Ondřej Michálek 1) Introduction to venom & venom in spiders 2) Venom apparatus morphology 3) Venom chemistry 4) Venom evolution 5) Venom ecology 6) Methods to study venoms & venom applications 7) Sum-up What is venom? • physical warfare -> chemical warfare • „A biological substance produced by an organism that contains molecules (“toxins”) which interfere with physiological or biochemical processes in another organism, which has evolved in the venomous organism to provide a benefit to itself once introduced to the other organism. The venom is produced and/or stored in a specialised structure and actively transferred to another organism through an injury by means of a specialised delivery system.” (Arbuckle, 2017) What is venom? • Key aspects of venom: • Toxins • Venom gland/tissue • Specialised venom apparatus • Transfer to target animal through injury • Alter physiological or biochemical processes in target animal (paralysis) • A benefit to venomous animal Koua et al., 2020 Lüddecke et al., 2019 Venom vs poison Venom vs poison vs toxungen Nelsen et al., 2014 Photo: Wolfgang Wuster Venom function • Predation • Defence Venom function – not just predation and/or defence… Schendel et al., 2019 How widespread is venom? • Evolved independently more than 100 times • Eight separate phyla (Schendel et al., 2019) How widespread is venom? • Evolved independently more than 100 times • Eight separate phyla (Schendel et al., 2019) • Arthropods and arachnids: (Lüddecke et al., 2021) V V V V Gereral morphology • Venom glands or organelles • Venom apparatus Walker al., 2018 Insect venom apparatus Nematocyst of cnidarians Gereral morphology Schendel et al., 2019 Spider venom apparatus morphology Lüddecke et al., 2021 Spider venom apparatus morphology Lüddecke et al., 2021 Type of toxins • Haemotoxins - disrupt haemostatic system - not common in spiders • Cytotoxins - impair the structure and function of cell membranes - not common in spiders Type of toxins • Haemotoxins - disrupt haemostatic system - not common in spiders • Cytotoxins - impair the structure and function of cell membranes - not common in spiders recluse spiders (Loxosceles spp.) Malaque et al., 2016 Type of toxins • Haemotoxins - disrupt haemostatic system - not common in spiders • Cytotoxins - impair the structure and function of cell membranes - not common in spiders • Neurotoxins - presynaptically or postsynaptically affect neurotransmission - main components of spider venom Neurotoxins Venom biochemistry • Tens to thousands of components in single species • Hereafter emphasis on spider venom • Types of molecules: • Small molecules • Peptides • Antimicrobial Peptides • Cysteine-rich Peptides • Proteins Pineda et al., 2020 Hadronyche infensa Photo: David Wilson Small molecules • < 1 kDa • various compounds: ions, salts, organic acids, nucleotides, nucleosides, amino acids, amines, alkaloids and polyamines • various functions: - neurotransmitters - co-factors facilitating the folding and activity of toxins - insecticidal neurotoxins reviewed in Lüddecke et al., 2021 examples of spider acylpolyamines Peptides • < 10 kDa • Antimicrobial Peptides • linear, α-helical peptides without disulfide bonds • dual function: antimicrobial activity or lytic peptides • most of those identified AMPs found in Lycosidae and Theraphosidae; also Zodariidae or Oxyopidae • more than 50 such peptides in the venom of Lycosa sinensis reviewed in Lüddecke et al., 2021 Peptides • < 10 kDa • Antimicrobial Peptides Langenegger, Nentwig & Kuhn-Nentwig, 2019 Peptides • < 10 kDa • Antimicrobial Peptides • Cysteine-rich Peptides • functionally most important group of components • principal neurotoxic components • typically with molecular masses below 10 kDa • rich in disulfide bonds • different families: Kunitz peptides, HAND peptides, DDH peptides, ICK peptides reviewed in Lüddecke et al., 2021 Peptides • ICK (inhibitor cysteine knot) peptides • structure: (triple-stranded) antiparallel β-sheets at least 6 cysteine residues (forming 3 disulfide bridges) -> pseudoknot motif • expanded cysteine scaffolds and/or double ICK (dICK) motifs • exceptional stability • mode of action: stable complexes with prey receptors, disrupting their normal function • targets: voltage-gated sodium, potassium and calcium channels acid-sensing ion channels, glutamate receptors transient receptor potential channels Inhibitor cystine knot (ICK) Herzig & King, 2015 reviewed in Lüddecke et al., 2021 Proteins • > 10 kDa • key venom components in some taxa (e.g., black widows) • latrotoxins • homotetrameric pores in the presynaptic neuronal membranes • phospholipase D • highly cytotoxic sphingomyelin-hydrolysing enzyme • neprilysin metalloproteases, CAP proteins • unclear function α-latrotoxin reviewed in Lüddecke et al., 2021 Mode of action - synergistic effects of venom compounds • temporally and spatially regulated interactions • dual prey inactivation strategy (Kuhn-Nentwig et al., 2019) reviewed in Lüddecke et al., 2021 Mode of action - synergistic effects of venom compounds • temporally and spatially regulated interactions • dual prey inactivation strategy (Kuhn-Nentwig et al., 2019) Some of the large proteins immediately disrupt prey physiology and metabolism, while others act to spread the neurotoxins and thus trigger a subsequent wave of paralysis. • some peptides mediate or enhance the bioactivity of others • rapid paralysis followed by long-term immobilization • neurotransmitters making binding sites accessible for other toxins reviewed in Lüddecke et al., 2021 Pineda et al., 2020 Evolution of the delivery system in spiders • origin not clear • salivary glands, similar to those of ticks • silk-producing glands present in early chelicerates • the size and placement of the venom apparatus • modern araneomorphs vs Mesothelae and Mygalomorphae • migration of the venom glands from the basal part of the chelicerae into the prosoma • reflected during ontogenesis reviewed in Lüddecke et al., 2021 Recruitment and neofunctionalization Evolution of venom compounds • recruitment and weaponization • signalling molecules into unregulated agonists or inhibitors • frequent duplications • existing toxins can also undergo neofunctionalization • new activities and functions reviewed in Casewell et al., 2012; Lüddecke et al., 2021 Gene duplication • ICK peptides • pseudoknot motif - amino acid substitutions can accumulate with little impact on structure • descendants of a single weaponized ICK lineage • duplication and structural diversification • domain duplication - dICK peptides • also proposed for latrotoxins reviewed in Lüddecke et al., 2021 Overview of spider ICK peptides evolution Pineda et al., 2020 Horizontal gene transfer • recruitment of toxins from bacterial and fungal donors • phospholipase D • in the family Sicariidae • a single proteobacterial ancestor • also proposed to explain the origin of αLTX of Parasteatoda tepidariorum • otherwise documented in other venomous taxa, such as centipedes (Undheim & Jenner, 2021) reviewed in Lüddecke et al., 2021 Molecular mechanisms of venom evolution - overview Senji Laxme et al. 2019 Selection pressures acting on venom • strong positive selection • ‘Red Queen hypothesis’ – predator vs prey • but older lineages such as spiders show signatures of purifying selection • a two-speed model of venom gene evolution • positive selection mostly acts during the early stages of ecological specialization • followed by an extended stage of purifying selection reviewed in Lüddecke et al., 2021 Ontogeny • venom properties linked to life-history stages • venom yield increases as the spider ages • venom yield declines prior to ecdysis • compositional alterations as the spider ages reviewed in Lüddecke et al., 2021 Geograpic variation • the variability of venom profiles between allopatric populations of the same species • well documented in other venomous taxa, such as snakes • little attention in spiders (contrasting results) • Eratigena agrestis (Agelenidae) - Europe vs North America - no differences • Loxosceles rufescens (Sicariidae) Latrodectus spp. (Theridiidae) - differences in venom profiles and toxicity Photo: Rudolf Macek reviewed in Lüddecke et al., 2021 Eratigena agrestis Loxosceles rufescens Photo: David Wilson Hadronyche infensa Sexual dimorphism • well documented in spiders • australian funnel-web spiders - fatal males bites in humans, females much less toxic • Phoneutria nigriventer (Ctenidae) Loxosceles intermedia (Sicariidae) - females more toxic than males - sex-specific components Photo: Graham Wise reviewed in Lüddecke et al., 2021 Phoneutria nigriventer Individual variability • well documented in other venomous taxa, less attention in spiders • only recently documented in Hadronyche valida Photo: David Wilson reviewed in Lüddecke et al., 2021 Envenomation strategies • envenomation as major strategy to incapacitate prey in most spiders - some exceptions: Araneidae, Uloboridae, Scytotidae • toxins on the silk strands of the web (Esteves et al., 2020) • venom in defence - escape prioritized - dry bites - aposematism (supported by pain-inducing components) reviewed in Lüddecke et al., 2021 Photo: Fritz Geller-Grimm Scytodes thoracica Photo: Christine Hanrahan Argiope bruennichi Uloborus walckenaerius Venom optimization • venom - physiologically expensive • venom optimisation (or venom metering) - conservation of venom resources - economical delivery of venom - weak prey -> small amount of venom - resistant/dangerous prey -> larger ammount of venom • trophic specialisation - highly effective, but simpler venom - dispensable venom components purged from the venom to save resources Lüddecke et al., 2021; Morgenstern & King 2013; Pekár et al., 2018a,b Behavioural experiments • laboratory experiments with living specimens • for example, observation of prey paralysis after bite (e.g., Pekár et al, 2018b) Venom composition - venomics • integration of transcriptomic, proteomic (and genomic) approaches • transcriptomics of the venom-producing tissue • mass spectrometry (MS) based proteomics • bioinformatic integration of the data dissected glands RNA extraction sequencing crude venom reduction, alkylation, trypsin digestion mass spectrometer sequence annotation toxin names toxin families Structure of venom compotents • X-ray crystallography - rarely used for spider toxins - larger proteins • nuclear magnetic resonance (NMR) - most used for spider toxins - peptides • cryoelectron microscopy (EM) - rarely used for spider toxins Venom physiology bioassays • venom efficiency – venom injection bioassays – crude venom, venom fractions, isolated recombinant toxins • venom physiology – patch clamp technique – target Ion Channels synthesized and expressed in Xenopus oocytes – toxins added (recombinant toxins) – electrophysiological two-electrode voltage-clamp recordings Venom applications Venom applications - pesticides • eco-friendly bioinsecticides Venom applications - pesticides Venom applications - drug leads Ma, Mahadevappa & Kwok, 2015 Venom applications - drug leads Photo: David Wilson funnel-web spider Hadronyche infensa prof. Glenn King -> cure for stroke Glenn King: Deadly cures: a spider-venom peptide for treating ischemic injuries of the heart and brain (1st EUVEN CONGRESS) Glenn King: Deadly cures: a spider-venom peptide for treating ischemic injuries of the heart and brain (1st EUVEN CONGRESS) Glenn King: Deadly cures: a spider-venom peptide for treating ischemic injuries of the heart and brain (1st EUVEN CONGRESS) Glenn King: Deadly cures: a spider-venom peptide for treating ischemic injuries of the heart and brain (1st EUVEN CONGRESS) Summarization • one of the key traits of spiders • mainly for predation and defence • hundreds to thousand compounds per species • neurotoxins, mainly ICK peptides • evolved through duplication and neofunctionalization • positive selection followed by purifying selection • sexual dimorphism, perhaps some geographic and individual variability • costly substance – venom optimisation • various methods to study venoms; promising applications (drugs and pesticides) Arbuckle, K. 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