A new translucent mutant silkworm was discovered in the genetic stocks of the univoltine Hime'nichi Japanese race of Bombyx mori held by the National Institute of Agrobiological Sciences. In some egg batches of this mutant, many embryos died immediately after pigmentation. Additionally, larvae died frequently at the 4th and 5th larval stages. These observations revealed the weak constitution of the mutant insect. The degree of the translucency in the mutants was considered intermediate in comparison with other translucent mutants. Interestingly, in addition to mutant progeny resulting from sibmating of mutants, the F1 generation also segregated several normal phenotypes. Genetic analysis showed that the mutant gene was recessive and different from other translucent genes reported previously. The mutant was given the name, Hime'nichi translucent (ohi). Linkage analysis showed that ohi was linked to the 16th chromosome. The ohi locus was determined to be at the 30.5 cM position using a three-point test with two typical genetic markers, cts and lu, on the 16th chromosome.
In this article, we tried a newly developed condition for long-term preservation of fertilized silkworm eggs. The “Modified 2-years preservation method of silkworm eggs” was applied to some varieties of commercial silkworm strains. The condition is based on the “2-years preservation” reported by Shimizu et al. (1994), in which we altered the 6th intermediate care at 15°C for 1 day into 10°C for 10 days. Hybrid eggs preserved by this method showed practical hatchability as high as 65% without causing any defects on larval growth. In the case of original strains, however, hatchabilities were relatively low and a serious inbalance was often observed in the sexual ratio of the hatched larvae even if the “Modified 2-years preservation of the eggs” was applied. However, it was possible to maintain the eggs in next generation for another 2 years, showing certain improvement of hatchabilities in many strains. Good larval growth was obtained less than 1 year after the preservation even if they were suppressed by 2 years preservation. Thus, we consider that the “Modified 2-years preservation” is useful in maintaining original silkworm strains efficiently. In order to improve the hatchability of long-term preserved eggs, it was effective to keep a sufficient air supply by inserting spacers between each egg card.
Bombyx mori cultured cell line NIAS-Bm-Ke1 (Ke1 cells) that adjusted to serum-free medium was established. Ke1 cells showed moderate cell growth in serum-free media, but the cells showed low susceptibility to infection with BmPTLNPV, a recombinant BmNPV expressing the luciferase gene. Addition of heat-treated silkworm hemolymph in serum-free medium led to enhance the susceptibility of Ke1 cells to BmPTLNPV infection. By infection with a hybrid baculovirus, which is infectious to both Ke1 and Sf9 cells and encodes the luciferase gene, Ke1 cells cultured in KBM700 serum-free medium containing heat-treated silkworm hemolymph showed higher luciferase activity than Sf9 cells cultured in SF900II serum-free medium containing 10% of FBS. Ke1 cells were adaptable to suspension culture using KBM700 serum-free media. Luciferase gene expression by BmPTLNPV infection was observed in the cells cultured in KBM700 serum-free medium without heat-treated silkworm hemolymph only if the infected cells were cultured in the medium containing heat-treated silkworm hemolymph for a certain period just after infection. Based on these results, we concluded that Ke1 cells are suitable for the expression of recombinant protein in the baculovirus gene expression system.