Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Population dynamics of the sea snake Emydocephalus annulatus (Elapidae, Hydrophiinae).

For sea snakes as for many types of animals, long-term studies on population biology are rare and hence, we do not understand the degree to which annual variation in population sizes is driven by density-dependent regulation versus by stochastic abiotic factors. We monitored three populations of turtle-headed sea snakes (Emydocephalus annulatus) in New Caledonia over an 18-year period. Annual recruitment (% change in numbers) showed negative density-dependence: that is, recruitment increased when population densities were low, and decreased when densities were high. Windy weather during winter increased survival of neonates, perhaps by shielding them from predation; but those same weather conditions reduced body condition and the reproductive output of adult snakes. The role for density-dependence in annual dynamics of these populations is consistent with the slow, K-selected life-history attributes of the species; and the influence of weather conditions on reproductive output suggests that females adjust their allocation to reproduction based on food availability during vitellogenesis.

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.

Slow life history leaves endangered snake vulnerable to illegal collecting.

Global wildlife trade is a multibillion-dollar industry and a significant driver of vertebrate extinction risk. Yet, few studies have quantified the impact of wild harvesting for the illicit pet trade on populations. Long-lived species, by virtue of their slow life history characteristics, may be unable to sustain even low levels of collecting. Here, we assessed the impact of illegal collecting on populations of endangered broad-headed snakes (Hoplocephalus bungaroides) at gated (protected) and ungated (unprotected) sites. Because broad-headed snakes are long-lived, grow slowly and reproduce infrequently, populations are likely vulnerable to increases in adult mortality. Long-term data revealed that annual survival rates of snakes were significantly lower in the ungated population than the gated population, consistent with the hypothesis of human removal of snakes for the pet trade. Population viability analysis showed that the ungated population has a strongly negative population growth rate and is only prevented from ultimate extinction by dispersal of small numbers of individuals from the gated population. Sensitivity analyses showed that the removal of a small number of adult females was sufficient to impose negative population growth and suggests that threatened species with slow life histories are likely to be especially vulnerable to illegal collecting.

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

The behaviour of sea snakes (Emydocephalus annulatus) shifts with the tides.

Tidal cycles are known to affect the ecology of many marine animals, but logistical obstacles have discouraged behavioural studies on sea snakes in the wild. Here, we analyse a large dataset (1,445 observations of 126 individuals) to explore tidally-driven shifts in the behaviour of free-ranging turtle-headed sea snakes (Emydocephalus annulatus, Hydrophiinae) in the Baie des Citrons, New Caledonia. Snakes tended to move into newly-inundated areas with the rising tide, and became more active (e.g. switched from inactivity to mate-searching and courting) as water levels rose. However, the relative use of alternative habitat types was largely unaffected by tidal phase.

Spectral Diversification and Trans-Species Allelic Polymorphism during the Land-to-Sea Transition in Snakes.

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.

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.

Carbon monoxide inhibits hemotoxic activity of Elapidae venoms: potential role of heme.

Envenomation by hemotoxic enzymes continues to be a major cause of morbidity and mortality throughout the world. With regard to treatment, the gold standard to abrogate coagulopathy caused by these venoms is still the administration of antivenom; however, despite antivenom therapy, coagulopathy still occurs and recurs. Of interest, this laboratory has demonstrated in vitro and in vivo that coagulopathy inducing venom derived from snakes of the family Viperidae exposed to carbon monoxide (CO) is inhibited, potentially by an attached heme. The present investigation sought to determine if venoms derived from snakes of the Elapidae family (taipans and cobras) could also be inhibited with CO or with the metheme inducing agent, O-phenylhydroxylamine (PHA). Assessing changes in coagulation kinetics of human plasma with thrombelastography, venoms from Elapidae snakes were exposed in isolation to CO (five species) or PHA (one specie) and placed in human plasma to assess changes in procoagulant or anticoagulant activity. The procoagulant activity of two taipan venoms and anticoagulant activity of three cobra venoms were significantly inhibited by CO. The venom of the inland taipan was also inhibited by PHA. In sum, these data demonstrate indirectly that the biometal heme is likely bound to these disparate venoms as an intermediary modulatory molecule. In conclusion, CO may not just be a potential therapeutic agent to treat envenomation but also may be a potential modulator of heme as a protective mechanism for venomous snakes against injury from their own proteolytic venoms.

Burrowing with a kinetic snout in a snake (Elapidae: Aspidelaps scutatus).

Of the few elongate, fossorial vertebrates that have been examined for their burrowing mechanics, all were found to use an akinetic, reinforced skull to push into the soil, powered mostly by trunk muscles. Reinforced skulls were considered essential for head-first burrowing. In contrast, I found that the skull of the fossorial shield-nosed cobra (Aspidelaps scutatus) is not reinforced and retains the kinetic potential typical of many non-fossorial snakes. Aspidelaps scutatus burrows using a greatly enlarged rostral scale that is attached to a kinetic snout that is independently mobile with respect to the rest of the skull. Two mechanisms of burrowing are used: (1) anteriorly directed head thrusts from a loosely bent body that is anchored against the walls of the tunnel by friction, and (2) side-to-side shovelling using the head and rostral scale. The premaxilla, to which the rostral scale is attached, lacks any direct muscle attachments. Rostral scale movements are powered by, first, retractions of the palato-pterygoid bar, mediated by a ligament that connects the anterior end of the palatine to the transverse process of the premaxilla and, second, by contraction of a previously undescribed muscle slip of the m. retractor pterygoidei that inserts on the skin at the edge of the rostral scale. In derived snakes, palatomaxillary movements are highly conserved and power prey capture and transport behaviors. Aspidelaps scutatus has co-opted those mechanisms for the unrelated function of burrowing without compromising the original feeding functions, showing the potential for evolution of functional innovations in highly conserved systems.

Catch a tiger snake by its tail: Differential toxicity, co-factor dependence and antivenom efficacy in a procoagulant clade of Australian venomous snakes.

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.

Correlation between ontogenetic dietary shifts and venom variation in Australian brown snakes (Pseudonaja).

Venom is a key evolutionary trait, as evidenced by its widespread convergent evolution across the animal kingdom. In an escalating prey-predator arms race, venoms evolve rapidly to guarantee predatory or defensive success. Variation in venom composition is ubiquitous among snakes. Here, we tested variation in venom activity on substrates relevant to blood coagulation among Pseudonaja (brown snake) species, Australian elapids responsible for the majority of medically important human envenomations in Australia. A functional approach was employed to elucidate interspecific variation in venom activity in all nine currently recognised species of Pseudonaja. Fluorometric enzymatic activity assays were performed to test variation in whole venom procoagulant activity among species. Analyses confirmed the previously documented ontogenetic shift from non-coagulopathic venom in juveniles to coagulopathic venom as adults, except for the case of P. modesta, which retains non-coagulopathic venom as an adult. These shifts in venom activity correlate with documented ontogenetic shifts in diet among brown snakes from specialisation on reptilian prey as juveniles (and throughout the life cycle of P. modesta), to a more generalised diet in adults that includes mammals. The results of this study bring to light findings relevant to both clinical and evolutionary toxinology.

How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting.

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.

Bites by the Monocled Cobra, Naja kaouthia, in Chittagong Division, Bangladesh: Epidemiology, Clinical Features of Envenoming and Management of 70 Identified Cases.

AbstractWe describe 70 cases of monocled cobra (Naja kaouthia) bite admitted to Chittagong Medical College Hospital, Bangladesh. The biting snakes were identified by examining the dead snake and/or detecting N. kaouthia venom antigens in patients' serum. Bites were most common in the early morning and evening during the monsoon (May-July). Ligatures were routinely applied to the bitten limb before admission. Thirty-seven patients consulted traditional healers, most of whom made incisions around the bite site. Fifty-eight patients experienced severe neurotoxicity and most suffered swelling and pain of the bitten limb. The use of an Indian polyvalent antivenom in patients exhibiting severe neurotoxicity resulted in clinical improvement but most patients experienced moderate-to-severe adverse reactions. Antivenom did not influence local blistering and necrosis appearing in 19 patients; 12 required debridement. Edrophonium significantly improved the ability of patients to open the eyes, endurance of upward gaze, and peak expiratory flow rate suggesting that a longer-acting anticholinesterase drug (neostigmine) could be recommended for first aid. The study suggested that regionally appropriate antivenom should be raised against the venoms of the major envenoming species of Bangladesh and highlighted the need to improve the training of staff of local medical centers and to invest in the basic health infrastructure in rural communities.

Rapid Radiations and the Race to Redundancy: An Investigation of the Evolution of Australian Elapid Snake Venoms.

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.

Coming up for air: thermal dependence of dive behaviours and metabolism in sea snakes.

Cutaneous gas exchange allows some air-breathing diving ectotherms to supplement their pulmonary oxygen uptake, which may allow prolongation of dives and an increased capacity to withstand anthropogenic and natural threatening processes that increase submergence times. However, little is known of the interplay between metabolism, bimodal oxygen uptake and activity levels across thermal environments in diving ectotherms. Here, we show in two species of sea snake (spine-bellied sea snake, Hydrophis curtus; and elegant sea snake, Hydrophis elegans) that increasing temperature elevates surfacing rate, increases total oxygen consumption and decreases dive duration. The majority of dives observed in both species remained within estimated maximal aerobic limits. While cutaneous gas exchange accounted for a substantial proportion of total oxygen consumption (up to 23%), unexpectedly it was independent of water temperature and activity levels, suggesting a diffusion-limited mechanism. Our findings demonstrate that rising water temperature and a limited capability to up-regulate cutaneous oxygen uptake may compromise the proficiency with which sea snakes perform prolonged dives. This may hinder their capacity to withstand ongoing anthropogenic activities like trawl fishing, and increase their susceptibility to surface predation as their natural environments continue to warm.

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.

Stabilising the Integrity of Snake Venom mRNA Stored under Tropical Field Conditions Expands Research Horizons.

BACKGROUND: Snake venoms contain many proteinaceous toxins that can cause severe pathology and mortality in snakebite victims. Interestingly, mRNA encoding such toxins can be recovered directly from venom, although yields are low and quality is unknown. It also remains unclear whether such RNA contains information about toxin isoforms and whether it is representative of mRNA recovered from conventional sources, such as the venom gland. Answering these questions will address the feasibility of using venom-derived RNA for future research relevant to biomedical and antivenom applications. METHODOLOGY/PRINCIPAL FINDINGS: Venom was extracted from several species of snake, including both members of the Viperidae and Elapidae, and either lyophilized or immediately added to TRIzol reagent. TRIzol-treated venom was incubated at a range of temperatures (4-37°C) for a range of durations (0-48 hours), followed by subsequent RNA isolation and assessments of RNA quantity and quality. Subsequently, full-length toxin transcripts were targeted for PCR amplification and Sanger sequencing. TRIzol-treated venom yielded total RNA of greater quantity and quality than lyophilized venom, and with quality comparable to venom gland-derived RNA. Full-length sequences from multiple Viperidae and Elapidae toxin families were successfully PCR amplified from TRIzol-treated venom RNA. We demonstrated that venom can be stored in TRIzol for 48 hours at 4-19°C, and 8 hours at 37°C, at minimal cost to RNA quality, and found that venom RNA encoded multiple toxin isoforms that seemed homologous (98-99% identity) to those found in the venom gland. CONCLUSIONS/SIGNIFICANCE: The non-invasive experimental modifications we propose will facilitate the future investigation of venom composition by using venom as an alternative source to venom gland tissue for RNA-based studies, thus obviating the undesirable need to sacrifice snakes for such research purposes. In addition, they expand research horizons to rare, endangered or protected snake species and provide more flexibility to performing fieldwork on venomous snakes in tropical conditions.

Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Trocarin D (TroD), a venom prothrombin activator from Tropidechis carinatus, shares similar structure and function with blood coagulation factor Xa [Tropidechis carinatus FX (TrFX) a]. Their distinct physiologic roles are due to their distinct expression patterns. The genes of TroD and TrFX are highly similar, except for promoter and intron 1, indicating that TroD has probably evolved by duplication of FX, the plasma counterpart. The promoter insertion in TroD accounts for the elevated but not venom gland-specific expression. Here we examined the roles of 3 insertions and 2 deletions in intron 1 of TroD in the regulation of expression using luciferase as a reporter. By systematic deletions, we showed that a 209 bp region within the second insertion silences expression in mammalian and unmilked venom gland cells. Through bioinformatics analysis, we identified 5 AG-rich motifs in this region. All except the 5th motif are important for silencing function. YY1, Sp3 and HMGB2 were identified to bind these AG-rich motifs and silence gene expression in mammalian cells. Similar AG-rich motif clusters are also found in other toxin genes but not in their physiologic counterparts. Thus, AG-rich motifs contribute to regulation of expression of TroD, and probably other toxin genes.-Han, S. X., Kwong, S., Ge, R., Kolatkar, P. R., Woods, A. E., Blanchet, G., Kini, R. M. Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Fatal presumed tiger snake (Notechis scutatus) envenomation in a cat with measurement of venom and antivenom concentration.

A fatal outcome of a presumed tiger snake (Notechis scutatus) envenomation in a cat is described. Detectable venom components and antivenom concentrations in serum from clotted and centrifuged whole blood and urine were measured using a sensitive and specific ELISA. The cat presented in a paralysed state with a markedly elevated serum CK but with normal clotting times. The cat was treated with intravenous fluids and received two vials of equine whole IgG bivalent (tiger and brown snake) antivenom. Despite treatment the cat's condition did not improve and it died 36 h post-presentation. Serum concentration of detectable tiger snake venom components at initial presentation was 311 ng/mL and urine 832 ng/mL, this declined to non-detectable levels in serum 15-min after intravenous antivenom. Urine concentration of detectable tiger snake venom components declined to 22 ng/mL at post-mortem. Measurement of equine anti-tiger snake venom specific antibody demonstrated a concentration of 7.2 Units/mL in serum at post-mortem which had declined from an initial high of 13 Units/mL at 15-min post-antivenom. The ELISA data demonstrated the complete clearance of detectable venom components from serum with no recurrence in the post-mortem samples. Antivenom concentrations in serum at initial presentation were at least 100-fold higher than theoretically required to neutralise the circulating concentrations of venom. Despite the fatal outcome in this case it was concluded that this was unlikely that is was due to insufficient antivenom.