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Meghana

Grade 11 | New York

The Effects of Heat-Shock Therapy on the Immunity of Hard Clams With Quahog Parasite Unknown Disease

Introduction
As I graze my hand over the corrugated shell of a hard clam that had washed ashore on the Long Island Sound, I thought of its significance in all aspects of life, from its cultural significance in the indigenous traditions of the Native Americans to being a vital component in the aquatic food chain as suspension feeders. I thought about how these multifaceted creatures are often taken for granted, despite how significant they truly are. As my interest in these mollusks increased, I learned about a potent parasite that has had devastating effects on the New England clam population. Shocked by this information, I started to investigate more about this disease and soon found out that it is called quahog parasite unknown (QPX) disease, and that a lab at Stony Brook University, which I contacted, was investigating prime mitigation strategies for it. I was ecstatic about my placement as a summer intern at this lab, and my amazing investigation began.

Background
Hard clams, Mercenaria mercenaria, are found off the North American Atlantic Coast from Canada all the way to Florida. The clams’ ubiquity is due to their tolerance for the broad ranges in salinity and temperature in their environment. Because of their wide distribution, hard clams are one of the most commercially important mollusks for the local and national fishing industries (Barnes, Allam and Gall 2009). Not just a source of food for humans, clams are a significant component of the aquatic food chain; organisms such as snails, crabs and shrimp depend on the sustainability of the clam for their own survival (Tarnowski). However, in recent years the clam population in New England has deteriorated. The great decline in the hard clam population is a result of quahog parasite unknown (QPX) disease activity, which has adversely affected clam populations on a national scale, indicating a formidable epizootic. With prevalence rates as high as 70% in areas such as Raritan Bay, one of Long Island’s primary sources of hard clams, QPX disease has significantly compromised the potential growth and vitality of hard clams (Barnes et al. 2009).

From previous studies, QPX has been deemed an opportunistic parasite; the presence of the parasite alone is not enough to initiate a thorough infection. Therefore, other environmental factors must play an influential role in the development of the disease. External factors such as water temperature have been shown to affect disease development (Dahl et al. 2011). Because many marine invertebrates, including the hard clam, are poikilothermic—the temperature of their tissues rises and falls with the temperature of the water—the environment can affect the development of any infection in these types of organisms. For instance, a previous study showed a significant reduction in QPX-infected clam survival rates when water temperatures exceeded 23ºC and that the parasite was eradicated at 32ºC (Perrigaul, Bugge and Allam 2010). Additionally, previous investigations have shown evidence of healing and a significant reduction in mortality rates among QPX-infected clams when maintained at temperatures greater than or equal to 21ºC, showing that not only does water temperature play a role in the development of the infection, but also that is is an influential factor in the recovery of the clam from the infection (Dahl and Allam 2007).

There have been several initiatives to mitigate the QPX infection, including transplant operations of clams from colder to warmer waters, using the temperature change to cause a decline in QPX prevalence (Allam 2009). However, because of the evidence showing cold water temperature to be one of the key factors in QPX growth, an even more direct use of temperature to control QPX disease was initiated in this study: to utilize acute heat-shock therapy as a component of the clam transplant process. This study aims to foster effective mitigation and management strategies by determining the optimal duration and temperature of heat-shock therapy that would promote the greatest remission of QPX disease in clams.

 
Questions
1. How will short-term “heat shock” treatments on exposed clams affect their pre-existing QPX infections?

2. What are the optimal short-term “heat-shock” conditions that would induce the greatest decline in QPX prevalence rates in infected clams?

 
Hypotheses
1. I hypothesized that short-term heat-shock treatments would promote further remission and a reduction in QPX infections, because the clams would be exposed to higher water temperatures in the treatments that would lower the prevalence of QPX disease.

2. I hypothesized that optimal heat-shock conditions would range from 21ºC to 27ºC for a duration of less than eight hours, because previous research has shown that temperatures in this range are effective.

Materials

  • 540 hard clams obtained from Massachusetts Bay (not all the clams were used in the experiment because some died off before the experiment began due to elevated QPX infection rates)
  • 22 treatment tanks (two for each heat-shock treatment and two for the controls)
  • Incubation ovens
  • Centrifuge
  • Planktonic microalgae (food for maintaining the hard clams)
  • Homogenizer
  • DNA extraction kits (including the various buffers needed)
  • Micropipettes
  • QPCR assay machine
  • Nanodrop spectrophotometer
  • -80ºC freezers
  • Safety equipment: lab coats, gloves

Procedure and Design
Five hundred and forty clams, collected from a highly infected wild clam population in Cape Cod, Massachusetts, were transferred to the lab and put through a range of heat-shock treatments (Figure 1). The variables tested in this experiment were the water temperature and the duration of the treatment, and the dependent variable was the population of clams surviving for 10 weeks after heat-shock treatment.

Figure 1. Experimental design for lab trials. An X designates the specific treatment condition tested.
  18°C 21°C 27°C 32°C 37°C
18 hours X X X    
8 hours   X X    
4 hours     X X  
2 hours     X X X

A total of 10 heat-shock treatments were conducted, with a control treatment left at 18ºC and exposed to the air for 18 hours (Figure 1). Each treatment was composed of approximately 40 clams divided into two

The clams undergoing the various heat-shock treatments


identical tanks, for a total of two trials for each treatment. Upon being transferred from the field, the clams were maintained in incubation ovens until the target water temperature was reached, with the exception of the 18ºC  treatment and control, which were maintained in a cold room. Each manipulated treatment tank consisted of temperatures ranging from 18ºC to 37ºC and left for durations ranging from 2 to 18 hours. After their heat-shock treatment, the clams were transferred to separate seawater tanks maintained at 18ºC, simulating the lowest temperatures at typical transplant sites.

Throughout the treatments and after, the clams were fed and monitored daily for mortality. Fourteen clams were collected approximately one week after their heat-shock treatment to assess the prevalence of QPX infection, which would represent the initial prevalence of QPX in the sample population, since the effects of the heat-shock treatment would not yet have ended a QPX infection in the clams (Dahl et al. 2011). The remaining clams in each treatment group were collected from their seawater tanks and assessed for QPX 10 weeks after their treatment. The clam samples

In the midst of preparing for DNA extraction process by micro pipetting buffer solution in the a homogenized clam sample.


were bled and dissected for homogenization. Once homogenized, the clam samples were labeled and stored in -80ºC freezers for preservation until they were ready for DNA extraction.

Once all the samples were homogenized and ready to be diagnosed for QPX, they were centrifuged, which allowed the separation of plasma from the cell pallet. The DNA of the tissue was then extracted and processed to determine the prevalence of QPX by using a real-time polymerase chain reaction (QPCR) assay (Perrigault et al. 2009). The QPX prevalence rate for the clam samples were recorded and assessed. The prevalence and intensity of the QPX infection were determined by the nanodrop spectrophotometer. The results of the QPCR analysis were then analyzed for statistical significance with ANOVA and a T-test, using Statview.

Data/Results
The results from the 10 different treatments and the controls are shown in Figures 2 and 3. The numbers on Days 1 and 7 and at Week 10 after the heat-shock treatment are the percentage of clams collected during that time period that were QPX-positive when tested under QPCR settings. Because each heat-shock treatment was conducted in two separate tanks, the data for each kind of treatment is divided into A and B.

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Figure 2: (left) This table shows the before prevalence rates of the hard clams in each treatment and tank.

Figure 3: (right) This table shows the after prevalence rates of the hard clams in each treatment and tank.


Figure 2 describes the prevalence of QPX-infected clams collected on Days 1 and 7, broken down by the type of heat-shock treatment and tank, either A or B. The mean column describes the mean number of QPX-infected clams in both Tank A and Tank B for each treatment, respectively. These columns represents the prevalence of QPX infection in the original clam samples, which will be compared to the prevalence of infected clams 10 weeks after their heat-shock treatments.

Figure 3 shows the prevalance of QPX infection in the clams that survived and were collected 10 weeks after their heat shock treatments. It should be noted that Treatment A at 3ºC for two hours does not have any data because all the clams that underwent the treatment died off before the 10-week collection date.

Statistical Analyses
Figure 4 shows a means plot from ANOVA of all the treatments tested, displaying the differences in QPX prevalence levels before and after the heat-shock treatments. The means plot shows explicitly how treatments with a duration of two hours had the greatest declines in prevalence levels. I ran a T-test between the treatments that were two hours long and the treatments that were longer than two hours. Figure 5 shows a highly significant (P-value < .01) difference in disease prevalence levels between treatments of two hours and those that were longer.

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Figure 4: shows a means plot of the difference in the before and after prevalence rates of each treatment


Figure 5: A t-test showing a significant (p=&lt;.01) difference in change in prevalence levels of clams in treatments with durations of 2 hours and in treatment with durations greater than 2 hours.
  t=test for Equality of Means
t df Sig. (2-tailed) Mean Difference Std. Error Difference 95% Confidence Interval of the Difference
Lower Upper
Difference in prevalence after 10 weeks Equal variances assumed 4.695 20 .000 .3793354 .0807942 .2108016 .5478692
2 Hour Treatments 3.765 6.478 .008 .3793354 .1007573 .1371306 .6215402

Discussion
This study provides a comprehensive assessment of favorable conditions for heat-shock implementation. The results show the effects of higher water temperatures on the prevalence of QPX disease: clams in three of the tested treatments showed a decline in disease prevalence. As indicated by Figure 6, treatments at 27ºC for two hours, at 32ºC for two hours, and at 37ºC for two hours showed reductions in disease prevalence. It can also be observed that in a few of the treatments—at 27ºC for eight hours and at 21ºC for all durations—the clams showed an increase in QPX prevalence after 10 weeks. However, the difference was not statistically significant and could be the result of small sample size, as only 14 clams from the original sample were assessed for the rate of QPX infection because of the limited number of hard clams available for this experiment.

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Figure 6: Shows the before and after QPX prevalence rates of the clams in each treatment.


While there was a decline in disease prevalence after treatment at 37ºC for two hours, a careful look at the number of clam mortalities before the 10-week collection period shows that the effectiveness of this treatment is questionable. In Tank A with this treatment, 14 clams died before collection at 10 weeks, showing an alarming 70% mortality rate, and six clams died in Tank B, a 30% mortality rate (Figure 7). These mortality rates were the highest of all the treatments tested. QPX diagnostics from the dead clams show that many of the clams were not heavily infected with QPX and more likely died as a result of intolerable heat stress (Figure 7). The stress on these clams can be substantiated by previous studies that showed an increase in mortality rates and physiological stress in clams exposed to temperatures higher than 32ºC. In addition, it has been shown that clams are more likely to show signs of stress when temperature changes are rapid compared to gradual acclimation (Weber et al.). Because the clams in this experiment were placed into 18ºC tanks shortly after their heat treatments, the clams in the 37ºC water had to withstand the greatest change in temperature in the shortest amount of time, resulting in stress and higher mortality rates.

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Figure 7: shows the mortality rates of each treatment and the proportion the clams that had died off that were QPX negative or positive in each treatment


Treatments at 32ºC for two hours showed a decline in QPX prevalence rates similar to treatments at 27ºC for two hours (Figure 3). Even though 32ºC is significantly warmer and might cause more stress on the clams, there was no significant difference in mortality rates. The discrepancy between my results and previous reports on the physiological effects of high temperatures on clams can be explained by the relatively short duration of the treatments. Previous reports have identified hard clams’ tolerance of temperature stress if it lasts for hours rather than days, as long as the temperatures are within their naturally tolerable limits (Weber et al.). From a pragmatic perspective, treatment at 27ºC is better not only because the temperature is in the clam’s tolerable temperature range but also because it is the range of the clams’ optimal viability (Roegner and Mann). Treatments at 27 ºC reduced QPX prevalence as well as treatments at 32ºC, but the additional stress on the clam was minimal.

Also notable in my study was the effect of treatment duration on QPX prevalence rates. Interestingly, a two-hour treatment was shown to reduce QPX prevalence rates more effectively than longer durations. For example, for treatments at 27ºC, the two-hour treatments showed the greatest decline in prevalence when compared to clams treated for up to 18 hours. Therefore, my study shows that the efficacy of higher temperatures is highest in short durations. The treatments at 27ºC show that both warmer temperatures and the duration of treatment are important to effectively reduce QPX prevalence rates. These results are contrary to previous studies that suggested that keeping clams at warmer temperatures—27ºC and 32ºC—for longer time periods was beneficial in reducing QPX prevalence (Dahl, Thiel and Allam, 2010).

Conclusion
In conclusion, this study demonstrated that using a short-term heat-shock treatment during transplant operations could increase the effectiveness of the QPX mitigation strategy. Along with the establishing that heat-shock treatment is beneficial, this research has shown that the optimal conditions for heat-shock treatment are at 27ºC for two hours. This experiment also showed that the short duration of the heat shock is an important factor in reducing QPX prevalence levels. This study sheds light on how to enhance mitigation strategies for QPX in clams, and introduces a new treatment plan to bolster the efficiency of current mitigation operations.

Further Research
For future investigation, it would be quite interesting to test additional factors in order to substantiate and further understand the true effects of these heat-shock treatments. For example, this study focused on the effects of heat-shock treatments on the prevalence of QPX in the clams. However, a more in-depth study on how treatments affect clam physiology by studying the expression of certain proteins resulting from each treatment would further elucidate which treatment conditions are optimal. The effectiveness of the treatment depends not only on its effectiveness in decreasing QPX prevalence rates but also on avoiding excessive stress on the hard clams undergoing the treatment. Also, for future experiments, a large sample size of hard clams in the experiment would be preferable; if the sample size were more than 30, then its statistical significance would be more certain. 

Acknowledgements
I would like to thank Dr. Bassem Allam, associate professor at Stony Brook University, for his guidance and advice, and for creating this amazing opportunity at his lab. I would also like to thank Kailai Wang, a Ph.D. student at Stony Brook University, for guiding me with this project and sharing all the technical skills necessary to make this project feasible. Finally, I would like to thank Ms. Veronica Ade and Dr. Hendrickson, the science research teachers at my high school, for their guidance and for encouraging me to submit my research to the Young Naturalist Awards. And thanks to Ms. Levin for helping me run a statistical analysis on the data. 

Bibliography
Barnes, D., B. Allam, and K. Gall. (2009). “QPX Disease in Hard Clams.” New York Sea Grant (2003). Retrieved from the World Wide Web. http://www.seagrant.sunysb.edu/seafood/pdfs/QPX-Brochure03.pdf

Dahl, S.F., and B. Allam. “Laboratory transmission studies of QPX disease in the northern quahog (hard clam): Development of an infection procedure.” Journal of Shellfish Research 26(2) (2007): 383-389.

Dahl, S., M. Perrigault, Q. Liu, J. Collier, D. Barnes, and B. Allam. “Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (quahog parasite unknown) disease development: I. Dynamics of QPX  disease.” Journal of Invertebrate Pathology 106 (2011): 314-321.

Dahl, S., J. Thiel, and B. Allam. “Field Performance and QPX Disease Progress in Cultured and Wild-Type Strains of Mercenaria mercenaria in New York Waters.” Journal of Shellfish Research 29.1 (2010): 83-90.

Gast, R.J., et al. “Environmental distribution and persistence of quahog parasite unknown (QPX).” Diseases of Aquatic Organisms 81 (2008): 219–229.

Perrigault, M., S. Dahl, E. Espinosa, L. Gambino, and B. Allam. “Effects of temperature on hard clam (Mercenaria mercenaria) immunity and qpx (quahog parasite unknown) disease development: 11. Defense parameters.” Journal of Invertebrate Pathology 106 (2011): 322-332.

Perrigault, M., Bugge, D., & Allam, B. (2010). “Effect of environmental factors on survival and growth of quahog parasite unknown (QPX) in vitro.” Journal of Invertebrate Pathology 104 (2010): 83-89.

Perrigault, M., and B. Allam. “Cytotoxicity of quahog parasite unknown (QPX) toward hard clam (Mercenaria mercenaria) haemocytes and interactions between different pathogen isolates and host strains.” Parasitology 136.11 (Sep. 2009).

Roegner, G.C., and R. Mann. “Hard Clam: Mercenaria mercenaria.” Maryland Department of Natural Resources.Retrieved from the World Wide Web. http://www.dnr.state.md.us/irc/docs/00000260_05.pdf

Tarnowski, Mitchell. “Fish Facts: Hard Shell Clam.” Maryland Department of Natural Resources. Retrieved from the World Wide Web. http://www.dnr.state.md.us/fisheries/fishfacts/hardshell_clam.asp

Weber, K., L. Sturmer, E. Hoover, and S. Baker. “The Role of Water Temperature in Hard Clam Aquaculture.” University of Florida, IFAS Extension. Retrieved from  the World Wide Web. http://edis.ifas.ufl.edu/fa151

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