Enterobacter Strain IPPBiotE33 Displays a Synergistic Effect with Bacillus thuringiensis Bt185.

The discovery and isolation of new non-Bt insecticidal bacteria and genes are significant for the development of new biopesticides against coleopteran pests. In this study, we evaluated the insecticidal activity of non-Bt insecticidal bacteria, PPBiotE33, IPPBiotC41, IPPBiotA42 and IPPBiotC43, isolated from the peanut rhizosphere. All these strains showed insecticidal activity against first- and third-instar larvae of Holotrichia parallela, Holotrichia oblita, Anomala corpulenta and Potosia brevitarsis. IPPBiotE33 showed the highest toxicity among the four strains and exhibited virulence against Colaphellus bowringi. The genome of IPPBiotE33 was sequenced, and a new protein, 03673, with growth inhibition effects on C. bowringi was obtained. In addition, IPPBiotE33 had a synergistic effect with Bacillus thuringiensis Bt185 against H. parallela in bioassays and back-inoculation experiments with peanut seedlings. IPPBiotE33 induced a decrease in hemocytes and an increase in phenol oxidase activity in H. parallela hemolymph, known as the immunosuppressive effect, which mediated synergistic activity with Bt185. This study increased our knowledge of the new insecticidal strain IPPBiotE33 and shed new light on the research on new insecticidal coaction mechanisms and new blended pesticides.

The Influence of Environmental Values on Consumer Intentions to Participate in Agritourism-A Model to Extend TPB.

This study examines the influence of environmental values on consumer intentions to participate in agritourism through the theory of planned behaviour (TPB) and value-belief-norm (VBN) theory. It proposes an integrative model by adding two variables, i.e., environmental benefits and the human-nature coordination concept, to the TPB. The study employs a questionnaire survey method and a sample of 640, which was statistically analysed through structural equation modeling (SEM). The results reveal that the "environmental values-attitudes-behavioural intentions" framework has scientific applicability in agritourism. Environmental values, measured through the variables environmental benefits and the human-nature coordination concept, are positively correlated directly or indirectly with agritourism consumption intentions, while attitudes and subjective norms serve as mediators. However, the mediating effect of perceived behavioural control is not statistically significant, indicating making efforts to influence attitudes and subjective norms is more useful for and effective in stimulating the public's intentions towards agritourism. As this study tests the hypotheses with empirical data, it provides practical implications for policy-makers and programme managers.

The effect of Bt Cry9Ee toxin on honey bee brood and adults reared in vitro, Apis mellifera (Hymenoptera: Apidae).

The effects of Bt Cry9Ee toxin on honey bee, Apis mellifera L., survival, developmental rate, larval weight, pollen consumption, and midgut bacterial diversity were tested in the laboratory. Honey bee larvae and adults were reared in vitro and fed a diet that contained Cry9Ee toxin at 0.01, 0.1, 1, and 10 mg/L. Cry9Ee toxin 0.01, 0.1, and 1 mg/L in diet used in this study may represent a value closer to field relevance and the highest concentration is unlikely to be encountered in the field and thus represent a worst case scenario. The dependent variables were compared for groups of honey bees feeding on treated diet and those feeding on negative control (no addition of a test substance), solvent control (0.01 mM Na2CO3), and positive control diet (dimethoate 45 mg/L). Bt Cry9Ee toxin did not affect survival or larval weight, and the result was great confidence in accepting the null hypothesis by power analysis. The effect on development rates and pollen consumption were the inconclusive results because the post-hoc power was less than 0.8. Furthermore, the midgut bacterial structure and compositions were determined using high-throughput sequencing targeting the V3-V4 regions of the 16S rDNA. All core honey bee intestinal bacterial class such as γ-Proteobacteria, Actinobacteria, α-Proteobacteria, Bacilli, ß-Proteobacteria, and Bacteroidia were detected, and no significant changes were found in the species diversity and richness between Cry9Ee treatments and laboratory control.

Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis.

Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.

Mining tissue-specific contigs from peanut (Arachis hypogaea L.) for promoter cloning by deep transcriptome sequencing.

Peanut (Arachis hypogaea L.), one of the most important oil legumes in the world, is heavily damaged by white grubs. Tissue-specific promoters are needed to incorporate insect resistance genes into peanut by genetic transformation to control the subterranean pests. Transcriptome sequencing is the most effective way to analyze differential gene expression in this non-model species and contribute to promoter cloning. The transcriptomes of the roots, seeds and leaves of peanut were sequenced using Illumina technology. A simple digital expression profile was established based on number of transcripts per million clean tags (TPM) from different tissues. Subsequently, 584 root-specific candidate transcript assembly contigs (TACs) and 316 seed-specific candidate TACs were identified. Among these candidate TACs, 55.3% were root-specific and 64.6% were seed-specific by semi-quantitative RT-PCR analysis. Moreover, the consistency of semi-quantitative RT-PCR with the simple digital expression profile was correlated with the length and TPM value of TACs. The results of gene ontology showed that some root-specific TACs are involved in stress resistance and respond to auxin stimulus, whereas, seed-specific candidate TACs are involved in embryo development, lipid storage and long-chain fatty acid biosynthesis. One root-specific promoter was cloned and characterized. We developed a high-yield screening system in peanut by establishing a simple digital expression profile based on Illumina sequencing. The feasible and rapid method presented by this study can be used for other non-model crops to explore tissue-specific or spatially specific promoters.

In Vivo and In Vitro Interactions between Exopolysaccharides from Bacillus thuringensis HD270 and Vip3Aa11 Protein.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

Characterization and evaluation of actinomycete from the Protaetia brevitarsis Larva Frass.

Protaetia brevitarsis larvae (PBL) are soil insects important for the soil organic carbon cycle, and PBL frass not only contains a large amount of humic acid but also affects the diversity, novelty, and potential functions of actinomycetes. Here, we characterized and assessed the actinomycete. The operational taxonomic unit (OTU) data showed that 90% of the actinomycetes cannot be annotated to species, and pure culture and genome analysis showed that 35% of the strains had the potential to be new species, indicating the novelty of PBL frass actinomycetes. Additionally, genome annotation showed that many gene clusters related to antifungal, antibacterial and insecticidal compound synthesis were identified, and confrontation culture confirmed the antifungal activities of the actinomycetes against soil-borne plant pathogenic fungi. The incubation experiment results showed that all isolates were able to thrive on media composed of straw powder and alkaline lignin. These results indicated that PBL hindgut-enriched actinomycetes could survive in soil by using the residual lignocellulose organic matter from plant residues, and the antibiotics produced not only give them a competitive advantage among soil microflora but also have a certain inhibitory effect on plant diseases and pests. This study suggests that the application of PBL frass can not only supplement soil humic acid but also potentially affect the soil microbiota of cultivated land, which is beneficial for the healthy growth of crops.

Co-production of 1,3-propanediol and phage phiKpS2 from the glycerol fermentation by Klebsiella pneumoniae.

As an alternative to antibiotics in response to antimicrobial-resistant infections, bacteriophages (phages) are garnering renewed interest in recent years. However, the massive preparation of phage is restricted using traditional pathogens as host cells, which incurs additional costs and contamination. In this study, an opportunistic pathogen, Klebsiella pneumoniae used to convert glycerol to 1,3-propanediol (1,3-PDO), was reused to prepare phage after fermentation. The phage infection showed that the fed-batch fermentation broth containing 71.6 g/L 1,3-PDO can be directly used for preparation of phage with a titer of 1 × 108 pfu/mL. Then, the two-step salting-out extraction was adopted to remove most impurities, e.g. acetic acid (93.5%), ethanol (91.5%) and cells (99.4%) at the first step, and obtain 1,3-PDO (56.6%) in the top phase as well as phage (97.4%) in the middle phase at the second step. This integrated process provides a cheap and environment-friendly manner for coproduction of 1,3-PDO and phage.

Identification of a biomarker for Bacillus thuringiensis strains with high toxicity against Spodoptera frugiperda based on insecticidal gene linkage analysis.

BACKGROUND: Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces various insecticidal proteins used to control insect pests. Spodoptera frugiperda is a global insect pest which causes serious damage to crops, but bio-insecticides currently available to control this pest have limited activity and so new ones are always being sought. In this study we have tested the hypothesis that a biomarker for strain toxicity could be found that would greatly facilitate the identification of new potential products. RESULTS: Using genomic sequencing data we constructed a linkage network of insecticidal genes from 1957 Bt genomes and found that four gene families, namely cry1A, cry1I, cry2A and vip3A, showed strong linkage. For 95 strains isolated from soil samples we assayed them for toxicity towards S. frugiperda and for the presence of the above gene families. All of the strains that showed high toxicity also contained a member of the vip3A gene family. Two of them were more toxic than a commercially available strain and genomic sequencing identified a number of potentially novel toxin-encoding genes. CONCLUSIONS: The presence of a vip3A gene in the genome of a Bt strain proved to be a strong indicator of toxicity towards S. frugiperda validating this biomarker approach as a strategy for future discovery programs. © 2024 Society of Chemical Industry.

Plants recruit insecticidal bacteria to defend against herbivore attacks.

Pest feeding affects the rhizobacteria community. The rhizomicrobiota activates salicylic acid and jasmonic acid signaling pathways to help plants deal with pest infestation. However, whether plants can recruit special pesticidal microorganisms to deal with attack from herbivores is unclear. A system composed of peanuts and first-instar larvae of Holotrichia parallela were used to analyze whether peanuts truly enrich the insecticidal bacteria after feeding by larvae, and whether inoculation of the enriched bacteria promotes the resistance of plants to herbivore. In this study, high-throughput sequencing of 16 S rRNA gene amplicons was used to demonstrate that infestation of the subterranean pest H. parallela quickly changed the rhizosphere bacterial community structure within 24 h, and the abundance of Enterobacteriaceae, especially Enterobacter, was manifestly enriched. Root feeding induced rhizobacteria to form a more complex co-occurrence network than the control. Rhizosphere bacteria were isolated, and 4 isolates with high toxicity against H. parallela larvae were obtained by random forest analysis. In a back-inoculation experiment using a split-root system, green fluorescent protein (GFP)-labeled Enterobacter sp. IPPBiotE33 was observed to be enriched in uneaten peanut roots. Additionally, supplementation with IPPBiotE33 alleviated the adverse effects of H. parallela on peanuts. Our findings indicated that herbivore infestation could induce plants to assemble bacteria with specific larvicidal activity to address threats.

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela.

KEY MESSAGE: Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times. White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.

Identification and functional characterization of eight novel tpp family genes from Bacillus thuringiensis.

BACKGROUND: Bacillus thuringiensis (Bt) and its transgenic crops are widely used as biological control agents for agricultural pests. The tpp family is a branch of Bt insecticidal genes and consists of a few members. Research on the Tpp family proteins has focused on the binary toxins Gpp34Ab/Tpp35Ab and Tpp1/Tpp2, which need to function together to achieve insecticidal activity. However, only a few tpp family genes have been reported to exert insecticidal activity independently. This study aimed to identify and characterize tpp family genes that independently perform insecticidal functions. RESULTS: A total of 162 nucleotide sequences homologous to the single component Bt insecticidal gene tpp78Aa were obtained from the genome data of 1368 wild-type Bt strains, and 25 new full-length tpp family genes were identified. Eight new tpp family genes were successfully cloned and expressed, and bioassays of the expressed products were performed against five different pests. Bioassay results showed that these proteins exerted high insecticidal activity only against Laodelphax striatellus, a globally important rice pest, and were named Tpp78Ab1, Tpp78Bb1, Tpp78Ca1, Tpp78Da1, Tpp80Aa3, Tpp80Ac1, Tpp80Ad1, and Tpp80Ae1. The LC50 values of Tpp78Ab1, Tpp78Bb1, Tpp78Ca1, and Tpp80Ae1 against L. striatum were 8.1, 8.6, 10.1, and 9.6 µg mL-1 , respectively. The phylogenetic tree and conserved motifs indicated that the Tpp family had a common evolutionary ancestor. During evolution, the C-terminal pore-forming domain of the Tpp family adopted a similar arrangement; however, the N-terminal conserved motif showed high variability. CONCLUSION: Twenty-five full-length tpp family genes were identified. Eight new tpp family genes were cloned successfully, which could independently achieve insecticidal activity against L. striatellus. This provides abundant genetic resources for the biological control of important rice pests. In this study, we found that the relative conservation of the Tpp family proteins in the lengthy evolutionary process and the diversity generated for adapting to the environment can lay a theoretical foundation for an in-depth analysis of the function and evolution of the Tpp family. © 2023 Society of Chemical Industry.

Analysis of Synergism between Extracellular Polysaccharide from Bacillus thuringensis subsp. kurstaki HD270 and Insecticidal Proteins.

Bacillus thuringiensis (Bt) is the most widely used biopesticide worldwide and can produce several insecticidal crystal proteins and vegetative insecticidal proteins (Vips) at different growth stages. In our previous study, extracellular polysaccharides (EPSs) of Bt strain HD270 were found to enhance the insecticidal activity of Cry1Ac protoxin against Plutella xylostella (L.) and promote the binding of Cry1Ac to the intestinal brush border membrane vesicles (BBMVs). Whether the synergistic activity of Bt EPSs is common to other Cry1-type or Vip proteins is unclear, as is the potential synergistic mechanism. In this study, crude EPS-HD270 was found to increase the toxicity of Cry1-type toxins and Vip3Aa11 against different lepidopteran pests by approximately 2-fold. The purified EPS-HD270 also possessed synergistic activity against the toxicity of Cry1Ac and Vip3Aa11 against Spodoptera frugiperda (J.E. Smith) and Helicoverpa armigera (Hübner). Furthermore, we found that EPS-HD270 had a strong binding ability with Vip3Aa11 and promoted the binding of Vip3Aa11 to the BBMVs of H. armigera and S. frugiperda. Bt EPS-HD270 also protected Vip3Aa11 from proteolytic processing in larval midgut juice. Bt EPSs had universal synergistic effects on Cry1-type or Vip toxins against S. frugiperda and H. armigera. Bt EPS-HD270 exhibited synergistic activity with Vip3Aa through promotion of binding to BBMVs and protection from digestion by midgut protease. The results indicated that synergistic activity with Bt toxins was an important function of Bt EPSs, which was very different from other Bacillus spp.

Greater wax moth control in apiaries can be improved by combining Bacillus thuringiensis and entrapments.

The greater wax moth (GWM), Galleria mellonella (Lepidoptera: Pyralidae), is a major bee pest that causes significant damage to beehives and results in economic losses. Bacillus thuringiensis (Bt) appears as a potential sustainable solution to control this pest. Here, we develop a novel Bt strain (designated BiotGm) that exhibits insecticidal activity against GWM larvae with a LC50 value lower than 2 µg/g, and low toxicity levels to honey bee with a LC50 = 20598.78 µg/mL for larvae and no observed adverse effect concentration = 100 µg/mL for adults. We design an entrapment method consisting of a lure for GWM larvae, BiotGm, and a trapping device that prevents bees from contacting the lure. We find that this method reduces the population of GWM larvae in both laboratory and field trials. Overall, these results provide a promising direction for the application of Bt-based biological control of GWM in beehives, although further optimization remain necessary.

CEA cell adhesion molecule 5 enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment.

Hematopoietic stem cell (HSC) surface markers improve the understanding of cell identity and function. Here, we report that human HSCs can be distinguished by their expression of the CEA Cell Adhesion Molecule 5 (CEACAM5, CD66e), which serves as a marker and a regulator of HSC function. CD66e+ cells exhibited a 5.5-fold enrichment for functional long term HSCs compared to CD66e- cells. CD66e+CD34+CD90+CD45RA- cells displayed robust multi-lineage repopulation and serial reconstitution ability in immunodeficient mice compared to CD66e-CD34+CD90+CD45RA-cells. CD66e expression also identified almost all repopulating HSCs within the CD34+CD90+CD45RA- population. Together, these results indicated that CEACAM5 is a marker that enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment.

Dual color multiplex TTF-1 + Napsin A and p63 + CK5 immunostaining for subcategorizing of poorly differentiated pulmonary non-small carcinomas into adenocarcinoma and squamous cell carcinoma in fine needle aspiration specimens.

BACKGROUND: The distinction of lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) has important therapeutic implications. Napsin A is a recently developed marker, which has shown high specificity for lung tissue in the surgical pathology specimens. In this study, we have evaluated whether the use of a panel of novel multiplex cocktails of TTF-1 + Napsin A and p63 + CK5 for dual color immunostaining will improve the diagnostic accuracy of lung adenocarcinoma and squamous cell carcinoma in fine needle aspiration (FNA) specimens, usually with relatively scant microfragments of diagnostic material. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded, adequately cellular FNA cell blocks with a confirmed diagnosis of either ADC (n = 22), SCC (n = 20) or poorly differentiated carcinoma (PDC; n = 7), from a total of 49 consecutive cases, were studied. All these cases had subsequently confirmed diagnosis in biopsies or resection specimens. The sections were immunostained with two color methods of TTF-1 + Napsin A and p63 + CK5 multiplex cocktails. The presence of one or more unequivocal individual tumor cells with convincing brown nuclear TTF-1 and red cytoplasmic Napsin A staining, and cells with brown nuclear p63 and membranous / cytoplasmic CK5 staining were interpreted as 'positive'. RESULTS: All 20 FNA cell blocks from SCC cases were positive for dual stain p63 + CK5 and negative for dual stain TTF-1 + Napsin A. The sensitivity and specificity of the dual immunoexpressions of p63 + CK5 for SCC of lung FNAs were both 100%. All 22 ADC cases were positive with dual stain of TTF-1 + Napsin A and negative for dual stain of p63 + CK5. On follow-up of the surgical pathology specimens, 22 cases were confirmed as ADC. The sensitivity of the dual immunoexpression of TTF-1 + Napsin A for ADC of lung FNAs was 100% and the specificity was also 100%. Of the seven PDC cases, five cases that were positive for dual stain p63 + CK5 and negative for dual stain TTF-1 + Napsin A could be categorized as SCC. Two of the seven (2 / 7) PDC cases were positive for dual stain TTF-1 + Napsin A and negative for dual stain p63 + CK5, consistent with ADC. CONCLUSIONS: Simultaneous coordinate or individual immunostaining for Napsin A / TTF-1 in ADC and p63 / CK5 in SCC demonstrated high sensitivity and specificity. The panel with multiplex Napsin A / TTF-1 and p63 / CK5 dual color immunostains could specifically subcategorize PDC into ADC and SCC in lung FNA specimens. Multiplex dual color Napsin A / TTF-1 and p63 / CK5 immunostaining is especially recommended for evaluation of FNA specimens with relatively scant cellularity.

The Bethesda System thyroid diagnostic categories in the African-American population in conjunction with surgical pathology follow-up.

BACKGROUND: It has been reported that African-Americans (AA) have a higher prevalence of overall malignancy compared to Caucasians, in the United States, yet the incidence of thyroid malignancy is half. The aim of this study is to assess the rate of malignant versus benign thyroid disease in AA from an urban-based hospital with an academic setting. Our study analyzed the AA population with respect to fine needle aspiration (FNA) of thyroid lesions, in correlation with final surgical pathology. This is the first study of its kind to our knowledge. DESIGN: We retrospectively reviewed thyroid FNA cytology between January 2005 and February 2011. Consecutive FNA specimens with corresponding follow-up surgical pathology were included. The patients were categorized as African- American (AA) and Non-African-American (NAA), which included Caucasians (C), Hispanics (H), and Others (O). The FNA results were classified using the latest edition of The Bethesda System for Reporting Thyroid Cytopathology (TBS-Thy) and the follow-up surgical pathology was used for the final categorization. RESULTS: We studied 258 cases: 144 AA (56%) and 114 NAA [43 C (17%), 3 H (1%), and 68 O (28%)]. The average age for AA was 51 years (range 20 - 88) and for NAA was 53 years (range 25 - 86). There were more females than males in the AA versus the NAA group (85 vs. 75%). The incidence of thyroid lesions in the FNA specimens was similar between these two populations. The distribution of benign versus malignant diagnosis on follow-up surgical pathology was examined across TBS-Thy class. CONCLUSION: Our data suggest that distribution of benign versus malignant lesions in the thyroid FNA of AA versus NAA, with follow-up surgical pathology, is comparable for TBS-Thy classes, non-diagnostic (I), benign (II), suspicious for malignancy (V), and malignant (VI) in AA versus NAA.

Lignocellulose degradation in Protaetia brevitarsis larvae digestive tract: refining on a tightly designed microbial fermentation production line.

BACKGROUND: The Scarabaeidae insect Protaetia brevitarsis (PB) has recently gained increasing research interest as a resource insect because its larvae can effectively convert decaying organic matter to plant growth-promoting frass with a high humic acid content and produce healthy, nutritional insect protein sources. Lignocellulose is the main component of PB larvae (PBL) feed, but PB genome annotation shows that PBL carbohydrate-active enzymes are not able to complete the lignocellulose degradation process. Thus, the mechanism by which PBL efficiently degrade lignocellulose is worthy of further study. RESULTS: Herein, we used combined host genomic and gut metagenomic datasets to investigate the lignocellulose degradation activity of PBL, and a comprehensive reference catalog of gut microbial genes and host gut transcriptomic genes was first established. We characterized a gene repertoire comprising highly abundant and diversified lignocellulose-degrading enzymes and demonstrated that there was unique teamwork between PBL and their gut bacterial microbiota for efficient lignocellulose degradation. PBL selectively enriched lignocellulose-degrading microbial species, mainly from Firmicutes and Bacteroidetes, which are capable of producing a broad array of cellulases and hemicellulases, thus playing a major role in lignocellulosic biomass degradation. In addition, most of the lignocellulose degradation-related module sequences in the PBL microbiome were novel. PBL provide organic functional complementarity for lignocellulose degradation via their evolved strong mouthparts, alkaline midgut, and mild stable hindgut microenvironment to facilitate lignocellulosic biomass grinding, dissolving, and symbiotic microbial fermentation, respectively. CONCLUSIONS: This work shows that PBL are a promising model to study lignocellulose degradation, which can provide highly abundant novel enzymes and relevant lignocellulose-degrading bacterial strains for biotechnological biomass conversion industries. The unique teamwork between PBL and their gut symbiotic bacterial microbiota for efficient lignocellulose degradation will expand the knowledge of holobionts and open a new beginning in the theory of holobionts. Video Abstract.

Vip3Aa domain IV and V mutants confer higher insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera.

BACKGROUND: The fall armyworm Spodoptera frugiperda and cotton bollworm Helicoverpa armigera are major insect pests of corn and cotton worldwide. Genetically engineered crops producing Vip3Aa, a potent endotoxin, from the bacterium Bacillus thuringiensis (Bt) are effective in controlling these two harmful pests. However, Vip3Aa efficacy is relatively weak compared to that of other Bt proteins such as Cry1A and Cry1F. This study sought to modify Vip3Aa for increased insecticidal activity and determine the cause of elevated activity. RESULTS: The two triple Vip3Aa mutants in domains IV and V (Vip3Aa-S543N/I544L/E627A and Vip3Aa-S543N/I544L/S686R) exhibited 7.3-fold and 2.8-fold increased toxicity against S. frugiperda, respectively, compared with the wild type while the toxicity of Vip3Aa-S543N/I544L/S686R was 3.2 times that of wild-type protein in H. armigera. The mutants had enhanced stability in midgut juice and 2.6-5.1 times higher binding affinity against S. frugiperda and H. armigera compared with wild type protein. CONCLUSIONS: The enhanced toxicity of Vip3Aa mutants was due to increased stability and binding affinity during infection. The amino acids S543 and I544 combined with E627 or S686 in domains IV and V of Vip3Aa are important for maintaining structural stability and receptor binding. The results match insecticidal activity (LC50 ) with binding activity (Kd ), which provides novel clues for the rational design of Bt insecticidal proteins. © 2022 Society of Chemical Industry.

One-step salting-out extraction of bacteriophage from its infection broth of Acinetobacter baumannii.

Phages as a potential alternative antibiotic for multidrug-resistant bacterial infections are receiving great attention worldwide. However, the traditional separation and purification of phage are cumbersome, time-consuming, costly and inefficient. In this study, phage phiAB9 for multidrug-resistant Acinetobacter baumannii was separated and purified by a simple and cost-saving one-step salting-out extraction (SOE). Several kinds of salts and organic solvents without effect on phage survival were chosen to form the SOE systems, and the composition of SOE systems were optimized according to the phage recovery rate and impurity removal rate. After one-step SOE by an optimal system composed of 18% (w/w) ammonium citrate and 40% (w/w) ethyl acetate, the recovery rate of phage in the middle phase could reach to 90.82%, and most proteins (99.57%), cells (97.98%) and endotoxin (84.08%) were well removed, with a concentration factor of 210 and the purification factors of phage to proteins, cells and endotoxin were 303.64, 133.55 and 5.36, respectively. Comparing with two-step SOE and traditional aqueous two-phase extraction, one-step SOE may be an available method for the separation and purification of phages.