RNA-seq of Rice Yellow Stem Borer Scirpophaga incertulas Reveals Molecular Insights During Four Larval Developmental Stages

Abstract

The yellow stem borer (YSB), Scirpophaga incertulas, is a prominent pest in rice cultivation causing serious yield losses. The larval stage is an important stage in YSB, responsible for maximum infestation. However, limited knowledge exists on the biology and mechanisms underlying the growth and differentiation of YSB. To understand and identify the genes involved in YSB development and infestation, so as to design pest control strategies, we performed de novo transcriptome analysis at the first, third, fifth, and seventh larval developmental stages employing Illumina Hi-seq. High-quality reads (HQR) of ∼229 Mb were assembled into 24,775 transcripts with an average size of 1485 bp. Genes associated with various metabolic processes, i.e., detoxification mechanism [CYP450, GSTs, and carboxylesterases (CarEs)], RNA interference (RNAi) machinery (Dcr-1, Dcr-2, Ago-1, Ago-2, Sid-1, Sid-2, Sid-3, and Sid-1-related gene), chemoreception (CSPs, GRs, OBPs, and ORs), and regulators [transcription factors (TFs) and hormones] were differentially regulated during the developmental stages. Identification of stage-specific transcripts made it possible to determine the essential processes of larval development. Comparative transcriptome analysis revealed that YSB has not evolved much with respect to the detoxification mechanism, but showed the presence of distinct RNAi machinery. The presence of strong specific visual recognition coupled with chemosensory mechanisms supports the monophagous nature of YSB. Designed expressed sequenced tags-simple-sequence repeats (EST-SSRs) will facilitate accurate estimation of the genetic diversity of YSB. This is the first report on characterization of the YSB transcriptome and the identification of genes involved in key processes, which will help researchers and industry to devise novel pest control strategies. This study also opens up a new avenue to develop next-generation resistant rice using RNAi or genome editing approaches.

Keywords: RNAi; Scirpophaga incertulas; de novo transcriptome; detoxification mechanism; growth and development; insect.

Figure 1

Schematic representation of S. incertulas (yellow stem borer) transcriptome sequencing. EST-SSR, expressed sequenced tags-simple-sequence repeats; HQ, high-quality; MISA, MIcroSAtellite identification tool.

Figure 2

Venn diagram depicting the number of S. incertulas transcripts in each larval developmental stage. L₁, first instar larvae; L₃, third instar larvae; L₅, fifth instar larvae; L₇, seventh instar larvae.

Figure 3

Gene Ontology classification of S. incertulas transcripts. YSB transcripts were classified into biological process, cellular component, and molecular function. The left and right y-axes denote separately the percent and number of genes in the particular category. YSB, yellow stem borer.

Figure 4

Top 10 metabolic pathways represented in S. incertulas transcriptome through KEGG pathway analysis. KEGG, Kyoto Encyclopedia of Genes and Genomes; TCA, tricarboxylic acid cycle.

Figure 5

Phylogenetic tree of S. incertulas transcripts encoding insecticide mechanism along with other lepidopteran insects. (a) Cytochrome P450 (CYP450); (b) Glutathione S-transferases (GSTs); and (c) Carboxylesterases (CarEs). The tree was constructed from the multiple alignments using MEGA 5.0 software and generated with 1000 bootstrap trials using the Neighbor-Joining method. The numbers at the top of each node indicate bootstrap confidence values obtained for each node after 1000 repetitions. Bm, B. mori; Cm, C. medinalis; Cs, C. suppressalis; Hm, Heliconius melpomene; Ms, M. sexta; Of, O. furnacalis; Px, Plutella xylostella; Se, S. exigua; Si, S. incertulas; Sl, S. litura.

Figure 6

Phylogenetic tree for annotated sequences of chemoreception mechanism among S. incertulas (Si), lepidopteran, and hemipteran insects. Bm, B. mori; Cm, Cn. medinalis; Cs, C. suppressalis; Ha, H. armigera; Hv, He. virescens; Ms, M. sexta; Nl, N. lugens; On, O. nubilalis; Of, O. furnacalis; Px, P. xylostella; Se, S. exigua;.

Figure 7

Phylogenetic tree of RNA interference machinery from S. incertulas (Si) with lepidopteran and coleopteran insects. Bm, B. mori; Sl, S. litura; Tc, T. castenum.

Figure 8

Differentially expressed transcripts between four larval developmental stages of S. incertulas. Up- (red) and downregulated (green) YSB transcripts between the four developmental stages. L₁, first instar larvae; L₃, third instar larvae; L₅, fifth instar larvae; L₇, seventh instar larvae; YSB, yellow stem borer.

Figure 9

Significantly up- and downregulated and exclusively expressed transcripts at each stage of S. incertulas larval development.

Figure 10

Expression analysis of YSB transcripts at four developmental stages based on their relative FPKM values. Transcripts were hierarchically cluster based on average Pearson distance, complete linkage method. (a) Transcripts in major TF families; (b) Transcripts in RNAi machinery; and (c) Transcripts in chemoreception. Green indicates the lowest level of expression, black indicates the intermediate level of expression, and red indicates the highest level of expression. FPKM, Fragments Per Kilobase of transcript per Million mapped reads; L₁, first instar larvae; L₃, third instar larvae; L₅, fifth instar larvae; L₇, seventh instar larvae; RNAi, RNA interference; TF, transcription factor; YSB, yellow stem borer.

Figure 11

Validation of the RNA-sequencing (RNA-seq) data using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). All data were normalized to the reference gene, yellow stem borer β-actin. The transcripts validated were ecdysteroid-regulated protein (ERP), cytochrome p450 (CYP6AB51), neuropeptide receptor A10 (NPR) aminopeptidase N (APN), zinc finger DNA-binding protein (ZFDB), nicotinic acetylcholine receptor (nAChR), carboxylesterase (CarE), chemosensory protein (CSP), seminal fluid protein (SFP), and helix-loop-helix protein (HLH). Error bars indicates statistical significance of data.

Figure 12

Comparative analysis of yellow stem borer transcriptome with lepidopteran and hemipteran insect protein sequences at a cut-off E-value 10⁻⁶ and ≤ 80% similarity.