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.

Detection of polymorphisms of the mtDNA control region of Caretta caretta (Testudines: Cheloniidae) by PCR-SSCP

Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra-and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.

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.

Modulation of the adrenocortical stress response in marine turtles (Cheloniidae): evidence for a hormonal tactic maximizing maternal reproductive investment

The relationships between reproductive condition, level of reproductive investment and adrenocortical modulation to capture stress in marine turtles form the basis of this study. When subjected to either capture or ecological stressors, nesting marine turtles have demonstrated adrenocortical responses that are both small in magnitude, and slow in responsiveness. These observations were further investigated to determine whether this minimal stress response was a physiological strategy to maximize reproductive investment in adult green Chelonia mydas and hawksbill Eretmochelys imbricata turtles. Female green and hawksbill turtles exhibited a decrease in adrenocortical responsiveness with progressive reproductive condition. Breeding turtles exhibited most suppression of their adrenocortical response to capture compared to both non-breeding and pre-breeding female counterparts. Nesting green turtles maintained a suppressed adrenocortical response to capture throughout the nesting season despite decreased reproductive investment. In contrast, male green and hawksbill turtles were less able to modulate their corticosterone (B) response to acute capture stress. During breeding, male turtles possessed significantly greater adrenocortical responses to capture than females. These results could indicate that the large reproductive investment necessary for female marine turtle reproduction might underlie the marked decrease in adrenocortical responsiveness. This hormonal mechanism could function as one strategy by which female marine turtles maximize their current reproductive event, even though under certain situations this mechanism could entail costs to female survival.

Notas sobre neonatos de Lepidochelys Olivácea (Testudines: Cheloniidae) en playa nueva esperanza, Tumbes, Perú

Informa sobre la nidada y neonatos de "tortuga pico de loro" observados en marzo de 2008 en la playa de Nueva Esperanza.

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.

Aspectos da biologia reprodutiva de Eretmochelys imbricata (testudines, cheloniidae) no litoral sul do Rio Grande do Norte, Brasil

Female hawksbill turtles (Eretmochelys imbricata) nesting along the southeastcoastline of Rio Grande do Norte State, Brazil (6º13'40"S, 35º03'05"W) were captured and weighed during the four months from January to April 2007, in the course of the annual egglaying season, which extended from 06 rd November 2006 to 30 rd May 2007. In all, 99 weight measurements were performed. On first contact the females exhibited an average post-oviposition weight of 79.1 kg (range 56.2-98.9 kg, SD = 10.9 kg, n = 44 females). Those individuals which were subsequently recaptured showed a mean weight loss of 1.7 kg (range 0.7-4.5 kg, SD = 1.0 kg, n = 39 sets of measurements on 20 females) in the interval between two consecutive post-ovipositions, separated by a maximum time interval of 17 days. In the cases where the female aborted the nesting process, the pre-oviposition weight was measured. The clutch weight, that is to say, the weight loss between consecutive pre-oviposition and post-oviposition measurements (separated by a maximum time interval of 3 days), was found to be 5.2 kg (range 4.3-6.0 kg, SD = 0.9 kg, n = 6 sets of measurements). This value is significantly higher (t-test, p<0.001) than the loss between two consecutive post-oviposition measurements with the same female. The mean recovery in body weight, that is to say, the average gain in weight between successive post-oviposition and pre-oviposition captures of the same individual (separated by a time interval of 12 to 17 days), was found to be 3.0 kg (range 1.9-4.3 kg, SD = 1.0 kg, n = 4 sets of measurements) Although the small sample size makes it unwise to generalise, the recovery in body weight was found to be always significantly lower (t-test, p<0.005) than the clutch weight. This fact is in agreement with the observed weight loss tendency throughout the breeding season for this species. Considering the clutch weight and the internidal recovery in body weight we found that the total weight loss of the adult hawksbill females after three to five nesting events varied from 10.4% (range 8.7-11.9%, SD = 1.6%, n = 3) to 14.1% (range 11.8-15.4%, SD = 1.3%, n = 6) in relation to their initial pre-oviposition weight. If there were no body weight recovery during the internesting interval we estimate that a female that nests three to five times in the course of the season would lose from 19% to 31% of its initial weight. We emphasise that our clutch weight estimate was performed by weighing the females and not by multiplying the number of eggs in the nest by their average unit weight. In this way, our measurements take into account the loss of liquid during the oviposition. Despite the unequivocal evidence of body weight recovery during the internidal interval, it is not clear if the cause of this process is rehydration or feeding

Contribuição à citogenética de testudines e análise de assimetria flutuante em Eretmochelys imbricata, Cheloniidae

This work has contributed to knowledge of the order Testudines from cytogenetic and morphological point of view. With regard to the aspects proposed cytogenetic characterization of the species Mesoclemmys tuberculata (n = 5), endemic to the Caatinga biomes, through conventional techniques of cytogenetics and molecular levels. This species presented 2n = 58, NF = 64, the first submetacentric pair, the second metacentric and third subtelocentric, and the other microchromosome telocentric. This species showed a nucleolar bearing pair, coincident with the 18S ribosomal rDNA and that proved to be heterochromatic. Small heterochromatic blocks were also found in the centromeres of the largest chromosomes, as well as terminal regions in most other chromosomes of the complement, that were GC +. Telomeric sequences showed variable patterns of signal intensity, with some repeats more intense in microchromosomes and subtly in the larger ones. When compared with other species of the genus, the G-banding patterns showed a marked similarity between them. The first karyotypic description of the species will aid in future studies and the understanding of evolutionary aspects of this family. From the morphological point of view, we carried out studies of fluctuating asymmetry in sea turtle Eretmochelys imbricata, using methods of benchmarking between hatchlings and adults and their implications for natural selection. Data were collected at two different times: first during the spawning female and the second during the outbreak and birth of the nest. The analyzed characteristics consisted of measurements of length and width of front and rear flippers (CANT, LANT, CPOS and LPOS) also collected data on the number of hull plates, side plates (NPL), the surrounding plates (NPCIRC), and plastron; plates power plants (NPP), inframarginais plates (NPIM). With the values of asymmetry we calculated the value of strict heritability for these traits, the calculation was based on only one parent. A nonparametric analysis Mann-Whitneywas performed to compare the groups (females X hatchlings, newborn hatchlings X dead hatchlings). Adult females showed no bilateral fluctuating asymmetry (FA = 0) on the number plates of the hull and plastron, while offspring, living and dead, showed a greater level of variation in these meristic parameters. In the analysis of females x hatchlings we found a significant difference between the levels of asymmetry in hoof plates (p=0.006) an the width of hindlimbs (p=0.001). Levels of FA suggest an accurate indicator as to the viability or maintenance of the individual to the reproductive phase. The coefficient of heritability (h2) of FA , obtained from the regression analysis, showed that both have low and not statistically significant values(p> 0.1). In the case of exclusion of the effective role of genetics in the generation of FA, reproductive strategies based on high number of subsidiaries products, such as those observed in E. imbricata seems to implicate the production of individuals with high level of developmental instability

Occurrence of Amphiorchis solus (Simha & Chattopadhyaya, 1970) (Digenea: Spirorchiidae) Infecting the Green Turtle Chelonia mydas Linnaeus, 1758 (Testudines: Cheloniidae) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Occurrence of Amphiorchis indicus Mehrotra, 1973 (Digenea, Spirorchiidae) infecting Green turtle Chelonia mydas Linnaeus, 1758 (Testudines, Cheloniidae) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Helminth parasites of juvenile green turtles Chelonia mydas (Testudines, Cheloniidae) in Brazil

The present study offers a parasitological analysis of juvenile individuals of the green turtle (Chelonia mydas) found on the Brazilian coast between 2004 and 2011. Helminths were found in 90 out of 136 individuals (66.2%, CI= 57.7 - 74.0). In total, 29,411 helminths were collected, belonging to the families Brachycoeliidae, Cladorchiidae, Microscaphidiidae, Pronocephalidae, Rhytidodidae, and Spirorchiidae. Mean species richness was 4.74 (CI= 4.03 - 5.46), the mean intensity was 327 (CI= 223 - 489) the mean abundance was 216 (CI= 146 - 339). This study also reports new geographical records for: Angiodictyum longum, Angiodictyum parallelum, Rameshwarotrema uterocrescens, Pyelosomum cochlear, Schizamphistomum scleroporum, Cymatocarpus solearis and Neospirorchis sp. This is the first analysis of helminths composition in juveniles of green turtles.

Aspectos da biologia reprodutiva de Eretmochelys imbricata (testudines, cheloniidae) no litoral sul do Rio Grande do Norte, Brasil

Female hawksbill turtles (Eretmochelys imbricata) nesting along the southeastcoastline of Rio Grande do Norte State, Brazil (6º13'40"S, 35º03'05"W) were captured and weighed during the four months from January to April 2007, in the course of the annual egglaying season, which extended from 06 rd November 2006 to 30 rd May 2007. In all, 99 weight measurements were performed. On first contact the females exhibited an average post-oviposition weight of 79.1 kg (range 56.2-98.9 kg, SD = 10.9 kg, n = 44 females). Those individuals which were subsequently recaptured showed a mean weight loss of 1.7 kg (range 0.7-4.5 kg, SD = 1.0 kg, n = 39 sets of measurements on 20 females) in the interval between two consecutive post-ovipositions, separated by a maximum time interval of 17 days. In the cases where the female aborted the nesting process, the pre-oviposition weight was measured. The clutch weight, that is to say, the weight loss between consecutive pre-oviposition and post-oviposition measurements (separated by a maximum time interval of 3 days), was found to be 5.2 kg (range 4.3-6.0 kg, SD = 0.9 kg, n = 6 sets of measurements). This value is significantly higher (t-test, p<0.001) than the loss between two consecutive post-oviposition measurements with the same female. The mean recovery in body weight, that is to say, the average gain in weight between successive post-oviposition and pre-oviposition captures of the same individual (separated by a time interval of 12 to 17 days), was found to be 3.0 kg (range 1.9-4.3 kg, SD = 1.0 kg, n = 4 sets of measurements) Although the small sample size makes it unwise to generalise, the recovery in body weight was found to be always significantly lower (t-test, p<0.005) than the clutch weight. This fact is in agreement with the observed weight loss tendency throughout the breeding season for this species. Considering the clutch weight and the internidal recovery in body weight we found that the total weight loss of the adult hawksbill females after three to five nesting events varied from 10.4% (range 8.7-11.9%, SD = 1.6%, n = 3) to 14.1% (range 11.8-15.4%, SD = 1.3%, n = 6) in relation to their initial pre-oviposition weight. If there were no body weight recovery during the internesting interval we estimate that a female that nests three to five times in the course of the season would lose from 19% to 31% of its initial weight. We emphasise that our clutch weight estimate was performed by weighing the females and not by multiplying the number of eggs in the nest by their average unit weight. In this way, our measurements take into account the loss of liquid during the oviposition. Despite the unequivocal evidence of body weight recovery during the internidal interval, it is not clear if the cause of this process is rehydration or feeding

The effects of tourism, beachfront development and increased light pollution on nesting Loggerhead turtles Caretta caretta (Linnaeus, 1758) on Sal, Cape Verde Islands

Loggerhead Caretta caretta is now the only species of marine turtle nesting on the island of Sal, Cape Verde Islands. Since 2008, ADTMA - SOS Tartarugas has patrolled all the southern beaches of the island in order to protect nesting females and to collect nesting data. Although hunting is still a major issue, with 90 turtles killed in 2009, habitat loss and light pollution are becoming an ever more serious threat. Construction sites, hotels, apartment buildings and restaurants close to beaches, bright lights and illegal removal of sand are contributing to a marked decrease in the total number of nesting turtles on some beaches. In 2009, beaches on Sal experienced an average increase in nests of 200%, while the beach most affected by construction (Tortuga Beach) saw a decrease of nests of 7.3% (from 19.1% of total number of nests in 2008 to 11.8% in 2010). This beach also recorded a much lower nest to emergence ratio than normal (17.6% of emergences resulting in nests compared to 29.9% in other areas), indicating reluctance to nest due to light pollution and other disturbances.