Sex-linked pheromone receptor genes of the European corn borer, Ostrinia nubilalis, are in tandem arrays

Abstract

Background: Tuning of the olfactory system of male moths to conspecific female sex pheromones is crucial for correct species recognition; however, little is known about the genetic changes that drive speciation in this system. Moths of the genus Ostrinia are good models to elucidate this question, since significant differences in pheromone blends are observed within and among species. Odorant receptors (ORs) play a critical role in recognition of female sex pheromones; eight types of OR genes expressed in male antennae were previously reported in Ostrinia moths.

Methodology/principal findings: We screened an O. nubilalis bacterial artificial chromosome (BAC) library by PCR, and constructed three contigs from isolated clones containing the reported OR genes. Fluorescence in situ hybridization (FISH) analysis using these clones as probes demonstrated that the largest contig, which contained eight OR genes, was located on the Z chromosome; two others harboring two and one OR genes were found on two autosomes. Sequence determination of BAC clones revealed the Z-linked OR genes were closely related and tandemly arrayed; moreover, four of them shared 181-bp direct repeats spanning exon 7 and intron 7.

Conclusions/significance: This is the first report of tandemly arrayed sex pheromone receptor genes in Lepidoptera. The localization of an OR gene cluster on the Z chromosome agrees with previous findings for a Z-linked locus responsible for O. nubilalis male behavioral response to sex pheromone. The 181-bp direct repeats might enhance gene duplications by unequal crossovers. An autosomal locus responsible for male response to sex pheromone in Heliothis virescens and H. subflexa was recently reported to contain at least four OR genes. Taken together, these findings support the hypothesis that generation of additional copies of OR genes can increase the potential for male moths to acquire altered specificity for pheromone components, and accordingly, facilitate differentiation of sex pheromones.

Figure 1. Identification of the chromosomal position of O. nubilalis OR genes by BAC-FISH analysis.

BAC probe codes are shown on the right of each chromosome image (for details, see Table S2). Lines indicate correspondence of orthologous genes between B. mori and O. nubilalis. A) Comparison of the Z chromosomes. A vertical bar on the left side represents the B. mori Z chromosome drawn to relative scale taken from Kaikobase (http://sgp.dna.affrc.go.jp/KAIKObase/). B,C) Images of O. nubilalis chromosomes orthologous to B. mori chromosomes 16(B) and 23(C).

Figure 2. Structure of O. nubilalis OR genes determined in this study.

Numbers under bars indicate distance from putative transcriptional start sites (kbp) when all the exons and introns are identified. Blue squares represent exons and dotted squares indicate unidentified exons. Dotted lines show the correspondence of conserved sequences between the OnubOR5a/b, 7a/b genes and red squares indicate sequences solely appearing in one of the genes. Blue arrows represent putative repetitive elements found in O. nubilalis ESTs and genomic sequences.

Figure 3. Phylogenetic relationship of lepidopteran OR genes belonging to the sex pheromone receptor subfamily.

Blue and red boxes in panel A indicate regions separately shown in panels B and C, detailed phylogenetic trees of OR4, 5, 8 and OnOr6 genes (B) and OR7 genes (C) of Ostrinia moths. Detailed information of genes used for comparison is listed in Table S4.

Figure 4. The structure of the 181-bp repeat sequences.

Yellow squares indicate exon 7 sequences not included in the repeat. Blue squares indicate sequences shared with exon7 and repeats in intron 7. Pink squares indicate intron7-specific sequences. Green squares indicate sequences conserved among the OnubOR5a,b genes. Red vertical lines show base substitutions. Black vertical line indicates a putative deletion in the last truncated repeat of the OnubOR8 gene.

Figure 5. The order of O. nubilalis OR genes on the Z chromosome.

Pentagons represent the position, length and transcriptional orientation of OR genes. Bars under the pentagons indicate the sequenced contigs of clones 11K16 and 01I19.