20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.

Metabonomic analysis of Bombyx mori (Heterocera: Bombysidae) treated with acetaminophen.

The feasibility of using Bombyx mori as model animal is attracting more attention. Whether the effect of drugs on the metabolite profiling was consistent with those in mammals was an aspect to evaluate the feasibility of B. mori as model animal. In this study, we used acetaminophen to treat Dazao fifth-instar B. mori, and its metabolites in hemolymph were detected by gas chromatography-mass spectrometry. The corresponding data were processed and analyzed by total model analysis, principal component analysis, partial least squares-discriminant analysis, orthogonal partial least squares-discriminant analysis, and finally, the difference metabolites between acetaminophen group and control group were selected and identified by our reference material database and the National Institute of Standard and Technology database. The results showed that acetaminophen administration induced elevation of metabolites related to energy source, the intermediate of cholesterol synthesis, and the metabolites related to melanization and also induced the decrease of metabolites in pathway of Krebs cycle, the cholesterol, and sitosterol, which suggested that acetaminophen administration inhibited energy metabolism and promoted the expenditure and imbalance of hormone and melanization.

Functional analysis of 30K proteins during silk gland degeneration by a caspase-dependent pathway in Bombyx.

The 30K proteins are involved with important functions in the growth and development of Bombyx mori. In this study, the synthesis and regulation of 30K proteins were examined during the degeneration of Bombyx silk glands. On day 3 of the fifth instar, the protein level of 30Kc19 was low, whereas the silk proteins were rapidly synthesized. However, synthesis and accumulation of the 30Kc19 protein significantly increased at the prepupal stage and on day 1 of the pupal stage. At this stage, the silk gland cells were filled with 30Kc19 and genomic DNA. Moreover, the transcript levels of the 30K-encoding genes, including 30Kc6, 30Kc12, 30Kc19 and 30Kc23 were up-regulated during the degeneration of the Bombyx silk glands. During the time that the levels of the 30Kc19 protein were significantly up-regulated, it is notable that the transcript levels of the BmAtg8, BmAtg6 and BmDronc genes dramatically increased to regulate the programmed cell death of this gland. On day 1 of the pupal stage, intense fragmentation of genomic DNA occurred in the silk gland cells, and the putative active form of caspase was detected in the cytoplasm, showing the complete degradation of the silk glands in one day. In conclusion, the 30K proteins are synthesized in high concentrations, while proteolysis mediates silk gland degeneration in Bombyx by a caspase-dependent pathway. We propose that the 30K proteins may be nutrients and energy vectors to be absorbed by the developing tissues of pupae or moths.

Parental environmental exposure leads to glycometabolic disturbances that affect fertilization of eggs in the silkworm Bombyx mori: the parental transcript legacy.

Parental transcript legacy plays an important role in fertilization and development of the early embryo. Parental environmental exposure affects the fertilization of eggs, but the underlying biochemical mechanism is largely unresolved. In this study, the parental environmental effects on fertilization of eggs were explored in the silkworm Bombyx mori (B. mori), an ideal lepidopteran animal model. The results showed that the rate of fertilization decreased after the parents were exposed to a poor environment at 32 °C with continuous illumination for 72 h on days 6-9 of the pupal stage, which is a key period for germ cell maturation. This was likely attributable to lower energy charge values, obstructed nicotinamide adenine dinucleotide (NAD(+)) regeneration and inactive tricarboxylic acid cycle (TCA), leading to accumulation of large amounts of pyruvic acid and lactic acid. This effect was related to energy metabolism via glycolysis; in particular disruption of pyruvate metabolism. In conclusion, this study showed parental exposure to an abnormal environment during germ cell maturation affected glycolysis and the subsequent fertilization of eggs via the parental transcript legacy in B. mori.