BACKGROUND Many insect pests rely on microbial symbionts to obtain nutrients or for defence, thereby allowing them to exploit novel food sources and degrade environmental xenobiotics, including pesticides. Although Lepidoptera is one of the most diverse insect taxa and includes important agricultural pests, lepidopteran microbiotas, particularly functional traits, have not been studied widely. Here, we provide a comprehensive characterization of the gut microbiota across multiple mulberry‐feeding lepidopteran species, resolving both community structure and metabolic potential. RESULTS Our results indicate abundant bacteria inside the gut of larval Lepidoptera. However, even though they were fed the same diet, the structures of the bacterial communities differed in four major mulberry pest species, suggesting host‐specific effects on microbial associations. Community‐level metabolic reconstructions further showed that although taxonomic composition varied greatly, carbohydrate and amino acid metabolism and membrane transporter were key functional capabilities of the gut bacteria in all samples, which may play basic roles in the larval gut. In addition, principal coordinate analysis (PCoA) of gut bacterial‐predicted gene ontologies revealed specialized features of the microbiota associated with these mulberry pests, which were divided into two distinct clusters (macrolepidopterans and microlepidopterans). This pattern became even more prominent when further Lepidoptera species were involved. CONCLUSIONS A suite of gut microbiota metabolic functions significantly correlated with larval size; the metabolism of terpenoids and polyketides, xenobiotics biodegradation and metabolism were specifically enriched in large species, while small larvae had enhanced nucleotide metabolism. Our report paves the way for uncovering the correlation between host phenotype and microbial symbiosis in this notorious insect pest group. © 2019 Society of Chemical Industry

BACKGROUND: Microsporidia, a group of obligate intracellular fungal-related parasites, have been used as efficient biocontrol agents for agriculture and forestry pests due to their host specificity and transovarial transmission. They mainly infect insect pests through the intestinal tract, but the interactions between microsporidia and the gut microbiota of the host have not been well demonstrated.RESULTS: Based on the microsporidia-Bombyx mori model, we report that the susceptibility of silkworms to exposure to the microsporidium Nosema bombycis was both dose and time dependent. Comparative analyses of the silkworm gut microbiome revealed substantially increased abundance of Enterococcus belonging to Firmicutes after N. bombycis infection. Furthermore, a bacterial strain (LX10) was obtained from the gut of B. mori and identified as Enterococcus faecalis based on 16S rRNA sequence analysis. E. faecalis LX10 reduced the N. bombycis spore germination rate and the infection efficiency in vitro and in vivo, as confirmed by bioassay tests and histopathological analyses. In addition, after simultaneous oral feeding with E. faecalis LX10 and N. bombycis, gene (Akirin, Cecropin A, Mesh, Ssk, DUOX and NOS) expression, hydrogen peroxide and nitric oxide levels, and glutathione S-transferase (GST) activity showed different degrees of recovery and correction compared with those under N. bombycis infection alone. Finally, the enterococcin LX protein was identified from sterile LX10 fermentation liquid based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.CONCLUSION: Altogether, the results revealed that E. faecalis LX10 with anti-N. bombycis activity might play an important role in protecting silkworms from microsporidia. Removal of these specific commensal bacteria with antibiotics and utilization of transgenic symbiotic systems may effectively improve the biocontrol value of microsporidia.

The complex gut microbiome is a malleable microbial community that can undergo remodeling in response to many factors, including the gut environment and microbial properties. Enterococcus has emerged as one of the predominant gut commensal bacterial and plays a fundamental role in the host physiology and health of the major economic agricultural insect, Bombyx mori. Although extensive research on gut structure and microbiome diversity has been carried out, how these microbial consortia are established in multifarious niches within the gut has not been well characterized to date. Here, an Enterococcus species that was stably associated with its host, the model organism B. mori, was identified in the larval gut. GFP–tagged E. faecalis LX10 was constructed as a model bacterium to track the colonization mechanism in the intestine of B. mori. The results revealed that the minimum and optimum colonization results were obtained by feeding at doses of 105 CFU/silkworm and 107 CFU/silkworm, respectively, as confirmed by bioassays and fluorescence-activated cell sorting analyses (FACS). Furthermore, a comprehensive genome-wide exploration of signal sequences provided insight into the relevant colonization properties of E. faecalis LX10. E. faecalis LX10 grew well under alkaline conditions and stably reduced the intestinal pH through lactic acid production. Additionally, the genomic features responsible for lactic acid fermentation were characterized. We further expressed and purified E. faecalis bacteriocin and found that it was particularly effective against other gut bacteria, including Enterococcus casselifavus, Enterococcus mundtii, Serratia marcescens, Bacillus amyloliquefaciens, and Escherichia coli. In addition, the successful colonization of E. faecalis LX10 led to drastically increased expression of all adhesion genes (znuA, lepB, hssA, adhE, EbpA, and Lap), defense genes (cspp, tagF, and esp), regulation gene (BfmRS), secretion gene (prkC) and immune evasion genes (patA and patB), while the expression of iron acquisition genes (ddpD and metN) was largely unchanged or decreased. This work establishes an unprecedented conceptual model for understanding B. mori–gut microbiota interactions in an ecological context. Moreover, these results shed light on the molecular mechanisms of gut microbiota proliferation and colonization in the intestinal tract of this insect.