Toward an Integrated Understanding of the Lepidoptera Microbiome.

Research over the past 30 years has led to a widespread acceptance that insects establish widespread and diverse associations with microorganisms. More recently, microbiome research has been accelerating in lepidopteran systems, leading to a greater understanding of both endosymbiont and gut microorganisms and how they contribute to integral aspects of the host. Lepidoptera are associated with a robust assemblage of microorganisms, some of which may be stable and routinely detected in larval and adult hosts, while others are ephemeral and transient. Certain microorganisms that populate Lepidoptera can contribute significantly to the hosts' performance and fitness, while others are inconsequential. We emphasize the context-dependent nature of the interactions between players. While our review discusses the contemporary literature, there are major avenues yet to be explored to determine both the fundamental aspects of host-microbe interactions and potential applications for the lepidopteran microbiome; we describe these avenues after our synthesis.

Probiotic potentials of the silkworm gut symbiont Enterococcus casseliflavus ECB140, a promising L-tryptophan producer living inside the host.

AIMS: L-tryptophan is an essential aromatic amino acid for the growth and development of animals. Studies about enteric L-tryptophan-producing bacteria are scarce. In this report, we characterized the probiotic potential of Enterococcus casseliflavus ECB140, focusing on its L-tryptophan production abilities. METHODS AND RESULTS: ECB140 strain was isolated from the silkworm gut and can survive under strong alkaline environmental conditions. Bacterial colonization traits (motility and biofilm) were examined and showed that only ECB140 produced flagellum and strong biofilms compared with other Enterococcus strains. Comparative genome sequence analyses showed that only ECB140 possessed a complete route for L-tryptophan synthesis among all 15 strains. High-performance liquid chromatography and qRT-PCR confirmed the capability of ECB140 to produce L-tryptophan. Besides, the genome also contains the biosynthesis pathways of several other essential amino acids, such as phenylalanine, threonine, valine, leucine, isoleucine and lysine. These results indicate that ECB140 has the ability to survive passage through the gut and could act as a candidate probiotic. CONCLUSIONS: The study describes a novel, natural silkworm gut symbiont capable of producing L-tryptophan. Enterococcus casseliflavus ECB140 physical and genomic attributes offer possibilities for its colonization and provide L-tryptophan for lepidopteran insects.

CRISPR/Cas9-Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant-Associated Isolates.

As one of the most widespread groups of Gram-negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.

Insect microbial symbionts as a novel source for biotechnology.

Insecta is the most diverse and largest class of animals on Earth, appearing together with the emergence of the first terrestrial ecosystem. Owing to this great diversity and long-term coexistence, an amazing variety of symbiotic microorganisms have adapted specifically to insects as hosts. Insect symbionts not only participate in many relationships with the hosts but also represent a novel resource for biotechnological applications. The exploitation of mutualistic symbiosis represents a promising area to search for bioactive compounds and new enzymes for potential clinical, industrial or environmental applications. Moreover, the manipulation of parasitic symbiosis has particular potential to solve practical problems for the control of agricultural pests and disease vectors. Although the study of microbial symbionts has been impaired by the unculturability of most symbionts, the rapidly growing catalogue of microbial genomes and the application of modern genetic techniques provide an alternative approach to using these microbes. This minireview presents examples of microbial symbionts isolated from insects for emerging biotechnological use and illuminates new ways for discovering microorganisms of applied value from a particularly promising source.

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.

Ricin-B-lectin enhances microsporidia Nosema bombycis infection in BmN cells from silkworm Bombyx mori.

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion.

The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses.

BACKGROUND: Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years. RESULTS: Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host. CONCLUSIONS: We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.

Assessing the quality and eco-beneficial microbes in the use of silkworm excrement compost.

Sericulture has become widespread globally, and the utilization of artificial diets produces a substantial quantity of silkworm excrement. Although silkworm excrement can be composted for environmentally friendly disposal, the potential utility of the resulting compost remains underexplored. The aim of this study was to assess the quality of this unique compost and screen for eco-beneficial microbes, providing a new perspective on microbial research in waste management, especially in sustainable agriculture. The low-concentration compost application exhibited a greater plant growth-promoting effect, which was attributed to an appropriate nutritional value (N, P, K, and dissolved organic matter) and the presence of plant growth-promoting bacteria (PGPB) within the compost. Encouraged by the "One Health" concept, the eco-benefits of potent PGPB, namely, Klebsiella pneumoniae and Bacillus licheniformis, in sericulture were further evaluated. For plants, K. pneumoniae and B. licheniformis increased plant weight by 152.44 % and 130.91 %, respectively. We also found that even a simple synthetic community composed of the two bacteria performed better than any single bacterium. For animals, K. pneumoniae significantly increased the silkworm (Qiufeng × Baiyu strain) cocoon shell weight by 111.94 %, which could increase sericulture profitability. We also elucidated the mechanism by which K. pneumoniae assisted silkworms in degrading tannic acid, a common plant-derived antifeedant, thereby increasing silkworm feed efficiency. Overall, these findings provide the first data revealing multiple beneficial interactions among silkworm excrement-derived microbes, plants, and animals, highlighting the importance of focusing on microbes in sustainable agriculture.

Multiomics analysis reveals the molecular basis for increased body weight in silkworms (Bombyx mori) exposed to environmental concentrations of polystyrene micro- and nanoplastics.

INTRODUCTION: Micro- and nanoplastics (MNPs) are emerging environmental pollutants that have raised serious concerns about their potential impact on ecosystem and organism health. Despite increasing efforts to investigate the impacts of micro- and nanoplastics (MNPs) on biota little is known about their potential impacts on terrestrial organisms, especially insects, at environmental concentrations. OBJECTIVES: To address this gap, we used an insect model, silkworm Bombyx mori to examine the potential long-term impacts of different sizes of polystyrene (PS) MNPs at environmentally realistic concentrations (0.25 to 1.0 µg/mL). METHODS: After exposure to PS-MNPs over most of the larval lifetime (from second to last instar), the endpoints were examined by an integrated physiological (growth and survival) and multiomics approach (metabolomics, 16S rRNA, and transcriptomics). RESULTS: Our results indicated that dietary exposures to PS-MNPs had no lethal effect on survivorship, but interestingly, increased host body weight. Multiomics analysis revealed that PS-MNPs exposure significantly altered multiple pathways, particularly lipid metabolism, leading to enriched energy reserves. Furthermore, the exposure changed the structure and composition of the gut microbiome and increased the abundance of gut bacteria Acinetobacter and Enterococcus. Notably, the predicted functional profiles and metabolite expressions were significantly correlated with bacterial abundance. Importantly, these observed effects were particle size-dependent and were ranked as PS-S (91.92 nm) > PS-M (5.69 µm) > PS-L (9.7 µm). CONCLUSION: Overall, PS-MNPs at environmentally realistic concentrations exerted stimulatory effects on energy metabolism that subsequently enhanced body weight in silkworms, suggesting that chronic PS-MNPs exposure might trigger weight gain in animals and humans by influencing host energy and microbiota homeostasis.

Fitness effects of synthetic and natural diet preservatives on the edible insect Bombyx mori.

Silkworm pupae as widely consumed insect products are good biosources of protein and micronutrients. Silkworm rearing throughout the year can be achieved by feeding them an artificial diet instead of native plants, facilitating extensive pupa production. However, artificial diets are prone to spoilage caused by bacterial contamination. Here, we evaluated the antiseptic effect of ethylparaben (EP, chemical preservative) and medium-chain fatty acids (MCFA, natural preservative) in a silkworm artificial diet. Results showed that both preservatives effectively inhibited pathogenic bacterial growth. Furthermore, the addition of EP or MCFA did not negatively impact the production capacity of silkworms and the homeostasis of gut microbiota. However, the expression of genes involved in detoxification such as Ugt2, and immune response such as Cecropin B, were upregulated after EP consumption. Therefore, natural preservative MCFA emerges as a suitable option from a safety perspective. These findings highlight future directions for improving insect artificial diet formulation.

Insect Insights at the Single-Cell Level: Technologies and Applications.

Single-cell techniques are a promising way to unravel the complexity and heterogeneity of transcripts at the cellular level and to reveal the composition of different cell types and functions in a tissue or organ. In recent years, advances in single-cell RNA sequencing (scRNA-seq) have further changed our view of biological systems. The application of scRNA-seq in insects enables the comprehensive characterization of both common and rare cell types and cell states, the discovery of new cell types, and revealing how cell types relate to each other. The recent application of scRNA-seq techniques to insect tissues has led to a number of exciting discoveries. Here we provide an overview of scRNA-seq and its application in insect research, focusing on biological applications, current challenges, and future opportunities to make new discoveries with scRNA-seq in insects.

Primer selection impacts the evaluation of microecological patterns in environmental microbiomes.

This study revealed that primer selection substantially influences the taxonomic and predicted functional composition and the characterization of microecological patterns, which was not alleviated by close-reference clustering. Biases were relatively consistent across different habitats in community profiling but not in microecological patterns. These primer biases could be attributed to multiple aspects, including taxa specificity, regional hypervariability, and amplification efficiency.

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1.

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and ß subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; ß subunit was a tetramer that had sarcosine oxidase and L: -proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αß)(4) form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and ß subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αß)(4) complex may separately exist as a function enzyme in different conditions.

Smartphones as an Ecological Niche of Microorganisms: Microbial Activities, Assembly, and Opportunistic Pathogens.

Smartphone usage and contact frequency are unprecedentedly high in this era, and they affect humans mentally and physically. However, the characteristics of the microorganisms associated with smartphones and smartphone hygiene habits remain unclear. In this study, using various culture-independent techniques, including high-throughput sequencing, real-time quantitative PCR (RT-qPCR), the ATP bioluminescence system, and electron microscopy, we investigated the structure, assembly, quantity, and dynamic metabolic activity of the bacterial community on smartphone surfaces and the user's dominant and nondominant hands. We found that smartphone microbiotas are more similar to the nondominant hand microbiotas than the dominant hand microbiotas and show significantly decreased phylogenetic diversity and stronger deterministic processes than the hand microbiota. Significant interindividual microbiota differences were observed, contributing to an average owner identification accuracy of 70.6% using smartphone microbiota. Furthermore, it is estimated that approximately 1.75 × 106 bacteria (2.24 × 104/cm2) exist on the touchscreen of a single smartphone, and microbial activities remain stable for at least 48 h. Scanning electron microscopy detected large fragments harboring microorganisms, suggesting that smartphone microbiotas live on the secreta or other substances, e.g., human cell debris and food debris. Fortunately, simple smartphone cleaning/hygiene could significantly reduce the bacterial load. Taken together, our results demonstrate that smartphone surfaces not only are a reservoir of microbes but also provide an ecological niche in which microbiotas, particularly opportunistic pathogens, can survive, be active, and even grow. IMPORTANCE Currently, people spend an average of 4.2 h per day on their smartphones. Due to the COVID-19 pandemic, this figure may still be increasing. The high frequency of smartphone usage may allow microbes, particularly pathogens, to attach to-and even survive on-phone surfaces, potentially causing adverse effects on humans. We employed various culture-independent techniques in this study to evaluate the microbiological features and hygiene of smartphones, including community assembly, bacterial load, and activity. Our data showed that deterministic processes drive smartphone microbiota assembly and that approximately 1.75 × 106 bacteria exist on a single smartphone touchscreen, with activities being stable for at least 48 h. Fortunately, simple smartphone cleaning/hygiene could significantly reduce the bacterial load. This work expands our understanding of the microbial ecology of smartphone surfaces and might facilitate the development of electronic device cleaning/hygiene guidelines to support public health.

Deciphering environmental resistome and mobilome risks on the stone monument: A reservoir of antimicrobial resistance genes.

Antimicrobial resistance (AMR) in the environment has attracted increasing attention as an emerging global threat to public health. Stone is an essential ecosystem in nature and also an important material for human society, having architectural and aesthetic values. However, little is known about the AMR in stone ecosystems, particularly in the stone monument, where antimicrobials are often applied against biodeterioration. Here, we provide the first detailed metagenomic study of AMR genes across different types of biodeteriorated stone monuments, which revealed abundant and diverse AMR genes conferring resistance to drugs (antibiotics), biocides, and metals. Totally, 132 AMR subtypes belonging to 27 AMR types were detected including copper-, rifampin-, and aminocoumarins-resistance genes, of which diversity was mainly explained by the spatial turnover (replacement of genes between samples) rather than nestedness (loss of nested genes between samples). Source track analysis confirms that stone resistomes are likely driven by anthropogenic activities across stone heritage areas. We also detected various mobile genetic elements (namely mobilome, e.g., prophages, plasmids, and insertion sequences) that could accelerate replication and horizontal transfer of AMR genes. Host-tracking analysis further identified multiple biodeterioration-related bacterial genera such as Pseudonocardia, Sphingmonas, and Streptomyces as the major hosts of resistome. Taken together, these findings highlight that stone microbiota is one of the natural reservoirs of antimicrobial-resistant hazards, and the diverse resistome and mobilome carried by active biodeteriogens may improve their adaptation on stone and even deactivate the antimicrobials applied against biodeterioration. This enhanced knowledge may also provide novel and specific avenues for environmental management and stone heritage protection.

Dietary exposure of copper and zinc oxides nanoparticles affect the fitness, enzyme activity, and microbial community of the model insect, silkworm Bombyx mori.

Copper and Zinc oxides nanoparticles (CuO and ZnO NPs, respectively) are among the most produced and commonly used engineered nanomaterials. They can be released into the environment, thereby causing health concerns and risks to biodiversity that indicate a need to evaluate their toxicological effects in a complex situation. Here, we used the insect model organism silkworm Bombyx mori to address the concerns about the biological effects associated with dietary exposure of CuO and ZnO NPs. ICP-MS analysis revealed significant accumulation of Cu and Zn (the latter being more accumulated) in silkworms' tissues (gut, fat body, silk gland, and malpighian tubule), and some elimination through feces in the respective NPs-exposed groups. NPs-exposures led to a decrease in larval body mass, survivorship, and cocoon production, where the effects of ZnO NPs were more pronounced. We also found that NPs-exposure induced gene expression changes (Attacin, lysozyme, SOD, and Dronc) and altered the activities of antioxidant enzymes (SOD, GST, and CAT), as well as impaired nutrient metabolism (alpha-amylase). Given their antibacterial property, CuO and ZnO NPs decreased species richness and diversity of the gut bacterial community and shifted their configuration to overt microbiome i.e., decreased abundance of probiotics (e.g., Acetobacter) and increased pathobionts (e.g., Pseudomonas, Bacillus, Escherichia, Enterococcus, Ralstonia, etc.) proportions. Overall, this integrated study revealed the unintended negative effects of CuO and ZnO NPs on silkworms and highlighted the potential to inevitably affect all living things due to intensive and possible mishandling of nanomaterials.

From surviving to thriving, the assembly processes of microbial communities in stone biodeterioration: A case study of the West Lake UNESCO World Heritage area in China.

Serious concerns regarding stone biodeterioration have been raised due to the loss of aesthetic value and hidden dangers in stone cultural heritages and buildings. Stone biodeterioration involves a complex ecological interplay among organisms, however, the ecological mechanisms (deterministic or stochastic processes) that determine the microbial community on stone remain poorly understood. Here, using both amplicon and shotgun metagenomic sequencing approaches, we comprehensively investigated the biodiversity, assembly, and function of communities (including prokaryotes, fungi, microfauna, and plants) on various types of deteriorating limestone across different habitats in Feilaifeng. By generalizing classic ecological models to stone habitats, we further uncovered and quantified the mechanisms underlying microbial community assembly processes and microbial interactions within the biodeteriorated limestone. Community profiling revealed stable ecosystem functional potential despite high taxonomic variation across different biodeterioration types, suggesting non-random community assembly. Increased niche differentiation occurred in prokaryotes and fungi but not in microfauna and plant during biodeterioration. Certain microbial groups such as nitrifying archaea and bacteria showed wider niche breadth and likely contributing to the initiation, succession and expansion of stone biodeterioration. Consistently, prokaryotes were more strongly structured by selection-based deterministic processes, while micro-eukaryotes were more influenced by dispersal and drift-based stochastic processes. Importantly, microbial coexistence maintains network robustness within stone microbiotas, highlighting mutual cooperation among functional microorganisms. These results provide new insights into microbial community assembly mechanisms in stone ecosystems and may aid in the sustainable conservation of stone materials of interest.

The gut commensal bacterium Enterococcus faecalis LX10 contributes to defending against Nosema bombycis infection in Bombyx mori.

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.

Enterococcal symbionts of caterpillars facilitate the utilization of a suboptimal diet.

Bacterial gut symbionts of insect herbivores can impact their host through different mechanisms. However, in most lepidopteran systems we lack experimental examples to explain how specific members of the gut bacterial community influence their host. We used fall armyworm (Spodoptera frugiperda) as a model system to address this objective. We implemented axenic and gnotobiotic techniques using two semi-artificial diets with pinto bean and wheat germ-based components. Following an initial screen of bacterial isolates representing different genera, larvae inoculated with Enterococcus FAW 2-1 exhibited increased body mass on the pinto bean diet, but not on the wheat germ diet. We conducted a systematic bioassay screening of Enterococcus isolated from fall armyworm, revealing they had divergent effects on the hosts' usage pinto bean diet, even among phylogenetically similar isolates. Dilution of the pinto bean diet revealed that larvae performed better on less-concentrated diets, suggesting the presence of a potential toxin. Collectively, these results demonstrate that some gut microorganisms of lepidopterans can benefit the host, but the dietary context is key towards understanding the direction of the response and magnitude of the effect. We provide evidence that gut microorganisms may play a wider role in mediating feeding breadth in lepidopteran pests, but overall impacts could be related to the environmental stress and the metabolic potentials of the microorganisms inhabiting the gut.

Contribution of sample processing to gut microbiome analysis in the model Lepidoptera, silkworm Bombyx mori.

Microbes that live inside insects play various roles in host biology, ranging from nutrient supplementation to host defense. Although Lepidoptera (butterflies and moths) are one of the most diverse insect taxa and important in natural ecosystems, their microbiotas are little-studied, and to understand their structure and function, it is necessary to identify potential factors that affect microbiome analysis. Using a model organism, the silkworm Bombyx mori, we investigated the effects of different sample types (whole gut, gut content, gut tissue, starvation, or frass) and metagenomic DNA extraction methodologies (small-scale versus large-scale) on the composition and diversity of the caterpillar gut microbial communities. High-throughput 16S rRNA gene sequencing and computational analysis of the resulting data unraveled that DNA extraction has a large effect on the outcome of metagenomic analysis: significant biases were observed in estimates of community diversity and in the ratio between Gram-positive and Gram-negative bacteria. Furthermore, bacterial communities differed significantly among sample types. The gut content and whole gut samples differed least, both had a higher percentage of Enterococcus and Acinetobacter species; whereas the frass and starvation samples differed substantially from the whole gut and were poor representatives of the gut microbiome. Thus, we recommend a small-scale DNA extraction methodology for sampling the whole gut under normal insect rearing conditions whenever possible, as this approach provides the most accurate assessment of the gut microbiome. Our study highlights that evaluation of the optimal sample-processing approach should be the first step taken to confidently assess the contributions of microbiota to Lepidoptera.