Crowdsourcing snake identification with online communities of professional herpetologists and avocational snake enthusiasts.

Species identification can be challenging for biologists, healthcare practitioners and members of the general public. Snakes are no exception, and the potential medical consequences of venomous snake misidentification can be significant. Here, we collected data on identification of 100 snake species by building a week-long online citizen science challenge which attracted more than 1000 participants from around the world. We show that a large community including both professional herpetologists and skilled avocational snake enthusiasts with the potential to quickly (less than 2 min) and accurately (69-90%; see text) identify snakes is active online around the clock, but that only a small fraction of community members are proficient at identifying snakes to the species level, even when provided with the snake's geographical origin. Nevertheless, participants showed great enthusiasm and engagement, and our study provides evidence that innovative citizen science/crowdsourcing approaches can play significant roles in training and building capacity. Although identification by an expert familiar with the local snake fauna will always be the gold standard, we suggest that healthcare workers, clinicians, epidemiologists and other parties interested in snakebite could become more connected to these communities, and that professional herpetologists and skilled avocational snake enthusiasts could organize ways to help connect medical professionals to crowdsourcing platforms. Involving skilled avocational snake enthusiasts in decision making could build the capacity of healthcare workers to identify snakes more quickly, specifically and accurately, and ultimately improve snakebite treatment data and outcomes.

Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Venom Complexity in a Pitviper Produced by Facultative Parthenogenesis.

Facultative parthenogenesis (FP) is asexual reproduction in plant and animal species that would otherwise reproduce sexually. This process in vertebrates typically results from automictic development (likely terminal fusion) and is phylogenetically widespread. In squamate reptiles and chondrichthyan fishes, FP has been reported to occur in nature and can result in the production of reproductively viable offspring; suggesting that it is of ecological and evolutionary significance. However, terminal fusion automixis is believed to result in near genome-wide reductions in heterozygosity; thus, FP seems likely to affect key phenotypic characters, yet this remains almost completely unstudied. Snake venom is a complex phenotypic character primarily used to subjugate prey and is thus tightly linked to individual fitness. Surprisingly, the composition and function of venom produced by a parthenogenetic pitviper exhibits a high degree of similarity to that of its mother and conspecifics from the same population. Therefore, the apparent loss of allelic diversity caused by FP appears unlikely to have a significant impact on the prey-capturing ability of this snake. Accordingly, the pitviper offspring produced by FP retained complex phenotypic characteristics associated with fitness. This result reinforces the potential ecological and evolutionary importance of FP and questions our understanding of the inheritance of venom-associated genes.

Venom lethality and diet: differential responses of natural prey and model organisms to the venom of the saw-scaled vipers (Echis).

The composition of snake venoms shows a high degree of variation at all taxonomic levels, and natural selection for diet has been implicated as a potential cause. Saw-scaled vipers (Echis) provide a good model for studying this phenomenon. The venoms of arthropod feeding species of Echis are significantly more toxic to natural scorpion prey than those of species which feed predominantly upon vertebrate prey. Although testing venom activity on natural prey is important for our understanding of the evolution of venom, natural prey species are often difficult to obtain in sufficient numbers for toxinological work. In order to test the viability of using cheaper and more easily available model organisms for toxicity assessments in evolutionary research, and the extent to which toxicity of arthropod-eating Echis venoms is increased to arthropods in general or targeted to certain groups, we conducted median lethal dosage (LD(50)) and time to death trials using the desert locust (Schistocerca gregaria) as a model arthropod, rarely consumed by wild Echis. The venoms of arthropod specialist Echis were found to be significantly more toxic to locusts than the venom of a vertebrate feeding outgroup (Bitis arietans), and one arthropod specialist venom was found to be more toxic than those species which feed upon arthropods infrequently or not at all. The venoms of arthropod specialists were also found to cause death and incapacitation faster than the vertebrate feeding outgroup. Despite some similarity of trends, there are considerable differences between the response of natural prey (scorpions) and a model arthropod (locust) to the venoms of Echis species. This suggests that when possible, natural prey rather than convenient model organisms should be used to gain an understanding of the functional significance of variation in venom composition in snakes.

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.

Molecular evolution and phylogeny of elapid snake venom three-finger toxins.

Animal venom components are of considerable interest to researchers across a wide variety of disciplines, including molecular biology, biochemistry, medicine, and evolutionary genetics. The three-finger family of snake venom peptides is a particularly interesting and biochemically complex group of venom peptides, because they are encoded by a large multigene family and display a diverse array of functional activities. In addition, understanding how this complex and highly varied multigene family evolved is an interesting question to researchers investigating the biochemical diversity of these peptides and their impact on human health. Therefore, the purpose of our study was to investigate the long-term evolutionary patterns exhibited by these snake venom toxins to understand the mechanisms by which they diversified into a large, biochemically diverse, multigene family. Our results show a much greater diversity of family members than was previously known, including a number of subfamilies that did not fall within any previously identified groups with characterized activities. In addition, we found that the long-term evolutionary processes that gave rise to the diversity of three-finger toxins are consistent with the birth-and-death model of multigene family evolution. It is anticipated that this "three-finger toxin toolkit" will prove to be useful in providing a clearer picture of the diversity of investigational ligands or potential therapeutics available within this important family.

The conserved structure of snake venom toxins confers extensive immunological cross-reactivity to toxin-specific antibody.

We have demonstrated previously that antisera from mice immunised with DNA encoding the carboxy-terminal domain (JD9) of a potent haemorrhagic metalloproteinase, jararhagin, neutralised over 70% of the haemorrhagic activity of the whole Bothrops jararaca venom. Here, we demonstrate that the JD9-specific antibody possesses extensive immunological reactivity to venom components in snakes of distinct species and genera. The polyspecific immunological reactivity of the antibody showed a correlation with amino acid sequence identity and with predicted antigenic domains of JD9-analogues in venoms of snakes with closest phylogenetic links to B. jararaca. This study further promotes the potential of DNA immunisation to generate toxin-specific antibodies with polyspecific cover. An analysis of the reactivity of the JD9-specific antisera to B. atrox complex venoms that exhibited intraspecific variation in the venom proteome revealed, however, that the toxin-specific approach to antivenom development requires a more in-depth knowledge of the target molecules than is required for conventional antivenoms.

Historical biogeography of the Western Rattlesnake (Serpentes: viperidae: Crotalus viridis), inferred from mitochondrial DNA sequence information.

We infer the phylogeography of the Western Rattlesnake (Crotalus viridis) using phylogenetic analysis of mitochondrial DNA sequences from 1345 bp of the genes for cytochrome b and NADH dehydrogenase subunit 4. Two main clades are revealed: one includes populations from east and south of the Rocky Mountains (conventionally referred to as Crotalus viridis viridis and C. v. nuntius), and the other consists of populations west of the Rocky Mountains. Within the western clade, a population from southern Arizona (C. v. cerberus) represents the sister taxon to the remaining western populations. The conventional subspecies recognized in this species do not fully correspond to the phylogenetic pattern, and a review of the systematic status of several populations is needed. Our data allow the inferences that small body size evolved twice and that the ability of one population (C. v. concolor) to secrete highly lethal toxins related to Mojave toxin arose within the complex. Our phylogeny should represent the basis for further studies on the causes of geographical variation in this complex.

Synopsis of recent developments in venomous snake systematics, No. 3.

We present recent findings in the systematics of venomous snakes, with emphasis on those which affect the nomenclature and our understanding of species limits in these animals. Changes in systematics reviewed here include particularly the genera Acanthophis, Elapsoidea, Bitis, Lachesis, Porthidium, Trimeresurus/Tropidolaemus and Vipera. Other new publications of more general interest to toxinologists are also presented.

Synopsis of recent developments in venomous snake systematics, No. 2.

Developments in our understanding of the systematics of venomous snakes since the beginning of 1996 are discussed and reviewed with special emphasis on their relevance and implications for toxinologists and clinicians. Groups of snakes affected by recent developments include the genera Elapomorphus, Rhabdophis, Vermicella, Atheris, Daboia, Agkistrodon/Gloydius, Bothrops/Bothriopsis and Trimeresurus. Other important publications on venomous snakes are noted.

Synopsis of recent developments in venomous snake systematics.

Changes to our understanding of venomous snake systematics, and the consequent changes in the nomenclature of these animals, have traditionally been a great source of confusion among biomedical researchers. This paper aims to facilitate access to the taxonomic literature by presenting a synopsis of the changes in venomous snake systematics that have taken place recently (primarily since 1992), together with some comments on the implications of these changes for toxinologists and clinicians. Some long-standing problems in venomous snake taxonomy receive special attention. This includes Asiatic Naja, Asiatic Agkistrodon/Gloydius, Bothrops and related genera, Trimeresurus, Echis, Daboia (including Daboia russellii) and Vipera. It is hoped that this synopsis will result in the use of a more up-to-date and interpretable nomenclature for venomous snakes in the toxinological literature.

Taxonomic changes and toxinology: systematic revisions of the Asiatic cobras (Naja naja species complex)

Until recently, all Asiatic cobra populations were regarded as belonging to one single species, Naja naja. Recent revisions have shown that there are in fact at least 10 full species of Asiatic Naja. In order to allow the existing literature to be reconciled with these recent discoveries, an interpretation of the older nomenclature is provided. Problematic areas, especially concerning the species N. sumatrana and N. siamensis, are highlighted.