Can Predator-Induced Stress be Detected in the Protein Expression Profile of the Blue Crab (Callinectes sapidus)?

MaryRose K. Hall, Sarah E. Beland, Daphne N. Pariser, Terri J. Seron Department of Natural Sciences, Flagler College, St. Augustine Florida

The proteins of interest are not limited to myosin, hemocyanin and polymeric immunoglobulin receptor but also glyceraldehyde-3-phosphate dehydrogenase (GAPDH), projectin, and smooth endoplasmic reticulum calcium ATPase (SERCA), among others that were expressing proteins related to stress. Three key physiological traits that are affected by stress, and potentially predation, are heat shock protein 70 (Hsp 70), energy storage, and oxidative stress.5

It seems that the proteins are related to flight muscle response, cellular growth, or an immune response. Meaning that the crabs were possibly expressing these proteins more for protective measures during predation.

Also, it seems that there is a difference in the proteins present and abundance through time. It is possible that in the preliminary 30-minute interaction with the alligator that the crabs needed proteins that would allow them to have a quick response (to flee) and protect against infection, whereas after 150-minutes it seems that there is more of a long-term response (repair-like mechanisms that are occurring). The results from this experiment exemplify that prey are affected by predation on a molecular level. Organisms will change behavior to avoid predation and in the case of some fish they will sacrifice growth and reproduction, so predation is then a function of the presence of the predator and the behavior of the prey.2 Not only do predators affect the behavior and physiology of prey organisms, but also apex predators can have top-down control on ecosystems.6 Nifong (2013) recently confirmed that apex predators directly impact their prey by affecting density, behavior, and foraging. Certain predators can largely contribute to trophic cascading in ecosystems.7

Even though these proteins were being produced at the time of the predation, there exact purpose is unclear. It is important to understand the stress related reaction such as lowered immune response and protein expression levels, since they are an important food source and are important to marsh ecosystems. Furthermore, this can potentially have an affect on trophic cascading. If the crabs are more susceptible to infection or death after exposure to predation then this could result in negative effects for population numbers.

250 kDa

150

100

75

50

37

25

20

1510

Hemocyanin

Myosin ImmunoglobulinReceptor

Name Size Function SpeciesMyosin 257 kDA a motor and transfer protein Water Flea

(Daphnia pulex)

Hemocyanin 77 kDA carries oxygen to the blood, and help fight off infections

Blue Crab (Callinectes sapidus)

Polymeric Immunoglobulin Receptor

83 kDA found in muscle and connective tissues, and will help fight off infection in those areas

Sumatran Orangutan (Pongo abeii)

1 2 3 4

Stress?Disease?

5

INTRODUCTION RESULTS DISCUSSION

Can the stress induced by the presence of a predator be measured? 

Predation can directly lower the abundance of prey through consumption but can also indirectly lower abundance by inducing stress which may lead to disease and subsequent early death. Predation has also previously been shown to reduce prey survival by reducing habitat availability and altering trophic structures.2

The blue crab is both economically and environmentally important. Stress in blue crabs has been implicated in leading to bacterial infections such as pepper disease. Infections like these greatly reduce the marketability of the crabs and therefore decrease profits. Blue crabs are a main predator of snails and therefore vital to a healthy, balanced marshland ecosystem. Snails have been shown to decimate marsh grasses thereby increasing erosion.3

Stress in blue crabs can result from fluctuations in the physical environment such as oxygen levels, water temperature, or salinity. However, in the local St. Augustine, Florida area, blue crabs can be found in freshwater rivers and in the open ocean, as well as in the estuaries in between. This species is therefore highly adapted and tolerant of changing physical conditions. Another potential form of stress on blue crabs is predation. Recently a new predator has been identified, which may play an additional role in the stress of blue crabs. The american alligator has been found to journey from freshwater inland ponds to the estuaries and feed on blue crabs. Presumably, the blue crab will have a markedly different response to dealing with the stress of having a predatory alligator in its presence than it does to the stress of quickly changing physical parameters of their surrounding water habitat.

Previous studies have shown that prey can experience an increase in cellular respiration due to the increase in energy expenditure during a predation event.5 Similarly, an increased metabolism has been shown to increase the production of stress proteins.8 This study has limited the type of stress to only predator- induced stress by use of a controlled environment. Studying stress on a molecular level will determine precisely which genes are affected by predator-induced stress and shed light on the physiological pathways that are coupled to stress.

METHODSSixty adult blue crabs were utilized as well as two American alligators. The

blue crabs were exposed to the alligators for 30 minutes or 150 minutes.

• After the allotted time the crabs were removed from the tanks, and tissue was taken immediately from the right claw.

• The tissue was immediately put into an SDS solution and frozen on dry ice.• Gel electrophoresis was used to separate and visualize any differences in the

expressed proteins while mass spectrometry was used to identify the specific proteins that were expressed during predation stress.

• Each sample was standardized to a total protein concentration of 0.9 mg/mL• 25µl were loaded into each well of the 4-15% Mini-Protean Polyacrylamide Protein

Gel (Bio-Rad).• Gel electrophoresis was performed for 30 minutes at 200 volts.

Contact Daphne Pariser for further questions at: DP1666@NYUMC.ORG

Acknowledgements: James Nifong, Mark Rowden , JohnWooldridge, Greg Territo, Barbara Blonder, Brandon Storm, Wayne Riggs, Flagler College Undergraduate Dissemination Research Grant

REFERENCES1. Asano, M., & Komiyama, K. (2011). Polymeric immunoglobulin receptor.Journal of oral science, 53(2). 2. Bax, N. J. "The significance and prediction of predation in marine fisheries." ICES Journal of Marine Science: Journal du Conseil 55.6 (1998): 997-1030. 3. Bax, N. J. "The significance and prediction of predation in marine fisheries." ICES Journal of Marine Science: Journal du Conseil 55.6 (1998): 997-1030. ; Terwilliger, Nora B. "Hemocyanins and the immune response: defense against the dark arts." Integrative and comparative biology 47.4 (2007): 662-665.; Nifong, James C., and Brian R. Silliman. "Impacts of a large-bodied, apex predator (< i> Alligator mississippiensis</i> Daudin 1801) on salt marsh food webs." Journal of Experimental Marine Biology and Ecology 440 (2013): 185-191., 4. Bell, G. W., Eggleston, D. B., & Noga, E. J. (2010). Molecular keys unlock the mysteries of variable survival responses of blue crabs to hypoxia. Oecologia,163(1), 57-68.,5. Janssens, L., & Stoks, R. (2013). Predation risk causes oxidative damage in prey. Biology letters, 9(4), 201303506. Leopold, A. (1949). The Land Ethic, in a Sand County Almanac, and Sketches Here and There7. Silliman, Brian Reed, and Mark D. Bertness. "A trophic cascade regulates salt marsh primary production." Proceedings of the national Academy of Sciences 99.16 (2002): 10500-10505. 8. Slos, Stefanie, and Robby Stoks. "Predation risk induces stress proteins and reduces antioxidant defense." Functional Ecology 22.4 (2008): 637-642., 9. Zhang, X., Huang, C., & Qin, Q. (2004). Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon. Antiviral research, 61(2), 93-99. , 10. Zhang, J. R., Mostov, K. E., Lamm, M. E., Nanno, M., Shimida, S. I., Ohwaki, M., & Tuomanen, E. (2000). The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. Cell,102(6), 827-837.  

1. No alligator 30 min, 2. Alligator 30 min, 3. No alligator 150 min, 4. Alligator 150min, 5. Standard

Bright blue bands are indicative of the proteins being expressed in the blue crab’s claw tissue. The above three proteins, hemocyanin, polymeric immunoglobulin receptor, and myosin, were verified by mass spectrometry. The literature suggests that these proteins play a role in stress.

When comparing myosin at 257 kDa, it appears that in the alligator to no alligator, there are more proteins in a crab exposed to the alligator. Also indicated by mass spectrometer data. This potential finding could suggest for crabs to have a need for up regulation in myosin. One of the functions of myosin is a fight or flight response and it has to do with muscle contraction and motor movement. To further verify the findings quantification is needed.

Data also indicates that hemocyanin is up regulated in the alligator condition. In the 150min alligator condition there is a larger increase in the hemocyanin than the 150min no alligator. There are more proteins being expressed in the 150 than in the 30min alligator experiment. During up regulation oxygen to the blood is needed to help.8 When these conditions become unfavorable (high salinity and low DO) for the blue crab the amount of Hcy concentration is increased, which raises the carrying capacity of O2 in their hemolymph .4 It has been shown that Hcy has non-specific antiviral properties and there seems no cytotoxicity against host cells.9

Polymeric immunoglobulin receptor is a protein in the immunoglobulin super family. This family of proteins are involved in recognition, adhesion and the binding processes in the cell. 10 Polymeric immunoglobulin receptor is directly involved with the immune system. It is produced by intestinal epithelial cells and it has a protective effect that can help prevent infections in the body. 1 Polymeric immunoglobulin receptor can prevent harmful infections from getting into certain parts of the body, which is helpful after a stressful situation because an organism is more prone to infections after being exposed to lots of stress.