Physiological Characterization of the Chitin Synthase A Gene Responsible for Biosynthesis of Cuticle Chitin in Culex Pipiens Pallens (Diptera: Culicidae)

Physiological Characterization of the Chitin Synthase A Gene Responsible for Biosynthesis of Cuticle Chitin in Culex Pipiens Pallens (Diptera: Culicidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Physiological Characterization of the Chitin Synthase A Gene Responsible for Biosynthesis of Cuticle Chitin in Culex Pipiens Pallens (Diptera: Culicidae) Xiaoshan Yang, Yang Xu, Qi Yin, Hongbo Zhang, Haitao Yin, Yan Sun, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-139574/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2021 Read the published version in Parasites & Vectors → Version 1 posted 12 You are reading this latest preprint version Abstract Background: The pathogens transmitted by mosquitoes ( Culex pipiens pallens ) to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) represents a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines the chitin content might help with novel vector control strategies. Results: The injection of small interfering RNA targeting CHSA (siCHSA) to knock down CHSA transcripts of in larval, pupal, and adult stages, showed different lethal phenotypes. In the larval and pupal stages, CHSA knockdown prevented larval molting, pupation, and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. In the adult stage, it also affected the laminar organization of mesoderm and the formation of pseudo orthogonally large fibers of the endoderm. Conclusion: The present study provides a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results showed that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our result further showed that CHSA is important for the structural integrity of the adult mosquito cuticle. Parasitology Chitin Chitin synthase molting mosquito insect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Mosquitoes are the vectors for transmission of many infectious diseases, causing hundreds of thousands of deaths every year [ 1-5 ]. Currently, chemical insecticides are the main agents used to control insect vectors and reduce disease transmission. However, the widespread use of chemical control has led to the development of insect resistance, the resurgence of target pests, food and environment pollution, and the destruction of non-target insects [ 6-8 ]. The environmental and health impacts of the toxicity of conventional insecticides have become increasingly unacceptable [ 9 ]. Therefore, there is an urgent need to develop novel, safer bioinsecticides with low toxicity [ 10 ]. The normal growth and development of insect is inseparable from the process of molting [ 11 ]. Many researchers investigated growth metabolic regulation and metabolism strategies in insects to identify novel targets for insect control [ 12-17 ]. The process of insect molting involves the biosynthesis, transformation, and modification of chitin [ 18 ]. Chitin, a linear polysaccharide of the amino sugar N-acetyl glucosamine, is the main component of insects’ extracellular barrier, such as the cuticle and the peritrophic matrix (PM). Chitin is also an important component in the tracheal system, reproductive ducts, and the ducts of various dermal glands in the internal structures of many insects [ 6 , 9 ]. Chitin plays an important role in supporting muscle attachment for movement, preventing chemical and physical damage, and preventing infectious diseases, representing the first line of defense in challenging environments [ 19 , 20 ]. Chitin biosynthesis plays an irreplaceable role in the growth of insects. Chitin biosynthesis requires modification and physiological regulation at developmental stages. Chitin is completely absent from vertebrates and higher plants[ 21 , 22 ], and thus has generated interest as a potential target for ecologically friendly insecticides; therefore, an understanding of chitin biosynthetic pathways could provide additional strategies for pest control. Chitin synthesis inhibitors (CSI) can prevent insects from molting by interfering with chitin biosynthesis [ 8 ]. Among them, the Benzylphenolurea (BPU) insecticides have shown great potential to inhibit chitin biosynthesis; however, the non-targeting effects of BPUs can adversely affect beneficial species, such as bees, making them a controversial group of insecticides [ 7 ]. Therefore, it is necessary to study chitin synthase to find new and effective target sites to combat insect pests. The chitin biosynthesis pathway involves eight key regulatory enzymes. The last step is executed by the chitin synthases (CHSs, UDP-N-acetyl-D- glucosamine: chitin 4-beta-N-acetylglucosaminyltransferase), which form a large group of plasma membrane proteins belonging to family 2 of the glycosyltransferases [ 23 ]. Insects commonly possess two chitin synthase genes: class A CHSs (CHSA) are primarily involved in chitin synthesis for the exoskeleton cuticle [ 24-28 ]; while class B CHSs (CHSB) play a major role in the synthesis of chitin in the intestinal PM [ 9 , 29 ]. Currently available data of biochemically characterized insect CHSs show that insect CHSA is associated with insect molting [ 30 , 31 ], and is a crucial enzyme that balances growth and development. RNA interference (RNAi) to silence CHSA in Manduca sexta [ 32 ] and Spodopetera exigua resulted in disruption of the cuticular exoskeleton and tracheal ectodermis [ 33 ]. In Tribolium castaneum and Locusta migratoria manilensis , CHSA is required for larval-larval processes, and for larval-pupal and pupal-adult processes [ 34 ]. In Tribolium castaneum , CHSA plays a prominent role in embryo development and oviposition [ 31 ]. In Anopheles gambiae , CHSA was detected in newly formed compound eyes [ 26 ]. However, those studies were performed in different developmental stages of different species, and the mechanism by which CHSA affects the molting process in mosquitoes remains unclear. Studying CHSA function in mosquitoes is crucial to acquire a full understanding of the regulatory processes of mosquito growth and reproduction, and might lead to the utilization of the CHSA gene in new approaches to insect control. Culex pipiens pallens ( C. pipiens pallens ) is a common house mosquito and is a vector of West Nile virus (WNV), epidemic encephalitis, Wucheraria bancrofti, and Brugia malavi [ 35 ]. In the present study, we aimed to identify and characterize the CHSA gene from C. pipiens pallens ( CpCHSA ). We used RNAi to identify whether CpCHSA is essential for growth and development at different development stages and in different tissues of C. pipiens pallens . The results indicated that CpCHSA affects chitin synthesis and degradation, which is necessary for molting and ecdysis processes. CpCHSA is also important for cuticle formation in adult stages, playing critical roles in endocuticle development. Materials And Methods Mosquito Rearing The C. pipiens pallens were reared at 28 ± 1 °C and 70–80% relative humidity with a 12 h light and 12 h dark photoperiod. The larvae were fed with rat chow. Adult mosquitoes were maintained on 7% sucrose solution. The female mosquitoes were fed fresh mouse blood to induce egg laying. Mosquitoes were not treated with insecticides or other chemicals. CpCHSA cDNA cloning The RNAiso Plus reagent (Takara, Tokyo, Japan) was used to isolate RNA. The full‑length sequence of CpCHSA cDNA was determined from seven overlapping PCR fragments (Additional file 1: Table S1). The 5'- and 3'- end fragments were obtained using rapid amplification of cDNA ends (RACE) using a SMARTer RACE 5'/3' Kit (Takara). PCR amplification products were analyzed using agarose gels and then purified (TIANGEN, Beijing, China). Purified DNA was ligated into vector pClone007 (TSINGKE, Nanjing, China) and sequenced. The obtained full-length cDNA of CpCHS A was submitted to the NCBI and received the accession number MH013352. Analysis of gene expression patterns Total RNA was isolated from the larval stage (1st, 2nd, 3 rd , and 4th), pupal stage (0 and 24 h) and adult stage (1–3 posteclosion (PE) and 1–3 post-blood meal (PBM)) to investigate the C. pipiens pallens developmental expression profile. The head, foregut, midgut, hindgut, Malpighian tubules, and carcass were dissected from 4th larvae or 3‑d old adult mosquitoes for tissue-specific expression analysis. Total RNA was isolated from the whole bodies of five mosquitoes and the tissues of ten mosquitos for biological replicates. First stand cDNA was synthesized using PrimeScript RT Master Mix (Takara). A LightCycler® 96 Instrument was used for quantitative real-time PCR (qPCR) analysis (Roche, Basel, Switzerland) with the BrightGreen 2*qPCR MasterMix-No Dye (Applied Biological Materials, Vancouver, Canada). The specific primers are shown in Additional file 2: Table S2. The qPCR reaction volume (10 μL) contained the Power SYBR Green PCR Master Mix, specific primer sequences, and diluted cDNA (1 mg/mL). The relative expression levels were normalized to the internal control ACTB (encoding β-actin) using the 2 −ΔΔCt method [ 36 ]. All experiments were performed with three biological replicates. Microinjection RNAi was used to knockdown the expression of CpCHSA . The small interfering RNA sequences used to silence the CpCHSA gene (siCHSA) and negative control (NC) are shown in Additional file 3: Table S3, both of which were designed and manufactured by Gene Pharma (Shanghai, China). siCHSA or NC (0.30 μg) was injected into the 3rd larvae abdomen and the adult female mosquitoe thorax. In the pupal stage, they were injected into the dorsal cuticle between the thorax and the abdomen. Both the wild-type (WT) group and NC group served as controls. Total RNA was isolated from whole mosquitoes (n ≥ 5), and CpCHSA transcript levels were analyzed after RNAi using qPCR. Immunofluorescence staining The chitin content of the abdominal integuments was quantified after siCHSA or NC injection using chitin staining. Pupae (0–1 h) were selected to be injected with siRNA, and at 24 h after injection, the pupal abdomen was immediately dissected. Slides of paraffin-embedded tissue were deparaffinized in xylene and rehydrated using an ethanol gradient. Fluorescent Brightener 28 (Sigma-Aldrich, Hamburg, Germany) was used to stain the sample, propidium iodide was used as a counterstain, anti-fluorescent stain was added, and the samples were observed under an AXIO confocal fluorescence microscope (Zeiss, Oberkochen, Germany)[ 37 ]. For the immunofluorescence experiment, rabbit polyclonal antibodies were prepared against CpCHSA . We designed the peptide antigen of CpCHSA by analyzing the cDNA and protein sequences (Additional file 4: Table S4). The peptide was synthesized, and then subcloned into pET-28a-sumo and PGEX-4T-AB1 transfer plasmids. (ABclonal Wuhan, China) synthesized the gene and produced polyclonal antibody. The acquired antibodies were tested to ensure that they met the experimental requirements. To analyze the localization of the CpCHSA protein, paraffin sections were made from pupae treated with siCHSA or NC. The tissues were fixed in 4% paraformaldehyde at 4 °C overnight. Sections were acquired and then deparaffinized using xylene with two washes of 15 min, rehydrated through successive baths of ethanol (100, 96, and 70% in water, 15 min each), washed twice for 5 min, and then once with PBST (0.01 M phosphate-buffered saline (PBS), pH 7.4 containing 0.1% Tween 20) for 10 min. The sections were blocked with 2% bovine serum albumin for 30 min, followed by incubation by anti-CpCHSA antibodies (1:100) at 4 °C overnight. The sections were washed and then incubated with Alexa Fluor @ 594-conjugated donkey anti-rabbit IgG (Abcam, Cambridge, UK) secondary antibody (1:200 in blocking buffer) for 50 min, in the dark. After three washes in PBST for 5 min each, the nuclei were stained with 4' ,6‑diamidino-2-phenylindole (DAPI) for 10 min in the dark. The sections were then wash with PBST and observed under a fluorescence microscope (Zeiss)[ 26 ]. Electron microscopy Pupae (0–1 h) and adults (12–24 h PE) were injected with siCHSA or NC. At 24 h after injection of pupae and at 72 h after injection of the PE adults, tissues were collected and fixed in 4% paraformaldehyde at 4 °C, and then washed with PBS three times for 15 min each. 1% OsO 4 in 0.1 M PBS (pH 7.4) was used to post-fix the samples for 2 h at room temperature. The OsO 4 was removed and the samples were rinsed in PBS (0.1 M, pH 7.4) three times for 15 min each. The samples were then dehydrated through successive concentrations of ethanol (50, 70, 80, 90, 95, and 100%, for 15 min), and finally through two changes of acetone for 15 min, and then infiltrated with 1:1 acetone: EMBed 812 for 3 h, 2:1 acetone: EMBed 812 overnight, and pure EMBed 812 for 7 h. The samples were kept at 37 °C overnight and then baked at 60 °C for 48 h. Sections were cut and stained with uranyl acetate for 15 min followed by lead citrate staining for 15 min. The sections were air dried overnight. The ultrastructure of the cuticles was then analyzed using transmission electron microscopy (TEM)[ 38 ]. Western blotting Western blotting was used to evaluate the specificity of the anti-CpCHSA antiserum and to verify the knockdown efficiency of RNAi. Total proteins were extracted from the whole body of larval, pupal, or adult mosquitoes using Radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Thermo, Rockford, IL, USA), and then centrifuged at 12,000 × g at 4 °C for 30 min. A bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) was used for protein quantification. The proteins were fractionated using 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were probed using anti‑CpCHSA (1:1000) and β-actin (Abclonal 1:7000) antibodies, followed by incubation with labeled secondary antibodies and visualization of the immunoreactive protein bands. The bands were analyzed using Image J software (NIH, Bethesda, MA, USA). Statistical analysis SPSS 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) were used for statistical analyses[ 37 ]. The statistical significance of the RNAi knockdown efficiency and the survival rate were analyzed using an unpaired Student’s t test. * p < 0.05, ** p < 0.01, and *** p <0.001. All experiments were performed using at least three independent cohorts. Results CpCHSA cDNA The CpCHSA cDNA was isolated using RACE-PCR to amplify the 5' and 3' regions (Figure 1). The full-length CpCHSA cDNA (GenBank ID: MH013352) comprises 5396 nucleotides, of which 4740 nucleotides encode a putative protein of 1579 amino acid residues with calculated molecular mass of about 179.54 KDa. The CpCHSA protein contains three predicted domains; an N-terminal domain with seven transmembrane helices, a highly conserved central domain, and a C-terminal domain with an additional seven transmembrane helices. Ten potential N-glycosylation sites were predicted using NetNGlyc 1.0 software from the ExPASy Proteomics website. CpCHSA expression pattern To confirm the role of CpCHSA in C. pipiens pallens , we first examined the mRNA expression of CpCHSA in whole eggs (at 0 and 12 h), larval instars (1st, 2nd, 3rd, and 4th), pupae (at 0 and 24 h), and adults (1–3 d) PE. The qPCR results indicated that CpCHSA is expressed in all these different developmental stages, with predominant expression in the pupae and adult (Figure 2a). We also examined the tissue-specific expression patterns of CpCHSA . CpCHSA transcripts were significantly enriched in the head, body, and hindgut of the fourth instar larvae (Figure 2b); and in the foregut, leg, wing, and body of 3-d PE female mosquitoes (Figure 2c). We then examined the pattern of CpCHSA protein expression. Immunohistochemical analysis of paraffin-embedded samples was performed using 12 h and 24 h pupa (Figure 3). In the pupal stage, the CpCHSA protein in RNAi-NC (control) mosquitoes was detected in the eyes and exoskeleton; however, there was almost no expression in the siCHSA group. RNAi of CpCHSA hinders molting of C. pipiens pallens To investigate the impact of CpCHSA on the molting process, we first monitored molting after knockdown of CpCHSA in third instar larvae (n = 50) and pupae (n = 50). The larvae (Figure 4) and pupae (Figure 5) injected with siCHSA displayed a molting defect phenotype at the third to fourth instar, the fourth instar to pupal stage, and the pupal stage to adult stage. Specifically, the old cuticle was incompletely separated from the mosquito body. Adult mosquitoes exhibited prominent deformities of the leg, abdomen, and wings. CpCHSA -deficient adults had difficulty righting themselves and taking off compared with control adults. In the third instar larvae (Figure 4), RNAi of CpCHSA led to a substantial decrease of 62.9%, (unpaired Student’s t test; p < 0.0001) in the expression of the CpCHSA gene at the mRNA level (Figure 4a). Among 50 larvae injected with siCHSA, 23.5% (unpaired Student’s t test; p = 0.0035) died during the molting process from the third instar to the fourth stage (Figure 4c), 31.71% (unpaired Student’s t test; p = 0.0071) of the survivors died during the molting process from the fourth instar to the pupal stage (Figure 4d), 48.7% (unpaired Student’s t test; p = 0.00127) of the survivors died during the molting process from pupal to adult (Figure 4e), and 20.1% of the survivors (unpaired Student’s t test; p = 0.00127) died during the PE (Figure 4f). In the pupal stage (Figure 5), RNAi of CpCHSA led to a 58.3% decrease (unpaired Student’s t test; p < 0.0001) in CpCHSA expression at the mRNA level (Figure 5a). Among 50 pupae injected with siCHSA, 90% (unpaired Student’s t test; p < 0.0001) died during the molting process from the pupal stage to the adult stage (Figure 5c) and 75% (unpaired Student’s t test; p = 0.00103) of the surviving adult mosquitoes exhibited prominent deformities (Figure 5d). Effect of CpCHSA on chitin metabolism To further determine the effect of knockdown of CpCHSA expression on chitin content and the cuticle, we injected siRNA into 0–1 h pupae, and performed chitin staining (Figure 6) and transmission electron microscopy (TEM) (Figure 7) on the abdominal integument at 12 and 24 h after injection. At 12 and 24 h after injection, the old cuticle and chitin were separated from the epithelial cell layer in the NC group, while in the siCHSA group it was not. The results showed that siCHSA hindered the separation of old chitin from the epithelial cell layer (Figure 6b), and prevented the separation of the old cuticle from the epithelial cell layer (Figure 7b, and c). At 24 h after injection, we observed that new cuticle and chitin were formed, and the structure of the old cuticle and chitin was incomplete in the NC group. In the siCHSA group, the formation of new cuticle and chitin was inhibited, and the structure of old cuticle and chitin was complete (Figure 7d, and e). The results showed that knockdown of CpCHSA transcription inhibited the degradation of the old cuticle and the formation of new cuticle, which was caused by diminished chitin synthesis and degradation. RNAi resulted in the rigid structure of the old cuticle being more complete than that of the NC group, which would obstruct shedding of the old cuticle during molting. CpCHSA is required for the cuticle The cuticle of insect consists of the envelope, epicuticle, and procuticle [ 39 ]. The procuticle can be further divided into the exocuticle, endocuticle and mesocuticle, with horizontally-aligned chitin-protein rich laminae [ 40 , 41 ]. We observed the effect of siCHSA on the ultrastructure of the cuticle using TEM (Figure 8). Injection of siCHSA into third instar larval and 0–1 h pupal mosquitoes did not prevent all pupae from molting. The structure of the cuticle in the abdomen of RNAi mosquitoes had looser and less compact laminae compared with that of the control at 1-day posteclosion (Figure 8 b, and c). In addition, knockdown of CpCHSA resulted in an irregular leg structure and indistinct boundaries in the procuticle, whereas the leg of NC exhibited a normally organized, complete structure with uniform thickness (Figure 8c). CpCHSA- deficiency results in abnormal adult cuticle To further assess the function of CpCHSA in the adult mosquito, we injected siCHSA into one day PE mosquitoes. However, no significant morphological abnormalities nor a different survival rate were observed in either the siCHSA or NC group (Figure 9). In contrast, at 3 d PE, the ultrastructure of endocuticle of leg from siCHSA-treated insects was abnormal. As reported in several other insects, two distinct layers of chitin have been reported in the epidermis after adult molting. One, called the “mesocuticle”, forms underneath the exocuticle after adult eclosion, and the other, called the “endocuticle”, continues to be deposited below the mesocuticle. To analyze whether CpCHSA deficiency affects the ultrastructure of the mesocuticle and endocuticle, the leg cuticle microstructure of adults aged 1 d and 3 d were detected. At 3 days after eclosion, the cuticle of the leg of the WT insect contained a mesocuticle and endocuticle with a normal appearance. In contrast, the mosquitoes treated with siCHSA had only mesoderm at the corresponding stage, with no apparent endoderm structure (Figure 10). Discussion Research on the relevance of chitin to molting been documented in many insect species of insects; however, related information in mosquitoes is limited [ 6 , 26 , 32 ]. CHSA is a key enzyme in the synthesis of chitin, which is important for the development and growth of insects. In the present study, knockdown CpCHSA in C. pipiens pallens resulted in failure of the molting process, and failure of the cuticle structural integrity in adult mosquitoes, which suggested that CHSA is essential for the growth and development of mosquitoes. CpCHSA is expressed in the cuticle during molting, which has been well documented in insects [ 22 ]. In our study, CpCHSA expression was not limited to the molting process and cuticle, but was detected at all developmental stages and in all tissues. The expression of CpCHSA was highest in the pupal stage, followed by that in the adult mosquito. Further analysis showed that the expression of CpCHSA was higher mainly in the exoskeletons of larvae and non-blood meal fed female mosquitoes. Immunohistochemistry showed that CpCHSA was mainly expressed in the cuticle. Taken together, these results prompted us to hypothesize that, in addition to a role in the cuticle during molting, CpCHSA might also be required to ensure normal growth and development of the body and physiological functions in mosquitoes. RNA interference was applied at different developmental stages of C. pipiens pallens to study the function of CpCHSA systematically. Lethal phenotypes were observed in mosquitoes treated with RNAi for CpCHSA at most developmental stages. CpCHSA appears to be indispensable in the process of molting. Treatment with siCHSA led to the death of mosquitoes at the time of molting, and the death rate was highest in the pupal-adult stage, in which almost all pupae died as pharate adults entrapped in the old pupal cuticle, and defects in surviving adult mosquitoes were also observed. These results suggested that that CpCHSA has an essential role in molting. It is generally believed that the absence of CHSA causes a chitin synthesis disorder, which affects the formation of new cuticle and leads to the death of the insects in the molting stage [ 39 ]. However, by chitin staining, we observed a reduction of the chitin content in the new cuticle and increased chitin in the old cuticle of the abdomen cuticle after RNAi of CpCHSA compared with that in the controls. The old chitin could not be separated from the epidermis after RNAi of CpCHSA , and the old chitin was thicker after injection of siCHSA compared with the controls. A TEM study of the pupal abdominal cuticle also showed the same phenomena. The cuticle consists of many thin layers with alternating dense layers. In the siCHSA group, the old cuticle was compact and intact. It extended directly from the apical membrane of the basal epidermal cells to the epidermis. In contrast, the old cuticle ultrastructure was disrupted in the NC group insects. The inner and intermediate areas of the cuticle showed thin, unorganized sections with low compactness. The siCHSA group showed thinner new cuticle, and the new and old epidermis could not be separated. Chitin degradation disorders result in dense and hard old epidermis, which inhibits shedding during molting. During development, insects must periodically molt to accommodate growth and overcome the rigid constraints imposed by the chitin exoskeleton. The molting process begins when the epidermis secretes the outer layer of the new cuticle, separating the epidermis from the overlying old cuticle. A "dissolution space" is then formed to separate the new (internal) cuticle from the old (external) cuticle. The old cuticle is qualitatively digested by chitinases, and then falls off to promote the molting process as the exuvia during a molt; the chitin in the new cuticle is promoted by chitin synthetase-mediated synthesis, which facilitates insect survival [ 37 ]. These results suggested that CpCHSA is essential for the degradation of the old chitin and old epidermis and the formation of the new chitin and new epidermis during molting. This unexpected finding suggests that CHSA is not only involved in the process of molting, but may also affects chitin degradation. Chitin synthases are involved in chitin synthesis, and chitinases are involved in chitin degradation, which are two different processes. One possibility is that CHSA can affect the degradation of chitin; however, further proof is needed. RNAi for CpCHSA did not result in 100% molting failure and approximately 10% the adults survived. The surviving adult insects had malformed wings, legs, and abdomens. We also observed a reduction in the thickness and a loss of organization of both the abdomen and leg after RNAi for CpCHSA compared with those in the controls, which would affect cuticle rigidity. Taken together, CpCHSA is essential for the molting process. Therefore, we could control the adult mosquito population and reduce the spread of disease by inhibiting the expression of CHSA in the larval or pupal stages. The application of siCHSA in larval and pupal stages significantly reduced the thickness and density of the cuticle layer in the abdomen and legs of adult mosquitoes compared with those in the control group. In the qPCR results, CpCHSA was observed to be highly expressed in the wings and legs of adult mosquitoes. Therefore, we investigated the function of CpCHSA in the adult stage. Silencing CpCHSA did not result in mortality and deformity during adult development. We hypothesized that knockdown CpCHSA would cause changes in the microstructures of the adult cuticle. As reported in several other species of beetles [ 39 ], two distinct layers of chitin have been reported in the cuticle after adult molting. One is called the "mesocuticle", which forms beneath the exocuticle one day after adult ecdysis, and the other is called the "endocuticle", which continues to deposit beneath the mesocuticle two days after adult ecdysis. To analyze the structure and composition of the cuticle of adult mosquitoes, the leg anatomy of wild-type 1 d- and 3 d-old adult mosquitoes was observed using TEM. We observed that the cuticle of the leg of the 3-d PE mosquito had obvious mesocuticle and endocuticle. In contrast, the siCHSA-treated insects lacked the endocuticle structure, and the exocuticle contained more pore canals, such that the cuticle cannot form a dense layered structure. The thickness and structure of the epidermis have been reported to be related to adult development. An intact cuticle structure helps the insect resist external pressure [ 42 , 43 ]. The results of the present study showed that CpCHSA is essential for the formation of intact and dense cuticle structures of adult mosquitoes. Conclusion CpCHSA defects lead not only to fatal developmental malformation, but also affect cuticle development in the adult stage. In the present study, we provided a description of the effect of CpCHSA in the larvae, pupae, and adults of C. pipiens pallens, suggesting that CHSA has a broader effect on insects than previously thought. Chitin inhibitors are controversial because of their non-specificity, thus CHSA could be used as a new target to kill mosquitoes more efficiently and safely throughout their growth cycle. If successful, these approaches could eventually reduce the spread of mosquito‑borne viral infections. Abbreviations CHSA, chitin synthase A; CHSB, chitin synthase B; RNAi, RNA interference; siRNA, small interfering RNA; WT, wild-type; NC, negative control; qPCR, quantitative real time PCR; PU, pupae; PE, posteclosion; AD, adult; L3, the third instar larvae; L4, the fourth instar larvae; SEM standard error of the mean; TEM, transmission electron microscopy; PBM, post-blood meal; WT, wild-type; C. pipiens pallens , Culex pipiens pallens . Declarations Acknowledgments Not applicable Funding This work was supported by the National Natural Science Foundation of China (grant No. 81772227, 81672056 and 81672058) and the National S & T Major Program (grant No. 2017ZX10303404-002-006). Competing interests The authors declare that they have no competing interests. Ethics approval All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University for the Use of Laboratory Animals (Protocol No. 582/2017). Consent for publication Not applicable. Availability of data and materials All data are fully available without restriction. Author Contributions BS, DZ performed the conceptualization; BS, DZ, YS, LM responsible for data curation; XSY, YX, QY, HBZ, HTN provided for data acquisition; DZ and XSY carried out date analysis and manuscript editing. All authors have read and approved the manuscript for submission. References Castilho CJ, Li D, Liu M, Liu Y, Gao H, Hurt RH: Mosquito bite prevention through graphene barrier layers. Proc Natl Acad Sci USA 2019, 116(37):18304-18309. https://doi.org/ 10.1073/pnas.1906612116 . PubMed PMID: 31451645. Ling L, Raikhel AS: Serotonin signaling regulates insulin-like peptides for growth, reproduction, and metabolism in the disease vector. Proc Natl Acad Sci USA 2018, 115(42):E9822-E9831. 2018;115(42): E9822-E31. https://doi.org/ 10.1073/pnas.1808243115 . PubMed PMID: 30275337. 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Supplementary Files SupplementryTables.doc GraphicalAbstract.tif Cite Share Download PDF Status: Published Journal Publication published 01 May, 2021 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Major Revision 13 Feb, 2021 Review # 3 received at journal 10 Feb, 2021 Review # 2 received at journal 01 Feb, 2021 Review # 1 received at journal 25 Jan, 2021 Reviewer # 3 agreed at journal 21 Jan, 2021 Reviewer # 2 agreed at journal 19 Jan, 2021 Reviewer # 1 agreed at journal 12 Jan, 2021 Reviewers invited by journal 11 Jan, 2021 Editor invited by journal 01 Jan, 2021 Editor assigned by journal 01 Jan, 2021 Submission checks completed at journal 01 Jan, 2021 First submitted to journal 30 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-139574","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":7386224,"identity":"2d7b3783-db38-4b43-826d-c4b5c90c6e9b","order_by":0,"name":"Xiaoshan Yang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoshan","middleName":"","lastName":"Yang","suffix":""},{"id":7386225,"identity":"2ded3602-f85d-4468-981a-26f2f57ab7ab","order_by":1,"name":"Yang Xu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xu","suffix":""},{"id":7386226,"identity":"5b8be691-c6a6-49f4-b790-7324ecafe6b3","order_by":2,"name":"Qi Yin","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Yin","suffix":""},{"id":7386227,"identity":"4e520496-4ffa-4a8e-b60a-24a5277d02f6","order_by":3,"name":"Hongbo Zhang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Zhang","suffix":""},{"id":7386228,"identity":"1838098d-5c7b-4f86-9688-635687cd426a","order_by":4,"name":"Haitao Yin","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Yin","suffix":""},{"id":7386229,"identity":"2616171e-2115-4ed4-8beb-53aa27cdb007","order_by":5,"name":"Yan Sun","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Sun","suffix":""},{"id":7386230,"identity":"6304e8c0-54d9-4588-9177-441246efe533","order_by":6,"name":"Lei Ma","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Ma","suffix":""},{"id":7386231,"identity":"df6d0a97-55b7-494f-a6b6-1e15333b5d5a","order_by":7,"name":"Dan Zhou","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Zhou","suffix":""},{"id":7386232,"identity":"c4f294bd-44ad-44d4-89a1-571c30f87a6c","order_by":8,"name":"Bo Shen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACCQYGZgYGGwbGBhCPjXgtaaRrOQzlEaNFfnbzsccFFefzmKedMWD4UHaYgX92A34tBneOpRvPOHO7mHF2jgHjjHOHGSTuHCCgRSLHTJq37XZiI1ALM2/bYaBIAgGHzcj/BtRyDqLlLzFaGG7ksAG1HIBoYSRGi8GNNDPpGWeSgVrSCg72nEvnkbhB0GHJz6QLKuwSN85O3vjgR5m1HP8MQg6DAcMGBoYDQJqHSPUg64hXOgpGwSgYBSMNAADw/0HqsLjxCAAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Shen","suffix":""}],"badges":[],"createdAt":"2021-01-02 13:26:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-139574/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-139574/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-021-04741-2","type":"published","date":"2021-05-01T09:06:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4758507,"identity":"5a6ed928-6fee-4a2e-a9d6-711d926ee66a","added_by":"auto","created_at":"2021-01-06 18:50:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":293355,"visible":true,"origin":"","legend":"Nucleotide and deduced amino acid sequences of CpCHSA from Culex pipiens pallens (MH013352). The stop codon (TAA) is indicated by an asterisk (*) and marked in black. The putative polyadenylation signal (AATAA) is marked in black. The amino acid sequence of the putative catalytic domain is colored gray with a black background. The signature motifs (EDR and QRRRW) have a black background, and the putative N-glycosylation sites are underlined ","description":"","filename":"Onlinefig1.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/2b5607d6cc71607ab51ed357.png"},{"id":4758508,"identity":"1b31a303-3bfd-4e80-a662-71a88bc8319d","added_by":"auto","created_at":"2021-01-06 18:50:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43748,"visible":true,"origin":"","legend":"CpCHSA gene expression patterns. Relative expression levels of CpCHSA in different stages and different tissues, as assessed using qPCR. (a) Different stages include egg (EG), larvae (L1–4), pupae (PU), posteclosion (PE), and post-blood meal (PBM). Tissues include the head (HE), foregut (FG), midgut (MG), hindgut (HG), Malpighian tubules (MT), ovary (OV), leg (LE), wing (WI), and carcass (CA). Relative expression level of CpCHSA in (b) fourth instar larvae, (c) 72 h PE stage. The ACTB (β-actin) gene was used as an internal reference. Relative expression levels were calculated based on the lowest expression value, which was ascribed an arbitrary value of 1. Data are the means ± SEM of three biological replicates","description":"","filename":"Onlinefig2.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/f4384383e052abf3a5e74292.png"},{"id":4758504,"identity":"abca9f44-b640-4812-94f8-04f6a5f4f98a","added_by":"auto","created_at":"2021-01-06 18:50:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106582,"visible":true,"origin":"","legend":"CpCHSA protein localization. Immunofluorescence analysis was performed to determine the location of CpCHSA in pupal stages. (a) RNAi of CpCHSA in the pupal stage. (b) Cryosections of pupae that had been injected with siCHSA or NC were incubated with the anti-CpCHSA antibody (red) ","description":"","filename":"Onlinefig3.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/02df094a9ba9bcaa9c68f8ca.png"},{"id":4759035,"identity":"aac524db-63f8-498c-b33d-d1a3724389e5","added_by":"auto","created_at":"2021-01-06 18:56:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":158754,"visible":true,"origin":"","legend":"Phenotypes produced by RNAi of CpCHSA in the larvae stage. (a) Schematic diagram of the effect of siCHSA treatment on the moulting phenotype. The red arrow indicates the shedding old cuticle in the defective moulting process. (b) RNAi of CpCHSA in third instar larvae. Expression levels of CpCHSA at 72 h after injecting siCHSA, as assessed using western blotting. (c) siCHSA injection into third instar larvae reduce the survival rate of fourth instar larvae. (d) The survival rate of fourth instar larvae to the pupal stage. (e) The eclosion rate of pupae. (f) The survival rate of PE mosquitoes. Data represent three biological replicates (50 individuals in each replicate) with three technical replicates, and results are shown as the mean ± SEM (*p \u003c0.05, **p \u003c0.01, ***p \u003c0.001). PU, pupae; AD, adult; L3, the third instar larvae; L4, the fourth instar larvae; O-cuticle, old cuticle; PE, posteclosion","description":"","filename":"Onlinefig4change.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/910fd08e1d4ec712dc1d9c55.png"},{"id":4758513,"identity":"a7225f75-a673-4020-8daa-f1e08e53ce92","added_by":"auto","created_at":"2021-01-06 18:50:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81500,"visible":true,"origin":"","legend":"Phenotypes produced by RNAi of CpCHSA in the pupal stage.\n(a) Schematic diagram of siCHSA treatment in the 0–1 h pupae. (b) Levels of CpCHSA at 24 h after injecting siCHSA; CpCHSA protein levels were determined using western blotting analysis with a CpCHSA-specific polyclonal antibody. (c) siCHSA injection into pupae reduced the eclosion rate; (d) The survival rate of PE mosquitoes. All surviving individuals were used for measurement. Data represent three biological replicates (50 individuals in each replicate) with three technical replicates, and the results are shown as the mean ± SEM (*p \u003c0.05, **p \u003c0.01, ***p \u003c0.001). PU, pupae; AD, adult; O-cuticle, old cuticle; PE, posteclosion","description":"","filename":"Onlinefig5.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/d881778a8ca3daaa88ac2cd3.png"},{"id":4758514,"identity":"5d8d64a8-cc9e-432a-9022-1e09e73c69b7","added_by":"auto","created_at":"2021-01-06 18:50:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":211694,"visible":true,"origin":"","legend":"Effect of CpCHSA on the chitin metabolism of the new and old chitin. \n(a) RNAi of CpCHSA in the pupal stage; the abdomen of pupa was detected. The red arrow indicates the chitin position. The chitin staining experiment in the integument was performed by injecting siCHSA or NC into the pupae after 12 h (b), and 24 h (c). O-chitin, old chitin; N-chitin, new chitin","description":"","filename":"Onlinefig6.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/aed6d3436fb0ed43fc9664fa.png"},{"id":4759036,"identity":"a6a766ff-c7c7-48f5-b0f4-688a9b6ae0ba","added_by":"auto","created_at":"2021-01-06 18:56:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":585892,"visible":true,"origin":"","legend":"Ultrastructure of pupal cuticle from CpCHSA-deficient mosquitoes.\n(a) We analyzed the results using TEM. The pupal abdomen cuticles in the NC (b), and the siCHSA groups (c) at 12 h after injection. The pupal abdomen cuticles in the NC (d), and the siCHSA groups (e) at 24 h after injection. O-cuticle, old cuticle; N cuticle, new cuticle; PC, pore canal; EC, epithelial cell ","description":"","filename":"Onlinefig7.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/0e66f6a5ccf5ecd86b73befe.png"},{"id":4758860,"identity":"47fb5361-68c9-45b8-928f-82f14b385240","added_by":"auto","created_at":"2021-01-06 18:53:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":819094,"visible":true,"origin":"","legend":"Ultrastructure of leg and abdominal cuticles from CpCHSA-deficient adults. (a) NC or siCHSA was injected into 0–1 h pupae (300 ng per pupae). (b) The ultrastructure of 24 h PE insects was analyzed using TEM. Insects injected with NC or siCHSA. In the NC and siCHSA groups, (c) there were differences in the thickness and structure of the abdominal cuticle, (d) there were differences in the leg cuticle between the NC and the siCHSA groups. PC, pore canal; EXO, exocuticle; MESO, mesocuticle ","description":"","filename":"Onlinefig8.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/629c9fac860be7c3013478bb.png"},{"id":4758861,"identity":"43e13ffd-a46a-4ef3-a558-b994617d009d","added_by":"auto","created_at":"2021-01-06 18:53:55","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":175840,"visible":true,"origin":"","legend":"Phenotypes produced by RNAi of CpCHSA in the adult stage. (a) The 12–24 h PE mosquitoes were injected with siCHSA (300 ng per mosquito) or NC (controls). (b) At the 72 h PE stage, CpCHSA protein levels were determined using western blotting analysis with a CpCHSA-specific polyclonal antibody. (c) Injection of siCHSA had no effect on the adult phenotype. (d) Injection of siCHSA had no effect on the adult survival rate within 14 days. The results are shown as the mean ± SEM (*p \u003c 0.05, **p \u003c 0.01, ***p \u003c 0.001)","description":"","filename":"Onlinefig9.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/cf968a76dfc39c245cf03312.png"},{"id":4758511,"identity":"ea8f8cdd-22eb-4e71-9632-78ee9e7e8a64","added_by":"auto","created_at":"2021-01-06 18:50:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":277434,"visible":true,"origin":"","legend":"Analysis of the ultrastructure of mosquito leg cuticles. (a) The 12–24 h PE mosquitoes were injected with siCHSA (300 ng per mosquito) or NC (controls). After 72 h, the PE mosquito legs were collected. (b) Leg cuticles were collected from wild type (WT) PE mosquitoes (at 24, 48, and 72 h). (c) Representative transmission electron microscopy (TEM) images from NC, and siCHSA. PE, posteclosion; EXO, exocuticle; ENDO, endocuticle; MESO, mesocuticle ","description":"","filename":"Onlinefig10.png","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/eca3c2d00a86aa6080243662.png"},{"id":15671239,"identity":"393fac76-564d-4e37-826b-7bd0dbbd276d","added_by":"auto","created_at":"2021-11-18 14:05:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4079398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/667436d5-6ab1-4ae3-ae2a-b6fece3fa487.pdf"},{"id":4758856,"identity":"9baf1683-cc5e-449c-acc3-91a13f0db78b","added_by":"auto","created_at":"2021-01-06 18:53:55","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49664,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementryTables.doc","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/fcaa910fb8621697a668b94e.doc"},{"id":4758858,"identity":"e63d50d2-7432-4184-9e01-0b86a3760891","added_by":"auto","created_at":"2021-01-06 18:53:55","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5362324,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-139574/v1/af8d2148afe250f0f25ff0cd.tif"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePhysiological Characterization of the \u003cem\u003eChitin Synthase A\u003c/em\u003e Gene Responsible for Biosynthesis of Cuticle Chitin in \u003cem\u003eCulex Pipiens Pallens\u003c/em\u003e (Diptera: Culicidae)\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eMosquitoes are the vectors for transmission of many infectious diseases, causing hundreds of thousands of deaths every year [\u003ca href=\"#_ENREF_1\"\u003e1-5\u003c/a\u003e]. Currently, chemical insecticides are the main agents used to control insect vectors and reduce disease transmission. However, the widespread use of chemical control has led to the development of insect resistance, the resurgence of target pests, food and environment pollution, and the destruction of non-target insects [\u003ca href=\"#_ENREF_6\"\u003e6-8\u003c/a\u003e]. The environmental and health impacts of the toxicity of conventional insecticides have become increasingly unacceptable [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. Therefore, there is an urgent need to develop novel, safer bioinsecticides with low toxicity [\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. The normal growth and development of insect is inseparable from the process of molting [\u003ca href=\"#_ENREF_11\"\u003e11\u003c/a\u003e]. Many researchers investigated growth metabolic regulation and metabolism strategies in insects to identify novel targets for insect control [\u003ca href=\"#_ENREF_12\"\u003e12-17\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThe process of insect molting involves the biosynthesis, transformation, and modification of chitin [\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. Chitin, a linear polysaccharide of the amino sugar N-acetyl glucosamine, is the main component of insects\u0026rsquo; extracellular barrier, such as the cuticle and the peritrophic matrix (PM).\u0026nbsp;Chitin is also an important component in the tracheal system, reproductive ducts, and the ducts of various dermal glands in the internal structures of many insects [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e, \u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. Chitin plays an important role in supporting muscle attachment for movement, preventing chemical and physical damage, and preventing infectious diseases, representing the first line of defense in challenging environments [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e, \u003ca href=\"#_ENREF_20\"\u003e20\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eChitin biosynthesis plays an irreplaceable role in the growth of insects. Chitin biosynthesis requires modification and physiological regulation at developmental stages. Chitin is completely absent from vertebrates and higher plants[\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e, \u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e], and thus has generated interest as a potential target for ecologically friendly insecticides; therefore, an understanding of chitin biosynthetic pathways could provide additional strategies for pest control. Chitin synthesis inhibitors (CSI) can prevent insects from molting by interfering with chitin biosynthesis [\u003ca href=\"#_ENREF_8\"\u003e8\u003c/a\u003e]. Among them, the Benzylphenolurea (BPU) insecticides have shown great potential to inhibit chitin biosynthesis; however, the non-targeting effects of BPUs can adversely affect beneficial species, such as bees, making them a controversial group of insecticides [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. Therefore, it is necessary to study chitin synthase to find new and effective target sites to combat insect pests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The chitin biosynthesis pathway involves eight key regulatory enzymes. The last step is executed by the chitin synthases (CHSs, UDP-N-acetyl-D- glucosamine: chitin 4-beta-N-acetylglucosaminyltransferase), which form a large group of plasma membrane proteins belonging to family 2 of the glycosyltransferases [\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e]. Insects commonly possess two chitin synthase genes: class A CHSs (CHSA) are primarily involved in chitin synthesis for the exoskeleton cuticle [\u003ca href=\"#_ENREF_24\"\u003e24-28\u003c/a\u003e]; while class B CHSs (CHSB) play a major role in the synthesis of chitin in the intestinal PM [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e, \u003ca href=\"#_ENREF_29\"\u003e29\u003c/a\u003e]. Currently available data of biochemically characterized insect CHSs show that insect CHSA is associated with insect molting [\u003ca href=\"#_ENREF_30\"\u003e30\u003c/a\u003e, \u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e], and is a crucial enzyme that balances growth and development. RNA interference (RNAi) to silence \u003cem\u003eCHSA\u003c/em\u003e in \u003cem\u003eManduca sexta\u003c/em\u003e [\u003ca href=\"#_ENREF_32\"\u003e32\u003c/a\u003e] and \u003cem\u003eSpodopetera exigua\u003c/em\u003e resulted in disruption of the cuticular exoskeleton and tracheal ectodermis [\u003ca href=\"#_ENREF_33\"\u003e33\u003c/a\u003e]. In \u003cem\u003eTribolium\u003c/em\u003e\u003cem\u003ecastaneum\u003c/em\u003e and\u003cem\u003e Locusta migratoria manilensis\u003c/em\u003e, CHSA is required for larval-larval processes, and for larval-pupal and pupal-adult processes [\u003ca href=\"#_ENREF_34\"\u003e34\u003c/a\u003e]. In \u003cem\u003eTribolium castaneum\u003c/em\u003e, CHSA plays a prominent role in embryo development and oviposition [\u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e]. In \u003cem\u003eAnopheles gambiae\u003c/em\u003e, CHSA was detected in newly formed compound eyes [\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e]. However, those studies were performed in different developmental stages of different species, and the mechanism by which CHSA affects the molting process in mosquitoes remains unclear. Studying CHSA function in mosquitoes is crucial to acquire a full understanding of the regulatory processes of mosquito growth and reproduction, and might lead to the utilization of the \u003cem\u003eCHSA\u003c/em\u003e gene in new approaches to insect control.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCulex pipiens pallens\u003c/em\u003e (\u003cem\u003eC. pipiens pallens\u003c/em\u003e) is a common house mosquito and is a vector of West Nile virus (WNV), epidemic encephalitis, Wucheraria bancrofti, and Brugia malavi [\u003ca href=\"#_ENREF_35\"\u003e35\u003c/a\u003e]. In the present study, we aimed to identify and characterize the \u003cem\u003eCHSA \u003c/em\u003egene from \u003cem\u003eC. pipiens pallens\u003c/em\u003e (\u003cem\u003eCpCHSA\u003c/em\u003e). We used RNAi to identify whether \u003cem\u003eCpCHSA \u003c/em\u003eis essential for growth and development at different development stages and in different tissues of \u003cem\u003eC. pipiens pallens\u003c/em\u003e. The results indicated that \u003cem\u003eCpCHSA\u003c/em\u003e affects chitin synthesis and degradation, which is necessary for molting and ecdysis processes. \u003cem\u003eCpCHSA\u003c/em\u003e is also important for cuticle formation in adult stages, playing critical roles in endocuticle development.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003eMosquito Rearing\u003c/h2\u003e\n\u003cp\u003eThe\u003cem\u003e C. pipiens pallens\u003c/em\u003e were reared at 28\u0026nbsp;\u0026plusmn;\u0026nbsp;1 \u0026deg;C and 70\u0026ndash;80% relative humidity with a 12 h light and 12 h dark photoperiod. The larvae were fed with rat chow. Adult mosquitoes were maintained on 7% sucrose solution. The female mosquitoes were fed fresh mouse blood to induce egg laying. Mosquitoes were not treated with insecticides or other chemicals.\u003c/p\u003e\n\u003ch2\u003eCpCHSA cDNA cloning\u003c/h2\u003e\n\u003cp\u003eThe RNAiso Plus reagent (Takara, Tokyo, Japan) was used to isolate RNA. The full‑length sequence of \u003cem\u003eCpCHSA\u003c/em\u003e\u0026nbsp;cDNA was determined from seven overlapping PCR fragments (Additional file 1: Table S1). The 5'- and 3'- end fragments were obtained using rapid amplification of cDNA ends (RACE) using a SMARTer\u0026nbsp;RACE 5'/3' Kit (Takara). PCR amplification products were analyzed using agarose gels and then purified (TIANGEN, Beijing, China). Purified DNA was ligated into\u0026nbsp;vector pClone007 (TSINGKE, Nanjing, China) and sequenced. The obtained full-length cDNA of \u003cem\u003eCpCHS\u003c/em\u003eA was submitted to the NCBI and received the accession number MH013352.\u003c/p\u003e\n\u003ch2\u003eAnalysis of gene expression patterns\u003c/h2\u003e\n\u003cp\u003eTotal RNA was isolated from the larval stage (1st, 2nd, 3\u003csup\u003erd\u003c/sup\u003e, and 4th), pupal stage (0 and 24 h) and adult stage (1\u0026ndash;3 posteclosion (PE) and 1\u0026ndash;3 post-blood meal (PBM)) to investigate the \u003cem\u003eC. pipiens pallens\u003c/em\u003e developmental expression profile. The head, foregut, midgut, hindgut, Malpighian tubules, and carcass were dissected from 4th larvae or 3‑d old adult mosquitoes for tissue-specific expression analysis. Total RNA was isolated from the whole bodies of five mosquitoes and the tissues of ten mosquitos for biological replicates. First stand cDNA was synthesized using PrimeScript RT Master Mix (Takara). A LightCycler\u0026reg; 96 Instrument was used for quantitative real-time PCR (qPCR) analysis (Roche, Basel, Switzerland) with the BrightGreen 2*qPCR MasterMix-No Dye (Applied Biological Materials, Vancouver, Canada). The specific primers are shown in Additional file 2: Table S2. The qPCR reaction volume (10 \u0026mu;L) contained the Power SYBR Green PCR Master Mix, specific primer sequences, and diluted cDNA (1 mg/mL). The relative expression levels were normalized to the internal control \u003cem\u003eACTB\u003c/em\u003e (encoding \u0026beta;-actin) using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method [\u003ca href=\"#_ENREF_36\"\u003e36\u003c/a\u003e]. All experiments were performed with three biological replicates.\u003c/p\u003e\n\u003ch2\u003eMicroinjection\u003c/h2\u003e\n\u003cp\u003eRNAi was used to knockdown the expression of\u003cem\u003e CpCHSA\u003c/em\u003e. The small interfering RNA sequences used to silence the \u003cem\u003eCpCHSA \u003c/em\u003egene (siCHSA) and negative control (NC) are shown in Additional file 3: Table S3, both of which were designed and manufactured by Gene Pharma (Shanghai, China). siCHSA or NC (0.30 \u0026mu;g) was injected into the 3rd larvae abdomen and the adult female mosquitoe thorax. In the pupal stage, they were injected into the dorsal cuticle between the thorax and the abdomen. Both the wild-type (WT) group and NC group served as controls. Total RNA was isolated from whole mosquitoes (n \u0026ge; 5), and\u003cem\u003e CpCHSA \u003c/em\u003etranscript levels were analyzed after RNAi using qPCR.\u003c/p\u003e\n\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\n\u003cp\u003eThe chitin content of the abdominal integuments was quantified after siCHSA or NC injection using chitin staining. Pupae (0\u0026ndash;1 h) were selected to be injected with siRNA, and at 24 h after injection, the pupal abdomen was immediately dissected. Slides of paraffin-embedded tissue were deparaffinized in xylene and rehydrated using an ethanol gradient. Fluorescent Brightener 28 (Sigma-Aldrich, Hamburg, Germany) was used to stain the sample, propidium iodide was used as a counterstain, anti-fluorescent stain was added, and the samples were observed under an AXIO confocal fluorescence microscope (Zeiss, Oberkochen, Germany)[\u003ca href=\"#_ENREF_37\"\u003e37\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eFor the immunofluorescence experiment, rabbit polyclonal antibodies were prepared against \u003cem\u003eCpCHSA\u003c/em\u003e. We designed the peptide antigen of \u003cem\u003eCpCHSA\u003c/em\u003e by analyzing the cDNA and protein sequences (Additional file 4: Table S4). The peptide was synthesized, and then subcloned into pET-28a-sumo and PGEX-4T-AB1 transfer plasmids. (ABclonal Wuhan, China) synthesized the gene and produced polyclonal antibody. The acquired antibodies were tested to ensure that they met the experimental requirements.\u003c/p\u003e\n\u003cp\u003eTo analyze the localization of the CpCHSA protein, paraffin sections were made from pupae treated with siCHSA or NC. The tissues were fixed in 4% paraformaldehyde at 4 \u0026deg;C overnight. Sections were acquired and then deparaffinized using xylene with two washes of 15 min, rehydrated through successive baths of ethanol (100, 96, and 70% in water, 15 min each), washed twice for 5\u0026nbsp;min, and then once with PBST (0.01 M phosphate-buffered saline (PBS), pH 7.4 containing 0.1% Tween 20) for 10 min. The sections were blocked with 2% bovine serum albumin for 30 min, followed by incubation by anti-CpCHSA antibodies (1:100) at 4 \u0026deg;C overnight. The sections were washed and then incubated with Alexa Fluor\u003csup\u003e@ \u003c/sup\u003e594-conjugated donkey anti-rabbit IgG (Abcam, Cambridge, UK) secondary antibody (1:200 in blocking buffer) for 50 min, in the dark. After three washes in PBST for 5 min each, the nuclei were stained with 4' ,6‑diamidino-2-phenylindole (DAPI) for 10 min in the dark. The sections were then wash with PBST and observed under a fluorescence microscope (Zeiss)[\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e].\u003c/p\u003e\n\u003ch2\u003eElectron microscopy\u003c/h2\u003e\n\u003cp\u003ePupae (0\u0026ndash;1 h) and adults (12\u0026ndash;24 h PE) were injected with siCHSA or NC. At 24 h after injection of pupae and at 72 h after injection of the PE adults, tissues were collected and fixed in 4% paraformaldehyde at 4 \u0026deg;C, and then washed with PBS three times for 15 min each. 1% OsO\u003csub\u003e4\u003c/sub\u003e in 0.1 M PBS (pH 7.4) was used to post-fix the samples for 2 h at room temperature. The OsO\u003csub\u003e4 \u003c/sub\u003ewas removed and the samples were rinsed in PBS (0.1 M, pH 7.4) three times for 15 min each. The samples were then dehydrated through successive concentrations of ethanol (50, 70, 80, 90, 95, and 100%, for 15 min), and finally through two changes of acetone for 15 min, and then infiltrated with 1:1 acetone: EMBed 812 for 3 h, 2:1 acetone: EMBed 812 overnight, and pure EMBed 812 for 7 h. The samples were kept at 37 \u0026deg;C overnight and then baked at 60 \u0026deg;C for 48 h. Sections were cut and stained with uranyl acetate for 15 min followed by lead citrate staining for 15 min. The sections were air dried overnight. The ultrastructure of the cuticles was then analyzed using transmission electron microscopy (TEM)[\u003ca href=\"#_ENREF_38\"\u003e38\u003c/a\u003e].\u003c/p\u003e\n\u003ch2\u003eWestern blotting\u003c/h2\u003e\n\u003cp\u003eWestern blotting was used to evaluate the specificity of the anti-CpCHSA antiserum and to verify the knockdown efficiency of RNAi. Total proteins were extracted from the whole body of larval, pupal, or adult mosquitoes using Radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Thermo, Rockford, IL, USA), and then centrifuged at 12,000\u0026nbsp;\u0026times; \u003cem\u003eg\u003c/em\u003e\u0026nbsp;at 4 \u0026deg;C for 30 min. A bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) was used for protein quantification. The proteins were fractionated using 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were probed using anti‑CpCHSA (1:1000) and \u0026beta;-actin (Abclonal 1:7000) antibodies, followed by incubation with labeled secondary antibodies and visualization of the immunoreactive protein bands. The bands were analyzed using Image J software (NIH, Bethesda, MA, USA).\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eSPSS 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) were used for statistical analyses[\u003ca href=\"#_ENREF_37\"\u003e37\u003c/a\u003e]. The statistical significance of the RNAi knockdown efficiency and the survival rate were analyzed using an unpaired Student\u0026rsquo;s\u003cem\u003e t\u003c/em\u003e test. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003ep \u003c/em\u003e\u0026lt;0.001. All experiments were performed using at least three independent cohorts.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cem\u003eCpCHSA \u003c/em\u003ecDNA\u003c/h2\u003e\n\u003cp\u003eThe \u003cem\u003eCpCHSA\u003c/em\u003e cDNA was isolated using RACE-PCR to amplify the 5' and 3' regions (Figure 1). The full-length \u003cem\u003eCpCHSA\u003c/em\u003e cDNA (GenBank ID: MH013352) comprises 5396 nucleotides, of which 4740 nucleotides encode a putative protein of 1579 amino acid residues with calculated molecular mass of about 179.54 KDa. The CpCHSA protein contains three predicted domains; an N-terminal domain with seven transmembrane helices, a highly conserved central domain, and a C-terminal domain with an additional seven transmembrane helices. Ten potential N-glycosylation sites were predicted using NetNGlyc 1.0 software from the ExPASy Proteomics website.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCpCHSA\u003c/em\u003e expression pattern\u003c/h2\u003e\n\u003cp\u003eTo confirm the role of \u003cem\u003eCpCHSA\u003c/em\u003e in \u003cem\u003eC. pipiens pallens\u003c/em\u003e, we first examined the mRNA expression of \u003cem\u003eCpCHSA\u003c/em\u003e in whole eggs (at 0 and 12 h), larval instars (1st, 2nd, 3rd, and 4th), pupae (at 0 and 24 h), and adults (1\u0026ndash;3 d) PE. The qPCR results indicated that \u003cem\u003eCpCHSA\u003c/em\u003e is expressed in all these different developmental stages, with predominant expression in the pupae and adult (Figure 2a). We also examined the tissue-specific expression patterns of \u003cem\u003eCpCHSA\u003c/em\u003e. \u003cem\u003eCpCHSA\u003c/em\u003e transcripts were significantly enriched in the head, body, and hindgut of the fourth instar larvae (Figure 2b); and in the foregut, leg, wing, and body of 3-d PE female mosquitoes (Figure 2c).\u003c/p\u003e\n\u003cp\u003eWe then examined the pattern of CpCHSA protein expression. Immunohistochemical analysis of paraffin-embedded samples was performed using 12 h and 24 h pupa (Figure 3). In the pupal stage, the CpCHSA protein in RNAi-NC (control) mosquitoes was detected in the eyes and exoskeleton; however, there was almost no expression in the siCHSA group.\u003c/p\u003e\n\u003ch2\u003eRNAi of \u003cem\u003eCpCHSA \u003c/em\u003ehinders molting of C. pipiens pallens\u003c/h2\u003e\n\u003cp\u003eTo investigate the impact of \u003cem\u003eCpCHSA\u003c/em\u003e on the molting process, we first monitored molting after knockdown of \u003cem\u003eCpCHSA \u003c/em\u003ein third instar larvae (n = 50) and pupae (n = 50). The larvae (Figure 4) and pupae (Figure 5) injected with siCHSA displayed a molting defect phenotype at the third to fourth instar, the fourth instar to pupal stage, and the pupal stage to adult stage. Specifically, the old cuticle was incompletely separated from the mosquito body. Adult mosquitoes exhibited prominent deformities of the leg, abdomen, and wings. \u003cem\u003eCpCHSA\u003c/em\u003e-deficient adults had difficulty righting themselves and taking off compared with control adults.\u003c/p\u003e\n\u003cp\u003eIn the third instar larvae (Figure 4), RNAi of\u003cem\u003e CpCHSA\u003c/em\u003e led to a substantial decrease of 62.9%, (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) in the expression of the \u003cem\u003eCpCHSA\u003c/em\u003e gene at the mRNA level (Figure 4a). Among 50 larvae injected with siCHSA, 23.5% (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e = 0.0035) died during the molting process from the third instar to the fourth stage (Figure 4c), 31.71% (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e = 0.0071) of the survivors died during the molting process from the fourth instar to the pupal stage (Figure 4d), 48.7% (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e = 0.00127) of the survivors died during the molting process from pupal to adult (Figure 4e), and 20.1% of the survivors (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e = 0.00127) died during the PE (Figure 4f). In the pupal stage (Figure 5), RNAi of \u003cem\u003eCpCHSA\u003c/em\u003e led to a 58.3% decrease (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) in \u003cem\u003eCpCHSA\u003c/em\u003e expression at the mRNA level (Figure 5a). Among 50 pupae injected with siCHSA, 90% (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) died during the molting process from the pupal stage to the adult stage (Figure 5c) and 75% (unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003ep\u003c/em\u003e = 0.00103) of the surviving adult mosquitoes exhibited prominent deformities (Figure 5d).\u003c/p\u003e\n\u003ch2\u003eEffect of\u003cem\u003e CpCHSA\u003c/em\u003e on chitin metabolism\u003c/h2\u003e\n\u003cp\u003eTo further determine the effect of knockdown of \u003cem\u003eCpCHSA\u003c/em\u003e expression on chitin content and the cuticle, we injected siRNA into 0\u0026ndash;1 h pupae, and performed chitin staining (Figure 6) and transmission electron microscopy (TEM) (Figure 7) on the abdominal integument at 12 and 24 h after injection. At 12 and 24 h after injection, the old cuticle and chitin were separated from the epithelial cell layer in the NC group, while in the siCHSA group it was not. The results showed that siCHSA hindered the separation of old chitin from the epithelial cell layer (Figure 6b), and prevented the separation of the old cuticle from the epithelial cell layer (Figure 7b, and c). At 24 h after injection, we observed that new cuticle and chitin were formed, and the structure of the old cuticle and chitin was incomplete in the NC group. In the siCHSA group, the formation of new cuticle and chitin was inhibited, and the structure of old cuticle and chitin was complete (Figure 7d, and e). The results showed that knockdown of \u003cem\u003eCpCHSA\u003c/em\u003e transcription inhibited the degradation of the old cuticle and the formation of new cuticle, which was caused by diminished chitin synthesis and degradation. RNAi resulted in the rigid structure of the old cuticle being more complete than that of the NC group, which would obstruct shedding of the old cuticle during molting.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCpCHSA \u003c/em\u003eis required for the cuticle\u003c/h2\u003e\n\u003cp\u003eThe cuticle of insect consists of the envelope, epicuticle, and procuticle [\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e]. The procuticle can be further divided into the exocuticle, endocuticle and mesocuticle, with horizontally-aligned chitin-protein rich laminae [\u003ca href=\"#_ENREF_40\"\u003e40\u003c/a\u003e, \u003ca href=\"#_ENREF_41\"\u003e41\u003c/a\u003e]. We observed the effect of siCHSA on the ultrastructure of the cuticle using TEM (Figure 8). Injection of siCHSA into third instar larval and 0\u0026ndash;1 h pupal mosquitoes did not prevent all pupae from molting. The structure of the cuticle in the abdomen of RNAi mosquitoes had looser and less compact laminae compared with that of the control at 1-day posteclosion (Figure 8 b, and c). In addition, knockdown of \u003cem\u003eCpCHSA\u003c/em\u003e resulted in an irregular leg structure and indistinct boundaries in the procuticle, whereas the leg of NC exhibited a normally organized, complete structure with uniform thickness (Figure 8c).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCpCHSA-\u003c/em\u003edeficiency results in abnormal adult cuticle\u003c/h2\u003e\n\u003cp\u003eTo further assess the function of \u003cem\u003eCpCHSA\u003c/em\u003e in the adult mosquito, we injected siCHSA into one day PE mosquitoes. However, no significant morphological abnormalities nor a different survival rate were observed in either the siCHSA or NC group (Figure 9). In contrast, at 3 d PE, the ultrastructure of endocuticle of leg from siCHSA-treated insects was abnormal. As reported in several other insects, two distinct layers of chitin have been reported in the epidermis after adult molting. One, called the \u0026ldquo;mesocuticle\u0026rdquo;, forms underneath the exocuticle after adult eclosion, and the other, called the \u0026ldquo;endocuticle\u0026rdquo;, continues to be deposited below the mesocuticle. To analyze whether\u003cem\u003e CpCHSA \u003c/em\u003edeficiency affects the ultrastructure of the mesocuticle and endocuticle, the leg cuticle microstructure of adults aged 1 d and 3 d were detected. At 3 days after eclosion, the cuticle of the leg of the WT insect contained a mesocuticle and endocuticle with a normal appearance. In contrast, the mosquitoes treated with siCHSA had only mesoderm at the corresponding stage, with no apparent endoderm structure (Figure 10).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eResearch on the relevance of chitin to molting been documented in many insect species of insects; however, related information in mosquitoes is limited [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e, \u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e, \u003ca href=\"#_ENREF_32\"\u003e32\u003c/a\u003e]. CHSA is a key enzyme in the synthesis of chitin, which is important for the development and growth of insects. In the present study, knockdown \u003cem\u003eCpCHSA \u003c/em\u003ein \u003cem\u003eC. pipiens pallens\u003c/em\u003e resulted in failure of the molting process, and failure of the cuticle structural integrity in adult mosquitoes, which suggested that CHSA is essential for the growth and development of mosquitoes. \u003cem\u003eCpCHSA\u003c/em\u003e is expressed in the cuticle during molting, which has been well documented in insects [\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e]. In our study, \u003cem\u003eCpCHSA\u003c/em\u003e expression was not limited to the molting process and cuticle, but was detected at all developmental stages and in all tissues. The expression of\u003cem\u003e CpCHSA\u003c/em\u003e was highest in the pupal stage, followed by that in the adult mosquito. Further analysis showed that the expression of \u003cem\u003eCpCHSA\u003c/em\u003e was higher mainly in the exoskeletons of larvae and non-blood meal fed female mosquitoes. Immunohistochemistry showed that CpCHSA was mainly expressed in the cuticle. Taken together, these results prompted us to hypothesize that, in addition to a role in the cuticle during molting, CpCHSA might also be required to ensure normal growth and development of the body and physiological functions in mosquitoes.\u003c/p\u003e\n\u003cp\u003eRNA interference was applied at different developmental stages of \u003cem\u003eC. pipiens pallens\u003c/em\u003e to study the function of CpCHSA systematically. Lethal phenotypes were observed in mosquitoes treated with RNAi for \u003cem\u003eCpCHSA\u003c/em\u003e at most developmental stages. \u003cem\u003eCpCHSA\u003c/em\u003e appears to be indispensable in the process of molting. Treatment with siCHSA led to the death of mosquitoes at the time of molting, and the death rate was highest in the pupal-adult stage, in which almost all pupae died as pharate adults entrapped in the old pupal cuticle, and defects in surviving adult mosquitoes were also observed. These results suggested that that CpCHSA has an essential role in molting. It is generally believed that the absence of CHSA causes a chitin synthesis disorder, which affects the formation of new cuticle and leads to the death of the insects in the molting stage [\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e]. However, by chitin staining, we observed a reduction of the chitin content in the new cuticle and increased chitin in the old cuticle of the abdomen cuticle after RNAi of \u003cem\u003eCpCHSA \u003c/em\u003ecompared with that in the controls. The old chitin could not be separated from the epidermis after RNAi of\u003cem\u003e CpCHSA\u003c/em\u003e, and the old chitin was thicker after injection of siCHSA compared with the controls. A TEM study of the pupal abdominal cuticle also showed the same phenomena. The cuticle consists of many thin layers with alternating dense layers. In the siCHSA group, the old cuticle was compact and intact. It extended directly from the apical membrane of the basal epidermal cells to the epidermis. In contrast, the old cuticle ultrastructure was disrupted in the NC group insects. The inner and intermediate areas of the cuticle showed thin, unorganized sections with low compactness. The siCHSA group showed thinner new cuticle, and the new and old epidermis could not be separated. Chitin degradation disorders result in dense and hard old epidermis, which inhibits shedding during molting. During development, insects must periodically molt to accommodate growth and overcome the rigid constraints imposed by the chitin exoskeleton. The molting process begins when the epidermis secretes the outer layer of the new cuticle, separating the epidermis from the overlying old cuticle. A \"dissolution space\" is then formed to separate the new (internal) cuticle from the old (external) cuticle. The old cuticle is qualitatively digested by chitinases, and then falls off to promote the molting process as the exuvia during a molt; the chitin in the new cuticle is promoted by chitin synthetase-mediated synthesis, which facilitates insect survival [\u003ca href=\"#_ENREF_37\"\u003e37\u003c/a\u003e]. These results suggested that CpCHSA is essential for the degradation of the old chitin and old epidermis and the formation of the new chitin and new epidermis during molting. This unexpected finding suggests that CHSA is not only involved in the process of molting, but may also affects chitin degradation. Chitin synthases are involved in chitin synthesis, and chitinases are involved in chitin degradation, which are two different processes. One possibility is that CHSA can affect the degradation of chitin; however, further proof is needed. RNAi for \u003cem\u003eCpCHSA \u003c/em\u003edid not result in 100% molting failure and approximately 10% the adults survived. The surviving adult insects had malformed wings, legs, and abdomens. We also observed a reduction in the thickness and a loss of organization of both the abdomen and leg after RNAi for \u003cem\u003eCpCHSA\u003c/em\u003e compared with those in the controls, which would affect cuticle rigidity. Taken together, CpCHSA is essential for the molting process. Therefore, we could control the adult mosquito population and reduce the spread of disease by inhibiting the expression of CHSA in the larval or pupal stages.\u003c/p\u003e\n\u003cp\u003eThe application of siCHSA in larval and pupal stages significantly reduced the thickness and density of the cuticle layer in the abdomen and legs of adult mosquitoes compared with those in the control group. In the qPCR results, \u003cem\u003eCpCHSA\u003c/em\u003e was observed to be highly expressed in the wings and legs of adult mosquitoes. Therefore, we investigated the function of CpCHSA in the adult stage. Silencing \u003cem\u003eCpCHSA\u003c/em\u003e did not result in mortality and deformity during adult development. We hypothesized that knockdown \u003cem\u003eCpCHSA \u003c/em\u003ewould cause changes in the microstructures of the adult cuticle. As reported in several other species of beetles [\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e], two distinct layers of chitin have been reported in the cuticle after adult molting. One is called the \"mesocuticle\", which forms beneath the exocuticle one day after adult ecdysis, and the other is called the \"endocuticle\", which continues to deposit beneath the mesocuticle two days after adult ecdysis. To analyze the structure and composition of the\u0026nbsp;cuticle of adult mosquitoes, the leg anatomy of wild-type 1 d- and 3 d-old adult mosquitoes was observed using TEM. We observed that the cuticle of the leg of the 3-d PE mosquito had obvious mesocuticle and endocuticle. In contrast, the siCHSA-treated insects lacked the endocuticle structure, and the exocuticle contained more pore canals, such that the cuticle cannot form a dense layered structure. The thickness and structure of the epidermis have been reported to be related to adult development. An intact cuticle structure helps the insect resist external pressure [\u003ca href=\"#_ENREF_42\"\u003e42\u003c/a\u003e, \u003ca href=\"#_ENREF_43\"\u003e43\u003c/a\u003e]. The results of the present study showed that CpCHSA is essential for the formation of intact and dense cuticle structures of adult mosquitoes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCpCHSA defects lead not only to fatal developmental malformation, but also affect cuticle development in the adult stage. In the present study, we provided a description of the effect of CpCHSA in the larvae, pupae, and adults of \u003cem\u003eC. pipiens pallens, \u003c/em\u003esuggesting that CHSA has a broader effect on insects than previously thought. Chitin inhibitors are controversial because of their non-specificity, thus CHSA could be used as a new target to kill mosquitoes more efficiently and safely throughout their growth cycle. If successful, these approaches could eventually reduce the spread of mosquito‑borne viral infections.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCHSA, chitin synthase A; CHSB, chitin synthase B; RNAi, RNA interference; siRNA, small interfering RNA; WT, wild-type; NC, negative control; qPCR, quantitative real time PCR; PU, pupae; PE, posteclosion; AD, adult; L3, the third instar larvae; L4, the fourth instar larvae; SEM standard error of the mean; TEM, transmission electron microscopy; PBM, post-blood meal; WT, wild-type; \u003cem\u003eC. pipiens pallens\u003c/em\u003e, \u003cem\u003eCulex pipiens pallens\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant No. 81772227, 81672056 and 81672058) and the National S \u0026amp; T Major Program (grant No. 2017ZX10303404-002-006).\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University for the Use of Laboratory Animals (Protocol No. 582/2017).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data are fully available without restriction.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eBS, DZ performed the conceptualization; BS, DZ, YS, LM responsible for data curation; XSY, YX, QY, HBZ, HTN provided for data acquisition; DZ and XSY carried out date analysis and manuscript editing. All authors have read and approved the manuscript for submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCastilho CJ, Li D, Liu M, Liu Y, Gao H, Hurt RH: Mosquito bite prevention through graphene barrier layers. Proc Natl Acad Sci USA 2019, 116(37):18304-18309. https://doi.org/\u003ca href=\"https://doi.org/10.1073/pnas.1906612116\"\u003e10.1073/pnas.1906612116\u003c/a\u003e. PubMed PMID: 31451645.\u003c/li\u003e\n\u003cli\u003eLing L, Raikhel AS: Serotonin signaling regulates insulin-like peptides for growth, reproduction, and metabolism in the disease vector. Proc Natl Acad Sci USA 2018, 115(42):E9822-E9831. 2018;115(42): E9822-E31. https://doi.org/\u003ca href=\"https://doi.org/10.1073/pnas.1808243115\"\u003e10.1073/pnas.1808243115\u003c/a\u003e. 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Pubmed Central PMCID: 6794787.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chitin, Chitin synthase, molting, mosquito, insect","lastPublishedDoi":"10.21203/rs.3.rs-139574/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-139574/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe pathogens transmitted by mosquitoes (\u003cem\u003eCulex pipiens pallens\u003c/em\u003e) to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) represents a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines the chitin content might help with novel vector control strategies. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The injection of small interfering RNA targeting \u003cem\u003eCHSA \u003c/em\u003e(siCHSA) to knock down \u003cem\u003eCHSA\u003c/em\u003e transcripts of in larval, pupal, and adult stages, showed different lethal phenotypes. In the larval and pupal stages, \u003cem\u003eCHSA\u003c/em\u003e knockdown prevented larval molting, pupation, and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. In the adult stage, it also affected the laminar organization of mesoderm and the formation of pseudo orthogonally large fibers of the endoderm. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe present study provides a systematic and comprehensive description of the effects of \u003cem\u003eCHSA\u003c/em\u003e on morphogenesis and metamorphosis. The results showed that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our result further showed that CHSA is important for the structural integrity of the adult mosquito cuticle.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Physiological Characterization of the Chitin Synthase A Gene Responsible for Biosynthesis of Cuticle Chitin in Culex Pipiens Pallens (Diptera: Culicidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-06 18:50:53","doi":"10.21203/rs.3.rs-139574/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2021-02-14T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-11T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-02-02T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-01-26T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-01-22T00:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-01-20T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-01-13T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-12T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-01-02T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-02T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-01-01T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-12-31T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"efdfc76e-4038-4be8-a486-9c75fd12efad","owner":[],"postedDate":"January 6th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1732750,"name":"Parasitology"}],"tags":[],"updatedAt":"2021-11-12T09:06:02+00:00","versionOfRecord":{"articleIdentity":"rs-139574","link":"https://doi.org/10.1186/s13071-021-04741-2","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2021-05-01 09:06:02","publishedOnDateReadable":"May 1st, 2021"},"versionCreatedAt":"2021-01-06 18:50:53","video":"","vorDoi":"10.1186/s13071-021-04741-2","vorDoiUrl":"https://doi.org/10.1186/s13071-021-04741-2","workflowStages":[]},"version":"v1","identity":"rs-139574","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-139574","identity":"rs-139574","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}