Changes in behavior often drive rapid adaptive evolution and speciation. However, the mechanistic basis for behavioral 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 utilize specific odors to locate fruit, and because they mate only on or near host fruit, changes in odor 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 odors 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 behavior, catalyzing the genesis of new biodiversity.

Uploaded as part of this dataset are raw files collected in pursuit of results part b (antennal lobe input), part c (activity in the antennal lobe), and part d (antennal lobe output) of our manuscript. All three of these types of files include both apple and hawthorn race flies, as we were interested in how they diverged at these different levels of olfactory processing, and are labeled by host race and, usually, odor stimulus given to cause response captured in the particular file.

The data for part b, antennal lobe input, consists of 19 ".oib" files, labeled with "partB", then numerically as "OSN_1" through "OSN_19", according to supplementary table s1 in the manuscript, and with whether they came from an "Apple" or "Hawthorn" race fly. They are the raw stacks of images obtained via confocal microscopy, consisting of one channel capturing the neuropil marker at the 647 wavelength and one channel capturing the neurobiotin tracer backfilling an OSN that responded to 3MB and BH at the 488 wavelength. Magnification was 40x, using an oil objective. Files can by opened in FIJI (Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–82). These files are raw files, before all processing. To replicate the results shown in the manuscript in Figure 3 and Supplementary Figure S2, one should consult the methods and supplementary methods of the manuscript, or the uploaded ReadMe file.

The data for part c, activity in the antennal lobe, consists of 26 ".tif" files, labeled "partC", then the numeric file name, followed by "Apple" or "Hawthorn" and then further followed by the odor stimulus during that particular trace "BH" or "3MB". Focused on the antennal lobe after it had been incubated with Calcium Green AM-ester, these traces show 10 seconds, recorded at 4 Hz on a CCD camera with a 20x water-immersion objective, wherein the given odor stimulus (BH or 3MB) was introduced at the 2 second time point. A peak of activity shown by the calcium-sensitive fluorescent dye can be seen for each of these traces within 2.5 seconds after the 2 second introduction of odor. Again, these are raw data files. To replicate results, corrections for movement and bleaching were undertaken, again using FIJI, as well as multi-step manual landmark registration, to produce Figure 4 and Supplementary Figures S3 and S4. For details on processing and analysis, one should consult the methods and supplementary methods of the manuscript or the uploaded ReadMe file.

The data for part d, antennal lobe output, consists of 10 ".oib" or ".oir" files, labeled with "partD", then the numerical file name, followed by "Apple" or "Hawthorn", then further described by the odor stimulus that the filled antennal lobe neuron responded to, "BH", or "3MB" and the glomerulus/i filled in that case, either "DM1", "VP1", or "VP2" or some combination thereof. This data for all 10 of these images is summarized by Supplementary Figures S5 and S6, with detailed analyses shown in Figure 5. Uploaded here are the raw data files, taken via confocal microscopy, showing one channel capturing the neuropil marker at the 647 wavelength and one channel capturing the neurobiotin tracer filling the ALN at the 488 wavelength. Magnification was 40x, using an oil objective. For details on the processing and analysis of these raw files, one should consult the methods and supplementary methods in the manuscript or the uploaded ReadMe file.

Uploaded with these files is a master ReadMe file (ReadMe_Tait_2021) that describes all three main experimental parts included in this publication: part b, c, and d. This file includes all methods from the manuscript, pulled from both the main paper and the supplementary methods, which were used to collect and analyze the data, as well as a listing of each associated file name.