Integrative and Comparative Biology Integrative and Comparative Biology, volume 56, number 5, pp. 962–972 doi:10.1093/icb/icw063 Society for Integrative and Comparative Biology SYMPOSIUM Small Packages, Big Returns: Uncovering the Venom Diversity of Small Invertebrate Conoidean Snails J. Gorson*,†,‡ and M. Holford*,†,‡,1 *Department of Chemistry, Hunter College, The City University of New York, Belfer Research Building, NY, 10021 USA; †Departments of Biology, Chemistry, and Biochemistry, The Graduate City, The City University of New York, NY, 10016 USA; ‡Invertebrate Zoology, Sackler Institute of Comparative Genomics, American Museum of Natural History, NY, 10024 USA From the symposium ‘‘Integrative and Comparative Biology of Venom’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2016 at Portland, Oregon. 1E-mail: [email protected] Synopsis Venomous organisms used in research were historically chosen based on size and availability. This opportunity- driven strategy created a species bias in which snakes, scorpions, and spiders became the primary subjects of venom research. Increasing technological advancements have enabled interdisciplinary studies using genomics, transcriptomics, and proteomics to expand venom investigation to animals that produce small amounts of venom or lack traditional venom producing organs. One group of non-traditional venomous organisms that have benefitted from the rise of -omic tech- nologies is the Conoideans. The Conoidean superfamily of venomous marine snails includes, the Terebridae, Turridae (s.l), and Conidae. Conoidea venom is used for both predation and defense, and therefore under strong selection pressures. The need for conoidean venom peptides to be potent and specific to their molecular targets has made them important tools for investigating cellular physiology and bioactive compounds that are beneficial to improving human health. A convincing case for the potential of Conoidean venom is made with the first commercially available conoidean venom peptide drug Ziconotide (PrialtÕ), an analgesic derived from Conus magus venom that is used to treat chronic pain in HIV and cancer patients. Investigation of conoidean venom using -omics technology provides significant insights into predator-driven diversification in biodiversity and identifies novel compounds for manipulating cellular communication, especially as it pertains to disease and disorders. Introduction venom toxins (3FTxs) sequenced. Only three studies Venom is defined as any exogenous substance that is have used harder to milk and less studied, non- used to elicit an adverse effect in its target, and as a front-fanged snakes to investigate 3FTx bioactivity result a wide range of organisms from notorious (Fry et al. 2003; Pawlak et al. 2006, 2009; King snakes to lesser known leeches and bees are consid- 2015). The venom research strategy of size and ac- ered venomous (Fig. 1; Escoubas and King 2009; cessibility can neglect the ecology, morphology, or Casewell et al. 2013; King 2015; Petras et al. 2015). evolutionary relatedness between organisms, resulting Historically, organisms used in venom research were in a diversity of venomous animals, such as inverte- chosen opportunistically, based on size and ease of brates, being effectively ignored (Modica and collection, which largely focused on vertebrates, spe- Holford 2010; Puillandre and Holford 2010; von cifically snakes. Two genera of snakes account for Reumont et al. 2014b). almost 40% of all published venom toxin sequences Invertebrates are underrepresented in venom re- in elapid snake venom research (Fry et al. 2008). search. Spiders, which are the most diverse group Remarkably, one easy to collect genus (Naja) has of venomous animals, with about 45,000 species, been used to identify 40% of all three-finger snake make up less than 5% of all venom research studies Advanced Access publication July 1, 2016 ß The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licen- ses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Venom diversity of Conoidean snails 963 Fig. 1. Biodiversity of venomous taxa. Phylogenetic reconstruction of the tree of life highlighting venomous organisms. Grey bars represent the clades that include venomous organisms. Highlighted clades represent the traditionally studied venomous taxa (scorpions, spiders, snakes, and lizards). (Nentwig 2013). Analogous to the within taxa bias diversification, predator–prey interactions, and to de- seen in snakes, of the 17,000 species of scorpions scribe the immense biodiversity of animals found on described, only 50 species have had their venom Earth (Fig. 1). investigated (King 2015). It can be argued that the venom research bias existed largely due to lack of Rise of -omics technological methods for effectively collecting and characterizing small quantities of venom. The defi- Early research on venom relied heavily on identifying ciency of invertebrates has led to a dearth of infor- proteins using Edman degradation and mass spectro- mation that has hindered venom research. However, metry (MS; Perkins et al. 1993a, 1993b). In conjunc- recent technological advancements in the field of tion with fractionation, MS allowed for the separation molecular biology and proteomics has increased the and identification of individual venom components. representation of marine cone snails, sea anemones, Development of soft ionization methods in the late bees, and ants in venom studies (Norton and Olivera 1980s, such as electrospray ionization (ESI; Fenn 2006; Moran et al. 2008; Barlow et al. 2009; Casewell 2003), and matrix-assisted laser desorption (Karas et al. 2013; Sanggaard et al. 2014; Zhang et al. 2015). and Hillenkamp 1988; Tanaka 2003) have revolution- Extensive research on a broad range of organisms is ized biological research. In particular, the ability to imperative in order to effectively derive and test hy- identify proteins directly from MS data is a powerful potheses about venom as it relates to species capability that soon demonstrated to be crucial for 964 J. Gorson and M. Holford analyzing venoms. Quickly thereafter, research groups The honey bee, Apis mellifera, was the first venom- started to implement MS techniques to characterize ous organism to have a fully sequenced genome using snake venom (Perkins et al. 1993a, 1993b). MS Sanger sequencing (Weinstock et al. 2006). Since then, approaches also enable the identification of isoforms the development of next generation sequencing (NGS) of venom peptides, slight variations in sequences, and high throughput techniques has allowed rapid sequenc- post translational modifications (Craig et al. 1999; ing of other venomous organisms. The genomes of Escoubas et al. 2008; Safavi-Hemami et al. 2014; tarantula Acanthoscurria geniculate (Sanggaard et al. Petras et al. 2015). Recent advancements in MS pro- 2014), scorpion Mesobuthus martensii (Cao et al. tocols have produced what are referred to as top- 2013), velvet spider Stegodyphus mimosarum down methods, in which whole intact venom com- (Sanggaard et al. 2014), fire ant Soenopsis invicta ponents can be identified (Breuker et al. 2008; (Wurm et al. 2011), and king cobra Ophiophagus Ueberheide et al. 2009; Anand et al. 2014; Sunagar hannah (Vonk et al. 2013) have all been sequenced et al. 2016). Although groundbreaking, in some or- using NGS technologies. With multiple platforms avail- ganisms a proteome-only MS approach can be prob- able, such as Illumina (Illumina, Inc., San Diego, lematic. MS requires extraction of venom, which is California), 454 (Roche Applied Science, Penzberg, impractical for organisms that do not readily store Germany), SOLiD (ThermoFisher Scientific, Waltham, venom for delivery and other organisms that have Massachusetts), and Ion Torrent (ThermoFisher hard-to-access venom delivery systems that prevent Scientific, Waltham, Massachusetts), genome sequenc- stimulation or extraction (von Reumont et al. ing of venomous organisms is becoming both accessi- 2014a). Additionally, MS methods for determining ble and affordable. However, genomics alone does not primary venom peptide sequences are largely depen- provide enough information for determining the exact dent on downstream data analyses of source data- mode and tempo of gene expression and does not give bases, such as Mascot or Genbank (Perkins et al. significant insight into differential gene expression 1999; Bhatia et al. 2012). For model organisms with within various tissue types (Sunagar et al. 2016). a rich complement of sequence databases, such as While genomics is the study of the complete DNA humans, mice, or drosophila, this is not an issue. composition of an organism, venom gland transcrip- In the case of non-model organisms, the application tomics is the sequencing of mRNA specific to the of MS methods for primary sequencing is severely venom gland or secretory tissue of a venomous or- limited by the database used. Non-model systems ganism and therefore a glimpse at the specific venom generally require de novo sequence assembly and cocktail being used at the time by the animal source databases that are either missing or deficient. (Durban et al. 2011; Dutertre et al. 2014; Gorson As a result, an