{"paper_id":"0b4e72cb-952b-49a2-9f86-a4dd15d20504","body_text":"1 \n \nIdentification and functional investigation of Octopus 1 \nvulgaris TRPV channels as potential nociceptors in 2 \ncephalopods 3 \nEleonora Maria Pieroni 1,2, Howard Baylis 3, Vincent O'Connor 1, Lindy Holden-Dye1, Luis Alfonso Yañez-4 \nGuerra1, Pamela Imperadore4, Graziano Fiorito4, James Dillon1* 5 \n1 School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK  6 \n2 Association for Cephalopod Research ‘CephRes’ ETS, Napoli, Italy  7 \n3 Department of Zoology, University of Cambridge, Cambridge, UK  8 \n4 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Napoli, Italy  9 \n*Author for correspondence: jcd@soton.ac.uk 10 \n  11 \n 12 \n 13 \n 14 \n 15 \n 16 \n 17 \n 18 \n 19 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n2 \n \nAbstract  20 \nNociception is an essential response for organisms to avoid potential harm and promote survival. Its 21 \nmolecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion 22 \nchannels exhibiting selective peripheral expression and functional coupling that underpins nociception 23 \nand pain modulation in complex organisms. However, the execution of protective behaviours triggered 24 \nby TRPVs is also found in species with a simpler nervous organisation, thus encouraging their 25 \ninvestigation in invertebrate model organisms to increase understanding of animal nociception. 26 \nCephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous 27 \nsystem, whose complexity suggests it might support pain -like states that exist in vertebrates. This 28 \npossibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. 29 \nDespite this, there is poor characterisation of cephalopod molecular nociceptors. 30 \nFor this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome 31 \n(Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent 32 \nexpression in sensory tissues. We investigated the functional competence of these TRPVs by 33 \nheterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the 34 \northologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response 35 \nto chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are 36 \npolymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes 37 \nshowed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by  nicotinamide. This study 38 \nhighlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods. 39 \nKeywords: C. elegans, animal welfare, pain, nociception, model hopping, octopus, evolution 40 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n3 \n \nBackground 41 \nGeneral nociception executes function by activating a reflexive withdrawal response , protecting 42 \norganisms from potentially threatening stimuli. Various avoidance behaviours operate across animal 43 \nphyla and advantages organismal survival (Smith and Lewin, 2009). Nociception is characterised by a 44 \nconserved specialised neuronal architecture and shared molecular determinants that persist across 45 \ndistinct nervous system organisations and largely encompasses a core reflex that triggers withdrawal 46 \navoidance responses (Julius and Basbaum, 2001). In more complex organisms, however, the reflexive 47 \nnociceptive responses are modulated by top -down neuronal signalling leading to the elaboration of  48 \nlearnt and emotionally encoded pain states (Basbaum et al., 2009). 49 \nCephalopods are invertebrate species characterised by well described withdrawal responses following 50 \nexposure to noxious challenges (Crook, Hanlon and Walters, 2013; Hague, Florini and Andrews, 2013; 51 \nCrook, 2021) . Furthermore, these molluscs possess a central nervous system that can act in a 52 \nhierarchical fashion, allowing them to express complex behaviours (Amodio et al., 2019; Schnell et al., 53 \n2021). The details of this anatomy are different from organisms that are unequivocally known to express 54 \nthe emotional states defined by pain, nonetheless cephalopod s’ central brain mass complexity 55 \nprovokes the possibility that their neural architecture is organised to allow brain states with 56 \ncomponents that resemble pain (Andrews et al., 2013; Smith  et al., 2013; Shigeno  et al., 2018). This 57 \nhighlights potential ethical issue s and associated welfare constraints about the experimental use of 58 \ncephalopods that have seen these animals included in research legislations (UK S.I. 1993/2103, 1993; 59 \nEuropean Parliament and Union, 2010). 60 \nThis raises the scientific value of detailing the biological organisation of nociception reflexes and their 61 \nmodulation in this important biological and societally utilised taxon.  62 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n4 \n \nIn our previous study, we identified O. vulgaris orthologues of genes with a defined role in nociception 63 \nand pain to enrich understanding of the molecular determinants of these phenomena in cephalopods. 64 \nWe coupled this to functional studies in C. elegans loss of function mutants of the corresponding genes 65 \nof interest and identified 19 potential candidates warranting more detailed investigation for their role in 66 \nO. vulgaris  nociception (Pieroni et al. , 2026) . Among our candidates, the previously investigated 67 \nvanilloid transient receptor potential (TRPV) emerged as a priority candidate for further investigation 68 \n(Caterina et al., 1997).  69 \nTRP receptors are a large family of ion channels highly conserved across animal species due to their 70 \nextensive role in different physiological functions (Pedersen, Owsianik and Nilius, 2005; Ramsey, 71 \nDelling and Clapham, 2006; Venkatachalam and Montell, 2007) . Among the seven major TRP  72 \nsubfamilies, the vanilloid sensitive homologues, include receptors that detect aversive stimuli (Colton, 73 \n2006; Radresa  et al. , 2012; Shibasaki, 2024) . The best characterised representative of TRPVs in 74 \nmammals is the capsaicin receptor TRPV1 (Caterina et al. , 1997) . TRPV1 is a polymodal nociceptor 75 \nwhich, in addition to vanilloid compounds, responds to cell damaging pH and temperature changes 76 \n(Venkatachalam and Montell, 2007; Dhaka  et al. , 2009; Julius, 2013) . TRPVs are typically tetrameric 77 \nmembrane receptors in which individual subunits have six transmembrane domains (TMs), cytosolic N- 78 \nand C - terminals and a hydrophobic short P -loop between the fifth and sixth TMs that creates the 79 \nselectivity filter (Rosasco and Gordon, 2017) . This structure and the sensory related functions  it 80 \nunderpins, are conserved across vertebrate and invertebrate species. This includes Hirudo medicinalis, 81 \nC. elegans and Drosophila melanogaster, where their contribution to nociception is well-documented 82 \n(Colbert, Smith and Bargmann, 1997; Tobin et al., 2002; Gong et al., 2004; Summers, Holec and Burrell, 83 \n2014; Ohnishi et al., 2020). 84 \nIn this study, we characterised two TRPV channel representatives ( Ovtrpv1 and Ovtrpv2) from O. 85 \nvulgaris. These are orthologues of C. elegans oc r and osm-9 receptors respectively, enabling us to 86 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n5 \n \nconfirm function by heterologous expression in C. elegans null mutants and successful rescue of the 87 \naversive response in these strains. We used in silico and experimental analyses encompassing PCR to 88 \nlocalise Ovtrpvs expression to distinct tissues, and Xenopus oocytes expression to characterise 89 \nchannel function. Taken together these data validate d two bona fide  TRPV channel representatives 90 \nfrom O. vulgaris which have the functional properties and anatomical localisation to act as polymodal 91 \nsensory receptors with a key role in detecting and classifying environmental stimuli.  92 \nResults  93 \nIn silico identification of O. vulgaris trpv transcript and its experimental 94 \nvalidation  95 \nWhen interrogating the previously available O. vulgaris transcriptome (Petrosino, 2015; Petrosino et al., 96 \n2022) using sequences of curated TRPV channels (e.g., H. sapiens  TRPV1), we retrieved two hits 97 \n(c32354_g7_i1 and c32354_g6_i1). However, in silico translation of these sequences revealed that the 98 \nretrieved hits corresponded to incomplete fragments of a larger transcript. We therefore performed an 99 \nin silico analysis reiterating the sequences between O. vulgaris, O. bimaculoides and Aplysia californica 100 \ntranscriptome databases. This led to the identification of three additional overlapping fragments 101 \n(c32354_g14_i1, c32354_g12_i1, c32354_g2_i1) generating a contig transcript encoding a putative full 102 \nlength TRPV channel subunit (transcript accession number: PV164572, Pieroni et al., 2026). Secondary 103 \nstructure prediction tools identified intracellular N - and C -terminals, six TMs and several ankyrin 104 \nrepeats in the N-terminal region (Figure 1). Using 3D modelling with AlphaFold 3 (Abramson et al., 2024) 105 \nand simulation of the protein structure within the membrane with PPM3 server  (Lomize, Todd and 106 \nPogozheva, 2022), we revealed the presence of a P-loop between TM 5 and TM 6, another key signature 107 \nof these channels that was not detectable with secondary structure prediction tools ( Figure 1 ). We 108 \ndesigned primers that flank the predicted  full length sequence and used PCR amplification from O. 109 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n6 \n \nvulgaris reverse transcribed mRNA combined with cDNA sequencing to authenticate it as a bona fide 110 \nsequence (accession number: PX926345). 111 \nOvtrpv1 is located on chromosome 22 of O. vulgaris genome 112 \nThe validated sequence was subsequently blasted against the most recently available O. vulgaris  113 \ngenome (Destanović et al. , 2023)  and this led to the identification of a genomic fragment on 114 \nchromosome 22 (OX597835.1). The translated protein (CAI9738794.1, OctVul6B016571P1) with an 115 \nautomated annotation of “transient receptor potential cation channel subfamily V member 5-like”, was 116 \nshorter than the sequence predicted and validated. We therefore analysed the putative Ovtrpv1 117 \ntranscript against the genomic fragment OX597835.1 using NCBI Magic -BLAST (Boratyn et al., 2019). 118 \nThe result confirmed the presence of the transcript distributed in 15 exons (Figure 2A), with an identity 119 \nof 99.6% over 100% coverage. Only nine mismatches, corresponding to single nucleotide 120 \npolymorphisms were detected, but these did not affect the translated amino acids. This analysis 121 \nhighlights that the sequence previously tagged by the PV164572 submission and experimentally verified 122 \n(accession number: PX926345) represents the correct annotation. 123 \nO. vulgaris genome analysis of Ovtrpv1 revealed a second TRPV sequence 124 \nDuring our interrogation of the most recent O. vulgaris predicted transcriptome (Destanović et al., 2023) 125 \nwith Ovtrpv1, we identified a distinct sequence located on chromosome 3 (OctVul6B028372T1). This 126 \nwas also automatically annotated based on sequence similarities as “transient receptor potential 127 \ncation channel subfamily V member 5 -like” but was not present in the previously available annotated 128 \ntranscriptome (Petrosino, 2015). Secondary structure prediction and AlphaFold3 modelling utilising the 129 \npredicted protein sequence, showed again typical TRPV canonical features (Figure 1). However, NCBI 130 \nconserved domains tool  (Lu et al. , 2020) highlighted homology to the bacterial “Spore_III_AF” super 131 \nfamily (cl17562; e-value = 5.81 × 10-3) in the predicted protein sequence. To resolve this, we blasted the 132 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n7 \n \npredicted sequence against other cephalopod  transcriptomes and identified a high homology to 133 \npredicted DNA sequences from O. sinensis  and O. bimaculoides . However, when we aligned the 134 \nproteins predicted from these DNA sequences, the apparent “Spore_III_AF ” super family  homology 135 \nappeared not to be conserved. Therefore, we blasted both O. vulgaris  Ovtrpv and O. sinensis  (the 136 \nclosest relative) Ostrpv against O. vulgaris genome using NCBI Magic -BLAST tool. Both  transcripts 137 \nmatched regions of chromosome 3 of O. vulgaris. However this matching was not as first envisaged and 138 \nannotated (Figure 2B). The original Ovtrpv2 predicted transcript was found to be distributed to 14 exons 139 \nwhilst the subsequent matching listed above found that Ostrpv2 aligned with an additional exon. 140 \nFurthermore, exons encoding the N-terminal region of the predicted protein were differently detected, 141 \nsuggesting a potential misprediction of the start codon ( Figure 2 B). We authenticated this matured 142 \nprediction using PCR primers designed to flank the ends of the new prediction (Supplementary Table 143 \nS4). This amplified a PCR product from mRNA  derived cDNA of 2799 bp. Sequencing this product 144 \nconfirmed a bona fide sequence that does not contain the unexpected bacterial functional domain 145 \n(Accession number: PX926346). The depiction of the physiological gene structure of Ovtrpv2 is 146 \nrepresented in Figure 2B. 147 \nCluster analysis and phylogenetic investigation confirmed OvTRPV1 and 148 \nOvTRPV2 belong to the vanilloid TRPV subfamily 149 \nWe next asked how these newly identified TRPV sequences related to the other TRP subfamilies. We 150 \nperformed a cluster analysis by blasting canonical curated TRP channel amino acid sequences against 151 \nthe proteome of 13 representative species (Supplementary Table S2). Our CLANS analysis showed the 152 \nexpected distinction among the different subfamilies of TRP channels and from other cationic ion 153 \nchannels (i.e., voltage-gated sodium, calcium and potassium ion channels, Figure 3A). The two newly 154 \nidentified sequences from O. vulgaris  were found within the same cluster that included other well -155 \ncharacterised vertebrate and invertebrate TRPVs. Our cluster analysis did not identify any additional O. 156 \nvulgaris TRPV candidate. All the sequences belonging to the TRPV cluster, were then used to build a 157 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n8 \n \nphylogenetic tree to reveal the relationship between the sequences ( Figure 3B ). The phylogeny 158 \nsuggested invertebrate and vertebrate TRPV receptors share a common ancestor receptor. When 159 \nlooking at the invertebrates, two main branches seemed to have evolved from a duplication event that 160 \ngave rise to “OCR-like” and “OSM-9-like” TRPV receptor branches, based on the homology with the C. 161 \nelegans TRPV channels. Interestingly, the two OvTRPV sequences were found to belong to distinct 162 \nbranches, with OvTRPV1 belonging to the “OCR-like” and OvTRPV2 belonging to the “OSM-9 like ” 163 \nbranch respectively (Figure 3B). 164 \nOvtrpv1 and Ovtrpv2 are expressed in sensory tissues 165 \nOnce established that the identified sequences are the only readily detected TRPV channel 166 \nrepresentatives in O. vulgaris genome, we investigated their relative  tissue distribution using PCR of 167 \ncDNA synthesised from regionally dissected O. vulgaris tissues (Figure 4A). As a comparison, we used 168 \nan orthologue of the previously identified class of chemotactile receptors (Ovcrt, OctVul6B024555T3), 169 \nfound to be selectively expressed in the sensory epithelium of the sucker (van Giesen et al., 2020). Our 170 \nresults showed a prevalent expression of both Ovtrpvs in the sensory tissues, such as the tip of the arm 171 \nand the sucker, and in the central brain mass, in a similar fashion to the Ovcrt (Figure 4B). Based on the 172 \nintensity of the genes of interest relative to the control, we evidenced a lower expression in the intestine, 173 \nwhite bodies, gill and kidney (Figure 4B).  174 \nOvtrpvs show polymodal sensory functions when heterologously expressed 175 \nin C. elegans 176 \nThe tissue distribution described above supports the notion that Ovtrpv1 and Ovtrpv2 act as 177 \ndeterminants for sensory detection. To investigate this, we took a model hopping approach in C. 178 \nelegans. We previously reported that a functional characterisation of O. vulgaris putative nociceptive 179 \ngenes can be carried out in C. elegans using behavioural readouts (Pieroni et al., 2026). We found high 180 \nsequence homology to  the nociceptive-related TRPV  channels Celeocr-2 and Celeosm-9. OvTRPV1 181 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n9 \n \nshares 55.0% amino acid similarity and 39.8% amino acid identity with the sensory OCR receptor 182 \nCeleOCR-2, while OvTRPV2 shares 54.7% amino acid similarity, and 40.5% amino acid identity with 183 \nCeleOSM-9. For this reason, we heterologously expressed Ovtrpv1 and Ovtrpv2 cDNA sequence in the 184 \ncorresponding C. elegans ocr-2 (ak47) and osm-9 (ky10) loss of function mutants under the control of 185 \ntheir putative respective worm promoters and tested the resulting lines for rescue of avoidance to 186 \ndifferent nociceptive modalities. 187 \nWe first investigated the ability of Ovtrpv expression to restore avoidance to low pH, a cue previously 188 \nidentified as relevant to cephalopod nociception (Hague, Florini and Andrews, 2013; Crook, 2021).  189 \nAversion to low pH was measured using a classical acute aversion assay, namely the drop assay.  WT 190 \nN2 worms respond ed around 70% of the time by displaying 3 or more backward movements once in 191 \ncontact with the drop of nociceptive cue (Figure 5A and B). The ocr-2 (A) and osm-9 (B) mutants showed 192 \nreduced backing response when the worms were  exposed to p H 3, consistent with previous data  193 \n(Sambongi et al., 2000). This was also observed in the mutant strains carrying the gfp transgene which 194 \nwere used as a reference for the rescue ( ocr-2 (ak47) vs ocr-2 (ak47) Pmyo -3::gfp, p= 0.9773;  osm-9 195 \n(ky10) vs osm-9 (ky10) Pmyo-3::gfp, p= 0.9878). When we reintroduced the C. elegans gene as a fosmid 196 \n(ocr-2 (WRM0634bB10) Pmyo -3::gfp vs ocr-2 (ak47) Pmyo -3::gfp, p<0.0001; osm-9 (WRM066bG12) 197 \nPmyo-2::gfp vs osm-9 (ky10) Pmyo-3::gfp, p<0.0001) or cDNA (Pocr-2::Celeocr-2 Pmyo-3::gfp vs ocr-2 198 \n(ak47) Pmyo -3::gfp, p<0.0001 ; Posm-9::Celeosm-9 Pmyo -3::gfp vs osm-9 (ky10) Pmyo -3::gfp, p= 199 \n0.0006) under the respective ocr-2 and osm-9 promoter, we restored the reduced pH sensitivity (Figure 200 \n5A and B).  201 \nAgainst this background we repeated the analysis with cDNA encoding OvTRPV1 and OvTRPV2. 202 \nWorm lines containing the Ovtrpvs cDNA showed a significant rescue of the response in the mutant 203 \nlines ( Pocr-2::Ovtrpv1 Pmyo -3::gfp vs ocr -2 (ak47) Pmyo -3::gfp, p<0.0001; Posm -9::Ovtrpv2 Pmyo -204 \n3::gfp vs osm-9 (ky10) Pmyo-3::gfp, p<0.0001).  205 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n10 \n \nIn detail, Ovtrpv1 led to a full rescue of the phenotype (Pocr-2::Ovtrpv1 Pmyo-3::gfp vs WT N2 p= 0.6235, 206 \nFigure 5 A).Ovtrpv2 showed a significant difference to the WT performance  (Posm-9::Ovtrpv2 Pmyo -207 \n3::gfp vs osm-9 (ky10) Pmyo-3::gfp, p<0.0001) suggesting a clear but incomplete rescue. However, this 208 \nwas also true for the corresponding C. elegans cDNA (Posm-9::Celeosm-9 Pmyo-3::gfp vs WT N2, p= 209 \n0.0004) and genomic ( osm-9 (WRM066bG12) Pmyo -2::gfp vs WT N2, p = 0.0068) rescue constructs 210 \n(Figure 5B). Thus, the partial rescue may reflect methodological confounds of the transgenic expression 211 \n(e.g., mosaic expression of the transgene, missing additional regulatory elements in the promoter 212 \nregion), rather than a reduced ability of the orthologue to substitute function. 213 \nNext, mechanical aversion was investigated in C. elegans using a nose touch assay, another classical 214 \nacute aversion test (Figure 6). Similarly to low pH aversion, WT worms showed an halted and/or backing 215 \nbehaviour when in contact with the eyebrow (Figure 6A and B).  216 \nAgain, both mutant lines showed an impaired response to the mechanical insult ( ocr-2 (ak47) vs ocr-2 217 \n(ak47) Pmyo -3::gfp, p>0.9999;  osm-9 (ky10)  vs osm-9 (ky10) Pmyo -3::gfp, p= 0.5753) a defective 218 \nresponse already reported for both mutant strains (Tobin et al., 2002). 219 \nThe introduction of the fosmid for both ocr-2 and osm-9 genes (Figure 6A and B) was able to recover the 220 \nlost aversive response in both mutant strains (ocr-2 (WRM0634bB10) Pmyo-3::gfp vs ocr-2 (ak47) Pmyo-221 \n3::gfp, p<0.0032; osm-9 (WRM066bG12) Pmyo -2::gfp vs osm-9 (ky10) Pmyo -3::gfp, p<0.0001). 222 \nSimilarly, C. elegans  ocr-2 and osm-9 cDNA showed a successful rescue ( Pocr-2::Celeocr-2 Pmyo-223 \n3::gfp vs ocr-2 (ak47) Pmyo-3::gfp, p= 0.0005; Posm-9::Celeosm-9 Pmyo-3::gfp vs osm-9 (ky10) Pmyo-224 \n3::gfp, p= 0.0002). 225 \nWhen introducing the O. vulgaris trpv cDNA, both constructs were able to rescue the gene function by 226 \nrecovering aversion to mechanical insults. Ovtrpv1 exhibited a modest rescue in ocr-2 (ak47)  227 \nbackground ( Pocr-2::Ovtrpv1 Pmyo -3::gfp vs ocr -2 (ak47) Pmyo -3::gfp, p= 0.0131, Figure 6 A) while  228 \nOvtrpv2 showed a full rescue of the behavioural phenotype of osm-9 (ky10) with more than 70% of the 229 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n11 \n \nlines tested (12 out of 17) efficiently responding to nose touch (Posm-9::Ovtrpv2 Pmyo-3::gfp vs WT N2, 230 \np= 0.1497, Figure 6B). 231 \nWe additionally tested rescue to another chemical noxious compound, the volatile repulsive cue 1 -232 \noctanol by measuring the latency to start a reversal behaviour when exposed to the airborne compound 233 \n(Figure 7). The impairment of the mutant strains was striking (average latency of ocr-2 (ak47) Pmyo -234 \n3::gfp of 11.65 s vs average latency of 4.13 s of WT N2; average latency of osm-9 (ky10) Pmyo-3::gfp of 235 \n11.06 s vs average latency of 2.25 s of WT N2, Figure 7A and B)  and has been previously reported in C. 236 \nelegans TRPV mutants (Thies et al., 2016). 237 \nRescue was achieved with both genomic ( ocr-2 (WRM0634bB10) Pmyo -3::gfp vs ocr-2 (ak47) Pmyo -238 \n3::gfp, p= 0.0016; osm-9 (WRM066bG12) Pmyo -2::gfp vs osm-9 (ky10) Pmyo -3::gfp, p<0.0001) and 239 \ncDNA construct (Pocr-2::Celeocr-2 Pmyo-3::gfp vs ocr-2 (ak47) Pmyo -3::gfp, p<0.0009 ; Posm-240 \n9::Celeosm-9 Pmyo-3::gfp vs osm-9 (ky10) Pmyo-3::gfp, p= 0.0172). 241 \nWhen heterologously expressing octopus cDNA, o nly a trend towards a reduced latency to initiate 242 \nreversals to diluted (30%) 1 -octanol was shown by Ovtrpv1-expressing lines (Pocr-2::Ovtrpv1 Pmyo-243 \n3::gfp vs ocr -2 (ak47) Pmyo -3::gfp, p= 0.0679, Figure 7 A), while osm-9 (ky10) mutants expressing 244 \nOvtrpv2 were successfully rescued  (Posm-9::Ovtrpv2 Pmyo -3::gfp vs osm -9 (ky10) Pmyo -3::gfp, p=  245 \n0.0002, Figure 7B). 246 \nInvestigation of OvTRPVs function using recombinant systems 247 \nThe successful rescue of different modalities and cues  through expression of Ovtrpv channels in 248 \nmutant C. elegans  strains, suggested they share sufficient structural similarity with their worm 249 \northologues to function in a similar fashion to the missing endogenous receptors in the intact organism. 250 \nHowever, whether these act upstream as direct activators or downstream as final effectors or 251 \nmodulators of the primary sensory signalling is not known. To address this, we heterologously 252 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n12 \n \nexpressed the receptors in recombinant systems, as it was previously shown in C. elegans that OSM-253 \n9/OCR-2 are both required to be correctly localised at the ciliated sensory neurons to exert a functional 254 \nresponse (Tobin et al., 2002; Ohnishi et al., 2020). We used Xenopus oocytes to test for direct receptor 255 \nactivation. We verified the ability to reconstitute a ligand -activated response from the Homo sapiens 256 \nHstrpv1 control using capsaicin , but Ovtrpvs-expressing oocytes did not respond to this vanilloid 257 \ncompound, suggesting this might not be an ecologically relevant stimulus in cephalopods  (Figure 8A). 258 \nHowever, a long-sustained response to 100 μM nicotinamide (NAM) was observed when Ovtrpv1 and 259 \nOvtrpv2 were co-expressed but not when either receptor was expressed alone (Figure 8B), suggesting 260 \nthat OvTRPV subunits require co -assembly of the two subunits to reconstitute a response in a 261 \nrecombinant system. 262 \nDiscussion 263 \nO. vulgaris has two distinct TRPV channel representatives 264 \nIn a previous in silico  analysis of the conserved molecular determinants putatively involved in O. 265 \nvulgaris nociception, we highlighted candidates that could underpin octopus  response to noxious 266 \nstimuli (Pieroni et al., 2026). 267 \nIn this study, we focussed on the nociceptive molecular determinant TRPV that belongs to the vanilloid 268 \nsubfamily of TRP channels (Himmel and Cox, 2020).  269 \nThe TRPV subfamily, and particularly the TRPV1 member in vertebrates, is known to be gated by several 270 \nnociceptive cues including capsaicin, low pH and high temperature, thus classifying it as a polymodal 271 \nreceptor (Caterina et al., 1997; Immke and Gavva, 2006; Dhaka  et al., 2009). An important aspect of 272 \nTRPV1 and related proteins is that this molecular sensing and signalling is placed in discrete cells in the 273 \nperipheral part of the body  so they can act as sensory receptors that initiate complex downstream 274 \nsignalling that allows the embedded circuits to drive judicious behavioural responses (Caterina et al., 275 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n13 \n \n1997). Consistent with this, a prevalent expression of TRPV1 in peripheral sensory neurons that interact 276 \nwith the environment has been extensively reported (Caterina et al., 1997; Nakagawa and Hiura, 2006; 277 \nSzigeti et al., 2012). In addition, a wider expression in the central brain implying distinct contributions 278 \nto nociception or other functions has also been found (Yuan and Burrell, 2010; Higgins  et al., 2013; 279 \nMarrone et al., 2017).  280 \nThe sensory function and the anatomical distribution of TRPV channels also pertain to invertebrate and 281 \nsimpler organisms (Colbert, Smith and Bargmann, 1997; Summers, Holec and Burrell, 2014) . This 282 \nprompted us to investigate the TRPV subfamily in O. vulgaris in which its complex nervous system and 283 \narticulated behaviours are speculated to allow elaboration of nociception into pain-like states (Birch et 284 \nal., 2021). Our work exploited the emerging access to octopus transcriptome and genome databases 285 \nand matured earlier designation of a single TRPV gene. Cluster analysis and phylogenetic investigation 286 \nrefined the designation of  OvTRPV1 and identified a second receptor,  OvTRPV2. Our analysis 287 \nauthenticated these genes and highlighted they are the only representatives of vanilloid TRP channels 288 \nfound in O. vulgaris  and represent two discrete  receptor channel  subunits ( Figure 3 A). Two TRPV 289 \nrepresentatives were also found in other cephalopods such as O. bimaculoides and O. sinensis and 290 \nalso in other species such as D. melanogaster or A. californica  (Figure 3B). This indicates potential 291 \nstructural conservation across distinct invertebrate genera which may well reflect strong functional 292 \nconservation of sensory specialisation. 293 \nAn interesting exception to the generalisation above is represented by C. elegans, which shows a total 294 \nof 5 TRPV representatives, suggesting a specific one-to-many orthologues expansion of these receptors 295 \ncompared to other invertebrate phyla (Kuzniar et al., 2008). We can identify an invertebrate TRPV group 296 \nreferred to as “OCR-like” TRPV branch. This grouping includes all the C. elegans OCR receptors (OCR 297 \n1-4) to which OvTRPV1 and D. melanogaster  Nanchung (NAN) are more closely related (55.0% 298 \nsimilarity). A second group, referred to as “OSM-9 like” branch, includes C. elegans OSM-9 to which 299 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n14 \n \nOvTRPV2 and D. melanogaster  Inactive (IAV) are related (54.7% similarity, Figure 3 B). The analysis 300 \nshows the i nvertebrate TRPVs share a common ancestor with the vertebrate TRPVs . This analysis 301 \nsupports a scenario in which this ancestor receptor underwent a duplication that gave rise to two 302 \ndistinct mammalian branches. One led to the TRPV5 and 6 ion channels which , despite retaining 303 \nsensitivity to pH, do not have any sensory related functions but act to regulate calcium homeostasis in 304 \nthe kidney and small intestine (Yeh et al., 2003; Nijenhuis, Hoenderop and Bindels, 2005; Lambers  et 305 \nal., 2007) . This subgroup is separated from the TRPV1 -4 channels  branch, in which further internal 306 \nduplications likely led to a specialisation of these receptors in compounds and stimuli detection (Peng, 307 \nShi and Kadowaki, 2015; Morini et al., 2022).  308 \nTissue expression supports the hypothesis that Ovtrpvs are sensory 309 \nmolecular determinants in octopus 310 \nAs indicated, the tissue expression of the mammalian TRPV1 justifies key function in sensory signalling 311 \n(Caterina et al. , 1997) . In the case of the Ovtrpvs, gross analysis of their mRNA expression across 312 \ndifferent tissues supports an important role in sensory signalling ( Figure 4). Both subunits appear to 313 \nshow relatively low expression but robust and reproducible amplification from tissues associated with 314 \nsensory signalling in cephalopods. This is reinforced when we compare the expression in the sensory 315 \ntissues (arm, tip of the arm and sucker) to a range of tissues encapsulating immune-related (e.g., white 316 \nbodies, haemocytes) and feeding and digestion -related (e.g., stomach, intestine) tissues ( Figure 4B). 317 \nAlthough tissue distribution cannot define specificity of function per se, we identified that Ovtrpv1 and 318 \nOvtrpv2 have overlapping expression with the selective chemotactile receptor orthologue Ovcrt (Figure 319 \n4B). This gene is reported to be enriched in the sensory epithelium of the sucker of O. bimaculoides with 320 \nreported related sensory functions  (van Giesen  et al. , 2020) . This enrichment is interesting as 321 \ncephalopods, and in particular octopus es, use their arms to sample the environment and classify 322 \nstimuli including those that evoke aversion. These structures which express sensory receptors are 323 \nprimary organisers of reflexive and complex responses (Rossi and Graziadei, 1956; Zullo, Fossati and 324 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n15 \n \nBenfenati, 2011; Hague, Florini and Andrews, 2013; Gutnick et al., 2020). The prevalence of Ovtrpv1 and 325 \nOvtrpv2 expression in the distal part of the arm, such as the tip and more specifically the suckers, 326 \nencourages the hypothesis these receptors modulate sensory detection in O. vulgaris. The comparison 327 \nwith Ovcrt expression suggests these receptors are low abundant, and this seems in line, at least for 328 \nOvtrpv1, with the RNA sequencing analysis of its fragment s performed in the previously assembled O. 329 \nvulgaris transcriptome (Petrosino, 2015; Petrosino et al., 2022).  330 \nIn a similar fashion to vertebrate TRPVs we showed that, Ovtrpvs expressed in the central brain mass of 331 \nO. vulgaris  (Figure 4 B), support s a potential role in  central control of  sensory and nocicepti ve 332 \nmodulation. 333 \nWe additionally detected expression of both Ovtrpvs in the intestine and, exclusively for Ovtrpv1, in the 334 \nstomach, digestive gland, white bodies, gill and kidney ( Figure 4 B). This broader expression in non -335 \nneuronal tissues for Ovtrpv1 is interesting when considering it belongs to the “OCR-like” branch. In C. 336 \nelegans, OCRs show a more heterogenous expression including the rectal gland cells and intestine 337 \nwhich play an important role in C. elegans digestive system (Tobin et al., 2002; Packer  et al., 2019). 338 \nAdditionally, ocr-3 and ocr-4 seem to be co-expressed in neuroendocrine cells with ocr-2, to regulate 339 \negg-laying (Jose et al., 2007).  340 \nFunctional characterisation through model hopping in C. elegans suggests 341 \nOvtrpvs are polymodal nociceptors  342 \nThe prevalent expression in sensory tissues of both receptors, encouraged us to functionally test them 343 \nfor their role in nociception.  344 \nAs we have already utilised C. elegans as a suitable model to perform functional characterisation of 345 \noctopus molecular nociceptor candidates  (Pieroni et al. , 2026) , we resorted to a model hopping 346 \napproach by expressing octopus receptors in the mutant strains for the orthologue genes ocr-2 and 347 \nosm-9. The phylogenetic distinction between the two receptors into “OCR-like” receptors for Ovtrpv1 348 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n16 \n \nand “OSM-9-like” for Ovtrpv2, justified our approach of heterologously expressing Ovtrpv1 under the 349 \nocr-2 promoter in an ocr-2 (ak47) mutant background and Ovtrpv2 under the osm-9 promoter in an osm-350 \n9 (ky10) mutant background.  351 \nThe null mutants osm-9 (ky10) and ocr-2 (ak47) show defects in different sensory modalities, including 352 \nchemical and mechanical aversion, effectively highlighting OSM-9 and OCR-2 as polymodal receptors 353 \n(Colbert, Smith and Bargmann, 1997; Tobin  et al., 2002; Liedtke  et al. , 2003; Thies  et al. , 2016). We 354 \ntherefore subjected our transgenic lines expressing Ovtrpv1 and Ovtrpv2 in the C. elegans mutants for 355 \nthe orthologous genes to acetic acid (pH 3), mechanical insults (i.e. nose touch) and volatile repellents 356 \nsuch as 1-octanol. 357 \nOvtrpv1 and Ovtrpv2 successfully rescued low pH ( Figure 5 ) and mechanical avoidance ( Figure 6 ) 358 \nsuggesting these receptors act as polymodal nociceptors in O. vulgaris. These two cues induce aversive 359 \nresponses in cephalopods. Low pH has been tested in ex vivo octopus arm preparations, showing a 360 \nquick withdrawal when in contact with an acidic solution, and in vivo experiments, in which injection of 361 \nacetic acid triggered grooming and protective behaviours (Hague, Florini and Andrews, 2013; Crook, 362 \n2021). Mechanical cues are also an important trigger for cephalopod sensory responses as von Frey 363 \nfilaments induce nociceptive responses and also trigger post -injury sensitisation in squids (Crook, 364 \nHanlon and Walters, 2013; Alupay, Hadjisolomou and Crook, 2014; Crook et al., 2014). 365 \nHowever, the molecular determinants of such responses have not been investigated in O. vulgaris and 366 \nthe TRPV channels we describe here are potential candidates. Whether OvTRPVs  act as channels 367 \ndirectly gated by pH or as indirect modulators of pH behavioural responses is not known  and the 368 \nproposed key residues for proton gating in mammalian TRPV1  (Jordt and Julius, 2002; Ryu et al., 2007; 369 \nAneiros et al., 2011), are not conserved in OvTRPVs or in CeleTRPVs. Therefore, either OvTRPV channels 370 \nhave a distinct mechanism for pH detection, or perhaps they possess a downstream modulatory role 371 \nfor this cue. As for mechanosensation, a key molecular determinant has been found in O. bimaculoides 372 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n17 \n \nas part of the TRPN family, which also includes the mechanosensory D. melanogaster NompC (Yan et 373 \nal., 2013; van Giesen et al., 2020). However, it is not uncommon in organisms to have multiple receptors 374 \nresponding to the same cue. This allows resolution of the stimulus detection and its texture, that leads 375 \nto the distinction between a soft touch and a nociceptive harmful input. C. elegans represents a good 376 \nexample of this, as osm-9/ocr-2 are not uniquely required for aversive nose touch (Kindt et al., 2007; Li 377 \net al., 2011), and different molecular interactions give rise to distinct sensitivities (e.g., mec-10/mec-4 378 \nfor gentle touch and mec-10/degt-1 for harsh touch, Chatzigeorgiou et al., 2010; Li et al., 2011). 379 \nOur C. elegans model hopping suggests that homologue expression gives the most potent rescue with 380 \nocr-2 promoter driving Ovtrpv1 and osm-9 promoter driving Ovtrpv2 expressions in nematode  loss of 381 \nfunction mutants of the orthologue trpv genes. However, these functional experiments preclude insight 382 \ninto their transduction localisation (upstream or downstream) or the required stoichiometry that makes 383 \nthe receptor functional in sensory cellular signalling. The evolutionary relationship between Ovtrpv1 384 \nand Ovtrpv2, consistent with other invertebrates, suggests important divergence in function (Figure 3B). 385 \nFurthermore, previous experiments suggested co -assembly of osm-9 and ocr-2 to exert their role 386 \n(Ohnishi et al., 2020; Griffin et al., 2025). Finally, the shared overlapping tissue expression of Ovtrpv1 387 \nand Ovtrpv2 suggests they might interact together to convey sensory detection . Altogether, these 388 \npieces of evidence, encouraged our approach of using recombinant assays. 389 \nHeterologous co-expression of Ovtrpv1 and Ovtrpv2 in Xenopus oocytes produced a sustained, slow 390 \nresponse to 100 μM NAM, supporting the hypothesis of co-assembly between Ovtrpv1 and Ovtrpv2 into 391 \nan active heteromeric channel that is directly gated by aversive substances (Figure 8). This finding is in 392 \nline with previously characterised invertebrate TRPV receptors. In both C. elegans and D. melanogaster, 393 \nco-expression of the pair osm-9/ocr-2 (but more robustly of  osm-9/ocr-4) and Nan/Iav respectively, 394 \nproduced dose-dependent responses to NAM (Upadhyay et al., 2016; Griffin et al., 2025). Behavioural 395 \nand cell -studies revealed this substance is a noxious bitter cue in invertebrates, eliciting avoidance 396 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n18 \n \nresponses and leading to a cell-death inducing accumulation of NAM through desensitisation of TRPV 397 \nchannels (Upadhyay et al. , 2016; Ishikawa  et al. , 2023) . Our data are therefore consistent with a 398 \nconserved TRPV role in invertebrate sensory detection and cell metabolic regulation  that are worth 399 \npursuing in the future. 400 \nConclusions 401 \nAltogether, this work identified two functional TRPV channels in O. vulgaris and contributed to providing 402 \nprecise annotation and experimental validation of two important molecular determinants of 403 \nnociception in cephalopods . Heterologous functional characterisation of these octopus TRPV 404 \nchannels in C. elegans and in the Xenopus oocyte recombinant system, suggests they are polymodal 405 \nand may have a physiological role in the octopus response to aversive chemical and mechanical cues.  406 \nThe sensory role of Ovtrpvs is supported by our endpoint PCR analysis of a wide set of tissues, 407 \ndemonstrating expression in sensory and nervous tissues. However, future analysis should focus on 408 \nqPCR and in situ hybridisation to confirm the potential selective expression in the tip of the arm and 409 \nsucker relative to other non-sensory tissues. 410 \nFinally, the established phylogenetic relationship, locating OvTRPV1 and OvTRPV2 in distinct 411 \nevolutionary branches of the vanilloid TRP channel subfamily, as well as the recombinant experiments 412 \nshowing the requirement for co-assembly of OvTRPV1 and OvTRPV2 to generate a functional channel, 413 \nprovides a route to experimentally investigate the pathways and the modalities in which Ovtrpvs 414 \noperate. At the same time it fosters the development of experimental platforms that might identify 415 \nimportant environmental sensory cues. Recent advances have in fact shown octopus evolved species-416 \nspecific receptors but that they also exploit conserved ones (van Giesen et al., 2020), showing the 417 \nimportance for cephalopods to recognise and classify stimuli in the environment through an elaborated 418 \nsensory system able to detect, integrate and select against nociceptive cues. 419 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n19 \n \nMaterials and Methods 420 \nIn silico analysis 421 \nBLAST search criteria to identify O. vulgaris transcripts 422 \nThe protein sequence of Homo sapiens TRPV1 (Hstrpv1) that encodes the human capsaicin receptor 423 \nwas retrieved from UniProtKB/Swiss -Prot (release 2021_04) and blasted against the previously 424 \npublished O. vulgaris de novo assembly transcriptome (Petrosino, 2015; Petrosino et al., 2022) using 425 \nthe TBLASTN algorithm (BLAST+ v2.10.0+) with a stand ard threshold of 10e -5 used as a cut -off. The 426 \nreference sequences were also used in a BLASTp search against the predicted proteome obtained from 427 \nthe most recent sequenced O. vulgaris genome (Destanović et al., 2023), using the same parameters 428 \ndescribed above. The hits retrieved from both searches, were blasted and compared against Octopus 429 \nbimaculoides (ASM119413v2), Octopus sinensis  (GCA_006345805.1) and Aplysia californica 430 \n(PRJNA13635) assemblies to facilitate assessment of completeness.  431 \nIn the case of the O. vulgaris chemotactile receptor (CRTs) orthologues, O. bimaculoides published 432 \nsequences were retrieved from NCBI and used for a BLASTp search against O. vulgaris genome using 433 \nthe parameters mentioned above (Supplementary Table S1) . All the resulting candidates were then 434 \naligned against the H. sapiens  acetylcholine receptor α7 subunit (NP_000737.1)  prior selection, to 435 \ncheck for the lack of the classical vicinal cysteines involved in the neurotransmitter binding, which 436 \nsignatures cephalopod CRTs (van Giesen et al., 2020). 437 \nExon-intron boundaries detection 438 \nThe reconstructed Ovtrpv transcript sequences were blasted against the assembled O. vulgaris  439 \ngenome (Destanović et al. , 2023)  to localise their chromosomal location. The closest match was 440 \ndownloaded and converted into a BLAST custom database through the ncbi/suite tool container in 441 \nDocker (Merkel, 2014) . Alignment between the Ovtrpv1 and Ovtrpv2 cDNA from O. vulgaris or other 442 \nspecies (i.e. O. sinensis ) and the genomic sequence was performed using ncbi/magicblast tool 443 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n20 \n \ncontainer (Boratyn et al., 2019). The results were sorted using biocontainers/samtools (Danecek et al., 444 \n2021) and the data were processed and visualised using Tablet software (The James Hutton Institute, 445 \nv1.21.02.08, Milne et al., 2013).  446 \nSecondary and 3D structure prediction and modelling 447 \nThe assembled contig cDNA was translated to its predicted amino acid sequence through Expasy 448 \ntranslate (Gasteiger et al. , 2003) . Each octopus predicted protein of interest was analysed using 449 \nDeepTMHMM - 1.0 to obtain a prediction of the secondary structure (Hallgren et al. , 2022) . A 3D 450 \nreconstruction of the proteins’ single subunit was obtained using AlphaFold 3 (Abramson et al., 2024) 451 \nand their representation within the cell membrane was modelled through PPM3 Server (Lomize, Todd 452 \nand Pogozheva, 2022). Analysis of the conserved protein functional domains was performed using NCBI 453 \nconserved domain (Lu et al., 2020). 454 \nAlignment with other species orthologue proteins was carried out using Clustal Omega (Madeira et al., 455 \n2022) and identity and similarities among the sequences were analysed with EMBOSS Water Pairwise 456 \nSequence Alignment (Madeira et al., 2024) using a BLOSUM62 matrix.  457 \nCluster analysis and phylogenetic investigation of OvTRPVs 458 \nReference protein sequences for TRP channels from H. sapiens, C. elegans and Drosophila 459 \nmelanogaster as well as other cationic ion channel sequences such as voltage -gated sodium, 460 \npotassium and calcium ion channels were used to perform a BLAST search (e -value 1e -10 and 40 461 \nmaximum target sequences) against the complete proteome of 13 representative species from 462 \nMammalia, Cephalopoda, Gastropoda,  Bivalvia, Insecta, Crustacea and Nematoda. This proteome 463 \nselection was based on the best Benchmarking Universal Single -Copy Orthologs (BUSCO) value 464 \n(Supplementary Table S2). All the results from each species were sorted and processed through CD-hit 465 \nusing a 0.95 similarity threshold to remove shorter isoforms or redundant sequences (Li and Godzik, 466 \n2006; Fu et al., 2012). The resulting unique sequences from all the species (1102 sequences) were then 467 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n21 \n \nused as a query to perform a CLuster ANalysis of Sequences (CLANS) (Zimmermann et al., 2018) with 468 \nthe BLAST HSPs e -value of 1e -12 and BLOSUM62 scoring matrix (Supplementary File S1). Visualisation 469 \nof the sequences relationship and cluster organisation was performed with the CLANS toolkit with a set 470 \np-value threshold of 1e -60 (Zimmermann et al., 2018). All the sequences of the cluster which included 471 \nthe target Ovtrpv1 and Ovtrpv2 sequences (50 sequences, Supplementary File S2) were selected and 472 \nused to generate a phylogenetic tree. Alignment of the sequences was performed using MAFFT v7.526, 473 \nE-INS-i method (Katoh and Standley, 2013) . The results were manually curated to exclude poorly 474 \naligned sequences (2) and then trimmed with TrimAl (Supplementary File S3) using gappy-out mode and 475 \ndefault parameters (Capella-Gutiérrez, Silla -Martínez and Gabaldón, 2009) . The best -fit model of 476 \nevolution was selected with ModelFinder (LG+I+G4 chosen according to the Bayesian Information 477 \nCriterion) in IQ-TREE3 v3.0.1 (Wong et al., 2025). Tree branches were obtained using aLRT-SH with 1,000 478 \nreplicates and ultrafast bootstrap method. The final phylogeny was visualised using FigTree v1.4.4 479 \n(http://tree.bio.ed.ac.uk/software/figtree/). The tree was rooted using the vertebrate TRPV channel 480 \nsubfamilies. 481 \nAnimal samples 482 \nO. vulgaris samples collection 483 \nYoung adults of O. vulgaris were caught by local artisanal fishermen in the Bay of Naples, Italy  and 484 \nhumanely killed for tissue collection. The following tissues were dissected and stored in 50 μL RNA later 485 \nfor subsequent analysis : supra -oesophageal mass (SEM), sub -oesophageal mass (SUB), optic lobe 486 \n(OL), stellate ganglion (StG), gastric ganglion (GG), arm (muscle + axial nerve cord, at 50% of its length), 487 \ntip of the arm (TIP), sucker (Su), intestine (Int), stomach (Stom), white body (WB), anterior salivary gland 488 \n(ASG), posterior salivary gland (PSG), digestive gland (DG), haemocytes (Hc), kidney (Kid), branchial 489 \nheart (BrH), skin, mantle (Man) and gill (Supplementary Table S3).  490 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n22 \n \nC. elegans strains and husbandry 491 \nThe following nematode strains were utilised in this study: CX10 osm-9 (ky10); CX4544 ocr-2 (ak47); the 492 \nBristol N2 was used as the wild -type (WT) strain . The strain genotype of the osm-9 (ky10) and ocr-2 493 \n(ak47) mutants was confirmed by designing a pair of primers flanking the region of mutations 494 \n(Supplementary Table S4) and sequencing (Eurofins Genomics).  495 \nC. elegans were cultured and maintained as described in Brenner (1974). Three days prior to any assay, 496 \ngravid adult worms were put on culture plates to lay eggs for 4h and then removed to produce a 497 \nsynchronised population to be tested at the L4+1-day old (young adult) stage. In the case of transgenic 498 \nlines, L4 worms expressing GFP derived from co-marker plasmid were selected from the synchronised 499 \npopulation and incubated overnight at 20 °C 24h prior the experiment. 500 \nMolecular biology  501 \nRNA extraction and cDNA synthesis from O. vulgaris tissues 502 \nSmall resections (7 -40 mg) of the indicated frozen tissues were collected in 2 mL Eppendorf tubes 503 \ncontaining 500 µl TRIzol® (Invitrogen ™), snap frozen and homogenized using a Handheld Homogeniser 504 \nSHM1 (Cole -Palmer). After 5 min at room temperature, 200 µl of chloroform (Sigma -Aldrich) were 505 \nadded, mixed, and incubated on ice for 15 min. Samples were centrifuged (12,000 x g, 4° C) and the 506 \ntotal RNA upper aqueous layer purified using PureLink® RNA Mini Kit (Invitrogen TM). DNase I treatment 507 \n(InvitrogenTM) was performed to remove potential contaminating genomic DNA. Quality (260/280 ≈ 2.0) 508 \nand quantity of extracted RNA were assessed through UV -visible absorption measurements 509 \n(NanoDrop™ 2000/2000c Spectrophotometers). For each sample,  1 µg of RNA was used for reverse 510 \ntranscription following the manufacturer’s protocol (SuperScript ™ III Reverse Transcriptase, 511 \nInvitrogenTM). The cDNA samples were stored at -20°C until further use. As previously reported, the 512 \nmollusc ‘hidden break’ does not allow a sufficient gel resolution to assess the RNA quality due to the 513 \nco-migration of the 18S and 28S rRNA fragments (Natsidis et al., 2019; Adema, 2021). Therefore, only 514 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n23 \n \nthe tissues from which we successfully amplified a control gene , cullin 1 (cul1) , were selected for 515 \nfurther use in PCR amplification of the genes of interest (Supplementary Table S3). 516 \nPrimer design and end point PCR  517 \nPrimer sequences of the genes of interests were designed using ApE-A plasmid Editor© v3.1.3 software 518 \nand purchased from Eurofins Genomics (Supplementary Table S4). Once diluted to a working 519 \nconcentration of 25 pmol/µL, the primers were used to amplify the sequence in the tissues described 520 \nabove using 1 µL of cDNA as template in a PCR Phusion ™ High-Fidelity DNA Polymerase (Thermo 521 \nScientific™) following manufacturer’s instructions.  522 \nPromoter and genes synthesis for cloning 523 \nThe promoter region of the osm-9 gene (Posm-9, approximately 1.6 kb – Colbert, Smith and Bargmann, 524 \n1997), the C. elegans osm-9 (Celeosm-9) and the Hstrpv1 cDNA sequences were synthesised using the 525 \nIntegrated DNA Technologies Gene synthesis service (Integrated DNA Technologies, USA ). The 526 \npromoter region of the ocr-2 gene (Pocr-2, approximately 2.5 kB - Sokolchik et al., 2005) and the O. 527 \nvulgaris trpv2 (Ovtrpv2) were synthesised using the Genscript gene synthesis service and subcloned 528 \ninto the pcDNA3.1 vector (Genscript Biotech, UK). The pcDNA3.0 plasmid containing the C. elegans 529 \nocr-2 cDNA sequence (Celeocr-2) was a kind gift from Prof. Cornelia Bargmann (Rockefeller University, 530 \nNY, USA). All the plasmid sequences received, synthesised and cloned were verified for their 531 \nauthenticity using the Oxford Nanopore Whole Plasmid Sequencing (WPS) service from Eurofins 532 \nGenomics. A missense mutation (AAC to AGC) in ocr-2 cDNA leading to N695S in the protein sequence 533 \nwas identified in the original pcDNA3-ocr-2 plasmid. However, the cDNA still showed a successful 534 \nbehavioural rescue when expressed in ocr-2 (ak47) mutant worms suggesting it is functionally silent, 535 \nconsistently with previous investigations (Tobin et al., 2002; Ohnishi et al., 2020). 536 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n24 \n \nCloning for expression in C. elegans 537 \nThe Pocr-2/Posm-9 sequences were cloned (using HindIII/XhoI) into the pWormgate2 vector (Johnson, 538 \nBehm and Trowell, 2005) to produce a pDEST -Pocr-2 or pDEST-Posm-9 vector for suitable expression 539 \nin C. elegans. The full -length transcript sequences of Ovtrpv1, Ovtrpv2 and Celeocr-2 were amplified 540 \n(Supplementary Table S4) using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific ™) and the 541 \nA’ overhangs were added using GoTaq® G2 DNA Polymerase (Promega; 30 min at 72 °C) prior to ligation 542 \ninto pCR ™8/GW/TOPO™ TA (Invitrogen ™). This sequence was then transferred into the pWormgate2, 543 \ndownstream of the corresponding promoter via attR/attL recombination using a Gateway™ LR Clonase™ 544 \nII Enzyme mix (Invitrogen™). This produced the final constructs: pDEST-Posm-9::Ovtrpv2, pDEST-Pocr-545 \n2::Ovtrpv1, pDEST -Pocr-2::Celeocr-2. The Celeosm-9 cDNA sequence was cloned via KpnI/SacI 546 \ndownstream of Posm-9 to produce pDEST-Posm-9::Celeosm-9 final construct (Supplementary File S4). 547 \nThe plasmids were transformed into chemically competent bacterial cells (Thermo Scientific™) and the 548 \namplified plasmids purified from overnight cultures. These were then purified using the Monarch® Spin 549 \nPlasmid Miniprep Kit (New England Biolabs®, UK)  and authenticated by sequencing (Eurofins 550 \nGenomics). 551 \nCloning for expression in Xenopus laevis oocytes 552 \nThe sequences of Ovtrpv1, Ovtrpv2, were cloned into the multiple cloning site of the oocyte expression 553 \nvector pTB207 via HindIII/NotI. The sequence of Hstrpv1 was cloned via PCR amplification to introduce 554 \nthe compatible cutting sites EcoRI-NotI (Supplementary Table S4). The pTB207 vector was a kind gift 555 \nfrom Jean -Louis Bessereau’s Lab (Claude Bernard University, Lyon, France). These plasmids were 556 \ntransformed into chemically competent bacterial cells (Thermo Scientific™) and the amplified plasmids 557 \npurified from overnight cultures. These were then purified using the QIAGEN Plasmid Maxi Kit (QIAGEN) 558 \nand authenticated by sequencing (Eurofins Genomics). The final construct sequences are available in 559 \nSupplementary File S5. 560 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n25 \n \nC. elegans fosmids purification and validation 561 \nThe fosmids B0212.3 osm-9 (clone ID: WRM066bG12) and C07G1 ocr-2 (cloneID: WRM0634bB10) were 562 \npurchased from K.K. DNAFORM (Yokohama, Japan). The amplified fosmids were purified using the 563 \nQIAGEN Plasmid Maxi Kit (QIAGEN). PCR verification was performed using primers designed to amplify 564 \nan internal region of osm-9/ocr-2 which was authenticated by sequencing (Eurofins Genomics). The 565 \nconstruct sequences are available in Supplementary File S6. 566 \nMicroinjections 567 \nThe purified pWormgate2 plasmid (Johnson, Behm and Trowell, 2005)  containing the indicated genes 568 \nwas microinjected (40 ng/ µl of plasmid,10 ng/ µl in the case of fosmid) and 30 ng/ µl of Pmyo-2::gfp or 569 \nPmyo-3::gfp (Addgene). These co -injection markers highlight the pharyngeal or the body wall muscle 570 \nrespectively. The microinjection was performed following the protocol described in Mello et al. (1991) 571 \nand Mello and Fire (1991) with aluminosilicate glass capillaries ( 1.0 mm  OD, 0.78 mm  ID, Harvard 572 \nApparatus). Injections were conducted as previously described and injected worms were transferred 573 \nonto a new seeded plate (Calahorro et al., 2022). Progeny from the injected worm was selected based 574 \non the fluorescence and transferred onto separate seeded plates to establish stable transgenic lines 575 \nfor propagation (F2 generation). The following transgenic lines were obtained: ocr-2 (ak47) Pmyo-3::gfp; 576 \nocr-2 ( WRM0634bB10) Pmyo -3::gfp; pDEST-Pocr-2::Ovtrpv1 Pmyo -3::gfp; pDEST -Pocr-2::Celeocr-2 577 \nPmyo-3::gfp in ocr-2(ak47) mutant background;  osm-9(ky10) Pmyo -3::gfp; osm -9 (WRM066bG12) 578 \nPmyo-2::gfp; pDEST -Posm-9::Celeosm-9,Pmyo-3::gfp, pDEST -Posm-9::Ovtrpv2 in osm-9 (ky10)  579 \nmutant background. 580 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n26 \n \nBehavioural assays 581 \nDrop assay to test chemoaversion in C. elegans 582 \nThe behavioural analyses were conducted in experimental arenas in which a microscope digital camera 583 \n(MU1403B, AmScopeTM) was mounted and worm behaviour in response to the indicated treatments was 584 \nrecorded. The captured videos were analysed through manual scoring using AmScope Software 585 \nv.10.11.2024. The assays were time stamped by the digital camera and the speed and quality of the 586 \nresponses assessed using the criteria listed below. The experimenter and observer were blind to the  587 \ngenotype of the strains under investigation. 588 \nChemical aversion in  C. elegans was performed using an adapted version of the classical acute drop 589 \nassay (Hilliard, Bargmann and Bazzicalupo, 2002; Hilliard  et al., 2004). Ten L4+1-day old worms from 590 \nindicated lines were transferred onto a 9 cm unseeded NGM plate and left undisturbed for at least 20 591 \nmin. This allows the worms to transition to roaming behaviour encompassing periods of extended 592 \nforward runs (Gray, Hill and Bargmann, 2005) . A small drop of noxious cue was delivered in front of a 593 \nmoving worm through a small glass capillary (1.0 mm OD, 0.78 mm ID, Harvard Apparatus) attached to 594 \na syringe. A binary score was assigned with a positive response (1) or negative response (0) for each 595 \ncompound tested within 5 s of exposure to the cue and was based on the average number of reversals 596 \nbeing equal or higher to the average number showed by N2s. The worms for each condition were tested 597 \nby exposing them to the drop only once. To trigger low pH response, acetic acid (CH 3COOH; Fisher 598 \nchemical) was dissolved in M9 buffer at a final pH of 3 (M9, pH 3). M9 buffer (pH 7) was checked not to 599 \ncause any response to N2s when administered alone.  600 \nNose touch assay 601 \nBriefly, an eyebrow was placed perpendicular to the front of a moving L4+1 -day old  worm until the 602 \nanimal hit the hair (Kaplan and Horvitz, 1993). Worms were given a binary score indicating nose touch 603 \nsensitivity (1) or insensitivity (0). The sensitive worms halted or/and reversed following the collision. The 604 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n27 \n \ninsensitive worms kept moving forward and tried to climb or cross the hair. The experiment was 605 \nperformed in a single set of 10 consecutive touches on 5 worms per strain. The final ratio of 606 \nsensitive/insensitive responses per worm was calculated and compared between N2, mutant and 607 \ntransgenic strains. 608 \nVolatile aversive response  609 \nFollowing the same husbandry described for the drop assay, 10 worms expressing long forward runs 610 \nwere individually exposed to 30% 1 -octanol (Sigma-Aldrich) solution only once. A thin platinum wire 611 \n(Ø 0.1 mm, Agar Scientific) was dipped into the solution and then waived in front of a moving worm until 612 \nthe animal stopped and initiated a backward movement. The average latency to start a reversal (s) was 613 \nrecorded with a cut-off of 15 s. 614 \nData analysis and statistics 615 \nIn the behavioural assays the transgenic lines that showed a performance that reached two standard 616 \ndeviations from the mean of the mutant line were considered rescued and included in the analysis.  617 \nData were analysed using either one -way or two -way parametric analysis of variance (ANOVA). Post -618 \nhoc comparisons were performed using the Dunnett’s multiple comparisons test. A level of probability 619 \nset at p<0.05 was used as statistically significant. Statistics were performed with GraphPad Prism 620 \nversion 10 for Windows (GraphPad Software, Boston, Massachusetts, USA). 621 \nTRPVs ligand activation 622 \nElectrophysiology of Xenopus Oocytes 623 \nDefolliculated Xenopus laevis  oocytes were obtained from EcoCyte Bioscience and maintained in 624 \nND96 solution as follows (in mM): 96 NaCl, 1 MgCl 2 , 5 HEPES, 1.8 CaCl 2 , and 2 KCl adjusted to pH 7.4. 625 \nThe plasmids of interests, pTB207-Hstrpv1, pTB207 -Ovtrpv1, pTB207 -Ovtrpv2, were linearised 626 \nusing PacI, followed by DNA purification (Zymo) and elution in RNAase free water. cRNA was 627 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n28 \n \nsynthesised using the T7 mMessage mMachine transcription kit (Thermo Fisher Scientific) according to 628 \nthe manufacturer's protocol (with incubation for 6h at 37 °C). RNA was purified using the GeneJET RNA 629 \npurification kit (Thermo Fisher Scientific) and quantified using a NanoDrop spectrophotometer. 630 \nOocytes were injected with 50 nl of 500 ng/µl RNA individually or co -expressed ( pTB207-631 \nOvtrpv1/pTB207-Ovtrpv2) using the Roboinject system (Multi Channel Systems). Injected oocytes were 632 \nincubated at 18 °C in ND96 solution until the day of recording, 4 days post-injection. 633 \nTwo-electrode voltage-clamp (TEVC) recordings were conducted using the Roboocyte2 System (Multi 634 \nChannel Systems). Measuring head electrode resistance was approximately 400-1200 kΩ, pulled on a 635 \nP-97 Micropipette Puller (Sutter Instrument). Electrodes contained AgCl wires backfilled with a 1 M KCl 636 \nand 1.5 M KAc mixture. Oocytes were clamped at -60 mV during continuous recording at 500 Hz. 637 \nCompounds applications (capsaicin, nicotinamide, ND96) lasted for 20 s for capsaicin followed by a 60 638 \ns wash with ND96, and 600 s for nicotinamide followed by a 200 s or 1200 s wash with ND96. Perfusion 639 \nspeed was set to approximately 3 ml/min throughout. Uninjected oocytes were also tested and did not 640 \nrespond to any of the compound tested. At least 7 oocytes were tested for each condition. Data were 641 \nanalysed using the Roboocyte2+ software. 642 \nCompounds and stimuli tested 643 \nThe following compounds were utilised in this study: capsaicin natural (Biosynth), acetic acid glacial 644 \n(FisherChemical), 1-octanol (Sigma-Aldrich), nicotinamide (NAM, Sigma-Aldrich). Stock solutions were 645 \nprepared either in ethanol or DMSO/distilled water, and the final working concentration was reached by 646 \ndissolving the solution in ND96 medium prior to performing the assay. Saline -injected oocytes did not 647 \nrespond in any case tested. 648 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n29 \n \nDeclarations 649 \nEthics approval 650 \nO. vulgaris  specimens were manipulated for the sole scope of tissue collection following  the local 651 \nAnimal Welfare Body authorisation (Ethical Clearance: ecACR -2302ts36). Animals were humanely 652 \nkilled adopting the principles described in Annex IV of Directive 2010/63/EU and following 653 \nrecommendations from Andrews et al. (2013), Fiorito et al. (2015), Butler-Struben et al. (2018) to ensure 654 \nresponsible and ethical use of animal -derived materials and to adhere to the principles of 655 \nReplacement, Reduction, and Refinement (3Rs) . The t issues were collected by a FELASA certified 656 \n(function D) competent person. All the experiments have been carried out in compliance with the Ethics 657 \nand Research Governance Online II (ERGO II) policy (nr 79739) in place at the University of 658 \nSouthampton. 659 \nConsent for publication 660 \nNot applicable 661 \nAvailability of data and materials 662 \nThe datasets (Supplementary Tables S1-S4 and Supplementary Files S1-S6) supporting the 663 \nconclusions of this article are available in the Zenodo repository at the following link: 664 \n10.5281/zenodo.18377710. 665 \nCompeting interests 666 \nThe authors declare no conflict of interest 667 \nFunding 668 \nEMP was supported by the HSA -Ceph 1/2019 grant to the Association for Cephalopod Research 669 \n‘CephRes’ ETS, Napoli, Italy, and The Gerald Kerkut Charitable Trust, UK. 670 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n30 \n \nAuthors' contributions 671 \nEMP: conceptualisation, data curation, in silico  analysis, experimental investigation, methodology,  672 \nvalidation, visualization and writing–original draft 673 \nHB: conceptualisation, data curation, experimental investigation, methodology,  visualization and 674 \nwriting– review and editing, resources 675 \nVOC: conceptualisation, methodology, writing –original draft, writing –review and editing, funding 676 \nacquisition and supervision, resources 677 \nLHD: conceptualisation, methodology, writing –original draft, writing –review and editing, funding 678 \nacquisition and supervision, resources 679 \nLAYG: methodology, validation, visualization and writing– review and editing, resources  680 \nPI: writing–review and editing, resources 681 \nGF: conceptualisation, writing–review and editing, funding acquisition 682 \nJD: conceptualisation, methodology, writing –original draft, writing –review and editing, funding 683 \nacquisition and supervision, resources 684 \nAcknowledgements 685 \nStrains were provided by the Caenorhabditis Genetic Center (CGC), funded by NIH Office of Research 686 \nInfrastructure Programs (P40 OD010440). We thank Prof Cori Bargmann for kindly providing us with 687 \nthe pcDNA3.1-ocr-2 construct. We thank Jean-Louis Bessereau’s Lab (Claude Bernard University, 688 \nLyon, France) for kindly providing the pTB207 vector. We thank Dr Iris Hardege and Tom Reynoldson 689 \nfor advice and support with electrophysiology. We thank Eng Marco Pieroni who kindly helped with the 690 \nsetup and use of the described Docker containers. 691 \n 692 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n31 \n \n 693 \nFigure 1 Schematic of the reconstructed Ovtrpv1 translated protein following secondary and 3D modelling. Typical TRPV 694 \nchannel secondary structure key signatures can be recognised , such as intracellular N - and C- terminals, a variable number 695 \nof ankyrin repeats in the N -terminal region, six transmembrane elements and a cytosolic P -loop between TM5-TM6 which is 696 \nresponsible for the pore formation and selectivity filter in the assembled tetrameric structure. 697 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n32 \n \n 698 \nFigure 2 Schematic representation of Octopus vulgaris trpv1  and trpv2 gene. A Ovtrpv1 was found to be located on chromosome 22 with 100% coverage and 99.6% identity. B The 699 \nsecond TRPV candidate gene, Ovtrpv2, is located on chromosome 3 and was initially mispredicted  (red framed rectangle) , requiring experimental validation. Striped pattern rectangles 700 \nrepresent initially absent exons. 701 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n33 \n \n 702 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n34 \n \nFigure 3 OvTRPV1 and OvTRPV2 are two distinct vanilloid transient receptor potential ion channels. A Visual 703 \nrepresentation of the relationship among the TRP channel subfamilies. The OvTRPVs were identified as part of the TRPV 704 \nsubfamily cluster (dashed red line). The two OvTRPV receptors (red circles with a black star) segregate into two distinct 705 \nsubgroups of the cluster, thus showing some structural distinctions between each other. The same division is observed with 706 \nthe C. elegans TRPVs (see legend in the top left corner). CACNA: voltage -gated calcium channels subunit alpha; KCN: 707 \npotassium voltage -gated channels; TPCN: Two pore calcium channel; TRPA: Transient Receptor Potential cation channel 708 \nsubfamily A; TRPC: transient receptor potential cation channel subfamily C; TRPM: transient receptor potential cation channel 709 \nsubfamily M; TRPML: mucolipin TRP cation channel; TRPP: polycystin transient receptor potential channel interacting; TRPV: 710 \ntransient receptor potential cation channel subfamily V; Unc: uncharacterised. B Phylogenetic analysis of OvTRPVs. 711 \nPhylogenetic tree showing the relationships among TRPV sequences from 13 representative species. The two O. vulgaris TRPV 712 \nsequences (OvTRPV1 and OvTRPV2) are highlighted in bold red. C. elegans OCR-like sequences are shown in bold cyan and 713 \nC. elegans OSM-9 in bold purple, while human representative TRPV sequences are indicated in bold black. The tree reveals 714 \ntwo distinct TRPV lineages in protostomes, corresponding to “OCR-like” and “OSM-9-like” channels, with OvTRPV1 and 715 \nOvTRPV2 clustering within these respective groups. 716 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n35 \n \n 717 \nFigure 4 Tissue distribution of Ovtrpv1 and Ovtrpv2 receptors in O. vulgaris. A Schematic representation of O. vulgaris tissues localisation in the body. B PCR amplification of Ovtrpv1 718 \nand Ovtrpv2 from a different set of tissue mRNA, grouped in the central brain mass, peripheral nervous system, sensory-, immune- and digestive-related tissues. The data are indicative of 719 \nthe PCR performed on mRNA extracted from the indicated tissue of 4 animals (Supplementary Table S3). Arm: muscle + axial nerve cord, at 50% of its length, TIP: tip of the arm, Su: sucker, 720 \nOL: optic lobe, SEM: supra-oesophageal mass, SUB: sub-oesophageal mass, StG: stellate ganglion, GG: gastric ganglion, Kid: Kidney, Stom: stomach, Int: intestine, DG: digestive gla nd, 721 \nASG: anterior salivary gland, WB: white bodies, Hc: haemocytes, PSG: posterior salivary gland, BrH: branchial heart, Man: man tle (muscle). Ovcrt1: O. vulgaris chemotactile receptor 1 722 \n(OctVul6B024555T3), Ovcul1: O. vulgaris cullin 1 (c28856_g1_i2), Ovtrpv1: O. vulgaris trpv1 (PX926345), Ovtrpv2: O. vulgaris trpv2 (PX926346). 723 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n36 \n \n 724 \nFigure 5 Ovtrpv1 and Ovtrpv2 rescue pH3-evoked avoidance in C. elegans ocr-2 (A) and osm-9 (B) mutant strains. The bar graphs represent the ratio of worms responding ± s.e.m. for 725 \nWT N2, ocr-2 (ak47), osm -9 (ky10), ocr -2 (ak47) Pmyo -3::gfp, osm-9 (ky10) Pmyo -3::gfp and fosmid lines. Each dot represents a replicate experiment in which we tested 10 worms per 726 \ncondition. For the C. elegans and O. vulgaris  cDNA constructs, each dot represents a rescue line (10 worms each), which was selected according to the threshold set at 2 st andard 727 \ndeviations above the mean of the reference mutant line ocr-2 (ak47) Pmyo-3::gfp (A) or osm-9 (ky10) Pmyo-3::gfp (B). All the average performances are here compared to the reference 728 \nmutant lines. Data were analysed using one-way ANOVA and Post-hoc comparisons have been performed with Dunnett’s multiple comparisons test. ***p<0.001, **** p<0.0001.  729 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n37 \n \n 730 \nFigure 6 Ovtrpv1 and Ovtrpv2 rescue mechanical aversion in C. elegans ocr-2 (A) and osm-9 (B) mutant strains. The bar graphs represent the ratio of worms responding ± s.e.m. for 731 \nWT N2, ocr-2 (ak47), osm-9 (ky10), ocr-2 (ak47) Pmyo-3::gfp, osm-9 (ky10) Pmyo-3::gfp, fosmid lines and Ovtrpv expressing lines. Each dot represents a replicate experiment in which we 732 \ntested 5 worms in a set of 10 trials. For the C. elegans and O. vulgaris cDNA constructs, each dot represents a rescue line (5 worms per 10 trial each), which was selected according to the 733 \nthreshold set at 2 standard deviations above the mean of the reference mutant line ocr-2 (ak47) Pmyo-3::gfp (A) or osm-9 (ky10) Pmyo-3::gfp (B). All the average performances are here 734 \ncompared to the reference mutant lines. Data were analysed using one -way ANOVA and Post-hoc comparisons have been performed with Dunnett’s multiple comparisons test *p<0.05, 735 \n**p<0.01, ***p<0.001, **** p<0.0001. 736 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n38 \n \n 737 \nFigure 7 Investigation of volatile aversion in C. elegans ocr-2 (A) and osm-9 (B) mutant strains. 738 \nThe bar graphs represent the average latency to start a reversal ± s.e.m. for WT N2, ocr-2 (ak47), osm-9 (ky10), ocr-2 (ak47) Pmyo-3::gfp, osm-9 (ky10) Pmyo-3::gfp, fosmid lines and Ovtrpv 739 \nexpressing lines. Each dot represents a replicate experiment in which we tested 5 worms in a set of 10 trials . For the C. elegans and O. vulgaris cDNA constructs, each dot represents a 740 \nrescue line (10 worms each), which was selected according to the threshold set at 2 standard deviations below the mean of the reference mutant line ocr-2 (ak47) Pmyo-3::gfp (A) or osm-741 \n9 (ky10) Pmyo-3::gfp (B). All the average performances are here compared to the reference mutant lines. Data were analysed using one-way ANOVA and Post-hoc comparisons have been 742 \nperformed with Dunnett’s multiple comparisons test *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001. 743 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint \n\n39 \n \n 744 \nFigure 8 Heterologous expression of O. vulgaris TRPV channels in Xenopus oocytes. A. Current traces from Xenopus oocytes injected with RNA encoding the human  TRPV1 channel  745 \nshows response to capsaicin at 100µM. The solid bars represent the period of capsaicin perfusion. Oocytes injected with RNA for O. vulgaris trpv1 and trpv2 separately or together show 746 \nno response to capsaicin at 100µM. Oocytes injected with water show no response. N ≥ 7 oocytes for each condition.  B. O. vulgaris TRPV1 and TRPV2 form a heteromeric channel which 747 \nresponds to nicotinamide. Xenopus oocytes injected with RNA for Ovtrpv1, Ovtrpv2 or water show no responses to nicotinamide at 100µM. Oocytes expressing Ovtrpv1 and Ovtrpv2 748 \ntogether show strong responses to nicotinamide 100µM. N ≥ 7 oocytes for each condition.749 \n.CC-BY 4.0 International licenseperpetuity. 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