Pigmentation plays a vital role in insect survival and reproduction. Many melanin pathway genes have been studied in holometabolous insects; however, they have only been studied in two hemimetabolous insect genera, Oncopeltus and Periplaneta. Here we analyzed three melanin pathway genes (TH, yellow, and aaNAT) using RNA interference (RNAi) in another hemimetabolous insect, namely the twin-spotted assassin bug, Platymeris biguttatus. TH was highly expressed in freshly molted nymphs and adults. TH RNAi resulted in a complete loss of black pigment, with yellow coloration maintained. Therefore, black pigment in this assassin bug is solely generated from the melanin pathway, whereas yellow pigment is generated from other unknown pigmentation pathways. yellow and aaNAT were highly expressed in the white spot of the hemelytra. Downregulation of yellow caused a brown phenotype with high mortality, indicating an important role of yellow functions in cuticle formation and in the process of converting melanin from brown to black. Interestingly, aaNAT RNAi caused not only loss of white pigment, but also loss of yellow and red pigments. This phenotype of aaNAT has not been reported in other insects. Our results provide new information for understanding the melanin pathway in which aaNAT is essential for the formation of colorless patterns.

Assassin bugs use their salivary venoms for various purposes, including defense, prey paralyzation, and extra-oral digestion, but the mechanisms underlying the functional complexity of the venom remain largely unclear. Since venom glands are composed of several chambers, it is suggested that individual chambers may be specialized to produce chemically distinct venoms to exert different functions. The current study assesses this hypothesis by performing toxicity assays and transcriptomic and proteomic analysis on components from three major venom gland chambers including the anterior main gland (AMG), the posterior main gland (PMG), and the accessory gland (AG) of the assassin bug Platymeris biguttatus. Proteotranscriptomic analysis reveals that AMG and PMG extracts are rich in hemolytic proteins and serine proteases, respectively, whereas transferrin and apolipophorin are dominant in the AG. Toxicity assays reveal that secretions from different gland chambers have distinct effects on the prey, with that from AG compromising prey mobility, that from PMG causing prey death and liquifying the corpse, and that from AMG showing no significant physiological effects. Our study reveals a functional cooperation among venom gland chambers of assassin bugs and provides new insights into physiological adaptations to venom-based predation and defense in venomous predatory bugs.

Acanthaspis cincticrus (Stål) is an assassin bug with a specialized camouflaging behavior to ambush ants in the nymphal stages. In this study, we comprehensively sequenced all the life stages of A. cincticrus, including the eggs, five nymph instars, female and male adults using Illumina HiSeq technology. We obtained 176 million clean sequence reads. The assembled 84,055 unigenes were annotated and classified functionally based on protein databases. Among the unigenes, 29.03% were annotated by one or more databases, suggesting their well-conserved functions. Comparison of the gene expression profiles in the egg, nymph and adult stages revealed certain bias. Functional enrichment analysis of significantly differentially expressed genes (SDEGs) showed positive correlation with specific physiological processes within each stage, including venom, aggression, olfactory recognition as well as growth and development. Relative expression of ten SDEGs involved in predation process was validated using quantitative real-time PCR (qRT-PCR).