Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics

Abstract

BackgroundAnimal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution.ResultsWe estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression.ConclusionsQuantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-366) contains supplementary material, which is available to authorized users.

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

“…However, the collection of venom, particularly from dangerous or small animals, is a challenging task. This protocol demonstrates how venom and venom glands can be collected from black widow spiders, and confirms the success of this approach via a combination of MuDPIT analysis of the venom and a protein database derived from cDNAs cloned from venom glands 21 . While this protocol works well for black widows and medium sized spiders, other venom collection techniques have been employed for larger mygalomorph (tarantula-like) spiders, such as direct aspiration of venom from fangs into glass pipettes e.g., 24 .…”

“…The collection of venom will stimulate venom gland gene expression, thereby facilitating the cloning of specific toxin transcripts via RT-PCR 10,11 . Identification of venom proteins can also be accomplished in a high-throughput manner by integrating standard mass spectrometry techniques with sequence databases generated from venom gland cDNA libraries 15,21 . An example of such work, starting from venom and glands collected using the protocol detailed above, demonstrating its effectiveness, is illustrated in Figure 1.…”

“…Here the MuDPIT analysis was performed by the University of Arizona Proteomics Consortium. Masses of detected peptides and their dissociated fragments (daughter ions, inferred from spectra) were compared to peptide sequences theoretically predicted from translations of cDNA sequences obtained from a gene expression library constructed from L. hesperus venom glands (glands were acquired by the dissection protocol described above) 21 . In this experiment, 36 proteins were detected from all collected spectra, at the 99.9% probability threshold, and contained at least one detected peptide.…”

Transcriptome Analysis to Understand the Toxicity of Latrodectus tredecimguttatus Eggs

“…For example, the vast majority of peptides containing ICK motif in spider venom have neurotoxic activities [23,24]. To date, the venoms of two kinds of black widow spiders ( L. tredecimguttatus and L. hesperus ) have been reported to contain such neurotoxic peptides [8,25]. Therefore, it is reasonable to speculate that there are ICK motif peptides in the L. tredecimguttatus eggs.…”

Transcriptome Profiling Analysis of Wolf Spider Pardosa pseudoannulata (Araneae: Lycosidae) after Cadmium Exposure

“…In recent years, the increasing use of high-throughput sequencing has provided us with an efficient and powerful platform to further study arachnids at molecular level [16,17,18]. Though the genes related to Cd response have been widely studied in many vertebrate and invertebrate [19,20,21], only a few genes of spider responding to heavy metals have been reported previously.…”