Chitin Synthesis Research Articles

Transcriptomic and Metabolomic Analysis Reveals Multifaceted Impact of Gcn5 Knockdown in Drosophila Development

Background: General control nonderepressible 5 (Gcn5) is a lysine acetyltransferase (KAT) that is evolutionarily conserved across eukaryotes, with two homologs (Kat2a and Kat2b) identified in humans and one (Gcn5) in Drosophila. Gcn5 contains a P300/CBP-associated factor (PCAF) domain, a Gcn5-N-acetyltransferase (GNAT) domain, and a Bromodomain, allowing it to regulate gene expression through the acetylation of both histone and non-histone proteins. In Drosophila, Gcn5 is crucial for embryonic development, with maternal Gcn5 supporting early development. However, the functional mechanisms of Gcn5 after the depletion of maternal deposits remain unclear. Methods: Our study employed the Gal4/UAS-RNAi system to achieve whole-body or heart-specific Gcn5 knockdown in Drosophila and selected 96-hour-old surviving larvae for transcriptomic and metabolomic analyses. Results: Omics results revealed that Gcn5 knockdown significantly impacts various metabolic pathways, as well as lysosomes, non-homologous end-joining, Toll and Imd signaling pathways, and circadian rhythms, among others. Furthermore, defects in chitin synthesis may be associated with impaired pupation. Additionally, heart-specific Gcn5 knockdown affected cardiac physiology but appeared to have a potential protective effect against age-related cardiac decline. Conclusions: These findings deepen our understanding of Gcn5’s roles in Drosophila development and provide valuable insights for developing Gcn5-targeted therapies, particularly considering its involvement in various human diseases.

Insights into the antifungal and anti-aflatoxin B1 mechanisms of carvone on Aspergillus flavus

Aspergillus flavus (A. flavus) and the carcinogen it produces, aflatoxin B1 (AFB1), often contaminate essential fat-containing crops and cereals, posing a significant risk to food safety and human health, and resulting in substantial economic losses. The development of green, safe, and environmentally friendly chemical fumigants for controlling A. flavus and AFB1 contamination is urgently needed. The effects and mechanisms of the natural compound carvone on A. flavus growth and AFB1 biosynthesis were therefore investigated. Carvone efficiently inhibited A. flavus growth, spore formation, and AFB1 production. The minimum inhibitory concentration (MIC) was 0.8 μL/mL. RNA-seq analysis showed that carvone inhibited spore formation by suppressing the transcription of spore development-related genes, including fluG, flbA, flbD, brlA, abaA, con6, con10, rodA, rodB, and dit2. Physiological and biochemical parameters showed that carvone disrupted the integrity of the cell wall and membrane, induced reactive oxygen species (ROS) accumulation, caused DNA damage, triggered cell autophagy, reduced ATP levels, which ultimately led to cell death. RNA-seq analysis confirmed these results, which found that genes related to chitin and ergosterol synthesis, and DNA replication were downregulated, and genes involved in antioxidants and cell autophagy were upregulated. In addition, carvone downregulated several transcription factors (CreA, SrrA, NsdD, and Crz1) and 11 AFB1 cluster genes, which inhibited AFB1 production. Together, the results provided new insights into the antifungal and anti-AFB1 mechanisms of carvone, as well as providing theoretical support for the development of new fumigants to control A. flavus and AFB1 contamination.

Chlorbenzuron downregulated HcLCP-17 expression by depressing two 20E-responsive transcription factors Br-C and βFTZ-F1 in Hyphantria cunea (Lepidoptera: Erebidae) larvae.

Cuticular proteins (CPs) play essential roles in forming cuticular structures in insects. However, the specific functions and regulatory mechanisms of CPs remain largely unexplored. In this study, the Larval cuticular protein 17 (HcLCP-17) gene was identified from Hyphantria cunea, a highly destructive and polyphagous forest pest. To investigate the role of HcLCP-17 in cuticular function and transcriptional regulation mediated by 20E-responsive transcription factors (ERTFs), we employed RNA interference (RNAi) and yeast one-hybrid assay techniques. Additionally, we examined the molecular mechanism by which chlorbenzuron, a type of benzoylphenylurea (BPU) that functions as a chitin synthesis inhibitor (CSI), affects the 20E signaling pathway and ultimately regulates HcLCP-17 expression. HcLCP-17 encodes a polypeptide consisting of 393 amino acids, which includes a chitin-binding domain. Silencing HcLCP-17 resulted in a disturbance in the structural organization of the larval cuticle and a notable reduction in chitin levels. HcLCP-17 expression was controlled by the interaction between Broad-Complex (Br-C)and beta Fushi Tarazu Factor-1 (βFTZ-F1) with its promoter fragment. Furthermore, the inhibitory effect of chlorbenzuron on HcLCP-17 expression was found to be potentially mediated by Br-C and βFTZ-F1. The study presents a novel mode of action for the 20E signaling pathway in regulating the expression of CPs and reveals the potential mode-of-action of BPUs in insect cuticles. These findings provide a theoretical basis for future utilization of LCP-17 as a pesticide target making a significant contribution to the development of effective pest management strategies. © 2024 Society of Chemical Industry.

Enhancing Mycoprotein Yield: Metabolic Modulation of Chitin Synthase in Fusarium venenatum.

Fusarium venenatum is being extensively utilized for microbial protein production. However, its high dietary fiber content results in substantial carbon loss. Inhibition of chitin biosynthesis presents a promising strategy to improve the mycoprotein yield. Through transcriptomic and bioinformatics analyses, chitin synthase gene FvChs3 was identified as crucial for chitin synthesis in F. venenatum. Knockout of the FvChs3 gene resulted in mycelial expansion and a 26% reduction in the chitin content of strain ΔFvChs3. Ethanol production from fermentation decreased by 47%, while the carbon conversion efficiency and protein conversion increased by 16% and 36%, respectively. Transcriptomic analysis revealed an upregulation of nitrogen metabolism in ΔFvChs3, while genes related to the glycolysis pathway for ethanol synthesis were downregulated. Further knockout of pyruvate decarboxylase gene FvPDC6 in ΔFvChs3 accelerated growth, leading to improvements in carbon and protein conversion of 29% and 40%, respectively. This research lays the foundation for enhancing fungal protein production.

Challenges in baiting to manage fungus-growing termite colonies.

Fungus-growing termites (Termitidae: Macrotermitinae) exhibit significant diversity and abundance in tropical and subtropical ecosystems. Fungus-growing termites consume a wide range of materials, including leaf litter, woody debris, agricultural crops, and wooden structures, including houses. Their presence in termite baiting stations can impede the control of other residential termite pests, such as Coptotermes spp. Current control practices for managing fungus-growing termites are both costly and time-consuming, rendering them impractical for large-scale application. In our review, we analyzed recent biological and control studies on fungus-growing termites to identify key challenges and suggest improvements in termite management strategies. We identified 5 primary factors contributing to the low efficacy of current control measures for fungus-growing termites: (i) sporadic foraging behavior, which diminishes consistent bait intake; (ii) naturally small colony sizes; (iii) the nonmolting nature of the worker caste, reducing the effectiveness of chitin synthesis inhibitors; (iv) fungal cultivation, which delays and mitigates pesticide toxicity; and (v) wide-range of feeding niche, which lessens their consumption of cellulose-based baits. These insights are critical for developing more effective termite control strategies.

Investigation of the effectiveness and molecular mechanisms of thiamin priming to control early blight disease in potato.

In several plant species, thiamin foliar application primes plant immunity and can be effective in controlling various diseases. However, the effectiveness of thiamin against potato pathogens has seldom been investigated. Additionally, the transcriptomics and metabolomics of immune priming by thiamin have not previously been investigated. Here, we tested the effect of thiamin application against Alternaria solani, the causal agent of early blight in potato, and identified associated changes in gene expression and metabolite content. Thiamin applied on foliage at an optimal concentration of 10 mM reduced lesion size by ~33%. However, prevention of lesion growth was temporally limited, as a reduction of lesion size occurred when leaves were inoculated 4 h, but not 24 h, following thiamin treatment. Additionally, the effect of thiamin on lesion size was restricted to the application site and was not systemic. RNA-seq analysis showed that thiamin affected the expression of 308 genes involved in the synthesis of salicylic acid, secondary metabolites, fatty acid, chitin, and primary metabolism, and photosynthesis, which were also amongst the thousands of genes differentially regulated in the response to pathogen alone. Several of these genes and pathways were more differentially expressed and enriched when thiamin and the pathogen were combined. Thiamin also delayed the downregulation of photosynthesis-associated genes in plants inoculated with A. solani. Metabolite analyses revealed that thiamin treatment in the absence of pathogen decreased the amounts of several organic compounds involved in the citric acid cycle. We hypothesize that thiamin primes plant defenses through perturbation of primary metabolism.

Crystal structure and Hirshfeld-surface analysis of an etoxazole metabolite designated R13

The etoxazole metabolite R13, systematic name 4-(4-tert-butyl-2-ethoxyphenyl)-2-(2,6-difluorophenyl)oxazole (C21H21F2NO2), results from the oxidation of etoxazole, a chitin synthesis inhibitor belonging to the oxazoline class, widely used as an insecticide/acaricide since 1998. The structure of R13 features a central oxazole ring with attached 2,6-difluorophenyl and 4-t-butyl-2-ethoxyphenyl moieties. The overall conformation gives dihedral angles between these rings and the oxazole of 24.91 (5)° (with difluorophenyl) and 15.30 (6)° (with t-butyl-ethoxyphenyl), indicating an overall deviation from planarity. Additionally, torsion angles of the ethoxy and t-butyl groups define the orientation of these substituents relative to their benzene ring. In the crystal packing, no significant hydrogen bonds are present, but a Hirshfeld surface analysis highlights weak intermolecular contacts leading to π–π-stacked dimers linked by weak C—H...N contacts. The packing analysis confirms that most intermolecular interactions involve hydrogen atoms.

Physiological Biochemistry and Multi-omics Reveal the Underlying Mechanism of Citral in Controlling Colletotrichum scovillei in Postharvest Chili

Colletotrichum scovillei is the dominant pathogen in postharvest chili. Citral (CI) exhibits extensive inhibitory effects on pathogenic fungi, but the effect on C. scovillei is still unclear. This study suggested that CI inhibited C. scovillei mycelial growth with minimum inhibitory concentration (MIC) 0.30 mg/mL and spore germination at 44.79±1.40%. Physiological biochemistry studies demonstrated that CI destroyed spore cell wall and membrane integrity in a dose-dependent. Transcriptome and metabolome combined analysis suggested that MIC CI inhibited chitin synthesis by down-regulated the expression of five chitin synthetase genes (CHS1-1, CHS1-2, CHS1-3, CHS1-4, CHS1-5) and up-regulated the expression of four chitinase genes (CTS-1, CTS-2, CTS-3, CTS-5), and also inhibited ergosterol synthesis by down-regulated the expression of ERG1, ERG3, ERG24-1, ERG24-2, ERG24-3 genes and up-regulated the expression of ERG4, ERG6-1, ERG6-2, ERG25-1, ERG26, ERG27 genes, resulting in the increase of substrate UDP-GlcNAc content and the decrease of ergosterol content, respectively. Furthermore, CI fumigation induced resistance of chili against C. scovillei by increasing the activities of antioxidant enzymes (CAT, SOD, POD, APX), and the incidence of postharvest chili was significantly decreased by 30.61% and 32.14% in MIC and 2×MIC CI treatment groups, respectively. This study provided technical support for CI application as chili postharvest preservative.

Nickel phytoremediation potential of Plantago major L.: Transcriptome analysis

Plantago major L, a ubiquitous perennial weed thriving in diverse harsh environments, possesses a substantial reservoir of resistance genes, particularly those conferring resistance to heavy metals. Despite its prevalence, the intricate molecular mechanisms underpinning its exceptional ability to endure heavy metal pollution remain largely unexplored. Through transcriptome analysis, this study intended to reveal the mechanisms behind the enrichment of heavy metal Ni (nickel) in P. major and its potential for phytoremediation. In total, 7848 differentially expressed genes (DEGs) exhibited dynamic changes in tissues treated with different Ni concentrations. It was discovered that the root of P. major exhibited a more pronounced and significant response when exposed to higher concentrations of Ni. Furthermore, the upregulated genes associated with adversity stress were significantly observed in response to Ni stress. The majority of pathways related to plant growth and photosynthesis were significantly reduced; however, pathways related to metabolite synthesis, chitin synthesis, and adversity signal transduction were stimulated, and pathways related to root cell wall organization or biogenesis were suppressed. We identified that the rate-limiting enzyme PmHISN1A/B in the histidine synthesis pathway significantly enhanced Ni tolerance of the transgenic Arabidopsis without side effects, which was different to its Arabidopsis homologs. The study uncovered a molecular basis for the Ni tolerance of P. major, a heavy metal remediation plant, and provided potential genetic resources to cultivate novel P. major varieties or breed stress resilience crops.

Chitin Translocation Is Functionally Coupled with Synthesis in Chitin Synthase.

Chitin, an extracellular polysaccharide, is synthesized by membrane-embedded chitin synthase (CHS) utilizing intracellular substrates. The mechanism of the translocation of synthesized chitin across the membrane to extracellular locations remains unresolved. We prove that the chitin synthase from Phytophthora sojae (PsCHS) is a processive glycosyltransferase, which can rapidly produce and tightly bind with the highly polymerized chitin. We further demonstrate that PsCHS is a bifunctional enzyme, which is necessary and sufficient to translocate the synthesized chitin. PsCHS was purified and then reconstituted into proteoliposomes (PLs). The nascent chitin is generated and protected from chitinase degradation unless detergent solubilizes the PLs, showing that PsCHS translocates the newly produced chitin into the lumen of the PLs. We also attempted to resolve the PsCHS structure of the synthesized chitin-bound state, although it was not successful; the obtained high-resolution structure of the UDP/Mn2+-bound state could still assist in describing the characterization of the PsCHS's transmembrane channel. Consistently, we demonstrate that PsCHS is indispensable and capable of translocating chitin in a process that is tightly coupled to chitin synthesis.

Fgk3, a Glycogen Synthase Kinase, Regulates Chitin Synthesis through the Carbon Catabolite Repressor FgCreA in Fusarium graminearum.

The glycogen synthase kinase-3 (GSK3) orthologs are well-conserved in eukaryotic organisms. However, their functions remain poorly characterized in filamentous fungi. In our previous study, we unveiled the function of Fgk3, the GSK3 ortholog, in glycogen metabolism in Fusarium graminearum, the causal agent of Fusarium head blight. Interestingly, the fgk3 mutant was unstable and tended to produce fast-growing suppressors, including secondary suppressors. Using whole-genome sequencing, we identified suppressor mutations in FgCHS5, FgFKS1, FgCREA, FgSSN6, FgRGR1, and FgPP2A in nine primary and four secondary suppressors. Subsequently, we validated that deletion of FgCHS5 or FgCREAΔH253 mutation partially suppressed the defects of fgk3 in vegetative growth and cell wall integrity, suggesting that Fgk3 may regulate the chitin synthesis through FgCreA-mediated transcriptional regulation in F. graminearum. Accordingly, the FGK3 deletion led to hyphal swelling with abnormal chitin deposition, and deletion of FGK3 or FgCREA caused the upregulation of the expression of chitin synthases FgCHS5 and FgCHS6. The interaction between Fgk3 and FgCreA was verified by Yeast two-hybrid and Co-Immunoprecipitation assays. More importantly, we verified that the nuclear localization and protein stability of FgCreA relies on the Fgk3 kinase, while the H253 deletion facilitated the re-localization of FgCreA to the nucleus in the fgk3 mutant background, potentially contributing to the suppression of the fgk3 mutant's defects. Intriguingly, the ΔH253 mutation of FgCreA, identified in suppressor mutant S3, is adjacent to a conserved phosphorylation site, S254, suggesting that this mutation may inhibit the S254 phosphorylation and promote the nuclear localization of FgCreA. Collectively, our findings indicate that the glycogen synthase kinase Fgk3 regulates the chitin synthesis through the carbon catabolite repressor FgCreA in F. graminearum.

How do termite baits work? implication of subterranean termite colony demography on the successful implementation of baits.

In 1995, the launch of the first commercial chitin synthesis inhibitor (CSI) bait led to the transformation of the subterranean termite control industry around the world. Their slow mode of action, which relies on both their ability to be transferred among nestmates and termite molting biology, has made them cost-effective solutions for subterranean termite colony elimination while minimizing the introduction of pesticides into the soil toward an environmentally sustainable strategy. However, despite successful commercial implementations, the acceptance of their use varies within the pest control industry around the world. Notably, the nuanced complexity of how CSI baits lead to colony elimination upon feeding by termite foragers has, in part, remained elusive for the past 3 decades, allowing for long-lasting misconceptions to persist. A recent series of studies has since provided complementary elements of understanding how CSI baits utilize termites' inherent colony demography, behavior, and physiology to trigger colony elimination after a characteristic succession of events within the colony collapse process. I here provide a synthetic overview of subterranean termite colony characteristics when exposed to CSI baits using Coptotermes (Wasmann) (Blattodea: Heterotermitidae) as a primary model system. The changes in colony demography through the colony collapse reflect how the mode of action of CSI baits makes them a prime solution for sustainable subterranean termite pest management. Following decades of innovation, ongoing interactions among termite researchers, bait product manufacturers, and pest management providers must continue to bring solutions to existing and emerging termite pest problems around the world.

Characterization of CYP303A1 and its potential application based on ZIF-8 nanoparticle-wrapped dsRNA in Nilaparvata lugens (Stål).

RNA interference (RNAi) technology has been put forward as a promising method for pest control and resistance management. Mining highly efficient lethal genes and constructing stable double-stranded RNA (dsRNA) delivery systems are of great significance to improve the application potential of RNAi technology. In this study, we characterized a molting-related gene, NlCYP303A1, in Nilaparvata lugens that was highly expressed in the cuticle and at the end stages of each instar in nymphs. Silencing the expression of NlCYP303A1 in N. lugens resulted in a deformed phenotype and a significant increase in mortality. Furthermore, interfering with NlCYP303A1 changed the relative expression of key genes in the chitin synthesis and degradation pathway. Finally, we used the nanocarrier zeolitic imidazolate framework-8 (ZIF-8) to load dsNlCYP303A1, forming a complex denoted as dsNlCYP303A1@ZIF-8. The results of both feeding and rice-seedling dip experiments indicated that the expression of NlCYP303A1 was dramatically and persistently suppressed by the dsNlCYP303A1@ZIF-8 treatment, compared with treatment with dsNlCYP303A1, suggesting that ZIF-8 can enhance the interference efficiency as well as the stability of dsNlCYP303A1. Our results demonstrate that the lethal gene NlCYP303A1 can be employed as an excellent target for RNAi technology by loading onto a nano-delivery system, and provide new insights into the creation of innovative pest control approaches. © 2024 Society of Chemical Industry.

Putative APSES family transcription factor mbp1 plays an essential role in regulating cell wall synthesis in the agaricomycete Pleurotus ostreatus

The clade A APSES family transcription factors (Mbp1, Swi4, and Swi6) contribute to cell wall synthesis regulation in fungi. Herein, evolutionary relationships among these proteins were clarified by phylogenetic analysis using various ascomycetes and basidiomycetes, and then the detailed function of Mbp1 in cell wall synthesis regulation was analyzed in Pleurotus ostreatus. Our phylogenetic analysis revealed that Mbp1 and Swi6 are widely conserved among various fungi, whereas Swi4 is a protein specific for Saccharomycotina. In P. ostreatus, two putative clade A APSES family transcription factors, protein ID 83192 and 134090, were found and identified as Mbp1 and Swi6, respectively. The mbp1 gene was then disrupted through homologous recombination using P. ostreatus 20b strain (Δku80) as a host to obtain mbp1 disruption strains (Δmbp1). Disruption of mbp1 significantly decreased the growth rate and shortened aerial hyphae, suggesting that Mbp1 is involved in mycelial growth, especially aerial hyphal growth. Furthermore, thinner cell walls, decreased relative percentage of β-glucan, and downregulation of all β-glucan synthase genes were observed in Δmbp1 strains. Therefore, Mbp1 plays an essential role in β-glucan synthesis regulation in P. ostreatus. Disruption of mbp1 also impacted the expression profiles of chitin synthase genes, septum formation, and sensitivity to a chitin synthesis inhibitor, suggesting that Mbp1 also regulates chitin synthesis. In conclusion, Mbp1 is responsible for normal mycelial growth and regulates β-glucan and chitin synthesis in P. ostreatus. To the best of our knowledge, this is the first report on the detailed function of Mbp1 in cell wall synthesis regulation in fungi.

Role of chitin synthases CHS1 and CHS2 in biosynthesis of the cyst wall of Cryptocaryon irritans

Cryptocaryon irritans, a protozoan parasite that infects marine fish, is characterized by a complex life cycle that includes a cyst-forming reproductive phase. However, the composition of the cyst wall and mechanism of its formation remain unclear. In this study, we identified chitin as a key component of the cyst wall using calcofluor white and wheat germ agglutinin, with Fourier-transform infrared spectroscopy confirming its β-form structure. Two chitin synthase genes, CHS1 and CHS2, were identified as being expressed throughout the life cycle and show close phylogenetic relationships with chitin synthase from ciliates. Incubation with specific anti-CHS1 and -CHS2 antibodies significantly reduced both the thickness and chitin content of the cyst wall, highlighting the critical role of these enzymes in chitin biosynthesis. Treatment with benzoylureas, which inhibit chitin synthesis, caused thinning of the cyst wall and downregulation of CHS gene expression, resulting in an 84 % reduction in the hatching rate after treatment with 0.01 mM CuSO4 compared with control tomonts. Western blot analysis demonstrated that recombinant CHS proteins are immunogenic, and tomonts from CHS-immunized grouper exhibited reduced size. These findings bridge a crucial knowledge gap in understanding of the C. irritans cyst wall and highlight promising targets for infection prevention and control strategies.

Effects of Dietary Zinc Chloride and Zinc Sulfate on Life History Performance and Hemolymph Metabolism of Spodoptera litura (Lepidoptera: Noctuidae).

Zinc is an essential micronutrient crucial in various biological processes of an organism. However, the effects of zinc vary depending on its chemical form. Therefore, the aim of this study was to conduct a comparative analysis of the life history performances and hemolymph metabolism of Spodoptera litura exposed to different concentrations of dietary zinc chloride (ZnCl2) and zinc sulfate (ZnSO4), utilizing two-sex life tables and untargeted metabolomics. The preadult survival rate of S. litura significantly decreased, while the preadult developmental period of S. litura was prolonged as the dietary ZnCl2 concentration increased. However, the fecundity of S. litura at 50 mg/kg dietary ZnCl2 was significantly increased. The intrinsic rate of increase (r) and the finite rate of increase (λ) in S. litura in the control group (CK, no exogenous ZnCl2 or ZnSO4 added) and with 50 mg/kg dietary ZnCl2 were significantly higher than those at 100 mg/kg, 200 mg/kg, and 300 mg/kg. Dietary ZnSO4 exerts a devastating effect on the survival of S. litura. Even at the lowest concentration of 50 mg/kg dietary ZnSO4, only 1% of S. litura could complete the entire life cycle. Furthermore, as the dietary ZnSO4 concentration increased, the developmental stage achievable by the S. litura larvae declined. High-throughput untargeted metabolomics demonstrated that both 100 mg/kg dietary ZnCl2 and ZnSO4 decreased the hemolymph vitamins levels and increased the vitamin C content, thereby helping S. litura larvae to counteract the stress induced by ZnCl2 and ZnSO4. Simultaneously, dietary ZnCl2 obstructed the chitin synthesis pathway in the hemolymph of S. litura, thus extending the developmental period of S. litura larvae. These results indicate that low concentrations of Zn2+ positively impact populations of S. litura, but the effectiveness and toxicity of Zn depend on its chemical form and concentration.

Characterization of larval gut microbiota of two endoparasitoid wasps associated with their common host, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae).

Insect gut microbes play important roles in digestion, metabolism, development, and environmental adaptation. Parasitoid wasps are one of the most important biological control agents in pest control, while the gut microbial species compositions and the associated functions have been poorly investigated. Two endoparasitoid wasps, Cotesia vestalis and Diadromus collaris, parasitize the larval stage and pupal stage of the diamondback moth, Plutella xylostella, respectively. Using whole-genome shotgun metagenomic sequencing, we characterized the gut microbial composition, diversity, and potential functional roles associated with the two parasitoid wasp larvae. The results reveal that Proteobacteria and Firmicutes are the dominant phyla in the gut of C. vestalis and D. collaris larvae, with Rhizobium and Enterococcus being the dominant genera. The putative microbial functions associated with the two parasitoid wasps might play a virtual role in assisting in consuming the host's nutritional composition. The enriched CAZymes family genes are primarily involved in the degradation and synthesis of chitin. Despite the richness of microbial species and communities, the microbes species and the microbial community structure exhibit significant similarity between the two parasitoid wasps and between the parasitoid wasp and the host P. xylostella. Notably, the prevalence of the genus Enterococcus shared among them suggests a possible link of gut microbes between the host and their associated parasitoids. Our study offers insights into the gut microbe-based interactions between the host and parasitoid wasps for the first time, potentially paving the way for the development of an ecologically friendly biocontrol strategy against the pest P. xylostella.IMPORTANCEEndoparasitoid wasps spend the majority of their lifespan within their host and heavily rely on the host's nutrition for survival. There is limited understanding regarding the composition and physiological impacts of gut microbial communities in parasitoid wasps, particularly during the larval stage, which is directly linked to the host. Based on a thorough characterization of the gut microbe and comprehensive comparative analysis, we found the microbial species of the larval parasitoid wasp Cotesia vestalis and the pupal parasitoid wasp Diadromus collaris were similar, sharing 159 genera and 277 species, as were the microbial community structure. Certain of the dominant microbial strains of the two parasitoid wasps were similar to that of their host Plutella xylostella larvae, revealing host insect may affect the microbial community of the parasitoid wasps. The putative microbial functions associated with the parasitoid wasp larvae play an important role in dietary consumption.

Knockdown of the glucosamine-6-phosphate N-acetyltransferase gene by RNA interference enhances the virulence of entomopathogenic fungi against rice leaffolder Cnaphalocrocis medinalis

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

Regulation of trehalose metabolism mediated by validamycin on chitin synthesis in Spodoptera frugiperda

AbstractTrehalase (TRE) is a key enzyme for degrading trehalose, which plays a vital role in the growth and development of insects. Although validamycin, a compound belonging to a class of efficient antibiotics and fungicides, can control pests by suppressing TRE activities, it remains unknown whether it acts on both trehalose and chitin metabolism in Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), a major pest of maize (Zea mays L., Poaceae). This study investigated the changes in trehalose metabolism after validamycin treatment in S. frugiperda and its effects on the downstream chitin synthesis pathway. Compared with the control, S. frugiperda exhibited varying degrees of mortality after treatment with four concentrations of validamycin, showing a dose‐dependent increase in mortality rate. The mortality rates 24 and 48 h after treatment with 0.07 mg μL−1 validamycin were 35.6% and 42.2%, respectively, indicating the effective lethal concentration. Treatment with 0.07 mg μL−1 validamycin led to developmental delay, abnormal molting, and death in S. frugiperda, but it exerted no lasting effects on the survival rate, pupal weight, and phenotype during its subsequent developmental stages. At 24 h after validamycin treatment, TRE1 and TRE2 activities and glucose content decreased significantly, whereas the trehalose content increased significantly. Treatment with validamycin significantly upregulated TRE1 and TRE2 expression after 24 and 48 h and downregulated the mRNA expression of chitin synthase A and B genes. However, after 72 h, chitin content was not significantly affected. Hence, validamycin can destroy the dynamic transformation balance of trehalose and glucose by inhibiting the activities of the two TREs, and further affect the expression of downstream chitin synthase genes. These findings provide a theoretical basis for using TRE inhibitors to control S. frugiperda.

Candida albicans Mannosidases, Dfg5 and Dcw1, Are Required for Cell Wall Integrity and Pathogenesis.

Candida albicans is an oral mucosal commensal fungus that transforms into an opportunistic pathogen under specific conditions, including immunosuppression. It causes oral and systemic candidiasis, which results in a significant health burden. Furthermore, an alarming rise in antifungal drug resistance in Candida species raises the urgent need for novel drugs and drug targets. C. albicans Dfg5 and Dcw1 are homologous cell wall alpha-1,6-mannosidases with critical functions and represent potential new drug targets. Our past studies have shown that Dfg5 and Dcw1 function in cell wall biogenesis through the cross-linking of glycoproteins into the cell wall, thus playing a key role in cell wall integrity. Additionally, Dfg5 and Dcw1 are required for hyphal morphogenesis. However, the exact functions of Dfg5 and Dcw1 in cell wall integrity, hyphal morphogenesis, and pathogenesis are not known. In this study, we determined the relation of Dfg5 and Dcw1 with Hog1 MAPK, which plays a key role in cell wall integrity via the regulation of chitin synthesis in C. albicans. Additionally, we also determined the effects of dfg5 and dcw1 mutations on the gene expression of transcriptional regulators of hyphal morphogenesis. Furthermore, we determined the effects of dfg5 and dcw1 mutations on pathogenesis in a mouse model of oral candidiasis. Our results demonstrate that dfg5 and dcw1 mutations, as well as a hog1 knockout mutation, result in the dysregulation of chitin synthesis, resulting in a cell separation defect. Heterozygous and conditional mutations in dfg5 and dcw1 resulted in decreased transcriptional levels of cst20, a positive regulator of hyphal morphogenesis. However, dfg5 and dcw1 mutations resulted in increased levels of all the five negative regulators of hyphal morphogenesis-Tup1, Nrg1, Mig1, Rbf1, and Rfg1. Additionally, Tup1 levels were significantly higher than other negative regulators, indicating that Dfg5 and Dcw1 function in hyphal morphogenesis by repressing Tup1. Finally, dfg5 and dcw1 mutations affected the ability of C. albicans to cause oral candidiasis in mice. Thus, the cell wall glycosidases Dfg5 and Dcw1 are required for virulence and pathogenesis and represent novel drug targets.