Comparative venom gland transcriptome surveys of the saw-scaled vipers (Viperidae: Echis) reveal substantial intra-family gene diversity and novel venom transcripts

Abstract

BackgroundVenom variation occurs at all taxonomical levels and can impact significantly upon the clinical manifestations and efficacy of antivenom therapy following snakebite. Variation in snake venom composition is thought to be subject to strong natural selection as a result of adaptation towards specific diets. Members of the medically important genus Echis exhibit considerable variation in venom composition, which has been demonstrated to co-evolve with evolutionary shifts in diet. We adopt a venom gland transcriptome approach in order to investigate the diversity of toxins in the genus and elucidate the mechanisms which result in prey-specific adaptations of venom composition.ResultsVenom gland transcriptomes were created for E. pyramidum leakeyi, E. coloratus and E. carinatus sochureki by sequencing ~1000 expressed sequence tags from venom gland cDNA libraries. A standardised methodology allowed a comprehensive intra-genus comparison of the venom gland profiles to be undertaken, including the previously described E. ocellatus transcriptome. Blast annotation revealed the presence of snake venom metalloproteinases, C-type lectins, group II phopholipases A2, serine proteases, L-amino oxidases and growth factors in all transcriptomes throughout the genus. Transcripts encoding disintegrins, cysteine-rich secretory proteins and hyaluronidases were obtained from at least one, but not all, species. A representative group of novel venom transcripts exhibiting similarity to lysosomal acid lipase were identified from the E. coloratus transcriptome, whilst novel metallopeptidases exhibiting similarity to neprilysin and dipeptidyl peptidase III were identified from E. p. leakeyi and E. coloratus respectively.ConclusionThe comparison of Echis venom gland transcriptomes revealed substantial intrageneric venom variation in representations and cluster numbers of the most abundant venom toxin families. The expression profiles of established toxin groups exhibit little obvious association with venom-related adaptations to diet described from this genus. We suggest therefore that alterations in isoform diversity or transcript expression levels within the major venom protein families are likely to be responsible for prey specificity, rather than differences in the representation of entire toxin families or the recruitment of novel toxin families, although the recruitment of lysosomal acid lipase as a response to vertebrate feeding cannot be excluded. Evidence of marked intrageneric venom variation within the medically important genus Echis strongly advocates further investigations into the medical significance of venom variation in this genus and its impact upon antivenom therapy.

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms

“…The high abundance of SVMPs in the venom proteomes (>50% of all toxins) of the four Echis species correlates with our previous reports that these toxins are transcribed at the highest level in the venom glands (26). The SVMPs, alongside the SPs, represent the majority of toxin genes transcribed in the venom glands of B. arietans and C. cerastes, and these are also the most abundant proteins secreted in their venoms.…”

“…These species were selected based on their medical importance in Africa and our previous descriptions of interspecific variation in venom composition, dietary preference, and prey lethality (12,20,25,26). We used our previously constructed and assembled venom gland transcriptomes for the four Echis species (26,27) and prepared venom gland trancriptomes for B. arietans and C. cerastes by using identical protocols. We generated proteomes from venom extracted from each of these species (28) and used translations of the transcriptomic datasets to facilitate protein identification (SI Appendix, Figs.…”

“…Venom gland transcriptomes for B. arietans (Nigeria) and C. cerastes (Egypt) were constructed using the protocols previously described for E. ocellatus (Nigeria), E. p. leakeyi (Kenya), E. coloratus (Egypt), and E. c. sochureki (United Arab Emirates) (26,27). Briefly, libraries were constructed by using mRNA extracted from venom glands pooled from 10 individuals of each species by using the CloneMiner method, with ∼1,000 clones sequenced by using Sanger sequencing for each library.…”

“…Briefly, libraries were constructed by using mRNA extracted from venom glands pooled from 10 individuals of each species by using the CloneMiner method, with ∼1,000 clones sequenced by using Sanger sequencing for each library. Resulting ESTs were assembled into contigs (putative gene products) and annotated, and their transcription level was quantified as previously described (26,27). Each dataset was then subjected to six frame translations and used as a reference database to facilitate proteomic identification.…”

Testing the Toxicofera: comparative reptile transcriptomics casts doubt on the single, early evolution of the reptile venom system

“…Snake venom is frequently cited as being highly complex or diverse (Li et al, 2005;Wagstaff 48 et al, 2006;Kini and Doley, 2010) and a large number of venom toxin genes and gene 49 families have been identified, predominantly from EST-based studies of gene expression 50 during the re-synthesis of venom in the venom glands following manually-induced emptying 51 ("milking") of extracted venom (Pahari et al, 2007;Casewell et al, 2009;Siang et al, 2010;52 that lies outside of the proposed Toxicofera clade (Figure 1). We have also taken advantage 111 of available transcriptomes or RNA-Seq data for corn snake vomeronasal organ 112 (Brykczynska et al, 2013) and brain (Tzika et al, 2011), garter snake (Thamnophis elegans) 113 liver (Schwartz and Bronikowski, 2013) and pooled tissues (brain, gonads, heart, kidney, 114 liver, spleen and blood of males and females (Schwartz et al, 2010)), Eastern diamondback 115 rattlesnake (Crotalus adamanteus) and eastern coral snake (Micrurus fulvius) venom glands 116 (Rokyta et al, 2011;Rokyta et al, 2012;Margres et al, 2013), king cobra (Ophiophagus 117 hannah) venom gland, accessory gland and pooled tissues (heart, lung, spleen, brain, testes, 118 gall bladder, pancreas, small intestine, kidney, liver, eye, tongue and stomach) (Vonk et al,119 2013), Burmese python (Python molurus) pooled liver and heart (Castoe et al, 2011), green 120 anole (Anolis carolinensis) pooled tissue (liver, tongue, gallbladder, spleen, heart, kidney and 121 lung), testis and ovary (Eckalbar et al, 2013) and bearded dragon (Pogona vitticeps), Nile 122 crocodile (Crocodylus niloticus) and chicken (Gallus gallus) brains (Tzika et al, 2011), as 123 well as whole genome sequences for the Burmese python and king cobra (Castoe et al, 2013; 124 .…”