Proteome Decomplexation of Trimeresurus erythrurus Venom from Mizoram, India.

Green pit vipers are the largest group of venomous vipers in tropical and subtropical Asia, which are responsible for most of the bite cases across this region. Among the green pit vipers of the Indian subcontinent, Trimeresurus erythrurus is the most prevalent; however, limited knowledge is available about its venomics. Proteome decomplexation of T. erythrurus venom using mass spectrometry revealed a blend of 53 different proteins/peptides belonging to 10 snake venom protein families. Phospholipase A2 and snake venom serine proteases were found to be the major enzymatic families, and Snaclec was the major nonenzymatic family in this venom. These protein families might be responsible for consumptive coagulopathy in victims. Along with these, snake venom metalloproteases, l-amino acid oxidases, disintegrins, and cysteine-rich secretory proteins were also found, which might be responsible for inducing painful edema, tissue necrosis, blistering, and defibrination in patients. Protein belonging to C-type lectins, C-type natriuretic peptides, and glutaminyl-peptide cyclotransfreases were also observed as trace proteins. The crude venom shows platelet aggregation in the absence of any agonist, suggesting their role in alterations in platelet functions. This study is the first proteomic analysis of T. erythrurus venom, contributing an overview of different snake venom proteins/peptides responsible for various pathophysiological disorders obtained in patients. Data are available via ProteomeXchange with the identifier PXD038311.

Reproduction technologies for the sustainable management of Caudata (salamander) and Gymnophiona (caecilian) biodiversity.

We review the use of reproduction technologies (RTs) to support the sustainable management of threatened Caudata (salamanders) and Gymnophiona (caecilian) biodiversity in conservation breeding programs (CBPs) or through biobanking alone. The Caudata include ∼760 species with ∼55% threatened, the Gymnophiona include ∼215 species with an undetermined but substantial number threatened, with 80% of Caudata and 65% of Gymnophiona habitat unprotected. Reproduction technologies include: (1) the exogenous hormonal induction of spermatozoa, eggs, or mating, (2) in vitro fertilisation, (3) intracytoplasmic sperm injection (ICSI), (4) the refrigerated storage of spermatozoa, (5) the cryopreservation of sperm, cell or tissues, (6) cloning, and (7) gonadal tissue or cell transplantation into living amphibians to eventually produce gametes and then individuals. Exogenous hormone regimens have been applied to 11 Caudata species to stimulate mating and to 14 species to enable the collection of spermatozoa or eggs. In vitro fertilisation has been successful in eight species, spermatozoa have been cryopreserved in seven species, and in two species in vitro fertilisation with cryopreserved spermatozoa has resulted in mature reproductive adults. However, the application of RTs to Caudata needs research and development over a broader range of species. Reproduction technologies are only now being developed for Gymnophiona, with many discoveries and pioneering achievement to be made. Species with the potential for repopulation are the focus of the few currently available amphibian CBPs. As Caudata and Gymnophiona eggs or larvae cannot be cryopreserved, and the capacity of CBPs is limited, the perpetuation of the biodiversity of an increasing number of species depends on the development of RTs to recover female individuals from cryopreserved and biobanked cells or tissues.

Bacterial Community in the Skin Microbiome of Frogs in a Coldspot of Chytridiomycosis Infection.

Chytridiomycosis is a fungal disease caused by the pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), which has caused declines in amphibian populations worldwide. Asia is considered as a coldspot of infection, since adult frogs are less susceptible to Bd-induced mortality or morbidity. Using the next-generation sequencing approach, we assessed the cutaneous bacterial community composition and presence of anti-Bd bacteria in six frog species from India using DNA isolated from skin swabs. All the six frog species sampled were tested using nested PCR and found Bd negative. We found a total of 551 OTUs on frog skin, of which the bacterial phyla such as Proteobacteria (56.15% average relative abundance) was dominated followed by Actinobacteria (21.98% average relative abundance) and Firmicutes (13.7% average relative abundance). The contribution of Proteobacteria in the anti-Bd community was highest and represented by 175 OTUs. Overall, the anti-Bd bacterial community dominated (51.7% anti-Bd OTUs) the skin microbiome of the frogs. The study highlights the putative role of frog skin microbiome in affording resistance to Bd infections in coldspots of infection.

The devil is in the detail: estimating species richness, density, and relative abundance of tropical island herpetofauna.

BACKGROUND: One of the basic premises of drawing samples from populations is that the samples are representative of the populations. However, error in sampling is poorly recognized, and it goes unnoticed especially in community ecology. By combining traditional open quadrats used for sampling forest floor herpetofauna with intensive bounded quadrats, we explore the effect of sampling error on estimates of species richness, diversity, and density in the Andaman Islands. RESULTS: Fisher's α measure of species diversity and second order jackknife estimate of species richness were not sensitive to number of individuals sampled. Sampling error resulted in underestimation of density in both frogs and lizards. It influenced relative abundance of individual species resulting in underestimation of abundance of small or camouflaged species; and also resulted in low precision in lizard species richness estimates. CONCLUSIONS: Sampling error resulted in underestimation of abundance of small, fossorial or camouflaged species. Imperfect detection from less intensive sampling method results incorrect estimates of abundance of herpetofauna. Fisher's α for species diversity and second order jackknife for species richness were robust measures. These have strong implications on inferences made from previous studies as well as sampling strategies for future studies. It is essential that these shortfalls are accounted for while communities are sampled or when datasets are compared.

Proteome analysis of Daboia russelii venom, a medically important snake from the Indian sub-continent.

Daboia russelii is a category-I medically important snake throughout the Indian sub-continent contributing to majority of snakebite incidences in this part of the world. As such, extensive studies on its venom composition and search of efficient and appropriate interventions for its treatment become crucial. In this study, the proteome of Daboia russelii venom from Tanore, Rajshahi, Bangladesh was profiled using a combination of chromatographic and mass spectrometric techniques. A total of 37 different proteins belonging to 11 different snake venom protein families were detected. Proteomics analysis revealed the presence of major phospholipase A2 toxins. Daboiatoxin (both A and B subunits), the main lethal PLA2 toxin in the venom of Daboia siamensis (Myanmar viper) which is neurotoxic, myotoxic and cytotoxic was detected. Presence of Daboxin P, which is a major protein in the venom of Indian Daboia russelii with strong anticoagulant activity, was also observed. Inconsistent distribution of such lethal toxins in the venom of same species calls for more investigations of snake venoms from lesser explored regions and formulation of better alternatives to the current antivenom therapy for efficient treatment.

Nuclear and Mitochondrial Genome Assemblies of the Beetle, Zygogramma bicolorata, a Globally Important Biocontrol Agent of Invasive Weed Parthenium hysterophorus.

Implementing a genetic-based approach to achieve the full potential of classical biocontrol programs has been advocated for decades. The availability of genome-level information brings the opportunity to scrutinize biocontrol traits for their efficacy and evolvability. However, implementation of this advocacy remains limited to few instances. Biocontrol of a globally noxious weed, Parthenium hysterophorus, by the leaf-feeding beetle, Zygogramma bicolorata, has been in place for more than four decades now, with varying levels of success. As the first step in providing genetic-based improvement to this biocontrol program, we describe the nuclear and mitochondrial assemblies of Z. bicolorata. We assembled the genome from the long-read sequence data, error corrected with high-throughput short reads and checked for contaminants and sequence duplication to produce a 936 Mb nuclear genome. With 96.5% Benchmarking Universal Single-Copy Orthologs completeness and the long terminal repeat assembly index 12.91, we present a reference-quality assembly that appeared to be repeat rich at 62.7% genome-wide and consists of 29,437 protein-coding regions. We detected signature of nuclear insertion of mitochondrial fragments in 80 nuclear positions comprising 13 kb out of 17.9 kb mitochondria genome sequence. This genome, along with its annotations, provides a valuable resource to gain further insights into the biocontrol traits of Z. bicolorata for improving the control of the invasive weed P. hysterophorus.

Evaluating Antivenom Efficacy against Echis carinatus Venoms-Screening for In Vitro Alternatives.

In India, polyvalent antivenom is the mainstay treatment for snakebite envenoming. Due to batch-to-batch variation in antivenom production, manufacturers have to estimate its efficacy at each stage of IgG purification using the median effective dose which involves 100-120 mice for each batch. There is an urgent need to replace the excessive use of animals in snake antivenom production using in vitro alternatives. We tested the efficacy of a single batch of polyvalent antivenom from VINS bioproducts limited on Echis carinatus venom collected from three different locations-Tamil Nadu (ECVTN), Goa (ECVGO) and Rajasthan (ECVRAJ)-using different in vitro assays. Firstly, size-exclusion chromatography (SEC-HPLC) was used to quantify antivenom-venom complexes to assess the binding efficiency of the antivenom. Secondly, clotting, proteolytic and PLA2 activity assays were performed to quantify the ability of the antivenom to neutralize venom effects. The use of both binding and functional assays allowed us to measure the efficacy of the antivenom, as they represent multiple impacts of snake envenomation. The response from the assays was recorded for different antivenom-venom ratios and the dose-response curves were plotted. Based on the parameters that explained the curves, the efficacy scores (ES) of antivenom were computed. The binding assay revealed that ECVTN had more antivenom-venom complexes formed compared to the other venoms. The capacity of antivenom to neutralize proteolytic and PLA2 effects was lowest against ECVRAJ. The mean efficacy score of antivenom against ECVTN was the greatest, which was expected, as ECVTN is mainly used by antivenom manufacturers. These findings pave a way for the development of in vitro alternatives in antivenom efficacy assessment.

Chytridiomycosis in Asian Amphibians, a Global Resource for Batrachochytrium dendrobatidis (Bd) Research.

Chytridiomycosis is an emerging infectious disease affecting amphibians globally and it is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Chytridiomycosis has caused dramatic declines and even extinctions in wild amphibian populations in Europe, Australia, Central and North America. Spanning over two and a half decades, extensive research has led to discovery of epizootic and enzootic lineages of this pathogen. However, the Bd-amphibian system had garnered less attention in Asia until recently when an ancestral Bd lineage was identified in the Korean peninsula. Amphibians co-exist with the pathogen in Asia, only sub-lethal effects have been documented on hosts. Such regions are 'coldspots' of infection and are an important resource to understand the dynamics between the enzootic pathogen-Bd and its obligate host-amphibians. Insights into the biology of infection have provided new knowledge on the multi-faceted interaction of Bd in a hyperdiverse Asian amphibian community. We present the findings and highlight the knowledge gap that exists, and propose the ways to bridge them. We emphasize that chytridiomycosis in Asia is an important wildlife disease and it needs focussed research, as it is a dynamic front of pathogen diversity and virulence.

Immunorecognition capacity of Indian polyvalent antivenom against venom toxins from two populations of Echis carinatus.

Clinicians report low efficacy of Indian polyvalent antivenom (PAV), with >20 vials required for treatment of a snakebite envenoming. We hypothesize that the antivenom efficacy could be reduced due to insufficient antibodies against some venom toxins. To test this, we used third-generation antivenomics to reveal bound and unbound venom toxins of Echis carinatus venom from Goa (ECVGO) and Tamil Nadu (ECVTN). We used 60, 120, 180, 240, 300, and 360 µg of venom and passed through mini-columns containing ~5 mg Antivenom bound to CNBr beads. The non-retained (unbound) and retained (bound) toxins were identified using reverse-phase HPLC and tandem mass spectrometry. Low molecular weight toxins - Short disintegrins (5.3 kDa) and DIS domain of P-II SVMP from ECVGO and ECVTN showed poor binding with antivenom. The immunorecognition sites of antivenom saturated at the lower antivenom-venom ratio for ECVGO than for ECVTN. The immunoretained capacity of antivenom against ECVTN was 140.6 µg and ECVGO was 125.1 µg. The amount of immunoretained toxins quantified can further be used to estimate the efficacy of antivenom by correlating it with in-vivo studies. The unbound toxins identified from this study could be targeted to improve the effectiveness of antivenom.

A practical method for storage, preservation and transportation of anuran urine samples using filter paper for hormone analysis.

Anurans (frogs and toads) expelled urine when handled and it could provide insights into their physiological status. However, storage, preservation and transportation are often challenging. The study aimed to standardize and validate a field method for short-term storage and preserve of anuran urine samples using Whatman filter papers. To examine the efficacy of storage conditions and type of papers, urinary based enzyme immunoassays were used to measure progesterone and testosterone hormone metabolites. High-Performance Liquid Chromatography was performed and revealed immunoreactive progesterone and testosterone metabolites in the urine samples. Urinary hormone metabolites concentration stored in filter paper at room temperature and control samples stored in -20°C for the same period were similar. Whatman grade 50 was found to be more suitable for storage of hormones than grade 3 paper for the experiments performed.•A cheap and simple storage method for storage of anuran urine in field conditions using filter papers.•Anuran urine could be preserved and transported under ambient conditions without significant changes and loss of hormones.•This method would facilitate the endocrine monitoring of anurans in remote areas where limited logistics are available.

Comparative proteomics of geographically distinct saw-scaled viper (Echis carinatus) venoms from India.

Snakebite is a socio-economic problem in tropical countries and it is exacerbated by geographical venom variation of snakes. We investigated on venom variation in geographically distinct populations of Echis carinatus from three ecologically distinct regions: Tamil Nadu (ECVTN), Goa (ECVGO), and Rajasthan (ECVRAJ). Venom was fractionated by RP-HPLC, combined with SDS-PAGE, and subjected to tandem mass spectrometry. Toxins were identified, and their relative abundance was estimated. Using NCBI database of Echis genus, we queried the MS/MS spectra, and found 69, 38 and 38 proteins in ECVTN, ECVGO and ECVRAJ respectively, belonging to 8-10 different toxin families. The differences in the venom profiles were due to change in the relative composition of the toxin families. Snake venom metalloproteinase (svMP), Snaclecs and Phospholipase A2 (PLA2) were the major venom components in all the venoms. Heteromeric Disintegrins were found in ECVTN and absent in other venoms. ECVRAJ showed higher abundance of low-molecular-weight (>30 kDa) proteins than ECVTN and ECVGO. Cysteine-rich venom protein (CRISP) was highest in ECVRAJ (7.34%), followed by ECVTN (0.01%) and in ECVGO, it was not detected. These findings highlight the need for evaluating the efficacy of the polyvalent anti-venom to neutralize the toxins from geographically distinct venoms of E. carinatus.

Niche dissociated assembly drives insular lizard community organization.

Interspecific competition for resources leading to niche partitioning is considered as one of the major drivers of community assembly. Competitive niche partitioning is diagnosed from species co-occurrence, species abundance distributions (SADs), and body size distributions of species. For several decades, studies have explored these patterns for the relative significance of interspecific competition in shaping communities. We explored these patterns in a finite assemblage of insectivorous lizards in the Andaman & Nicobar Islands, both at the level of archipelago and individual islands. Negative geographic co-occurrences occurred only between species pairs in islands separated by deep ocean channels. Ecologically similar species did not show positive co-occurrence in guild co-occurrence analyses, indicating that the negative geographical co-occurrences between species in islands were due to historical allopatry. Species abundance distribution was best explained by a Pareto distribution in both metacommunity and local communities. There was no predictable spacing of body sizes among co-existing species in local communities. The empirical data on insular lizard community on species co-occurrence, SADs, and body size ratios does not lend support to assortment of species in islands caused by niche subdivision. Such niche-dissociated assembly of species in islands might be an important factor in formation of biological communities, regardless of geographic scale.

Enzootic frog pathogen Batrachochytrium dendrobatidis in Asian tropics reveals high ITS haplotype diversity and low prevalence.

Emerging Infectious Diseases (EIDs) are a major threat to wildlife and a key player in the declining amphibian populations worldwide. One such EID is chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd), a fungal pathogen. Aetiology of Bd infection is poorly known from tropical frogs in Asian biodiversity hotspots. Surveys were carried out in four biodiversity hotspots to ascertain the status of Bd fungus. We collected a total of 1870 swab samples from frogs representing 32 genera and 111 species. Nested PCRs revealed low prevalence (8.4%) and high Bd haplotype richness was revealed after sequencing. We document 57 Bd Internal Transcribed Spacer region (ITS) haplotypes, of which 46 were unique to the global database. Bd ITS region showed indels at the Taqman binding site and qPCR reverse primer binding site, suggesting qPCR is unsuitable for diagnosis in Asian Bd coldspots. Our median-joining network and Bayesian tree analyses reveal that the Asian haplotypes, with the exception of Korea, formed a separate clade along with pandemic BdGPL (Bd Global Panzootic Lineage) haplotype. We hypothesise that the frog populations in Asian tropics might harbour several endemic strains of Bd, and the high levels of diversity and uniqueness of Bd haplotypes in the region, probably resulted from historical host-pathogen co-evolution.

Watch out where you sleep: nocturnal sleeping behaviour of Bay Island lizards.

Sleeping exposes lizards to predation. Therefore, sleeping strategies must be directed towards avoiding predation and might vary among syntopic species. We studied sleeping site characteristics of two syntopic, congeneric lizards-the Bay Island forest lizard, Coryphophylax subcristatus and the short-tailed Bay Island lizard, C. brevicaudus and evaluated inter-specific differences. We measured structural, microclimatic and potential predator avoidance at the sleeping perches of 386 C. subcristatus and 185 C. brevicaudus. Contrary to our expectation, we found similar perch use in both species. The lizards appeared to use narrow girth perch plants and accessed perches by moving both vertically and horizontally. Most lizards slept on leaves, with their heads directed towards the potential path of a predator approaching from the plant base. There was no inter-specific competition in the choices of sleeping perches. These choices indicate an anti-predator strategy involving both tactile and visual cues. This study provides insight into a rarely studied behaviour in reptiles and its adaptive significance.

A new genus and species of arboreal toad with phytotelmonous larvae, from the Andaman Islands, India (Lissamphibia, Anura, Bufonidae).

A new bufonid amphibian, belonging to a new monotypic genus, is described from the Andaman Islands, in the Bay of Bengal, Republic of India, based on unique external morphological and skeletal characters which are compared with those of known Oriental and other relevant bufonid genera. Blythophryne gen. n. is distinguished from other bufonid genera by its small adult size (mean SVL 24.02 mm), the presence of six presacral vertebrae, an absence of coccygeal expansions, presence of an elongated pair of parotoid glands, expanded discs at digit tips and phytotelmonous tadpoles that lack oral denticles. The taxonomic and phylogenetic position of the new taxon (that we named as Blythophryne beryet gen. et sp. n.) was ascertained by comparing its 12S and 16S partial genes with those of Oriental and other relevant bufonid lineages. Resulting molecular phylogeny supports the erection of a novel monotypic genus for this lineage from the Andaman Islands of India.

Molecular evolution of vertebrate neurotrophins: co-option of the highly conserved nerve growth factor gene into the advanced snake venom arsenalf.

Neurotrophins are a diverse class of structurally related proteins, essential for neuronal development, survival, plasticity and regeneration. They are characterized by major family members, such as the nerve growth factors (NGF), brain-derived neurotrophic factors (BDNF) and neurotrophin-3 (NT-3), which have been demonstrated here to lack coding sequence variations and follow the regime of negative selection, highlighting their extremely important conserved role in vertebrate homeostasis. However, in stark contrast, venom NGF secreted as part of the chemical arsenal of the venomous advanced snake family Elapidae (and to a lesser extent Viperidae) have characteristics consistent with the typical accelerated molecular evolution of venom components. This includes a rapid rate of diversification under the significant influence of positive-selection, with the majority of positively-selected sites found in the secreted ß-polypeptide chain (74%) and on the molecular surface of the protein (92%), while the core structural and functional residues remain highly constrained. Such focal mutagenesis generates active residues on the toxin molecular surface, which are capable of interacting with novel biological targets in prey to induce a myriad of pharmacological effects. We propose that caenophidian NGFs could participate in prey-envenoming by causing a massive release of chemical mediators from mast cells to mount inflammatory reactions and increase vascular permeability, thereby aiding the spread of other toxins and/or by acting as proapoptotic factors. Despite their presence in reptilian venom having been known for over 60 years, this is the first evidence that venom-secreted NGF follows the molecular evolutionary pattern of other venom components, and thus likely participates in prey-envenomation.

Literature based species occurrence data of birds of northeast India.

The northeast region of India is one of the world's most significant biodiversity hotspots. One of the richest bird areas in India, it is an important route for migratory birds and home to many endemic bird species. This paper describes a literature-based dataset of species occurrences of birds of northeast India. The occurrence records documented in the dataset are distributed across eleven states of India, viz.: Arunachal Pradesh, Assam, Bihar, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim, Tripura, Uttar Pradesh and West Bengal. The geospatial scope of the dataset represents 24 to 29 degree North latitude and 78 to 94 degree East longitude, and it comprises over 2400 occurrence records. These records have been collated from scholarly literature published between1915 and 2008, especially from the Journal of the Bombay Natural History Society (JBNHS). The temporal scale of the dataset represents bird observations recorded between 1909 and 2007. The dataset has been developed by employing MS Excel. The key elements in the database are scientific name, taxonomic classification, temporal and geospatial details including geo-coordinate precision, data collector, basis of record and primary source of the data record. The temporal and geospatial quality of more than 50% of the data records has been enhanced retrospectively. Where possible, data records are annotated with geospatial coordinate precision to the nearest minute. This dataset is being constantly updated with the addition of new data records, and quality enhancement of documented occurrences. The dataset can be used in species distribution and niche modeling studies. It is planned to expand the scope of the dataset to collate bird species occurrences across the Indian peninsula.