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》

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 《-》

Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron.

Farnesi LC ，Brito JM ，Linss JG ，Pelajo-Machado M ，Valle D ，Rezende GL ... -  《PLoS One》

Diversity of midgut bacteria in larvae and females of Aedes aegypti and Aedes albopictus from Gampaha District, Sri Lanka.

The midgut microbiota of mosquitoes maintain basal immune activity and immune priming. In recent years, scientists have focused on the use of microbial communities for vector control interventions. In the present study, the midgut bacteria of larvae and adults of Aedes aegypti and Ae. albopictus were assessed using both field-collected and laboratory-reared mosquitoes from Sri Lanka. Adults and larvae of Ae. aegypti and Ae. albopictus were collected from three selected areas in Gampaha Medical Officer of Health area, Gampaha District, Western Province, Sri Lanka. Bacterial colonies isolated from mosquito midgut dissections were identified by PCR amplification and sequencing of partial 16S rRNA gene fragments. Adults and larvae of Ae. aegypti and Ae. albopictus harbored 25 bacterial species. Bacillus endophyticus and Pantoea dispersa were found more frequently in field-collected Ae. aegypti and Ae. albopictus adults, respectively. The midgut bacteria of Ae. aegypti and Ae. albopictus adults (X2 = 556.167, df = 72, P < 0.001) and larvae (X2 = 633.11, df = 66, P < 0.001) were significantly different. There was a significant difference among the bacterial communities between field-collected adults (X2 = 48.974, df = 10, P < 0.001) and larvae (X2 = 84.981, df = 10, P < 0.001). Lysinibacillus sphaericus was a common species in adults and larvae of laboratory-reared Ae. aegypti. Only P. dispersa occurred in the field-collected adults of Ae. aegypti and Ae. albopictus. Species belonging to genera Terribacillus, Lysinibacillus, Agromyces and Kocuria were recorded from Aedes mosquitoes, in accordance with previously reported results. This study generated a comprehensive database on the culturable bacterial community found in the midgut of field-collected (Ae. aegypti and Ae. albopictus) and laboratory-reared (Ae. aegypti) mosquito larvae and adults from Sri Lanka. Data confirm that the midgut bacterial diversity in the studied mosquitoes varies according to species, developmental stage and strain (field vs laboratory).

Ranasinghe K ，Gunathilaka N ，Amarasinghe D ，Rodrigo W ，Udayanga L ... -  《Parasites & Vectors》

Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut.

In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.

Kelkenberg M ，Odman-Naresh J ，Muthukrishnan S ，Merzendorfer H ... -  《-》