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., assassins: crawling biochemists with diverse toxin pharmacopeias, Toxicon, https://doi.org/10.1016/j.toxicon.2018.11.312.

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1 Editorial 2 Arthropod assassins: crawling biochemists with diverse toxin pharmacopeias 3 Volker Herzig 1 4 5 6 1 Institute for Molecular Bioscience, The University of Queensland, St. Lucia QLD 4072, 7 Australia 8 9 10 11 *Address correspondence to: Volker Herzig, Institute for Molecular Bioscience, The University 12 of Queensland, St. Lucia QLD 4072, Australia; Phone: +61 7 3346 2018, Fax: +61 7 3346 2101, 13 Email: [email protected] 14 15 16 MANUSCRIPT 17 18 19 20 21 22 23 24 25 26 ACCEPTED

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27 Abstract 28 The millions of extant arthropod are testament to their evolutionary success that can at 29 least partially be attributed to usage, which evolved independently in at least 19 arthropod 30 lineages. While some primarily use venom for predation (e.g., and centipedes) 31 or defense (e.g., bees and caterpillars), it can also have more specialised functions (e.g. in 32 parasitoid to paralyse arthropods for their brood to feed on) or even a combination of 33 functions (e.g. the transvaalicus can deliver a prevenom for predator 34 deterrence and a venom for predation). Most arthropod are complex cocktails of water, 35 salts, small bioactive molecules, peptides, enzymes and larger proteins, with peptides usually 36 comprising the majority of toxins. Some venoms have been reported to contain > 1,000 37 peptide toxins, which function as combinatorial libraries to provide an evolutionary advantage. 38 The astounding diversity of venomous arthropods multiplied by their enormous toxin arsenals 39 results in an almost infinite resource for novel bioactive molecules. The main challenge for 40 exploiting this resource is the small size of most arthropods, which can be a limitation for current 41 venom extraction techniques. Fortunately, recent decades have seen an incredible improvement in 42 transcriptomic and proteomic techniques that have provided increasing sensitivity while reducing 43 sample requirements. In turn, this has provided a much larger variety of arthropod venom 44 compounds for potential applications such as therapeutics, molecular probes for basic research, 45 bioinsecticides or anti-parasitic drugs. This special issue of Toxicon aims to cover the breadth of 46 arthropod venom research, including toxin evolution, pharmacology, toxin discovery and 47 characterisation, toxin structures, clinical aspects, and potential applications. 48 49 50 1. Diversity of arthropod venom systems 51 Invertebrates within the phylum Arthropoda that MANUSCRIPT are characterised by segmented bodies and 52 paired jointed appendages are classified into the four extant subphyla , Myriapoda, 53 Crustacea and Hexapoda. Arthropods have been extremely successful over the course of 54 evolution and it is estimated that they comprise nearly 85% of all extant species (Giribet 55 and Edgecombe, 2012). Their evolutionary success story can partially be attributed to the use of 56 venoms, which have independently developed in all extant arthropod subphyla. Based on their 57 independent evolutionary origin (even multiple times within some lineages), the venom delivery 58 systems in arthropods can be localised in very different body regions (Fig. 1); for example, they 59 can be combined with, or close to, mouth parts (dipterans, bugs, spiders, ticks), present as 60 modified legs (centipedes, remipedes), in the palpal pincers (pseudoscorpions), in the antennae 61 (coleopterans - Cerambycidae), at the distal end of the body ( and hymenopterans) or as 62 toxic hairs covering parts of the body (lepidopteran larvae). 63 64 ACCEPTED

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

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

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124 125 126 2. Complexity of arthropod venom compositions 127 Venomous arthropods dwarf all other venomous organisms in both number and diversity, and 128 they also have some of the most complex venom compositions. Most arthropod venoms are 129 complex cocktails made up of water, salts, small bioactive molecules, peptides, enzymes and 130 larger proteins, with peptides or proteins usually comprising the majority of venom toxins. Some 131 spider venoms for example have been reported to contain > 1,000 peptide toxins (Escoubas et al., 132 2006). There are a number of reasons that might explain this incredible complexity of arthropod 133 venoms as summarized in Table 1. One reason is that venom is used for multiple purposes such 134 as predation and defense. Scorpions and assassin bugs can even secrete distinctly different 135 defensive and predatory venoms independently (Inceoglu et al., 2003; Walker et al., 2018b). 136 Another reason underlying their chemical complexity is the constant evolutionary race between 137 venomous arthropods and their prey or predators. Mutations in the envenomated victims can lead 138 to resistance to some toxins. Thus, as a built-in mechanism to anticipate and counteract possible 139 mutations of the molecular targets, arthropods employ combinatorial chemistry, gene duplication, 140 focal hypermutations as well as posttranslational modifications to dramatically increase their 141 pharmacological toxin diversity (Palma and Nakajima, 2005; Escoubas, 2006). In case the prey or 142 predator acquires resistance to one of the major toxins in the venom, other homologous toxins are 143 already present (if not in an individual, then most likely in an entire population) that retain 144 activity on the target. Thereby, those individuals that express the modified toxins gain an 145 evolutionary advantage, which leads to an evolutionary selection towards these homologous 146 toxins that have retained the activity on the respective molecular target. Another strategy of 147 venomous arthropods to counteract resistance is the presence of toxins that act on various 148 molecular targets, which also explains part of the MANUSCRIPTvenom complexity. Even if the envenomated 149 victim becomes resistant to one of the toxins, it might still be sensitive to some of the other toxins 150 present in the venom that act on different molecular targets. Synergistic toxins (Wullschleger et 151 al., 2004) and toxins that are active on different time-scales further contribute to the 152 pharmacological complexity of arthropod venoms. Fast-acting toxins (which can be reversible) 153 for example ensure that prey can be quickly overcome (Sousa et al., 2017), whereas slow-acting 154 (irreversible) toxins ensure that the prey remains paralysed while being consumed by the 155 venomous predator (Ikonomopoulou et al., 2016). Also, in addition to paralytic toxins, some 156 arthropods like assassin bugs contain enzymes in the venom that help in digesting the prey 157 (Walker et al., 2018a). The astounding diversity of venomous species multiplied by their 158 enormous toxin arsenals renders arthropod venoms an excellent source of novel bioactive 159 molecules. The main challenge to exploit this resource is the mostly small size of arthropods. 160 While few spiders, scorpions, wasps and centipedes can reach formidable sizes of 5-30 cm, the 161 great majority of arthropods are smaller than 1 cm. Most venom extraction techniques, which are 162 employed for larger arthropods, have limitations once the specimen becomes too small. However, 163 recent decades haveACCEPTED seen an incredible improvement in transcriptomic and proteomic techniques, 164 which has enabled the collection of detailed data from smaller sample quantities as exemplified 165 by the recently published first venom gland transcriptome from a pseudoscorpion (Santibanez- 166 Lopez et al., 2018). 167 168 169

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170 171 172 173 Table 1 : Possible reasons underlying the complexity of arthropod venoms 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 174 175 176 3. Research on venomous arthropods 177 Despite their evolutionary success and incredible diversity, venomous arthropods are still under- 178 represented in toxinological research. Based on articles published in Toxicon (Fig. 2, left box), 179 the proportional representation of research on venomous arthropods has not changed in the past 180 50 years and remains at about 19% of all articles. Nevertheless, the overall number of 181 publications on arthropod venoms has increased significantly. While 92 articles on arthropod 182 venoms were published in Toxicon in the 5-year periodMANUSCRIPT from 1962 to 1966, 102 articles were 183 published within the 6-month period from April to September 2017, representing an 11-fold 184 increase in the annual number of articles. Interestingly, there is also some geographical bias in 185 global arthropod venom research. A higher percentage of arthropod venom research (26%, see 186 Fig. 2, right box) was for example presented at the recent Venoms to Drugs (V2D) Meeting in 187 Noosa (Australia), which had a strong attendance of toxinologists from the region. The 188 knowledge that has been created over the years has provided a better understanding of how 189 arthropod venoms evolved, what their biological purpose is, the physiological effects that are 190 caused by envenomations, and the structure-activity relationships between arthropod toxins and 191 their molecular targets. This improved basic knowledge has helped to apply arthropod venom 192 compounds in basic research, agriculture or medicine. Nevertheless, our current knowledge on 193 arthropod venoms is still far from complete and represents more like the snowflake on the tip of 194 an iceberg, considering that the vast majority of venomous arthropods have not even been 195 studied. Thus, many new findings on arthropod venoms still await discovery by the next 196 generation of toxinologists. Some of these findings are presented in this special issue of Toxicon . 197 198 ACCEPTED

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Fish Plants Amphibians Fish Amphibians Cnidarian 2.9% 2.7% 4.2% 1% 2.7% Other topics Other topics Other topics 3.9% Molluscs 11.5% 8.8% 19.2% Reptiles Fungi 1% 30.4% 27.4% Plants Amphibians 3.1% 1.4% Cnidarian 1% Bacteria 1.5%13.7% Toxicon 4.1% Fish Toxicon V2D Molluscs 6.3% 53.1% 04-09/ 1962-66 Reptiles 2017 2017 Arthropods 1% 2.9% general Fungi 15.1% Reptiles 17.7% 8.8% 11.8% 5.5% Plants 2.9% 6.9% 5.9% Insects Arachnids Arachnids 20.5% Cnidarian 1% Insects Molluscs Arthropods general Arachnids 18.7% Arthropods 18.7% Arthropods 26.0 % Arthropods 200 Fig.2: Proportional representation of different venomous lineages in toxinological research. Left 201 box: Comparison of research articles published in the journal Toxicon during two different time 202 periods (1962–1966 with 96 publications, and April to September 2017 with 102 publications). 203 Right box: Talks and posters presented at the Venoms to Drugs conference in Noosa, Australia, 204 9.-14.10.2017 (with n=73 presentations; data analysis courtesy of Sabah Ul-Hasan, UC Merced, 205 USA). 206 207 4. Content of this special Toxicon issue on arthropod venoms 208 I am delighted that so many experts in the field have contributed to this special issue of Toxicon 209 on arthropod venoms, and I would like to thank all authors and reviewers for their contributions. 210 A total of 18 contributions (12 reviews, 5 original research articles and 1 case report) cover a 211 wide breadth of research from a taxonomically diverMANUSCRIPTse range of venomous arthropods, including 212 three of the four venomous arthropod subphyla (see Fig. 1). A series of reviews cover the clinical 213 importance of spiders (Vetter, 2018) and scorpions (Ward et al., 2018b), but also some of the 214 more neglected lineages such as hymenopterans (Schmidt, 2018), centipedes (Ombati et al., 215 2018a) and lepidopterans (Villas-Boas et al., 2018). In addition, the latter study also reviewed the 216 inoculation apparatus as well as the composition and function of lepidopteran venoms (Villas- 217 Boas et al., 2018). The contribution by Walker et al. (2018c) provides a general review on the 218 evolution, biology and biochemistry of insect venoms. Venomous insects are also the topic of a 219 review about the diversity of toxins from social hymenopterans (Dos Santos-Pinto et al., 2018) 220 and a research article on a membrane-disrupting wasp toxin responsible for tissue damage 221 (Ombati et al., 2018b). Besides insects, venomous arachnids are strongly represented in this 222 special issue. Potential applications of spider venom peptides for therapeutic or bioinsecticide 223 applications are reviewed by Saez & Herzig (2018), whereas Guo et al. examined the oral 224 insecticidal activity of venoms and peptide toxins in two dipteran toxicity assays (Guo 225 et al., 2018). In addition, Jerusalem & Lleti (2018) report on a rare case of loxoscelism in Europe 226 and another two contributions focus on the infamous Brazilian 227 nigriventer : PeigneurACCEPTED et al. reviewed the diversity of toxins isolated from this species (Peigneur et 228 al., 2018), whereas da Silva et al. studied the effects of the toxin PnTx2-6 on neurotransmitter 229 release and on its effects on proteins of the blood brain barrier (da Silva et al., 2018). Besides the 230 review on the medically significant scorpions (Ward et al., 2018b), scorpion venom research is 231 represented by two contributions from Lourival Possani's group and a research article from Darin 232 Rokyta’s group. Ortiz & Possani review the interaction of scorpion toxins with ion channels 233 (Ortiz and Possani, 2018), whereas Cid-Uribe et al. provide data on the proteomic and

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

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280 other arthropod venom peptides that are considered to be useful for treating human CNS 281 disorders. 282 283 I hope that readers of Toxicon will enjoy this special issue as much as I did, and that young 284 toxinologists might be inspired to shift their research focus to arthropod venoms and contribute to 285 uncovering the many secrets of these venoms that remain to be discovered. 286 287 288 5. References 289 Berkov, A., Rodriguez, N., and Centeno, P., 2008. Convergent evolution in the antennae of a 290 cerambycid beetle, Onychocerus albitarsis , and the sting of a scorpion. 291 Naturwissenschaften 95, 257-261. 292 Cabezas-Cruz, A., and Valdes, J.J., 2014. Are ticks venomous animals? Front. Zool. 11, 1-18. 293 Cid-Uribe, J.I., Santibanez-Lopez, C.E., Meneses, E.P., Batista, C.V.F., Jimenez-Vargas, J.M., 294 Ortiz, E., and Possani, L.D., 2018. The diversity of venom components of the scorpion 295 species Paravaejovis schwenkmeyeri (Scorpiones: Vaejovidae) revealed by transcriptome 296 and proteome analyses. Toxicon 151, 47-62. 297 da Silva, C.N.D., Lomeo, R.S., Torres, F.S., Borges, M.H., Nascimento, M.C., Rodrigues 298 Mesquita-Britto, M.H., Raposo, C., Pimenta, A.M.C., da Cruz-Hofling, M.A., Gomes, 299 D.A., and de Lima, M.E., 2018. PnTx2-6 (or δ-CNTX-Pn2a), a toxin from Phoneutria 300 nigriventer spider venom, releases L-glutamate from rat brain synaptosomes involving Na + 301 and Ca 2+ channels and changes protein expression at the blood-brain barrier. Toxicon 150, 302 280-288. 303 Daly, N.L., and Wilson, D., 2018. Structural diversity of arthropod venom toxins. Toxicon 152, 304 46-56. MANUSCRIPT 305 Dos Santos-Pinto, J.R.A., Perez-Riverol, A., Lasa, A.M., and Palma, M.S., 2018. Diversity of 306 peptidic and proteinaceous toxins from social Hymenoptera venoms. Toxicon 148, 172- 307 196. 308 Escoubas, P., 2006. Molecular diversification in spider venoms: A web of combinatorial peptide 309 libraries. Mol. Diversity 10, 545-554. 310 Escoubas, P., Sollod, B., and King, G.F., 2006. Venom landscapes: Mining the complexity of 311 spider venoms via a combined cDNA and mass spectrometric approach. Toxicon 47, 650- 312 663. 313 Foelix, R., 1992. Biologie der Spinnen. Thieme. 314 Giribet, G., and Edgecombe, G.D., 2012. Reevaluating the arthropod tree of life. Annu. Rev. 315 Entomol. 57, 167-186. 316 Guo, S., Herzig, V., and King, G.F., 2018. Dipteran toxicity assays for determining the oral 317 insecticidal activity of venoms and toxins. Toxicon 150, 297-303. 318 Hauke, T.J., and Herzig, V., 2017. Dangerous arachnids-Fake news or reality? Toxicon 138, 173- 319 183. 320 Herzig, V. Editorial-ACCEPTED Arthropod assassins: crawling biochemists with diverse toxin 321 pharmacopeias. Toxicon (update details) 322 Ikonomopoulou, M.P., Smith, J.J., Herzig, V., Pineda, S.S., Dziemborowicz, S., Er, S.Y., Durek, 323 T., Gilchrist, J., Alewood, P.F., Nicholson, G.M., Bosmans, F., and King, G.F., 2016. 324 Isolation of two insecticidal toxins from venom of the Australian theraphosid spider 325 Coremiocnemis tropix . Toxicon 123, 62-70.

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326 Inceoglu, B., Lango, J., Jing, J., Chen, L., Doymaz, F., Pessah, I.N., and Hammock, B.D., 2003. 327 One scorpion, two venoms: prevenom of acts as an alternative 328 type of venom with distinct mechanism of action. Proc. Natl. Acad. Sci. USA 100, 922- 329 927. 330 Jerusalem, K., and Salavert Lleti, M., 2018. Probable cutaneous loxoscelism with mild systemic 331 symptoms: A case report from Spain. Toxicon 156, 7-12. 332 Laxme R.R.S., Suranse V., Sunagar, K. Biochemistry, ecology and evolution of arthropod 333 venoms. Toxicon (update details) 334 Moreau, S.J., and Asgari, S., 2015. Venom proteins from parasitoid wasps and their biological 335 functions. Toxins 7, 2385-2412. 336 Murienne, J., Harvey, M.S., and Giribet, G., 2008. First molecular phylogeny of the major clades 337 of Pseudoscorpiones (Arthropoda: Chelicerata). Mol. Phylogenet. Evol. 49, 170-184. 338 Ombati, R., Luo, L., Yang, S., and Lai, R., 2018a. Centipede envenomation: Clinical importance 339 and the underlying molecular mechanisms. Toxicon 154, 60-68. 340 Ombati, R., Wang, Y., Du, C., Lu, X., Li, B., Nyachieo, A., Li, Y., Yang, S., and Lai, R., 2018b. 341 A membrane disrupting toxin from wasp venom underlies the molecular mechanism of 342 tissue damage. Toxicon 148, 56-63. 343 Ortiz, E., Gurrola, G.B., Schwartz, E.F., and Possani, L.D., 2015. Scorpion venom components as 344 potential candidates for drug development. Toxicon 93, 125-135. 345 Ortiz, E., and Possani, L.D., 2018. Scorpion toxins to unravel the conundrum of ion channel 346 structure and functioning. Toxicon 150, 17-27. 347 Palma, M.S., and Nakajima, T., 2005. A natural combinatorial chemistry strategy in 348 acylpolyamine toxins from Nephilinae orb-web spiders. Toxin Rev. 24, 209-234. 349 Peigneur, S., de Lima, M.E., and Tytgat, J., 2018. Phoneutria nigriventer venom: A 350 pharmacological treasure. Toxicon 151, 96-110.MANUSCRIPT 351 Perez-Riverol, A., Dos Santos-Pinto, J.R.A., Lasa, A.M., Palma, M.S., and Brochetto-Braga, 352 M.R., 2017. Wasp venomic: Unravelling the toxins arsenal of paulista venom and 353 its potential pharmaceutical applications. J. Proteomics 161, 88-103. 354 Pienaar, R., Neitz, A.W.H., and Mans, B.J., 2018. Tick paralysis: Solving an enigma. Vet. Sci. 5. 355 Polis, G.A., and McCormick, S.J., 1986. Scorpions, spiders and solpugids: predation and 356 competition among distantly related taxa. Oecologia 71, 111-116. 357 Rein, J.O., 2018. The Scorpion Files ( www.ntnu.no/ub/scorpion-files ). 358 Robinson, S.D., Undheim, E.A.B., Ueberheide, B., and King, G.F., 2017. Venom peptides as 359 therapeutics: advances, challenges and the future of venom-peptide discovery. Expert 360 Review of Proteomics 14, 931-939. 361 Robinson, S.D., Mueller, A., Clayton, D., Starobova, H., Hamilton, B.R., Payne, R.J., Vetter, I., 362 King, G.F., and Undheim, E.A.B., 2018. A comprehensive portrait of the venom of the 363 giant red bull ant, Myrmecia gulosa , reveals a hyperdiverse hymenopteran toxin gene 364 family. Sci. Adv. 4, eaau4640. 365 Saez, N., Herzig, V. Versatile spider venom peptides and their medical and agricultural 366 applications.ACCEPTED Toxicon (update details) 367 Santibanez-Lopez, C.E., Ontano, A.Z., Harvey, M.S., and Sharma, P.P., 2018. Transcriptomic 368 analysis of pseudoscorpion venom reveals a unique cocktail dominated by enzymes and 369 protease Inhibitors. Toxins 10. 370 Schmidt, J.O., 2018. Clinical consequences of toxic envenomations by Hymenoptera. Toxicon 371 150, 96-104.

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