The terpenoid backbone biosynthesis pathway is responsible for the synthesis of different backbones for terpenoids; (E)-β-farnesene (EβF), a sesquiterpene, is the major component of aphid alarm pheromone. Our previous studies eliminated the possibility of host plants and endosymbionts as the sources of EβF, and we thus speculate that the terpenoid pathway might affect the biosynthesis of EβF in aphids. First, the transcriptional responses of four genes encoding farnesyl diphosphate synthase (FPPS), geranylgeranyl diphosphate synthase (GGPPS) and decaprenyl diphosphate synthase in the cotton aphid Aphis gossypii to simulated stimulation were analysed using quantitative real-time PCR, showing an immediate decrease in the transcript abundances of the four genes. Next, RNA-interference-mediated gene knockdown was performed, indicating that fpps knockdown caused a significant cost in terms of body size and fecundity. Finally, an association analysis of gene knockdown with the amount of EβF was conducted, revealing that the concentration of EβF per milligram of aphid was drastically decreased in response to fpps knockdown, whereas ggpps knockdown significantly raised the concentration of EβF. Our data support a peculiar mode of biosynthesis and storage of the aphid alarm pheromone that relies directly on the terpenoid backbone biosynthesis pathway in the aphid.

The major component of aphid alarm pheromone is (E)-β-farnesene (EβF), but the molecular mechanisms of EβF synthesis are poorly understood. Here we established a biological model to study the modulation of EβF synthesis in the bird cherry-oat aphid Rhopalosiphum padi by using quantitative polymerase chain reaction, gas chromatography/mass spectrometry and RNA interference. Our results showed that the rearing conditions significantly affected the weight of adult and modulated EβF synthesis in a transgenerational manner. Specifically, the quantity of EβF per milligram of aphid was significantly reduced in the individually reared adult or 1st-instar nymphs derived from 1-day-old adult reared individually, but EβF in the nymph derived from 2-day-old adult that experienced collective conditions returned to normal. Further study revealed that the production of EβF started in embryo and was extended to early nymphal stage, which was modulated by farnesyl diphosphate synthase genes (RpFPPS1 and RpFPPS2) and rearing conditions. Knockdown of RpFPPS1 and RpFPPS2 confirmed the role played by FPPS in the biosynthesis of aphid alarm pheromone. Our results suggested that the production of EβF starts at the embryo stage and is modulated by FPPS and rearing conditions in R. padi, which sheds lights on the modulatory mechanisms of EβF in the aphid.

The alarm behavior plays a key role in the ecology of aphids, but the site and molecular mechanism for the biosynthesis of aphid alarm pheromone are largely unknown. Farnesyl diphosphate synthase (FPPS) catalyzes the synthesis of FPP, providing the precursor for the alarm pheromone (E)-β-farnesene (EβF), and we speculate that FPPS is closely associated with the biosynthetic pathway of EβF. We firstly analyzed the spatiotemporal expression of FPPS genes by using quantitative reverse transcription-polymerase chain reaction, showing that they were expressed uninterruptedly from the embryonic stage to adult stage, with an obvious increasing trend from embryo to 4th-instar in the green peach aphid Myzus persicae, but FPPS1 had an overall significantly higher expression level than FPPS2; both FPPS1 and FPPS2 exhibited the highest expression in the cornicle area. This expression pattern was verified in Acyrthosiphon pisum, suggesting that FPPS1 may play a more important role in aphids and the cornicle area is most likely the site for EβF biosynthesis. We thus conducted a quantitative measurement of EβF in M. persicae by gas chromatography-mass spectrometry. The data obtained were used to perform an association analysis with the expression data, revealing that the content of EβF per aphid was significantly correlated with the mean weight per aphid (r = 0.8534, P = 0.0307) and the expression level of FPPS1 (r = 0.9134, P = 0.0109), but not with that of FPPS2 (r = 0.4113, P = 0.4179); the concentration of EβF per milligram of aphid was not correlated with the mean weight per aphid or the expression level of FPPS genes. These data suggest that FPPS1 may play a key role in the biosynthesis of aphid alarm pheromone.