Gene-silencing reveals the functional significance of pheromone biosynthesis activating neuropeptide receptor (PBAN-R) in a male moth

Abstract

The role of pheromone biosynthesis activating neuropeptide (PBAN) in the regulation of pheromone biosynthesis of several female moth species is well elucidated, but its role in the males has been a mystery for over two decades since its discovery from both male and female central nervous systems. In previous studies we have identified the presence of the gene transcript for the PBAN-G-protein coupled receptor (PBAN-R) in Helicoverpa armigera male hair-pencil-aedaegus complexes (male complexes), a tissue structurally homologous to the female pheromone gland. Moreover, we showed that this transcript is up-regulated during pupal-adult development, analogous to its regulation in the female pheromone-glands, thereby indicating a likely functional gene. Here we argue in favor of PBAN's role in regulating the free fatty-acid components (myristic, palmitic, stearic, and oleic acids) and alcohol components (hexadecanol, cis-11 hexadecanol, and octadecanol) in male complexes. We demonstrate the diel periodicity in levels of male components, with peak titers occurring during the 7th-9th h in the scotophase, coincident with female pheromone production. In addition, we show significant stimulation of component levels by synthetic HezPBAN. Furthermore, we confirm PBAN's function in this tissue through knockdown of the PBAN-R gene using RNAi-mediated gene-silencing. Injections of PBAN-R dsRNA into the male hemocoel significantly inhibited levels of the various male components by 58%-74%. In conclusion, through gain and loss of function we revealed the functionality of the PBAN-R and the key components that are up-regulated by PBAN.

Fig. 1.

Levels of components from 3 d-old male complexes during the scotophase. (A) Illustration of male complexes showing valves (V) with hair-pencils (HP) on either side of the aedaegus (Ae). Dotted line represents dissection cut line. (B) Points represent the means ± s.e.m. of 4-11 replicates. Statistically significant differences (one-way ANOVA) were found at p < 0.005 in levels of palmitic acid [16∶COOH], stearic acid [18∶COOH], myristic acid [14∶COOH], octadecanol [18∶OH], tetradecanol [14∶OH], and at p < 0.05 in levels of oleic acid [(9)18∶COOH] and hexadecanol plus cis-11 hexadecanol [C-16 Alcohols]. No significant differences were found in the acetate components: tetradecenyl acetate [14: Ac], hexadecenyl acetate [16∶Ac], and octadecenyl acetate [18∶Ac]. (C) Percentage of volatile components at onset of scotophase compared to peak scotophase.

Fig. 2.

Effect of HezPBAN on levels of components from male complexes of 3 d-old decapitated males. Bars represent the means ± s.e.m. of 4-11 replicates. Statistically significant differences (Student t-test) are indicated with ** for p < 0.005 and * for p < 0.05. 16∶COOH = palmitic acid, (9)18∶COOH = oleic acid, 18∶COOH = stearic acid, 14∶COOH = myristic acid, C-16 Alcohols = hexadecanol plus cis-11 hexadecanol, 18∶OH = octadecanol.

Fig. 3.

The effect of silencing the PBAN-R by injection of dsRNA in vivo on male component levels. Males that were 1 d-old were injected with RNAase-free water, D. nautilus dsRNA, or PBAN-R dsRNA. After 24 h the males were decapitated and kept in moist containers for a further 24 h for endogenous PBAN depletion and they were injected a second time with either water (as a control) or Hez-PBAN for component production analysis. The different treatments are indicated in the table below the figure. All the results are expressed as percentage stimulation relative to component levels obtained in nonRNA interference-treated males following injection of HezPBAN (10 pmol/male). The control bars represent the relative percentage levels of components from nonRNA interference-treated males in the absence of PBAN. Bars represent means ± s.e.m. of 7-8 replicates. Different letters denote a statistically significant difference for each component (one-way ANOVA, p < 0.0001). 14∶COOH = myristic acid, C-16 Alcohols = hexadecanol plus cis-11 hexadecanol, 16∶COOH = palmitic acid, 18∶OH = octadecanol, (9)18∶COOH = oleic acid, 18∶COOH = stearic acid.