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De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator.

ABSTRACT: The brown widow spider, Latrodectus geometricus, is a predator of a variety of agricultural insects and is also hazardous for humans. Its venom is a true pharmacopeia representing neurotoxic peptides targeting the ion channels and/or receptors of both vertebrates and invertebrates. The lack of transcriptomic information, however, limits our knowledge of the diversity of components present in its venom. The purpose of this study was two-fold: (1) carry out a transcriptomic analysis of the venom, and (2) investigate the bioactivity of the venom using an electrophysiological bioassay. From 32,505 assembled transcripts, 8 toxin families were classified, and the ankyrin repeats (ANK), agatoxin, centipede toxin, ctenitoxin, lycotoxin, scorpion toxin-like, and SCP families were reported in the L. geometricus venom gland. The diversity of L. geometricus venom was also uncovered by the transcriptomics approach with the presence of defensins, chitinases, translationally controlled tumor proteins (TCTPs), leucine-rich proteins, serine proteases, and other important venom components. The venom was also chromatographically purified, and the activity contained in the fractions was investigated using an electrophysiological bioassay with the use of a voltage clamp on ion channels in order to find if the neurotoxic effects of the spider venom could be linked to a particular molecular target. The findings show that U24-ctenitoxin-Pn1a involves the inhibition of the insect sodium (Na_v) channels, BgNa_v and DmNa_v. This study provides an overview of the molecular diversity of L. geometricus venom, which can be used as a reference for the venom of other spider species. The venom composition profile also increases our knowledge for the development of novel insecticides targeting voltage-gated sodium channels.

SUBMITTER: Khamtorn P

PROVIDER: S-EPMC8746590 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.

Project description:Venom-gland transcriptomics is a key tool in the study of the evolution, ecology, function, and pharmacology of animal venoms. In particular, gene-expression variation and coding sequences gained through transcriptomics provide key information for explaining functional venom variation over both ecological and evolutionary timescales. The accuracy and usefulness of inferences made through transcriptomics, however, is limited by the accuracy of the transcriptome assembly, which is a bioinformatic problem with several possible solutions. Several methods have been employed to assemble venom-gland transcriptomes, with the Trinity assembler being the most commonly applied among them. Although previous evidence of variation in performance among assembly software exists, particularly regarding recovery of difficult-to-assemble multigene families such as snake venom metalloproteinases, much work to date still employs a single assembly method. We evaluated the performance of several commonly used de novo assembly methods for the recovery of both nontoxin transcripts and complete, high-quality venom-gene transcripts across eleven snake and four scorpion transcriptomes. We varied k-mer sizes used by some assemblers to evaluate the impact of k-mer length on transcript recovery. We showed that the recovery of nontoxin transcripts and toxin transcripts is best accomplished through different assembly software, with SDT at smaller k-mer lengths and Trinity being best for nontoxin recovery and a combination of SeqMan NGen and a seed-and-extend approach implemented in Extender as the best means of recovering a complete set of toxin transcripts. In particular, Extender was the only means tested capable of assembling multiple isoforms of the diverse snake venom metalloproteinase family, while traditional approaches such as Trinity recovered at most one metalloproteinase transcript. Our work demonstrated that traditional metrics of assembly performance are not predictive of performance in the recovery of complete and high quality toxin genes. Instead, effective venom-gland transcriptomic studies should combine and quality-filter the results of several assemblers with varying algorithmic strategies.

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De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator.

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