Does size matter? Venom proteomic and functional comparison between night adder species (Viperidae: Causus) with short and long venom glands.

Night adders (Causus species within the Viperidae family) are amphibian specialists and a common source of snakebite in Africa. Some species are unique in that they have the longest venom glands of any viper, extending approximately 10% of the body length. Despite their potential medical importance and evolutionary novelty, their venom has received almost no research attention. In this study, venoms from a short-glanded species (C. lichtensteinii) and from a long-glanded species (C. rhombeatus) were compared using a series of proteomic and bioactivity testing techniques to investigate and compare the toxin composition and functioning of the venoms of these two species. Both C. rhombeatus and C. lichtensteinii were similar in overall venom composition and inhibition of blood coagulation through non-clotting proteolytic cleavage of fibrinogen. While the 1D gel profiles were very similar to each other in the toxin types present, 2D gel analyses revealed isoformic differences within each toxin classes. This variation was congruent with differential efficacy of South African Institute for Medical Research snake polyvalent antivenom, with C. lichtensteinii unaffected at the dose tested while C. rhombeatus was moderately but significantly neutralized. Despite the variation within toxin classes, the similarity in overall venom biochemistry suggests that the selection pressure for the evolution of long glands served to increase venom yield in order to subjugate proportionally large anurans as a unique form of niche partitioning, and is not linked to significant changes in venom function. These results not only contribute to the body of venom evolution knowledge but also highlight the limited clinical management outcomes for Causus envenomations.

Multilocus phylogeny of the Asian Lance-headed pitvipers (Squamata, Viperidae, Protobothrops).

Despite the public health significance and potential applications to medical research, the evolutionary history of the Asian Lance-headed pitvipers (genus Protobothrops) remains inadequately studied. Previous research generally focused on a few selected species with no comprehensive molecular study of Protobothrops. Here, we conduct the first study to infer the phylogenetic relationships of all currently recognized species in this genus based on four mitochondrial DNA fragments and four nuclear genes. Bayesian Inference and Maximum Likelihood analyses show that within Protobothrops there are four strongly supported clades forming distinct subgroups. The first subgroup, which is sister to the other three, consists of three species, P. himalayanus, P. kaulbacki, and P. sieversorum. The second subgroup contains only P. mangshanensis. The final two subgroups, which are sister groups, include the other four and six Protobothrops species. Although our findings provide additional information on the phylogenetic relationships of the genus Protobothrops, the placement of P. dabieshanensis and P. elegans remains problematic. In addition, our molecular results indicate that P. trungkhanhensis, currently considered endemic to Vietnam, should be added to the species known from China. Our ancestral area estimation indicated that Protobothrops likely originated in southwestern China. This study improves our understanding of the evolutionary relationships among species of Asian Lance-headed pitvipers, providing a greater framework for future studies.

A cryptic palm-pitviper species (Squamata: Viperidae: Bothriechis) from the Costa Rican highlands, with notes on the variation within B. nigroviridis.

Middle America is one of the most biodiverse regions in the world, harboring an exceptional number of rare and endemic species. This is especially true of Middle American cloud forests, where montane specialists occupy restricted, high-elevation ranges making them attractive candidates for investigating historical biogeography and speciation. One such highland-restricted species, the black speckled palm-pitviper (Bothriechis nigroviridis), occupies the Central, Tilarán, and Talamanca Cordilleras in Costa Rica and Panama. In this study, we investigate the genetic and morphological variation among populations of B. nigroviridis by inferring a multilocus phylogeny (21 individuals) and analyzing meristic scale characters with a principal component analysis (64 individuals). We find B. nigroviridis sensu stricto to be composed of two deeply divergent lineages, one with a restricted range in the northern and central Cordillera Talamanca and the other ranging throughout the Central, Tilarán, and Talamanca Cordilleras. Furthermore, these two lineages are morphologically distinct, with previously unrecognized differences in several characters allowing us to name and diagnose a new species B. nubestris sp. nov. We also examine the genetic and morphological variation within B. nigroviridis and discuss biogeographic hypotheses that may have led to the diversification of Bothriechis lineages.

Water deprivation increases maternal corticosterone levels and enhances offspring growth in the snake Vipera aspis.

Circulating glucocorticoid (GC) levels may increase as a result of reproductive effort or in response to unpredictable events. However, GC secretion can vary with the availability of vital trophic resources such as energy. While water represents another critical resource, the impact of water deprivation on GC secretion during reproduction has not yet been thoroughly investigated. Here, we examined the effects of water deprivation on plasma corticosterone (CORT) concentrations of female aspic vipers (Vipera aspis), and determined the impacts of water deprivation on offspring traits. We exposed both pregnant and non-reproductive females to a 20-day water deprivation and compared their pre- and post-deprivation CORT levels with those of control females. At the end of the treatment, only water-deprived pregnant females showed a significant increase in CORT levels. In pregnant females, changes in baseline CORT level were correlated with changes in female hydration state. Changes in baseline CORT levels were also negatively influenced by maternal reproductive effort in pregnant control females, while such a relationship was not apparent in pregnant water-deprived females. Finally, we found that offspring from water-deprived females had higher growth rates than offspring from control females. Offspring growth was also positively correlated with changes in both maternal osmolality and baseline CORT levels. Together, our results suggest that dehydration increases maternal CORT levels, which may subsequently influence offspring development. Further long-term field studies are therefore required to assess whether there is an adaptive significance of this response.

Neonate-to-adult transition of snake venomics in the short-tailed pit viper, Gloydius brevicaudus.

Snake venoms undergo ontogenetic shifts in biochemical and pharmacological activities. This may be related to variation in venom components associated with the ontogenetic shift in diet. We used the short-tailed pit viper Gloydius brevicaudus that displays ontogenetic shifts in diet to examine whether the species displays a neonate-to-adult transition of snake venomics. Venoms from neonates and adults were pooled separately and then analyzed by 2-DE, MALDI-TOF-MS/MS and iTRAQ technologies. The 2-DE profiles showed that the main components in both types of venoms were acidic proteins, and that neonates and adults differed in snake venomics. The proteins with molecular masses/pI of ~12-39kDa/4.0-4.6, ~36-57kDa/5.6-7.0 and ~65-92kDa/4.5-5.8 were more abundant in the neonate venom, while the proteins with molecular masses/pI of ~12-19kDa/4.6-6.4, ~23-30kDa/5.4-6.3 and ~35-62kDa/4.6-5.4 were more abundant in the adult venom. The iTRAQ analysis showed quantitative changes in various toxin families, including mainly metalloproteinases, serine proteinases, phospholipase A2s and C-type lectins. The N-deglycosylation analysis demonstrated that glycosylation was an important post-translational modification of snake venom. Our results show a neonate-to-adult transition of snake venomics in G. brevicaudus. Such a transition might be driven by the divergence in dietary habits between neonates and adults. BIOLOGICAL SIGNIFICANCE: This study is first to demonstrate a neonate-to-adult transition of snake venomics in G. brevicaudus, and the results will be helpful in predicting and treating clinical pathologic symptoms caused by the snake at different developmental stages.

Effect of ambient temperature in neonate aspic vipers: growth, locomotor performance and defensive behaviors.

The impact of temperature during incubation and gestation has been tested in various reptiles; the postnatal period has been rarely investigated however. Three groups of newborn aspic vipers (Vipera aspis) were placed under contrasted thermal regimes during 7 months: (1) a cool 23°C constant regime, (2) a warm 28°C constant regime, and (3) an optimal regime with free-access to a wide range of temperatures. Later, all the snakes were placed under hibernation conditions (6°C) during 3 months. Finally all the snakes were placed in the optimal thermal regime during 2 additional months. The total duration of the experiment was of 12 months. Body mass and feeding rates were recorded weekly, body size was measured monthly. We also assessed locomotor performance and recorded several behavioral traits (e.g., defensive and predatory behaviors). As expected, snakes raised under cool temperatures exhibited low feeding rate, growth rate, body condition, and they exhibited poor locomotor performance; they also displayed marked defensive behaviors (e.g., high number of defensive bites) whilst hesitating during longer periods to bite a prey. Such behavioral effects were detected at the end of the experiment (i.e., 5 months after exposure to contrasted thermal treatments [3 months of hibernation plus 2 months of optimal regime]), revealing long term effects. Surprisingly, growth rate and locomotor performance were not different between the two other groups, warm constant 28°C versus optimal regimes (albeit several behavioral traits differed), suggesting that the access to a wide range of ambient temperatures was not a crucial factor.

Snake venomics across genus Lachesis. Ontogenetic changes in the venom composition of Lachesis stenophrys and comparative proteomics of the venoms of adult Lachesis melanocephala and Lachesis acrochorda.

We report the proteomic analysis of ontogenetic changes in venom composition of the Central American bushmaster, Lachesis stenophrys, and the characterization of the venom proteomes of two congeneric pitvipers, Lachesis melanocephala (black-headed bushmaster) and Lachesis acrochorda (Chochoan bushmaster). Along with the previous characterization of the venom proteome of Lachesis muta muta (from Bolivia), our present outcome enables a comparative overview of the composition and distribution of the toxic proteins across genus Lachesis. Comparative venomics revealed the close kinship of Central American L. stenophrys and L. melanocephala and support the elevation of L. acrochorda to species status. Major ontogenetic changes in the toxin composition of L. stenophrys venom involves quantitative changes in the concentration of vasoactive peptides and serine proteinases, which steadily decrease from birth to adulthood, and age-dependent de novo biosynthesis of Gal-lectin and snake venom metalloproteinases (SVMPs). The net result is a shift from a bradykinin-potentiating and C-type natriuretic peptide (BPP/C-NP)-rich and serine proteinase-rich venom in newborns and 2-years-old juveniles to a (PI>PIII) SVMP-rich venom in adults. Notwithstanding minor qualitative and quantitative differences, the venom arsenals of L. melanocephala and L. acrochorda are broadly similar between themselves and also closely mirror those of adult L. stenophrys and L. muta venoms. The high conservation of the overall composition of Central and South American bushmaster venoms provides the ground for rationalizing the "Lachesis syndrome", characterized by vagal syntomatology, sensorial disorders, hematologic, and cardiovascular manifestations, documented in envenomings by different species of this wide-ranging genus. This finding let us predict that monospecific Lachesic antivenoms may exhibit paraspecificity against all congeneric species.

Proteomic analysis of ontogenetic and diet-related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri).

Snake venom proteins show high levels of variation at the level of the individual yet the environmental and molecular mechanisms that generate this diversity remain unclear. Here we report the results of a controlled feeding experiment combined with proteomic analyses of periodically collected venom samples to assess the roles of ontogenetic and diet-related effects on venom composition of captive juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri). Juvenile snakes fed from birth with mice, lizards, or frogs showed little evidence for an ontogenetic shift in venom composition from 5 to 26 months in terms of substantial changes in the relative abundance of major classes of venom toxins. However, there were fine-scale changes in the relative abundance of D49-PLA2 15, PI-SVMPs, and PIII-SVMP 28, and a decline in the abundance of other PIII-SVMPs. Although juveniles raised on different prey exhibited distinct relative toxin compositional change rates, at 26 months old, their venoms showed similar patterns of protein composition suggesting little effect of diet on the overall make-up of venom in snakes this age or younger. In contrast, adult females raised on different prey over a 26 month period showed prey-related changes in the relative abundance of major protein families from initial to final samples. Specifically, mouse-fed females showed substantial increases in the relative abundance of total PLA2s and serine proteinases of 95% and >100%, respectively, whereas comparable values for lizard- (42% and -22%) and frog-fed females (2% and 11%) were distinctly smaller in magnitude. Venom from adult snakes fed on different prey also showed distinct changes in the abundance of PLA2 molecules 15, 19a, and 19, which were, respectively, (>100%, 33%, 63%), (>100%, 0%, 35%), and (71%, 20%, -4%) for the mice-, lizard-, and frog-diet. Venom from snakes raised on frogs contained a small (1.1%) but consistent amount of a PLA2 molecule (15a) not present in snakes fed on mice or lizards. This work provides evidence that venom composition is somewhat plastic in both juvenile and adult S. m. barbouri and that, at least in adults, prey consumed may influence the relative abundance of possibly functionally-distinct classes of venom proteins.

Within-clutch variation in venoms from hatchlings of Deinagkistrodon acutus (Viperidae).

We used 17 hatchling five-paced pit-vipers snakes (Deinagkistrodon acutus) to study within-clutch variation in snake venoms. We measured venom yield and total protein content, and examined the correlations between venom yield and hatchling size [snout-vent length (SVL) and body mass]. We also analyzed the electrophoretic profiles and enzymatic activities of venoms from hatchlings. Lyophilized venom mass was not correlated with SVL, nor with body mass. Liquid venom mass and total protein content were not correlated with body mass, but were positively correlated with SVL. Venom composition, as shown in SDS-PAGE chromatograms did vary among individuals but there were biochemical differences in activity which had to be due to subtle venom composition differences between the sexes. Female hatchlings showed higher esterolytic and fibrinogenolytic activities but lower proteolytic, collagenolytic, phosphomonoesterase and fibrinolytic activities than male hatchlings. We did not find sexual differences in 5' nucleotidase, phospholipase A(2) and hyaluronidase activities, and l-amino acid oxidase activities in either female or male hatchlings. Within-clutch variation in venoms from D. acutus hatchlings should be attributed to the individual-based differences in presence or absence, and the relative amount of the protein components, and might have a genetic basis.

História natural das serpentes da região de Munhoz, sul de Minas Gerais, Serra da Mantiqueira / Natural History of the snakes from the Munhoz region, south of Minas Gerais, Serra da Mantiqueira

Pouco se sabe sobre a herpetofauna das regioes mais elevadas da Serra da Mantiqueira, uma das porcoes mais altas e acidentadas do territorio brasileiro. Este estudo foi desenvolvido em uma area ao sul de Minas Gerais , na Serra da Mantiqueira, região com predominio das florestas Ombrofilas Mistas ( Floresta de Araucarias) e Campos de Altitude.Foram estudados aspectos biologicos e cologicos de uma comunidade de serpentes, caracterizando a riqueza, a abundancia e o uso do ambiente, alem da historia natural das especies por meio da caracterizacao do ciclo e das estrategias reprodutivas reprodutivas, padroes de atividade, comportamento, dieta, e analises de morfometria de adultos e filhotes. ...

Little is know about the snakes populaion in the higher elevations of the Serra da Mantiqueira, one of the higgest and most rugged portion in Brazilian territory. This study was conducted in the Serra da Mantiqueira, south of Minas Gerais in an area dominated by Atlantic Forest and altitude grasslands. The study show biological and ecological aspects within community of snakes by analizing the richness, the abundance, as how the environment was used and into the natural history of species thought characterization of reproductive strategies, activity patterns, behaviour, diet and morphometric analyses of young and adult snakes. ..

Does gestation or feeding affect the body temperature of the golden lancehead, Bothrops insularis (Squamata: Viperidae)under field conditions?

Temperature affects physiological performance in reptiles and, therefore, body temperature (Tb) control isargued to have an important adaptive value. Alterations in Tb due to transient changes in physiological state, as duringdigestion or gestation, are often linked to the potential benefits of a more precise Tb regulation. However, such thermoregulatory responses in nature remain controversial, particularly for tropical snakes. Herein, we measured Tb of the golden lanceheads, Bothrops insularis (Amaral, 1921), at Queimada Grande Island, southeastern Brazil, to test for alteration in selected body temperatures associated with feeding or gestation. We found no evidence that postprandial or gravid snakes selected for higher Tb indicating that, under natural conditions, body temperature regulation in B. insularis apparently encompasses other ecological factors beyond physiological state per se.

Contrasted thermal regimes do not influence digestion and growth rates in a snake from a temperate climate.

Temperature influences almost all life-history traits. For a period of 3 mo, we placed four groups of snakes under four contrasted thermal treatments: (1) a natural regime (NR), based on daily variations (24-h cycle); (2) an accelerated regime (AR), where the thermoperiod fluctuated rapidly (12-h cycle); (3) a slow regime (SR; 48-h cycle); and (4) a cool stable regime (ZR; no fluctuation). The mean temperature, set at 23°C, was identical for the four groups. For the first three groups (NR, AR, SR), ambient temperature fluctuated between 18°C and 28°C. Relative humidity and photoperiod were constant. We recorded feeding success, digestion efficiency, growth rate, activity, and ecdysis events. Differences between groups were expected because of varied exposure to the optimal temperatures, most notably in the ZR group, where the preferred body temperature for digestion (approximately 30°C) would not be reached. Surprisingly, there was no significant effect of the experimental treatment on feeding rate, digestion, body mass increase, and growth rate. Our results do not conform to the paradigm stipulating that maximal body temperature selected by ectotherms necessarily corresponds to the most efficient for resource assimilation and that temperature fluctuations are essential. We propose that increasing the digestive tract's performance through body-temperature elevation trades off against elevated (parasite) energy expenditure from the rest of the body. The main advantage of high body temperatures would be to reduce the amount of time necessary to assimilate prey rather than to improve the net mass gain during digestion.

Tail Luring by the Golden Lancehead (Bothrops insularis), an Island EndemicSnake from South-Eastern Brazil

Herein, we report on direct and indirect evidence of tail luring by the golden lancehead, Bothrops insularis (Viperidae), a threatened snake species endemic to a small island off the coast of South-eastern Brazil. Tail luring was recorded for a juvenile male in nature and circumstantial evidence indicates that it was directed to ectothermic prey. Indeed, B. insularis juveniles feed on small lizards and frogs and tail luring is probably an important hunting strategy of this species. Adult golden lanceheads prey mostly on birds. A frequently seen characteristic ambush posture, coiled with the conspicuous tail tip exposed close to the head, indicates that they use tail luring as well. Lured birds may peck at a snake's tail, and the high frequency of tail tip injuries in B. insularis (20.9%) compared to a continental sister species that rarely preys on birds (B. jararaca, 7.3%) strengthens the view that B. insularis adults lure birds.

The postnatal development of the optic nerve of a reptile (Vipera aspis): A quantitative ultrastructural study.

The number of axons in the optic nerve of the ovoviviparous reptile Vipera aspis was estimated from electron micrographs taken during the first 5 weeks of postnatal life. One to two days after birth, the optic nerve contains about 170,000 fibres, of which about 9% are myelinated. At the end of the fifth postnatal week, the number of optic fibres has fallen to about 100,000, of which about 42% are myelinated. This fibre loss continues after the fifth postnatal week, since in the adult viper the nerve contains about 60,000 fibres, of which 85% are myelinated; overall, about 65% of the optic nerve fibres present at birth disappear before the number of axons stabilises at the adult level. This study shows, for the first time, that the mode of development of the visual axons of reptiles is not that of anamniote vertebrates but similar to that of birds and mammals.

Age-dependent variations in the venom proteins of Vipera xanthina (Gray, 1849) (Ophidia: Viperidae).

In this study, polyacrylamide disc gel electrophoresis and densitometry analysis methods were used to analyze venom extracts of Vipera xanthina specimens of different lengths (35, 47 and 88 cm) collected from the same locality. The electropherograms of the venom protein samples showed age-dependent qualitative and quantitative variations.