Characterization of a Chitin Synthase Encoding Gene and Effect of Diflubenzuron in Soybean Aphid, Aphis Glycines

Abstract

Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®), a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.

Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths

“…However, in these three hemipterans only the ChSA were present while genes for ChSB were absent, which is consistent with the lack of PM in these hemipterans (Bansal et al 2012;Wang et al 2012).…”

“…Bactrocera dorsalis (Yang et al 2013); the moths Manduca sexta (Zhu et al 2002;Hogenkamp et al 2005), Ostrinia furnacalis Qu and Yang 2012), Plutella xylostella (Ashfaq et al 2007), Spodoptera exigua (Chen et al 2007; Kumar et al 2008), S. frugiperda (Bolognesi et al 2005); the beetle, Tribolium castaneum (Arakane et al 2004); the locust, Locusta migratoria manilensis (Zhang et al 2010); and the hemipterans, Aphis glycines (Bansal et al 2012), Laodelphax striatellus (Wang et al 2012) and Nilaparvata lugens (Wang et al 2012). However, in these three hemipterans only the ChSA were present while genes for ChSB were absent, which is consistent with the lack of PM in these hemipterans (Bansal et al 2012;Wang et al 2012).…”

Identification, mRNA Expression, and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly, Bactrocera dorsalis

“…Interestingly, no alternate forms had been identified the genome of pea aphid; only exon b was found. Whether the function of exon a has been taken over by exon b or whether exon a is not necessary for chitin synthesis in pea aphid is unknown 12. In this paper, our finding of the two alternative splicing exons of CHS1 in B. dorsalis supports the presence of only one splicing site in the middle of the ORF.…”

“…CHS1 is responsible for production of chitin required in cuticle and cuticular lining of the foregut, hindgut and trachea, whereas CHS2 specializes in chitin synthesis in the PM 1, 10. However, only CHS1 has been found in hemipterans such as pea aphids, triatomine bugs, and brown planthoppers, while the CHS2 gene was absent, consistent with the lack of PM in these insects 11, 12. The hemipterans may have lost CHS2 during their evolution.…”

Core RNAi Machinery and Sid1, a Component for Systemic RNAi, in the Hemipteran Insect, Aphis glycines

“…To determine the expression in different developmental stages, all four nymphal and adult (whole body) samples were collected from insects feeding on susceptible soybean plants (variety SD76R). Total RNA extraction, DNase treatment, cDNA synthesis, and qPCR reactions were performed as described previously [60–62]. Gene-specific qPCR primers were designed using Beacon Designer version 7.0 (Palo Alto, CA, USA), and are listed in Table S3.…”