Physiological characterization of chitin synthase A responsible for the biosynthesis of cuticle chitin in Culex pipiens pallens (Diptera: Culicidae)

Abstract

Background: The pathogens transmitted by mosquitoes 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) is a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines chitin content may contribute to the development of novel vector control strategies.

Results: The injection of small interfering RNA targeting CHSA (siCHSA) to knockdown CHSA transcripts in larval, pupal and adult stages of Culex pipiens pallens resulted in the appearance of different lethal phenotypes. When larval and pupal stages were injected with siCHSA, 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. When siCHSA was injected into mosquitoes in the adult stage, CHSA knockdown also affected the laminar organization of the mesoderm and the formation of pseudo-orthogonal patterns of the large fibers of the endoderm.

Conclusion: We provide a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results show that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our results further show that CHSA is important for the structural integrity of the adult mosquito cuticle.

Keywords: Chitin; Chitin synthase; Insect; Molting; Mosquito.

Fig. 1

CpCHSA gene expression patterns. Relative expression levels of CpCHSA in different stages and different tissues, as assessed using quantitative real-time PCR (qPCR). a Relative expression at different stages: egg (EG), larvae (L1–4), pupae (PU), adult post-eclosion (PE) and adult post-blood meal (PBM). Five mosquitoes were collected from each group for RNA extraction. Relative expression levels were calculated in comparison with that at 0 h EG, which was ascribed an arbitrary value of 1. b, c Relative expression level of CpCHSA in L4 (b) and adult mosquito at 72 h PE (c). Tissues included the head (HE), foregut (FG), midgut (MG), hindgut (HG), Malpighian tubules (MT), ovary (OV), leg (LE), wing (WI) and carcass (CA). Tissues from ten mosquitoes were collected from each group for RNA extraction. Relative expression levels were calculated in comparison with expression in MT (b) and HE (c), which were ascribed an arbitrary value of 1. The ACTB (β-actin) gene was used as an internal reference. Data are from three independent experiments with three biological replicates. Different lowercase letters above the bars indicate a statistically significant difference at P < 0.05 (analysis of variance [ANOVA]); the same letter indicates data are not significantly different. CpCHSA Chitin synthase A gene (CHSA) from Culex pipiens pallens. L1, L2, L3, L4 First-, second-, third-, fourth-instar larvae, respectively

Fig. 2

Phenotypes produced by RNA interference (RNAi) of CpCHSA in the larvae stage (n = 150). The red arrow (c, d) indicates the shedding of old cuticle (O-cuticle) in the defective molting process. a, b RNAi of CpCHSA in L3. Expression levels of CpCHSA at 72 h after injection of siCHSA, as assessed using western blotting (a) and qPCR (b). Five mosquitoes were collected from each group for protein or RNA extraction. c siCHSA injection into L3 reduce the survival rate of L4. d The survival rate of L4 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 the results are shown as the mean ± standard error of the mean (SEM). Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, according to Student’s t-test. NC Negative control, siCHSA short interfering RNA sequences used to silence the CpCHSA gene, WT wild type

Fig. 3

Phenotypes produced by RNAi of CpCHSA in the pupal stage (n ≥ 50). siCHSA treatment in the 0- to 1-h pupae. a CpCHSA protein levels were determined using western blotting analysis with CpCHSA-specific polyclonal antibodies at 24 h after injection of siCHSA. b qPCR analysis of CpCHSA gene expression at 24 h after the injection of siCHSA. 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. Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, according to Student’s t-test

Fig. 4

CpCHSA protein localization. Immunofluorescence analysis was performed to determine the location of CpCHSA protein expression in the pupal stages. a RNAi of CpCHSA in the pupal stage. b Cryosections of pupae that had been injected with siCHSA or the NC were incubated with the anti-CpCHSA antibody (red). The fluorescence was quantified using ImageJ software and expressed as the mean ± SEM (n = 5). Asterisk indicates a significant difference at *P < 0.05, according to Student’s t-test

Fig. 5

Effect of CpCHSA on chitin metabolism of the new and old chitin. a RNAi of CpCHSA in the pupal stage (n ≥ 50) The red arrow indicates the position of the collected samples. 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). N-chitin New chitin, O-chitin old chitin

Fig. 6

Ultrastructure of pupal cuticle from CpCHSA-deficient mosquitoes, as determined by transmission electron microscopy (TEM). a, b The pupal abdomen cuticles in the NC (a) and the siCHSA groups (b) at 12 h after injection. c, d The pupal abdomen cuticles in the NC (c), and the siCHSA groups (d) at 24 h after injection. EC Epithelial cell, PC pore canal

Fig. 7

Ultrastructure of leg and abdominal cuticles from CpCHSA-deficient adults. NC or siCHSA was injected into 0- to 1-h pupae (300 ng per pupae). a The ultrastructure of adults at 24 h PE was analyzed using TEM. Insects were injected with NC or siCHSA. The red arrow indicates the leg and abdomen of the adult. b–c Between the NC and siCHSA groups, there were differences in the thickness and structure of the abdominal cuticle (b) and differences in the leg cuticle (c). EXO Exocuticle, MESO mesocuticle

Fig. 8

Phenotypes produced by RNAi of CpCHSA in the adult stage. The 12- to 24-h PE mosquitoes were injected with siCHSA (300 ng per mosquito) or NC. a At the 72-h PE stage, CpCHSA protein levels were determined using western blotting analysis with a CpCHSA-specific polyclonal antibody. b qPCR analysis of CpCHSA gene expression level at the 72-h PE stage. c, e Injection of siCHSA had no effect on the adult phenotype. d, f Kaplan–Meier survival curves were used to determine the female and male adult survival rates. The results are shown as the mean ± SEM. Asterisks indicate a significant difference at ***P < 0.001, according to Student’s t-test

Fig. 9

Analysis of the ultrastructure of mosquito leg cuticles. a The 12- to 24-h PE mosquitoes were injected with siCHSA (300 ng per mosquito) or NC (controls). After 72 h, legs were collected from the PE mosquitoes. The black arrow indicates the location at which the sample was collected. b Representative TEM images of leg cuticles from WT, NC and siCHSAL PE mosquitoes at 24, 48 and 72 h. ENDO Endocuticle