Mulberry leaf powder is the most expensive among the ingredients in the artificial diets of the silkworm, yet it is essential for the growth of normal silkworm strains. In an effort to reduce the cost of artificial diets, green coffee bean extract was considered as the substitute for mulberry leaf powder. The analysis using HPLC revealed that commercial green coffee bean extracts contain more than 20% chlorogenic acid (5-CQA), a feeding stimulant of the silkworm identified from mulberry leaves. Test diets, with defatted soybean meal and corn meal as the main ingredients and varying amounts of green coffee bean extract, were fed to larvae of the normal silkworm strains during the 4th instar. As a result, diets containing more than 0.3% green coffee bean extract were found to be sufficient for larval growth. Based on these findings, a diet for grown silkworms (4th and 5th instar larvae) was developed. The growth performances of silkworm larvae fed the diet was comparable to that of larvae fed a regular commercial diet, suggesting the potential of green coffee bean extracts as a useful and cost-effective feed ingredient for the silkworm.
Ameiotic and meiotic parthenogenesis induced by carbonated water was investigated using ovarian eggs of the silkworm, Bombyx mori. We used B. mori females in which the red egg gene locus is heterozygous (+/re) in order to facilitate detection of parthenogenesis via egg pigmentation. More than 80% of the ovarian eggs from virgin females exhibited parthenogenesis when maintained at 17゚C for 1 day, soaked in carbonated water for 1 h at 17゚C, and subsequently maintained at 17゚C or 3 days. When ovarian eggs were soaked in carbonated water adjusted to different pH values, parthenogenesis was significantly increased in the acidic pH range where CO2 bubbles were generated in the water. Because eggs soaked in acidified non-carbonated mineral water exhibited a significantly lower induction rate, CO2 gas in carbonated water, rather than hydrogen ions, was suggested to play more important role in parthenogenesis induction. Sealing the front pole, back pole, lateral side, whole part, and no part of ovarian eggs with nail varnish (topcoat) to inhibit CO2 diffusion into the eggs resulted in 79.1%, 93.1%, 82.4%, 0%, and 84.4% egg parthenogenesis, respectively. These findings suggest that gaseous CO2 from carbonated water, penetrating eggs via unspecified surface areas, induces ameiotic parthenogenesis. In general, the hatchability of parthenogenetic eggs was very low. We hypothesize that embryonic lethality arises from haploid cells in parthenogenetic embryos.
Counting cell number in cultured cells is a simple but important task in the first step of the research. Pv11, which is a cultured cell line from the sleeping chironomid, Polypedilum vanderplanki, is smaller than typical insect cultured cells and it takes a lot of time and effort for cell counting. To solve this issue, we suggest a method using Python and OpenCV in this report. The cells were stained with two color fluorescent dyes (blue and red) and images were taken for counting. The one counting sample consisted of two images, from which it was possible to reduce counting errors. The time it took to do counting for one sample was 0.3 seconds and 8 seconds for 25 samples. We consider effective to write a simple code using Python adapted to the characteristics of target cultured cells.