Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae

Abstract

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

Keywords: Drosophila melanogaster; Scaptomyza flava; Brassicales; Or67b; SSR; chemoreceptor; evolution; gene duplication; herbivory; isothiocyanate; neofunctionalization; olfaction; olfactory receptor; olfactory specialization; specialization; subfunctionalization; wasabi.

Fig. 1.

Phylogeny of Scaptomyza and the orthologs of the odorant receptor Or67b (A) Time-calibrated Bayesian chronogram of Scaptomyza and Drosophila spp. inferred from nine protein coding and two ribosomal genes. Bars indicate 95% highest posterior density (HPD) age estimates in millions of years ago (Ma). Tree topology labeled as follows: Posterior Probability (PP):ML BS: Parsimony BS on branches. Scale bar proportional to Ma. (B) ML phylogeny reconstructed based on the coding sequence of Or67b orthologs from 12 Drosophila species, S. pallida, S. hsui, S. graminum, and S. flava. Source species for the Or67b coding sequences crossed into the empty neuron systems in subsequent figures are indicated. Support values are only indicated for bifurcations with less than 100% support in any analysis, labeled as in (A). Branches with significant support (FDR P-value < 0.05) for d_N/d_S values different from the background rate are indicated with colored branch labels (blue where the foreground rate is less than the background, and red/enlarged fonts where d_N/d_S is greater than the background). Only S. flava and D. mojavensis branches have significantly elevated d_N/d_S according to branch model tests. Scaptomyza flava, S. pallida, and D. melanogaster labeled identically to (A). Scale bar units are substitutions per site.

Fig. 2.

Responses of homologous Or67bs from D. melanogaster, S. pallida, and S. flava expressed in the D. melanogaster empty neuron systems to stimulation with natural odor blends. (A) Schematic representation of the single sensillum recording (SSR) using two “empty neuron systems”. Or67b proteins (X_Or67b, where X refers to the fly species) were expressed in a D. melanogaster mutant that lacks its endogenous Or22a in ab3A (antennal basiconic 3A) OSNs (Hallem and Carlson 2006; left), or Or67d in at1 (antennal trichoid 1) OSNs (Kurtovic et al. 2007; right). Note that the at1 empty neuron system was used only for expression of Sfla Or67b2, as this Or was not functional in the ab3A empty neuron system. Although comparison between the responses of this Or with other Or67b proteins should be interpreted cautiously (Syed et al. 2010), Or tuning (but not response intensity) is independent from the empty neuron system expressed for Or expression (supplementary fig. 5, Supplementary Material online). The antennal basiconic sensilla houses the A (which expresses one of the Or67b proteins) and the native intact B neuron; the A neuron has larger amplitude spikes than the B neuron, allowing discrimination of spikes originating from either neuron. The antennal trichoid sensilla houses a single OSN expressing Sfla Or67b2; arrows indicate spikes (see also supplementary fig. 4, Supplementary Material online). Calibration bars (vertical lines to the right of traces): 10 mV throughout all figures unless noted. (B) Representative electrophysiological recordings obtained from the targeted sensilla of flies expressing Or67b in OSNs in response to stimulation with apple cider vinegar, crushed rosette leaves of wild-type Col-0 Arabidopsis thaliana, and phytoalexin deficient 3 (PAD3) and aliphatic and indolic Glucosinolate Knock Out (GKO; myb28 myb29 cyp79b2 cyp79b3) A. thaliana mutants. Although all three A. thaliana genotypes have the same genetic background (Glazebrook and Ausubel 1994; Beekwilder et al. 2008), PAD3 plants are a more appropriate control for this study than GKO or Col-0, because PAD3 is deficient (as is GKO) in the production of camalexin but still produces normal levels of aliphatic or indolic glucosinolates. The bars above records indicate the onset and duration (1 s) of the stimulation throughout all figures unless otherwise noted. (C) Responses (net number of spikes/second, control subtracted, n = 6–9 obtained from 2 to 4 animals) evoked by stimulation with apple cider vinegar, odors from homogenized mustard leaves (arugula and A. thaliana), grated mustard root odors (wasabi, horseradish, turnip, and daikon), homogenized nonmustard leaf odors (tomato), and grated nonmustard root odors (beet, control). The outer edges of the horizontal bars represent the 25% and 75% quartiles, the vertical line inside the bars represents the median, and the whiskers represent 10% and 90% quartiles; each dot represents an individual response. Blue, gray, and green rectangles, respectively, show responses by stimulation with the Drosophila melanogaster attractant apple cider vinegar, non-ITC-bearing plants, and ITC-bearing plants. Asterisks indicate significant differences between the control-subtracted net number of spikes and a threshold value for responsiveness (10 spikes/second), as explained in Materials and Methods (one-sample signed rank tests; *P < 0.05, **P < 0.01). Neurons expressing Dmel Or67b and Spal Or67b, but not those expressing any of the S. flava Or67b paralogs, responded to stimulation with apple cider vinegar. Conversely, only neurons expressing S. flava Or67b paralogs responded to arugula odors (which bear ITCs). None of the S. flava paralogs responded to stimulation with the nonhost (non-ITC bearing) tomato. Dmel Or67b responded to all A. thaliana genotypes, whereas Sfla Or67b1-2 responded only to ITC-bearing A. thaliana, indicating that the presence of ITCs within plants is necessary to evoke responses from these two S. flava paralogs. Stimulation with wasabi root odors evoked responses from all Sfla Or67b paralogs but not from the Dmel or the Spal paralogs.

Fig. 3.

Responses of homologs Or67bs from D. melanogaster, S. pallida, and S. flava expressed in the D. melanogaster empty neuron systems to stimulation with single odorants. Experiments were conducted and analyzed as in figure 2. As before, the at1 empty neuron system was only used for expressing Sfla Or67b2 (see supplementary fig. 5, Supplementary Material online for responses to ITC from at1 OSNs expressing S. pallida and S. flava proteins). (A) Representative electrophysiological recordings obtained from the targeted sensilla of flies expressing Or67b genes in the empty neuron systems in response to stimulation with acetophenone and BITC at 1:100 vol/vol. (B) Responses evoked by stimulation with single odorants (tested at 1:100 vol/vol) categorized as follows: Dmel Or67b activators (Database of Odor Responses; Münch and Galizia 2016; blue), GLVs (gray), ITCs (green), benzyl thiocyanate (yellow), nitrile (pink), and TrpA1 activators (purple). OSNs expressing any of the Sfla Or67b paralogs respond strongly and selectively to ITCs (see also supplementary fig. 5, Supplementary Material online); these OSNs do not respond to benzyl thiocyanate stimulation (yellow bars), indicating that the ITC functional group (–N = C=S) is necessary for evoking responses from the S. flava paralogs. Spal Or67b and Dmel Or67b have similar odor-response profiles, responding mostly to stimulation with D. melanogaster activators and GLVs (*P < 0.05, **P < 0.01, one-sample signed rank tests performed as explained in fig. 2). Most odorants were diluted in mineral oil but some were diluted in other solvents as needed (see Materials and Methods). Responses to the appropriate solvent control were subtracted from odorant-evoked responses. (C) Tuning curves for each Or67b, showing the distribution of median responses to the 42 odorants tested (color coded as in A). The odorants are displayed along the horizontal axis according to the net responses they elicit from each Or. The odorants (numbers) eliciting the strongest responses for each Or are located at the center of the distribution and weaker activators are distributed along the edges (Hallem and Carlson 2006). Note that the strongest responses (center of the distribution) from Dmel Or67b and Spal Or67b are evoked by D. melanogaster activators and GLVs (blue and gray bars), whereas the strongest responses from all Sfla Or67b paralogs are evoked by ITCs (green bars). The tuning breadth of each Or is quantified by the kurtosis value (k) of the distribution (Willmore and Tolhurst 2001), with higher values indicating narrower odor-response profiles. The chemical structure of the top seven Sfla Or67b3 activators, as well as AITC, citronellal, acetophenone, cis-3-hexenyl butyrate, and benzyl thiocyanate (BTC) are shown in bottom.

Fig. 4.

Sfla Or67b1-3 have distinct ITC selectivity. (A) Dose responses of OSNs expressing Sfla Or67b1, Sfla Or67b2, and Sfla Or67b3 (abbreviated as b1, b2, and b3) to stimulation with increasing concentrations (vol/vol) of eight different ITCs (categorized according to molecular structure, top boxes; odorant abbreviations are as in fig. 3). As before, the at1 empty neuron system was used for expressing Sfla Or67b2 (see also supplementary fig. 5, Supplementary Material online). Data represent the control-subtracted net number of spikes (average ± SE; n = 6–8, obtained from 2 to 3 animals). (B) Heatmap of dose-responses (median, color coded) from the three Sfla paralog normalized (to allow comparisons across paralogs) by each paralog’s median response to 1:100 vol/vol of BITC (the strongest ITC activator across all paralogs). Asterisks indicate significant differences as explained in Materials and Methods (one-sample signed rank tests; * P < 0.05; ** P < 0.01). The strongest responses were evoked by the highest ITC concentration, with many compounds evoking responses from all paralogs, particularly in the case of Sfla Or67b1 and Or67b3. The number of stimuli that evoked responses decreased with decreasing odorant concentration. Overall, three ITCs evoked responses from two paralogs down to 10⁻⁴ vol/vol. See supplementary figure 6, Supplementary Material online for analysis of responses in odor space.

Fig. 5.

Olfactory behavioral responses of S. flava and its microbe-feeding relatives S. pallida and D. melanogaster to ecologically relevant odors and ITCs. (A) Schematic representation of the dual choice y-maze used to test the olfactory responses of flies (see details in supplementary fig. 8, Supplementary Material online). One arm of the maze offered constant odor/odorant airflow (apple cider vinegar, arugula, tomato, or single ITC compounds at 1:100 vol/vol), whereas the control arm offered a constant odorless airflow (controls: water for odor sources, and mineral oil for single odorants). In each test, a group of nonstarved flies (n = 3–4) was released at the base of the maze and allowed to choose between the two arms of the maze. Each test (maximum duration = 5 min) ended when the first insect (out of all released) made a choice. (B) Olfactory behavioral responses of D. melanogaster, S. pallida, and S. flava to apple cider vinegar odors and VOCs from leaves of arugula and tomato plants. Data represent the percentage of tests in which animals choose the odorous or odorless arms of the maze; numbers between parentheses indicate the number of tests with choices for one or the other arm. For each fly species and odor/odorant compound, data were analyzed using two-tailed Binomial tests (**P < 0.01, ****P < 0.001, *****P < 0.0001; gray-shaded bars serve to visualize that the proportion of tests with flies orienting toward that arm of the y-maze is significantly different from random). Scaptomyza flava was attracted to mustard (arugula) VOCs but not to nonmustard (tomato) VOCs. Scaptomyza flava tended to avoid apple cider vinegar odors although differences were not statistically significant (P = 0.054). Drosophila melanogaster was strongly attracted to apple cider vinegar but not to arugula or tomato leaf VOCs. Scaptomyza pallida was only attracted to tomato leaf VOCs. See supplementary figure 9, Supplementary Material online for responses to A. thaliana volatiles. (C) Olfactory behavioral responses of flies from the three species to single ITC compounds (20 µl of a 1:100 vol/vol dilution in mineral oil loaded in filter paper); the control arm had 20 µl of mineral oil loaded in filter paper. Assays were conducted as described in (B). Scaptomyza flava was strongly attracted to both ITC compounds tested, S. pallida was strongly repelled by BITC, and D. melanogaster was indifferent to either ITC.

Fig. 6.

Ectopic expression of Sfla Or67b3 in Or22a OSNs or Or67b OSNs conferred behavioral responses to BITC in D. melanogaster. The behavioral responses of D. melanogaster flies expressing different Or67b transgenes were tested in dual-choice assays (odorant vs. mineral oil) and experiments were conducted and analyzed as in figure 5 but flies were starved 24 h previous to testing. (A) Responses of flies expressing Dmel Or67b or Sfla Or67b3 in Or22a OSNs lacking its cognate Or and tested with BITC 1:1,000 vol/vol versus mineral oil. The two parental control lines (first two groups) and flies expressing Dmel Or67b (third group) were not attracted or repelled by BITC (binomial tests, P > 0.05 in all cases), whereas flies expressing Sfla Or67b3 were attracted to the odorant (*P < 0.05). Importantly, parental genetic control lines retain olfactory attraction toward apple cider vinegar, a potent D. melanogaster olfactory attractant and activator (supplementary fig. 10, Supplementary Material online). These results show that ITCs can evoke olfactory behavioral responses when Sfla Or67b3 is expressed in the D. melanogaster Or22a olfactory pathway, an important circuit for detection of host odorants in many drosophilid species (Dekker et al. 2006; Linz et al. 2013; Mansourian et al. 2018). (B) Same as (A), but flies expressed Dmel Or67b or Sfla Or67b3 in Or67b OSNs. Note that flies have the endogenous Or67b expressed in OSNs, in addition to the transgene. As in (A), only flies carrying the Sfla Or 67b3 transgene were attracted to BITC (*P < 0.05, third group). (C) Responses of D. melanogaster expressing a silencer of synaptic activity (Kir2.1) in Or67b OSNs (third group), along with the responses of the two parental genetic control lines (first and second group) in tests with acetophenone 1:50,000 vol/vol, a strong Dmel Or67b activator (Münch and Galizia 2016; fig. 3). Genetic control flies showed a trend for attraction (0.05 < P < 0.08) to low concentrations of acetophenone, whereas flies with Or67b OSNs silenced lost attraction and were instead repelled by the odorant. These behaviors mirrored those of wild-type D. melanogaster flies tested with low and high concentrations of acetophenone, respectively (supplementary fig. S11, Supplementary Material online; Strutz et al. 2014). In (A–C), transgenes are indicated to the left; gray-shaded bars serve to visualize that the proportion of tests with flies orienting toward that arm of the y-maze is significantly different (P < 0.05) from random.

Fig. 7.

A model for the evolution of Or67b and comparison with the known evolution of other Or and Ir orthologs in D. melanogaster and specialist drosophilid species. (A) Model for the evolution of Scaptomyza Or67b. The evolution of this Or begins with a shift in the ligand specificity of an ancestral Or67b (a) tuned to Dmel activators, GLVs, and ITCs (neofunctionalization, b). Subsequent gene triplication of Sfla Or67b gave rise to two additional paralogous Or67b genes (Sfla Or67b1, Sfla Or67b2, and Sfla Or67b3; c), each of the paralogs has different but overlapping ITC odorant-receptive ranges (figs. 3 and 4). (B) Evolution of drosophilid orthologs with known ligand specificities (Or22a, Ir75a, Ir75b, top; and Or67b, bottom). The Or22a protein orthologs from D. melanogaster (Dmel Or22a), D. sechellia (Dsec Or22a), D. erecta (Der Or22a), and D. suzukii (Dsuz Or22a) are all strongly activated by species-specific host-derived esters (compounds 1–4; top left; Dekker et al. 2006; Linz et al. 2013; Mansourian et al. 2018; Keesey et al. 2019). The Ir75a protein orthologs from D. melanogaster (Dmel Ir75a), and D. sechellia (Dsec Ir75a) are strongly activated respectively by the acid compounds 5 and 6 (Prieto-Godino et al. 2017). Similarly, the Ir75b protein orthologs from D. melanogaster (Dmel Ir75b) and D. sechellia (Dsec Ir75b) are respectively activated by the acids 7 and 8 (top right; Prieto-Godino et al. 2016). Dmel Or67b and Spal Or67b are strongly activated respectively by acetophenone and cis-3-hexenyl butyrate (compounds 9 and 10), whereas Sfla Or67b copies are activated by ITCs only (bottom, paralog-specific activation by compounds 11–13, abbreviations as in fig. 3). Note that Or22a, Ir75a, and Ir75b orthologs are all divergent but activated by ligands belonging to a single chemical class (whether esters or acids). On the other hand, the ligands of orthologs Or67b from Dmel and Spal are responsive to a variety of chemical classes which include alcohols, aldehydes, and ketones, whereas Sfla Or67b orthologs are responsive to ITCs, an entirely different compound chemical class. Notably, copies of Or22a, Or42b, and Or85d have been evolutionarily lost in S. flava. These Ors are important in mediating attraction to canonical microbe (especially yeast)-associated odors (Goldman-Huertas et al. 2015).