CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura.

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

Functional characterization of pheromone receptors in the moth Athetis dissimilis (Lepidoptera: Noctuidae).

Sex pheromones are crucial for communication between females and males in moths, and pheromone receptors (PRs) play a key role in peripheral coding of sex pheromones. During the last decade, many PR candidates have been identified based on transcriptome sequencing and bioinformatic analysis, but their detailed functions remain mostly unknown. Here, focusing on four PR candidates of Athetis dissimilis (AdisOR1, AdisOR6, AdisOR11 and AdisOR14) identified in a previous study, we first cloned the full-length cDNAs and determined the tissue expression profiles by quantitative real-time PCR (qPCR). The results revealed that expression of three of these genes were male antennae-specific, while AdisOR11 was similar in expression between male and female antennae. Furthermore, the expression level of AdisOR1 was much higher than those of the other three genes. Then, functional analysis was conducted using Xenopus oocyte system. AdisOR1 responded strongly to the sex pheromone component Z9-14:OH and the potential pheromone component Z9,E12-14:OH, suggesting its important role in the sex pheromone perception; AdisOR14 showed specificity for Z9,E12-14:OH; while AdisOR6 and AdisOR11 did not respond to any of the pheromone components and analogs tested. Taken together, this study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A. dissimilis.

CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.

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.

Expression Profile and Functional Characterization Suggesting the Involvement of Three Chemosensory Proteins in Perception of Host Plant Volatiles in Chilo suppressalis (Lepidoptera: Pyralidae).

The high sensitivity of the olfactory system is essential for feeding and oviposition in moth insects, and some chemosensory proteins (CSPs) are thought to play roles in this system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. In this study, to identify the olfactory CSPs from a repertoire of 21 CSP members in the notorious rice pest Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), tissue expression patterns were firstly examined by quantitative real-time polymerase chain reaction (qPCR). It showed that CSP2 was antennae specific and seven more CSPs (CSP1, 3, 4, 6, 15, 16, and 17) were antennae biased in expression, suggesting their olfactory roles; while other CSPs were multiple-tissue expressed and non-antennae biased, suggesting other functions for these genes. To further determine the ligand binding specificity, three putative olfactory genes (CSP1-3) were expressed in Escherichia coli cells, and binding affinity of these three recombinant CSP proteins were measured for 35 plant volatiles by the ligand binding assays. CSP1 and CSP2 exhibited high binding affinities (Ki ≤ 10.00 µM) for four (2-tridecanone, benzaldehyde, laurinaldehyde and 2-pentadecanone) and two (2-heptanol and (+)-cedrol) host plant volatiles, respectively; the three CSPs also showed moderate binding affinity (Ki = 10.01-20.00 µM) for 16 plant volatiles. Our study suggests that the three CSPs play essential roles in the perception of host plant volatiles, providing bases for the elucidation of olfactory mechanisms in this important pyralid pest.

Identification and Field Evaluation of the Sex Pheromone of Orthaga achatina (Lepidoptera: Pyralidae).

Orthaga achatina (Lepidoptera: Pyralidae) is the most serious pest in south China of camphor trees, Cinnamomum camphora (L.) Presl, an important urban tree species. Gas chromatography-electroantennographic detection (GC-EAD) of the sex pheromone of O. achatina showed three EAD-active components. Coupled gas chromatography/mass spectrometry analyses identified these as (Z)-11-hexadecenol (Z11-16:OH), (Z)-11-hexadecenyl acetate (Z11-16:OAc), and (3Z,6Z,9Z,12Z,15Z)-tricosapentaene (Z3,Z6,Z9,Z12,Z15-23:H). In field tests using different combinations of the three compounds, male moths were attracted to a mixture of Z11-16:OAc and Z3,Z6,Z9,Z12,Z15-23:H, but less attracted to other blends. Further field tests with different ratios of the two compounds determined the optimal ratio of the binary blend as 500:250. The addition of Z11-16:OH to Z11-16:OAc, or to the binary mixture of Z11-16: OAc and the pentaene did not yield higher catches. This shows that O. achatina uses a mixture of Type I and Type II sex pheromone components. Orthaga achatina is the third Pyraloidea species found to utilize Z3,Z6,Z9,Z12,Z15-23:H as a sex pheromone component.