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Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2–6 toxin classes that account for 91–99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs) and phospholipases A2 (PLA2s) comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA2s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1–2.0%) are found in all venoms except that of M. s. spixii. Other toxin families are present in all six venoms at trace levels (<0.005%). Minor and trace venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6–9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen previously, appear to have arisen in three species by gene duplication and fusion. Four species have transcripts homologous to the nociceptive toxin, (MitTx) α-subunit, but all six species had homologs to the β-subunit. The first non-neurotoxic, non-catalytic elapid phospholipase A2s are reported. All are probably myonecrotic. Phylogenetic analysis indicates that the six taxa diverged 15–35 million years ago and that they split from their last common ancestor with Old World elapines nearly 55 million years ago. Given their early diversification, many cryptic micrurine taxa are anticipated.

The Common Krait (Bungarus caeruleus) shares a distribution range with many other ‘phenotypically-similar’ kraits across the Indian subcontinent. Despite several reports of fatal envenomings by other Bungarus species, commercial Indian antivenoms are only manufactured against B. caeruleus. It is, therefore, imperative to understand the distribution of genetically distinct lineages of kraits, the compositional differences in their venoms, and the consequent impact of venom variation on the (pre)clinical effectiveness of antivenom therapy. To address this knowledge gap, we conducted phylogenetic and comparative venomics investigations of kraits in Southern and Western India. Phylogenetic reconstructions using mitochondrial markers revealed a new species of krait, Romulus’ krait (Bungarus romulusi sp. nov.), in Southern India. Additionally, we found that kraits with 17 mid-body dorsal scale rows in Western India do not represent a subspecies of the Sind Krait (B. sindanus walli) as previously believed, but are genetically very similar to B. sindanus in Pakistan. Furthermore, venom proteomics and comparative transcriptomics revealed completely contrasting venom profiles. While the venom gland transcriptomes of all three species were highly similar, venom proteomes and toxicity profiles differed significantly, suggesting the prominent role of post-genomic regulatory mechanisms in shaping the venoms of these cryptic kraits. In vitro venom recognition and in vivo neutralisation experiments revealed a strong negative impact of venom variability on the preclinical performance of commercial antivenoms. While the venom of B. caeruleus was neutralised as per the manufacturer’s claim, performance against the venoms of B. sindanus and B. romulusi was poor, highlighting the need for regionally-effective antivenoms in India.

Kunitz-type peptide expression has been described in the venom of snakes of the Viperidae, Elapidae and Colubridae families. This work aimed to identify these peptides in the venom gland transcriptome of the coral snake Micrurus mipartitus. Transcriptomic analysis revealed a high diversity of venom-associated Kunitz serine protease inhibitor proteins (KSPIs). A total of eight copies of KSPIs were predicted and grouped into four distinctive types, including short KSPI, long KSPI, Kunitz–Waprin (Ku-WAP) proteins, and a multi-domain Kunitz-type protein. From these, one short KSPI showed high identity with Micrurus tener and Austrelaps superbus. The long KSPI group exhibited similarity within the Micrurus genus and showed homology with various elapid snakes and even with the colubrid Pantherophis guttatus. A third group suggested the presence of Kunitz domains in addition to a whey-acidic-protein-type four-disulfide core domain. Finally, the fourth group corresponded to a transcript copy with a putative 511 amino acid protein, formerly annotated as KSPI, which UniProt classified as SPINT1. In conclusion, this study showed the diversity of Kunitz-type proteins expressed in the venom gland transcriptome of M. mipartitus.

Scorpion venom is a rich source of biological active peptides and proteins. Transcriptome analysis of the venom gland provides detailed insights about peptide and protein venom components. Following the transcriptome analysis of different species in our previous studies, our research team has focused on the as one of the endemic scorpions of Iran to obtain information about its venom proteins, in order to develop biological research focusing on medicinal applications of scorpion venom components and antivenom production. To gain insights into the protein composition of this scorpion venom, we performed transcriptomic analysis. Transcriptomic analysis of the venom gland of H. prepared from the Khuzestan province, was performed through Illumina paired-end sequencing (RNA-Seq), Trinity assembly, CD-Hit-EST clustering, and annotation of identified primary structures using bioinformatics approaches. Transcriptome analysis showed the presence of 96.4% of complete arthropod BUSCOs, indicating a high-quality assembly. From total of 45,795,108 paired-end 150 bp trimmed reads, the clustering step resulted in the generation of 101,180 assembled transcripts with N size of 1,149 bp. 96,071 Unigenes and 131,235 transcripts had a significant similarity (E-value 1e-3) with known proteins from UniProt, Swissprot, Animal toxin annotation project, and the Pfam database. The results were validated using InterProScan. These mainly correspond to ion channel inhibitors, metalloproteinases, neurotoxins, protease inhibitors, protease activators, Cysteine-rich secretory proteins, phospholipase A enzymes, antimicrobial peptides, growth factors, lipolysis-activating peptides, hyaluronidase, and, phospholipase D. Our venom gland transcriptomic approach identified several biologically active peptides including five LVP1-alpha and LVP1-beta isoforms, which we named HzLVP1_alpha1, HzLVP1_alpha2, HzLVP1_alpha3, HzLVP1_beta1, and HzLVP1_beta and have extremely characterized here. Except for HzLVP1_beta1, all other identified LVP1s are predicted to be stable proteins (instability index <40). Moreover, all isoform of LVP1s alpha and beta subunits are thermostable, with the most stability for HzLVP1_alpha2 (aliphatic index = 71.38). HzLVP1_alpha2 has also the highest half-life. Three-dimensional structure of all identified proteins compacts with three disulfide bridges. The extra cysteine residue may allow the proteins to form a hetero- or homodimer. LVP1 subunits of potentially interact with adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), two key enzymes in regulation of lipolysis in adipocytes, suggesting pharmacological properties of these identified proteins.

The Russell’s viper (Daboia siamensis) is a medically important venomous snake in Myanmar. Next-generation sequencing (NGS) shows potential to investigate the venom complexity, giving deeper insights into snakebite pathogenesis and possible drug discoveries. mRNA from venom gland tissue was extracted and sequenced on the Illumina HiSeq platform and de novo assembled by Trinity. The candidate toxin genes were identified via the Venomix pipeline. Protein sequences of identified toxin candidates were compared with the previously described venom proteins using Clustal Omega to assess the positional homology among candidates. Candidate venom transcripts were classified into 23 toxin gene families including 53 unique full-length transcripts. C-type lectins (CTLs) were the most highly expressed, followed by Kunitz-type serine protease inhibitors, disintegrins and Bradykinin potentiating peptide/C-type natriuretic peptide (BPP-CNP) precursors. Phospholipase A2, snake venom serine proteases, metalloproteinases, vascular endothelial growth factors, L-amino acid oxidases and cysteine-rich secretory proteins were under-represented within the transcriptomes. Several isoforms of transcripts which had not been previously reported in this species were discovered and described. Myanmar Russell’s viper venom glands displayed unique sex-specific transcriptome profiles which were correlated with clinical manifestation of envenoming. Our results show that NGS is a useful tool to comprehensively examine understudied venomous snakes.

The monocled cobra ( ) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology. The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T. The toxin transcripts showed high redundancy (41-82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin's fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from which have not been reported hitherto; these include -specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

Cone snails are carnivorous marine predators that prey on mollusks, worms, or fish. They purposefully inject a highly diversified and peptide-rich venom, which can vary according to the predatory or defensive intended use. Previous studies have shown some correlations between the predation- and defense-evoked venoms and specific sections of the venom gland. In this study, we focus on the characterization of the venom of Cylinder canonicus, a molluscivorous species collected from Mayotte Island. Integrated proteomics and transcriptomics studies allowed for the identification of 108 conotoxin sequences from 24 gene superfamilies, with the most represented sequences belonging to the O1, O2, M, and conkunitzin superfamilies. A comparison of the predatory injected venom and the distal, central, and proximal sections of the venom duct suggests mostly distal origin. Identified conotoxins will contribute to a better understanding of venom–ecology relationships in cone snails and provide a novel resource for potential drug discovery.

Ants (hymenoptera: Formicidae) have adapted to many different environments and have become some of the most prolific and successful insects. To date, 13,258 ant species have been reported. They have been classified into 333 genera and 17 subfamilies. Except for a few Formicinae, Dolichoderinae, and members of other subfamilies, most ant species have a sting with venom. The venoms are composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Unlike the venoms of other animals such as snakes and spiders, ant venoms have seldom been analyzed comprehensively, and their compositions are not yet completely known. In this study, we used both transcriptomic and peptidomic analyses to study the composition of the venom produced by the predatory ant species Odontomachus monticola. The transcriptome analysis yielded 49,639 contigs, of which 92 encoded toxin-like peptides and proteins with 18,106,338 mapped reads. We identified six pilosulin-like peptides by transcriptomic analysis in the venom gland. Further, we found intact pilosulin-like peptide 1 and truncated pilosulin-like peptides 2 and 3 by peptidomic analysis in the venom. Our findings related to ant venom peptides and proteins may lead the way towards development and application of novel pharmaceutical and biopesticidal resources.

Toll-like receptor 4 (TLR4) is a crucial inflammatory signaling pathway that can serve as a potential treatment target for various disorders. A number of inhibitors have been developed for the TLR4 pathway, and although no inhibitors have been approved for clinical use, most have been screened against the TLR4-MD2 conformation. The venom gland is the organ of venomous snakes that secretes substances that are toxic to other animals. The level of gene transcription in venom glands is different from that in other tissues, includes a large number of biologically active ingredients, and is an important natural resource for the development of new drugs. We constructed a T7 phage display library using the cobra (Naja atra) venom gland from the Guangdong Snake Breeding Plant and performed three rounds of screening with TLR4 as the target, randomly selecting monoclonal phage spots for PCR followed by Sanger sequencing. The obtained sequences were subjected to length analysis, molecular docking, solubility prediction, and stability prediction, and a peptide containing 39 amino acids (NA39) was finally screened out. The BLAST results indicated that NA39 was a sequence in RPL19 (Ribosomal Protein L19). After peptide synthesis, the binding ability of NA39 to TLR4 was verified by the surface plasmon resonance (SPR) technique. In this study, a new peptide that can specifically bind TLR4 was successfully screened from the cobra venom gland cDNA library, further demonstrating the effectiveness of phage display technology in the field of drug discovery.

Due to some similarity of innate immunity between insects and mammals, the study of the molecular mechanism of innate immunity in insects has become a focus of research. However, the exact molecular and cellular basis of immune system in insect remains poorly understood. Characterization of the transcriptomic response to Cd of spider is an effective approach to understanding the innate immunity mechanisms. In this study, we carried out transcriptome sequencing and gene expression analyses to develop molecular resources for Pardosa pseudoannulata venom glands with and without Cd treatments. A total of 92,778 assembled unigenes and 237 Cd stress-associated differentially expressed genes between the Cd-treated and control groups were obtained. Expression profile analysis demonstrated that immunity-related genes involved in bacterial invasion of epithelial cells, leukocyte transendothelial migration, platelet activation, apoptosis, phagosome, and Rap1 signaling pathway were upregulated by Cd exposure, except the genes involved in PPAR signaling pathway were downregulated. Our results provide the first comprehensive transcriptome dataset of venom glands in P. pseudoannulata response to Cd, which is valuable for throws light on the immunotoxicity mechanism of Cd, and the innate immunity complexity.