| The silkworm(Bombyx mori)is a model insect of lepidopteran that is economically important.Titanium dioxide nanoparticles(TiO2 NPs)was widely used as feed additives,and their biological effects have attracted widespread attention.Low-dose TiO2 NPs can promote the growth and development of silkworms,thus increasing cocoon yields and improving silkworm immunity.Phoxim is a classic organophosphorus pesticide characterized by its broad insecticidal spectrum and long residual period.Consumption of phoxim-contaminated mulberry leaves by silkworms can result in poisoning,posing a threat to cocoon production.Insects’ intestines are abundant in microbial communities that play an important role in the development of host insects.Exogenous substances can affect the structure and composition of the intestinal microbiota,and changes in the microbiota are known to alter the physiological functions of the intestine.Our team has conducted enormous research about the promoting effects of low-dose TiO2 NPs on the growth and development of silkworms,and the toxic effects of phoxim on silkworms.However,the mechanism of the influence of two exogenous substances on intestinal microbes has not been studied yet.To study the effects of TiO2 NPs and phoxim on intestinal microbes,the silkworm "Jingsong×Haoyue" was fed with mulberry leaves treated with TiO2 NPs or phoxim,and then phoximresistant strains were screened.The main research results are as follows:1.Effects of TiO2 NP exposure on the intestinal microorganisms of silkworms5th instar larvae of silkworm were continuously fed with low-dose TiO2 NPs(5 mg/L).At 72 h and 96 h,the larvae body weight in the TiO2 NPs treatment group was significantly higher than that in the control group(P≤0.01);the total cocoon weight increased,and both the cocoon shell weight and the ratio of cocoon shell increased significantly(P≤0.001).Notably,the ratio of cocoon shell increased by 16.96%and 9.67%in the male and female larvae,respectively.These data indicate that low-dose TiO2 NPs can significantly promote the growth and development of silkworms.The 16S rRNA gene sequencing results of intestinal microbes in the silkworm larvae from TiO2 NPs treatment group and the control group at 96 h were analyzed.It was found that the number of operational taxonomic units(OTU)was 615 in TiO2 NPs treatment group and 308 in the control group.The Alpha diversity index indicated that the richness and diversity of silkworm intestinal flora were increased.The samples of the TiO2 NPs treatment group were clustered separately,which was significantly different from the control samples(P≤0.05).Analysis of the number of bacterial genera showed that the types of dominant bacterial groups did not changed significantly,but their abundances changed significantly.The comparison results of abundance at the level of bacterial genera showed that:ⅰ)the abundances of Pseudomonas,Sphingomonas,Lachnospiraceae_NK4A 136 and Bacteroidales_S24-7 in the TiO2 NPs treatment group were significantly higher than those in the control group(P≤0.01 or P ≤0.001),ⅱ)meanwhile,the abundance of conditional pathogenic bacteria Serratia decreased.The biological effects of TiO2 NPs on the silkworm,including promotion of growth and development,immune enhancement and mediation of insecticide resistance,may be closely related to the changes in the abundance of the dominant intestinal flora of the silkworm after TiO2 NPs treatment.2.Effects of phoxim exposure on the intestinal microbes of the silkworm5th instar larvae of silkworm were continuously fed with mulberry leaves treated with 1.0 μg/mL phoxim(24 h LC50=7.86 μg/mL)for 96 hours,and symptoms of mild poisoning such as body shrinkage and head tremor appeared;body weight was significantly reduced after 72 h and 96 h(P≤0.05);the total cocoon weight of the phoxim treatment group was significantly reduced(P≤0.05).The results showed that phoxim treatment affected the growth and development of silkworm larvae.The total OTUs obtained from the two groups of intestinal samples was 633,of which 306 was in the control group and 462 in the phoxim treatment group.Shannon’s Index of the phoxim treatment group was significantly increased(P≤0.05),while the Simpson’s Index was significantly decreased(P≤0.05),indicating that the exposure of phoxim caused an increase in the diversity of the intestinal bacterial community in silkworm larvae.The principal component analysis showed that the samples from the silkworm larvae in the phoxim treatment group were obviously clustered and separated from the control samples.The abundances of Methylobacterium and Aureimonas in the intestine of silkworm larvae in the phoxim treatment group were significantly reduced(P≤0.05),while the abundance of Staphylococcus was significantly increased(P≤0.05).Phoxim treatment inhibited the transcription level of antimicrobial peptides(AMPs)related genes and reduced the silkworm’s resistance to Enterobacter cloacae,indicating that the immune system of silkworm larvae was suppressed after phoxim treatment,and that the changes in the composition and structure of intestinal microbes induced by phoxim treatment may affect the normal functions of the silkworm intestine.A total of 7 species of phoxim-tolerant bacteria was obtained after screening.We recolonized these 7 species of bacteria into the intestine of sterile silkworms,and found that Staphylococcus sciuri spp.,Sphingomonas spp.,and Klebsiella michiganensis spp.can effectively degrade phoxim and reduce the mortality of silkworms.In this paper,from the perspective of intestinal microbes,we analyzed the changes in the microbial composition and structure of the silkworm after feeding with two exogenous substances,studied the biological mechanism by which low-dose TiO2 NPs promote the growth and development of silkworms,and explored the new mechanism of damage by phoxim to silkworm,and screened and identified phoxim-resistant bacteria.This research provides a reference for in-depth studying of ⅰ)the complex relationship between exogenous chemical substances and the intestinal microbes of the silkworm,and ⅱ)the potential functions of the intestinal flora on the host silkworm.Moreover,it has important guiding significance for strengthening the resistance of non-target economic insects,preventing and controlling agricultural pests. |
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