Mechanism Of TiO₂ Nanoparticles Alleviating Damage Induced By High Temperature Stress In Silkworm,Bombyx Mori

Silkworm(Bombyx mori)is an important economic insect with a domestication history of about 8500 years.Long-term indoor feeding reduces the resistance of silkworm to adverse environments.Silkworm is a temperature-changing animal that is extremely sensitive to changes in environment temperature.The 5th instar larva are susceptible to high temperature stress during rearing which reducing the vitality and silk production of the silkworm and reducing the economic benefits of the silk industry.The regions of sericulture production have gradually shifted to the southwest and southern in China,but the southern region has a subtropical climate with high temperatures,where the summer and autumn temperatures are often higher than 30? or even 40?,which seriously impedes the development of silk industry.High temperature stress has become an important factor affecting the silk production in summer and autumn.At present,there are still no effective measures to cope with high temperature stress.Titanium dioxide nanoparticles(TiO2 NPs)are excellent nanomaterials with particle sizes ranging from 1 nm to 100 nm.Due to their small size effect,surface effect and quantum size effect,were widely used in light industry,environmental treatment and antibacterial disinfection,medicine and agriculture.Studies have shown that low-dose TiO2 NPs can promote the growth and development of silkworm larvae,increase the expressions of silk protein genes,improve the antioxidant capacity of silkworm fat body and alleviate the oxidative stress damage caused by phoxim pesticide poisoning.In order to investigate whether the treatment of TiO2 NPs can alleviate the damage and improve the vitality and silk yield of silkworm caused by high temperature stress,silkworm variety"Jingsong×Haoyue" was used as experimental materials to study the effects of TiO2 NPs on the silkworm vitality and cocoons under high temperature stress.1.Effects of TiO2 NPs on vitality of silkworm under high temperature stressIn this study,the effects of TiO2 NPs treatment on the vitality of silkworm larvae under different high temperature stress were studied by using the 5th instar larvae of silkworm as experiment material.The results showed that the body mass and survival rate of silkworm larvae were decreased significantly under high temperature stress at 36?(P<0.05).36?+TiO2 NPs treatment could not significantly alleviate the effect of high temperature on silkworm body weight and vitality(P>0.05).Under 30? high temperature stress,the body weight and vitality of silkworm larvae decreased significantly(P<0.05).The body weight and survival rate of silkworm larvae increased significantly after TiO2 NPs treatment(P<0.05).It indicated that the treatment of TiO2 NPs could effectively alleviate the effect of 30?high temperature on silkworm,and the alleviate effect of 36? was not obvious.In order to study the reason for the improvement of the vitality of silkworm under 30? treatment,this study examined the oxidation level in fat body.The results showed that ROS(Reactive oxygen species)and H2O2 levels decreased significantly(P<0.05).The anti-oxidation pathway BmCncC/Keap1 was activated,and the transcription levels of downstream CAT(Catalase)and superoxide dismutase(SOD)were significantly increased(P<0.05).After treatment with TiO2 NPs,the transcription of heat shock protein genes Hsp70 and Hsp90 were significantly up-regulated(P<0.05).The results showed that under 30? temperature,TiO2 NPs treatment reduced the oxidation level of fat body,promoted the transcription of antioxidant enzymes and heat shock protein genes,and improved the vitality of silkworm larvae.2.Effects of TiO2 NPs on silkworm cocoon under high temperature stressThe results showed that under 30? high temperature stress,the cocoon shell mass and the cocoon shell ratio were significantly decreased(P<0.05),and the irregular silkworm cocoon appeared,indicating that high temperature stress adversely affect the ability of cocooning of silkworm.The level of oxidation in the posterior silk gland was significantly increased(P<0.05),and the levels of ROS,H2O2 and MDA(Malondialdehyde)were significantly up-regulated by 1.56,1.76 and 2.13-fold.After Ti02 NPs treatment,the oxidative levels in posterior silk gland were decreased,and the transcription of fibroin genes Fib-H and P25 were increased significantly(P<0.05).The transcription of Bmdimm was significantly up-regulated(P<0.05).After treatment with TiO2 NPs,the transcription of heat shock protein genes Hsp70,Hsp90 and sHsp21.1 were significantly up-regulated(P<0.05).The results showed that TiO2 NPs treatment reduced the level of oxidation in silk gland,promoted the transcription of silk fibroin-related genes,and alleviated the effects of high temperature stress on silkworm cocoons.3.TiO2 NPs promote the transcriptions of Bm GrpE under thermal stressThe high temperature stress gene GrpE plays an important role in coping with high temperature stress.In this study,the ORF sequence of the coding region of BmGrpE gene was amplified by using the cDNA of silkworm fat body as template,which was 644 bp,and the molecular weight of the encoded protein was 24.1kd.The gene has a binding site for the heat shock transcription factor(Hsfl)at-1440 bp upstream of the promoter which indicated that it responses to the high temperature stress.The transcription level of BmGrpE gene was lower in silkworm egg and pupa stage,and higher in the 5th instar larvae stages.The transcription levels were higher in fat body,silk gland and midgut,lower in hemolymph in the 3th day of 5th instar larvae.After dsRNA treatment,the cell viability of BmN cells were significantly decreased under 34? and H2O2 stress(P<0.05).Mutation of amino acid of BmGrpE(H163L)enhances high temperature resistance of BmN cells.Under 30? stress,TiO2 NPs treatment promoted the transcription of BmGrpE gene in fat body and silk gland of silkworm.These results provide a reference for elucidating the anti-high temperature mechanism of silkworm and studying of the effect of BmGrpE gene on high temperature resistance of silkworm.This study clarified the mechanism of TiO2 NPs mediated improvement of the viability and cocooning rate of silkworm under high temperature stress,and the function of high temperature stress gene BmGrpE and its role under high temperature stress were identified.This study provides a way to improve the viability and cocoons quality of silkworm under high temperature stress and provide support for the industrial application of Ti02 NPs.