Abstract
Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via fatty acid synthesis (FAS). Biosynthesis of moth sex pheromones is usually regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-aa peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets (LDs), which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells prior to eclosion. PBAN activation of the PBAN receptor stimulates lipolysis of the stored LD triacylglycerols (TAGs) resulting in release of the bombykol precursor for final modification. While we have previously characterized a number of molecules involved in bombykol biosynthesis, little is known about the mechanisms of PBAN signaling that regulate the TAG lipolysis in PG cells. In the current study, we sought to further identify genes involved in bombykol biosynthesis as well as PBAN signaling, by using a subset of 312 expressed-sequence tag (EST) clones that are in either our B. mori PG cDNA library or the public B. mori EST databases, SilkBase and CYBERGATE, and which are preferentially expressed in the PG. Using RT-PCR expression analysis and an RNAi screening approach, we have identified another eight EST clones involved in bombykol biosynthesis. Furthermore, we have determined the functional role of a clone designated BmACP that encodes B. mori acyl carrier protein (ACP). Our results indicate that BmACP plays an essential role in the biosynthesis of the bombykol precursor fatty acid via the canonical FAS pathway during pheromonogenesis.
Highlights
In most moth species, sex pheromone biosynthesis is regulated by the pheromone biosynthesis activating neuropeptide (PBAN), a neurohormone consisting of 33 amino acid peptide that originates in the subesophageal ganglion and that is characterized by the core C-terminal FSPRLamide sequence
Because no decrease in bombykol production was observed in control pupae injected with DOUBLE-STRANDED RNA (dsRNA) for unrelated proteins including enhanced green fluorescent protein (EGFP), these findings suggest that disruption of bombykol production results from specific knockdown of the target gene sequences (Figure 1)
We have developed a method to facilitate identification of the genes involved in bombykol biosynthesis, using RT-PCR expression analysis and RNAi-mediated in vivo gene silencing in conjunction with expressed-sequence tag (EST) databases
Summary
Sex pheromone biosynthesis is regulated by the pheromone biosynthesis activating neuropeptide (PBAN), a neurohormone consisting of 33 amino acid peptide that originates in the subesophageal ganglion and that is characterized by the core C-terminal FSPRLamide sequence. Bombyx mori, the sex pheromone, bombykol (E, Z -10, 12-hexadecadien-1-ol), is synthesized de novo within PG cells from acetyl-CoA via conventional long chain fatty acid synthesis (FAS; Bjostad et al, 1987; Jurenka, 2003). On the day before adult emergence, B. mori PG cells rapidly accumulate numerous lipid droplets (LDs) within the cytoplasm (Fónagy et al, 2001). These LDs play an essential role in bombykol biosynthesis by acting as a reservoir for the de novo synthesized bombykol precursor, 10,12-hexadecadienoate, which is deposited in the LDs in the form of triacylglycerols (TAGs) with the precursor predominantly sequestered at the sn-1 and sn-3 positions of the glycerides (Matsumoto et al, 2002). The stored fatty acid is cleaved and converted to bombykol in response to PBAN stimulation (Matsumoto et al, 2007, 2010)
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