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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Toxic effects of acute exposure to polystyrene microplastics and nanoplastics on the model insect, silkworm Bombyx mori.

Microplastics and nanoplastics (MPs and NPs, respectively) are major contaminants of environmental concern due to their potentially detrimental effects on aquatic and terrestrial ecosystems. However, little is known about their potential toxicity in terrestrial organisms. Here, we used the model insect silkworm (Bombyx mori) to evaluate the potential hazardous effects of acute exposure (72 h) to polystyrene (PS) MPs and NPs at physiological, molecular, and biochemical levels as well as their impact on pathogen infection. Our results revealed no significant changes in larval body mass or survival. Nevertheless, exposure led to significant alterations in the expression of immunity-related genes (Cecropin A, Lysozyme, SOD, and GST) and antioxidant-mediated protective response (SOD, GST, and CAT enzymes) which differed in the PS-MP and PS-NP groups. Interestingly, PS-MPs induced a stronger immune response (higher expressions of Lysozyme, SOD, and GST genes along with increased activities of SOD, GST, and CAT enzymes) while the PS-NP response was more that of an inhibitory nature (decreased SOD activity and expression). As a result, upon infection with the natural pathogen Serratia marcescens Bm1, the PS-MP-exposed individuals survived the infection better whereas, PS-NP-exposed individuals exhibited significantly higher mortality. Thus, we infer that PS-MPs/NPs present ecological toxicity, which is closely related to their size, and that their exposure may render the organisms vulnerable or confer resistance to pathogen infections and ecotoxicants. Given the suitability of silkworm as a model organism, this study may promote its application for further investigation of the mechanism of adverse outcome pathways and in studies on bio-nano interactions.

Gut bacteria of the silkworm Bombyx mori facilitate host resistance against the toxic effects of organophosphate insecticides.

Organophosphate insecticides that are heavily used in agriculture for pest control have caused growing environmental problems and public health concerns worldwide. Ironically, insecticide resistance develops quickly in major lepidopteran pests, partially via their microbial symbionts. To investigate the possible mechanisms by which the microbiota confers insecticide resistance to Lepidoptera, the model organism silkworm Bombyx mori (Lepidoptera: Bombycidae) was fed different antibiotics to induce gut dysbiosis (microbiota imbalance). Larvae treated with polymyxin showed a significantly lower survival rate when exposed to chlorpyrifos. Through high-throughput sequencing, we found that the abundances of Stenotrophomonas and Enterococcus spp. changed substantially after treatment. To assess the roles played by these two groups of bacteria in chlorpyrifos resistance, a germ-free (GF) silkworm rearing protocol was established to avoid the influence of natural microbiota and antibiotics. Monoassociation of GF silkworms with Stenotrophomonas enhanced host resistance to chlorpyrifos, but not in Enterococcus-fed larvae, consistent with larval detoxification activity. GC-µECD detection of chlorpyrifos residues in feces indicated that neither Stenotrophomonas nor Enterococcus degraded chlorpyrifos directly in the gut. However, gut metabolomics analysis revealed a highly species-specific pattern, with higher levels of essential amino acid produced in the gut of silkworm larvae monoassociated with Stenotrophomonas. This critical nutrient provisioning significantly increased host fitness and thereby allowed larvae to circumvent the deleterious effects of these toxic chemicals more efficiently. Altogether, our study not only suggests a new mechanism for insecticide resistance in notorious lepidopteran pests but also provides a useful template for investigating the interplay between host and gut bacteria in complex environmental systems.

Gut microbiota metabolic potential correlates with body size between mulberry-feeding lepidopteran pest species.

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.

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.

Biocontrol Potential of a Novel Endophytic Bacterium From Mulberry (Morus) Tree.

Mulberry (Morus) is an economically important woody tree that is suitable for use in sericulture as forage and in medicine. However, this broad-leaved tree is facing multiple threats ranging from phytopathogens to insect pests. Here, a Gram-positive, endospore-forming bacterium (ZJU1) was frequently isolated from healthy mulberry plants by screening for foliar endophytes showing antagonism against pathogens and pests. Whole-genome sequencing and annotation resulted in a genome size of 4.06 Mb and classified the bacterium as a novel strain of Bacillus amyloliquefaciens that has rarely been identified from tree leaves. An integrative approach combining traditional natural product chemistry, activity bioassays, and high-resolution mass spectrometry confirmed that strain ZJU1 uses a blend of antimicrobials including peptides and volatile organic compounds to oppose Botrytis cinerea, a major phytopathogenic fungus causing mulberry gray mold disease. We showed that the inoculation of endophyte-free plants with ZJU1 significantly decreased both leaf necrosis and mortality under field conditions. In addition to the direct interactions of endophytes with foliar pathogens, in planta studies suggested that the inoculation of endophytes also induced plant systemic defense, including high expression levels of mulberry disease resistance genes. Moreover, when applied to the generalist herbivore Spodoptera litura, ZJU1 was sufficient to reduce the pest survival rate below 50%. A previously undiscovered crystal toxin (Cry10Aa) could contribute to this insecticidal effect against notorious lepidopteran pests. These unique traits clearly demonstrate that B. amyloliquefaciens ZJU1 is promising for the development of successful strategies for biocontrol applications. The search for new plant-beneficial microbes and engineering microbiomes is therefore of great significance for sustainably improving plant performance.