Titanium nanoparticles influence the Akt/Tor signal pathway in the silkworm, Bombyx mori, silk gland.

Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO2 NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO2 NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO2 NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO2 NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO2 NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6-, 12.1-, and 4.8-fold. Dietary inhibitors led to 2.6- to 4-fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO2 NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor-linked downstream proteins. These findings support our hypothesis that TiO2 NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO2 NPs actions.

Apoptosis of posterior silk gland of Bombyx mori during spinning period and the role of PI3K/Akt pathway.

Bombyx mori is an economic insect of the Lepidoptera. Its posterior silk gland (PSG) is an important organ for fibroin synthesis. In order to study the occurrence of apoptosis in PSG and the role of PI3K/Akt signaling pathway during spinning period, changes in morphology of silk gland, expressions of fibroin components Fib-H, Fib-L and P25 and Akt, TOR2, P70S6K and S6 in PI3K/Akt pathway, expressions of apoptosis related genes caspase-3, caspase-9 and activity of caspase-3 were explored. The results showed that the morphology of silk gland dramatically degenerated; transcription of Fib-H, Fib-L, and P25 gradually declined with time; and Fib-L protein level reduced by 0.6-fold at 72 h. Moreover, the transcription levels of Akt, TOR2, P70S6K, and S6 also decreased by 0.3-, 0.8-, 0.7-, and 0.1-fold, respectively, indicating that the downregulation of PI3K/Akt signaling pathway could lead to reduction in fibroin synthesis. In addition, the transcription levels of caspase-3 and caspase-9 increased by 1.3- and 3.6-fold, respectively, and the enzyme activity of caspase-3 grew at a maximum of 1.6-fold. The results showed the occurrence of apoptosis in PSG during spinning period. In conclusion, the present study indicated that both the decline in fibroin components and the increase in apoptosis-related genes were regulated by PI3K/Akt signaling pathway during spinning period, which shed new light on the functions of PI3K/Akt signaling pathway.

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS.

The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP- or CB-resistant strains have been generated. In this study, wild-type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21-fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.