to the gill filaments. •The fish gills secrete mucus in response to the irritation of the parasite. •Gill filaments loose a significant amount of surface area causing the increased venting rates of the fish. Gills no longer function at a maximum efficiency impairing oxygen exchange. •Adult males and females migrate to oral cavity to reproduce. This is why there is a greater preponderance of parasites toward the back of the oral cavity. •Parasites visible in the oral cavity only represent a fraction of parasites on fish •As the fish grows respiration demands increase in proportion to body size of fish. Fish becomes more susceptible to environmental stresses due to increased respiration demands. •At a certain size fish cannot maintain respiration requirements – this is why we have lost large fish infected in the lake •Fish eventually die from asphyxiation due loss of gas exchange. Further analysis of capture live fish from Smith Mountain Lake have confirmed the following. Developmental stages of the parasite are confined to the gill arches and rakers (Figure 1.4). We thoroughly examined the entire oral cavity make this determination. It is essential that live fish are used for this analysis. These findings were only possible on recently captured fish. The attachment site is the gill arch. Many stages of development are found within the arch (Figures 1.5 and 1.6). It appears the parasite attaches to the fish along this area and throughout the mouth. It develops and spends its entire life at the site of attachment. We have no evidence to suggest the parasites are migrating throughout the mouth to find suitable locations for growth. This evidence suggests that severity of infection is due to contact with the nauplii stages of the parasite. We do not have any evidence of the parasite embedding into the gill filaments. While our experiments demonstrate the increased oxygen uptake of the fish other mechanisms must be at work.

14

Achtheres- Morone Relationships in Reservoirs

Figure 1.5. Developmental Stage on Gill Arch.

Figure 1.6. Chalimus Stage Removed From Gill Arch.

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Achtheres- Morone Relationships in Reservoirs

Figure 1.7. Developing Young on Gill Arch.

1.3.

Impact of Parasite on Fish Respiration

It is possible under intense infestations the gill rakers could be damaged. Heavy infestations of the larvae on the gills can cause mortality (Hoffman 1977). Males may damage the gill filaments. Achtheres may speed the sedimentation of red blood cells and impact the number of red blood cells in host fish (Stepanova and Vjuskova 1985). Prevalence and intensity of infections of parasitic copepods are affected by long-term interactions with the host (Shulman 1958). Prevalence of infection has a positive correlation to size and age of fish (Hudson et. al. 1995). Initial Results 2002-2003 In our study we able to briefly establish a culture of five (5) uninfected striped bass from Claytor Reservoir in Pulaski Va. and three (3) infected fish from Smith Mountain Lake. Although the Claytor fish were larger in size than Smith Mountain fish (Table 3), we were able to collect excellent information and make inferences concerning the potential impact of the parasite on striped bass health and survivorship. We know from the existing studies and literature (Muzzall et. al 1995), infected fish tend to have increased respiration rates measured though venting. As soon as our fish were in culture, we made repeated measures (ten trials of one min each) on each fish recording the vents. Results appear in Table 1.3. Clearly infected fish respire at a higher rate than uninfected. Trends were significantly different. We need additional work to develop this

16

Achtheres- Morone Relationships in Reservoirs

relationship and deal with issues such as size of fish, temperature and oxygen content of the water. Table 1.3. Respiration rates (measured as gill vents) of experiment fish. Infected fish from Smith Mountain Lake, uninfected fish from Claytor Lake. Fish Type - Size

Respiration Rate - Vents per min (hr) [day]

21 “ Infected

54.1 (3249) [77976]

25 “ Infected

53.1 (3186) [76464]

28 “ Uninfected

44.3 (2659) [63816]

30 “ Uninfected

44.9 (2694) [64656]

32 “ Uninfected

42.1 (2526) [60624]

Because larger fish will vent (respire) at lower rates than smaller fish, we extrapolated our results with linear regression lines to estimate respiration according to size. Figure 9 shows the differences in slope of both lines suggesting that larger infected fish will still respire at much higher rates than non infected fish.

Vents per min.

Respiration Models 56 54 52 50 48 46 44 42 40 20

22.5

25

27.5

30

32.5

35

Fish Size (inches) Figure 1.8. Extrapolated respiration rates based on measured results (Table 1.3) and extrapolated using a linear model. Yellow line infected fish and purple line uninfected.

17

Achtheres- Morone Relationships in Reservoirs

Active Respiration Studies We established a culture of striped bass from Brookneal Hatchery beginning June 2006 through June 2007. Pond reared fingerlings arrived from the hatchery on June 28, 2006, were divided, and placed in 8 recirculating tanks at Claytor Nature Center in Bedford Virginia (Figure 10). We immediately began training the fish to eat pelleted food using bread and later 1 mm Ziegler crumble for striped bass. Temperature of the facility was maintained at 68 F (19C) throughout all experimentation and culture. To facilitate the growth of fish, larger pelleted food was used beginning with 3mm and then later 5mm pellets. Fish were fed twice each day using an automated feeding system. Tanks were cleaned weekly removing all settled solids and any dead fish.

Figure 1.9. Culture facilities at the Claytor Nature Center in Bedford Virginia. One line of tanks was infected with Achtheres parasite. This was done by removing gravid parasites from infected fish thus suspending each in small mesh baskets in each tank. We placed between 20-30 live gravid females in each basket. We believed similar to Conley and Curtis (1994) that mature female Achtheres egg sacs would hatch into nauplii, develop and attach to fish in our culture. Infection was determined by extracting fish from one of the 4 tanks and visually assessing the fish for parasites. Deceased fish were also examined for the presence of parasites or obvious physical parasitic/disease activity as further described by Schaperclaus (1991). Fish were infected at the time of experimentation. Transferring process from holding tank to Static Respirometer Dissolved oxygen consumption of the fish was determined in a basic static respirometer (Cech 1990). The static respirometer was designed using three separate 10-gallon tanks. One tank was used to measure infected fish, one for uninfected, and one for a control tank measuring natural oxygen consumption. Tanks were elevated on bricks with a single stir plate and stir bar under each tank. Plexiglas lids and duct tape were used as sealants for the tanks. In order to reduce stress to the fish water was used from their holding tanks. We prevented light from entering tanks during each experiment. An YSI meter was used

18

Achtheres- Morone Relationships in Reservoirs

to measure O2 before and after each experiment. Experimental time was set to one hour. Oxygen consumption was calculated using the following formula from Cech 1990 {MO2 = [(A-B) V]/T}

1

Where: MO2 = amount of oxygen consumed (mg/l) per hour A = Initial Dissolved O2 (mg/L) B = Final O2 measurement (mg/L) V = Volume of water used in respirometer (10 gallons = 37.85 Liters) T = Time (in hours) between initial and final O2 readings. RESULTS We tested 60 total fish throughout a 4 month period. Fish were matched from each of the infected and uninfected lines to produce similar weights (Table 1). Table 1.4. Summary of Respiration Experiments (N = 60) Fish Weight Oxygen Consumption Grams mg/O2/hour mg/Kg/hour Uninfected 30.04 7.79 Infected 29.98 11.18

272 378

We compared each group using ANOVA finding the respiration increase demonstrated by infected fish was significant (p = 0.022). This demonstrated the increased respiration rates of the infected fish line. Table 1.5. Individual oxygen consumption for each tested fish. Uninfected (mg/kg/hr) 628.3636364 113.8554217 455.3846154 378.5365854 107.038835 401.9457014 176.4444444 353.0172414 59.6 400.3461538 109.0384615 558.2375479 121.969697 245.0184502 189.9283154

Infected (mg/kg/hr) 90.65868263 566.4670659 845.3807107 203.9215686 166.6666667 447.3181818 75.55555556 600.8583691 473.015873 530.1945525 339.3129771 346.7175573 401.4393939 427.4074074 381.2454212

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Achtheres- Morone Relationships in Reservoirs

178.2312925 248.1818182 174.3092105 303.9344262 279.8076923 685.6707317 426.231454 42.5915493 359.9728261 240.3174603 189.7097625 114.1732283 231.5529412 329.638009 70.0617284

268.5810811 350.5050505 408.6378738 426.7973856 313.3116883 430.7692308 155.8529412 754.7752809 266.6937669 189.1891892 437.1128609 317.0680628 289.7607656 589.6860987 238.0434783

272.4369746

377.7648246

Indirect impact of Achtheres on Respiration In order for a fish to uphold its homeostatic state when impacted by a stress, Barton (2002) suggests that secondary physiological responses, such as respiratory functions, may change. Our results suggest that infected fish have a higher level of O2 consumption. Dissolved oxygen plays a major role in how striped bass select their habitat (Hill et al. 1981). During summer months these conditions are reduced, causing stress to the fish. When Achtheres parasite is added to this environment, the fish uses more energy than it normally would for respiration, suggesting that a combination of environmental and parasitic-respiratory stressors have a major impact on fish. Previous studies have indicated that environmental stress can greatly affect the impact parasites have on their host (Lafferty & Kuris 1999). In our research facility, we experienced periods where several fish unexplainably deceased, but did not find evidence that the parasite had direct lethal affects to its host. When combined with environmental stressors, it is possible that this parasite-host relationship can have lethal consequences to fish infected by Achtheres. Valtonen et al. (1993) suggests that age and size dependencies of Achtheres may only occur in warmer months of the year, which may also explain greater impacts and stressors to larger fish during this time of the year in reservoir environments infected with this parasitic copepod. We are certain that the parasite in our study is affecting the respiration and oxygen consumption of striped bass. Achtheres parasites tend to be highly prevalent in the mouth and gill area. We have evidence to suggest that the parasite found in the fish is affecting the oxygen consumption of the fish; specifically forcing the fish to consume more oxygen than it normally would.

20

Achtheres- Morone Relationships in Reservoirs

1.4

Effect of parasite on blood of Striped Bass

Achtheres infection has been linked to anemia, low red blood cell RBC numbers, in fish (Stepanova and Vjuskova 1985). To determine whether Striped Bass with the Achtheres parasite were anemic, between April 21, 2007 and May 10, 2007, Stripped Bass blood samples were obtained for comparison of Achtheres infected fish from Smith Mountain Lake to samples from uninfected fish from Claytor Lake. Samples were obtained through the assistance of the Brookneal Hatchery. On 3 separate occasions the Hatchery provided 4 or more fish captured by electric shock, for their propagation program, transported to the hatchery, and maintained in a holding tank about 6 hours until sampling. Stripers from the hatchery consisted of both males and females with an average length of 24.8 in., and size range of 21 to 29 inches. The Achtheres parasite was observed on oral cavity and gill rakers of all fish sampled from Smith Mountain Lake. Uninfected fish from Claytor Lake were obtained courtesy of Dan Wilson and John Copeland of the Virginia Department of Game and Inland Fisheries. Fish from Claytor Lake were collected primarily on a single outing using electroshock and capture by anglers. Following capture fish were placed in a circulating holding tank on the boat and blood samples were obtained within one hour of capture. The average length of Stripers from Clayter Lake was 15 in and the range was 8 to 29 inches. Of the Stripers from Clayter Lake 3 were greater than 20 inches and 5 were less than 10 inches. No Achtheres parasites were observed on the fish from Claytor Lake. To determine whether there was a difference in the number of blood cells in fish from the 2 groups, packed cell volume (PCV) was determined by hematocrit. Fresh blood samples were drawn into heparinized microcapillary, hematocrit tubes, and immediately centrifuged at 8000 rpm for 5 minutes to separate plasma and cell fractions of blood. The data are presented in table 3. Table 1.6. Effect of parasite on % PCV of wild Striped Bass. Source Number of Fish % PCV + SD Smith Mountain Lake 14 33.1 + 3.73 Claytor Lake 8 39.3 + 3.58 The data indicate that Stripers from Claytor Lake have more blood cells than those from Smith Mountain Lake (fish infected with Achtheres were anemic compared to uninfected fish). Data was analyzed with a 2 sample t-test comparison which revealed that the difference was significant (0.005 significance level). To address concerns that the difference might be due to the number of small fish in the Claytor Lake sample, we did statistical analysis of PCV data from small versus large fish from Claytor Lake and found that there was no statistically significant difference between PCV values of fish greater than 20 inches (n = 3; PCV = 39.0+0.91) and fish less than 10 inches (n = 5; PCV = 39.38+4.69) at the 0.05 level of significance.

21

Achtheres- Morone Relationships in Reservoirs

There was also concern that the transport and longer holding time of the Smith Mountain Lake fish at the Brookneal Hatchery may have affected PCV results. Though we can not rule out possible effects of transport and holding on Smith Mountain Lake fish, a previous study (Lebelo S.L., Saunders D.K., and Crawford T.G. 2001) has shown that the PCV of blood from Striped bass in hypoxic conditions and conditions designed to simulate transport did not vary from normal controls, suggesting minimal effects due to transport and holding in the current comparison. The finding that Stripers infected with the Achtheres parasite are anemic further supports our theory that infected fish are energetically compromised making them susceptible to other life threatening challenges. The anemia coupled with a thickening of the gill exchange surface described in the background section of this report, would mean that the ability of infected fish to supply oxygen to active tissue in time of need would be compromised. The finding of anemia would contribute to the need for infected fish to increase their respiratory rate as described above. In addition to the hematocrit samples to determine PCV, at the time of collection a sample of blood was smeared on a microscope slide for analysis of the percentage of red and white blood cells. Blood smears from Claytor Lake uninfected, and Smith Mountain Lake infected Striped Bass were stained with Wright’s Stain solution which allows red blood cells to be distinguished from white blood cells under microscopic observation. Between 200 and 400 cells per smear were counted to determine percentage white blood cells. A sample preparation appears in figure 14 below.

22

Achtheres- Morone Relationships in Reservoirs

WBC

RBC

Figure 1.10. A typical blood smear from Striped Bass consisting of mainly red blood cells (RBC) with scattered white blood cells (WBC). White blood cells are the cells responsible for fighting infection, in humans an elevated white blood cell count is used routinely to diagnose infection (Tortora & Grabowski, 2000). Fish have been shown to respond to infections with an increase in the percentage of white blood cells (Chistiakov, Hellemans, and Volckaert, 2007). We were surprised to find that Striped Bass infected with the parasite had no statistically significant difference in the percentage of white blood cells compared to uninfected Striped Bass. The results of white blood cell analysis are presented in Table 4. Table 1.7. Effect of parasite on %WBC of Wild Striped Bass. Source Number of Fish Average %WBC + SD Smith Mountain Lake 6 1.78 + 0.88 Claytor Lake 6 2.50 + 0.92

A t-test at the 0.1 level of significance revealed no difference between the percent WBC in infected fish from Smith Mountain Lake compared to uninfected fish from Claytor Lake. At first this appears to be a surprising result, but the literature reveals that the immune system of fish can be depressed in times of stress (Wendelaar Bonga 1997) as has been observed in humans (Tortora & Grabowski, 2000). It is possible that infected fish mount an immune response to the parasite when they are first infected that includes

23

Achtheres- Morone Relationships in Reservoirs

increase blood white blood cells, but with time the white blood cell counts normalize, or alternatively the parasite may be able to fool the fish immune system into not fighting it. Further studies in a more controlled environment will be needed to determine this. We believe that the parasite constitutes a prolonged stress for infected animals which leads to energy depletion. The state of energy depletion could result from decreased respiratory capacity due to gill damage coupled with red blood cell anemia. The increased respiratory rate would be evidence that infected fish are trying to compensate for decreased gill respiratory efficiency, and anemia. The anemia that we have currently observed may have resulted from inadequate energy to maintain normal blood cell numbers, or from a direct effect by the parasite to kill blood cells of the fish. In any event having fewer red blood cells would lead to further reductions in energy of infected fish. Taken together, the compromised respiration and anemia would suggest that infected fish have serious energy depletion which would almost certainly mean that their ability to cope with change such as temperature, oxygen levels, food availability, infections, or meeting the energy requirement for growth, is severely hampered. Ultimately this translates to a diminished population of smaller less hardy fish, with a higher death rate. In support of this hypothesis Dan Wilson has observed a decrease in the population of large, citation, Striped Bass in Smith Mountain Lake (figure 15 below).

24

Achtheres- Morone Relationships in Reservoirs

Section 2. Bioenergetics Model Smith Mountain Lake Citations 200 180

Number of Citations

160 140 120 100 80

Bass Striper Crappie

60 40 20 0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

Figure 2.1. Since the copepod (Achtheres) began affecting the fish of Smith Mountain Lake in 2003 the number of citations of striped bass, largemouth bass, and crappies have significantly decreased.

The goal of this portion of the project was to create a bioenergetics growth model for striped bass (Morone saxatilis) in Smith Mountain Lake. This growth model should produce a information for growth that mimics actual data to manipulate. By producing a working model we can examine the impact of impacted respiration rate on fish growth. Maple 6.0 was used to create the model of striped bass growth. See Appendices for full Maple 6.0 code. The growth model allows for the respiration of and temperature of the water to be varied. The basic differential used for the growth model is: dG  C  R  F U dt

where dG/dt is the growth of the fish over time, C is the consumption that the fish takes in each day, R is the respiration, F is the egestion, and U is the excretion. The first variable in the differential equation for growth is consumption. The equation that gives this variable is: C  C max* P * f (T )

25

Achtheres- Morone Relationships in Reservoirs

Cmax is the maximum daily consumption for striped bass at its optimum temperature. P is the “proportion of maximum ration” and is called the P value. P can be from 0  P  1 and it can be changed so the predicted growth curve of striped bass can match the observed growth curve of striped bass. f(T) is a function dependent on temperature that is directly proportional to the consumption rate. This function is based on the maximum feeding temperature of striped bass. The basic equation for Cmax is:

C max  C *W C A

B

CA is a constant of the intercept of the mass dependence function for 1g Morone saxatilis at 0oC and CB is the mass dependence coefficient. W is the weight of the Morone saxatilis in grams.

f (T )  KA * KB Where KA and K are the increasing and decreasing parts of the function f(T) respectively.

KA 

(CK 1 * L1) (1  CK 1 * ( L1  1))

L1 e

G1*(T CQ))

  1  .98 * (1  CK 1)   * ln  G1     CK 1 * 0.02   (CTO  CQ) 

KB 

CK 4 * L2 1  CK 4 * ( L2  1)

(G *(C T )) L  e 2 TL 2  1 G2    CTL  CTM

  0.98 * (1  CK 4 )   * ln     CK 4 * 0.02 

Where CK1 is a given variable value in the chart represents the water temperature at which the fraction of dependence on temperature is a small fraction of the maximum rate CQ. “The CTO is a given variable in the chart that represents the water temperature when the consumption rate is 98% of the maximum. The upper temperature at which dependence is some reduced Fraction CK4 of the maximum rate CTM.”

26

Achtheres- Morone Relationships in Reservoirs

The form for the respiration function: R  R A *W RB * f 2 * ACT

where R is the respiration of the striped bass. This respiration depends on the striped bass weight water temperature and level of activity. W is the mass of the striped bass in grams. RA is the intercept of the growth mass function and RB is the slope of this same function. F(2) is again the temperature dependence function and T is the water temperature in C0. Also, ACT is the activity.

f2e

( RQ *T )

T

ACT  e RTO A Where RQ is value given in the chart and it approximates the Q10. This is the rate at which f(2) increases at low temperatures. The RTO is the coefficient for how swimming speed depends on metabolism.

U  UA * (C  F ) F  FA * C

Where excretion (U) and egestion (F) are the processes of the striped bass discharging of waste from its body. Table 2.1. The species specific parameters used for striped bass in this model (Hartman and Brandt, 1995). Source YOY Age-1 Age-2 Adult Species Age Consumption CA 0.3021 0.3021 0.3021 0.3021 CB -0.2523 -0.2523 -0.2523 -0.2523 CQ 2.6 6.6 6.6 7.4 CTO 21.6 19 18 15 CTM 22.7 28 29 28 CTL 28.3 30 32 30 CK1 0.047 0.262 0.255 0.323 CK4 0.713 0.85 0.9 0.85 Respiration RA 0.00146 0.0028 0.0028 0.0028 RB -0.2702 -0.218 -0.218 -0.218 RQ 0.08339 0.076 0.076 0.076 RTO 0.9014 0.5002 0.5002 0.05002

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Achtheres- Morone Relationships in Reservoirs

Egestion/Excretion FA UA 2.7.

0.104 0.068

0.104 0.068

0.104 0.068

0.104 0.068

Sensitivity

High sensitivity means that as the individual parameters of the differential equation are changed by a small amount it affects the end results and graphs to a high degree. Low sensitivity means that as the individual parameters are changed by a small amount the end result and graphs are affected minimally. Each parameter will have a different level of sensitivity. It is ideal for the growth model to have as many parameters having low sensitivity as possible. Thus the parameters can be changed to account for changes in the real world environment. The parameters RA, W, and T were tested to determine individual sensitivity to changing their values in this growth model. It is desirable to have RA and W be sensitive variable with resulting graphs change dramatically when RA and W are altered. Conversely, temperature should not be a sensitive variable. Its graphs should have little variation. When testing RA three graphs were produced for three varying values of RA while leaving the rest of the parameters the same. Originally RA was .0028. We will multiply this number by 2 and 4 to determine its sensitivity.

Change in Respiration 12

10

Weight

8

Normal x2 x$

6

4

2

0 10

20

30

40

50

60

Temperature

Figure 2.6. Change of Respiration Dependent on Weight and Temperature.

28

Achtheres- Morone Relationships in Reservoirs

This graph indicated that the higher the water temperature, the more sensitive the data becomes to changes in respiration. Since the resulting growth rate graphs change greatly it is determined that the respiration of the striped bass is sensitive. When testing the sensitivity of the weight (W) variable three graphs were produced for three varying values of W while leaving the rest of the parameters the same. Graphs were produced from the starting weights of a striped bass at 10g, 100g, and 1000g and showed the weight change over one day at various temperatures.

Figure 2.7.

Figure 2.8.

29

Achtheres- Morone Relationships in Reservoirs

Figure 2.9. These three graphs show that as the starting weight decreases the variation of the resulting graphs become more significant. The max growth over a one day period at any temperature for a striped bass from a starting weight of 1000g was about .34g. From a starting weight of 100g the max growth over a day was about double at .617g. Finally from a starting weight of 10g the max growth rate was about 1.13g. The graphs of the striped bass growth over one day changed dramatically over varying temperatures. Thus weight is a sensitive parameter. 2.8.

Stability

The stability of a model is analyzed on the steady state version of any model. The steady state of this growth model would be a resulting graph of a zero slope. Thus the weight of striped bass over time would be constant. This would be the unchangeable stable steady state form of the model. It would be ideal to have a model when it became unbalanced slightly from its steady state it would result in an unsteady state that eventually settles back into its original steady state. Although when some models become unbalanced from their original steady state they change dramatically and eventually settle into a new steady state. This is not ideal for a growth model because if the parameters of the model were changed to produce an unbalanced steady state it would be difficult to analyze results if the resulting steady state was different from the original before the changes were made. This entire process can be visualized by a ball rolling from a mountain peak to the valley. Stability of this growth model is not as important as its degree of sensitivity to parameter changes because bioenergetics models are typically very stable with respect to the initial conditions described in section 3.2. The slope field from this bioenergetics model of striped bass growth takes the form of:

30

Achtheres- Morone Relationships in Reservoirs

Figure 2.10. Slope Field of Bioenergetics Model of Striped Bass Growth. This graph provides evidence that this model is stable because the slope is the same for any weight at a specific temperature. 2.9.

Results and Discussion

The three factors that were altered or looked at over a range were weight (W), temperature (T), and respiration rate (RA). First the model was used to measure how changes in striped bass starting weights affected the fish’s growth at varied temperatures and a constant normal respiration. These graphs can also show at which temperatures striped bass will experience the most growth. The first starting weight run in the model was 10g at normal respiration.

Figure 2.11. – Daily growth of a 10 gram fish over a range of temperature. At 20oC the weight of the striped bass was 10.628g. The maximum growth for one day was about 1.2g for a 10 gram fish. 31

Achtheres- Morone Relationships in Reservoirs

Next the starting weight run in the model was 1000g at normal respiration.

Figure 2.12 – Growth of 1000 gram fish over a varied range of temperature. At 20oC the weight of the striped bass was 1000.190g. The maximum growth for one day was about 0.3grams. Finally a starting weight of 10,000g for a striped bass was run using the model.

Figure 2.13. At 20oC the weight of the striped bass was 10000.103g. The maximum growth for one day was 0.2 grams.

32

Achtheres- Morone Relationships in Reservoirs

The results of the first set of tests on the model showed good stability and predictability of growth over a range of temperatures. Growth rate declines rapidly with age and this is demonstrated by our model. Growth rates of smaller fish (1.3 grams per day) are closer to fish found in reservoirs than rates demonstrated by larger fish in our model. Yet this model is still useful to present impacts due to respiration changes. At this point we used the model to make predictions on growth with varied respiration rates. Varied respiration rates represented impact of parasite. Striped bass are considered to be adult fish when they reach their third year of life. We assumed the average weight of striped bass at the beginning of their third year of life was 1814 grams. Thus this is the starting weight used to produce a graph that grows the fish out over a period of eight months (assumed growing season). This is done because the starting parameters for striped bass under three years of age are different than those for adult striped bass. In order to grow a three year old striped bass a loop version of the model was used. The purpose of varying the respiration is to figure out at what degree of respiration rate that striped bass begin to experience a negative growth rate. The starting temperature was kept at 25oC.

First an 1814g striped bass was grown at normal respiration rate for eight months.

Figure 2.14. Model predictions of growth for 1814 gram fish over eight month period at optimal conditions.

The resulting graph (figure 2.14) predicted a positive growth rate with fish gaining 104 grams of weight. The same 1814g striped bass was run in the model at five times the normal respiration. Evidence for these elevated respiration rates were derived from our laboratory studies.

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Achtheres- Morone Relationships in Reservoirs

Figure 2.15. Growth of 1814 gram fish with 5X elevated respiration rates over eight months. The resulting graph shows a positive growth rate with the weight of a striped bass after eight month gaining 46 grams of weight. The same 1814g fish at nine times the normal respiration:

Figure 2.17. Growth Rate of 1814g Fish at 9X Normal Respiration over eight months. This fish lost 18 grams of weight over 8 month period. This run of the model is more suggestive of death as loss of weight is not a condition expected. 2.10.

Conclusion

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Achtheres- Morone Relationships in Reservoirs

We believe the growth model did produce results that help understand impacts related to Achtheres. Increased respiration for striped bass directly decreases their growth. This would suggest that varied levels of parasite load on the gills thus impacting respiration will slow growth of the fish. This coupled with stress associated with temperature and oxygen levels in could create observed fish kills. Thus, this parasite may be responsible indirectly for observed fish kills. Additionally, growth rates of striped bass may be inhibited by the parasite. Fish may not achieve trophy size due to additional costs of respiration brought on by Achtheres. Size and age relationships of fish from infected and uninfected reservoirs may add evidence to this hypothesis. Yet other environmental variables such as temperature and forage will always confound this result.

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Achtheres- Morone Relationships in Reservoirs

Section 3. Work in Progress and Future Directions 3.1

Histology Studies

At the time we collected blood samples from Smith Mountain Lake-infected, and Claytor Lake-uninfected fish we also collected tissue samples for microscopic analysis of gill tissue from infected and uninfected fish. This tissue was collected so that we might determine whether gill filament structure was damaged by the presence of the parasite as has been reported in the literature sited above. We anticipate being able to measure the respiratory membrane, which is the actual tissue barrier between blood and water which oxygen must cross for respiration to occur at the gill surface. We will also assess local immune reaction, inflammatory response, around the site of parasite attachment for correlation with blood white blood cell counts in determining the immune status of infected fish. Currently 2 students, Adrian Wright, and Amy Rosso from the Biology/BioMed programs, are processing that tissue for microscopic analysis and we hope to begin making observations in the coming weeks.

3.2

Parasite/Striped Bass aquaculture model

Because field observations made on uninfected and infected Striped Bass from Claytor and Smith Mountain Lake reservoirs, respectively, could have been influenced by other outside factors -such as temperature, oxygen tension, and food availability- we wanted to study the relationship between parasite and host under more controlled conditions. For these studies we established Striped Bass populations at the Clayter Nature Center as described above in the respiration studies. To date the fish have attained a length of 8 to 12 inches. 3.3

Validation of blood parameters for Striped Bass in aquaculture

To determine whether uninfected fish in the aquaculture center were representative of uninfected fish in the wild, we determined packed cell volume (PCV) and percentage white blood cell counts on blood samples (3 samples on 3 different days for a total of 9) from uninfected cultured fish and compared our results to those obtained for uninfected wild fish from Claytor Lake reservoir. The results are presented in Table 5, below.

Table 3.1. Effect of aquaculture on % PCV. Source Number of Fish Aquaculture Center 9 Claytor Lake 8

% PCV + SD 38.7 + 5.24 39.3 + 3.58

A t-test at the 0.1 level of significance revealed no difference between the %PCV of aquaculture fish and uninfected fish from Claytor Lake reservoir. The findings for % PCV suggest that uninfected aquaculture fish are representative of wild uninfected Striped Bass.

36

Achtheres- Morone Relationships in Reservoirs

We also analyzed blood samples from uninfected aquaculture fish for % WBC, and compared our findings to those from uninfected Claytor Lake fish. The results are presented in Table 6, below. Table 3.2. Effect of aquaculture on % WBC. Source Number of Fish Claytor Lake 6 Aquaculture Center 6

% WBC + SD 2.50 + 0.92 3.65 + 1.04

Even though it looks as if fish from the aquaculture center had a greater % WBC than fish from Claytor Lake t-test analysis revealed that the difference was not significant at the 0.05 level of significance. However, we have observed that fish in aquaculture have a tendency to have higher white blood cell counts than wild fish, this is perhaps a measure of good health or age differences. In conclusion, blood analysis indicates that uninfected fish in the controlled conditions of aquaculture are representative of uninfected fish in the wild. 3.4

Effects of Achtheres exposure on Striped Bass in aquaculture

The next crucial step in our aquaculture studies is to establish visible parasite infections in an experimental group of fish. We hope to do this in the coming weeks as the temperature drops allowing capture of infected fish from Smith Mountain Lake. In the past we have established infected groups of Striped Bass at the Claytor Center by placing infected fish in tanks on the same line as cultured fish to be infected. Once we have an infected group in aquaculture we will make the following comparisons: 1) hematocrit 2) % white blood cells 3) gill tissue histology 4) respiratory rate 5) oxygen consumption studies These comparisons will allow us to validate the model in relation to past results from fish in aquaculture, and the field observations we have made on Smith Mountain Lakeinfected and Claytor Lake-uninfected fish. We plan on extending our study to include: 1) blood gas analysis 2) differential white blood cell analysis 3) blood protein analysis 4) red blood cell size analysis 5) Achtheres developmental stages 6) ability of infected fish to handle stress 7) mortality 8) growth

37

Achtheres- Morone Relationships in Reservoirs

3.5

Field studies of fish infected with the parasite in the wild.

We would like to continue our collaboration with the Virginia Department of Game and Inland Fisheries to: 1. Develop a model to monitor infection density of fish in Smith Mountain Lake over time using digital images of parasites on gill rakers to estimate density. We propose to acquire these images at times when Striped Bass are being surveyed by the Virginia Department of Game and Inland Fisheries. This would allow use to correlate parasite infection density with any dramatic Striped Bass population changes in the future. 2. Continue to study parasite life cycle stages on gill rakers during different seasons of the year, with an attempt to quantify different stages at different seasons 3. Continue blood work on wild type fish with special emphasis on seasonal changes.

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Kabata, Z. 1969. Revision of the genus Salmincola Wilson, 1915 (Copepoda: Lernaeopodidae). Journal of Fisheries Research Board of Canada. 26: 29873041. Kabata, Z. and Cousens, B. 1973. Life cycle of Salmincola californiensis (Dana 1852) (Copepoda: Lernaeopodidae). Journal of the Fisheries Research Board of Canada. 30: 881-903. Lafferty, K. D., and Kuris, A. M. 1999. How environmental stress affects the impacts of parasites. Limnology and Oceanography Part 2: The Effects of Multiple Stressors on Freshwater and Marine Ecosystems. 44(3): 925-931. Lebelo, SL, DK Saunders, TG Crawford. 2001. Observations on Blood Viscosity in Striped Bass, Morone saxatilis (Walbaum) Associated with Fish Hatchery Conditions. Transactions of the Kansas Academy of Science. 104(3): 183-194. Levinton, J. 2001. Marine biology; function, biodiversity, ecology. 2nd edition. Oxford University Press. Oxford. New York. Muzzall, P. M., Peebles, C. R., and Thomas, M. V. 1995. Parasites of the round goby, Neogobius melanostomus, and tubenose goby, Proterorhinus marmoratus (Perciformes: Gobiidae), from the St. Clair River and Lake St. Clair, Michigan. Journal of Helminthological Society of Washington 62:226-228. Patriarche, M.H., and R.S. Campbell. 1957. The development of the fish population in a new flood-control reservoir in Missouri, 1948 to 1954. Transactions of the American Fisheries Society 87: 240-258. Piasecki, W., A.E. Goodwin, J.C. Erias, and B.F. Nowak. 2004. Importance of Copepods in freshwater aquaculture. Zoological Studies 43:193-205. Schaffler, JJ and JJ Isley. 2002. Habitat Use by Striped Bass in Relation to Seasonal Changes in Water Quality in a Southern Reservoir. Trans. Am. Fish. Soc. 131:817-827. Schaperclaus, W. 1991. Fish Diseases. 1st edition. New Delhi: Amerind Publishing Company: 1-38 Shelton, W.L., W.D. Davies, T.A. King, and T.J. Timmons. 1979. Variation in the growth of the initial year class of largemouth bass in West Point Reservoir, Alabama and Georgia. Transactions of the American Fisheries Society 108: 142-149. Shahady, T.D. and J.S. Ashwell, Lynchburg College, unpub. Data. Stepanova GA and LA. Vjuskova 1985. Achtheres percarum infection in pike-perch

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from Volga river delta. In: Proceedings of a symposium on parasitic fish diseases (O.N. Bauer Editor). pp. 133. Lenigrad, Nauka Thatcher, V.E. 1998. Copepods and fishers in the Brazilian Amazon. Journal of Marine Systems. 15(1-4): 97-112 Tracy, C.R., and K.A. Christian. 1986. Ecological Relations Among Space, Time, and Thermal Niche Axes. Ecology. 67(3): 609-615. Valtonen, ET, H Tuuha, ON Pugachev. 1993. Seasonal studies of the biology of Actheres percarum in perch, Perca fluviatilis from four Finnish lakes over a three year period. J. Fish. Biol. 43:621-632. Walburg, C.H. 1977. Lake Francis Case, a Missouri River reservoir: changes in the fish population in 1954-75, and suggestions for management. Technical Papers of the Bureau of Sport Fisheries and Wildlife Research Report 95, Washington, D.C., USA. Wendelaar Bonga, SE. 1997. The Stress Response In Fish. Physiol Rev. 77(3):591-625 Wilson, CB. 1915. North American parasitic Copepoda belonging to the Lernaeopodidae, with a revision of the entire family. Proc. U.S. Natl. Mus. 47:565-729.

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43