Identification and Expression Analyses of Olfactory Gene Families in the Rice Grasshopper, Oxya chinensis, From Antennal Transcriptomes

Abstract

The rice grasshopper Oxya chinensis is an important agricultural pest of rice and other gramineous plants. Chemosensory genes are crucial factors in direct interactions with odorants in the olfactory process. Here we identified genes encoding 18 odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 94 olfactory receptors (ORs), 12 ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs) from O. chinensis using an transcriptomic approach. Semi-quantitative RT-PCR assays revealed that six OBP-encoding genes (OchiOBP4, 5, 8, 9, 10, and 14), one CSP gene (OchiOBP10) and two IR genes (OchiIR28 and 29) were exclusively expressed in antennae, suggesting their roles in olfaction. Real-time quantitative PCR analyses revealed that genes expressed exclusively or predominantly in antennae also displayed significant differences in expression levels between males and females. Among the differentially expressed genes, 17 OR-encoding genes, one CSP- and one SNMP-gene showed female-biased expression, suggesting that they may be involved in some female-specific behaviors such as seeking oviposition site; whereas the three remaining OR-encoding genes showed male-biased expression, indicating their possible roles in sensing female sex pheromones. Our results laid a solid foundation for future studies to reveal olfactory mechanisms as well as designing strategies for controlling this rice pest.

Keywords: Oxya chinensis; antennal transcriptome; expression analysis; identification; olfactory gene.

FIGURE 1

Phylogenetic tree of putative odorant-binding proteins (OBPs). Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated on the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site. Classic OBPs are in red; plus-C OBPs type-A in green; plus-C OBPs type-B in blue; and atypical OBPs in purple.

FIGURE 2

Phylogenetic tree of putative chemosensory proteins (CSPs). Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated at the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site.

FIGURE 3

Phylogenetic tree of putative odorant receptors (ORs). The distance tree was rooted by the conservative ORco gene orthologs (red). The species- specific clades and sister pairs are labeled with red dots. Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated at the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site. The clade I is in orange; clade II in green; and clade III in blue.

FIGURE 4

Phylogenetic tree of putative ionotropic receptors (IRs). Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated at the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site.

FIGURE 5

Phylogenetic tree of putative sensory neuron membrane proteins (SNMPs). Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated at the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site.

FIGURE 6

Tissue-specific expression of OchiOBPs, OchiCSPs, and OchiIRs. (A) OBPs. (B) CSPs. (C) IRs. Genes exclusively or predominantly expressed in antennae are labeled in red dots. The β-actin (Ochiβ-actin) and ribosomal protein 49 (Ochirp49) were used as internal references to test the integrity of each cDNA template. An, antennae; M, maxillary palps; T, foreleg tarsus; W, wings; G, genitals.

FIGURE 7

Relative expression levels of the olfactory-related genes in the female antennae, male antennae, and other body parts from Oxya chinensis. (A) ORs. (B) SNMPs and the antenna-predominant candidates (OBPs, CSPs, and IRs). FA, female antennae; MA, male antennae; Bo, other body parts. The expression levels were estimated using the 2^–ΔΔCT method. Bars represent standard error of three independent biological replicates with three technical duplicates for each replicate. Different small letters indicate statistically significant difference between tissues (p < 0.05, ANOVA, HSD).