Functional plasticity of the venom delivery system in snakes with a focus on the poststrike prey release behavior

We explored variations in the morphology and function of the envenomation system in the four families of snakes comprising the Colubroidea (Viperidae, Elapidae, Atractaspididae, and Colubridae) using our own prey capture records and those from the literature. We first described the current knowledge of the morphology and function of venom delivery systems and then explored the functional plasticity found in those systems, focusing on how the propensity of snakes to release prey after the strike is influenced by various ecological parameters. Front-fanged families (Viperidae, Elapidae, and Atractaspididae) differ in the morphology and topographical relationships of the maxilla as well as in the lengths of their dorsal constrictor muscles (retractor vomeris; protractor, retractor, and levator pterygoidei; protractor quadrati), which move the bones comprising the upper jaw, giving some viperids relatively greater maxillary mobility compared to that of other colubroids. Rear-fanged colubrids vary in maxillary rotation capabilities, but most have a relatively unmodified palatal morphology compared to non-venomous colubrids. Viperids launch rapid strikes at prey, whereas elapids and colubrids use a variety of behaviors to grab prey. Viperids and elapids envenomate prey by opening their mouth and rotating both maxillae to erect their fangs. Both fangs are embedded in the prey by a bite that often results in some retraction of the maxilla. In contrast, Atractaspis (Atractaspididae) envenomates prey by extruding a fang unilaterally from its closed mouth and stabbing it into the prey by a downward-backwards jerk of its head. Rear-fanged colubrids envenomate prey by repeated unilateral or bilateral raking motions of one or both maxillae, some aspects of which are kinematically similar to the envenomation behavior in Atractaspis. The envenomation behavior, including the strike and prey release behaviors, varies within families as a function of prey size and habitat preference. Rear-fanged colubrids, arboreal viperids, and elapids tend to hold on to their prey after striking it, whereas atractaspidids and many terrestrial viperids release their prey after striking it. Larger prey are more frequently released than smaller prey by terrestrial front-fanged species. Venom delivery systems demonstrate a range of kinematic patterns that are correlated to sometimes only minor modifications of a common morphology of the jaw apparatus. The kinematics of the jaw apparatus are correlated with phylogeny, but also show functional plasticity relating to habitat and prey.     

Bioweapons synthesis and storage: The venom gland of front-fanged snakes

A paradoxical task of the venom gland of snakes is the synthesis and storage of an instantly available suite of toxins to immobilize prey and the protection of the snake against its own venom components. Furthermore, autolysis of the venom constituents due to the action of venom metalloproteases is an additional problem, particularly among viperid venoms, which are typically rich in lytic enzymatic proteins. To address questions concerning these problems, the structure of the venom gland was investigated using light microscopy, SEM and TEM. The composition of the venom originating from the intact venom apparatus or from the main venom gland alone was analyzed by electrophoresis, and the pH of freshly expressed venom as well as pH optima of several representative enzymes was evaluated. Results from several species of rattlesnakes demonstrated that the venom gland is structurally complex, particularly in its small rostral portion called the accessory gland, which may be a site of activation of venom components. Secreted venom is stable in extremes of temperature and dilution, and several proximate mechanisms, including pH and endogenous inhibitors, exist which inhibit enzymatic activity of the venom during storage within the venom gland but allow for spontaneous activation upon injection into prey. Whereas acid secretion by the parietal cells activates digestive enzymes in the stomach, within the venom gland acidification inhibits venom enzymes. We propose that the mitochondria-rich cells of the main venom gland, which are morphologically and histochemically very similar to the parietal cells of the mammalian gastric pit, play a central role in the stabilization of the venom by secreting acidic compounds into the venom and maintaining the stored venom at pH 5.4. Hence, our results indicate yet another trophic link between the processes of venom production and of digestion, and demonstrate that the venom glands of snakes may represent an excellent model for the study of protein stability and maintenance of toxic proteins.     

The effect of trap spacing on the capture of brown tree snakes on Guam

Trapping is central to the integrated control programme to deter brown tree snakes from entering the outbound cargo flow from Guam. Trapping brown tree snakes is effective, but labour intensive. Increasing inter-trap spacings without loss of efficacy could substantially increase the efficiency of efforts to prevent dispersal of this species. Inter-trap spacings of 20, 30 and 40 m along perimeter trap lines were compared using recaptures of tagged brown tree snakes. No differences were found among the different spacing distances for the distribution of recapture times. The results indicate that for some situations, snake trapping may be extended or made more efficient by increasing the distances between traps.     

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.     

Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences

Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species.     

Partial consumption of prey: the significance of prey water loss on estimates of biomass intake

Summary A number of studies on the feeding behaviour of sucking predators have estimated the weight of biomass the predator extracts from the prey by measuring the weight change occurring in the prey. This method does not consider that a proportion of the prey weight change is lost to the immediate environment. I examined the spider Diaea sp. feeding on the fruit fly Drosophila immigrans and found that the prey lost approximately 28% more weight than the predator gained. This difference was largely explained by water loss from the prey. My results suggest that water loss, which is not available to the predator, is an important part of prey weight loss. To avoid overestimating predator biomass gain it is necessary to measure the predator weight gain directly or take into account water loss as a component of prey weight change.     

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-³H]thymidine into proliferating Epstein–Barr transformed (EBV) B-cells. The LD₅₀ was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an α-helical secondary structure.     

不同地理单元尖吻蝮蛇毒蛋白组分比较研究

目的探讨不同地理单元尖吻蝮蛇毒蛋白组分有无差异性。方法采用非变性聚丙烯酰胺凝胶电泳(NativePAGE)方法,比较采自安徽黄山(黄山单元)和贵州梵净山(西部单元)尖吻蝮蛇毒组分。结果梵净山尖吻蝮的蛇毒蛋白表达量和条带数目均高于黄山尖吻蝮。结论黄山单元和西部单元的尖吻蝮蛇毒蛋白组分具有差异性。     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

Structural and Functional Characterization of a Novel Homodimeric Three-finger Neurotoxin from the Venom of Ophiophagus hannah (King Cobra)

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (αβγδ) and neuronal (α₇, α₃β₂, and α₄β₂) nicotinic acetylcholine receptors (nAChRs) with highest affinity for α₇-nAChRs. The high resolution (1.5 Å) crystal structure revealed haditoxin to be a homodimer, like κ-neurotoxins, which target neuronal α₃β₂- and α₄β₂-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain α-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain α-neurotoxins and κ-neurotoxins notwithstanding, haditoxin exhibited unique blockade of α₇-nAChRs (IC₅₀ 180 nm), which is recognized by neither short-chain α-neurotoxins nor κ-neurotoxins. This is the first report of a dimeric short-chain α-neurotoxin interacting with neuronal α₇-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Adenosine in the venoms from viperinae snakes of the genus Bitis: identification and quantitation using LC/MS and CE/MS

Snake venoms are rich sources of toxic proteins and small molecules. This study was directed at molecules of molecular mass below 1 kDa. Thirty different venoms, of either neurotoxic or haemorrhagic type, were fractionated using size-exclusion chromatography. Only venoms of the Puff adder (Bitis arietans), Gaboon viper (Bitis gabonica), and Rhinoceros viper (Bitis nasicornis) exhibited large absorbance peaks at lambda(280 nm) in the total volume range of the chromatographic column indicating the presence of abundant low molecular mass material. Analysis of fractions containing this material using both HPLC and capillary electrophoresis interfaced with electrospray ion-trap mass spectrometry unequivocally established that the bioactive nucleoside, adenosine, was the major component. The concentrations of adenosine found (Puff adder--97.7 x 10(-6) mol L(-1); Gaboon viper--28.0 x 10(-6) mol L(-1); and Rhinoceros viper-56.8 x 10(-6) mol L(-1)) were above those required to activate all known sub-types of adenosine receptors. Adenosine may thus act at the site of envenomation causing local vasodilatation and may play a role in the subsequent systemic hypotension observed.     

Feeding behavior modulation in the leopard lizard (Gambelia wislizenii): effects of noxious versus innocuous prey

Feeding, a fundamentally rhythmic behavior in many animals, is expected to exhibit modulation with respect to prey type. Using high-speed videography (200 frames s⁻¹) and kinematic analysis, we investigated prey-processing behavior in the long-nosed leopard lizard (Gambelia wislizenii). The effects of two prey types were examined, innocuous immature crickets (Acheta domesticus) and noxious stinging hymenopterans (honeybees [Apis mellifer] and yellow jackets [Vespula sp.]). Stinging hymenopterans are processed more extensively, with higher gape-cycling frequencies, and for a greater duration than are crickets. Generalized tetrapod feeding models were used as a framework to test the hypothesis that gape profile characteristics are modulated in response to prey noxiousness. Cricket processing generally fits the models, but hymenopteran processing departs from typical model parameters. In particular, the SO phase is absent to barely detectable during hymenopteran processing. This likely represents an effect of extrinsic neural input on a centrally directed rhythmic motor pattern, possibly to avoid being stung. Differences in the capture behavior of crickets versus hymenopterans indicate that G. wislizenii assesses prey noxiousness before physical contact with prey and modifies its capture behavior accordingly. These results add to the growing body of evidence that sensory information can play a critical role in shaping stereotyped rhythmic behaviors in non-mammalian tetrapods.     

Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor

Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.     

Allometry and selection in a novel predator–prey system: Australian snakes and the invading cane toad

Because many organismal traits vary with body size, interactions between species can be affected by the respective body sizes of the participants. We focus on a novel predator–prey system involving an introduced, highly toxic anuran (the cane toad, Bufo marinus ) and native Australian snakes. The chance of a snake dying after ingesting a toad depends on the size of the snake and the size of the toad, and ultimately reflects the effect of four allometries: (1) physiological tolerance (the rate that physiological tolerance to toad toxin changes with snake size); (2) swallowing ability (the rate that maximal ingestible toad size (i.e. snake head size) increases with snake body size); (3) prey size (the rate that prey size taken by snakes increases with snake head size) and (4) toad toxicity (the rate that toxicity increases with toad size). We measured these allometries, and combined them to estimate the rate at which a snake's resistance changes with toad toxicity. The parotoid glands (and thus, toxicity) of toads increased disproportionately with toad size (i.e. relative to body size, larger toads were more toxic) but simultaneously, head size relative to body size (and thus, maximal ingestible prey size relative to predator size) declined with increasing body size in snakes. Thus, these two allometries tended to cancel each other out. Physiological tolerance to toxins did not vary with snake body size. The end result was that across snake species, mean adult body size did not affect vulnerability. Within species, however, smaller predators were more vulnerable, because the intraspecific rate of decrease in relative head size of snakes was steeper than the rate of increase in toxicity of toads. Thus, toad invasion may cause disproportionate mortality of juvenile snakes, and adults of the sex with smaller mean adult body sizes.     

The venom and venom apparatus of the sea snake Lapemis hardwicki Gray

The venom apparatus of Lapemis hardwicki , consisting of two functional fangs, their venom glands, and associated musculature, are described. The yield of venom per snake ranged from 2.4-5.2 mg. The LD₅₀ of the crude venom varied from 0.7-1.4 mg/kg intravenously in mice. The toxicological, chemical and immunological properties of the venom are discussed.     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

Immunolocalization of venom metalloproteases in venom glands of adult and of newborn snakes of Bothrops jararaca

Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.