Paraspecificity of Mexican Antivipmyn TRI antivenom in envenomation by Chinese Protobothrops mangshanensis (Mangshan pit viper) in France: a case report and experimental neutralization of venom procoagulant effect.

This case report presents an exotic envenomation by a Chinese snake, Protobothrops mangshanensis. Its venom exhibited potent activity against plasma and fibrinogen, among other enzymatic activities. The patient initially presented with edema of the right upper limb, without tissue necrosis. There were no signs of bleeding; however, severe hypofibrinogenemia was observed (nadir value at 0.4 g/L), with a marked increase in fibrinogen degradation products and D-dimers, without any other coagulation disturbances. In the absence of a specific antivenom available against Asian Crotalinae venoms, the patient was treated at the 29th hour after bite with six vials of Antivipmyn™ TRI (Instituto Bioclon, Mexico, Mexico), a Mexican antivenom initially intended for American Crotalinae venoms, i.e., Bothrops asper, Lachesis muta and Crotalus durissus. Fibrinogen began to rise 6 hours after the antivenom infusion and was within the normal range 38 hours later. The report also underscores the utility of ClotPro® (Haemonetics ®USA), a viscoelastic test, for real-time monitoring of the snakebite-related coagulopathy. The clotting time was extended to 188 seconds on the EX-test while the MCF was decreased to 31 mm on the EX-test and the AP-test and was not measurable on the FIB-test, confirming severe hypofibrinogenemia. In order to confirm the paraspecificity of antivenom on the venom of P. mangshanensis, we studied the experimental neutralization of the venom procoagulant effect by Antivipmyn TRI and Green Pit Viper antivenom, which has been used in previous published clinical cases of P. mangshanensis envenomation. Both Antivipmyn™ TRI and Green Pit Viper antivenom corrected the procoagulant effect induced by P. mangshanensis venom. These findings suggest that Antivipmyn™ TRI cross-reacts with Protobothrops mangshanensis venom. In the absence of antivenom covering Asian Crotalinae, Antivipmyn TRI should be considered to treat an envenomation by Protobothrops spp.

Phylogenomic discordance is driven by wide-spread introgression and incomplete lineage sorting during rapid species diversification within rattlesnakes (Viperidae: Crotalus and Sistrurus).

Phylogenomics allows us to uncover the historical signal of evolutionary processes through time and estimate phylogenetic networks accounting for these signals. Insight from genome-wide data further allows us to pinpoint the contributions to phylogenetic signal from hybridization, introgression, and ancestral polymorphism across the genome. Here we focus on how these processes have contributed to phylogenetic discordance among rattlesnakes (genera Crotalus and Sistrurus), a group for which there are numerous conflicting phylogenetic hypotheses based on a diverse array of molecular datasets and analytical methods. We address the instability of the rattlesnake phylogeny using genomic data generated from transcriptomes sampled from nearly all known species. These genomic data, analyzed with coalescent and network-based approaches, reveal numerous instances of rapid speciation where individual gene trees conflict with the species tree. Moreover, the evolutionary history of rattlesnakes is dominated by incomplete speciation and frequent hybridization, both of which have likely influenced past interpretations of phylogeny. We present a new framework in which the evolutionary relationships of this group can only be understood in light of genome-wide data and network-based analytical methods. Our data suggest that network radiations, like seen within the rattlesnakes, can only be understood in a phylogenomic context, necessitating similar approaches in our attempts to understand evolutionary history in other rapidly radiating species.

Embryonic development of the neotropical pit viper Bothrops atrox (Serpentes: Viperidae: Crotalinae), with emphasis on pit organ morphogenesis and its evolution in snakes.

BACKGROUND: Bothrops atrox is a pit viper with a loreal pit organ, and its embryological development remains undescribed. Here, we provide a comprehensive description of the embryology of B. atrox, focusing on the loreal pit organ and cephalic scales. RESULTS: We characterized 13 developmental stages of B. atrox based on external features consistent with the embryogenesis of previously described snake species. The loreal pit organ originates from the circumorbital region and migrates to its final position. In Crotalinae, the pit organ first becomes visible at stage 28, whereas in Pythonidae labial, pit organs appear at Stage 35. Pit organs evolved independently three times in Serpentes, encompassing Boidae, Pythonidae, and Crotalinae. Boidae lacks embryological information for pit organs. Furthermore, we observed that head scalation onset occurs at Stage 33 in B. atrox, with fusion of scales surrounding the loreal pit organ. CONCLUSIONS: The embryology of pit organs in Pythonidae and Boidae species remains poorly understood. Our detailed embryological descriptions are critical for proposing developmental scenarios for pit organs and guiding future research on these structures.

Phylogenomic discordance is driven by wide-spread introgression and incomplete lineage sorting during rapid species diversification within rattlesnakes (Viperidae: Crotalus and Sistrurus)

Phylogenomics allows us to uncover the historical signal of evolutionary processes through time and estimate phylogenetic networks accounting for these signals. Insight from genome-wide data further allows us to pinpoint the contributions to phylogenetic signal from hybridization, introgression, and ancestral polymorphism across the genome. Here we focus on how these processes have contributed to phylogenetic discordance among rattlesnakes (genera Crotalus and Sistrurus), a group for which there are numerous conflicting phylogenetic hypotheses based on a diverse array of molecular datasets and analytical methods. We address the instability of the rattlesnake phylogeny using genomic data generated from transcriptomes sampled from nearly all known species. These genomic data, analyzed with coalescent and network-based approaches, reveal numerous instances of rapid speciation where individual gene trees conflict with the species tree. Moreover, the evolutionary history of rattlesnakes is dominated by incomplete speciation and frequent hybridization, both of which have likely influenced past interpretations of phylogeny. We present a new framework in which the evolutionary relationships of this group can only be understood in light of genome-wide data and network-based analytical methods. Our data suggest that network radiations, like seen within the rattlesnakes, can only be understood in a phylogenomic context, necessitating similar approaches in our attempts to understand evolutionary history in other rapidly radiating species.

Safe administration of Crotalidae equine immune F(ab')2 antivenom in a patient who suffered anaphylaxis from Crotalidae polyvalent immune Fab antivenom.

Allergic reactions to Crotalidae polyvalent immune Fab and Crotalidae immune F(ab')2 are uncommon but potentially life-threatening. It is unknown whether cross-reactivity reactions exist between these two antivenoms. We report a case of a patient who suffered anaphylaxis from Crotalidae polyvalent immune Fab but subsequently was safely administered Crotalidae immune F(ab')2 after a presumed Agkistrodon contortix (copperhead) envenomation. This single case supports the safety of Crotalidae immune F(ab')2 administration in patients with a history of anaphylaxis to Crotalidae polyvalent immune Fab treatment.

Análise da variação morfológica em Bothrops moojeni e espécies associadas

The genus Bothrops comprises a group of venomous snakes widely distributed throughout the Neotropics and presents a complex pattern of morphological variation. Among Bothrops species, the atrox group is composed by: B. atrox Linnaeu, 1758; B. leucurus Wagler, 1824; B. moojeni Hoge, 1966; B. marajoensis Hoge, 1966; B. asper Garman, 1883; B. isabelae Sandner Montilla, 1979; B. colombienses Hallowell; B. caribbaeus Garman, 1887 and B. lanceolatus Bonnaterre, 1790. The difficulty of morphological characterization of the species of this group has stimulated several discussions among researchers about the taxonomic validity of some representatives and their real geographic limits. This paper brings a taxonomic revision of the species of B. moojeni through the use of morphological evidence. Populations of B. atrox and B. leucurus historically associated with B. moojeni were also analyzed. Morphometric, meristic characters and drawing and coloration patterns of 362 specimens were analyzed, and statistical analyses were performed among the species. The study was conducted at the Laboratory of Zoological Collections of the Instituto Butantan. The described characteristics of the studied species showed similarities, demonstrating the recurring difficulty in identifying them. Furthermore, overlapping populations were recorded in the Cerrado region between B. atrox and B. moojeni, and the latter with B. leucurus in areas near the Atlantic Forest. A Principal Component Analysis (PCA) was used to show sexual dimorphism among species, and Boxplot, Plot and Histogram charts were used to observe the analyses. The morphological and meristic analyses of B. moojeni, B. atrox and B. leucurus in this study showed significant dimorphism in twelve of the 32 variables analyzed. We observed significant differences in B. atrox in the amount of ventral and sub-caudal scales, as well as in head width. The combination of these results demonstrates that the diversity of the atrox group is complex and its characters overlap across the three species, making morphological relationships among them similar. However, to better understand the differentiation between the species analyzed further research through phylogenetic and molecular analyses and to analyze more specimens will be necessary.

O gênero Bothrops compreende um grupo de serpentes peçonhentas amplamente distribuídas pela região neotropical e apresenta um complexo padrão de variação morfológica. Dentre as espécies de Bothrops, o grupo atrox é composto por: B. atrox Linnaeu, 1758; B. leucurus Wagler, 1824; B. moojeni Hoge, 1966; B. marajoensis Hoge, 1966; B. asper Garman, 1883; B. isabelae Sandner Montilla, 1979; B. colombienses Hallowell; B. caribbaeus Garman, 1887 e B. lanceolatus Bonnaterre, 1790. A dificuldade de caracterização morfológica das espécies deste grupo impulsiona várias discussões entre pesquisadores sobre a validade taxonômica de alguns representantes e seus reais limites geográficos. Este trabalho traz uma revisão taxonômica da espécie de B. moojeni através do uso de evidências morfológicas. Também foram analisadas populações de B. atrox e B. leucurus historicamente associadas a B. moojeni. Foram analisados caracteres morfométricos, merísticos e padrão de desenho e coloração de 362 espécimes, efetuando as análises estatísticas entre as espécies. O estudo foi conduzido no Laboratório de Coleções Zoológicas do Instituto Butantan. As características descritas das espécies estudadas apresentaram semelhanças, demonstrando a dificuldade recorrente na identificação das mesmas. Além disso, foram registradas sobreposições de populações na região de Cerrado entre B. atrox e B. moojeni e esta última com B. leucurus em áreas próximas da Mata Atlântica. Uma Análise de Componentes Principais (PCA) foi utilizada para evidenciar o dimorfismo sexual dentre as espécies, além de serem utilizados gráficos de Boxplot, Plotagem e Histogramas para a observação das análises. As análises morfológicas e merísticas de B. moojeni, B. atrox e B. leucurus deste estudo apresentaram dimorfismos significativos em doze das 32 variáveis analisadas. Observamos diferenças significativas em B. atrox na quantidade de escamas ventrais e subcaudais, assim como na largura da cabeça. A combinação destes resultados demonstra que a diversidade do grupo atrox é complexa e seus caracteres se sobrepõem às três espécies, tornando relações morfológicas entre elas semelhantes. No entanto, para compreender melhor a diferenciação entre as espécies analisadas serão necessárias novas pesquisas através de análises filogenéticas e moleculares e analisar mais espécimes.

Análise de ninhadas de Bothrops atrox (Serpente: Viperidae): razão sexual, tamanho de ninhada e investimento materno

To obtain reproductive success, snakes from the Viperidae family have developed several reproductive strategies and behaviors, such as: fecundity, reproductive period and mode, size and number of offspring in a litter, average adult body size, age and size at sexual maturity, reproductive frequency and sperm storage. An important feature within the reproductive strategies of snakes is the body size, which establishes several significant reproductive parameters. The common lancehead (Bothrops atrox) is a snake with wide distribution, mainly in northern Brazil and of great medical importance, due to the number of snakebites in the Amazon region. Since the reproductive biology of snakes varies, the objective of this work was to analyze litter size, offspring sex ratio and maternal investment in litters of B. atrox females, considering the morphometric data of mothers and offspring. In total, 31 litters of 26 females from the Herpetology Laboratory at Instituto Butantan, were evaluated. However, as a criterion for statistical analysis, only litters in which the number of offspring was greater than the number of atresic eggs, in the period from 2010 to 2021, were considered. Data were analyzed using BioEstat 5.3 software. D'Agostino normality test, t test, Pearson's Linear Correlation and Chi-square test were performed using independent data. The number of offspring born in each litter ranged from 5 to 31. There was no significant difference between the birth ratio of males and females in the litters. It was found that the morphometric measures of the mother do not interfere with the measures of the offspring, since they presented a pattern. There was a positive correlation between the morphometric measurements of females and the number of snakes in the litter, respectively, (p)0.0019 for CRC and (p)< 0.0001 for mass. The percentage of maternal investment was 20.1%, considering the prepartum and postpartum mass of the female, while the standard deviation (sd) was 10.07%. Finally, the correlation between the percentage of maternal investment and the mothers’ length did not suggest a correlation between the variables, (p0.0993), thus explaining that, regardless of the size of the mothers, they proportionally invest the same in their litters, and that larger females have agreater number of off spring.

Para obtenção do sucesso reprodutivo, as serpentes da família Viperidae desenvolveram diversas estratégias e comportamentos reprodutivos, como: fecundidade, período e modo reprodutivo, tamanho e número de filhotes em uma ninhada, tamanho médio do corpo adulto, idade e tamanho na maturidade sexual, frequência reprodutiva e estocagem de espermatozoides. Uma característica importante dentro das estratégias reprodutivas de serpentes é o tamanho do corpo, que estabelece diversos parâmetros reprodutivos significativos. A Jararaca-da- Amazonia (Bothrops atrox) é uma serpente de ampla distribuição, principalmente no norte do Brasil e de grande importância médica, devido ao número de acidentes ofídicos na região Amazônica. Visto que a biologia reprodutiva de serpentes varia, o objetivo deste trabalho foi analisar o tamanho da ninhada, razão sexual dos filhotes e o investimento materno em ninhadas de fêmeas de B. atrox, considerando os dados morfométricos das mães e dos filhotes. Ao total, foram avaliadas 31 fichas de ninhadas, de 26 fêmeas, contudo, como critério para as análises estatísticas, foram consideradas apenas ninhadas nas quais o número de filhotes foi superior ao número de ovos atrésicos, provenientes do Laboratório de Herpetologia do Instituto Butantan, do ano de 2010 a 2021. Os dados foram analisados através do software BioEstat 5.3. Foram realizados teste de normalidade D’Agostino, teste t, Correlação Linear de Pearson e Qui-quadrado, mediante o uso de dados independentes. A quantidade de filhotes nascidos na ninhada variou de 5 a 31. Não houve diferença significativa entre a razão de nascimento de machos e fêmeas nas ninhadas. Constatou-se que as medidas morfométricas da mãe, não interferem nas medidas da prole, uma vez que estes apresentaram um padrão. Há correlação positiva entre as medidas morfométricas das fêmeas e a quantidade de filhotes na ninhada, sendo respectivamente, (p)=0.0019 para o CRC e (p)=< 0.0001 para a massa. A porcentagem de investimento materno, foi de 20,1% na massa das mães, relacionando a massa pré-parto e pós-parto, e o desvio padrão (DP) foi de 10,07%. Por fim, a correlação entre a porcentagem do investimento materno e o comprimento das mães, não sugeriu correlação entres as variáveis (p=0.0993), explicando assim, que independentemente do tamanho das mães, elas investem proporcionalmente o mesmo em suas ninhadas, e que fêmeas maiores possuem um maior número de filhotes.

Do pit vipers assess their venom? Defensive tactics of Deinagkistrodon acutus shift with changed venom reserve.

To maximize survival probability, animals must assess predation risks and adopt flexible defensive strategies based on specific conditions. Pit vipers utilize venom for predation and self-defense, and venom status significantly influences its effectiveness. Thus, pit vipers may evaluate their venom reserve and adopt corresponding defensive tactics. Twenty-three sharp-snouted pit vipers (Deinagkistrodon acutus) were grouped by different venom status and were subjected to eight behavior trials. Subjects' defensive behaviors were recorded and analyzed. Results showed that the normal venom group displayed stable responses across the trials. The low venom group showed fewer strikes and more fleeing behaviors at the end of experiments. After given prolonged intervals for replenishing the venom, significant increases of strike behaviors were observed in the replenishing venom group. These results demonstrated the capability of adopting flexible defensive tactics based on varied venom reserve and provided new evidence for venom-status-recognition.

Failure of a Mexican antivenom on recovery from snakebite-related coagulopathy in French Guiana.

INTRODUCTION: In French Guiana, most snakebites are caused by crotalids, with the main signs being tissue damage and bleeding due to venom-induced coagulopathy. Since December 2014 the Western Guiana Hospital (WGH) has used Antivipmyn Tri TM, a Mexican polyvalent antivenom. The aim of the study was to assess its benefit on the correction of snakebite-related coagulopathy. METHODS: This retrospective study included patients hospitalized at the WGH with snakebite and a coagulopathy defined by: a prothrombin rate (PR) lower than 45%, an activated partial thromboplastin time ratio (aPTTr) greater than 2 or a fibrinogen lower than 100 mg.dL-1. The antivenom group included patients receiving Antivipmyn Tri TM from December 2014 to September 2017. The control group included patients admitted between January 2013 and November 2014 (when antivenom was unavailable) or admitted between December 2014 and September 2017 during times of antivenom shortage. We graphically compared the time courses of PR, aPTTr and fibrinogen between groups. Other endpoints were the length of hospital stay and the need for surgery or dialysis. RESULTS: 84 patients were included: 42 in the antivenom group, 42 in the control group. Both groups were similar for age, sex-ratio, proportion of bleedings, necrosis, and severity. Most patients in the antivenom group received 3 vials. There were no significant differences in recovery of PR, aPTTr and fibrinogen through the first 24 h. Fibrinogen declined again in the control group at 30 h and showed a slower rise to normal concentration. There were no significant differences in any secondary endpoint. CONCLUSION: Antivipmyn Tri TM as currently used did not show any benefit in recovery from coagulopathy.

Comparison of F(ab')2 and Fab antivenoms in rattlesnake envenomation: First year's post-marketing experience with F(ab')2 in New Mexico.

Two antivenoms are available for rattlesnake envenomations in the U.S., Fab (CroFab®, BTG, UK), and F(ab')2 (Anavip®, Bioclon, Mexico) antivenom (AV) with F(ab')2AV released in October 2018. The F(ab')2AV Phase 3 comparative clinical trial demonstrated similar efficacy in treating venom-caused hematologic toxicity, similar rates of Types I and III hypersensitivity reactions, and a lower rate of recurrent hematological effects than FabAV. We hypothesized that a post-marketing, comparative study of effectiveness and rates of hypersensitivity reactions in treating rattlesnake envenomations in New Mexico would demonstrate similar outcomes. Patients eligible for the study presented to a New Mexico healthcare facility between May and October 2019 and were known/suspected to have a rattlesnake bite. Exclusion criteria for antivenom comparison were those with a dry bite, lost to follow-up, or late presentation. All cases were included for patient/bite demographics, initial local control, hematological control, number of maintenance/control doses, development of persistent, recurrent or late-, new-onset hematologic effects, and hypersensitivity reactions. We used Fisher's exact tests for analysis and 0.05 cutoff to determine significance. There were 54 rattlesnake-bitten patients in New Mexico with 17 excluded for comparison of antivenom because of dry bites, loss to follow-up, and one case of late presentation. Thirty-seven patients remained for comparative analysis between F(ab')2AV (n = 11) and FabAV (n = 26). There were no significant demographic differences between F(ab')2 and Fab-treated patients. No patient had a Type I hypersensitivity reaction. No rescue doses were given. The rate of recurrent, persistent or late-, new-onset of hematologic effects was 0% with F(ab')2AV and 29% with FabAV. No patient was readmitted. No patient had bleeding complications. Type III hypersensitivity reactions were similar between F(ab')2AV (36%) and FabAV (25%). The results of our study are consistent with the Phase 3 clinical comparative trial and indicate no significant differences in safety or effectiveness between FabAV and F(ab')2AV. F(ab')2AV offers the advantages of not requiring maintenance doses and may have a lower rate of late hematologic effects in treating rattlesnake envenomations.

Evolutionary Interpretations of Nicotinic Acetylcholine Receptor Targeting Venom Effects by a Clade of Asian Viperidae Snakes.

Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

Rain forest shifts through time and riverine barriers shaped the diversification of South American terrestrial pit vipers (Bothrops jararacussu species group)

Aim To investigate (a) historical biogeographical connections and species interchange among rain forest habitats and (b) the role of riverine barriers on population divergence and speciation in the Neotropical region. Location Amazonia and Atlantic Forest in South America. Taxon Bothrops jararacussu species group (Serpentes: Viperidae). Methods We inferred phylogenetic relationships within Bothrops with an emphasis on the jararacussu species group under a Bayesian framework based on six molecular loci. We also used genetic coalescent simulations and approximate Bayesian computation to infer historical demography within the jararacussu group based on tests of alternative scenarios. Results We found the jararacussu species group to be monophyletic. The Atlantic Forest species B. pirajai and B. muriciensis were inferred nested within this group, closely related to B. jararacussu, confirming that Atlantic Forest species form a clade. The historical demographic analyses support vicariant separation between populations of B. brazili north and south of the Amazon River during the Miocene–Pliocene border, as well as colonization of the Atlantic Forest by a northern Amazonian ancestor in the Pleistocene. Main Conclusion The evolutionary history of the jararacussu species group sheds light on the dynamism of Neotropical rain forests over time, with at least one event of forest expansion leading to faunal interchange between Amazonian and Atlantic forests in the Pleistocene. Moreover, tests of alternative demographic scenarios suggest that the populations of B. brazili from north and south of the Amazon River originated from a vicariant event during the Miocene–Pliocene border, in agreement with the proposed age of establishment of the modern Amazon River drainage. Our results also have taxonomic implications for these medically important venomous snakes, supporting unrecognized diversity at the species level.

Do New World pitvipers "scale-down" at high elevations? Macroecological patterns of scale characters and body size.

Bergmann's rule describes the macroecological pattern of increasing body size in response to higher latitudes and elevations. This pattern is extensively documented in endothermic vertebrates, within and among species; however, studies involving ectotherms are less common and suggest no consistent pattern for amphibians and reptiles. Moreover, adaptive traits, such as epidermal features like scales, have not been widely examined in conjunction with Bergmann's rule, even though these traits affect physiological processes, such as thermoregulation, which are hypothesized as underlying mechanisms for the pattern. Here, we investigate how scale characters correlate with elevation among 122 New World pitviper species, representing 15 genera. We found a contra-Bergmann's pattern, where body size is smaller at higher elevations. This pattern was mainly driven by the presence of small-bodied clades at high elevations and large-bodied clades at low elevations, emphasizing the importance of taxonomic scope in studying macroecological patterns. Within a subset of speciose clades, we found that only Crotalus demonstrated a significant negative relationship between body size and elevation, perhaps because of its wide elevational range. In addition, we found a positive correlation between scale counts and body size but no independent effect of elevation on scale numbers. Our study increases our knowledge of Bergmann's rule in reptiles by specifically examining characters of squamation and suggests a need to reexamine macroecological patterns for this group.

Multilocus phylogeography of the brown-spotted pitviper Protobothrops mucrosquamatus (Reptilia: Serpentes: Viperidae) sheds a new light on the diversification pattern in Asia.

Understanding the influence of geographical events and climate changes on genetic diversity is essential in explaining current patterns of genetic structure and geographic distribution of organisms. We inferred phylogenetic relationships, investigated historical demography, explored the evolutionary history, and clarified intraspecific taxonomy of Protobothrops mucrosquamatus, which is one of the commonest and most wide-ranging Asian pitvipers. A total of 184 samples from 54 localities were sequenced and analyzed for two mitochondrial gene fragments and two nuclear genes. Phylogenetic reconstruction based on mtDNA sequences revealed the existence of a minimum of five geographically structured and well-supported lineages within P. mucrosquamatus. Based on the mtDNA gene tree, and the geographic relationship between populations allied by matrilineal lineages, a complex longitudinal and latitudinal diversification pattern was uncovered in P. mucrosquamatus. The estimated date of the origin of the species (about 5.3 Ma) and divergence of the intraspecific lineages match the rapid uplifting of Qinghai-Xizang Plateau, and is also consistent with those of some other co-distributed Asian pitvipers. Formation of the two island lineages (Taiwan and Hainan) was generally congruent with the first isolation of the islands, but the two lineages showed different relationships with the continental Asian populations in comparison with some other pitvipers. Population historical demographic analyses, based on three methods, showed that all lineages have experienced slight population expansion in and around the Dali Glacial. Tests of intraspecific taxonomy indicated that no cryptic taxon is present within this widely distributed snake.

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake, the Eastern Massasauga (Sistrurus catenatus).

Genetic diversity is fundamental to maintaining the long-term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.

Complex longitudinal diversification across South China and Vietnam in Stejneger's pit viper, Viridovipera stejnegeri (Schmidt, 1925) (Reptilia: Serpentes: Viperidae).

Viridovipera stejnegeri is one of the most common pit vipers in Asia, with a wide distribution in southern China and Vietnam. We investigated historical demography and explored how the environment and climatic factors have shaped genetic diversity and the evolutionary history of this venomous snake. A total of 171 samples from 47 localities were sequenced and analysed for two mitochondrial gene fragments and three nuclear genes. Gene trees reveal the existence of two well-supported clades (Southwest China and Southeast China) with seven distinct and strongly supported, geographically structured subclades within V. stejnegeri. Estimation of divergence time and ancestral area suggests that V. stejnegeri originated at ~6.0 Ma in the late Miocene on the Yunnan-Guizhou Plateau. The estimated date of origin and divergence of the island populations of Taiwan and Hainan closely matches the geological origin of the both islands. The mtDNA gene tree reveals the presence of west-east diversification in V. stejnegeri populations. Complex orogenesis and heterogeneous habitats, as well as climate-mediated habitat differentiation including glacial cycles, all have influenced population structure and the distribution of this taxon. The validity of V. stejnegeri chenbihuii is questionable, and this subspecies most probably represents an invalid taxon.

Snakebite! Crotalinae Envenomation of a Man in Rhode Island.

The incidence of poisonous snakebites has regional variance. Health care providers' knowledge and comfort in treating these envenomated patients depends on the density of poisonous snakes in their environment, with practitioners in the southern U.S. typically treating more exposed patients than those in colder regions in the North. We present a rare case of a confirmed copperhead snakebite that occurred in Rhode Island. We will review Copperhead bites, clinical management and treatment options.

The importance of being genomic: Non-coding and coding sequences suggest different models of toxin multi-gene family evolution.

Studies of multi-gene protein families, including many toxins, are crucial for understanding the role of gene duplication in generating protein diversity in general. However, many evolutionary analyses of gene families are based on coding sequences, and do not take into account many potentially confounding evolutionary factors, such as recombination and convergence due to selection. We illustrate this using snake venom gene sequences from the Phospholipase A2 (PLA2) subfamily. Novel gene sequences from 20 species of understudied Asian pitvipers were analyzed alongside available genomic PLA2 sequences from another four crotaline and several viperine species. In contrast to previous analyses of this toxin family based on cDNA sequences, we find that duplication events are concentrated at the tips of the tree, suggesting that major functions such as presynaptic neurotoxicity have evolved convergently multiple times in pitvipers. We provide evidence that this discrepancy is due to differing evolutionary patterns between introns and exons. The effects of several well-known sources of bias on the phylogeny were small, compared to the effect of analyses based on different partitions of the gene (whole gene sequence, non-coding regions, cDNA sequence). Switches of function were found to be largely associated with strong selection, and with duplication events. Use of coding sequences for phylogeny estimation potentially produces incorrect inferences about the action of selection on individual lineages and sites. Our results have major implications for phylogenomic methods of functional inference as well as for our understanding of the evolution of multigene families.

Rebound coagulopathy in patients with snakebite presenting with marked initial coagulopathy.

OBJECTIVE: An estimated 70% of patients with pit viper snakebites require antivenom to treat serious complications such as coagulopathy. Evidence-based guidance is limited for the appropriate administration of Crotalinae Polyvalent Immune Fab (FabAV) and the duration of laboratory follow-up. The objective of our study was to assess the incidence of marked and recurrent envenomation coagulopathy at our trauma center and identify practice patterns that may prevent serious complications. METHODS: A retrospective case review was conducted over a 3-year period on patients treated for symptomatic snakebite injury. Case records were reviewed for the inclusion criteria of international normalized ratio (INR) greater than 2.0. The exclusion criterion was limited to patients receiving anticoagulant therapy. RESULTS: In all, 61 patients were identified on retrospective chart review and 3 patients (4.9%) met inclusion criteria. Two of the 3 patients had marked rebound coagulopathy requiring readmission and additional treatment. In our small series, 2 patients presenting after crotaline envenomation with increased INR (>6.0), decreased fibrinogen (<60 mg/dL), and decreased platelet count (<100,000/mL) had recurrent coagulopathy and were asymptomatic, and recurrence was noted only with follow-up laboratory testing. All patients responded positively within a matter of hours to repeat FabAV administration, with resolution of rebound coagulopathy. CONCLUSIONS: We recommend periodic monitoring of patients with increased INR, decreased fibrinogen, and decreased platelet count. Patients should be monitored for 10 to 14 days after envenomation to identify asymptomatic rebound coagulopathy. Prompt readministration of FabAV appears to correct the coagulopathy.