Sperm competition in squamate reptiles.

Multiple paternity is ubiquitous within the polyphyletic group called 'reptiles', especially within the lizards and snakes. Therefore, the probability of sperm competition occurring, and being intense, is high. Squamates exhibit a diversity of tactics to ensure fertilization success in the face of sperm competition. The duration of female sperm storage, which can be many months and even years in some species, remains an enigma. Here, we emphasize some mechanisms that might affect patterns of paternity, the source and function of ejaculates and features of the female reproductive tract that may aid in long-term sperm storage. In doing so, we present a new analysis of the relationship between sperm size, the strength of sperm competition and the duration of female sperm storage. Lizards and snakes are a diverse group that has provided many excellent models for the study of an array of life-history strategies. However, when it comes to postcopulatory sexual selection, there is much left to discover. This article is part of the theme issue 'Fifty years of sperm competition'.

Puncture versus capture: which stresses animals the most?

The prerogative of animal welfare science includes wild species and ecological studies. Yet, guidance enshrined in legislation is narrowly derived from studies involving laboratory rodents; legitimacy for non-mammalian free-ranging species is thus debatable. The European directive 2010/63/EU illustrates this problem. It includes this key statement: "Practices not likely to cause pain, suffering, distress or lasting harm equivalent to, or higher than, that caused by the introduction of a needle…" which determines if the directive shall apply. Protocols involving surgery clearly fall within the scope of the directive: procedures are scrutinized, investigators and technicians must be qualified and various agreements are required (e.g. issued by an ethical committee). By contrast, non-invasive procedures, like mark-recapture population studies, merely need a permit from wildlife authorities (at least in most countries). Yet, blood sampling that implies the introduction of a needle-one of the most common practices in animals-could shift any study on the constraining-side of the directive, on the grounds that puncture impacts individuals more severely than capture. We examined the validity of the needle-threshold using the stress response of free-ranging snakes. Our results based on physiological markers show that blood sampling does not add any stress to that triggered by capture, and thus questions the usefulness of the needle-threshold to gauge welfare in wild animals. The specificities of studying wild species should be considered to redress captivity biased animal welfare policy.

Non-venomous snakebites in the Western Brazilian Amazon

Abstract INTRODUCTION: In this study, we examined the clinical manifestations, laboratory evidence, and the circumstances of snakebites caused by non-venomous snakes, which were treated at the Regional Hospital of Juruá in Cruzeiro do Sul. METHODS: Data were collected through patient interviews, identification of the species that were taken to the hospital, and the clinical manifestations. RESULTS: Eight confirmed and four probable cases of non-venomous snakebites were recorded. CONCLUSIONS: The symptoms produced by the snakes Helicops angulatus and Philodryas viridissima, combined with their coloration can be confused with venomous snakes (Bothrops atrox and Bothrops bilineatus), thus resulting in incorrect bothropic snakebite diagnosis.

TGF-ß1 polymorphism in American tegumentary leishmaniasis in a Southern Brazilian population

Abstract INTRODUCTION: Genetic polymorphisms define the cytokine production leading to susceptibility or resistance to diseases. We studied the cytokine polymorphism in the development of tegumentary leishmaniasis (TL). METHODS: Genotyping of TNF-α, TGF-β1, IFN-γ, IL-6, and IL-10 were performed by polymerase chain reaction assay. RESULTS: G and C alleles of TGF- β1 (codon 25) were the most common in controls and patients, respectively. G/G was the most frequent genotype in controls, and G/C and C/C in patients. CONCLUSIONS: G/G genotype of codon 25 in TGF-β1 appeared to confer resistance, and G/C and C/C genotypes, susceptibility to TL in this population.

Monitoring the reproductive activity in captive bred female ball pythons (P. regius) by ultrasound evaluation and noninvasive analysis of faecal reproductive hormone (progesterone and 17ß-estradiol) metabolites trends.

The royal python (Python regius) is commonly bred in captivity. To have a successful breeding season, accurate monitoring of the reproductive activity is necessary. The use of non-invasive monitoring methods in exotics is important in order to minimize stress. For this purpose ultrasound has been anecdotally used to monitor royal python reproductive activity. However, there is limited information regarding the reproductive cycle of this species. The aim of the present study is to monitor the female reproductive cycle of the royal python using ultrasonography and gonadal steroid metabolite measurements in the faeces. The reproductive activity of one hundred twenty-nine adult female P. regius was examined during two consecutive years. We performed brief scans on non-anaesthetized snakes using a portable ultrasound system and a 10-12 MHz linear array transducer (MyLab™ 30 Gold, Esaote). Ultrasound features, dimension and echogenicity of the reproductive structures were determined. During the second reproductive cycle, the hormonal profiles of 30 animals were also evaluated, with a monthly collection of faecal samples. These samples were classified according to reproductive stage, as identified by ultrasonographic examination, and the mean faecal progesterone and 17ß-estradiol levels were calculated using the results from an enzyme-linked immunosorbent assay (ELISA). Progesterone levels increased during the reproductive cycle. Estradiol levels showed greater variability, although they appeared to increase before coupling when compared to the levels between coupling and egg laying. The present study suggests that it is possible to identify different phases in the female royal python reproductive cycle: anovulatory phase, transition, folliculogenesis and embryogenesis. Ultrasound is also useful for identifying follicular regression or slugs. Gonadal steroid metabolite measurements from the faeces could help integrate reproductive information. The use of ultrasonography in addition to the steroid metabolite measurement in the faeces gives an accurate picture of ovarian activity in captive adult female royal pythons.

Discovery of an old, archipelago-wide, endemic radiation of Philippine snakes.

The extraordinarily rich land vertebrate biodiversity of the Philippines includes at least 112 species of terrestrial snakes (74% of which are endemic to the archipelago) in 41 genera (12% endemic). Endemic Philippine snake genera include Cyclocorus (two species), Hemibungarus (three species), Hologerrhum (two species), Oxyrhabdium (two species), and Myersophis (monotypic). Although Hemibungarus and Oxyrhabdium have been included in previous species-level phylogenies, the affinities of the other three Philippine endemic genera are completely unknown. We generated novel DNA sequences for six species from four genera and analyzed these in conjunction with data from earlier studies to infer a phylogeny for the group containing Colubridae, Elapoidea (Elapidae + Lamprophiidae), and Homalopsidae. We present a novel phylogenetic result that strongly supports the existence of an entirely endemic Philippine radiation of elapoid snakes that originated 35-25 million years ago. We provide a revised, phylogeny-based classification to accommodate the new clade, transfer Cyclocorus, Hologerrhum, and Myersophis to Lamprophiidae, and provide the first estimate of the evolutionary relationships among these genera and the related Oxyrhabdium, setting the stage for future investigation of this entirely endemic, novel Philippine elapoid radiation.

Snakes exhibit tissue-specific variation in cardiotonic steroid sensitivity of Na+/K+-ATPase.

Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na+/K+-ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na+/K+-ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na+/K+-ATPases of snakes react to the toxins. Some tissues might have Na+/K+-ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na+/K+-ATPase substitutions and observed resistance using actual snake Na+/K+-ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na+/K+-ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na+/K+-ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa.

Clinical and laboratory features distinguishing between Deinagkistrodon acutus and Daboia siamensis envenomation

There are 6 species of venomous snakes in Taiwan. Two of them, Deinagkistrodon acutus (D. acutus) and Daboia siamensis (D. siamensis), can cause significant coagulopathy. However, a significant proportion of patients with snakebites cannot identify the correct snake species after envenomation, which hampers the application of antivenom. Hence, the differential diagnosis between the two snakebites by clinical presentations is important. This study aims to compare their clinical and laboratory features for the purpose of differential diagnosis between the two snakebites. Methods: We retrospectively reviewed the medical records of patients who arrived at the emergency department due to D. acutus or D. siamensis envenomation, between 2003 and 2016, in one medical center in eastern Taiwan. Since these snakebites are rare, we also included 3 cases reported from another hospital in central Taiwan. Results: In total, 15 patients bitten by D. acutus and 12 patients by D. siamensis were analyzed. Hemorrhagic bulla formation and the need for surgical intervention only presented for D. acutus envenomation cases (Both 53.3% vs. 0.0%, P= 0.003). As to laboratory features, lower platelet counts (20.0 × 103/µL [interquartile range, 14-66 × 103/µL] vs. 149.0 × 103/µL [102.3-274.3 × 103/µL], P = 0.001), lower D-dimer level (1423.4 µg/L [713.4-4212.3 µg/L] vs. 12,500.0 µg/L [2351.4-200,000 µg/L], P = 0.008), higher proportion of patients with moderate-to-severe thrombocytopenia (platelet count < 100 × 103/µL) (80% vs. 16.7%, odds ratio (OR) = 20.0, 95% CI, 2.77-144.31; P = 0.002), and lower proportion of patients with extremely high D-dimer (> 5000 ng/mL) (16.7% vs. 66.7%, adjusted OR = 0.1 (95% CI, 0.01-0.69; P = 0.036) were found among cases of D. acutus envenomation compared to D. siamensis envenomation. The combination of hemorrhagic bulla, thrombocytopenia, and a lack of extremely high D-dimer had good discriminatory power (area under the curve (AUC) = 0.965; 95% CI, 0.904-1.00) for distinguishing D. acutus from D. siamensis envenomation. Conclusions: The presentation of moderate to severe thrombocytopenia (platelet count < 100 × 103/µL) and hemorrhagic bulla formation may indicate D. acutus envenomation. However, the envenomed patient with extremely high D-dimer levels may indicate a D. siamensis envenomation. These findings may help diagnose and select the right antivenom in patients with unknown snakebites who present significant coagulopathy.(AU)

Evaluation of sperm quality of Erythrolamprus poecilogyrus sublineatus (Cope, 1860) (Serpentes, Dipsadidae) / Avaliação da qualidade seminal de serpentes Erythrolamprus poecilogyrus sublineatus (Cope, 1860) (Serpentes, Dipsadidae)

Abstract Erythrolamprus poecilogyrus sublineatus (Cope, 1860), is a species widely distributed in the Pampa Domain, occurring in Rio Grande do Sul, Argentina and Uruguay, mainlyin the pampa region. In the coastal region of southern Brazil this is serpent is considered one of the most abundant. The purpose of the present study is to describe the techniques of sperm evaluation in vitro for E. poecilogyrus sublineatus in the coastal plain of Rio Grande do Sul, Brazil. After laparatomy the efferent vases were collected and the semen was diluted in 1ml Beltsville Thawing Solution. The characteristics of motility, membrane integrity, mitochondria, acrosome, DNA, cell viability and cellular functionality were evaluated. Fluorescent probes were used for the evaluation of sperm structure in epifluorescence microscope. With the techniques described, it was possible to identify intact and injured cells, enabling the determination of cell characteristics for the spring season (October and November). It was observed in the analyses that 80% of sperm cells were mobile and that 84.1 ± 8.0% of sperm membranes were intact. The standards found were of 48 ± 13.8% of intact acrosome, 73.6 ± 6.0 of perfect DNA and of 91.8 ± 4.0 of functional mitochondria. Thus, these values from the sperm analysis can be used as standards for the species Erythrolamprus poecilogyrus sublineatus.

Resumo Erythrolamprus poecilogyrus sublineatus (Cope, 1860), é uma espécie amplamente distribuída no Domínio Pampa, ocorrendo no Rio Grande do Sul, Argentina e Uruguai, principalmente na região dos pampas. Na região costeira do extremo sul do Brasil essa é uma das serpentes consideradas mais abundantes. O objetivo deste estudo é descrever as técnicas de avaliação espermática in vitro para E. poecilogyrus sublineatus da planície costeira do Rio Grande do Sul, Brasil. Após laparatomia os vasos eferentes foram coletados e o sêmen diluído em 1ml Beltsville Thawing Solution. Foram avaliadas as características de motilidade, integridade de membrana, mitocôndria, acrossoma, DNA, viabilidade celular e funcionalidade celular. Foram utilizadas sondas fluorescentes para avaliação das estruturas espermática em microscópio de epifluoescência. Com as técnicas descritas foram possível identificar células integras e lesadas, podendo determinar as características celulares para o período de primavera (outubro e novembro). Nas análises foi observado que 80% das células espermáticas estavam móveis e que 84,1 ± 8,0% das membranas espermáticas estavam íntegras. Os padrões encontrados para foram de 48 ± 13,8% de acrossoma íntegro, 73,6 ± 6,0 de DNA íntegro e de 91,8 ± 4,0 de mitocôndria funcional. Desta forma, esses valores das análises espermáticas podem ser utilizados como padrão para a espécie Erythrolamprus poecilogyrus sublineatus.

Reproductive Disorders in Snakes.

Reproduction of snakes is one of the challenging aspects of herpetology medicine. Due to the complexity of reproduction, several disorders may present before, during, or after this process. This article describes the physical examination, and radiographic, ultrasonographic, and endoscopic findings associated with reproductive disorders in snakes. Surgical techniques used to resolve reproductive disorders in snakes are described. Finally, common reproductive disorders in snakes are individually discussed.

Reproductive biology of the Green Ground Snake Erythrolamprus poecilogyrus sublineatus (Serpentes: Dipsadidae) in Subtropical Brazil

ABSTRACT We investigated reproductive features of the dipsadid snake Erythrolamprus poecilogyrus sublineatus in the southernmost Brazilian coast, a subtropical region characterized by well-marked seasons. Females are significantly smaller than males, have a shorter tail, and reach sexual maturity at later times along their development. In contrast to tropical subspecies, E. p. sublineatus females presented a seasonal pattern, with secondary follicles occurring from late winter to early autumn and egg production restricted to the whole spring and early summer. Males presented seasonal variation in testes volume (increase in autumn and decrease in winter) while no significant seasonal variation was found in ductus deferens width. The number of oviductal eggs varied from two to nine, thus, real fecundity is also inferior than that observed in tropical E. poecilogyrus forms. Considering the thermal requirements for reproduction, it is possible that the colder climatic conditions of the southern Brazilian coast have shaped the seasonal reproductive pattern in E. p. sublineatus. The shorter body size of this subspecies may also represent a conditioning factor of low fecundity.

Evaluación de análisis de endemicidad y PAE para identificar trazos generalizados, a partir de las serpientes del estado de Hidalgo, México / Evaluation of PAE and endemicity analysis for identifying generalized tracks using snakes in Hidalgo, Mexico

Resumen:Uno de los conceptos centrales en la Panbiogeografía es el trazo generalizado, el cual representa una biota ancestral que se ha fragmentado por eventos geológicos y puede recuperarse a través de distintos métodos, que incluyen el análisis de simplicidad de endemismos (PAE) y el análisis de endemicidad (AE). El PAE es un método frecuentemente utilizado para la identificación de trazos generalizados, mientras que el AE fue diseñado para encontrar áreas de endemismo, pero recientemente se ha usado también para identificar trazos generalizados. En este trabajo se evaluaron ambos métodos para la identificación de los trazos generalizados a partir de los datos de 84 serpientes distribuidas en el estado de Hidalgo, México. Con el PAE se obtuvo un trazo generalizado formado por tres trazos individuales (Agkistrodon taylori, Crotalus totonacus y Pliocercus elapoides), que corresponden a sinapomorfías que apoyan al clado principal. La prueba de robustez estadística mostró un soporte del 89 %, mientras que con el AE se identificaron dos áreas de endemismo con valores del índice de endemicidad de 2.71-2.96 y 2.84- 3.09 respectivamente, las cuales fueron transformadas a trazos generalizados; el primero formado por tres trazos individuales (Micrurus bernadi, Rhadinaea marcellae y R. quinquelineata) y el segundo formado por dos trazos individuales (Geophis mutitorques y Thamnophis sumichrasti). Estos trazos generalizados pueden considerarse un solo patrón de distribución, dada la cercanía geográfica y su topología. Al compararse los métodos se observó que ambos son útiles para la identificación de trazos generalizados, y aunque son independientes, se sugiere su uso de manera complementaria. Sin embargo, para una correcta aplicación e interpretación de los resultados, es necesario considerar las bases teóricas de cada método, así como la correcta elección del tamaño de las unidades de estudio. En el caso particular del AE si se utilizan tamaños de celda pequeños éstos pueden resultar ideales para la búsqueda de patrones biogeográficos a través de límites geopolíticos y esta información puede favorecer la realización de pro- puestas de conservación a nivel estatal que consideren a las especies con distribuciones restringidas, pues la pérdida de éstas representaría la extinción de linajes únicos.

Abstract:One of the most important concepts in Panbiogeography is the generalized track, which represents an ancestral biota fragmented by geological events that can be recovered through several methods, including Parsimony analysis of endemicity (PAE) and endemicity analysis (EA). PAE has been frequently used to identify generalized tracks, while EA is primarily designed to find areas of endemicity, but has been recently proposed for identifying generalized tracks as well. In this study we evaluated these methods to find generalized tracks using the distribution of the 84 snake species of Hidalgo. PAE found one generalized track from three individual tracks (Agkistrodon taylori, Crotalus totonacus and Pliocercus elapoides), supported by 89 % of Bootstrap, and EA identified two generalized tracks, with endemicity index values of 2.71-2.96 and 2.84-3.09, respectively. Those areas were transformed to generalized tracks. The first generalized track was retrieved from three individual tracks (Micrurus bernadi, Rhadinaea marcellae and R. quinquelineata), and the second was recovered from two individual tracks (Geophis mutitorques and Thamnophis sumichrasti). These generalized tracks can be considered a unique distribution pattern, because they resembled each other and agreed in shape. When comparing both methods, we noted that both are useful for identifying generalized tracks, and although they can be used independently, we suggest their complementary use. Nevertheless, to obtain accurate results, it is useful to consider theoretical bases of both methods, along with an appropriate choice of the size of the area. Results using small-grid size in EA are ideal for searching biogeographical patterns within geopolitical limits. Furthermore, they can be used for conservation proposals at state level where endemic species become irreplaceable, and where losing them would imply the extinction of unique lineages. Rev. Biol. Trop. 64 (4): 1611-1624. Epub 2016 December 01.

Trophic divergence despite morphological convergence in a continental radiation of snakes.

Ecological and phenotypic convergence is a potential outcome of adaptive radiation in response to ecological opportunity. However, a number of factors may limit convergence during evolutionary radiations, including interregional differences in biogeographic history and clade-specific constraints on form and function. Here, we demonstrate that a single clade of terrestrial snakes from Australia--the oxyuranine elapids--exhibits widespread morphological convergence with a phylogenetically diverse and distantly related assemblage of snakes from North America. Australian elapids have evolved nearly the full spectrum of phenotypic modalities that occurs among North American snakes. Much of the convergence appears to involve the recurrent evolution of stereotyped morphologies associated with foraging mode, locomotion and habitat use. By contrast, analysis of snake diets indicates striking divergence in feeding ecology between these faunas, partially reflecting regional differences in ecological allometry between Australia and North America. Widespread phenotypic convergence with the North American snake fauna coupled with divergence in feeding ecology are clear examples of how independent continental radiations may converge along some ecological axes yet differ profoundly along others.

Evidence for determinism in species diversification and contingency in phenotypic evolution during adaptive radiation.

Adaptive radiation (AR) theory predicts that groups sharing the same source of ecological opportunity (EO) will experience deterministic species diversification and morphological evolution. Thus, deterministic ecological and morphological evolution should be correlated with deterministic patterns in the tempo and mode of speciation for groups in similar habitats and time periods. We test this hypothesis using well-sampled phylogenies of four squamate groups that colonized the New World (NW) in the Late Oligocene. We use both standard and coalescent models to assess species diversification, as well as likelihood models to examine morphological evolution. All squamate groups show similar early pulses of speciation, as well as diversity-dependent ecological limits on clade size at a continental scale. In contrast, processes of morphological evolution are not easily predictable and do not show similar pulses of early and rapid change. Patterns of morphological and species diversification thus appear uncoupled across these groups. This indicates that the processes that drive diversification and disparification are not mechanistically linked, even among similar groups of taxa experiencing the same sources of EO. It also suggests that processes of phenotypic diversification cannot be predicted solely from the existence of an AR or knowledge of the process of diversification.

Morphology and putative function of the colon and cloaca of marine and freshwater snakes.

Among tetrapods, evidence for postrenal modification of the urine by the distal digestive tract (including the colon and cloaca) is highly variable. Birds and bladderless reptiles are of interest because the colon and cloaca represent the only sites from which water and ions can be reclaimed from the urine secreted by the kidney. For animals occupying desiccating environments (e.g., deserts and marine environments), postrenal modification of the urine may directly contribute to the maintenance of hypo-osmotic body fluids. We compared the morphology and distribution of key proteins in the colon, cloaca, and urogenital ducts of watersnakes from marine (Nerodia clarkii clarkii) and freshwater (Nerodia fasciata) habitats. Specifically, we examined the epithelia of each tissue for evidence of mucus production by examining the distribution of mucopolysaccharides, and for evidence of water/ion regulation by examining the distribution of Na(+) /K(+) -ATPase (NKA), Na(+) /K(+) /Cl(-) cotransporter (NKCC), and aquaporin 3 (AQP3). NKCC localized to the basolateral epithelium of the colon, urodeal sphincter, and proctodeum, consistent with a role in secretion of Na(+), Cl(-) , and K(+) from the tissue, but NKA was not detected in the colon or any compartment of the cloaca. Interestingly, NKA was detected in the basolateral epithelium of the ureters, suggesting the urothelium may play a role in active ion transport. AQP3 was detected in the ureters and coprodeal complex, consistent with a role in urinary and fecal dehydration or, potentially, in the production of the watery component of the mucus secreted by the coprodeal complex. Since no differences in general cloacal morphology, production of mucus, or the distribution of ion transporters/water channels were detected between the two species, cloacal osmoregulation may either be regulated by proteins not examined in this study or may not be responsible for the differential success of N. c. clarkii and N. fasciata in marine habitats.

Innate immune performance and steroid hormone profiles of pregnant versus nonpregnant cottonmouth snakes (Agkistrodon piscivorus).

Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates.

Bioenergetic components of reproductive effort in viviparous snakes: costs of vitellogenesis exceed costs of pregnancy.

Reproductive effort has been defined as the proportion of an organism's energy budget that is allocated to reproduction over a biologically meaningful time period. Historically, studies of reproductive bioenergetics considered energy content of gametes, but not costs of gamete production. Although metabolic costs of vitellogenesis (MCV) fundamentally reflect the primary bioenergetic cost of reproductive allocation in female reptiles, the few investigations that have considered costs of reproductive allocation have focused on metabolic costs of pregnancy (MCP) in viviparous species. We define MCP as energetic costs incurred by pregnant females, including all costs of maintaining gestation conditions necessary for embryogenesis. MCP by our definition do not include fetal costs of embryogenesis. We measured metabolic rates in five species of viviparous snakes (Agkistrodon contortrix, Boa constrictor, Eryx colubrinus, Nerodia sipedon, and Thamnophis sirtalis) during vitellogenesis and pregnancy in order to estimate MCV and MCP. Across all species, MCV were responsible for 30% increases in maternal metabolism. Phylogenetically-independent contrasts showed that MCV were significantly greater in B. constrictor than in other species, likely because B. constrictor yolk energy content was greater than that of other species. Estimates of MCP were not significantly different from zero in any species. In viviparous snakes, MCV appear to represent significant bioenergetic expenditures, while MCP do not. We suggest that MCV, together with yolk energy content, represent the most significant component of reptilian reproductive effort, and therefore deserve greater attention than MCP in studies of reptilian reproductive bioenergetics.

Insights into the adaptive significance of vertical pupil shape in snakes.

Pupil shape in vertebrates ranges from circular to vertical, with multiple phylogenetic shifts in this trait. Our analyses challenge the widely held view that the vertical pupil evolved as an adaptation to enhance night vision. On functional grounds, a variable-aperture vertical pupil (i) allows a nocturnal species to have a sensitive retina for night vision but avoid dazzle by day by adjusting pupil closure, and (ii) increases visual acuity by day, because a narrow vertical pupil can project a sharper image onto the retina in the horizontal plane. Detection of horizontal movement may be critical for predators that wait in ambush for moving prey, suggesting that foraging mode (ambush predation) as well as polyphasic activity may favour the evolution of vertical pupil shape. Camouflage (disruption of the circular outline of the eye) also may be beneficial for ambush predators. A comparative analysis in snakes reveals significant functional links between pupil shape and foraging mode, as well as between pupil shape and diel timing of activity. Similar associations between ambush predation and vertically slit pupils occur in lizards and mammals also, suggesting that foraging mode has exerted major selective forces on visual systems in vertebrates.

Molecular cloning and characterization of cDNAs encoding metalloproteinases from snake venom glands.

Snake venom metalloproteinases (SVMPs) are a superfamily of zinc-dependent proteases and participate in a number of important biological, physiological and pathophysiological processes. In this work, we simultaneously amplified nine cDNAs encoding different classes of metalloproteinases from glands of four different snake species (Agkistrodon contortrix laticinctus, Crotalus atrox, Crotalus viridis viridis and Agkistrodon piscivorus leucostoma) by RT-PCR with a pair of primers. Among the encoded metalloproteinases, two enzymes (AclVMP-I and AplVMP-I), three enzymes (CaVMP-II, CvvVMP-II and AplVMP-II) and four enzymes (AclVMP-III, CaVMP-III, CvvVMP-III and AplVMP-III) with the characteristic motif (HEXXHXXGXXH) of metalloproteinase belong to type P-I, P-II and P-III enzymes, respectively. Disintegrin domains of CaVMP-II and CvvVMP-II from two Crotatus snakes contain RGD-motif whereas AplVMP-II from Agkistrodon snake has KGD-motif. Instead of R/KGD-motif within disintegrin domain of SVMP-II enzyme, CaVMP-III, CvvVMP-III and AplVMP-III enzymes contain SECD-motif, while AclVMP-III has DDCD-motif in their corresponding position of disintegrin-like domains. There are 12 Cys amino acids in cysterin-rich domains of each P-III enzyme. Moreover, a disintegrin precursor (AplDis) with RGD-motif also simultaneously amplified from the glands of A.p. leucostoma while amplifying AplVMP-II and AplVMP-III, which indicated that different types of SVMPs and related genes are present in a single species of snake and share a consensus sequence at the 3' and 5' untranslated regions. RT-PCR result also showed that P-III is highly expressed in Crotalus snakes than in Agkistrodon snakes. Aligning the deduced amino acid sequence of these enzymes with other SVMPs from GenBank database indicated that this is the first report on the isolation of cDNAs encoding P-II and P-III enzymes from C.v. viridis and A.p. leucostoma snakes. The availability of these SVMP sequences directly facilitated further studies of structure characterization and diversified function analysis.

Voluntary exposure of some western-hemisphere snake and lizard species to ultraviolet-B radiation in the field: how much ultraviolet-B should a lizard or snake receive in captivity?

Studies of voluntary exposure to ultraviolet-B (UVB) radiation from the sun in the field were conducted in the southern US and Jamaica for 15 species of lizards and snakes occupying various habitats. Species were sorted into four zones of UVB exposure ranging from a median UV index of 0.35 for zone 1 to 3.1 for zone 4. Guidelines for UVB exposure in captivity of these and species occupying similar light environments are presented. Data for most species were collected during mid-day during the spring breeding season, which appeared to be the time of maximum exposure. For two species of Sceloporus studied more intensively there was significant variation of exposure among times of the day and among seasons. So, all-day studies over the entire active season are necessary to fully understand the pattern of natural exposure for a particular diurnal species. Environmental and body temperature and thermoregulation as well as UVB/vitamin D photoregulation influences exposure to UVB. Regressions allowing the inter-conversion of readings among some meters with different detector sensitivities are presented. Readings of natural sunlight predict the same photobiosynthetic potential for vitamin D as the same reading from artificial sources whose wavelength distribution within the UVB band of the source is comparable to that of sunlight. Research approaches to further increase our understanding of vitamin D and UVB use and requirements for squamate reptiles in captivity are outlined.