Fig 1.
Loss of sex determination pathway genes impairs male courtship and mating behaviors.
(A) Diagram of the sex determination cascade in the silkworm. (B) Diagram of the behavioral test setup. The adult male (M-M) is placed at a distance of 10 cm or 20 cm from a wild-type adult female (WT-F, which releases pheromones) or from a wild-type male control, and behavior is monitored. (C and D) The courtship and mating behavior indexes for wild-type males (WT) and BmMasc, BmPSI, Bmdsx, and Bmfru mutant males. The results are expressed as percentage from 90 pairs tests with Fisher exact test. An index of 0% indicates the absence of courtship behavior or failed mating behavior.
Fig 2.
Silkworms with mutations in BmMasc, BmPSI, and Bmdsx have abnormal antennal structures.
(A) Gross morphology of antennae of wild-type and mutant males (M) and females (F). Scale bars: 1 mm. (B) SEM images of the sensilla trichoidea structures in the middle of the antennae of wild-type and mutant males. Scale bars: 50 μm. (C) Antennal lengths of wild-type and mutant adults. The results are expressed as the means ± SEM of 10 independent biological replicates. *** represent significant difference at the 0.001 level (one- way ANOVA), compared with the WT-F and WT-M; n.s. indicates that the difference is not statistically significant. (D) The number of sensilla trichoidea in one SEM scan field in wild-type and mutant adults. The results are expressed as the means ± SEM of 5 insects per group. ** and *** represents significant differences at the 0.01 and 0.001 level (one- way ANOVA) compared with the control; n.s. indicates that the difference is not statistically significant.
Fig 3.
Electrophysiological analyses reveal abnormalities in responses to pheromone components of male silkworms with mutations in sex determination genes.
(A) Representative EAGs of wild-type and mutant BmMasc, BmPSI, Bmdsx, and Bmfru male moths in response to hexane (upper panel), 10 μg bombykol (middle panel), and 10 μg bombykal (lower panel). (B) Representative single sensillum recording (SSR) from wild-type and mutant BmMasc, BmPSI, Bmdsx, and Bmfru males in response to hexane (upper panel), 10 μg bombykol (middle panel), and 10 μg bombykal (lower panel). The stimulus was applied for 300 ms, indicated by a red line under the trace. (C and D) Mean responses of male antennae to C) 10 μg of bombykol and D) 10 μg bombykal. The statistical significance between WT (n = 10) and BmMasc (n = 7), BmPSI (n = 5), Bmdsx (n = 8), and Bmfru (n = 11) mutant responses was analyzed with one-way ANOVA. Data are shown as means ± SEM; *, **, and *** represent significant differences at the 0.05, 0.01, and 0.001 levels, respectively, compared with the WT-M. (E and F) Mean responses of neurons in male sensillum trichodea to E) 10 μg of bombykol and F) 10 μg bombykal. The statistical significance between WT (n = 30) and BmMasc (n = 50), BmPSI (n = 44), Bmdsx (n = 30), and Bmfru (n = 76) mutants was analyzed with one-way ANOVA. Data are shown as means ± SEM; ** and *** indicates p < 0.01 and p < 0.001, compared with the WT-M, and n.s. indicates no significance.
Fig 4.
Expression change of olfactory sensory system genes in sex determination gene mutants.
(A) Relative mRNA expression levels of BmPBP1, BmPBP2, BmPBP3, BmOR1, BmOR2, and BmOR3 in WT and mutant males. Three individual biological replicates were performed with real-time quantitative PCR (qPCR). Plotted are means ± SEM. *, ** and *** represent significant differences at the 0.05, 0.01 and 0.001 levels with one- way ANOVA, comparing each gene was with the corresponding WT-M; n.s. represents not significant. (B) Summary of the expression change of olfactory system genes in each mutant. ‘-’ represents no significant change compare to WT, ‘↓’, ‘↓↓’, ‘↓↓↓’ represent significant decreased (*, **, ***) compare to WT, ‘↑↑’, ‘↑↑↑’ represent significant increased (**, ***) compare to WT.
Fig 5.
Loss of BmOR3 expression extends mating time.
(A) Structure of the BmOR3 gene with nine exons indicated by boxes (black boxes, 5’- and 3’-UTRs; white boxes, coding exons). Target sites 1 and 2 are binding sites for sgRNAs. (B) The mean transcript levels (± SEM) of BmOR3 are down-regulated significantly compared to wild-type levels in the three BmOR3 mutant male (M) and female (F) lines. At least five males with mixed antenna were examined for each line. *** indicates p < 0.001 compared with the relevant control using one-way ANOVA. (C) Somatic mutations were induced in the F1 founder animals following crosses of nos-Cas9 with U6-sgRNA strains. PCR analyses with primers to amplify a region of 600 bp revealed deletion mutations in the G0 mutants. The red arrowhead indicates the deleted region. (D) Deletion mutation in the heterozygous offspring after crossing nos-Cas9 and U6-BmOR3sgRNA transgenic silkworm lines. The targeting sequence is shown in black, and the PAM sequence is in red. The deletion size in nucleotides is indicated above the red arrow at the site of the deletion. (E and F) Courtship and mating behavior indexes of BmOR3 mutant and WT males. Data are shown as percentage from 150 pairs tests with chi-squared test. ***p < 0.001. (G) Percentage of autosegregated WT and mutant males *** indicates significant difference at the 0.001 level with chi-squared test.
Fig 6.
BmOR3 mutant male silk moths have nearly normal electrophysiological responses to bombykol but not bombykal.
(A) Representative EAGs from wild-type and BmOR3 mutant male moths in response to hexane (upper panel), 10 μg bombykol (middle panel), and 10 μg bombykal (lower panel). (B and C) Mean responses of WT (n = 9) and BmOR3 mutant (n = 9) male antennae to B) 10 μg of bombykol and C) 10 μg of bombykal. Data are means ± SEM; n.s. indicates no significant difference and ** represents a significant difference at the 0.01 level as determined by Student’s t-test. (D) Representative single sensillum recording (SSR) of wild-type and BmOR3 mutant male moths in response to hexane (upper panel), 10 μg bombykol (middle panel), and 10 μg bombykal (lower panel). The stimulus was applied for 300 ms as indicated with a red line under the trace. (E and F) Mean (± SEM) responses of neurons in male sensillum trichodea to E) 10 μg of bombykol or F) 10 μg of bombykal in WT (n = 26) and BmOR3 mutant (n = 40) male moths. *** indicates p < 0.001 and n.s. indicates no significant difference as determined by Student’s t-test.
Fig 7.
Proposed genetic regulation pathway of sexual behavior in B. mori.
Sex determination pathway factors, olfactory sensory factors, and sex pheromones influence courtship and mating behavior. The Bmdsx-BmOR1/3-Bombykol and Bmfru-BmOR3-Bombykal cascades are the two primary pathways involved in olfactory-based sexual behavior. Disruption of the sex pathway gene Bmdsx blocks the expression of both BmOR1 and BmOR3, whereas disruption of Bmfru mainly inhibits expression of BmOR3. Thus, mutation of Bmdsx leads to an abnormal response to bombykol and bombykal and inhibits courtship and mating by male moths, but mutation of Bmfru disrupts termination of mating due to an abnormal response to bombykal.