Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Hematological profile of Chelonia mydas (Testudines, Cheloniidae) according to the severity of fibropapillomatosis or its absence

The green turtle Chelonia mydas feeds and nests in the Brazilian coastal area and is considered an endangered species by the World Conservation Union (IUCN 2009) and threatened by the Red List of Brazilian Fauna (Ministério do Meio Ambiente 2009). Fibropapillomatosis is a disease characterized by benign skin tumors (fibropapillomas), and it is one of the main threats to the survival of this species. Studies suggest the involvement of viruses as infectious agents associated with environmental and genetic factors. Blood samples were collected from 45 turtles captured in the coastal area of the state of Sao Paulo, Brazil. From these, 27 were affected by fibropapillomas and 18 were tumor free. Biometrical data on the turtles, size, location and quantity of tumors were recorded. The area occupied by fibropapillomas per animal was calculated and four groups were determined according to severity of the disease or its absence. The objective of the study was to compare hemogram results of the sea turtles classified in these four groups. The lowest hematocrit value was observed in severely affected animals. In the hemoglobin assay, the highest value was observed in the group of tumor free turtles and the lowest, in animals severely affected. Lymphocyte counts and curved carapace length were on the verge of statistical significance.

Lesions caused by cardiovascular flukes (Digenea : Spirorchidae) in stranded green turtles (Chelonia mydas)

Evidence of infection with spirorchid flukes (Digenea: Spirorchidae) was sought at necropsy of 96 stranded green turtles, Chelonia mydas, that were examined during the course of a survey of marine turtle mortality in southeastern Queensland, Australia. Three species of spirorchid (Hapalotrema mehrai, H. postorchis, and Neospirorchis schistosomatoides) were identified. Severe disease due to spirorchid fluke infection (spirorchidiasis) was implicated as the principal cause of mortality in 10 turtles (10%), and appeared to be one of multiple severe problems in an additional 29 turtles (30%). Although flukes were observed in only 45% of stranded C. mydas in this study, presumed spirorchid fluke infection was diagnosed in an additional 53% of turtles, based principally on characteristic necropsy lesions and to a lesser extent on the histopathological detection of spirorchid eggs. Characteristic necropsy lesions included miliary spirorchid egg granulomas, which were observed most readily on serosal surfaces, particularly of the small intestine. Cardiovascular lesions included mural endocarditis, arteritis, and thrombosis, frequently accompanied by aneurysm formation. Resolution of thrombi was observed to occur via a combination of granuloma formation about indigestible components (spirorchid fluke egg shells) and exteriorization through the vessel wall, which resulted in granulomatous nodules on the adventitial surface. Septic aortic thrombosis complicated by disseminated bacterial infection, observed in five turtles, was recorded for the first time. Egg granulomas were ubiquitous in turtle tissues throughout this study. Although they generally appeared to be mild or incidental lesions, they were occasionally associated with severe multifocal granulomatous pneumonia or meningitis.

Hapalotrema (Digenea : Spirorchidae) in the green turtle (Chelonia mydas) in Australia

Hapalotrema mehrai Rao, 1976 and Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchidae) are redescribed from the heart and pulmonary arteries of the green turtle, Chelonia mydas, from Moreton Bay in south-eastern Queensland. Hapalotrema pambanensis Gupta and Mehrotra, 1981 from C. mydas in India is made a synonym of H. mehrai. Hapalotrema dorsopora Dailey, Fast and Balazs, 1993 from C. mydas from Hawaii was described with a dorsally opening uterine pore, but this is found to be the opening of Laurer's canal; therefore H. dorsopora is also made a synonym of H. mehrai. In addition to differences in the numbers of testes and general dimensions, H. mehrai and H. postorchis differ in the development of Laurer's canal and in the absence of a canalicular seminal receptacle in H. postorchis.

Development of the pulmonary surfactant system in the green sea turtle, Chelonia mydas

This study describes the developmental changes in pulmonary surfactant (PS) lipids throughout incubation in the sea turtle, Chelonia mydas. Total phospholipid (PL), disaturated phospholipid (DSP) and cholesterol (Chol) harvested from lung washings increased with advancing incubation, where secretion was maximal at pipping, coincident with the onset of pulmonary ventilation. The DSP/PL ratio increased, whereas the Chol/PL and the Chol/DSP ratio declined throughout development. The phospholipids, therefore, are independently regulated from Chol and their development matches that of mammals. To explore whether hypoxia could elicit an effect on the development of the PS system, embryos were exposed to a chronic dose of 17% O-2 for the final similar to 40% of incubation. Hypoxia did not affect incubation time, absolute, nor relative abundance of the surfactant lipids, demonstrating that the development of the system is robust and that embryonic development continues unabated under mild hypoxia. Hypoxia-incubated hatchlings had lighter wet lung weights than those from normoxia, inferring that mild hypoxia facilitates lung clearance in this species. (C) 2001 Elsevier Science B.V. All rights reserved.

Temperature variation within and between nests of the green sea turtle, Chelonia mydas (Chelonia : Cheloniidae) on Heron Island, Great Barrier Reef

Four temperature data-loggers were placed in each of five green sea turtle nests on Heron Island in the 1998-99 nesting season. Temperatures in all nests increased as incubation progressed due to general sand heating and increased metabolic heat production of the developing embryos. Even at the top of nests no daily diurnal fluctuation in temperature was evident. The temperature of eggs in the middle of the nest increased above those in the nest periphery during the last third of incubation. However, this metabolic nest heating would have little effect on hatchling sex ratio because it occurred after the sex-determining period. Small differences in temperature between regions of a nest persisted throughout incubation and may be important in ensuring the production of at least some individuals of the opposite sex in nests that have temperatures close to either the all-male or all-female determining temperatures. Location and degree of shading of nests had little effect on mean nest temperature, but deeper nests were generally cooler and therefore were predicted to produce a higher proportion of males than were shallower nests. Nest temperature profile data indicated that the 1998-99 nesting season on Heron Island would have produced a strongly female-biased sex ratio amongst hatchlings.

Incubation temperature, energy expenditure and hatchling size in the green turtle (Chelonia mydas), a species with temperature-sensitive sex determination

Eggs from the Heron Island, Great Barrier Reef, nesting population of green turtles (Chelonia mydas) were incubated at all-male-determining (26 degreesC) and all-female-determining (30 degreesC) temperatures. Oxygen consumption and embryonic growth were monitored throughout incubation, and hatchling masses and body dimensions were measured from both temperatures. Eggs hatched after 79 and 53 days incubation at 26 degreesC and 30 degreesC respectively. Oxygen consumption at both temperatures increased to a peak several days before hatching, a pattern typical of turtle embryos, and the rate of oxygen was higher at 30 degreesC than 26 degreesC. The total amount of energy consumed during incubation, and hatchling dimensions, were similar at both temperatures, but hatchlings from 26 degreesC had larger mass, larger yolk-free mass and smaller residual yolks than hatchlings from 30 degreesC. Because of the difference in mass of hatchlings, hatchlings from 30 degreesC had a higher production cost.

Patterns of lipid storage and mobilisation in the female green sea turtle (Chelonia mydas)

Reproductive data from southern Queensland indicate that vitellogenesis in female Chelonia mydas takes approximately 8 months and is followed by a migration to a breeding area. At Heron Island, females lay multiple clutches over approximately 3 months. To investigate how females mobilise and store lipid during the breeding season we collected plasma, yolk, and fat tissue samples from females at a variety of stages during the nesting season. In breeding females, concentrations of plasma triglyceride increased seasonally. They reached peak concentrations during vitellogenesis and courtship, remained high throughout the nesting season, and then declined to a nadir after the last clutch. Plasma protein concentration increased throughout the breeding season, peaking following the last clutch for the season. Yolk lipids were highest during courtship and were similar throughout the nesting season, suggesting that uptake of lipid by ovarian follicles is completed prior to the beginning of the nesting season. Plasma triglyceride decreases in females with prolonged periods of unsuccessful nesting, and total lipid levels in adipose tissue and follicle yolks were significantly lower in atretic females. It appears that: (1) endogenous energy reserves can be reduced by stochastic environmental events (such as those reducing nesting success), and (2) a metabolic shift signalling the end of the nesting season is characterised by a drop in plasma triglycerides and slight increase in total plasma protein.

Variação na dieta da tartaruga verde, Chelonia mydas, e o impacto da ingestão de lixo ao longo da costa brasileira

Neste trabalho nós estudamos a dieta da tartaruga verde, Chelonia mydas, e os fatores envolvidos na variação de sua ecologia alimentar. Avaliamos também o impacto da ingestão de lixo, e os fatores que podem explicar a elevada ingestão destes resíduos entre os animais marinhos. No estudo da ecologia alimentar, nós avaliamos mais de 400 indivíduos, entre dados originais e da literatura, distribuídos ao longo de um gradiente latitudinal e diversos ambientes. As tartarugas se alimentaram majoritariamente de macroalgas, porém apresentaram uma grande plasticidade alimentar, tanto em relação à estratégia de forrageamento quanto à dieta. Nas regiões mais frias e com menor disponibilidade de algas, as tartarugas mudaram de uma dieta herbívora, para uma dieta baseada em matéria animal. Esta mudança de dieta acarretou também em uma mudança de estratégia de forrageamento, saindo da alimentação bentônica para uma alimentação pelágica. Estratégia esta que também foi encontrada nas áreas estuarinas. A plasticidade alimentar se deve à interação de fatores intrínsecos (restrições fisiológicas) e extrínsecos (regionais e locais). As diferenças nas estratégias de forragenamento acarretam também em diferenças na exposição a ameaças. Um exemplo disso é a ingestão de lixo, que apesar de ter sido registrada em mais de 70% das tartarugas (N = 265), representou uma ameaça maior aos animais com estratégia de forrageamento pelágica. O plástico foi o material mais ingerido, tendo como principal fonte itens relacionados à alimentação e sacolas plásticas. O estudo também mostrou que uma quantidade pequena de lixo (0,5 g) é suficiente para causar a morte. Este resultado revelou que o potencial de letalidade por ingestão de lixo é muito maior que a mortalidade observada. A verdadeira ameaça da ingestão de lixo está sendo mascarada pela elevada mortalidade relacionada às atividades pesqueiras. A ingestão de lixo é normalmente atribuída à confusão de um item alimentar específico com o resíduo, como águas-vivas e sacolas plásticas. Porém, nós mostramos que se trata de uma questão mais ampla, e usamos a tartaruga verde, aves marinhas e peixes para ressaltar a importância de outros fatores como: abundância do lixo no ambiente, estratégia de forrageamento, capacidade de detecção do resíduo e amplitude da dieta. Nós acreditamos que a ingestão de lixo ocorre devido a uma armadilha evolutiva muito mais ampla do que a previamente sugerida, e que deve afetar muito mais espécies que as que foram até hoje reportadas. Desarmar esta armadilha será particularmente difícil devido ao contínuo e crescente despejo de plástico no ambiente marinho e sua alta persistência no ambiente.

Hematological profile of Chelonia mydas (Testudines, Cheloniidae) according to the severity of fibropapillomatosis or its absence

The green turtle Chelonia mydas feeds and nests in the Brazilian coastal area and is considered an endangered species by the World Conservation Union (IUCN 2009) and threatened by the Red List of Brazilian Fauna (Ministério do Meio Ambiente 2009). Fibropapillomatosis is a disease characterized by benign skin tumors (fibropapillomas), and it is one of the main threats to the survival of this species. Studies suggest the involvement of viruses as infectious agents associated with environmental and genetic factors. Blood samples were collected from 45 turtles captured in the coastal area of the state of Sao Paulo, Brazil. From these, 27 were affected by fibropapillomas and 18 were tumor free. Biometrical data on the turtles, size, location and quantity of tumors were recorded. The area occupied by fibropapillomas per animal was calculated and four groups were determined according to severity of the disease or its absence. The objective of the study was to compare hemogram results of the sea turtles classified in these four groups. The lowest hematocrit value was observed in severely affected animals. In the hemoglobin assay, the highest value was observed in the group of tumor free turtles and the lowest, in animals severely affected. Lymphocyte counts and curved carapace length were on the verge of statistical significance.

Morfologia do tubo digestório da tartaruga verde (Chelonia mydas)

Para avaliar a morfologia do tubo digestório da tartaruga verde (Chelonia mydas) 10 animais, 9 juvenis e 1 adulto, foram analisados. A retirada dos órgãos digestórios procedeu-se após abertura do plastrão. A descrição e a medida do comprimento de cada órgão [esôfago, estômago, intestino delgado (ID) e intestino grosso (IG)] foram realizadas com o tubo digestório aberto. Os resultados mostraram que tanto nos animais juvenis como no adulto, o esôfago foi marcado pela presença de papilas pontiagudas em sua mucosa interna. O estômago apresentou aspecto saculiforme com fundo cego. No ID foi observado pregas reticulares na mucosa duodenal, enquanto o jejuno e do íleo mostrou pregas retilíneas longitudinais. O IG foi marcado pela alternância de regiões abauladas (haustros ou saculações) e estreitamentos. A microscopia do tudo digestório em C. mydas revelou esôfago com mucosa pregueada revestida por epitélio estratificado pavimentoso queratinizado. O estômago mostrou-se dividido em regiões: cárdica, fúndica, pilórica, as quais diferiam quanto ao número de glândulas e disposição da camada muscular. O ID apresentou-se marcado por vilosidades, e o IG por mucosa pregueada contendo glândulas na lâmina própria. A morfologia do tubo digestório da tartaruga verde mostrou-se adaptada ao seu hábito alimentar, possuindo especializações e um longo trato digestório que promove o aumento da superfície de absorção, já que seu alimento é de difícil digestão.

Morfometria do coração e dos vasos da base e sua implicação no mergulho em Chelonia mydas

Objetivou-se caracterizar a morfologia das câmaras cardíacas e das artérias aortas e pulmonares da espécie Chelonia mydas. Foram avaliados 11 espécimes de C. mydas mortas coletadas no litoral do estado do Rio Grande do Norte, Brasil. Os animais foram necropsiados para a obtenção do coração, fragmentos das artérias aorta e pulmonares direita e esquerda. Os vasos adquiridos foram fixados em formol e submetidos ao processamento histológico de rotina e coloração com Técnica de Verhoff modificada. Enquanto, do coração, os parâmetros largura, altura base-ápice e a circunferência ventricular foram mensurados por meio do paquímetro. Nessa espécie a microscopia das artérias pulmonares e artérias aortas variaram de acordo com o antímero. A maior espessura relativa do Cavum Venosum (CV) auxilia no bombeamento cardíaco durante o mergulho e sua menor espessura direita é uma vantagem para a dilatação ventricular durante a imersão profunda enquanto que a quantificação das lâminas elásticas e fibras musculares da túnica média das artérias aortas e pulmonares direita e esquerda comprovaram que a túnica média das aortas predomina o componente elástico vs. muscular, entretanto, nas artérias pulmonares o componente elástico não-predomina. Essa angioarquitetura pode estar relacionada com a capacidade de mergulho, favorecendo um maior aproveitamento do sangue oxigenado armazenado previamente durante o período de apneia.

Hematological and histopathological evaluation of wildlife green turtles (Chelonia mydas) with and without fibropapilloma from the north coast of São Paulo State, Brazil

Blood profiles were determined in 47 juvenile green turtles, Chelonia mydas, from São Paulo northern coast, Brazil. Twenty-nine were affected by fibropapillomas and 18 were tumor free. Complete gross and histopathologic examinations of the fibropapillo were performed in 21 green turtles. Biometrical data, size, location and amount of tumors were recorded. The papillomas varied in morphology, location, size, color and texture. We found hyperplastic stroma, rich in blood vessels and connective tissue with increase in thickness of the dermis. The tumors w0ere classified as papillomas or fibropapillomas according to their epithelial and/or stromal proliferation. The lowest Mean Corpuscular Hemoglobin (HCM) values were observed in affected turtles.