Identification of Genes Putatively Involved in Chitin Metabolism and Insecticide Detoxification in the Rice Leaf Folder (Cnaphalocrocis medinalis) Larvae through Transcriptomic Analysis

Abstract

The rice leaf roller (Cnaphalocrocis medinalis) is one of the most destructive agricultural pests. Due to its migratory behavior, it is difficult to control worldwide. To date, little is known about major genes of C. medinalis involved in chitin metabolism and insecticide detoxification. In order to obtain a comprehensive genome dataset of C. medinalis, we conducted de novo transcriptome sequencing which focused on the major feeding stage of fourth-instar larvae, and our work revealed useful information on chitin metabolism and insecticide detoxification and target genes of C. medinalis. We acquired 29,367,797 Illumina reads and assembled these reads into 63,174 unigenes with an average length of 753 bp. Among these unigenes, 31,810 were annotated against the National Center for Biotechnology Information non-redundant (NCBI nr) protein database, resulting in 24,246, 8669 and 18,176 assigned to Swiss-Prot, clusters of orthologous group (COG), and gene ontology (GO), respectively. We were able to map 10,043 unigenes into 285 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Specifically, 16 genes, including five chitin deacetylases, two chitin synthases, five chitinases and four other related enzymes, were identified to be putatively involved in chitin biosynthesis and degradation, whereas 360 genes, including cytochrome P450s, glutathione S-transferases, esterases, and acetylcholinesterases, were found to be potentially involved in insecticide detoxification or as insecticide targets. The reliability of the transcriptome data was determined by reverse transcription quantitative PCR (RT-qPCR) for the selected genes. Our data serves as a new and valuable sequence resource for genomic studies on C. medinalis. The findings should improve our understanding of C. medinalis genetics and contribute to management of this important agricultural pest.

Keywords: Cnaphalocrocis medinalis; chitin metabolism; insecticide detoxification; insecticide target; transcriptome.

Figure 1

Characteristics of homology search of Illumina sequences against the non-redundant (nr) database. (A) Species distribution is shown as the percentage of the total homologous sequences (with an E-value ≤1.0 × 10⁻⁵); (B) E-value distribution of BLAST hits for each unique sequence with a cut-off E-value of 1.0 × 10⁻⁵. We used all insect proteins in the National Center for Biotechnology Information (NCBI) nr database for homology search and extracted the best hit of each sequence for analysis.

Figure 2

Functional annotation of assembled sequences based on gene ontology (GO) categorization. GO analysis was performed at the level two for three main categories (cellular component, molecular function, and biological process).

Figure 3

Clusters of orthologous group (COG) classification. A total of 8669 unigenes with non-redundant database hits were grouped into 25 COG classifications.

Figure 4

Phylogenetic relationships of chitin-related enzymes deduced from the open reading frame (ORF) among C. medinalis and other insect species. The tree was constructed from the multiple alignments using MEGA 6.0 program (1000 bootstrap replications). Bootstrap values >50% are shown. The C. medinalis enzymes are indicated by red, green or pink markers. Cm, Cnaphalocrocis medinalis; Bm, Bombyx mori; Of, Ostrinia furnacalis; Px, Plutella xylostella; Se, Spodoptera exiqua; Eo, Ectropis oblique; Ms, Manduca sexta; Ag, Anopheles gambiae; Dm, Drosophila melanogaster; Bd, Bactrocera dorsalis; Tc, Tribolium castaneum; Mb, Mamestra brassicae; Dp, Danaus plexippus; Cf, Choristoneura fumiferana; Nv, Nasonia vitripennis; Md, Microplitis demolitor; Px, Papilio xuthus; Pp, Papilio polytes.

Figure 5

Real-time PCR analysis of the transcripts putatively encoding chitin metabolism enzymes and detoxification-related enzymes in C. medinalis. (A to E refer to relative expression level of C. medinalis chitin synthase 1 (CmCHS1), chitinase 1 (CmCHT1), chitin deacetylase 1 (CmCDA1), cytochrome P450 monooxygenase CYP9A79 (CmCYP9A79) and glutathionine S-transferase (CmGST). * p < 0.05, ** p < 0.01).

Figure 6

(A) Interaction networks of chitin enzymes associated with chitin biosynthesis based on the STRING website (focused on a specific protein network in Drosophila melanogaster); (B) Chitin metabolic pathway and related genes in C. medinalis. The solid arrow indicates chitin biosynthetic pathway; The dotted arrow and chain line arrow indicates chitin degradation pathway; CDA: chitin deacetylase, CHT: chitinase, Hex: β-l-N-acetylhexosaminidase, CHS: chitin synthase, UAP: UDP-N-acetylglucosamine pyrophosphorylase, PAGM: phosphoacetylglucosamine mutase, NAGK: N-acetyl-d-glucosamine kinase.