A reversal in sensory processing accompanies ongoing ecological divergence and speciation in Rhagoletis pomonella

Abstract

Changes in behaviour often drive rapid adaptive evolution and speciation. However, the mechanistic basis for behavioural shifts is largely unknown. The tephritid fruit fly Rhagoletis pomonella is an example of ecological specialization and speciation in action via a recent host plant shift from hawthorn to apple. These flies primarily use specific odours to locate fruit, and because they mate only on or near host fruit, changes in odour preference for apples versus hawthorns translate directly to prezygotic reproductive isolation, initiating speciation. Using a variety of techniques, we found a reversal between apple and hawthorn flies in the sensory processing of key odours associated with host fruit preference at the first olfactory synapse, linking changes in the antennal lobe of the brain with ongoing ecological divergence. Indeed, changes to specific neural pathways of any sensory modality may be a broad mechanism for changes in animal behaviour, catalysing the genesis of new biodiversity.

Keywords: antennal lobe; host choice behaviour; olfaction; sensory processing; speciation.

Figure 1.

Rhagoletis pomonella host fidelity. (a) Both sexes of the ancestral hawthorn race are attracted to volatiles from hawthorn fruit. 3MB is essential for hawthorn fly attraction, while BH, a key apple volatile, is an antagonist. (b) The same volatiles exhibit opposite valences for the apple race, which uses BH to locate apples and is antagonized by 3MB. The mating flies image was adapted from a photo by Joseph Berger, Bugwood.org, licensed under CC BY 3.0. (Online version in colour.)

Figure 2.

Overview of R. pomonella olfaction. (a) OSNs are located in stereotyped groupings within sensilla on antennae, where odorants including BH and 3MB interact with protein receptors. (b) The antennal lobe (AL) is the first olfactory synapse, where OSNs from the periphery provide input to specific glomeruli (Gl). (c) The projection neurons (PNs), a specific type of ALN, bring olfactory information from the AL to higher brain centres such as the mushroom bodies and the lateral horn. (Online version in colour.)

Figure 3.

Rhagoletis pomonella brain anatomy and identification of glomeruli innervated by host volatile responsive OSNs. (a) Confocal Z-projection micrograph (75 µm depth) showing synapsin (red) and FMRFamide-like neuron immunoreactivity (green). Anatomical regions are identified: superior medial protocerebrum (SMP), superior lateral protocerebrum (SLP), lateral horn (LH), anterior venterolateral protocerebrum (AVLP), mushroom body (MB), antennal lobe (AL) and gnathal ganglia (GNG). (b) Three-dimensional reconstruction based on histological sections showing the optic lobe (OL), mushroom body calyx (CA), mushroom body ventral lobe (VL), mushroom body medial lobe (ML), central complex (CX) and AL. (c) Three-dimensional reconstruction of the AL and the MB indicating the spur (SPU) overlaid onto a Z-projection micrograph (300 µm depth). The glomeruli (GL) are labelled with different colours. (d) Three-dimensional reconstruction with glomeruli (DM1, VP1 and VP2) targeted by OSNs responsive to the volatiles BH and 3MB in black. (e,f) Confocal Z-projection of the AL following neurobiotin backfilling of OSNs (green), targeting glomerulus DM1 (e, arrowhead) and targeting glomeruli VP1 and VP2 (f, arrowheads). The synaptic marker nc82 (red) labels background neuropil. (Online version in colour.)

Figure 4.

Differences in brain activity while responding to fruit volatiles. (a, top) Greyscale image of right antennal lobe (AL) with antennal nerve (AN) and oesophagus (ES), and with a small ROI encompassing the DM1, VP1 and VP2 glomeruli in red. (bottom) False colour-coded responses of an individual hawthorn fly to the solvent control, 1-octen-3-ol, 3MB and BH, normalized to the highest response within that animal. (b) Fluorescence change over time in response to 3MB (orange) and BH (green) in the same hawthorn fly as in a. (c) Anatomical greyscale images overlaid with averaged false colour-coded responses of hawthorn flies and (d), the same in apple flies, responding to 3MB (orange box) and BH (green box). (e) Boxplots summarizing hawthorn fly peak neural activity and (f) the same in apple flies. (g) Boxplot showing the normalized difference index for each race (significantly different, Welch's t-test, d.f. = 8, t = 2.707, p = 0.0268). (Online version in colour.)

Figure 5.

Targeting of ALNs responding to key volatiles. (a,b,d,e) (top) Paired 5 s intracellular recording traces for ALNs responding to BH (top) or 3MB (bottom). A volatile prompted a response if the volatile's name is in colour. Grey lines below indicate the 0.5 s stimulus. (bottom) Maximum confocal Z-projections (10–20 µm anterior, left, and 10–20 µm posterior, right) of labelled ALNs corresponding to the spike recordings, using neurobiotin (green), counter-labelled with synaptic marker nc82 (red). Scale bars, 50 µm. ALNs targeting DM1, VP1 or VP2 glomeruli indicated with white arrowheads; glomeruli lacking arbourization are outlined by dotted lines. Insets for each panel summarize glomeruli targeted by ALNs responding to BH (green; a,d) or 3MB (orange; b,e). (c,f) Scatter plots showing the normalized difference index of average fluorescence intensity within the three target glomeruli (ΔF DM1 – ΔF VP1and2)/(ΔF DM1 + ΔF VP1and2) for confocal Z-projections of the ALNs responding to either BH or 3MB in each race. (Online version in colour.)