Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae)

Abstract

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sapand phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10 -16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera. Venoms are chemical arsenals injected by one animal into another to disrupt the homeostasis of the injected animal in ways that assist predation, defense, or feeding by the injecting animal (1). Typically, venoms are composed of multiple toxins, including peptides, enzymes, and small molecules, such as polyamines, that bind to and affect the function of multiple molecular targets in the injected animal. Because of their key role governing life-or-death interactions between animals, venom toxins are subject to selection pressures that have resulted in unique evolutionary patterns such as massive duplication and accelerated evolution of toxin-encoding genes (2-5). In addition, the properties that ensure that toxins confer a fitness advantage to the animals that produce them, including high stability and potency, make them well suited for use as insecticides, therapeutics, and pharmacological tools (6 -11). However, our understanding of the factors shaping venom evolution, and our ability to repurpose venom toxins for biotechnological use, is limited ...

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms

“…[6,38] Other venoms, especially those of snakes, jellyfish, sea anemones, centipedes, assassin bugs, and robber flies also contain abundant larger polypeptide toxins (>10 kDa) such as pore-forming toxins; enzymes such hyaluronidase, proteases, and phospholipase A 2 ; and cysteinerich secretory proteins (CRiSPs). [39][40][41][42][43] Since polypeptides are the dominant components of most venoms, these chemical arsenals are ideally suited to characteriza-tion using mass spectrometry (MS)-based proteomics. [44,45] However, there are numerous challenges that must be addressed in MS studies of venom.…”

“…For the many venom peptides that fall in the 1-4 kDa mass range, [54] matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) in reflectron positive mode is a convenient method and offers resolution sufficient to determine toxin monoisotopic masses. [43,55,56] Toxins larger than 5 kDa are often difficult to measure in reflectron-positive mode, but approximate average masses can be obtained using linear mode MALDI-TOF. [57,58] Application of MALDI-TOF MS to whole venom has allowed researchers to rapidly generate venom massprofiles.…”

“…LC-MS/MS using DDA is the most common method for obtaining venom peptide fragmentation data in many laboratories, [22,25,[48][49][50][51][52]57,[65][66][67][68][69][70][71][72][73][74][75][76][77][78][79] including ours. [29,42,43,53,61,[80][81][82][83] These data are typically searched using an automated algorithm against a database of protein sequences to identify peptide primary structures. However the non-matched data from these experiments are also well suited to de novo sequencing, particularly for mass spectra collected from high mass resolving instruments.…”

“…It should be noted that in many groups of venomous animals, most or all polypeptide toxin precursors begin with a signal sequence. [6,43] However, some animals produce toxin precursors without signal sequences-a classic example being -latrotoxin produced by black widow spiders (Latrodectus sp. ), which has no signal peptide, is translated in the cytoplasm, and released into the gland lumen by holocrine secretion (disruption of the secretory cell membrane).…”

“…B) Total ion chromatogram from LC-MS/MS of untrypsinized venom, with peaks labeled with peptide identities determined from MS 1 and MS 2 spectra. Panels C and D, assassin bug venom proteome, adapted from Walker et al [43] with permission. 2017, ASB&MB.…”