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.
Highlights
Introduction in published maps and institutionalAssassin bugs belong to the hemipteran family Reduviidae
Since it has previously been reported that electrostimulation and harassment of assassin bugs can yield different venoms from different gland lumens [4,12,13], we investigated if this was true for H. rufovarius (Figure 1)
Venom could be obtained by either stimulus, venom obtained by electrostimulation and harassment of H. rufovarius yielded similar mass profiles using matrix-assisted laser desorption/ionisation time-offlight mass spectrometry (MALDI-TOF-MS) using a 5800 MALDI-TOF instrument (SCIEX, Framingham, MA, USA) (Figure 2)
Summary
Introduction in published maps and institutionalAssassin bugs belong to the hemipteran family Reduviidae. Most assassin bugs use their proboscis to pierce prey, inject venom, and feed from the envenomated prey. Entomophagous assassin bugs are descended from ancestors that have retained predation as their principle trophic strategy for >200 million years [2]. Over this time, evolution has honed the insecticidal capability of assassin bug venom to contain potent and quick-acting insecticidal compounds. In 1961, Edwards [3] showed that the first instar larva of the assassin bug Rhynocoris carmelita could paralyse an insect greater than 400 times its weight (a first instar Mediterranean flour moth, Ephestia kuehniella) in under 10 s. The red spot assassin bug Platymeris rhadamanthus can paralyse the American cockroach Periplaneta americana sufficiently to stop its struggling affiliations
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