Identification of Odorant-Binding and Chemosensory Protein Genes in Mythimna separata Adult Brains Using Transcriptome Analyses

Abstract

Large numbers of chemosensory genes have been identified in the peripheral sensory organs of the pest Mythimna separata (Walker) to increase our understanding of chemoreception-related molecular mechanisms and to identify molecular targets for pest control. Chemosensory-related genes are expressed in various tissues, including non-sensory organs, and they play diverse roles. To better understand the functions of chemosensory-related genes in non-sensory organs, transcriptomic analyses of M. separata brains were performed. In total, 29 odorant-binding proteins (OBPs) and 16 chemosensory proteins (CSPs) putative genes were identified in the transcriptomic data set. The further examination of sex- and tissue-specific expression using RT-PCR suggested that eight OBPs (OBP5, -7, -11, -13, -16, -18, -21, and -24) and eight CSPs (CSP2-4, -8, CSP10-12, and -15) genes were expressed in the brain. Furthermore, bands representing most OBPs and CSPs could be detected in antennae, except for a few that underwent sex-biased expression in abdomens, legs, or wings. An RT-qPCR analysis of the expression profiles of six OBPs (OBP3-5, -9, -10, and -16) and two CSPs (CSP3 and CSP4) in different tissues and sexes indicated that OBP16 was highly expressed in male brain, and CSP3 and CSP4 were female-biased and highly expressed in brain. The expression levels of OBP5 and OBP10 in brain were not significantly different between the sexes. The findings expand our current understanding of the expression patterns of OBPs and CSPs in M. separata sensory and non-sensory tissues. These results provide valuable reference data for exploring novel functions of OBPs and CSPs in M. separata and may help in developing effective biological control strategies for managing this pest by exploring novel molecular targets.

Keywords: Mythimna separata; brain transcriptome; chemosensory genes; chemosensory protein; non-sensory organ; odorant binding protein.

FIGURE 1

Proportional homology distribution among other insect species based on the best BLAST hits against the NR database for the assembled unigenes from the Mythimna separata brain transcriptomes.

FIGURE 2

Histograms of gene ontology (GO) classifications (A) and clusters of orthologous groups of proteins (KOG) (B). (A) The GO classifications are summarized into three main categories: biological processes, cellular component, and molecular function. The right y-axis indicates the number of genes in a category, and the left y-axis indicates the percentage of genes in a specific term in that main category. (B) The x-axis indicates 26 categories. The left y-axis indicates the percentage of a specific gene classification in that main category, and the right y-axis indicates the number of genes in a category.

FIGURE 3

Multiple alignment of amino acid sequences of OBPs from M. separata. In the sequence alignments, only the proteins with full-length ORFs were selected. Conserved cysteine residues are highlighted, and signal peptides are boxed in red.

FIGURE 4

Multiple alignment of amino acid sequences of CSPs from M. separata. In the sequence alignments, the C-terminus of CSP15 and N-terminus of CSP16 are truncated. Four conserved residues are highlighted, and signal peptides are boxed in red.

FIGURE 5

Phylogenetic tree of putative OBPs from lepidopteran species. This tree was constructed using MEGA5.2 based on alignment results of MAFFT. Msep: Mythimna separata (black); Harm: Helicoverpa armigera (cyan); Hass: Helicoverpa assulta (green); Bmor: Bombyx mori (red); Hvir: Heliothis virescens (dark violet); Sexi: Spodoptera exigua (blue) Slit: Spodoptera litura (sandy brown). The clades in violet and light cyan represent general odorant-binding proteins and pheromone-binding proteins, respectively.

FIGURE 6

Phylogenetic tree of putative CSPs from lepidopteran species. This tree was constructed using MEGA5.2 based on alignment results of MAFFT. Msep: Mythimna separata (blue); Aips: Agrotis ipsilon (brown); Harm: Helicoverpa armigera (red); Hvir: Heliothis virescens (dark orchid); Hass: Helicoverpa assulta (cyan); Bmor: Bombyx mori (green); Slit: Spodoptera litura (orange); Sexi: Spodoptera exigua (black).

FIGURE 7

Mythimna separata OBPs and CSPs transcript levels in different tissues of male and female adults as evaluated by RT-PCR. MB: male brains; FB: female brains; MA: male antennae; FA: female antennae; MAb: male abdomens; FAb: female abdomens; ML: male legs; FL: female legs; MW: male wings; FW: female wings.

FIGURE 8

Six OBPs and two CSPs transcript levels in different tissues of both sexes as evaluated by RT-qPCR. The internal controls β-actin and gapdh were used to normalize transcript levels in each sample. The standard error is represented by the error bar, and the different letters above each bar denote significant differences (p < 0.05).