Accepted Manuscript

Arthropod assassins: crawling biochemists with diverse toxin pharmacopeias

Volker Herzig

PII: S0041-0101(18)31041-9

DOI: https://doi.org/10.1016/j.toxicon.2018.11.312

Reference: TOXCON 6038

To appear in: Toxicon

Please cite this article as: Herzig, V., Arthropod assassins: crawling biochemists with diverse toxinpharmacopeias, Toxicon, https://doi.org/10.1016/j.toxicon.2018.11.312.

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Editorial 1

Arthropod assassins: crawling biochemists with diverse toxin pharmacopeias 2

Volker Herzig1 3 4

5 1 Institute for Molecular Bioscience, The University of Queensland, St. Lucia QLD 4072, 6

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*Address correspondence to: Volker Herzig, Institute for Molecular Bioscience, The University 11

of Queensland, St. Lucia QLD 4072, Australia; Phone: +61 7 3346 2018, Fax: +61 7 3346 2101, 12

Email: v.herzig@uq.edu.au 13

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Abstract 27 The millions of extant arthropod species are testament to their evolutionary success that can at 28 least partially be attributed to venom usage, which evolved independently in at least 19 arthropod 29 lineages. While some arthropods primarily use venom for predation (e.g., spiders and centipedes) 30 or defense (e.g., bees and caterpillars), it can also have more specialised functions (e.g. in 31 parasitoid wasps to paralyse arthropods for their brood to feed on) or even a combination of 32 functions (e.g. the scorpion Parabuthus transvaalicus can deliver a prevenom for predator 33 deterrence and a venom for predation). Most arthropod venoms are complex cocktails of water, 34 salts, small bioactive molecules, peptides, enzymes and larger proteins, with peptides usually 35 comprising the majority of toxins. Some spider venoms have been reported to contain > 1,000 36 peptide toxins, which function as combinatorial libraries to provide an evolutionary advantage. 37 The astounding diversity of venomous arthropods multiplied by their enormous toxin arsenals 38 results in an almost infinite resource for novel bioactive molecules. The main challenge for 39 exploiting this resource is the small size of most arthropods, which can be a limitation for current 40 venom extraction techniques. Fortunately, recent decades have seen an incredible improvement in 41 transcriptomic and proteomic techniques that have provided increasing sensitivity while reducing 42 sample requirements. In turn, this has provided a much larger variety of arthropod venom 43 compounds for potential applications such as therapeutics, molecular probes for basic research, 44 bioinsecticides or anti-parasitic drugs. This special issue of Toxicon aims to cover the breadth of 45 arthropod venom research, including toxin evolution, pharmacology, toxin discovery and 46 characterisation, toxin structures, clinical aspects, and potential applications. 47 48 49 1. Diversity of arthropod venom systems 50

Invertebrates within the phylum Arthropoda that are characterised by segmented bodies and 51 paired jointed appendages are classified into the four extant subphyla Chelicerata, Myriapoda, 52 Crustacea and Hexapoda. Arthropods have been extremely successful over the course of 53 evolution and it is estimated that they comprise nearly 85% of all extant animal species (Giribet 54 and Edgecombe, 2012). Their evolutionary success story can partially be attributed to the use of 55 venoms, which have independently developed in all extant arthropod subphyla. Based on their 56 independent evolutionary origin (even multiple times within some lineages), the venom delivery 57 systems in arthropods can be localised in very different body regions (Fig. 1); for example, they 58 can be combined with, or close to, mouth parts (dipterans, bugs, spiders, ticks), present as 59 modified legs (centipedes, remipedes), in the palpal pincers (pseudoscorpions), in the antennae 60 (coleopterans - Cerambycidae), at the distal end of the body (scorpions and hymenopterans) or as 61 toxic hairs covering parts of the body (lepidopteran larvae). 62 63 64

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66 Fig.1: Venomous representatives of the four arthropod subphyla with the red arrows and the 67 zoomed images indicating the anatomic location of the venom delivery system. Photographs 68 authored by Tobias Hauke (Germany; spider, scorpion), Kriton Kunz (Germany; 69 pseudoscorpion), Mario Sergio Palma (Brazil; wasp, ant), Gavin Rice (Australia; caterpillar), 70 Ivo Muniz (Brazil; beetle), Eivind A.B. Undheim (Australia; centipede, assassin bug), Ingo Wendt 71 (Germany; robber fly, pseudoscorpion chelae, centipede focipules), Björn von Reumont 72 (Germany; remipede). 73 74 75 Crustaceans are the least well-studied arthropods in terms of venoms and only a single example 76 of a venomous crustacean, the cave-dwelling remipede Xibalbanus tulumensis, has been reported 77

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so far (von Reumont et al., 2014a). Remipedes have paired venom glands located in the first 78 segments of the cephalothorax and they use their paired fang-like maxillulae for venom delivery 79 (von Reumont et al., 2014a). 80 In contrast, venomous species in the other arthropod subphyla are common or in some lineages 81 even the most abundant representatives. Within the Myriapoda, the class Chilopoda contains 82 about 3500 species of venomous centipedes; these arthropods date back to at least 430 mya, 83 making their venom systems one of the oldest among terrestrial animals (Undheim et al., 2015). 84 For venom delivery, centipedes use their paired forcipules ("poison claws") containing the venom 85 glands, which evolved from the first pair of walking legs (Undheim et al., 2015). Within the 86 Chelicerata, venomous lineages have only developed in the class Arachnida, which comprises 87 four orders that have independently evolved venom systems: Acari (ticks), Araneae (true 88 spiders), Scorpiones (scorpions) and Pseudoscorpiones (pseudoscorpions). Within the Acari, ticks 89 have been recently considered as venomous ectoparasites due to the composition and function of 90 their saliva/venom (Cabezas-Cruz and Valdes, 2014), although this classification is not 91 unanimously accepted and might depend of the actual definition of the term “venom” that is used 92 (Pienaar et al., 2018). Tick saliva/venom is produced in the salivary glands and injected into the 93 host via a structure called the hypostome, which is used to penetrate the host’s epidermis and 94 helps in anchoring the tick while feeding (Pienaar et al., 2018). Pseudoscorpiones with their 95 venom glands being located in the fixed and/or the movable finger of their pedipalpal pincers 96 comprise about 3300 species worldwide (Murienne et al., 2008), but their small size has so far 97 limited research on their venoms. A little less diverse are the scorpions, with 2336 species known 98 to date (Rein, 2018) and all of them use venom. Their paired venom glands are located in the last 99 segment of the metasoma ("tail") and connected to a single stinger for venom injection (Yigit and 100 Benli, 2008). The most diverse of all arachnids are the spiders, which currently comprise over 101 47,000 species (World Spider Catalog, 2018). All spiders, with the exception of the family 102 Uloboridae (~ 0.6% of all spiders) are venomous, although in contrast to public opinion the vast 103 majority are not dangerous to humans (Hauke and Herzig, 2017). In mygalomorph spiders, the 104 paired venom glands are located in the basal part of the chelicerae, whereas in araneomorph 105 spiders they can extend into the prosoma (Foelix, 1992). Finally, the Hexapoda contain six 106 venomous orders of insects, including the hemimetabolous Hemiptera (bugs) and the 107 holometabolous Neuroptera (e.g. antlions), Hymenoptera (bees, wasps and ants), Diptera (flies), 108 Lepidoptera (butterflies and moths) and Coleoptera (beetles). Despite (or maybe because of) their 109 incredible diversity, insects are extremely under-represented in venom research. The best studied 110 order of venomous insects by far is Hymenoptera, although other hymenopteran venoms from 111 ants and wasps have also been studied (Moreau and Asgari, 2015; Touchard et al., 2016; Perez-112 Riverol et al., 2017; Robinson et al., 2018). Dipterans are another insect order with several 113 different venomous lineages, but they have received far less attention compared to 114 hymenopterans. Venom in dipterans is used by adult members of the family Asilidae (robber 115 flies) and by larval Tabanidae (horse flies), Sciomyzidae (marsh flies), Cecidomyiidae (gall 116 midges) and Vermileonidae (von Reumont et al., 2014b). In coleopterans, a single cerambycid 117 species has been described in which the adult beetle delivers (a possibly defensive) venom with 118 the tip of the antennae, which has been modified into a scorpion-like stinger (Berkov et al., 119 2008). In addition, some larval beetles also have venom glands that are used for prey capture (for 120 details see Walker et al., 2018c). 121 122 123

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124 125 2. Complexity of arthropod venom compositions 126

Venomous arthropods dwarf all other venomous organisms in both number and diversity, and 127 they also have some of the most complex venom compositions. Most arthropod venoms are 128 complex cocktails made up of water, salts, small bioactive molecules, peptides, enzymes and 129 larger proteins, with peptides or proteins usually comprising the majority of venom toxins. Some 130 spider venoms for example have been reported to contain > 1,000 peptide toxins (Escoubas et al., 131 2006). There are a number of reasons that might explain this incredible complexity of arthropod 132 venoms as summarized in Table 1. One reason is that venom is used for multiple purposes such 133 as predation and defense. Scorpions and assassin bugs can even secrete distinctly different 134 defensive and predatory venoms independently (Inceoglu et al., 2003; Walker et al., 2018b). 135 Another reason underlying their chemical complexity is the constant evolutionary race between 136 venomous arthropods and their prey or predators. Mutations in the envenomated victims can lead 137 to resistance to some toxins. Thus, as a built-in mechanism to anticipate and counteract possible 138 mutations of the molecular targets, arthropods employ combinatorial chemistry, gene duplication, 139 focal hypermutations as well as posttranslational modifications to dramatically increase their 140 pharmacological toxin diversity (Palma and Nakajima, 2005; Escoubas, 2006). In case the prey or 141 predator acquires resistance to one of the major toxins in the venom, other homologous toxins are 142 already present (if not in an individual, then most likely in an entire population) that retain 143 activity on the target. Thereby, those individuals that express the modified toxins gain an 144 evolutionary advantage, which leads to an evolutionary selection towards these homologous 145 toxins that have retained the activity on the respective molecular target. Another strategy of 146 venomous arthropods to counteract resistance is the presence of toxins that act on various 147 molecular targets, which also explains part of the venom complexity. Even if the envenomated 148 victim becomes resistant to one of the toxins, it might still be sensitive to some of the other toxins 149 present in the venom that act on different molecular targets. Synergistic toxins (Wullschleger et 150 al., 2004) and toxins that are active on different time-scales further contribute to the 151 pharmacological complexity of arthropod venoms. Fast-acting toxins (which can be reversible) 152 for example ensure that prey can be quickly overcome (Sousa et al., 2017), whereas slow-acting 153 (irreversible) toxins ensure that the prey remains paralysed while being consumed by the 154 venomous predator (Ikonomopoulou et al., 2016). Also, in addition to paralytic toxins, some 155 arthropods like assassin bugs contain enzymes in the venom that help in digesting the prey 156 (Walker et al., 2018a). The astounding diversity of venomous species multiplied by their 157 enormous toxin arsenals renders arthropod venoms an excellent source of novel bioactive 158 molecules. The main challenge to exploit this resource is the mostly small size of arthropods. 159 While few spiders, scorpions, wasps and centipedes can reach formidable sizes of 5-30 cm, the 160 great majority of arthropods are smaller than 1 cm. Most venom extraction techniques, which are 161 employed for larger arthropods, have limitations once the specimen becomes too small. However, 162 recent decades have seen an incredible improvement in transcriptomic and proteomic techniques, 163 which has enabled the collection of detailed data from smaller sample quantities as exemplified 164 by the recently published first venom gland transcriptome from a pseudoscorpion (Santibanez-165 Lopez et al., 2018). 166 167 168 169

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170 171 172 Table 1: Possible reasons underlying the complexity of arthropod venoms 173

Reasons for venom complexity Details Different purposes of venom usage Predation, defense, conservation of victims as a food source

for parasitic larvae, digestion Gaining evolutionary advantage Presence of a multitude of homologous toxins anticipates

potential mutations in molecular targets Toxins acting on different targets If prey or predator acquires resistance to one toxin, other

toxins with different modes of action might still be active Toxins acting on different time scales Fast acting toxins cause rapid immobilisation, whereas slow-

acting toxins ensure that the victim remains paralysed Synergistic toxins Interaction of several venom components can enhance the

overall pharmacological effects in the envenomated victim 174 175 3. Research on venomous arthropods 176

Despite their evolutionary success and incredible diversity, venomous arthropods are still under-177 represented in toxinological research. Based on articles published in Toxicon (Fig. 2, left box), 178 the proportional representation of research on venomous arthropods has not changed in the past 179 50 years and remains at about 19% of all articles. Nevertheless, the overall number of 180 publications on arthropod venoms has increased significantly. While 92 articles on arthropod 181 venoms were published in Toxicon in the 5-year period from 1962 to 1966, 102 articles were 182 published within the 6-month period from April to September 2017, representing an 11-fold 183 increase in the annual number of articles. Interestingly, there is also some geographical bias in 184 global arthropod venom research. A higher percentage of arthropod venom research (26%, see 185 Fig. 2, right box) was for example presented at the recent Venoms to Drugs (V2D) Meeting in 186 Noosa (Australia), which had a strong attendance of toxinologists from the region. The 187 knowledge that has been created over the years has provided a better understanding of how 188 arthropod venoms evolved, what their biological purpose is, the physiological effects that are 189 caused by envenomations, and the structure-activity relationships between arthropod toxins and 190 their molecular targets. This improved basic knowledge has helped to apply arthropod venom 191 compounds in basic research, agriculture or medicine. Nevertheless, our current knowledge on 192 arthropod venoms is still far from complete and represents more like the snowflake on the tip of 193 an iceberg, considering that the vast majority of venomous arthropods have not even been 194 studied. Thus, many new findings on arthropod venoms still await discovery by the next 195 generation of toxinologists. Some of these findings are presented in this special issue of Toxicon. 196 197 198

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Fig.2: Proportional representation of different venomous lineages in toxinological research. Left 200 box: Comparison of research articles published in the journal Toxicon during two different time 201 periods (1962–1966 with 96 publications, and April to September 2017 with 102 publications). 202 Right box: Talks and posters presented at the Venoms to Drugs conference in Noosa, Australia, 203 9.-14.10.2017 (with n=73 presentations; data analysis courtesy of Sabah Ul-Hasan, UC Merced, 204 USA). 205 206 4. Content of this special Toxicon issue on arthropod venoms 207

I am delighted that so many experts in the field have contributed to this special issue of Toxicon 208 on arthropod venoms, and I would like to thank all authors and reviewers for their contributions. 209 A total of 18 contributions (12 reviews, 5 original research articles and 1 case report) cover a 210 wide breadth of research from a taxonomically diverse range of venomous arthropods, including 211 three of the four venomous arthropod subphyla (see Fig. 1). A series of reviews cover the clinical 212 importance of spiders (Vetter, 2018) and scorpions (Ward et al., 2018b), but also some of the 213 more neglected lineages such as hymenopterans (Schmidt, 2018), centipedes (Ombati et al., 214 2018a) and lepidopterans (Villas-Boas et al., 2018). In addition, the latter study also reviewed the 215 inoculation apparatus as well as the composition and function of lepidopteran venoms (Villas-216 Boas et al., 2018). The contribution by Walker et al. (2018c) provides a general review on the 217 evolution, biology and biochemistry of insect venoms. Venomous insects are also the topic of a 218 review about the diversity of toxins from social hymenopterans (Dos Santos-Pinto et al., 2018) 219 and a research article on a membrane-disrupting wasp toxin responsible for tissue damage 220 (Ombati et al., 2018b). Besides insects, venomous arachnids are strongly represented in this 221 special issue. Potential applications of spider venom peptides for therapeutic or bioinsecticide 222 applications are reviewed by Saez & Herzig (2018), whereas Guo et al. examined the oral 223 insecticidal activity of arachnid venoms and peptide toxins in two dipteran toxicity assays (Guo 224 et al., 2018). In addition, Jerusalem & Lleti (2018) report on a rare case of loxoscelism in Europe 225 and another two contributions focus on the infamous Brazilian wandering spider Phoneutria 226 nigriventer: Peigneur et al. reviewed the diversity of toxins isolated from this species (Peigneur et 227 al., 2018), whereas da Silva et al. studied the effects of the toxin PnTx2-6 on neurotransmitter 228 release and on its effects on proteins of the blood brain barrier (da Silva et al., 2018). Besides the 229 review on the medically significant scorpions (Ward et al., 2018b), scorpion venom research is 230 represented by two contributions from Lourival Possani's group and a research article from Darin 231 Rokyta’s group. Ortiz & Possani review the interaction of scorpion toxins with ion channels 232 (Ortiz and Possani, 2018), whereas Cid-Uribe et al. provide data on the proteomic and 233

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transcriptomic study of a non-buthid scorpion belonging to the family Vaejovidae (Cid-Uribe et 234 al., 2018) and Ward et al. examines sex-related differences in the venom composition of the 235 Hentz striped scorpion (Ward et al., 2018a). The remaining reviews do not focus on specific 236 arthropod lineages, but rather provide a general overview of the biochemistry and evolution of 237 arthropod venoms (Laxme et al., 2018) or the structural diversity of arthropod toxins (Daly and 238 Wilson, 2018). 239 240 In this editorial (Herzig, 2018), I also want to point out some personal highlights of this special 241 issue. In the clinically-focused contributions, I found it quite interesting to read that there is a 242 generally low prevalence of species that can be considered potentially dangerous to humans (e.g., 243 0.5% for spiders (Hauke and Herzig, 2017), 0.5% for centipedes (Ombati et al., 2018a), and 244 0.03% for hymenopterans (Schmidt, 2018)), with scorpions being the exception with 23% 245 (Hauke and Herzig, 2017). This raises the question why a significantly larger percentage of 246 scorpions have evolved venoms that can harm humans. While I cannot provide a definitive 247 answer, one might speculate that environmental factors (e.g. increased pressures from vertebrate 248 predators of scorpions in mostly arid habitats) could have played a role. The possibility that the 249 prey spectrum of scorpions includes more vertebrate species on the other hand seems unlikely, as 250 both scorpions and spiders are generalist predators with the majority of prey being other 251 arthropods (Polis and McCormick, 1986). A fact on scorpion envenomation that concerned me 252 quite a bit is the apparent lack of verified case reports for a large number of scorpions that are 253 considered as medically significant, even for those species that cause frequent envenomations 254 (Ward et al., 2018b). This lack of data makes it rather difficult to identify those species that are 255 potentially dangerous to humans, which in turn might for example hamper the development of 256 efficient antivenoms. What I also found quite interesting from the clinically-focused 257 contributions are the tables comparing cases of multiple stings in humans caused by honeybees or 258 hornets, indicating that deaths in children or elderly/sick people can be caused by a few hundred 259 bee stings or a few dozen hornet stings, respectively, whereas some adults even survived > 2,000 260 bee stings or > 100 hornet stings (Schmidt, 2018). 261 262 Another striking observation as extracted from the reviews by Walker et al. (2018c) and Laxme 263 et al. (2018) is that venom systems have at least evolved 19 times independently within various 264 arthropod lineages (or 29 times, if secretions that facilitate parasitism through hemolymph or 265 blood feeding are also included), which highlights the evolutionary advantage gained by venom 266 usage. A rather surprising finding on the other hand was the high percentage of orally insecticidal 267 arachnid venoms (Guo et al., 2018), given that arachnids inject their venoms and therefore lack 268 the evolutionary pressure to develop oral activity. Although the mechanism underlying the oral 269 activity of arachnid venom peptides remains poorly understood, the potential of oral application 270 in insects will certainly be beneficial towards their use as bioinsecticides. 271 272 Treatment of human disorders affecting the central nervous system is another frequently 273 discussed application of arthropod venom peptides (Ortiz et al., 2015; Undheim et al., 2016; 274 Walker et al., 2016; Robinson et al., 2017; Saez and Herzig, 2018;). Unfortunately, many of these 275 efforts have been hampered by the fact that most venom peptides do not cross the blood-brain-276 barrier. A striking exception is the venom peptide PnTx2-6 from the spider Phoneutria 277 nigriventer (da Silva et al., 2018). It will therefore be of much interest in future to decipher the 278 mechanism by which PnTx2-6 crosses the blood-brain-barrier and to apply this knowledge to 279

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other arthropod venom peptides that are considered to be useful for treating human CNS 280 disorders. 281 282 I hope that readers of Toxicon will enjoy this special issue as much as I did, and that young 283 toxinologists might be inspired to shift their research focus to arthropod venoms and contribute to 284 uncovering the many secrets of these venoms that remain to be discovered. 285 286 287 5. References 288 Berkov, A., Rodriguez, N., and Centeno, P., 2008. Convergent evolution in the antennae of a 289

cerambycid beetle, Onychocerus albitarsis, and the sting of a scorpion. 290 Naturwissenschaften 95, 257-261. 291

Cabezas-Cruz, A., and Valdes, J.J., 2014. Are ticks venomous animals? Front. Zool. 11, 1-18. 292 Cid-Uribe, J.I., Santibanez-Lopez, C.E., Meneses, E.P., Batista, C.V.F., Jimenez-Vargas, J.M., 293

Ortiz, E., and Possani, L.D., 2018. The diversity of venom components of the scorpion 294 species Paravaejovis schwenkmeyeri (Scorpiones: Vaejovidae) revealed by transcriptome 295 and proteome analyses. Toxicon 151, 47-62. 296

da Silva, C.N.D., Lomeo, R.S., Torres, F.S., Borges, M.H., Nascimento, M.C., Rodrigues 297 Mesquita-Britto, M.H., Raposo, C., Pimenta, A.M.C., da Cruz-Hofling, M.A., Gomes, 298 D.A., and de Lima, M.E., 2018. PnTx2-6 (or δ-CNTX-Pn2a), a toxin from Phoneutria 299 nigriventer spider venom, releases L-glutamate from rat brain synaptosomes involving Na+ 300 and Ca2+ channels and changes protein expression at the blood-brain barrier. Toxicon 150, 301 280-288. 302

Daly, N.L., and Wilson, D., 2018. Structural diversity of arthropod venom toxins. Toxicon 152, 303 46-56. 304

Dos Santos-Pinto, J.R.A., Perez-Riverol, A., Lasa, A.M., and Palma, M.S., 2018. Diversity of 305 peptidic and proteinaceous toxins from social Hymenoptera venoms. Toxicon 148, 172-306 196. 307

Escoubas, P., 2006. Molecular diversification in spider venoms: A web of combinatorial peptide 308 libraries. Mol. Diversity 10, 545-554. 309

Escoubas, P., Sollod, B., and King, G.F., 2006. Venom landscapes: Mining the complexity of 310 spider venoms via a combined cDNA and mass spectrometric approach. Toxicon 47, 650-311 663. 312

Foelix, R., 1992. Biologie der Spinnen. Thieme. 313 Giribet, G., and Edgecombe, G.D., 2012. Reevaluating the arthropod tree of life. Annu. Rev. 314

Entomol. 57, 167-186. 315 Guo, S., Herzig, V., and King, G.F., 2018. Dipteran toxicity assays for determining the oral 316

insecticidal activity of venoms and toxins. Toxicon 150, 297-303. 317 Hauke, T.J., and Herzig, V., 2017. Dangerous arachnids-Fake news or reality? Toxicon 138, 173-318

183. 319 Herzig, V. Editorial- Arthropod assassins: crawling biochemists with diverse toxin 320

pharmacopeias. Toxicon (update details) 321 Ikonomopoulou, M.P., Smith, J.J., Herzig, V., Pineda, S.S., Dziemborowicz, S., Er, S.Y., Durek, 322

T., Gilchrist, J., Alewood, P.F., Nicholson, G.M., Bosmans, F., and King, G.F., 2016. 323 Isolation of two insecticidal toxins from venom of the Australian theraphosid spider 324 Coremiocnemis tropix. Toxicon 123, 62-70. 325

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Inceoglu, B., Lango, J., Jing, J., Chen, L., Doymaz, F., Pessah, I.N., and Hammock, B.D., 2003. 326 One scorpion, two venoms: prevenom of Parabuthus transvaalicus acts as an alternative 327 type of venom with distinct mechanism of action. Proc. Natl. Acad. Sci. USA 100, 922-328 927. 329

Jerusalem, K., and Salavert Lleti, M., 2018. Probable cutaneous loxoscelism with mild systemic 330 symptoms: A case report from Spain. Toxicon 156, 7-12. 331

Laxme R.R.S., Suranse V., Sunagar, K. Biochemistry, ecology and evolution of arthropod 332 venoms. Toxicon (update details) 333

Moreau, S.J., and Asgari, S., 2015. Venom proteins from parasitoid wasps and their biological 334 functions. Toxins 7, 2385-2412. 335

Murienne, J., Harvey, M.S., and Giribet, G., 2008. First molecular phylogeny of the major clades 336 of Pseudoscorpiones (Arthropoda: Chelicerata). Mol. Phylogenet. Evol. 49, 170-184. 337

Ombati, R., Luo, L., Yang, S., and Lai, R., 2018a. Centipede envenomation: Clinical importance 338 and the underlying molecular mechanisms. Toxicon 154, 60-68. 339

Ombati, R., Wang, Y., Du, C., Lu, X., Li, B., Nyachieo, A., Li, Y., Yang, S., and Lai, R., 2018b. 340 A membrane disrupting toxin from wasp venom underlies the molecular mechanism of 341 tissue damage. Toxicon 148, 56-63. 342

Ortiz, E., Gurrola, G.B., Schwartz, E.F., and Possani, L.D., 2015. Scorpion venom components as 343 potential candidates for drug development. Toxicon 93, 125-135. 344

Ortiz, E., and Possani, L.D., 2018. Scorpion toxins to unravel the conundrum of ion channel 345 structure and functioning. Toxicon 150, 17-27. 346

Palma, M.S., and Nakajima, T., 2005. A natural combinatorial chemistry strategy in 347 acylpolyamine toxins from Nephilinae orb-web spiders. Toxin Rev. 24, 209-234. 348

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Perez-Riverol, A., Dos Santos-Pinto, J.R.A., Lasa, A.M., Palma, M.S., and Brochetto-Braga, 351 M.R., 2017. Wasp venomic: Unravelling the toxins arsenal of Polybia paulista venom and 352 its potential pharmaceutical applications. J. Proteomics 161, 88-103. 353

Pienaar, R., Neitz, A.W.H., and Mans, B.J., 2018. Tick paralysis: Solving an enigma. Vet. Sci. 5. 354 Polis, G.A., and McCormick, S.J., 1986. Scorpions, spiders and solpugids: predation and 355

competition among distantly related taxa. Oecologia 71, 111-116. 356 Rein, J.O., 2018. The Scorpion Files (www.ntnu.no/ub/scorpion-files). 357 Robinson, S.D., Undheim, E.A.B., Ueberheide, B., and King, G.F., 2017. Venom peptides as 358

therapeutics: advances, challenges and the future of venom-peptide discovery. Expert 359 Review of Proteomics 14, 931-939. 360

Robinson, S.D., Mueller, A., Clayton, D., Starobova, H., Hamilton, B.R., Payne, R.J., Vetter, I., 361 King, G.F., and Undheim, E.A.B., 2018. A comprehensive portrait of the venom of the 362 giant red bull ant, Myrmecia gulosa, reveals a hyperdiverse hymenopteran toxin gene 363 family. Sci. Adv. 4, eaau4640. 364

Saez, N., Herzig, V. Versatile spider venom peptides and their medical and agricultural 365 applications. Toxicon (update details) 366

Santibanez-Lopez, C.E., Ontano, A.Z., Harvey, M.S., and Sharma, P.P., 2018. Transcriptomic 367 analysis of pseudoscorpion venom reveals a unique cocktail dominated by enzymes and 368 protease Inhibitors. Toxins 10. 369

Schmidt, J.O., 2018. Clinical consequences of toxic envenomations by Hymenoptera. Toxicon 370 150, 96-104. 371

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