Quantification and visualization of Dippu-AST mRNA in the brain of adult Diploptera punctata: mated females vs. virgin females vs. males

Abstract

YXFGLa-allatostatins (ASTs) are a family of neuropeptides that inhibit juvenile hormone biosynthesis in vitro by the corpora allata of cockroaches and crickets. We have investigated the levels of AST expression in the brain of adult mated and virgin females, and adult males of the cockroach Diploptera punctata, for 11 days following emergence. To quantify Dippu-AST mRNA we have used an optimized, sensitive and specific quantitative competitive reverse-transcriptase polymerase chain reaction (QC–RT–PCR) method. Using this technique, we show that Dippu-AST is expressed at higher levels in the brains of mated females than in the brains of virgin females. Specifically, levels of Dippu-AST mRNA in day 0 mated females are 2.5× higher than levels in day 0 virgin females. By day 6–7, levels in mated females are 20× higher than those in virgin females. However, the general pattern of expression is preserved in mated and virgin females; levels of Dippu-AST mRNA are relatively high in newly eclosed females, fall steadily to significantly lower levels on day 5 (mated) and day 5–7 (virgins), and then increase and remain relatively stable until at least day 10. No significant changes in Dippu-AST mRNA are apparent in male brains during the observation period. However, the general pattern of expression is virtually the inverse of that seen in females. Dippu-AST mRNA in male brains is relatively low following emergence, increases to maximum levels on day 5, following which, levels steadily fall. In situ hybridization suggests that fluctuations in Dippu-AST mRNA expression are a result of differential transcription within medial and lateral cells of the pars intercerebralis in both males and females. In addition to yielding insights into Dippu-AST function, the quantification of Dippu-AST expression under normal conditions is useful in providing a basis for comparison of Dippu-AST expression following various physiological manipulations.