Natural Variation in Odorant Recognition Among Odorant-Binding Proteins in Drosophila melanogaster

Chemical recognition is essential for survival and reproduction. Adaptive evolution has resulted in diverse chemoreceptor families, in which polymorphisms contribute to individual variation in chemosensation. To gain insights into the genetic determinants of individual variation in odorant recognition, we measured olfactory responses to two structurally similar odorants in a population of wild-derived inbred lines of Drosophila melanogaster. Odorant-binding proteins (OBPs) are the first components of the insect olfactory system to encounter odorants. Previously four single-nucleotide polymorphisms (SNPs) in the Obp99 group were associated with variation in olfactory responses to benzaldehyde. Here, we identify six different SNPs that are associated with variation in responses to a structurally similar odorant, acetophenone, in the same Obp genes. Five SNPs are in coding regions of Obp99b and Obp99d and one SNP is in the 3′-untranslated region of Obp99a (A610G). We found that the 610G allele is associated with higher response scores to acetophenone than the 610A allele, but with lower expression of Obp99a, suggesting that binding of acetophenone to Opb99a might limit rather than facilitate access to odorant receptors. Our results show that overlapping sets of OBPs contribute to odorant recognition for structurally similar odorants, but that different SNPs are associated with odorant-specific individual variation. Thus, dual olfactory recognition where OBPs regulate odorant access to receptors may enhance olfactory discrimination.ADAPTIVE evolution in diverse chemical environments has resulted in large multigene chemoreceptor families, including odorant-binding protein (Obp) genes, odorant receptor (Or) genes, and gustatory receptor (Gr) genes (Hekmat-Scafe et al. 2002; Robertson et al. 2003; Nozawa and Nei 2007; Nei et al. 2008; Su et al. 2009). Polymorphisms in these chemoreceptor genes contribute to individual variation in chemosensory behavior (Keller et al. 2007; Wang et al. 2007). At the same time, combinatorial recognition of odorants may contribute functional redundancy, which allows individual variation without compromising overall olfactory ability. This may be the reason why segregating null alleles of chemoreceptor genes can be maintained within a population (Takahashi and Takano-Shimizu 2005; Wang et al. 2007). Drosophila melanogaster presents a favorable model for investigating the genetic basis of individual variation in olfactory discrimination, because the genome can be manipulated readily. Furthermore, flies can be inbred, which enables repeated behavioral measurements on identical genotypes under controlled environmental conditions. In addition, both the olfactory and the gustatory systems of Drosophila have been well characterized (Su et al. 2009; Yarmolinsky et al. 2009). Convergent projections of olfactory neurons expressing distinct odorant receptors have been mapped to specific glomeruli in the antennal lobe (Gao et al. 2000; Vosshall et al. 2000), and detailed electrophysiological studies on transgenic flies have identified molecular response profiles of a large fraction of the odorant receptor repertoire (de Bruyne et al. 2001). Surprisingly, however, behavioral responses to odorants do not necessarily conform to predictions based on electrophysiological response profiles (Keller and Vosshall 2007).Whereas Drosophila odorant receptors have been studied extensively, less is known about the function of odorant-binding proteins (OBPs) in mediating odor recognition and olfactory discrimination. OBPs are secreted by support cells in olfactory sensilla into the aqueous perilymph that surrounds olfactory dendrites and are thought to facilitate solubilization and transport of hydrophobic odorants, thereby either promoting or limiting access of odorants to odorant receptors (Steinbrecht 1998). For example, the pheromone-binding protein of the silk moth, Bombyx mori, binds and releases bombykol in a pH-dependent manner at the membrane interface (Wojtasek and Leal 1999; Sakurai et al. 2004). In D. melanogaster, an OBP, Lush, is essential for activation of the Or67d receptor by the pheromone cis-vaccenyl acetate in trichoid sensilla of the Drosophila third antennal segment (Ha and Smith 2006; Kurtovic et al. 2007). Binding of the pheromone causes a conformational transition in Lush, which enables this OBP to activate the Or67d receptor (Laughlin et al. 2008). Lush also interacts with short chain alcohols (Kim et al. 1998), but recognition of alcohols by Lush does not involve a conformation change and, thus, proceeds via a different mechanism (Stower and Logan 2008).Polymorphisms in Obp genes can serve as a substrate for natural selection and contribute to speciation. A polymorphism in Obp57e is responsible for differences in host plant preference between D. sechellia and D. melanogaster. D. melanogaster flies lacking the Obp57e and Obp57d genes were no longer repelled by hexanoic and octanoic acid, toxins produced by Morinda citrifolia, the host plant for D. sechellia. Here, inactivation of an Obp gene has enabled D. sechellia to occupy a specialist evolutionary niche (Matsuo et al. 2007). Differences in expression levels between Ors and Obps between D. sechellia and D. simulans have also been reported and postulated to contribute to the evolution of host plant preferences (Kopp et al. 2008).Despite the demonstrated importance of OBPs in pheromone and host plant recognition, little is known about how naturally occurring allelic variation in Obp genes affects individual variation in olfactory behavior. Previously, we identified polymorphisms associated with natural variation in olfactory behavior in response to benzaldehyde in Obp99a, Obp99c, and Obp99d in a population of wild-derived inbred lines of D. melanogaster (Wang et al. 2007). These studies indicated that these OBPs are likely to recognize benzaldehyde in a combinatorial manner, similar to odorant recognition by mammalian odorant receptors (Malnic et al. 1999). This observation enables us to begin to explore OBP odorant response profiles using a population genetics approach that capitalizes on naturally occurring mutations that affect behavior. As a first step, we asked whether variation in responses to odorants that are chemically similar would be associated with the same or overlapping sets of OBPs and, if so, whether the same or different polymorphisms in these OBPs would contribute to individual variation for olfactory behavior in response to these odorants. We focused on genes of the Obp99 group, previously associated with phenotypic variation in response to benzaldehyde. We obtained complete sequences of these genes from 297 inbred lines from the same wild-derived inbred population of D. melanogaster and measured variation in olfactory behavior in response to acetophenone, which is structurally similar to benzaldehyde. These odorants occur in fruits from host plants on which flies from the Raleigh population feed (e.g., apples and peaches). We find that overlapping sets of OBPs contribute to recognition of these two odorants, but that different SNPs are associated with odorant-specific individual variation.