Molecular cloning and characterization of the allatostatin receptor in the cockroach Diploptera punctata
Allatostatin , Allatostatin receptor , Juvenile hormone , RNAi
Allatostatins (ASTs) are neuropeptides that inhibit the biosynthesis and release of juvenile hormone from the corpora allata (CA) of various insects including the cockroach Diploptera punctata. We hypothesized that a similar allatostatin receptor would exist in the cockroach D. punctata that may regulate the numerous physiological effects that this family of peptides exerts on a range of target tissues. Using polymerase chain reaction (PCR) strategies successful in isolating other insect allatostatin receptor sequences utilized primers designed to known mammalian somatostatin receptors and Drosophila allatostatin receptor (AlstR). Once an internal PCR fragment was proven to be consistent with the sequence of an allatostatin receptor (AstR) then the sequence was completed by rapid amplification of cDNA ends (RACE). The putative allatostatin-like receptor sequence encoding 425 amino acid residues was isolated from a cDNA library prepared from corpora allata of D. punctata. We show that dsRNA targeting the allatostatin receptor gene of D. punctata injected into freshly moulted adult cockroaches produced a long-lasting reduction in the mRNA levels in midgut tissues. The effect lasted up to 6 days. Following dsRNA injection, the juvenile hormone (JH) titers in the corpora allata were clearly raised suggest that the putative inhibition of receptor RNA expression may increase JH production. The receptor is expressed in brains, corpora allata, abdominal ganglion, midguts, ovaries, and testes. We have examined these same tissues with regard to changes in expression levels of Dippu-AstR. JH biosynthesis peaks on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA is expressed at the highest levels on day 6 post-emergence in brain and corpora allata and day 2 post-emergence in midgut. Dippu-AstR is likely responsible for the decline in JH biosynthesis after day 5 post-emergence. Virgin females midgut and CA Dippu-AstR mRNA expression dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR was similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declines to low levels by day 4. Testes also express peaks of Dippu-AstR expression on days 4 and 7. A role for Dippu-AST in testes is yet unknown.