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Snake venoms are complex mixtures of toxic proteins that hold significant medical, pharmacological and evolutionary interest. To better understand the genetic diversity underlying snake venoms, we developed VenomCap, a novel exon-capture probe set targeting toxin-coding genes from a wide range of elapid snakes, with a particular focus on the ecologically diverse and medically important subfamily Hydrophiinae. We tested the capture success of VenomCap across 24 species, representing all major elapid lineages. We included snake phylogenomic probes in the VenomCap capture set, allowing us to compare capture performance between venom and phylogenomic loci and to infer elapid phylogenetic relationships. We demonstrated VenomCap's ability to recover exons from ~1500 target markers, representing a total of 24 known venom gene families, which includes the dominant gene families found in elapid venoms. We find that VenomCap's capture results are robust across all elapids sampled, and especially among hydrophiines, with respect to measures of target capture success (target loci matched, sensitivity, specificity and missing data). As a cost-effective and efficient alternative to full genome sequencing, VenomCap can dramatically accelerate the sequencing and analysis of venom gene families. Overall, our tool offers a model for genomic studies on snake venom gene diversity and evolution that can be expanded for comprehensive comparisons across the other families of venomous snakes., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey., (© 2024. The Author(s).)

Background: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris)., Results: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis., Conclusions: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype., (© 2024. The Author(s).)

The location of female-specific/linked loci identified in Siamese cobra (Naja kaouthia) previously has been determined through in silico chromosome mapping of the Indian cobra genome (N. naja) as a reference genome. In the present study, we used in silico chromosome mapping to identify sex-specific and linked loci in Siamese cobra. Many sex-specific and sex-linked loci were successfully mapped on the Z sex chromosome, with 227 of the 475 specific loci frequently mapped in a region covering 57 Mb and positioned at 38,992,675-95,561,177 bp of the Indian cobra genome (N. naja). This suggested the existence of a putative sex-determining region (SDR), with one specific locus (PA100000600) homologous to the TOPBP1 gene. The involvement of TOPBP1 gene may lead to abnormal synaptonemal complexes and meiotic chromosomal defects, resulting in male infertility. These findings offer valuable insights into the genetic basis and functional aspects of sex-specific traits in the Siamese cobra, which will contribute to our understanding of snake genetics and evolutionary biology., (© 2023. The Author(s) under exclusive licence to The Genetics Society of Korea.)

Background: Sea snakes underwent a complete transition from land to sea within the last ~ 15 million years, yet they remain a conspicuous gap in molecular studies of marine adaptation in vertebrates., Results: Here, we generate four new annotated sea snake genomes, three of these at chromosome-scale (Hydrophis major, H. ornatus and H. curtus), and perform detailed comparative genomic analyses of sea snakes and their closest terrestrial relatives. Phylogenomic analyses highlight the possibility of near-simultaneous speciation at the root of Hydrophis, and synteny maps show intra-chromosomal variations that will be important targets for future adaptation and speciation genomic studies of this system. We then used a strict screen for positive selection in sea snakes (against a background of seven terrestrial snake genomes) to identify genes over-represented in hypoxia adaptation, sensory perception, immune response and morphological development., Conclusions: We provide the best reference genomes currently available for the prolific and medically important elapid snake radiation. Our analyses highlight the phylogenetic complexity and conserved genome structure within Hydrophis. Positively selected marine-associated genes provide promising candidates for future, functional studies linking genetic signatures to the marine phenotypes of sea snakes and other vertebrates., (© 2023. The Author(s).)

With one exception, the only known hemiurid trematodes that do not use teleost fishes as definitive hosts instead occur in marine elapid snakes. These comprise six species across four genera and three subfamilies, and so presumably indicate at least three independent invasions of marine snakes from teleost fishes. Here, one of these taxa, Tubulovesicula laticaudi Parukhin, 1969 (= T. orientalis Chattopadhyaya, 1970 n. syn.) is reported from Sri Lanka, collected from Shaw's sea snake Hydrophis curtus (Shaw) (Elapidae: Hydrophiinae: Hydrophinii), the annulated sea snake H. cyanocinctus Daudin and the yellow sea snake H. spiralis (Shaw) off Nayaru in the Bay of Bengal, and from H. spiralis in Portugal Bay, Gulf of Mannar. Novel molecular data, for COI mtDNA and ITS2 and 28S rDNA, are the first for a species of Tubulovesicula Yamaguti, 1934. Nominally, Tubulovesicula belongs in the Dinurinae Looss, 1907, but in phylogenetic analyses based on 28S rDNA, our sequences for T. laticaudi resolved relatively distant from that for representatives of Dinurus Looss, 1907, the type-genus, rendering the subfamily polyphyletic. Tubulovesicula laticaudi resolved closest to data for the type-species of the Plerurinae Gibson & Bray, 1979, but that subfamily is also polyphyletic. These findings lead us to re-evaluate an alternative classification considered by Gibson & Bray (1979). We propose restricting the Dinurinae for forms with a permanent sinus-organ (Dinurus, Ectenurus Looss, 1907; Erilepturus Woolcock, 1935; Paradinurus Vigueras, 1958; Qadriana Bilqees, 1971) and resurrect the Mecoderinae Skrjabin & Guschanskaja, 1954 for forms with a temporary sinus-organ (Mecoderus Manter, 1940, Allostomachicola Yamaguti, 1958, Stomachicola Yamaguti, 1934 and Tubulovesicula)., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The genus Demansia Günther is the most diverse genus of Australian terrestrial elapids. A phylogenetic framework for the familiar but problematic taxa D. psammophis and D. reticulata (Gray) has been long overdue to ascertain interspecific relationships and resolve unclear taxonomic issues. Here we present an integrated molecular phylogenetic and morphological analyses to review species delineation, resulting in confirmation that both D. psammophis and D. reticulata are full species and that some populations referred to D. r. cupreiceps Storr are not distinguishable from more typical D. reticulata. We also find the widespread central Australian population (treated by most authors as part of cupreiceps) to be specifically distinct. We redescribe D. psammophis and D. reticulata to clarify morphological and geographical boundaries and describe D. cyanochasma sp. nov. based on a combination of molecular genetic markers, details of colour and pattern, adult total length and a few morphometric attributes. We also designate a lectotype for D. psammophis from the original syntype series and comment on the necessity for further taxonomic refinement of this distinctive group.

Snake venoms are complex mixtures of toxins that differ on interspecific (between species) and intraspecific (within species) levels. Whether venom variation within a group of closely related species is explained by the presence, absence and/or relative abundances of venom toxins remains largely unknown. Taipans ( Oxyuranus spp.) and brown snakes ( Pseudonaja spp.) represent medically relevant species of snakes across the Australasian region and provide an excellent model clade for studying interspecific and intraspecific venom variation. Using liquid chromatography with ultraviolet and mass spectrometry detection, we analyzed a total of 31 venoms covering all species of this monophyletic clade, including widespread localities. Our results reveal major interspecific and intraspecific venom variation in Oxyuranus and Pseudonaja species, partially corresponding with their geographical regions and phylogenetic relationships. This extensive venom variability is generated by a combination of the absence/presence and differential abundance of venom toxins. Our study highlights that venom systems can be highly dynamical on the interspecific and intraspecific levels and underscores that the rapid toxin evolvability potentially causes major impacts on neglected tropical snakebites.

Mitochondrial genomes of four elapid snakes (three marine species [ Emydocephalus ijimae , Hydrophis ornatus , and Hydrophis melanocephalus ], and one terrestrial species [ Sinomicrurus japonicus ]) were completely sequenced by a combination of Sanger sequencing, next-generation sequencing and Nanopore sequencing. Nanopore sequencing was especially effective in accurately reading through long tandem repeats in these genomes. This led us to show that major noncoding regions in the mitochondrial genomes of those three sea snakes contain considerably long tandem duplications, unlike the mitochondrial genomes previously reported for same and other sea snake species. We also found a transposition of the light-strand replication origin within a tRNA gene cluster for the three sea snakes. This change can be explained by the Tandem Duplication-Random Loss model, which was further supported by remnant intervening sequences between tRNA genes. Mitochondrial genomes of true snakes (Alethinophidia) have been shown to contain duplicate major noncoding regions, each of which includes the control region necessary for regulating the heavy-strand replication and transcription from both strands. However, the control region completely disappeared from one of the two major noncoding regions for two Hydrophis sea snakes, posing evolutionary questions on the roles of duplicate control regions in snake mitochondrial genomes. The timing and molecular mechanisms for these changes are discussed based on the elapid phylogeny.

Bungarus multicinctus is a widely distributed and medically important elapid snake that produces lethal neurotoxic venom. To study and enhance existing antivenom, we explore the complete repertoire of its toxin genes based on de novo chromosome-level assembly and multi-tissue transcriptome data. Comparative genomic analyses suggest that the three-finger toxin family (3FTX) may evolve through the neofunctionalization of flanking LY6E. A long-neglected 3FTX subfamily (i.e., MKA-3FTX) is also investigated. Only one MKA-3FTX gene, which evolves a different protein conformation, is under positive selection and actively transcribed in the venom gland, functioning as a major toxin effector together with MKT-3FTX subfamily homologs. Furthermore, this lethal snake may acquire self-resistance to its β-bungarotoxin via amino acid replacements on fast-evolving KCNA2. This study provides valuable resources for further evolutionary and structure-function studies of snake toxins, which are fundamental for the development of effective antivenoms and drug candidates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Specimens of Dolichoperoides macalpini (Nicoll, 1914) (Digenea: Dolichoperoididae) were collected from Australian venomous snakes (Elapidae): Notechis scutatus Peters, 1861 and Austrelaps superbus (Günther, 1858) from Tasmania and surrounding islands and N. s. occidentalis Glauert, 1948 from wetlands near Perth, Western Australia. Despite variation in morphological measurements, genetic analysis showed that the one species of digeneans infected the snakes from all locations. This study presents the first DNA sequences for D. macalpini (internal transcribed spacer, 18S, 28S), confirming its placement in a family separate from the Reniferidae and Telorchiidae. Analysis of the infection dynamics of infection in Western Australian snakes showed significant differences in levels of infection between wetland locations, season and year of collection. Infection of D. macalpini was reported in the gastrointestinal tract, including the mouth, in freshly euthanised snakes in Western Australia, and in the lung in Tasmanian snakes, consistent with earlier reports. Differences in morphology and site of infection are suggested to be due to a combination of season and maturity of the digenean, with infection potentially occurring early in the season, as the snakes emerge from torpor. The need for research on the seasonal dynamics of infection with this parasite is discussed., (© 2022. The Author(s).)

Transposable elements (TEs), also known as jumping genes, are sequences able to move or copy themselves within a genome. As TEs move throughout genomes they often act as a source of genetic novelty, hence understanding TE evolution within lineages may help in understanding environmental adaptation. Studies into the TE content of lineages of mammals such as bats have uncovered horizontal transposon transfer (HTT) into these lineages, with squamates often also containing the same TEs. Despite the repeated finding of HTT into squamates, little comparative research has examined the evolution of TEs within squamates. Here we examine a diverse family of Australo-Melanesian snakes (Hydrophiinae) to examine if the previously identified, order-wide pattern of variable TE content and activity holds true on a smaller scale. Hydrophiinae diverged from Asian elapids ~30 Mya and have since rapidly diversified into six amphibious, ~60 marine and ~100 terrestrial species that fill a broad range of ecological niches. We find TE diversity and expansion differs between hydrophiines and their Asian relatives and identify multiple HTTs into Hydrophiinae, including three likely transferred into the ancestral hydrophiine from fish. These HTT events provide the first tangible evidence that Hydrophiinae reached Australia from Asia via a marine route.

Background: The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes., Results: Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus, Heterodon nasicus, Rhabdophis subminiatus; Homalopsidae - Homalopsis buccata; Lamprophiidae - Malpolon monspessulanus, Psammophis schokari, Psammophis subtaeniatus, Rhamphiophis oxyrhynchus; and Viperidae - Bitis atropos, Pseudocerastes urarachnoides, Tropidolaeumus subannulatus, Vipera transcaucasiana. These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms., Conclusions: We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator-prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline., (© 2021. The Author(s).)

Urbanisation alters landscapes, introduces wildlife to novel stressors, and fragments habitats into remnant 'islands'. Within these islands, isolated wildlife populations can experience genetic drift and subsequently suffer from inbreeding depression and reduced adaptive potential. The Western tiger snake (Notechis scutatus occidentalis) is a predator of wetlands in the Swan Coastal Plain, a unique bioregion that has suffered substantial degradation through the development of the city of Perth, Western Australia. Within the urban matrix, tiger snakes now only persist in a handful of wetlands where they are known to bioaccumulate a suite of contaminants, and have recently been suggested as a relevant bioindicator of ecosystem health. Here, we used genome-wide single nucleotide polymorphism (SNP) data to explore the contemporary population genomics of seven tiger snake populations across the urban matrix. Specifically, we used population genomic structure and diversity, effective population sizes (Ne), and heterozygosity-fitness correlations to assess fitness of each population with respect to urbanisation. We found that population genomic structure was strongest across the northern and southern sides of a major river system, with the northern cluster of populations exhibiting lower heterozygosities than the southern cluster, likely due to a lack of historical gene flow. We also observed an increasing signal of inbreeding and genetic drift with increasing geographic isolation due to urbanisation. Effective population sizes (Ne) at most sites were small (< 100), with Ne appearing to reflect the area of available habitat rather than the degree of adjacent urbanisation. This suggests that ecosystem management and restoration may be the best method to buffer the further loss of genetic diversity in urban wetlands. If tiger snake populations continue to decline in urban areas, our results provide a baseline measure of genomic diversity, as well as highlighting which 'islands' of habitat are most in need of management and protection., Competing Interests: No authors have competing interests.

Cobra cytotoxins (CTs), the three-fingered proteins, feature high amino acid sequence homology in the beta-strands and variations in the loop regions. We selected a pair of cytotoxins from Naja kaouthia crude venom to clarify the sequence-structure relationships. Using chromatography and mass spectroscopy, we separated and identified the mixture of cytotoxins 2 and 3, differentiated by the only Val 41/Ala 41 substitution. Here, using natural abundance 13 C, 15 N NMR-spectroscopy we performed chemical shift assignments of the signals of the both toxins in aqueous solution in the major and minor forms. Combining NOE and chemical shift data, the toxins' spatial structure was determined. Finally, we proved that the tip of the "finger"-2, or the loop-2 of cytotoxins adopts the shape of an omega-loop with a tightly-bound water molecule in its cavity. Comparison with other NMR and X-ray structures of cytotoxins possessing different amino acid sequences reveals spatial similarity in this family of proteins, including the loop-2 region, previously considered to be flexible., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The genus Calliophis is the most basal branch of the family Elapidae and several species in it have developed highly elongated venom glands. Recent research has shown that C. bivirgatus has evolved a seemingly unique toxin (calliotoxin) that produces spastic paralysis in their prey by acting on the voltage-gated sodium (Na V ) channels. We assembled a transcriptome from C. bivirgatus to investigate the molecular characteristics of these toxins and the venom as a whole. We find strong confirmation that this genus produces the classic elapid eight-cysteine three-finger toxins, that δδ-elapitoxins (toxins that resemble calliotoxin) are responsible for a substantial portion of the venom composition, and that these toxins form a distinct clade within a larger, more diverse clade of C. bivirgatus three-finger toxins. This broader clade of C. bivirgatus toxins also contains the previously named maticotoxins and is somewhat closely related to cytotoxins from other elapids. However, the toxins from this clade that have been characterized are not themselves cytotoxic. No other toxins show clear relationships to toxins of known function from other species.

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 A 2 (PLA 2 ) 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., (Copyright © 2021, American Association for the Advancement of Science.)

The question of whether spatial aspects of evolution differ in marine versus terrestrial realms has endured since Ernst Mayr's 1954 essay on marine speciation. Marine systems are often suggested to support larger and more highly connected populations, but quantitative comparisons with terrestrial systems have been lacking. Here, we compared the population histories of marine and terrestrial elapid snakes using the pairwise sequentially Markovian coalescent (PSMC) model to track historical fluctuations in species' effective population sizes (N e ) from individual whole-genome sequences. To do this we generated a draft genome for the olive sea snake (Aiysurus laevis) and analysed this alongside six published elapid genomes and their sequence reads (marine species Hydrophis curtus, H. melanocephalus and Laticauda laticaudata; terrestrial species Pseudonaja textilis, Naja Naja and Notechis scutatus). Counter to the expectation that marine species should show higher overall N e and less pronounced fluctuations in N e , our analyses reveal demographic patterns that are highly variable among species and do not clearly correspond to major ecological divisions. At deeper time intervals, the four marine elapids appear to have experienced relatively stable N e , while each terrestrial species shows a prominent upturn in N e starting at ~4 million years ago (Ma) followed by an equally strong decline. However, over the last million years, all seven species show strong and divergent fluctuations. Estimates of N e in the most recent intervals (~10 kya) are lowest in two of four marine species (H. melanocephalus and Laticauda), and do not correspond to contemporary range sizes in marine or terrestrial taxa., (© 2020 John Wiley & Sons Ltd.)

Geographical variation of animal venom is common among venomous animals. This kind of intraspecific variation based on geographical location mainly concerned from envenomation cases and brought new problems in animal venom studies, including venom components regulatory mechanisms, differentiation of venom activities, and clinical treatment methods. At present, food is considered as the most related factor influencing venom development. Related research defined the variational venomous animal species by the comparison of venom components and activities in snakes, jellyfish, scorpions, cone snails, ants, parasitoid wasps, spiders and toads. In snake venom studies, researchers found that antivenom effectiveness was variated to different located venom samples. As described in some snake venom research, developing region-specific antivenom is the development trend. The difficulties of developing region-specific antivenom and theoretical solutions have been discussed. This review summarized biological studies of animal venom geographical variation by species, compared venom components and major biological activities of the vary venom from the same species, and listed the basic methods in comparing venom protein compositions and major toxicity differences to provide a comprehensive reference., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The red-bellied form of Calliophis intestinalis (Laurenti, 1768) sensu lato was originally reported from Pahang, west Malaysia. To determine the taxonomic status of this form, we examined the type specimens of Elaps sumatranus Lidth De Jeude, 1890, Calliophis intestinalis everetti (Boulenger, 1896), and Callophis furcatus var. nigrotaeniatus Peters, 1863. The results indicated that the red-bellied form of C. intestinalis should be named as Calliophis nigrotaeniatus comb. nov., whose valid species status was based on morphological and molecular analyses. We designate a lectotype and redescribe the species, which is genetically close to Calliophis bilineatus (Peters, 1881) from the Philippines, and is clearly distinguishable from other congeners by possessing a pair of gray or dark blue lateral stripes and by being bright red on the ventrum. Elaps sumatranus and C. i. everetti are relegated to subjective junior synonyms of C. nigrotaeniatus .

Intra-specific venom variation has the potential to provide important insights into the evolution of snake venom, but remains a relatively neglected aspect of snake venom studies. We investigated the venom from 13 individual coastal taipans Oxyuranus scutellatus from four localities on the north-east coast of Australia, spanning a distance of 2000 km. The intra-specific variation in taipan venom was considerably less than the inter-specific variation between it and the other Australian elapids to which it was compared. The electrophoretic venom profile of O. scutellatus was visually different to six other genera of Australian elapids, but not to its congener inland taipan O. microlepidotus. There was minimal geographical variation in taipan venom, as the intra-population variation exceeded the inter-population variation for enzymatic activity, procoagulant activity, and the abundance of neurotoxins. The pre-synaptic neurotoxin (taipoxin) was more abundant than the post-synaptic neurotoxins (3FTx), with a median of 11.0% (interquartile range (IQR): 9.7% to 18.3%; range: 6.7% to 23.6%) vs. a median of 3.4% (IQR: 0.4% to 6.7%; range: 0% to 8.1%). Three taipan individuals almost completely lacked post-synaptic neurotoxins, which was not associated with geography and occurred within two populations. We found no evidence of sexual dimorphism in taipan venom. Our study provides a basis for evaluating the significance of intra-specific venom variation within a phylogenetic context by comparing it to the inter-specific and inter-generic variation. The considerable intra-population variation we observed supports the use of several unpooled individuals from each population when making inter-specific comparisons.

Snakes are descended from highly visual lizards [1] but have limited (probably dichromatic) color vision attributed to a dim-light lifestyle of early snakes [2-4]. The living species of front-fanged elapids, however, are ecologically very diverse, with ∼300 terrestrial species (cobras, taipans, etc.) and ∼60 fully marine sea snakes, plus eight independently marine, amphibious sea kraits [1]. Here, we investigate the evolution of spectral sensitivity in elapids by analyzing their opsin genes (which are responsible for sensitivity to UV and visible light), retinal photoreceptors, and ocular lenses. We found that sea snakes underwent rapid adaptive diversification of their visual pigments when compared with their terrestrial and amphibious relatives. The three opsins present in snakes (SWS1, LWS, and RH1) have evolved under positive selection in elapids, and in sea snakes they have undergone multiple shifts in spectral sensitivity toward the longer wavelengths that dominate below the sea surface. Several relatively distantly related Hydrophis sea snakes are polymorphic for shortwave sensitive visual pigment encoded by alleles of SWS1. This spectral site polymorphism is expected to confer expanded "UV-blue" spectral sensitivity and is estimated to have persisted twice as long as the predicted survival time for selectively neutral nuclear alleles. We suggest that this polymorphism is adaptively maintained across Hydrophis species via balancing selection, similarly to the LWS polymorphism that confers allelic trichromacy in some primates. Diving sea snakes thus appear to share parallel mechanisms of color vision diversification with fruit-eating primates., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The relative influence of diet and phylogeny on snake venom activity is a poorly understood aspect of snake venom evolution. We measured the activity of two enzyme toxin groups - phospholipase A 2 (PLA 2 ), and L-amino acid oxidase (LAAO) - in the venom of 39 species of Australian elapids (40% of terrestrial species diversity) and used linear parsimony and BayesTraits to investigate any correlation between enzyme activity and phylogeny or diet., Results: PLA 2 activity ranged from 0 to 481 nmol/min/mg of venom, and LAAO activity ranged from 0 to 351 nmol/min/mg. Phylogenetic comparative methods, implemented in BayesTraits showed that enzyme activity was strongly correlated with phylogeny, more so for LAAO activity. For example, LAAO activity was absent in both the Vermicella and Pseudonaja/Oxyuranus clade, supporting previously proposed relationships among these disparate taxa. There was no association between broad dietary categories and either enzyme activity. There was strong evidence for faster initial rates of change over evolutionary time for LAAO (delta parameter mean 0.2), but no such pattern in PLA 2 (delta parameter mean 0.64). There were some exceptions to the phylogenetic patterns of enzyme activity: different PLA 2 activity in the ecologically similar sister-species Denisonia devisi and D. maculata; large inter-specific differences in PLA 2 activity in Hoplocephalus and Austrelaps., Conclusions: We have shown that phylogeny is a stronger influence on venom enzyme activity than diet for two of the four major enzyme families present in snake venoms. PLA 2 and LAAO activities had contrasting evolutionary dynamics with the higher delta value for PLA 2 Some species/individuals lacked activity in one protein family suggesting that the loss of single protein family may not incur a significant fitness cost.

Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the blood clotting factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. Cofactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immunizing horses with ScNtx can successfully neutralize the lethality of pure recombinant and native short-chain α-neurotoxins, as well as whole neurotoxic elapid venoms from diverse genera such as Micrurus, Dendroaspis, Naja, Walterinnesia, Ophiophagus and Hydrophis. These results provide a proof-of-principle for using recombinant proteins with rationally designed consensus sequences as universal immunogens for developing next-generation antivenoms with higher effectiveness and broader neutralizing capacity.

Background: Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes., Results: We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG) n and six microsatellite repeat motifs ((AAGG) 8 , (AGAT) 8 , (AAAC) 8 , (ACAG) 8 , (AATC) 8 , and (AAAAT) 6 ) were observed on NKAW, and most of these were also found on other amniote sex chromosomes., Conclusions: The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.

Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by 1 H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).

To investigate the phylogenetic relationships of the genus Laticauda to related higher taxa, we compared the sequences of four mitochondrial genes (12S rRNA, 16S rRNA, ND4, Cytb) from three Laticauda species (L. colubrina, L. laticaudata, and L. semifasciata) with those of 55 Asian and Australo-Melanesian elapid species. We also characterized the complete mitogenomes of the three Laticauda species and compared the sequences of 13 mitochondrial genes from Laticauda species with five terrestrial elapid and one viperid species to estimate phylogenetic relationships and divergence times. Our results showed that the genus Laticauda is paraphyletic to terrestrial elapids and diverged from the Asian elapids approximately 16.23 Mya. The mitogenomes of the three Laticauda species commonly encoded 13 proteins, 22 tRNAs, 12S and 16S rRNAs and two control regions and ranged from 17,170 and 17,450 bp in size. The L. colubrina mitogenome was more similar to that of L. laticaudata than that of L. semifasciata. The divergence time among the three Laticauda clades was estimated at 8-10 Mya, and a close phylogenetic relationship between L. colubrina and L. laticaudata was found. Our results contribute to our understanding of the evolutionary history of sea kraits.

Coral snakes, most notably the genus Micrurus, are the only terrestrial elapid snakes in the Americas. Elapid venoms are generally known for their potent neurotoxicity which is usually caused by Three-Finger Toxin (3FTx) proteins. These toxins can have a wide array of functions that have been characterized from the venom of other elapids. We examined publicly available sequences from Micrurus 3FTx to show that they belong to 8 monophyletic clades that diverged as deep in the 3FTx phylogenetic tree as the other clades with characterized functions. Functional residues from previously characterized clades of 3FTx are not well conserved in most of the Micrurus toxin clades. We also analyzed the patterns of selection on these toxins and find that they have been diversifying at different rates, with some having undergone extreme diversifying selection. This suggests that Micrurus 3FTx may contain a previously underappreciated functional diversity that has implications for the clinical outcomes of bite victims, the evolution and ecology of the genus, as well as the potential for biodiscovery efforts focusing on these toxins.

A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This study examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels of cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This article reports five cases of colonic adenocarcinomas in a family group of captive Amur rat snake (Elaphe schrenckii) from the Réserve Africaine de Sigean, France. This tumor was detected in three females and two males, all adults, and accounted for 16% of causes of death of adults of this species at this institution from 1986 to 2013. Grossly, mild to marked thickening of the intestinal wall cranially to the cloaca was found in four cases; tan to yellow firm masses were noted in the distal intestinal wall in the other case. Microscopically, neoplasms were characterized by infiltrating, poorly circumscribed, and unencapsulated nests of epithelial cells. Marked anisokaryosis and anisocytosis were seen in all neoplasms. The etiology of the neoplasms was not determined, but the familial clustering suggests a common etiologic factor.

Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13 C, 15 N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genetic analyses of Australasian organisms have resulted in the identification of extensive cryptic diversity across the continent. The venomous elapid snakes are among the best-studied organismal groups in this region, but many knowledge gaps persist: for instance, despite their iconic status, the species-level diversity among Australo-Papuan blacksnakes (Pseudechis) has remained poorly understood due to the existence of a group of cryptic species within the P. australis species complex, collectively termed "pygmy mulga snakes". Using two mitochondrial and three nuclear loci we assess species boundaries within the genus using Bayesian species delimitation methods and reconstruct their phylogenetic history using multispecies coalescent approaches. Our analyses support the recognition of 10 species, including all of the currently described pygmy mulga snakes and one undescribed species from the Northern Territory of Australia. Phylogenetic relationships within the genus are broadly consistent with previous work, with the recognition of three major groups, the viviparous red-bellied black snake P. porphyriacus forming the sister species to two clades consisting of ovoviviparous species., (Copyright © 2016. Published by Elsevier Inc.)

We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in Pichia pastoris. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90 °C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p < 0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Australia is the stronghold of the front-fanged venomous snake family Elapidae. The Australasian elapid snake radiation, which includes approximately 100 terrestrial species in Australia, as well as Melanesian species and all the world's sea snakes, is less than 12 million years old. The incredible phenotypic and ecological diversity of the clade is matched by considerable diversity in venom composition. The clade's evolutionary youth and dynamic evolution should make it of particular interest to toxinologists, however, the majority of species, which are small, typically inoffensive, and seldom encountered by non-herpetologists, have been almost completely neglected by researchers. The present study investigates the venom composition of 28 species proteomically, revealing several interesting trends in venom composition, and reports, for the first time in elapid snakes, the existence of an ontogenetic shift in the venom composition and activity of brown snakes ( Pseudonaja sp.). Trends in venom composition are compared to the snakes' feeding ecology and the paper concludes with an extended discussion of the selection pressures shaping the evolution of snake venom., Competing Interests: The authors declare no conflict of interest.

Strong spatial sorting of genetic variation in contiguous populations is often explained by local adaptation or secondary contact following allopatric divergence. A third explanation, spatial sorting by stochastic effects of range expansion, has been considered less often though theoretical models suggest it should be widespread, if ephemeral. In a study designed to delimit species within a clade of venomous coralsnakes, we identified an unusual pattern within the Texas coral snake (Micrurus tener): strong spatial sorting of divergent mitochondrial (mtDNA) lineages over a portion of its range, but weak sorting of these lineages elsewhere. We tested three alternative hypotheses to explain this pattern-local adaptation, secondary contact following allopatric divergence, and range expansion. Collectively, near panmixia of nuclear DNA, the signal of range expansion associated sampling drift, expansion origins in the Gulf Coast of Mexico, and species distribution modeling suggest that the spatial sorting of divergent mtDNA lineages within M. tener has resulted from genetic surfing of standing mtDNA variation-not local adaptation or allopatric divergence. Our findings highlight the potential for the stochastic effects of recent range expansion to mislead estimations of population divergence made from mtDNA, which may be exacerbated in systems with low vagility, ancestral mtDNA polymorphism, and male-biased dispersal., (© 2016 The Author(s).)

Antaioserpens warro sensu lato is known from two populations, one in north-eastern Queensland (Qld), the other from south central Qld. Morphological and genetic assessments demonstrate that these widely separated populations represent two species. A re-examination of museum specimens and the type descriptions show that the name A. warro (De Vis) has been erroneously applied to the north-eastern Qld species. The type specimen of A. warro, from the Gladstone district in south-east Qld, is badly faded but the colour pattern as described by De Vis (1884a) is consistent with that of recently collected specimens from south central Qld and it is this species to which the name applies. The earliest available name for the species from north-eastern Qld is A. albiceps (Boulenger, 1898). Both A. warro and A. albiceps are redescribed herein.

Four sea snakes (two Hydrophis major, one Hydrophis platurus, one Hydrophis elegans) were found washed ashore on different beaches in the Sunshine Coast region and Fraser Island in Queensland, Australia between 2007-2013. Each snake had multiple granulomas and locally extensive regions of pallor evident in the hypaxial and intercostal musculature along the body. Lesions in two individuals were also associated with vertebral and rib fractures. Histological examination revealed granulomas scattered throughout skeletal muscle, subcutaneous adipose tissue and fractured bone. These were composed of dense aggregates of microsporidian spores surrounded by a mantle of macrophages. Sequences (ssrRNA) were obtained from lesions in three sea snakes and all revealed 99% similarity with Heterosporis anguillarum from the Japanese eel (Anguillarum japonica). However, ultrastructural characteristics of the organism were not consistent with those of previous descriptions. Electron microscopic examination of skeletal muscle revealed large cysts (not xenomas) bound by walls of fibrillar material (Heterosporis-like sporophorocyst walls were not detected). The cysts contained numerous mature microsporidian spores arranged in small clusters, sometimes apparently within sporophorous vesicles. The microspores were monomorphic, oval and measured 2.5-3.0 μm by 1.6-1.8 μm. They contained isofilar polar filaments with 11 (infrequently 9-12) coils arranged in two ranks. This is the first published report of a microsporidian infection in hydrophiid sea snakes. This discovery shows microsporidia with molecular affinities to Heterosporis anguillarum but ultrastructural characters most consistent with the genus Pleistophora (but no hitherto described species). Further studies are required to determine whether the microsporidian presented here belongs to the genus Heterosporis, or to a polymorphic species group as suggested by the recognition of a robust Pleistophora/Heterosporis clade by molecular studies. The gross and histological pathology associated with these infections are described.

Background: Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage., Methods and Findings: In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay., Conclusion: Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.

Australian elapid venom remains an under-investigated resource of novel bioactive peptides. In this study, the venom gland transcriptomes and proteomes of the Australian western brown snakes, Pseudonaja aspidorhyncha and Pseudonaja nuchalis, were compared to Pseudonaja textilis. A deep venomics strategy incorporating high throughput 454 pyrosequencing gave a total of 200,911 raw reads for the three venoms. Subsequent annotation identified 5716 transcripts from 20 different toxin families with inter-specific variation between species observed in eight of the less abundant families. Integration of each venom proteome with the corresponding annotated reads identified 65 isoforms from six toxin families; high sequence coverage highlighted subtle differences between sequences and intra and inter-specific variation between species. High quality MS/MS data identified unusual glycoforms with natriuretic peptides from P. aspidorhyncha and P. nuchaliscontaining O-linked trisaccharides with high homology to the glycosylated region of TNPc. Molecular evolutionary assessments indicated the accelerated evolution of all toxin families with the exception of both natriuretic peptides and P. aspidorhyncha PLA2s that were found to be evolutionarily constrained under purifying selection pressures. This study has revealed a wide range of novel peptide sequences from six bioactive peptide families and highlights the subtle differences between toxins in these closely related species., Biological Significance: Mining Australia's vastly untapped source of toxins from its venomous creatures has been significantly advanced by employing deep venomics methodology. Technological advances in transcriptome analysis using next generation sequencing platforms and proteome analysis by highly sensitive tandem mass spectrometry allowed a more comprehensive interrogation of three underinvestigated brown snake (Pseudonaja) venoms uncovering many novel peptide sequences that are unique to these closely related species. This generic strategy will provide invaluable information when applied to other venomous snakes for a deeper understanding of venom composition, envenomation, venom evolution, as well as identifying research tools and drug leads., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Phospholipase A2 (PLA2) is the most abundant protein found in snake venom. PLA2 induces a variety of pharmacological effects such as neurotoxicity, myotoxicity and cardiotoxicity as well as anticoagulant, hemolytic, anti-platelet, hypertensive, hemorrhagic and edema inducing effects. In this study, the three dimensional structure of PLA2 of Naja sputatrix (Malayan spitting cobra) was modeled by I-TASSER, SWISS-MODEL, PRIME and MODELLER programs. The best model was selected based on overall stereo-chemical quality. Further, molecular dynamics simulation was performed to know the stability of the modeled protein using Gromacs software. Average structure was generated during the simulation period of 10 ns. High throughput virtual screening was employed through different databases (Asinex, Hit finder, Maybridge, TOSLab and ZINC databases) against PLA2. The top seven compounds were selected based on the docking score and free energy binding calculations. These compounds were analyzed by quantum polarized ligand docking, induced fit docking and density functional theory calculation. Furthermore, the stability of lead molecules in the active site of PLA2 was employed by MD simulation. The results show that selected lead molecules were highly stable in the active site of PLA2.

This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 μg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic β-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

Background: The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS., Results: Transcriptomic results reveal high redundancy of toxin transcripts (3357.36 FPKM/transcript) despite small cluster numbers, implying gene duplication and diversification within restricted protein families. Among the 23 toxin families identified, three-finger toxins (3FTxs) and snake-venom metalloproteases (SVMPs) have the most diverse isoforms. These 2 toxin families are also the most abundantly transcribed, followed in descending order by phospholipases A2 (PLA2s), cysteine-rich secretory proteins (CRISPs), Kunitz-type inhibitors (KUNs), and L-amino acid oxidases (LAAOs). Seventeen toxin families exhibited low mRNA expression, including hyaluronidase, DPP-IV and 5'-nucleotidase that were not previously reported in the venom-gland transcriptome of a Balinese O. hannah. On the other hand, the MOh proteome includes 3FTxs, the most abundantly expressed proteins in the venom (43 % toxin sbundance). Within this toxin family, there are 6 long-chain, 5 short-chain and 2 non-conventional 3FTx. Neurotoxins comprise the major 3FTxs in the MOh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. The presence of toxic enzymes such as LAAOs, SVMPs and PLA2 would explain tissue inflammation and necrotising destruction in local envenoming. Dissimilarities in the subtypes and sequences between the neurotoxins of MOh and Naja kaouthia (monocled cobra) are in agreement with the poor cross-neutralization activity of N. kaouthia antivenom used against MOh venom. Besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5'-nucleotidase, and DPP-IV in the venom proteome suggests its probable hypotensive action in subduing prey., Conclusion: This study reports the diversity and abundance of toxins in the venom of the Malaysian king cobra (MOh). The results correlate with the pathophysiological actions of MOh venom, and dispute the use of Naja cobra antivenoms to treat MOh envenomation. The findings also provide a deeper insight into venom variations due to geography, which is crucial for the development of a useful pan-regional antivenom.

Hydrophidae, one of the precious traditional Chinese medicines, is generally drily preserved to prevent corruption, but it is hard to identify the species of Hydrophidae through the appearance because of the change due to the drying process. The identification through analysis on gene barcode, a new technique in species identification, can avoid the problem. The gene barcodes of the 6 species of Hydrophidae like Lapemis hardwickii were aquired through DNA extraction and gene sequencing. These barcodes were then in sequence alignment and test the identification efficency by BLAST. Our results revealed that the barcode sequences performed high identification efficiency, and had obvious difference between intra- and inter-species. These all indicated that Cyt b DNA barcoding can confirm the Hydrophidae identification.

Cathelicidins are a family of gene-encoded peptide effectors of innate immunity found exclusively in vertebrates. They play pivotal roles in host immune defense against microbial invasions. Dozens of cathelicidins have been identified from several vertebrate species. However, no cathelicidin from marine reptiles has been characterized previously. Here we report the identification and characterization of a novel cathelicidin (Hc-CATH) from the sea snake Hydrophis cyanocinctus. Hc-CATH is composed of 30 amino acids, and the sequence is KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL. Circular dichroism spectroscopy and structure modeling analysis indicated that Hc-CATH mainly assumes an amphipathic α-helical conformation in bacterial membrane-mimetic solutions. It possesses potent broad-spectrum and rapid antimicrobial activity. Meanwhile, it is highly stable and shows low cytotoxicity toward mammalian cells. The microbial killing activity of Hc-CATH is executed through the disruption of cell membrane and lysis of bacterial cells. In addition, Hc-CATH exhibited potent anti-inflammatory activity by inhibiting the LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Hc-CATH directly binds with LPS to neutralize its toxicity, and it also binds to Toll-like receptor 4 (TLR4/MD2 complex), which therefore inhibits the binding of LPS to TLR4/MD2 complex and the subsequent activation of LPS-induced inflammatory response pathways. Taken together, our study demonstrates that Hc-CATH, the first cathelicidin from sea snake discovered to have both antimicrobial and anti-inflammatory activity, is a potent candidate for the development of peptide antibiotics., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

This study presents the first set of comprehensive reference intervals (RIs) for plasma biochemistry and haematology for three species of sea snakes common to the Indo-Pacific waters of Australia. In total 98 snakes, composed of Hydrophis curtus (n= 60), H. elegans (n = 27) and H. peronii (n = 11), were captured, clinically examined and had venous blood samples collected. All snakes were deemed healthy and in good to excellent body condition with snout to vent lengths of 40.7-73.9 cm (H. curtus), 68.9-131.4 cm (H. elegans) and 55.0-83.0 cm (H. peronii), respectively. Lymphocyte numbers, alkaline phosphatase, alanine aminotransferase, creatine kinase and lactate dehydrogenase levels were species-dependent. All other parameters are presented as a single range for the three species. Gender ratio was evenly distributed for H. curtus and H. elegans, but 8/11 (73%) of H. peronii were males. No significant differences were detected between males and females for any of the measured blood parameters. Lymph contamination was considered and accounted for. Although only three species of sea snakes are represented in this study, the RIs generated may be useful in the clinical assessment of other sea snake species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The critically endangered leaf-scaled (Aipysurus foliosquamaI) and short-nosed (A. apraefrontalis) sea snakes are currently recognised only from Ashmore and Hibernia reefs ~600km off the northwest Australian coast. Steep population declines in both species were documented over 15 years and neither has been sighted on dedicated surveys of Ashmore and Hibernia since 2001. We examine specimens of these species that were collected from coastal northwest Australian habitats up until 2010 (A.foliosquama) and 2012 (A. apraefrontalis) and were either overlooked or treated as vagrants in conservation assessments. Morphological variation and mitochondrial sequence data confirm the assignment of these coastal specimens to A. foliosquama (Barrow Island, and offshore from Port Hedland) and A.apraefrontalis (Exmouth Gulf, and offshore from Roebourne and Broome). Collection dates, and molecular and morphological variation between coastal and offshore specimens, suggest that the coastal specimens are not vagrants as previously suspected, but instead represent separate breeding populations. The newly recognised populations present another chance for leaf-scaled and short-nosed sea snakes, but coastal habitats in northwest Australia are widely threatened by infrastructure developments and sea snakes are presently omitted from environmental impact assessments for industry. Further studies are urgently needed to assess these species' remaining distributions, population structure, and extent of occurrence in protected areas.

Selection is predicted to drive diversification within species and lead to local adaptation, but understanding the mechanistic details underlying this process and thus the genetic basis of adaptive evolution requires the mapping of genotype to phenotype. Venom is complex and involves many genes, but the specialization of the venom gland toward toxin production allows specific transcripts to be correlated with specific toxic proteins, establishing a direct link from genotype to phenotype. To determine the extent of expression variation and identify the processes driving patterns of phenotypic diversity, we constructed genotype-phenotype maps and compared range-wide toxin-protein expression variation for two species of snake with nearly identical ranges: the eastern diamondback rattlesnake (Crotalus adamanteus) and the eastern coral snake (Micrurus fulvius). We detected significant expression variation in C. adamanteus, identified the specific loci associated with population differentiation, and found that loci expressed at all levels contributed to this divergence. Contrary to expectations, we found no expression variation in M. fulvius, suggesting that M. fulvius populations are not locally adapted. Our results not only linked expression variation at specific loci to divergence in a polygenic, complex trait but also have extensive conservation and biomedical implications. C. adamanteus is currently a candidate for federal listing under the Endangered Species Act, and the loss of any major population would result in the irrevocable loss of a unique venom phenotype. The lack of variation in M. fulvius has significant biomedical application because our data will assist in the development of effective antivenom for this species., (Copyright © 2015 by the Genetics Society of America.)

The venom arsenal of the New Guinea small-eyed snake, Micropechis ikaheka, was investigated by a joint cDNA sequencing and venomics approach. Twenty-seven full-length DNA sequences encoding novel venom proteins were recovered in this study. Using this cDNA dataset we achieved locus-specific resolution for 19 out of the approximately 50 reverse-phase- and SDS-PAGE-separated venom proteins. The venom proteome of M. ikaheka is dominated by at least 29 D49-phospholipase A₂ (PLA₂) and 14 short and long neurotoxins of the three-finger toxin (3FTx) family. These protein classes represent, respectively, 80% and 9.2% of the total venom proteins. Two PIII-metalloproteinase (SVMP) molecules (7.6%), three CRISP isoforms (1.8%), and a single Kunitz-type inhibitor, vespryn, 5'-nucleotidase, serine proteinase and LAO molecules, none of which represents more than 0.7% of the total venom proteome, complete the protein arsenal of M. ikaheka. In concordance with clinical observations, this venom composition points to a central role for post-synaptically-acting neurotoxic toxins in the envenomation strategy developed by this species. PLA₂ molecules represent the main myotoxic components of M. ikaheka venom. In addition, the estimated LD₅₀ for mice of the reverse-phase-isolated 3FTx (0.22 mg/kg) and PLA₂ (1.62 mg/kg) enriched fractions, strongly suggests that these two toxin classes contribute synergistically to venom lethality, with the 3FTxs playing a dominant role. The high structural and functional conservation exhibited by M. ikaheka and Australian elapid venoms may underlay the positive clinical outcomes of envenoming resulting from bites by M. ikaheka that have been documented through the use of bioCSL polyvalent antivenom., Biological Significance: The poorly understood venom proteome of the New Guinea small-eyed snake, Micropechis ikaheka, a large and powerfully built elapid endemic to Papua New Guinea and Indonesian West Papua province, was investigated through a combined venomics and venom gland transcriptomics approach. Although M. ikaheka accounts for only a small proportion of snakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites by this snake. Major effects of envenomings include life-threatening post-synaptic neuromuscular blockade resulting in respiratory paralysis, myotoxicity, severe bleeding, hypotension and cardiovascular abnormalities. We have investigated the contribution of 3FTxs and PLA₂ molecules in venom lethality, myotoxicity, and cardiovascular function. Our work provides important correlations between venom composition and its pharmacological activity. In conjunction with the antivenomics work reported in the companion paper, our study may contribute to improve treatment outcomes for snakebite victims of M. ikaheka., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The Australian Elapidae spotted black snake Pseudechis guttatus venom proteome composition was analyzed by high throughput mass spectrometry. The crude venom proteins were decomplexed by 2D-PAGE and in-gel digestion peptides from 66 spot samples and analyzed by tandem mass spectrometry-LC-ESI-ion trap. Protein identification was performed combining PEAKS studio 7.0 and Mascot software. The analysis identified l-amino-acid oxidases, phospholipases A2, metalloproteases, nerve growth factors and ecto-5'-nucleotidases, and for the first time in this venom the components cysteine-rich secretory proteins similar to pseudechetoxin, phospholipase B and transferrin-like protein. The envenomation symptoms are in agreement with the identified components, but the present limitations of database information might impair the detection of toxin families, protein species and still unknown toxins. From the qualitative point of view, the similarity of this venom with the ones from other Pseudechis species could be assigned to recent speciation events., Biological Significance: Studies on the proteome of Australian Elapidae (Ancanthophiinae) are quite rare. In the present work we performed, using classic proteomic methods, a qualitative and partial analysis of the proteic components of Pseudechis guttatus venom. Although previous studies contributed to the knowledge of the major components of this venom, our study revealed some yet undescribed protein species, as well as new toxins, such as CRiSPs, phospholipase B, transferrin-like protein and ecto 5'-nucleotidase., (Copyright © 2014 Elsevier B.V. All rights reserved.)