Chitin synthase genes of Aedes albopictus and their effects on development of pupae.

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.

Ruiling Z ，Sha A ，Zhong Z 《-》

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

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). 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. 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. 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.

Zhang C ，Ding Y ，Zhou M ，Tang Y ，Chen R ，Chen Y ，Wen Y ，Wang S ... -  《Parasites & Vectors》

MicroRNA-989 controls Aedes albopictus pupal-adult transition process by influencing cuticle chitin metabolism in pupae.

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

Zhang R ，Liu W ，Fu J ，Zhang Z ... -  《Parasites & Vectors》

miR-306-5p is involved in chitin metabolism in Aedes albopictus pupae via linc8338-miR-306-5p-XM_019678125.2 axis.

Aedes albopictus can transmit several lethal arboviruses. This mosquito has become a sever public health threat due to its rapidly changing global distribution. Chitin, which is the major component of the cuticle and peritrophic membrane (PM), is crucial for the growth and development of insect. microRNAs (miRNAs) play important roles in the posttranscriptional level regulation of gene expression, thereby influencing many biological processes in insects. In this study, an attempt was made to evaluate the role of miR-306-5p in regulating chitin metabolism in Ae. albopictus pupae. Overexpression of miR-306-5p resulted in a significantly reduced survival rate in pupae and an increased malformation rate in adults. Both in vivo and in vitro evidence confirmed the presence of the competing endogenous RNA (ceRNA) regulatory axis (linc8338-miR-306-5p-XM_019678125.2). RNAi of linc8338 and XM_019678125.2 had effects on pupae similar to those of miR-306-5p. The highest expression level of miR-306-5p was found in the midgut, and alteration in the expression of miR-306-5p, XM_019678125.2 and linc8338 induced increased transcript levels of chitin synthase 2 (AaCHS2) and decreased chitinase 10 (AaCht10); as well as increased thickness of the midgut and enlarged midgut epithelial cells. The results of this study highlight the potential of miR-306-5p as a prospective target in mosquito control and confirm that the ceRNA mechanism is involved in chitin metabolism. These findings will provide a basis for further studies to uncover the molecular mechanisms through which ncRNAs regulate chitin metabolism.

Zhang R ，Liu W ，Zhang Z 《-》

De novo characterization of transcriptome and gene expression dynamics in epidermis during the larval-pupal metamorphosis of common cutworm.

Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, β-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and β-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects.

Gu J ，Huang LX ，Gong YJ ，Zheng SC ，Liu L ，Huang LH ，Feng QL ... -  《-》