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. 2012;8(10):1323-34.
doi: 10.7150/ijbs.4189. Epub 2012 Oct 25.

Characterization of a chitin synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines

Raman Bansal  1 , M A Rouf Mian, Omprakash Mittapalli, Andy P Michel
Affiliations

Characterization of a chitin synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines

Raman Bansal et al. Int J Biol Sci. 2012.

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.

Keywords: Aphis glycines; Chitin synthase; Diflubenzuron.; Embryo; Integument.

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Conflict of interest statement

Competing Interests: The author(s) declare that they have no competing interests.

Figures

Figure 1
Figure 1
Nucleotide and deduced amino acid sequences of AyCHS cDNA from Aphis glycines. The start codon (ATG), stop codon (TGA) and putative polyadenylation signal (AATAAA) are highlighted in black. The 16 hydrophobic, membrane-spanning α-helices predicted by TMHMM Server v. 2.0 are highlighted in gray. The amino acid sequence of the putative catalytic domain predicted by SMART tool is boxed. The 12 putative N-glycosylation sites predicted by PROSCAN are underlined. The cDNA sequence was deposited in the GenBank with accession number JQ246352.
Figure 1
Figure 1
Nucleotide and deduced amino acid sequences of AyCHS cDNA from Aphis glycines. The start codon (ATG), stop codon (TGA) and putative polyadenylation signal (AATAAA) are highlighted in black. The 16 hydrophobic, membrane-spanning α-helices predicted by TMHMM Server v. 2.0 are highlighted in gray. The amino acid sequence of the putative catalytic domain predicted by SMART tool is boxed. The 12 putative N-glycosylation sites predicted by PROSCAN are underlined. The cDNA sequence was deposited in the GenBank with accession number JQ246352.
Figure 2
Figure 2
Alignment of putative catalytic domain of chitin synthase gene in different hemipteran insects. The conserved and similar amino acid residues are labeled in black and grey backgrounds respectively. Red boxed amino acid residues represent highly conserved regions in glycosyltransferases (family 2) enzymes (based on 3). Sequence alignment was performed using Clustalw2 program -.The following insect chitin synthase sequences were used in the alignment: ApCHS: Acyrthosiphon pisum, XP_003247517.1; AyCHS: Aphis glycines, JQ246352; LsCHS: Laodelphax striatellus, ADR73029.1; NlCHS: Nilaparvata lugens, AEL88648.1.
Figure 3
Figure 3
Phylogeny of insect chitin synthases. A Phylogenetic tree constructed from amino acid sequences of various insect chitin synthases is shown. The percentages of replicate trees in which the sequences clustered together in the bootstrap test (10000 replicates) are shown (only above 50%) next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The scale bar represents 0.1 expected substitutions per amino acid position. The phylogenetic analysis was conducted in MEGA5.05. Chitin synthases were from Aphis glycines (Ay), Acyrthosiphon pisum (Ap), Laodelphax striatellus (Ls), Nilaparvata lugens (Nl), Pediculus humanus (Ph), Locusta migratoria (Lm), Manduca sexta (Ms), Aedes aegypti (Aa), Anopheles gambiae (Ag), Apis mellifera (Am), Drosophila melanogaster (Dm), Tribolium castaneum (Tc), Plutella xylostella (Px), and Caenorhabditis elegans (Ce). The accession numbers for various chitin synthases used in the phylogenetic analysis are provided in the Materials and Methods section.
Figure 4
Figure 4
Relative expression levels of chitin synthase gene (AyCHS) in different tissues and developmental stages of A. glycines as determined by real time PCR. Bars were generated after measuring the relative mRNA level of AyCHS gene in A. glycines. The mean (± S.E) expression level is represented for two biological replicates for developmental profiles and two biological replicates for tissue expression profiles. EF1α gene was used as an internal reference gene. The relative expression was calculated based on the value of the lowest expression which was ascribed an arbitrary value of 1. Different letters on the bars of the histogram indicate significant difference in gene expression compared to the treatment with lowest expression at P value < 0.05 (t-test). Different tissues dissected from A. glycines adults are A. gut, fat body and integument B. integument and embryo. Different developmental stages are C. N1-1st instar nymph, N2-2nd instar nymph, N3- 3rd instar nymph, N4- 4th instar nymph, A-adult.
Figure 5
Figure 5
Effect on diflubenzuron on A. glycines survival. A. Dead nymphal instar of A. glycines after being fed with soybean leaves that were treated earlier with 50ppm DFB solution. B. Alive and successfully molted nymphal instar of A. glycines after being fed with soybean leaves that were dipped earlier in water (control). C. Mortality of A. glycines nymphs following their feeding upon soybean leaves that were treated earlier with different dosages of DFB. Different data points in the figure represent the percent mortality (± S.E) as calculated from 3 replicates (n=3), each with 30 freshly hatched nymphs. D. Relative expression of the AyCHS in A. glycines feeding upon soybean leaves that were treated earlier with DFB @500ppm (treated) and water (control). Bars were generated after measuring the relative mRNA lelvel of AyCHS in A. glycines nymphs at day 3 and day 6. The mean (± S.D) expression level is represented for three biological replicates (n=3). Asterisk (*) indicates the significant difference at P value < 0.05 (t-test).
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