Twenty-hydroxyecdysone produced by dephosphorylation and ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori.

Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

Using silkworms as a laboratory animal to evaluate medicines and foods.

For this special issue, we, the Editors of Drug Discoveries & Therapeutics, have asked researchers who are using silkworms to actively develop drugs and study foods to summarize their recent work. Our profound hope is that this special issue encourages researchers who are helping to develop the new field of "using silkworms as a laboratory animal to evaluate medicines and foods".

A hyperglycemic silkworm model for evaluating hypoglycemic activity of Rehmanniae Radix, an herbal medicine.

Silkworm shows hyperglycemia after intake of diet containing large amount of glucose. The hyperglycemic silkworm model is useful for evaluation of anti-diabetic drugs. A hot water extract of Rehmanniae Radix, an herbal medicine, showed hypoglycemic effect against the hyperglycemic silkworms. This method is applicable for quick and simple evaluation of the hypoglycemic activities of different batches of Rehmanniae Radix. Our findings suggest that silkworms have a lot of merit as experimental animals for evaluation of various herbal medicines.

Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression and function.

To better understand the molecular mechanisms of diapause initiation, we used the sensitive cDNA subtraction (selective amplification via biotin- and restriction-mediated enrichment) method and isolated a novel gene expressed abundantly in diapause eggs of the silkworm, Bombyx mori, which encodes a homolog of the human oxidation resistance 1 (OXR1) protein. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses confirmed that BmOXR1 mRNA and its 140-kDa protein were differentially expressed in diapause eggs compared to non-diapause eggs. OXR1 double-stranded RNA (dsRNA) was injected into diapause-destined eggs before the cellular blastoderm stage, and 4days later, when untreated eggs reached the diapause stage, the OXR1 protein disappeared; however, these eggs remained in diapause, suggesting that BmOXR1 is not essential for diapause initiation and/or maintenance. To further investigate the in vivo function of BmOXR1 apart from its role in diapause, we overexpressed BmOXR1 in Drosophila melanogaster. The fruit fly male adult life-span was significantly extended in the 50%-survival time when adults were reared on diets both with and without H2O2 solution under 25°C incubation. These results suggest that BmOXR1 functions in D. melanogaster via a possible antioxidant effect. As BmOXR1 was expressed mainly in the nuclei of D. melanogaster cells, the mechanism underlying its antioxidation effect appears to be different from that in humans where it is expressed mainly in the mitochondria. Taken together, these results suggest that BmOXR1 might serve as an antioxidant regulator during the early diapause stage.

Use of silkworms to evaluate the pathogenicity of bacteria attached to cedar pollen.

Injection of a Japanese cedar pollen suspension into silkworm hemolymph kills the silkworms. A certain species of bacteria proliferated in the hemolymph of the dead silkworms. A 16S rDNA analysis demonstrated that the proliferating bacteria were Bacillus cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus amyloliquefaciens. Among them, B. cereus, B. thuringiensis, and B. weihenstephanensis exhibited hemolysis against sheep red blood cells and were lethal to mice. A culture filtrate of B. amyloliquefaciens showed enzyme activity toward the pectic membrane of cedar pollen. These results suggest that silkworms as an animal model are useful for evaluating the pathogenicity of bacteria attached to cedar pollen.