Gene 536 (2014) 366–375

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Gene

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Recruitment and diversiﬁcation of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression☆

Caryn McCowan 1, Jessica E. Garb ⁎

Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA 01854, USA article info abstract

Article history: Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, Accepted 21 November 2013 including the convergent recruitment of homologous genes for venom expression. Here we provide novel evi- Available online 5 December 2013 dence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin Keywords: peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown Molecular evolution widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these se- Phylogeny Latrodectin quences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, Venom show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homo- Latrodectus logs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's 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 im- proved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods. © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction arisen in several animal lineages including cnidarians, spiders, myria- pods, scorpions, cone snails, cephalopods, snakes and mammals (Fry Venoms are protein-rich secretions that have evolved in predatory et al., 2009; Kordis and Gubensek, 2000; Wong and Belov, 2012). The animals for the purpose of prey immobilization and defense (Casewell venom produced by each species is often a complex cocktail of protein et al., 2013; Fry et al., 2009). Venom production has independently 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 Abbreviations: aa, amino acid(s); AIC, Akaike Information Criterion; α-Latrotoxin taxa (Casewell et al., 2013; Fry et al., 2009). Many venom toxins origi- LMWPs, α-latrotoxin associated low molecular weight proteins; BEB, Bayes Empirical nate from a gene duplicate encoding a structurally stable, cysteine- Bayes; bp, base pair(s); cDNA, DNA complementary to RNA; CHH, Crustacean rich protein involved in a rapidly acting physiological process (Fry Hyperglycemic Hormone; CPP, Clade Posterior Probability; dN, nonsynonymous substitutions per nonsynonymous site; ds, double strand(ed); dS, synonymous substitutions per et al., 2009). If such gene duplicates experience relaxed selection, muta- synonymous site; EST, Expressed Sequence Tag(s); FEL, ﬁxed effects likelihood; FUBAR, tions may accumulate in their coding and regulatory regions, causing Fast Unbiased Bayesian AppRoximation; GTR+I+G, Generalized Time Reversible plus them to be expressed in venom tissue. Further duplication of venom- Gamma plus Invariant model; ICK, Inhibitor Cystine Knot; ITP, Ion Transport Peptide; LRT, likelihood ratio test; MEME, Mixed Effects Model of Episodic Diversifying Selection; expressed genes, coupled with high mutation rates, generates a ML, maximum likelihood; NCBI, National Center for Biotechnology Information; RTA, multigene venom toxin family targeting a variety of receptors in diverse Random Taxon Additions; RTA clade, Retrolateral Tibial Apophysis clade; SLAC, single- prey species (Duda et al., 2009; Fry et al., 2009; Sollod et al., 2005). likelihood ancestor counting; TPM3uf+I+G, three parameter with unequal base frequen- Numerous studies have focused on the activity and composition of cies plus gamma plus Invariant model. black widow spider venom (genus Latrodectus), but few have consid- ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commer- ered its evolution (Garb and Hayashi, 2013; Holz and Habener, 1998; cial use, distribution, and reproduction in any medium, provided the original author and Ushkaryov et al., 2004). Black widow spider venom has a potent neuro- source are credited. toxic effect on mammals, and bite symptoms may include nausea, ⁎ Corresponding author at: Department of Biological Sciences, University of vomiting and intense pain (Grishin, 1998; Nicholson and Graudins, Massachusetts Lowell, 198 Riverside Street, Olsen Hall 520, Lowell, MA 01854, USA. Tel.: +1 978 934 2899; fax: +1 978 934 3044. 2002; Ushkaryov et al., 2004; Vetter and Isbister, 2008). Latrodectus E-mail address: [email protected] (J.E. Garb). venom has largely been characterized from the Eurasian species 1 Present address: Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Latrodectus tredecimguttatus, though the genus contains 31 species

(Platnick, 2013). L. tredecimguttatus' venom is dominated by latrotoxins, in the pZErO™-2 plasmid (Invitrogen Corp). Top10 Escherichia coli which are large polypeptides ~1200 amino acids long (Ushkaryov et al., was transformed with recombinant plasmids via electroporation and 2004). Of the four latrotoxins, α-latrotoxin (α-LTX) is the only verte- grown on agar plates. Clones were arrayed on 12-18 96-well micro- brate neurotoxin and is responsible for the effects associated with plates, which were screened for cDNA size using electrophoresis (Garb widow bites (Ushkaryov et al., 2004). α-LTX acts as a calcium ion chan- and Hayashi, 2005). nel in the presynaptic nerve terminal membrane and causes massive neurotransmitter release (Orlova et al., 2000; Ushkaryov et al., 2004). 2.2. cDNA sequencing and bioinformatic analyses Latrodectins or α-latrotoxin associated low molecular weight proteins (α-latrotoxin LMWPs), are a second family of venom peptides cDNA inserts ≥200 bp were sequenced using the universal SP6 from L. tredecimguttatus venom, only known from two cDNA sequences primer. Latrodectin cDNAs were also sequenced in the reverse direction (Kiyatkin et al., 1992; Pescatori et al., 1995). Latrodectins are peptides of using the T7 primer. Sequences were edited using Sequencher 4.8 (Gene ~70 amino acids that cannot be separated from latrotoxins using stan- Codes, Ann Arbor, MI), and assembled into contiguous sequences dard protein purification (Kiyatkin et al., 1990, 1992; Pescatori et al., (contigs). Highly similar sequences were identified using the 1995; Volkova et al., 1995). Multiple studies have demonstrated that BLASTclust program (http://toolkit.tuebingen.mpg.de/blastclust/). All purified latrodectin is not toxic in insects and mammals (Gasparini cDNAs were used as BLASTx queries against the NCBI nr protein data- et al., 1994; Grishin et al., 1993; Kiyatkin et al., 1995; Volkova et al., base (Altschul et al., 1990). Latrodectin cDNAs were translated based 1995). However, latrodectins may function as subunits of a latrotoxin on the predicted frame of the top BLASTx hit. A sampling of CHH/ITP ho- complex (Kiyatkin et al., 1992), even though latrotoxins do not require mologs from arthropod and nematode taxa were included in subse- latrodectins for neurotransmitter release (Dulubova et al., 1996; Grishin quent analyses (Table 1) from phylogenetically distinct lineages of the et al., 1993; Kiyatkin et al., 1995; Volynski et al., 1999). CHH/ITP superfamily as determined by Montagne et al. (2010). Follow- Gasparini et al. (1994) noted that latrodectins have sequence simi- ing Montagne et al. (2010), phylogenetic analyses across the CHH/ITP larities to the Crustacean Hyperglycemic Hormone (CHH) family, family were restricted to sequences representing the short, amidated which contains neuropeptides from crustaceans that includes Type I forms of these peptides. Additional long forms of CHH/ITP peptides peptides involved in ionic metabolism and osmoregulation and Type II exist in crustaceans and insects, which are the result of 1–2additional peptides, comprising more specialized developmental hormones exons that are alternatively spliced. These additional exons appear to (Montagne et al., 2010). The CHH family exists in insects as the Ion be unique to Pancrustacea and present problems for phylogenetic anal- Transport Peptides (ITPs), and CHH/ITP homologs have also been iden- yses with sequences from other taxa, as these additional exons do not tified in ticks and nematodes (Montagne et al., 2010). The latrodectins, exist in arachnid and nematode ITP homologs (Montagne et al., 2010). CHHs, and ITPs are similar in length, share six conserved cysteines in the Additional arachnid latrodectin homologs were identified with tBLASTx mature peptide that adopt the same disulfide bond pairing, and have a searches of NCBI's EST database (dbEST) using L. tredecimguttatus similar alpha-helical structure (Gasparini et al., 1994). It is likely that latrodectins 1 and 2 as queries. Putative signal peptides in sequences latrodectins were recruited for venom gland expression from a broadly were predicted using SignalP 4.0 (http://www.cbs.dtu.dk/services/ expressed spider CHH/ITP homolog. However, the diversity of SignalP/). Potential cystine inhibitor knot motif structure was predicted latrodectins or their relationships to the CHH/ITP neuropeptide super- with the Knoter1d program (http://knottin.cbs.cnrs.fr/Tools_1D.php). family has not been explored in a phylogenetic framework. Sequences were submitted to the ClanTox (http://www.clantox.cs. We investigated the expression and evolution of latrodectin se- huji.ac.il/index.php)(Naamati et al., 2009) classifier of animal toxins. quences across widow spiders using venom gland cDNA libraries from Toxin prediction by ClanTox for the query sequence is highly dependent the Western black widow spider (Latrodectus hesperus), the brown on the presence and distribution of cysteines, based on a training set of widow spider (Latrodectus geometricus), and the cupboard spider true and false ion channel toxin inhibitors (Naamati et al., 2009). (Steatoda grossa), in the putative sister genus to Latrodectus (Agnarsson, 2004; Arnedo et al., 2004). We examined these sequences 2.3. Latrodectin-2 gene characterization with putative homologs identified from database searches using phylogenetic and molecular evolutionary analyses to determine patterns of An alignment of latrodectin cDNAs was used to develop primers to selection on and diversification among latrodectins. We also character- PCR-amplify latrodectin 1 and 2 from genomic DNA. Only primers for ized the partial structure of latrodectin genes, which provides novel latrodectin 2 (LD2-F 5′-GATGCTTAAGCTTATCTGCATTG-3′ and LD2-R support for their derivation from CHH/ITP neuropeptides. Our results 5′-GGATATTGTGTAGTAAAGCAATTC-3′) produced amplifications from advance the understanding of the evolutionary origins and diversity of Latrodectus species. PCR fragments were ligated into the pCR 2.1 TOPO venom proteins, as well as the function of latrodectins in black widow vector (Invitrogen Corp.) and electroporated into Top10 E. coli. Clones venom. were selected for plasmid purification and sequenced with M13F and M13R primers. Intron position and sequence was inferred from 2. Materials and methods interrupted coding sequence determined from cDNAs, and identifying putative 5′GU and AG 3′ splice sites. 2.1. cDNA library construction and screening 2.4. Phylogenetic and molecular evolutionary analysis L. hesperus and L. geometricus were collected in California (Riverside and San Diego, respectively). S. grossa were purchased from Phylogenetic trees were constructed from the sequences listed in SpiderPharm (Yarnell, Arizona). 42 L. hesperus,27L. geometricus, and Table 1. Amino acid sequences were aligned using the MUSCLE program 25 S. grossa adult females were used to make separate venom gland (Edgar, 2004), followed by manual adjustment. Amino acid alignments cDNA libraries from each species. Total RNA was extracted from homog- were used to guide an alignment of corresponding nucleotides using enized venom glands using Trizol™ and purified using the RNeasy Kit tranalign (http://emboss.bioinformatics.nl/cgi-bin/emboss/tranalign); (Qiagen Inc., Valencia, CA). mRNA was isolated from total RNA using only unique sequences were included in analyses. Phylogenetic trees the Dynabeads™ mRNA purification kit (Invitrogen Corp., Carlsbad, were computed from the nucleotide alignment with maximum likeli- CA). cDNA was synthesized using the protocol in Garb and Hayashi hood (ML) and Bayesian methods, using the substitution model deter- (2005). cDNAs were size-selected for transcripts ≥1000 bp in length mined with the Akaike Information Criterion (AIC) in jModelTest with a Chromaspin 1000 column. This retains many fragments (Posada, 2008). ML trees were constructed using PAUP 4b10 b1000 bp, but reduces their overrepresentation. cDNAs were ligated (Swofford, 2003). A heuristic search was performed using 100 random 368 C. McCowan, J.E. Garb / Gene 536 (2014) 366–375

taxon addition (RTA) replicates with gaps as missing data. 100 bootstrap replicates were performed using three RTAs per replicate. Bayes- ian trees were estimated with Mr.Bayes 3.2.1 (Ronquist et al., 2012), Table 1 Sequences used in analyses with protein names and GenBank accession numbers. using the model selected by the AIC by MrModeltest 2.3 (Nylander, 2004). The program was run for 5 × 106 generations; trees were a Species Protein descriptor Protein Nucleotide sampled every 1000 generations. The first 25% of trees were removed accession accession as “burn-in” after standard deviation of split frequencies fell below Sequences obtained from GenBank 0.01. The post burn-in trees were used to construct a 50% majority- Latrodectus Latrodectin 1; α-latrotoxin- CAA44830 X63116 tredecimgutt- associated low molecular rule consensus tree, from which all Clade Posterior Probability (CPP) atus weight protein (LMWP) values were computed. The two nematode (Caenorhabditis elegans) Latrodectus Latrodectin 2; α-latrotoxin- AAY33774 DQ011856 sequences were used to root the tree, which represent basal sequences tredecimgutt- associated low molecular in the CHH/ITP superfamily (Montagne et al., 2010). atus weight protein 2 (LMWP2) Tests of selection on codons and along branches of the phylogeny Tegenaria TaITX-1; U1-agatoxin-Ta1a; CAA11839 AJ224127 agrestis Paralytic insecticidal toxin 1 were performed using the codeml package of PAML 4.3 (Yang, 2007) Tegenaria TaITX-2; U1-agatoxin-Ta1b; CAA11840 AJ224128 and the HyPhy package via the www.datamonkey.org web server agrestis Paralytic insecticidal toxin 2 (Delport et al., 2010). Estimates of nonsynonymous substitutions per Tegenaria TaITX-3U1-agatoxin-Ta1c; CAA11841 AJ224129 nonsynonymous site (dN) over synonymous substitutions per synony- agrestis Paralytic insecticidal toxin 3 ω Mesobuthus Venom cDNA clone Inferred from CB334130.1 mous site (dS), or , for branches and sites in the latrodectin phylogeny, gibbosus Mg_AFT_30E12 EST as well as for pairwise sequence comparisons were determined using Loxosceles laeta EY188575.1; LLAE0203C L. Inferred from EY188575 codeml. These analyses were based on an ML phylogeny restricted to laeta venom cDNA EST a clade of spider latrodectin sequences, and limited to single representa- Loxosceles HO003697; EST1080 L. Inferred from HO003697 tives from clades of nearly identical sequences, and all analyses used the intermedia intermedia venom cDNA EST Dermacentor ITP; Prepro ion transport-like ACC99599 EU620224 cleandata = 1 option (Yang, 2007). In codeml the MO, M1a, M2a, M3, variabilis peptide M7 and M8 site models were used to estimate ω under the assumption Ixodes scapularis ITP; CHH (PO-type) variant 1 XP_002400720 XM_002400676 of no site variation (M0) in comparison to variable ω among sites (M3); precursor and to compare models to test for positive selection (M1a vs. M2a and Caenorhabditis ITP1; Uncharacterized protein ZC168.2 Z70312.1 M7 vs. M8; Yang et al., 2000). We also tested for variable ω across elegans ZC168.2 positions 9773- 10008 branches, comparing the free-ratio to the one-ratio model, as well as Caenorhabditis ITP2; Protein C05E11.6 CCD63262 NM_076381 the free-ratio model in comparison to when ω was fixed to 1. Statistical elegans differences among models were estimated using a likelihood ratio test Daphnia magna ITP; Putative Ion Transport ABO43963 EF178503 (LRT). We also used the HyPhy package in the www.datamonkey.org Peptide-like Aedes aegypti ITP; Ion-Transport Peptide AAY29661 AY950504 server (Delport et al., 2010) to perform the single-likelihood ancestor Drosophila ITP; Ion-Transport Peptide ABZ88142 NM_001169822 counting (SLAC) method, the fixed effects likelihood (FEL) method, melanogaster positions 1080- and the Fast Unbiased Bayesian AppRoximation (FUBAR) method to 1406 detect positive and negative selection on codons, as well as the Mixed Schistocerca ITP; Ion-Transport Peptide AAB16822 U36919 Effects Model of Episodic Diversifying Selection (MEME) method to de- gregaria Homarus CHH; Hyperglycemic hormone ABA42180 DQ181792 tect diversifying episodic selection on codons. The GA-Branch module in gammarus B datamonkey was used to fit dN/dS rate classes to branches of the tree. Homarus VIH; Vitellogenesis inhibiting ABA42181 DQ181793 Analyses used the nucleotide evolution model selected by the AIC in gammarus hormone the datamonkey model selection tool, the ML tree from the correspond- Microctonus Venom protein 10 ABY19395 EU249359 fi hyperodae ing alignment, and were conducted using default signi cance levels.

Sequences characterized in this study 3. Results Latrodectus LHV117 KF751506 hesperus Latrodectus LHV218 KF751507 3.1. Sequence features and variability of latrodectins and their homologs hesperus Latrodectus LHV45 KF751508 We sequenced 1002 venom gland cDNAs from L. hesperus (355 hesperus ESTs), L. geometricus (281), and S. grossa (366). BLASTx results revealed Latrodectus LHV319 KF751509 hesperus that 30 cDNAs were most similar to latrodectins from L. tredecimguttatus Latrodectus LHV238 KF751510 (7 from L. hesperus, 11 from L. geometricus and 12 from S. grossa). Nine- hesperus teen of the 30 sequences were unique at the nucleotide level (6 from Latrodectus LHV229 KF751511 L. hesperus, 4 from L. geometricus and 9 from S. grossa; Table 1). Both hesperus Latrodectus LGV89 KF751512 L. hesperus and L. geometricus had latrodectins that clustered into two geometricus groups based on BLASTclust results, with the two groups being most L. geometricus LGV361 KF751513 similar to latrodectin 1 or 2 from L. tredecimguttatus. S. grossa sequences L. geometricus LGV332 KF751514 contained three distinct latrodectin clusters, suggesting the presence of L. geometricus LGV382 KF751515 three paralogs. Across these species, translated latrodectins ranged in Steatoda grossa SGV242 KF751516 Steatoda grossa SGV282 KF751517 length from 88 to 97 amino acids, and shared a minimum of 22.9% Steatoda grossa SGV150 KF751518 pair-wise identity, with an average of 41.4% pair-wise identity. Steatoda grossa SGV152 KF751519 After the top BLASTx hits of latrodectin 1 or 2 from Steatoda grossa SGV23 KF751520 L. tredecimguttatus, BLASTx searches with latrodectin cDNAs included Steatoda grossa SGV335 KF751521 Steatoda grossa SGV311 KF751522 many hits to Crustacean Hyperglycemic Hormone (CHH) peptides and Steatoda grossa SGV81 KF751523 Ion Transport Peptides (ITP). Translated BLAST searches of NCBI's Steatoda grossa SGV41 KF751524 dbEST database also identiﬁed hits to L. tredecimguttatus latrodectins a Alternative protein names provided where available and separated by semicolons; in four other arachnid species. These included three insecticidal protein descriptors derived from UniProtKB database or NCBI. venom neurotoxins TaITX 1–3 from the hobo spider Tegenaria agrestis C. McCowan, J.E. Garb / Gene 536 (2014) 366–375 369

Fig. 1. Alignment of latrodectin venom peptides with ecdysozoan CHH/ITP neuropeptide homologs and conserved intron position. (A) Alignment of latrodectin venom peptides from Latrodectus and Steatoda with putative homologs from arthropod venom cDNAs and from the ecdysozoan CHH/ITP neuropeptide superfamily. Numbers 1–6 on top indicate six cysteine residues conserved across all sequences. Predicted signal peptides are underlined and dashes represent hypothesized insertions/deletions. Arrow indicates position of conserved phase 2 intron shared among sequences; boxed region detailed in part b. Additional signal sequence MHHQKQQQQQKQQGEAP found in the Schistocerca ITP peptide (but absent in all other sequences), is upstream of alignment position 1 and not shown. (B) (Top) Schematic of primary structure for CHH/ITP/latrodectin peptides marking approximate position of six conserved cysteine residues, the fourth cysteine and the following two residues, the boxed region corresponds to the boxed region in part a; (middle) gene structure illustrating the conserved phase 2 intron for the L. hesperus latrodectin 2 peptide; (bottom) gene structure of Ixodes Ion Transport Peptide showing conserved phase 2 intron. Nucleotides in square brackets represent intronic sequence, only showing canonical splice sites. 370 C. McCowan, J.E. Garb / Gene 536 (2014) 366–375

(Family Agelenidae), venom cDNAs from recluse spiders Loxosceles laeta latrodectin 1 clade, the sequences are largely arranged based on expec- and Loxosceles intermedia (Family Sicariidae), and a venom gland EST tations from species relationships (Fig. 3), with L. hesperus being more from the scorpion Mesobuthus gibbosus. In addition, venom protein closely related to L. tredecimguttatus than it is to L. geometricus (Garb 10 from the wasp Microctonus hyperodae was also identified with and Hayashi, 2013; Garb et al., 2004). However, the latrodectin 2 clade PSIBLAST searches of NCBI's nr database as having similarity to united L. geometricus and S. grossa sequences. An ML tree of the spider latrodectins (Table 1). All sequences used in phylogenetic analyses sequences alone, sampling one sequence per paralog per species, have six cysteines in their predicted mature peptides that are 100% con- showed a topology identical to that seen for the Steatoda/Latrodectus served (Fig. 1A). All examined sequences, excepting the Daphnia magna sub-clades within the larger tree (−ln L = 2870.97520; Fig. 4). The putative ITP and the Schistocerca gregaria ITP, were predicted to have a reduced tree showed a strongly supported clade of Steatoda and signal peptide by SignalP (Fig. 1A). The Knoter1d program predicted Latrodectus latrodectin paralogs including latrodectin 1 (BS = 98%), that none of the examined sequences were inhibitor cystine knot and moderate support for a clade including latrodectin 2 orthologs (ICK) toxins. The ClanTox server classified all Latrodectus and Steatoda and the third S. grossa paralog (BS = 57%). mature latrodectins, as well as the Loxoceles, Tegenaria,andMesobuthus homologs as “possibly toxin-like”, rather than “probably toxin-like” or 3.4. Analyses of selection on latrodectins “toxin-like”. All other CHH/ITP homologs were classified as “probably not toxin-like”. Codeml estimates of ω across branches in the reduced latrodectin tree using the free ratio model ranged from 0.0034 to infinity (∞), the 3.2. Latrodectin gene structure latter value being for branches where 0 synonymous substitutions were estimated (Fig. 4). Several of the branches where ω = ∞ had esti- Genomic PCR products of latrodectin 2 from L. hesperus and mates of relatively high numbers of nonsynonymous substitutions (e.g., L. geometricus (GenBank Accessions: KF751525–KF751526) contained 14.6, 32.3, 19.4, 5.1; Fig. 4), and some branches had ω values exceeding two exons, the sequences of which agree with their corresponding 1 (e.g., 13.6, 4.1; Fig. 4). The one ratio model estimated an overall ω cDNA sequences, excepting 1–2 nucleotide differences that likely reflect value for all branches as 0.3597, and this model was a significantly allelic differences, as cDNAs and genomic DNA were derived from differ- worse fit than the free ratio model (Table 2). However, the one ratio ent individuals. The two exons were interrupted by a phase 2 intron in model was significantly a better fit to the data than fixing ω to 1. This the codon of the residue following the fourth cysteine in each mature suggests an overall pattern of purifying selection on latrodectin lineages peptide (Fig. 1B). A phase 2 intron interrupting the codon of the residue over time, with potential evidence for positive selection on several following the fourth cysteine is also found in all CHH/ITP family branches where ω exceeds 1. LRT comparisons rejected the M0 over members, except ITP2 genes from Caenorhabditis and the ITP gene of the M3 model, suggesting variable ω across sites. However, the M2a Trichinella spiralis (Montagne et al., 2010), and some CHH/ITP members model was not significantly different from M1a, nor was the M8 have more introns in addition to the one in this position (Montagne model significantly different from M7, and the Bayes Empirical Bayes et al., 2010). It is possible that there are additional introns in genomic (BEB) procedure found no evidence of positive selection on specific latrodectin 2, as the genomic sequence we obtained was restricted to sites within latrodectin (i.e., Pr (ω N 1) = 0.90 or higher; Table 2). the first 71 out of 88 total residues. However, our cDNA sequences do HyPhy analyses of positive selection on latrodectin codons identified not suggest the potential for alternatively spliced forms of latrodectin seven positively selected sites, all of which were detected with the genes. The L. geometricus latrodectin 2 intron is 1777 bp in length, MEME method, which identifies episodic positive selection. Two of 241 bp longer than the intron in L. hesperus.Moreover,theL. geometricus these sites were also significant for the FEL method analysis (Table 3). intron sequence is extremely divergent from the intron in L. hesperus Two of the seven positively selected sites are located in the predicted (50.4% nucleotide identity), whereas the exons share 82% identity. signal peptide region, while the rest are equally distributed along the mature peptide. Fourteen codons were identified as negatively selected 3.3. Phylogenetic analyses of latrodectins and CHH/ITP homologs with significance in at least one HyPhy analysis, including five of the six

conserved cysteine residues (Table 3). The average dN/dS value over the Maximum likelihood (ML) and Bayesian tree searches were per- entire alignment estimated by the SLAC method was 0.475. The GA- formed on a nucleotide alignment of latrodectins and CHH/ITP homo- Branch analysis ﬁts all branches in the tree to two dN/dS rate classes: logs in Table 1. Model selection for this alignment using the AIC in dN/dS of 0.257 to 66% of the branches and dN/dS of 0.867 to 34% of the jModelTest selected the TPM3uf+I+G model of nucleotide substitution branches. While the branch rate classes do not exceed 1, this result is with model parameters as follows: [A–C] = 1.5771; [A–G] = 2.2915; consistent with variable rates of selection across the tree. [A–T] = 1.0000; [C–G] = 1.5771; [C–T] = 2.2915; freqA = 0.2861; freqC = 0.2256; freqG = 0.2419; freqT = 0.2464; I = 0.039; 4. Discussion G = 3.6720. The AIC in MrModeltest 2.3 selected the GTR+I+G substitution model. The Bayesian consensus tree from this model contained a 4.1. Multiple recruitments of CHH/ITP neuropeptides for venom gland clade including all spider venom cDNAs (CPP = 1.00, Bootstrap expression Support (BS) = 67%), which is sister to the scorpion venom cDNA (CPP = 0.92; Fig. 2). The ML searches recovered a single tree of −ln A particularly fascinating aspect of venom evolution is the observa- L = 8174.43986, which was identical in topology to the Bayesian tree tion that many similar proteins and peptides are expressed in the in Fig. 2, but resolved the two unresolved nodes. Tick (Ixodes + venom of animal lineages that have independently evolved venom pro- Dermacentor) ITPs appear more closely related to crustacean and insect duction (Casewell et al., 2013; Fry et al., 2009; Wong and Belov, 2012). CHH and ITPs than to the arachnid venom cDNAs (CPP = 0.56); howev- These multiple recruitments suggest that certain classes of proteins that er, this relationship did not have bootstrap support in the ML analysis. are largely expressed in another body tissue are more likely to be re- Within the spider clade, the latrodectin cDNAs from Latrodectus and cruited and retained for venom expression, and that while the recruit- Steatoda are united with limited support (CPP = 0.63) and this clade ment event is convergent, the proteins involved may be homologous is sister to a grouping of Loxoceles and Tegenaria venom cDNAs at some level, though they likely involve paralogs. In this study we pro- (CPP = 0.86). These relationships, though identical, are not well sup- vide an example of a widespread family of arthropod peptides, the CHH/ ported in the ML tree. Two clades containing either latrodectin 1 and 2 ITP neuropeptides, which may represent another example of conver- were recovered, each with strong support (CPP = 1.00 for both; gent recruitment for venom gland expression. Our ﬁnding of a phase 2 latrodectins 1 BS = 62%, latrodectins 2 BS = 84%). Within the intron in the latrodectin codon following the fourth cysteine codon in C. McCowan, J.E. Garb / Gene 536 (2014) 366–375 371

Fig. 2. Phylogenetic relationships of latrodectins and ecdysozoan CHH/ITP neuropeptide homologs. Phylogram of Bayesian consensus tree from analysis of nucleotides encoding latrodectins and putative CHH/ITP superfamily peptides. Values above nodes indicate Clade Posterior Probability values (CPP). Numbers below nodes indicate maximum likelihood bootstrap values from 100 replicates. The tree is rooted with C. elegans homologs. Hatched lines indicate shortened branch for ﬁgure quality.

Latrodectus mactans clade Latrodectus hesperus (Theridiidae)

Latrodectus tredecimguttatus (Theridiidae)

Latrodectus geometricus (Theridiidae) 229.5 MYA Entelegynae Steatoda grossa (Theridiidae)

375.5 MYA Orbiculariae:Araneoidea Tegenaria agrestis (Agelenidae) RTA clade

Loxosceles laeta (Sicariidae) Araneae) (Arachnida: Haplogynae Loxosceles intermedia (Sicariidae)

Fig. 3. Species relationships and divergence dates for spiders expressing latrodectins and homologous venom peptides. Phylogenetic relationships for spider species from which latrodectin peptides or their homologs were obtained and analyzed in this study. Divergence dates of major lineages at nodes as estimated by Ayoub et al. (2007) in millions of years ago (MYA). Spe- cies relationships are summarized from species phylogenies in Coddington et al. (2004) and Garb and Hayashi (2013). 372 C. McCowan, J.E. Garb / Gene 536 (2014) 366–375

Fig. 4. Branch-specific patterns of selection on the spider phylogeny of latrodectins and homologous spider venom peptides. Maximum likelihood phylogenetic tree of latrodectin sequences and homologous spider venom peptides. Numbers above branches show ML estimates of ω (nonsynonymous/synonymous substitution rates) determined from the branch free-ratio model of codeml in PAML. Values of infinity are appended with estimates of nonsynonymous differences over synonymous differences for that branch. Numbers under the branches indicate ML bootstrap values for that node from 100 replicates. its mature peptide, which is found in nearly all other CHH/ITP genes, re- may have happened at least three times: once in Hymenoptera, once inforces the claim that latrodectins are derived from this ecdysozoan in scorpions, and at least once in spiders. Although the hymenopteran, family of neuropeptide hormones (Gasparini et al., 1994). CHH/ITP neu- scorpion and spider CHH/ITP/latrodectin peptides are grouped as a ropeptides share some of the characteristics of other protein families clade in our phylogenetic trees, we suggest as many as three venom re- that have been independently recruited for venom expression, such as cruitments, because each lineage independently evolved venom pro- a signal peptide and multiple cysteine residues that participate in disul- duction. This is a parsimony-based argument, as each lineage is more fide bonds (Gasparini et al., 1994). closely related to non-venomous species, and because their venoms The functions of CHH/ITP neuropeptides are varied, including roles are produced in non-homologous glands (cheliceral glands in spiders in ionic metabolism, regulation of molting and reproduction, and osmo- vs. abdominal venom glands in scorpions and Hymenoptera). This regulation (Montagne et al., 2010), and their widespread expression in scenario of three recruitment events can be tested in future studies by various body tissues may further predispose them for venom recruit- performing whole-genome sequencing and expression analyses of ment. Recruitment of CHH/ITP neuropeptides for venom expression each group, to confirm whether genes encoding venom-specific peptides are derived from duplicates of CHH/ITP genes that are maintained

Table 2 for expression in other body tissues. Codon substitution models for spider latrodectin ML tree (Fig. 4) with ω estimates and probabilities. 4.2. Widespread distribution of CHH/ITP/latrodectins in spider venoms Model −ln L Parameter estimates χ2 df P α Branches Free −1893.66 Previous sequences of the latrodectin/ -latrotoxin LMWP venom ratio peptides were known from L. tredecimguttatus (Kiyatkin et al., 1992; One −1913.90 ω = 0.3597 40.49 21 0.0064 Pescatori et al., 1995), and our results conﬁrm that these peptides are ratio expressed in the venoms of other Latrodectus and Steatoda species. − ω b Fixed 1937.048 = 1 46.29 1 0.0001 α ratio Our bioinformatic searches further suggest that latrodectin/ - Sites M0 −1913.90 ω =0.3597 latrotoxin LMWP homologs are also present in the venoms of two M3 −1877.97 p(0,1,2): 0.1061 0.5314 71.86 4 b0.0001 additional spider genera in very distantly related families: Tegenaria 0.3625 (Agelenidae) and Loxosceles (Sicariidae). Agelenidae, Sicariidae and ω(0,1,2): 0.0060 0.2675 Theridiidae (the family of Latrodectus and Steatoda), are representatives 0.8830 M1a −1887.75 p(0,1): 0.5740 0.4261 of three deep lineages (RTA clade, Haplogynae and Orbiculariae, respec- ω(0,1): w: 0.2122 1.0000 tively; Fig. 3) within the suborder Araneomorphae (true spiders) M2a −1887.75 p(0,1,2): 0.5740 0.3680 001 (Coddington et al., 2004). Assuming a single recruitment of CHH/ITP/ 0.0580 latrodectins for venom expression in spiders, we estimate that this ω(0,1,2): 0.2122 1.0000 1.0000 event took place at least 375 million years ago, the age of the common M7 −1882.28 p = 0.8329 q = 1.0868 ancestor of these families (Ayoub et al., 2007). Further characterization M8 −1882.03 p0 = 0.9085 p = 0.9452 0.51 2 0.7765 of spider venoms is needed to determine the greater distribution of q = 1.5070 (p1 = 0.0916) latrodectins, and to test whether a single ancient recruitment for ω =1.2587 venom expression was followed by numerous losses, or if multiple C. McCowan, J.E. Garb / Gene 536 (2014) 366–375 373

Table 3 Summary of results from HyPhy analyses of codon-speciﬁc selection in the latrodectin alignment.

Codona SLAC SLAC FEL FEL FUBAR FUBAR MEME MEME b c d +f e dN-dS p-value dN-dS p-value dN-dS Post. Pr. ω p-value Positively selected codons 2 1.155 0.157 0.269 0.030 0.97 0.884 N100 0.041 7 0.040 0.655 0.164 0.089 −0.016 0.634 N100 0.086 12 −0.002 0.650 0.301 0.353 0.118 0.755 31.863 0.033 24 −0.035 0.664 −0.209 0.453 −1.352 0.275 N100 0.048 31 −0.376 0.794 −0.233 0.523 −1.243 0.265 N100 0.022 51 0.448 0.505 −0.017 0.939 −0.070 0.477 94.614 0.053 54 0.076 0.622 −0.150 0.684 −0.201 0.456 N100 0.062

Negatively selected codons 4 −0.088 0.583 −0.594 0.068 −0.225 0.761 –– 15 −0.576 0.331 −0.286 0.110 −0.427 0.937 –– 17 −0.592 0.347 −3.026 0.004 −3.339 0.980 –– 19 (C-1) −0.737 0.183 −0.074 0.084 −0.121 0.959 –– 22 −1.239 0.125 −0.313 0.075 −0.379 0.894 –– 23 −0.800 0.318 −1.178 0.057 −1.649 0.865 –– 25 −1.680 0.073 −0.383 0.044 −0.438 0.915 –– 26 −1.645 0.080 −0.965 0.026 −0.657 0.898 –– 33 (C-2) −2.272 0.006 −1.514 0.001 −1.934 0.998 –– 36 (C-3) −2.515 0.002 −0.597 0.000 −1.414 0.999 –– 47 (C-5) −1.298 0.041 −0.289 0.006 −0.487 0.993 –– 48 −1.223 0.079 −0.197 0.073 −0.275 0.951 –– 56 (C-6) −1.163 0.048 −345.342 0.003 −2.134 0.995 –– 60 −1.227 0.056 −0.223 0.027 −0.134 0.948 –– a. Letter C in parentheses, followed by number indicates conserved cysteine according to positions defined in Fig. 1; note that C-4 has the same codon for all sequences in this alignment, as well as in a larger alignment except the Microctonus and one C. elegans ITP sequence. b–d, e. Statistical support for positive or negative selection by each method is indicated by bold p- values or bold posterior probability values (Post. Pr.); f. values indicate inferred ω (β+/α), values where they exceed 100 include sites where α is 0. recruitments took place in spiders. For species having only venom vertebrate neurotoxin α-latrotoxin preceding the origin of the cDNAs (but not cDNAs in the same protein family from other tissues) Latrodectus genus, as opposed to the mactans clade (Garb and available for phylogenetic analyses, venom cDNAs are likely to form a Hayashi, 2013). The venoms of species from the mactans clade (which monophyletic clade, potentially erroneously implying a single recruit- contains both the black widow and red-back spiders) exhibit greater ment for venom expression (Casewell et al., 2012). toxicity relative to members of the geometricus clade as well as to spe- The denser sampling of latrodectin sequences we provide allows for cies outside of Latrodectus (Graudins et al., 2002, 2012; Müller et al., more detailed evolutionary analyses. The phylogenetic arrangement of 1992). This increased toxicity of black widow venom has largely been the Latrodectus and Steatoda sequences strongly supports the expres- attributed to α-latrotoxin sequence identity and expression levels sion of two-three latrodectin paralogs in the venom of each species. (Garb and Hayashi, 2013; Graudins et al., 2012), but venom proteins Relationships within the latrodectin 1 clade are largely consistent with such as latrodectins that may work synergistically with α-latrotoxin expected relationships for Latrodectus species, e.g., (((L. hesperus + could also play an important role in explaining venom functional L. tredecimguttatus) L. geometricus) S. grossa); (Garb and Hayashi, variation. 2013; Garb et al., 2004). However, there is evidence for additional While codeml analyses did not find statistical support for positive lineage-specific gene duplication within S. grossa. Given the available selection at specific codons, HyPhy analyses found some statistical sup- data, a lineage-specific duplication in Steatoda is more plausible than port of positive selection on 7 sites (largely due to episodic diversifying this third paralog arising in the common ancestor of all four species, selection), as well as negative selection on 14 sites (over a total of 62 co- but only being expressed in S. grossa. In contrast to the latrodectin 1 dons considered when excluding gapped regions). This included sup- clade, the latrodectin 2 clade does not agree with expected phylogenetic port for negative selection at 5 of the 6 conserved cysteine residues, relationships, as the S. grossa latrodectin 2 sequence is joined to the all of which are expected to be critical for maintaining the disulfide L. geometricus sequence, although by a much longer branch. While this bonds that shape the overall fold of this peptide (Gasparini et al., relationship may be an artifact of long-branch attraction, there is also 1994). Five of these six conserved cysteine residues (all but cysteine the possibility of additional paralogs in this clade that were not sampled 4) had dN b dS values, where the difference was significant in multiple from the other species. HyPhy modules (Table 3). The fourth cysteine has the same codon Additional evolutionary analyses suggest evidence for positive and (TGC) for all sequences in this alignment (as well as in the Fig. 1Aalign- negative selection acting on particular latrodectin sequences and ment excepting the Microctonus and one C. elegans ITP sequence), codons. Specifically, codeml analyses identified four branches in the explaining why this codon was not detected as negatively selected. latrodectin phylogeny where branch-specific estimates of ω exceeded This high degree of conservation of cysteine codons is similar to the 1, as well as four additional branches where only nonsynonymous sub- pattern observed in conotoxins, where there are high rates of stitutions (ranging from 5.1 to 32.3 substitutions) were estimated nonsynonymous substitutions in most codons, but a striking uniformity (Fig. 4). Branches where ω exceed 1 sometimes precede gene duplica- of cysteine codons across sequences (Conticello et al., 2001; Duda and tion events, but are also associated with nodes connecting orthologs. Palumbi, 1999). Both latrodectins 1 and 2 appear to have experienced positive selection in the most recent common ancestor of the Latrodectus mactans clade 4.3. Function of latrodectins in black widow venom (L. geometricus and Latrodectus rhodesiensis form the geometricus clade, all other species form the mactans clade within Latrodectus) Previous biochemical studies of black widow spider venom which (Garb and Hayashi, 2013; Garb et al., 2004), which suggests a shift in indicated latrodectins constitute a major fraction of the venom, sug- its functional activity coincident with the origin of black widow species. gested latrodectin 1 was in approximately equal quantities with α- Similar analyses indicated accelerated substitution rates in the latrotoxin (Grishin et al., 1993; Kiyatkin et al., 1992; Volkova et al., 374 C. McCowan, J.E. Garb / Gene 536 (2014) 366–375

1995). Latrodectins only comprised 3–4% of all ESTs we cloned from homologous genes for venom expression. These findings should be in- Latrodectus and Steatoda venom gland cDNA libraries, but they were vestigated as comprehensive transcriptomic and genomic resources among the few transcript types represented by multiple copies in the are developed for neglected venomous taxa. libraries, consistent with their relatively high expression in venom glands. Given the abundance of latrodectins in black widow spider Conflict of interest venom, and evidence of paralogs that experienced negative and positive selection, it is likely that latrodectins plays some key role in prey acqui- The authors declare no conflicts of interest. sition. However, past functional studies indicated that purified latrodectins are not toxic to mammals or insects (Kiyatkin et al., 1995; Volkova et al., 1995). For example, purified latrodectin 1 and 2 from Acknowledgements L. tredecimguttatus is non-toxic to cockroaches (Periplaneta americana) when injected at doses of up to 80 μg/g, having not caused lethality or We thank Nadia Ayoub, Kanaka Varun Bhere, Cheryl Hayashi, paralysis (Volkova et al., 1995). Purified L. tredecimguttatus latrodectins Konrad Zinsmaier, Adele Zhou for their help in collecting data for this were also not toxic to mice at concentrations up to 2.3 mg/kg intrave- study. Some spiders were kindly supplied by Chuck Kristensen and nously, and 0.8 mg/kg intracerebroventricularly (Volkova et al., 1995). Marshal Hedin. We thank Nadia Ayoub, Rujuta Gadjil, Peter Gaines, Interestingly, TaITX-1–3, proposed latrodectin homologs from T. agrestis Robert Haney, and Alex Lancaster for their helpful comments on this (hobo spider) venom, are characterized as highly potent insect neuro- manuscript. This work was supported by a University of Massachusetts toxins that kill via paralysis and cause elevated firing of neurons from Lowell Faculty-Student Collaborative Research Project Grant to Caryn the central nervous system, although the mechanism of insecticidal ac- McCowan and Jessica Garb, and grants 1F32GM083661-01 and tivity is unclear (Johnson et al., 1998). This suggests that spider venom 1R15GM097714-01 from the National Institutes of Health (NIGMS) to peptides derived from the CHH/ITP neuropeptide family exhibit sub- Jessica Garb. stantial functional diversity. Latrodectins typically co-purify with latrotoxins and are biochemi- References cally challenging to separate (Grishin et al., 1993; Kiyatkin et al., 1992; Volkova et al., 1995). Latrodectin 1 was first isolated in an attempt to Agnarsson, I., 2004. Morphological phylogeny of cobweb spiders and their relatives – isolate α-latrotoxin (Kiyatkin et al., 1992), and latrodectin 2 was isolat- (Araneae, Araneoidea, Theridiidae). Zool. J. Linn. Soc. 141, 447 626. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment ed with latroinsectotoxin fractions (Volkova et al., 1995). This suggested search tool. J. Mol. Biol. 215, 403–410. that latrodectins interact with latrotoxins as heteromeric complexes Arnedo, M.A., Coddington, J., Agnarsson, I., Gillespie, R.G., 2004. From a comb to a tree: (Kiyatkin et al., 1992). Subsequent work showing that recombinant α- phylogenetic relationships of the comb-footed spiders (Araneae, Theridiidae) inferred from nuclear and mitochondrial genes. Mol. Phylogenet. Evol. 31, 225–245. latrotoxin can recapitulate the effects of whole venom in causing neuro- Audsley, N., Mcintosh, C., Phillips, J.E., 1992. Actions of ion-transport peptide from locust transmitter release in the absence of latrodectin 1 (Volynski et al., corpus cardiacum on several hindgut transport processes. J. Exp. Biol. 173, 275–288. 1999), casts some doubt on whether α-latrotoxin and latrodectin 1 Ayoub, N.A., Garb, J.E., Hedin, M., Hayashi, C.Y., 2007. Utility of the nuclear protein-coding gene, elongation factor-1 gamma (EF-1gamma), for spider systematics, emphasizing function together. Nevertheless, Grishin et al. (1993) used latrodectin family level relationships of tarantulas and their kin (Araneae: Mygalomorphae). 1-specific antibodies to show that inhibition of latrodectin 1 in the pres- Mol. Phylogenet. Evol. 42, 394–409. ence of α-latrotoxin resulted in a decrease in calcium ion entry in syn- Casewell, N.R., Huttley, G.A., Wüster, W., 2012. Dynamic evolution of venom proteins in α squamate reptiles. Nat. Commun. 3, 1066. aptosomes relative to -latrotoxin alone, but latrodectin 1 inhibition Casewell, N.R., Wüster, W., Vonk, F.J., Harrison, R.A., Fry, B.G., 2013. Complex cocktails: the also resulted in an increase in neurotransmitter release in a separate ex- evolutionary novelty of venoms. Trends Ecol. Evol. 28, 219–229. periment. Despite these somewhat mixed results, Grishin et al. (1993) Coddington, J.A., Giribet, G., Harvey, M.S., Prendini, L., Walter, D.E., 2004. Arachnida. In: α Cracraft, J., Donoghue, M. (Eds.), Assembling the tree of life. Oxford University suggested that latrodectins work to enhance -latrotoxin function by Press, pp. 296–318. promoting calcium influx and neurotransmitter release. The derivation Conticello, S.G., Gilad, Y., Avidan, N., Ben-Asher, E., Levy, Z., Fainzilber, M., 2001. Mecha- of latrodectins from Ion Transport Peptides (ITP) suggests that their nisms for evolving hypervariability: the case of conopeptides. Mol. Biol. Evol. 18, – function in black widow venom may involve modulating the transport 120 131. Delport, W., Poon, A.F.Y., Frost, S.D.W., Kosakovsky Pond, S.L., 2010. Datamonkey 2010: a of calcium ions around or near nerve cells, potentially to enhance the suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26, toxicity of α-latrotoxin. For example, locust Ion Transport Peptide 2455–2457. fi (ITP) functions to control the movement of chloride anions across cellu- Duda, T.F., Palumbi, S.R., 1999. Molecular genetics of ecological diversi cation: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc. Natl. Acad. lar membranes of the hindgut (Audsley et al., 1992). Moreover, Sci. U. S. A. 96, 6820–6823. latrotoxins and latrodectins could form a complex that in vivo negative- Duda Jr., T.F., Chang, D., Lewis, B.D., Lee, T., 2009. Geographic variation in venom allelic ly affects neurons by offsetting ion balance near different channel types, composition and diets of the widespread predatory marine gastropod Conus ebraeus. Plos One 4, e6245. which may or may not contribute to neurotransmitter release. While Dulubova, I.E., et al., 1996. Cloning and structure of delta-latroinsectotoxin, a novel insect- the evolutionary analyses we present reinforces the functional impor- specific member of the latrotoxin family: functional expression requires C-terminal tance of latrodectins in black widow spider venom, more extensive neu- truncation. J. Biol. Chem. 271, 7535–7543. Edgar, R.C., 2004. MUSCLE: a multiple sequence alignment method with reduced time rophysiological and biochemical studies of their structure and activities and space complexity. BMC Bioinformatics 5, 113. in relation to the better understood latrotoxins are clearly needed to Escoubas, P., Sollod, B., King, G.F., 2006. Venom landscapes: mining the complexity of spi- determine their function in whole venom. der venoms via a combined cDNA and mass spectrometric approach. Toxicon 47, 650–663. Fry, B.G., et al., 2009. The toxicogenomic multiverse: convergent recruitment of proteins 4.4. Conclusions into animal venoms. Annu. Rev. Genomics Hum. Genet. 10, 483–511. Garb, J.E., Hayashi, C.Y., 2005. Modular evolution of egg case silk genes across orb- – Using phylogenetic analyses and evidence from gene structure, we weaving spider superfamilies. Proc. Natl. Acad. Sci. U. S. A. 102, 11379 11384. Garb, J.E., Hayashi, C.Y., 2013. Molecular evolution of α-latrotoxin, the exceptionally po- show that latrodectin peptides from black widow spider venom are tent vertebrate neurotoxin in black widow spider venom. Mol. Biol. Evol. 30, derived from the ecdysozoan superfamily of neuropeptides containing 999–1014. Crustacean Hyperglycemic Hormones (CHH) and Ion Transport Pep- Garb, J.E., Gonzalez, A., Gillespie, R.G., 2004. The black widow spider genus Latrodectus (Araneae: Theridiidae): phylogeny, biogeography, and invasion history. Mol. tides (ITP). Evidence of positive and negative selection operating on Phylogenet. Evol. 31, 1127–1142. latrodectin sequences supports an important functional role of these Gasparini, S., et al., 1994. The low molecular weight protein which co-purifies with α- peptides in black widow venom. Our phylogenetic evidence also sug- latrotoxin is structurally related to crustacean hyperglycemic hormones. J. Biol. Chem. 269, 19803–19809. gests the deployment of CHH/ITP homologs for venom expression in ad- Graudins, A., Gunja, N., Broady, K.W., Nicholson, G.M., 2002. Clinical and in vitro evidence ditional arthropod taxa, consistent with the independent recruitment of for the efficacy of Australian red-back spider (Latrodectus hasselti) antivenom in the C. McCowan, J.E. Garb / Gene 536 (2014) 366–375 375

treatment of envenomation by a Cupboard spider (Steatoda grossa). Toxicon 40, Nylander, J.A.A., 2004. MrModeltest v2. Program distributed by the author. (Evolutionary 767–775. Biology Centre, Uppsala University) (online at https://github.com/nylander/ Graudins, A., et al., 2012. Cloning and activity of a novel α-latrotoxin from red-back spider MrModeltest2 (Accessed 15 July 2013)). venom. Biochem. Pharmacol. 83, 170–183. Orlova, E.V., et al., 2000. Structure of α-latrotoxin oligomers reveals that divalent cation- Grishin, E.V., 1998. Black widow spider toxins: the present and the future. Toxicon 36, dependent tetramers form membrane pores. Nat. Struct. Biol. 7, 48–53. 1693–1701. Pescatori, M., Bradbury, A., Bouet, F., Gargano, N., Mastrogiacomo, A., Grasso, A., 1995. The Grishin, E.V., et al., 1993. Modulation of functional activities of the neurotoxin from black cloning of a cDNA encoding a protein (latrodectin) which co-purifies with the α- widow spider venom. FEBS Lett. 336, 205–207. latrotoxin from the black widow spider Latrodectus tredecimguttatus (Theridiidae). Holz, G.G., Habener, J.F., 1998. Black widow spider α-latrotoxin: a presynaptic neurotoxin Eur. J. Biochem. FEBS 230, 322–328. that shares structural homology with the glucagon-like peptide-1 family of insulin Platnick, N.I., 2013. The world spider catalog, version 13.5. American Museum of Natural secretagogic hormones. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 121, 177–184. History (online at http://research.amnh.org/iz/spiders/catalog (Accessed 15 July Johnson, J.H., et al., 1998. Novel insecticidal peptides from Tegenaria agrestis spider venom 2013)). may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Posada, D., 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, Physiol. 38, 19–31. 1253–1256. Kiyatkin, N.I., Dulubova, I.E., Chekhovskaya, I.A., Grishin, E.V., 1990. Cloning and structure Ronquist, F., et al., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and of cDNA encoding alpha-latrotoxin from black widow spider venom. FEBS Lett. 270, model choice across a large model space. Syst. Biol. 61, 539–542. 127–131. Sollod, B.L., Wilson, D., Zhaxybayeva, O., Gogarten, J.P., Drinkwater, R., King, G.F., 2005. Kiyatkin, N., Dulubova, I., Chekhovskaya, I., Lipkin, A., Grishin, E., 1992. Structure of the Were arachnids the first to use combinatorial peptide libraries? Peptides 26, low molecular weight protein copurified with α-latrotoxin. Toxicon 30, 771–774. 131–139. Kiyatkin, N.I., Kulikovskaya, I.M., Grishin, E.V., Beadle, D.J., King, L.A., 1995. Functional Swofford, D.L., 2003. PAUP*. Phylogenetic analysis using parsimony (*and other characterization of black widow spider neurotoxins synthesised in insect cells. Eur. methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. J. Biochem. FEBS 230, 854–859. Ushkaryov, Y.A., Volynski, K.E., Ashton, A.C., 2004. The multiple actions of black Kordis, D., Gubensek, F., 2000. Adaptive evolution of animal toxin multigene families. widow spider toxins and their selective use in neurosecretion studies. Toxicon Gene 261, 43–52. 43, 527–542. Montagne, N., Desdevises, Y., Soyez, D., Toullec, J.-Y., 2010. Molecular evolution of the Vetter, R.S., Isbister, G.K., 2008. Medical aspects of spider bites. Annu. Rev. Entomol. 53, crustacean hyperglycemic hormone family in ecdysozoans. BMC Evol. Biol. 10, 62. 409–429. Müller, G., Kriegler, A., Van Zyl, J., van der Walt, B., Dippenaar, van Jaarsveld, P., 1992. Volkova, T.M., Pluzhnikov, K.A., Woll, P.G., Grishin, E.V., 1995. Low molecular weight com- Comparison of the toxicity, neurotransmitter releasing potency and polypeptide ponents from black widow spider venom. Toxicon 33, 483–489. composition of the venoms from Stetoda foravae, Latrodectus indistinctus,and Volynski, K.E., Nosyreva, E.D., Ushkaryov, Y.A., Grishin, E.V., 1999. Functional expression of L. geometricus (Araneae: Theridiidae). S. Afr. J. Sci. 88, 113–116. α-latrotoxin in baculovirus system. FEBS Lett. 442, 25–28. Naamati, G., Askenazi, M., Linial, M., 2009. ClanTox: a classifier of short animal toxins. Wong, E.S.W., Belov, K., 2012. Venom evolution through gene duplications. Gene 496, 1–7. Nucleic Acids Res. 37, W363–W368. Yang, Z., 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, Nicholson, G.M., Graudins, A., 2002. Spiders of medical importance in the Asia-Pacific: 1586–1591. atracotoxin, latrotoxin and related spider neurotoxins. Clin. Exp. Pharmacol. Physiol. Yang, Z., Nielsen, R., Goldman, N., Pedersen, A.M., 2000. Codon-substitution models for 29, 785–794. heterogeneous selection pressure at amino acid sites. Genetics 155, 431–449.