Abstract
Pheromone‐binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP‐single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target‐gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1‐sgRNA injected eggs and 19.5% for PBP3‐sgRNA injected eggs. Second, by an in‐crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA‐injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.
Highlights
Moths have evolved a highly sophisticated and sensitive olfactory system to guide various important behaviors, such as locating food sources and finding mating part-ners
With four different Pheromone-binding proteins (PBPs) genes found in C. suppressalis (Cao et al, 2014), functional differentiation among them comes as an important point for better understanding the complexity and sensitivity of the sex pheromone olfactory system
We for the first time provide direct in vivo evidence that both CsupPBP1 and 3 function in the sex pheromone perception, and that CsupPBP1 is more
Summary
Moths have evolved a highly sophisticated and sensitive olfactory system to guide various important behaviors, such as locating food sources and finding mating part-ners. The major proteins involved in the olfactory system at the peripheral level includes odorant-binding proteins (OBPs), odorant receptors (ORs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs) and odorant-degrading enzymes (ODEs) (Leal, 2013). Among these proteins, OBP is a large family of small (ß15 kDa) hydrophilic carrier proteins that contain two-four disulphide bridges. OBPs primarily function as solubilizers and carriers of the hydrophobic odorants in the aqueous sensillum lymph, protecting and transporting hydrophobic odorants to ORs on the dendritic membrane (Pelosi & Maida, 1995; Leal, 2005).
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