Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera

Abstract

Background: Diabrotica virgifera virgifera, western corn rootworm, is one of the most devastating species in North America. D. v. virgifera neonates crawl through the soil to locate the roots on which they feed. Carbon dioxide (CO2) is one of the important volatile cues that attract D. v. virgifera larvae to roots.

Results: In this study, we identified three putative D. v. virgifera gustatory receptor genes (Dvv_Gr1, Dvv_Gr2, and Dvv_Gr3). Phylogenetic analyses confirmed their orthologous relationships with known insect CO2 receptor genes from Drosophila, mosquitoes, and Tribolium. The phylogenetic reconstruction of insect CO2 receptor proteins and the gene expression profiles were analyzed. Quantitative analysis of gene expression indicated that the patterns of expression of these three candidate genes vary among larval tissues (i.e., head, integument, fat body, and midgut) and different development stages (i.e., egg, three larval stages, adult male and female).

Conclusion: The Dvv_Gr2 gene exhibited highest expression in heads and neonates, suggesting its importance in allowing neonate larvae to orient to its host plant. Similar expression patterns across tissues and developmental stages for Dvv_Gr1 and Dvv_Gr3 suggest a potentially different role. Findings from this study will allow further exploration of the functional role of specific CO2 receptor proteins in D. v. virgifera.

Fig. 1

The maximum-likelihood phylogeny of insect CO₂ receptor proteins. Three CO₂ receptor proteins identified from D. v. virgifera (Dvv) were compared against orthologous proteins from T. castaneum (Tcas), D. melanogaster (Dmel), and A. gambiae (Agam). For the T. castaneum proteins, the naming convention proposed by Robertson and Kent [11] is used. The phylogeny is the consensus tree based on bootstrap analysis with 1000 pseudoreplicates. The numbers at nodes show the bootstrap supporting values (%)

Fig. 2

Comparison of CO₂ receptor gene structures between D. v. virgifera and T. castaneum. Exons and introns located within the coding regions are depicted by boxes and peaks with their lengths (bp), respectively. The total length (bp) of each coding region is shown in square brackets. D. v. virgifera gene structures were determined by comparing transcript sequences and the draft genome sequence (Hugh Robertson, personal communication). Exon–intron structures for T. cantaneum CO₂ receptor genes are based on the annotations available in the BeetleBase [39]: TC030102 (TcasGr1), TC030103 (TcasGr2), and TC030104 (TcasGr3)

Fig. 3

Expression of CO₂ receptors (a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different tissues of Diabrotica v. virgifera. For qRT-PCR, relative expression of Dvv_Gr genes in different tissues was measured and normalized to an endogenous control (EF1a) as described in the “Methods” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P < 0.050)

Fig. 4

Expression of CO₂ receptors (a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different development stages of Diabrotica v. virgifera. For qRT-PCR, relative expression of Dvv_Gr genes in different stages was measured and normalized to an endogenous control (actin) as described in the “Methods” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P < 0.050)