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Trehalase (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. [ABSTRACT FROM AUTHOR]

Background: Cell wall integrity (CWI) is crucial for fungal growth, pathogenesis, and adaptation to extracellular environments. Calcofluor white (CFW) is a cell wall perturbant that inhibits fungal growth, yet little is known about how phytopathogenic fungi respond to the CFW-induced stress. Results: In this study, we unveiled a significant discovery that CFW triggered the translocation of the transcription factor CgCrzA from the cytoplasm to the nucleus in Colletotrichum gloeosporioides. This translocation was regulated by an interacting protein, CgMkk1, a mitogen-activated protein kinase involved in the CWI pathway. Further analysis revealed that CgMkk1 facilitated nuclear translocation by phosphorylating CgCrzA at the Ser280 residue. Using chromatin immunoprecipitation sequencing, we identified two downstream targets of CgCrzA, namely CgCHS5 and CgCHS6, which are critical for growth, cell wall integrity, and pathogenicity as chitin synthase genes. Conclusions: These findings provide a novel insight into the regulatory mechanism of CgMkk1-CgCrzA-CgChs5/6, which enables response of the cell wall inhibitor CFW and facilitates infectious growth for C. gloeosporioides. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) using double‐stranded RNA (dsRNA) against fungal pathogens is an emerging field of crop disease control. We aimed to evaluate RNAi against the fungus Cercospora zeina causing grey leaf spot (GLS) disease on maize. Orthologues of Dicer‐like 1, Dicer‐like 2, RNA‐dependent RNA polymerase and two copies of Argonaute were identified in the C. zeina genome and were shown to be expressed in vitro and in planta. Confocal microscopy showed that C. zeina took up exogenously applied dsRNA labelled with fluorescein. GFP‐transgenic C. zeina was treated with GFP‐specific dsRNA, and GFP mRNA expression and protein fluorescence were reduced by 57% and 61%, respectively. A Cz3‐dsRNA targeting C. zeina chitin synthase D (CHSD), phosphatidylserine decarboxylase proenzyme 3 (PSD3) and extracellular protein 2 (ECP2) was constructed. Treatment of C. zeina cultures with the Cz3‐dsRNA reduced CHSD expression by 47% and reduced cell viability by 34%. Maize leaves were inoculated with C. zeina conidia, and Cz3‐dsRNA was applied either with the conidia or 16 h later. GLS disease was significantly reduced compared to the water control for the 16 h post‐inoculation (hpi) treatment with Cz3‐dsRNA, but not for the GFP‐dsRNA specificity control or treatments at 0 hpi. We hypothesized that germination of C. zeina conidia was required for effective dsRNA‐mediated control, and this was borne out by microscopy observations that most of the C. zeina conidia (70%) germinated successfully on the maize leaf surface within 16 hpi. This work lays the groundwork for a dsRNA‐based fungicide against this foliar pathogen. [ABSTRACT FROM AUTHOR]

The globally prevalent pests, Diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) and Beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), pose significant threats to cruciferous vegetables. They have rapidly developed resistance to a wide range of insecticides, leading to significant yield losses and increased control expenses. In this study, we have established an efficient approach utilizing amplicon sequencing to detect the frequency of 15 target resistance mutant sites in 6 molecular targets, acetylcholinesterase 1 (ACE1), chitin synthase 1 (CHS1), the γ-aminobutyric acid receptor (GABAR), glutamate-gated chloride channel (GluCl), voltage-gated sodium channels (NaV), and ryanodine receptor (RyR) in P. xylostella and the frequency of 11 mutations in 5 molecular targets (except GluCl) in S. exigua in China. Our findings indicate that P. xylostella exhibits remarkably high frequency (over 88.67%) in pyrethroid resistance-related mutations T929I and L1014F of NaV. In S. exigua , the frequencies of L659F mutation were ranging from 41.92% to 74.89%. In addition, the organophosphorus resistance-related mutations A298S and G324A of ACE1 were detected at frequencies ranging from 34.29% to 75.66%, and these 2 mutations occurred simultaneously (from 29.22% to 65.79%) in P. xylostella. An interannual variation in mutation frequency from 2019 to 2021 was found for P. xylostella in HNCS. The frequency of A298S and G324A mutations steadily increased while the frequency of G4946E and I4790M mutations continuously decreased. These results unveil a worrisome scenario of multiple resistance sites in these 2 pests in China and provide valuable insights for the practical application of pesticides in the field. [ABSTRACT FROM AUTHOR]

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa. The insecticidal protein Vip3Aa is used in genetically engineered crops to overcome pest resistance to other insecticidal transgenes. This study shows that insertion of a transposable element into a gut-specific chitin synthase gene causes Vip3Aa resistance in field-collected individuals of the lepidopteran pest Spodoptera frugiperda (fall armyworm). [ABSTRACT FROM AUTHOR]

There is a high interest in utilizing natural bioactive products derived from plants as a substitute for synthetic chemicals in the industry. This research focuses on the phytochemical composition of essential oils (EOs) of Ammi visnaga L. and Trachyspermum ammi L and their insecticidal activity against Sitophilus oryzae (L.), a common pest found in stored cereals. The EOs were extracted through steam distillation and analyzed using gas chromatography coupled with mass spectrometry (GC-MS). The EOs of A. visnaga consisted of twenty-four components, with Abietadiene (41.23%) being the most abundant, followed by linalool (25.54%) and limonene (19.04%). On the other hand, the EOs of T. ammi consisted of twenty-eight main components, with isothymol being the most abundant (51.88%). The results revealed that the EOs of T. Ammi (DL50 = 0.1 µL EOs/L of air) were more toxic than A. visnaga (0.38 µL EOs/L of air), with the toxicity varying based on doses and exposure periods. To further understand the molecular mechanisms underlying this activity, molecular docking and dynamic simulations were performed using the major chemical constituents of the oils. The simulation results indicated that the major compounds, Abietadiene and isothymol, interact with the catalytic sites of the target proteins, inhibiting acetylcholinesterase and chitin synthase. These interactions form energetically favorable systems that remain stable throughout the molecular dynamic period. This research provides valuable insights into the potential of these EOs as natural insecticides and highlights the importance of molecular modeling in understanding the biological activities of plant-derived compounds. [ABSTRACT FROM AUTHOR]

Abstract Background This study sheds light on the genetic blueprints of chaetogenesis (bristle formation), a complex biomineralization process essential not only for the diverse group of bristle worms (annelids) but also for other spiralians. We explore the complex genetic mechanisms behind chaetae formation in Osedax japonicus, the bone-devouring deep-sea worm known for its unique ecological niche and morphological adaptations. Results We characterized the chaetal structure and musculature using electron microscopy and immunohistochemistry, and combined RNAseq of larval stages with in-situ hybridization chain reaction (HCR) to reveal gene expression patterns integral to chaetogenesis. Our findings pinpoint a distinct surge in gene expression during the larval stage of active chaetogenesis, identifying specific genes and cells involved. Conclusions Our research underscores the value of studying on non-model, "aberrant" organisms like Osedax, whose unique, temporally restricted chaetogenesis provided insights into elevated gene expression across specific larval stages and led to the identification of genes critical for chaetae formation. The genes identified as directly involved in chaetogenesis lay the groundwork for future comparative studies across Annelida and Spiralia, potentially elucidating the homology of chaetae-like chitinous structures and their evolution.

Chitin is widely found in fungi, insects and crustaceans, but not in plants and vertebrates. It is an important component of insect exoskeleton, trachea and midgut peritrophic membrane. During the molting process of insects, chitin-rich structures need to be reconstructed to complete the expansion of the insect body, and the synthesis of chitin needs to be strictly controlled. Therefore, chitin biosynthesis has been an important target for pest control. With the rapid development of pesticide resistance in insect populations, pest prevention and control is facing new challenges, and it is necessary to constantly search for new pest control targets to develop new insecticides and achieve effective pest control. Chitin synthase (CHS) is a key enzyme in insect chitin synthesis pathway and plays an important role in chitin synthesis. There are two kinds of chitin synthases, CHS1 and CHS2. CHS1 is expressed in the exoskeleton and trachea of insects and catalyzes chitin synthesis, while CHS2 is mainly responsible for chitin synthesis in the midgut peritrophic membrane. Interference of CHS1 can lead to defects in insect epidermis and abnormal development of dorsal trunk of tracheae, while the inhibition of CHS2 often results in shorter midgut and weight loss. Down-regulation of both CHS genes in insects could cause a large number of deaths. Insects have complex transcriptional regulation mechanisms of CHS to ensure their normal growth and development, and respond to external stimuli. Based on domestic and foreign studies, we reviewed the research progress in transcriptional regulation of insect CHS, including the effects of insect hormones, transcription factors, epidermal injury and feeding stimulation, genes and substances related to chitin metabolism, microRNA, and inhibitors of chitin synthesis on CHS mRNA levels in insects, with an aim to provide a theoretical basis for the development and utilization of green pesticides targeting CHS for pest control in the future.

Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSVII were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. Key points: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development. [ABSTRACT FROM AUTHOR]

Background: This study sheds light on the genetic blueprints of chaetogenesis (bristle formation), a complex biomineralization process essential not only for the diverse group of bristle worms (annelids) but also for other spiralians. We explore the complex genetic mechanisms behind chaetae formation in Osedax japonicus, the bone-devouring deep-sea worm known for its unique ecological niche and morphological adaptations. Results: We characterized the chaetal structure and musculature using electron microscopy and immunohistochemistry, and combined RNAseq of larval stages with in-situ hybridization chain reaction (HCR) to reveal gene expression patterns integral to chaetogenesis. Our findings pinpoint a distinct surge in gene expression during the larval stage of active chaetogenesis, identifying specific genes and cells involved. Conclusions: Our research underscores the value of studying on non-model, "aberrant" organisms like Osedax, whose unique, temporally restricted chaetogenesis provided insights into elevated gene expression across specific larval stages and led to the identification of genes critical for chaetae formation. The genes identified as directly involved in chaetogenesis lay the groundwork for future comparative studies across Annelida and Spiralia, potentially elucidating the homology of chaetae-like chitinous structures and their evolution. [ABSTRACT FROM AUTHOR]

Phaseolus vulgaris L. , commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), β-tub (β-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha- l -Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF , GAPDH , and β-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes. • Gene expression investigations are hampered by the lack of reference genes in C. lindemuthianum. • We provide the list of qPCR genes that can be used to perform expression analysis of genes in C. lindemuthianum. • We identified EF and GAPDH as the most stable genes. • Our results will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes. [ABSTRACT FROM AUTHOR]

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions. [ABSTRACT FROM AUTHOR]

Chitin is widely found in fungi, insects and crustaceans, but not in plants and vertebrates. It is an important component of insect exoskeleton, trachea and midgut peritrophic membrane. During the molting process of insects, chitin-rich structures need to be reconstructed to complete the expansion of the insect body, and the synthesis of chitin needs to be strictly controlled. Therefore, chitin biosynthesis has been an important target for pest control. With the rapid development of pesticide resistance in insect populations, pest prevention and control is facing new challenges, and it is necessary to constantly search for new pest control targets to develop new insecticides and achieve effective pest control. Chitin synthase (CHS) is a key enzyme in insect chitin synthesis pathway and plays an important role in chitin synthesis. There are two kinds of chitin synthases, CHS1 and CHS2. CHS1 is expressed in the exoskeleton and trachea of insects and catalyzes chitin synthesis, while CHS2 is mainly responsible for chitin synthesis in the midgut peritrophic membrane. Interference of CHS1 can lead to defects in insect epidermis and abnormal development of dorsal trunk of tracheae, while the inhibition of CHS2 often results in shorter midgut and weight loss. Down-regulation of both CHS genes in insects could cause a large number of deaths. Insects have complex transcriptional regulation mechanisms of CHS to ensure their normal growth and development, and respond to external stimuli. Based on domestic and foreign studies, we reviewed the research progress in transcriptional regulation of insect CHS, including the effects of insect hormones, transcription factors, epidermal injury and feeding stimulation, genes and substances related to chitin metabolism, microRNA, and inhibitors of chitin synthesis on CHS mRNA levels in insects, with an aim to provide a theoretical basis for the development and utilization of green pesticides targeting CHS for pest control in the future. [ABSTRACT FROM AUTHOR]

Abstract Background Encystment is an important survival strategy extensively employed by microbial organisms to survive unfavorable conditions. Single-celled ciliated protists (ciliates) are popular model eukaryotes for studying encystment, whereby these cells degenerate their ciliary structures and develop cyst walls, then reverse the process under more favorable conditions. However, to date, the evolutionary basis and mechanism for encystment in ciliates is largely unknown. With the rapid development of high-throughput sequencing technologies, genome sequencing and comparative genomics of ciliates have become effective methods to provide insights into above questions. Results Here, we profiled the MAC genome of Pseudourostyla cristata, a model hypotrich ciliate for encystment studies. Like other hypotrich MAC genomes, the P. cristata MAC genome is extremely fragmented with a single gene on most chromosomes, and encodes introns that are generally small and lack a conserved branch point for pre-mRNA splicing. Gene family expansion analyses indicate that multiple gene families involved in the encystment are expanded during the evolution of P. cristata. Furthermore, genomic comparisons with other five representative hypotrichs indicate that gene families of phosphorelay sensor kinase, which play a role in the two-component signal transduction system that is related to encystment, show significant expansion among all six hypotrichs. Additionally, cyst wall-related chitin synthase genes have experienced structural changes that increase them from single-exon to multi-exon genes during evolution. These genomic features potentially promote the encystment in hypotrichs and enhance their ability to survive in adverse environments during evolution. Conclusions We systematically investigated the genomic structure of hypotrichs and key evolutionary phenomenon, gene family expansion, for encystment promotion in ciliates. In summary, our results provided insights into the evolutionary mechanism of encystment in ciliates.

Background: Encystment is an important survival strategy extensively employed by microbial organisms to survive unfavorable conditions. Single-celled ciliated protists (ciliates) are popular model eukaryotes for studying encystment, whereby these cells degenerate their ciliary structures and develop cyst walls, then reverse the process under more favorable conditions. However, to date, the evolutionary basis and mechanism for encystment in ciliates is largely unknown. With the rapid development of high-throughput sequencing technologies, genome sequencing and comparative genomics of ciliates have become effective methods to provide insights into above questions. Results: Here, we profiled the MAC genome of Pseudourostyla cristata, a model hypotrich ciliate for encystment studies. Like other hypotrich MAC genomes, the P. cristata MAC genome is extremely fragmented with a single gene on most chromosomes, and encodes introns that are generally small and lack a conserved branch point for pre-mRNA splicing. Gene family expansion analyses indicate that multiple gene families involved in the encystment are expanded during the evolution of P. cristata. Furthermore, genomic comparisons with other five representative hypotrichs indicate that gene families of phosphorelay sensor kinase, which play a role in the two-component signal transduction system that is related to encystment, show significant expansion among all six hypotrichs. Additionally, cyst wall-related chitin synthase genes have experienced structural changes that increase them from single-exon to multi-exon genes during evolution. These genomic features potentially promote the encystment in hypotrichs and enhance their ability to survive in adverse environments during evolution. Conclusions: We systematically investigated the genomic structure of hypotrichs and key evolutionary phenomenon, gene family expansion, for encystment promotion in ciliates. In summary, our results provided insights into the evolutionary mechanism of encystment in ciliates. [ABSTRACT FROM AUTHOR]

Soybean phytophthora blight is a severe menace to global agriculture, causing annual losses surpassing USD 1 billion. Present crop loss mitigation strategies primarily rely on chemical pesticides and disease-resistant breeding, frequently surpassed by the pathogens' quick adaptive evolution. In this urgent scenario, our research delves into innovative antimicrobial peptides characterized by low drug resistance and environmental friendliness. Inhibiting chitin synthase gene activity in Phytophthora sojae impairs vital functions such as growth and sporulation, presenting an effective method to reduce its pathogenic impact. In our study, we screened 16 previously tested peptides to evaluate their antimicrobial effects against Phytophthora using structure-guided drug design, which involves molecular docking, saturation mutagenesis, molecular dynamics, and toxicity prediction. The in silico analysis identified AMP_04 with potential inhibitory activity against Phytophthora sojae's chitin synthase. Through three rounds of saturation mutagenesis, we pin-pointed the most effective triple mutant, TP (D10K, G11I, S14L). Molecular dynamic simulations revealed TP's stability in the chitin synthase-TP complex and its transmembrane mechanism, employing an all-atom force field. Our findings demonstrate the efficacy of TP in occupying the substrate-binding pocket and translocation catalytic channel. Effective inhibition of the chitin synthase enzyme can be achieved. Specifically, the triple mutant demonstrates enhanced antimicrobial potency and decreased toxicity relative to the wild-type AMP_04, utilizing a mechanism akin to the barrel-stave model during membrane translocation. Collectively, our study provides a new strategy that could be used as a potent antimicrobial agent in combatting soybean blight, contributing to sustainable agricultural practices. [ABSTRACT FROM AUTHOR]

Chitin synthase is an indispensable enzyme that is involved in the synthesis of chitin, a key component of the insect cuticle during insect development, which has long been considered a potential target for pest control. The German cockroach Blattella germanica is a notorious urban pest that has developed resistance to more than one class of insecticide. Therefore, there is an urgent need to explore novel green molecular approaches for sustainable insect pest control. In this study, two chitin synthase (CHS) genes (BgCHS1 and BgCHS2) were identified for the first time in B. germanica: Phylogenetic relationships, motif patterns, and conserved domain regions of all the predicted CHS genes were identified. Furthermore, the function characteristic of the two BgCHS genes was studied using a microinjection-based RNAi system. The results showed that silencing of BgCHS1 and BgCHS2 both resulted in high mortality. Knockdown of the BgCHS1 gene resulted in impaired molting and abnormal development, while knockdown of the BgCHS2 gene expression resulted in abnormal development. Additionally, a large-scale dsRNA production system via Escherichia coli was built and a nanoparticle-based (Star Polymers, SPc) oral delivery system of an SPc-wrapped two dsRNA of dsBgCHS1 and dsBgCHS2 was evaluated, which resulted in a high mortality and ultrastructural changes in the integument and midgut of cockroach nymphs, respectively. This study illustrates the functions of the two novel identified CHS genes in B. germanica, which can serve as potential gene targets for controlling one of the most serious urban health pests. [ABSTRACT FROM AUTHOR]

Background: Armenia is considered particularly vulnerable to life-threatening vector-borne diseases (VBDs) including malaria, West Nile virus disease and leishmaniasis. However, information relevant for the control of the vectors of these diseases, such as their insecticide resistance profile, is scarce. The present study was conducted to provide the first evidence on insecticide resistance mechanisms circulating in major mosquito and sand fly populations in Armenia. Methods: Sampling sites were targeted based mainly on previous historical records of VBD occurrences in humans and vertebrate hosts. Initially, molecular species identification on the collected vector samples was performed. Subsequently, molecular diagnostic assays [polymerase chain reaction (PCR), Sanger sequencing, PCR-restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR)] were performed to profile for major insecticide resistance mechanisms, i.e. target site insensitivity in voltage-gated sodium channel (vgsc) associated with pyrethroid resistance, acetylcholinesterase (ace-1) target site mutations linked to organophosphate (OP) and carbamate (CRB) resistance, chitin synthase (chs-1) target site mutations associated with diflubenzuron (DFB) resistance and gene amplification of carboxylesterases (CCEs) associated with resistance to the OP temephos. Results: Anopheles mosquitoes were principally represented by Anopheles sacharovi, a well-known malaria vector in Armenia, which showed no signs of resistance mechanisms. Contrarily, the knockdown resistance (kdr) mutations V1016G and L1014F/C in the vgsc gene were detected in the arboviral mosquito vectors Aedes albopictus and Culex pipiens, respectively. The kdr mutation L1014S was also detected in the sand fly, vectors of leishmaniasis, Phlebotomus papatasi and P. tobbi, whereas no mutations were found in the remaining collected sand fly species, P. sergenti, P. perfiliewi and P. caucasicus. Conclusions: This is the first study to report on molecular mechanisms of insecticide resistance circulating in major mosquito and sand fly disease vectors in Armenia and highlights the need for the establishment of systematic resistance monitoring practices for the implementation of evidence-based control applications. [ABSTRACT FROM AUTHOR]

The cotton bollworm, Helicoverpa armigera, stands as a major agricultural pest, displaying resistance to a diverse range of insecticides. Diflubenzuron, a member of the benzoylurea class of insecticides, serve by suppressing the synthesis of chitin in insects. This disruption hampers their typical metamorphic processes like molting and pupation, ultimately culminating in death. In this study, the toxicity of diflubenzuron against H. armigera was investigated. The results showed that the LC50 of diflubenzuron on 3rd instar bollworm was 109 mg/kg after 7-day exposure. Most importantly, diflubenzuron treatment led to a change in the chitin synthesis pathway, accompanied by a significant surge in chitinase (HaChi) expression. The feasibility of combining with RNA interference (RNAi) technology to control cotton bollworm was further explored. We demonstrated that three chitin synthesis pathway genes Chitin synthase 1 (HaCHS1), chitin synthase 2 (HaCHS2) and HaChi were significantly inhibited following exposure to dsRNAs, leading to the death of cotton bollworm. Among these, the dsHaCHi treatment group manifested the most significant effect. Subsequent combined treatment of 3rd instar larvae with dsHaChi and diflubenzuron reduced survival rates of cotton bollworm by 24% and 15.33%, respectively. Simultaneously, there were recognized effects on larval weight, pupal weight, and pupation rate to varying degrees. This study underscores the potential of combining RNAi technology with conventional chemical pesticides, this combination can improve pest control efficiency, and offers a novel strategy to compensate for the low RNAi efficiency in lepidopteran insects, consequently reducing the dependency on chemical pesticides. [ABSTRACT FROM AUTHOR]

The "Wattle and Daub" model of cyst wall formation in Entamoeba invadens has been used to explain encystment in Entamoeba histolytica, the causal agent of amoebiasis, and this process could be a potential target for new antiamoebic drugs. In this study, we studied the morphological stages of chitin wall formation in E. invadens in more detail using fluorescent chitin-binding dyes and the immunolocalization of cyst wall proteins. It was found that chitin deposition was mainly initiated on the cell surface at a specific point or at different points at the same time. The cystic wall grew outward and gradually covered the entire surface of the cyst over time, following the model of Wattle and Daub. The onset of chitin deposition was guided by the localization of chitin synthase 1 to the plasma membrane, occurring on the basis of the Jacob lectin in the cell membrane. During encystation, F-actin was reorganized into the cortical region within the early stages of encystation and remained intact until the completion of the chitin wall. The disruption of actin polymerization in the cortical region inhibited proper wall formation, producing wall-less cysts or cysts with defective chitin walls, indicating the importance of the cortical actin cytoskeleton for proper cyst wall formation. [ABSTRACT FROM AUTHOR]

ABSTRACT Cryptococcus neoformans causes cryptococcal meningoencephalitis, a disease that kills more than 180,000 people annually. Contributing to its success as a fungal pathogen is its cell wall surrounded by a capsule. When the cryptococcal cell wall is compromised, exposed pathogen-associated molecular pattern molecules (PAMPs) could trigger host recognition and initiate attack against this fungus. Thus, cell wall composition and structure are tightly regulated. The cryptococcal cell wall is unusual in that chitosan, the acetylated form of chitin, is predominant over chitin and is essential for virulence. Recently, it was shown that acidic pH weakens the cell wall and increases exposure of PAMPs partly due to decreased chitosan levels. However, the molecular mechanism responsible for the cell wall remodeling in acidic pH is unknown. In this study, by screening for genes involved in cryptococcal tolerance to high levels of CO2, we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO2 due to acidification of unbuffered media. Overexpression of MAY1 increases the cryptococcal cell size and elevates PAMP exposure, causing a hyper-inflammatory response in the host while MAY1 deletion does the opposite. We discovered that May1 weakens the cell wall and reduces the chitosan level, partly due to its involvement in the degradation of Chs3, the sole chitin synthase that supplies chitin to be converted to chitosan. Consistently, overexpression of CHS3 largely rescues the phenotype of MAY1oe in acidic media. Collectively, we demonstrate that May1 remodels the cryptococcal cell wall in acidic pH by reducing chitosan levels through its influence on Chs3.IMPORTANCEThe fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

Due to the adverse environmental impacts of toxic heavy metal–based antifoulants, the screening of environmentally friendly antifoulants has become important for the development of marine antifouling technology. Compared with the traditional lengthy and costly screening method, computer-aided drug design (CADD) offers a promising and efficient solution that can accelerate the screening process of green antifoulants. In this study, we selected barnacle chitin synthase (CHS, an important enzyme for barnacle settlement and development) as the target protein for docking screening. Three CHS genes were identified in the barnacle Amphibalanus amphitrite, and their encoded proteins were found to share a conserved glycosyltransferase domain. Molecular docking of 31,561 marine natural products with AaCHSs revealed that zoanthamine alkaloids had the best binding affinity (−11.8 to −12.6 kcal/mol) to AaCHSs. Considering that the low abundance of zoanthamine alkaloids in marine organisms would limit their application as antifoulants, a marine fungal–derived natural product, mycoepoxydiene (MED), which has a similar chemical structure to zoanthamine alkaloids and the potential for large-scale production by fermentation, was selected and validated for stable binding to AaCHS2L2 using molecular docking and molecular dynamics simulations. Finally, the efficacy of MED in inhibiting cyprid settlement of A. amphitrite was confirmed by a bioassay that demonstrated an EC50 of 1.97 μg/mL, suggesting its potential as an antifoulant candidate. Our research confirmed the reliability of using AaCHSs as antifouling targets and has provided insights for the efficient discovery of green antifoulants by CADD.

Litchi (Litchi chinensis Sonn.) is an evergreen subtropical fruit tree native to southern China. Litchi is vulnerable to a wide range of diseases affecting yield and fruit quality. Anthracnose is one of the main diseases during the period of growth and storage, which has a serious impact on the quality and production of litchi. In December 2020 to May 2021, typical anthracnose symptoms were observed on litchi leaves in different orchards in Qinzhou City, Guangxi Province, Southern China. According to colony features, conidial and appressorial morphology, and sequence analysis of several genomic regions (internal transcribed spacer (ITS) region, chitin synthase (chs‐1), actin (act), calmodulin (cal), glyceraldehyde‐3‐phosphate dehydrogenase (gapdh), β‐tubulin (tub2) and the intergenic region of apn2 and MAT1‐2‐1 (ApMat)), 44 isolates were obtained, and 26 were identified as three Colletotrichum species: C. fructicola (50%), C. siamense (42.31%), C. gigasporum (7.69%). The pathogenicity tests were performed with conidial suspension and mycelia plugs to inoculate wounded litchi seedlings. The results of pathogenicity tests showed that the virulence of C. gigasporum was the weakest, and the virulence of C. fructicola was the strongest. This is the first report of C. gigasporum causing anthracnose of litchi worldwide. [ABSTRACT FROM AUTHOR]

Anthracnose of oak (Quercus) caused by Colletotrichum spp. is one of the most common diseases in oak forests. To investigate the species diversity of Colletotrichum associated with oak anthracnose, symptomatic leaf samples of three oak species (Q. acutissima, Q. mongolica and Q. variabilis) were collected from Anhui, Hainan, Henan, Shaanxi and Shandong Provinces, Inner Mongolia Autonomous Region, and Beijing City in China from 2019 to 2022. A total of 219 Colletotrichum isolates were obtained and identified by morphological and phylogenetic analyses of the rDNA internal transcribed spacer (ITS), glyceraldehyde‐3‐phosphate dehydrogenase (gapdh), chitin synthase 1 (chs‐1), actin (act) and β‐tubulin (tub2) sequences. Nine species were identified: C. fioriniae (two isolates, 0.9%), C. camellia‐japonicae (two isolates, 0.9%), C. karstii (three isolates, 1.4%), C. quercicola (two isolates, 0.9%), C. aenigma (three isolates, 1.4%), C. endophyticum (two isolates, 0.9%), C. fructicola (68 isolates, 31.1%), C. gloeosporioides sensu stricto (74 isolates, 33.8%) and C. siamense (63 isolates, 28.8%). Pathogenicity was confirmed using Koch's postulates, which showed that five species (C. camellia‐japonicae, C. endophyticum, C. fructicola, C. gloeosporioides s. s. and C. siamense) caused Q. acutissima anthracnose, four species (C. karstii, C. fructicola, C. gloeosporioides s. s. and C. siamense) caused Q. mongolica anthracnose and six species (C. fioriniae, C. quercicola, C. aenigma, C. fructicola, C. gloeosporioides s. s. and C. siamense) caused Q. variabilis anthracnose. This study demonstrates the pathogenic species diversity of Colletotrichum on Q. acutissima, Q. mongolica and Q. variabilis. [ABSTRACT FROM AUTHOR]

Synthesis of chitin is a subject of great interest in the fields of physiology and immunology of crustaceans. Chitinous tissues include not only the carapace, but also an acellular membrane in the intestine called the peritrophic membrane (PM). Here, we describe the first report of chitin synthase (CHS) of a penaeid shrimp, kuruma shrimp Penaeus japonicus. Histological observations showed that fecal matter in the midgut of kuruma shrimp was wrapped with a PM, which physically separated it from the midgut epithelium. Subsequently, the chitin synthase transcript was amplified from the midgut of the shrimp. The chitin synthase gene of kuruma shrimp (MjCHS) encodes 1,523 amino acid residues. Structural prediction analysis showed that the N-terminal region of MjCHS protein included nine transmembrane helices, the middle region included the catalytic region with several conserved motifs which are found in CHSs from other arthropods, and the C-terminal region included seven transmembrane helices. Although insects have distinct exoskeletal and intestinal chitin synthases, the phylogenetic analysis suggested that crustaceans have a single CHS. MjCHS mRNA was constantly detected in the digestive tract, including the midgut and hepatopancreas of both juvenile and adult kuruma shrimp, suggesting a stable synthesis of chitin in those organs. In contrast, MjCHS mRNA was also detected in the hindgut and uropod of juvenile shrimp. After molting, the mRNA levels of MjCHS in the stomach and uropod were higher than other molting cycles. These results suggest that MjCHS contributes to chitin synthesis in both the digestive tract and the epidermis, providing fundamental insights into chitin synthesis of crustaceans. [ABSTRACT FROM AUTHOR]

Background: The microbiota of multicellular organisms undergoes considerable changes during host ontogeny but the general mechanisms that control community assembly and succession are poorly understood. Here, we use bacterial recolonization experiments in Nematostella vectensis as a model to understand general mechanisms determining bacterial establishment and succession. We compared the dynamic establishment of the microbiome on the germfree host and on inert silicone tubes. Results: Following the dynamic reconstruction of microbial communities on both substrates, we show that the initial colonization events are strongly influenced by the host but not by the silicone tube, while the subsequent bacteria-bacteria interactions are the main driver of bacterial succession. Interestingly, the recolonization pattern on adult hosts resembles the ontogenetic colonization succession. This process occurs independently of the bacterial composition of the inoculum and can be followed at the level of individual bacteria. To identify potential metabolic traits associated with initial colonization success and potential metabolic interactions among bacteria associated with bacterial succession, we reconstructed the metabolic networks of bacterial colonizers based on their genomes. These analyses revealed that bacterial metabolic capabilities reflect the recolonization pattern, and the degradation of chitin might be a selection factor during early recolonization of the animal. Concurrently, transcriptomic analyses revealed that Nematostella possesses two chitin synthase genes, one of which is upregulated during early recolonization. Conclusions: Our results show that early recolonization events are strongly controlled by the host while subsequent colonization depends on metabolic bacteria-bacteria interactions largely independent of host ontogeny. -JTs9knfCQzALX-coLcUGo Video Abstract [ABSTRACT FROM AUTHOR]

Background: Aedes albopictus is a vector of numerous devastating arboviruses and places heavy burdens on global public health. Chitin is one of the important components of cuticles and targeting chitin metabolism is a promising strategy for preventing mosquito dispersal and mosquito-borne diseases. Increasing evidence suggests that microRNAs (miRNAs) play crucial roles in various physiological processes of insects. Methods: A previous analysis suggested that the microRNA miR-989 is potentially involved in chitin metabolism in Ae. albopictus pupae. In the present study, we found that the expression level of miR-989 was significantly overexpressed after injection of agomir. A dual-luciferase assay was used to determine the direct target of miR-989. Survival rate, eclosion rate and malformation rate were statistically analyzed to evaluate the potential effect of miR-989. Hematoxylin–eosin staining and chitin staining were used to evaluate the microstructural changes in the cuticles of Ae. albopictus pupae. Results: Overexpression of miR-989 resulted in a significantly reduced survival rate and eclosion rate of pupae and an elevated malformation rate of adults. The results suggested that miR-989 acted as a regulator of chitin metabolism in Ae. albopictus pupae by affecting the transcript levels of the Ae. albopictus genes encoding chitin synthase 1 (AaCHS1) and chitinase 10 (AaCht10). The altered expression levels of the two chitin metabolism-related enzymes (CHS1 and Cht10, respectively) caused the structural changes in cuticles and further affected the pupal-adult transition process of Ae. albopictus. XM_029863591.1 was proven to be the target gene of miR-989 and displayed similar effects on pupae as miR-989. Conclusions: The microRNA miR-989 was found to be essential for chitin metabolism in old and new cuticles of Ae. albopictus pupae. The results of the current study suggested that miR-989 could be used as a potential target to control Ae. albopictus. [ABSTRACT FROM AUTHOR]

Background: Current mosquito‐borne disease vector control strategies, largely based on chemical insecticides, are seriously threatened by increasing resistance worldwide. There is also growing concerned about the adverse effects of insecticides on nontarget organisms and the environment, therefore effective and ecologically friendly alternative approaches are urgently needed. Targeting critical steps of reproduction is considered a potential way to control mosquito populations. Herein, we focused on the roles of chitin synthase A (encoded by chsa) in the reproduction of female mosquitoes. Results: The injection of small interfering RNA targeting Cpchsa in female Culex pipiens pallens (Diptera: Culicidae) had antireproductive effects, including decreased follicle numbers, egg‐laying, and hatching rate. Scanning electron microscopy observations showed that Cpchsa silencing caused a defective egg envelope, including absence of the vitelline membrane and cracked chorion layers, which resulted in abnormal permeability. Widely distributed nurse cell apoptosis and follicular epithelial cell autophagy were observed in Cpchsa‐silenced ovaries during the vitellogenesis phase. Consistent with the detective egg envelope formation during oogenesis, the exochorionic eggshell structures were also affected in eggs deposited by Cpchsa‐silenced mosquitoes. Conclusion: This study provided fundamental evidence for the role of chitin synthase A in the female reproductive process of mosquitoes and might result in a novel alternative strategy for mosquito control. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Aucuba japonica Thunb is an evergreen woody ornamental plant with significant economic and ecological values. It also produces aucubin, showing a variety of biological activities. It is widely planted in the southwest region of China, including karst landscape areas in Guizhou Province. In January 2022, a serious leaf blight disease was observed on the leaves of A. japonica in the outdoor gardens of Guizhou University, Guiyang, Guizhou, China. The causal agent was identified as Colletotrichum aenigma through amplification and sequencing of the internal transcribed spacer (ITS) region, translation of the chitin synthase (CHS) and actin (ACT) genes, and morphological characterizations. Koch's postulates were confirmed by its pathogenicity on healthy leaves, including re-isolation and identification. To our knowledge, this is the first report of C. aenigma causing leaf blight on A. japonica worldwide. To identify pathogen characteristics that could be utilized for future disease management, the effects of temperature and light on mycelial growth, conidia production, and conidial germination, and the effects of humidity on conidial germination were studied. Optimal temperatures for mycelial growth of C. aenigma BY827 were 25-30℃, while 15℃ and 35℃ were favorable for conidia production. Concurrently, alternating 10-h light and 14-h dark, proved to be beneficial for mycelial growth and conidial germination. Additionally, conidial germination was enhanced at 90% humidity. In vitro screenings of ten chemical pesticides to assess their efficacy in suppressing C. aenigma representative strain BY827. Among them, difenoconazole showed the best inhibition rate, with an EC50 (concentration for 50% of maximal effect) value of 0.0148 μg/ml. Subsequently, field experiment results showed that difenoconazole had the highest control efficiency on A. japonica leaf blight (the decreasing rate of disease incidence and decreasing rate of disease index were 44.60 and 47.75%, respectively). Interestingly, we discovered that C. aenigma BY827 may develop resistance to mancozeb, which is not reported yet among Colletotrichum spp.strains. In conclusion, our study provided new insights into the causal agent of A. japonica leaf blight, and the effective fungicides evaluated provided an important basis and potential resource for the sustainable control of A. japonica leaf blight caused by C. aenigma in the field. [ABSTRACT FROM AUTHOR]

In this study, Sargassum latifolium (S. latifolium) n-hexane extract as a novel source for insecticidal bioactive compounds was investigated. The composition of the extract was identified by using GC-MS analysis. We performed the study on four types of insects and larvae sp (Sarcophaga bullata, Tribolium castaneum, Musca domestica, and Solenopsis invicta) which cause a deleterious impact on the crops and human health. We used scanning electron microscopy to show the destructive effects of S. latifolium extract on the exoskeleton. The treatment exhibited severe morphological changes and a high mortality rate. Also, different bioinformatic approaches were utilized to investigate the possible insecticidal mechanism of the extract. Molecular docking showed the potential of the compounds to be chitin synthase inhibitors. Besides, the toxicokinetic parameters of the most promising compounds were evaluated to assess the impact of using the extract as an insecticidal agent on human health. S. latifolium extract exhibited promising insecticidal activity and acceptable toxicokinetic properties as compared to other known pesticide groups. [Display omitted] • Sargassum latifolium extract has a promising insecticidal activity. • Utilization of the extract showed sever damage on the exoskeleton of the insects. • The compounds in the extract exhibited promising binding affinities with chitin synthase. • The identified compounds in the extract have safe toxicokinetic properties. [ABSTRACT FROM AUTHOR]

Viburnum odoratissimum Ker-Gawl is native to Asia and is usually used as a garden ornamental. In September 2022, a leaf blotch on V. odoratissimum was observed in Nanjing, Jiangsu, China. The disease causes the leaves of the plants to curl and dry up and defoliate early. It not only seriously affects the growth of the plants but also greatly reduces the ornamental value. The pathogenic fungus was isolated from the diseased leaves, and the fungus was identified to be Colletotrichum siamense based on morphological features and multilocus phylogenetic analyses of the internal transcribed spacer (ITS) region, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. Pathogenicity tests were performed by inoculating healthy leaves with conidia. C. siamense can grow at 15–35 °C, with an optimal growth temperature at 25–30 °C. The results of sensitivity to nine fungicides showed that C. siamense was the most sensitive to prochloraz in the concentration range of 0.01 μg/mL to 100 μg/mL. Therefore, spraying prochloraz before the optimum growth temperature of pathogenic fungus can achieve effective control. It provided useful information for future studies on the prevention and treatment strategies of C. siamense. This is the first report of leaf blotch caused by C. siamense on V. odoratissimum in China and worldwide. [ABSTRACT FROM AUTHOR]

In the literature, RNA-based interference methods have been proposed for the control of pathogenic fungi in plants, such as virus-mediated silencing (VIGS). The objective of this work was to evaluate the functionality and efficiency of the VIGS EuMV-YPΔCHS4 vector, which contains a fragment of the Fusarium sp. chitin synthase 4 (CHS4) gene sequence, and was constructed from the EuMV-YP vector:ΔAV1. The VIGS vector was evaluated under two conditions in Nicotiana benthamiana plants previously inoculated with Fusarium sp. and in plants with inoculation after the inoculation of the VIGS vector. The results showed that in both treatments with the EuMV-YPΔCHS4, a delay in leaf fall during the first days of infection and a decrease in the stem damage of almost one centimeter, compared to the control infected with the fungus. In addition, a reduction of up to 30% in the relative expression of the CHS4 gene was observed. These results suggest that the application of VIGS vectors may be efficient as a strategy to protect plants against Fusarium sp. [ABSTRACT FROM AUTHOR]

BACKGROUND: Previous studies have shown that fungicides have insecticidal activity that can potentially be used as an insecticide resistance management strategy in the brown planthopper Nilaparvata lugens (Stål). However, the mechanism that induces mortality of N. lugens remains elusive. RESULTS: In the present study, the insecticidal activities of 14 fungicides against N. lugens were determined, of which tebuconazole had the highest insecticidal activity compared with the other fungicides. Furthermore, tebuconazole significantly inhibited the expression of the chitin synthase gene NlCHS1; the chitinase genes NlCht1, NlCht5, NlCht7, NlCht9, and NlCht10; and the β‐N‐acetylhexosaminidase genes NlHex3, NlHex4, NlHex5 and NlHex6; it significantly suppressed the expression of ecdysteroid biosynthetic genes as well, including SDR, CYP307A2, CYP307B1, CYP306A2, CYP302A1, CYP315A1 and CYP314A1 of N. lugens. Additionally, tebuconazole affected the diversity, structure, composition, and function of the symbiotic fungi of N. lugens, as well as the relative abundance of saprophytes and pathogens, suggesting that tebuconazole reshapes the diversity and function of symbiotic fungi of N. lugens. CONCLUSION: Our findings illustrate the insecticidal mechanism of tebuconazole, possibly by inhibiting normal molting or disrupting microbial homeostasis in N. lugens, and provide an important rationale for developing novel insect management strategies to delay escalating insecticide resistance. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Chitin is one of the most abundant natural biopolymers and serves as a critical structural component of extracellular matrices, including fungal cell walls and insect exoskeletons. As a linear polymer of β-(1,4)-linked N-acetylglucosamine, chitin is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. In this study, we determine seven different cryo-electron microscopy structures of a Saccharomyces cerevisiae chitin synthase in the absence and presence of glycosyl donor, acceptor, product, or peptidyl nucleoside inhibitors. Combined with functional analyses, these structures show how the donor and acceptor substrates bind in the active site, how substrate hydrolysis drives self-priming, how a chitin-conducting transmembrane channel opens, and how peptidyl nucleoside inhibitors inhibit chitin synthase. Our work provides a structural basis for understanding the function and inhibition of chitin synthase. Chitin, the second most abundant natural polysaccharide in nature, is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. Here the authors determine cryo-EM structures of the chitin synthase, which reveal its activation, catalytic and inhibitory mechanisms [ABSTRACT FROM AUTHOR]

Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental adaptation, virulence, and metabolism in fungi. However, except for ChsC, a class III CHS, little is known about the functions of CHSs in Aspergillus niger, an important fungus that is widely applied in the fermentation industry and food processing, as well as a spoilage fungus of food and a human pathogen. This study showed the important functions of ChsA, a class II CHS, in A. niger using multi-phenotypic and transcriptional analyses under various conditions. The deletion of chsA led to severe defects in conidiation on different media and resulted in the formation of smaller and less compact pellets with less septa in hyphal cells during submerged fermentation. Compared with the WT, the ΔchsA mutants exhibited less chitin content, reduced growth under the stresses of cell wall-disturbing and oxidative agents, more released protoplasts, a thicker conidial wall, decreased production of amylases, pectinases, cellulases, and malic acid, and increased citric acid production. However, ΔchsA mutants displayed insignificant changes in their sensitivity to osmotic agents and infection ability on apple. These findings concurred with the alteration in the transcript levels and enzymatic activities of some phenotype-related genes. Conclusively, ChsA is important for cell wall integrity and mycelial morphology, and acts as a positive regulator of conidiation, cellular responses to oxidative stresses, and the production of malic acid and some enzymes, but negatively regulates the citric acid production in A. niger. [ABSTRACT FROM AUTHOR]

The burrowing nematode Radopholus similis is considered a major problem of intensive banana cultivation. It can cause extensive root damage resulting in the toppling disease of banana, which means that plants fall to the ground. Soaking R. similis in double-stranded (ds) RNA of the nematode genes Rps13, chitin synthase (Chs-2), Unc-87, Pat-10 or beta-1,4-endoglucanase (Eng1a) suppressed reproduction on carrot discs, from 2.8-fold (Chs-2) to 7-fold (Rps13). The East African Highland Banana cultivar Nakitembe was then transformed with constructs for expression of dsRNA against the same genes, and for each construct, 30 independent transformants were tested with nematode infection. Four months after transfer from in vitro culture to the greenhouse, the banana plants were transferred to a screenhouse and inoculated with 2000 nematodes per plant, and thirteen weeks later, they were analyzed for several parameters including plant growth, root necrosis and final nematode population. Plants with dsRNA constructs against the nematode genes were on average showing lower nematode multiplication and root damage than the nontransformed controls or the banana plants expressing dsRNA against the nonendogenous gene. In conclusion, RNAi seems to efficiently protect banana against damage caused by R. similis, opening perspectives to control this pest. [ABSTRACT FROM AUTHOR]

Papaya fruit cv. Maradol showing typical symptoms of anthracnose were observed in a commercial orchard located in the municipality of Pinotepa Nacional at the coastal region of Oaxaca, Mexico in September 2020. The incidence rate of anthracnose reached 60%. The symptoms consisted of sunken, water-soaked lesions with salmon-coloured conidia masses on the surface of papaya fruit. The aim of this study was to identify the causal agent of papaya anthracnose based on the phylogenetic analysis of four concatenated datasets. The isolate exhibited sparse white to greyish aerial mycelium and clearly shaped concentric rings with exudate of orange conidia on potato dextrose agar. The fungus was identified as Colletotrichum brevisporum using sequence data of the internal transcribed spacer (ITS) region, and part of the actin (act), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and chitin synthase (chs-1) genes. The fungus isolated from diseased fruit produced lesions on wounded fruit and unwounded fruit. The pathogen was re-isolated from lesions on inoculated fruit to fulfill Koch's postulates. To our knowledge, this is the first report of C. brevisporum causing anthracnose disease on papaya fruit in Mexico. [ABSTRACT FROM AUTHOR]

It is interesting to identify factors involved in the regulation of the encystation of Entamoeba histolytica that differentiate trophozoites into cysts. Evolutionarily conserved three amino acid loop extension (TALE) homeodomain proteins act as transcription factors and execute a variety of functions that are essential for life. A TALE homeodomain (EhHbox) protein‐encoding gene has been identified in E. histolytica (Eh) that is highly upregulated during heat shock, glucose, and serum starvation. Its ortholog, EiHbox1, a putative homeobox protein in E. invadens (Ei), is also highly upregulated during the early hours of encystation, glucose starvation, and heat shock. They belong to the PBX family of TALE homeobox proteins and have conserved residues in the homeodomain that are essential for DNA binding. Both are localized in the nucleus during encystation and under different stress conditions. The electrophoretic mobility shift assay confirmed that the recombinant GST‐EhHbox binds to the reported TGACAG and TGATTGAT motifs. Down‐regulation of EiHbox1 by gene silencing reduced Chitin synthase, Jacob, and increased Jessie gene expression, resulting in defective cysts and decreased encystation efficiency and viability. Overall, our results suggest that the TALE homeobox family has been conserved during evolution and acts as a transcription factor to control the differentiation of Entamoeba by regulating the key encystation‐induced genes. [ABSTRACT FROM AUTHOR]

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) affects Aspergillus fumigatus Af293's growth in vitro, iron metabolism, resistance in intermicrobial competition with Pseudomonas aeruginosa, resistance to osmotic stress, and resistance to the chitin synthase inhibitor nikkomycin Z. Here, we show that response to high temperature, Congo Red-induced stress, and hydrogen peroxide are also dependent on the viral infection status of A. fumigatus. AfuPmV-1- infected Af293 was more susceptible than virus-free Af293 to growth inhibition by high temperature, hydrogen peroxide, Congo Red exposure, and nutrient restriction. Increased resistance of virus-free fungus was observed when cultures were started from conidia but, in the case of high temperature and hydrogen peroxide, not when cultures were started from hyphae. This indicates that the virus impairs the stress response during the growth phase of germination of conidia and development into hyphae. In conclusion, our work indicates that AfuPmV-1 infection in A. fumigatus impairs host responses to stress, as shown by exposure to high temperature, oxidative stress such as hydrogen peroxide, and some cell wall stresses, as shown by exposure to Congo Red (in agreement with our previous observations using nikkomycin Z) and nutrient restriction. [ABSTRACT FROM AUTHOR]

Several Colletotrichum species are able to cause anthracnose disease in coffee (Coffea arabica L.) and occur in all coffee production areas worldwide. A planned investigation of coffee plantations was carried out in Southwest Saudi Arabia in October, November, and December 2022. Various patterns of symptoms were observed in all 23 surveyed coffee plantations due to unknown causal agents. Isolation from symptomatic fresh samples was performed on a PDA medium supplemented with streptomycin sulfate (300 mg L−1) and copper hydroxide (42.5 mg L−1). Twenty-seven pure isolates of Colletotrichum-like fungi were obtained using a spore suspension method. The taxonomic placements of Colletotrichum-like fungi were performed based on the sequence dataset of multi-loci of internal transcribed spacer region rDNA (ITS), chitin synthase I (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB2), and partial mating type (Mat1–2) (ApMat) genes. The novel species are described in detail, including comprehensive morphological characteristics and colored illustrations. The pathogenicity of the isolated Colletotrichum species was assessed on detached coffee leaves as well as green and red fruit under laboratory conditions. The multi-locus phylogenetic analyses of the six-loci, ITS, ACT, CHS-1, TUB2, GAPDH and ApMat, revealed that 25 isolates were allocated within the C. gloeosporioides complex, while the remaining two isolates were assigned to the C. boninense complex. Six species were recognized, four of them, C. aeschynomenes, C. siamense, C. phyllanthi, and C. karstii, had been previously described. Based on molecular analyses and morphological examination comparisons, C. saudianum and C. coffeae-arabicae represent novel members within the C. gloeosporioides complex. Pathogenicity investigation confirmed that the Colletotrichum species could induce disease in coffee leaves as well as green and red fruits with variations. Based on the available literature and research, this is the first documentation for C. aeschynomenes, C. siamense, C. karstii, C. phyllanthi, C. saudianum, and C. coffeae-arabicae to cause anthracnose on coffee in Saudi Arabia. [ABSTRACT FROM AUTHOR]

Simple Summary: Heortia vitessoides Moore is a leaf-eating pest that affects Aquilaria sinensis. In the outbreak period, the leaves of Aquilaria sinensis can be eaten up in a short time, resulting in the death of trees and great economic losses. Chitin is the main component of insect cuticle, peritrophic membrane, and tracheal intima. Chitin synthesis in insects is a complex process that requires the cooperation of many enzymes. Chitin synthase is one of the key enzymes in the process. Chitin synthase is divided into two types: chitin synthase A gene (Chsa) and chitin synthase B gene (Chsb). Chsb is mainly responsible for the tissue synthesis of chitin in midgut peritrophic membrane. It was found that the expression of HvChsb was inhibited, the growth and development were abnormal, and the mortality rate was increased. These findings provide a reference for the prevention and control of the pest from the perspective of gene manipulation. The chitin synthase B gene is a key enzyme in the chitin synthesis of insect peritrophic matrix (PM), which affects insects' feeding behavior. The chitin synthase B gene was cloned from the transcription library of Heortia vitessoides Moore. RT-qPCR showed that HvChsb was highly expressed in the larval stage of H. vitessoides, especially on the first day of the pre-pupal stage, as well as in the midgut of larvae and the abdomen of adults. After starvation treatment, HvChsb was found to be significantly inhibited over time. After 48 h of starvation, the feeding experiment showed that HvChsb increased with the prolongation of the re-feeding time. The experimental data showed that feeding affected the expression of HvChsb. HvChsb was effectively silenced via RNA interference; thus, its function was lost, significantly decreasing the survival rate of H. vitessoides. The survival rate from larval-to-pupal stages was only 43.33%, and this rate was accompanied by abnormal phenotypes. It can be seen that HvChsb plays a key role in the average growth and development of H. vitessoides. [ABSTRACT FROM AUTHOR]

Fungal infections, such as those caused by Aspergillus, Alternaria, Colletotrichum, Penicillium, and Rhizopus, are considered the major global threat to human life. In seeking a treatment, we synthesized and characterized copper nanoparticles (CuNPs) using Curcuma longa extract. C. longa plant extract has been previously studied and validated for its strong antimicrobial properties. Novel green particles were synthesized in this study using C. longa and copper nitrate. We also investigated antifungal activity through inhibition studies and real-time expression of gene members belonging to the chitin synthase family. Synthesized CuNPs were characterized by ultraviolet-visible spectroscopy, Fourier transform-infrared spectroscopy, X-ray diffraction, and nanoparticle tracking analysis. Gene expression was determined by real-time PCR. The results visibly confirmed the antifungal activity of the synthesized CuNPs against Aspergillus through zones of growth inhibition. The zone diameters were comparable to those of the positive control used in the study. The synthesized CuNPs were 60 nm in diameter and with a resonance peak at 535 nm. The observations of the downregulation of chitin synthase gene members 1, 2, and 3 suggest significant antifungal activity of the synthesized CuNPs. The collective findings indicate the potential value of green-synthesized CuNPs as antifungal agents.

The rice stem borer, Chilo suppressalis, has developed a high level of resistance to many of the compounds currently used for control. There is therefore an urgent need to develop novel control methods for C. suppressalis. Insect chitin synthases (CHS) have attracted interest as a potential target for insect pest management. However, to date, CHS have not been characterized in C. suppressalis. Two CHS genes (CsCHS1 and CsCHS2) were identified and cloned from C. suppressalis. Two transcript variants were identified for CsCHS1, CsCHS1a and CsCHS1b. Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument. In contrast, CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut. Knockdown of CsCHS1 by RNA interference significantly inhibited the molting and pupation of C. suppressalis, and knockdown of CsCHS2 significantly affected growth during the larval stage, but had no significant effect on the pupation. Moreover, knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate, larval survivorship, pupation rate, and eclosion rate, but only larval survivorship at the G0 generation was lowered after the knockout of CsCHS2. These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C. suppressalis, and so have potential as insecticidal targets for the control of this highly damaging pest. [ABSTRACT FROM AUTHOR]

Camellia oleifera, a woody plant that produces edible oil, is indigenous to China. The devastating disease of anthracnose inflicts significant financial losses on Ca. oleifera. The primary causative agent of anthracnose on Ca. oleifera is Colletotrichum fructicola. Chitin, a pivotal constituent of fungal cell walls, assumes a critical function in their proliferation and maturation. To study the biological functions of chitin synthase 1(Chs1) in C. fructicola, the CfCHS1 gene knockout mutants, ∆Cfchs1-1 and ∆Cfchs1-2, and their complementary strain, ∆Cfchs1/CfCHS1, of C. fructicola were generated. Our results showed that the colony diameters of wild-type and complement-strain ∆Cfchs1/CfCHS1, mutant ∆Cfchs1-1 and ∆Cfchs1-2 cultured on the CM and MM medium were 5.2, 5.0, 2.2 and 2.4 cm and 4.0, 4.0, 2.1 and 2.6 cm, respectively, which were significantly smaller for the mutant than for the wild type and complement strain; the inhibition rates on the CM medium supplemented with H2O2, DTT, SDS and CR were 87.0% and 88.5%, 29.6% and 27.1%, 88.0% and 89.4%, and 41.7% and 28.7%, respectively, for the mutant strains, ∆Cfchs1-1 and ∆Cfchs1-2, which were significantly higher than those for the other two strains; the rate of hyphal tips with CFW fluorescence in ∆Cfchs1-1 and ∆Cfchs1-2 was 13.3% and 15.0%, which was significantly lower than those for the other two strains; the mutant strains, ∆Cfchs1-1 and ∆Cfchs1-2, lost the ability to produce conidia; the mutant strains showed weaker pathogenicity on wounded and unwounded Ca. oleifera leaves than the wild type and complement strain. The findings of this study suggest that CfChs1 plays a crucial role in the growth and development, stress responses, and pathogenicity of C. fructicola. Thus, this gene could be a potential target for developing novel fungicide. [ABSTRACT FROM AUTHOR]

In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region. [ABSTRACT FROM AUTHOR]

The spotted bollworm Earias vittella (Lepidoptera: Nolidae) is a polyphagous pest with enormous economic significance, primarily affecting cotton and okra. However, the lack of gene sequence information on this pest has a significant constraint on molecular investigations and the formulation of superior pest management strategies. An RNA-seq-based transcriptome study was conducted to alleviate such limitations, and de novo assembly was performed to obtain transcript sequences of this pest. Reference gene identification across E. vittella developmental stages and RNAi treatments were conducted using its sequence information, which resulted in identifying transcription elongation factor (TEF), V-type proton ATPase (V-ATPase), and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as the most suitable reference genes for normalization in RT-qPCR-based gene expression studies. The present study also identified important developmental, RNAi pathway, and RNAi target genes and performed life-stage developmental expression analysis using RT-qPCR to select the optimal targets for RNAi. We found that naked dsRNA degradation in the E. vittella hemolymph is the primary reason for poor RNAi. A total of six genes including Juvenile hormone methyl transferase (JHAMT), Chitin synthase (CHS), Aminopeptidase (AMN), Cadherin (CAD), Alpha-amylase (AMY), and V-type proton ATPase (V-ATPase) were selected and knocked down significantly with three different nanoparticles encapsulated dsRNA conjugates, i.e., Chitosan-dsRNA, carbon quantum dots-dsRNA (CQD-dsRNA), and Lipofectamine-dsRNA conjugate. These results demonstrate that feeding nanoparticle-shielded dsRNA silences target genes and suggests that nanoparticle-based RNAi can efficiently manage this pest. [ABSTRACT FROM AUTHOR]

Bletilla striata is a traditional Chinese medicine plant. Guizhou Province is the largest producer of B. striata, with the biggest cultivation area and highest yield in China. Anthracnose is the main disease affecting the crop. In July 2021, an anthracnose outbreak occurred in Shibing County, Guizhou Province, seriously affecting the yield and quality of B. striata. Thirty samples of B. striata anthracnose were collected from Shuangjing, Niudachang, and Yangliutang towns in three main planting areas of Shibing County to clarify the causal agent of B. striata anthracnose. Fifty anthracnose strains were isolated by tissue isolation and single-conidium culture. Through morphological observation and multigene phylogenetic analysis of the internal transcribed spacer, actin, beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase and chitin synthase genes, the isolated strains were identified as Colletotrichum spaethianum. The infection process of C. spaethianum on B. striata leaves was observed by cell tissue staining. The results showed that at 25°C, the conidia of C. spaethianum on B. striata leaves began to germinate after 3 h, and the peak period of conidia germination was 8 h. Appressoria formation peaked after 24 h. During 48–72 h, the hyphae randomly expanded horizontally and vertically on the host surface, gradually forming a reticular distribution. This study is the first report of C. spaethianum causing anthracnose in B. striata. Our study clarifies the infection and development process of C. spaethianum on B. striata leaves, thus providing a theoretical basis for further study on the monitoring of B. striata anthracnose. [ABSTRACT FROM AUTHOR]

Verticillium wilt (VW) of cotton poses a serious threat to the quality and yield of cotton. Verticillium dahliae is the primary causal agent of cotton VW. Moreover, V. dahliae can infect more than 200 species of dicotyledonous plants. The fungal cell wall plays a crucial role in its growth, development and pathogenicity. However, the mechanism of cell wall synthesis in V. dahliae and its role in pathogenesis remains unclear. In this study, we identified two chitin synthase (CHS) genes VdChs5 and VdChs7 containing myosin motor-like domain (MMD) and characterized their role in virulence of V. dahliae. The results showed that the functions of VdChs5 and VdChs7 were largely redundant, and target deletion of both VdChs5 and VdChs7 in V. dahliae did not affect vegetative growth, but reduced conidial production. ΔVdChs5Chs7 deletion mutant failed to colonize and proliferate in cotton vascular tissue, and exhibited significantly reduced virulence on cotton, suggesting that VdChs5 and VdChs7 are necessary for pathogenesis. In addition, the thickness of the cell wall in ΔVdChs5Chs7 showed significantly decreased, and ΔVdChs5Chs7 mutant exhibited hypersensitivity to cell wall perturbing agents and reactive oxygen species (ROS), indicating that VdChs5 and VdChs7 play key roles in cell wall integrity. Further, host-induced gene silencing (HIGS) silenced transcripts of VdChs5 and VdChs7 in susceptible cotton (Gossypium hirsutum L. acc. TM-1) enhanced resistance to cotton VW. Taken together, our data demonstrated that VdChs5 and VdChs7 play pivotal roles in proliferation, cell wall integrity, and pathogenicity, and provided a novel strategy to improve Verticillium wilt resistance in cotton and other susceptible host plants. [ABSTRACT FROM AUTHOR]

Chitin is one of the most abundant polysaccharides in nature, forming important structures in insects, crustaceans, and fungal cell walls. Vertebrates on the other hand are generally considered "nonchitinous" organisms, despite having highly conserved chitin metabolismassociated genes. Recent work has revealed that the largest group of vertebrates, the teleosts, have the potential to both synthesize and degrade endogenous chitin. Yet, little is known about the genes and proteins responsible for these dynamic processes. Here, we used comparative genomics, transcriptomics, and chromatin accessibility data to characterize the repertoire, evolution, and regulation of genes involved in chitin metabolism in teleosts, with a particular focus on Atlantic salmon. Reconstruction of gene family phylogenies provides evidence for an expansion of teleost and salmonid chitinase and chitin synthase genes after multiple whole-genome duplications. Analyses of multi-tissue gene expression data demonstrated a strong bias of gastrointestinal tract expression for chitin metabolism genes, but with different spatial and temporal tissue specificities. Finally, we integrated transcriptomes from a developmental time series of the gastrointestinal tract with chromatin accessibility data to identify putative transcription factors responsible for regulating chitin metabolism gene expression (CDX1 and CDX2) as well as tissue-specific divergence in the regulation of gene duplicates (FOXJ2). The findings presented here support the hypothesis that chitin metabolism genes in teleosts play a role in developing and maintaining a chitin-based barrier in the teleost gut and provide a basis for further investigations into the molecular basis of this barrier. [ABSTRACT FROM AUTHOR]