Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina

The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.     

Thrombin-like serine proteases in Cerasted venoms (Cerasted cerastes and Cerastes vipera)

Cerastes cerastes and Cerastes vipera snake venoms are rich of thrombin-like, serine-protease polypeptides. Many proteins have been isolated, purified and characterized from these vipers. These proteins act in various way on blood coagulation pathway and platelet function.     

Immunological study and hemolytic activity of the venom of the horned viper, Cerastes cerastes (Linne, 1758) (Reptilia, Viperidae)

The immunological study of the venom of the Horned Viper, Cerastes cerastes (Linné, 1758) points out eight fractions from which the components have various hemolyticus actions.     

Comparative morphology of the skin of Natrix tessellata (family: Colubridae) and Cerastes vipera (family: Viperidae)

We studied beneficial difference of the skin of two snakes. Two snakes were chosen from two different habitats and two families: Colubridae (Natrix tessellata) and Viperidae (Cerastes vipera). The investigations were performed by light and electron microscopy. Histologically, the skin of the studied species show pronounced modifications that correlated with functional demands. The scales in Natrix tessellata overlapped slightly, while in Cerastes vipera they were highly overlapped. SEM shows that scales of Natrix tessellata had bidentate tips while the scales of Cerastes vipera were keeled. Histochemically, in both studied species, melanocytes and collagenous fibres were distributed throughout the dermis. Polysaccharides were highly concentrated in the epidermis and dermis of both species while proteins were highly concentrated only in the epidermis. Transmission electron microscopy (TEM) showed that the skin of both snakes consisted of keratins located in the epidermis. Some lipids and mucus were incorporated into the outer scale surfaces such that lipids were part of the fully keratinised hard layer of the snakes' skins. Lipids are probably responsible for limiting water loss and ion movements across the skin. Melanosomes from epidermal melanocytes were present only in Cerastes vipera. In aggregate, these results indicate that snakeskin may provide an ecological indicator whereby epidermal and integumentary specializations may be ecologically correlated.     

Activity budgets, habitat use and body size in two coexisting species of sand wasps (Microbembex: Sphecidae, Hymenoptera)

ABSTRACT. 1. Two superficially similar species of Microbembex, M.monodonta (Say) and M.cubana Bohart (Hymenoptera, Sphecidae, Nyssoninae), occur syntopically on Staniel Cay, a small Bahamian island (15 km²).
2. Nesting (female) and territorial (male) aggregations of M.cubana were smaller and more numerous than those of M.monodonta in the study site and on the island as a whole.
3. Both species had regular patterns of daily activities that required them to use a variety of microhabitats in the study area.
4. Males of both species defended territories from c. 09.00 to 12.00 hours EST (in May) and the left to feed on nectar until they retired to sleeping burrows, typically from 13.00 to 15.00 hours. Male M.monodonta were active longer each day and intermixed feeding and territorial defence more than did male M.cubana.
5. Females of both species were active for longer each day, from c. 08.30 to 15.30 hours, and intermixed their respective activities – hunting for provisions, feeding, and nest digging and maintenance – during each day more than did males. Female M.monodonta were active longer each day, rising earlier and retiring later, than female M.cubana.
6. In M.monodonta , females were larger (length, X =11.00 mm) than males (10.5 mm). In M.cubana , males were larger (11.1 mm) than females (10.4 mm). Although mean differences were small, they were highly significant ( F =22.42; P <10⁻⁴) for the species-sex interaction.
7. Differences in activity budgets, habitat use, and sexual size dimorphism appear to be related. A combination of factors, particularly thermal constraints, possible competition, and different mating systems, could explain these patterns.     

Molecular cloning and expression of a functional snake venom serine proteinase,with platelet aggregating activity,from the Cerastes cerastes viper

The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.     

Water detection in the desert sand scorpion,Paruroctonus mesaensis (Scorpionida,Vaejovidae)

Summary For the sand scorpion, Paruroctonus mesaensis, substrate moisture is a powerful and fast-acting stimulus of discrete behaviors related to localization and imbibitory uptake of water. These behaviors are readily observed in the field and quantified in the laboratory when free-roaming animals encounter sand substrates dampened by small amounts of water. Of 10 behaviors we monitored in laboratory tests, 5 (pedipalp-pull, rototiller-digging, prolonged stops, headstand, and backingup) occurred only after contact with a moistened substrate. These water-stimulated behaviors were selectively blocked when all 8 tarsal leg segments were coated with wax; coverings of the chemosensory pectine appendages had little to no effect. Electrophysiological recordings from chemoreceptor organs on the tarsi showed that neurons innervating the dorsal tarsal organ, were highly sensitive to humid air stimuli while the numerous, poretipped hairs on the ventral surface were responsive to aqueous solutions applied directly to their tips. Selective blocking of the 8 tarsal organs had no effect on water sensitive behavior indicating that the chemosensory hairs mediate detection of substrate moisture. Such localized, sensory triggering of a robust and directed behavior presents a useful model for further neuroethological studies.Abbreviations H headstand - PP pedipalp-pull - RD rototillerdigging - B backing-up - C wall-climbing - P pause - W walk-through - S stop - T turn - R rest - PST pacific standard time - SEM scanning electron microscopy     

Promiscuity in sand lizards (Lacerta agilis) and adder snakes (Vipera berus): causes and consequences

We review postcopulatory phenomena in the Swedish sand lizard (Lacerta agilis) and adder (Vipera berus), and in particular, links between female promiscuity, determinants of paternity, and offspring viability. In both species, females mate multiply and exhibit a positive relationship between the number of partners and offspring viability. We conclude that this relationship is most likely the result of variable genetic compatibility between mates arising from postcopulatory phenomena, predominantly assortative fertilization with respect to parental genotypes. However, males who were more successful at mate acquisition were also more successful in situations of sperm competition, suggesting a possible link between male (diploid and haploid) genetic quality per se and probability of fertilization. Neither the number of partners nor the number of matings influenced the risk of infertility in sand lizards, suggesting that selection for reduced risk of infertility is not a sufficient explanation for maintaining female promiscuity in this population. Finally, we conclude that the relatively low genetic variability exhibited by our study populations may have facilitated detection of genetic benefits compared to more outbred ones. However, recent work derived from outbred populations in other taxa suggest a greater generality of the principles we discuss than previously may have been appreciated.     

Malformed offspring,sibling matings,and selection against inbreeding in the sand lizard (Lacerta agilis)

We demonstrated that sand lizards (Lacerta agilis) are more likely to have malformed offspring when they mate with siblings. Offspring with malformations, such as deformed limbs and heads, have zero survival in a natural population. Normal-looking siblings of malformed hatchlings also had a reduced survival in the wild, compared to offspring from clutches in which all siblings appeared normal. The proportion of malformed hatchlings in the natural population was ca. 10%, in spite of differences in juvenile dispersal between males and females. Male juveniles disperse significantly further from their natal sites than do female juveniles.     

Moving in two worlds: aquatic and terrestrial locomotion in sea snakes (Laticauda colubrina,Laticaudidae)

Yellow‐lipped sea kraits (Laticauda colubrina) are amphibious in their habits. We measured their locomotor speeds in water and on land to investigate two topics: (1) to what degree have adaptations to increase swimming speed (paddle‐like tail etc.) reduced terrestrial locomotor ability in sea kraits?; and (2) do a sea krait’s sex and body size influence its locomotor ability in these two habitats, as might be expected from the fact that different age and sex classes of sea kraits use the marine and terrestrial environments in different ways? To estimate ancestral states for locomotor performance, we measured speeds of three species of Australian terrestrial elapids that spend part of their time foraging in water. The evolutionary modifications of Laticauda for marine life have enhanced their swimming speeds by about 60%, but decreased their terrestrial locomotor speed by about 80%. Larger snakes moved faster than smaller individuals in absolute terms but were slower in terms of body lengths travelled per second, especially on land. Male sea kraits were faster than females (independent of the body‐size effect), especially on land. Prey items in the gut reduced locomotor speeds both on land and in water. Proteroglyphous snakes may offer exceptional opportunities to study phylogenetic shifts in locomotor ability, because (1) they display multiple independent evolutionary shifts from terrestrial to aquatic habits, and (2) one proteroglyph lineage (the laticaudids) displays considerable intraspecific and interspecific diversity in terms of the degree to which they use terrestrial vs. aquatic habitats.     

Prey and feeding phenology of the desert sand scorpion Pamroctonus mesaensis (Scorpionidae: Vaejovidae)

The diet and feeding of the vaejovid scorpion Pamroctonus mesaensis Stahnke was investigated during a five-year study. Foraging and feeding behaviour are described. This scorpion is a "sit and wait" predator that eats a wide variety of cursorial, fossorial and aerial prey. Ninety-five prey species were recorded. Three year classes of scorpions exist. Each class captures a different proportion of major prey species and a significantly different average size of prey.
The proportion of each major taxon of prey in the diet is as follows: tenebrionid beetles (42%), Orthoptera (17%), other scorpions (16%) and Hymenoptera (12%). When analysed in relation to biomass, larger prey species assume more importance while smaller species are relatively less important. Although P. mesaensis ranks as the fourth most numerous prey species, they represent the most important diet item in terms of ingested biomass.
When observed, 3·75% of all scorpions were feeding. This percentage varied seasonally from a high in spring (7.0%) to a low in November and December (0.5 %). The proportions of major prey taxa in the diet also varied seasonally. Prey species were classified into three phenological categories: pulsed, seasonal and annual. Examples of each are given.     

The snake family Psammophiidae (Reptilia: Serpentes): phylogenetics and species delimitation in the African sand snakes (Psammophis Boie, 1825) and allied genera

This study constitutes the first evolutionary investigation of the snake family Psammophiidae--the most widespread, most clearly defined, yet perhaps the taxonomically most problematic of Africa's family-level snake lineages. Little is known of psammophiid evolutionary relationships, and the type genus Psammophis is one of the largest and taxonomically most complex of the African snake genera. Our aims were to reconstruct psammophiid phylogenetic relationships and to improve characterisation of species boundaries in problematic Psammophis species complexes. We used approximately 2500 bases of DNA sequence from the mitochondrial and nuclear genomes, and 114 terminals covering all psammophiid genera and incorporating approximately 75% of recognised species and subspecies. Phylogenetic reconstructions were conducted primarily in a Bayesian framework and we used the Wiens/Penkrot protocol to aid species delimitation. Rhamphiophis is diphyletic, with Rhamphiophis acutus emerging sister to Psammophylax. Consequently we transfer the three subspecies of Rhamphiophis acutus to the genus Psammophylax. The monotypic genus Dipsina is sister to Psammophis. The two species of Dromophis occupy divergent positions deeply nested within Psammophis, and we therefore relegate Dromophis to the synonymy of Psammophis. Our results allow division of the taxonomically problematic Psammophis 'sibilans' species complex into two monophyletic entities, provisionally named the 'phillipsii' and 'subtaeniatus' complexes. Within these two clades we found support for the status of many existing species, but not for a distinction between P.p. phillipsii and P. mossambicus. Additionally, P. cf. phillipsii occidentalis deserves species status as the sister taxon of P. brevirostris.     

Influence of extrinsic variables on activity and habitat selection of lowland tapirs (Tapirus terrestris) in the coastal sand plain shrub,southern Brazil

The objectives of this research were to: 1. evaluate the circadian activity patterns of lowland tapirs (Tapirus terrestris) throughout the seasons and 2. study the influence of moonlight, temperature and rainfall on the activity patterns and habitat selection of this species, in the coastal sand shrub in southern Brazil. From June 2005 to June 2006, eight tapirs were monitored in a large enclosure containing open and vegetation-covered areas, using four camera traps. Differences in activity patterns within seasons were found. Tapir predominately presented nocturnal-crepuscular activity; however, they differed in the winter, with cathemeral activity patterns. Covered areas were mostly used during periods of extreme temperatures, with less diurnal and more nocturnal activities within these areas, on hotter days. Activity in open areas mainly occurred during periods of intermediate temperatures, both during the day and in the night. Moonlight intensity did not influence nocturnal activities. On days of precipitation of 34 mm or more, there was no record of open-area activities, despite constant activity in covered-area.     

Sexual selection, stabilizing selection and fluctuating asymmetry in two populations of pupfish (Cyprinodon pecosensis)

Fluctuating asymmetry of morphological traits is thought to reflect the capacity of a genotype to produce an integrated, functional phenotype. I tested three predictions. (1) In a polygynous breeding system, under intense sexual selection on males, breeding males should show greater symmetry in bilaterally symmetrical traits than non-breeding males or females. (2) If these traits are under stabilizing selection, highly symmetrical individuals also should be modal phenotypes, thus near the mean value for that trait, whereas individuals with increased asymmetry should represent marginal phenotypes, near the extremes of the distribution for that trait. (3) Differences in the intensity of sexual selection should be reflected in differences in the degree of fluctuating asymmetry between sexes among populations. I examined the relationship between male breeding status and the degree of fluctuating asymmetry of four bilaterally symmetrical- traits, preorbital and preopercular pores and pectoral and pelvic fin rays, in two populations of Pecos pupfish which differed in the intensity of sexual selection. These traits do not function in male-male competition or female choice, thus are not directly affected by sexual selection. In Mirror Lake breeding males, as a group, were most symmetrical for all four traits, while non-breeding males and females showed higher levels of fluctuating asymmetry. Similarly, symmetrical individuals also represented modal phenotypes for four traits (breeding males), and for three traits (non-breeding males and females). These patterns were not seen in the Lake Francis population, where breeding males were as asymmetrical as non-breeding males and females, and the degree of fluctuating symmetry did not differ between modal and marginal phenotypes for any of the four traits. When ecological conditions favour intense sexual selection, either through female choice, male-male competition, or both, breeding males represent the most fit phenotypes. Thus sexual selection reinforces the effects of stabilizing selection on characters that do not function as secondary sexual traits. However, when sexual selection is relaxed, differences between sexes disappear.     

Temporal variation in diets and trophic partitioning by coexisting lizards (Ctenotus: Scincidae) in central Australia

Summary The diets of five syntopic species of Ctenotus were examined over a two-year period on a 60 ha spinifex grassland site in central Australia. The aims of the study were to test predictions that termites were an important part of the food web for syntopic Ctenotus in spinifex grasslands, and to examine seasonal changes in prey use and dietary overlap between the species. Environmental conditions during the first season of the study were dry resulting in generally low invertebrate abundance. In contrast the second season was relatively moist and overall invertebrate abundance was higher than in the first season. Diets of five species of Ctenotus contained a range of terrestrial prey although one species (C. pantherinus) was relatively termite-specialized at all times. Dietary overlap at the ordinal level between the species was generally higher during dry periods when prey abundance was low, and higher for species-pairs that were similar in body size. During the driest period of the study most species of Ctenotus ate a high proportion of termite prey which accounted for the high dietary overlap. However, each species of Ctenotus consumed different genera or foraging guilds of termites. The results suggest that most of these lizards were opportunistic in their selection of prey but that during dry periods when prey are scarce, termites may play a significant role in supporting a high -diversity of Ctenotus.     

Relationship between floral traits and floral visitors in two coexisting Tecoma species (Bignoniaceae)

In this paper, we studied the floral biology of Tecoma fulva ssp. garrocha and T.?stans in Vaqueros (Salta, Argentina), where both species coexist. We tested the idea that floral traits are associated with the pollinator types that visit them. According to our results, T.?fulva ssp. garrocha presented traits common to bird flowers and were visited by two species of Trochilidae (Colibri serrirostris and Chlorostilbon lucidus). In addition, T.?stans exhibited traits common to hymenoptera flowers and were visited principally by Apis mellifera and Bombus atratus. This study showed that floral traits are predictive of animal visitors.     

Genetic analysis of the mating system and opportunity for sexual selection in northern water snakes (Nerodia sipedon)

We used data collected over 3 years at two study sites to quantifythe rates and consequences of multiple paternity and to determinethe opportunity for selection on male and female northern watersnakes (Nerodia sipedon). We sampled litters from 45 femalesthat gave birth to 811 offspring. Using eight microsatelliteDNA loci (probability of exclusion of nonparental males >0.99), we assigned paternity to 93% of neonates from one studypopulation and 69% of neonates from the other population. Observationsof participation in mating aggregations predicted individual reproductive success poorly for two reasons. First, males regularlycourted nonreproductive females. Second, more than half ofall sexually mature males obtained no reproductive successeach year, despite the fact that many of them participatedin mating aggregations. The number of sires per litter ranged from one to five, with 58% of all litters sired by more thanone male. Multiple paternity increased with female size, apparentlyboth because bigger females mated with more males and becausethe larger litters of big females provide paternity opportunitiesto more males. Multiple paternity was also more prevalent inyears with shorter mating seasons. We detected no advantage to multiple paternity in reducing either the number of unfertilizedovules or stillborn young. Despite the majority of males siringno young each year, some males fathered young with as manyas three different females in one year. Male reproductive successincreased by more than 10 offspring for each additional mate,whereas female success increased by fewer than 2 offspring foreach additional mate. The opportunity for sexual selectionwas more than five times higher in males than females.     

Temporal variation in spatial organization of a rodent community in the southwestern Kyzylkum desert (Middle Asia)

The distribution of niches in resource space and the niche patterns of a 14-species community of Middle Asian desert rodents were studied during two years - at low and high rodent density - using discriminant function analysis Nineteen quantitative environmental parameters (soil structure and vegetation), measured in 550 plots within 22 1 -ha grids, were considered The first three canonical axes of resource space account for 72% of the variance The first two axes represent complex environmental gradients the first axis represents a general landscape gradient from sand to clay soils, the second axis reflects a gradient of in creasing productivity The third axis reflects with in-habitat environmental variation All community parameters, as well as parameters of individual species niches, were unstable between years At the same time, different parameters vary in different extent Position of niche centroids along macro-habitat axes, as. well as macrohabitat niche breadth, were relatively stable between years, but these parameters for microhabitat axis and values of niche overlap were much more variable A strong correlation between changes m relative between-habitat niche breadth and differences in average niche overlap with relative changes in species abundances indicate density dependence of these parameters Changes in niche overlap is a consequence of between-year differences in guild patterns Guild structure was pronounced at high density when the level of niche overlap was intermediate At low density, when the level of niche overlap decreased, guild structure was incon-spicous Different levels of diversity differed in their sensitivity to density changes α-diversity was relatively constant as a result of between-year stability of niche centroid positions However level of ß-diversity varied significantly between years reflecting changes in the level of niche overlap, because a decrease in niche overlap leads to an increase in the rate of species turnover