toxins Article Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug (Havinthus rufovarius) Laura C. Wait , Andrew A. Walker * and Glenn F. King * Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Rd, St Lucia, QLD 4072, Australia; [email protected] * Correspondence: [email protected] (A.A.W.); [email protected] (G.F.K.); Tel.: +61-7-3346-2011 (A.A.W.); +61-7-3346-2025 (G.F.K.) Abstract: Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug (Havinthus rufovarius), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies (Lucilia cuprina). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with diverse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug Pristhesancus plagipennis, their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments Citation: Wait, L.C.; Walker, A.A.; revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured King, G.F. Crouching Tiger, Hidden a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may Protein: Searching for Insecticidal contribute to the insecticidal activity of assassin bug venom. Toxins in Venom of the Red Tiger Assassin Bug (Havinthus Keywords: venom; toxin; assassin bug; Reduviidae; insecticidal; CUB domain; cystatin rufovarius). Toxins 2021, 13, 3. https:// dx.doi.org/10.3390/toxins13010003 Key Contribution: Insecticidal fractions of red tiger assassin bug (Havinthus rufovarius) venom contained a solitary CUB domain protein and cystatin; suggesting an insecticidal function for Received: 17 November 2020 Accepted: 18 December 2020 these proteins. Published: 22 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims 1. Introduction in published maps and institutional Assassin bugs belong to the hemipteran family Reduviidae. Most assassin bugs affiliations. use their proboscis to pierce prey, inject venom, and feed from the envenomated prey. An exception is the kissing bugs, which are blood-feeding ectoparasites, whose venom disrupts host haemostatic defences [1]. Unlike kissing bugs, entomophagous assassin bugs Copyright: © 2020 by the authors. Li- are descended from ancestors that have retained predation as their principle trophic strategy censee MDPI, Basel, Switzerland. This for >200 million years [2]. Over this time, evolution has honed the insecticidal capability of article is an open access article distributed assassin bug venom to contain potent and quick-acting insecticidal compounds. In 1961, under the terms and conditions of the Edwards [3] showed that the first instar larva of the assassin bug Rhynocoris carmelita could Creative Commons Attribution (CC BY) paralyse an insect greater than 400 times its weight (a first instar Mediterranean flour moth, license (https://creativecommons.org/ Ephestia kuehniella) in under 10 s. The red spot assassin bug Platymeris rhadamanthus can licenses/by/4.0/). paralyse the American cockroach Periplaneta americana sufficiently to stop its struggling Toxins 2021, 13, 3. https://dx.doi.org/10.3390/toxins13010003 https://www.mdpi.com/journal/toxins Toxins 2021, 13, x FOR PEER REVIEW 2 of 12 Toxins 2021, 13, 3 bug Platymeris rhadamanthus can paralyse the American cockroach Periplaneta americana2 of 12 sufficiently to stop its struggling within 3–5 s, and completely paralyse it within 15 s. In- jection of 0.1 µl of venom from the bee-killer assassin bug Pristhesancus plagipennis ob- withintained 3–5 by s,electrostimulation, and completely paralyse which is it secreted within 15 by s. the Injection large posterior of 0.1 µl ofmain venom venom from gland, the bee-killerparalyses assassin third-instar bug Pristhesancuscrickets within plagipennis seconds obtained[4]. However, by electrostimulation, compared to snake, which cone is secretedsnail, and by arachnid the large venoms, posterior the main toxins venom underlying gland, paralyses these effects third-instar in assassin crickets bug venoms within secondsare poorly [4]. characterised. However, compared to snake, cone snail, and arachnid venoms, the toxins underlyingThe ability these effectsof arachnid in assassin venoms bug to venoms induce are prey poorly paralysis characterised. is due to their ability to modulateThe ability the function of arachnid of ion venoms channels to induceon the surface prey paralysis of excitable is due cells to [5,6]. their abilityThe major to modulatebioactive thecomponents function ofof ionmost channels spider venom on the are surface disulfide of excitable-rich peptide cells [ 5neurotoxins,6]. The major that bioactivebind to and components modulate of presynaptic most spider or venom postsynaptic are disulfide-rich neuronal ion peptide channels. neurotoxins Assassin that bug bindvenoms to and also modulate contain presynapticdisulfide-rich or peptide postsynaptic neurotoxins, neuronal such ion channels.Ptu1 from Assassin the peiratine bug venomsassassin also bug containPeirates disulfide-richturpis, which inhibits peptide the neurotoxins, voltage-gated such calcium Ptu1 fromchannel the Ca peiratineV.2.2 [7]. assassinPtu1 has bug an Peiratesinhibitor turpis cystine, which knot inhibits(ICK) fold the [8], voltage-gated which has been calcium independently channel Ca Vrecruited.2.2 [7]. Ptu1into hasvenom an inhibitors produced cystine by phylogenetically knot (ICK) fold [8diverse], which animal has been groups, independently including recruitedarachnids intoand venoms cone snails produced [9]. Previously, by phylogenetically we reported diversethat insecticidal animal groups, venom includingof the assassin arachnids bug P. andplagipennis cone snails contained [9]. Previously, multiple wepeptides reported with that primary insecticidal structures venom suggesting of the assassin that bugthey P.adopt plagipennis an ICKcontained fold [10]. multipleHowever, peptides in that study, with primarywe also reported structures that suggesting such peptides that theymade adoptup a ansmall ICK proportion fold [10]. However,(1–3%) of inP. thatplagipennis study, we venom, also reported whereas that many such of peptidesthe most made abun- updant a small venom proportion components (1–3%) were of P. of plagipennis unknownvenom, function. whereas Moreover, many injection of the most of Ptu abundant family venompeptide componentss from assassin were ofbug unknown venom into function. prey Moreover,species has injection been performed, of Ptu family but peptides did not fromresult assassin in paralysis bug venom[7,11]. This into suggests prey species that hasother been components performed, underlie but did the not paralytic result inef- paralysisfects of assassin [7,11]. bug This venom. suggests that other components underlie the paralytic effects of assassinIn bugthis venom.study, we aim to better understand the venom components responsible for the paralysingIn this study, and we lethal aim toeffects better of understand reduviid venoms the venom by isolating components insecticidal responsible compounds for the paralysingfrom the venom and lethal of the effects red of tiger reduviid assassin venoms bug byHavinthus isolating rufovarius insecticidal (Figure compounds 1). We fromshow thethat venom two of of the redmost tiger potent assassin paralytic bug Havinthus venom fractions rufovarius contain(Figure a1 ).solitary We show CUB that domain two ofprotein the most and potent a cystatin, paralytic neither venom of which fractions has contain previously a solitary been CUBreported domain to possess protein insecti- and a cystatin, neither of which has previously been reported to possess insecticidal activity. cidal activity. Figure 1. Red tiger assassin bug H. rufovarius. This harpactorine species is common under the bark Figure 1. Red tiger assassin bug H. rufovarius. This harpactorine species is common under the bark of Eucalyptus sp. trees and is widespread in Australia. The individual shown is displaying a threat of Eucalyptus sp. trees and is widespread in Australia. The individual shown is displaying a threat posture with proboscis extended in response to provocation with tweezers. Photo © Jiayi Jin. posture with proboscis extended in response to provocation with tweezers. Photo © Jiayi Jin. 2. Results 2.1. Electrostimulation and Harassment Yield Similar Venom Since it has previously been reported that electrostimulation and harassment of assas- sin bugs can yield different venoms from different gland lumens [4,12,13],