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geometricusandL. CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectins placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin Onalespib (AT13387) development informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods. Keywords:molecular development, phylogeny, latrodectin, venom,Latrodectus == 1. Introduction == Venoms are protein-rich secretions that have developed in predatory animals for the purpose of prey immobilization and defense (Casewell et al., 2013;Fry et al., 2009). Venom production has independently arisen in several animal lineages including cnidarians, spiders, myriapods, scorpions, cone snails, cephalopods, snakes and mammals (Fry et al., 2009; Kordis and Gubensek, 2000;Wong and Belov, 2012). The venom produced by each species is often a complex cocktail of protein neurotoxins, hemotoxins and proteases (Escoubas et al., 2006;Fry et al., 2009;Sollod et al., 2005). Moreover, it appears that homologous genes were convergently recruited for venom expression in divergent taxa (Casewell et al., 2013;Fry et al., 2009). Many venom toxins originate from a gene duplicate encoding a structurally stable, cysteine-rich protein involved in a rapidly acting physiological Onalespib (AT13387) process (Fry et al., 2009). If such gene duplicates experience relaxed selection, mutations may accumulate in their coding and regulatory regions, causing them to be expressed in venom tissue. Further duplication of venom-expressed genes, coupled with high mutation rates, generates a multigene venom toxin family targeting a variety of receptors in diverse prey species (Duda et al., 2009;Fry et al., 2009;Sollod et al., 2005). Numerous studies have focused on the activity and composition of black widow spider venom (genusLatrodectus), but few have considered its development (Garb and Hayashi, 2013;Holz and Habener, 1998;Ushkaryov et al., 2004). Black widow spider venom has a potent neurotoxic effect on mammals, and bite symptoms may include nausea, vomiting and intense pain (Grishin, 1998;Nicholson and Graudins, 2002;Ushkaryov et al., 2004;Vetter and Isbister, 2008).Latrodectusvenom has largely been characterized from your Eurasian speciesL. tredecimguttatus, though the genus contains 31 species (Platnick, 2013).L. tredecimguttatusvenom is usually dominated by latrotoxins, which are large polypeptides ~1200 amino acids long (Ushkaryov et al., 2004). Of the four latrotoxins, -latrotoxin (-LTX) is the only vertebrate neurotoxin and is responsible for the effects associated with widow bites (Ushkaryov et al., 2004). -LTX functions as a calcium ion channel in the presynaptic nerve terminal membrane and causes massive neurotransmitter release (Orlova et al., 2000;Ushkaryov et al., 2004). Latrodectins or -latrotoxin associated Low Molecular Excess weight Proteins (-latrotoxin LMWPs), are a second family of venom peptides fromL. tredecimguttatusvenom, only known from two cDNA sequences (Kiyatkin et al., 1992;Pescatori et al., 1995). Latrodectins are peptides of ~70 amino acids that cannot be separated from latrotoxins using standard protein purification (Kiyatkin et al., 1992,1990;Pescatori et al., 1995;Volkova et al., 1995). Multiple studies have exhibited that purified latrodectin is not toxic in insects and mammals (Gasparini et al., 1994;Grishin et al., 1993;Kiyatkin et al., 1995;Volkova et al., 1995). However, latrodectins may function as subunits of a latrotoxin complex (Kiyatkin et al., 1992), even though latrotoxins do not require latrodectins for neurotransmitter release (Dulubova et al., 1996;Grishin et al., 1993;Kiyatkin et al., 1995;Volynski et al., 1999). Gasparini et al. (1994)noted that latrodectins have sequence similarities to the Crustacean Hyperglycemic Hormone (CHH) family, which contains neuropeptides from crustaceans that includes Type I peptides involved in ionic metabolism and osmoregulation and Type II peptides, comprising more specialized developmental hormones (Montagne et al., 2010). The CHH family exists in insects as the Ion Transport Peptides (ITPs), and CHH/ITP homologs have also GGT1 been recognized in ticks and Onalespib (AT13387) nematodes (Montagne et al., 2010). The latrodectins, CHHs, and ITPs are comparable in length, share six conserved cysteines in the mature peptide that adopt the same disulfide bond pairing, and have a similar alpha-helical structure (Gasparini et al., 1994). It is likely that latrodectins were recruited for venom gland expression from a broadly expressed spider CHH/ITP homolog. However, the diversity of latrodectins or their associations to the CHH/ITP neuropeptide superfamily Onalespib (AT13387) has not been explored in a phylogenetic framework. We investigated the expression and development of latrodectin sequences across widow spiders using venom gland cDNA libraries from your Western black widow spider (L. hesperus), the brown.