A peer-reviewed version of this preprint was published in PeerJ on 1 June 2018. View the peer-reviewed version (peerj.com/articles/4691), which is the preferred citable publication unless you specifically need to cite this preprint. Zobel-Thropp PA, Bulger EA, Cordes MHJ, Binford GJ, Gillespie RG, Brewer MS. 2018. Sexually dimorphic venom proteins in long-jawed orb-weaving spiders (Tetragnatha) comprise novel gene families. PeerJ 6:e4691 https://doi.org/10.7717/peerj.4691 Sexually dimorphic venom proteins in long-jawed orb-weaving spiders (Tetragnatha) with potential roles in sexual interactions Pamela A Zobel-Thropp 1 , Emily A Bulger 2 , Matthew H J Cordes 3 , Greta J Binford 1 , Rosemary G Gillespie 4 , Michael S Brewer Corresp. 5 1 Department of Biology, Lewis and Clark College, Portland, OR, United States 2 Division of Biological Sciences, University of California, San Diego, San Diego, CA, United States 3 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, United States 4 Department of Environmental Science, Policy, and Management, University of California, Berkeley, Berkeley, CA, United States 5 Department of Biology, East Carolina University, Greenville, NC, United States Corresponding Author: Michael S Brewer Email address: [email protected] Venom has been associated with the ecological success of many groups of organisms, most notably reptiles, gastropods, and arachnids. In some cases, diversification has been directly linked to tailoring of venoms for dietary specialization. Spiders in particular are known for their diverse venoms and wide range of predatory behaviors, although there is much to learn about scales of variation in venom composition and function. The current study focuses on venom characteristics in different sexes within a species of spider. We chose the genus Tetragnatha (Tetragnathidae) because of its unusual courtship behavior involving interlocking of the venom delivering chelicerae (i.e., the jaws), and several species in the genus are already known to have sexually dimorphic venoms. Here, we use transcriptome and proteome analyses to identify venom components that are dimorphic in Tetragnatha versicolor. We present cDNA sequences of unique high molecular weight proteins that are only present in males and that have remote, if any, detectable similarity to known venom components in spiders or other venomous lineages and several have no detectable homologs in existing databases. While the function of these proteins is not known, their presence in association with the cheliceral locking mechanism during mating together with the presence of prolonged male-male mating attempts in a related, cheliceral-locking species (Doryonychus raptor) lacking the dimorphism suggests potential for a role in sexual communication. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3358v1 | CC BY 4.0 Open Access | rec: 20 Oct 2017, publ: 20 Oct 2017 1 2 3 4 Sexually dimorphic venom proteins in long-jawed orb-weaving spiders (Tetragnatha) with 5 potential roles in sexual interactions 6 7 Pamela A. Zobel-Thropp1, Emily A. Bulger2, Mathew H.J. Cordes3, Greta J. Binford1, Rosemary 8 G. Gillespie4, Michael S. Brewer5 9 10 1Department of Biology, Lewis and Clard College, Portland, OR, USA 11 2Division of Biological Sciences, University of California, San Diego, San Diego, CA, USA 12 3Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA 13 4Deparment of Environmental Science, Policy, and Management, University of California, 14 Berkeley, Berkeley, CA, USA 15 5Department of Biology, East Carolina University, Greenville, NC, USA 16 17 Corresponding Author: 18 Michael S. Brewer5 19 20 Email address: [email protected] 21 22 23 24 ABSTRACT 25 Venom has been associated with the ecological success of many groups of organisms, most 26 notably reptiles, gastropods, and arachnids. In some cases, diversification has been directly 27 linked to tailoring of venoms for dietary specialization. Spiders in particular are known for their 28 diverse venoms and wide range of predatory behaviors, although there is much to learn about 29 scales of variation in venom composition and function. The current study focuses on venom 30 characteristics in different sexes within a species of spider. We chose the genus Tetragnatha 31 (Tetragnathidae) because of its unusual courtship behavior involving interlocking of the venom 32 delivering chelicerae (i.e., the jaws), and several species in the genus are already known to have 33 sexually dimorphic venoms. Here, we use transcriptome and proteome analyses to identify 34 venom components that are dimorphic in Tetragnatha versicolor. We present cDNA sequences 35 of unique high molecular weight proteins that are only present in males and that have remote, if 36 any, detectable similarity to known venom components in spiders or other venomous lineages 37 and several have no detectable homologs in existing databases. While the function of these 38 proteins is not known, their presence in association with the cheliceral locking mechanism during 39 mating together with the presence of prolonged male-male mating attempts in a related, 40 cheliceral-locking species (Doryonychus raptor) lacking the dimorphism suggests potential for a 41 role in sexual communication. 42 43 INTRODUCTION 44 Phenotypic differences between sexes are widespread among animals, and can be attributed to 45 sexual and/or natural selection. Sexual selection is the most common explanation for 46 morphological and behavioral differences, which are frequently attributed to intra- or intersexual 47 competition for mates. However, natural selection may also lead to differences as a result of 48 bimodal or dimorphic niche separation between the sexes (Berns, 2013). Recent work has 49 highlighted the importance of additional modalities that differ between sexes, including acoustic 50 (Elias & Mason, 2014) and chemical (Wyatt, 2014) traits. Here we focus on sexual differences in 51 chemical traits, and more specifically on venom, in which several studies have highlighted 52 variation according to sex. We examine sexual differences in venom composition of spiders, and 53 consider mechanisms of selection that may have given rise to these differences. 54 Venoms are complex chemical cocktails that attract research attention in both applied and 55 basic sciences and have been characterized in many animals, including mammals (shrews, 56 platypus), toxicoferan reptiles (lizards and snakes), fish, sea anemones, cephalopods, cone snails, 57 insects, centipedes, scorpions, shrews, and arachnids (Fry et al., 2009). They can be impressively 58 complex; among spiders in particular, individual venoms can have 1000s of components 59 (Escoubas, 2006). The complexity typically consists of related sets of molecules within which 60 are components with exquisite functional specificity, and novel activities (reviews in (Kuhn- 61 Nentwig & Stöcklin, 2011; Smith et al., 2013; King, 2015). Activities involve manipulation of 62 physiological processes, particularly neurological, thus they are a source of discovery of 63 components with human applications in pharmaceuticals or insecticides (King, 2015). 64 Venoms frequently exhibit sexual dimorphism (snakes, (Menezes et al., 2006); scorpions, 65 (D'Suze, Sandoval & Sevcik, 2015; Miller et al., 2016); spiders, (Herzig et al., 2008; Binford, 66 Gillespie & Maddison, 2016b)). Because the primary functional roles of venoms in spiders are 67 thought to be predation and defense, in most cases sexual differences in venom composition 68 between adults of many species are hypothesized to result from natural selection optimizing 69 composition based on differences in feeding biology and associated differences in diet 70 composition and/or vulnerability to predation. Thus, hypotheses to explain sexual dimorphism in 71 spider venoms typically center on differential optimization of chemical pools for divergent adult 72 niches. Though largely untested, these hypotheses provide plausible explanations for many 73 known dimorphisms. For example, in Sydney funnel-web spiders (Atrax robustus), male venoms 74 are more toxic to mammals than are those of females (Gray & Sutherland, 1978), potentially 75 associated with male Atrax being found more often wandering outside of burrows than females 76 (Isbister & Gray, 2004). In contrast, in the theridiid spiders Latrodectus mactans and Steatoda 77 paykulliana, female venoms have higher mammalian neurotoxic activity than male venoms 78 {Maretic:1964um}. These spiders eat vertebrates and bite humans defensively, so the general 79 pattern of increased female potency has been attributed to the shorter lifespan of male spiders 80 and reduced adult foraging of males (Rash, King & Hodgson, 2000). 81 While sexual differences in venom composition driven by adult niche are likely, venoms 82 are also known to play a role in sexual biology (Polis & Sissom, 1990) and thus may be under 83 the influence of sexual selection. As secreted molecules with intra-individual functionality, they 84 have potential for biological roles in sex (Binford, Gillespie & Maddison, 2016b). The possibility 85 that different components within an individual’s venom have different roles and/or contexts of 86 usage, sets up exciting potential for considering competing or complementary evolutionary 87 mechanisms influencing venom phenotypes. 88 The long jawed orb-weaving spiders (Araneae: Tetragnathidae: Tetragnatha) provide a 89 compelling context to explore the nature and potential cause of sexual dimorphism in venoms. 90 Members of the genus Tetragnatha are broadly distributed with ca. 347 species worldwide 91 (World Spider Catalog). The majority of species worldwide are remarkably uniform in 92 appearance, dull brown or olive in color, with long first and second legs, typically long jaws in 93 adulthood, and an elongate opisthosoma (Levi, 1981). Their behavior and ecology is also fairly 94 homogeneous as they generally construct a light and fragile orb web with an open center and 95 build the web over water or in other wet places (Gillespie, 1987), although they have undergone 96 adaptive radiation in the Hawaiian Islands, associated with marked shifts in ecology and 97 behavior (Gillespie, 2004a; Blackledge & Gillespie, 2004). One striking aspect of this genus of 98 spiders is their very unusual sexual behavior: While courtship in most spiders involves an 99 elaborate and extended period of vibrational or visual communication, in most Tetragnatha there 100 is little evident communication prior to the male and female approaching each other. They 101 connect physically by spreading the chelicerae wide and locking fangs (Figure 1A), involving a 102 dorsal spur on the male chelicerae. The cheliceral locking mechanism apparently precludes the 103 need for epigynal coupling and is associated with secondary loss of a sclerotized epigynum (Levi, 104 1981). 105 Comparisons of crude venoms between sexes of Tetragnatha using 1-D protein 106 electrophoresis have identified a particularly striking sexual dimorphism in which males have an 107 abundance of high molecular weight components that are not in females (Binford, Gillespie & 108 Maddison, 2016b). Phylogenetic comparisons across species indicate that these high molecular 109 weight components persist across an evolutionary transition in feeding biology that reduces the 110 differences in adult feeding niches. Specifically, adult males of orb-weaving Tetragnatha species 111 do not typically build webs and are functionally wandering predators. However, a lineage of 112 Tetragnatha in Hawaii has lost orb-weaving behavior (Gillespie, 2004b; 2005) and both males 113 and females wander in search of prey, thus reducing dimorphism in feeding biology. Males could 114 be more prone to predation and have unique components that function in defense, but if so, 115 increased vulnerability would also affect female wandering Tetragnatha. So a defensive role 116 does not seem likely. This pattern, combined with the cheliceral locking behaviors during 117 copulation, lead to a hypothesis that the unique male components in venom play some as yet 118 undescribed role in mating biology (Binford, Gillespie & Maddison, 2016b). 119 A starting place for testing our hypothesis that the unique components in male 120 Tetragnatha venoms function in sexual biology, is to characterize the chemicals that contribute 121 to the differences. The goal of this study is to identify the molecules that are sexually dimorphic 122 in venoms of a readily accessible “model” species, Tetragnatha versicolor. Using comparative 123 venom gland transcriptomes and proteomes of adult males and females, we identify sequence 124 characteristics of dimorphic components, with particular attention to those unique to males. We 125 infer function preliminarily using homology searching and report the discovery of divergent 126 highly expressed male-specific proteins that support the potential for functioning in mating 127 biology. 128 129 MATERIAL AND METHODS 130 Collection. Individuals of Tetragnatha versicolor were collected by hand from three populations: 131 along the southern fork of Strawberry Creek on the UC Berkeley Campus (UCB) (37.872ºN, 132 122.262ºW), Little Sugar Creek, Binford Farm (BF), Crawfordsville, Indiana (40.061ºN, 133 86.853ºW), and Greenville, North Carolina (ECU) (35.626ºN, 77.409ºW). 134 Venom Extraction. Live specimens were transported to Lewis & Clark College where venom 135 was extracted by electrostimulation (Binford, 2001). We obtained venom samples from 19 136 females and 16 males from UCB, nine females and two males from BF, and nine females and 137 five males from ECU. To compile sufficient protein amounts for proteomics analyses, venom 138 was pooled within sexes for each of these three populations. 139 RNA Isolation Trancsriptomic analyses were performed using RNA isolated only from Indiana 140 (BF) specimens. To capture some breadth of transcriptional timing after emptying venom glands, 141 surviving spiders (10 females and two males) were divided into two groups within each sex, and 142 venom glands were isolated from five females and one male two and three days after venom 143 extraction. The glands extracted two and three days after milking were pooled within sexes, and 144 processed and analyzed separately between sexes for all subsequent analyses. To extract glands, 145 spiders were anesthetized with CO and venom glands were removed by dissection and flash- 2 146 frozen immediately in liquid nitrogen. Total RNA was isolated by grinding tissues in TRIzol® 147 reagent (Life Technologies, Carlsbad, CA), adding chloroform (200 μL per mL of TRIzol®), 148 mixing by inversion, and incubating for 20 min at 4° C. The tube was centrifuged at 4° C for 15 149 min at 14,000 rpm. An equal volume of cold 100% ethanol was added to the RNA-containing 150 upper aqueous phase. The solution was then passed through an RNeasy® Mini Spin Column 151 (Qiagen, Chatsworth, CA) and purified, according to RNeasy® protocols. 152 Illumina RNA Sequencing and Quality Control. RNA extractions from the BF population of T. 153 versicolor males (2) and females (10) were shipped to the Genomic Services Lab at 154 HudsonAlpha (Huntsville, AL), where cDNA libraries were prepared from total RNA (poly-A 155 isolation, Illumina TruSeq RNA Library Prep Kit v2), and 50 bp paired-end Illumina HiSeq 2500 156 RNA-seq was used to generate sequence reads. All QC trimming and assemblies were done 157 using a pipeline provided by the UC Berkeley Museum of Vertebrate Zoology 158 (https://github.com/MVZSEQ). Quality and GC content of the resulting paired-end reads was 159 assessed using the FastQC v0.10.0 program 160 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). TRIMMOMATIC v0.36 (Bolger, 161 Lohse & Usadel, 2014) and CUTADAPT v1.7.1 (Martin, 2011) were used to clean up the 162 sequence data: nucleotides below a quality threshold of 20 were trimmed from the ends of 163 sequences, and sequences shorter than 36 nucleotides (after trimming) were discarded. The reads 164 were aligned to a custom library of bacterial sequences to remove prokaryotic contamination 165 using BOWTIE2 v2.1.0 (Langmead & Salzberg, 2012). Individual paired-end files were 166 resynchronized, removing any paired-end sequences only present in one of the two files. Left and 167 right reads that overlap were combined into a single longer read to aid in downstream assembly 168 using FLASH v1.2.7 (Magoč & Salzberg, 2011). 169 Transcriptome Assembly and ORF Prediction. The resulting male and female files were 170 assembled separately, by sex, and combined for a general T. versicolor venom transcriptome. 171 The processed reads for each sex and the combined read files were assembled using the 172 TRINITY pipeline v2.0.6 (http://trinityrnaseq.sourceforge.net/) with default parameters except 173 the following, group_pairs_distance=999 and min_kmer_cov=2. High-confidence open reading 174 frames (ORFs) (i.e., likely coding sequences), were obtained for each gene in the transcriptome 175 using TRANSDECODER r20140704 (Haas et al., 2013). A minimum protein length of 30 amino 176 acids was used in ORF predictions. The completeness of each assembly was assessed via 177 BUSCO v1.1 (Simão et al., 2015). 178 Read mapping to identify transcriptome dimorphisms. The processed reads from each sex were 179 mapped against the combined assembly to identify genes that are unique to either sex, with 180 particular emphasis placed on male-only transcripts. The mapping was performed using STAR 181 v2.4.2a (Dobin et al., 2012) and default parameters. A custom python script was used to generate 182 a BED file from the combined transcriptome and BEDTOOLS v2.18.1 “multicov” (Quinlan & 183 Hall, 2010) was used to generate counts of reads from each sex mapping to combined assembly 184 transcripts. An additional round of mapping with BOWTIE v1.1.1 (Langmead et al., 2009)