The utility of reptile blood transcriptomes in molecular ecology.

Reptiles and other nonmammalian vertebrates have transcriptionally active nucleated red blood cells. If blood transcriptomes can provide quantitative data to address questions relevant to molecular ecology, this could circumvent the need to euthanize animals to assay tissues. This would allow longitudinal sampling of animals' responses to treatments, as well as sampling of protected taxa. We developed and annotated blood transcriptomes from six reptile species and found on average 25,000 proteins are being transcribed in the blood, and there is a CORE group of 9,282 orthogroups that are found in at least four of six species. In comparison to liver transcriptomes from the same taxa, approximately two-thirds of the orthogroups were found in both blood and liver; and a similar percentage of ecologically relevant gene groups (insulin and insulin-like signalling, electron transport chain, oxidative stress, glucocorticoid receptors) were found transcribed in both blood and liver. As a resource, we provide a user-friendly database of gene ids identified in each blood transcriptome. Although on average 37% of reads mapped to haemoglobin, importantly, the majority of nonhaemoglobin transcripts had sufficient depth (e.g., 97% at ≥10 reads) to be included in differential gene expression analysis. Thus, we demonstrate that RNAseq blood transcriptomes from a very small blood sample (<10 µl) is a minimally invasive option in nonmammalian vertebrates for quantifying expression of a large number of ecologically relevant genes that would allow longitudinal sampling and sampling of protected populations.

Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?

There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild animals respond to environmental challenges. Blood is the best medium for obtaining information about recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 min of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone (CORT), in four reptile species for which blood was taken within a range of times 11 min or less after first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 s), and rock iguanas (Cyclura cychlura; 2 min 44 s), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-min sampling period. In both snake species, samples taken up to 3-7 min after CORT began to increase still had lower CORT concentrations than after exposure to a standard restraint stressor. The "3-min rule" appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others (cottonmouths, fence lizards).

Effects of Chlorella vulgaris polysaccharides accumulation on growth characteristics of Trachemys scripta elegans.

The present study investigated the effects of the accumulated polysaccharides in Chlorella vulgaris microalgae on the growth characteristics of Trachemys scripta elegans. Sodium alginate was used to prepare immobilized C. vulgaris, and the antioxidant effects of the accumulated polysaccharides in it were determined using Caenorhabditis elegans as a model. We determined the specific growth rates of T. s. elegans (10 in each group) and their levels of non-specific immune-related indexes (including alkaline phosphatase; total superoxide dismutase; catalase; malondialdehyde). Under optimal culturing conditions, the accumulated polysaccharide content in C. vulgaris reached 32.7% (dry weight). Polysaccharides from C. vulgaris significantly improved the hydrogen peroxide-induced oxidative stress resistance and resulted in the enhancement of stress resistance-related antioxidant enzymes, including total superoxide dismutase and catalase (p < 0.05). The accumulated polysaccharides in C. vulgaris were heteropolysaccharides comprising rhamnose, ribose, arabinose, xylose, 2-deoxy-D-glucose, mannose, glucose, galactose, and glucosamine with a molar ratio of 0.26: 0.62: 0.21: 0.10: 0.08: 0.18: 1.00: 0.42: 0.17. Compared with the control group with common feeds, suspended and immobilized C. vulgaris with higher accumulated polysaccharide levels had a positive effect on the specific growth rate of the T. s. elegans (p < 0.05). Further, the suspended and immobilized C. vulgaris with higher accumulated polysaccharide levels significantly increased serum alkaline phosphatase, total superoxide dismutase and catalase activity (p < 0.05) and decreased serum malondialdehyde levels of T. s. elegans (p < 0.05).

Molecular identification of blood meals in mosquitoes (Diptera, Culicidae) in urban and forested habitats in southern Brazil.

The study of host associations of mosquitoes (Diptera, Culicidae) provides valuable information to assist in our understanding of a variety of related issues, from their life-history to the entomological surveillance of pathogens. In this study, we identified and characterized mosquito blood meals from both urban and forested areas in the city of Paranaguá, state of Paraná, Brazil, by analyzing the amplification of host DNA ingested by mosquitoes under different storage conditions and digestion levels. Host DNA preservation was evaluated in fresh blood meals according to storage duration (30 to 180 days) and temperature (-20°C / -80°C) and, in digested blood, according the degree of digestion classified on the Sella scale. Molecular analysis of blood meals was based on DNA extraction and amplification of a fragment of the mitochondrial COI gene. We determined that, up to180 days of storage, the evaluated temperatures did not influence the preservation of fresh blood meals DNA, whereas the amplification success was increasingly reduced over the course of the digestion process. The species Anopheles cruzii, Aedes fluviatilis, Aedes scapularis, Psorophora ferox, Culex quinquefasciatus, Culex mollis, and Culex intrincatus, together with specimens representing four subgenera and one genus of Culicidae [Ae. (Ochlerotatus), Cx. (Culex), Cx. (Melanoconion), Cx. (Microculex), and Limatus, respectively] had their blood meals identified. Their diverse host use was evidenced by the identification of 19 species of vertebrate host, namely two amphibians, three mammals and 14 birds. Birds were the most commonly identified host in blood meals. These results not only show the diversity of mosquito hosts, but also underscore the challenges involved in monitoring arboviruses of public health importance, given potential combinations of host use for each mosquito species.

Unraveling avian and reptilian hematology: An optical and electron microscopic study of the buffy coat.

BACKGROUND: Blood centrifugation and buffy coats are at the cornerstone of hematology. In mammals, the buffy coat has a layered disposition (from bottom to top) with neutrophils on top of erythrocytes, followed by monocytes/lymphocytes, and platelets. In nonmammals, this distribution is unknown. Recently, the cell tube block (CTB) technique was developed to study the buffy coat, but it was never applied to nonmammal buffy coats. OBJECTIVES: This study aimed to evaluate using the CTB technique to study reptilian and avian buffy coats and to propose its use for clinical applications. METHODS: Blood from five birds and eight reptiles of different species was obtained to make CTBs that were processed for optical/electron microscopy. H&E, Sirius red, and immunohistochemistry staining against CD3 (to label T lymphocytes) were applied to the CTBs. RESULTS: In birds, the buffy coat had a layered appearance with the granulocyte layer containing granulocytes (heterophils and eosinophils) and nucleated erythrocytes followed by a mononuclear cell layer containing lymphocytes, monocytes, and thrombocytes. In some animals, a nucleated erythrocyte layer was observed admixed with the granulocyte/mononuclear cell layer. A small clot within the buffy coat was seen in seven reptiles, and less definition of layers occurred in reptiles, with only one or two layers. Lymphocytes appeared toward the top of the buffy coat. CONCLUSIONS: From a comparative hematology perspective, the buffy coat of mammals differs from that of birds and more from that of reptiles. The CTB technique can be used to study these differences in avian and reptilian hematology, especially to study atypical circulating cells, hemoparasites, or blood cell proportions in health and disease.

Biometria corporal e parâmetros hematológicos de Trachemys scripta elegans e Trachemys dorbignyi (Testudines: Emydidae) criadas em cativeiro em Petrolina, Pernambuco / Body biometry and hematological parameters of Trachemys scripta elegans and Trachemys dorbignyi (Testudines: Emydidae) reared in captivity in Petrolina/PE, Brazil

Este estudo objetivou avaliar a biometria corporal e o perfil hematológico de Trachemys scripta elegans (N=28) e de Trachemys dorbignyi (N=22) criadas em cativeiro na região do submédio do Vale do São Francisco, semiárido nordestino brasileiro, visando estabelecer valores sanguíneos básicos de saúde e gerar dados úteis na fisiologia comparativa de Testudines. Após 120 dias de adaptação e jejum de 24 horas, 2,5 mL de sangue foram coletados do seio occipital dorsal e depositados em tubo com heparina sódica para a avaliação, na sequência, dos níveis hematologicos. A contagem total de eritrócitos (CTE) e global de leucócitos (CGL) foi realizada em câmara de Neubauer; a dosagem de hemoglobina (HGB) pelo método da método da cianometahemoglobina e o hematócrito (HCT) através da técnica do microhematócrito. A partir da CTE estabeleceram-se matematicamente os índices hematimétricos. A biometria corporal também foi avaliada: a) massa corporal (MC, g); b) dimensões máximas da carapaça [comprimento (CMC, cm) e largura (LMC, cm)];c) dimensões máximas do plastrão [comprimento (CMP, cm) e largura (LMP, cm)]; d) comprimento total da cauda (CTC, cm); e) comprimento linear da base da cauda ao orifício cloacal (CprC, cm); f) comprimento linear do orifício cloacal a extremidade da cauda(CpoC, cm). T. scripta elegans apresentaram valores maiores (P < 0,05) para a biometria corporal, enquanto que o CTC e CprC foram maiores (P<0,05) em T dorbignyi. Os níveis hematológicos não diferiram (P>0,05) entre as espécies. Os resultados demostram que a maior parte da variação observada entre T. scripta elegans e T. dorbignyi é explicada pelas variáveis biométricas e que algumas correlações hematológicas caracterizam diferenças interespecíficas. Conclui-se que os resultados lançam luz sobre valores de referência para estas espécies mantidas em cativeiro na região do semiárido e servem como um modelo para a fisiologia comparativa intra e interespécies.(AU)

This study aimed to evaluate the body biometry and hematological profile of Trachemys scripta elegans (N=28) and Trachemys dorbignyi (N=22) reared in captivity in the Brazilian submedium northeastern semi-arid region in the Valley of the São Francisco river. It aimed to establish basic health blood values and generate useful data on the comparative physiology of Testudines. After 120-day adaptation and 24-hour fasting, 2.5mL of blood were collected from the dorsal occipital sinus and deposited into a tube with sodium heparin for evaluation, following, of hematological levels. The red blood cell count (RBC) and GLC was conducted in a Neubauer chamber, the hemoglobin level (HGB) was supplied by the cyanmethemoglobin method and the hematocrit (HCT) was obtained by the microhematocrit technique. Based on the RBC, the hematimetric were mathematically established. Body biometry were also evaluated: a) body mass (BM, g); b) maximum dimensions of the carapace [length (MLC, cm) and width (MWC, cm)]; c) maximum dimensions of plastron [length (MLP, cm) and width (MWP, cm)]; d) total length of tail (TLT, cm); e) linear length from the base of the tail to the cloacal orifice (LPrC, cm); f) linear length from the cloacal orifice to the extremity of the tail (LPoC, cm). T. scripta elegans showed higher values (P<0.05) for biometrics, while TLT and LPrC were higher (P<0.05) in T. dorbignyi. The hematological values did not differ (P>0.05) among species. The results show that most of the variation found between T. scripta elegans and T. dorbignyi is explained by the biometric variables and that some hematologic correlations characterize interspecies differences. It was conclude that the results shed light on benchmarks for these species kept in captivity in the northeastern semi-arid region and serve as a model for intra and interspecies comparative physiology.(AU)

Glucocorticoids in tuatara (Sphenodon punctatus): Some influential factors, and applications in conservation management.

Glucocorticoid (GC) hormones promote basic life processes, regulate life-history transitions, and help individuals cope with challenges and stressors, thereby playing an important fitness role. Here, we review recent evidence for several factors that influence plasma concentrations of corticosterone (CORT), the main GC in tuatara (Sphenodon punctatus), and discuss the application of CORT as a physiological tool to monitor conservation efforts. Observational studies show an association between CORT concentrations and seasonal reproductive activity, ambient temperature, and ecological habitat parameters (including presence of rats/seabird abundance, sex-ratio, and genetic diversity), and experimental studies show a positive influence of acute temperature increase on the CORT response. Recently, CORT physiology has been applied as a monitoring tool in tuatara translocation programmes. No signs of chronic stress in CORT profiles were observed during standard short- and long-term translocation and rat eradication procedures, giving confidence that current conservation efforts are supportive in population recovery. These results provide a foundation for comparative understanding of stress physiology in reptiles, and will be critical for managing future population viability of tuatara in a changing environment.

Right-to-left shunt has modest effects on CO2 delivery to the gut during digestion, but compromises oxygen delivery.

By virtue of their cardiovascular anatomy, reptiles and amphibians can shunt blood away from the pulmonary or systemic circuits, but the functional role of this characteristic trait remains unclear. It has been suggested that right-to-left (R-L) shunt (recirculation of systemic blood within the body) fuels the gastric mucosa with acidified and CO2-rich blood to facilitate gastric acid secretion during digestion. However, in addition to elevating PCO2 , R-L shunt also reduces arterial O2 levels and would compromise O2 delivery during the increased metabolic state of digestion. Conversely, arterial PCO2  can also be elevated by lowering ventilation relative to metabolism (i.e. reducing the air convection requirement, ACR). Based on a mathematical analysis of the relative roles of ACR and R-L shunt on O2 and CO2 levels, we predict that ventilatory modifications are much more effective for gastric CO2 supply with only modest effects on O2 delivery. Conversely, elevating CO2 levels by means of R-L shunt would come at a cost of significant reductions in O2 levels. The different effects of altering ACR and R-L shunt on O2 and CO2 levels are explained by the differences in the effective blood capacitance coefficients.