Prevalence of the symbiont Lagenophrys callinectes in blue crabs Callinectes sapidus

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1 Prevalence of the symbiont Lagenophrys callinectes in blue crabs Callinectes sapidus Abstract Megan Arias School of Renewable Natural Resources, Louisiana State University Blue crabs (Callinectes sapidus Rathbun 1896) range from Argentina to Nova Scotia tolerating a variety of salinities, temperatures, and water depth depending on the life history stage requirements. Louisiana is currently the largest blue crab fishery in the Gulf of Mexico landing approximately $43.1 million in 2012 out of the Gulf s approximate total $52.9 million (NOAA 2012). These landing values stress the importance of understanding the health of Louisiana s blue crab population because, as for all marine organisms, diseases and parasites can be major threats. Lagenophrys callinectes is a symbiont identified in the Gulf that targets blue crab gills and is locally known as the black gill parasite. To determine prevalence of this symbiont in Louisiana, gills of 587 blue crabs were collected and examined during winter, spring, summer, and fall in 2013 from four sites: Grand Isle, Rockefeller, Lake Pontchartrain, and Cocodrie (near LUMCON facility). Prevalence of L. callinectes was highest at Lake Pontchartrain (98.6%) and lowest at Rockefeller (87.5%). Spring samples collectively resulted in lowest prevalence (85%) of L. callinectes and winter resulted in the highest prevalence (95.83%). Statistical models comparing L. callinectes prevalence per site should include season as a factor and models for prevalence during a season should include site, season, and site-season interaction. The overall high prevalence (92.16%) of L. callinectes for 2013 may seem alarming; however, if high prevalence continues to be recorded, these infestations could be considered normal, even expected. Introduction Blue crabs (Callinectes sapidus) range from Argentina to Nova Scotia and can tolerate a variety of salinities from freshwater to full strength seawater (0 to 32+ ppt). Habitat selection depends on the requirements of each life history stage. The seven larval stages are spent in open ocean waters followed by migration of megalopae to more shallow, less saline estuarine waters (Darnell 1959). Though mating occurs in low salinity river and estuarine systems, adult blue crabs can be found in nearshore to offshore habitats of varying salinities. In Louisiana, blue crabs play an integral role in the ecosystem and economy. In estuarine food webs, this r-selected species functions as predator and prey. Predominant food items vary among blue crabs; they have been described as opportunistic benthic detritivores, primary carnivores, cannibals, and general scavengers; fish, crustaceans, bivalves, vascular plant material, algae, insects, organic debris and foraminiferans are typical diet components (Guillory et al. 2001). As the largest blue crab fishery in the Gulf of Mexico, Louisiana landed approximately $43.1 million of blue crabs (dockside value) in 2012 out of the Gulf s approximate total $52.9 million (NOAA 2012). Florida, the next largest Gulf blue crab fishery, landed about $38 million less than Louisiana in These amounts stress the importance of understanding the health of Louisiana s blue crab population because, as for all marine organisms, diseases and parasites can be major threats.

2 An important system in the blue crab often targeted by diseases and parasites is the respiratory system, specifically the gills. The blue crab has eight gills that function in respiration, ion regulation and excretion. One gill symbiont, Lagenophrys callinectes, has been identified in the Gulf, though prevalence remains unknown. L. callinectes, locally known as black gill parasite, is an ectocommensal loricate ciliate that shows distinct host specificity and peaks in the summer months (Guillory 2001). It is recognizable by its protective yellowish lorica (Fig.1), or shell, which cements to flat gill lamellae surfaces. This ciliate feeds on bacteria and derives no nutrition from its host. However, blue crabs can be harmed or killed by this symbiont as it can potentially interfere with gill function, such as respiration and excretion (Shields and Overstreet 2007). If heavily infested, the crab can be asphyxiated. Mortalities due to L. callinectes infestation are typically observed in crab holding or shedding tanks (Couch 1966). The ciliate can also cause reproductive and foraging limitations with heavy infestation (Schuwerack et al. 2001). Blue crab growth is determinate and occurs during molting, or ecdysis. In early life stages, crabs molt every few days. As crab size increases, molt frequency decreases eventually leading to a terminal molt which is assumed to be about the 25 th molt (Guillory 2001). When the crab molts, the ciliate is shed with the old shell, ending the symbiosis. These ciliates may respond to upcoming molts by producing reproductive stages that could re-infest fresh gill cuticle (Shields and Overstreet 2007). Figure 1 Lagenophrys callinectes forms dark circular structures (loricae) on this blue crab gill. The majority of research on blue crab disease and parasite prevalence focuses on outbreaks in specific bays (e.g., Chesapeake Bay) and areas along the Atlantic Coast where widespread epizootics have been correlated to large declines in blue crab landings (Shields 2003). Similar research is limited and sporadic for the Gulf of Mexico. Though L. callinectes appears ubiquitous, Atlantic Coast data may not be transferrable to the Gulf Coast due to different water conditions. Without consistent, population specific prevalence data in the Gulf, determining a correlation between increased disease and parasite prevalence and decreased commercial landings is nearly impossible. Recording the prevalence of L. callinectes in Louisiana blue crabs will create preliminary data to be used in comparisons with future detections of L. callinectes.

3 Methods Blue crab collection Blue crabs (Callinectes sapidus) with a carapace length of 110 mm or greater were collected from four sample sites in Louisiana (Fig. 2): Lake Pontchartrain, Cocodrie near the LUMCON facility, Grand Isle, and Rockefeller Wildlife Management Area. All crabs were collected in During the summer, 60 crabs were collected from each site totaling 240; 244 were collected during the fall: 60 each from Lake Pontchartrain, LUMCON, and Grand Isle and 64 from Rockefeller; 80 were collected during the spring: 24 from Lake Pontchartrain, 13 from Grand Isle, and 43 from Rockefeller; 24 crabs were collected during the winter: 7 from Grand Isle and 17 from Rockefeller. Techniques used for capturing the blue crabs included seining, baited line, trawling, and baited traps. Figure 2 Map of southern Louisiana with locations pinpointed (red circles). Gill tissue Gill tissue was removed during dissection (Fig. 3) and stored at -20 C until further analysis. To avoid ciliate loss, which can occur during standard histological tissue fixation and staining, only frozen tissue samples were examined. Figure 3. This sample was collected at Rockefeller in January Arrows point to gills for collection.

4 Diagnostic techniques Coloration of gills was noted for some, but not all, crabs placing them into two categories: 1) discolored (Fig. 4) and 2) non-discolored (Fig. 5). Light microscopy of both discolored and nondiscolored gill tissue was used to determine presence or absence of the L. callinectes lorica. Presence of the lorica was recorded regardless of whether it was broken or occupied. Figure 4. Discolored black gill Figure 5. Normal colored gill Statistical methods Following the completion of gill diagnoses, prevalence frequencies within and between sites was determined. In R-Studio (R 2013), a generalized linear model (GLM) was run with different factors of interest (site and season) to analyze significant models. An analysis of deviance with a chi-square test statistic was used to analyze model fit. Alpha was set at 0.05 for all statistical analyses. Results A total of 587 gill samples were examined and presence or absence of L. callinectes was recorded. Positive and negative recordings of L. callinectes were 541 and 46, respectively (see Table 1 and Table 2). Springtime, with 80 examined crabs, resulted in the lowest recorded prevalence. Lake Pontchartrain exhibited the highest overall prevalence (98.6%) of L. callinectes whereas Rockefeller showed the lowest overall prevalence (87.5%). Grand Isle during the spring had a notably low prevalence of 23%, the lowest of any site during any month (Fig. 6b.). Excluding spring Grand Isle results, at Rockefeller and Lake Pontchartrain, overall prevalence of L. callinectes during the spring was 97%. Prevalence for each season ranged from 85% to 95.83% (Table 1). Individual site prevalence ranged from 87.5% to 98.6% (Table 2). Overall there was 92.16% prevalence for the sampling year. Table. 1 Results of blue crab gill examinations per season Season Positive Negative Total Prevalence (%) Winter Spring Summer Fall Total

5 Table. 2 Results of blue crab gill examinations per site Site Positive Negative Total Prevalence (%) Rockefeller Grand Isle LUMCON Pontchartrain Total Fig. 6 a-d. a) Grand Isle prevalence during winter, spring, summer, and fall. b) Rockefeller prevalence during winter, spring, summer, and fall. c) Lake Pontchartrain prevalence during spring, summer, and fall. d) LUMCON prevalence during summer and fall. For Grand Isle, the GLM model with season as an explanatory variable for presence or absence of L. callinectes resulted in a p-value of p <0.001 for spring. The Grand Isle model including season had an AIC of and excluding season had a larger AIC of (Deviance = , p <0.001). Rockefeller p-values during the fall and summer were p <0.001 and p = , respectively. A model including season as an explanatory variable for prevalence at Rockefeller had an AIC of and the model excluding season had an AIC of (Deviance = 9.85, p = ). P-values for each season at Lake Pontchartrain were all p > The AIC for the model including season at Lake Pontchartrain was and for excluding season was (Deviance = , p = ). Each season at LUMCON had a p-value of p > The AIC for the LUMCON model including season as an explanatory variable for prevalence was and for excluding season was (Deviance = ,

6 p = ). For winter, the GLM model with site as an explanatory variable for presence or absence of L. callinectes resulted in p-values for each site (Rockefeller and Grand Isle) of p >0.05. P-values for Grand Isle and Lake Pontchartrain during the spring were p >0.05 and Rockefeller was p < Summer and fall models with site as an explanatory variable for presence or absence of the ciliate resulted in p-values of p >0.05 for each site. Discussion and Management Implications Lagenophrys callinectes was present at each site for all four seasons. There was a significant seasonal effect on L. callinectes prevalence at Rockefeller during summer and fall. Sample sizes at Rockefeller for winter (17) and spring (43) were smaller than summer (60) and fall (64). Significant seasonal effects could change or lose significance with increased samples collected over a longer period of time. In particular, summer at this site could be the result of an odd year since this ciliate s peak prevalence is typically during the summer. Further sampling can determine if this was a summer of high, low, or average prevalence at Rockefeller. Lake Pontchartrain was successfully sampled during the spring, summer, and fall. Including these seasons, the GLM model resulted in no significant seasonal effect. Winter sampling could significantly affect prevalence at this site, though it is unlikely based on the similar high prevalence shared by summer, spring, and fall. Grand Isle s low L. callinectes prevalence during the spring is hypothesized to be attributable to the crab s molting rate. As water temperatures warm into the spring, molting rates increase significantly then continue at a slower rate during warmer summer temperatures (Guillory et al. 2001). Spring resulted in the lowest overall prevalence of the ciliate, influenced by the low springtime value at Grand Isle. Grand Isle also provided the lowest number of samples during spring, 13 out of 80 total samples, possibly lowering the importance of spring s overall prevalence. This small sample size also could have influenced the significant seasonal effect at Grand Isle. For LUMCON, the GLM model with season as a variable suggested there was no significant seasonal effect. Sampling at this site was successful only during summer and fall. When comparing the LUMCON models including season and excluding season as a variable for L. callinectes prevalence, the model including season was a better fit. With more seasons of data, it is possible that season could explain variation in prevalence at LUMCON. Frequent molting in the spring allows for shedding of the ciliate. At that point, crabs are susceptible to re-infestation; noticeable in high summer prevalence when molting is less frequent. Crabs also molt less frequently in cold winter temperatures. Due to slow molting rates, L. callinectes should have highest prevalence during the summer and winter compared to spring and fall. Summer and winter did have a higher prevalence of the ciliate than spring; however, fall has a higher prevalence than summer. Models for both summer and fall suggested no site variation. However, repeated sampling over more years could yield results favoring higher summer and winter prevalence. Based on the data from this study, models comparing L. callinectes prevalence at a site should include season as a factor. Models for prevalence during a season should include site, season, and site-season interaction.

7 The overall high prevalence of a potentially, albeit unlikely, life-threatening blue crab parasite may seem alarming. If high prevalence continues to be recorded, Lagenophrys callinectes infestations could be considered normal, even expected. They seem to cause little noticeable harm to the crabs, something that may be seen more during seasons of slower molting rates, and have been studied less than incontestably threatening diseases and parasites. Considering the blue crab s heavy influence on Louisiana s commercial fishery, research may be needed to determine mortality caused by L. callinectes and the impact an increased prevalence would have on the economy, if any. Future research could also consider the effect of various infestation intensity levels on gill function and color and whether this parasite warrants the name black gill. Acknowledgements Funding for this research was provided by the Louisiana Sea Grant Undergraduate Research Opportunities Program. I would like to extend a special thank you to Dr. Julie Anderson, Holly Rogers, and Nikki Anderson for providing invaluable assistance and guidance along the way.

8 Literature cited Couch, J. A Two peritrichous ciliates from the gills of the blue crab. Chesapeake Science 7: Darnell, R. M Studies of the life history of the blue crab (Callinectes sapidus Rathbun) in Louisiana waters. Transactions of the American Fisheries Society 88: Guillory, V., H. Perry, P. Steele, T. Wagner, W. Keithly, B. Pellegrin, J. Petterson, T. Floyd, B. Buckson, L. Hartman, E. Holder, and C. Moss The blue crab fishery of the Gulf of Mexico, United States: A regional management plan. No. 96. Gulf States Marine Fisheries Commission. NOAA Fisheries: Office of Science and Technology. Annual Commercial Landing Statistics. NOAA, Silver Springs Pelton, Tom and Bill Goldsborough. Chesapeake Bay Foundation Saving a National Treasure. Bad water and the decline of blue crabs in the Chesapeake Bay. Chesapeake Bay Foundation, n.d. Web. 10 Dec < R Core Team R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria < Schuwerack, P. M. M., J. W. Lewis, and P. W. Jones Pathological and physiological changes in the South African freshwater crab Potamonautes warreni Calman induced by microbial gill infestations. Journal of Invertebrate Pathology 77: Shields, J. D Research priorities for diseases of the blue crab Callinectes sapidus. Bulletin of Marine Science 72: Shields, J. D. and R. M. Overstreet Diseases, Parasites, and Other Symbionts. Pages in V. S. Kennedy and L. E. Cronin, editors. The Blue Crab: Callinectes sapidus. Maryland Sea Grant College, College Park.