Drosophila melanogaster larvae excrete amylase and perform external digestion of their food. In this study, to investigate whether their external digestion ability varies in response to changes in the external environment, we measured the relative amount of amylase excreted by larvae using a new method: the iodine starch agar method (ISAM). Analysis using this method revealed that the amount of amylase excreted by larvae increased in accordance with the increase in the agar concentration. In addition, we investigated the effect on the larval growth rate of adding amylase to the diet. Pupation occurred 24 h later in food containing 1% amylase than in food containing no amylase. These results suggest that the larvae adjust their amylase excretion in response to changes in the external environment, and that its level has a marked influence on the larval growth rate.

In Drosophila melanogaster, transformer-2 (tra-2) is essential for female differentiation and is known to induce female-specific splicing of doublesex (dsx). The function of Bmtra-2, the Bombyx mori homolog of tra-2, on the other hand remains to be elucidated. As an initial step to learn about the biological function of Bmtra-2, we determined whether Bmtra-2 is capable of inducing the female-specific splicing of Drosophila dsx. RNAi-mediated knockdown of Bmtra-2 using Bombyx cultured cells transiently transfected with a dsx minigene revealed that Bmtra-2 can induce female-specific splicing of Drosophila dsx. To examine the role Bmtra-2 plays in regulating sex-specific splicing of Bmdsx pre-mRNA, we used an RNAi approach to reduce BmTra-2 expression in the early embryo. Embryos injected with dsRNAs or siRNAs targeted to Bmtra-2 showed no variation in the sex-specific splicing pattern of Bmdsx pre-mRNA. RNAi knockdown of Bmtra-2 in the early embryo caused abnormal testis formation. Taken together, these results indicate that Bmtra-2 is required for normal testis development, but is not involved in regulating the sex-specific splicing of Bmdsx pre-mRNA, even though it is capable of inducing the female-specific splicing of Drosophila dsx.

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (Imp^M) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori. In the silkworm, Bombyx mori, a W-chromosome-linked gene Feminizer (Fem) determines femaleness. Fem transcript yields a piRNA (Fem piRNA) and Fem- piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masculinizer (Masc). Masc is required for male-specific expression of Bmdsx, which is an important regulatory gene for sexual differentiation, and therefore, Masc is considered to be essential for maleness. However, there has been no direct evidence that Masc indeed causes maleness in sexually dimorphic structures. To clarify this point, we established silkworm strains carrying a transgene that expresses Fem-piRNA-resistant Masc gene (Masc-R). Transgenic expression of the Masc-R induced male mode of expressions of downstream sex-determining genes in females. Notably, ovaries in these females exhibited testis-like structures that contained sperm bundles. Homozygous expression of the Masc-R caused formation of the male-specific abdominal segment in adult females and induced partial male differentiation in female genitalia. Thus, Masc can induce maleness at the morphological level and is sufficient for spermatogenesis. This is the first report to our knowledge on a gene that can masculinize a wide variety of sexual characteristics in lepidopteran insects.

Sexual fate of the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae) is determined by the complementary sex determination (CSD) mechanism as is the case in honeybees. However, to date, genes involved in sex determination have not been identified in this species. In this study, we attempted to identify orthologs of complementary sex-determiner (csd), feminizer (fem), and doublesex (dsx) from the A. rosae genome, all of which are crucial components of the sex determination cascade in the honeybee. As a result, we identified a sawfly ortholog of dsx (designated as Ardsx). Rapid amplification of cDNA ends (RACE) using total RNA extracted from male and female larvae identified three male-specific variants and three female-specific variants. Comparison between the full-length Ardsx cDNAs and the genomic sequence revealed that exon 5 was differentially spliced between the male- and female-specific variants. RT-PCR analysis demonstrated that Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner at almost all the developmental stages. RNAi-mediated knockdown of Ardsx in males caused severe defects in the reproductive organs and, notably, induced development of the ovipository apparatus containing the dorsal pair of blades and the sheath. These males also showed abnormalities in testes and seminal vesicles and lacked mature sperm. The present study provides the first direct evidence that dsx is essential for sexual development in hymenopteran species.