George H. Balazs

Global population structure and natural history of the green turtle chelonia mydas in terms of matriarchal phylogeny

To address aspects of the evolution and natural history of green turtles, we assayed mitochondrial (mt) DNA genotypes from 226 specimens representing 15 major rookeries around the world. Phylogenetic analyses of these data revealed (1) a comparatively low level of mtDNA variability and a slow mtDNA evolutionary rate (relative to estimates for many other vertebrates); (2) a fundamental phylogenetic split distinguishing all green turtles in the Atlantic‐Mediterranean from those in the Indian‐Pacific Oceans; (3) no evidence for matrilineal distinctiveness of a commonly recognized taxonomic form in the East Pacific (the black turtle C.m. agassizi or C. agassizi); (4) in opposition to published hypotheses, a recent origin for the Ascension Island rookery, and its close genetic relationship to a geographically proximate rookery in Brazil; and (5) a geographic population substructure within each ocean basin (typically involving fixed or nearly fixed genotypic differences between nesting populations) that suggests a strong propensity for natal homing by females. Overall, the global matriarchal phylogeny of Chelonia mydas appears to have been shaped by both geography (ocean basin separations) and behavior (natal homing on regional or rookery‐specific scales). The shallow evolutionary population structure within ocean basins likely results from demographic turnover (extinction and colonization) of rookeries over time frames that are short by evolutionary standards but long by ecological standards.

Adrenal and Hematological Responses to Stress in Juvenile Green Turtles (Chelonia mydas) with and without Fibropapillomas

This study reports baseline adrenocortical, hematological, and plasma biochemical values for clinically healthy juvenile green turtles from a discrete population at Kaneohe Bay, island of Oahu, Hawaii. Using a general linear modeling program, we compared mean values for these parameters with mean values of a group afflicted with green turtle fibropapillomas (GTFP). Turtles of similar size classes from both groups were collected under the same conditions in the same study area and season at the same time of the day. Corticosterone, hematological, and enzymatic responses to acute and chronic stress were characterized for each group at four different sampling periods: 0 h (within 2 min of capture), 1 h, 3-4 h, and 24 h postcapture. On the basis of the differences identified between groups and times within a group, we conclude that turtles with GTFP are chronically stressed and immunosuppressed.

Organic contaminants and trace metals in the tissues of green turtles (Chelonia mydas) afflicted with fibropapillomas in the Hawaiian islands

Environmental contaminants have been listed as a possible cause of green turtle fibropapillomas (GTFP). Brain, fat, liver, and kidney tissues from 10 juvenile green turtles (Chelonia mydas) afflicted with GTFP, were tested to determine exposure to selected environmental pollutants and any possible relation to GTFP. One juvenile green turtle free of the disease, one pelagic green turtle, and one pelagic loggerhead turtle (Caretta caretta) served as controls. Egg shells and tissues from three green turtle hatchlings were also tested. The tissues and shells analysed in this study indicated that none contained any of the listed organochlorine, polychlorinated biphenyl, organophosphate, or carbamate insecticides in concentrations above the stated method of detection limits. Most of the concentrations of selenium and heavy metals were also considered to be below levels reported normal in other animal species. No correlation was found between the contaminants tested and GTFP because of the low levels detected. Trace metals and other pollutants tested in this study play a minor role in the aetiology of GTFP in a discrete green turtle population at Kaneohe Bay, Island of Oahu, Hawaii.

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.

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 fibropapillomatosis (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.

Daily movements, habitat use, and submergence intervals of normal and tumor-bearing juvenile green turtles (Chelonia mydas L.) within a foraging area in the Hawaiian islands

Depth-sensitive ultrasonic transmitters monitored the horizontal and vertical movements of 12 juvenile (<65 cm carapace length) green turtles (Chelonia mydas L.) in Kaneohe Bay, Oahu (Hawaii, USA). This site was chosen because of its accessibility, its importance as a foraging area, and the high incidence (≈50%) of fibropapillomatosis, a tumor disease of unknown etiology. Our objectives were to determine the daily movements, habitat use, and submergence intervals of normal and tumor-bearing animals. The presence of tumors had no obvious effects on movement patterns or habitat use. All turtles remained within a small portion of the bay where patch reefs and shallow coral-covered areas are common, and algal growth most abundant. During daylight, two normal and two tumor-bearing animals remained within known feeding areas, all other turtles studied stayed within deep mud bottom channels or within crevices on the sides of reefs. All, except one tumor-bearing turtle, moved up on to shallow patch reefs or shallow coral-covered areas at night. Submergence intervals for both groups were short (over 90% were 33 min or less and none exceeded 66 min) compared to maximum breath-hold times (up to 5 h) measured in the laboratory by earlier workers. Juvenile green turtles in Hawaii, therefore, most likely maintain aerobic metabolism while submerged and surface before oxygen stores are significantly depleted. Tumor-bearing turtles had a higher frequency of longer submergence intervals during the night, indicating they may have been somewhat less active at night. Normal turtles showed no such day-night difference.

The Potential Role of Natural Tumor Promoters in Marine Turtle Fibropapillomatosis

Abstract Fibropapillomatosis (FP) in green turtles Chelonia mydas is a debilitating, neoplastic disease that has reached worldwide epizootic levels. The etiology of FP is unknown but has been linked to oncogenic viruses. Toxic benthic dinoflagellates (Prorocentrum spp.) are not typically considered tumorigenic agents, yet they have a worldwide distribution and produce a tumor promoter, okadaic acid (OA). Prorocentrum spp. are epiphytic on macroalgae and seagrasses that are normal components of green turtle diets. Here we show that green turtles in the Hawaiian Islands consume Prorocentrum and that high-risk FP areas are associated with areas where P. lima and P. concavum are both highly prevalent and abundant. The presence of presumptive OA in the tissues of Hawaiian green turtles further suggests exposure and a potential role for this tumor promoter in the etiology of FP.

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 parenchyrna 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.

Land Use, Macroalgae, and a Tumor-Forming Disease in Marine Turtles

Wildlife diseases are an increasing concern for endangered species conservation, but their occurrence, causes, and human influences are often unknown. We analyzed 3,939 records of stranded Hawaiian green sea turtles (Chelonia mydas) over 28 years to understand fibropapillomatosis, a tumor-forming disease linked to a herpesvirus. Turtle size is a consistent risk factor and size-standardized models revealed considerable spatial and temporal variability. The disease peaked in some areas in the 1990s, in some regions rates remained constant, and elsewhere rates increased. Land use, onshore of where the turtles feed, may play a role. Elevated disease rates were clustered in watersheds with high nitrogen-footprints; an index of natural and anthropogenic factors that affect coastal eutrophication. Further analysis shows strong epidemiological links between disease rates, nitrogen-footprints, and invasive macroalgae and points to foraging ecology. These turtles now forage on invasive macroalgae, which can dominate nutrient rich waters and sequester environmental N in the amino acid arginine. Arginine is known to regulate immune activity, promote herpesviruses, and contribute to tumor formation. Our results have implications for understanding diseases in aquatic organisms, eutrophication, herpesviruses, and tumor formation.

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.