Effect Of Nanoparticulate Anatase Titanium Dioxide On The Growth,Development And Protein Synthesis Of Silkworm (Bombyx Mori)

Bombyx mori（B. mori） is an important economic insect. It was domesticated from Bombyx mandarina over 5 700 years ago. Silk industry is the traditional industry of China, China is a major silk production country and its annual production of raw silk accounts for more than 78% of the total raw silk in the world. To increase the output of cocoon and improve the quality of raw silk is the central task of the development of silk industry, through of improving B. mori variety, developing efficient breeding technology and new technology of silk reeling can improve the yield and quality of cocoon. In recent years, the development of nanomaterials is widely used in feed additive of livestock production with the rapid development of nano biotechnology, there no related reports on the nanomaterials used in sericultural production.Titanium dioxide nanoparticles（TiO₂ NPs） based on its surface effect, volume effect and quantum size effect, macroscopic quantum tunneling effect, and so on, is widely applied in biological field. The biological effect of TiO₂ NPs is closely related to its concentration, high concentrations of TiO₂ NPs is toxic to biology, low concentration of TiO₂ NPs is used as additive can promotes the growth of livestock and protein synthesis. The ability of silk protein synthesis of B. mori directly affects the silk production, low concentration of TiO₂ NPs feed B. mori whether can promote the growth of B. mori and protein synthesis, no relevant report can be found at present.In order to study the TiO₂ NPs whether can promote the growth, development and protein synthesis of B. mori. This study used 5 mg/L TiO₂ NPs feed B. mori, B. mori body weight and feed efficiency（the amount of ingested food, amount of digestion, and percentage of ingested food） are surveyed, the results showed that the body weight of TiO₂ NP-fed group increased by 4.64% than control, the amount of ingested food increased by 5.82%, the amount of digestion increased by 18.63%, and percentage of ingested food is increased by 17.58%, above of all showed that TiO₂ NPs fed can promote the growth and development of B. mori. Bm NPFR is orphan G protein-coupled receptor of B. mori, and its specific endogenous ligands are Bm NPF1 a and Bm NPF1 b. Bm NPFR regulates food intake and growth of B. mori by its specific and highly efficient binding to Bm NPF1 a and Bm NPF1 b, mediate the internalization of Bm NPFR, activate ERK1/2 signaling pathway, and participate in the regulation of the expression of the physiological development-related genes to exert their roles of promoting food intake and growth in B. mori. In order to further study the mechanism of TiO₂ NPs promotes the growth and development of B. mori, apply RNAi to B. mori neuropeptide receptor Bm NPFR, using Real-time quantitative Polymerase Chain Reaction（q RT-PCR） to detect the transcription level of neuropeptide receptor Bm NPFR, neuropeptide ligand Bm NPF1 a and Bm NPF1b; used western blotting to test the regulation of feeding and growth of ERK phosphorylation protein MAPK, and research the combination of the neuropeptide receptor Bm NPFR and neuropeptide ligands Bm NPF1 a, Bm NPF1 b. The results showed that after TiO₂ NPs treatment for 72 h, the transcriptional levels of Bm NPFR, Bm NPF1 a, and Bm NPF1 b in the midgut were 1.58, 1.43, and 1.34 folds, respectively, of those of the control, but 1.99, 2.26, and 2.19 folds, respectively, of the Bm NPFR-ds RNA injection group; the phosphorylation level of MAPK was 24.03% higher than the control, while the phosphorylation level of Bm NPFR-ds RNA injection group was 71.00% of control. The results showed that TiO₂ NPs increase Bm NPFR expression, enhance the interactions between Bm NPFR with Bm NPF1 a and Bm NPF1 b, increase the phosphorylation of ERK protein, activate ERK1/2 phosphorylation signaling pathway, promote B. mori growth and development.The one of main influence of biological growth and development is the protein synthesis and accumulation, the main expression is made eggs and the change of silk protein synthesis in the B. mori. Silk protein synthesis is an important part of B. mori protein synthesis, in order to study the TiO₂ NPs whether affect the silk protein synthesis of B. mori, investigated the cocoon quality（the cocoon mass, cocoon shell mass and ratio of cocoon shell）, used the q RT-PCR, Semi quantitative PCR（RT-PCR） and western blotting to test the level of transcription and expression of Fib-L gene and transcription of SGF-1. The results showed that after feeding with TiO₂ NPs, cocoon mass was an increase of 4.74 %, as compared to the control group. The cocoon shell mass and ratio of cocoon shell were increased by 9.30% and 4.51%, respectively, compared with those of the control group. The results showed that the m RNA levels of Fib-L were 1.30, 1.29, 3.17, and 5.93 folds higher at 48, 96, 144 and 192 h after feeding with TiO₂ NPs as compared those at the corresponding time points in the control group; the m RNA levels of SGF-1 were 1.39, 2.21, 5.79, and 3.29 folds higher at 48, 96, 144 and 192 h after feeding with TiO₂ NPs as compared those at the corresponding time points in the control group; the two PCR and western blotting methods give the consistent results. Together, these data indicate that the TiO₂ NPs feeding can increase the synthesis of silk proteins.In order to further research the mechanism of TiO₂ NPs increased silk protein synthesis, through of the results of Digital Gene Expression Profiling（DGE） about the transcription and expression profile data, to analysis the molecular mechanism of TiO₂ NPs increased silk protein synthesis. The DGE results analysis showed that, a total of 4 741 expressed genes were detected in the samples. Among them, 306 genes were differentially expressed, of which, 137 were up-regulated and 169 were down-regulated. Among the genes involved in fibroin synthesis, 106 genes were significantly altered, 97 genes were up-regulated and 9 genes were down-regulated, at the same time, through of the GO and KEGG categorize differentially expressed genes. In order to detect the reliability of DGE data, eight genes which are closely related to protein synthesis and hydrolysis in the silk gland were further analyzed by q RT-PCR. Three of them, aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, the enzymes involved in protein hydrolysis and degradation. Five of them, Lysyl-t RNA synthetase, cuticular protein glycine-rich 10, splicing factor arginine/serine-rich 6, serine protease inhibitor 28 and aspartate aminotransferase are protein synthetase and modifying enzymes, the results were consistent with those with DGE. The results demonstrate that feeding B. mori with TiO₂ NPs could increase the transcription of genes related to fibroin synthesis, reduce transcription of genes related to the protein hydrolysis and degradation enzyme, maybe this is associated with TiO₂ NPs feeding lead to increased fibroin synthesis in silk gland.To study the TiO₂ NPs treatment whether there is a cumulative effect on nutrition absorption and material use for silk protein synthesis, the protease activity in midgut and the contents of these four amino acids in the hemolymph of the 5th instar were assayed after being fed with TiO₂ NPs at 48, 96, 144 and 192 h. The results showed that the protease activities in the TiO₂ NPs-fed larvae at 48, 96, 144 and 192 h were 2.44, 1.77, 2.46, and 1.33 folds higher than those at the corresponding time points of the control group, respectively. Fibroin mainly contains four amino acids, namely glycine（GLY）, alanine（ALA）, serine（SER） and tyrosine（TYR）, TiO₂ NPs treatment group compared with control group, it has been showed that the contents of main amino acids for the synthesis of fibroin were increased in the earlier period of the 5th instar, thereafter, the contents of these amino acids declined in the later period. These data indicated that TiO₂ NPs treatment not only can increase protease activities in the midgut of B. mori for improving the absorption and utilization of nutrition in the mulberry leaves, but also can enhance the transport of these amino acids promote the utilization of material for silk protein synthesis.To reseach the effect of TiO₂ NPs on laying eggs related protein synthesis, oviposition, mass per egg and gonadosomatic index（GSI） were investigated. 51 more eggs per insect and 0.14×10-4 g more mass per egg were found after TiO₂ NPs feeding, after TiO₂ NPs treatment, GSI were significantly higher than in the control. It showed that the TiO₂ NPs treatment increase oviposition and improve the quality of eggs. To further study the possible mechanism of TiO₂ NPs effect B. mori laying eggs, the transcript levels of gonad development-related genes in the ovaries and testes were determined by the q RT-PCR method. The TiO₂ NPs-induced up-regulated genes in the ovaries, including Vg, Ovo, Ovo-781, Ovo-498, Otu, Sxl-S and Sxl-L, with 6.77, 2.00, 5.33, 2.99, 2.43, 1.73 and 2.30 folds increase, respectively. The TiO₂ NPs-induced up-regulated genes in the testes, including Achi-L, Aly, and Vlg with 3.55, 5.78 and 2.36 folds increase, respectively. The above results show that TiO₂ NPs can up-regulate the transcription of genes related to reproductive development of gonads, and could be responsible for the change of oviposition after TiO₂ NPs treatment.The results showed that the low concentration of TiO₂ NPs feed B. mori to enter midgut at first, increase neuropeptide receptor Bm NPFR and neuropeptide ligands Bm NPF1 a, Bm NPF1 b transcription, expression, and promote the activation of ERK1/2 signaling pathways of B. mori, promote the growth and development of B. mori. At the same time, TiO₂ NPs up-regulated related genes in silk protein synthesis, down-regulated related genes in proteolytic enzyme genes, increase the protease activity in the midgut, enhance the transport ability of amino acid, promote the transcription regulation of silk protein synthesis, increase the synthesis of silk protein and yield of silk. In addition, after TiO₂ NPs treatment, the transcription level of B. mori gonad development related gene increase, promote the growth and development of the gonads, with transport and supply of adequate nutrition, increase the oviposition. This study extends the application of TiO₂ NPs in the field of sericulture, as a new additive for TiO₂ NPs promote the development of sericulture provide a reference.