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Spodoptera frugiperda is a significant agricultural pest, severely impacting the yield and quality of grain. Chitin is the momentous component of exoskeletons, which has a significant impact on the growth and development of insects. Our previous study found that exposure to lufenuron can reduce the expression of chitinase gene (SfCHT5) and increase the expression of chitin synthase gene (SfCHSB), two key genes for chitin synthesis in S. frugiperda. However, the post-transcriptional regulatory mechanisms of these key genes in S. frugiperda remain unclear. With miRNA as the entry point, target site prediction, dual luciferase reporter assays, and mimics/inhibitors injection were used to explore the post-transcriptional regulatory mechanism of SfCHSB and SfCHT5. The combined results confirm that miR-10482-5p targets SfCHT5 and regulates its expression, while miR-9a targets and regulates the expression of both SfCHT5 and SfCHSB. Additionally, injection of miR-10482-5p and miR-9a mimic significantly reduced the mortality rates of S. frugiperda treated with lufenuron, increased pupation rate, and emergence rate. These findings underscore the critical regulatory role of miR-10482-5p and miR-9a in modulating the expression of key genes involved in chitin synthesis, thereby enhancing the tolerance of S. frugiperda to lufenuron and influencing its growth and development. In summary, this study preliminarily elucidates the post-transcriptional regulatory mechanisms of miRNA-mediated SfCHSB and SfCHT5 expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Clade A APSES family transcription factor Swi6 functions alongside Mbp1 to form the MBF (MluI cell cycle box-binding factor) complex in ascomycetes. In the agaricomycete Pleurotus ostreatus, Mbp1 plays a crucial role in regulating β-glucan and chitin synthesis; however, the role of Swi6 has not been explored in this fungus. In this study, its involvement in cell wall synthesis regulation was analysed using swi6 disruption strains in P. ostreatus. The Δswi6 strains exhibited reduced growth rates and shorter aerial hyphae formation in both agar and liquid media, suggesting an essential role of Swi6 in normal vegetative growth. Furthermore, swi6 disruption affected cell wall thickness distribution, the expression of specific chitin synthase genes, the relative percentage of chitin, and sensitivity to calcofluor white, suggesting that Swi6 is required for normal chitin synthesis regulation in P. ostreatus. In contrast, no significant differences were observed between the wild-type and Δswi6 strains in the relative percentage of α- and β-glucan and the expression of α- and β-glucan synthase genes, suggesting its unimportant role in α- and β-glucan synthesis regulation. In conclusion, Swi6 is necessary for normal mycelial growth and chitin synthesis regulation in P. ostreatus. To the best of our knowledge, this study is the first report on the functional differences and overlaps between Mbp1 and Swi6 in the regulation of cell wall synthesis in agaricomycetes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tetranychus urticae , a highly destructive global pest, infests a wide range of plant species, including crucial food crops and ornamental plants. Effective control methods for this pest remain limited. Chitin synthase (CHS) is a key enzyme in the biosynthesis of chitin, which is essential for the growth and development of arthropods. However, the lack of detailed research on arthropod CHS proteins has hindered the development of targeted pesticides. In this study, we successfully expressed and purified the full-length Tu CHS protein, which exhibited significant enzymatic activity in vitro . Utilizing this protein, we developed a reliable screening method to identify inhibitors targeting Tu CHS. Two inhibitors, ZHZ-ZI-11 and SUY-SC-15, were identified. These compounds interfere with chitin translocation within the cell rather than inhibiting CHS enzyme activity. Both inhibitors demonstrated significant acaricidal efficacy, with improved performance when formulated as nanoemulsions. This study presents the first use of arthropod CHS protein for screening potential insecticides targeting chitin biosynthesis during mite development. Our findings provide a solid foundation for the development of novel, environmentally friendly pesticides aimed at CHS.

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 Δ FvChs3 . 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 Δ FvChs3 , while genes related to the glycolysis pathway for ethanol synthesis were downregulated. Further knockout of pyruvate decarboxylase gene FvPDC6 in Δ FvChs3 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.

Chitin-synthase (CHS) is found in most eukaryotes and has a complex evolutionary history. Research into CHS has mainly been in the context of biomineralization of mollusc shells an area of high interest due to the consequences of ocean acidification. Exploration of CHS at the genomic level in molluscs, the evolution of isoforms, their tissue distribution, and response to environmental challenges are largely unknown. Exploiting the extensive molecular resources for mollusc species it is revealed that bivalves possess the largest number of CHS genes (12-22) reported to date in eukaryotes. The evolutionary tree constructed at the class level of molluscs indicates four CHS Type II isoforms (A-D) probably existed in the most recent common ancestor, and Type II-A (Type II-A-1/Type II-A-2) and Type II-C (Type II-C-1/Type II-C-2) underwent further differentiation. Non-specific loss of CHS isoforms occurred at the class level, and in some Type II (B-D groups) isoforms the myosin head domain, which is associated with shell formation, was not preserved and highly species-specific tissue expression of CHS isoforms occurred. These observations strongly support the idea of CHS functional diversification with shell biomineralization being one of several important functions. Analysis of transcriptome data uncovered the species-specific potential of CHS isoforms in shell formation and a species-specific response to ocean acidification (OA). The impact of OA was not CHS isoform-dependent although in Mytilus, Type I-B and Type II-D gene expression was down-regulated in both M. galloprovincialis and M. coruscus. In summary, during CHS evolution the gene family expanded in bivalves generating a large diversity of isoforms with different structures and with a ubiquitous tissue distribution suggesting that chitin is involved in many biological functions. These findings provide insight into CHS evolution in molluscs and lay the foundation for research into their function and response to environmental changes., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 CuSO 4 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., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 ( Ps CHS) is a processive glycosyltransferase, which can rapidly produce and tightly bind with the highly polymerized chitin. We further demonstrate that Ps CHS is a bifunctional enzyme, which is necessary and sufficient to translocate the synthesized chitin. Ps CHS 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 Ps CHS translocates the newly produced chitin into the lumen of the PLs. We also attempted to resolve the Ps CHS structure of the synthesized chitin-bound state, although it was not successful; the obtained high-resolution structure of the UDP/Mn 2+ -bound state could still assist in describing the characterization of the Ps CHS's transmembrane channel. Consistently, we demonstrate that Ps CHS is indispensable and capable of translocating chitin in a process that is tightly coupled to chitin synthesis.

Background: Browning is the key problem hindering the industrialization of pear wine. The use of high-yield glutathione Saccharomyces cerevisiae in the fermentation of pear wine can inhibit browning. Glutathione reductase (GR) can ensure the reduction of glutathione. Spore immobilization of enzymes is a new technology. It is a new attempt to apply spore-immobilized GR in combination with high-yield glutathione S. cerevisiae to inhibit browning of pear wine., Results: Saccharomyces cerevisiae spore immobilization enzyme technology was used to immobilize GR in the spores of mutant S. cerevisiae dit1∆, osw2∆ and chs3∆ and wild-type S. cerevisiae. The enzyme activity of GR immobilized by chs3∆ spores was the highest of 3.08 U mg -1  min -1 . The chs3∆ spore-immobilized GR had certain resistance to ethanol, citric acid, sucrose, glucose and proteinase K. Electron microscopy analysis showed that the spore wall of chs3∆ had moderate size holes, which might be the main reason why it immobilized GR with the highest enzyme activity. And the GR was immobilized between the prespore membrane and mannoprotein layer of the spore wall. When chs3∆ spore-immobilized GR (chs3∆-GR) was added to Dangshan pear wine fermented by high-yield glutathione S. cerevisiae JN32-9, the presence of chs3∆-GR could further protect amino acids, polyphenols and glucose from oxidation, thereby reducing the browning of the pear wine during storage by 47.32%., Conclusion: GR immobilized by S. cerevisiae spores was effective in inhibiting the browning of pear wine. The method was simple, green and effective and did not increase the production cost of pear wine. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The invasive species Aedes albopictus is a major vector of several arboviruses. The global spread of this species seriously threatens human health. Insecticide resistance is an increasing problem worldwide that limits the efficacy of mosquito control. As the major structural component of cuticles, chitin is indispensable to insects. Chitin synthase (CHS) is the enzyme that catalyzes the biosynthesis of chitin at the final step. In this study, two CHS genes of Aedes albopictus (AaCHS1 and AaCHS2) were identified and their basic characteristics were evaluated via bioinformatics analysis. The highest abundance of AaCHS1 transcripts was detected in pupae, whereas that of AaCHS2 transcripts was detected in females; the highest expression levels of AaCHS1 and AaCHS2 were found in the epidermis and the midgut of pupae, respectively. The survival and emergence rates of pupae were significantly reduced after the injection of double-stranded RNA of AaCHS1 or AaCHS2, indicating that both AaCHS1 and AaCHS2 play crucial roles in the pupal development. In addition, the chitin content of pupae was obviously decreased after the suppression of AaCHS1 expression by RNA interference (RNAi) treatment. This influence of the RNAi treatment was further supported by the reduced chitin thickness and weakened chitin fluorescence signal in the new cuticle. The midgut of pupae presented a reduced intensity of the chitin fluorescence signal along with RNAi treatment specific to AaCHS2 expression. The results of this study indicate that CHS genes may be suitable as molecular targets used for controlling mosquitoes., (© 2024 Wiley Periodicals LLC.)

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., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chitin is an essential structural component of fungal cell walls composed of transmembrane proteins called chitin synthases (CHSs), which have a large range of reported effects in ascomycetes; however, are poorly understood in agaricomycetes. In this study, evolutionary and molecular genetic analyses of chs genes were conducted using genomic information from nine ascomycete and six basidiomycete species. The results support the existence of seven previously classified chs clades and the discovery of three novel basidiomycete-specific clades (BI-BIII). The agaricomycete fungus Pleurotus ostreatus was observed to have nine putative chs genes, four of which were basidiomycete-specific. Three of these basidiomycete specific genes were disrupted in the P. ostreatus 20b strain (ku80 disruptant) through homologous recombination and transformants were obtained (Δchsb2, Δchsb3, and Δchsb4). Despite numerous transformations Δchsb1 was unobtainable, suggesting disruption of this gene causes a crucial negative effect in P. ostreatus. Disruption of these chsb2-4 genes caused sparser mycelia with rougher surfaces and shorter aerial hyphae. They also caused increased sensitivity to cell wall and membrane stress, thinner cell walls, and overexpression of other chitin and glucan synthases. These genes have distinct roles in the structural formation of aerial hyphae and cell walls, which are important for understanding basidiomycete evolution in filamentous fungi., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely elucidated, nor has its disturbing effect on chitin synthesis genes. In this study, bioassay results demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC 25 lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that five chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increase in larval susceptibilities to LC 25 lufenuron by 75.48%, 65.00%, 68.42% and 28.00%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and proved that the combination of RNAi and lufenuron would improve the control effect of this pest., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023. Published by Elsevier Inc.)

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The mycelium of the plant pathogenic fungus Fusarium graminearum exhibits distinct structures for vegetative growth, asexual sporulation, sexual development, virulence, and chlamydospore formation. These structures are vital for the survival and pathogenicity of the fungus, necessitating precise regulation based on environmental cues. Initially identified in Magnaporthe oryzae , the transcription factor Con7p regulates conidiation and infection-related morphogenesis, but not vegetative growth. We characterized the Con7p ortholog Fg Con7, and deletion of FgCON7 resulted in severe defects in conidium production, virulence, sexual development, and vegetative growth. The mycelia of the deletion mutant transformed into chlamydospore-like structures with high chitin level accumulation. Notably, boosting FgABAA expression partially alleviated developmental issues in the FgCON7 deletion mutant. Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis confirmed a direct genetic link between FgABAA and FgCON7 . Furthermore, the chitin synthase gene Fg6550 (FGSG_06550) showed significant upregulation in the FgCON7 deletion mutant, and altering FgCON7 expression affected cell wall integrity. Further research will focus on understanding the behavior of the chitin synthase gene and its regulation by Fg Con7 in F. graminearum . This study contributes significantly to our understanding of the genetic pathways that regulate hyphal differentiation and conidiation in this plant pathogenic fungus., Importance: The ascomycete fungus Fusarium graminearum is the primary cause of head blight disease in wheat and barley, as well as ear and stalk rot in maize. Given the importance of conidia and ascospores in the disease cycle of F. graminearum , precise spatiotemporal regulation of these biological processes is crucial. In this study, we characterized the Magnaporthe oryzae Con7p ortholog and discovered that Fg Con7 significantly influences various crucial aspects of fungal development and pathogenicity. Notably, overexpression of FgABAA partially restored developmental defects in the FgCON7 deletion mutant. ChIP-qPCR analysis confirmed a direct genetic link between FgABAA and FgCON7 . Furthermore, our research revealed a clear correlation between Fg Con7 and chitin accumulation and the expression of chitin synthase genes. These findings offer valuable insights into the genetic mechanisms regulating conidiation and the significance of mycelial differentiation in this plant pathogenic fungus., Competing Interests: The authors declare no conflict of interest.

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 EC 50 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.

Spiders, the major predatory enemies of insect pests in fields, are vulnerable to insecticides. In this study, we observed that the recommended dose of buprofezin delayed the molting of the pond wolf spider Pardosa pseudoannulata, although it had no lethal effect on the spiders. Since buprofezin is an insect chitin biosynthesis inhibitor, we identified two chitin synthase genes (PpCHS1 and PpCHS2) in P. pseudoannulata. Tissue-specific expression profiling showed that PpCHS1 was most highly expressed in cuticle. In contrast, PpCHS2 showed highest mRNA levels in the midgut and fat body. RNAi knockdown of PpCHS1 significantly delayed the molting of 12-days old spiderlings, whereas no significant effect on the molting was observed in the PpCHS2-silencing spiderlings. The expression of PpCHS1 was significantly suppressed in the spiderlings treated with buprofezin, but rescued by exogenous ecdysteroid ponasterone A (PA). Consistent with this result, the molting delay caused by buprofezin was also rescued by PA. The results revealed that buprofezin delayed the molting of spiders by suppressing PpCHS1 expression, which will benefit the protection of P. pseudoannulate and related spider species., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Canine demodicosis is a parasitic condition affecting the skin of dogs. The present study was designed to characterize chitin synthase gene of Demodex canis. The molecular technique was used for better understanding of this gene. A total of 75 dogs which are reared as pets with or without showing any skin lesions were examined during the study period. Skin scrapings were examined by indirect method using 10% potassium hydroxide solution under 10 × microscope. DNA samples were extracted from positive skin samples and were subjected to PCR for molecular identification. A total of 25 dogs irrespective of age, sex, breed or coat showed positive result for D. canis. The PCR revealed a single amplified product of 339 bp length which exactly matched with D. canis. The chitin synthase gene was amplified by PCR, subsequently cloned, sequenced, and compared with available data in GenBank for the particular gene of D. canis. Only one single nucleotide polymorphism (SNP) was noticed at 231 position of the chitin synthase gene sequence when compared to other isolates. The molecular technique confirms with the morphological identity of D. canis. This report signifies the value of peculiar tool to identify 'follicular mite' even from apparently healthy skin. [ABSTRACT FROM AUTHOR]

During growth and development of insects, the steroid hormone 20-Hydroxyecdysone (20E) regulates the molting process through activation of a series of genes including E74 , E75 and HR3 by the 20E receptor EcR. Here, we analyzed the function of LmHR3 in the migratory locust Locusta migratoria . By sequence comparison, we first identified and characterized the putative nuclear receptor protein (LmHR3) based on L. migratoria transcriptome data. The full length cDNA is 2272 bp long encoding a protein of 455 amino acids that contains a DNA binding domain (zinc finger) and a ligand binding domain. Phylogenetic analyses showed that LmHR3 has a high homology with the ortholog from Blattaria . RT-qPCR results revealed that LmHR3 has a low level expression in the early days of 5th instar nymphs, and then increases and peaks at day 6, followed by a decrease to low levels before ecdysis. The LmHR3 , hence, coincides with the profile of circulating 20E levels. Indeed, we show that transcription of LmHR3 is induced by 20E in vivo , and significantly suppressed by successfully knocking down expression of LmEcR . After injection of dsRNA for LmHR3 (ds LmHR3 ) at day 2 of earlier instar nymphs (3rd and 4th instar) and final instar nymphs (5th instar), none of the nymphs were able to molt normally, and eventually died. Chitin staining and ultra-structural analysis showed that both the synthesis of the new cuticle and the degradation of the old cuticle were blocked in the ds LmHR3 treated nymphs. Especially, chitin synthesis genes ( LmUAP1 and LmCHS1 ) and chitinase genes ( LmCHT5 and LmCHT10 ) were significantly down-regulated in the ds LmHR3 treatment group. Together, our results suggest that LmHR3 is involved in the control of chitin synthesis and degradation during L. migratoria molting. [ABSTRACT FROM AUTHOR]

We previously reported that the moderate knockdown of chitin synthase 1 gene of the model beetle Tribolium castaneum impairs the host defense against entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae , which infect host insects via the direct penetration of cuticular integuments (Hayakawa et al., 2017). In this study, we focused on the antifungal roles of laccase2 ( Lac2 ) as well as yellow-e ( Y-e ) genes, both of which are shown to be important to the establishment of stable cuticular structures in this beetle species. The expression profiles of the two genes somewhat resembled each other, peaking in late prepupae and mid to late pupae, while the transcript levels of Lac2 were higher than Y-e throughout. The knockdown of Lac2 gene at the prepupal and pupal peaks with relatively small amounts of dsRNA resulted in pupae with a lighter color and adults with a lighter color and dimpled/wrinkled elytra, respectively. Meanwhile, similar gene knockdown of Y-e but with 10 times more dsRNA compared to Lac2 resulted in pupae having a normal appearance and adults with a darker color. We conducted fungal infection assays with B. bassiana and M. anisopliae using these knockdown animals. The knockdown of Y-e gene had no or limited effects in both pupae and adults in terms of the antifungal host defense. Similarly, the knockdown of Lac2 gene did not change significantly the defense phenotypes of the resulting pupae. By sharp contrast, the host defense of the adult beetles against the two fungal species was almost totally destroyed by the moderate knockdown of Lac2 gene, suggesting its indispensable role in antifungal host defense presumably through the construction of sound cuticles of the adults. Finally, we investigated the maturation of host defense against fungal infection in the Lac2 knockdown adults and found that while the day 10 adults were still susceptible to M. anisopliae infection with some delay of death in comparison with day 1 adults, they exhibited complete refractoriness to B. bassiana . [ABSTRACT FROM AUTHOR]

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery. In addition, the interaction of Chs3 with its chaperone Chs7 seems to mask additional cytosolic degrons, thereby avoiding their recognition by the ERAD-C pathway. On top of that, Chs3 molecules that are not degraded by conventional ERAD can move along the ER membrane to reach the inner nuclear membrane, where they are degraded by the inner nuclear membrane-associated degradation (INMAD) system, which contributes to the intracellular homeostasis of Chs3. These results indicate that Chs3 is an excellent model to study quality control mechanisms in the cell and reinforce its role as a paradigm in intracellular trafficking research.

We applied a new RNA interference (RNAi) system using rolling circle transcription (RCT) technology to generate RNA microspheres (RMS) for targeting two key chitin synthetic pathway genes [chitin synthase A (CHSA), chitin synthase B (CHSB)] in the larvae of the oriental armyworm (Mythimna separate), a RNAi-unsusceptible agriculturally important lepidopteran pest. Feeding the third-instar larvae with the RMS-CHSA- or RMS-CHSB-treated corn leaf discs suppressed the expression of CHSA by 81.7% or CHSB by 88.1%, respectively, at 72 h. The silencing of CHSA consequently affected the larval development, including the reduced body weight (54.0%) and length (41.3%), as evaluated on the 7th day, and caused significant larval mortalities (51.1%) as evaluated on the 14th day. Similar results were obtained with the larvae fed RMS-CHSB. We also compared RNAi efficiencies among different strategies: 1) two multi-target RMS [i.e., RMS-(CHSA + CHSB), RMS-CHSA + RMS-CHSB], and 2) multi-target RMS and single-target RMS (i.e., either RMS-CHSA or RMS-CHSB) and found no significant differences in RNAi efficiency. By using Cy3-labeled RMS, we confirmed that RMS can be rapidly internalized into Sf9 cells (<6 h). The rapid cellular uptake of RMS accompanied with significant RNAi efficiency through larval feeding suggests that the RCT-based RNAi system can be readily applied to study the gene functions and further developed as bio-pesticides for insect pest management. Additionally, our new RNAi system takes the advantage of the microRNA (miRNA)-mediated RNAi pathway using miRNA duplexes generated in vivo from the RMS by the target insect. The system can be used for RNAi in a wide range of insect species, including lepidopteran insects which often exhibit extremely low RNAi efficiency using other RNAi approaches., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 Society of Chemical Industry.)

Glycosyltransferases (GTs) are a large and diverse group of enzymes responsible for catalyzing the formation of a glycosidic bond between a donor molecule, usually a monosaccharide, and a wide range of acceptor molecules, thus, playing critical roles in various essential biological processes. Chitin and cellulose synthases are two inverting processive integral membrane GTs, belonging to the type-2 family involved in the biosynthesis of chitin and cellulose, respectively. Herein, we report that bacterial cellulose and chitin synthases share an E-D-D-ED-QRW-TK active site common motif that is spatially co-localized. This motif is conserved among distant bacterial evolutionary species despite their low amino acid sequence and structural similarities between them. This theoretical framework offers a new perspective to the current view that bacterial cellulose and chitin synthases are substrate specific and that chitin and cellulose are organism specific. It lays the ground for future in vivo and in silico experimental assessment of cellulose synthase catalytic promiscuity against uridine diphosphate N-acetylglucosamine and chitin synthase against uridine diphosphate glucose, respectively., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Background: Mosquitoes are an important vector of viral transmission, and due to the complexity of the pathogens they transmit, vector control may be the most effective strategy to control mosquito-borne diseases. Chitin is required for insect growth and development and is absent in higher animals and plants, so regulating the chitin synthesis pathway can serve as a potentially effective means to control vector insects. Most of the current research on the chitin synthase (CHS) gene is focused on chitin synthase-1 (CHS-1), while relatively little is known about chitin synthase-2 (CHS-2)., Results: The CHS-2 gene of Ae. albopictus is highly conserved and closely related to that of Aedes aegypti. The expression of CHS-2 in the third-instar larvae and pupal stage of Ae. albopictus was relatively high, and CHS-2 expression in adult mosquitoes reached the highest value 24 h after blood-feeding. In the fourth-instar larvae of Ae. albopictus, CHS-2 expression was significantly higher in the midgut than in the epidermis. Silencing CHS-2 in Ae. albopictus larvae had no effect on larval survival and emergence. The expression of four genes related to chitin synthesis enzymes was significantly upregulated, the expression level of three genes was unchanged, and only the expression level of GFAT was significantly downregulated. The expression of chitin metabolism-related genes was also upregulated after silencing. The level of chitin in the midgut of Ae. albopictus larvae was significantly decreased, while the chitinase activity was unchanged. The epithelium of the midgut showed vacuolization, cell invagination and partial cell rupture, and the structure of the peritrophic membrane was destroyed or even absent., Methods: The expression of CHS-2 in different developmental stages and tissues of Aedes albopictus was detected by real-time fluorescence quantitative PCR (qPCR). After silencing CHS-2 of the fourth-instar larvae of Ae. albopictus by RNA interference (RNAi), the expression levels of genes related to chitin metabolism, chitin content and chitinase activity in the larvae were detected. The structure of peritrophic membrane in the midgut of the fourth-instar larvae after silencing was observed by paraffin section and hematoxylin-eosin (HE) staining., Conclusion: CHS-2 can affect midgut chitin synthesis and breakdown by regulating chitin metabolic pathway-related genes and is involved in the formation of the midgut peritrophic membrane in Ae. albopictus, playing an important role in growth and development. It may be a potential target for enhancing other control methods., (© 2023. The Author(s).)

Chitin synthase (CHS) is a crucial enzyme involved in the final step of the insect chitin biosynthetic pathway. In this study, we cloned the full-length cDNA sequence of a chitin synthase gene (TCiCHS) from the brown citrus aphid, Toxoptera citricida, an important citrus pest and the main vector of citrus tristeza virus worldwide. TCiCHS was expressed during the entire lifecycle and in all insect tissues examined. Expression was highest in first-second-instar nymphs, nymph-adult transitions and in the abdomen (6.7-fold higher than head). Embryos had a higher expression level than the integument. Fourthinstar nymphs were exposed to 5 and 500 mg/l concentrations of the chitin synthesis inhibitor diflubenzuron (DFB) for 48 h and had the highest mortality at the 500 mg/l concentration. The mRNA expression levels of TCiCHS were significantly enhanced upon the exposure of nymphs to both low and high DFB concentrations. Silencing of TCiCHS occurred through plant-mediated double-stranded RNA (dsRNA) feeding. Most dsRNA-fed nymphs were unable to moult to the next stage, and the expression of TCiCHS decreased 48% compared with controls. These results demonstrate that TCiCHS plays an important role in nymph to adult development, is possibly help identify molecular targets for To. citricida control. [ABSTRACT FROM AUTHOR]

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., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

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 metabolism-associated 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., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., (© 2023. The Author(s).)

The classical dermatophytes diagnosis is based on mycological culture and microscopy observation both human and animal hair, skin, and nail samples. The aim of this work was to develop the new in-house real-time PCR with pan-dematophyte reaction for detection and identification of the main dermatophytes directly from hair samples, providing a simple and rapid diagnosis of dermatophytosis in dogs and cats. An in-house SYBR-Green real-time PCR was designed and used for detecting a DNA fragment encoding chitin synthase 1 (CHS1). A total of 287 samples were processed by culture, microscopic examination with KOH 10%, and real-time PCR (qPCR) analysis. Melting curve analysis of the CHS1 fragment revealed to be reproducible, showing a single distinct peak for each species of dermatophyte, namely Trichophyton mentagrophytes, T. verrucosum, Microsporum canis, and Nannizzia gypsea (formerly M. gypseum). Then, out of the 287 clinically suspected cases of dermatophytosis, 50% were positive for dermatophytes by qPCR, 44% by mycological culture, and 25% by microscopic examination. Microsporum canis was identified in 117 samples tested by culture and 134 samples tested by qPCR, followed by N. gypsea in 5 samples (either tested by culture or qPCR) and T. mentagrophytes detected in 4 and 5 samples when tested by culture or qPCR, respectively. Overall, qPCR allowed the diagnosis of dermatophytosis in clinical samples. The results suggest this newly proposed in-house real-time PCR assay can be used as alternative diagnosis and rapid identification of dermatophytes frequently associated to clinical hair samples of dogs and cats., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Cnidarians are commonly recognized as sea jellies, corals, or complex colonies such as the Portuguese man-of-war. While some cnidarians possess rigid internal calcareous skeletons (e.g., corals), many are soft-bodied. Intriguingly, genes coding for the chitin-biosynthetic enzyme, chitin synthase (CHS) , were recently identified in the model anemone Nematostella vectensis, a species lacking hard structures. Here we report the prevalence and diversity of CHS across Cnidaria and show that cnidarian chitin synthase genes display diverse protein domain organizations. We found that CHS is expressed in cnidarian species and/or developmental stages with no reported chitinous or rigid morphological structures. Chitin affinity histochemistry indicates that chitin is present in soft tissues of some scyphozoan and hydrozoan medusae. To further elucidate the biology of chitin in cnidarian soft tissues, we focused on CHS expression in N. vectensis . Spatial expression data show that three CHS orthologs are differentially expressed in Nematostella embryos and larvae during development, suggesting that chitin has an integral role in the biology of this species. Understanding how a non-bilaterian lineage such as Cnidaria employs chitin may provide new insight into hitherto unknown functions of polysaccharides in animals, as well as their role in the evolution of biological novelty.

β-Chitin has important ecological and physiological roles and potential for widespread applications, but the characterization of chitin-related enzymes from β-chitin producers was rarely reported. Querying against the Tara Oceans Gene Atlas, 4,939 chitin-related unique sequences from 12 Pfam accessions were found in Bacillariophyta metatranscriptomes. Putative chitin synthase (CHS) sequences are decreasingly present in Crustacea (39%), Stramenopiles (16%) and Insecta (14%) from the Marine Atlas of Tara Oceans Unigenes version 1 Metatranscriptomes (MATOUv1+T) database. A CHS gene from the model diatom Thalassiosira pseudonana (Thaps3&#95;J4413, designated Tp CHS1 ) was identified. Homology analysis of Tp CHS1 in Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP), PhycoCosm, and the PLAZA diatom omics data set showed that Mediophyceae and Thalassionemales species were potential new β-chitin producers besides Thalassiosirales. Tp CHS1 was overexpressed in Saccharomyces cerevisiae and Phaeodactylum tricornutum. In transgenic P. tricornutum lines, TpCHS1-eGFP localizes to the Golgi apparatus and plasma membrane and predominantly accumulates in the cleavage furrow during cell division. Enhanced Tp CHS1 expression could induce abnormal cell morphology and reduce growth rates in P. tricornutum , which might be ascribed to the inhibition of the G2/M phase. S. cerevisiae was proved to be a better system for expressing large amounts of active TpCHS1, which effectively incorporates UDP- N -acetylglucosamine in radiometric in vitro assays. Our study expands the knowledge on chitin synthase taxonomic distribution in marine eukaryotic microbes, and is the first to collectively characterize an active marine diatom CHS which may play an important role during cell division. IMPORTANCE As the most abundant biopolymer in the oceans, the significance of chitin and its biosynthesis is rarely demonstrated in diatoms, which are the main contributors to the primary productivity of the oceans, ascribed to their huge biomass and efficient photosynthesis. We retrieved genes involved in chitin-based metabolism against the Tara Oceans Gene Atlas to expand our knowledge about their diversity and distribution in the marine environment. Potential new producers of chitin were found from the analysis of various algal transcriptome and genome databases. Heterologous expression confirms that Thalassiosira pseudonana contains an active chitin synthase (CHS) which may play an important role in the cell division process of diatoms. This study provides new insight into CHS geographic and taxonomic distribution in marine eukaryotic microbes, as well as into a new CHS functioning in the biosynthesis of β-chitin in diatoms.

Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, we use Drosophila as a model. We previously showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. Here, we carry out a cellular and molecular analysis of chitin deposition, and we show that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. We propose a model in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 De Giorgio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lufenuron is an effective benzoylurea insecticide that inhibits the synthesis of chitin and regulates the growth of insects. However, little is known about the effects of lufenuron treatment on the development of Spodoptera frugiperda (J. E. Smith). In this study, we assessed the toxicity of lufenuron on S. frugiperda and evaluated the effects of lufenuron treatment on the growth and development of S. frugiperda. The results showed that lufenuron exhibits high insecticidal activity against S. frugiperda, with the LC 50 value of 0.99 mg L -1 . Lufenuron treatments can significantly prolong the larval developmental duration and reduce the rates of pupation and emergence. To further explore the underlying mechanism of this observation, the expression profiles of the chitin synthase gene (SfCHS) and chitinase gene (SfCHT), two key enzyme genes involved in the molting of S. frugiperda, were determined after exposure to lufenuron for 96 h. The results of qRT-PCR demonstrated that lufenuron treatments can significantly reduce the expression of SfCHT, while the expression of SfCHS remained relatively stable. Furthermore, we found that lufenuron strongly interacted with chitinase (SfCHT) (-10.8 kcal/mol) and chitin synthase (SfCHS) (R1: -9.7 kcal/mol; R2: -10.2 kcal/mol). Our results indicated that lufenuron has significant effects on the development of S. frugiperda that might be attributed to the differential expression of SfCHT and SfCHS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In a previous study, we found that the embryonic cuticle of Rhodnius prolixus is a chitin-based structure that helps the first instar nymph to hatch from the chorion. Here, we investigated how the reduction of transcripts induced by CHS dsRNA injection affects R. prolixus embryogenesis and eclosion. Deposition of chitin in the embryonic cuticle begins later at embryogenesis, around day 8, and ends approximately at day 15, when the insects are ready for eclosion. In R. prolixus, chitin deposition follows pari passu with the synthesis of the chitin synthase mRNA, indicating a regulation at the transcriptional level. The reduction of the chitin synthase gene transcripts by the injection of CHS dRNA prevented chitin deposition during embryonic cuticle formation, being lethal to hatching nymphs, which end up dying while stuck in the chorionic border trying to leave the chorion. The successful eclosion rates were reduced by 60% in animals treated with CHS dsRNA when compared to animals injected with a control (dsRNA no related gene or water). We found that the harmful effects on oviposition and eclosion are possibly due to changes in the structure of the embryonic cuticle, as observed by directly comparing the morphology of control and chitin-deficient embryonic cuticles under the transmission electron microscope. The lack of chitin and changes in its morphological characteristics appears to alter the embryonic cuticle physiology and functionality. Additionally, we observed that the effects of CHS dRNA treatment on R. prolixus females lasted up to 3 egg-laying cycles (∼100 days), pointing to R. prolixus as a useful model for developmental studies. [ABSTRACT FROM AUTHOR]

Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB. [ABSTRACT FROM AUTHOR]

The article presents a study that aims at identifying seven chitin synthase-encoding genes in the genome of dimorphic ascomycete yeast Yarrowia (Y.) lipolytica. The study revealed that class II chitin synthase plays an important role in septum formation in Y. lipolytica. It also suggests that class V and VI chitin synthases with myosin motor-like domain (MMD) are involved in the maintenance of cell wall integrity in Y. lipolytica.

Trafficking of the chitin synthase Chs2p from the endoplasmic reticulum (ER) to the bud-neck in late mitosis is tightly regulated by the cell cycle via phosphorylation of serine residues in the N-terminus of the protein. Here, we describe the effects of Chs2p phosphorylation on the interaction with coat protein complex II (COPII). Identification of a cdc5ts mutant, which fails to transport Chs2p-3xGFP to the bud-neck and instead accumulates the protein in intracellular puncta, led us to discover that Chs2p-3xGFP accumulates at ER exit sites in metaphase-arrested wild-type cells. Using an in vitro ER vesicle formation assay we showed that phosphorylation of Chs2p by the cyclin-dependent kinase CDK1 prevents packaging into COPII vesicles, whereas dephosphorylation of Chs2p by the phosphatase Cdc14p stimulates selection into the vesicles. We found that the cytoplasmic N-terminal domain of Chs2p, which contains the CDK1 phosphorylation sites, interacts with the COPII component Sec24p in a yeast two-hybrid assay and that phosphomimetic substitutions of serines at the CDK1 consensus sites reduces the interaction. Our data suggest that dephosphorylation functions as a molecular switch for regulated ER exit of Chs2p. [ABSTRACT FROM AUTHOR]

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and β-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-β-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect., (© 2022 Wiley Periodicals LLC.)

The biosynthesis of citric acid (CA) using Aspergillus niger as a carrier is influenced by mycelium morphology, which is determined by the expression level of morphology-related genes. As a key component of the fungal cell wall, chitin content has an important effect on morphogenesis, and to investigate the effects of this on fermentation performance, we used RNA interference to knockdown chitin synthase C (CHSC) and chitin synthase activator (CHS3) to obtain the single-gene mutant strains A. niger chs3 and chsC and the double mutant A. niger chs3C. We found that the CA fermentation performance of the two single mutants was significantly better than that of the double mutant. The mutant A. niger chs3-4 exhibited CA production potential compared to that of the parent strain in scale-up fermentation; we determined certain characteristics of CA high-yielding strain fermentation pellets. In addition, when chsC alone was silenced, there was very little change in chs3 mRNA levels, whereas those of chsC were significantly reduced when only chs3 was silenced. As this may be because of a synergistic effect between chsC and chs3, and we speculated that the latent activation target of CHS3 is CHSC, our results confirmed this hypothesis. This study is the first application of a separation and combination silence strategy of chitin synthase and chitin synthase activator in the morphology of A. niger CA fermentation. Furthermore, it provides new insights into the method for the morphological study of A. niger fermentation and the interaction of homologous genes. KEY POINTS: • The function of chitin synthase C (chsC) and chitin synthase activator (chs3) is tightly interrelated. • Mycelial morphology was optimized by knockdown of CHS3, resulting in the overproduction of citric acid. • The separation and combination silence strategies are promising tools for the interaction of homologous housekeeping genes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chitin synthesis has attracted scientific interest for decades as an essential part of fungal biology and for its potential as a target for antifungal therapies. While this interest remains, three decades ago, pioneering molecular studies on chitin synthesis regulation identified the major chitin synthase in yeast, Chs3, as an authentic paradigm in the field of the intracellular trafficking of integral membrane proteins. Over the years, researchers have shown how the intracellular trafficking of Chs3 recapitulates all the steps in the intracellular trafficking of integral membrane proteins, from their synthesis in the endoplasmic reticulum to their degradation in the vacuole. This trafficking includes specific mechanisms for sorting in the trans -Golgi network, regulated endocytosis, and endosomal recycling at different levels. This review summarizes the work carried out on chitin synthesis regulation, mostly focusing on Chs3 as a molecular model to study the mechanisms involved in the control of the intracellular trafficking of proteins.

Background: High-levels of etoxazole resistance have not yet been frequently reported in Panonychus citri. Although a highly resistant strain was discovered in 2014, etoxazole resistance has not become a significant problem in areas of citrus production in Japan. A target site mutation in chitin synthase 1 (CHS1), I1017F, is a major etoxazole-resistance factor in Tetranychus urticae. To investigate the mechanisms of etoxazole resistance and the dispersal of resistance genes, we analyzed target-site mutations in a highly resistant strain and their geographical distribution in Japan., Results: High-level etoxazole resistance was completely recessive. The I1017F mutation was detected in CHS1 of the highly resistant strain, and its frequency was correlated with the hatchability of eggs treated with etoxazole. Sequencing and variant frequency analyses of local populations by quantitative polymerase chain reaction revealed that I1017F is restricted to the Ariake Sea area of Kyushu Island. Although a new nonsynonymous substitution, S1016L, accompanied by I1017F was found in CHS1 of the highly resistant strain, CRISPR/Cas9 engineering of flies showed that S1016L had no effect on the etoxazole resistance conferred by I1017F., Conclusion: I1017F is a major target site mutation that confers high-level etoxazole resistance on P. citri. Dispersion of I1017F possibly was suppressed as a result of the completely recessive inheritance of resistance together with low gene flow between local populations. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound β-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Here, using split-ubiquitin membrane yeast two-hybrid and pull-down assays, we have demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Abstract: Chitin synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulié et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI chitin synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target. [Copyright &y& Elsevier]

Abstract: Chitin, an essential component of peritrophic matrix (PM), is produced by a series of biochemical reactions. Chitin synthase plays a crucial role in chitin polymerization in chitin biosynthetic pathway. In this study, we identified and characterized a full-length cDNA of chitin synthase 2 gene (LmCHS2) from Locusta migratoria. The cDNA contains an open reading frame of 4569 nucleotides that encode 1523 amino acid residues, and 76- and 373-nucleotides for 5′- and 3′-noncoding regions, respectively. Analysis of LmCHS2 transcript in different tissues of the locust by using real-time quantitative PCR indicated that LmCHS2 was exclusively expressed in midgut and gastric caeca (a part of the midgut). The highest expression was found in the anterior midgut with a decline of the transcript level from the anterior to posterior regions. During growth and development of locusts, there was only a slight expression in eggs, but the expression gradually increased from nymphs to adults. In situ hybridization further revealed that LmCHS2 transcript mainly presented in the apical regions of brush border forming columnar cells of gastric caeca. LmCHS2 dsRNA was injected to fifth-instar nymphs to further explore biological functions of LmCHS2. Significantly down-regulated transcript of LmCHS2 resulted in a cessation of feeding and a high mortality of the insect. However, no visible abnormal morphological change of locusts was observed until insects molted to adults. After dissection, we found that the average length of midguts from the LmCHS2 dsRNA-injected locusts was shorter than that of the control insects that were injected with dsGFP. Furthermore, microsection of midguts showed that the PM of the LmCHS2 dsRNA-injected nymphs was amorphous and thin as compared with the controls. Our results demonstrate that LmCHS2 is responsible for the biosynthesis of chitin associated with PM and plays an essential role in locust growth and development. [Copyright &y& Elsevier]

Abstract: Chitin synthase (CHS) represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely tuned chitin biosynthesis and this pathway is absent in humans and other vertebrates. Current knowledge on CHS in insects, especially their structures, functions, and regulations is still very limited. We report the identification and characterization of two chitin synthase genes, AgCHS1 and AgCHS2, in African malaria mosquito, Anopheles gambiae. AgCHS1 and AgCHS2 were predicted to encode proteins of 1,578 and 1,586 amino acid residues, respectively. Their deduced amino acid sequences show high similarities to other insect chitin synthases. Transcriptional analysis indicated that AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription (RT)-PCR and real-time quantitative PCR. Relatively high expression was detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut (cardia included) followed by the midgut for AgCHS2. Fluorescence in situ hybridization (FISH) and immunohistochemical analysis revealed new information including the localization of the two enzymes in the ommatidia of the compound eyes, and AgCHS2 in the thoracic and abdominal inter-segmental regions of pupal integument. [Copyright &y& Elsevier]

Abstract: Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family. Most insects possess two CHS genes (CHS1 and CHS2); however, genome and transcriptome searches showed that N. lugens possibly possess only CHS1 in both databases. Two transcript variants (CHS1a and CHS1b) resulting from exclusively alternative splicing (exon 19a or 19b in N. lugens) were identified for each of the two rice planthopper CHS1s. Gene structure comparison using the genomes that are currently sequenced showed that the CHS1 genes in all insects except Acyrthosiphon pisum have two transcript variants. Transcription of NlCHS1a reached its highest level just after molting, whereas NlCHS1b reached its highest expression level 1–2 days before molting. Injection of the N. lugens nymphs with double-strand RNA (dsRNA) of CHS1, CHS1a and CHS1b reduced the corresponding variant transcript levels and exhibited subsequent phenotypes. Silencing of CHS1 and CHS1a resulted in elongated distal wing pads and the “wasp-waisted” or crimpled cuticle phenotypes and eventually died, whereas the phenotypes caused by injection of NlCHS1b dsRNA seem not so obvious although slightly increased mortality was observed. Our results suggest that N. lugens likely lacks CHS2 and CHS1 may be efficient target gene for RNAi-based N. lugens control. [Copyright &y& Elsevier]

Abstract: In this study, we carried out the isolation and characterization of chitin synthase genes (CHS) of the main citrus fruit postharvest pathogen Penicillium digitatum. Using distinct sets of degenerate primers designed from conserved regions of CHS genes of yeast and filamentous fungi, PCR methods, and a DNA genomic library, five putative CHS genes (PdigCHSI, PdigCHSII, PdigCHSIII, PdigCHSV, and PdigCHSVII) were identified, isolated, sequenced, and characterized. Phylogenetic analyses, sequence identity, and domain conservation support the annotation as CHS. A very high sequence identity and strong synteny were found with corresponding regions from the genome of Penicillium chrysogenum. Gene expression of P. digitatum CHS genes during mycelium axenic growth, under oxidative and osmotic stress conditions, and during infection of citrus fruits was confirmed and quantified using quantitative RT-PCR (qRT-PCR). PdigCHSIII had the highest expression among the five genes by one order of magnitude, while PdigCHSII had the lowest. However, PdigCHSII was strongly induced coincident with conidial production, suggesting a role in conidiogenesis. The expression of PdigCHSI, PdigCHSIII, PdigCHSV, and PdigCHSVII was upregulated during infection of citrus fruit. PdigCHSV and PdigCHSVII coexpressed in most of the experiments carried out, and they are separated by a 1.77kb intergenic region and arranged in opposite directions. [Copyright &y& Elsevier]