Host plant-driven sensory specialization in Drosophila erecta

Abstract

Finding appropriate feeding and breeding sites is crucial for all insects. To fulfil this vital task, many insects rely on their sense of smell. Alterations in the habitat--or in lifestyle--should accordingly also be reflected in the olfactory system. Solid functional evidence for direct adaptations in the olfactory system is however scarce. We have, therefore, examined the sense of smell of Drosophila erecta, a close relative of Drosophila melanogaster and specialist on screw pine fruits (Pandanus spp.). In comparison with three sympatric sibling species, D. erecta shows specific alterations in its olfactory system towards detection and processing of a characteristic Pandanus volatile (3-methyl-2-butenyl acetate, 3M2BA). We show that D. erecta is more sensitive towards this substance, and that the increased sensitivity derives from a numerical increase of one olfactory sensory neuron (OSN) class. We also show that axons from these OSNs form a complex of enlarged glomeruli in the antennal lobe, the first olfactory brain centre, of D. erecta. Finally, we show that 3M2BA induces oviposition in D. erecta, but not in D. melanogaster. The presumed adaptations observed here follow to a remarkable degree those found in Drosophila sechellia, a specialist upon noni fruit, and suggest a general principle for how specialization affects the sense of smell.

Figure 1.

Geographical distribution of D. erecta, its sympatric siblings and the genus Pandanus in tropical Africa. The four melanogaster sibling species differ in their ecology, distribution and phylogenetic relationship. Drosophila erecta occurs in swampy and coastal habitats of western-central Africa, and uses fresh Pandanus spp. fruits as a food source and breeding site. Its closest relative, Drosophila orena (known from a single collection event on Mount Lefo, Cameroon), has an unknown ecology. Drosophila melanogaster (cosmopolitan) and D. yakuba (endemic to tropical Africa) are generalists. Map by courtesy of Wikipedia; modified from ([15,16]; http://www.mobot.org). Pandanus candelabrum image adapted from an original illustration. Reproduced with the kind permission of the Director and the Board of Trustees, Royal Botanic Gardens, Kew.

Figure 2.

Antennal response spectra of the four melanogaster sibling species—evoked by the Pandanus sp. fruit headspace volatiles. (a) Headspace odour of Pandanus sp. fruits (upper part) and EAD responses (lower part). Bar stands for 1 mV of EAD response. Active compounds are coded as follows: 2,3-butanediol (no. 1), ethyl butyrate (no. 2), ethyl isovalerate (no. 3), isoamyl acetate (no. 4), 2-heptanone (no. 5), styrene (no. 6), 3M2BA (no. 7), ethyl tiglate (no. 8), 6-methyl-5-heptene-2one (no. 9), ethyl hexanoate (no. 10), linalool (no. 13), phenethyl alcohol (no. 14), benzyl acetate (no. 15), ethyl benzoate (no. 16), isopentyl hexanoate (no. 17), β-phenethyl acetate (no. 18). Peaks (no. 11), (no. 12) and (no. 19) are unidentified. Emphasized in pink is the novel Drosophila ligand 3M2BA (no. 7). (b) PCA (variance–covariance matrix) of quantified GC-EAD responses. PC1, 2 and 3 are plotted in three-dimensional space (79% cumulative variance). Species differ significantly from each other (one-way ANOSIM; Bray–Curtis distance; R = 0.78; p < 0.001). Isoamyl acetate (no. 4), 3M2BA (no. 7) and phenethyl alcohol (no. 14) most supported to the observed dissimilarity (SIMPER; all groups pooled; cumulative contribution 30.8%). (c) EAG dose–response curves of 3M2BA from the four species. Drosophila erecta is highly sensitive to 3M2BA, starting at 10⁻⁵ (paired t-test, **p < 0.01, emphasized). Drosophila erecta differs from its siblings (one-way ANOVA; Turkey's post hoc test; p > 0.05 n.s.; *p < 0.05; **p < 0.01). Mean ± s.e. In all graphs, species abbreviation and colour-code is as follows: D. melanogaster (Dmel, red), D. yakuba (Dyak, green), D. orena (Dore, violet) and D. erecta (Dere, blue), and n = 5 per species. Pandanus candelabrum image adapted from an original illustration. Reproduced with the kind permission of the Director and the Board of Trustees, Royal Botanic Gardens, Kew.

Figure 3.

Identification of ORs activated by 3-methyl-2-butenyl acetate via functional imaging and single sensillum recordings. (a) Functional imaging in D. melanogaster. Representative recording of both ALs performed with 3M2BA (i), and activity traces of the DM2 glomerulus (ii) in response to the same set of odours as used in (c). Error bars represent s.d. (b) Glomerular atlas of the AL. (c) Representative recordings performed with different reference stimuli (EHX, ethyl hexanoate; BEA, benzaldehyde; and MOL, mineral oil) and different 3M2BA concentrations illustrate the strong activation of DM2 glomerulus by 3M2BA (i). Images are individually scaled to the strongest activated glomeruli. Values below the ΔF/F threshold are omitted to illustrate the specificity of the signals, as well as the glomerular arrangement as visualized by the intrinsic fluorescence. Corresponding odour-induced activity (average % ΔF/F) plotted on schematic ALs (ii). (d) Single sensillum recording (SSR) measurements of the large basiconic sensilla in female D. erecta illustrate strong activation of the Or22a receptor (ab3A neuron) by 3M2BA. At higher concentrations, Or59b (ab2A neuron) is also activated. (e) SSR dose–response experiments performed on ab2 and ab3 sensilla in D. erecta (blue: solid line, ab3A Dere; dashed line, ab2A Dere) and D. melanogaster (red: solid line, ab3A Dmel; dashed line, ab2A Dmel) females with 3M2BA. Mean ± s.e.

Figure 4.

Morphological changes towards a complex of enlarged glomeruli. (a) Reconstruction of female antennal lobes (ALs) of the specialist D. erecta (i) and the generalist D. melanogaster (iii). Glomeruli terminology according to Couto [34]. ALs are viewed from medial to lateral. Comparison of the relative volume of the complex of enlarged glomeruli (based on the corresponding antennal input—large basiconic sensilla type ab1, ab2 and ab3) in D. erecta (blue), and D. melanogaster (red) (ii). The four glomeruli were up to 2.5 times enlarged in D. erecta, compared to the morphological structures in D. melanogaster (DM2×1.73; DM4×1.71; DM5×1.6; VM5d×2.5). n = 3 for D. melanogaster, n = 4 for D. erecta. Scale bar = 10 µm. Dorsal (D), lateral (L). (b) Neuronal backfill of ab3 sensilla in female D. erecta, viewed in three different planes of the AL. Labelled axons converge into the region of enlarged glomeruli (upper part). The corresponding planes are displayed in the reconstructed AL. Numbers in the paranthesis correspond to the plane (lower part). (c) Relative number of large basiconic sensilla of the four species under investigation [4]. Species name abbreviations according to the first three species letters. Total numbers of sensilla are given in parentheses. (d) Influence of 3M2BA in combination with spatial information (vertical structures) in oviposition site preference in the specialist D. erecta (i) and the generalist D. melanogaster (ii). Transparent bars represent relative number of eggs counted on the plates in total (control, light blue; 3M2BA, pink); solid bars include spatial information (vertical surface; horizontal surface; and vertical surface around odour cup). Mean ± s.d. Spatial preference of D. erecta and D. melanogaster: vertical medium surface > vertical gap around the odour cup > horizontal medium surface (D. erecta, p = 0.003; D. melanogaster, p = 0.002). 3M2BA significantly triggered oviposition in D. erecta, but not in D. melanogaster (D. erecta, p = 0.003; D. melanogaster, p > 0.05). Combination 3M2BA and spatial: D. erecta laid significantly more eggs inside the vertical gap around the odour cup > vertical medium structure (D. erecta, p < 0.001; D. melanogaster, p > 0.05). Per species, n = 6 cage of 30 flies.