Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron

Abstract

Chitin synthase (EC 2.4.1.16) is a crucial enzyme responsible for chitin biosynthesis in all chitin-containing organisms. This paper reports a complete cDNA encoding chitin synthase 1 ( AqCHS1), change of AqCHS1 mRNA level in response to diflubenzuron exposure, and concentration-dependent effect of diflubenzuron on chitin synthesis in the common malaria mosquito ( Anopheles quadrimaculatus). The cDNA consists of 5723 nucleotides, including an open reading frame (ORF) of 4734 nucleotides that encode 1578 amino acid residues and a non-translated region of 989 nucleotides. The deduced amino acid sequence contains all the chitin synthase signature motifs (EDR, QRRRW and SWGTR) and shows 97% identity to that of An. gambiae ( AgCHS1, XM_321337). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 μg/L. As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 μg/L for 24 h. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 μg/L for 48 h resulted in decreases of chitin content by 9.0%, 43%, 58% and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased chitin synthesis may imply possible inhibition of chitin synthase, or abnormal chitin synthase translocation or chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. Further studies are necessary to elucidate the relationship between reduced chitin synthesis and increased expression of AqCHS1 in order to shed new light on trafficking and regulation of chitin biosynthesis in the mosquito affected by diflubenzuron.