Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug (Havinthus rufovarius)

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 revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.

An Assassin’s Secret: Multifunctional Cytotoxic Compounds in the Predation Venom of the Assassin Bug Psytalla horrida (Reduviidae, Hemiptera)

“…−50% in less than a minute, probably representing the effects of fast-acting compounds such as neurotoxins. Venom peptides from assassin bugs that have been suggested to act as neurotoxins include ptu1like peptides, cystatins, and CUB domain proteins [16,30]. The fast decrease in [Ca 2+ ] i was followed by a gradual increase over several minutes.…”

“…A recent study attempted to identify the insecticidal venom components of the red tiger assassin bug Havinthus rufovarius (Bergroth) by determining the toxic activity of venom fractions. The fractions with the strongest paralytic and lethal effects on sheep blowflies contained primarily a CUB domain protein and a cystatin, suggesting that one or both proteins contribute to the venom's insecticidal activity [16]. In the hematophagous bug Triatoma infestans (Klug), trialysin has been identified as a major toxic component that exerts cytotoxic effects against bacteria, protozoa, and mammalian cells by disrupting cell membranes.…”

The Predatory Stink Bug Arma custos (Hemiptera: Pentatomidae) Produces a Complex Proteinaceous Venom to Overcome Caterpillar Prey

“…The identification of compounds in the salivary venom of predatory heteropterans is essential for assessing their potential for biological pest management. Recently, with the development of transcriptome and proteome analyses, the composition of predatory heteropteran venoms has been studied in several species, including assassin bugs (the family Reduviidae) [8,9], minute pirate bugs (the family Anthocoridae) [10], and true water bugs (the infraorder Nepomorpha) [11,12] (Figure S1). These studies revealed that venoms are typically complex cocktails of organic and inorganic compounds, with numerous proteinaceous components [13][14][15].…”

Identification and functional analysis of serine protease inhibitor gene family of Eocanthecona furcellata (Wolff)

“…The saliva of predatory bugs has been identified as a source of toxins, including some digestive enzymes, proteins, peptides, etc. ( Beak and Lee, 2014 ), which have the functions of paralyzing prey, defense, digesting nutrients, antibacterial, and lysing cells ( Walker et al, 2016 ; Tonk et al, 2020 ; Wait et al, 2020 ). Currently, the research on the application of predatory bug venom mainly focuses on the Reduviidae bugs.…”

“…The venom of Pristhesancus plagipennis was injected into Lucilia cuprina , and paralysis and death could be produced after 15 min of application, while injection into crickets could immediately produce paralysis ( Walker et al, 2018a ). Four components isolated from the salivary glands of Havinthus rufovarius were injected into L. cuprina , resulting in paralysis and death ( Wait et al, 2020 ). Previously, our laboratory found that the salivary gland secretion of E. furcellata had a lethal effect on the fourth instar larvae of Spodoptera litura and affected its detoxification enzyme activity.…”