Toxicon 142 (2018) 14e29 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Venom-gland transcriptomics and venom proteomics of the Hentz striped scorpion (Centruroides hentzi; Buthidae) reveal high toxin diversity in a harmless member of a lethal family * Micaiah J. Ward, Schyler A. Ellsworth, Darin R. Rokyta Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA article info abstract Article history: Of the 14 extant scorpion families, Buthidae has the most thoroughly characterized venoms. Most of this Received 9 November 2017 characterization, however, has been limited to species with medically significant stings, including Received in revised form members of the Centruroides genus, which have caused human deaths (e.g., Centruroides sculpturatus). To 7 December 2017 understand the origin and evolution of highly toxic venoms, we should also characterize the more Accepted 11 December 2017 harmless venoms of close relatives. We used Illumina sequencing to separately characterize the venom- Available online 14 December 2017 gland transcriptomes of a male and female Hentz striped scorpion (Centruroides hentzi) and performed independent quantitative mass-spectrometry analysis of the venom from each individual, providing the Keywords: fi Scorpion rst full venom characterization of a Centruroides species that poses no serious threat to humans. We fi fi Venom identi ed 59 venom proteins that were proteomically con rmed, 63 additional transcripts that were Transcriptome identified on the basis of homology to known toxins, and 355 nontoxins expressed in the venom-glands. þ þ Proteome The most abundant toxins belonged to the Na and K -channel toxin classes. Antimicrobial peptides and peptidases were also identified, along with a large group of venom proteins that could not be classified based on homology, suggesting C. hentzi is a source of previously untapped toxin diversity. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction representation of scorpions in the literature by focusing only on scorpions that cause severe physiological symptoms in humans, Scorpions are one of the most ancient extant venomous line- while neglecting those that are much less harmful. To better un- ages, originating approximately 430 million years ago, and have derstand why some species are harmful to humans, we should also since diversified into 14 recognized families and over 1700 investigate why some of their close relatives are not. described species (Soleglad and Fet, 2003; Stockmann and Ythier, In addition to buthids, venom characterizations (either full or 2010). Only approximately 30 scorpion species are known to be partial) have been completed for seven other scorpion families: harmful to humans, and all but one of these (Hemiscorpius lepturus Caraboctonidae (Schwartz et al., 2007; Rokyta and Ward, 2017), of the family Scorpionidae) are members of the family Buthidae Chaerilidae (He et al., 2013), Euscorpiidae (Ma et al., 2009, 2012; (Pipelzadeh et al., 2007; Chippaux and Goyffon, 2008), which are Santibanez-L~ opez et al., 2017), Scorpionidae (Ma et al., 2010; known for their rich diversity and high abundance of ion-channel Diego-García et al., 2012), Superstitioniidae (Santibanez-L~ opez toxins (Fet et al., 2003). The impact on human populations has et al., 2016), Urodacidae (Luna-Ramírez et al., 2015), and Vaejovi- resulted in buthids being the most well characterized scorpion dae (Quintero-Hernandez et al., 2015). Only a handful of these family in terms of venom (Ruiming et al., 2010; Ma et al., 2012; characterizations have used high-throughput transcriptomic Alvarenga et al., 2012; Rendon-Anaya et al., 2012; Valdez- methods (Rendon-Anaya et al., 2012; Luna-Ramírez et al., 2015; de Velazquez et al., 2013; Diego-García et al., 2014; Mille et al., 2014; Oliveira et al., 2015; Santibanez-L~ opez et al., 2016). The first com- de Oliveira et al., 2015), but has also led to a biased bined high-coverage venom-gland transcriptomic and venom proteomic analysis for a scorpion species was recently completed for Hadrurus spadix (Rokyta and Ward, 2017), a member of the * Corresponding author. Florida State University, Department of Biological Sci- family Caraboctonidae. These venom characterizations revealed ence, 319 Stadium Dr., Tallahassee, FL 32306-4295, USA. many previously unknown and medically significant venom E-mail address: [email protected] (D.R. Rokyta). https://doi.org/10.1016/j.toxicon.2017.12.042 0041-0101/© 2017 Elsevier Ltd. All rights reserved. M.J. Ward et al. / Toxicon 142 (2018) 14e29 15 components, such as antimicrobial peptides and anticancer pep- tides (Zeng et al., 2005; Harrison et al., 2014; Ortiz et al., 2015), as well as a plethora of ion-channel toxins with mammal and insect specificity (de la Vega et al., 2010; Quintero-Hernandez et al., 2013). In H. spadix, 66 of 148 putative toxins identified could not be assigned a tentative function on the basis of homology to toxins with known functions (Rokyta and Ward, 2017). Previous work on non-buthids has demonstrated the biodiscovery potential that could result from shifting the emphasis of scorpion venom research from the well-characterized harmful species to the generally ignored harmless species. Centruroides are members of the family Buthidae that are widely distributed throughout North, South, and Central America and are often referred to as bark scorpions. A partial venom-gland tran- scriptome and venom proteome characterization was described for Centruroides tecomanus (Valdez-Velazquez et al., 2013, 2016), and high-throughput 454 transcriptome sequencing was completed for Centruroides noxius (Rendon-Anaya et al., 2012). A small number of þ individual toxins, primarily Na -channel toxins, have also been purified and characterized from Centruroides sculpturatus (Wang and Strichartz, 1983), Centruroides noxius (Possani et al., 1985), Fig. 1. Dorsal view of a male (A) and female (B) C. hentzi clearly illustrates sexual dimorphism in this species. Males have smaller, more slender bodies and longer Centruroides suffusus (Martin et al., 1987), and Centruroides mar- metasomal segments in comparison to the females, which have larger bodies and garitatus (Garcia-Calvo et al., 1993). All of these species are shorter metasomal segments. Individual male and female C. hentzi shown here are considered medically significant within their ranges, and all are representative only and do no correspond with individuals C0136 and C0148 discussed potentially deadly to humans (Chippaux and Goyffon, 2008). throughout the manuscript. Venom from the Arizona bark scorpion (C. sculpturatus), for example, can give rise to symptoms of methamphetamine overdose Venom and venom-glands were extracted using methods pre- and even lead to death (Skolnik and Ewald, 2013; Strommen and viously described in detail (Rokyta and Ward, 2017). After a 5 min Shirazi, 2015). Some Centruroides species, however, are not anesthetization with CO , electrical stimulation was applied to the considered harmful to humans. Centruroides vittatus, which is 2 base of the telson (stinger) to induce a muscle contraction and native primarily to the south central United States (Shelley and expel the venom. Venom was then lyophilized and stored at À80C Sissom, 1995), has been reported to have mild stings (More et al., until later use. Four days after venom extraction, venom-glands 2004; Kang and Brooks, 2017). Centruroides hentzi, which is were removed under a stereoscopic microscope with micro- native to the southeastern United States (Shelley and Sissom, 1995; surgical dissection instruments. Prior to gland removal, scorpions Stevenson et al., 2012), has no known records of envenomations were fully anesthetized with CO for approximately 15 min. The requiring hospitalization, or even treatment, in medical literature 2 metasoma and intact telson were then cut from the scorpion body (Kang and Brooks, 2017), and stings that have been reported were and taped to a sterile surface such that the telson was visible under said to be painful, but short-lived and with no medical conse- the microscope. Using a micro-surgical blade, the telson was gently quences (Stevenson et al., 2012). To begin to address differences cut open and tweezers were used to peel back each side of the between the deadly Centruroides species and their significantly telson to access the venom-glands. Venom-glands were removed less-harmful relatives, we completed the first high-throughput by scraping out the inside of the telson using curved surgical venom-gland transcriptomic and proteomic characterization for a tweezers, and this tissue was immediately transferred to 100 mLof member of this genus, the Hentz striped scorpion (C. hentzi). We RNAlater. The gland tissue in RNAlater was stored at 4 C overnight independently sequenced venom-gland transcriptomes from a and transferred to À80C until further use. The scorpion body and male and female C. hentzi and conducted a parallel LC-MS/MS remaining metasoma of each specimen were preserved in 95% analysis of their venoms, providing a complete venom character- ethanol and stored at À80C. ization for a member of the most well-studied, medically important scorpion families. 2.2. Transcriptome sequencing 2. Materials and methods Scorpion venom-gland RNA extraction was performed as pre- 2.1. Scorpions, venoms, and venom-glands viously described (Rokyta and Ward, 2017). Briefly, 500 mL of Trizol (Invitrogen) was mixed with the 100 mL RNAlater containing the