1 Collateral death—Australian funnel-web evolved human-lethal 2 δ-hexatoxins for defense against vertebrate predators 3 4 Volker Herzig1,2*@, Kartik Sunagar3*, David T.R. Wilson4*, Sandy S. Pineda1,5*, Mathilde R. 5 Israel1, Sebastien Dutertre6, Brianna Sollod McFarland7, Eivind A.B. Undheim1,8,9, Wayne C. 6 Hodgson10, Paul F. Alewood1, Richard J. Lewis1, Frank Bosmans11, Irina Vetter1,12, Glenn F. 7 King1@, Bryan G. Fry13@ 8 9 1. Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia 10 2. School of Science & Engineering, University of the Sunshine Coast, Sippy Downs, QLD 4556, Australia 11 3. Evolutionary Venomics Lab, Centre for Ecological Sciences, Indian Institute of Science, Bangalore 12 560012. 13 4. Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook 14 University, Smithfield, QLD 4878; Australia 15 5. Brain and Mind Centre, University of Sydney, Camperdown, NSW 2052, Australia 16 6. Institut des Biomolécules Max Mousseron, UMR 5247, Université Montpellier - CNRS, Place Eugène 17 Bataillon, 34095 Montpellier Cedex 5, France 18 7. Sollod Scientific Analysis, Ft. Collins, CO, USA 19 8. Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD 4072, Australia 20 9. Centre for Ecology and Evolutionary Synthesis, Department of Biosciences, University of Oslo, 0316 21 Oslo, Norway 22 10. Monash Venom Group, Department of Pharmacology, Monash University, Building 13E, Clayton 23 Campus, Clayton, VIC 3800, Australia 24 11. Basic and Applied Medical Sciences Department, Faculty of Medicine, Ghent University, 9000 Ghent, 25 Belgium 26 12. School of Pharmacy, The University of Queensland, Woolloongabba QLD 4102, Australia 27 13. Venom Evolution Laboratory, School of Biological Sciences, The University of Queensland, St. Lucia, 28 QLD 4072, Australia 29 * equal contribution 30 @ Address correspondence to [email protected], [email protected] or [email protected] 31 32 Classification: BIOLOGICAL SCIENCES: Evolution 33 Keywords: venom; Australian funnel-web spider; lethal toxin; predation; predator deterrence; toxin 34 evolution

1 35 Abstract (250 words max; currently 248) 36 Australian funnel-web spiders are infamous for causing human fatalities, which are induced by 37 venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in 38 the prey or predator spectrum during evolution of these spiders, and consequently the primate 39 lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male 40 spiders that wander from their burrow in search of females during the mating season, which 41 suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. 42 Although 35 species of funnel-web spiders have been described, only eight δ-HXTXs from five 43 species have been characterized, resulting in a lack of understanding of the ecological roles and 44 molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of ten funnel- 45 web species, we report 24 novel δ-HXTXs. Phylogenetic and evolutionary assessments reveal a 46 remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within 47 funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal 48 toxin from the Sydney funnel-web spider robustus, induces pain in mice by inhibiting

49 inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signalling. δ-HXTX-

50 Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. 51 Considering their algogenic effects in mice, potent insecticidal effects, and high-levels of sequence 52 conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory 53 function to a role in defending against non-human vertebrate predators by male spiders, with their 54 lethal effects on humans being an unfortunate evolutionary coincidence. 55 56 Significance Statement (120 words max; currently 120) 57 The venom of Australian funnel-web spiders contains δ-hexatoxins (δ-HXTXs) that exert fatal 58 neurotoxic effects in humans by inhibiting inactivation of voltage-gated sodium channels, but their 59 precise ecological role remains unclear. Sequencing of venom-gland transcriptomes from ten 60 funnel-web species uncovered 24 novel δ-HXTXs. Evolutionary analysis revealed extreme 61 conservation of these toxins, despite their ancient origin. We isolated the lethal δ-HXTX from 62 venom of the Sydney funnel-web spider and showed that it induces pain in mice, suggesting a role 63 in predator deterrence. Although humans are not the target of δ-HXTXs, these toxins likely evolved 64 to deter vertebrate predators commonly encountered by these spiders, such as bandicoots, birds and 65 lizards. Thus, the lethal potency of δ-HXTXs against humans is an unfortunate evolutionary

2 66 coincidence.

3 67 Introduction 68 Despite their fearsome reputation, only a few species of spiders can cause death or serious harm to 69 humans (1). An infamous exception is the Australian funnel-web spider, arguably the world’s 70 deadliest spider (2). These spiders produce extraordinarily complex venoms, with each venom 71 containing up to several thousand peptide toxins (3, 4). Despite this chemical complexity, a single 72 family of toxins known as the δ-hexatoxins (δ-HXTXs) are responsible for the human 73 envenomation syndrome (5). There are currently 35 described species of Australian funnel-web 74 spiders and 33 species of related non-Australian spiders in the , but to date only 75 eight δ-HXTX sequences have been reported from five species within this broad clade. A 76 homologous δ-actinopoditoxin (δ-AOTX) is present in venom of the related Australian mouse 77 spider bradleyi (6), which can cause serious human envenomations with symptoms 78 resembling those from funnel-web spider bites (7). 79 80 δ-HXTXs and δ-AOTX comprise 42–44 residues and contain four disulfide bonds, three of which 81 are arranged in an inhibitor cystine knot (ICK) motif (8, 9). δ-HXTXs slow the inactivation of

82 vertebrate tetrodotoxin (TTX)-sensitive voltage-gated sodium (NaV) channels and insect NaV 83 channels by binding to the voltage-sensor in channel domain IV (10, 11). In human bite victims, δ- 84 HXTXs cause disturbances in respiration, blood pressure and heart rate, followed by severe 85 hypotension. Without treatment with commercial antivenom (5), fatalities can occur by respiratory 86 and circulatory failure within a few hours of the bite (12). Interestingly, in striking contrast to 87 humans and other primates, some vertebrates such as dogs and cats are insensitive to funnel-web 88 envenomation (13). 89 90 Humans did not feature in the prey or predator spectrum during evolution of funnel-web spiders as 91 primates were not present 150–200 MYA when these spiders originated (14). Thus, the underlying 92 reason for the peculiar susceptibility of humans to δ-HXTXs and the ecological role of these toxins 93 remains enigmatic. The δ-HXTXs are insecticidal (15, 16), which might suggest a role in prey 94 capture. However, in some species, these toxins are secreted in very low abundance in the venoms 95 of female spiders and immature males, consistent with the fact that only sexually mature male 96 spiders cause severe or lethal human envenomations (17). Moreover, it is hard to reconcile a role 97 for these toxins in predation given that sexually mature males, in whose venom the toxins are most

4 98 abundant, rarely feed during the mating season. Rather, the fact that adult males leave the safety of 99 their burrows to search for female spiders (2), making them more susceptible to predators, suggests 100 a role for the δ-HXTXs in predator deterrence. A well-documented strategy for defensive toxins is 101 to induce pain (18-20), and pain is a common symptom following funnel-web envenomation (2, 102 17). Consistent with the idea that the δ-HXTXs serve a defensive role by inducing pain in vertebrate 103 predators, Magi 4 from the venom of a Japanese funnel-web spider potentiates the activity of

104 NaV1.1 and NaV1.6 (21), which are involved in pain signalling (19, 22). 105 106 In the current study, we identified 24 novel δ-HXTX sequences from ten species of Australian 107 funnel-web spiders, and evaluated their molecular evolution, phylogenetic histories, insecticidal

108 activity, and potency against human NaV channels involved in pain signalling. Taken together, our 109 data provides strong evidence that the δ-HXTXs were recruited by funnel-web spiders as a weapon 110 to deter vertebrate predators, and that their lethal effects on humans is an unfortunate evolutionary 111 coincidence. 112 113 RESULTS 114 δ-HXTX sequences 115 δ-HXTXs from seven funnel-web spider species ( infensa, H. valida, H. venenata, 116 H. versuta, Atrax robustus, A. sutherlandi and Illawarra wisharti) were sequenced via Rapid 117 Amplification of cDNA Ends (RACE) (see Supporting information for details). In addition, we 118 generated cDNA libraries for H. infensa, H. modesta, H. cerberea and H. formidabilis. Our 119 complete dataset (see Supplementary Excel Table 1), based on our RACE and transcriptomic data 120 plus sequences available from the literature, comprised 169 mature peptide sequences and 167 121 corresponding nucleotide sequences (the latter were only missing for δ-HXTX-Hv1b and δ-AOTX- 122 Mb1a). Our dataset contained 132 δ-HXTXs, 1 δ-AOTX, and 18 homologous U-HXTXs from 123 H. infensa, 17 related U-BATXs from Trittame loki, and 1 μ-HXTX from Macrothele gigas. 124 Removal of 53 duplicate or incomplete sequences and the two peptide sequences that lacked a 125 corresponding nucleotide sequence yielded a total of 114 nucleotide sequences for phylogenetic 126 analysis. The data revealed 24 new mature δ-HXTXs sequences to complement the eight published 127 δ-HXTX sequences. 128

5 129 Phylogenetic history and molecular evolution of δ-HXTXs 130 The identification of homologous sequences from the barychelid spider T. loki in this study reveals 131 that these U-BATXs, μ-HXTX-Mg1c and the δ-HXTXs were probably derived from a common 132 ancestral toxin. However, there are also important differences between the U-BATXs and μ- 133 HXTX-Mg1c in comparison to all δ-HXTXs from atracid and macrothelid funnel-web spiders. 134 Firstly, U-BATXs and μ-HXTX-Mg1c have only six Cys residues that form an ICK motif, whereas 135 the δ-HXTXs usually contain eight Cys residues, with one an additional disulfide bond. Secondly, 136 the δ-HXTXs comprise a rare triplet of Cys residues in positions 14–16 (based on δ-HXTX-Ar1a), 137 which is not present in μ-HXTX-Mg1c or any of the U-BATXs. Moreover, in the Bayesian 138 phylogenetic analyses, all funnel-web spider sequences clustered together and were well separated 139 from the T. loki sequences (Fig. 1). All of these considerations taken together highlight the 140 divergent evolution of δ-HXTXs in atracid and macrothelid funnel-web spiders following their 141 phylogenetic separation from other mygalomorph lineages (Fig. 1). 142 143 In contrast to the massive sequence variations observed in most venom proteins, the δ-HXTXs are 144 surprisingly well-conserved (Fig. 2). The signal peptide, propeptide and mature peptide coding 145 regions in the δ-HXTXs were characterized by extremely low omega (ω) values (i.e., the ratio of 146 non-synonymous to synonymous substitutions) (Fig. 2A; Table 1), indicative of strong sequence 147 conservation despite being recruited into the venoms of funnel-web spiders around 150–200 MYA 148 (14). Consistent with the findings of the maximum-likelihood method, the Bayesian approaches 149 identified only three positively selected sites (2.6% of sites)—positions that rapidly diversify across 150 time—while many sites (15.6%) were found to be evolving under the pervasive influence of 151 negative selection, which ensures sequence conservation across time. A few amino acid sites 152 (n = 7) were found to be experiencing diversifying selection, albeit in an episodic fashion (i.e., in 153 short bursts across time) (Fig. 2B-D). 154 155 Cys derivations and codon usage 156 The three-dimensional structure of many venom peptides, including the δ-HXTXs, relies heavily 157 on the formation of disulfide bonds. Analysis of the arrangement of eight Cys residues in the δ- 158 HXTXs (Fig. 3A) indicates that Cys residues 1–5 are extremely well-conserved in all funnel-web 159 spiders. However, many subtypes were discovered with either missing or novel cysteines,

6 160 indicating the probable evolution of novel forms and functions. For instance, while one derivation 161 each was found with a missing cysteine at positions 6 and 7, δ-HXTXs lacking Cys 8 were more 162 common with 11 derivations in δ-HXTXs from H. infensa, with one of them even possessing a 163 novel cysteine residue (which we refer to as position 9). The precursor peptides with the 8th Cys 164 residue missing correspond to four mature toxin sequences and comparison with combined MALDI 165 and Orbitrap mass spectrometry data for H. infensa, venom (4) reveals closely matching molecular 166 masses for three of the monomers, one homodimer and four heterodimers (Fig. 3B). Analysis of 167 the codons used for Cys residues also revealed some genus-specific variations (Fig. 3C). In all 168 funnel-web δ-HXTXs, Cys 2 is encoded by TGT, whereas Cys 3 is encoded by TGC. In Atrax and 169 Illawarra, all other Cys are encoded only by TGT (except for Cys 8 in Illawarra being solely 170 encoded by TGC). In Hadronyche, Cys 1, 5, 6, 7, and 8 are dominated by TGT and Cys 4 is 171 preferentially encoded by TGC. In contrast to all other funnel-web spider genera, in Macrothele all 172 Cys (except for Cys 2) are dominantly encoded by TGC. The observed codon bias could be 173 important for the rapid expression of toxins in the venom gland. 174

175 NaV channel subtype selectivity of δ-HXTXs-Ar1a 176 In order to examine the biological role of the δ-HXTXs, we used reversed-phase HPLC to isolate 177 δ-HXTX-Ar1a (hereafter Ar1a) from the venom of A. robustus (Fig. 4). We then used a FLIPR-

178 based fluorescent assay to assess the ability of Ar1a to potentiate currents from human NaV1.1–

179 NaV1.8 channels stably expressed in HEK293 cells. This assay was previously used to determine

180 the pharmacological activity of the scorpion venom peptide OD1. OD1 activates several NaV 181 channel subtypes and the potency and subtype selectivity of OD1 as determined by FLIPR was 182 found to be comparable to data obtained using electrophysiological assays (23). 183

184 Ar1a had no effect on NaV1.4, NaV1.5, NaV1.7 and NaV1.8 at concentrations up to 150 nM, whereas 185 a concentration-dependent potentiation of veratridine-evoked responses was observed in cells

186 expressing NaV1.1, NaV1.2, NaV1.3 and NaV1.6 (Fig 5A). Ar1a was an equipotent potentiator of

187 NaV1.1 (EC50 30.2 nM), NaV1.2 (EC50 38.9 nM), NaV1.3 (EC50 38.9 nM) and NaV1.6 (EC50 91.2

188 nM) with comparable mean pEC50 (i.e. negative logarithm of the EC50) values of 7.52 ± 0.24, 7.41 189 ± 0.09, 7.41 ± 0.15, and 7.04 ± 0.08, respectively. 190

7 191 Mammalian nocifensive responses 192 Administration of Ar1a (100 nM, 20 µl) by shallow subcutaneous (intraplantar) injection into the 193 foot pad of male C57BL/6 mice elicited mild, transient nocifensive responses consisting of 194 flinching, lifting, licking, and shaking of the hind paw. This nocifensive behaviour was 195 characterized by relatively slow onset (5 min post-injection) to a peak at 15 min post-injection with 196 10.5 ± 1.5 flinches per 5 min. No systemic effects, such as the muscle fasciculations, salivation or 197 other effects associated with the human envenomation syndrome, were observed at this dose. 198 199 Toxicity of Ar1a in blowflies

200 Injection of Ar1a into sheep blowflies (L. cuprina) caused contractile paralysis with a PD50 of 201 319 ± 42 pmol/g at 1 h post-injection (Fig: 5B). However, even at the highest dose tested (which 202 was limited by the amount of native δ-HXTX-Ar1a available), all flies fully recovered within 24 h, 203 indicating that the toxin's insecticidal effects in blowflies are reversible. 204

205 Effect of Ar1a on BgNaV1

206 Given its activity on human NaV channels, we decided to examine if the insecticidal effects of Ar1a

207 are due to potentiation of the activity of insect NaV channels. For this purpose, we examined the

208 effects of Ar1a on the BgNaV1 channel from the German cockroach Blattella germanica expressed

209 in Xenopus oocytes. At 1 nM, Ar1a had no effect on BgNaV1 currents whereas 10 nM Ar1a induced

210 a substantial persistent current (Fig. 5C). BgNaV1 fast inactivation was completely inhibited by

211 100 nM Ar1a. Boltzmann fits of the normalized conductance–voltage relationships (G/Gmax) and

212 steady-state inactivation relationships (I/Imax) revealed no significant difference in V1/2 before and 213 after addition of 1 or 10 nM Ar1a (t-test: p ≤ 0.01) but 100 nM Ar1a caused substantial

214 hyperpolarising shifts in the V1/2 of both channel activation (from –37.2 ± 0.6 to –44.9 ± 0.7 mV) 215 and steady-state inactivation (from –50.3 ± 0.1 to –63.3 ± 0.7 mV) (Fig. 5D). 216

8 217 DISCUSSION 218 In the present study, we employed a multi-pronged approach involving venom gland 219 transcriptomics, molecular and phylogenetic analyses, and functional assays to determine the role 220 of the lethal δ-HXTXs in the ecology of funnel-web spiders. 221 222 Enigmatic evolutionary conservation of δ-HXTXs 223 Molecular evolutionary assessments revealed that the genes encoding for δ-HXTXs have remained 224 nearly unchanged despite originating in the common ancestor of atracid and macrothelid funnel- 225 web spiders 150-200 MYA (14). Together with our phylogenetic analysis, this indicates that 226 despite the single early origin of δ-HXTXs in funnel-web spiders, they have diversified at a much 227 slower evolutionary rate than many other spider toxins (24-28). The increased level of sequence 228 conservation is consistent with a role for the δ-HXTXs in defense. Due to their relatively limited 229 use, and consequent exclusion from the typical Red Queen mode of competitive evolution, 230 defensive toxins are theorized to evolve slower than their predatory counterparts (31). 231 232 Our phylogenetic analysis demonstrates that all δ-HXTX sequences evolved from a common 233 ancestral toxin scaffold, with early gene duplications and diversification present before the recently 234 proposed split into the families Macrothelidae and Atracidae (32). Further support of a split 235 between these families is provided by the nucleotide sequences used to encode the Cys residues, 236 with Atracidae being dominated by TGT and Macrothelidae being dominated by TGC. Our 237 phylogeny also provides evidence for at least two neofunctionalisation events. The first event 238 occurred during the early evolution of δ-HXTXs. μ-HXTX-Mg1c, the most basal of the funnel- 239 web spider sequences included in our analysis, has an ICK motif but lacks the two Cys residues 240 involved in the formation of the 4th disulfide bond (33). μ-HXTX-Mg1c is a homolog of μ-HXTX- 241 Mg1a and μ-HXTX-Mg1b, which are both known to be insecticidal, but not active against 242 vertebrates (34). On the other hand, δ-HXTX-Mg1a, which is sister to the remaining δ-HXTX 243 sequences, has both vertebrate and insecticidal activities (34). These activities would be consistent 244 with a repurposing of δ-HXTXs from the purely insecticidal activity of their ancestral plesiotypic 245 form to dual activity against both mammals and insects, which occurred around 150-200 MYA 246 (14). Given the absence of primates in Australia at the time when δ-HXTX originated (humans first 247 populated Australia 65,000 years ago (35), and Australia lacks indigenous non-human primates),

9 248 the primate toxicity of δ-HXTXs can only be regarded as coincidental. The second 249 neofunctionalisation event is apparent in the genus Hadronyche with multiple convergent losses of 250 the last Cys residue. Such convergence points towards a strong selection pressure, possibly 251 resulting in a change in selectivity or potency. The fact that an odd number of Cys residues is 252 energetically unfavoured led us to investigate whether these 7 Cys residue derivations of δ-HXTXs 253 form dimers. Mass-spectrometric analysis of H. infensa venom (4) revealed the presence of masses 254 corresponding to monomers, homo- and heterodimers. Unfortunately, nothing is yet known about 255 the activities of these δ-HXTX derivations or the dimers that are formed, which provides an 256 exciting area for future investigations. 257 258 Clues from the activity of Ar1a 259 δ-HXTXs were previously demonstrated to inflict potent but reversible paralysis in blowfly larvae

260 and crickets (16). We found that Ar1a potently inhibits fast inactivation of the cockroach BgNaV 261 channel, which is consistent with the contractile paralysis induced in blowflies and reminiscent of

262 the effects of other toxins that target insect NaV channels [reviewed in (36)]. 263 264 Employing venom components to cause pain is a common evolutionary strategy for self-defense 265 in venomous (19, 31, 37-39). Strong support for a defensive role of δ-HXTXs is therefore 266 provided by the nocifensive response that Ar1a induced following intraplantar injections in mice. 267 Although the lethal dose of δ-HXTXs varies considerably within vertebrates (13), we presume that 268 high local tissue concentrations of δ-HXTXs resulting from a funnel-web spider bite will induce

269 algogenic effects in a much wider range of vertebrates than LD50 experiments might indicate. 270 271 With regard to the subtype selectivity of δ-HXTXs, δ-HXTX-Mg1a (Magi 4) from the Japanese

272 funnel-web spider preferentially activated rat NaV1.1, NaV1.3 and mouse NaV1.6, whilst also

273 showing weak activity at rat NaV1.2 channels [3]. For Ar1a, we observed equipotent activity across

274 NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Overall, this is consistent with a defensive role, as both

275 NaV1.1 and NaV1.6 are known to be involved in pain signalling (19, 22, 40). The activity of Ar1a

276 at NaV1.6 is further consistent with the observed effects of Australian funnel-web venoms in the

277 chick biventer assay (Fig. S1 and (41)), as NaV1.6 is the predominant isoform at the nodes of

278 Ranvier in motor neurons. Thus, inhibition of the inactivation of this NaV channel isoform could

10 279 contribute to both sensory and motor effects in envenomed individuals, making δ-HXTXs a 280 powerful weapon to deter predators. 281 282 Differential expression of δ-HXTXs 283 The clinical syndrome resulting from funnel-web spider envenomation of vertebrates is driven by 284 the δ-HXTXs (5). Male A. robustus venom was reported to be at least six-times more potent than 285 the female venom (42). In addition, the venoms of six male funnel-web species were found to be 286 more potent than females in inducing toxic effects in the chick biventer nerve-muscle preparation 287 (41), consistent with increased expression of δ-HXTXs in male venoms. Male funnel-web spiders 288 are more exposed to vertebrate predation once they leave the safety of their burrows to search for 289 female mates, so increased expression of a defensive toxin would make ecological sense to allow 290 adult males to defend against these predators. Moreover, since adult male mygalomorph spiders 291 consume less food than females (43), the increased expression of δ-HXTXs in mature male spiders 292 is inconsistent with a role for these toxins in prey capture. 293 294 Conclusion 295 In summary, our data suggest that the δ-HXTXs likely evolved from having an ancestral role in 296 predation to a primary role in defense against ecologically important vertebrate predators, with 297 their lethal potency against humans being an unfortunate evolutionarily coincidence. 298 299 Materials and methods 300 Australian funnel-web spiders were collected from various locations and states across Australia, as 301 summarized in Table 1. The spiders were individually housed at ~23–25˚C in dark cabinets until 302 venom and venom-glands were dissected. 303 304 Nomenclature 305 Toxins were named according to the rational nomenclature described previously (44). Spider 306 was taken from the World Spider Catalog version 21.0 (45). 307 308 Data deposition

11 309 Metadata and annotated nucleotide sequences generated were deposited in the European 310 Nucleotide Archive under project accessions PRJEB6062 for H. infensa, PRJEB14734 for 311 H. cerberea and PRJEB14965 for H. formidabilis. All UniProt and ArachnoServer accessions for 312 sequences used for phylogenetic analysis are listed in the Supplementary Excel Table 1. 313 314 Table 1: 315 Collection sites and species used for venom-gland library construction Species Collection site Number and sex of Collector specimens* Atrax robustus Sydney, NSW 1 M/1 F Graham Nicholson Atrax sutherlandi Gerringong, NSW 1 M David Wilson Hadronyche infensa Fraser Island and Toowoomba, QLD 5 F (RACE) David Wilson Hadronyche infensa Fraser Island and Toowoomba, QLD 3 F (454) Sandy Pineda Hadronyche modesta Kalorama, Dandenong Ranges, VIC 4 F Bryan Fry Hadronyche valida Binna Burra, QLD 1 juvenile M David Wilson Hadronyche venenata Tooms Lake, TAS 1 F Glenn Gregg Hadronyche versuta Blue Mountains, NSW 1 F Graham Nicholson Hadronyche formidabilis South Tamborine, QLD 1 F/4 juveniles Sebastien Dutertre Hadronyche cerberea Blue Mountains, NSW 2 F Sebastien Dutertre Illawara wisharti Gerringong, NSW 1 M David Wilson 316 *F= female; M= male 317 318 mRNA isolation and cDNA library construction 319 mRNA and cDNA libraries were isolated and constructed using the protocols summarized in 320 Supplementary Table 1. For details of RACE, Sanger and next-generation-sequencing see 321 Supporting information.

322 Phylogenetics and selection analyses 323 Reconstruction of the phylogenetic history and the molecular evolution of δ-HXTXs was 324 performed as detailed in the Supporting information. 325 326 RP-HPLC purification of Ar1a 327 Milked lyophilized venom from male A. robustus specimens was supplied by the Australian Reptile 328 Park (Gosford, NSW). The venom was reconstituted in MilliQ water to a concentration of

12 329 ~5 mg/mL and Ar1a was purified using RP-HPLC as outlined in Supporting Information. 330

331 Determination of the NaV subtype selectivity of Ar1a

332 The activity of Ar1a on human NaV channels stably expressed in HEK293 cells was determined 333 using a FLIPRTetra assay (23) as described in Supporting Information. 334 335 Algogenic effects Ar1a 336 Ethical approval for in vivo experiments was obtained from The University of Queensland 337 Ethics Committee (PHARM/512/12/RAMACIOTTI) and they were conducted in accordance with 338 the Queensland Animal Care and Protection Act (2002), the Australian Code of Practice for the 339 Care and Use of Animals for Scientific Purposes (8th edition, 2013), and the International 340 Association for the Study of Pain Guidelines for the Use of Animals in Research. Details of 341 intraplantar administration of Ar1a and assessment of induced nocifensive behaviour is detailed in 342 Supporting Information. 343 344 Insecticidal effects of Ar1a 345 Native Ar1a was tested for insecticidal toxicity by injection into sheep blowflies (46) as described 346 in the Supporting Information. 347

348 Activity of Ar1a on BgNaV1 349 Two-electrode voltage-clamp electrophysiology was used to determine the activity of Ar1a on

350 BgNaV1 heterologously expressed in Xenopus oocytes, as outlined in the Supporting information. 351 352 Competing interest 353 The authors have no competing interests to declare. 354 355 Acknowledgements 356 We acknowledge support from the Australian National Health & Medical Research Council 357 (Principal Research Fellowship APP1136889 and Program Grant APP1072113 to GFK, Career 358 Development Fellowship APP1162503 to IV), the Australian Research Council (Discovery Grant 359 DP190100304 to BGF, Future Fellowship FT190100482 to VH). KS was supported by the

13 360 Department of Science and Technology (DST) INSPIRE Faculty Award 361 (DST/INSPIRE/04/2017/000071), DST-FIST (SR/FST/LS-II/2018/233) and the DBT-IISc 362 Partnership Program. IV was supported by an ECR grant from The Clive & Vera Ramaciotti 363 Foundation. The authors thank Dr Roger Drinkwater for assistance with sequencing, Dr Robert 364 Raven (Queensland Museum) and Mr Graham Wishart for specimen collection and identification, 365 Mr Glenn Gregg and Prof. Graham Nicholson for providing spiders, the Australian Reptile Park 366 for provision of A. robustus venom, Geoff Brown (Department of Agriculture and Fisheries,

367 Queensland) for blowflies, and Ke Dong (Michigan State University) for sharing BgNaV1/TipE 368 clones. 369

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17 482 483 Fig. 1: Phylogenetic reconstruction of δ-HXTXs. Tree representing the phylogenetic history of δ-HXTXs as 484 estimated by maximum-likelihood inference. The thickness of branches corresponds to node supports (thick branch: 485 bootstrap ≥ 75; thin branch: bootstrap < 75), and various species are presented in distinct colors. The cysteine pattern 486 (black lines) and activities (red lines) are annotated on the right. Fly and mouse icons indicate insecticidal and 487 vertebrate activity, respectively. The underscore sequence numbers refer to the unique preprotoxin nomenclature used 488 in the ArachnoServer database (47).

18 489 490 Fig. 2: Molecular evolution of δ-HXTXs. (A) Toxin precursor domains and their rate of evolution, indicated as ω 491 values (i.e., non-synonymous-to-synonymous substitution rate ratio). (B) Structure of δ-HXTX-Hv1a (PDB accession 492 code 1VTX) (8), depicting the locations of positively selected sites. (C) Sequence alignment of δ-HXTXs from 493 representative species. The positions of the four disulfide bonds are indicated by lines above and below the sequence 494 alignment, while the extent of evolutionary conservation of amino acids (calculated from the complete alignment of 495 114 sequences) is denoted by the illustrated color code. Note: Loop IV is not present in the plesiotypic T. loki sequence 496 and is instead characteristic of the apotypic δ-HXTX sequences. (D) Statistics associated with selection analyses. eFast 497 Unconstrained Bayesian AppRoximation; fsites experiencing episodic diversifying selection (0.05 significance) by the 498 Mixed Effects Model Evolution (MEME); gPositively selected sites detected by the Bayes Empirical Bayes approach 499 implemented in M8; hNumber of sites under pervasive diversifying selection at the posterior probability ≥0.95 500 (FUBAR); iNumber of sites under pervasive purifying selection at the posterior probability ≥0.95 (FUBAR). 501

19 502 503 Fig. 3: Cys derivations and codon usage in δ-HXTXs. (A) Alignment of δ-HXTXs representing major changes in 504 the Cys framework. (B) Expected and experimentally determined molecular masses for the 7 Cys residue-derivations 505 of δ-HXTX homologs from H. infensa. Several masses closely matching either monomers or homo/heterodimers were 506 observed using MALDI or Orbitrap mass spectrometry. (C) Codon usage for Cys residues in δ-HXTXs from different 507 funnel-web genera. Lines at top represent the four disulfide bonds in the prototypical Ar1a toxin. 508

20 509 510 Fig. 4: Isolation and purification of Ar1a from A. robustus venom. (A) Chromatogram from C18 RP-HPLC 511 fractionation of A. robustus venom with peak containing Ar1a highlighted in red. Inset is a photo of female A. robustus. 512 (B) Chromatogram from hydrophobic interaction chromatography of Ar1a-containing peak from panel (A). Inset 513 shows MALDI-MS spectrum of purified Ar1a. The dashed line indicates the solvent gradient, with percentage of 514 solvent B (90% acetonitrile/0.045% TFA) indicated on the right ordinate axis. 515

21 516 517 Fig. 5: Biological characterization of Ar1a. (A) NaV subtype selectivity of Ar1a. The effect of Ar1a on NaV1.1– 518 NaV1.8 heterologously expressed in HEK293 cells in combination with the human b1 subunit was assessed using a 519 membrane potential assay. Ar1a elicited a concentration-dependent increase in membrane potential in the presence of 520 veratridine (5 µM) in cells expressing NaV1.1 (pEC50 7.52 ± 0.24), NaV1.2 (pEC50 7.41 ± 0.09), NaV1.3 (pEC50 7.41 ± 521 0.15) and NaV1.6 (pEC50 7.04 ± 0.08). Data are presented as mean ± SEM (n = 3). (B) Dose-response curve for paralysis 522 of L. cuprina blowflies injected with Ar1a. Paralysis was assessed at 1 h post-injection. Error bars indicate SEM. The 523 median paralytic dose (PD50) was determined as the mean ± SEM of three independent experiments. All paralytic 524 effects were reversible within 24 h and no lethal effects were observed. (C) Dose-dependent inhibition of BgNaV1 fast 525 inactivation by Ar1a. Representative sodium currents were elicited by a depolarization to −20mV before (black) and 526 after (red) addition of toxin from a holding potential of −90 mV. (D) Normalized conductance–voltage relationships 527 (G/Gmax; black filled circles) and steady-state inactivation relationships (I/Imax; black open circles) of BgNaV1 before 528 (black circles) and after (red circles) Ar1a application. Channel-expressing oocytes were depolarized in 5-mV steps 529 from a holding potential of −90 mV. n = 3–5; error bars represent SEM.

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