Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009-2021.

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

PULMONARY AND COELOMIC MYCOSES DUE TO METARHIZIUM AND BEAUVERIA SPECIES IN REPTILES.

This report documents cases of fatal pulmonary mycosis caused by entomopathogenic fungi in the genera Metarhizium and Beauveria (Order Hypocreales) in a loggerhead sea turtle (Caretta caretta), a Chinese alligator (Alligator sinensis), two gopher tortoises (Gopherus polyphemus), a Cuvier's dwarf caiman (Paleosuchus palpebrosus), a false gharial (Tomistoma schlegelii), a green sea turtle (Chelonia mydas), and a Kemp's ridley sea turtle (Lepidochelys kempii), and a case of granulomatous coelomitis in a hawksbill sea turtle (Eretmochelys imbricata). Fungi identified in these cases included Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, Metarhizium robertsii, and one case of infection by a novel Metarhizium species. The animals were either housed at zoos or brought into rehabilitation from the wild. Although the majority of animals had comorbidities, the fungal infections were believed to be the primary cause of death. Fungal susceptibility testing was performed on two Beauveria spp. isolates, and revealed lower minimum inhibitory concentrations for itraconazole and voriconazole when compared to terbinafine and fluconazole. This case series demonstrates that a variety of reptile species from different orders are vulnerable to infection with Metarhizium, and multiple species of sea turtle are susceptible to infection with Beauveria.

Identification of a novel Neorickettsia species in a Kemp's ridley sea turtle with granulomatous nephritis and development of a quantitative PCR assay.

An adult male Kemp's ridley turtle was found dead on the coast of Kenedy County, Texas, in August 2019 with bilateral severe, diffuse granulomatous nephritis. Pan-bacterial 16S rRNA gene polymerase chain reaction (PCR) and amplicon sequencing of affected tissue indicated the presence of a Neorickettsia. Neorickettsia is a genus of obligate intracellular Alphaproteobacteria that are transmitted by digenean trematodes. For further characterization, primers were designed to amplify and sequence the groEL gene. Phylogenetic analysis found that the organism was distinct from other known species to a degree consistent with a novel species. Immunohistochemistry using an antibody directed against a Neorickettsia surface protein showed bacterial clusters within the renal granulomas. A species-specific quantitative PCR was designed and detected the organism within the liver and colon of the index case. A quantitative PCR survey of grossly normal kidneys opportunistically collected from additional stranded sea turtle kidneys detected this organism in five of 15 Kemp's ridley turtles, two of nine green turtles, and neither of two loggerhead turtles. Recognition of this novel organism in an endangered species is concerning; additional work is underway to further characterize the potential of this organism as a pathogen of sea turtles.

Correction: Turner et al. Steatitis in Cold-Stunned Kemp's Ridley Sea Turtles (Lepidochelys kempii). Animals 2021, 11, 898.

In the original publication [...].

A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

Osteomyelitis in cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) hospitalized for rehabilitation: 25 cases (2008-2018).

OBJECTIVE: To characterize osteolytic lesions in cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) hospitalized for rehabilitation and describe methods used for the management of such lesions. ANIMALS: 25 stranded, cold-stunned Kemp's ridley sea turtles hospitalized between 2008 and 2018. PROCEDURES: Medical records of sea turtles with a diagnosis of osteolytic lesions were reviewed retrospectively to obtain the date of diagnosis, clinical signs, radiographic findings, microbial culture results, hematologic and plasma biochemical data, cytologic and histologic findings, antimicrobial history, time to first negative culture result, treatment duration, and outcome. RESULTS: Lesions were identified radiographically a median of 50 days after admission and were located within epiphyses or metaphyses of various appendicular joints. Lesions were associated with periarticular swelling (n = 24), lameness (16), lethargy (2), and hyporexia (2). Bacterial culture yielded growth of single organisms (n = 16), multiple organisms (2), or no growth (6). Significant differences in hematologic and biochemical data were detected between the times of diagnosis and convalescence. Cytologic and histologic findings characterized the lesions as osteomyelitis leading to septic arthritis. Sixteen sea turtles were managed medically, and 8 were managed medically and surgically. Surgery resulted in rapid improvement in joint mobility and overall clinical status. Most (22/25 [88%]) sea turtles survived and were released after long-term management. CONCLUSIONS AND CLINICAL RELEVANCE: During rehabilitation, cold-stunned Kemp's ridley sea turtles may be affected by osteomyelitis. Medical management based on antimicrobial susceptibility testing was effective for most turtles. Long term management efforts in turtles are justified by high survival rate.

Fibropapillomatosis and Chelonid Alphaherpesvirus 5 Infection in Kemp's Ridley Sea Turtles (Lepidochelys kempii).

Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006-2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation.

A Baseline Model For Estimating the Risk of Gas Embolism in Sea Turtles During Routine Dives.

Sea turtles, like other air-breathing diving vertebrates, commonly experience significant gas embolism (GE) when incidentally caught at depth in fishing gear and brought to the surface. To better understand why sea turtles develop GE, we built a mathematical model to estimate partial pressures of N2 (PN2), O2 (PO2), and CO2 (PCO2) in the major body-compartments of diving loggerheads (Caretta caretta), leatherbacks (Dermochelys coriacea), and green turtles (Chelonia mydas). This model was adapted from a published model for estimating gas dynamics in marine mammals and penguins. To parameterize the sea turtle model, we used values gleaned from previously published literature and 22 necropsies. Next, we applied this model to data collected from free-roaming individuals of the three study species. Finally, we varied body-condition and cardiac output within the model to see how these factors affected the risk of GE. Our model suggests that cardiac output likely plays a significant role in the modulation of GE, especially in the deeper diving leatherback turtles. This baseline model also indicates that even during routine diving behavior, sea turtles are at high risk of GE. This likely means that turtles have additional behavioral, anatomical, and/or physiologic adaptions that serve to reduce the probability of GE but were not incorporated in this model. Identifying these adaptations and incorporating them into future iterations of this model will further reveal the factors driving GE in sea turtles.

Steatitis in Cold-Stunned Kemp's Ridley Sea Turtles (Lepidochelys kempii).

The pathogenesis of steatitis that infrequently occurs in cold-stunned Kemp's ridley sea turtles (KRT; Lepidochelys kempii) has been undetermined. The objectives of this study were to investigate the clinical (n = 23) and histologic findings (n = 11) in cold-stunned KRT, and to compare plasma concentrations of α-tocopherol (vitamin E), thiobarbituric acid reactive substances (TBARS), and the TBARS to vitamin E (T/E) ratio (an assessment of oxidative stress) between cold-stunned KRT with clinically and/or histologically confirmed steatitis (n = 10) and free-ranging KRT (n = 9). None of the cold-stunned turtles had clinically detectable steatitis at admission, and the median number of days to diagnosis of steatitis was 71 (range 33­469). Histologic findings of affected adipose tissue included heterophilic (n = 9) and/or histiocytic (n = 5) steatitis, fat necrosis (n = 7), myonecrosis (n = 2), and intralesional bacteria (n = 6). Cold-stunned KRT had significantly lower plasma vitamin E concentrations (median = 3.5 nmol/g), lower plasma TBARS concentrations (median = 1.6 nmol/g), and higher T/E ratios (median = 0.50), than controls (62.3 nmol/g; 2.1 nmol/g; 0.03, respectively). These results suggest a multifactorial etiology for the development of steatitis in KRT during rehabilitation, including tissue injury, septicemia, and various factors resulting in imbalances of anti-/oxidative status. By highlighting the need to provide more effective vitamin E supplementation, and the need to re-assess specific components of the diet, this study may lead to reduced incidence and improved medical management of steatitis in cold-stunned sea turtles.

Transcriptomic Profiling of Fibropapillomatosis in Green Sea Turtles (Chelonia mydas) From South Texas.

Sea turtle fibropapillomatosis (FP) is a tumor promoting disease that is one of several threats globally to endangered sea turtle populations. The prevalence of FP is highest in green sea turtle (Chelonia mydas) populations, and historically has shown considerable temporal growth. FP tumors can significantly affect the ability of turtles to forage for food and avoid predation and can grow to debilitating sizes. In the current study, based in South Texas, we have applied transcriptome sequencing to FP tumors and healthy control tissue to study the gene expression profiles of FP. By identifying differentially expressed turtle genes in FP, and matching these genes to their closest human ortholog we draw on the wealth of human based knowledge, specifically human cancer, to identify new insights into the biology of sea turtle FP. We show that several genes aberrantly expressed in FP tumors have known tumor promoting biology in humans, including CTHRC1 and NLRC5, and provide support that disruption of the Wnt signaling pathway is a feature of FP. Further, we profiled the expression of current targets of immune checkpoint inhibitors from human oncology in FP tumors and identified potential candidates for future studies.

Scavenging versus predation: shark-bite injuries in stranded sea turtles in the southeastern USA.

Injuries inflicted by sharks are a frequent observation in stranded sea turtles. Sharks prey on live turtles and scavenge carcasses, which can create uncertainty as to the cause of stranding when sea turtles are found dead with shark-bite wounds. Consequently, attributing the cause of stranding to a shark attack based purely on the presence of the characteristic wounds can overestimate predation by sharks as a cause of mortality. To better characterize the timing of shark-bite wounds relative to death of sea turtles in the southeastern USA, we performed necropsies on 70 stranded turtles that were found dead in which the predominant observation was bite wounds without any grossly evident vital responses (inflammation or healing). Postmortem examination included assessment for evidence of exsanguination and histopathological evaluation of skeletal muscle comprising wound margins. We characterized wounds as antemortem, perimortem, or postmortem based on specific criteria related to the presence or absence of supravital and intravital responses. Most (80%) shark-bite wounds were postmortem, 10% were antemortem, and 10% were perimortem. We found that antemortem and postmortem wounds were similar in extent and location except for wounds that primarily involved the shell, which were never found in cases of scavenging. For sea turtles found dead in the southeastern USA, our findings suggest that most shark-bite wounds without externally evident vital responses are due to scavenging. Additionally, this scavenging can significantly damage a carcass, potentially obscuring the detection of other causes of mortality. These findings should be considered when using data derived from stranded sea turtles to conduct mortality assessments.

Gas embolism and massive blunt force trauma to sea turtles entrained in hopper dredges in North and South Carolina, USA.

Decompression sickness (DCS) has been described mainly in loggerhead turtles Caretta caretta bycaught in trawls and gillnets. Here we present cases of gas emboli (GE) in 8 green turtles Chelonia mydas and 2 Kemp's ridleys Lepidochelys kempii entrained in hopper dredges that were working at 8.8-15.2 m depths during shipping channel maintenance or beach renourishment activities. Turtle weights ranged from 2.2 to 6.7 kg. All were found alive with blunt force injuries from passage through the dredge and were taken to rehabilitation facilities. Four green turtles died or were euthanized within 24 h. Six turtles survived. Radiographic or ultrasonographic evidence of GE was detected in 4 turtles, including 3 mortalities. Computed tomography (CT) revealed perirenal and cervical GE in 4 turtles, including 1 mortality. No GE were detected in 2 of the survivors. Upon necropsy, GE were found in mesenteric vessels, the right atrium, and kidneys. Histopathology confirmed that tissues were in a good state of preservation without evidence of bacterial overgrowth or putrefactive gas formation. Death likely resulted primarily from massive tissue trauma from the dredge, but moderate GE could have led to DCS and complicated recovery. The surviving turtles weighed less than those that did not survive. Besides hypothesized stress/exercise-induced circulatory changes of blood through the lungs and pressure reduction of forced surfacing from depth, drastic pressure change within the dredge pipes before and after the pump could contribute to GE. Hopper dredge entrainment is an additional cause of GE and potential DCS in sea turtles.

Caryospora-Like Coccidia Infecting Green Turtles (Chelonia mydas): An Emerging Disease With Evidence of Interoceanic Dissemination.

Protozoa morphologically consistent with Caryospora sp. are one of the few pathogens associated with episodic mass mortality events involving free-ranging sea turtles. Parasitism of green turtles (Chelonia mydas) by these coccidia and associated mortality was first reported in maricultured turtles in the Caribbean during the 1970s. Years later, epizootics affecting wild green turtles in Australia occurred in 1991 and 2014. The first clinical cases of Caryospora-like infections reported elsewhere in free-ranging turtles were from the southeastern US in 2012. Following these initial individual cases in this region, we documented an epizootic and mass mortality of green turtles along the Atlantic coast of southern Florida from November 2014 through April 2015 and continued to detect additional, sporadic cases in the southeastern US in subsequent years. No cases of coccidial disease were recorded in the southeastern US prior to 2012 despite clinical evaluation and necropsy of stranded sea turtles in this region since the 1980s, suggesting that the frequency of clinical coccidiosis has increased here. Moreover, we also recorded the first stranding associated with infection by a Caryospora-like organism in Hawai'i in 2018. To further characterize the coccidia, we sequenced part of the 18S ribosomal and mitochondrial cytochrome oxidase I genes of coccidia collected from 62 green turtles found in the southeastern US and from one green turtle found in Hawai'i. We also sequenced the ribosomal internal transcribed spacer regions from selected cases and compared all results with those obtained from Caryospora-like coccidia collected from green turtles found in Australia. Eight distinct genotypes were represented in green turtles from the southeastern US. One genotype predominated and was identical to that of coccidia collected from the green turtle found in Hawai'i. We also found a coccidian genotype in green turtles from Florida and Australia with identical 18S and mitochondrial sequences, and only slight inter-regional differences in the internal transcribed spacer 2. We found no evidence of geographical structuring based on phylogenetic analysis. Low genetic variability among the coccidia found in green turtle populations with minimal natural connectivity suggests recent interoceanic dissemination of these parasites, which could pose a risk to sea turtle populations.

A novel host-adapted strain of Salmonella Typhimurium causes renal disease in olive ridley turtles (Lepidochelys olivacea) in the Pacific.

Salmonella spp. are frequently shed by wildlife including turtles, but S. enterica subsp. enterica serovar Typhimurium or lesions associated with Salmonella are rare in turtles. Between 1996 and 2016, we necropsied 127 apparently healthy pelagic olive ridley turtles (Lepidochelys olivacea) that died from drowning bycatch in fisheries and 44 live or freshly dead stranded turtles from the west coast of North and Central America and Hawaii. Seven percent (9/127) of pelagic and 47% (21/44) of stranded turtles had renal granulomas associated with S. Typhimurium. Stranded animals were 12 times more likely than pelagic animals to have Salmonella-induced nephritis suggesting that Salmonella may have been a contributing cause of stranding. S. Typhimurium was the only Salmonella serovar detected in L. olivacea, and phylogenetic analysis from whole genome sequencing showed that the isolates from L. olivacea formed a single clade distinct from other S. Typhimurium. Molecular clock analysis revealed that this novel clade may have originated as recently as a few decades ago. The phylogenetic lineage leading to this group is enriched for non-synonymous changes within the genomic area of Salmonella pathogenicity island 1 suggesting that these genes are important for host adaptation.

Assessing Karenia brevis red tide as a mortality factor of sea turtles in Florida, USA.

Data on Karenia brevis red tides (≥105 cells l-1) and on dead or debilitated (i.e. stranded) Kemp's ridleys Lepidochelys kempii, loggerheads Caretta caretta, green turtles Chelonia mydas, hawksbills Eretmochelys imbricata, and leatherbacks Dermochelys coriacea documented in Florida during 1986-2013 were evaluated to assess red tides as a sea turtle mortality factor. Unusually large numbers of stranded sea turtles were found coincident with red tides primarily along Florida's Gulf coast but also along a portion of Florida's Atlantic coast. These strandings were mainly adult and large immature loggerheads and Kemp's ridleys, and small immature green turtles and hawksbills. Unusually large numbers of stranded leatherbacks never coincided with red tide. For the 3 most common species, results of stranding data modeling, and of investigations that included determining brevetoxin concentrations in samples collected from stranded turtles, all indicated that red tides were associated with greater and more frequent increases in the numbers of stranded loggerheads and Kemp's ridleys than in the number of stranded green turtles. The mean annual number of stranded sea turtles attributed to K. brevis red tide was 80 (SE = 21.6, range = 2-338). Considering typical stranding probabilities, the overall mortality was probably 5-10 times greater. Red tide accounted for a substantial portion of all stranded loggerheads (7.1%) and Kemp's ridleys (17.7%), and a smaller portion of all stranded green turtles (1.6%). Even though K. brevis red tides occur naturally, the mortality they cause needs to be considered when managing these threatened and endangered species.

Saxitoxin Poisoning in Green Turtles (Chelonia mydas) Linked to Scavenging on Mass Mortality of Caribbean Sharpnose Puffer Fish (Canthigaster rostrata-Tetraodontidae).

Fish within the family Tetraodontidae are potential sources of both endogenous tetrodotoxins (TTXs) and dietary derived saxitoxins (STXs). Ingestion of fish tissues containing these toxins by other vertebrates can lead to severe illness and death. The Caribbean sharpnose puffer (Canthigaster rostrata) is a widespread tetraodontid species within the western Atlantic. Mass settlement of juveniles into foraging habitats have been associated with large-scale puffer fish mortality events. In 2013, 2014, and 2017, puffer mortality events on the southern Caribbean coast of Costa Rica were also associated with strandings of green turtles (Chelonia mydas) found to have fed on C. rostrata. Stranded sea turtles were found dead without apparent cause or alive with severe neurological signs that resolved during short periods of captivity. Puffer fish and turtle organ samples were analyzed for both TTXs and STXs. Concentrations of TTXs were extremely low in the fish (0.5-0.7 µg/g) and undetectable in turtle stomach contents. However, concentrations of STXs in whole fish (16.6-47.5 µg STX-eq/g) exceeded the 0.8 µg STX-eq/g human seafood safety threshold for STXs by orders of magnitude. Saxitoxins were also detected in samples of stomach contents (ingested fish), brain, lung, kidney, and serum from three affected turtles. Study results indicate that saxitoxicosis resulting from opportunistic foraging on C. rostrata during fish mortality events may be a significant factor in episodic stranding of green sea turtles in this region.

Brevetoxin exposure in sea turtles in south Texas (USA) during Karenia brevis red tide.

Five green (Chelonia mydas) and 11 Kemp's ridley (Lepidochelys kempii) sea turtles found dead, or that died soon after stranding, on the southern Texas (USA) coast during 2 Karenia brevis blooms (October 2015, September-October 2016) were tested for exposure to brevetoxins (PbTx). Tissues (liver, kidney) and digesta (stomach and intestinal contents) were analyzed by ELISA. Three green turtles found alive during the 2015 event and 2 Kemp's ridley turtles found alive during the 2016 event exhibited signs of PbTx exposure, including lethargy and/or convulsions of the head and neck. PbTx were detected in 1 or more tissues or digesta in all 16 stranded turtles. Detected PbTx concentrations ranged from 2 to >2000 ng g-1. Necropsy examination and results of PbTx analysis indicated that 10 of the Kemp's ridleys and 2 of the green turtles died from brevetoxicosis via ingestion. This is the first documentation of sea turtle mortality in Texas attributed to brevetoxicosis.

Evidence of Diversity, Site, and Host Specificity of Sea Turtle Blood Flukes (Digenea: Schistosomatoidea: "Spirorchiidae"): A Molecular Prospecting Study.

Neospirorchis (Digenea: "Spirorchiidae") are blood flukes of sea turtles. Trematodes tentatively identified as Neospirorchis sp. infect various sites within sea turtles inhabiting waters of the southeastern United States, but efforts to obtain specimens adequate for morphologic study has proven difficult. Two genetic targets, the internal transcribed spacer region of the ribosomal RNA gene and the partial mitochondrial cytochrome c oxidase subunit I gene, were used to investigate potential diversity among parasite specimens collected from stranded sea turtles. Sequence data were obtained from 215 trematode and egg specimens collected from 92 individual free-ranging cheloniid sea turtles comprising 4 host species. Molecular analysis yielded more than 20 different genotypes. We were able to assign 1 genotype to 1 of the 2 recognized species, Neospirorchis pricei Manter and Larson, 1950 . In many examples, genotypes exhibited host and site specificity. Our findings indicate considerable diversity of parasites resembling Neospirorchis with evidence of a number of uncharacterized blood flukes that require additional study.

Defining risk variables causing gas embolism in loggerhead sea turtles (Caretta caretta) caught in trawls and gillnets.

Incidental capture, or 'bycatch' in fishing gear is a major global threat to sea turtle populations. A recent study showed that underwater entrapment in fishing gear followed by rapid decompression may cause gas bubble formation within the blood stream (embolism) and tissues leading to organ injury, impairment, and even mortality in some bycaught individuals. We analyzed data from 128 capture events using logistic and ordinal regression to examine risk factors associated with gas embolism in sea turtles captured in trawls and gillnets. Likelihood of fatal decompression increases with increasing depth of gear deployment. A direct relationship was found between depth, risk and severity of embolism, which has not been previously demonstrated in any breath-hold diving species. For the trawl fishery in this study, an average trawl depth of 65 m was estimated to result in 50% mortality in by-caught turtles throughout the year. This finding is critical for a more accurate estimation of sea turtle mortality rates resulting from different fisheries and for devising efforts to avoid or minimize the harmful effects of capture.

Elucidation of the first definitively identified life cycle for a marine turtle blood fluke (Trematoda: Spirorchiidae) enables informed control.

Blood flukes of the family Spirorchiidae are significant pathogens of both free-ranging and captive marine turtles. Despite a significant proportion of marine turtle mortality being attributable to spirorchiid infections, details of their life cycles remain almost entirely unknown. Here we report on the molecular elucidation of the complete life cycle of a marine spirorchiid, identified as Amphiorchis sp., infecting vermetid gastropods and captive hatched neonate Caretta caretta in the Oceanogràfic Aquarium, in Valencia, Spain. Specimens of a vermetid gastropod, Thylaeodus cf. rugulosus (Monterosato, 1878), collected from the aquarium filtration system housing diseased C. caretta, were infected with sporocysts and cercariae consistent with the family Spirorchiidae. We generated rDNA sequence data [internal transcribed spacer 2 (ITS2) and partial 28S rDNA] from infections from the vermetid which were identical to sequences generated from eggs from the serosa of the intestine of neonate C. caretta, and an adult spirorchiid from the liver of a C. caretta from Florida, USA. Given the reliability of these markers in the delineation of trematode species, we consider all three stages to represent the same species and tentatively identify it as a species of Amphiorchis Price, 1934. The source of infection at the Oceanogràfic Foundation Rehabilitation Centre, Valencia, Spain, is inferred to be an adult C. caretta from the western Mediterranean being rehabilitated in the same facility. Phylogenetic analysis suggests that this Amphiorchis sp. is closely related to other spirorchiids of marine turtles (species of Carettacola Manter & Larson, 1950, Hapalotrema Looss, 1899 and Learedius Price, 1934). We discuss implications of the present findings for the control of spirorchiidiasis in captivity, for the better understanding of epidemiology in wild individuals, and the elucidation of further life cycles.