Wolfgang Wüster

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 king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

Significance Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This diverse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey. Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution

The processes that drive the evolution of snake venom variability, particularly the role of diet, have been a topic of intense recent research interest. Here, we test whether extensive variation in venom composition in the medically important viper genus Echis is associated with shifts in diet. Examination of stomach and hindgut contents revealed extreme variation between the major clades of Echis in the proportion of arthropod prey consumed. The toxicity (median lethal dose, LD50) of representative Echis venoms to a natural scorpion prey species was found to be strongly associated with the degree of arthropod feeding. Mapping the results onto a novel Echis phylogeny generated from nuclear and mitochondrial sequence data revealed two independent instances of coevolution of venom toxicity and diet. Unlike venom LD50, the speed with which venoms incapacitated and killed scorpions was not associated with the degree of arthropod feeding. The prey-specific venom toxicity of arthropod-feeding Echis may thus be adaptive primarily by reducing venom expenditure. Overall, our results provide strong evidence that variation in snake venom composition results from adaptive evolution driven by natural selection for different diets, and underscores the need for a multi-faceted, integrative approach to the study of the causes of venom evolution.

Tracing an invasion: landbridges, refugia, and the phylogeography of the Neotropical rattlesnake (Serpentes: Viperidae: Crotalus durissus)

Pleistocene fragmentation of the Amazonian rainforest has been hypothesized to be a major cause of Neotropical speciation and diversity. However, the role and even the reality of Pleistocene forest refugia have attracted much scepticism. In Amazonia, previous phylogeographical studies have focused mostly on organisms found in the forests themselves, and generally found speciation events to have predated the Pleistocene. However, molecular studies of open‐formation taxa found both north and south of the Amazonian forests, probably because of vicariance resulting from expansion of the rainforests, may provide novel insights into the age of continuous forest cover across the Amazon basin. Here, we analyse three mitochondrial genes to infer the phylogeography of one such trans‐Amazonian vicariant, the Neotropical rattlesnake (Crotalus durissus), which occupies primarily seasonal formations from Mexico to Argentina, but avoids the rainforests of Central and tropical South America. The phylogeographical pattern is consistent with gradual dispersal along the Central American Isthmus, followed by more rapid dispersal into and across South America after the uplift of the Isthmus of Panamá. Low sequence divergence between populations from north and south of the Amazon rainforest is consistent with mid‐Pleistocene divergence, approximately 1.1 million years ago (Ma). This suggests that the Amazonian rainforests must have become fragmented or at least shrunk considerably during that period, lending support to the Pleistocene refugia theory as an important cause of distribution patterns, if not necessarily speciation, in Amazonian forest organisms. These results highlight the potential of nonforest species to contribute to an understanding of the history of the Amazonian rainforests themselves.

A nesting of vipers: Phylogeny and historical biogeography of the Viperidae (Squamata: Serpentes)

Despite their medical interest, the phylogeny of the snake family Viperidae remains inadequately understood. Previous studies have generally focused either on the pitvipers (Crotalinae) or on the Old World vipers (Viperinae), but there has been no comprehensive molecular study of the Viperidae as a whole, leaving the affinities of key taxa unresolved. Here, we infer the phylogenetic relationships among the extant genera of the Viperidae from the sequences of four mitochondrial genes (cytochrome b, NADH subunit 4, 16S and 12S rRNA). The results confirm Azemiops as the sister group of the Crotalinae, whereas Causus is nested within the Viperinae, and thus not a basal viperid or viperine. Relationships among the major clades of Viperinae remain poorly resolved despite increased sequence information compared to previous studies. Bayesian molecular dating in conjunction with dispersal-vicariance analysis suggests an early Tertiary origin in Asia for the crown group Viperidae, and rejects suggestions of a relatively recent, early to mid-Tertiary origin of the Caenophidia.

Ending the drought: New strategies for improving the flow of affordable, effective antivenoms in Asia and Africa

The development of snake antivenoms more than a century ago should have heralded effective treatment of the scourge of snakebite envenoming in impoverished, mostly rural populations around the world. That snakebite still exists today, as a widely untreated illness that maims, kills and terrifies men, women and children in vulnerable communities, is a cruel anachronism. Antivenom can be an effective, safe and affordable treatment for snakebites, but apathy, inaction and the politicisation of public health have marginalised both the problem (making snakebite arguably the most neglected of all neglected tropical diseases) and its solution. For lack of any coordinated approach, provision of antivenoms has been pushed off the public health agenda, leading to an incongruous decline in demand for these crucial antidotes, excused and fed by new priorities, an absence of epidemiological data, and a poor regulatory framework. These factors facilitated the infiltration of poor quality products that degrade user confidence and undermine legitimate producers. The result is that tens of thousands are denied an essential life-saving medicine, allowing a toll of human suffering that is a summation of many individual catastrophes. No strategy has been developed to address this problem and to overcome the intransigence and inaction responsible for the global tragedy of snakebite. Attempts to engage with the broader public health community through the World Health Organisation (WHO), GAVI, and other agencies have failed. Consequently, the toxinology community has taken on a leadership role in a new approach, the Global Snakebite Initiative, which seeks to mobilise the resources, skills and experience of scientists and clinicians for whom venoms, toxins, antivenoms, snakes and snakebites are already fields of interest. Proteomics is one such discipline, which has embraced the potential of using venoms in bio-discovery and systems biology. The fields of venomics and antivenomics have recently evolved from this discipline, offering fresh hope for the victims of snakebites by providing an exciting insight into the complexities, nature, fundamental properties and significance of venom constituents. Such a rational approach brings with it the potential to design new immunising mixtures from which to raise potent antivenoms with wider therapeutic ranges. This addresses a major practical limitation in antivenom use recognised since the beginning of the 20th century: the restriction of therapeutic effectiveness to the specific venom immunogen used in production. Antivenomic techniques enable the interactions between venoms and antivenoms to be examined in detail, and if combined with functional assays of specific activity and followed up by clinical trials of effectiveness and safety, can be powerful tools with which to evaluate the suitability of current and new antivenoms for meeting urgent regional needs. We propose two mechanisms through which the Global Snakebite Initiative might seek to end the antivenom drought in Africa and Asia: first by establishing a multidisciplinary, multicentre, international collaboration to evaluate currently available antivenoms against the venoms of medically important snakes from specific nations in Africa and Asia using a combination of proteomic, antivenomic and WHO-endorsed preclinical assessment protocols, to provide a validated evidence base for either recommending or rejecting individual products; and secondly by bringing the power of proteomics to bear on the design of new immunising mixtures to raise Pan-African and Pan-Asian polyvalent antivenoms of improved potency and quality. These products will be subject to rigorous clinical assessment. We propose radically to change the basis upon which antivenoms are produced and supplied for the developing world. Donor funding and strategic public health alliances will be sought to make it possible not only to sustain the financial viability of antivenom production partnerships, but also to ensure that patients are relieved of the costs of antivenom so that poverty is no longer a barrier to the treatment of this important, but grossly neglected public health emergency.

Isolation of a Neurotoxin (α-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 α-colubritoxin, isolated from the Asian ratsnake Coelognathusradiatus (formerly known as Elapheradiata), 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.

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms

Significance The toxic composition of snake venom varies between species. Such variation can have major medical implications for the treatment of human snakebite victims. Venom variation is largely attributed to differences in toxin-encoding genes present in the genome or venom gland of snakes. Here, we demonstrate that mechanisms affecting the transcription, translation, and posttranslational modification of toxins also significantly contribute to the diversity of venom protein composition. Venom variation observed between related snake species is therefore the result of a complex interaction between a variety of genetic and postgenomic factors acting on toxin genes. Ultimately, this variation results in significant differences in venom-induced pathology and lethality and can undermine the efficacy of antivenom therapies used to treat human snakebite victims. Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite.

Comparative venom gland transcriptome surveys of the saw-scaled vipers (Viperidae: Echis) reveal substantial intra-family gene diversity and novel venom transcripts

BackgroundVenom variation occurs at all taxonomical levels and can impact significantly upon the clinical manifestations and efficacy of antivenom therapy following snakebite. Variation in snake venom composition is thought to be subject to strong natural selection as a result of adaptation towards specific diets. Members of the medically important genus Echis exhibit considerable variation in venom composition, which has been demonstrated to co-evolve with evolutionary shifts in diet. We adopt a venom gland transcriptome approach in order to investigate the diversity of toxins in the genus and elucidate the mechanisms which result in prey-specific adaptations of venom composition.ResultsVenom gland transcriptomes were created for E. pyramidum leakeyi, E. coloratus and E. carinatus sochureki by sequencing ~1000 expressed sequence tags from venom gland cDNA libraries. A standardised methodology allowed a comprehensive intra-genus comparison of the venom gland profiles to be undertaken, including the previously described E. ocellatus transcriptome. Blast annotation revealed the presence of snake venom metalloproteinases, C-type lectins, group II phopholipases A2, serine proteases, L-amino oxidases and growth factors in all transcriptomes throughout the genus. Transcripts encoding disintegrins, cysteine-rich secretory proteins and hyaluronidases were obtained from at least one, but not all, species. A representative group of novel venom transcripts exhibiting similarity to lysosomal acid lipase were identified from the E. coloratus transcriptome, whilst novel metallopeptidases exhibiting similarity to neprilysin and dipeptidyl peptidase III were identified from E. p. leakeyi and E. coloratus respectively.ConclusionThe comparison of Echis venom gland transcriptomes revealed substantial intrageneric venom variation in representations and cluster numbers of the most abundant venom toxin families. The expression profiles of established toxin groups exhibit little obvious association with venom-related adaptations to diet described from this genus. We suggest therefore that alterations in isoform diversity or transcript expression levels within the major venom protein families are likely to be responsible for prey specificity, rather than differences in the representation of entire toxin families or the recruitment of novel toxin families, although the recruitment of lysosomal acid lipase as a response to vertebrate feeding cannot be excluded. Evidence of marked intrageneric venom variation within the medically important genus Echis strongly advocates further investigations into the medical significance of venom variation in this genus and its impact upon antivenom therapy.

Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest, and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae)

Phylogeographic studies using mitochondrial DNA sequence information are frequently used as the principal source of evidence to infer species boundaries. However, a critical analysis of further evidence is essential to test whether different haplotype clades identify different species. We demonstrate a hypothesis‐testing approach, using a combination of phylogeographic methods, multivariate morphometrics and matrix association tests, to investigate species boundaries in eastern Brazilian pitvipers conventionally assigned to the species Bothrops leucurus and B. pradoi. Two basal haplotype clades with partly overlapping geographical distributions are identified, which could either represent two partly sympatric species, or multiple haplotypes within one organismal lineage. We use partial Mantel matrix association tests to verify whether generalized morphology, or any of four supposedly diagnostic characters for the two species, show any association with mtDNA variation. Negative results lead to the conclusion that the haplotype clades do not denote independently evolving organismal lineages, and do not constitute separate species under any criterion.