In vivo functional characterisation of pheromone binding protein-1 in the silkmoth, Bombyx mori

Abstract

Male moths detect sex pheromones emitted by conspecific females with high sensitivity and specificity by the olfactory sensilla on their antennae. Pheromone binding proteins (PBPs) are highly enriched in the sensillum lymph of pheromone sensitive olfactory sensilla and are supposed to contribute to the sensitivity and selectivity of pheromone detection in moths. However, the functional role of PBPs in moth sex pheromone detection in vivo remains obscure. In the silkmoth, Bombyx mori, female moths emit bombykol as a single attractive sex pheromone component along with a small amount of bombykal that negatively modulates the behavioural responses to bombykol. A pair of olfactory receptor neurons, specifically tuned to bombykol or bombykal, co-localise in the trichodeum sensilla, the sensillum lymph of which contains a single PBP, namely, BmPBP1. We analysed the roles of BmPBP1 using BmPBP1-knockout silkmoth lines generated by transcription activator-like effector nuclease-mediated gene targeting. Electroantennogram analysis revealed that the peak response amplitudes of BmPBP1-knockout male antennae to bombykol and bombykal were significantly reduced by a similar percentage when compared with those of the wild-type males. Our results indicate that BmPBP1 plays a crucial role in enhancing the sensitivity, but not the selectivity, of sex pheromone detection in silkmoths.

Figure 1

Generation of BmPBP1-knockout silkmoths. (a) Schematic representation of the genomic structure of BmPBP1 (top) and target sequences of transcription activator-like effector nucleases (TALENs; bottom). Exons are indicated by blue boxes and the start/stop codon locations are shown. TALENs were constructed to target sequences in the second exon. The sequences of TALEN recognition sites are shown at the bottom of the genomic structure. (b) TALEN-induced mutant alleles generated in this study. The wild-type sequence is aligned with the deletion mutant sequences of BmPBP1. The deletions are indicated by a dashed line. The red box in the 4-bp deletion sequence indicates the position of a frame shift. Right and left TALEN recognition sequences are highlighted in red and blue characters, respectively. The black arrow under the sequences indicates the 3′ primer site used for genotyping by using genomic PCR. (c) A representative genomic PCR analysis of the 4-bp deletion allele is shown. The PCR products obtained using genomic DNA isolated from the wings of a mutation-homozygous individual (4del/4del), or a mutation-heterozygous individual (WT/4del), and a wild-type individual were separated by electrophoresis. The PCR primers corresponding to sequences flanking the deletion regions (left) or PCR primers with the 3′ primer designed to anneal to the deleted sequence (the black arrow in (b))(right) were used. (d) Deduced amino acid sequences of wild-type (top) and 4-bp deletion BmPBP1 moths (bottom). The black arrowhead on the sequences indicates a signal peptide cleavage site, and the red arrow indicates the position of a frame shift caused by the deletions. Red boxes indicate amino acids that are identical between the two sequences.

Figure 2

Electroantennogram (EAG) analyses of the response of BmPBP1 mutants to sex pheromone components. (a) Representative EAG of the antennae from BmPBP1−/BmPBP1− and wild-type male moths in response to 1000 ng bombykol (left) and bombykal (right). The stimulus was applied for 200 ms, as indicated by the solid line on the trace. (b) Dose-dependent increase in bombykol- (left) or bombykal- (right) induced peak EAG amplitudes in BmPBP1−/BmPBP1− (red; n = 11) and wild-type (blue; n = 5) male moth antennae. Error bars represent ± SEM. The asterisks indicate significant differences between the groups (**p < 0.01), as determined using Student’s t-test for comparing pairs of data. (c) Comparison of linalool and citral-induced peak EAG amplitudes of BmPBP1−/BmPBP1− (red; n = 5 for linalool, n = 6 for citral) and wild-type (blue; n = 5 for linalool, n = 7 for citral) male moths. Error bars represent ± SEM. No significant difference was detected between the two groups (Student’s t-test; p = 0.700 for linalool, p = 0.529 for citral).

Figure 3

Behavioural response of BmPBP1-knockout males to pheromones. The behavioural response percentages of BmPBP1−/BmPBP1− (red) and wild-type (blue) male moths to different doses of bombykol (a) or bombykal (b) are plotted. The asterisk indicates significant differences between the groups (*p < 0.05), as determined using Fisher’s exact probability test for comparing pairs of data.