RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach, Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha-amylase Blattella germanica Tergal Gland protein-1 (BGTG-1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG-1 protein. BGTG-1 gene expression profiles were determined by using quantitative real-time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG-1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG-1 gene expression by injecting BGTG-1 homologous double-stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG-1 dsRNA showed ∼90% lower BGTG-1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.

Characterization of five CYP4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus-infected and uninfected psyllids.

Previously, we reported that Candidatus Liberibacter asiaticus (Las)-infected Diaphorina citri are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In the present study, we investigated expression levels of family 4 cytochrome P450 (CYP4) genes in Las-infected and uninfected D.citri adults. Five novel CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) were identified. Four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las-infected males, whereas only one was expressed at significantly higher levels in uninfected than Las-infected females. These results suggest that levels of cytochrome P450 monooxygenases in D.citri may be linked to expression levels of these CYP4 genes. Expression of all five CYP4 genes was induced by exposure of D.citri to imidacloprid, suggesting their possible involvement in metabolism of this toxin. Higher expression of the five CYP4 genes was found in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA-interference to silence overexpression of genes associated with insecticide resistance in D.citri. These newly identified genes may also serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of D.citri.