1 Collateral death—Australian funnel-web spiders 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. India 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: spider 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 Atrax 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 genus Macrothele, 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 Missulena 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 (Hadronyche 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.