Lepidopteran insects are one of the most widespread and speciose lineages on Earth, with many common pests and beneficial insect species. The evolutionary success of their diversification depends on the essential functions of gut microorganisms. This diverse gut microbiota of lepidopteran insects provides benefits in nutrition and reproductive regulation and plays an important role in the defence against pathogens, enhancing host immune homeostasis. In addition, gut symbionts have shown promising applications in the development of novel tools for biological control, biodegradation of waste, and blocking the transmission of insect-borne diseases. Even though most microbial symbionts are unculturable, the rapidly expanding catalogue of microbial genomes and the application of modern genetic techniques offer a viable alternative for studying these microbes. Here, we discuss the gut structure and microbial diversity of lepidopteran insects, as well as advances in the understanding of symbiotic relationships and interactions between hosts and symbionts. Furthermore, we provide an overview of the function of the gut microbiota, including in host nutrition and metabolism, immune defence, and potential mechanisms of detoxification. Due to the relevance of lepidopteran pests in agricultural production, it can be expected that the research on the interactions between lepidopteran insects and their gut microbiota will be used for biological pest control and protection of beneficial insects in the future.

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. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH >10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for Microsporidia. Here we show, using an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated respectively by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analyses in vitro and in vivo. Furthermore the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape of N. bombycis spores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general.

Nosema bombycis (N. bombycis, Nb) is a fungus-related and obligate intracellular parasite that causes chronic pebrine disease in the silkworm. After infecting the host, spores obtain energy from host cells and survive for several days. This symbiosis between the pathogen and the host cell suggests that N. bombycis prevents apoptosis and reactive oxygen species (ROS) production of host cells to create the optimal environmental conditions for its growth and development. In this study, different methods were used to prove that N. bombycis suppressed apoptosis in BmN cells. Flow cytometry analysis results showed that spores suppressed apoptosis of BmN cells at 2 and 5 days after infection (P < 0.05). Compared with actinomycin D (ActD) treatment, apoptosis of BmN cells was apparently reduced after spore infection (P < 0.01). Forty-eight hours after infection, the ROS production of BmN cells was down-regulated compared with that after ActD treatment for 6 h. Furthermore, N. bombycis prevented the formation of apoptosomes by down-regulating the expression of apaf-1 and cytochrome C. In addition, N. bombycis also up-regulated the expression of buffy. Western blot analysis demonstrated that spores decreased the level of host cytochrome C at 48 and 98 h post infection. Thus, our results suggested that N. bombycis inhibited the mitochondrial apoptotic pathway of the host cells to create an optimal environment for its own survival. FullText for HTML: https://doi.org/10.1093/abbs/gmv062

SUMMARY: Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.