Whip it into shape: Revision of the Demansia psammophis (Schlegel, 1837) complex (Squamata: Elapidae), with a description of a new species from central Australia.

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.

Morphology, Diet, and Reproduction of Coastal Hydrophis Sea Snakes (Elapidae: Hydrophiinae) at Their Northern Distribution Limit.

The Ryukyu Archipelago represents the northern distribution limit for hydrophiine sea snakes, the largest group of marine reptiles. Ryukyuan sea snakes may have developed distinct local adaptations in morphology and ecology, but they have been poorly studied. We examined preserved specimens of 111 Hydrophismelanocephalusand 61 Hydrophis ornatusfrom the Ryukyu Archipelago to obtain data on morphology, diet, and reproduction. Sexual size dimorphism was detected in H. melanocephalus (mean ± standard deviation of adult snout-vent length: SVL, females 1062 ± 141 mm vs. males 959 ± 96 mm) but not in H. ornatus. Female H. melanocephalus had larger head widths and shorter tail lengths relative to SVL compared to males. Relative girth was low in neonates of both species (1.0-1.3), but increased in adults to about 1.7-2.6 in H. melanocephalus and 1.3-1.8 in female H. ornatus. Stomach contents of H. melanocephalus consisted of ophichthid and congrid eels, a sand diver, and gobies, whereas in H. ornatus, gobies and a goat fish were found. Litter size of three reproductive H. melanocephalus ranged from five to seven, and parturition seems to occur from August to October. Litter size of six H. ornatus ranged from two to seven, and was correlated with maternal SVL. Parturition in H. ornatus probably occurs around November. Different selective forces related to locomotion, feeding and predation risk, which influence the pregnant mother and neonates, may have resulted in having few, long but slender offspring that show positive allometric growth in hind-body girth.

An evaluation of the nomina for death adders (Acanthophis Daudin, 1803) proposed by Wells amp; Wellington (1985), and confirmation of A. cryptamydros Maddock et al., 2015 as the valid name for the Kimberley death adder.

We assess the availability of four names proposed by Wells Wellington (1985) for Australian death adders (Acanthophis). In agreement with previous literature, A. hawkei is an available name, whereas A. armstrongi, A. lancasteri, and A. schistos are not described in conformity with the requirements of Articles 13.1.1 or 13.1.2 of the International Code of Zoological Nomenclature and are therefore considered nomina nuda. Consequently, A. cryptamydros Maddock et al., 2015, is confirmed as the valid name for the Kimberley death adder of Western Australia. We comment on the need for greater clarity in the Code, and emphasise that the responsibility for establishing the availability of new nomina rests with their authors, not subsequent researchers.

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

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

Systematics of Calliophis intestinalis with the Resurrection of Calliophis nigrotaeniatus (Elapidae, Serpentes).

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.

First Authenticated Case of a Bite by Rare and Elusive Blood-Bellied Coral Snake (Calliophis haematoetron).

Blood-bellied coral snake (Calliophis haematoetron) is a recently discovered forest-dwelling species of elapid hitherto known from 3 specimens found from central Sri Lanka. Herein we describe the first authenticated case of blood-bellied coral snakebite. The victim, an 11-mo-old infant who received the bite while handling the snake at her home, had mild transient swelling at the bite site. The patient had no clinical or laboratory evidence of systemic envenoming. We highlight the importance of clinicians being aware of the occurrence of this potentially medically important elapid snake in anthropogenic habitats.

An Appetite for Destruction: Detecting Prey-Selective Binding of α-Neurotoxins in the Venom of Afro-Asian Elapids.

Prey-selective venoms and toxins have been documented across only a few species of snakes. The lack of research in this area has been due to the absence of suitably flexible testing platforms. In order to test more species for prey specificity of their venom, we used an innovative taxonomically flexible, high-throughput biolayer interferometry approach to ascertain the relative binding of 29 α-neurotoxic venoms from African and Asian elapid representatives (26 Naja spp., Aspidelaps scutatus, Elapsoidea boulengeri, and four locales of Ophiophagus hannah) to the alpha-1 nicotinic acetylcholine receptor orthosteric (active) site for amphibian, lizard, snake, bird, and rodent targets. Our results detected prey-selective, intraspecific, and geographical differences of α-neurotoxic binding. The results also suggest that crude venom that shows prey selectivity is likely driven by the proportions of prey-specific α-neurotoxins with differential selectivity within the crude venom. Our results also suggest that since the α-neurotoxic prey targeting does not always account for the full dietary breadth of a species, other toxin classes with a different pathophysiological function likely play an equally important role in prey immobilisation of the crude venom depending on the prey type envenomated. The use of this innovative and taxonomically flexible diverse assay in functional venom testing can be key in attempting to understanding the evolution and ecology of α-neurotoxic snake venoms, as well as opening up biochemical and pharmacological avenues to explore other venom effects.

Interspecific and intraspecific venom enzymatic variation among cobras (Naja sp. and Ophiophagus hannah).

The genera Ophiophagus and Naja comprise part of a clade of snakes referred to as cobras, dangerously venomous front-fanged snakes in the family Elapidae responsible for significant human mortality and morbidity throughout Asia and Africa. We evaluated venom enzyme variation for eleven cobra species and three N. kaouthia populations using SDS-PAGE venom fingerprinting and numerous enzyme assays. Acetylcholinesterase and PLA2 activities were the most variable between species, and PLA2 activity was significantly different between Malaysian and Thailand N. kaouthia populations. Venom metalloproteinase activity was low and significantly different among most species, but levels were identical for N. kaouthia populations; minor variation in venom L-amino acid oxidase and phosphodiesterase activities were seen between cobra species. Naja siamensis venom lacked the α-fibrinogenolytic activity common to other cobra venoms. In addition, venom from N. siamensis had no detectable metalloproteinase activity and exhibited an SDS-PAGE profile with reduced abundance of higher mass proteins. Venom profiles from spitting cobras (N. siamensis, N. pallida, and N. mossambica) exhibited similar reductions in higher mass proteins, suggesting the evolution of venoms of reduced complexity and decreased enzymatic activity among spitting cobras. Generally, the venom proteomes of cobras show highly abundant three-finger toxin diversity, followed by large quantities of PLA2s. However, PLA2 bands and activity were very reduced for N. haje, N. annulifera and N. nivea. Venom compositionalenzy analysis provides insight into the evolution, diversification and distribution of different venom phenotypes that complements venomic data, and this information is critical for the development of effective antivenoms and snakebite treatment.

A new species of the genus Sinomicrurus (Serpentes: Elapidae) from China and Vietnam.

A new species of Sinomicrurus Slowinski, Boundy, and Lawson, 2001 is described herein based on a series of specimens. The new species, Sinomicrurus peinani sp. nov., occurs in southern China and northern Vietnam. Sinomicrurus peinani sp. nov. is distinguished from its congeners by the following combination of characters: (1) 30-35 black cross-bands on body and tail; (2) 13 dorsal scale rows throughout, all smooth; (3) white belly with black cross-bands or irregular spots; (4) broad white transverse bar on top of head with inverted V-shaped anterior margin, white bar wider than anterior black bar; and (5) frontal V-like, 1.3 times as long as wide. In addition, new occurrences of S. houi in Guangxi, China, and Vietnam are discussed.

Activity of two key toxin groups in Australian elapid venoms show a strong correlation to phylogeny but not to diet.

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 A2 (PLA2), 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: PLA2 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 PLA2 (delta parameter mean 0.64). There were some exceptions to the phylogenetic patterns of enzyme activity: different PLA2 activity in the ecologically similar sister-species Denisonia devisi and D. maculata; large inter-specific differences in PLA2 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. PLA2 and LAAO activities had contrasting evolutionary dynamics with the higher delta value for PLA2 Some species/individuals lacked activity in one protein family suggesting that the loss of single protein family may not incur a significant fitness cost.

Clinical implications of convergent procoagulant toxicity and differential antivenom efficacy in Australian elapid snake venoms.

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.

Primary structures and partial toxicological characterization of two phospholipases A2 from Micrurus mipartitus and Micrurus dumerilii coral snake venoms.

Snake venom phospholipases A2 (PLA2) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA2s have been purified and characterized from coral snake (Micrurus spp.) venoms, and their role in envenomation remains largely unknown. In this report, we describe the isolation, sequencing and partial functional characterization of two Micrurus PLA2s: MmipPLA2 from Micrurus mipartitus and MdumPLA2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA2 consisted of 117 residues with a pI of 5.6. Both PLA2s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA2 was not lethal (up to 3 µg/g body weight) but strongly myotoxic. MdumPLA2 displayed higher anticoagulant activity than MmipPLA2in vitro and caused more sustained edema in the mouse footpad assay. Neither of these enzymes was cytolytic to cultured skeletal muscle C2C12 myotubes. Based on their structural differences, the two enzymes were placed in separate lineages in a partial phylogeny of Micrurus venom PLA2s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA2s.

Multi-locus phylogeny and species delimitation of Australo-Papuan blacksnakes (Pseudechis Wagler, 1830: Elapidae: Serpentes).

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.

Two snakes from eastern Australia (Serpentes: Elapidae); a revised concept of Antaioserpens warro (De Vis) and a redescription of A. albiceps (Boulenger).

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.

The evolution of scale sensilla in the transition from land to sea in elapid snakes.

Scale sensilla are small tactile mechanosensory organs located on the head scales of many squamate reptiles (lizards and snakes). In sea snakes and sea kraits (Elapidae: Hydrophiinae), these scale organs are presumptive scale sensilla that purportedly function as both tactile mechanoreceptors and potentially as hydrodynamic receptors capable of sensing the displacement of water. We combined scanning electron microscopy, silicone casting of the skin and quadrate sampling with a phylogenetic analysis to assess morphological variation in sensilla on the postocular head scale(s) across four terrestrial, 13 fully aquatic and two semi-aquatic species of elapids. Substantial variation exists in the overall coverage of sensilla (0.8-6.5%) among the species sampled and is broadly overlapping in aquatic and terrestrial lineages. However, two observations suggest a divergent, possibly hydrodynamic sensory role of sensilla in sea snake and sea krait species. First, scale sensilla are more protruding (dome-shaped) in aquatic species than in their terrestrial counterparts. Second, exceptionally high overall coverage of sensilla is found only in the fully aquatic sea snakes, and this attribute appears to have evolved multiple times within this group. Our quantification of coverage as a proxy for relative 'sensitivity' represents the first analysis of the evolution of sensilla in the transition from terrestrial to marine habitats. However, evidence from physiological and behavioural studies is needed to confirm the functional role of scale sensilla in sea snakes and sea kraits.

Ultrastructural and Molecular Characterisation of an Heterosporis-Like Microsporidian in Australian Sea Snakes (Hydrophiinae).

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.

A comparative study of venomics of Naja naja from India and Sri Lanka, clinical manifestations and antivenomics of an Indian polyspecific antivenom.

Naja naja (Indian cobra) from Sri Lanka and India is the WHO Category 1 medically important snakes in both countries. Some antivenom produced against Indian N. naja (NNi) were less effective against Sri Lankan N. naja (NNsl). Proteomes of NNi and NNsl venoms were studied by RP-HPLC, SDS-PAGE and LC/MS/MS. Six protein families were identified in both venoms with the most abundant were the 3 finger toxins (3FTs) where cytotoxins (CTX) subtype predominated, followed by phospholipase A2, cysteine-rich venom protein, snake venom metalloproteases, venom growth factors, and protease inhibitors. Qualitative and quantitative differences in the venomics profiles were observed. Some proteins were isolated from either NNi or NNsl venom. Postsynaptic neurotoxins (NTX) were identified for the first time in NNsl venom. Thus, there are geographic intra-specific variations of venom composition of the two N. naja. The relative abundance of CTX and NTX explained well the clinical manifestations of these venoms. Antivenomics study of an Indian antivenom (Vins) showed the antibodies effectively bound all venom toxins from both snakes but more avidly to the Indian venom proteins. The lower antibody affinity towards the 'heterologous' venom was the likely cause of poor efficacy of the Indian antivenom used to treat NNsl envenoming.

A new species of death adder (Acanthophis: Serpentes: Elapidae) from north-western Australia.

Australian death adders (genus Acanthophis) are highly venomous snakes with conservative morphology and sit-and-wait predatory habits, with only moderate taxonomic diversity that nevertheless remains incompletely understood. Analyses of mitochondrial and nuclear gene sequences and morphological characteristics of death adders in northern Australia reveal the existence of a new species from the Kimberley region of Western Australia and the Northern Territory, which we describe as Acanthophis cryptamydros sp. nov. Although populations from the Kimberley were previously considered conspecific with Northern Territory death adders of the A. rugosus complex, our mtDNA analysis indicates that its closest relatives are desert death adders, A. pyrrhus. We found that A. cryptamydros sp. nov. is distinct in both mtDNA and nDNA analysis, and possesses multiple morphological characteristics that allow it to be distinguished from all other Acanthophis species. This study further supports the Kimberley region as an area with high endemic biodiversity.

Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

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 ß-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.

A new species of monadal coral snake of the genus Micrurus (Serpentes, Elapidae) from western Amazon.

We described a new species of monadal coral snake of the genus Micrurus from the region of Tabatinga and Leticia, along the boundaries of Brazil, Colombia, and Peru. The new species can be distinguished from the other congeners by the combination of the following characters: absence of a pale nuchal collar; black cephalic-cap extending from rostral to firstdorsal scale and enclosing white tipped prefrontal scales; upper half of first to four supralabials and postoculars black; tricolor body coloration, with 27-31 black rings bordered by narrower white rings and 27-31 red rings; tail coloration similar to body, with alternating black rings bordered by irregular narrow white rings, red rings of the same width as the black rings; ventral scales 205-225; subcaudal scales 39-47.