Resistance to the entomopathogenic fungus Beauveria brongniartii (Sacc.) is controlled by a dominant gene in the silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae). We determined the linkage group by which the resistance gene links through linkage analysis with the linkage map of the silkworm based on single nucleotide polymorphisms (SNPs). Taking advantage of the lack of crossing over in females of the silkworm, backcrossed F₁ progenies (BC₁) made by crossing an F₁ female (susceptibility-developed strain AkakoS×resistance-developed strain p50TR) and a male AkakoS were used. The resistant BC₁ individuals were selected by screening of injection with conidial suspension of B. brongniartii. We selected SNP markers that were able to distinguish the genotypes of AkakoS and p50TR on each of the 28 linkage groups and analyzed the genotypes of the resistant BC₁ individuals on each linkage group with the SNP markers. There are two genotypes in BC₁, homo (AkakoS) type and hetero (F₁) type, but only hetero type BC₁ individuals can be resistant to B. brongniartii on the linkage group to which the resistance gene links. All resistant BC₁ individuals showed hetero type only at the 14th linkage group, suggesting that the resistance gene of the silkworm to B. brongniartii exists on the 14th linkage group. We designated this gene as “resistant to B. brongniartii” with the abbreviation Rbbr.

View full abstract

Cultured cell lines maintained in different laboratories often show different phenotypes despite originating from the same cell line. In this study, we compared Bombyx mori nucleopolyhedrovirus (BmNPV) infectivity in B. mori ovary-derived BmN4 cell lines that had been cultured for decades at three independent laboratories. These were designated BmT (maintained at University of Tokyo), BmY (maintained at Yamaguchi University) and BmQ (maintained at Kyushu University). Infection studies revealed that the BmY cells produced and released many more occlusion bodies than the other two cell lines. Luciferase assay showed that the activity of the polyhedrin promoter was higher in the BmY and BmQ cells than in BmT cells. Budded virus production was lower in the BmQ cells than in the other two lines, and the BmQ cells were not able to form distinct plaques of BmNPV. Quantitative RT-PCR experiments revealed that BmNPV early gene expression was higher and earlier in the BmT cells than in the BmY or BmQ cells. Collectively, BmN4 cell lines subcultured under different conditions for years exhibit different properties, including virus infectivity. We can exploit these differences and had better take advantage of each cell line depending on the intended use.

View full abstract

Promoter activity of the nitric oxide synthase (NOS) gene from the silkworm, Bombyx mori, was analyzed to identify cis-regulatory elements that contribute to activation of this gene upon bacterial infection. We first cloned and sequenced the 5’-upstream region of B. mori (Bm) NOS1 genes. Two types of 5’-upstream regions were detected, namely an L-type and an S-type lacking a portion of the L-type, and both types contained the κB site and three C/EBP binding sites which were located in the proximal upstream region near the κB site. Reporter assays showed that only the S-type was strongly activated by BmRelB, but not BmRelA, and mutation of the κB site resulted in a significant reduction in promoter activity. Deletion of C/EBP binding sites strongly diminished the BmRelB-dependent promoter activity, suggesting an important role for these sites in the activation of the BmNOS1 gene. Moreover, co-transfection experiments with BmRelB and BmC/EBP expression vectors demonstrated that the relative ratio of these transcription factors affects S-type promoter activity.

View full abstract

The presence of large amounts of free D-serine in Bombyx mori was first established by Corrigan and Srinivasan (1966). In this study, we investigated D-serine localization and serine racemase and D-serine dehydratase (DSD) activities in various organs with respect to age and developmental stage to determine the metabolism and role of D-serine in the silkworms. d-Serine and both enzymes were coincidently found in the midgut, fat body, testis, and ovary. The highest D-serine levels and both enzyme activities were also coincidently observed in the day-3 and day-5 pupal stages. The highest D-serine levels were found in the hemolymph and midgut contents, although no serine racemase and DSD activities were detected there. These results indicate that intrinsic D-serine production is mediated by serine racemase, and that D-serine degradation to pyruvate is due to the catabolic action of DSD in the midgut, fat body, testis, and ovary. Large amounts of D-serine were reduced prior to silkworm emergence, and some D-serine may be used for ATP synthesis in the testis.

View full abstract

We analyzed 3633 Japanese Bombyx mandarina COI sequences from 37 locations in Japan, found 94 segregating sites in 714-bp long COI nucleotide sequences and eventually identified 121 haplotypes. We further confirmed that the 121 haplotypes were different from the 8 B. mori haplotypes reported by Yukuhiro et al. (2011), and the result strongly suggested little gene flow from B. mori to Japanese B. mandarina. We discuss the properties of the mutational differences between the two species.

View full abstract