(a) Physiologically active compounds
The results from the broad screening of compounds for EAG activity in C. clypeatus established physiological activity for several short-chained carboxylic acids and aldehydes, several mono- and diamines, as well as to methanol, ethanol, benzaldehyde, ammonium hydroxide and water vapour (a). Odourants from the other chemical classes, i.e. esters, lactones, ketones, aromatics (besides benzaldehyde), cyclic terpenes, ethers, furans, sulphur compounds, thiocyanates and amino acids, were not physiologically active (for inactive compounds, see electronic supplementary material). Carbon dioxide was scored as EAG-active, having an average response of −131 µV (i.e. less than or equal to −30 µV), but activity was only noted in two out of 10 preparations and must therefore be considered unreliable. The active compounds could be grouped into two categories, where acids, aldehydes and water vapour generate negative EAG responses, while amines, ammonium hydroxide and alcohols generate inverted, positive EAG responses (a,b).
Figure 1. 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 (more ...)
To visualize the representation of all compounds screened for EAG response in C. clypeatus in a multi-dimensional physico-chemical odour space, we projected it into two dimensions via a PCA (see electronic supplementary material, figure S1a). The investigated compounds cover a large part of odour space, with no significant separation of physiologically active and inactive compounds (one-way AnoSim for Euclidean distances, p = 0.8). Within the active odourants, however, compounds eliciting negative and positive EAG responses were clearly separated in their odour space representation (one-way AnoSim for Euclidean distances, p < 0.0001; electronic supplementary material, figure S1b), demonstrating that they differ in chemical properties.
As water vapour greatly influenced the physiological and behavioural responses (see §3b,d,e), we checked for the water solubility of all compounds screened for EAG activity in C. clypeatus. Here, we found a highly significant difference in water solubility, where physiologically active compounds were found to be more water-soluble, whereas inactive compounds can be of any solubility (Mann–Whitney U-test, Z = −4.58, p < 0.001; electronic supplementary material, figure S2).
The EAG responses to acids and amines were clearly dose-dependent in C. clypeatus (a). Relatively high thresholds were noted, with responses from 10−2 dilution (equivalent to 7.5–9.4 µg stimulus load) for isovaleric and valeric acid and trimethylamine, whereas triethylamine and butyric, propionic and acetic acid were active at 1 : 500 dilutions (1.5–2.1 µg stimulus load) and diethylamine already at 10−3 dilution (0.7 µg stimulus load). Also C. compressus showed dose-dependent responses to these odourants although the magnitude of the responses was about 10-fold lower (see electronic supplementary material, figure S3). Furthermore, the reversed EAG response pattern for acids and amines held true also in this species, and notably also in the marine hermit crab P. bernhardus (b).
Figure 2. 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 (more ...)
(b) Humidity level greatly affects the physiological response in Coenobita clypeatus
Motivated by increased behavioural responses when water was added to the stimuli (see §3d,e), we repeated EAG recordings under different humidity levels. Our results confirm the importance of water vapour for olfactory performance in C. clypeatus: At 10−1 and 10−2 dilutions, the EAG responses to all tested odourants were significantly larger at high humidity as compared with responses at low humidity (three up to more than 10 times larger depending on odourant and concentration tested; a) while at 10−3 dilutions, humidity significantly affected the EAG response only for propionic acid, triethylamine and propylamine (a). Since 10−4 dilutions did not elicit EAG activity (i.e. mean response was never greater than 30 µV or less than or equal to 30 µV), this concentration was not analysed. Generally, the EAG response recovered and was as high at a repeated second high humidity test as it had been during the initial high humidity recording, confirming that the decrease in response at the low humidity level was not due to a deteriorating antennular preparation. In contrast, in the vinegar fly D. melanogaster, no difference in EAG response was found at different humidity levels (b).
Figure 3. 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 (more ...)
None of the compounds defined as EAG inactive in the initial screening experiment were found active when re-tested at higher humidity, which confirms that the inactivity was not due to a too low humidity level (for compounds, see electronic supplementary material).
(c) Behavioural response to odours from food and water
In nature, Coenobita
spp. use odours to find food [7
]. By establishing a reliable two-choice bioassay in the laboratory, we wanted to repeat these experiments under more controlled conditions. We could show that C. clypeatus
is significantly attracted to natural, complex odours from fruit and seawater (). While the distribution of crabs between two empty pit-fall traps was random, almost all crabs (26 out of 30, i.e. 87%) selected a trap with banana over an empty trap and similarly, 21 out of 26 (81%) chose the apple treatment over an empty control. However, the crabs were not attracted to odours from all food items provided, since neither overripe banana nor raw salmon were chosen over the controls. Furthermore, the crabs were significantly attracted to ASW, but not to distilled water.
Figure 4. 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, (more ...)
(d) Water vapour greatly affects the behavioural response to natural, complex odours
To test the hypothesis that water vapour influences the attractiveness of natural, complex food odours, we used ground peanut snacks, being a dry food item that is highly preferred by the crabs when provided during rearing, as well as banana extract. When tested as a dry stimulus, C. clypeatus was not attracted to peanut snacks, but when presented in combination with water, this food item was significantly attractive to the crabs (). Similarly, banana extract was found to be significantly attractive to the crabs only when presented in combination with water ().
(e) Behavioural response to physiologically active odourants
To test if the crabs also respond behaviourally to physiologically active odours, some representative synthetic compounds were tested in the bioassay. When tested as pure stimuli, neither propionic acid, the aldehyde propanal nor triethylamine elicited a behavioural reaction in the crabs, but propylamine was significantly avoided (). However, when tested in combination with water, both amines were significantly repulsive, while both propionic acid and propanal were significantly attractive to the crabs (). In line with the physiological results, isoamyl acetate did not elicit any behavioural response, neither when tested alone, nor in combination with water ().