Elsevier

Aquatic Toxicology

Volume 97, Issue 4, 10 May 2010, Pages 293-303
Aquatic Toxicology

Effects of brevetoxin exposure on the immune system of loggerhead sea turtles

https://doi.org/10.1016/j.aquatox.2009.12.014Get rights and content

Abstract

Blooms of the toxic dinoflagellate, Karenia brevis, occur almost annually off the Florida coast. These blooms, commonly called “red tides”, produce a group of neurotoxins collectively termed brevetoxins. Many species of sealife, including sea turtles, are severely impacted by brevetoxin exposure. Effects of brevetoxins on immune cells were investigated in rescued loggerhead sea turtles, Caretta caretta, as well as through in vitro experiments using peripheral blood leukocytes (PBL) collected from captive sea turtles. In rescued animals, plasma brevetoxin concentrations were measured using a competitive ELISA. Plasma lysozyme activity was measured using a turbidity assay. Lysozyme activity correlated positively with plasma brevetoxin concentrations. Differential expression of genes affected by brevetoxin exposure was determined using two separate suppression subtractive hybridization experiments. In one experiment, genes from PBL collected from sea turtles rescued from red tide toxin exposure were compared to genes from PBL collected from healthy captive loggerhead sea turtles. In the second experiment, PBL from healthy captive loggerhead sea turtles were exposed to brevetoxin (500 ng PbTx-2/ml) in vitro for 18 h and compared to unexposed PBL. Results from the subtraction hybridization experiment conducted with red tide rescued sea turtle PBL indicated that genes involved in oxidative stress or xenobiotic metabolism were up-regulated. Using quantitative real-time PCR, a greater than 2-fold increase in superoxide dismutase and thioredoxin and greater than 10-fold increase in expression of thiopurine S-methyltransferase were observed. Results from the in vitro subtraction hybridization experiment indicated that genes coding for cytochrome c oxidases were the major up-regulated genes. Using quantitative real-time PCR, a greater than 8-fold increase in expression of β-tubulin and greater than 3-fold increase in expression of ubiquinol were observed. Brevetoxin exposure may have significant implications for immune function in loggerhead sea turtles.

Introduction

The loggerhead sea turtle, Caretta caretta, is protected as a threatened species under the U.S. Endangered Species Act (Pritchard, 1997). These marine turtles inhabit waters off the southwestern coast of Florida, with over 90% of loggerhead sea turtles in the U.S. nesting on Florida beaches (Florida Fish and Wildlife Research Institute, 2008). In general, sea turtles are exposed to many threats in their habitat, including loss of habitat from coastal development, disorientation of hatchlings by beachfront lighting, nest predation, watercraft strikes, incidental take from commercial fishing activities, and marine pollution and debris (U.S. Fish and Wildlife Service, 2009). One of the major threats to the sea turtle population off the southwestern coast of Florida is the frequent, almost annual, occurrence of blooms of the toxic dinoflagellate, Karenia brevis. These blooms are often referred to as “red tide” and produce a suite of cyclic polyether neurotoxins, collectively termed brevetoxins, that result in massive fish kills, large numbers of mortalities in sea turtles and marine mammals, contamination of shellfish, and severe respiratory effects in humans. Beyond mortality, the effects of brevetoxins on sea turtle health are not well-characterized. Although sea turtles are physically robust and able to accommodate severe physical damage, they appear to be surprisingly susceptible to biological and chemical insults (Lutcavage and Lutz, 1997). The effects of many stressors, such as the long-term effects of toxin exposure, are unknown. Sea turtles are exposed to brevetoxins through both inhalation of aerosolized toxins and ingestion of prey items that contain accumulated toxin (Flewelling et al., 2005). It is important to understand the risk that brevetoxin exposure poses to immune function in loggerhead sea turtles as these effects have the potential to affect sea turtle population survival.

Mortality associated with red tide toxin exposure has been documented in many species of sea life inhabiting the southwestern coast of Florida, including marine mammals, sea turtles, fish, and aquatic birds. Although mortality often results, it is frequently not observed until weeks or months after exposure, indicating a sublethal component associated with red tide toxin. Brevetoxin exposure was implicated in the deaths of over 150 manatees during an epizootic in 1996 (Bossart et al., 1998). The death of 107 bottlenose dolphins along the Florida Panhandle in 2004 was attributed to red tide toxin exposure through ingestion of contaminated prey (Flewelling et al., 2005). Loggerhead sea turtles have been impacted by red tide blooms as well, with at least 109 loggerhead sea turtle mortalities attributed to red tide toxins during 2005 (A. Foley, pers. comm.) and at least 70 during 2006 (D. Fauquier, pers. comm.). Other than mortality and general symptoms of lethargy and muscle weakness characteristic of brevetoxicosis, effects of red tide toxin exposure on sea turtle health are not well understood. A major concern is that brevetoxins can impact immune function in species naturally exposed in their habitat. Effects of red tide toxin exposure on immune function in sea life have been reported (Bossart et al., 1998, Benson et al., 1999, Benson et al., 2004, Benson et al., 2005, Walsh et al., 2005, Walsh et al., 2007, Walsh et al., 2009). Brevetoxins have been found within immune cells from a variety of species, including manatees (Bossart et al., 1998), cormorants (Kreuder et al., 2002), and following experimental exposure in laboratory animals (Benson et al., 1999, Benson et al., 2004, Benson et al., 2005). Additional reports indicated a potential inflammatory component associated with brevetoxin exposure in other species (Bossart et al., 1998, Walsh et al., 2007). In other systems, it was also documented that oxidative stress or xenobiotic metabolic pathways were activated in response to brevetoxin (Radwan and Ramsdell, 2006, Walsh et al., 2009). Based on these reports, we hypothesized that brevetoxin exposure may result in inflammatory responses and up-regulation of pathways related to oxidative stress or xenobiotic metabolism in peripheral blood leukocytes of loggerhead sea turtles.

In the study presented here, immune system effects of brevetoxin exposure were examined in both rescued animals and following in vitro treatment of sea turtle PBL from healthy captive animals. To document level of exposure, concentrations of brevetoxins in rescued sea turtle plasma were measured. To identify potential immune system factors affected by brevetoxins in sea turtles, plasma lysozyme activity was assessed in combination with a genomic approach to identify genes differentially regulated by brevetoxins. Using suppressive subtractive hybridization, genes differentially expressed by environmental exposure to brevetoxin in PBL from rescued sea turtles and in vitro exposure of PBL from healthy captive sea turtles to brevetoxin (PbTx-2) were identified. The effects of brevetoxins on loggerhead sea turtle immune function have not yet been addressed in any published study.

Section snippets

Animals

Blood samples from 18 adult loggerhead sea turtles (Caretta caretta) were utilized in various parts of this study (Table 1). All turtles sampled in this study were treated humanely in accordance with protocols approved by Mote Marine Laboratory Institutional Animal Care and Use Committee (MML IACUC Protocol #CW-2) and with required state and federal permits (FFWCC Marine Turtle Permit TP #126). The loggerhead sea turtles were divided into four categories (Table 1): healthy captive (HC; N = 3),

Animals

Rescued sea turtles were identified as suffering from red tide toxicity (brevetoxicosis) by experienced veterinarians at MML's STRH. On rescue, sea turtles displayed symptoms characteristic of brevetoxicosis, such as lethargy and muscle weakness (D. Fauquier, pers. comm.). Suspected red tide toxicity was confirmed by determining levels of red tide toxin in blood (D. Fauquier, pers. comm.).

Plasma brevetoxin levels

Concentrations of brevetoxins in the plasma of rescued, pre-release, and non-red tide exposed loggerhead

Discussion

Loggerhead sea turtles are classified as a threatened species (IUCN, 2008) and inhabit areas where frequent harmful algal blooms occur. Therefore, it is important to understand how marine toxins, specifically brevetoxins, potentially impact the health of loggerhead sea turtles. Sea turtles are long-lived animals, with a comparatively slow metabolic rate (Milton and Lutz, 2003); consequently, the cumulative effect of exposure to stressors such as brevetoxins may result in considerable impact.

Conclusion

The effects of brevetoxin exposure on functional aspects of sea turtle immunity have not yet been addressed in any published study. It is important to understand how harmful algal blooms affect marine life, particularly threatened or endangered species. In the study presented here, even the moderate correlations that were observed with brevetoxin and immune function parameters suggest that loggerhead sea turtles may be sensitive to immunomodulatory effects of brevetoxins. Such alterations in

Conflict of interest

None.

Roles of authors

Catherine J. Walsh is the primary author of this manuscript. She was responsible for project idea, designing experimentation, and overseeing all components of the research presented in this paper. She was also responsible for writing the paper.

Stephanie R. Leggett is a staff biologist working in the laboratory of Dr. Catherine Walsh. She was responsible for conducting brevetoxin ELISA on turtle plasma samples, archiving all samples, isolating sea turtle PBL, isolating RNA, and conducting in

Acknowledgments

This project was funded by Florida Fish and Wildlife Conservation Commission, Extended Red Tide Monitoring (FWC Agreement No. 06125). FWC played no role in study design or in the collection analysis and interpretation of data nor in writing this paper. FWC also played no role in the decision to submit this paper for publication. The authors would like to thank MML Sea Turtle Rehabilitation Hospital staff including C. Manire, E. Anderson, L. Byrd, and D. Fauquier. The authors would also like to

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