Short-term triphenyltin exposure alters microbial homeostasis in the silkworm (Bombyx mori) midgut

Triphenyltin (TPT) is a widespread synthetic chemical used in many fields and its potential risk to organisms has been comprehensively investigated using different animal models and species. Currently, little is known about the effects of TPT exposure on microbial midgut diversity, therefore we explored these effects in the lepidopterous silkworm model using 16S rDNA sequencing. In total, 5273 and 5065 operational taxonomic units (OTUs) were identified in control and TPT-exposure group samples, ranging from 424 to 728 OTUs/sample. Alpha-diversity analyses revealed that TPT exposure induced the fluctuations of gut microbial diversity and abundance while beta-diversity analyses identified a distinct impact on major gut microbiota components. In our microbiome analyses, 23 phyla and 353 genera were recognized in the control group, while 20 phyla and 358 genera were recognized in the TPT exposure group. At the genus level, midgut microbiota were composed of several predominant bacterial genera, including Muribaculaceae, Lactobacillus, and UCG-010. In the TPT exposure group, o__Bacillales, f__Bacillaceae, and f__Caldicoprobacteraceae abundance was relatively high, while f__Oscillospiraceae, f__Fusobacteriaceae, and f__SC_I_84 abundance was relatively high in the control group. Gene function analyses in silkworm microbiota after TPT exposure showed that biosynthesis of ansamycins, fructose and mannose metabolism, glycerolipid metabolism, type II diabetes mellitus, glycolysis/gluconeogenesis, lipid metabolism, translation proteins, atrazine degradation, DNA repair and recombination proteins, nicotinate and nicotinamide metabolism were significantly increased. Collectively, our silkworm model identified gut microbial diversity risks and the adverse effects from TPT exposure, which were similar to other aquatic animals. Therefore, TPT levels in environmental samples must be monitored to prevent ecological harm.

Data preprocessing and statistical analysis.Trimmomatic (Version 0.35) 19 and FLASH (Version 1.2.11) 20 programs were used to remove low-quality reads and assemble paired-end reads, respectively.Clean reads were clustered into operational taxonomic units (OTUs) using Vsearch (Version 2.4.2) 21 .All valid reads were blasted against the Silva Version 138 database (16s/18s rDNA) using the RDP classifier (Version 2.2) 22 and Unite database using BLAST 23 .The microbial diversity in the midgut content isolated from silkworm larvae was analyzed by the α-diversity, including species richness (Chao 1) and community diversity (Shannon, Simpson and PD whole tree).The Unifrac Principal coordinates analysis (PCoA) between the TPT exposure group and the control group was drawn with the QIIME software (version 1.8.0).To characterize the microbial differences between the different groups, linear discriminant analysis (LDA) effect size (LEfSe) analysis was employed using Galaxy online tools (http:// hutte nhower.sph.harva rd.edu/ lefse/).
Through the pathway enrichment analysis of differential metabolites, it is helpful to understand the mechanism of metabolic pathway change in different samples.Pathway enrichment analysis was performed using KEGG ID of differential metabolites, and the enrichment results of metabolic pathways were obtained.Hypergeometric tests were applied to identify pathway entries that were significantly enriched in significantly differentially expressed metabolites compared to the overall background.Its calculation formula is as follows: N is the total number of metabolites; n is the number of differentially expressed metabolites in N. M is the number of metabolites annotated in a particular pathway; m is the number of differential metabolites in a particular pathway.With p-value ≤ 0.05 as the threshold, the pathway that meets this condition is the pathway that is significantly enriched in differential metabolites.The smaller the p-value, the more significant the difference of the metabolic pathway.

Results
Data acquisition and analysis.In total, 805,966 and 810,383 raw sequences were collected from the control (CK) and TPT exposure groups (TPT), respectively (Table S1).After quality testing, 765,471 and 763,686 high-quality reads were respectively acquired with an average of 76,458 (range 75,112-78,588) clean reads/sample.Rarefaction and rank abundance curve results indicated that nearly all microbial species were recognized (Fig. 1a,b).Using Vsearch (Version 2.4.2) software to classify OTUs based on nucleotide-sequence similarity, 5273 and 5065 OTUs were identified in CK and TPT-exposure group samples, respectively, ranging from 424 to 728 OTUs/sample (Fig. 1c).Comparative analyses of diversity indices between two groups showed no significant differences in Chao1, Simpson, Shannon, and PD_whole_tree indices (Fig. 2a-d).Diversity index dilution curve showed that the curve will eventually flatten, indicating that the sequencing depth is large enough and the established libraries can truly and effectively reflect the diversity of bacterial in the samples with research significance and practical value (Fig. S1).Alpha-diversity analyses revealed that TPT exposure could induce the fluctuations of gut microbial diversity and abundance in silkworms.Beta-diversity was analyzed using principal coordinate analysis (PCoA) (Fig. 2e), which reflected gut microbial similarities and differences between groups, and showed that most of individuals in both groups were separated and minority individuals were clustered together, suggesting that TPT exposure had an obvious impact on major gut microbiota components.
Predicted functional potential of the altered microbiome.To explore altered genes in silkworm microbiota after TPT exposure, Kyoto Encyclopedia of Genes and Genomes 24,25 (KEGG, www.KEGG.jp/ KEGG/ kegg1.html) orthology level 3 pathways were identified using PICRUSt2, and showed that biosynthesis of ansamycins, fructose and mannose metabolism, glycerolipid metabolism, type II diabetes mellitus, glycolysis/ gluconeogenesis, lipid metabolism, translation proteins, atrazine degradation, DNA repair and recombination proteins, nicotinate and nicotinamide metabolism were significantly increased after TPT exposure (Fig. S2).

Discussion
TPT is a toxic chemical used in various industrial and agricultural applications.TPT exposure risk studies have reported harmful effects on human health, including reproductive toxicity, immunotoxicity, neurotoxicity, and endocrine disruption 7,8 .TPT exposure occurs via inhalation, ingestion, or skin contact with contaminated water, soil, or food 26,27 .Therefore, proper protective measures should be taken to minimize TPT exposure and limit environmental contamination in occupational settings.In this study, we examined TPT exposure on silkworm microbial diversity and showed that TPT exposure induced the gut microbial diversity and abundance fluctuations (alpha-diversity index analyses).Similarly, beta-diversity analyses showed that TPT exposure had an obvious impact on major gut microbiota components.
It is reported that TPT exposure exerts a negative impact on microbial diversity, including bacteria, fungi, and algae [28][29][30] .Exposure alters microbial community structures, thereby reducing bacterial abundance and diversity while promoting pathogen growth.In our study, we found that TPT can upregulate the abundance of pathogen in the midgut, including f__Campylobacteraceae, f__Mycoplasmataceae, and o__Mycoplasmatales (Fig. 3).Members of the family Campylobacteraceae neither ferment nor oxidize carbohydrates, instead they obtain energy from amino acids, or tricarboxylic acid cycle intermediates 31 .Members of the family Mycoplasmataceae can cause cell damage through different mechanisms, such as obtaining lipids and cholesterol on the cell membrane, www.nature.com/scientificreports/causing membrane damage, and releasing neurotoxins, phosphatases, and hydrogen peroxide 32,33 .Therefore, we infer that TPT can cause damage to midgut cells by upregulating pathogenic bacteria in the intestine.It was reported that the physiological stress of TPT has a negative impact on many intestinal bacteria, significantly reducing microbial diversity 34 .Furthermore, TPT could cause lipid metabolism abnormalities by affecting intestinal microbiome 34 .Our investigation found that TPT induced a significant increase in fructose and mannose metabolism, glycerolipid metabolism, glycolysis/gluconeogenesis, lipid metabolism, and translation proteins in the midgut microbiome of silkworms (Fig. S2).These changes may be related to the abnormality of carbohydrate, lipid, and amino acid metabolism in the midgut of silkworms caused by TPT exposure 18 .In our study, we used the silkworm model to explore the effects of TPT exposure on gut microbial diversity and observed that adverse effects were similar to other aquatic animals 34 .Therefore, TPT levels must be monitored in environmental samples to prevent potential ecological harm.

Figure 1 .
Figure 1.TPT exposure effects on microbial species and operational taxonomic units (OTUs).(a) Rarefaction and (b) rank abundance curves.(c) OTUs were identified between the control (CK) and TPT-exposure group (TPT) samples.

Figure 3 .
Figure 3. Comparative analysis of microbial taxonomic composition.(a) Annotation analysis of significantly different bacteria (SDB) based on the linear discriminant analysis coupled with effect size measurements (LEfSe).(b) Score plot of SDB based on LEfSe analysis.Green and red indicates a SDB that is more abundant in the TPT and CK group, respectively.