Effects Of Dietary TiO₂ NPs On Fat Body Physiological Function Of Bombyx Mori

Silkworm?Bombyx mori?is an important economic insect,belongs to Lepidoptera Bombycidae and has complete metamorphosis including four stages:egg,larva,pupa and adult.It is the larvae that uses the absorbed nutrients to provide energy for the growth,development and metamorphosis,while synthesizes and secretes a large amount of silk proteins.Fat body is silkworm's intermediate metabolic organ,playing important physiological roles in nutrient storage and transport,metabolic detoxification.TiO₂ NPs is nanomaterial widely used as environmental cleanup material,daily necessities,and chemicals.It has been shown that feeding silkworm with TiO₂ NPs can increase feed efficiency and promote silk protein synthesis.TiO₂ NPs can mediate the synthesis of ecdysterone to affect silkworm growth and development,and enhance silkworm's anti-viral ability.In this study,we profiled the gene expression in silkworm fat body after TiO₂ NPs treatment,validated the major RNA-seq findings,detected the expression of related proteins and determined the contents of trehalose and triglyceride,the activity of lipase,detoxifying enzyme and the amount of total proteins.RNA seq results showed that expression of 11268 genes were changed,including 5789up-regulated genes and 5479 down regulated genes with 341 differentially expressed genes?P?0.01?,including 138 up-regulated genes and 203 down regulated genes in fat body after TiO₂ NPs treatment.Gene ontology enrichment analysis showed that,in the biological process,differentially expressed genes mainly concentrated in biological regulation,cellular process,metabolic process;in the cellular component mainly concentrated in cell part,extracellular region,organelle;in the molecular function mainly concentrated in the binding,structural molecule activity,transporter activity.KEGG pathway enrichment results showed that significant differences in expression genes were mainly concentrated in the signaling channels,calcium signaling pathway,phagosome pathway.The results showed that the addition of TiO₂ NPs could affect the expression of fat body gene,GO analysis and KEGG pathway enrichment showed that the addition of TiO₂ NPs could significantly affect the metabolism of fat body.QRT-PCR results showed in the insulin signaling pathway,the target genes expression levels were increased by 2.36 and 1.48-fold,respectively.The expression levels of protein metabolism related target genes were increased by 42.38 and 6.20-fold,respectively,and that of threonine dehydratase gene was decreased by 3.09-fold.The expression level of fatty acid desaturase was increased by 2.63-fold while the expression levels fatty acid transport protein was decreased by 5.12-fold.The expression levels of carbohydrate metabolism related target genes were increased by 6.63 and 1.12-fold,respectively.The results indicated TiO₂ NPs could improve fat body nutrition metabolism in silkworm.After TiO₂ NPs treatment,the expression of insulin-like peptide?neuropeptide receptor B were up-regulated by 5.17 and 1.12 fold,the expression of hsp 68?hsp 70?hsp 1?hsp-16.1/hsp-16.11?hsp90?hsp 20.8?hsp 20.4 were up-regulated by 3.61?2.82?2.80?2.60?2.59?2.36 and 1.91-fold,respectively.In TiO₂ NPs group,p70S6K,ERK,RAS,Akt,4EBP2,S6,MEK,and eIF4E were up-regulated by 3.59,2.74,2.65,2.24,1.61,1.54,1.49,and 1.48-fold,respectively.In phoxim group,RAS,4EBP2,ERK,p70S6K,S6,Akt,eIF4E,and MEK were up-regulated by10.69,9.18,5.77,5.10,5.03,4.12,2.61,and 2.24-fold,respectively.Compared with that in phoxim group,the up-regulation of selected genes was smaller in TiO₂ NPs+phoxim group,with RAS,4EBP2,p70S6K,S6,Akt,eIF4E,MEK,and ERK being up-regulated by 6.30,5.77,4.62,4.46,3.92,2.59,1.83,and 1.51-fold,respectively.These results indicated TiO₂ NPs could promote the growth of silkworm and alleviate the damage of phoxim on the fat body of silkworm.Heat shock proteins maintain life activity under stress conditions,as a molecular partner to ensure that the protein is properly folded,involved in immune function.The heat shock proteins of insects are associated with diapause,injury repair,cell proliferation and development.The validation of heat shock protein expression in this study shows that the addition of TiO₂ NPs can enhance the resistance of silkworm to adverse conditions.The contents of lipase,trehalose and total protein were increased by 3.86,1.34 and1.29-fold,respectively while the triglyceride was decreased by 0.94-fold after TiO₂ NPs treatment.Compared with that in phoxim group,the up-regulation of total protein content,target proteins expression,detoxification activity was smaller in TiO₂ NPs+phoxim group.The results indicated that TiO₂ NPs can improve fat body nutrient metabolism and alleviate the damage of phoxim on fat body of silkworm,the study could be a new reference and support to research effects of TiO₂ NPs on functions of silkworm fat body.The results of this study showed that the feeding of TiO₂ NPs can significantly affect the gene expression,promote the nutritional metabolism,and alleviate the damage of phoxim to the fat body of silkworm.