Identification and functional investigation of Octopus vulgaris TRPV channels as potential nociceptors in cephalopods

20 Nociception is an essential response for organisms to avoid potential harm and promote survival. Its 21 molecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion 22 channels exhibiting selective peripheral expression and functional coupling that underpins nociception 23 and pain modulation in complex organisms. However, the execution of protective behaviours triggered 24 by TRPVs is also found in species with a simpler nervous organisation, thus encouraging their 25 investigation in invertebrate model organisms to increase understanding of animal nociception. 26 Cephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous 27 system, whose complexity suggests it might support pain -like states that exist in vertebrates. This 28 possibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. 29 Despite this, there is poor characterisation of cephalopod molecular nociceptors. 30 For this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome 31 (Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent 32 expression in sensory tissues. We investigated the functional competence of these TRPVs by 33 heterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the 34 orthologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response 35 to chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are 36 polymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes 37 showed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by nicotinamide. This study 38 highlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods. 39

C. elegans, animal welfare, pain, nociception, model hopping, octopus, evolution 40 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 3

41 General nociception executes function by activating a reflexive withdrawal response , protecting 42 organisms from potentially threatening stimuli. Various avoidance behaviours operate across animal 43 phyla and advantages organismal survival (Smith and Lewin, 2009). Nociception is characterised by a 44 conserved specialised neuronal architecture and shared molecular determinants that persist across 45 distinct nervous system organisations and largely encompasses a core reflex that triggers withdrawal 46 avoidance responses (Julius and Basbaum, 2001). In more complex organisms, however, the reflexive 47 nociceptive responses are modulated by top -down neuronal signalling leading to the elaboration of 48 learnt and emotionally encoded pain states (Basbaum et al., 2009). 49 Cephalopods are invertebrate species characterised by well described withdrawal responses following 50 exposure to noxious challenges (Crook, Hanlon and Walters, 2013; Hague, Florini and Andrews, 2013; 51 Crook, 2021) . Furthermore, these molluscs possess a central nervous system that can act in a 52 hierarchical fashion, allowing them to express complex behaviours (Amodio et al., 2019; Schnell et al., 53 2021). The details of this anatomy are different from organisms that are unequivocally known to express 54 the emotional states defined by pain, nonetheless cephalopod s’ central brain mass complexity 55 provokes the possibility that their neural architecture is organised to allow brain states with 56 components that resemble pain (Andrews et al., 2013; Smith et al., 2013; Shigeno et al., 2018). This 57 highlights potential ethical issue s and associated welfare constraints about the experimental use of 58 cephalopods that have seen these animals included in research legislations (UK S.I. 1993/2103, 1993; 59 European Parliament and Union, 2010). 60 This raises the scientific value of detailing the biological organisation of nociception reflexes and their 61 modulation in this important biological and societally utilised taxon. 62 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 4 In our previous study, we identified O. vulgaris orthologues of genes with a defined role in nociception 63 and pain to enrich understanding of the molecular determinants of these phenomena in cephalopods. 64 We coupled this to functional studies in C. elegans loss of function mutants of the corresponding genes 65 of interest and identified 19 potential candidates warranting more detailed investigation for their role in 66 O. vulgaris nociception (Pieroni et al. , 2026) . Among our candidates, the previously investigated 67 vanilloid transient receptor potential (TRPV) emerged as a priority candidate for further investigation 68 (Caterina et al., 1997). 69 TRP receptors are a large family of ion channels highly conserved across animal species due to their 70 extensive role in different physiological functions (Pedersen, Owsianik and Nilius, 2005; Ramsey, 71 Delling and Clapham, 2006; Venkatachalam and Montell, 2007) . Among the seven major TRP 72 subfamilies, the vanilloid sensitive homologues, include receptors that detect aversive stimuli (Colton, 73 2006; Radresa et al. , 2012; Shibasaki, 2024) . The best characterised representative of TRPVs in 74 mammals is the capsaicin receptor TRPV1 (Caterina et al. , 1997) . TRPV1 is a polymodal nociceptor 75 which, in addition to vanilloid compounds, responds to cell damaging pH and temperature changes 76 (Venkatachalam and Montell, 2007; Dhaka et al. , 2009; Julius, 2013) . TRPVs are typically tetrameric 77 membrane receptors in which individual subunits have six transmembrane domains (TMs), cytosolic N- 78 and C - terminals and a hydrophobic short P -loop between the fifth and sixth TMs that creates the 79 selectivity filter (Rosasco and Gordon, 2017) . This structure and the sensory related functions it 80 underpins, are conserved across vertebrate and invertebrate species. This includes Hirudo medicinalis, 81 C. elegans and Drosophila melanogaster, where their contribution to nociception is well-documented 82 (Colbert, Smith and Bargmann, 1997; Tobin et al., 2002; Gong et al., 2004; Summers, Holec and Burrell, 83 2014; Ohnishi et al., 2020). 84 In this study, we characterised two TRPV channel representatives ( Ovtrpv1 and Ovtrpv2) from O. 85 vulgaris. These are orthologues of C. elegans oc r and osm-9 receptors respectively, enabling us to 86 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 5 confirm function by heterologous expression in C. elegans null mutants and successful rescue of the 87 aversive response in these strains. We used in silico and experimental analyses encompassing PCR to 88 localise Ovtrpvs expression to distinct tissues, and Xenopus oocytes expression to characterise 89 channel function. Taken together these data validate d two bona fide TRPV channel representatives 90 from O. vulgaris which have the functional properties and anatomical localisation to act as polymodal 91 sensory receptors with a key role in detecting and classifying environmental stimuli. 92

93 In silico identification of O. vulgaris trpv transcript and its experimental 94 validation 95 When interrogating the previously available O. vulgaris transcriptome (Petrosino, 2015; Petrosino et al., 96 2022) using sequences of curated TRPV channels (e.g., H. sapiens TRPV1), we retrieved two hits 97 (c32354_g7_i1 and c32354_g6_i1). However, in silico translation of these sequences revealed that the 98 retrieved hits corresponded to incomplete fragments of a larger transcript. We therefore performed an 99 in silico analysis reiterating the sequences between O. vulgaris, O. bimaculoides and Aplysia californica 100 transcriptome databases. This led to the identification of three additional overlapping fragments 101 (c32354_g14_i1, c32354_g12_i1, c32354_g2_i1) generating a contig transcript encoding a putative full 102 length TRPV channel subunit (transcript accession number: PV164572, Pieroni et al., 2026). Secondary 103 structure prediction tools identified intracellular N - and C -terminals, six TMs and several ankyrin 104 repeats in the N-terminal region (Figure 1). Using 3D modelling with AlphaFold 3 (Abramson et al., 2024) 105 and simulation of the protein structure within the membrane with PPM3 server (Lomize, Todd and 106 Pogozheva, 2022), we revealed the presence of a P-loop between TM 5 and TM 6, another key signature 107 of these channels that was not detectable with secondary structure prediction tools ( Figure 1 ). We 108 designed primers that flank the predicted full length sequence and used PCR amplification from O. 109 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 6 vulgaris reverse transcribed mRNA combined with cDNA sequencing to authenticate it as a bona fide 110 sequence (accession number: PX926345). 111 Ovtrpv1 is located on chromosome 22 of O. vulgaris genome 112 The validated sequence was subsequently blasted against the most recently available O. vulgaris 113 genome (Destanović et al. , 2023) and this led to the identification of a genomic fragment on 114 chromosome 22 (OX597835.1). The translated protein (CAI9738794.1, OctVul6B016571P1) with an 115 automated annotation of “transient receptor potential cation channel subfamily V member 5-like”, was 116 shorter than the sequence predicted and validated. We therefore analysed the putative Ovtrpv1 117 transcript against the genomic fragment OX597835.1 using NCBI Magic -BLAST (Boratyn et al., 2019). 118 The result confirmed the presence of the transcript distributed in 15 exons (Figure 2A), with an identity 119 of 99.6% over 100% coverage. Only nine mismatches, corresponding to single nucleotide 120 polymorphisms were detected, but these did not affect the translated amino acids. This analysis 121 highlights that the sequence previously tagged by the PV164572 submission and experimentally verified 122 (accession number: PX926345) represents the correct annotation. 123 O. vulgaris genome analysis of Ovtrpv1 revealed a second TRPV sequence 124 During our interrogation of the most recent O. vulgaris predicted transcriptome (Destanović et al., 2023) 125 with Ovtrpv1, we identified a distinct sequence located on chromosome 3 (OctVul6B028372T1). This 126 was also automatically annotated based on sequence similarities as “transient receptor potential 127 cation channel subfamily V member 5 -like” but was not present in the previously available annotated 128 transcriptome (Petrosino, 2015). Secondary structure prediction and AlphaFold3 modelling utilising the 129 predicted protein sequence, showed again typical TRPV canonical features (Figure 1). However, NCBI 130 conserved domains tool (Lu et al. , 2020) highlighted homology to the bacterial “Spore_III_AF” super 131 family (cl17562; e-value = 5.81 × 10-3) in the predicted protein sequence. To resolve this, we blasted the 132 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 7 predicted sequence against other cephalopod transcriptomes and identified a high homology to 133 predicted DNA sequences from O. sinensis and O. bimaculoides . However, when we aligned the 134 proteins predicted from these DNA sequences, the apparent “Spore_III_AF ” super family homology 135 appeared not to be conserved. Therefore, we blasted both O. vulgaris Ovtrpv and O. sinensis (the 136 closest relative) Ostrpv against O. vulgaris genome using NCBI Magic -BLAST tool. Both transcripts 137 matched regions of chromosome 3 of O. vulgaris. However this matching was not as first envisaged and 138 annotated (Figure 2B). The original Ovtrpv2 predicted transcript was found to be distributed to 14 exons 139 whilst the subsequent matching listed above found that Ostrpv2 aligned with an additional exon. 140 Furthermore, exons encoding the N-terminal region of the predicted protein were differently detected, 141 suggesting a potential misprediction of the start codon ( Figure 2 B). We authenticated this matured 142 prediction using PCR primers designed to flank the ends of the new prediction (Supplementary Table 143 S4). This amplified a PCR product from mRNA derived cDNA of 2799 bp. Sequencing this product 144 confirmed a bona fide sequence that does not contain the unexpected bacterial functional domain 145 (Accession number: PX926346). The depiction of the physiological gene structure of Ovtrpv2 is 146 represented in Figure 2B. 147 Cluster analysis and phylogenetic investigation confirmed OvTRPV1 and 148 OvTRPV2 belong to the vanilloid TRPV subfamily 149 We next asked how these newly identified TRPV sequences related to the other TRP subfamilies. We 150 performed a cluster analysis by blasting canonical curated TRP channel amino acid sequences against 151 the proteome of 13 representative species (Supplementary Table S2). Our CLANS analysis showed the 152 expected distinction among the different subfamilies of TRP channels and from other cationic ion 153 channels (i.e., voltage-gated sodium, calcium and potassium ion channels, Figure 3A). The two newly 154 identified sequences from O. vulgaris were found within the same cluster that included other well -155 characterised vertebrate and invertebrate TRPVs. Our cluster analysis did not identify any additional O. 156 vulgaris TRPV candidate. All the sequences belonging to the TRPV cluster, were then used to build a 157 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 8 phylogenetic tree to reveal the relationship between the sequences ( Figure 3B ). The phylogeny 158 suggested invertebrate and vertebrate TRPV receptors share a common ancestor receptor. When 159 looking at the invertebrates, two main branches seemed to have evolved from a duplication event that 160 gave rise to “OCR-like” and “OSM-9-like” TRPV receptor branches, based on the homology with the C. 161 elegans TRPV channels. Interestingly, the two OvTRPV sequences were found to belong to distinct 162 branches, with OvTRPV1 belonging to the “OCR-like” and OvTRPV2 belonging to the “OSM-9 like ” 163 branch respectively (Figure 3B). 164 Ovtrpv1 and Ovtrpv2 are expressed in sensory tissues 165 Once established that the identified sequences are the only readily detected TRPV channel 166 representatives in O. vulgaris genome, we investigated their relative tissue distribution using PCR of 167 cDNA synthesised from regionally dissected O. vulgaris tissues (Figure 4A). As a comparison, we used 168 an orthologue of the previously identified class of chemotactile receptors (Ovcrt, OctVul6B024555T3), 169 found to be selectively expressed in the sensory epithelium of the sucker (van Giesen et al., 2020). Our 170

showed a prevalent expression of both Ovtrpvs in the sensory tissues, such as the tip of the arm 171 and the sucker, and in the central brain mass, in a similar fashion to the Ovcrt (Figure 4B). Based on the 172 intensity of the genes of interest relative to the control, we evidenced a lower expression in the intestine, 173 white bodies, gill and kidney (Figure 4B). 174 Ovtrpvs show polymodal sensory functions when heterologously expressed 175 in C. elegans 176 The tissue distribution described above supports the notion that Ovtrpv1 and Ovtrpv2 act as 177 determinants for sensory detection. To investigate this, we took a model hopping approach in C. 178 elegans. We previously reported that a functional characterisation of O. vulgaris putative nociceptive 179 genes can be carried out in C. elegans using behavioural readouts (Pieroni et al., 2026). We found high 180 sequence homology to the nociceptive-related TRPV channels Celeocr-2 and Celeosm-9. OvTRPV1 181 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 9 shares 55.0% amino acid similarity and 39.8% amino acid identity with the sensory OCR receptor 182 CeleOCR-2, while OvTRPV2 shares 54.7% amino acid similarity, and 40.5% amino acid identity with 183 CeleOSM-9. For this reason, we heterologously expressed Ovtrpv1 and Ovtrpv2 cDNA sequence in the 184 corresponding C. elegans ocr-2 (ak47) and osm-9 (ky10) loss of function mutants under the control of 185 their putative respective worm promoters and tested the resulting lines for rescue of avoidance to 186 different nociceptive modalities. 187 We first investigated the ability of Ovtrpv expression to restore avoidance to low pH, a cue previously 188 identified as relevant to cephalopod nociception (Hague, Florini and Andrews, 2013; Crook, 2021). 189 Aversion to low pH was measured using a classical acute aversion assay, namely the drop assay. WT 190 N2 worms respond ed around 70% of the time by displaying 3 or more backward movements once in 191 contact with the drop of nociceptive cue (Figure 5A and B). The ocr-2 (A) and osm-9 (B) mutants showed 192 reduced backing response when the worms were exposed to p H 3, consistent with previous data 193 (Sambongi et al., 2000). This was also observed in the mutant strains carrying the gfp transgene which 194 were used as a reference for the rescue ( ocr-2 (ak47) vs ocr-2 (ak47) Pmyo -3::gfp, p= 0.9773; osm-9 195 (ky10) vs osm-9 (ky10) Pmyo-3::gfp, p= 0.9878). When we reintroduced the C. elegans gene as a fosmid 196 (ocr-2 (WRM0634bB10) Pmyo -3::gfp vs ocr-2 (ak47) Pmyo -3::gfp, p<0.0001; osm-9 (WRM066bG12) 197 Pmyo-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 (ak47) Pmyo -3::gfp, p<0.0001 ; Posm-9::Celeosm-9 Pmyo -3::gfp vs osm-9 (ky10) Pmyo -3::gfp, p= 199 0.0006) under the respective ocr-2 and osm-9 promoter, we restored the reduced pH sensitivity (Figure 200 5A and B). 201 Against this background we repeated the analysis with cDNA encoding OvTRPV1 and OvTRPV2. 202 Worm lines containing the Ovtrpvs cDNA showed a significant rescue of the response in the mutant 203 lines ( Pocr-2::Ovtrpv1 Pmyo -3::gfp vs ocr -2 (ak47) Pmyo -3::gfp, p<0.0001; Posm -9::Ovtrpv2 Pmyo -204 3::gfp vs osm-9 (ky10) Pmyo-3::gfp, p<0.0001). 205 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 10 In detail, Ovtrpv1 led to a full rescue of the phenotype (Pocr-2::Ovtrpv1 Pmyo-3::gfp vs WT N2 p= 0.6235, 206 Figure 5 A).Ovtrpv2 showed a significant difference to the WT performance (Posm-9::Ovtrpv2 Pmyo -207 3::gfp vs osm-9 (ky10) Pmyo-3::gfp, p<0.0001) suggesting a clear but incomplete rescue. However, this 208 was also true for the corresponding C. elegans cDNA (Posm-9::Celeosm-9 Pmyo-3::gfp vs WT N2, p= 209 0.0004) and genomic ( osm-9 (WRM066bG12) Pmyo -2::gfp vs WT N2, p = 0.0068) rescue constructs 210 (Figure 5B). Thus, the partial rescue may reflect methodological confounds of the transgenic expression 211 (e.g., mosaic expression of the transgene, missing additional regulatory elements in the promoter 212 region), rather than a reduced ability of the orthologue to substitute function. 213 Next, mechanical aversion was investigated in C. elegans using a nose touch assay, another classical 214 acute aversion test (Figure 6). Similarly to low pH aversion, WT worms showed an halted and/or backing 215 behaviour when in contact with the eyebrow (Figure 6A and B). 216 Again, both mutant lines showed an impaired response to the mechanical insult ( ocr-2 (ak47) vs ocr-2 217 (ak47) Pmyo -3::gfp, p>0.9999; osm-9 (ky10) vs osm-9 (ky10) Pmyo -3::gfp, p= 0.5753) a defective 218 response already reported for both mutant strains (Tobin et al., 2002). 219 The introduction of the fosmid for both ocr-2 and osm-9 genes (Figure 6A and B) was able to recover the 220 lost aversive response in both mutant strains (ocr-2 (WRM0634bB10) Pmyo-3::gfp vs ocr-2 (ak47) Pmyo-221 3::gfp, p<0.0032; osm-9 (WRM066bG12) Pmyo -2::gfp vs osm-9 (ky10) Pmyo -3::gfp, p<0.0001). 222 Similarly, C. elegans ocr-2 and osm-9 cDNA showed a successful rescue ( Pocr-2::Celeocr-2 Pmyo-223 3::gfp vs ocr-2 (ak47) Pmyo-3::gfp, p= 0.0005; Posm-9::Celeosm-9 Pmyo-3::gfp vs osm-9 (ky10) Pmyo-224 3::gfp, p= 0.0002). 225 When introducing the O. vulgaris trpv cDNA, both constructs were able to rescue the gene function by 226 recovering aversion to mechanical insults. Ovtrpv1 exhibited a modest rescue in ocr-2 (ak47) 227

( Pocr-2::Ovtrpv1 Pmyo -3::gfp vs ocr -2 (ak47) Pmyo -3::gfp, p= 0.0131, Figure 6 A) while 228 Ovtrpv2 showed a full rescue of the behavioural phenotype of osm-9 (ky10) with more than 70% of the 229 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 11 lines tested (12 out of 17) efficiently responding to nose touch (Posm-9::Ovtrpv2 Pmyo-3::gfp vs WT N2, 230 p= 0.1497, Figure 6B). 231 We additionally tested rescue to another chemical noxious compound, the volatile repulsive cue 1 -232 octanol by measuring the latency to start a reversal behaviour when exposed to the airborne compound 233 (Figure 7). The impairment of the mutant strains was striking (average latency of ocr-2 (ak47) Pmyo -234 3::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 11.06 s vs average latency of 2.25 s of WT N2, Figure 7A and B) and has been previously reported in C. 236 elegans TRPV mutants (Thies et al., 2016). 237 Rescue was achieved with both genomic ( ocr-2 (WRM0634bB10) Pmyo -3::gfp vs ocr-2 (ak47) Pmyo -238 3::gfp, p= 0.0016; osm-9 (WRM066bG12) Pmyo -2::gfp vs osm-9 (ky10) Pmyo -3::gfp, p<0.0001) and 239 cDNA construct (Pocr-2::Celeocr-2 Pmyo-3::gfp vs ocr-2 (ak47) Pmyo -3::gfp, p<0.0009 ; Posm-240 9::Celeosm-9 Pmyo-3::gfp vs osm-9 (ky10) Pmyo-3::gfp, p= 0.0172). 241 When heterologously expressing octopus cDNA, o nly a trend towards a reduced latency to initiate 242 reversals to diluted (30%) 1 -octanol was shown by Ovtrpv1-expressing lines (Pocr-2::Ovtrpv1 Pmyo-243 3::gfp vs ocr -2 (ak47) Pmyo -3::gfp, p= 0.0679, Figure 7 A), while osm-9 (ky10) mutants expressing 244 Ovtrpv2 were successfully rescued (Posm-9::Ovtrpv2 Pmyo -3::gfp vs osm -9 (ky10) Pmyo -3::gfp, p= 245 0.0002, Figure 7B). 246 Investigation of OvTRPVs function using recombinant systems 247 The successful rescue of different modalities and cues through expression of Ovtrpv channels in 248 mutant C. elegans strains, suggested they share sufficient structural similarity with their worm 249 orthologues to function in a similar fashion to the missing endogenous receptors in the intact organism. 250 However, whether these act upstream as direct activators or downstream as final effectors or 251 modulators of the primary sensory signalling is not known. To address this, we heterologously 252 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 12 expressed the receptors in recombinant systems, as it was previously shown in C. elegans that OSM-253 9/OCR-2 are both required to be correctly localised at the ciliated sensory neurons to exert a functional 254 response (Tobin et al., 2002; Ohnishi et al., 2020). We used Xenopus oocytes to test for direct receptor 255 activation. We verified the ability to reconstitute a ligand -activated response from the Homo sapiens 256 Hstrpv1 control using capsaicin , but Ovtrpvs-expressing oocytes did not respond to this vanilloid 257 compound, suggesting this might not be an ecologically relevant stimulus in cephalopods (Figure 8A). 258 However, a long-sustained response to 100 μM nicotinamide (NAM) was observed when Ovtrpv1 and 259 Ovtrpv2 were co-expressed but not when either receptor was expressed alone (Figure 8B), suggesting 260 that OvTRPV subunits require co -assembly of the two subunits to reconstitute a response in a 261 recombinant system. 262

263 O. vulgaris has two distinct TRPV channel representatives 264 In a previous in silico analysis of the conserved molecular determinants putatively involved in O. 265 vulgaris nociception, we highlighted candidates that could underpin octopus response to noxious 266 stimuli (Pieroni et al., 2026). 267 In this study, we focussed on the nociceptive molecular determinant TRPV that belongs to the vanilloid 268 subfamily of TRP channels (Himmel and Cox, 2020). 269 The TRPV subfamily, and particularly the TRPV1 member in vertebrates, is known to be gated by several 270 nociceptive cues including capsaicin, low pH and high temperature, thus classifying it as a polymodal 271 receptor (Caterina et al., 1997; Immke and Gavva, 2006; Dhaka et al., 2009). An important aspect of 272 TRPV1 and related proteins is that this molecular sensing and signalling is placed in discrete cells in the 273 peripheral part of the body so they can act as sensory receptors that initiate complex downstream 274 signalling that allows the embedded circuits to drive judicious behavioural responses (Caterina et al., 275 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 13 1997). Consistent with this, a prevalent expression of TRPV1 in peripheral sensory neurons that interact 276 with the environment has been extensively reported (Caterina et al., 1997; Nakagawa and Hiura, 2006; 277 Szigeti et al., 2012). In addition, a wider expression in the central brain implying distinct contributions 278 to nociception or other functions has also been found (Yuan and Burrell, 2010; Higgins et al., 2013; 279 Marrone et al., 2017). 280 The sensory function and the anatomical distribution of TRPV channels also pertain to invertebrate and 281 simpler organisms (Colbert, Smith and Bargmann, 1997; Summers, Holec and Burrell, 2014) . This 282 prompted us to investigate the TRPV subfamily in O. vulgaris in which its complex nervous system and 283 articulated behaviours are speculated to allow elaboration of nociception into pain-like states (Birch et 284 al., 2021). Our work exploited the emerging access to octopus transcriptome and genome databases 285 and matured earlier designation of a single TRPV gene. Cluster analysis and phylogenetic investigation 286 refined the designation of OvTRPV1 and identified a second receptor, OvTRPV2. Our analysis 287 authenticated these genes and highlighted they are the only representatives of vanilloid TRP channels 288 found in O. vulgaris and represent two discrete receptor channel subunits ( Figure 3 A). Two TRPV 289 representatives were also found in other cephalopods such as O. bimaculoides and O. sinensis and 290 also in other species such as D. melanogaster or A. californica (Figure 3B). This indicates potential 291 structural conservation across distinct invertebrate genera which may well reflect strong functional 292 conservation of sensory specialisation. 293 An interesting exception to the generalisation above is represented by C. elegans, which shows a total 294 of 5 TRPV representatives, suggesting a specific one-to-many orthologues expansion of these receptors 295 compared to other invertebrate phyla (Kuzniar et al., 2008). We can identify an invertebrate TRPV group 296 referred to as “OCR-like” TRPV branch. This grouping includes all the C. elegans OCR receptors (OCR 297 1-4) to which OvTRPV1 and D. melanogaster Nanchung (NAN) are more closely related (55.0% 298 similarity). A second group, referred to as “OSM-9 like” branch, includes C. elegans OSM-9 to which 299 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 14 OvTRPV2 and D. melanogaster Inactive (IAV) are related (54.7% similarity, Figure 3 B). The analysis 300 shows the i nvertebrate TRPVs share a common ancestor with the vertebrate TRPVs . This analysis 301 supports a scenario in which this ancestor receptor underwent a duplication that gave rise to two 302 distinct mammalian branches. One led to the TRPV5 and 6 ion channels which , despite retaining 303 sensitivity to pH, do not have any sensory related functions but act to regulate calcium homeostasis in 304 the kidney and small intestine (Yeh et al., 2003; Nijenhuis, Hoenderop and Bindels, 2005; Lambers et 305 al., 2007) . This subgroup is separated from the TRPV1 -4 channels branch, in which further internal 306 duplications likely led to a specialisation of these receptors in compounds and stimuli detection (Peng, 307 Shi and Kadowaki, 2015; Morini et al., 2022). 308 Tissue expression supports the hypothesis that Ovtrpvs are sensory 309 molecular determinants in octopus 310 As indicated, the tissue expression of the mammalian TRPV1 justifies key function in sensory signalling 311 (Caterina et al. , 1997) . In the case of the Ovtrpvs, gross analysis of their mRNA expression across 312 different tissues supports an important role in sensory signalling ( Figure 4). Both subunits appear to 313 show relatively low expression but robust and reproducible amplification from tissues associated with 314 sensory signalling in cephalopods. This is reinforced when we compare the expression in the sensory 315 tissues (arm, tip of the arm and sucker) to a range of tissues encapsulating immune-related (e.g., white 316 bodies, haemocytes) and feeding and digestion -related (e.g., stomach, intestine) tissues ( Figure 4B). 317 Although tissue distribution cannot define specificity of function per se, we identified that Ovtrpv1 and 318 Ovtrpv2 have overlapping expression with the selective chemotactile receptor orthologue Ovcrt (Figure 319 4B). This gene is reported to be enriched in the sensory epithelium of the sucker of O. bimaculoides with 320 reported related sensory functions (van Giesen et al. , 2020) . This enrichment is interesting as 321 cephalopods, and in particular octopus es, use their arms to sample the environment and classify 322 stimuli including those that evoke aversion. These structures which express sensory receptors are 323 primary organisers of reflexive and complex responses (Rossi and Graziadei, 1956; Zullo, Fossati and 324 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 15 Benfenati, 2011; Hague, Florini and Andrews, 2013; Gutnick et al., 2020). The prevalence of Ovtrpv1 and 325 Ovtrpv2 expression in the distal part of the arm, such as the tip and more specifically the suckers, 326 encourages the hypothesis these receptors modulate sensory detection in O. vulgaris. The comparison 327 with Ovcrt expression suggests these receptors are low abundant, and this seems in line, at least for 328 Ovtrpv1, with the RNA sequencing analysis of its fragment s performed in the previously assembled O. 329 vulgaris transcriptome (Petrosino, 2015; Petrosino et al., 2022). 330 In a similar fashion to vertebrate TRPVs we showed that, Ovtrpvs expressed in the central brain mass of 331 O. vulgaris (Figure 4 B), support s a potential role in central control of sensory and nocicepti ve 332 modulation. 333 We additionally detected expression of both Ovtrpvs in the intestine and, exclusively for Ovtrpv1, in the 334 stomach, digestive gland, white bodies, gill and kidney ( Figure 4 B). This broader expression in non -335 neuronal tissues for Ovtrpv1 is interesting when considering it belongs to the “OCR-like” branch. In C. 336 elegans, OCRs show a more heterogenous expression including the rectal gland cells and intestine 337 which play an important role in C. elegans digestive system (Tobin et al., 2002; Packer et al., 2019). 338 Additionally, ocr-3 and ocr-4 seem to be co-expressed in neuroendocrine cells with ocr-2, to regulate 339 egg-laying (Jose et al., 2007). 340 Functional characterisation through model hopping in C. elegans suggests 341 Ovtrpvs are polymodal nociceptors 342 The prevalent expression in sensory tissues of both receptors, encouraged us to functionally test them 343 for their role in nociception. 344 As we have already utilised C. elegans as a suitable model to perform functional characterisation of 345 octopus molecular nociceptor candidates (Pieroni et al. , 2026) , we resorted to a model hopping 346 approach by expressing octopus receptors in the mutant strains for the orthologue genes ocr-2 and 347 osm-9. The phylogenetic distinction between the two receptors into “OCR-like” receptors for Ovtrpv1 348 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 16 and “OSM-9-like” for Ovtrpv2, justified our approach of heterologously expressing Ovtrpv1 under the 349 ocr-2 promoter in an ocr-2 (ak47) mutant background and Ovtrpv2 under the osm-9 promoter in an osm-350 9 (ky10) mutant background. 351 The null mutants osm-9 (ky10) and ocr-2 (ak47) show defects in different sensory modalities, including 352 chemical and mechanical aversion, effectively highlighting OSM-9 and OCR-2 as polymodal receptors 353 (Colbert, Smith and Bargmann, 1997; Tobin et al., 2002; Liedtke et al. , 2003; Thies et al. , 2016). We 354 therefore subjected our transgenic lines expressing Ovtrpv1 and Ovtrpv2 in the C. elegans mutants for 355 the orthologous genes to acetic acid (pH 3), mechanical insults (i.e. nose touch) and volatile repellents 356 such as 1-octanol. 357 Ovtrpv1 and Ovtrpv2 successfully rescued low pH ( Figure 5 ) and mechanical avoidance ( Figure 6 ) 358 suggesting these receptors act as polymodal nociceptors in O. vulgaris. These two cues induce aversive 359 responses in cephalopods. Low pH has been tested in ex vivo octopus arm preparations, showing a 360 quick withdrawal when in contact with an acidic solution, and in vivo experiments, in which injection of 361 acetic acid triggered grooming and protective behaviours (Hague, Florini and Andrews, 2013; Crook, 362 2021). Mechanical cues are also an important trigger for cephalopod sensory responses as von Frey 363 filaments induce nociceptive responses and also trigger post -injury sensitisation in squids (Crook, 364 Hanlon and Walters, 2013; Alupay, Hadjisolomou and Crook, 2014; Crook et al., 2014). 365 However, the molecular determinants of such responses have not been investigated in O. vulgaris and 366 the TRPV channels we describe here are potential candidates. Whether OvTRPVs act as channels 367 directly gated by pH or as indirect modulators of pH behavioural responses is not known and the 368 proposed key residues for proton gating in mammalian TRPV1 (Jordt and Julius, 2002; Ryu et al., 2007; 369 Aneiros et al., 2011), are not conserved in OvTRPVs or in CeleTRPVs. Therefore, either OvTRPV channels 370 have a distinct mechanism for pH detection, or perhaps they possess a downstream modulatory role 371 for this cue. As for mechanosensation, a key molecular determinant has been found in O. bimaculoides 372 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 17 as part of the TRPN family, which also includes the mechanosensory D. melanogaster NompC (Yan et 373 al., 2013; van Giesen et al., 2020). However, it is not uncommon in organisms to have multiple receptors 374 responding to the same cue. This allows resolution of the stimulus detection and its texture, that leads 375 to the distinction between a soft touch and a nociceptive harmful input. C. elegans represents a good 376 example of this, as osm-9/ocr-2 are not uniquely required for aversive nose touch (Kindt et al., 2007; Li 377 et al., 2011), and different molecular interactions give rise to distinct sensitivities (e.g., mec-10/mec-4 378 for gentle touch and mec-10/degt-1 for harsh touch, Chatzigeorgiou et al., 2010; Li et al., 2011). 379 Our C. elegans model hopping suggests that homologue expression gives the most potent rescue with 380 ocr-2 promoter driving Ovtrpv1 and osm-9 promoter driving Ovtrpv2 expressions in nematode loss of 381 function mutants of the orthologue trpv genes. However, these functional experiments preclude insight 382 into their transduction localisation (upstream or downstream) or the required stoichiometry that makes 383 the receptor functional in sensory cellular signalling. The evolutionary relationship between Ovtrpv1 384 and Ovtrpv2, consistent with other invertebrates, suggests important divergence in function (Figure 3B). 385 Furthermore, previous experiments suggested co -assembly of osm-9 and ocr-2 to exert their role 386 (Ohnishi et al., 2020; Griffin et al., 2025). Finally, the shared overlapping tissue expression of Ovtrpv1 387 and Ovtrpv2 suggests they might interact together to convey sensory detection . Altogether, these 388 pieces of evidence, encouraged our approach of using recombinant assays. 389 Heterologous co-expression of Ovtrpv1 and Ovtrpv2 in Xenopus oocytes produced a sustained, slow 390 response to 100 μM NAM, supporting the hypothesis of co-assembly between Ovtrpv1 and Ovtrpv2 into 391 an active heteromeric channel that is directly gated by aversive substances (Figure 8). This finding is in 392 line with previously characterised invertebrate TRPV receptors. In both C. elegans and D. melanogaster, 393 co-expression of the pair osm-9/ocr-2 (but more robustly of osm-9/ocr-4) and Nan/Iav respectively, 394 produced dose-dependent responses to NAM (Upadhyay et al., 2016; Griffin et al., 2025). Behavioural 395 and cell -studies revealed this substance is a noxious bitter cue in invertebrates, eliciting avoidance 396 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 18 responses and leading to a cell-death inducing accumulation of NAM through desensitisation of TRPV 397 channels (Upadhyay et al. , 2016; Ishikawa et al. , 2023) . Our data are therefore consistent with a 398 conserved TRPV role in invertebrate sensory detection and cell metabolic regulation that are worth 399 pursuing in the future. 400

401 Altogether, this work identified two functional TRPV channels in O. vulgaris and contributed to providing 402 precise annotation and experimental validation of two important molecular determinants of 403 nociception in cephalopods . Heterologous functional characterisation of these octopus TRPV 404 channels in C. elegans and in the Xenopus oocyte recombinant system, suggests they are polymodal 405 and may have a physiological role in the octopus response to aversive chemical and mechanical cues. 406 The sensory role of Ovtrpvs is supported by our endpoint PCR analysis of a wide set of tissues, 407 demonstrating expression in sensory and nervous tissues. However, future analysis should focus on 408 qPCR and in situ hybridisation to confirm the potential selective expression in the tip of the arm and 409 sucker relative to other non-sensory tissues. 410 Finally, the established phylogenetic relationship, locating OvTRPV1 and OvTRPV2 in distinct 411 evolutionary branches of the vanilloid TRP channel subfamily, as well as the recombinant experiments 412 showing the requirement for co-assembly of OvTRPV1 and OvTRPV2 to generate a functional channel, 413 provides a route to experimentally investigate the pathways and the modalities in which Ovtrpvs 414 operate. At the same time it fosters the development of experimental platforms that might identify 415 important environmental sensory cues. Recent advances have in fact shown octopus evolved species-416 specific receptors but that they also exploit conserved ones (van Giesen et al., 2020), showing the 417 importance for cephalopods to recognise and classify stimuli in the environment through an elaborated 418 sensory system able to detect, integrate and select against nociceptive cues. 419 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 19

420 In silico analysis 421 BLAST search criteria to identify O. vulgaris transcripts 422 The protein sequence of Homo sapiens TRPV1 (Hstrpv1) that encodes the human capsaicin receptor 423 was retrieved from UniProtKB/Swiss -Prot (release 2021_04) and blasted against the previously 424 published O. vulgaris de novo assembly transcriptome (Petrosino, 2015; Petrosino et al., 2022) using 425 the TBLASTN algorithm (BLAST+ v2.10.0+) with a stand ard threshold of 10e -5 used as a cut -off. The 426

sequences were also used in a BLASTp search against the predicted proteome obtained from 427 the most recent sequenced O. vulgaris genome (Destanović et al., 2023), using the same parameters 428 described above. The hits retrieved from both searches, were blasted and compared against Octopus 429 bimaculoides (ASM119413v2), Octopus sinensis (GCA_006345805.1) and Aplysia californica 430 (PRJNA13635) assemblies to facilitate assessment of completeness. 431 In the case of the O. vulgaris chemotactile receptor (CRTs) orthologues, O. bimaculoides published 432 sequences were retrieved from NCBI and used for a BLASTp search against O. vulgaris genome using 433 the parameters mentioned above (Supplementary Table S1) . All the resulting candidates were then 434 aligned against the H. sapiens acetylcholine receptor α7 subunit (NP_000737.1) prior selection, to 435 check for the lack of the classical vicinal cysteines involved in the neurotransmitter binding, which 436 signatures cephalopod CRTs (van Giesen et al., 2020). 437 Exon-intron boundaries detection 438 The reconstructed Ovtrpv transcript sequences were blasted against the assembled O. vulgaris 439 genome (Destanović et al. , 2023) to localise their chromosomal location. The closest match was 440 downloaded and converted into a BLAST custom database through the ncbi/suite tool container in 441 Docker (Merkel, 2014) . Alignment between the Ovtrpv1 and Ovtrpv2 cDNA from O. vulgaris or other 442 species (i.e. O. sinensis ) and the genomic sequence was performed using ncbi/magicblast tool 443 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 20 container (Boratyn et al., 2019). The results were sorted using biocontainers/samtools (Danecek et al., 444 2021) and the data were processed and visualised using Tablet software (The James Hutton Institute, 445 v1.21.02.08, Milne et al., 2013). 446 Secondary and 3D structure prediction and modelling 447 The assembled contig cDNA was translated to its predicted amino acid sequence through Expasy 448 translate (Gasteiger et al. , 2003) . Each octopus predicted protein of interest was analysed using 449 DeepTMHMM - 1.0 to obtain a prediction of the secondary structure (Hallgren et al. , 2022) . A 3D 450 reconstruction of the proteins’ single subunit was obtained using AlphaFold 3 (Abramson et al., 2024) 451 and their representation within the cell membrane was modelled through PPM3 Server (Lomize, Todd 452 and Pogozheva, 2022). Analysis of the conserved protein functional domains was performed using NCBI 453 conserved domain (Lu et al., 2020). 454 Alignment with other species orthologue proteins was carried out using Clustal Omega (Madeira et al., 455 2022) and identity and similarities among the sequences were analysed with EMBOSS Water Pairwise 456 Sequence Alignment (Madeira et al., 2024) using a BLOSUM62 matrix. 457 Cluster analysis and phylogenetic investigation of OvTRPVs 458

protein sequences for TRP channels from H. sapiens, C. elegans and Drosophila 459 melanogaster as well as other cationic ion channel sequences such as voltage -gated sodium, 460 potassium and calcium ion channels were used to perform a BLAST search (e -value 1e -10 and 40 461 maximum target sequences) against the complete proteome of 13 representative species from 462 Mammalia, Cephalopoda, Gastropoda, Bivalvia, Insecta, Crustacea and Nematoda. This proteome 463 selection was based on the best Benchmarking Universal Single -Copy Orthologs (BUSCO) value 464 (Supplementary Table S2). All the results from each species were sorted and processed through CD-hit 465 using a 0.95 similarity threshold to remove shorter isoforms or redundant sequences (Li and Godzik, 466 2006; Fu et al., 2012). The resulting unique sequences from all the species (1102 sequences) were then 467 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 21 used as a query to perform a CLuster ANalysis of Sequences (CLANS) (Zimmermann et al., 2018) with 468 the BLAST HSPs e -value of 1e -12 and BLOSUM62 scoring matrix (Supplementary File S1). Visualisation 469 of the sequences relationship and cluster organisation was performed with the CLANS toolkit with a set 470 p-value threshold of 1e -60 (Zimmermann et al., 2018). All the sequences of the cluster which included 471 the target Ovtrpv1 and Ovtrpv2 sequences (50 sequences, Supplementary File S2) were selected and 472 used to generate a phylogenetic tree. Alignment of the sequences was performed using MAFFT v7.526, 473 E-INS-i method (Katoh and Standley, 2013) . The results were manually curated to exclude poorly 474 aligned sequences (2) and then trimmed with TrimAl (Supplementary File S3) using gappy-out mode and 475 default parameters (Capella-Gutiérrez, Silla -Martínez and Gabaldón, 2009) . The best -fit model of 476 evolution was selected with ModelFinder (LG+I+G4 chosen according to the Bayesian Information 477 Criterion) in IQ-TREE3 v3.0.1 (Wong et al., 2025). Tree branches were obtained using aLRT-SH with 1,000 478 replicates and ultrafast bootstrap method. The final phylogeny was visualised using FigTree v1.4.4 479 (http://tree.bio.ed.ac.uk/software/figtree/). The tree was rooted using the vertebrate TRPV channel 480 subfamilies. 481 Animal samples 482 O. vulgaris samples collection 483 Young adults of O. vulgaris were caught by local artisanal fishermen in the Bay of Naples, Italy and 484 humanely killed for tissue collection. The following tissues were dissected and stored in 50 μL RNA later 485 for subsequent analysis : supra -oesophageal mass (SEM), sub -oesophageal mass (SUB), optic lobe 486 (OL), stellate ganglion (StG), gastric ganglion (GG), arm (muscle + axial nerve cord, at 50% of its length), 487 tip of the arm (TIP), sucker (Su), intestine (Int), stomach (Stom), white body (WB), anterior salivary gland 488 (ASG), posterior salivary gland (PSG), digestive gland (DG), haemocytes (Hc), kidney (Kid), branchial 489 heart (BrH), skin, mantle (Man) and gill (Supplementary Table S3). 490 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 22 C. elegans strains and husbandry 491 The following nematode strains were utilised in this study: CX10 osm-9 (ky10); CX4544 ocr-2 (ak47); the 492 Bristol N2 was used as the wild -type (WT) strain . The strain genotype of the osm-9 (ky10) and ocr-2 493 (ak47) mutants was confirmed by designing a pair of primers flanking the region of mutations 494 (Supplementary Table S4) and sequencing (Eurofins Genomics). 495 C. elegans were cultured and maintained as described in Brenner (1974). Three days prior to any assay, 496 gravid adult worms were put on culture plates to lay eggs for 4h and then removed to produce a 497 synchronised population to be tested at the L4+1-day old (young adult) stage. In the case of transgenic 498 lines, L4 worms expressing GFP derived from co-marker plasmid were selected from the synchronised 499 population and incubated overnight at 20 °C 24h prior the experiment. 500 Molecular biology 501 RNA extraction and cDNA synthesis from O. vulgaris tissues 502 Small resections (7 -40 mg) of the indicated frozen tissues were collected in 2 mL Eppendorf tubes 503 containing 500 µl TRIzol® (Invitrogen ™), snap frozen and homogenized using a Handheld Homogeniser 504 SHM1 (Cole -Palmer). After 5 min at room temperature, 200 µl of chloroform (Sigma -Aldrich) were 505 added, mixed, and incubated on ice for 15 min. Samples were centrifuged (12,000 x g, 4° C) and the 506 total RNA upper aqueous layer purified using PureLink® RNA Mini Kit (Invitrogen TM). DNase I treatment 507 (InvitrogenTM) was performed to remove potential contaminating genomic DNA. Quality (260/280 ≈ 2.0) 508 and quantity of extracted RNA were assessed through UV -visible absorption measurements 509 (NanoDrop™ 2000/2000c Spectrophotometers). For each sample, 1 µg of RNA was used for reverse 510 transcription following the manufacturer’s protocol (SuperScript ™ III Reverse Transcriptase, 511 InvitrogenTM). The cDNA samples were stored at -20°C until further use. As previously reported, the 512 mollusc ‘hidden break’ does not allow a sufficient gel resolution to assess the RNA quality due to the 513 co-migration of the 18S and 28S rRNA fragments (Natsidis et al., 2019; Adema, 2021). Therefore, only 514 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 23 the tissues from which we successfully amplified a control gene , cullin 1 (cul1) , were selected for 515 further use in PCR amplification of the genes of interest (Supplementary Table S3). 516 Primer design and end point PCR 517 Primer sequences of the genes of interests were designed using ApE-A plasmid Editor© v3.1.3 software 518 and purchased from Eurofins Genomics (Supplementary Table S4). Once diluted to a working 519 concentration of 25 pmol/µL, the primers were used to amplify the sequence in the tissues described 520 above using 1 µL of cDNA as template in a PCR Phusion ™ High-Fidelity DNA Polymerase (Thermo 521 Scientific™) following manufacturer’s instructions. 522 Promoter and genes synthesis for cloning 523 The promoter region of the osm-9 gene (Posm-9, approximately 1.6 kb – Colbert, Smith and Bargmann, 524 1997), the C. elegans osm-9 (Celeosm-9) and the Hstrpv1 cDNA sequences were synthesised using the 525 Integrated DNA Technologies Gene synthesis service (Integrated DNA Technologies, USA ). The 526 promoter region of the ocr-2 gene (Pocr-2, approximately 2.5 kB - Sokolchik et al., 2005) and the O. 527 vulgaris trpv2 (Ovtrpv2) were synthesised using the Genscript gene synthesis service and subcloned 528 into the pcDNA3.1 vector (Genscript Biotech, UK). The pcDNA3.0 plasmid containing the C. elegans 529 ocr-2 cDNA sequence (Celeocr-2) was a kind gift from Prof. Cornelia Bargmann (Rockefeller University, 530 NY, USA). All the plasmid sequences received, synthesised and cloned were verified for their 531 authenticity using the Oxford Nanopore Whole Plasmid Sequencing (WPS) service from Eurofins 532 Genomics. A missense mutation (AAC to AGC) in ocr-2 cDNA leading to N695S in the protein sequence 533 was identified in the original pcDNA3-ocr-2 plasmid. However, the cDNA still showed a successful 534 behavioural rescue when expressed in ocr-2 (ak47) mutant worms suggesting it is functionally silent, 535 consistently with previous investigations (Tobin et al., 2002; Ohnishi et al., 2020). 536 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 24 Cloning for expression in C. elegans 537 The Pocr-2/Posm-9 sequences were cloned (using HindIII/XhoI) into the pWormgate2 vector (Johnson, 538 Behm and Trowell, 2005) to produce a pDEST -Pocr-2 or pDEST-Posm-9 vector for suitable expression 539 in C. elegans. The full -length transcript sequences of Ovtrpv1, Ovtrpv2 and Celeocr-2 were amplified 540 (Supplementary Table S4) using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific ™) and the 541 A’ overhangs were added using GoTaq® G2 DNA Polymerase (Promega; 30 min at 72 °C) prior to ligation 542 into pCR ™8/GW/TOPO™ TA (Invitrogen ™). This sequence was then transferred into the pWormgate2, 543 downstream of the corresponding promoter via attR/attL recombination using a Gateway™ LR Clonase™ 544 II Enzyme mix (Invitrogen™). This produced the final constructs: pDEST-Posm-9::Ovtrpv2, pDEST-Pocr-545 2::Ovtrpv1, pDEST -Pocr-2::Celeocr-2. The Celeosm-9 cDNA sequence was cloned via KpnI/SacI 546 downstream of Posm-9 to produce pDEST-Posm-9::Celeosm-9 final construct (Supplementary File S4). 547 The plasmids were transformed into chemically competent bacterial cells (Thermo Scientific™) and the 548 amplified plasmids purified from overnight cultures. These were then purified using the Monarch® Spin 549 Plasmid Miniprep Kit (New England Biolabs®, UK) and authenticated by sequencing (Eurofins 550 Genomics). 551 Cloning for expression in Xenopus laevis oocytes 552 The sequences of Ovtrpv1, Ovtrpv2, were cloned into the multiple cloning site of the oocyte expression 553 vector pTB207 via HindIII/NotI. The sequence of Hstrpv1 was cloned via PCR amplification to introduce 554 the compatible cutting sites EcoRI-NotI (Supplementary Table S4). The pTB207 vector was a kind gift 555 from Jean -Louis Bessereau’s Lab (Claude Bernard University, Lyon, France). These plasmids were 556 transformed into chemically competent bacterial cells (Thermo Scientific™) and the amplified plasmids 557 purified from overnight cultures. These were then purified using the QIAGEN Plasmid Maxi Kit (QIAGEN) 558 and authenticated by sequencing (Eurofins Genomics). The final construct sequences are available in 559 Supplementary File S5. 560 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 25 C. elegans fosmids purification and validation 561 The fosmids B0212.3 osm-9 (clone ID: WRM066bG12) and C07G1 ocr-2 (cloneID: WRM0634bB10) were 562 purchased from K.K. DNAFORM (Yokohama, Japan). The amplified fosmids were purified using the 563 QIAGEN Plasmid Maxi Kit (QIAGEN). PCR verification was performed using primers designed to amplify 564 an internal region of osm-9/ocr-2 which was authenticated by sequencing (Eurofins Genomics). The 565 construct sequences are available in Supplementary File S6. 566 Microinjections 567 The purified pWormgate2 plasmid (Johnson, Behm and Trowell, 2005) containing the indicated genes 568 was microinjected (40 ng/ µl of plasmid,10 ng/ µl in the case of fosmid) and 30 ng/ µl of Pmyo-2::gfp or 569 Pmyo-3::gfp (Addgene). These co -injection markers highlight the pharyngeal or the body wall muscle 570 respectively. The microinjection was performed following the protocol described in Mello et al. (1991) 571 and Mello and Fire (1991) with aluminosilicate glass capillaries ( 1.0 mm OD, 0.78 mm ID, Harvard 572 Apparatus). Injections were conducted as previously described and injected worms were transferred 573 onto a new seeded plate (Calahorro et al., 2022). Progeny from the injected worm was selected based 574 on the fluorescence and transferred onto separate seeded plates to establish stable transgenic lines 575 for propagation (F2 generation). The following transgenic lines were obtained: ocr-2 (ak47) Pmyo-3::gfp; 576 ocr-2 ( WRM0634bB10) Pmyo -3::gfp; pDEST-Pocr-2::Ovtrpv1 Pmyo -3::gfp; pDEST -Pocr-2::Celeocr-2 577 Pmyo-3::gfp in ocr-2(ak47) mutant background; osm-9(ky10) Pmyo -3::gfp; osm -9 (WRM066bG12) 578 Pmyo-2::gfp; pDEST -Posm-9::Celeosm-9,Pmyo-3::gfp, pDEST -Posm-9::Ovtrpv2 in osm-9 (ky10) 579 mutant background. 580 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 26 Behavioural assays 581 Drop assay to test chemoaversion in C. elegans 582 The behavioural analyses were conducted in experimental arenas in which a microscope digital camera 583 (MU1403B, AmScopeTM) was mounted and worm behaviour in response to the indicated treatments was 584 recorded. The captured videos were analysed through manual scoring using AmScope Software 585 v.10.11.2024. The assays were time stamped by the digital camera and the speed and quality of the 586 responses assessed using the criteria listed below. The experimenter and observer were blind to the 587 genotype of the strains under investigation. 588 Chemical aversion in C. elegans was performed using an adapted version of the classical acute drop 589 assay (Hilliard, Bargmann and Bazzicalupo, 2002; Hilliard et al., 2004). Ten L4+1-day old worms from 590 indicated lines were transferred onto a 9 cm unseeded NGM plate and left undisturbed for at least 20 591 min. This allows the worms to transition to roaming behaviour encompassing periods of extended 592 forward runs (Gray, Hill and Bargmann, 2005) . A small drop of noxious cue was delivered in front of a 593 moving worm through a small glass capillary (1.0 mm OD, 0.78 mm ID, Harvard Apparatus) attached to 594 a syringe. A binary score was assigned with a positive response (1) or negative response (0) for each 595 compound tested within 5 s of exposure to the cue and was based on the average number of reversals 596 being equal or higher to the average number showed by N2s. The worms for each condition were tested 597 by exposing them to the drop only once. To trigger low pH response, acetic acid (CH 3COOH; Fisher 598 chemical) was dissolved in M9 buffer at a final pH of 3 (M9, pH 3). M9 buffer (pH 7) was checked not to 599 cause any response to N2s when administered alone. 600 Nose touch assay 601 Briefly, an eyebrow was placed perpendicular to the front of a moving L4+1 -day old worm until the 602 animal hit the hair (Kaplan and Horvitz, 1993). Worms were given a binary score indicating nose touch 603 sensitivity (1) or insensitivity (0). The sensitive worms halted or/and reversed following the collision. The 604 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 27 insensitive worms kept moving forward and tried to climb or cross the hair. The experiment was 605 performed in a single set of 10 consecutive touches on 5 worms per strain. The final ratio of 606 sensitive/insensitive responses per worm was calculated and compared between N2, mutant and 607 transgenic strains. 608 Volatile aversive response 609 Following the same husbandry described for the drop assay, 10 worms expressing long forward runs 610 were individually exposed to 30% 1 -octanol (Sigma-Aldrich) solution only once. A thin platinum wire 611 (Ø 0.1 mm, Agar Scientific) was dipped into the solution and then waived in front of a moving worm until 612 the animal stopped and initiated a backward movement. The average latency to start a reversal (s) was 613 recorded with a cut-off of 15 s. 614 Data analysis and statistics 615 In the behavioural assays the transgenic lines that showed a performance that reached two standard 616 deviations from the mean of the mutant line were considered rescued and included in the analysis. 617 Data were analysed using either one -way or two -way parametric analysis of variance (ANOVA). Post -618 hoc comparisons were performed using the Dunnett’s multiple comparisons test. A level of probability 619 set at p<0.05 was used as statistically significant. Statistics were performed with GraphPad Prism 620 version 10 for Windows (GraphPad Software, Boston, Massachusetts, USA). 621 TRPVs ligand activation 622 Electrophysiology of Xenopus Oocytes 623 Defolliculated Xenopus laevis oocytes were obtained from EcoCyte Bioscience and maintained in 624 ND96 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 The plasmids of interests, pTB207-Hstrpv1, pTB207 -Ovtrpv1, pTB207 -Ovtrpv2, were linearised 626 using PacI, followed by DNA purification (Zymo) and elution in RNAase free water. cRNA was 627 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 28 synthesised using the T7 mMessage mMachine transcription kit (Thermo Fisher Scientific) according to 628 the manufacturer's protocol (with incubation for 6h at 37 °C). RNA was purified using the GeneJET RNA 629 purification kit (Thermo Fisher Scientific) and quantified using a NanoDrop spectrophotometer. 630 Oocytes were injected with 50 nl of 500 ng/µl RNA individually or co -expressed ( pTB207-631 Ovtrpv1/pTB207-Ovtrpv2) using the Roboinject system (Multi Channel Systems). Injected oocytes were 632 incubated at 18 °C in ND96 solution until the day of recording, 4 days post-injection. 633 Two-electrode voltage-clamp (TEVC) recordings were conducted using the Roboocyte2 System (Multi 634 Channel Systems). Measuring head electrode resistance was approximately 400-1200 kΩ, pulled on a 635 P-97 Micropipette Puller (Sutter Instrument). Electrodes contained AgCl wires backfilled with a 1 M KCl 636 and 1.5 M KAc mixture. Oocytes were clamped at -60 mV during continuous recording at 500 Hz. 637 Compounds applications (capsaicin, nicotinamide, ND96) lasted for 20 s for capsaicin followed by a 60 638 s wash with ND96, and 600 s for nicotinamide followed by a 200 s or 1200 s wash with ND96. Perfusion 639 speed was set to approximately 3 ml/min throughout. Uninjected oocytes were also tested and did not 640 respond to any of the compound tested. At least 7 oocytes were tested for each condition. Data were 641 analysed using the Roboocyte2+ software. 642 Compounds and stimuli tested 643 The following compounds were utilised in this study: capsaicin natural (Biosynth), acetic acid glacial 644 (FisherChemical), 1-octanol (Sigma-Aldrich), nicotinamide (NAM, Sigma-Aldrich). Stock solutions were 645 prepared either in ethanol or DMSO/distilled water, and the final working concentration was reached by 646 dissolving the solution in ND96 medium prior to performing the assay. Saline -injected oocytes did not 647 respond in any case tested. 648 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 29 Declarations 649 Ethics approval 650 O. vulgaris specimens were manipulated for the sole scope of tissue collection following the local 651 Animal Welfare Body authorisation (Ethical Clearance: ecACR -2302ts36). Animals were humanely 652 killed adopting the principles described in Annex IV of Directive 2010/63/EU and following 653 recommendations from Andrews et al. (2013), Fiorito et al. (2015), Butler-Struben et al. (2018) to ensure 654 responsible and ethical use of animal -derived materials and to adhere to the principles of 655 Replacement, Reduction, and Refinement (3Rs) . The t issues were collected by a FELASA certified 656 (function D) competent person. All the experiments have been carried out in compliance with the Ethics 657 and Research Governance Online II (ERGO II) policy (nr 79739) in place at the University of 658 Southampton. 659 Consent for publication 660 Not applicable 661 Availability of data and materials 662 The datasets (Supplementary Tables S1-S4 and Supplementary Files S1-S6) supporting the 663

of this article are available in the Zenodo repository at the following link: 664 10.5281/zenodo.18377710. 665 Competing interests 666 The authors declare no conflict of interest 667 Funding 668 EMP was supported by the HSA -Ceph 1/2019 grant to the Association for Cephalopod Research 669 ‘CephRes’ ETS, Napoli, Italy, and The Gerald Kerkut Charitable Trust, UK. 670 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 30 Authors' contributions 671 EMP: conceptualisation, data curation, in silico analysis, experimental investigation, methodology, 672 validation, visualization and writing–original draft 673 HB: conceptualisation, data curation, experimental investigation, methodology, visualization and 674 writing– review and editing, resources 675 VOC: conceptualisation, methodology, writing –original draft, writing –review and editing, funding 676 acquisition and supervision, resources 677 LHD: conceptualisation, methodology, writing –original draft, writing –review and editing, funding 678 acquisition and supervision, resources 679 LAYG: methodology, validation, visualization and writing– review and editing, resources 680 PI: writing–review and editing, resources 681 GF: conceptualisation, writing–review and editing, funding acquisition 682 JD: conceptualisation, methodology, writing –original draft, writing –review and editing, funding 683 acquisition and supervision, resources 684

685 Strains were provided by the Caenorhabditis Genetic Center (CGC), funded by NIH Office of Research 686 Infrastructure Programs (P40 OD010440). We thank Prof Cori Bargmann for kindly providing us with 687 the pcDNA3.1-ocr-2 construct. We thank Jean-Louis Bessereau’s Lab (Claude Bernard University, 688 Lyon, France) for kindly providing the pTB207 vector. We thank Dr Iris Hardege and Tom Reynoldson 689 for advice and support with electrophysiology. We thank Eng Marco Pieroni who kindly helped with the 690 setup and use of the described Docker containers. 691 692 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 31 693 Figure 1 Schematic of the reconstructed Ovtrpv1 translated protein following secondary and 3D modelling. Typical TRPV 694 channel secondary structure key signatures can be recognised , such as intracellular N - and C- terminals, a variable number 695 of ankyrin repeats in the N -terminal region, six transmembrane elements and a cytosolic P -loop between TM5-TM6 which is 696 responsible for the pore formation and selectivity filter in the assembled tetrameric structure. 697 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 32 698 Figure 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 second TRPV candidate gene, Ovtrpv2, is located on chromosome 3 and was initially mispredicted (red framed rectangle) , requiring experimental validation. Striped pattern rectangles 700 represent initially absent exons. 701 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 33 702 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 34 Figure 3 OvTRPV1 and OvTRPV2 are two distinct vanilloid transient receptor potential ion channels. A Visual 703 representation of the relationship among the TRP channel subfamilies. The OvTRPVs were identified as part of the TRPV 704 subfamily cluster (dashed red line). The two OvTRPV receptors (red circles with a black star) segregate into two distinct 705 subgroups of the cluster, thus showing some structural distinctions between each other. The same division is observed with 706 the C. elegans TRPVs (see legend in the top left corner). CACNA: voltage -gated calcium channels subunit alpha; KCN: 707 potassium voltage -gated channels; TPCN: Two pore calcium channel; TRPA: Transient Receptor Potential cation channel 708 subfamily A; TRPC: transient receptor potential cation channel subfamily C; TRPM: transient receptor potential cation channel 709 subfamily M; TRPML: mucolipin TRP cation channel; TRPP: polycystin transient receptor potential channel interacting; TRPV: 710 transient receptor potential cation channel subfamily V; Unc: uncharacterised. B Phylogenetic analysis of OvTRPVs. 711 Phylogenetic tree showing the relationships among TRPV sequences from 13 representative species. The two O. vulgaris TRPV 712 sequences (OvTRPV1 and OvTRPV2) are highlighted in bold red. C. elegans OCR-like sequences are shown in bold cyan and 713 C. elegans OSM-9 in bold purple, while human representative TRPV sequences are indicated in bold black. The tree reveals 714 two distinct TRPV lineages in protostomes, corresponding to “OCR-like” and “OSM-9-like” channels, with OvTRPV1 and 715 OvTRPV2 clustering within these respective groups. 716 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 35 717 Figure 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 and 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 the 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 OL: 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 ASG: 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 (OctVul6B024555T3), Ovcul1: O. vulgaris cullin 1 (c28856_g1_i2), Ovtrpv1: O. vulgaris trpv1 (PX926345), Ovtrpv2: O. vulgaris trpv2 (PX926346). 723 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 36 724 Figure 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 WT 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 condition. 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 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 compared to the reference 728 mutant 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 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 37 730 Figure 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 WT 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 tested 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 threshold 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 compared 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 **p<0.01, ***p<0.001, **** p<0.0001. 736 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 38 737 Figure 7 Investigation of volatile aversion in C. elegans ocr-2 (A) and osm-9 (B) mutant strains. 738 The 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 expressing 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 rescue 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 9 (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 performed with Dunnett’s multiple comparisons test *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001. 743 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 39 744 Figure 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 shows 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 no 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 responds 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 together show strong responses to nicotinamide 100µM. N ≥ 7 oocytes for each condition.749 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 40

750 Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., 751 Ballard, A.J., Bambrick, J., Bodenstein, S.W., Evans, D.A., Hung, C.-C., O’Neill, M., Reiman, D., 752 Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., Beattie, C., Bertolli, O., Bridgland, A., 753 Cherepanov, A., Congreve, M., Cowen-Rivers, A.I., Cowie, A., Figurnov, M., Fuchs, F.B., Gladman, H., 754 Jain, R., Khan, Y.A., Low, C.M.R., Perlin, K., Potapenko, A., Savy, P., Singh, S., Stecula, A., 755 Thillaisundaram, A., Tong, C., Yakneen, S., Zhong, E.D., Zielinski, M., Žídek, A., Bapst, V., Kohli, P., 756 Jaderberg, M., Hassabis, D. and Jumper, J.M. (2024) 'Accurate structure prediction of biomolecular 757 interactions with AlphaFold 3', Nature, 630(8016), pp. 493-500. Available at: 758 https://doi.org/10.1038/s41586-024-07487-w 759 Adema, C.M. (2021) 'Sticky problems: extraction of nucleic acids from molluscs', Philosophical 760 Transactions of the Royal Society B, 376(1825), p. 20200162. Available at: 761 https://doi.org/https://doi.org/10.1098/rstb.2020.0162 762 Alupay, J.S., Hadjisolomou, S.P. and Crook, R.J. (2014) 'Arm injury produces long-term behavioral and 763 neural hypersensitivity in octopus', Neurosci Lett, 558, pp. 137-142. Available at: 764 https://doi.org/https://doi.org/10.1016/j.neulet.2013.11.002 765 Amodio, P., Boeckle, M., Schnell, A.K., Ostojíc, L., Fiorito, G. and Clayton, N.S. (2019) 'Grow smart and 766 die young: why did cephalopods evolve intelligence?', Trends in ecology & evolution, 34(1), pp. 45-56. 767 Available at: https://doi.org/https://doi.org/10.1016/j.tree.2018.10.010 768 Andrews, P.L.R., Darmaillacq, A.-S., Dennison, N., Gleadall, I.G., Hawkins, P., Messenger, J.B., 769 Osorio, D., Smith, V.J. and Smith, J.A. (2013) 'The identification and management of pain, suffering and 770 distress in cephalopods, including anaesthesia, analgesia and humane killing', Journal of 771 Experimental Marine Biology and Ecology, 447, pp. 46-64. Available at: 772 https://doi.org/https://doi.org/10.1016/j.jembe.2013.02.010 773 Aneiros, E., Cao, L., Papakosta, M., Stevens, E.B., Phillips, S. and Grimm, C. (2011) 'The biophysical 774 and molecular basis of TRPV1 proton gating', The EMBO journal, 30(6), pp. 994-1002. Available at: 775 https://doi.org/https://doi.org/10.1038/emboj.2011.19 776 Basbaum, A.I., Bautista, D.M., Scherrer, G. and Julius, D. (2009) 'Cellular and molecular mechanisms 777 of pain', Cell, 139(2), pp. 267-284. Available at: https://doi.org/10.1016/j.cell.2009.09.028 778 Birch, J., Burn, C., Schnell, A., Browning, H. and Crump, A. (2021) 'Review of the Evidence of Sentience 779 in Cephalopod Molluscs and Decapod Crustaceans', LSE Consulting. Available at: 780 https://doi.org/https://www.lse.ac.uk/News/News-Assets/PDFs/2021/Sentience-in-Cephalopod-781 Molluscs-and-Decapod-Crustaceans-Final-Report-November-2021.pdf 782 Boratyn, G.M., Thierry-Mieg, J., Thierry-Mieg, D., Busby, B. and Madden, T.L. (2019) 'Magic-BLAST, an 783 accurate RNA-seq aligner for long and short reads', BMC bioinformatics, 20, pp. 1-19. Available at: 784 https://doi.org/https://doi.org/10.1186/s12859-019-2996-x 785 Brenner, S. (1974) 'The genetics of Caenorhabditis elegans', Genetics, 77(1), pp. 71-94. Available at: 786 https://doi.org/https://doi.org/10.1093/genetics/77.1.71 787 Butler-Struben, H.M., Brophy, S.M., Johnson, N.A. and Crook, R.J. (2018) 'In vivo recording of neural 788 and behavioral correlates of anesthesia induction, reversal, and euthanasia in cephalopod molluscs', 789 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 41 Frontiers in physiology, 9, p. 109. Available at: 790 https://doi.org/https://doi.org/10.3389/fphys.2018.00109 791 Calahorro, F., Chapman, M., Dudkiewicz, K., Holden-Dye, L. and O'Connor, V. (2022) 792 'PharmacoGenetic targeting of a C. elegans essential neuron provides an in vivo screening for novel 793 modulators of nematode ion channel function', Pesticide Biochemistry and Physiology, 186, p. 794 105152. Available at: https://doi.org/https://doi.org/10.1016/j.pestbp.2022.105152 795 Capella-Gutiérrez, S., Silla-Martínez, J.M. and Gabaldón, T. (2009) 'trimAl: a tool for automated 796 alignment trimming in large-scale phylogenetic analyses', Bioinformatics, 25(15), pp. 1972-1973. 797 Available at: https://doi.org/https://doi.org/10.1093/bioinformatics/btp348 798 Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D. and Julius, D. (1997) 'The 799 capsaicin receptor: a heat-activated ion channel in the pain pathway', Nature, 389(6653), pp. 816-824. 800 Available at: https://doi.org/https://doi.org/10.1038/39807 801 Chatzigeorgiou, M., Yoo, S., Watson, J.D., Lee, W.-H., Spencer, W.C., Kindt, K.S., Hwang, S.W., Miller 802 Iii, D.M., Treinin, M. and Driscoll, M. (2010) 'Specific roles for DEG/ENaC and TRP channels in touch 803 and thermosensation in C. elegans nociceptors', Nat Neurosci, 13(7), pp. 861-868. Available at: 804 https://doi.org/https://doi.org/10.1038/nn.2581 805 Colbert, H.A., Smith, T.L. and Bargmann, C.I. (1997) 'OSM-9, A novel protein with structural similarity 806 to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis 807 elegans', J Neurosci, 17(21), pp. 8259-8269. Available at: 808 https://doi.org/https://doi.org/10.1523/JNEUROSCI.17-21-08259.1997 809 Colton, C.K. (2006) 'TRPV3 is a polymodal receptor'. Available at: 810 https://doi.org/http://rave.ohiolink.edu/etdc/view?acc_num=osu1164046830 811 Crook, R.J. (2021) 'Behavioral and neurophysiological evidence suggests affective pain experience in 812 octopus', iScience, 24(3), p. 102229. Available at: https://doi.org/10.1016/j.isci.2021.102229 813 Crook, R.J., Dickson, K., Hanlon, R.T. and Walters, E.T. (2014) 'Nociceptive sensitization reduces 814 predation risk', Curr Biol, 24(10), pp. 1121-1125. Available at: 815 https://doi.org/https://doi.org/10.1016/j.cub.2014.03.043 816 Crook, R.J., Hanlon, R.T. and Walters, E.T. (2013) 'Squid have nociceptors that display widespread 817 long-term sensitization and spontaneous activity after bodily injury', J Neurosci, 33(24), pp. 10021-818 10026. Available at: https://doi.org/https://doi.org/10.1523/JNEUROSCI.0646-13.2013 819 Danecek, P., Bonfield, J.K., Liddle, J., Marshall, J., Ohan, V., Pollard, M.O., Whitwham, A., Keane, T., 820 McCarthy, S.A., Davies, R.M. and Li, H. (2021) 'Twelve years of SAMtools and BCFtools', GigaScience, 821 10(2), p. giab008. Available at: https://doi.org/10.1093/gigascience/giab008 822 Destanović, D., Schultz, D.T., Styfhals, R., Cruz, F., Gómez-Garrido, J., Gut, M., Gut, I., Fiorito, G., 823 Simakov, O. and Alioto, T.S. (2023) 'A chromosome-level reference genome for the common octopus, 824 Octopus vulgaris (Cuvier, 1797)', G3-Genes Genom Genet, pp. 2023-05. Available at: 825 https://doi.org/https://doi.org/10.1093/g3journal/jkad220 826 Dhaka, A., Uzzell, V., Dubin, A.E., Mathur, J., Petrus, M., Bandell, M. and Patapoutian, A. (2009) 'TRPV1 827 is activated by both acidic and basic pH', J Neurosci, 29(1), pp. 153-158. Available at: 828 https://doi.org/https://doi.org/10.1523/JNEUROSCI.4901-08.2009 829 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 42 European Parliament and Union, C.o.t.E. (2010) Directive 2010/63/EU of the European Parliament and 830 of the Council of 22 September 2010 on the protection of animals used for scientific purposes. 831 Council of Europe. Available at: https://eur-lex.europa.eu/legal-832 content/EN/ALL/?uri=CELEX:32010L0063 2010). 833 Fiorito, G., Affuso, A., Basil, J., Cole, A., De Girolamo, P., D’angelo, L., Dickel, L., Gestal, C., Grasso, F. 834 and Kuba, M. (2015) 'Guidelines for the care and welfare of cephalopods in research–a consensus 835 based on an initiative by CephRes, FELASA and the Boyd Group', Laboratory animals, 49(2_suppl), pp. 836 1-90. Available at: https://doi.org/https://doi.org/10.1177/0023677215580006 837 Fu, L., Niu, B., Zhu, Z., Wu, S. and Li, W. (2012) 'CD-HIT: accelerated for clustering the next-generation 838 sequencing data', Bioinformatics, 28(23), pp. 3150-2. Available at: 839 https://doi.org/10.1093/bioinformatics/bts565 840 Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D. and Bairoch, A. (2003) 'ExPASy: The 841 proteomics server for in-depth protein knowledge and analysis', Nucleic Acids Res, 31(13), pp. 3784-842 8. Available at: https://doi.org/10.1093/nar/gkg563 843 Gong, Z., Son, W., Chung, Y.D., Kim, J., Shin, D.W., McClung, C.A., Lee, Y., Lee, H.W., Chang, D.-J. and 844 Kaang, B.-K. (2004) 'Two interdependent TRPV channel subunits, Inactive and Nanchung, mediate 845 hearing in Drosophila', Journal of Neuroscience, 24(41), pp. 9059-9066. Available at: 846 https://doi.org/https://doi.org/10.1523/JNEUROSCI.1645-04.2004 847 Gray, J.M., Hill, J.J. and Bargmann, C.I. (2005) 'A circuit for navigation in Caenorhabditis elegans', P 848 Natl Acad Sci USA, 102(9), pp. 3184-3191. Available at: 849 https://doi.org/https://doi.org/10.1073/pnas.0409009101 850 Griffin, S., Garbers, A., Banina, L., Perry, T. and Watson, G. (2025) 'Characterization of Caenorhabditis 851 elegans transient receptor potential vanilloid (TRPV) channels and their interactions with insecticidal 852 compounds', Pesticide Biochemistry and Physiology, 212, p. 106447. Available at: 853 https://doi.org/https://doi.org/10.1016/j.pestbp.2025.106447 854 Gutnick, T., Zullo, L., Hochner, B. and Kuba, M.J. (2020) 'Use of peripheral sensory information for 855 central nervous control of arm movement by Octopus vulgaris', Current Biology, 30(21), pp. 4322-856 4327. Available at: https://doi.org/10.1016/j.cub.2020.08.037 857 Hague, T., Florini, M. and Andrews, P.L.R. (2013) 'Preliminary in vitro functional evidence for reflex 858 responses to noxious stimuli in the arms of Octopus vulgaris', J Exp Mar Biol Ecol, 447, pp. 100-105. 859 Available at: https://doi.org/https://doi.org/10.1016/j.jembe.2013.02.016 860 Hallgren, J., Tsirigos, K.D., Pedersen, M.D., Almagro Armenteros, J.J., Marcatili, P., Nielsen, H., Krogh, 861 A. and Winther, O. (2022) 'DeepTMHMM predicts alpha and beta transmembrane proteins using deep 862 neural networks', BioRxiv, pp. 2022-04. Available at: 863 https://doi.org/https://doi.org/10.1101/2022.04.08.487609 864 Higgins, A., Yuan, S., Wang, Y. and Burrell, B.D. (2013) 'Differential Modulation of Nociceptive versus 865 Non-Nociceptive Synapses by Endocannabinoids', Molecular Pain, 9, pp. 1744-8069-9-26. Available 866 at: https://doi.org/10.1186/1744-8069-9-26 867 Hilliard, M.A., Bargmann, C.I. and Bazzicalupo, P. (2002) 'C. elegans responds to chemical repellents 868 by integrating sensory inputs from the head and the tail', Curr Biol, 12(9), pp. 730-4. Available at: 869 https://doi.org/10.1016/s0960-9822(02)00813-8 870 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 43 Hilliard, M.A., Bergamasco, C., Arbucci, S., Plasterk, R.H.A. and Bazzicalupo, P. (2004) 'Worms taste 871 bitter: ASH neurons, QUI‐1, GPA‐3 and ODR‐3 mediate quinine avoidance in Caenorhabditis elegans', 872 The EMBO journal, 23(5), pp. 1101-1111. Available at: https://doi.org/10.1038/sj.emboj.7600107 873 Himmel, N.J. and Cox, D.N. (2020) 'Transient receptor potential channels: current perspectives on 874 evolution, structure, function and nomenclature', Proceedings of the Royal Society B: Biological 875 Sciences, 287(1933), p. 20201309. Available at: https://doi.org/10.1098/rspb.2020.1309 876 Immke, D.C. and Gavva, N.R. (2006) 'The TRPV1 receptor and nociception', Semin Cell Dev Biol, 17, 877 pp. 582-591. Available at: https://doi.org/https://doi.org/10.1016/j.semcdb.2006.09.004 878 Ishikawa, S., Iida, C., Takezawa, Y., Chiba, K. and Kage-Nakadai, E. (2023) 'Avoidance behavior and 879 experience-dependent tolerance in response to bitter compounds in Caenorhabditis elegans', 880 Bioscience, Biotechnology, and Biochemistry, 87(3), pp. 314-319. Available at: 881 https://doi.org/10.1093/bbb/zbac200 882 Johnson, N.M., Behm, C.A. and Trowell, S.C. (2005) 'Heritable and inducible gene knockdown in C. 883 elegans using Wormgate and the ORFeome', Gene, 359, pp. 26-34. Available at: 884 https://doi.org/https://doi.org/10.1016/j.gene.2005.05.034 885 Jordt, S.-E. and Julius, D. (2002) 'Molecular basis for species-specific sensitivity to “hot” chili 886 peppers', Cell, 108(3), pp. 421-430. Available at: https://doi.org/10.1016/s0092-8674(02)00637-2 887 Jose, A.M., Bany, I.A., Chase, D.L. and Koelle, M.R. (2007) 'A specific subset of transient receptor 888 potential vanilloid-type channel subunits in Caenorhabditis elegans endocrine cells function as mixed 889 heteromers to promote neurotransmitter release', Genetics, 175(1), pp. 93-105. Available at: 890 https://doi.org/10.1534/genetics.106.065516 891 Julius, D. (2013) 'TRP channels and pain', Annu Rev Cell Dev Bi, 29, pp. 355-384. Available at: 892 https://doi.org/10.1146/annurev-cellbio-101011-155833 893 Julius, D. and Basbaum, A.I. (2001) 'Molecular mechanisms of nociception', Nature, 413(6852), pp. 894 203-210. Available at: https://doi.org/https://doi.org/10.1038/35093019 895 Kaplan, J.M. and Horvitz, H.R. (1993) 'A dual mechanosensory and chemosensory neuron in 896 Caenorhabditis elegans', Proceedings of the National Academy of Sciences, 90(6), pp. 2227-2231. 897 Available at: https://doi.org/https://doi.org/10.1073/pnas.90.6.2227 898 Katoh, K. and Standley, D.M. (2013) 'MAFFT multiple sequence alignment software version 7: 899 improvements in performance and usability', Molecular biology and evolution, 30(4), pp. 772-780. 900 Available at: https://doi.org/10.1093/molbev/mst010 901 Kindt, K.S., Viswanath, V., Macpherson, L., Quast, K., Hu, H., Patapoutian, A. and Schafer, W.R. (2007) 902 'Caenorhabditis elegans TRPA-1 functions in mechanosensation', Nat Neurosci, 10(5), pp. 568-577. 903 Available at: https://doi.org/https://doi.org/10.1038/nn1886 904 Kuzniar, A., van Ham, R.C.H.J., Pongor, S. and Leunissen, J.A.M. (2008) 'The quest for orthologs: 905 finding the corresponding gene across genomes', Trends in Genetics, 24(11), pp. 539-551. Available 906 at: https://doi.org/10.1016/j.tig.2008.08.009 907 Lambers, T.T., Oancea, E., de Groot, T., Topala, C.N., Hoenderop, J.G. and Bindels, R.J. (2007) 908 'Extracellular pH Dynamically Controls Cell Surface Delivery of Functional TRPV5 Channels', 909 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 44 Molecular and Cellular Biology, 27(4), pp. 1486-1494. Available at: 910 https://doi.org/10.1128/MCB.01468-06 911 Li, W. and Godzik, A. (2006) 'Cd-hit: a fast program for clustering and comparing large sets of protein 912 or nucleotide sequences', Bioinformatics, 22(13), pp. 1658-1659. Available at: 913 https://doi.org/10.1093/bioinformatics/btl158 914 Li, W., Kang, L., Piggott, B.J., Feng, Z. and Xu, X.Z. (2011) 'The neural circuits and sensory channels 915 mediating harsh touch sensation in Caenorhabditis elegans', Nat Commun, 2(1), pp. 1-9. Available at: 916 https://doi.org/https://doi.org/10.1038/ncomms1308 917 Liedtke, W., Tobin, D.M., Bargmann, C.I. and Friedman, J.M. (2003) 'Mammalian TRPV4 (VR-OAC) 918 directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans', 919 Proceedings of the National Academy of Sciences, 100(suppl_2), pp. 14531-14536. Available at: 920 https://doi.org/10.1073/pnas.2235619100 921 Lomize, A.L., Todd, S.C. and Pogozheva, I.D. (2022) 'Spatial arrangement of proteins in planar and 922 curved membranes by PPM 3.0', Protein Sci, 31(1), pp. 209-220. Available at: 923 https://doi.org/10.1002/pro.4219 924 Lu, S., Wang, J., Chitsaz, F., Derbyshire, M.K., Geer, R.C., Gonzales, N.R., Gwadz, M., Hurwitz, D.I., 925 Marchler, G.H., Song, J.S., Thanki, N., Yamashita, R.A., Yang, M., Zhang, D., Zheng, C., Lanczycki, C.J. 926 and Marchler-Bauer, A. (2020) 'CDD/SPARCLE: the conserved domain database in 2020', Nucleic 927 Acids Res, 48(D1), pp. D265-d268. Available at: https://doi.org/10.1093/nar/gkz991 928 Madeira, F., Madhusoodanan, N., Lee, J., Eusebi, A., Niewielska, A., Tivey, A.R.N., Lopez, R. and 929 Butcher, S. (2024) 'The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024', Nucleic 930 acids research, 52(W1), pp. W521-W525. Available at: https://doi.org/10.1093/nar/gkae241 931 Madeira, F., Pearce, M., Tivey, A.R.N., Basutkar, P., Lee, J., Edbali, O., Madhusoodanan, N., 932 Kolesnikov, A. and Lopez, R. (2022) 'Search and sequence analysis tools services from EMBL-EBI in 933 2022', Nucleic Acids Res, 50(W1), pp. W276-W279. Available at: https://doi.org/10.1093/nar/gkac240 934 Marrone, M.C., Morabito, A., Giustizieri, M., Chiurchiù, V., Leuti, A., Mattioli, M., Marinelli, S., Riganti, 935 L., Lombardi, M. and Murana, E. (2017) 'TRPV1 channels are critical brain inflammation detectors and 936 neuropathic pain biomarkers in mice', Nature communications, 8(1), p. 15292. Available at: 937 https://doi.org/https://doi.org/10.1038/ncomms15292 938 Mello, C.C., Kramer, J.M., Stinchcomb, D. and Ambros, V. (1991) 'Efficient gene transfer in C. elegans: 939 extrachromosomal maintenance and integration of transforming sequences', The EMBO journal, 940 10(12), pp. 3959-3970. Available at: https://doi.org/10.1002/j.1460-2075.1991.tb04966.x 941 Merkel, D. (2014) 'Docker: lightweight Linux containers for consistent development and deployment', 942 Linux J., 2014(239), p. Article 2. Available at: 943 https://doi.org/https://dl.acm.org/doi/10.5555/2600239.2600241 944 Milne, I., Stephen, G., Bayer, M., Cock, P.J.A., Pritchard, L., Cardle, L., Shaw, P.D. and Marshall, D. 945 (2013) 'Using Tablet for visual exploration of second-generation sequencing data', Briefings in 946 Bioinformatics, 14(2), pp. 193-202. Available at: https://doi.org/10.1093/bib/bbs012 947 Morini, M., Bergqvist, C.A., Asturiano, J.F., Larhammar, D. and Dufour, S. (2022) 'Dynamic evolution of 948 transient receptor potential vanilloid (TRPV) ion channel family with numerous gene duplications and 949 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 45 losses', Frontiers in Endocrinology, 13, p. 1013868. Available at: 950 https://doi.org/10.3389/fendo.2022.1013868 951 Nakagawa, H. and Hiura, A. (2006) 'Capsaicin, transient receptor potential (TRP) protein subfamilies 952 and the particular relationship between capsaicin receptors and small primary sensory neurons', 953 Anatomical science international, 81(3), pp. 135-155. 954 Natsidis, P., Schiffer, P.H., Salvador-Martínez, I. and Telford, M.J. (2019) 'Computational discovery of 955 hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity 956 Numbers', Scientific reports, 9(1), p. 19477. Available at: 957 https://doi.org/https://doi.org/10.1038/s41598-019-55573-1 958 Nijenhuis, T., Hoenderop, J.G.J. and Bindels, R.J.M. (2005) 'TRPV5 and TRPV6 in Ca2+ (re) absorption: 959 regulating Ca2+ entry at the gate', Pflügers Archiv, 451(1), pp. 181-192. Available at: 960 https://doi.org/10.1007/s00424-005-1430-6 961 Ohnishi, K., Saito, S., Miura, T., Ohta, A., Tominaga, M., Sokabe, T. and Kuhara, A. (2020) 'OSM-9 and 962 OCR-2 TRPV channels are accessorial warm receptors in Caenorhabditis elegans temperature 963 acclimatisation', Scientific Reports, 10(1), pp. 1-14. Available at: 964 https://doi.org/https://doi.org/10.1038/s41598-020-75302-3 965 Packer, J.S., Zhu, Q., Huynh, C., Sivaramakrishnan, P., Preston, E., Dueck, H., Stefanik, D., Tan, K., 966 Trapnell, C., Kim, J., Waterston, R.H. and Murray, J.I. (2019) 'A lineage-resolved molecular atlas of C. 967 elegans embryogenesis at single-cell resolution', Science, 365(6459), p. eaax1971. Available at: 968 https://doi.org/10.1126/science.aax1971 969 Pedersen, S.F., Owsianik, G. and Nilius, B. (2005) 'TRP channels: an overview', Cell calcium, 38(3-4), 970 pp. 233-252. Available at: https://doi.org/10.1016/j.ceca.2005.06.028 971 Peng, G., Shi, X. and Kadowaki, T. (2015) 'Evolution of TRP channels inferred by their classification in 972 diverse animal species', Mol Phylogenet Evol, 84, pp. 145-157. Available at: 973 https://doi.org/https://doi.org/10.1016/j.ympev.2014.06.016 974 Petrosino, G. (2015) The transcriptional landscape of the nervous system of Octopus vulgaris. 975 Doctoral Dissertation, Università degli Studi di Napoli Federico II Available at: 976 10.6092/UNINA/FEDOA/10212. 977 Petrosino, G., Ponte, G., Volpe, M., Zarrella, I., Ansaloni, F., Langella, C., Di Cristina, G., Finaurini, S., 978 Russo, M.T. and Basu, S. (2022) 'Identification of LINE retrotransposons and long non-coding RNAs 979 expressed in the octopus brain', BMC Biol, 20(1), p. 116. Available at: https://doi.org/10.1186/s12915-980 022-01303-5 981 Pieroni, E.M., O'Connor, V., Holden-Dye, L., Imperadore, P., Fiorito, G. and Dillon, J. (2026) 982 'Identification of molecular nociceptors in Octopus vulgaris through functional characterisation in 983 Caenorhabditis elegans', Biology Open, 15(1), p. bio062268. Available at: 984 https://doi.org/10.1242/bio.062268 985 Radresa, O., Zicha, S., Brown, W. and Laird, J.M.A. (2012) 'TRPV1 as a polymodal sensor: potential to 986 discover TRPV1 antagonists selective for specific activating modalities', TRP Channels in Drug 987 Discovery: Volume I, pp. 221-235. Available at: https://doi.org/https://doi.org/10.1007/978-1-62703-988 077-9_11 989 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 46 Ramsey, I.S., Delling, M. and Clapham, D.E. (2006) 'An introduction to TRP channels', Annu Rev 990 Physiol, 68, pp. 619-647. Available at: https://doi.org/10.1146/annurev.physiol.68.040204.100431 991 Rosasco, M.G. and Gordon, S.E. 2017. TRP Channels: What Do They Look Like? CRC Press/Taylor 992 & Francis, Boca Raton (FL). 993 Rossi, F. and Graziadei, P. (1956) 'Nouvelles Contributions à la connaissance du systême nerveux du 994 tentacule des céphalopodes. II', Cells Tissues Organs, 26(3), pp. 165-174. Available at: 995 https://doi.org/https://doi.org/10.1159/000141096 996 Ryu, S., Liu, B., Yao, J., Fu, Q. and Qin, F. (2007) 'Uncoupling proton activation of vanilloid receptor 997 TRPV1', Journal of Neuroscience, 27(47), pp. 12797-12807. Available at: 998 https://doi.org/https://doi.org/10.1523/JNEUROSCI.2324-07.2007 999 Sambongi, Y., Takeda, K., Wakabayashi, T., Ueda, I., Wada, Y. and Futai, M. (2000) 'Caenorhabditis 1000 elegans senses protons through amphid chemosensory neurons: proton signals elicit avoidance 1001 behavior', Neuroreport, 11(10), pp. 2229-2232. Available at: https://doi.org/10.1097/00001756-1002 200007140-00033 1003 Schnell, A.K., Amodio, P., Boeckle, M. and Clayton, N.S. (2021) 'How intelligent is a cephalopod? 1004 Lessons from comparative cognition', Biological Reviews, 96(1), pp. 162-178. Available at: 1005 https://doi.org/10.1111/brv.12651 1006 Shibasaki, K. (2024) 'TRPV4 activation by core body temperature has multimodal functions in the 1007 central nervous system', The Journal of Physiological Sciences, 74(1), p. 55. Available at: 1008 https://doi.org/10.1186/s12576-024-00948-x 1009 Shigeno, S., Andrews, P.L.R., Ponte, G. and Fiorito, G. (2018) 'Cephalopod brains: an overview of 1010 current knowledge to facilitate comparison with vertebrates', Frontiers in Physiology, 9, p. 952. 1011 Available at: https://doi.org/https://doi.org/10.3389/fphys.2018.00952 1012 Smith, E.S.J. and Lewin, G.R. (2009) 'Nociceptors: a phylogenetic view', J Comp Physiol A, 195(12), pp. 1013 1089-1106. Available at: https://doi.org/https://doi.org/10.1007/s00359-009-0482-z 1014 Smith, J.A., Andrews, P.L.R., Hawkins, P., Louhimies, S., Ponte, G. and Dickel, L. (2013) 'Cephalopod 1015 research and EU Directive 2010/63/EU: Requirements, impacts and ethical review', J Exp Mar Biol 1016 Ecol, 447, pp. 31-45. Available at: https://doi.org/https://doi.org/10.1016/j.jembe.2013.02.009 1017 Sokolchik, I., Tanabe, T., Baldi, P.F. and Sze, J.Y. (2005) 'Polymodal sensory function of the 1018 Caenorhabditis elegans OCR-2 channel arises from distinct intrinsic determinants within the protein 1019 and is selectively conserved in mammalian TRPV proteins', Journal of Neuroscience, 25(4), pp. 1015-1020 1023. Available at: https://doi.org/10.1523/JNEUROSCI.3107-04.2005 1021 Summers, T., Holec, S. and Burrell, B.D. (2014) 'Physiological and behavioral evidence of a capsaicin-1022 sensitive TRPV-like channel in the medicinal leech', Journal of Experimental Biology, 217(23), pp. 1023 4167-4173. Available at: https://doi.org/https://doi.org/10.1242/jeb.110049 1024 Szigeti, C., Sántha, P., Körtvély, E., Nyári, T., Horváth, V.J., Deák, É., Dux, M., Gulya, K. and Jancsó, G. 1025 (2012) 'Disparate changes in the expression of transient receptor potential vanilloid type 1 receptor 1026 mRNA and protein in dorsal root ganglion neurons following local capsaicin treatment of the sciatic 1027 nerve in the rat', Neuroscience, 201, pp. 320-330. 1028 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint 47 Thies, J., Neutzler, V., O'Leary, F. and Liu, H. (2016) 'Differential Effects of TRPA and TRPV Channels on 1029 Behaviors of Caenorhabditis elegans', Journal of Experimental Neuroscience, 10, pp. JEN-S32837. 1030 Available at: https://doi.org/10.4137/JEN.S32837 1031 Tobin, D.M., Madsen, D.M., Kahn-Kirby, A., Peckol, E.L., Moulder, G., Barstead, R., Maricq, A.V. and 1032 Bargmann, C.I. (2002) 'Combinatorial expression of TRPV channel proteins defines their sensory 1033 functions and subcellular localization in C. elegans neurons', Neuron, 35(2), pp. 307-318. Available at: 1034 https://doi.org/10.1016/s0896-6273(02)00757-2 1035 UK S.I. 1993/2103 (1993) The Animals (Scientific Procedures) Act(Amendment) Order 1993. Available 1036 at: https://www.legislation.gov.uk/uksi/1993/2103/made. 1037 Upadhyay, A., Pisupati, A., Jegla, T., Crook, M., Mickolajczyk, K.J., Shorey, M., Rohan, L.E., Billings, 1038 K.A., Rolls, M.M. and Hancock, W.O. (2016) 'Nicotinamide is an endogenous agonist for a C. elegans 1039 TRPV OSM-9 and OCR-4 channel', Nature communications, 7(1), p. 13135. Available at: 1040 https://doi.org/10.1038/ncomms13135 1041 van Giesen, L., Kilian, P.B., Allard, C.A.H. and Bellono, N.W. (2020) 'Molecular basis of chemotactile 1042 sensation in octopus', Cell, 183(3), pp. 594-604. Available at: 1043 https://doi.org/https://doi.org/10.1016/j.cell.2020.09.008 1044 Venkatachalam, K. and Montell, C. (2007) 'TRP channels', Annu. Rev. Biochem., 76, pp. 387-417. 1045 Available at: https://doi.org/10.1146/annurev.biochem.75.103004.142819 1046 Wong, T.K.F., Ly-Trong, N., Ren, H., Baños, H., Roger, A.J., Susko, E., Bielow, C., De Maio, N., 1047 Goldman, N. and Hahn, M.W. (2025) 'IQ-TREE 3: Phylogenomic Inference Software using Complex 1048 Evolutionary Models'. Available at: https://doi.org/https://doi.org/10.32942/x2p62n 1049 Yan, Z., Zhang, W., He, Y., Gorczyca, D., Xiang, Y., Cheng, L.E., Meltzer, S., Jan, L.Y. and Jan, Y.N. 1050 (2013) 'Drosophila NOMPC is a mechanotransduction channel subunit for gentle-touch sensation', 1051 Nature, 493(7431), pp. 221-225. Available at: https://doi.org/https://doi.org/10.1038/nature11685 1052 Yeh, B.-I., Sun, T.-J., Lee, J.Z., Chen, H.-H. and Huang, C.-L. (2003) 'Mechanism and molecular 1053 determinant for regulation of rabbit transient receptor potential type 5 (TRPV5) channel by 1054 extracellular pH', Journal of Biological Chemistry, 278(51), pp. 51044-51052. Available at: 1055 https://doi.org/10.1074/jbc.M306326200 1056 Yuan, S. and Burrell, B.D. (2010) 'Endocannabinoid-Dependent LTD in a Nociceptive Synapse Requires 1057 Activation of a Presynaptic TRPV-Like Receptor', Journal of Neurophysiology, 104(5), pp. 2766-2777. 1058 Available at: https://doi.org/10.1152/jn.00491.2010 1059 Zimmermann, L., Stephens, A., Nam, S.-Z., Rau, D., Kübler, J., Lozajic, M., Gabler, F., Söding, J., 1060 Lupas, A.N. and Alva, V. (2018) 'A Completely Reimplemented MPI Bioinformatics Toolkit with a New 1061 HHpred Server at its Core', Journal of Molecular Biology, 430(15), pp. 2237-2243. Available at: 1062 https://doi.org/https://doi.org/10.1016/j.jmb.2017.12.007 1063 Zullo, L., Fossati, S. and Benfenati, F. (2011) 'Transmission of sensory responses in the peripheral 1064 nervous system of the arm of Octopus vulgaris', Vie et milieu-life and enVironment, 61(4), pp. 197-201. 1065 Available at: 1066 https://doi.org/https://www.researchgate.net/publication/235932116_Transmission_of_sensory_resp1067 onses_in_the_peripheral_nervous_system_of_the_arm_of_Octopus_vulgaris 1068 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 28, 2026. ; https://doi.org/10.64898/2026.03.27.714695doi: bioRxiv preprint