Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

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.

Dissecting the manipulation of lufenuron on chitin synthesis in Helicoverpa armigera.

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 LC25 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 LC25 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.

Homologous Design and Three-Dimensional Quantitative Structure-Activity Relationship Study of Acaricidal 2,4-Diphenyloxazolines Containing Different Heteroatoms and Alkyl Chains.

In order to speculate the three-dimensional structure of the potential binding pocket of the chitin synthase inhibitor, a series of 2,4-diphenyloxazoline derivatives with different lengths of alkyl chains and heteroatoms were designed and synthesized by a homologous strategy. The bioassay results indicate that both the length of the alkyl chains and the type of substituents can affect the acaricidal activity against mite eggs. Compounds containing chloropropyl, alkoxyalkyl, and para-substituted phenoxyalkyl or phenylthioalkyl groups exhibit good activity, while those containing steric hindrance substituents or carbonyl substituents on the benzene ring exhibit reduced activity. Three-dimensional quantitative structure-activity relationship (3D-QSAR) study showed that there may be a narrow hydrophobic region deep in the pocket, and the steric effect plays a more important role than the electrostatic effect. The current work will provide assistance for future molecular design and target binding research.

Con7 is a key transcription regulator for conidiogenesis in the plant pathogenic fungus Fusarium graminearum.

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 FgCon7, 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 FgCon7 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 FgCon7 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 FgCon7 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.

Buprofezin delayed the molting of Pardosa pseudoannulata, a predatory enemy for insect pests, by suppressing chitin synthase 1 expression.

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.

Rapid discovery of a new antifoulant: From in silico studies targeting barnacle chitin synthase to efficacy against barnacle settlement.

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.

Paired plant immune CHS3-CSA1 receptor alleles form distinct hetero-oligomeric complexes.

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.

Antioxidant Dipeptides Enhance Osmotic Stress Tolerance by Regulating the Yeast Cell Wall and Membrane.

This study aimed to investigate the role of the yeast cell wall and membrane in enhancing osmotic tolerance by antioxidant dipeptides (ADs) including Ala-His (AH), Thr-Tyr (TY), and Phe-Cys (FC). Results revealed that ADs could improve the integrity of the cell wall by restructuring polysaccharide structures. Specifically, FC significantly (p < 0.05) reduced the leakage of nucleic acid and protein by 2.86% and 5.36%, respectively, compared to the control. In addition, membrane lipid composition played a crucial role in enhancing yeast tolerance by ADs, including the increase of cell membrane integrity and the decrease of permeability by regulating the ratio of unsaturated fatty acids. The up-regulation of gene expression associated with the cell wall integrity pathway (RLM1, SLT2, MNN9, FKS1, and CHS3) and fatty acid biosynthesis (ACC1, HFA1, OLE1, ERG1, and FAA1) further confirmed the positive impact of ADs on yeast tolerance against osmotic stress.

Potential molecular mechanism of reuterin on the inhibition of Aspergillus flavus conidial germination: An in silico study.

Reuterin is a natural antifungal agent derived from certain strains of Limosilactobacillus reuteri. Our previous study revealed that 6 mM reuterin inhibited completely the conidial germination of aflatoxigenic Aspergillus flavus. This study investigated the potential molecular mechanism of reuterin in inhibiting A. flavus conidial germination, which was pre-assumed that it correlated to the inhibition of some essential enzyme activity involved in conidial germination, specifically 1,3-ß-glucan synthase, chitin synthase, and catalases (catalase, bifunctional catalase-peroxidase, and spore-specific catalase). The complex of 1,3-ß-glucan synthase and chitin synthase with reuterin had a lower binding affinity than that with the substrate. Conversely, the complex of catalases with reuterin had a higher binding affinity than that with the substrate. It was suggested that 1,3-ß-glucan synthase and chitin synthase tended to bind the substrate rather than bind reuterin. In contrast, catalases tended to bind reuterin rather than bind the substrate. Therefore, reuterin could be a potential inhibitor of catalases but may not be an inhibitor of 1,3-ß-glucan synthase and chitin synthase. In this in silico study, we predicted that the potential molecular mechanism of reuterin in inhibiting A. flavus conidial germination was due to the inhibition of catalases activities by competitively binding to the enzymes active sites, thus resulting in the accumulation of reactive oxygen species in cells, leading to cells damage. PRACTICAL APPLICATION: This in silico study revealed that reuterin is a potential inhibitor of catalases in A. flavus, thereby interfering with the antioxidant system during conidial germination. This finding shows that reuterin can be used as an antifungal agent in food or agricultural products, inhibiting conidial germination completely.

Advances in understanding insect chitin biosynthesis.

Chitin, a natural polymer of N-acetylglucosamine chains, is a principal component of the apical extracellular matrix in arthropods. Chitin microfibrils serve as structural components of natural biocomposites present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes, fungi and arthropods. In this review, we summarize the frontier advances of insect chitin synthesis. More specifically, we focus on the chitin synthase (CHS), which catalyzes the key biosynthesis step. CHS is also known as an attractive insecticidal target in that this enzyme is absent in mammals, birds or plants. As no insect chitin synthase structure have been reported so far, we review recent studies on glycosyltransferase domain structures derived from fungi and oomycetes, which are conserved in CHS from all species containing chitin. Auxiliary proteins, which coordinate with CHS in chitin biosynthesis and assembly, are also discussed.

Structural dynamics and functional analysis of Saprolegnia parasitica chitin synthases 5 in a phospholipid bilayer.

Saprolegnia parasitica is an oomycete responsible for a fish disease called saprolegniosis, which poses an economic and environmental burden on aquaculture production. In Saprolegnia, CHS5 of S. parasitica (SpCHS5) contains an N-terminal domain, a catalytic domain of the glycosyltransferase -2 family containing a GT-A fold, and a C-terminal transmembrane domain. No three-dimensional structure of SpCHS5 is reported yet disclosing the structural details of this protein. We have developed a structural model of full-length SpCHS5 and validated it by molecular dynamics simulation technique. From the 1 microsecond simulations, we retrieved the stable RoseTTAFold model SpCHS5 protein to explain characteristics and structural features. Furthermore, from the analysis of the movement of chitin in the protein cavity, we assumed that ARG 482, GLN 527, PHE 529, PHE 530, LEU 540, SER 541, TYR 544, ASN 634, THR 641, TYR 645, THR 641, ASN 772 residues as a main cavity lining site. In SMD analysis, we investigated the opening of the transmembrane cavity required for chitin translocation. The pulling of chitin from the internal cavity to the extracellular region was observed through steered molecular dynamics simulations. A comparison of the initial and final structures of chitin complex showed that there's a transmembrane cavity opening in the simulations. Overall, this present work will help us understand the structural and functional basis of CHS5 and design inhibitors against SpCHS5.Communicated by Ramaswamy H. Sarma.

Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate).

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.

Multiple quality control mechanisms monitor yeast chitin synthase folding in the endoplasmic reticulum.

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.

Insights Into a Chitin Synthase of Kuruma Shrimp Penaeus japonicus and Its Role in Peritrophic Membrane and Cuticle Formation.

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.

RNAi-mediated CHS-2 silencing affects the synthesis of chitin and the formation of the peritrophic membrane in the midgut of Aedes albopictus larvae.

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.

Structure, catalysis, chitin transport, and selective inhibition of chitin synthase.

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 ß-(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.

Conserved Active Site Architecture Between Bacterial Cellulose and Chitin Synthases.

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.

A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila.

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.

POUM2 homeostasis regulates intimal remodeling and cells fate in the anterior silk gland of the silkworm.

Tissue/organ remodeling and cells fate determination play key roles in the life cycle of animals. However, they are still poorly understood in insects, especially in the silkworm. The anterior silk gland (ASG) of the silkworm is essential for the formation and performance of silk fibers, but the regulatory mechanism of ASG remodeling and cells fate determination is less known. Here we found that silencing of POUM2 caused shorter ASG length, intimal structural defects, silkworm spinning failure, and the resultant naked pupae death, but cells number was not affected. Cells staining showed that DNA endoreduplication was not affected in the ASG. Transmission electron microscopy and chitin staining showed cuticle proteins and chitin were greatly reduced in the ASG during the molting period. Transcriptional analysis showed the expression profiles of cuticle proteins and chitin synthase were similar to that of POUM2 during the molting period, and POUM2 down-regulation reduced the expression of cuticle proteins, chitin synthase, autophagy and apoptosis-related genes. While the phenotype resulting from POUM2 over-expression was similar to that of POUM2 down-regulation. Cells staining revealed marked cells apoptosis with cells number reduction and inhibition of DNA endoreduplication in the ASG. Transcriptional analysis showed the expression of autophagy and apoptosis-related genes, and some cuticle proteins and chitin synthase were significantly up-regulated. The results suggest that POUM2 homeostasis regulates ASG intimal remodeling and cells fate, thus affecting ASG development, silkworm spinning and metamorphosis. Our studies not only offer potential molecular targets for genetic improvement of silk performance and molecular breeding of the silkworm, but also provide new insights into POU factor-mediated tissue remodeling and cells fate determination in insects.

Molecular and functional analysis of chitin synthase genes in Chilo suppressalis (Lepidoptera: Crambidae).

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.