We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

We have previously reported that Bmdsx, a homologue of the sex-determining gene doublesex ( dsx), was sex-specifically expressed in various tissues of the silkworm. The primary transcript of Bmdsx is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, we expressed BmDSXF in males from a ubiquitous promoter and examined its regulatory activities. We show that BmDSXF functions as a positive regulator of the hexameric storage protein termed SP1 and vitellogenin genes that are predominantly expressed in females. We also show that expression of Bmdsx(F) in males results in the repression of the pheromone-binding protein gene that is preferentially expressed in males. Gel-mobility shift assays demonstrated that BmDSX proteins bind to the sequence (ACATTGT) between -95 and -89 nt relative to the transcriptional initiation site of the vitellogenin gene. These results strongly suggest that Bmdsx is a final regulatory gene in the hierarchy of regulatory genes controlling the expression of female-specific protein in Bombyx mori.

The sex determination pathway is different between Drosophila melanogaster and Bombyx mori in the initial signal. Here we show evidence that the sex determination pathway in B. mori is similar to that of D. melanogaster at the level of the terminal regulator, doublesex (dsx), which is essential for the proper differentiation of the sexually dimorphic somatic features of D. melanogaster. In B. mori, a homolog of dsx (Bmdsx) is expressed in various tissues, and its primary transcript is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, transgenic silkworms carrying a construct with a Bmdsx male cDNA placed under the control of either an hsp70 promoter or a Bombyx actin3 promoter were generated by piggyBac-mediated germline transformation. Ectopic expression of the male cDNA in females resulted in abnormal differentiation of certain female-specific genital organs and caused partial male differentiation in female genitalia. Transgenic analysis also revealed that the expression of BmDSXM in females caused repression of the female-specifically expressed gene, the vitellogenin gene, and also resulted in activation of the pheromone-binding protein gene that is dominantly expressed in males. These results provide evidence that the role of BmDSXM includes the activation of some aspects of male differentiation as well as the repression of female differentiation. Taken together with our previous data on the function of BmDSXF, we can conclude that Bmdsx is a double-switch gene at the final step in the sex-determination cascade of B. mori.

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Injection of double-stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild-type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10-19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid co-injected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence-specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects.