RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera

Abstract

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs β bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs β and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs β and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

“…In experiments for gene functional analysis, the delivery of dsRNA is often performed by microinjection because a lower amount of dsRNA is required and the RNAi response is faster when compared to ingestion of dsRNA. Moreover, microinjection is commonly used when the insects are refractory to RNAi by feeding . However, for the practical application of RNAi to control insect pests in the field it is necessary that the target insect be sensitive to RNAi by oral exposure.…”

“…Moreover, microinjection is commonly used when the insects are refractory to RNAi by feeding. [31][32][33][34][35] However, for the practical application of RNAi to control insect pests in the field it is necessary that the target insect be sensitive to RNAi by oral exposure. Therefore, we performed feeding bioassays to access whether ingestion of dsRNA could induce RNAi in SLW adults.…”

Hormonal control and target genes of ftz‐f1 expression in the honeybee Apis mellifera: a positive loop linking juvenile hormone, ftz‐f1, and vitellogenin

“…The amounts of applied hormone (20E and JH) were based on previous experiments in which we had examined their effects on regulating gene expression during the pharate‐adult stage (Barchuk et al ., ; Mello et al ., ; Costa et al . ).…”

“…cDNA was synthesized as described earlier, and transcription levels were assessed by qPCR. The amounts of applied hormone (20E and JH) were based on previous experiments in which we had examined their effects on regulating gene expression during the pharate-adult stage (Barchuk et al, 2002(Barchuk et al, , 2004Mello et al, 2014;Costa et al 2016).…”

Targeting the potassium ion channel genes SK and SH as a novel approach for control of insect pests: efficacy and biosafety

“…and Tijsterman and Plasterk indicated that activation of the RNAi machinery in the organism's cells requires the introduction of dsRNA matching around 21–25 bp of mRNA. To address this important concern, we selected the honeybee A. mellifera since not only are they important pollinators but they are highly sensitive to dsRNA, exhibiting a strong RNAi response …”