Loading ...
Terrapin ...
Other
20
0
Try Now
Log In
Pricing
<p>lable at ScienceDirect Toxicon 129 (2017) 36e43 Contents lists avai Toxicon journal homepage: www.elsevier.com/locate/toxicon The role of a PSP-producing Alexandrium bloom in an unprecedented diamondback terrapin (Malaclemys terrapin) mortality event in Flanders Bay, New York, USA Theresa K. Hattenrath-Lehmann a, Robert J. Ossiboff b, 1, Craig A. Burnell c, Carlton D. Rauschenberg c, Kevin Hynes d, Russell L. Burke e, Elizabeth M. Bunting b, Kim Durham f, Christopher J. Gobler a, * a Stony Brook University, School of Marine and Atmospheric Sciences, Southampton, NY, 11968, USA b Cornell University, College of Veterinary Medicine, Department of Population Medicine and Diagnostic Sciences, Ithaca, NY, 14853, USA c Bigelow Analytical Services, Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, 04544, USA d New York State Department of Environmental Conservation, Wildlife Health Unit, Delmar, NY, 12054, USA e Hofstra University, Department of Biology, Hempstead, NY, 11549, USA f Riverhead Foundation for Marine Research and Preservation, Riverhead, NY, 11901, USA a r t i c l e i n f o Article history: Received 11 November 2016 Received in revised form 3 February 2017 Accepted 11 February 2017 Available online 14 February 2017 Keywords: Alexandrium Diamondback terrapins PST Saxitoxin Turtle Chelonian * Corresponding author. E-mail address: christopher.gobler@stonybrook.ed 1 Present address: Zoological Pathology Program, Co University of Illinois, Brookfield, IL, USA. http://dx.doi.org/10.1016/j.toxicon.2017.02.006 0041-0101/© 2017 Elsevier Ltd. All rights reserved. a b s t r a c t Diamondback terrapins (Malaclemys terrapin) are a threatened or endangered species in much of their range along the U.S. Atlantic and Gulf coasts. Over an approximately three-week period from late April to mid-May 2015, hundreds of adult diamondback terrapins were found dead on the shores of Flanders Bay, Long Island, New York, USA. Concurrent with the mortality event, elevated densities of the paralytic shellfish toxin (PST)-producing dinoflagellate, Alexandrium fundyense (>104 cells L1) and high levels of PST in bivalves (maximal levels ¼ 540 mg STX eq. 100 g1 shellfish tissue) were observed in the Flanders Bay region, resulting in shellfish bed closures in regional tributaries. Gross and histologic postmortem examinations of terrapins revealed no physical trauma to individuals or a common, underlying disease process to explain the deaths. PST compounds (0.2e12.5 mg STX eq. 100 g1) were present in various M. terrapin tissues collected over the duration of the mortality event. High-throughput sequencing revealed that the ribbed mussel (Geukensia demissa, a PST vector) was present in the gastrointestinal tracks of all terrapin samples tested. While the potential of PST to cause mortality in chelonians has not been well-characterized, in the absence of other significant findings from necropsies and pathological analyses, we provide evidence that PST in shellfish was likely high enough to cause or contribute to the mortality in these small (<2.0 kg) animals. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction Several genera of phytoplankton can produce a variety of bio- toxins (i.e. saxitoxin, domoic acid, brevetoxins, microcystins) that can have serious health, ecosystem, and economic impacts (Anderson et al., 2008, 2012, 2002; Hoagland et al., 2002; Sunda et al., 2006; Van Dolah, 2000). Beyond the well-established ef- fects these harmful algal blooms (HABs) and their toxins can have u (C.J. Gobler). llege of Veterinary Medicine, on humans via shellfish poisoning events (Anderson et al., 2012; Van Dolah, 2000), many of these toxins are transferred through the food chain and thus can affect a variety of wildlife including fish (Landsberg et al., 2014), marine mammals (Geraci et al., 1989; Lefebvre et al., 2016; Miller et al., 2010; Van Dolah et al., 2003), seabirds (Shearn-Bochsler et al., 2014; Shumway et al., 2003), and sea turtles (Amaya et al., 2014; Capper et al., 2013; Fauquier et al., 2013; Licea et al., 2008). Paralytic shellfish toxins (PST) are composed of more than 20 different saxitoxin (STX) analogs (Rourke et al., 2008), a particularly potent class of algal toxins. Saxitoxin and associated analogs have a long history of causing seabird deaths (Shearn-Bochsler et al., 2014; Shumway et al., 2003) as well as marine mammal strandings and Fig. 1. Locations of Alexandrium sampling sites and terrapin collection sites. Black star ¼ Meetinghouse Creek, White star ¼ James Creek. Black diamond ¼ Terrapin sample CU1. Gray diamond ¼ Terrapin sample SB1. White diamond ¼ terrapin sample CU2. Black circle ¼ all other terrapin samples. Dead terrapins were found along the north and south shore of western Flanders Bay during late April and early May 2015. T.K. Hattenrath-Lehmann et al. / Toxicon 129 (2017) 36e43 37 mortalities (Geraci et al., 1989; Lefebvre et al., 2016; Van Dolah et al., 2003). Globally, dinoflagellates in the genus Alexandrium are the primary causative species of paralytic shellfish poisoning (PSP; Landsberg et al., 2014) although since the 1970s there have been a series of sea turtle deaths attributed to STX poisoning from dinoflagellate blooms caused by Pyrodinium spp. (Amaya et al., 2014; Licea et al., 2008; Maclean, 1975). To our knowledge, there have been no reports of STX poisoning in turtle species inhabiting freshwater or brackish environments. Diamondback terrapins (Malaclemys terrapin) are medium-sized emydid turtles with strong sexual dimorphism (carapace length of adult females to 23 cm, adult males to 14 cm) that inhabit brackish water habitats, marshes, and mangroves along the U.S. Atlantic and Gulf coasts (Ernst and Lovich, 2009). M. terrapin was heavily har- vested for food during the 19th and early 20th centuries (Schaffer et al., 2008) and more recently has suffered population declines from habitat loss, road mortality, and as by-catch in crab fishing gear (Bishop, 1983; Dorcas et al., 2007; Grosse et al., 2011; Ner and Burke, 2008; Roosenburg et al., 1997; Szerlag and McRobert, 2006). Unlike sea turtles, terrapins are more sedentary, showing strong fidelity to foraging, nesting, and overwintering habitats generally spanning no more than one km (Baldwin et al., 2005; Tucker et al., 2001). Terrapins eat a variety of marine invertebrates including hard shelled prey such as salt marsh periwinkles (Littorina irrorata; Tucker et al., 1995), Atlantic ribbed mussels (Geukensia demissa), and blue crabs (Callininectes sapidus) (Erazmus, 2012; Petrochic, 2009). Given that terrapins consume shellfish, exposure to STX is plausible via these known PST vectors. To date, no study has demonstrated the effects of- or the lethal dose (LD50) of- PSTs in terrapins or any other reptile. Here we report on aM. terrapinmortality event that occurred in Flanders Bay, NY, USA, that coincided with Alexandrium blooms in this system. This study presents Alexandrium bloom dynamics, toxicity in shellfish, and a description of terrapin deaths. Subsets of dead terrapins from different stages of the mortality event were necropsied, histologically examined, and various tissues were analyzed for the presence of PST. High-throughput sequencing of DNA isolated from intestinal contents was used to assess prey consumed by the terrapins. Additionally, we present a series of dose-response scenarios based on the range of known lethal doses for vertebrates. 2. Materials and methods 2.1. Alexandrium monitoring Field samples were collected on a weekly basis from April through June during 2015. Samples were collected from Meeting- house Creek (4056012.600N 7237006.300W) and James Creek (4059010.700N 7232005.600W), both tidal tributaries located in the Peconic Estuary, NY, USA (Fig. 1). Concentrated water samples were made by sieving 2 L of subsurface water through a 200 mmmesh (to eliminate large zooplankton) and then onto a 20 mm sieve that was backwashed into a 15 mL centrifuge tube. Alexandrium fundyense densities were enumerated using a highly sensitive molecular probe procedure described by Anderson et al. (2005). Briefly, ali- quots of phytoplankton concentrates (formalin and then methanol preserved) were hybridized with an oligonucleotide probe specific for the NA1 North American (Group I, recently revised as A. fundyense (John et al., 2014)) ribotype Alexandrium fundyense/ catenella/tamarense with Cy3 dye conjugated to the 50 terminus (50-/5Cy3/AGT GCA ACA CTC CCA CCA-30). Cells were enumerated using a Nikon epifluorescence microscope with a Cy3™ filter set (Anderson et al., 2005) as described in Hattenrath et al. (2010). Shellfish toxicity data was supplied by the New York State Department of Environmental Conservation (NYSDEC). Briefly, hundreds of bluemussels (Mytilus edulis) were collected from Stony Brook Harbor, NY, USA, where Alexandrium and PSP have never been detected (Hattenrath-Lehmann, Gobler, NYSDEC, unpub.). Mussels were placed in mesh bags in aliquots of 15 mussels per bag and were deployed within Meetinghouse Creek and James Creek in early April of 2015. Mussels were collected weekly from each site, shucked of their meat, and prepared for PST analysis using standard techniques (AOAC, 1990). Toxin levels in shellfish were quantified using standard mouse bioassays (AOAC, 1990) performed by FDA approved laboratories (NYSDEC lab and Resource Access Interna- tional, LLC, ME, USA). 2.2. Diamondback terrapin postmortem examinations and ranavirus testing Complete postmortem examinations were performed on 12 carcasses submitted to the New York State Veterinary Diagnostic Laboratory at Cornell University (Ithaca, NY, USA) and on three carcasses submitted to the New York State Department of Envi- ronmental Conservation Wildlife Health Unit at the Wildlife Re- sources Center (Delmar, NY, USA). Sample size during this study was based on the availability of carcasses and their condition upon collection. Samples of fresh liver, kidney and heart were collected and archived frozen (20 C); samples of gastrointestinal contents were collected and archived frozen (20 C). The choana and cloaca were swabbed with a single rayon-tipped, plastic shaft, fine tip swab (Medical Wire and Equipment, Wiltshire, England) and swabs were archived frozen (20 C). A complete set of tissues was collected, fixed and stored in 10% neutral buffered formalin at room temperature, and processed routinely for histologic examination. Paraffin sections were cut at 5 mm and stained with hematoxylin and eosin (HE). Choanal/cloacal swabs and gastrointestinal con- tents (when present) were collected from an additional 18 car- casses in poor postmortem condition and archived frozen (20 C). An additional three gross postmortem examinations were con- ducted by the Riverhead Foundation for Marine Research and T.K. Hattenrath-Lehmann et al. / Toxicon 129 (2017) 36e43 38 Preservation (Riverhead, NY, USA); samples of kidney, heart, liver and gastrointestinal contents from one, freshly killed individual were archived frozen (20 C). Swab nucleic acid extractionwas performedwith Prepman Ultra (Applied Biosystems, Foster City, California, USA) per the manu- facturer's recommendations. Extracts were diluted 1:10 in molec- ular grade water. Quantification of extracted nucleic acids was performed on a subset of samples using Qubit fluorometric quan- tification (Invitrogen, Carlsbad, California, USA). Nucleic acid ex- tracts were screened for ranavirus using a previously published hydrolysis probe quantitative polymerase chain reaction (qPCR) targeting a portion of the viral major capsid protein (Pallister et al., 2007). Briefly, qPCR reactions were composed of sense (50-CTC ATC GTT CTG GCC ATC AA-30) and antisense primers (50-TCC CAT CGA GCCGTT CA-30), a Taqman hydrolysis probe (50-/6FAM/CAC AAC ATT ATC CGC ATC/MGBNFQ/e30), mixed with TaqMan Fast-Virus 1-step Master Mix (Life Technologies, Carlsbad, CA, USA), TaqMan Exoge- nous Internal Positive Control (Life Technologies, Carlsbad, CA, USA), and 2 ml choanal-cloacal swab extracts. Plates were run on a StepOne Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) under the following conditions: 1 cycle at 50 C for 5 min; 1 cycle at 95 C for 20 s; 40 cycles of 95 C for 3 s followed by 60 C for 30 s and a data collection point. A commercially synthesized ssDNA oligomer including ranavirus primer and probe binding sites (In- tegrated DNA Technologies, Coralville, IA, USA) and a positive clinical sample extract from an eastern box turtle (Terrapene car- olina carolina) were used as positive controls. 2.3. Paralytic shellfish toxin measurements in diamondback terrapin tissues Paralytic shellfish toxin (PST) concentrations in terrapin tissues were investigated at Bigelow Analytical Services, Bigelow Labora- tory for Ocean Sciences (East Boothbay, Maine, USA). Lyophilized tissue samples were reconstituted in deionizedwater and extracted with 0.1 M acetic acid (Bricelj et al., 1991) using 1.65 mL g1 wet tissue and a Brinkmann Instruments Polytron homogenizer. Sam- ples were centrifuged at 4000g for 20 min at 5 C. Supernatants were loaded onto preconditioned Waters Sep-Pak C-18 Light single use cartridges and eluted by syringe pressure. Samples were analyzed via high performance liquid chromatography with post- column oxidation and fluorescence detection (HPLC-PCOX-FLD; Van de Riet et al., 2011). The total toxicity for each sample was calculated from the sum of the individual saxitoxin-normalized toxicities for all PSTs present (Van de Riet et al., 2009). Each sample was analyzed for: 1) carbamate (GTX4, GTX1, GTX3, GTX2, NEO, and STX), decarbamoyl (dcGTX3, dcGTX2, and dcSTX) and N-sulfocarbamoyl (GTX5) PST congeners; and 2) two N-sulfocarbamoyl congeners (C1 and C2). Certified calibration standards (Institute for Marine Biosciences, National Research Council of Canada) were used to identify and quantify the individual compounds. Checks were conducted to ensure identified peaks were not due to autofluorescence by non- PST compounds. The limit of quantitation (LOQ) for this approach is approximately 0.1 mg STX eq 100 g1, based on values from the analysis of terrapin tissues that were not exposed to saxitoxin. 2.4. Sequencing COI gene of diamondback terrapin intestinal contents Intestinal contents from three terrapins were extracted using the Qiagen Dneasy Blood and Tissue kit (Leray et al., 2013). Aliquots of 25 mg were lysed at 56 C for 1.5 h and extractions were con- ducted according to the manufacturers' tissue protocol. Following extraction, double-stranded DNA was quantified on a Qubit® fluo- rometer using a dsDNA BR (Broad-Range) Assay kit (Qubit®). All samples were normalized and sent to Molecular Research Labs (Shallowater, TX, USA) for amplicon sequencing. The mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified using miCOIintF and jgHCO2198 (50-TAI ACY TCI GGR TGI CCR AAR AAY CA-30) degenerate primers from Leray et al. (2013). The forward primer, however, was modified to allow for amplification of local shellfish species (miCOIintF modified: 50-GGW RSD GGR TGR ACW MTT TAY CCK CC-30), resulting in an ~300 bp product. In addition, annealing blocking primers were created to prevent the amplifi- cation of predator-specific DNA (M. terrapin) as to not mask the prey DNA signal (Leray et al., 2013). The blocking primer (50- TAY CCYCCATTAGCC GGA AAC CTA/3SpC3/-30) was created to anneal to the forward universal primer, was modified at the 30 end with a Spacer C3 CPG to prevent elongation, and was added at 10 the concentration of the universal primers (Leray et al., 2013). For each sample, an identifying barcode was placed on the forward primer and a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) was performed. The following PCR conditions were used: 94 C for 3 min, followed by 28 cycles of 94 C for 30 s, 53 C for 40 s and 72 C for 1 min, and a final elongation step at 72 C for 5 min. Successful amplification was determined by examining PCR products using a 2% agarose gel. Each bar-coded sample was pooled in equal proportions based on its molecular weight and DNA con- centration. Pooled samples were then purified using calibrated Ampure XP beads and subsequently used to prepare a DNA library by following Illumina TruSeq DNA library. Paired-end (2 300) sequencing was performed on an Illumina MiSeq following the manufacturer's guidelines. Sequence data was processed using the Quantitative Insights Into Microbial Ecology v1.9.1 pipeline (QIIME, http://qiime.org; Caporaso et al., 2010b). Raw sequences were depleted of barcodes, paired-end reads joined, depleted of primers, demultiplexed, and quality-filtered using the default parameters in QIIME v1.9.1. The resulting quality filtered sequences were then clustered into operational taxonomic units (OTUs) at 99% similarity with UCLUST (Edgar, 2010) using the open reference clustering protocol and a custom COI database as the reference set. A custom COI database was created from sequences of local fish and shellfish species deposited in GenBank (http://www.ncbi.nlm.nih.gov). The representative sequence set was aligned using PyNAST (Caporaso et al., 2010a), taxonomically classified using BLAST (Altschul et al., 1990), and an OTU table was generated. 3. Results 3.1. Alexandrium blooms and diamondback terrapin deaths Alexandrium cells were observed in Meetinghouse Creek beginning on 9 April 2015 with densities peaking at ~47,000 cells L1 on 29 April and the bloom ending two weeks later (12 May; 37 cells L1; Fig. 2). At the peak of the bloom, mussels deployed in Meetinghouse Creek had accumulated low levels of PST (44 mg STX eq 100g1) but became highly toxic, almost seven times greater than the federal closure limit (80 mg STX eq 100g1), one week later (5 May; 510 mg STX eq 100g1; Fig. 2) with toxin levels dissipating thereafter. Meetinghouse Creek, and the adjoining Terry Creek, were closed to all shellfish harvest on 6May and reopened on 1 July to harvest. In James Creek, Alexandrium densities increased from 91 cells L1 (20 April) to ~5100 cells L1 (4 May) and peaked at ~14,000 cells L1 on 15 May (Fig. 2). Concurrently, PST levels in mussels were 350 mg STX eq 100g1 on 16 May prompting the closure of shellfish beds in this region (Figs. 1 and 2). During spring 2015, hundreds of dead terrapins washed up in the western Fig. 2. (A) Meetinghouse Creek and (B) James Creek Alexandrium densities (cells per L) and corresponding shellfish toxicity of blue mussels measured via mouse bioassay during the spring of 2015. Gray box indicates the mortality period of terrapins collected during this study. Black dotted line indicates the first report of dead terrapins. T.K. Hattenrath-Lehmann et al. / Toxicon 129 (2017) 36e43 39 Flanders Bay region (Figs. 1 and 2). The first report of dead dia- mondback terrapins was on 21 April 2015 and continued through mid-May (Fig. 2, gray box), during which several sets of terrapins were collected for examination (Fig. 1; Table 1). During the mor- tality event, 27 terrapin carcasses were collected and submitted by a local turtle rescue center and 6 additional terrapin carcasses were collected by NYSDEC biologists. A singular survey during this three- week mortality event on 15 May 2015 quantified more than 100 dead terrapins along Iron Point, a peninsula representing a very small fraction of the Flanders Bay coastline (Fig. 1). Alexandrium densities have been historically monitored in the Flanders Bay region (including Reeves Bay, Terry Creek and East Creek, all of which are < 1 km from or are connected to Meeting- house Creek) in the late 1980s (Nuzzi and Waters, 1993) and have been monitored specifically in Meetinghouse Creek every spring since 2008 (Table 2), with peak bloom densities occurring between late-March to mid-May. To date, the 2015 bloom (~47,000 cells L1) achieved the highest Alexandrium densities ever observed in Table 1 PST concentrations (mg STX eq 100 g1) in the organs of- and condition of- diamondba University, Cornell and Delmar. GI ¼ Gastrointestinal. n/a ¼ not analyzed. * ¼ sample G (freshly collected), F ¼ fair postmortem condition (some autolysis), P ¼ poor postmortem Project ID # Mortality period PST in tissue (mg STX eq 10 Kidney Heart SB1 Late (5/15) 0 1.2 CU1 Early (4/24) 1 0 CU2 Early (4/24) 0 12.5 CU3 Middle (4/28e5/3) 0 n/a CU4 Middle (4/28e5/3) 0 n/a CU5 Middle (4/28e5/3) n/a n/a CU6 Middle (4/28e5/3) n/a n/a Del1 Middle (4/28e5/3) n/a 0 Del2 Middle (4/28e5/3) n/a 0 Del3 Middle (4/28e5/3) n/a n/a Meetinghouse Creek or the Peconic Bay region (Table 2). Similarly, PST levels measured in 2015 (540 mg STX eq 100g1) were the highest ever measured in the Meetinghouse Creek or the Peconic Estuary (Table 2). 3.2. Diamondback terrapin postmortem examinations and ranavirus testing During the mortality event, 33 fresh or previously frozen car- casses were submitted for postmortem examination. Fifteen car- casses (5 males, 10 females) were selected for complete postmortem examination based on the degree of postmortem autolysis and the date at which the carcass was found during the mortality event. All animals were mature, with males having a medianweight of 345 g (range 270e370 g) and a median carapacial length of 13.5 cm (range 11.5e15 cm) and females having a median weight of 1596 g (range 1323e1905 g) and a median carapacial length of 21.5 cm (range 20.5e23 cm). All females had active ck terrapins collected from the Flanders Bay region and examined at Stony Brook I contents used for high-throughput sequencing. G ¼ good postmortem condition condition (decay/autolysis). 0 g1) Condition of terrapins Liver GI contents 0 0 G 1.2 n/a G 0 n/a G 0 n/a P 0 n/a F n/a 0.2 P n/a 0.3 P 0 n/a F n/a n/a F n/a 0 P Table 2 Maximum Alexandrium densities (cells L1; peak date in parentheses) and maximum shellfish toxicity (mg STX eq 100 g1 shellfish tissue) measured via mouse bioassay in deployed blue mussels (Mytilus edulis) from Meetinghouse Creek, NY, USA. n.m. ¼ not monitored, and * denotes no closure implemented, although over the federal closure limit. a, b, c¼ data collected from Nuzzi andWaters 1993 and are from Reeves Bay, Terry's Creek and East Creek, respectively. Italics indicates year of terrapin mortalities. Year Maximum Alexandrium densities (cells L1) Maximum shellfish toxicity (mg STX eq 100 g1 shellfish tissue) 1986 14,000 (20-May)a 190a* 1987 500 (20-April)a 50a 1988 1600 (9-April)a 60a 1989 5700 (30-March)a 60a 1989 480 (4-May)b <40b 1989 1000 (4-May)c 58c 2008 4733 (29-Apr) n.m. 2009 19,868 (23-Apr) <40 2010 1982 (15-Apr) 57 2011 1166 (5-May) 48 2012 17,206 (11-Apr) 380* 2013 1058 (10-Apr) 40 2014 7480 (8-May) 53 2015 46,690 (29-April) 540 2016 550 (16-May) <40 T.K. Hattenrath-Lehmann et al. / Toxicon 129 (2017) 36e43 40 ovarian folliculogenesis at the time of death. Individuals not in an advanced state of decay had full GI tracts and an intact fat layer. No consistent, significant gross or histologic changes were present in the examined tissues and a cause of death could not be determined histologically. None of the terrapins exhibited any overt signs of physical trauma. A subset (n ¼ 4) of the terrapins had small to moderate amounts of red-tinged, clear pulmonary fluid; in other terrapins, the lungs were aerated and free of fluid. Other findings, including mononuclear infiltrates in the renal interstitium (n ¼ 3) and mild parenchymal hemorrhage (n ¼ 1) were considered mild and incidental to the mortality event. The degree of postmortem autolysis and freeze thaw in a number of carcasses limited histo- logic tissue interpretation. Although not previously documented in terrapins, amphibian- like ranaviruses have been associated with mortality in other chelonian species (Marschang et al., 1999; Sim et al., 2016;Winzeler et al., 2015). Combined choanal/cloacal swabs collected from 27 terrapins were all negative for the major capsid protein of amphibian-like ranaviruses by qPCR. 3.3. Paralytic shellfish toxins in diamondback terrapin tissue Initial analyses of terrapin gastrointestinal contents revealed the qualitative presence of PST via mouse bioassay (Darcie Couture, Research Access International, ME, USA; pers. comm.). Subsequent analyses quantified PST in various organs (kidney, heart, liver, gastrointestinal contents) of diamondback terrapins collected over the duration of the reported mortality event (Table 1). Individuals Table 3 PST analog concentrations (mg 100 g1) and toxicity equivalents (mg STX eq 100 g1) GI ¼ Gastrointestinal. Project ID # Organ PST concentration in tissue (and eq mg 100 g1 (mg STX eq. 100 g1) GTX5 NEO SB1 Heart CU1 Kidney CU1 Liver CU2 Heart 8.4 (7.8) CU5 GI contents 3.6 (0.2) CU6 GI contents 4.2 (0.3) that were collected and frozen immediately after death (CU1, CU2, SB1) had the highest levels and most consistent presence of PST (Table 1). Consistent with Alexandrium cultures isolated from the New York region (Hattenrath-Lehmann et al., 2015), C2 was the most abundant congener measured in terrapin tissue (mg 100 g1; Table 3). Heart tissues from these individuals had the highest PST con- centrations with individual CU2 and SB1 having 12.5 (consisting of NEO and STX) and 1.2 mg STX eq 100g1 (consisting of C2), respectively (Tables 1 and 3). Individual CU1 contained 1.2 mg STX eq 100g1 liver tissue and 1.0 mg STX eq 100g1 kidney tissue consisting of C2 (Tables 1 and 3). Individuals in advanced states of decay had either low levels of PST within gastrointestinal contents (0.2e0.3 mg STX eq 100g1) consisting of GTX5 or no detectable levels of PST in any tissue (Tables 1 and 3). 3.4. High throughput sequencing of diamondback terrapin intestinal contents Samples sequenced with primer sets targeting the mitochon- drial cytochrome c oxidase subunit I gene (COI) generated 440,776 paired-end reads with an amplicon size of ~300bp. After quality filtering and joining reads a total of 388,897 reads clustered at 99% identity into 259 OTUs. The majority (257) of OTUs were ‘unas- signed’ meaning that they did not match any sequences in the custom database of fish and local shellfish species that may represent potential PST vectors. The two OTUs that did match the custom database were both Geukensia demissa (ribbed mussel; 99% identity). G. demissa sequences were amplified from all three samples (CU5, CU6, and Del3; Table 1) representing <1% of total reads. Paralytic shellfish toxins were present in the intestinal con- tents of all but one (Del3) of the samples sequenced (Table 1). 4. Discussion Since the middle of the 20th century, harmful algal blooms have become more pervasive along coastlines world-wide (Anderson et al., 2008, 2012, 2002; Hoagland et al., 2002; Sunda et al., 2006; Van Dolah, 2000). Concurrently, diamondback terrapin pop- ulations, a species that is limited to shallow brackishwater marshes and mangrove swamps of the U.S. Atlantic and Gulf Coasts, have become threatened or endangered in parts of its range due to overharvesting, habitat loss, road mortality, and by-catch (Bishop, 1983; Dorcas et al., 2007; Grosse et al., 2011; Ner and Burke, 2008; Roosenburg et al., 1997; Schaffer et al., 2008; Szerlag and McRobert, 2006). While turtle deaths have been attributed to HABs in general and PST in particular in some tropical regions (Amaya et al., 2014; Licea et al., 2008; Maclean, 1975), such occur- rences have not been reported within temperate estuaries. This study documented the concurrent outbreak of Alexandrium blooms, PST accumulation in bivalves, terrapin mortality, PST accumulation in the organs of diamondback terrapins collected from the Flanders Bay region. . toxicity) Total toxicity mg STX eq. 100 g1 STX C2 12.5 (1.2) 1.2 10.3 (1) 1 12.1 (1.2) 1.2 4.7 (4.7) 12.5 0.2 0.3 </p>
