Impact Of TiO₂ NPs On Innate Immunity Mechanism In Silkworms, Bombyx Mori

Silkworm（Bombyx mori） is a model species for lepidopteran insects and an important economic insect. In the long process of evolution, the silkworm formed a unique innate immune system as other lepidoptera, including humoral immunity and cellular immunity. The humoral immunity includes a variety of recognition receptors, intracellular signaling factor, peptide, and protease enzyme cascade reactions. In order to overcome the impact of adverse external environment and resist to the exogenous microbial infection, the silkworm mainly depend on the innate immune system for the absence of acquired immune system. Therefore, how to improve the resistance of silkworm to pathogenic microorganisms has been a hot and difficult research area in sericulture. The results in mammals indicated that nanoparticles（NPs） could affect the innate immune system by changing the activity of immune cells, increasing or decreasing non-specific immune response to involve the immune regulation and activiting the complement system. However, whether nanoparticles can affect the silkworm innate immune system has not been reported.It has been reported that titanium dioxide nanoparticles（Ti O₂ NPs） has an antibacterial function with long-lasting antibacterial effect and a broad antibacterial spectrum. The anatase Ti O₂ NPs has a strong killing effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bud bacillus, Aspergillus and Salmonella etc. Low doses（5 mg/L） of Ti O₂ NPs can promote silkworm growth, increase food intake and the production and quality of raw silks. Moreover, it can improve silkworm’s resistance to organophosphate pesticides and Bm NPV. However, the effect of Ti O₂ NPs on silkworm innate immunity has not been reported. In this study, we performed research on silkworm that pretreatment with 5 mg/L Ti O₂ NPs, and the main results are as follows:1. The digital gene expression profiling of silkworm midgut pretreated with Ti O₂ NPsIn order to study the effect of Ti O₂ NPs on silkworm innate immunity and obtain the gene expression profiles, we performed digital gene expression profile（DGE） analysis for silkworm midgut, which revealed that pretreatment with Ti O₂ NPs caused significant changes in 15 pathways（P <0.05）. Of these pathways, 5 tyrosine metabolism-related genes showed 33.47- to 12739.00-fold changes in expression, while 4 melanogenesis-related genes were upregulated by 33.47- to 1148.00-fold. Results from ontology annotation and pathway analysis indicated that the differentially expressed genes mainly participate in immune response, defense response, energy metabolism, and apoptosis. The transcription level of the immune response-related gene, MAP kinse-ERK kinase, was upregulated by 65.80-fold; the transcription levels of defense response-related moricin I, peptidoglycan recognition protein S6, and beta-1,3-glucan-binding protein were increased by 97.68-, 2.68-, and 2.16-fold; the transcription level of apoptosis negative regulation gene GF12231 was increased by 48.84-fold.2. The inhibitory effect of Ti O₂ NPs on Bb in vitroIn order to investigate the effect of Ti O₂ NPs on Bb in vitro, this study measured the growth curve incubated with the different concentration of 0 mg/L, 5 mg/L, 50 mg/L and 100 mg/L Ti O₂ NPs. The results showed that Ti O₂ NPs could inhibit the proliferation of Bb with a dose effect, the higher the concentration of Ti O₂ NPs, the more significant effect of inhibition. Moreover, the virulence effect of Ti O₂ NPs incubation with Bb for different time showed that the inhibition of Ti O₂ NPs to Bb with a time-effect, and the longer time inhibition, the more obvious inhibiting effect.3. The impact on silkworm phenol oxidase activity and inhibiting effect on Bb pretreated with Ti O₂ NPsThe results of testing on phenol oxidase activity of silkworm hemolymph showed that Ti O₂ NPs treatment increased PPO enzyme activity by 10.4 %, as revealed by hemolymph phenol oxidase test. Ti O₂ NPs pretreatment caused varied degrees of upregulation in phenol oxidase cascade key genes Mapk-Erk, PGRP-S2 and Bm PPO1, Toll immune pathways key genes β-GRP3, PGRP-S2 and Rel, Imd immune pathways key genes β-GRP4, PGRP-S3 and Imd, and the antimicrobial peptide gene Bm Moricin I, as revealed by q RT-PCR; the highest increase was 525.43-fold, and the lowest was 2.00-fold. After infection by injection with Bacillus bombysepticus, silkworms pretreated with Ti O₂ NPs showed increased survival rate of 13.30%; q RT-PCR showed that Ti O₂ NPs reduced not only the phenol oxidase cascade but also the activation levels of Toll and Imd induced by B. bombysepticus. As revealed by RT-PCR analysis of Caspase3, Ti O₂ NPs also inhibited the apoptosis that occurred in control silkworms.4. Mechanism of enhanced Bm NPV-resistance by Ti O₂ NPs in silkwormsIn this study, Ti O₂ NPs’ effect on Bm NPV resistance was investigated by analyzing the characteristics of Bm NPV proliferation and transcriptional differences in silkworm midgut and the transcriptional changes of immunity related genes after feeding with Ti O₂ NPs. We found that low doses of Ti O₂ NPs improved the resistance of silkworm against Bm NPV by 14.88-fold, with the mortalities of the experimental group and control group being 0.56 % and 8.33 % at 144 h, respectively. The proliferation of Bm NPV in the midgut was significantly increased 72 h after infection in both experimental and control groups; the control group reached the peak at 120 h, while the experimental group took more than 24 hours to reach the maximal value that was 12.63 times lower than the control, indicating that Ti O₂ NPs can inhibit Bm NPV proliferation in the midgut. Consistently, the expression of the Bm NPV-resistant gene Bmlipase-1 had the same increase pattern as the proliferation changes. Immune signaling pathway analysis revealed that Ti O₂ NPs inhibited the proliferation of silkworm Bm NPV to reduce the activation levels of janus kinase/signal transducer and activator of transcription（JAK/STAT） and phosphatidylinositol 3-kinase（PI3K）-Akt signaling pathway, while promoting the expression of Bmakt to improve the immunity. Overall, our results demonstrated that Ti O₂ NPs increased silkworm resistance against Bm NPV by inhibiting virus proliferation and improving immunity in silkworms.5. The effect of Ti O₂ NPs on Bm N cell in silkwormIn this study, we detected the effect of the vary concentration of Ti O₂ NPs on the proliferation of Bm N cell. The results showed that Ti O₂ NPs promoted the proliferation of Bm N cell with a dose-dependent effect. When the concentration of Ti O₂ NPs lower than 100 mg/L, the higher the concentration, the more obvious effect on promotion. In addition, the study also examined the impact of Ti O₂ NPs on ROS level of Bm N cell in silkworm with the concentration of 5 mg/L, and found that Ti O₂ NPs significantly reduced the ROS level of Bm N cell.This study was the first to study the impact of Ti O₂ NPs on innate immune regulatory mechanism in silkworm. We obtained the gene expression profiling of silkworm midgut at the 3rd-day of fifth instar treated with Ti O₂ NPs. We proved that the mechanism of Ti O₂ Nps enhanced the resistance to Bb was by activating the phenol oxidase cascade, Toll and Imd pathway and the expression of antibacterial peptide Moricin I. In addition, the mechanism of Ti O₂ NPs enhanced the resistance to Bm NPV was by inhibiting the virus profiling and improving the silkworm immune.This research clarified the impact of Ti O₂ NPs on innate immune mechanism in silkworm and also provided a new ideas and theoretical basis for the application of Ti O₂ NPs to silkworm disease prevention.