Neonatal biometry and characterization of Amazonian Turtle nests (Podocnemis expansa)

The present study aimed to study the reproduction of the Amazon turtle (Podocnemis expansa) through the evaluation of the reproductive parameters of adult females. This study was carried out in the Crixás-Açu River, municipality of Mundo Novo, state of Goiás, Brazil. In September, the biometrics of 20 females were evaluated during the spawning period. Their nests were marked for subsequent evaluation of hatchlings, measuring the distances of each nest to the river and vegetation. The second stage consisted of the evaluation of hatchlings and characterization of nests after egg hatching. The datawere tested using the Pearson Correlation to measure the degree of linear correlation between the variables analyzed, such as the parameters of females, hatchlings, nests, and eggs. Nest depth was positively correlated with the number of eggshells found. We also found a correlation between the number of eggsshells and the distance of nests to the vegetation. Although moderate, this result indicates that as the distance to the vegetation increases, the number of eggshells found in nests decreases. The spawning site for Amazon turtle breeding is crucial to determine a higher number of eggshatching and better development of hatchlings. However, the literature is still very scarce regarding the choice of spawning grounds and the influence that vegetation may have on the development of freshwater turtle hatchlingsin Brazil.

Sex ratio of the green sea turtle Chelonia mydas (Testudines: Cheloniidae) hatchlings in the Guanahacabibes Peninsula, Cuba / Proporción sexual de las crías de la tortuga verde Chelonia mydas (Testudines: Cheloniidae) en la Península de Guanahacabibes, Cuba

Introduction: Sea turtles have temperature dependent sex determination. The increase in global temperature leads to higher nest temperatures that can cause a prevalence of females, threatening the future of these species. Objective: The present work aims to evaluate the trend of incubation temperatures and the incubation period, as well as to estimate the sex ratio in nests of Chelonia mydas at Antonio and La Barca beaches, Southwestern Cuba, during the seasons from 2012 to 2018. Methods: Temperature data loggers were placed in green turtle nests with a representativeness that varied between the years analyzed. To assess the temporal variation of temperatures and incubation periods, a Kruskal-Wallis test was performed in each case. Sex ratio was estimated from its relation with temperature and incubation duration. Results: At La Barca beach, there was a 1.5 °C increase in the mean nest temperature from 2012 to 2018, although no differences were found in the period from 2015 to 2018. At Antonio beach, there is no trend since no differences were found in the mean nest temperature except for the years 2013 and 2017, which had lower temperatures than the other seasons. In both beaches mean nest temperature exceeded 30 °C in most of the years. As a result, there was a predominance of nests with incubation periods shorter than 55 days. With these values, a female hatchling production over 90 % is expected in both study sites. Conclusions: In correspondence with the registered temperature and incubation period values, most of the years reflect a hatchling production biased towards females in both beaches.

Introducción: La determinación del sexo en tortugas marinas depende de la temperatura. El aumento de la temperatura global conlleva a temperaturas más altas en los nidos, lo cual puede provocar un predominio de las hembras, amenazando el futuro de estas especies. Objetivo: El presente trabajo busca evaluar la tendencia de las temperaturas y los períodos de incubación, así como estimar la proporción sexual en nidos de Chelonia mydas,en las playas Antonio y La Barca, suroeste de Cuba, durante las temporadas del 2012 al 2018. Métodos: Se colocaron sensores para registrar la temperatura en los nidos de esta especie con una representatividad que varió entre los años analizados. Para evaluar la variación temporal de las temperaturas y los periodos de incubación, se realizó una prueba de Kruskal-Wallis en cada caso. Las estimaciones de las proporciones sexuales se realizaron a partir de la temperatura y duración de la incubación. Resultados: En playa La Barca, hubo un incremento de 1.5 °C en la temperatura promedio del nido del 2012 al 2018, aunque no se encontraron diferencias en el periodo de 2015-2018. En playa Antonio, no hay tendencia ya que no se encontraron diferencias en la temperatura promedio del nido, excepto para el 2013 y 2017, los cuales tuvieron temperaturas menores que en las otras temporadas. En ambas playas la temperatura promedio del nido excedió 30 °C en la mayoría de los años. Como resultado, hubo un predominio de nidos con periodos de incubación inferiores a 55 días. Con estos valores, se espera una producción de hembras en las crías superior al 90 % en ambos sitios de estudio. Conclusiones: En correspondencia con los valores de temperatura y periodo de incubación registrados, la mayoría de los años refleja una producción de crías sesgada hacia las hembras en ambas playas.

Red-eared slider hatchlings (Trachemys scripta) show a seasonal shift in behavioral types.

Correlated and repeatable patterns of behavior, termed behavioral types, can affect individual fitness. The most advantageous behavioral type may differ across predictable environments (e.g., seasonally), and maternally mediated effects may match hatchling behavior to the environment. We measured righting response, an indicator of behavioral type, of juvenile red-eared slider turtles (Trachemys scripta) emerging from early and late season clutches to understand if the production of behavioral types differs across the nesting season. There was a significant effect of season, with early season hatchlings righting more quickly than late season hatchlings, and we explored two potential underlying mechanisms, maternal estrogens and maternal investment (e.g., yolk allocation). We dosed early season eggs with an estrogen mixture to mimic late season eggs and assayed hatchling righting response, but found no significant effect of this maternal effect. We assessed maternal investment by measuring egg, hatchling, and residual yolk masses. We found a seasonal pattern in yolk allocation, where early season eggs have more yolk than late season eggs. Early season hatchlings used more yolk for growth rather than maintenance of existing tissues, resulting in larger hatchlings. Interestingly, across both seasons, hatchlings that received less maternal yolk appeared to be more efficient at converting yolk to tissue, but we found no direct correlation with righting behavior. We demonstrate that the prevalence of behavioral types varies across the nesting season, creating correlated suites of seasonal phenotypes in turtle hatchlings, but it appears that neither maternal estrogens or investment in yolk directly underlie this shift in behavior.

Green turtle (Chelonia mydas) population demographics at three chemically distinct foraging areas in the northern Great Barrier Reef.

The catchments of the Great Barrier Reef (GBR) have experienced significant modifications in recent decades, leading to increases in sources of pollutants and declines in coastal water quality. As coastal waters of the GBR support some of the highest density green turtle (Chelonia mydas) foraging populations in the western Pacific Ocean, understanding the effects of contaminants on GBR green turtle populations is a priority. In 2012, elevated strandings of green turtles in the Upstart Bay region instigated the WWF's collaborative Rivers to Reef to Turtles (RRT) project to investigate if coastal pollutants are compromising green turtle health. Important to interpreting these investigations into toxicology and health is understanding the demographics of the green turtle populations being investigated. In three green turtle foraging grounds, Cleveland Bay (CLV), Upstart Bay (UPB) and the Howick Group of Reefs (HWK), this study explored population size, age class structure, sex ratio, growth rates, body condition and diet, as well as indices of turtle health, such as plastron barnacle loads and eye lesions. The three foraging populations had similar age class structure and adult sex ratios to other green turtle foraging populations in the GBR. Somatic growth rate was nonlinear, peaking in immature turtles, and was much slower in turtles foraging at HWK compared to the other two sites. This may have been due to differences in food source, which was supported by the observed dietary shifts between seagrass and algae in HWK turtles, compared to a consistently seagrass diet in CLV and UPB turtles. There were also small differences in body condition between sites, as well as differences in barnacle loads, eye lesions and occurrence of fibropapilloma tumors. This study provides important information on green turtle foraging ground population dynamics in the northern GBR, and context for the other papers in this special issue.

Influencia de la vigilancia en una zona rocosa dentro de un área marina protegida, Pacífico Central de Costa Rica / Surveillance influence in a rocky shore within a marine protected area, Central Pacific of Costa Rica

Resumen Los intermareales rocosos son sitios importantes para peces, aves, invertebrados y algas entre otros, que se utilizan para la extracción de recursos alimenticios para las comunidades costeras. Se presentan los resultados de la evaluación de una costa rocosa en el Pacífico Central de Costa Rica (Refugio Nacional de Vida Silvestre Playa Hermosa-Punta Mala), usando indicadores biológicos propuestos por el protocolo del Ministerio de Ambiente y Energía. Se analizó la influencia de la vigilancia en dos sitios dentro de un AMP sobre el intermareal rocoso. La recolección de datos se realizó durante la época seca y la época lluviosa de 2015. Los datos mostraron que Nerita scabricosta presentó muchas colonias formadas por pocos individuos. La cobertura de macroalgas y los invertebrados bajo las rocas mostraron una leve diferencia entre los niveles de vigilancia. La densidad de Tetraclita stalactifera no mostró variaciones. Se sugiere la hipótesis que el daño mecánico asociado al turismo, pesca y extracción, está influyendo sobre los organismos de la zona rocosa. Algunos de los bioindicadores utilizados mostraron ser influenciados por los niveles de vigilancia y aplicación de las medidas de manejo.

Abstract Rocky intertidal are important sites for fish, birds, invertebrates and algae, among others, which are used for the extraction of food resources for coastal communities. We present the results of conservation assessment of a rocky shore in the Central Pacific of Costa Rica (Playa Hermosa-Punta Mala National Wildlife Refuge) using the biological indicators proposed by the National Environmental Ministry. This study aims to analyze the influence of surveillance, between two sites, over the rocky shore habitat. The sampling was carried out during both the dry and rainy seasons in 2015. Our data shows that Nerita scabricosta -harvested in small-scale fisheries- presents a high number of colonies formed by few individuals. Moreover, the abundance varied between seasons, probably due to a storm surge. Macroalgae differed slightly among sites, where turf had higher percentage coverage in low surveillance areas, while brown algae had higher coverage in high surveillance areas. Mechanical damage (abrasion due to rocks overturning) associated with tourism and harvesting could be influencing the low surveillance area, explaining these differences. Macroalgae presented a seasonal change, probably related to the storm surge. The density of the Tetraclita stalactifera did not exhibit variations. The surveillance level and enforcement influenced some bioindicators of the rocky shore, and therefore could be used to assess the extractive pressure. Rev. Biol. Trop. 66(3): 984-995. Epub 2018 September 01.

Cadmium induces histopathological injuries and ultrastructural changes in the liver of freshwater turtle (Chinemys reevesii).

The study investigated the histopathological and ultrastructural lesions of liver of freshwater turtle Chinemys reevesii exposed to Cadmium (Cd). The animals were exposed to 0 mg kg-1 (0.85% normal saline (NS)), 7.5 mg kg-1, 15 mg kg-1, 30 mg kg-1 Cd chloride separately by intraperitoneal injection. Liver samples were collected for examination of lesions under light and electronic microscopes. Results showed that liver tissues from Cd -treated animals presented various degrees of histopathological lesions. Liver cells showed swollen, degeneration and necrosis with dose-dependent manner. Under electronic microscope, nucleus, mitochondria and rough endoplasmic reticulum presented various degrees of lesions with dose-dependent manner. In conclusion, Cd has significant toxicity on liver tissue of the freshwater turtle, which occurs in a dose-dependent manner.

De novo transcriptome analysis reveals insights into different mechanisms of growth and immunity in a Chinese soft-shelled turtle hybrid and the parental varieties.

The Chinese soft-shelled turtle (Pelodiscus sinensis) is a highly important freshwater aquaculture species in China. The molecular mechanisms underlying changes in immunity and growth in hybrid vigor are not well understood. In the present study, the transcriptomes from significantly different P. sinensis strains (Qingxi black turtle, B and Japanese strain, J) and the resulting hybrid (Zajiao-1, F) were sequenced using an Illumina sequencing platform. Differentially expressed genes (DEGs) between Zajiao-1 and the Qingxi black turtle were enriched mainly in the HTLV-I infection and Hippo signaling pathways, while DEGs between the Zajiao-1 and Japanese strain were enriched mainly in tryptophan metabolism, caner-associated pathways, transcriptional dysregulation in cancer, amebiasis, Fcγ-mediated phagocytosis and the peroxisome pathway. Highly expressed genes involved in the regulation of disorders of the fatty acid biosynthesis, immune and cardiovascular systems in P. sinensis were found among the DEGs. Enrichment categories for gene ontology included cellular processes, metabolic pathways, and the actin cytoskeleton pathway. The reliability of the sequencing data was verified through quantitative real-time polymerase chain reaction analysis of 20 immunity or growth-related genes. These findings offer new insights into heterosis of growth traits and resistance to stresses and potential strategies for selective breeding.

Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles (Chrysemys picta).

Environmental chemicals can disrupt endocrine signaling and adversely impact sexual differentiation in wildlife. Bisphenol A (BPA) is an estrogenic chemical commonly found in a variety of habitats. In this study, we used painted turtles (Chrysemys picta), which have temperature-dependent sex determination (TSD), as an animal model for ontogenetic endocrine disruption by BPA. We hypothesized that BPA would override TSD and disrupt sexual development. We incubated farm-raised turtle eggs at the male-producing temperature (26°C), randomly assigned individuals to treatment groups: control, vehicle control, 17ß-estradiol (E2, 20ng/g-egg) or 0.01, 1.0, 100µgBPA/g-egg and harvested tissues at hatch. Typical female gonads were present in 89% of the E2-treated "males", but in none of the control males (n=35). Gonads of BPA-exposed turtles had varying amounts of ovarian-like cortical (OLC) tissue and disorganized testicular tubules in the medulla. Although the percentage of males with OLCs increased with BPA dose (BPA-low=30%, BPA-medium=33%, BPA-high=39%), this difference was not significant (p=0.85). In all three BPA treatments, SOX9 patterns revealed disorganized medullary testicular tubules and ß-catenin expression in a thickened cortex. Liver vitellogenin, a female-specific liver protein commonly used as an exposure biomarker, was not induced by any of the treatments. Notably, these results suggest that developmental exposure to BPA disrupts sexual differentiation in painted turtles. Further examination is necessary to determine the underlying mechanisms of sex reversal in reptiles and how these translate to EDC exposure in wild populations.

Conservación y actividad reproductiva de tortuga lora ( Lepidochelys olivacea ) en la playa de anidación solitaria Punta Banco, Pacifico Sur de Costa Rica. Recomendaciones de manejo a través de dieciséis años de monitoreo / Conservation and reproductive activity of Olive Ridley sea turtles (Lepidochelys olivacea) in Punta Banco, a solitary nesting beach in South Pacific Costa Rica: Management recommendations after sixteen years of monitoring

Resumen Se realizó un monitoreo de la actividad de anidación de tortugas marinas en Punta Banco, Pacífico Sur de Costa Rica, de forma ininterrumpida por diez y seis años, acompañado por actividades de conservación como la relocalización de los nidos en viveros. Se realizaron análisis de éxito de eclosión para nidos "in situ" y en vivero. Evaluamos por este medio la biología reproductiva de la tortuga lora (Lepidochelys olivacea), por mucho la tortuga más común que anida en Punta Banco (98% de las tortugas observadas). Entre 1996 y 2011, se llevó a cabo el monitoreo diario de las actividades de anidación de julio a diciembre. Marcamos un total de 1 239 tortugas L. olivacea y registramos 4 130 eventos de anidación. El total de anidaciones para L. olivacea varió de 239 en el año 1996 a 402 en 1999, mientras que el número de hembras avistadas varió de 18 en 1996 a 146 en 1999. Las hembras presentaron un largo curvo del caparazón promedio de 66.8cm y ancho curvo de caparazón promedio de 70.7cm. La media para el tamaño de las nidadas fue de 96.7 huevos, con una frecuencia de puesta observada de 1.07 nidos/hembra y un intervalo de puesta observado de 19.95 días. El éxito de eclosión de nidos "in situ" fue de 61.38%, mientras que el éxito de nidos relocalizados en viveros fue de 77.9%. La población de L. olivacea en Punta Banca muestra una tendencia positiva.

Abstract Sea turtle nesting activity was monitored in Punta Banco, South Pacific Costa Rica uninterruptedly for sixteen years, accompanied by conservation activities such as nest relocation in hatcheries. Hatching success evaluations were held for "in situ" and relocated nests. We hereby evaluate the reproductive biology of the olive ridley sea turtle (Lepidochelys olivacea), by far the most common turtle sea turtle nesting in Punta Banco (98% of observed turtles). Daily monitoring of nesting activities was held every year from July to December, from 1996 to 2011. We tagged a total of 1 239 turtles and recorded 4 130 nesting events. The number of recorded nesting events per nesting season ranged from 239 in 1996 to 402 in 1999, whereas the number of observed females ranged from 18 in 1996 to 146 in 1999. Nesting females had an average curved carapace length of 66.8cm and an average curve carapace width of 70.7cm. The average clutch size was 96.7, with an observed frequency of 1.07 nests/female and an nesting interval of 19.95 days. Hatching success of "in situ" nests was 61.38%, whereas hatching success for nests relocated into hatcheries was 77.9%. The L. olivacea population in Punta Banco beach displays a a positive trend. Rev. Biol. Trop. 63 (Suppl. 1): 383-394. Epub 2015 April 01.

Selección de sitios de anidación de Lepidochelys olivacea (Testudines: Cheloniidae) en el Pacífico Sur de Costa Rica / Nest-site selection of Lepidochelys olivacea (Testudines: Cheloniidae) in the South Pacific region of Costa Rica

Resumen La selección del sitio de anidación en tortugas marinas es determinante para la sobrevivencia de la progenie, sin embargo para Lepidochelys olivacea existe poca información al respecto. Este trabajo se enfocó en determinar si L. olivacea selecciona sus sitios de anidación de acuerdo a un patrón o preferencia espacial determinada, o si por el contrario distribuye sus nidos aleatoriamente en la playa. La recolección de datos se realizó entre julio y setiembre del 2011, en las playas de Piro y Pejeperro, Península de Osa, Costa Rica. La ubicación del nido se evaluó midiendo las distancias entre la línea de marea y el nido, entre el nido y la vegetación, y determinando la posición del nido con respecto a los sectores marcados cada 100m en la playa. En ambas playas, las tortugas mostraron una marcada preferencia por anidar lo más lejos posible de la línea de marea, lo cual protege a sus nidos de inundación pero los expone a un mayor riesgo de depredación. Además, en playa Piro se detectó una mayor concentración de nidos cerca de la desembocadura del río, y en las cercanías de un estero en el caso de Pejeperro. Si estas zonas están asociadas a un mayor éxito de eclosión, es posible que las tortugas estén regresando y seleccionado esos sitios por impronta o filopatría. Futuras investigaciones deberán enfocarse en determinar si en eventos de anidación sucesivos, L. olivacea muestra repetibilidad y mantiene sus preferencias de anidación a nivel de individuo.

Abstract In sea turtles, nest-site selection plays a key role in the survival of the offspring, with a direct influence on the sex of the hatchlings, incubation period, embryonic mortality, and hatchling size. Nest-site choice has been studied for most sea turtle species, but the information for Lepidochelys olivacea, the most abundant sea turtle species, is scarce. The present work explores the spatial nesting patterns of L. olivacea and seeks to determine whether this species employs a scatter nesting strategy or chooses to clump its nests based on a specific spatial preference. Night patrols were conducted between July and September 2011 at the beaches of Piro and Pejeperro, located in the Osa Peninsula, in the South Pacific region of Costa Rica. Nest-site location was evaluated by measuring the distance from the tide line to the nest, distance from nest to vegetation, and by noting beach sector according to beach-marker setup. In total, 84 nesting turtles were observed. Sea turtles in both beaches showed a strong preference to locate their nests as far from the tide line as possible, crawling longer distances when presented with wider stretches of beach. Turtles nesting at Piro beach also favored areas that were close to the river mouth, while turtles at Pejeperro preferred to nest near an estuary. It seems that the main driving force that influences L. olivacea to choose a nest site is to protect the eggs from tidal inundation, even if this means a higher predation risk from nesting too close to the vegetation, in addition to a longer trip to the ocean for both the female adult and the hatchlings. Nests near rivers and estuaries may have higher hatching success, which could prompt females to return and select these areas due to imprinting or philopatry. Future research focused on recapture could provide valuable information on individual behavior, and prove whether or not L. olivacea shows strong nest-site fidelity and repeatability of nesting preferences at the individual level. Rev. Biol. Trop. 63 (Suppl. 1): 375-381. Epub 2015 April 01.

Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina.

Ambient temperatures during embryonic development determine gonadal sex in many reptiles. The temperature sensitive period for sex determination has been defined by shifting eggs between female- and male-producing temperatures in a few species. This phase spans 20-35% of embryogenesis in most species, which makes it difficult to define the mechanisms that transduce temperature into a signal for ovarian versus testicular development. We present an extensive set of studies that define a brief period when high temperature specifies, and then determines, ovarian fate in a northern population of snapping turtles, Chelydra serpentina. We shifted embryos from male to female temperatures, or vice versa, at various stages of development. Gonads in embryos incubated at female temperatures commit to ovarian fate earlier (by stage 18) than gonads in embryos incubated at male temperatures commit to testicular fate (by stages 19-21). In double shift studies, embryos were incubated at a female temperature, exposed to a male temperature for set times, and shifted back to the original temperature, or vice versa. The time required to induce ovarian development (≤6 days at female temperatures) was much shorter than the time required to induce testicular formation (>20 days at male temperatures). Differentiation of the gonads at the histological level occurred after the sex-determining period. Nevertheless, we found that a change in temperature rapidly (within 24h) influenced expression and splicing of WT1 mRNA: the absolute abundance of WT1 mRNA, the relative abundance of +KTS versus -KTS isoforms, as well as the ratio of +KTS:-KTS isoforms was higher in gonads at a male versus a female temperature. In conclusion, ovarian fate is more readily determined than testicular fate in snapping turtle embryos. The short sex-determining period in this species (6-8% of embryogenesis) will facilitate studies of molecular mechanisms for specification and determination of gonad fate by temperature.

Immunocytochemistry suggests that the prevalence of a sub-type of beta-proteins determines the hardness in the epidermis of the hard-shelled turtle.

The corneous layer of the epidermis in hard-shelled turtles largely derives from the accumulation of beta-proteins as indicated by microscopic, in situ hybridization, and immunocytochemical and Western blotting analysis. The expression of mRNAs of one of the most common type of beta-proteins shows higher expression in upper spinosus and pre-corneous keratinocytes of growing scutes. Two beta-proteins of 14-16 kDa, indicated as Tu2 and Tu17 and representing two subtypes of beta-proteins co-accumulate in the thick corneous layer of the epidermis in hard-shelled turtle. The two beta-proteins apparently mix in differentiating and mature corneocytes although Tu2 appears more prevalent than Tu17. The specific role of the different subtypes in the formation of the hard corneous material of the carapace and plastron is not clear. It is hypothesized that the relative amount of beta-proteins belonging to the two subclasses in relation to the alpha-keratin meshwork present in keratinocytes contributes to the formation of a variably resistant and inflexible corneous layer. Tu17 may have a more globular structure than Tu2 and is likely present in denser areas of the corneous layer containing also alpha-keratin. The increase of cysteine-glycine-rich beta-proteins in the matrix located among alpha-keratin filaments may allow the formation of a hard corneous material, probably through increase of cross-bridge formation and hydrophobicity.

In ovo inhibition of steroid metabolism by bisphenol-A as a potential mechanism of endocrine disruption.

During embryonic development, endogenous signals, for example steroid hormones, and exogenous signals, for example endocrine disrupting chemicals (EDCs), have the capacity to produce phenotypic effects that persist into adulthood. As the actions of steroids are mediated through the binding of steroid receptors, most studies of EDCs have assumed that they too elicit their effects by binding steroid receptors. We tested an alternative hypothesis, namely that EDCs elicit their effects during embryonic development by disrupting the metabolism of maternally derived steroids, thereby allowing maternally derived steroids to bind steroid receptors and elicit effects. Specifically, we examined the ability of the EDC, bisphenol-A (BPA) to inhibit the normal metabolism of oestradiol during the first nine days of embryonic development in the red-eared slider turtle (Trachemys scripta). We found that, when BPA was present, oestrogen metabolism was inhibited when compared to control eggs. In particular, the formation of oestrone sulfate was blocked in BPA-treated eggs. We postulate that the oestrogenic effects of EDCs may be driven, at least in part, by inappropriate oestrogen signalling. The retention of oestrogens at points of development when they would normally be metabolized to inactive forms might also help explain low-dose effects frequently reported for EDCs.

Effect of dietary cadmium on fitness, growth, genotoxicity and accumulation in the Yellow-spotted River Turtle, Podocnemis unifilis.

The aim of this study was to expose the Yellow-spotted River Turtle, Podocnemis unifilis, to dietary cadmium (Cd) contamination. The P. unifilis were fed with a Cd contaminated diet (590 µgg(-1)) or a control diet for 30 and 60 days. After the Cd feeding period, the locomotor performance and specific growth rate were assessed. Blood samples were drawn for micronuclei analysis and tissues were collected to analyze the Cd concentration. Dietary Cd influenced the fitness of turtles at 30 days (righting time 752s), but not after 60 days (righting time 43.67s). Micronuclei in erythrocytes (12 ± 5‰) were significantly greater in contaminated turtle at 60 days. Cd accumulation is found in gut, intestine, kidney, fat, liver and blood of animals from contaminated diet group and the Cd concentration of almost all the tissues had increased following the 30-60-day feeding period. Cd does not impair animal the fitness after sixty days of dietary treatment, but it does can cause an accumulation on P. unifilis.

Biological activity of oestradiol sulphate in an oviparous amniote: implications for maternal steroid effects.

Understanding the many factors that underlie phenotypic variation is of profound importance to evolutionary biologists. The embryonic endocrine environment is one such factor that has received much attention. In placental amniotes, the dynamic interaction of maternal and embryonic steroid production and metabolism is critical to regulating the endocrine environment. Less is known about how embryos of oviparous amniotes regulate their endocrine environment because most studies have focused on relating initial steroid levels in the yolk at oviposition to offspring phenotype. We tested the hypothesis that embryos of oviparous amniotes regulate their endocrine environment by conjugating maternal steroids and subsequently using the metabolites as precursors for steroid production later in development. Using the red-eared slider turtle (Trachemys scripta), we first characterized the conjugation of exogenous oestradiol to either oestradiol glucuronide or oestradiol sulphate (E(2)-S) in ovo during the first 15 days of development. Results show that oestradiol is primarily conjugated to E(2)-S. We then examined whether E(2)-S influenced sex determination and report that E(2)-S increases the production of female offspring. These data demonstrate that oviparous amniotes can both sulphonate steroids and respond to sulphonated steroids during embryonic development in a manner similar to placental amniotes.

Histología gonadal y criterios fenotípicos de maduración en las tortugas marinas Chelonia mydas y Eretmochelys imbricata (Testudines: Chelonidae) de Cuba

Gonadic histology and phenotypical maturation criteria in the marine turtles Chelonia mydas and Eretmochelys imbricata (Testudines: Chelonidae) from Cuba. Gonad maturity is usually evaluated through macroscopic analysis of the gonads. In sea turtles, the maturation stages are associated with body size, depending on the studied marine stock. Fishermen classify turtles bigger than 65.0cm as sexually mature. If they have secondary sex characters they are recorded as breeding males. We compared body size with macroscopic and microscopic gonad characteristics in two Cuban turtles. Eighteen individuals of C. mydas and twenty of E. imbricata was obtained from the legal fishery stock of Jardines del Rey Archipelago (Cuba), from October 2005 and 2006. In males, breeding condition (maximum spermiogenesis) was checked by histological analysis of the testes. In females, sexual maturity was identified by the presence of vitellogenic follicles or ovarian corpora. Most males were immature (C. mydas: 79.0cm; E. imbricata: 73.1±4.9cm, n=3) and lacked secondary sex characters. Some E. imbricata without a developed penis were in spermatogenic stages II to IV (i.e. pubescent). Most females were immature (C. mydas: 79.6±7.7cm, n=17; E. imbricata: 69.0±7.1cm, n=16; i.e.prepubescent and pubescent. The prepubescent females had ovaries with previtellogenic follicles near 1.0mm in a compact and yellowish stroma. The pubescent females had ovaries with previtellogenic follicles between 2.0 and 3.0mm. The stroma was more loosened and irrigated than in prepubescent turtles. The finding of spermatogenic activity in pubescent males indicates asynchrony between testicular and penial development in E. imbricata. The current phenotypical approach used by fishermen is not enough to determine sexual maturation in these turtles. The minimal size tentatively should be changed to: C. mydas: above 93.0cm and E. imbricata above 79.0cm. Rev. Biol. Trop. 58 (1): 287-298. Epub 2010 March 01.

La madurez gonadal se suele evaluar a través del análisis macroscópico de las gónadas. En las tortugas marinas, las etapas de maduración están asociadas con el tamaño del cuerpo, dependiendo de la población estudiada. Los pescadores clasifican las tortugas que miden más de 65.0cm como maduras sexualmente. Si tienen los caracteres sexuales secundarios se registran como machos en edad reproductora. Nosotros comparáramos el tamaño del cuerpo con las características gonadales macroscópicas y microscópicas de dos tortugas de Cuba. Se obtuvieron dieciocho individuos de C. mydas y veinte de E. imbricata en la pesquería legal del Archipiélago Jardines del Rey (Cuba), entre octubre de 2005 y 2006. En los machos, el estado reproductivo (máxima espermiogénesis) se comprobó mediante el análisis histológico de los testículos. En las hembras, la madurez sexual fue identificada por la presencia de folículos vitelogénicos u ovarios corpora. La mayoría de los machos eran inmaduros (C. mydas: 79.0cm; E. imbricata: 73.1±4.9cm, n=3) y carecían de los caracteres sexuales secundarios. Algunos E. imbricata que no tenían un pene desarrollado se encontraban en fases espermatogénicas entre II y IV (p. ej. pubescentes). La mayoría de las hembras eran inmaduras (C. mydas: 79.6±7.7cm, n=17; E. imbricata: 69.0±7.1cm, n=16; p. ej. prepubescente y pubescente). Las hembras prepubescentes tenían ovarios con folículos previtelogénicos de cerca de 1.0mm en un estroma compacto y amarillento. Las hembras pubescentes tenían ovarios con folículos previtelogénicos entre 2.0 y 3.0mm. El estroma fue más distendido e irrigado que en las tortugas pubescentes. El hallazgo de actividad espermatogénica en machos pubescentes indica la asincronía entre el desarrollo de testículos y pene en E. imbricata. El criterio fenotípico actual utilizado por los pescadores no es suficiente para determinar la madurez sexual de estas tortugas. La talla mínima tentativa debe cambiar a: C. mydas: por encima de 93.0cm y E. imbricata por encima de 79.0cm.

Características da reprodução de tartarugas marinhas (Testudines, Cheloniidae) no litoral sul da Bahia, Brasil / Reproduction of sea turtles (Testudines, Cheloniidae) in the Southern Coast of Bahia, Brazil

Poucos estudos foram realizados sobre a nidificação de tartarugas marinhas no litoral sul da Bahia, região que está passando por um processo rápido de ocupação turística que pode ameaçar ainda mais estas espécies. Por estes motivos, este estudo teve como objetivos identificar as espécies de tartarugas marinhas que desovam no local e descrever as principais características de suas ninhadas. Durante quatro temporadas reprodutivas, de 2004 a 2008, foram identificados e monitorados os ninhos encontrados nas praias localizadas entre Uruçuca e Itacaré no litoral sul da Bahia. As variáveis coletadas foram: tamanho de ninhada, duração da incubação, sucesso de eclosão e fases de mortalidade embrionária. Foi registrada uma média de 37 ± 6,2 ninhos por temporada, com maior freqüência de ninhos das espécies Caretta caretta e Eretmochelys imbricata, tendo sido registradas também desovas esporádicas de Chelonia mydas. A temporada de desova de C. caretta ocorre de outubro a janeiro e de E. imbricata de dezembro a março. Este deslocamento temporal das temporadas reprodutivas pode estar relacionado a uma adaptação para minimizar a competição interespecífica. Além da época de nidificação, as duas espécies diferiram no tamanho de suas ninhadas, mas não diferiram na duração da incubação e no sucesso de eclosão. Diante dos resultados, acredita-se que áreas com baixa concentração de ninhos também devem ser alvo de programas de conservação, devido à sua relevância para a ampliação da variação genotípica e, sobretudo, fenotípica, das espécies de tartarugas marinhas, de forma que as mesmas possam se adaptar e sobreviver às constantes ameaças às quais estão submetidas. Tais programas de conservação podem ser estabelecidos por meio de parcerias entre organizações não governamentais e instituições privadas e a instituição governamental responsável pela conservação das tartarugas marinhas (Centro TAMAR/ICMBio).

Few studies have been carried out on nesting of sea turtles in the southern coast of Bahia, an area that is experiencing a fast process of tourist occupation what can increase levels of threat to these species. Thus, this study aimed to identify sea turtle species that nest in the area and to describe the main characteristics of their clutches. During four reproductive seasons, from 2004 to 2008, we identified and monitored nests found at beaches located between the municipalities of Uruçuca and Itacaré, in the southern coast of Bahia. The collected variables were: clutch size, incubation duration, hatching success and stages of embryonic mortality. We registered an average of 37 ± 6.2 nests per season, with nests of Caretta caretta and Eretmochelys imbricata being more frequent, along with sporadic nests of Chelonia mydas. Nesting season of C. caretta extends from October to January and that of E. imbricata from December to March. This temporary displacement of reproductive seasons might be related to a selective adjustment to minimize competition among species. Besides the time of nesting, the two species differed also in clutch size, but they did not differ in incubation duration and in hatching success. According to our results, we believe that areas with low concentration of nests should also be a target of conservation programs, due to their relevance to the increase of genotypic, and specially fenotypic, variation of sea turtle species, so that Brazilian populations can adapt and survive to the constant threats. Those conservation programs can be established by partnerships between non-governmental organizations and private institutions and the Brazilian government institution for the conservation of sea turtles (Centro TAMAR/ICMBio).

A modified yolk biopsy technique improves survivorship of turtle eggs.

Nongenetic maternal contributions, such as steroid hormones, have received much attention in recent years because they have the potential to influence offspring phenotype. Research in oviparous taxa has demonstrated that there is among-species variability in their response to these maternal contributions. However, studies in chelonians and crocodilians have been hampered by the fact that techniques involving egg manipulations that breach the eggshell routinely result in massive egg mortality. In this study, we present an improved yolk manipulation technique that resulted in increased egg survival (in excess of 70% survival) in the red-eared slider turtle (Trachemys scripta) and that may be broadly applicable to other species. By elevating survival to a level on par with other oviparous taxa, this method permits a more thorough exploration of reptilian egg physiology and allows for studies that examine traits in both the egg and the resulting hatchling.

Steroid signaling and temperature-dependent sex determination-Reviewing the evidence for early action of estrogen during ovarian determination in turtles.

The developmental processes underlying gonadal differentiation are conserved across vertebrates, but the triggers initiating these trajectories are extremely variable. The red-eared slider turtle (Trachemys scripta elegans) exhibits temperature-dependent sex determination (TSD), a system where incubation temperature during a temperature-sensitive period of development determines offspring sex. However, gonadal sex is sensitive to both temperature and hormones during this period-particularly estrogen. We present a model for temperature-based differences in aromatase expression as a critical step in ovarian determination. Localized estrogen production facilitates ovarian development while inhibiting male-specific gene expression. At male-producing temperatures aromatase is not upregulated, thereby allowing testis development.

Growth-related changes in histology and immunolocalization of steroid hormone receptors in gonads of the immature male green turtle (Chelonia mydas).

Studies on the population dynamics of sea turtles require histological evaluation of the ontogenetic development and the activity of the gonads for reproduction. To investigate the growth-related changes of gonads in the immature male green turtle (Chelonia mydas), the histological changes of testes and epididymides and the localization of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor were examined. The testes were categorized histologically into six developmental stages, and a scarce relationship between straight carapace length and gonadal development was confirmed based on the histological analysis. Several kinds of steroid hormone receptors were examined to show distributions in both testes and epididymides, for which their immunoreactivities were enhanced according to the developmental stage of the testes. These results suggest that straight carapace length is not an adequate indicator of maturity determination, whereas histological and immunohistochemical evaluations are useful in identifying the growth stages of green turtles, owing to the higher sensitivity to steroid hormones that appear during growth.