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.

Heavy Metal Contamination in Phrynops geoffroanus (Schweigger, 1812) (Testudines: Chelidae) in a River Basin, So Paulo, Brazil

The Piracicaba River basin is considered the most disturbed river basin in the state of So Paulo. Considerable amounts of agricultural residues are seasonally drained into the river, and the region is also highly urbanized and industrialized with an incipient sewage treatment system. The presence of heavy metals has been previously reported for the water and riverbed in Piracicaba river basin. In this study we evaluated 13 heavy metals in the blood of 37 Geoffroy`s side-necked turtles, Phrynops geoffroanus, from Piracicaba River and Piracicamirim Creek, one of its tributaries. Blood levels of As, Co, Cr, Se and Pb varied among sites, whereas Sn varied between males and females. However, no obvious pathology was detected. Serum level of Cu (2,194 ng g(-1)) and Pb (1,150 ng g(-1)) found in this study are the highest ever described for any reptile; however, no clinical symptoms have been detected in the present study. There is no information about the time scale of such contamination, which could be currently subclinical and yet lead to a breakdown in the population reproductive success in a few years. Based on the present study, legal enforcement is urged in order to locate and extirpate heavy metal sources in the Piracicaba River basin. In addition, monitoring should include humans and commercial fish consumed in local markets.

Structure of a Population of Hydromedusa maximiliani (Testudines, Chelidae) from Parque Estadual da Serra do Mar, an Atlantic Rainforest Preserve in Southeastern Brazil

We studied the structure of a population of Hydromedusa maximiliani associated with a stream in Parque Estadual da Serra do Mar, Nucleo Itutinga-Piloes, southeastern Brazil, between October 2004 and October 2005. Twenty-five individuals were captured, and a population size of 43.72 +/- 23.7 individuals was estimated. This value is similar to that of the population of Parque Estadual Carlos Botelho, another Atlantic forest reserve of southeastern Brazil. Males were recaptured more frequently than females, suggesting higher activity and/or greater movement of males.

Cheirogaster sp. (O. Testudines, Fam. Testudinidae, Geochelone sl.) du Paléogène de Naia, Tondela et l’âge du gisement

A land tortoise from a new locality at Naia, Tondela, is described. It is to be reported either to an advanced form of the genus Hadrianus or to an archaic representative of Cheirogaster; it may be included in the comprehensive genus Geochelone s.l., excluding however Ergilemys and its descendants. There is a strong possibility in favour of Cheirogaster. Testudo must also be excluded. It is not possible to classify this specimen at species'level. Our specimen does agree best with Upper Eocene Testudinidae and with some Lower Oligocene ones. Its age is certainly not Upper Oligocene or later, nor Lower and Middle Eocene. This datation is not opposed to the age of the fossiliferous clays of Naia as supposed by correlation with another locality - Côja, about 30 km to the South - which yielded an assemblage of mammals whose Ludian (Upper Bartonian s.l.) age seems well established. Naia and Côja's fossil-bearing clays must be nearly synchronous; their origin is well in place among the phenomena related to the surrection of iberian Central Chain during paroxysmal phase of pyrenean orogenesis.

Anoplotherium (mammalia, artiodactyla) et geochelone (reptilia, testudines) à côja: les vertebras fossils et l’éocène supérieur au Portugal

A mammal (Anoplotherium cf. commune) and a land tortoise (Geochelone is. gen.] sp.) from the Ludian (Uppermost Eocene) locality of Côja have been identified. Age can be more accurately established now, from level 3 to level 5 in the Ludian stage, probably 4. Relationships between Côja's feldspathic sands, a correlative unit «Arenitos de Vale Furado», and the paroxysmal phase of pyrenean orogeny are confirmed.

Avaliação da predação de Podocnemis expansa e Podocnemis unifilis (Testudines, Podocnemididae) no rio Javaés, Tocantins

Podocnemis expansa e P. unifilis são animais de vida longa, com uma demorada maturação sexual, o que influencia uma baixa taxa de substituição de indivíduos. Suas populações são caracterizadas por uma pequena mortalidade dos animais adultos, mas alta taxa de mortalidade de filhotes e embriões. Sendo a predação natural de ninhos e filhotes um dos fatores mais importantes do baixo sucesso de eclosão dessas espécies. No rio Javaés, os ovos e recém-eclodidos podem ser predados por uma grande diversidade de animais: dentre as aves, urubus (Coragyps atratus e Cathartes aura), carcará (Polyborus plancus), jaburu (Jabiru mycteria); lagartos (Tupinambis teguixin) e mamíferos de pequeno porte, coati (Nasua nasua) e cachorro-do-mato (Cerdocyon thous). Do total anual de desovas de P. unifilis em média 65,98% são predadas, sendo 41,68% de forma total e 24,30% parcialmente. Enquanto que apenas 5,31% das ninhadas de P. expansa são sempre parcialmente predadas. Dentre os predadores aquáticos existem diversos peixes, principalmente piranhas (Serrasalmus nattereri) e jacarés (Melanosuchus niger e Caimam crocodilus). Os predadores das fêmeas de P. unifilis são: jacaré-açu (Melanosuchus niger), onça-pintada (Panthera onca) e onça-parda (Puma concolor). Enquanto que as fêmeas de P. expansa em postura, somente são predadas por P. onca. As fêmeas de P. unifilis em postura são predadas num total médio de 3,93% anualmente, enquanto que para P. expansa a média anual é 5,66% das fêmeas.

Avaliação de padrão irregular dos escudos do casco em Podocnemis expansa e Podocnemis unifilis (Testudines, Podocnemididae)

O casco de Podocnemis expansa (tartaruga-da-amazônia) e Podocnemis unifilis (tracajá), os maiores quelônios de água doce da América do Sul, é uma estrutura única que diferencia esse grupo dos vertebrados atuais e está associado a alguns padrões comportamentais. O padrão dos escudos que compõem o casco dos quelônios é bastante uniforme, mas variações já foram descritas e analisadas para um grande número de espécies em todas as famílias existentes atualmente. Averiguou-se a incidência de irregularidades do padrão de escutelação no casco dessas espécies, durante os anos de 1999 a 2003 no rio Javaés, entorno do Parque Nacional do Araguaia e da Área de Proteção Ambiental Bananal/ Cantão, oeste do estado do Tocantins. P. expansa se apresenta como uma espécie mais suscetível à ocorrência de anormalidades no casco em relação a P. unifilis. Sendo que, dos 14.378 filhotes avaliados, (13,32 %) apresentavam irregularidades no padrão de escutelação enquanto que dos 1.329 filhotes de P. unifilis apenas (4,44 %). Em P. expansa, os defeitos ocorreram quase que em sua totalidade na carapaça (98,64 %) e em menor número (83,05 %) em P. unifilis.

Description of behavioral patterns of Podocnemis erythrocephala (Spix, 1824) (Testudines: Podocnemididae) (Red-headed river turtle) in captivity, Manaus, Amazonas, Brazil

The biology and ecology of South American turtles is still poorly known, particularly, for the Brazilian species. Laboratory studies are essential to understand the life cycles of aquatic turtles species and to help in formulating management plans for their conservation. As a contribution to the knowledge of Podocnemis erythrocephala species, we give a description of its species-typical behaviors, categorized as: maintenance, locomotion, feeding, agonistic and reproduction, based on captives observations of four pairs of turtles in an aquarium in Manaus, Brazil. Similarities and differences with the repertoires of other turtle species are discussed, concluding that turtles have much more complex adaptative strategies and social life than was believed.

Ciclo ovárico y jerarquía folicular de Peltocephalus dumerilianus (Testudines: Podocnemididae)

Se analizaron cuarenta pares de ovarios de hembras adultas de Peltocephalus dumerilianus (Schweigger 1812), para establecer los cambios morfológicos mensuales producidos durante un año. Se llevaron a cabo exámenes macroscópicos de los oviductos y de los ovarios de especímenes que los habitantes ribereños del municipio de Barcelos, Amazonas, Brasil, utilizaron para su consumo, usando metodológicamente la medida del diámetro y conteo de los folículos ováricos y de los cuerpos lúteos. Los resultados muestran la presencia gradual de folículos acorde con la época reproductiva, resaltando que durante el último tercio del proceso, justo antes de la postura se hacen más evidentes; se analiza la relación existente entre el tamaño de la hembra y la masa ovárica, la cual es estadísticamente significativa (F (10,29)=3,655, p < 0,05). Se hace evidente la existencia de una jerarquía ovario folicular que demuestra que para esta especie solo es posible fisiológicamente una postura por año.

Histologia e histoquímica do tubo digestório de Phrynops geoffroanus (Testudines, Chelidae)

Phrynops geoffroanus é o quelônio onívoro com mais ampla distribuição geográfica na América do Sul. Este trabalho descreve a histologia e histoquímica do tubo digestório desta espécie, relacionando as características dos órgãos com seu hábito alimentar. O esôfago, estômago e intestino de quatro espécimes foram fixados em formol 10% e incluídos em parafina por técnica histológica de rotina. Depois, cortes de 5 µm de espessura foram corados com hematoxilina-eosina (HE), ácido periódico de Schiff (PAS) e alcian blue (AB) pH 0.4 e 2.5. O tubo é formado pelas camadas mucosa, submucosa, muscular e adventícia ou serosa. A mucosa do esôfago e do estômago é revestida pelo epitélio simples cilíndrico com células mucossecretoras, onde estão inseridas glândulas intraepiteliais na porção do esôfago e fossetas gástricas desembocando em glândulas no estômago. O estômago divide-se em anterior, médio e posterior, de acordo com a profundidade das fossetas e a concentração de glândulas gástricas. O intestino é revestido pelo epitélio simples cilíndrico com borda estriada e células caliciformes e divide-se em anterior e posterior, de acordo com o padrão de dobramentos da mucosa e o número de células caliciformes. Reatividade ao PAS e AB é observada em todo o tubo. Fibras musculares lisas estão presentes na camada mucosa de todos os segmentos. A camada muscular é formada por duas subcamadas de músculo liso, exceto na porção posterior do estômago. Este estudo ajudará no entendimento da fisiologia digestiva da espécie investigada e fornecerá dados para análises comparativas com outros quelônios.

Ontogenetic variation of plastral spotting pattern in Phrynops hilarii (Testudines, Chelidae)

The plastral spotting variation in the chelid turtle Phrynops hilarii (Duméril & Bibron, 1835) in relation to sex, size, and geographic procedence of individuals was analyzed. States for qualitative characters were analyzed using non-parametric tests. Quantitative characters (shell and scute measurements) were standardized for body size by linear regression against carapace length, and were subjected to principal components analysis and canonical discriminant function analysis. Results suggest that increased plastral spotting is a polymorphic ontogenetic trait in P. hilarii. Neither hatchlings nor juveniles have plastral pattern moderately or heavily pigmented. The simplest pattern, however, may persist without changes in some adults. There are no differences between sexes. The spatial distribution of the plastral pattern is not ordered latitudinally or longitudinally, showing no relationship with gradients of elevation, temperature, or precipitation. This pattern trait lacks of taxonomic significance. The morphometric analysis failed to reveal any character of diagnostic utility in the plastron to support the possibility that these patterns correspond to different sympatric taxa.

Os Testudines continentais do Rio Grande do Sul, Brasil: taxonomia, história natural e conservação

O Rio Grande do Sul é o estado mais meridional do Brasil, apresentando fauna e flora peculiares associadas às características morfoclimáticas da região. A diversidade de Testudines do Rio Grande do Sul é representada por seis espécies continentais e cinco marinhas. Este estudo apresenta comentários sobre a diversidade de quelônios continentais do Rio Grande do Sul, através de uma compilação de dados publicados e alguns inéditos sobre sua biologia e estado de conservação.

Temperature-sex determination in Podocnemis expansa (Testudines, Podocnemididae)

This study has been carried out at the central region of the Araguaia river on the border between the states of Goiás and Mato Grosso in the Brazilian Amazon Basin from September to December 2000. We recorded temperature fluctuation, clutch-size, incubation period and hatching success rate and hatchlings' sex ratio of five nests of Podocnemis expansa (Schweigger, 1812). Despite the relatively small sample size we infer that: a) nests of P. expansa in the central Araguaia river have a lower incubation temperature than nests located further south; however, incubation period is shorter, hatching success rate is lower and clutch-size is larger; b) Podocnemis expansa may present a female-male-female (FMF) pattern of temperature sex-determination (TSD); c) thermosensitive period of sex determination apparently occur at the last third of the incubation period; and, d) future studies should prioritize the relationship between temperature variation (i.e., range and cycle) and embryos development, survivorship and sex determination.