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Isolation of a neurotoxin (alpha-colubritoxin) from a nonvenomous colubrid: evidence for early origin of venom in snakes.

The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called alpha-colubritoxin, isolated from the Asian ratsnake Coelognathus radiatus (formerly known as Elaphe radiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.

Amino Acid Sequence↗

Complete mitochondrial DNA sequences of six snakes: phylogenetic relationships and molecular evolution of genomic features.

Complete mitochondrial DNA (mtDNA) sequences were determined for representative species from six snake families: the acrochordid little file snake, the bold boa constrictor, the cylindrophiid red pipe snake, the viperid himehabu, the pythonid ball python, and the xenopeltid sunbeam snake. Thirteen protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 control regions were identified in these mtDNAs. Duplication of the control region and translocation of the tRNALeu gene were two notable features of the snake mtDNAs. The duplicate control regions had nearly identical nucleotide sequences within species but they were divergent among species, suggesting concerted sequence evolution of the two control regions. In addition, the duplicate control regions appear to have facilitated an interchange of some flanking tRNA genes in the viperid lineage. Phylogenetic analyses were conducted using a large number of sites (9570 sites in total) derived from the complete mtDNA sequences. Our data strongly suggested a new phylogenetic relationship among the major families of snakes: ((((Viperidae, Colubridae), Acrochordidae), (((Pythonidae, Xenopeltidae), Cylindrophiidae), Boidae)), Leptotyphlopidae). This conclusion was distinct from a widely accepted view based on morphological characters in denying the sister-group relationship of boids and pythonids, as well as the basal divergence of nonmacrostomatan cylindrophiids. These results imply the significance to reconstruct the snake phylogeny with ample molecular data, such as those from complete mtDNA sequences.

Animals↗

Induced defences in an endangered amphibian in response to an introduced snake predator.

Introduced species have contributed significantly to the extinction of endemic species on islands. They also create new selection pressures on their prey that may result in modified life history strategies. Introduced viperine snakes ( Natrix maura) have been implicated in the decline of the endemic midwife toad of Mallorca ( Alytes muletensis). A comparison of A. muletensis tadpoles in natural pools with and without snakes showed that those populations subject to snake predation possessed longer tails with narrower tail fins but deeper tail muscles. Field and laboratory experiments showed that these changes in tail morphology could be induced by chemical and tactile cues from snakes. Populations of tadpoles that were subject to snake predation also displayed clear bimodal size-frequency distributions, with intermediate-sized tadpoles missing from the pools completely. Tadpoles in pools frequented by snakes developed faster in relation to their body size than those in pools without snakes. Variation in morphology between toad populations may therefore be caused by a combination of size-selective predation and tadpole plasticity. The results of this study indicate that the introduction of alien species can result in selection for induced defences, which may facilitate coexistence between predator and prey under certain conditions.

Analysis of Variance↗

Do lizards and snakes really differ in their ability to take large prey? A study of relative prey mass and feeding tactics in lizards.

Adaptations of snakes to overpower and ingest relatively large prey have attracted considerable research, whereas lizards generally are regarded as unable to subdue or ingest such large prey items. Our data challenge this assumption. On morphological grounds, most lizards lack the highly kinetic skulls that facilitate prey ingestion in macrostomate snakes, but (1) are capable of reducing large items into ingestible-sized pieces, and (2) have much larger heads relative to body length than do snakes. Thus, maximum ingestible prey size might be as high in some lizards as in snakes. Also, the willingness of lizards to tackle very large prey items may have been underestimated. Captive hatchling scincid lizards (Bassiana duperreyi) offered crickets of a range of relative prey masses (RPMs) attacked (and sometimes consumed parts of) crickets as large as or larger than their own body mass. RPM affected foraging responses: larger crickets were less likely to be attacked (especially on the abdomen), more likely to be avoided, and less likely to provide significant nutritional benefit to the predator. Nonetheless, lizards successfully attacked and consumed most crickets < or =35% of the predator's own body mass, representing RPM as high as for most prey taken by snakes. Thus, although lizards lack the impressive cranial kinesis or prey-subduction adaptations of snakes, at least some lizards are capable of overpowering and ingesting prey items as large as those consumed by snakes of similar body sizes.

Analysis of Variance↗

Element levels in snakes in South Carolina: differences between a control site and exposed site on the Savannah River site.

Levels of 18 elements, including lead, mercury, selenium, and uranium, were examined in three species of snakes from an exposed and reference site on the Department of Energy's Savannah River Site in South Carolina. We tested the hypotheses that there were no differences as a function of species, and there were no difference between the exposed and control site for blood and muscle (tail) samples for banded water snake (Nerodia fasciata), brown water snake (N. taxispilota) and cotton mouth (Akistrodon piscivorous). The banded water snakes collected were significantly smaller than the other two species. For blood, there were significant species differences only for barium, copper, selenium, uranium and zinc, while for muscle tissue there were significant interspecific differences in aluminum, arsenic, barium, cobalt, cesium, copper, iron, lead, mercury, manganese, strontium, vanadium and zinc, suggesting that muscle tissue in the tail is a better indicator of potential interspecific differences. It is also easier logistically to collect tail tissue than blood. Where one species had significantly higher levels than the other species in muscle tissue levels, cottonmouth had higher levels of five elements (aluminum, cobalt, lead, mercury, vanadium), brown water snake had two (lead, strontium), and banded water snake had only barium. There were few significant differences between the control and reference site for levels of blood, but several for muscle tissue. All three species had significantly higher levels of arsenic and manganese at Tim's Branch than the reference site, and nickel and uranium were significantly higher for banded water snake and cotton mouth, the larger species. Individuals with high exposure of one element were exposed to high levels of other elements.

Animals↗

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake--III. Induction.

1. 3-Methylcholanthrene administration increased levels of cytochrome P-450, benzo[a]pyrene hydroxylase activity and p-nitrophenetole O-deethylase activity in hepatic microsomes from the brown trout (Salmo trutta), leopard frog (Rana pipiens) and the garter snake (Thamnophis). The level of aminopyrine N-demethylase activity was increased in trout microsomes, but not in those from the frog, snake or rat. 2. The shift in the Soret maximum of the reduced carbon monoxide difference spectrum of cytochrome P-450 from 450 to 448 nm, which is observed when 3-methylcholanthrene is administered to rats, was not seen in microsomes from the trout, frog or snake. 3. Benzo[a]pyrene hydroxylase activity induced by 3-methylcholanthrene in the trout, frog and snake was inhibited by relatively low concentrations of SKF 525-A, but that induced in the rat was not. alpha-Naphthoflavone inhibited 3-methylcholanthrene-induced benzo[a]pyrene hydroxylase activity in the trout, frog and rat, but not in the snake. 4. 3-Methylcholanthrene induced an increase in the 455/430 nm peak height ratio of the reduced ethylisocyanide spectrum of microsomes in the rat and trout, but not in the frog or snake. 5. These observations (items 1--4 of the abstract) show that different species of cytochrome P-450 are induced by 3-methylcholanthrene in the four vertebrates. 6. Phenobarbital did not alter the components or the activities of hepatic monooxygenase systems in the trout, frog or snake, even though it was shown to accumulate in the livers of these vertebrates as readily as it does in the liver of the rat.

Animals↗

Biochemical characterization of phospholipase A2 (trimorphin) from the venom of the Sonoran Lyre Snake Trimorphodon biscutatus lambda (family Colubridae).

Phospholipases A(2) (PLA(2)), common venom components and bioregulatory enzymes, have been isolated and sequenced from many snake venoms, but never from the venom (Duvernoy's gland secretion) of colubrid snakes. We report for the first time the purification, biochemical characterization and partial sequence of a PLA(2) (trimorphin) from the venom of a colubrid snake, Trimorphodon biscutatus lambda (Sonoran Lyre Snake). Specific phospholipase activity of the purified PLA(2) was confirmed by enzyme assays. The molecular weight of the enzyme has been determined by SDS-PAGE and mass spectrometry to be 13,996 kDa. The sequence of 50 amino acid residues from the N-terminal has been identified and shows a high degree of sequence homology to the type IA PLA(2)s, especially the Asp-49 enzymes. The Cys-11 residue, characteristic of the group IA PLA(2)s, and the Ca(2+) binding loop residues (Tyr-28, Gly-30, Gly-32, and Asp-49) are conserved. In addition, the His-48 residue, a key component of the active site, is also conserved in trimorphin. The results of phylogenetic analysis on the basis of amino acid sequence homology demonstrate that trimorphin belongs to the type IA family, and it appears to share a close evolutionary relationship with the PLA(2)s from hydrophiine elapid snakes (sea snakes and Australian venomous snakes).

Amino Acid Sequence↗

Occupational injuries with captive lance-headed vipers (Bothrops moojeni): experience from a snake farm in Brazil.

We studied occupational injuries with captive lance-headed vipers (Bothrops moojeni) that occurred in a snake farm in south-eastern Brazil from February 1981 to May 1999. The risk of injury, taking into account 13 cases of snake-associated injuries (12 of them snake bites) was 2.73 per 10,000 person-days of work, and 3.51 per 100,000 venom extractions. Thirteen cases of injury occurred in seven workers, whereas 18 workers were never injured, suggesting that some individuals have a higher risk of injury than others perhaps due to lack of concentration or overconfidence. Eight episodes of occupational injuries occurring in four technicians, including a case of eye injury due to splashed venom during extraction, are reported. Assessment of whether envenoming occurred was facilitated by knowledge of the snake species and size, history of recent venom extraction and snake feeding, and examination of snake venom glands. Hypersensitivity reactions (anaphylaxis and serum sickness) to antivenom are a risk particularly to those workers who were bitten more than once and medicated previously. Antivenom therefore should not be administered to these individuals unless there is clear evidence that envenoming occurred or is likely to have occurred. Hypersensitivity to the venom is also a health concern for workers from snake farms.

Accidents, Occupational↗

The prevalence and morbidity of snake bite and treatment-seeking behaviour among a rural Kenyan population.

Snake-bite mortality among a rural population in Kenya was estimated to be 6.7/100,000 people each year, representing 0.7% of all deaths. A community-based retrospective survey of 4712 households provided estimates of the incidence of snake bite in this population. Although 151/100,000 people are bitten each year, only 19% of these are bitten by potentially venomous snakes. When those who had been bitten were shown photographs of a range of locally prevalent snakes, most indicated that both venomous and non-venomous snakes were capable of causing death. Most (68%) of bite cases sought treatment from a traditional healer who invariably used local herbal preparations applied to the bite site and/or in a ring around the bitten limb. Local skin incisions were also commonly practised. The use of traditional medicine for snake bite is a feature of most areas of the developing world where venomous snakes are prevalent. Improvements in early referral and appropriate care will only occur when traditional healers are integrated into primary health care and hospital-based health systems.

Adolescent↗

Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences.

We analyzed the origin and evolution of snake venom toxin families represented in both viperid and elapid snakes by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. Out of eight toxin families analyzed, five provided clear evidence of recruitment into the snake venom proteome before the diversification of the advanced snakes (Kunitz-type protease inhibitors, CRISP toxins, galactose-binding lectins, M12B peptidases, nerve growth factor toxins), and one was equivocal (cystatin toxins). In two others (phospholipase A(2) and natriuretic toxins), the nonmonophyly of venom toxins demonstrates that presence of these proteins in elapids and viperids results from independent recruitment events. The ANP/BNP natriuretic toxins are likely to be basal, whereas the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited twice. In contrast to the basal recruitment of the galactose-binding lectins, the C-type lectins were shown to be Viperidae only, with the alpha-chains and beta-chains resulting from an early duplication event. These results provide strong additional evidence that venom evolved once, at the base of the advanced snake radiation, rather than multiple times in different lineages, with these toxins also present in the venoms of the "colubrid" snake families. Moreover, they provide a first insight into the composition of the earliest ophidian venoms and point the way toward a research program that could elucidate the functional context of the evolution of the snake venom proteome.

Animals↗

Molecular evidence and marine snake origins.

A molecular phylogeny was used to refute the marine scenario for snake origins. Nuclear gene sequences suggested that snakes are not closely related to living varanid lizards, thus also apparently contradicting proposed relationships between snakes and marine mosasaurs (usually considered to be varanoids). However, mosasaurs share derived similarities with both snakes and living varanids. A reanalysis of the morphological data suggests that, if the relationships between living taxa are constrained to the proposed molecular tree, with fossil forms allowed to insert in their optimal positions within this framework, mosasaurs cluster with snakes rather than with varanids. Combined morphological and molecular analyses also still unite marine lizards with snakes. Thus, the molecular data do not refute the phylogenetic evidence for a marine origin of snakes.

Animals↗

Empirical evidence for an optimal body size in snakes.

The concept of optimal size has been invoked to explain patterns in body size of terrestrial mammals. However, the generality of this phenomenon has not been tested with similarly complete data from other taxonomic groups. In this study we describe three statistical patterns of body size in snakes, all of which indicate an optimal length of 1.0 m. First, a distribution of largest body lengths of 618 snake species had a single mode at 1.0 m. Second, we found a positive relationship between the size of the largest member of an island snake assemblage and island area and a negative relationship between the size of the smallest member of an island snake assemblage and island area. Best-fit lines through these data cross at a point corresponding to 1.0 m in body length, the presumed optimal size for a one-species island. Third, mainland snake species smaller than 1.0 m become larger on islands whereas those larger than 1.0 m become smaller on islands. The observation that all three analyses converge on a common body size is concordant with patterns observed in mammals and partial analyses of four other disparate animal clades. Because snakes differ so strikingly from mammals (ectotherms, gape-limited predators, elongate body shape) the concordant patterns of these two groups provide strong evidence for the evolution of an optimal body size within independent monophyletic groups. However, snakes differ from other taxonomic groups that have been studied in exhibiting a body size distribution that is not obviously skewed in either direction. We suggest that idiosyncratic features of the natural history of ectotherms allow relatively unconstrained distributions of body size whereas physiological limitations of endotherms constrain distributions of body size to a right skew.

Animals↗

Morphological integration and adaptation in the snake feeding system: a comparative phylogenetic study.

A long-standing hypothesis for the adaptive radiation of macrostomatan snakes is that their enlarged gape--compared to both lizards and basal snakes--enables them to consume "large" prey. At first glance, this hypothesis seems plausible, or even likely, given the wealth of studies showing a tight match between maximum consumed prey mass and head size in snakes. However, this hypothesis has never been tested within a comparative framework. We address this issue here by testing this hypothesis in 12 monophyletic clades of macrostomatan snakes using recently published phylogenies, published maximum consumed prey mass data and morphological measurements taken from a large sample of museum specimens. Our nonphylogenetically corrected analysis shows that head width--independent of body size--is significantly related to mean maximum consumed prey mass among these clades, and this relationship becomes even more significant when phylogeny is taken into account. Therefore, these data do support the hypothesis that head shape is adapted to prey size in snakes. Additionally, we calculated a phylogenetically corrected morphological variance-covariance matrix to examine the role of morphological integration during head shape evolution in snakes. This matrix shows that head width strongly covaries with both jaw length and out-lever length of the lower jaw. As a result, selection on head width will likely be associated with concomitant changes in jaw length and lower jaw out-lever length in snakes.

Adaptation, Physiological↗

Photoreceptor cell specific proteins of snake pineal.

The pineal body of lower vertebrates is saccular and directly photoreceptive. The pineal gland of mammals is parenchymal and not directly photoreceptive. The parenchymal morphology of snake pineal raises questions of direct photoreceptivity of snake pineal and of correspondence of molecular homology with morphological homology. S-antigen and rhodopsin are highly conserved photoreceptor cell specific proteins. We used site-specific monoclonal antibodies (MAb) to study S-antigen and rhodopsin of snake pineal. Immunohistochemical reactivity of snake retina and pineal was compared to that of trout, guinea pig, and rat. MAb's to S-antigen reacted with each pineal and retina tested, but reactivity of individual MAb's with snake tissue was more similar to that with trout than with rat or guinea pig tissue. MAb's to rhodopsin did not react with snake pineal, although they did react with the photoreceptive trout pineal body. MAb's to rhodopsin reacted with retina of each species. These results suggest that although snake pineal is morphologically similar to mammalian pineal, and like mammalian pineal is probably not directly photoreceptive, it does have S-antigen homology with lower vertebrates such as trout.

Animals↗

Control of arterial pressure in aquatic sea snakes.

Cardiovascular responses to head-up tilt, acutely graded hemorrhage, and pharmacologic stimulation by principal autonomic drugs were studied in four species of marine snakes, principally Aipysurus laevis (family Hydrophiidae). Arterial pressure varied inversely with tilt angle and blood volume deficit in conscious snakes outside of water, indicating that physiological regulation was poor or lacking. Calculated arterial pressures at head level typically diminished to zero in A. laevis tilted to angles greater than or equal to 30 degrees. Arterial pressure (corrected for external water pressure) did not change when these snakes were tilted in seawater. Changes of arterial pressure induced by tilt, blood loss, or autonomic drugs elicited reflex adjustments in heart activity, but the magnitude of these responses was less than that observed in terrestrial species of snake. It is concluded that baroreflexes are present but comparatively ineffective in sea snakes. Snakes tolerated large losses of blood volume, and extravascular fluids were absorbed into the circulation during hemorrhage; both hemorrhage and estimated hemodilution volumes exceeded 100% of the initial blood volume in Acalyptophis peronii. Thus, in marine snakes major fluid shifts between nonvascular and vascular compartments significantly compensate hypovolemia but, because of minor autonomic adjustments, do not result in a well-regulated arterial pressure.

Acetylcholine↗

The pathology of nephrotoxicity of gentamicin in snakes. A model for reptilian gout.

Two gopher snakes (Pitophis melanoleucus catenifer) each were given 5 mg/kg body weight gentamicin every 72 hours (group 1); two snakes each were given 5 mg/kg/day (group 2). Doses for both groups were given over a 2-week period. After the second week, the dose for one snake in each group was increased to 50 mg/kg/day for 2 more weeks and then discontinued. Weekly renal biopsies taken from snakes in group 1 showed no abnormalities by light microscopy during and at the completion of the experiment. Snakes in group 2 had cloudy swelling of the proximal tubules at 2 and 4 weeks after the gentamicin was administered. Snakes given the high dose of gentamicin had hydropic degeneration of the proximal tubules 2 weeks after the dose was raised to 50 mg/kg/day. This progressed to tubular necrosis 1 week after the gentamicin was discontinued. These snakes (high dose) also developed visceral gout, apparently as the result of the extensive tubular necrosis. Tophi were in the pericardium, serosal membranes and parenchyma of the kidneys, liver, spleen and lungs.

Animals↗

Partial purification of prolactin-like substance from snake (Ptyas mucosa) pituitaries.

Pituitary extract of the common rat snake (Ptyas mucosa) was found to be capable of displacing the binding of 125I-labelled ovine prolactin to female rat liver membranes, suggesting the presence of prolactin-like substance in snake pituitary. The snake prolactin-like substance was unadsorbed on Concanavalin A-Sepharose, but adsorbed on DEAE-cellulose. The partially purified snake prolactin-like substance was also capable of displacing the binding of 125I-labelled ovine prolactin to snake kidney and large intestine membranes. Chromatographic fractions derived from snake pituitary and which possessed potent growth hormone receptor binding activity were devoid of prolactin receptor binding activity, suggesting the existence of distinct prolactin-like and growth hormone-like substances in snake pituitary.

Animals↗

Snake venom metalloproteinase containing a disintegrin-like domain, its structure-activity relationships at interacting with integrins.

Snake venom disintegrins represent a family of RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp)-containing proteins which have been reported to be unique and potentially useful tools not only for investigating integrin-ligand interactions, but also for the development of anti-thrombotic agents in terms of their anti-platelet activities. Snake venom proteins containing a disintegrin-like domain represent another super-family of proteins in which many of them have been demonstrated to have similar ability to inhibit platelet aggregation and integrin-mediated cell adhesion as the disintegrins. This super-family includes a large number of snake venom metalloproteinases and disintegrin related, RGD-containing snake venom proteins (disintegrin-like proteins) such as dendroaspin. Recently, a family of homologues of the snake venom metalloproteinases have been found in a wide variety of mammalian tissues as well as in other eukaryotic organisms termed ADAM (a disintegrin-like and metalloproteinase) proteins. ADAMs are members of the metazincins that also include the related matrix metalloprotease (MMPs). Some of ADAM proteins have now shown to interact with integrins, and the disintegrin-like domain may be crucial part in their function as proteases. A description of structure-activity relationships of snake venom proteins containing a disintegrin-like domain is outlined in this review, along with reports of the modulation of protein activity by recombinant mutation. Comparison is also made of the structural and functional features of the metalloproteinases in snakes compared with those from other species. The review is intended to provide insights in which may assist the development of new therapeutic approaches.

Animals↗