Multi-year tracking reveals extensive pelagic phase of juvenile loggerhead sea turtles in the North Pacific.

BACKGROUND: The juvenile stage of loggerhead sea turtles (Caretta caretta) can last for decades. In the North Pacific Ocean, much is known about their seasonal movements in relation to pelagic habitat, yet understanding their multi-year, basin-scale movements has proven more difficult. Here, we categorize the large-scale movements of 231 turtles satellite tracked from 1997 to 2013 and explore the influence of biological and environmental drivers on basin-scale movement. RESULTS: Results show high residency of juvenile loggerheads within the Central North Pacific and a moderate influence of the Earth's magnetic field, but no real-time environmental driver to explain migratory behavior. CONCLUSIONS: We suggest the Central North Pacific acts as important developmental foraging grounds for young juvenile loggerhead sea turtles, rather than just a migratory corridor. We propose several hypotheses that may influence the connectivity between western and eastern juvenile loggerhead foraging grounds in the North Pacific Ocean.

Active dispersal in loggerhead sea turtles (Caretta caretta) during the 'lost years'.

Highly migratory marine species can travel long distances and across entire ocean basins to reach foraging and breeding grounds, yet gaps persist in our knowledge of oceanic dispersal and habitat use. This is especially true for sea turtles, whose complex life history and lengthy pelagic stage present unique conservation challenges. Few studies have explored how these young at-sea turtles navigate their environment, but advancements in satellite technology and numerical models have shown that active and passive movements are used in relation to open ocean features. Here, we provide the first study, to the best of our knowledge, to simultaneously combine a high-resolution physical forcing ocean circulation model with long-term multi-year tracking data of young, trans-oceanic North Pacific loggerhead sea turtles during their 'lost years' at sea. From 2010 to 2014, we compare simulated trajectories of passive transport with empirical data of 1-3 year old turtles released off Japan (29.7-37.5 straight carapace length cm). After several years, the at-sea distribution of simulated current-driven trajectories significantly differed from that of the observed turtle tracks. These results underscore current theories on active dispersal by young oceanic-stage sea turtles and give further weight to hypotheses of juvenile foraging strategies for this species. Such information can also provide critical geographical information for spatially explicit conservation approaches to this endangered population.

Dynamics of Virus Shedding and In Situ Confirmation of Chelonid Herpesvirus 5 in Hawaiian Green Turtles With Fibropapillomatosis.

Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

Immune status of free-ranging green turtles with fibropapillomatosis from Hawaii.

Cell-mediated and humoral immune status of free-ranging green turtles (Chelonia mydas) in Hawaii (USA) with and without fibropapillornatosis (FP) were assessed. Tumored and non-tumored turtles from Kaneohe Bay (KB) on the island of Oahu and from FP-free areas on the west (Kona/Kohala) coast of the island of Hawaii were sampled from April 1998 through February 1999. Turtles on Oahu were grouped (0-3) for severity of tumors with 0 for absence of tumors, 1 for light, 2 for moderate, and 3 for most severe. Turtles were weighed, straight carapace length measured and the regression slope of weight to straight carapace length compared between groups (KB0, KB1, KB2, KB3, Kona). Blood was assayed for differential white blood cell count, hematocrit, in vitro peripheral blood mononuclear cell (PBMC) proliferation in the presence of concanavalin A (ConA) and phytohaemagglutinin (PHA), and protein electrophoresis. On Oahu, heterophil/lymphocyte ratio increased while eosinophil/monocyte ratio decreased with increasing tumors score. Peripheral blood mononuclear cell proliferation indices for ConA and PHA were significantly lower for turtles with tumor scores 2 and 3. Tumor score 3 turtles (KB3) had significantly lower hematocrit, total protein, alpha 1, alpha 2, and gamma globulins than the other four groups. No significant differences in immune status were seen between non-tumored (or KB1) turtles from Oahu and Hawaii. There was no significant difference between groups in regression slopes of body condition to carapace length. We conclude that turtles with severe FP are imunosuppressed. Furthermore, the lack of significant difference in immune status between non-tumored (and KB1) turtles from Oahu and Kona/Kohala indicates that immunosuppression may not be a prerequisite for development of FP.

Quantitative analysis of herpesvirus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR.

Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogeneous (range 2-20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001-170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5-4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

Congener-specific profile and toxicity assessment of PCBs in green turtles (Chelonia mydas) from the Hawaiian Islands.

Chemical pollution may play a role in the etiology of fibropapillomatosis in green turtles (Chelonia mydas). In this preliminary study, polychlorinated biphenyls (PCBs) were measured in the livers and adipose fats of green turtles collected after they were stranded on Oahu Island, Hawaii in 1992-1993. Average concentrations of total PCBs were 45-58 ng/g dry weight and 73-665 ng/g in the liver and adipose tissues, respectively. Hexachlorobiphenyls were predominant homologues, PCBs 153 and 138 were dominant congeners in all the turtle tissues. Among the most toxic coplanar congeners, in the order of abundance, were PCB 77 > 126 > 169. Estimated toxic equivalents (TEQs) of PCBs to 2,3,7,8-tetrachlorodibenzo-p-dioxin were 8-15 pg/g in the livers and 13-48 pg/g in the adipose tissues. PCB 126 contributed 85-91% of the total TEQs.

Detection of herpesviral sequences in tissues of green turtles with fibropapilloma by polymerase chain reaction.

An alpha-herpesvirus has been associated recently with green turtle fibropapilloma (FP). To further clarify the role of this newfound green turtle herpesvirus (GTHV) in the pathogenesis of FP, various normal-appearing tissues and organs (including skin, eye, brain, heart, liver, spleen, intestine, lung, kidney, nerve, gonad, tongue, gall bladder, urinary bladder, thyroid and peripheral blood mononuclear cells (PBMC) from blood) and tumor tissues from 19 green turtles (Chelonia mydas) with FP, and tissues from three green turtles without FP, collected during 1997 to 1999 in the Hawaiian Islands, were tested for GTHV sequences by nested polymerase chain reaction (PCR), using GTHV-specific oligonuclotide primers. GTHV sequences were detected in all tumors (51/51) and most tissues (133/167) of tumored turtles. By contrast, such sequences were undetectable in tissues (0/28) of three non-tumored turtles. Analysis of GTHV sequences detected in different tissues and tumors revealed a low degree of genetic diversity (<1%). The wide distribution of this newfound herpesvirus in tumors and tissues of tumored green turtles and its absence in tissues of non-tumored turtles, argues for an etiologic role in FP.

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas.

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.

Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas.

Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 50+/-5 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species.

Comparative pathology and pathogenesis of spontaneous and experimentally induced fibropapillomas of green turtles (Chelonia mydas).

Tumor biopsy samples from 25 Floridian and 15 Hawaiian green turtles (Chelonia mydas) with spontaneous green turtle fibropapillomatosis (GTFP) and from 27 captive-reared green turtles with experimentally induced GTFP were examined microscopically to differentiate the histologic features that result from GTFP pathogenesis and those that result from incidental factors that may vary according to geographic region. Common histologic features for spontaneous and experimentally induced tumors included fibroblast proliferation in the superficial dermis, epidermal acanthosis and hyperkeratosis, epidermal basal cell degeneration with dermal-epidermal cleft formation, spinous layer degeneration with intraepidermal vesicle and pustule formation, and ulceration. Visceral tumors, found in eight of 10 (80%) free-ranging turtles with cutaneous disease that were examined after death, had extensive interstitial fibrous proliferation. The presence of spirorchid trematode eggs and associated foreign body granulomas, common secondary findings within spontaneous tumors, varied by geographic location, and these findings were not observed in experimentally induced tumors. Eosinophilic intranuclear inclusions and intranuclear herpesvirus-associated antigen immunoreactivity were found in 18 of 38 (47%) experimentally induced cutaneous tumors and nine of 119 (7.5%) spontaneous tumors from Floridian but not Hawaiian turtles. The possible involvement of GTFP-associated herpesvirus in the pathogenesis of epidermal degenerative changes and GTFP pathogenesis is discussed.

Relating tumor score to hematology in green turtles with fibropapillomatosis in Hawaii.

The relationship between hematologic status and severity of tumor affliction in green turtles (Chelonia mydas) with fibropapillomatosis (FP) was examined. During 1 wk periods in July 1997 and July 1998, we bled 108 free-ranging green turtles from Pala'au (Molokai, Hawaii, USA) where FP is endemic. Blood was analyzed for hematocrit, estimated total solids, total white blood cell (WBC) count and differential WBC count. Each turtle was assigned a subjective tumor score ranging from 0 (no visible external tumors) to 3 (heavily tumored) that indicated the severity of FP. There was a progressive increase in monocytes and a decrease in all other hematologic parameters except heterophils and total numbers of white blood cells as tumor score increased. These data indicate that tumor score can relate to physiologic status of green turtles afflicted with FP, and that tumor score is a useful field monitor of severity of FP in this species.

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas.

Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30 degrees C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles.

OBJECTIVE: To identify and characterize blood cells from free-ranging Hawaiian green turtles, Chelonia mydas. SAMPLE POPULATION: 26 green turtles from Puako on the island of Hawaii and Kaneohe Bay on the island of Oahu. PROCEDURE: Blood was examined, using light and electron microscopy and cytochemical stains that included benzidine peroxidase, chloroacetate esterase, alpha naphthyl butyrate esterase, acid phosphatase, Sudan black B, periodic acid-Schiff, and toluidine blue. RESULTS: 6 types of WBC were identified: lymphocytes, monocytes, thrombocytes, heterophils, basophils, and eosinophils (small and large). Morphologic characteristics of mononuclear cells and most granulocytes were similar to those of cells from other reptiles except that green turtles have both large and small eosinophils. CONCLUSIONS: Our classification of green turtle blood cells clarifies improper nomenclature reported previously and provides a reference for future hematologic studies in this species.

Three closely related herpesviruses are associated with fibropapillomatosis in marine turtles.

Green turtle fibropapillomatosis is a neoplastic disease of increasingly significant threat to the survivability of this species. Degenerate PCR primers that target highly conserved regions of genes encoding herpesvirus DNA polymerases were used to amplify a DNA sequence from fibropapillomas and fibromas from Hawaiian and Florida green turtles. All of the tumors tested (n = 23) were found to harbor viral DNA, whereas no viral DNA was detected in skin biopsies from tumor-negative turtles. The tissue distribution of the green turtle herpesvirus appears to be generally limited to tumors where viral DNA was found to accumulate at approximately two to five copies per cell and is occasionally detected, only by PCR, in some tissues normally associated with tumor development. In addition, herpesviral DNA was detected in fibropapillomas from two loggerhead and four olive ridley turtles. Nucleotide sequencing of a 483-bp fragment of the turtle herpesvirus DNA polymerase gene determined that the Florida green turtle and loggerhead turtle sequences are identical and differ from the Hawaiian green turtle sequence by five nucleotide changes, which results in two amino acid substitutions. The olive ridley sequence differs from the Florida and Hawaiian green turtle sequences by 15 and 16 nucleotide changes, respectively, resulting in four amino acid substitutions, three of which are unique to the olive ridley sequence. Our data suggest that these closely related turtle herpesviruses are intimately involved in the genesis of fibropapillomatosis.

Spirorchidiasis and fibropapillomatosis in green turtles from the Hawaiian Islands.

Pathologic examination of green turtles (Chelonia mydas) from the Hawaiian Islands (USA) was performed to determine the primary cause of mortality. Lesions were associated with fibropapillomatosis (FP) and/or spirorchidiasis (SP) in 16 of 17 green turtles examined. Gross lesions included moderate to severe emaciation, lobulated fibropapillomas of different size classes, serous atrophy of fat, and edema of subcutaneous tissue and muscle. Anasarca, hydropericardium and pulmonary edema were common findings. The neoplastic lesions observed in the gastrointestinal tract, lungs, liver, and kidneys of 29% of turtles examined were histologically characterized as fibromas. A generalized thickening and hardening of major vessels and thrombosis with partial or complete lumen occlusion were observed in turtles with FP and SP. Histologically, lymphoplasmocytic endarteritis was observed in vessels of turtles with both conditions. Multifocal granulomas were associated with trematode ova in the parenchyma of most organs of all turtles with FP and SP. Spirorchidiasis and FP were considered the primary causes of mortality in the turtles examined. Further studies should focus on the pathogenic interaction of both conditions and their synergism as debilitating and fatal diseases in this threatened species.

Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands.

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.

Detection by ELISA of circulating anti-blood fluke (Carettacola, Hapalotrema, and Learedius) immunoglobulins in Hawaiian green turtles (Chelonia mydas).

An enzyme-linked immunosorbent assay (ELISA) is described utilizing the surface glycocalyx crude antigen of adult blood trematodes Learedius learedi, Hapalotrema dorsopora, and Carettacola hawaiiensis, for the detection of circulating antibodies (Ab) in Hawaiian green turtles (Chelonia mydas) naturally infected with the parasites and with or without green turtle fibropapillomatosis (GTFP). A concentration of 10.0 micrograms/ml of antigen was optimal in terms of test specificity and sensitivity. A direct ELISA with anti-reptilian/amphibian phosphatase-labeled IgG identified C. mydas Ab at a dilution of 1/12,800. Utilizing indirect ELISA, it was possible to detect Ab to blood flukes at a dilution of 1/3,200. The gross lesions and histopathology were typical for cardiovascular spirorchidiasis in C. mydas. No significant relationship was found between the size of turtles and the degree of GTFP severity. Forty-seven of 59 (80%) samples, from 5 sites, gave a positive ELISA reaction; 6 of the 47 (13%) specimens gave significantly (P < 0.001) higher absorbance values, with 5 from the same location. All 12 (20%) ELISA-negative turtles originated from 1 site, and the absorbance values of the 41 animals from this location were significantly lower (P < 0.015) when compared with the other 4 sites. The proposed assay is fast, has the feature of visual scoring, and can be used for determination of exposure of C. mydas to the spirorchid trematodes.

Trans-Pacific migrations of the loggerhead turtle (Caretta caretta) demonstrated with mitochondrial DNA markers.

Juvenile loggerhead turtles (Caretta caretta) have recently been documented in the vicinity of Baja California and thousands of these animals have been captured in oceanic fisheries of the North Pacific. The presence of loggerhead turtles in the central and eastern North Pacific is a prominent enigma in marine turtle distribution because the nearest documented nesting concentrations for this species are in Australia and Japan, over 10,000 km from Baja California. To determine the origin of the Baja California feeding aggregate and North Pacific fishery mortalities, samples from nesting areas and pelagic feeding aggregates were compared with genetic markers derived from mtDNA control region sequences. Overall, 57 of 60 pelagic samples (95%) match haplotypes seen only in Japanese nesting areas, implicating Japan as the primary source of turtles in the North Pacific Current and around Baja California. Australian nesting colonies may contribute the remaining 5% of these pelagic feeding aggregates. Juvenile loggerhead turtles apparently traverse the entire Pacific Ocean, approximately one-third of the planet, in the course of developmental migrations, but mortality in high-seas fisheries raises concern over the future of this migratory population.

Evaluation of Hawaiian green turtles (Chelonia mydas) for potential pathogens associated with fibropapillomas.

Thirty-two juvenile green turtles (Chelonia mydas) were captured alive in Kaneohe Bay, Island of Oahu, Hawaii, during September 1991. Ten of the turtles sampled were afflicted with green turtle fibropapillomatosis (GTFP) in varying degrees of severity. Virus isolation attempts were negative in all individuals. Using nasopharyngeal and cloacal swabs, we isolated 28 Gram negative bacteria, five Gram positive cocci, Bacillus spp., and diphtheroids. The most common isolates included Pseudomonas fluorescens (68%), P. putrefaciens (66%), Vibrio alginolyticus (50%), non-hemolytic Streptococcus (50%), V. damsela (47%), and V. fluvialis (47%). Chlamydial antigen was detected in four of the turtles sampled. The primary lesions in animals with GTFP were hyperplasia of squamous epithelial cells and mesodermal proliferation with a marked degree of orthokeratotic hyperkeratosis. Mites, leeches, and other organisms were associated with the surface of papilloma lesions. The etiologic agent of GTFP was not isolated.

Carettacola hawaiiensis n. sp. (Trematoda: Spirorchidae) from the green turtle, Chelonia mydas, in Hawaii.

Carettacola hawaiiensis n. sp. (Trematoda: Spirorchidae) is described from the hepatic vessels of the green turtle, Chelonia mydas (L.), in Hawaii. The new species differs from any previously described species of Carettacola in size, placement of vitellaria, and shape and placement of Laurer's canal. The genus Haemoxenicon Martin and Bamberger, 1952, becomes a synonym of Carettacola Manter and Larson, 1950. Haemoxenicon stunkardi Martin and Bamberger, 1952, is transferred to the genus Carettacola Manter and Larson, 1950, and becomes Carettacola stunkardi n. comb. An emended generic diagnosis for Carettacola is given along with a key to the species.