Venom Delivery System Research Articles

Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins

Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III α-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: κ-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx.

Function of the Dufour’s gland in solitary and social Hymenoptera

The poison gland and Dufour's gland are the two glands associated with the sting apparatus in female Apocrita (Hymenoptera). While the poison gland usually functions as an integral part of the venom delivery system, the Dufour's gland has been found to differ in its function in various hymenopteran groups. Like all exocrine glands, the function of the Dufour's gland is to secrete chemicals, but the nature and function of the secretions varies in different taxa. Functions of the Dufour's gland secretions range from serving as a component of material used in nest building, larval food, and pheromones involved in communicative functions that are important for both solitary and social species. This review summarizes the different functions reported for the Dufour's gland in hymenopterans, illustrating how the Dufour's gland secretions can be adapted to give rise to various functions in response to different challenges posed by the ways of life followed by different taxa. Aspects of development, structure, chemistry and the evolution of different functions are also touched upon briefly.

Unusual stability of messenger RNA in snake venom reveals gene expression dynamics of venom replenishment.

Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3–7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies.

The structural and functional diversification of the Toxicofera reptile venom system

The evolutionary origin and diversification of the reptilian venom system is described. The resolution of higher-order molecular phylogenetics has clearly established that a venom system is ancestral to snakes. The diversification of the venom system within lizards is discussed, as is the role of venom delivery in the behavioural ecology of these taxa (particularly Varanus komodoensis). The more extensive diversification of the venom system in snakes is summarised, including its loss in some clades. Finally, we discuss the contentious issue of a definition for “venom”, supporting an evolutionary definition that recognises the homology of both the venom delivery systems and the toxins themselves.

A developmental staging series for the African house snake, Boaedon (Lamprophis) fuliginosus

Embryonic staging series are important tools in the study of morphological evolution as they establish a common standard for future studies. In this study, we describe the in ovo embryological development of the African house snake (Boaedon fuliginosus), a non-venomous, egg-laying species within the superfamily Elapoidea. We develop our staging series based on external morphology of the embryo including the head, eye, facial prominences, pharyngeal slits, heart, scales, and endolymphatic ducts. An analysis of embryonic growth in length and mass is presented, as well as preliminary data on craniofacial skeletal development. Our results indicate that B. fuliginosus embryos are well into organogenesis but lack well-defined facial prominences at the time of oviposition. Mandibular and maxillary processes extend rostrally within 8 days (stage 3), corresponding to the first appearance of Meckel's cartilages. Overall, the development of the craniofacial skeleton in B. fuliginosus appears similar to that of other snake species with intramembraneous bones (e.g., dentary and compound bones) ossifying before most of the endochondral bones, the first of which to ossify are the quadrate and the otic capsule. Our staging series is the first to describe the post-ovipositional development of a non-venomous elapoid based on external morphology. This species is an extremely tractable captive that can produce large clutches of eggs every 45 days throughout the year. As such, B. fuliginosus should be a good model for evolutionary developmental biologists focusing on the craniofacial skeleton, loss of limbs, generational teeth, and venom delivery systems.

Toxicity and metabolic costs of the Atlantic stingray ( Dasyatis sabina) venom delivery system in relation to its role in life history

The Atlantic stingray ( Dasyatis sabina) venomous spine is thought to be primarily a defensive adaptation, but may also play key roles in reproduction and conspecific social interactions. This study quantified metabolic costs associated with the venom delivery system, venom toxicity (LC 50), and routine resting metabolism for Atlantic stingrays. Respirometry trials estimated an average routine resting metabolism for Atlantic stingrays of 0.484 kcal g −1 h − 1 ; whereas the average metabolic rate of the venomous delivery system was 0.000162 kcal g −1 h − 1 , or 0.04% of the fish's resting rate. Ballistic bomb calorimetry revealed an average spine caloric density of 0.238 kcal per gram of spine. Acute toxicity tests using sheepshead minnow, Cyprinodon variegates, as a surrogate, determined a venom LC 50 value of 0.181 mg protein g fish − 1 . When compared with other venomous species, where venom is used primarily for defense and prey capture, the low toxicity and low metabolic cost of the Atlantic stingray's venomous system suggests that defense is not the primary purpose of the venomous spine.

EXTINCTION, ECOLOGICAL OPPORTUNITY, AND THE ORIGINS OF GLOBAL SNAKE DIVERSITY

Snake diversity varies by at least two orders of magnitude among extant lineages, with numerous groups containing only one or two species, and several young clades exhibiting exceptional richness (>700 taxa). With a phylogeny containing all known families and subfamilies, we find that these patterns cannot be explained by background rates of speciation and extinction. The majority of diversity appears to derive from a radiation within the superfamily Colubroidea, potentially stemming from the colonization of new areas and the evolution of advanced venom-delivery systems. In contrast, negative relationships between clade age, clade size, and diversification rate suggest the potential for possible bias in estimated diversification rates, interpreted by some recent authors as support for ecologically mediated limits on diversity. However, evidence from the fossil record indicates that numerous lineages were far more diverse in the past, and that extinction has had an important impact on extant diversity patterns. Thus, failure to adequately account for extinction appears to prevent both rate- and diversity-limited models from fully characterizing richness dynamics in snakes. We suggest that clade-level extinction may provide a key mechanism for explaining negative or hump-shaped relationships between clade age and diversity, and the prevalence of ancient, species-poor lineages in numerous groups.

Phylogenetic relationships among the Stiletto Snakes (genus Atractaspis) based on external morphology

Stiletto Snakes (genus Atractaspis) are the most poorly studied of the three clades of snakes with front-fanged venom-delivery systems. Fossorial and endemic to Africa and the Middle East, Atractaspis possess uniquely derived mobile fangs that allow them to perform a ‘side-stabbing’ method of envenomation, rather than the frontal strike of most other venomous snakes. While many studies have examined the functional anatomy of these unique fangs, the evolutionary history of Atractaspis has been comparatively neglected by researchers. While several evolutionary hypotheses exist for relationships within the Atractaspididae, no study has focused on the phylogenetic relationships within the genus Atractaspis itself. This study presents the first attempt to reconstruct the phylogenetic history of 14 species within this genus, using external morphological data. Using Macrelaps and Homoroselaps as alternate outgroups, We compare the results of two phylogenies and provide a starting point for an investigation of the evolutionary history of this remarkable yet relatively little-known genus.

Grooves to tubes: evolution of the venom delivery system in a Late Triassic “reptile”

Venom delivery systems occur in a wide range of extant and fossil vertebrates and are primarily based on oral adaptations. Teeth range from unmodified (Komodo dragons) to highly specialized fangs similar to hypodermic needles (protero- and solenoglyphous snakes). Developmental biologists have documented evidence for an infolding pathway of fang evolution, where the groove folds over to create the more derived condition. However, the oldest known members of venomous clades retain the same condition as their extant relatives, resulting in no fossil evidence for the transition. Based on a comparison of previously known specimens with newly discovered teeth from North Carolina, we describe a new species of the Late Triassic archosauriform Uatchitodon and provide detailed analyses that provide evidence for both venom conduction and document a complete structural series from shallow grooves to fully enclosed tubular canals. While known only from teeth, Uatchitodon is highly diagnostic in possessing compound serrations and for having two venom canals on each tooth in the dentition. Further, although not a snake, Uatchitodon sheds light on the evolutionary trajectory of venom delivery systems in amniotes and provide solid evidence for venom conduction in archosaur-line diapsids.

Evolution of non-homologous venom delivery systems in West Indian insectivores?

The venomous capabilities of certain mammal species have long been recognized by zoologists, as well as in local folklore (Pournelle, 1968; Dufton, 1992). The extratarsal spur present on the hind l...

A reassessment of the purported venom delivery system of the bird-like raptor Sinornithosaurus

Gong and colleagues recently described unusual traits in the dromaeosaurid Sinornithosaurus that were interpreted as the first evidence of a venomous dinosaur. This interpretation was based on extremely elongated maxillary teeth, morphologically similar to those present in poisonous snakes; labial grooves on maxillary and dentary tooth crowns; and an additional ornamented depression in the lateral surface of the maxillary bone (subfenestral fossa). A reappraisal of each of these morphological traits is provided here in light of comparisons with other theropod dinosaurs and previous discussions for inferring poisonous capabilities in fossil taxa. We fail to recognize unambiguous evidence supporting the presence of a venom delivery system in Sinornithosaurus. For example, the extremely elongated teeth seem to be a taphonomic artifact due to the displacement of teeth outside the alveoli; the labial grooves are present in a wide variety of theropods; and no strong evidence for the lodging of a venomous gland is recognized. In contrast, the cranial and dental anatomy of Sinornithosaurus is congruent with that of other dromaeosaurids. The weak support for a venomous Sinornithosaurus renders unlikely the ecological model proposed by Gong and colleagues for this predatory dinosaur.

Spitting versus Biting: Differential Venom Gland Contraction Regulates Venom Expenditure in the Black-Necked Spitting Cobra, Naja nigricollis nigricollis

According to the venom-metering hypothesis, snakes have the cognitive (decision-making) capacity to control, or meter, how much venom is ejected from the fangs. Critics of venom metering have argued, largely from absence of evidence, that differential venom gland contraction in snakes is trivial or nonexistent. To address this criticism, we videotaped the defensive bite so fNaja nigricollis nigricollis during routine venom extractions. Mean duration of venom flow during a single pulse from a fang, when biting (0.35 sec) was significantly longer than that reported previously for spitting (0.066 sec). Moreover, mean mass of venom expended per pulse from a fang during biting (juveniles: 14.2 mg; adults: 188 mg) significantly exceeded that reported for spitting (1.85 mg). During a single bite, both juveniles and adults delivered venom via pulses that were single, multiple (each associated with a jaw contraction), unilateral (from one fang), or bilateral (from both fangs more or less simultaneously). Although juveniles and adults exhibited similar venom flow duration, adults delivered significantly more venom during biting at significantly greater rates of venom flow through the fang. Because venom gland contraction provides the only propulsive force for venom expulsion, our results confirm that N. n. nigricollis meters larger quantities of venom during biting than spitting via differential venom gland contraction. Because of the high degree of functional convergence between venom delivery systems of elapids (including Spitting Cobras) and viperids (the other large family of venomous snakes), the capacity for differential venom gland contraction may be widespread among snakes.

Helodermatid Lizard from the Miocene of Florida, the Evolution of the Dentary in Helodermatidae, and Comments on Dentary Morphology in Varanoidea

A helodermatid dentary and osteoderms from the Miocene Thomas Farm locality in Gilchrist County, Florida, demonstrate the broad historical distribution of Helodermatidae in North America during the Tertiary. A comparative approach, taking into account variation in extant Heloderma, reveals that the dentary is intermediate in several characters between the plesiomorphically long, low, mediolaterally narrow dentary of Eurheloderma gallicum and the taller, wider dentary of extant Heloderma. An eminence upon the intramandibular septum in the Thomas Farm helodermatid and some extant Varanoidea is part of a previously unrecognized anatomical structure, a foramen formed by dentary and postdentary elements and normally covered by the splenial. The teeth of the Florida helodermatid are intermediate between the more numerous, shorter teeth of E. gallicum and the fewer, taller teeth of extant Heloderma. Certain dental structures whose evolution is here elucidated are part of the novel helodermatid venom delivery system. The evolution of the dentary and teeth in Varanoidea as a whole is a more complex and informative story than was previously appreciated. No living varanoid displays unmodified the primitive varanoid conical tooth morphology. Extant Heloderma show a dentition modified in association with venom delivery, whereas Varanidae show labiolingual compression of the tooth crown. Varanus in particular is highly modified and should not be used as a proxy for the varanoid ancestor in studies of the dentigerous elements and dentition. The informativeness of isolated squamate dentaries is evidenced by the derivation of 15 new phylogenetic characters from the anatomy of helodermatid dentaries and dentition.

The evolution of venom-conducting fangs: Insights from developmental biology

The present study of the origin of the various types of fang represented among colubroid snakes (i.e., tubular, grooved, and ungrooved) attempts to reconcile the morphology of adult fangs with current phylogenetic hypotheses. Observations of growth series of developing tubular fangs were hypothesised to shed light on the evolutionary origin of fangs in snakes. While molecular phylogenies and evolutionary studies of venom proteins and of other anatomical components of the venom-delivery system reconstruct a consistent evolutionary scenario, the character of a tubular venom-conducting fang does not fit in this scenario. The present review offers a series of possible scenarios to resolve this anomaly. Of these, a new idea argues that a heterochronic mechanism (alteration of the timing of developmental events) may provide the answer that the ungrooved and grooved teeth of colubrid snakes evolved from an ancestral tubular fang by means of attachment of replacement tubular fangs to the maxilla at an earlier developmental stage than usual (precocial ankylosis).

Mechanisms controlling venom expulsion in the western diamondback rattlesnake, Crotalus atrox

Although many studies have documented variation in the amount of venom expended during bites of venomous snakes, the mechanistic source of this variation remains uncertain. This study used experimental techniques to examine how two different features of the venom delivery system, the muscle surrounding the venom gland (the Compressor Glandulae in the rattlesnake) and the fang sheath, could influence venom flow in the western diamondback rattlesnake, Crotalus atrox. Differential contraction of the Compressor Glandulae explained only approximately 30% of the variation in venom flow. Lifting (compression) of the fang sheath as occurs during a normal strike produced marked increases in venom flow; these changes were closely correlated and exceed in magnitude by almost 10 x those recorded from the Compressor Glandulae alone. These results suggest that variation in these two aspects of the venom delivery system--both in terms of magnitude and temporal patterning--explain most of the observed variation in venom injection. The lack of functional or mechanical links between the Compressor Glandulae and the fang sheath, and the lack of skeletal or smooth muscle within the fang sheath, make it unlikely that variation in venom flow is under direct neural control. Instead, differential venom injection results from differences in the pressurization by the Compressor Glandulae, the gate keeping effects of the fang sheath and enclosed soft-tissue chambers, and by differences in the pressure returned by peripheral resistance of the target tissue.

Cost of Producing Venom in Three North American Pitviper Species

For over a century, hypotheses regarding the primary functional utility of snake venoms have been debated in literature. Researchers have speculated that the development of venom delivery systems has been a key innovation leading to the evolutionary radiation of venomous snakes over the past 25–30 million years. Interestingly, little is known about the energetic requirements involved in producing venoms in these animals. Here I examined the metabolic cost associated with venom production in three species of North American pitvipers. Immediately following venom extraction, snakes demonstrated an 11% increase in resting metabolic rates during the first 72 h of venom replenishment; this metabolic increase was apparently a result of metabolic costs involved with venom production and was an order of magnitude greater than that predicted for producing an identical mass of mixed body growth. Extracted liquid venom yield of snakes was allometrically correlated with snake body mass (4.77W0.60) and had mean moisture content of 70.9%. Venom yields (wet or dry) were not correlated with the magnitude of the metabolic increase measured during the first 72 h of venom replenishment, suggesting the cost of venom replenishment is independent of extracted mass. The significant post-extraction metabolic increases measured in these snakes support existing hypotheses about the metabolic cost of venom production and may help explain why these animals meter their venom conservatively.

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.

Influence of the venom delivery system on intraoral prey transport in snakes

We compared intraoral prey transport in venomous snake species from four families (two atractaspidids, nine elapids, three colubrids, 44 viperids) with that in eight non-venomous colubrid species, most feeding on similar mammalian prey. The morphology of the venom delivery system suggests that intraoral prey transport performance should be slightly decreased in atractaspidids, unmodified in most elapids and venomous colubrids, and increased or unmodified in vipers, as compared to that in non-venomous colubrid snakes. Our measurements of relative intraoral prey transport performance show that differences among families do not match expectations based on morphology or past studies. Decreased performance in Atractaspis results from reduction and loss of teeth on the medial palatal elements and dentaries, but affects only early phases of ingestion. Although joint and bone features of elapids and colubrids are similar, intraoral prey transport performance is significantly lower in elapids than in colubrids. Predicted enhancement of intraoral prey transport performance in vipers as compared to colubrids was not borne out by measurements, presumably because palatopterygoid movement during intraoral prey transport is reduced in many viper species to limit fang erection. Absence of significant performance differences between colubrids and viperids might suggest that evolution of the viperid venom delivery system was subject to little selection pressure from intraoral prey transport. Another possibility is that there are trade-offs between intraoral prey transport and strike performance in vipers related to relative skull mass and jaw fragility. Immobilizing prey prior to intraoral transport places less demand on transport performance in vipers. In this model, the conservative kinesis and greater robustness of the colubrid palate has greater potential for transporting live prey with less risk of injury.

Snake fangs from the Lower Miocene of Germany: evolutionary stability of perfect weapons

There is a general consensus that most of today's nonvenomous snakes are descendants of venomous snakes that lost their venomous capabilities secondarily. This implies that the evolutionary history of venomous snakes and their venom apparatus should be older than the current evidence from the fossil record. We compared some of the oldest-known fossil snake fangs from the Lower Miocene of Germany with those of modern viperids and elapids and found their morphology to be indistinguishable from the modern forms. The primary function of recent elapid and viperid snake fangs is to facilitate the extremely rapid, stab-like application of highly toxic venoms. Our findings therefore indicate that the other components of the venom-delivery system of Early Miocene vipers and elapids were also highly developed, and that these snakes used their venom in the same way as their modern relatives. Thus, the fossil record supports the view that snakes used their venoms to rapidly subdue prey long before the mid-Tertiary onset of the global environmental changes that seem to have supported the successful radiation of venomous snakes.

Early evolution of the venom system in lizards and snakes

Among extant reptiles only two lineages are known to have evolved venom delivery systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded Lizard). Evolution of the venom system is thought to underlie the impressive radiation of the advanced snakes (2,500 of 3,000 snake species). In contrast, the lizard venom system is thought to be restricted to just two species and to have evolved independently from the snake venom system. Here we report the presence of venom toxins in two additional lizard lineages (Monitor Lizards and Iguania) and show that all lineages possessing toxin-secreting oral glands form a clade, demonstrating a single early origin of the venom system in lizards and snakes. Construction of gland complementary-DNA libraries and phylogenetic analysis of transcripts revealed that nine toxin types are shared between lizards and snakes. Toxinological analyses of venom components from the Lace Monitor Varanus varius showed potent effects on blood pressure and clotting ability, bioactivities associated with a rapid loss of consciousness and extensive bleeding in prey. The iguanian lizard Pogona barbata retains characteristics of the ancestral venom system, namely serial, lobular non-compound venom-secreting glands on both the upper and lower jaws, whereas the advanced snakes and anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard) have more derived venom systems characterized by the loss of the mandibular (lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians and anguimorphs form a single clade provides overwhelming support for a single, early origin of the venom system in lizards and snakes. These results provide new insights into the evolution of the venom system in squamate reptiles and open new avenues for biomedical research and drug design using hitherto unexplored venom proteins.