Electroantennogram (EAG) responses in Coenobita clypeatus. (a) Active odourants elicited a negative (−) or a positive (+) EAG response. Stimuli were presented at 10⁻¹ dilutions, except the solvents hexane, dichloromethane, mineral oil and water that were presented pure, and carbon dioxide that was presented by extracting headspace over dry ice with a syringe and injecting this headspace into the constant airstream passing over the antennule. Responses were calculated by measuring the maximum response peak (negative or positive) following the 0.5 s odourant presentation and subtracting the response to pure solvent. For the solvents (hexane, dichloromethane, mineral oil and water), responses to a blank were subtracted, and for carbon dioxide, an equivalent air volume served as control. Symbols represent the average response (R) of at least 10 antennular preparations. Dots (•), no response: −30 µV < R < 30 µV; one minus symbol, −30 µV ≥ R > −100 µV; two minus symbols, −100 µV ≥ R > − 1000 µV; three minus symbols, −1000 µV ≥ R > −5000 µV; four minus symbols, R ≤ −5000 µV; one plus symbol, 30 µV ≤ R < 100 µV; two plus symbols, 100 µV ≤ R < 1000 µV; three plus symbols, 1000 µV ≤ R < 5000 µV; four plus symbols, R ≥ 5000 µV. Compounds are colour coded according to functional group: blue, acid; green, aldehyde; brown, aromatic; red, amine; orange, alcohol. (b) Examples of EAG responses to 10⁻¹ odourant dilutions, showing typical negative and positive responses to a carboxylic acid (propionic acid) and an amine (diethylamine). In the upper control trace, the pure solvent (hexane) served as stimulus (stimulus presentation indicated by bar above each trace).

EAG response to odourants tested at different humidity levels, for Coenobita clypeatus (a) and Drosophila melanogaster (b). A first recording session, including the odourants and concentrations to be tested, was performed at the initial high humidity (dark blue, 86–91% RH), a second at the low humidity (orange, 32–35% RH) and finally, a third at the high humidity level (final high humidity—light blue, 86–91% RH). (a) For C. clypeatus, four odourants were presented at dilutions from 10⁻⁴ to 10⁻¹ in each recording session (n = 5, error bars represent s.e.m. asterisk (*), p < 0.05-values based on repeated measurement ANOVA, followed by Tukey–Kramer multiple comparisons test). (b) For D. melanogaster, three odourants were presented at 10⁻¹ dilution in a recording session (no significant differences between responses at the different humidity levels, tested with repeated measurement ANOVA; error bars represent s.e.m., n = 6).

Dose-dependent EAG responses to selected odourants (a) in the land hermit crabs Coenobita clypeatus and (b) in the marine hermit crab Pagurus bernhardus. Odourants (1 µl) were presented to the antennule in ascending steps. For line colours and markers in (b), see (a) (error bars represent s.e.m.; n = 9–14 in (a); n = 5 for the lower and 10 for the higher concentration in (b)).

Behavioural response of C. clypeatus to natural and synthetic odourants in a two-choice bioassay. Different natural and synthetic odourants (light grey, crabs to treatment; with and without 10 ml distilled water, W) were tested against an empty control, water and/or solvent (S) (black, crabs to control). Two empty controls were used as negative control (top bars). Synthetic odourants were presented at 10⁻² dilutions in mineral oil. (G-test, * p < 0.05, ** p < 0.01, *** p < 0.001, n indicated to the right for each test.)