In the venom of spiders, linear peptides (LPs), also called cytolytical or antimicrobial peptides, represent a largely neglected group of mostly membrane active substances that contribute in some spider species considerably to the killing power of spider venom. By next-generation sequencing venom gland transcriptome analysis, we investigated 48 spider species from 23 spider families and detected LPs in 20 species, belonging to five spider families (Ctenidae, Lycosidae, Oxyopidae, Pisauridae, and Zodariidae). The structural diversity is extraordinary high in some species: the lynx spider Oxyopes heterophthalmus contains 62 and the lycosid Pardosa palustris 60 different LPs. In total, we identified 524 linear peptide structures and some of them are in lycosids identical on amino acid level. LPs are mainly encoded in complex precursor structures in which, after the signal peptide and propeptide, 13 or more LPs (Hogna radiata) are connected by linkers. Besides Cupiennius species, also in Oxyopidae, posttranslational modifications of some precursor structures result in the formation of two-chain peptides. It is obvious that complex precursor structures represent a very suitable and fast method to produce a high number and a high diversity of bioactive LPs as economically as possible. At least in Lycosidae, Oxyopidae, and in the genus Cupiennius, LPs reach very high Transcripts Per Kilobase Million values, indicating functional importance within the envenomation process.

Departamento de Ecología y Biología Evolutiva Instituto de Investigaciones Biologicas Clemente Estable Montevideo Uruguay

Departamento de Ecología y Evolución Facultad de Ciencias UdelaR Montevideo Uruguay

Department of Botany and Zoology Faculty of Science Masaryk University Brno Czech Republic

Dipartimento di Scienze della Vita e Biologia dei Sistemi University of Torino Torino Italy

Institute of Ecology and Evolution University of Bern Bern Switzerland

Interfaculty Bioinformatics Unit University of Bern Bern Switzerland

Swiss Institute of Bioinformatics Lausanne Switzerland

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Almagro Armenteros J. J., Tsirigos K. D., Sønderby C. K., Petersen T. N., Winther O., Brunak S., et al. (2019). SignalP 5.0 Improves Signal Peptide Predictions Using Deep Neural Networks. Nat. Biotechnol. 37, 420–423. 10.1038/s41587-019-0036-z PubMed DOI

Bayega A., Wang Y. C., Oikonomopoulos S., Djambazian H., Fahiminiya S., Ragoussis J. (2018). Transcript Profiling Using Long-Read Sequencing Technologies. Methods Mol. Biol. 1783, 121–147. 10.1007/978-1-4939-7834-2_6 PubMed DOI

Bray N. L., Pimentel H., Melsted P., Pachter L. (2016). Near-optimal Probabilistic RNA-Seq Quantification. Nat. Biotechnol. 34, 525–527. 10.1038/nbt.3519 PubMed DOI

Budnik B. A., Olsen J. V., Egorov T. A., Anisimova V. E., Galkina T. G., Musolyamov A. K., et al. (2004). De Novo sequencing of Antimicrobial Peptides Isolated from the Venom Glands of the Wolf Spider Lycosa singoriensis . J. Mass. Spectrom. 39, 193–201. 10.1002/jms.577 PubMed DOI

Cheng D.-Q., Piel W. H. (2018). The Origins of the Psechridae: Web-Building Lycosoid Spiders. Mol. Phylogenet. Evol. 125, 213–219. 10.1016/j.ympev.2018.03.035 PubMed DOI

Corzo G., Villegas E., Gómez-Lagunas F., Possani L. D., Belokoneva O. S., Nakajima T. (2002). Oxyopinins, Large Amphipathic Peptides Isolated from the Venom of the Wolf Spider Oxyopes kitabensis with Cytolytic Properties and Positive Insecticidal Cooperativity with Spider Neurotoxins. J. Biol. Chem. 277, 23627–23637. 10.1074/jbc.M200511200 PubMed DOI

Crooks G. E., Hon G., Chandonia J. M., Brenner S. E. (2004). WebLogo: a Sequence Logo Generator. Genome Res. 14, 1188–1190. 10.1101/gr.849004 PubMed DOI PMC

Diniz M. R. V., Paiva A. L. B., Guerra-Duarte C., Nishiyama M. Y., Jr., Mudadu M. A., Oliveira U. d., et al. (2018). An Overview of Phoneutria nigriventer Spider Venom Using Combined Transcriptomic and Proteomic Approaches. PLoS One 13, e0200628. 10.1371/journal.pone.0200628 PubMed DOI PMC

Dubovskii P. V., Vassilevski A. A., Kozlov S. A., Feofanov A. V., Grishin E. V., Efremov R. G. (2015). Latarcins: Versatile Spider Venom Peptides. Cell. Mol. Life Sci. 72, 4501–4522. 10.1007/s00018-015-2016-x PubMed DOI PMC

Dubovskii P. V., Vassilevski A. A., Samsonova O. V., Egorova N. S., Kozlov S. A., Feofanov A. V., et al. (2011). Novel Lynx Spider Toxin Shares Common Molecular Architecture with Defense Peptides from Frog Skin. FEBS J. 278, 4382–4393. 10.1111/j.1742-4658.2011.08361.x PubMed DOI

Fernández R., Kallal R. J., Dimitrov D., Ballesteros J. A., Arnedo M. A., Giribet G., et al. (2018). Phylogenomics, Diversification Dynamics, and Comparative Transcriptomics across the Spider Tree of Life. Curr. Biol. 28, 1489–1497. 10.1016/j.cub.2018.03.064 PubMed DOI

Garnier J., Gibrat J.-F., Robson B. (1996). [32] GOR Method for Predicting Protein Secondary Structure from Amino Acid Sequence. Methods Enzymol. 266, 540–553. 10.1016/s0076-6879(96)66034-0 PubMed DOI

Ghezellou P., Garikapati V., Kazemi S. M., Strupat K., Ghassempour A., Spengler B. (2018). A Perspective View of Top‐down Proteomics in Snake Venom Research. Rapid Commun. Mass. Spectrom. 33, 20–27. 10.1002/rcm.8255 PubMed DOI

Giordano F., Aigrain L., Quail M. A., Coupland P., Bonfield J. K., Davies R. M., et al. (2017). De Novo Yeast Genome Assemblies From MinION, PacBio and MiSeq Platforms. Sci. Rep. 7, 3935. 10.1038/s41598-017-03996-z PubMed DOI PMC

Grabherr M. G., Haas B. J., Yassour M., Levin J. Z., Thompson D. A., Amit I., et al. (2011). Full-length Transcriptome Assembly From RNA-Seq Data without a Reference Genome. Nat. Biotechnol. 29, 644–652. 10.1038/nbt.1883 PubMed DOI PMC

Jones D. T., Taylor W. R., Thornton J. M. (1992). The Rapid Generation of Mutation Data Matrices from Protein Sequences. Bioinformatics 8, 275–282. 10.1093/bioinformatics/8.3.275 PubMed DOI

Kozlov S. A., Grishin E. V. (2007). The Universal Algorithm of Maturation for Secretory and Excretory Protein Precursors. Toxicon 49, 721–726. 10.1016/j.toxicon.2006.11.007 PubMed DOI

Kozlov S. A., Vassilevski A. A., Feofanov A. V., Surovoy A. Y., Karpunin D. V., Grishin E. V. (2006). Latarcins, Antimicrobial and Cytolytic Peptides from the Venom of the Spider Lachesana tarabaevi (Zodariidae) that Exemplify Biomolecular Diversity. J. Biol. Chem. 281, 20983–20992. 10.1074/jbc.M602168200 PubMed DOI

Kuhn-Nentwig L. (2021). Complex Precursor Structures of Cytolytic Cupiennins Identified in Spider Venom Gland Transcriptomes. Sci. Rep. 11, 4009. 10.1038/s41598-021-83624-z PubMed DOI PMC

Kuhn-Nentwig L., Langenegger N., Heller M., Koua D., Nentwig W. (2019). The Dual Prey-Inactivation Strategy of Spiders-In-Depth Venomic Analysis of Cupiennius salei . Toxins 11, 167. 10.3390/toxins11030167 PubMed DOI PMC

Kuhn-Nentwig L., Müller J., Schaller J., Walz A., Dathe M., Nentwig W. (2002). Cupiennin 1, a New Family of Highly Basic Antimicrobial Peptides in the Venom of the Spider Cupiennius salei (Ctenidae). J. Biol. Chem. 277, 11208–11216. 10.1074/jbc.M111099200 PubMed DOI

Kuhn-Nentwig L., Stöcklin R., Nentwig W. (2011a). Venom Composition and Strategies in Spiders. Adv. Insect Physiol. 40, 1–86. 10.1016/B978-0-12-387668-3.00001-5 DOI

Kuhn-Nentwig L., Willems J., Seebeck T., Shalaby T., Kaiser M., Nentwig W. (2011b). Cupiennin 1a Exhibits a Remarkably Broad, Non-stereospecific Cytolytic Activity on Bacteria, Protozoan Parasites, Insects, and Human Cancer Cells. Amino Acids 40, 69–76. 10.1007/s00726-009-0471-0 PubMed DOI

Kumar S., Stecher G., Li M., Knyaz C., Tamura K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 35, 1547–1549. 10.1093/molbev/msy096 PubMed DOI PMC

Kuzmenkov A. I., Sachkova M. Y., Kovalchuk S. I., Grishin E. V., Vassilevski A. A. (2016). Lachesana tarabaevi, an Expert in Membrane-Active Toxins. Biochem. J. 473, 2495–2506. 10.1042/bcj20160436 PubMed DOI

Langenegger N., Koua D., Schürch S., Heller M., Nentwig W., Kuhn-Nentwig L. (2018). Identification of a Precursor Processing Protease from the Spider Cupiennius salei Essential for Venom Neurotoxin Maturation. J. Biol. Chem. 293, 2079–2090. 10.1074/jbc.M117.810911 PubMed DOI PMC

Langenegger N., Nentwig W., Kuhn-Nentwig L. (2019). Spider Venom: Components, Modes of Action, and Novel Strategies in Transcriptomic and Proteomic Analyses. Toxins 11, 611. 10.3390/toxins11100611 PubMed DOI PMC

Madeira F., Park Y. M., Lee J., Buso N., Gur T., Madhusoodanan N., et al. (2019). The EMBL-EBI Search and Sequence Analysis Tools APIs in 2019. Nucleic Acids Res. 47, W636–W641. 10.1093/nar/gkz268 PubMed DOI PMC

Melani R. D., Nogueira F. C. S., Domont G. B. (2017). It Is Time for Top-Down Venomics. J. Venom Anim. Toxins Incl Trop. Dis. 23, 44. 10.1186/s40409-017-0135-6 PubMed DOI PMC

Melo-Braga M. N., De Marco Almeida F., Dos Santos D. M., de Avelar Júnior J. T., Dos Reis P. V. M., De Lima M. E. (2020). Antimicrobial Peptides from Lycosidae (Sundevall, 1833) Spiders. Cpps 21, 527–541. 10.2174/1389203721666200116091911 PubMed DOI

Paiva A. L. B., Mudadu M. A., Pereira E. H. T., Marri C. A., Guerra-Duarte C., Diniz M. R. V. (2019). Transcriptome Analysis of the Spider Phoneutria pertyi Venom Glands Reveals Novel Venom Components for the Genus Phoneutria . Toxicon 163, 59–69. 10.1016/j.toxicon.2019.03.014 PubMed DOI

Pekár S., Brabec M. (2016). Modern Analysis of Biological Data: generalised Linear Models in R. (Brno, Czech: Masaryk University Press; ).

Pekár S., Petráková L., Šedo O., Korenko S., Zdráhal Z. (2018). Trophic Niche, Capture Efficiency and Venom Profiles of Six Sympatric Ant-Eating Spider Species (Araneae: Zodariidae). Mol. Ecol. 27, 1053–1064. 10.1111/mec.14485 PubMed DOI

Pekár S., Šedo O., Líznarová E., Korenko S., Zdráhal Z. (2014). David and Goliath: Potent Venom of an Ant-Eating Spider (Araneae) Enables Capture of a Giant Prey. Naturwissenschaften 101, 533–540. 10.1007/s00114-014-1189-8 PubMed DOI

Piacentini L. N., Ramírez M. J. (2019). Hunting the Wolf: A Molecular Phylogeny of the Wolf Spiders (Araneae, Lycosidae). Mol. Phylogenet. Evol. 136, 227–240. 10.1016/j.ympev.2019.04.004 PubMed DOI

Pimenta A. M. C., Rates B., Bloch C., Jr., Gomes P. C., Santoro M. M., De Lima M. E., et al. (2005). Electrospray Ionization Quadrupole Time-Of-Flight and Matrix-Assisted Laser Desorption/ionization Tandem Time-Of-Flight Mass Spectrometric Analyses to Solve Micro-heterogeneity in Post-translationally Modified Peptides from Phoneutria nigriventer (Aranea, Ctenidae) Venom. Rapid Commun. Mass. Spectrom. 19, 31–37. 10.1002/rcm.1751 PubMed DOI

Pineda S. S., Chin Y. K.-Y., Undheim E. A. B., Senff S., Mobli M., Dauly C., et al. (2020). Structural Venomics Reveals Evolution of a Complex Venom by Duplication and Diversification of an Ancient Peptide-Encoding Gene. Proc. Natl. Acad. Sci. USA 117, 11399–11408. 10.1073/pnas.1914536117 PubMed DOI PMC

R_Core_Team (2021). R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing.

Rádis-Baptista G. (2021). Cell-penetrating Peptides Derived from Animal Venoms and Toxins. Toxins 13, 147. 10.3390/toxins13020147 PubMed DOI PMC

Reis P. V. M., Boff D., Verly R. M., Melo-Braga M. N., Cortés M. E., Santos D. M., et al. (2018). LyeTxI-b, a Synthetic Peptide Derived from Lycosa erythrognatha Spider Venom, Shows Potent Antibiotic Activity In Vitro and In Vivo . Front. Microbiol. 9, 667. 10.3389/fmicb.2018.00667 PubMed DOI PMC

Robinson S. D., Undheim E. A. B., Ueberheide B., King G. F. (2017). Venom Peptides as Therapeutics: Advances, Challenges and the Future of Venom-Peptide Discovery. Expert Rev. Proteomics 14, 931–939. 10.1080/14789450.2017.1377613 PubMed DOI

Sachkova M. Y., Slavokhotova A. A., Grishin E. V., Vassilevski A. A. (2014). Genes and Evolution of Two-Domain Toxins from Lynx Spider Venom. FEBS Lett. 588, 740–745. 10.1016/j.febslet.2014.01.018 PubMed DOI

Saez N. J., Herzig V. (2019). Versatile Spider Venom Peptides and Their Medical and Agricultural Applications. Toxicon 158, 109–126. 10.1016/j.toxicon.2018.11.298 PubMed DOI

Sanggaard K. W., Bechsgaard J. S., Fang X., Duan J., Dyrlund T. F., Gupta V., et al. (2014). Spider Genomes Provide Insight into Composition and Evolution of Venom and Silk. Nat. Commun. 5, 3765. 10.1038/ncomms4765 PubMed DOI PMC

Selden P. A., Penney D. (2010). Fossil Spiders. Biol. Rev. Camb Philos. Soc. 85, 171–206. 10.1111/j.1469-185X.2009.00099.x PubMed DOI

Sollod B. L., Wilson D., Zhaxybayeva O., Gogarten J. P., Drinkwater R., King G. F. (2005). Were Arachnids the First to Use Combinatorial Peptide Libraries? Peptides 26, 131–139. 10.1016/j.peptides.2004.07.016 PubMed DOI

Undheim E. A. B., Grimm L. L., Low C.-F., Morgenstern D., Herzig V., Zobel-Thropp P., et al. (2015). Weaponization of a Hormone: Convergent Recruitment of Hyperglycemic Hormone into the Venom of Arthropod Predators. Structure 23, 1283–1292. 10.1016/j.str.2015.05.003 PubMed DOI

Vassilevski A. A., Kozlov S. A., Samsonova O. V., Egorova N. S., Karpunin D. V., Pluzhnikov K. A., et al. (2008). Cyto-insectotoxins, a Novel Class of Cytolytic and Insecticidal Peptides from Spider Venom. Biochem. J. 411, 687–696. 10.1042/bj20071123 PubMed DOI

Vassilevski A. A., Sachkova M. Y., Ignatova A. A., Kozlov S. A., Feofanov A. V., Grishin E. V. (2013). Spider Toxins Comprising Disulfide-Rich and Linear Amphipathic Domains: a New Class of Molecules Identified in the Lynx Spider Oxyopes takobius . FEBS J. 280, 6247–6261. 10.1111/febs.12547 PubMed DOI

Wagner G. P., Kin K., Lynch V. J. (2012). Measurement of mRNA Abundance Using RNA-Seq Data: RPKM Measure Is Inconsistent Among Samples. Theor. Biosci. 131, 281–285. 10.1007/s12064-012-0162-3 PubMed DOI

Waterhouse A. M., Procter J. B., Martin D. M. A., Clamp M., Barton G. J. (2009). Jalview Version 2--a Multiple Sequence Alignment Editor and Analysis Workbench. Bioinformatics 25, 1189–1191. 10.1093/bioinformatics/btp033 PubMed DOI PMC

Wheeler W. C., Coddington J. A., Crowley L. M., Dimitrov D., Goloboff P. A., Griswold C. E., et al. (2017). The Spider Tree of Life: Phylogeny of Araneae Based on Target‐gene Analyses from an Extensive Taxon Sampling. Cladistics 33, 574–616. 10.1111/cla.12182 PubMed DOI

WSC (2021). World Spider Catalog. Version 22.0. Natural History Museum Bern. Available at: http://wsc.nmbe.ch. (Accessed on 12.04.2021).

Wullschleger B., Nentwig W., Kuhn-Nentwig L. (2005). Spider Venom: Enhancement of Venom Efficacy Mediated by Different Synergistic Strategies in Cupiennius salei . J. Exp. Biol. 208, 2115–2121. 10.1242/jeb.01594 PubMed DOI

Yan L., Adams M. E. (1998). Lycotoxins, Antimicrobial Peptides from Venom of the Wolf Spider Lycosa carolinensis . J. Biol. Chem. 273, 2059–2066. 10.1074/jbc.273.4.2059 PubMed DOI