Molecular Basis of Chemotactile Sensation in Octopus

Abstract

Animals display wide-ranging evolutionary adaptations based on their ecological niche. Octopuses explore the seafloor with their flexible arms using a specialized "taste by touch" system to locally sense and respond to prey-derived chemicals and movement. How the peripherally distributed octopus nervous system mediates relatively autonomous arm behavior is unknown. Here, we report that octopus arms use a family of cephalopod-specific chemotactile receptors (CRs) to detect poorly soluble natural products, thereby defining a form of contact-dependent, aquatic chemosensation. CRs form discrete ion channel complexes that mediate the detection of diverse stimuli and transduction of specific ionic signals. Furthermore, distinct chemo- and mechanosensory cells exhibit specific receptor expression and electrical activities to support peripheral information coding and complex chemotactile behaviors. These findings demonstrate that the peripherally distributed octopus nervous system is a key site for signal processing and highlight how molecular and anatomical features synergistically evolve to suit an animal's environmental context.

Keywords: chemosensation; evolution; ion channels; neuroethology; neuroscience; octopus; sensory physiology; signal transduction.

Figure 1.. Octopus arms contain distinct chemo- and mechanoreceptor cells

(A) (Left) Octopus bimaculoides. (Middle) Putative nerve tracts from an octopus embryo sucker stained with anti-horseradish peroxidase antibody (HRP, green) and nuclear stain (DAPI, blue). (Right) HRP-positive cells in the sucker epithelium. (B) Morphologically-distinct cell types from the sensory epithelium were defined by their responses to discrete sensory stimuli. 1μm displacement of the dendritic ending activated mechanoreceptor cells, but not chemoreceptor or support cells. (C) Fish extract (<3kDa) elicited inwardly-rectifying currents from chemoreceptor cells, but not mechanoreceptor or support cells. (D) Quantification of responses in the 3 distinct cell types to mechanical and chemical stimulation. See also Figure S1 and Supplemental video 1.

Figure 2.. Chemoreceptor and mechanoreceptor cells transduce distinct electrical signals

(A) An identical current injection stimulus evoked tonic voltage spiking in chemoreceptor cells and phasic responses in mechanoreceptor cells, respectively. Support cells did not respond to similar stimuli (representative of n = 5). Identical stimuli evoked distinct spike frequencies in chemoreceptor and mechanoreceptor cells. n = 11 – 14, p < 0.0001 for > 20pA, multiple row two-tailed student’s t-test. Data represented as mean ± SEM. (B, C) Chemoreceptor and mechanoreceptor cells had similar voltage-gated inward currents, which were sensitive to tetrodotoxin (TTX, 1μM) and absent in non-excitable support cells. Chemoreceptor V_a1/2 = −6.71 ± 0.62mV, V_i1/2 = −37.70 ± 1.5mV, n = 7. Mechanoreceptor V_a1/2 = −5.66 ± 0.58mV, V_i1/2 = −43.57 ± 1.2mV, n = 7. (D, E) Chemoreceptor and mechanoreceptor cells had distinct 4-AP (1mM)-sensitive K⁺ currents: mechanoreceptor currents were activated at more negative voltages, had smaller amplitude, and exhibited more sensitive and complete voltage-dependent inactivation. Chemoreceptor V_a1/2 = 13.05 ± 0.99mV, V_i1/2 = −29.80 ± 0.95mV, n = 6. Mechanoreceptor V_a1/2 = −4.35 ± 1.4mV, V_i1/2 = −18.95 ± 0.95mV, n = 6. Data represented as mean ± SEM. See also Figure S1.

Figure 3.. NompC is a conserved mechanoreceptor in octopus arms

(A) Octopus tissue samples used for comparative transcriptomics. (B) Comparison of mRNA transcripts (normalized counts) revealed NompC (no mechanoreceptor potential C) as the sensory receptor orthologue enriched in the sucker epithelium. (C) Co-localization of NompC and neuronal marker Elav visualized by RNAscope in situ hybridization. (D) Comparison of mechanically-evoked currents (pipette displacement) in native mechanoreceptor cells (1.46 ± 0.18μm, n = 13) to Octopus bimaculoides NompC (4.75 ± 0.75μm, n = 4) and Drosophila melanogaster NompC (4.25 ± 0.47μm, n = 4) expressed in HEK293 cells. Untransfected cells did not respond to similar stimulation (n = 6). (E) Mechanically-evoked currents exhibited similar transient kinetics and gadolinium (Gd³⁺)-sensitivity (100 μM). n = 4 – 6 cells. Data represented as mean ± SEM. See also Figure S2.

Figure 4.. Chemotactile Receptors Are Enriched in Sucker Epithelium, Exhibit Combinatorial Expression, and Are Sensitive to Prey-derived Chemicals

(A) mRNA transcripts encoding CRs were enriched in the sucker sensory epithelium relative to other sampled tissues (brain, eye, statocyst, skin, olfactory organ, sucker, sensory epithelium of sucker cup). Scale: z-scaled normalized counts. (B) Phylogenetic analysis across 3 distinctdifferent octopus species revealed that CRs branch from canonical acetylcholine receptors, suggesting a common ancestor. Scale: branch length, Newick tree format. (C) CRs exhibited combinatorial expression patterns. 3 distinct CRs (CR840, CR518, and CR737) localized to the sensory epithelium, as visualized by RNAscope in situ hybridization. Nuclei stained with DAPI (blue). (D) Xenopus laevis oocytes expressing CRs were insensitive to acetylcholine (ACh, 1mM) but robustly responded to fish or crab extract with an inwardly-rectifying current similar to those observed in native chemoreceptor cells. Fish extract did not elicit currents from ACh-sensitive human α7 acetylcholine receptors (hα7) or uninjected oocytes. n = 4 – 8, p < 0.0001 for fish extract responses in CRs versus hα7, one-way ANOVA with post-hoc Bonferroni test. See also Figure S3 and S4.

Figure 5.. Discrete CRs and chemoreceptor cells are regulated by distinct compounds

(A) Native chemoreceptor cells were activated by fish extract and inhibited by octopus ink (<3kDa). n = 6, p < 0.0001, one-way ANOVA with post-hoc Bonferroni test. (B) Fish extract activated oocytes expressing CR518 or CR840, and CR518 was more sensitive to ink. n = 4 – 6, p < 0.0001 for CR518 inhibition, one-way ANOVA with post-hoc Bonferroni test. (C) HEK293 expressing CR840 responded to hydrophilic (HPL) but not hydrophobic (HPB) fish fractions while CR518 was only stimulated by HPB fractions. Responses normalized to total fish extract, n = 8 – 10, p < 0.0001 one-way ANOVA with post-hoc Bonferroni test. (D) Distinct native chemoreceptor cells were sensitive to HPB or both individually-applied HPL or HPB fractions. (E) Patch clamp screen in HEK293 cells expressing CR840 or CR518 quantified as log2 (current amplitude at −110mV), n = 3 – 10, 1mM of tested compound. Both CRs robustly responded to the bitter compound chloroquine and CR518 was sensitive to several terpenoids. (F) Distinct fish-sensitive chemoreceptor cells responded to independently-applied CR-sensitive compounds (25μM). (G) Current-voltage (I-V) relationships from a native chemoreceptor cell and HEK293 cells expressing CR840 or CR518. Similar to extracts, indicated compounds evoked inwardly-rectifying currents and CRs exhibited distinct pharmacological profiles in response to 30μM atractylon and nootkatone, 200μM carvacrol, and 25μM chloroquine. Data represented as mean ± SEM. See also Figure S5.

Figure 6.. CRs form heteromeric channel complexes that influence stimulus detection and transduction

(A) Fish extract elicited distinct responses from oocytes depending on CR co-expression (n = 2 – 14). (B) Co-immunoprecipitation CRs with indicated epitope tags using anti-FLAG beads showed that CR828 directly interacted with CR518 or CR840 when co-expressed in oocytes. Neither CR was immunoprecipitated on FLAG beads when both co-expressed CRs were HA-tagged (* = IgG) (C) HEK293 cells expressing heteromeric CR840–828 exhibited decreased chloroquine-sensitivity compared with CR840 alone. Currents normalized to maximal currents evoked by maximal concentration in the same cell. n = 6 – 8, p < 0.0001, two-tailed Student’s t-test. (D) CR518–828 expression caused increased nootakatone-sensitivity compared with CR518. n = 8 – 12, p < 0.0001, two-tailed student’s t-test. (E) (Top) In chemoreceptor cells, fish extract evoked inwardly-rectifying currents with two distinct ion selectivity profiles: cell type 1 was permeant to monovalent ions (n = 4) and type 2 also passed Ca²⁺ (n = 3). (Bottom) Quantification, p < 0.0001 for Ca²⁺ permeation, two-way ANOVA (n = 7) with post-hoc Bonferroni test, p < 0.001. (F) (Top) HEK293 expressing CR complexes showed distinct ion selectivity. (Bottom) p < 0.05 for differences in Ca²⁺ permeation in CR518 and CR840 and p < 0.0001 for Ca²⁺ in homo- and heteromeric CR518, two-way ANOVA with post-hoc Bonferroni test (n =4–5). Data represented as mean ± SEM. See also Figure S6 and S7.

Figure 7.. Chemotactile exploration integrates CR agonists and mechanical stimuli

(A) Octopuses explored agarose floors divided into chemical-containing and control sides. Example quantification of the number and duration of floor-touches over 10 minutes. Blue represents touches on compound-infused agarose and grey represents neutral agarose touches. (B) Control experiment in which agarose sides were independently-prepared and only contained seawater. Exploring octopuses showed no difference in the number or duration of agarose touches. (Left) Number of touches on both sides. (Right) Histogram with overlaid density plot showing the distribution of duration of touches for both sides. (C) Polygodial (100μM, n = 7) elicited increased touches (p < 0.05, paired Student’s t-test) with shorter duration compared to control side in the same experiments (p < 0.0001, Wilcoxon t-test). (D) Similar results were observed for nootkatone (500μM, n = 10, p < 0.05 for number of touches and p < 0.0001 for duration) and (E) carvacrol (500μM, n = 9, p < 0.01 for number of touches and p < 0.001 for duration). Bar graphs display mean ± SEM, histograms display counts for binned duration (boxes), median (dashed lines) and kernel density estimate (solid line). See also Supplemental video 2 and 3.