Surface fish exhibit approach to food and social odors, while avoiding alarm and death odors.

a. Cavefish populations are found across the Sierra de El Abra mountain range of Northeast Mexico. b. Odors were categorized by behavioral associations in previous fish literature: food (teal), social (purple), alarm (orange) and death associated (chrimson) odors. c. Surface fish did not exhibit approach or avoidance to water control scent (n=23), but did show significant approach to the addition of water soaked in bloodworms (food scent; n=16). d. Surface fish exhibit positive approach to food and social odors (teal and purple; n=24 and n=20), while displaying negative avoidance to alarm and death odors (orange and crimson; n=27 and n=24). Representational (ef) and population (g) ethograms for food (teal; bloodworm), social (purple; ammonia), alarm (orange; skin extract) and death (chrimson; decay extract) elicited swimming behaviors. Dotted lines denote neutral (swim, sw), negative (dart, da; dig, di; freeze, fr; spin, sp) and positive (bite, bi; circle, ci; shimmy, sh) swim bouts. Indices were compared using non-parametric ANOVA’s, followed by Tukey corrected p-value multiple comparisons. p-values; -p<0.05, -p<0.01, - p<0.001.

Odorants used in olfaction experiments

Behavioral bout definitions and characteristics

Behavioral pose pictographs for each bout type.

Fish depicting the beginning of a bout are transparent and end of bout (black). Red arrows depict x,y direction and speed (fast/slow) that occurs during the bout. Positive swim bouts associated with approach are swim, circle, bite and shimmy, while negative swim bouts associated with avoidance are dart, freeze, spin and dig. All bouts were observed and defined in surface fish, while cavefish populations have lost all negative bouts.

Light masks skin extract approach behaviors in cavefish.

a-c. Direct white light via acrylic diffusion revealed no difference between populations when exposed to skin extract (n=9, all populations). b. Box plots reveal negative odor indices in surface fish and neutral odor indices in cavefish. c. Ethograms reveal darting (da) and freezing (fr) in surface fish, while cavefish mostly perform routine swims. d-f Indirect white light via low lux projections on a back wall revealed (e) positive odor indices in cavefish, (f) an increase in cavefish circling behavior and the emergence of head spinning (sp) and digging (di) in surface fish (sfpa, n=15; moti, n=12). g-i. Direct IR-light via acrylic diffusion resulted in (h) higher positive odor indices in cavefish and (i) revealed biting (bi) behaviors in cavefish (n=10, all populations). Indices were compared using non-parametric ANOVA’s, followed by Tukey corrected p-value multiple comparisons. p-values; -p<0.05, -p<0.01, -p<0.001.

Cavefish exhibit approach behaviors when exposed to ancestrally negative alarm and decay odors.

a. Pairwise heatmap of odor index averages between odor and A. mexicanus population. Odor indices were encoded as positive (dark blue,1) to negative (dark red, -1) colors to help visualize attraction and repulsion to different odors (sample sizes in Table 3). b. Horizontal violin plots that exhibit positive (blue) and negative (red) odor index values for each population. Each row corresponds to the odor row and color values depicted in the heatmap. c-f. Representative and population ethograms for (cd) social odors and (ef) decay extract behaviors, where each row corresponds to a specific population. gh. Quantification of (g) circling and (h) bite bouts in surface fish and cavefish populations exposed to water, bloodworms, ammonia, skin and decay extract. Odor Indices were compared using non-parametric ANOVA’s, followed by Tukey corrected p-value multiple comparisons. p-values; -p<0.05, -p<0.01, -p<0.001.

Sample sizes by odor and fish population

Surface to cave F2 hybrid behavior suggests olfactory perception is genetically inherited.

a. Surface to Tinaja F2 hybrids were generated by crossing Surface to Tinaja F1 siblings. b. Hybrid population behavior for social (purple), alarm (orange) and death (crimson) odors span the full approach to avoidance value range (n=29). c-e. Hybrid population comparisons binned by (c) eyes, (d) pigment and (e) sex (male = 17, female = 12). f. Representative ethograms exhibiting female circling and shimmy bouts that are indicative of mating. g-i Pearson correlations for (g) decay versus ammonia, (h) skin versus ammonia and (i) skin versus decay. Indices were compared using non-parametric ANOVA’s, followed by Tukey corrected p-value multiple comparisons. p-values; -p<0.05, - p<0.01, -p<0.001.

Food availability impacts avoidance/attraction behaviors and hunger drives surface fish towards negative odors.

a. Pictograph demonstrating the paradigm for withholding food across 4 weeks and measuring odor perception at the end of each week. bc. Behavioral analysis of odor perception indices for (b) skin extract and (c) decay extract exposed fish across four weeks. de. ethogram charts displaying bout percentages for (d) skin extract and (e) decay extract exposed surface fish at the beginning and end of the experiment. Indices were compared using non-parametric ANOVA’s, followed by Tukey corrected p-value multiple comparisons. p-values; - p<0.05, - p<0.01, - p<0.001.

Brain mapping in the forebrain finds similar activity patterns in the cavefish thalamus and preoptic region when smelling food or decay extract.

a. schematic of experimental pipeline through two weeks of dissection, staining, clearing and imaging b. example of adult brain registration where autofluorescence (auto subject) is used to register to our reference brain (auto ref). The final product is a reformatted brain (subject reg) in the reference space (auto ref/ subj reg). c. example of normalized pERK in the pallium of a skin exposed fish (arrow pointing to dorsal pallium, e.g. amygdala correlate). d. atlas analysis key showing the four subregions that were analyzed for activity mapping. Dm, medial pallium; PO, preoptic region; Th, thalamus. e. Optical sections displaying normalized p-ERK signal in Pachon cavefish exposed to DMSO (control p-ERK), bloodworm water (food p-ERK), skin extract (p-ERK) and decay extract (decay p-ERK). Each column of images is derived from a single fish for each condition, top anterior to bottom posterior positions. Flat arrows point to pallial and preoptic regions of activity, while yellow asterisks flank high p-ERK in the thalamus of food and decay exposed fish f. Box plots comparing regional p-ERK intensities of Pachon cavefish exposed to four conditions, control (DMSO), food (bloodworm water), skin (skin extract) and decay (decay extract). Indices were compared using non-parametric ANOVA’s, followed by Tukey corrected p-value multiple comparisons. p-values; - p<0.05, - p<0.01, - p<0.001.

Time series depicting red dye in behavioral tank with and without DMSO.

Each image shows red dye with no DMSO (0%) versus red dye with DMSO (0.5%) across three time points, 10 seconds, 30 seconds and 1 minute.

Odor indices for the four control odors.

No stimulus = baseline movement, water = odor sham (pipette current), bloodworm water = positive (food) and mustard oil = negative (pain). Each graph represents 1 minute of behavior following the introduction of the title odor.

Food odor indices for all populations.

Bloodworms are in the previous graph (S3) as an established positive control group. Each graph represents 1 minute of behavior following the introduction of either bat guano or artemia water.

Social odor indices for all populations.

Each graph represents 1 minute of behavior following the introduction of either ammonium chloride, bile, urea or prostaglandin F2-alpha.

Aggregated odor indices of all social odors for each population.

All social data was reanalyzed by sex type and paired statistical comparisons.

Alarm odor indices for all populations.

Each graph represents 1 minute of behavior following the introduction of either skin extract, spermine, pyridine-n-oxide, chondroitin sulfate or bacteria (Staphylococcus saprophyticus).

Death odor indices for all populations.

Each graph represents 1 minute of behavior following the introduction of either decay extract, putrescine, diaminohexane, or cadaverine.