Chitin Synthesis Pathway Research Articles

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

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

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

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

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

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

Conserved microRNAs miR-8-3p and miR-2a-3 targeting chitin biosynthesis to regulate the molting process of Sogatella furcifera (Horváth)(Hemiptera: Delphacidae).

Molting is a key solution to growth restriction in insects. The periodic synthesis and degradation of chitin, one of the major components of the insect epidermis, is necessary for insect growth. MicroRNA (miRNA) have been implicated in molting regulation, yet their involvement in the interplay interaction between the chitin synthesis pathway and 20-hydroxyecdysone signaling remains poorly understood. In this study, soluble trehalase (Tre1) and phosphoacetylglucosamine mutase (PAGM) were identified as targets of conserved miR-8-3p and miR-2a-3, respectively. The expression profiles of miR-8-3p-SfTre1 and miR-2a-3-SfPAGM exhibited an opposite pattern during the different developmental stages, indicating a negative regulatory relationship between them. This relationship was confirmed by an in vitro dual-luciferase reporter system. Overexpression of miR-8-3p and miR-2a-3 by injection of mimics inhibited the expression of their respective target genes and increased mortality, leading to death in the pre-molting, and molting death phenomena. They also caused a decrease in chitin content and expression levels of key genes in the chitin synthesis pathway (SfTre1, SfTre2, SfHK, SfG6PI, SfGFAT, SfGNA, SfPAGM, SfUAP, SfCHS1, SfCHS1a, and SfCHS1b). Conversely, the injection of miRNA inhibitors resulted in the upregulation of the expression levels of these genes. Following 20E treatment, the expression levels of miR-8-3p and miR-2a-3 decreased significantly, while their corresponding target genes increased significantly. These results indicate that miR-8-3p and miR-2a-3 play a regulatory role in the molting of Sogatella furcifera by targeting SfTre1 and SfPAGM, respectively. These findings provide new potential targets for the development of subsequent new control strategies.

Silencing of Glutamine: Fructose-6-Phosphate Aminotransferase Impairs Growth and Development in Sogatella furcifera (Hemiptera: Delphacidae).

Glutamine: fructose-6-phosphate aminotransferase (GFAT), the fourth enzyme in the chitin synthesis pathway, exerts wide-ranging effects on the growth and development of organisms. However, the role of GFAT in Sogatella furcifera remains unknown. In this study, the functional significance of the GFAT gene of S. furcifera was analyzed using a reverse transcription-polymerase chain reaction and RNA interference (RNAi) analyses. The complementary DNA sequence of SfGFAT was 3162 bp in length and contained a 2067 bp open reading frame encoding 688 amino acid residues. Structural domain analysis indicated that the SfGFAT protein consisted of one glutamine aminotransferase class 2 domain and two sugar isomerase domains. Expression profile analysis revealed that SfGFAT was expressed throughout the egg, nymph, and adult phases and was strongly expressed on the first day of each nymph stage and in the integuments of five tissues. RNAi results revealed that SfGFAT gene silencing significantly inhibited the mRNA expression of the target gene and resulted in severe mortality among S. furcifera. In summary, these findings demonstrate that SfGFAT plays a critical role in the development of S. furcifera. Moreover, these results may aid in the development of methods to control the spread of S. furcifera.

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

BackgroundMosquitoes 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).ResultsThe 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.MethodsThe 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 chitinaseactivity in the larvae were detected. The structure of peritrophic membrane in the midgut of the fourth-instar larvaeafter silencing was observed by paraffin section and hematoxylin–eosin (HE) staining.ConclusionCHS-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.Graphical

FDP-Na-induced enhancement of glycolysis impacts larval growth and development and chitin biosynthesis in fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

Fructose 1, 6-diphosphate (FDP) is an endogenous intermediate in the glycolytic pathway, as well as an allosteric activator of phosphofructokinase (PFK). Based on the role in promoting glycolysis, FDP has been widely used as a therapeutic agent for mitigating the damage of endotoxemia and ischemia/reperfusion in clinical practice. However, the effect of exogenous FDP-induced glycolysis activation on insect carbohydrate metabolism and chitin synthesis remains largely unclear. Here, we investigated for the first time the effects of FDP-Na, an allosteric activator of PFK, on the growth and development of Hyphantria cunea larvae, a serious defoliator in agriculture and forestry, especially on glycolysis and chitin synthesis. The results showed that FDP-Na significantly restrained the growth and development of H. cunea larvae and resulted in larval lethality. After treatment with FDP-Na, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) were significantly activated, and HcHK2, HcPFK, HcPK were dramatically upregulated, which suggested that FDP-Na enhanced glycolysis in H. cunea larvae. Meanwhile, FDP-Na also distinctly impacted chitin biosynthesis by disturbing transcriptions of genes in the chitin synthesis pathway, resulting in changes of chitin contents in the midgut and epidermis of H. cunea larvae. Therefore, we considered that FDP-Na caused the growth and development arrest, and impacted chitin biosynthesis, probably by disturbing in vivo glycolysis and carbohydrate metabolism in H. cunea larvae. The findings provide a new perspective on the mechanism by which glycolysis regulates insect growth and development, and lay the foundation for exploring the potential application of glycolysis activators in pest control as well.

Trehalose-6-phosphate synthase regulates chitin synthesis in Mythimna separata.

Trehalose is a substrate for the chitin synthesis pathway in insects. Thus, it directly affects chitin synthesis and metabolism. Trehalose-6-phosphate synthase (TPS) is a crucial enzyme in the trehalose synthesis pathway in insects, but its functions in Mythimna separata remain unclear. In this study, a TPS-encoding sequence in M. separata (MsTPS) was cloned and characterized. Its expression patterns at different developmental stages and in diverse tissues were investigated. The results indicated that MsTPS was expressed at all analyzed developmental stages, with peak expression levels in the pupal stage. Moreover, MsTPS was expressed in the foregut, midgut, hindgut, fat body, salivary gland, Malpighian tubules, and integument, with the highest expression levels in the fat body. The inhibition of MsTPS expression via RNA interference (RNAi) resulted in significant decreases in the trehalose content and TPS activity. It also resulted in significant changes in Chitin synthase (MsCHSA and MsCHSB) expression, and significantly decrease the chitin content in the midgut and integument of M. separata. Additionally, the silencing of MsTPS was associated with a significant decrease in M. separata weight, larval feed intake, and ability to utilize food. It also induced abnormal phenotypic changes and increased the M. separata mortality and malformation rates. Hence, MsTPS is important for M. separata chitin synthesis. The results of this study also suggest RNAi technology may be useful for enhancing the methods used to control M. separata infestations.

Functional characterization of chitin synthesis pathway genes, HaAGM and HaUAP, reveal their crucial roles in ecdysis and survival of Helicoverpa armigera (Hübner)

The chitin metabolic pathway is one of the most lucrative targets for designing pest management regimes. Inhibition of the chitin synthesis pathway causes detrimental effects on the normal growth and development of insects. Phospho-N-acetylglucosamine mutase (AGM) and UDP-N-acetylglucosamine pyrophosphorylase (UAP) are two key chitin biosynthesis enzymes in insects including Helicoverpa armigera, a pest of global significance. In the present study, we have identified, cloned and recombinantly expressed AGM and UAP from H. armigera (HaAGM and HaUAP). Biochemical characterization of recombinant HaAGM and HaUAP exhibited high affinities for their natural substrates N-acetyl glucosamine-6-phosphate (Km 38.72 ± 2.41) and N-acetyl glucosamine-1-phosphate (Km 3.66 ± 0.13), respectively. In the coupled enzyme-catalytic assay, HaAGM and HaUAP yielded the end-products, inorganic pyrophosphate and UDP-GlcNAc, confirming their active participation in the chitin synthesis pathway of H. armigera. Gene expression profiling revealed that HaAGM and HaUAP genes were expressed in all developmental stages and key tissues. These genes also showed substantial responses towards the moulting hormone 20-hydroxyecdysone and chitin biosynthesis inhibitor, novaluron. Remarkably, the RNAi-mediated knockdown of either HaAGM or HaUAP led to severe developmental deformities and significant mortality ranging from 65.61 to 72.54%. Overall findings suggest that HaAGM and HaUAP play crucial roles in the ecdysis and survival of H. armigera. Further, these genes could serve as potential targets for designing pest management strategies for H. armigera.

Chitinase (CHI) of Spodoptera frugiperda affects molting development by regulating the metabolism of chitin and trehalose

Chitin is the main component of insect exoskeleton and midgut peritrophic membrane. Insect molting is the result of the balance and coordination of chitin synthesis and degradation in chitin metabolism under the action of hormones. In this study, a 678 bp dsRNA fragment was designed and synthesized according to the known CHI (Chitinase) sequence of Spodoptera frugiperda. It was injected into the larvae to observe the molting and development of S. frugiperda. At the same time, the activities of trehalase and chitinase, the contents of trehalose, chitin and other substances were detected, and the expression of related genes in the chitin synthesis pathway was determined. The results showed that CHI gene was highly expressed at the end of each instar, prepupa and pupal stage before molting; At 12 and 24 h after dsRNA injection of CHI gene of S. frugiperda, the expression of CHI gene decreased significantly, and the chitinase activity decreased significantly from 12 to 48 h. The expression of chitin synthase (CHSB) gene decreased significantly, and the chitin content increased significantly. Some larvae could not molt normally and complete development, leading to certain mortality. Secondly, after RNAi of CHI gene, the content of glucose and glycogen increased first and then decreased, while the content of trehalose decreased significantly or showed a downward trend. The activities of the two types of trehalase and the expression levels of trehalase genes decreased first and then increased, especially the trehalase activities increased significantly at 48 h after dsCHI injection. And trehalose-6-phosphate synthase (TPS), glutamine: fructose-6-phosphate amidotransferase (GFAT), UDP-N-acetylglucosamine pyrophosphorylases (UAP), hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and phosphoacetylglucosamine mutase (PAGM) all decreased significantly at 24 h, and then increased or significantly increased at 48 h. These results indicated that when the expression of chitinase gene of S. frugiperda was inhibited, it affected the degradation of chitin in the old epidermis and the formation of new epidermis, and the content of chitin increased, which led to the failure of larvae to molt normally. Moreover, the chitin synthesis pathway and trehalose metabolism were also regulated. The relevant results provide a theoretical basis for screening target genes and developing green insecticides to control pests by using the chitin metabolism pathway.

Knockdown of GFAT disrupts chitin synthesis in Hyphantria cunea larvae

Glutamine-fructose-6-phosphate transaminase (GFAT) has been reported to regulate the hexosamine biosynthetic pathway as the first rate-limiting enzyme. As a key enzyme that catalyzes the substrate of glycosylation modification, which has a wide-ranging effect on cellular functions. However, there are few studies on the relationship between GFAT and chitin metabolism in insects. In the present study, the GFAT gene from Hyphantria cunea was identified based on transcriptome and bioinformatic analysis. The role of HcGFAT in regulating development and chitin synthesis was analyzed by RNA interference (RNAi) in H. cunea larvae. The full-length HcGFAT gene (2028 bp) encodes a 676 amino acid (aa) polypeptide had typical structural features of the SIS and Gn_AT_II superfamily. Phylogenetic analyses showed that GFAT of H. cunea shares the highest homology and identity with GFAT of Ostrinia furnacalis. Expression profiles indicated that HcGFAT was expressed throughout larval, pupal and three tissues (midgut, fat body, epidermis), and highly expressed in the last instar of larvae and strongly expressed in epidermis among three tissues. Bioassay results showed that knockdown of HcGFAT repressed larval growth and development, resulting in a significant loss of larval body weight. Meanwhile, HcGFAT knockdown also significantly caused larval developmental deformity. Knockdown of HcGFAT regulated the expression of four other critical genes in the chitin synthesis pathway (HcGNA, HcPAGM, HcUAP, HcCHSA), and ultimately resulted in decreased chitin content in the epidermis. In summary, these findings indicated that GFAT plays a critical role in larval growth and development, as well as chitin synthesis in H. cunea.

Knockdown of hexokinase in Diaphorina citri Kuwayama (Hemiptera: Liviidae) by RNAi inhibits chitin synthesis and leads to abnormal phenotypes.

Silencing specific genes in pests using RNA interference (RNAi) technology is a promising new pest-control strategy. The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important citrus pest because it transmits Candidatus Liberibacter asiaticus, which causes huanglongbing. Chitin is essential for insect development, and enzymes in this pathway are attractive targets for pest control. The hexokinase gene DcHK was characterized from D.citri to impair proper growth and chitin synthesis through RNAi. The transcription of DcHK was more highly developed in third-instar nymphs, adults and the Malpighian tube. The RNAi needed for D.citri is dose-dependent, with 600 ng μl-1 dsDcHK sufficient to knockdown endogenous DcHK expression. The messenger RNA (mRNA) level was lowest at 36 h after dosing, and there were significant effects on the relative levels of mRNA in the chitin synthesis pathway (DcTre, DcG6PI, DcGNAT, DcGFAT, DcPGM, DcUAP and DcCHS), leading to mortality, reduced body weight and abnormal or lethal phenotypes. RNAi can be triggered by orally delivered double-stranded RNA in D.citri. These results can provide support for HK genes as a new potential target for citrus psyllid control. © 2022 Society of Chemical Industry.

Analysis of secondary metabolite gene clusters and chitin biosynthesis pathways of Monascus purpureus with high production of pigment and citrinin based on whole-genome sequencing.

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.

Identification and Phylogenetic Analysis of Chitin Synthase Genes from the Deep-Sea Polychaete Branchipolynoe onnuriensis Genome

Chitin, one of the most abundant biopolymers in nature, is a crucial material that provides sufficient rigidity to the exoskeleton. In addition, chitin is a valuable substance in both the medical and industrial fields. The synthesis of chitin is catalyzed by chitin synthase (CHS) enzymes. Although the chitin synthesis pathway is highly conserved from fungi to invertebrates, CHSs have mostly only been investigated in insects and crustaceans. Especially, little is known about annelids from hydrothermal vents. To understand chitin synthesis from the evolutionary view in a deep-sea environment, we first generated the whole-genome sequencing of the parasitic polychaete Branchipolynoe onnuriensis. We identified seven putative CHS genes (BonCHS1-BonCHS7) by domain searches and phylogenetic analyses. This study showed that most crustaceans have only a single copy or two gene copies, whereas at least two independent gene duplication events occur in B. onnuriensis. This is the first study of CHS obtained from a parasitic species inhabiting a hydrothermal vent and will provide insight into various organisms’ adaptation to the deep-sea hosts.

Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury

Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKβ1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKβ1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.

Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila.

Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr, and Abd-A showed that the potential complex mechanism of kkv transcription is regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis.

The Antifungal Effects of Citral on Magnaporthe oryzae Occur via Modulation of Chitin Content as Revealed by RNA-Seq Analysis.

The natural product citral has previously been demonstrated to possess antifungal activity against Magnaporthe oryzae. The purpose of this study was to screen and annotate genes that were differentially expressed (DEGs) in M. oryzae after treatment with citral using RNA sequencing (RNA-seq). Thereafter, samples were reprepared for quantitative real-time PCR (RT-qPCR) analysis verification of RNA-seq data. The results showed that 649 DEGs in M. oryzae were significantly affected after treatment with citral (100 μg/mL) for 24 h. Kyoto Encyclopedia of Genes and Genomes (KEGG) and a gene ontology (GO) analysis showed that DEGs were mainly enriched in amino sugar and nucleotide sugar metabolic pathways, including the chitin synthesis pathway and UDP sugar synthesis pathway. The results of the RT-qPCR analysis also showed that the chitin present in M. oryzae might be degraded to chitosan, chitobiose, N-acetyl-D-glucosamine, and β-D-fructose-6-phosphate following treatment with citral. Chitin degradation was indicated by damaged cell-wall integrity. Moreover, the UDP glucose synthesis pathway was involved in glycolysis and gluconeogenesis, providing precursors for the synthesis of polysaccharides. Galactose-1-phosphate uridylyltransferase, which is involved in the regulation of UDP-α-D-galactose and α-D-galactose-1-phosphate, was downregulated. This would result in the inhibition of UDP glucose (UDP-Glc) synthesis, a reduction in cell-wall glucan content, and the destruction of cell-wall integrity.

3-Bromopyruvate-induced glycolysis inhibition impacts larval growth and development and carbohydrate homeostasis in fall webworm, Hyphantria cunea Drury

As a typical glycolytic inhibitor, 3-bromopyruvate (3-BrPA) has been extensively studied in cancer therapy in recent decades. However, few studies focused on 3-BrPA in regulating the growth and development of insects, and the relationship and regulatory mechanism between glycolysis and chitin biosynthesis remain largely unknown. The Hyphantria cunea, named fall webworm, is a notorious defoliator, which caused a huge economic loss to agriculture and forestry. Here, we investigated the effects of 3-BrPA on the growth and development, glycolysis, carbohydrate homeostasis, as well as chitin synthesis in H. cunea larvae. To elucidate the action mechanism of 3-BrPA on H. cunea will provide a new insight for the control of this pest. The results showed that 3-BrPA dramatically restrained the growth and development of H. cunea larvae and resulted in larval lethality. Meanwhile, we confirmed that 3-BrPA caused a significant decrease in carbohydrate, adenosine triphosphate (ATP), pyruvic acid (PA), and triglyceride (TG) levels by inhibiting glycolysis in H. cunea larvae. Further studies indicated that 3-BrPA significantly affected the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and trehalase, as well as expressions of the genes related to glycolysis, resulting in carbohydrate homeostasis disorder. Moreover, it was found that 3-BrPA enhanced 20-hydroxyecdysone (20E) signaling by upregulating HcCYP306A1 and HcCYP314A1, two critical genes in 20E synthesis pathway, and accelerated chitin synthesis by upregulating transcriptional levels of genes in the chitin synthesis pathway in H. cunea larvae. Taken together, our findings provide a novel insight into the mechanism of glycolytic inhibitor in regulating the growth and development of insects, and lay a foundation for the potential application of glycolytic inhibitors in pest control as well.

Annotation of chitin biosynthesis genes in Diaphorina citri, the Asian citrus psyllid.

The polysaccharide chitin is critical for the formation of many insect structures, including the exoskeleton, and is required for normal development. Here we report the annotation of three genes from the chitin synthesis pathway in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), the vector of Huanglongbing (citrus greening disease). Most insects have two chitin synthase (CHS) genes but, like other hemipterans, D. citri has only one. In contrast, D. citri is unusual among insects in having two UDP-N-acetylglucosamine pyrophosphorylase (UAP) genes. One of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our work helps pave the way for potential utilization of these genes as pest control targets to reduce the spread of Huanglongbing.

Dsk2 involves in conidiation, multi‐stress tolerance and thermal adaptation in Beauveria bassiana

Dsk2 is a nuclear-enriched ubiquitin-like polyubiquitin-binding protein that regulates protein degradation in yeast but has not been explored yet in filamentous fungi, such as Beauveria bassiana. Here, we report Beauveria bassiana Dsk2 located both in the nucleus and in cytoplasm of hyphal cells. Deletion of Dsk2 resulted in mild growth defect on scant media with various carbon/nitrogen sources and dramatic attenuation in conidiation capability at optimal condition. Compared to the wild-type, ΔDsk2 strains are much more sensitive to high osmotic and oxidative pressure during vegetative growth. Meanwhile, the mutant strains showed an increased chemical tolerance to Congo red and calcofluor white, two cell wall perturbing agents. The transcriptional changes of genes involved in central development, superoxide dismutase and chitin synthesis pathway indicate that Dsk2 acts as a multi-functional regulator in adapting to environmental changes. Importantly, Dsk2 negatively regulated the ability of thermal resistance in B. bassiana, which makes it a potential target gene for constructing engineering anti-thermal strains in the circumstance of global warming. Altogether, our finding highlights novel roles of Dsk2 involved in the asexual cycle, multi-stress tolerance and pest control potential of B. bassiana.