Context-dependent venom deployment and protein composition in two assassin bugs

Abstract

The Heteroptera are a diverse suborder of phytophagous, hematophagous, and zoophagous insects. The shift to zoophagy can be traced back to the transformation of salivary glands into venom glands, but the venom is used not only to kill and digest invertebrate prey but also as a defense strategy, mainly against vertebrates. In this study, we used an integrated transcriptomics and proteomics approach to compare the composition of venoms from the anterior main gland (AMG) and posterior main gland (PMG) of the reduviid bugs Platymeris biguttatus L. and Psytalla horrida Stål. In both species, the AMG and PMG secreted distinct protein mixtures with few interspecific differences. PMG venom consisted mostly of S1 proteases, redulysins, Ptu1-like peptides, and uncharacterized proteins, whereas AMG venom contained hemolysins and cystatins. There was a remarkable difference in biological activity between the AMG and PMG venoms, with only PMG venom conferring digestive, neurotoxic, hemolytic, antibacterial, and cytotoxic effects. Proteomic analysis of venom samples revealed the context-dependent use of AMG and PMG venom. Although both species secreted PMG venom alone to overwhelm their prey and facilitate digestion, the deployment of defensive venom was species-dependent. P. biguttatus almost exclusively used PMG venom for defense, whereas P. horrida secreted PMG venom in response to mild harassment but AMG venom in response to more intense harassment. This intriguing context-dependent use of defensive venom indicates that future research should focus on species-dependent differences in venom composition and defense strategies among predatory Heteroptera.

Keywords: assassin bug; defense venom; prey‐killing venom; proteomics; transcriptomics; zoophagy.

Figure 1

Schematic workflow of an integrated transcriptomic, proteomic and assay‐based approach to identify the venom‐specific proteins and venom activity of P. biguttatus and P. horrida

Figure 2

Digestive effects of P. biguttatus PMG venom on G. mellonella larvae at different time points after venom injection

Figure 3

Hemolytic (a), antimicrobial (b), and cytotoxic (c) effects of PMG venom extracted from P. biguttatus and P. horrida. (a) AMG = 20 µg/µl anterior main gland venom; PMG = 100 µg/µl posterior main gland venom; + = 1% Triton X‐100. PMG venom generated large hemolytic zones in blood agar plates, indicating the presence of proteins with strong hemolytic activity. (b) AMG/PMG as above; + = 0.5 µg/µl gentamycin. PMG venom from either species caused the significant inhibition of bacterial growth in an E. coli inhibition zone assay. (c) Diluted PMG venom displayed cytotoxic activity against Sf9 cells, reducing the cell density and causing extensive cell death

Figure 4

SDS‐PAGE analysis of venom extracts from homogenized glandular tissue and venom collected without dissection from P. horrida and P. biguttatus. AMG = anterior main gland extract; PMG = posterior main gland extract; P1 = provocation venom (mild harassment); P2 = provocation venom (cold stress); P3 = provocation venom (strong harassment); I1 = injection venom (prey dummy) after 1.5 min; and PM = protein marker

Figure 5

Protein composition of the AMG, PMG, and gut secretions of P. biguttatus and P. horrida. Color‐coded blocks show the number of contigs identified in transcriptome datasets encoding specific classes of functional proteins. The venom protein families are shown separately in the inset box

Figure 6

Proteins of the P. horrida PMG and prey dummy venom identified by LC‐MS/MS. The Coomassie‐stained protein gel on the left yielded the PMG venom proteins shown on the right, including the predicted protein masses (kDa), the total score, number of assigned peptides and descriptions. The excised bands are indicated with numbers and lines on the right side of the protein gel. For the proteins identified by LC‐MS/MS, gene expression levels (log2 TPM) in the PMG, AMG, gut, and remaining body tissues are shown in the heat map. PM = protein marker. See Table S1 for the identity of matching predicted proteins in the P. horrida transcriptome

Figure 7

Proteins of the P. horrida AMG and defense venom (mild harassment) identified by LC‐MS/MS. The Coomassie‐stained protein gel on the left yielded the AMG venom proteins shown on the right, including the predicted protein masses (kDa), the total score, number of assigned peptides and descriptions. The excised bands are indicated with numbers and lines on the right side of the protein gel. For the proteins identified by LC‐MS/MS, gene expression levels (log2 TPM) in AMG, PMG, gut, and remaining body tissues are shown in the heat map. PM = protein marker. See Table S1 for the identity of matching predicted proteins in the P. horrida transcriptome

Figure 8

Gene expression levels (log2 TPM) of proteins from different venom protein families in the rest of body tissue (RB), gut, AMG, and PMG for P. biguttatus and P. horrida