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.

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.

SWATH-based quantitative proteomics reveals the mechanism of enhanced Bombyx mori nucleopolyhedrovirus-resistance in silkworm reared on UV-B treated mulberry leaves.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most acute infectious diseases in silkworm, which has led to great economic loss in sericulture. Previous study showed that the content of secondary metabolites in mulberry leaves, particularly for moracin N, was increased after UV-B irradiation. In this study, the BmNPV resistance of silkworms reared on UV-B treated and moracin N spread mulberry leaves was improved. To uncover the mechanism of enhanced BmNPV resistance, silkworm midguts from UV-B treated mulberry leaves (BUM) and moracin N (BNM) groups were analyzed by SWATH-based proteomic technique. Of note, the abundance of ribosomal proteins in BUM and BNM groups was significantly changed to maintain the synthesis of total protein levels and cell survival. While, cytochrome c oxidase subunit II, calcium ATPase and programmed cell death 4 involved in apoptotic process were up-regulated in BNM group. Expressions of lipase-1, serine protease precursor, Rab1 protein, and histone genes were increased significantly in BNM group. These results suggest that moracin N might be the main active component in UV-B treated mulberry leaves which could improve the BmNPV-resistance of silkworm through promoting apoptotic cell death, enhancing the organism immunity, and regulating the intercellular environment of cells in silkworm. It also presents an innovative process to reduce the mortality rate of silkworms infected with BmNPV.

Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector.

OBJECTIVE: To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein. RESULTS: The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE. CONCLUSIONS: The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.

Transcriptome sequencing and characterization of ungerminated and germinated spores of Nosema bombycis.

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori. Germination, an indispensible process through which microsporidia infect the host cells, is regarded as a key developmental turning point for microsporidia from dormant state to reproduction state. Thus, elucidating the transcriptome changes before and after germination is crucial for parasite control. However, the molecular basis of germination of microsporidia remains unknown. To investigate this germination process, the transcriptome of N. bombycis ungerminated spores and germinated spores were sequenced and analyzed. More than 60 million high-quality transcript reads were generated from these two groups using RNA-Seq technology. After assembly, 2756 and 2690 unigenes were identified, respectively, and subsequently annotated based on known proteins. After analysis of differentially expressed genes, 66 genes were identified to be differentially expressed (P ≤ 0.05) between these two groups. A protein phosphatase-associated gene was first identified to be significantly up-regulated as determined by RNA-Seq and immunoblot analysis, indicating that dephosphorylation might potentially contribute to microsporidia germination. The DEGs that encode proteins involved in glycometabolism, spore wall proteins and ricin B lectin of N. bombycis were also analyzed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed genes responsible for some specific biological functions and processes. The datasets generated in this study provide a basic characterization of the transcriptome changes in N. bombycis during germination. The analysis of transcriptome data and identification of certain functional genes which are robust candidate genes related to germination will help to provide a deep understanding of spore germination and invasion.

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.

Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5.

UNLABELLED: Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-ß) in DF-1 cells pretreated with IFN-α/ß. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-ß expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-ß expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. IMPORTANCE: MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-ß did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-ß expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.

Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous Colorado strain and the heterologous Minnesota strain.

UNLABELLED: Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2'-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2'-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. IMPORTANCE: Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV), human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus, and highly lethal emerging pathogens such as Nipah virus and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategies for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2'-O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacking 2'-O MTase were not only highly attenuated in turkeys but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other animal and human paramyxoviruses for rationally designing live attenuated vaccines.

Proteomic analysis of silkworm midgut cellular proteins interacting with the 5' end of infectious flacherie virus genomic RNA.

The flacherie disease in the silkworm is caused by the infectious flacherie virus (IFV). IFV relies on its 5' region of genomic RNA to recruit host-related factors to implement viral translation and replication. To identify host proteins bound to the 5'-region of IFV RNA and identify proteins important for its function, mass spectrometry was used to identify proteins from silkworm midgut extracts that were obtained using RNA aptamer-labeled 5' region of IFV RNA. We found 325 protein groups (unique peptide≥2) bound to the 5' region of IFV RNA including translation-related factors (16 ribosomal subunits, 3 eukaryotic initiation factor subunits, 1 elongation factor subunit and 6 potential internal ribosome entry site trans-acting factors), cytoskeleton-related proteins, membrane-related proteins, metabolism enzymes, and other proteins. These results can be used to study the translation and replication related factors of IFV interacting with host silkworm and to control flacherie disease in silkworm.

Nosema bombycis (Microsporidia) suppresses apoptosis in BmN cells (Bombyx mori).

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.

Proteomic analysis of BmN cells (Bombyx mori) in response to infection with Nosema bombycis.

Nosema bombycis (N. bombycis, Nb) is an obligate intracellular parasite, which can cause pebrine disease in the silkworm. To investigate the effects of N. bombycis infection on the host cells, proteomes from BmN cells that had or had not been infected with N. bombycis at different infection stages were characterized with two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry, which identified 24 differentially expressed host proteins with significant intensity differences (P < 0.05) at least at one time point in mock- and N. bombycis infected cells. Notably, gene ontology analyses showed that these proteins are involved in many important biological reactions. During the infection phase, proteins involved in energy metabolism and oxidative stress had up-regulated expression. Two proteins participated in ubiquitin-dependent protein catabolic process had down-regulated expression. Quantitative real-time polymerase chain reaction was used to analyze the transcriptional profiles of these identified proteins. Taken together, the abundance changes, putative functions, and participation in biological reactions for the identified proteins produce a host-responsive protein model in N. bombycis-infected BmN cells. These findings further our knowledge about the effect of energy defect parasites on the host cells.

Detection of an internal translation activity in the 5' region of Bombyx mori infectious flacherie virus.

The 5' untranslated region plays an important role in positive-sense single-stranded RNA virus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins. Infectious flacherie virus (IFV) is a positive-sense single-stranded RNA virus; however, the IRES function is still not proved. To investigate whether the sequences of IFV contain IRES activity, a series of bicistronic reporter (DsRed and enhanced green fluorescent protein) recombinant baculoviruses were constructed to infect the insect cells and silkworm using the Bombyx mori baculovirus expression system. Results showed that the upstream 311, 323, 383, 551, and 599 nt have IRES activity except for the 155-nt region in BmN cells. More importantly, the tetraloop structure containing region between 551 and 599 nt appeared to be responsible for the enhanced IRES activity in different insect cell lines and silkworm. These results indicated that the IRES activity is not species specific and tissue specific. Therefore, our findings may provide the basis for the simultaneous expression of two or various different genes under the same promoter in baculovirus expression system.

Diversity and Functional Roles of the Gut Microbiota in Lepidopteran Insects.

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.

Features and Colonization Strategies of Enterococcus faecalis in the Gut of Bombyx mori.

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.

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.

Identification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis.

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.

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.

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.

Gene analysis of an antiviral protein SP-2 from Chinese wild silkworm, Bombyx mandarina Moore and its bioactivity assay.

The cDNA encoding an antiviral protein SP-2 against BmNPV was cloned from the midgut of Chinese wild silkworm, Bombyx mandarina Moore (GenBank access AY945210) based on the available information of the domesticated silkworm. Its cDNA was 855 bp encoding 284 amino acids with predicted molecular weight of 29.6 kDa. Its full length in genomics was 1376 bp, including 5 exons and 4 introns. The expression analysis indicated that it was only expressed in midgut, and its expression level was higher during feeding stage of larval instars while very lower during the moltism and mature stages. The deduced amino acid sequence of this protein showed eight-amino-acid variation compared with the counterpart of domesticated silkworm. Its antiviral activity was assayed through in vitro test. The results indicated that it showed strong bioactivity against BmNPV, and its activity was 1.6 fold higher that the counterpart of domesticated silkworm.