Expression of ionotropic receptors in terrestrial hermit crab's olfactory sensory neurons

Abstract

Coenobitidae are one out of at least five crustacean lineages which independently succeeded in the transition from water to land. This change in lifestyle required adaptation of the peripheral olfactory organs, the antennules, in order to sense chemical cues in the new terrestrial habitat. Hermit crab olfactory aesthetascs are arranged in a field on the distal segment of the antennular flagellum. Aesthetascs house approximately 300 dendrites with their cell bodies arranged in spindle-like complexes of ca. 150 cell bodies each. While the aesthetascs of aquatic crustaceans have been shown to be the place of odor uptake and previous studies identified ionotropic receptors (IRs) as the putative chemosensory receptors expressed in decapod antennules, the expression of IRs besides the IR co-receptors IR25a and IR93a in olfactory sensory neurons (OSNs) has not been documented yet. Our goal was to reveal the expression and distribution pattern of non-co-receptor IRs in OSNs of Coenobita clypeatus, a terrestrial hermit crab, with RNA in situ hybridization. We expanded our previously published RNAseq dataset, and revealed 22 novel IR candidates in the Coenobita antennules. We then used RNA probes directed against three different IRs to visualize their expression within the OSN cell body complexes. Furthermore we aimed to characterize ligand spectra of single aesthetascs by recording local field potentials and responses from individual dendrites. This also allowed comparison to functional data from insect OSNs expressing antennal IRs. We show that this orphan receptor subgroup with presumably non-olfactory function in insects is likely the basis of olfaction in terrestrial hermit crabs.

Keywords: antennules; crustacea; electrophysiology; in situ hybridization; ionotropic receptors; olfaction.

Figure 1

GO term distribution after annotation by BLAST2GO. (A) Cellular component level 2, (B) cellular component level 3, (C) molecular function level 2, (D) molecular function level 3, (E) biological Process level 2, (F) biological process level 3.

Figure 2

Analysis of Ionotropic Receptor relationships between C. clypeatus, P. bernhardus, P. argus, D. pulex, P. humanus humanus and D. melanogaster. (Sequences of P. bernhardus from Groh et al., ; and C. clypeatus from this study and Groh et al., ; P. argus from Corey et al., ; D. pulex, P. humanus, and D. melanogaster from Croset et al., 2010). Alignment of aminoacid sequences using muscle (Edgar, 2004); dendrogram compilation by FastTree (Price et al., 2010); support values of branches indicated by color gradient (blue to black 0–0.5, black to red 0.5–1).

Figure 3

Distribution of IR co-receptor IR25a in the last antennular section, LSM scan of whole mount immunohistochemical assay, IR25a protein labeled in green, counterstain of cell core with SYTOX orange, (A) overview of OSN cell bodies expressing IR25a, (B) IR25a in the aesthetasc dendrites, (C) closeup of an exemplary aesthetasc, (D) control without primary antibody, (E) IR25a in dendrites (outlined arrowheads) from cell bodies (filled arrowheads) to the aesthetasc pad.

Figure 4

LSM scan of whole mount fluorescence in situ hybridization. Nuclear stain: SYTOX blue, Small letters refer to the respective region of higher magnification scan, asterisks to enlarged details; signals indicated by arrowheads, Boxplot: Signal count statistics and distribution significance of the respective regions (One-Way ANOVA and Tukey's test), ^***p < 0.001, (A) IR1, (B) IR6, (C) IR26.

Figure 5

Electrophysiological responses elicited by odor stimulation measured as changes of local field potentials (A,B) and changes in spike frequency (C). (A) Response spectra of single aesthetascs in the respective antennular regions. Numbers refer to individual preparations. +, Response;•, no response; nt, not tested; (B,C), responses to stimulation by ammonium hydroxide recorded as (B) local field potentials, (C) single sensillum recordings. Each trace represents an individual preparation. Time of odor application indicated by black squares.

Figure 6

Electrophysiological responses elicited by odor stimulation measured as changes of local field potentials of representative aesthetascs in the three aesthetasc field regions. Time of odor application indicated by black bars.