{"paper_id":"6b1b9dc5-75ea-4e2e-91c2-bd47ddb78f74","body_text":"Developmental priming increases copper-tolerance in a model fish 1 \nspecies via epigenetic-and microbiome-mediated mechanisms 2 \nTamsyn M. Uren Webster *1, Lauren V. Laing 2, Jemima Onime 2, Hannah Littler 2,3, Rob  J. 3 \nMcFarling2,3, Josie Paris 2, Jennifer A. Fitzgerald 2,3, Anke Lange 2, Audrey Farbos 2, Karen Moore 2, 4 \nMatthew D. Hitchings4, Ronny van Aerle3,5, Nic R. Bury6, Eduarda M. Santos*2,3 5 \n1. Biosciences, Faculty of Science & Engineering, Swansea University, Swansea, UK 6 \n2. Biosciences, Faculty of Health and Life Sciences, University of Exeter, Exeter, UK 7 \n3. Collaborative Centre for Sustainable Aquaculture Futures (SAF), Exeter, UK 8 \n4. Institute of Life Sciences, Swansea University, Swansea, UK 9 \n5. Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth, UK 10 \n6. Institute for Life Sciences, University of Southampton, Southampton, UK 11 \n* email: T.M.UrenWebster@swansea.ac.uk; E.M.Santos@exeter.ac.uk  12 \nAbstract  13 \nPollution is a significant t hreat to aquatic ecosystems globally and , in order to survive, natural 14 \npopulations depend upon their ability to rapidly develop tolerance to chemical stressors.  We 15 \nexamined whether early-life priming enhances life-long copper-tolerance in a model fish species 16 \nvia developmental plasticity. Stickleback (Gasterosteus aculeatus) embryos were pre -exposed 17 \nto a low concentration of copper (10 µg/L) during early development , reared in clean water for 18 \nnine months alongside a control group, and then exposed to copper (0,10 and 20 µg/L ) for 96 h 19 \nas adults . Priming markedly reduced evidence of copper-toxicity in adult gills at the 20 \ntranscriptional level (including reduced cellular stress response  (CSR) and disruption of ion -21 \nhomeostasis) and increased inducibility of the metal-binding protein, metallothionein. In 22 \nparallel, we identified epigenetic and microbiome-mediated mechanisms likely contributing to 23 \nthis tolerance. Pre -exposure induced persistent DNA methylation  changes, consistent with 24 \npriming of CSR and ion-homeostasis pathways. We identified enhanced copper-tolerance in the 25 \ngill microbiota of primed fish that likely also contributed to host tolerance. These findings provide 26 \ncritical evidence for developmental plasticity induced by chemical stressors in animals, highlight 27 \nthe importance of integrated microbiome and epigenetic responses,  and enhance our 28 \nunderstanding of how natural populations cope with pollution in their environment. 29 \nKey words:  phenotypic plasticity, conditioning, toxic metal, RNA-Seq, sensitivity, early -life 30 \nreprogramming, stress priming  31 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nIntroduction 32 \nAquatic ecosystems are threatened by unprecedented anthropogenic challenges, including 33 \nchemical pollution. To better understand, predict, and ultimately to mitigate, the impacts of 34 \npollution, it is important to consider the relative sensitivity of different species and populations, 35 \nincluding their ability to develop tolerance following chemical exposure. Although there is some 36 \nevidence of local adaptation to pollution in microorganisms, plants and metazoa following long-37 \nterm exposure1–4, for many species survival will ultimately depend on an ability to rapidly acquire 38 \ntolerance to acute and fluctuating stressors5,6.  39 \nPhenotypic plasticity allows organisms to rapidly adjust to environmental challenges within their 40 \nlifetime and encompasses a range of physiological, morphological, and behavioural 41 \nadjustments5,7,8. Acclimation is usually rapidly induced and reversible, with changes diminishing 42 \nafter stressor -removal5,9,10. In contrast, developmental plasticity occurs when environmental 43 \nconditions experienced during critical early -development windows induce persistent, often 44 \nirreversible, changes in phenotyp e10–12. For plants, there is good evidence that pre -exposure to 45 \nchemicals and other abiotic stressors during early life, or ‘priming’, can induce persistent 46 \ntolerance via stress memory 13–15. For animals, while temperature -induced developmental 47 \nplasticity has been documented in fish and other ectotherms 7,10,16,17, it is unclear whether this 48 \nphenomenon can similarly enhance tolerance to  environmental pollutants. Only a handful of 49 \nstudies have examined whether chemical exposure in early development alters subsequent 50 \nsensitivity18–21. This knowledge is critical to understand the sensitivity of natural populations 51 \nexperiencing fluctuating levels of pollution, a common feature of many natural environments.  52 \nEpigenetic mechanisms, regulating differences in gene expression, have been shown to underly 53 \ndevelopmental plasticity across diverse systems 8,22–25. I n plants, multiple  epigenetic 54 \nmechanisms, including chromatin remodelling, DNA methylation and ncRNAs,  are known to  55 \nfacilitate primed stress memor ies, conferring tolerance to environmental stressors, including  56 \ntoxic metals26,27. Similarly, for animals, epigenetic mechanisms contribute to increased thermal 57 \ntolerance following early life exposure17,25,28,29. Regarding chemical stressors in animals, research 58 \nhas so far focused only on epigenetic toxicity. Many classes of chemical pollutant s, including 59 \nmetals, pesticides  and endocrine disruptors , are known to induce epigenetic modifications 60 \nassociated with adverse health outcomes, dependent on the timing and nature of exposure30–33. 61 \nHowever, the potential for epigenetic mechanisms to contribute to enhanced  chemical 62 \ntolerance in animals remains unexplored.  63 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nHost-associated microbiota also play a critical role in influencing sensitivity to environmental 64 \nstressors by extending host adaptive phenotypic capacity 34,35. While microbiomes are often 65 \nsensitive to disruption by environmental stressors, they have an extensive capacity to rapidly 66 \ndevelop tolerance34,36,37. Crucially, microbial adaptive plasticity can enhance host tolerance to 67 \nenvironmental challenges38,39. Tolerant microbiota can, for example, limit adverse effects in the 68 \nhost associated with microbiome disruption, and/or confer specific benefits, such as enhanced 69 \nmetabolism or sequestration of toxins 36,40,41. As with the epigenome, the microbiome has 70 \nheightened environmental sensitivity during early development 42. While microbiome priming is 71 \nemerging as a technique to enhance agricultural productivity and stressor resilience 41, its 72 \npotential role in environmental chemical-tolerance is largely unexplored. 73 \nIt is unknown whether, in animals, tolerance to environmental chemicals can be acquired via 74 \ndevelopmental plasticity, the extent to which this occurs, or the specific mechanisms 75 \ncontributing to this effect. We aimed to address these questions by examining whether copper, 76 \na widespread aquatic pollutant, can induce developmental plasticity in three-spined stickleback 77 \n(Gasterosteus aculeatus), a well-established model in evolutionary ecology and ecotoxicology. 78 \nWe tested the hypothesis that priming would induce persistent, elevated tolerance to copper, 79 \nwith both epigenetic and microbiome -mediated mechanisms contributing to  this adaptive 80 \nresponse. To specifically examine the capacity for developmental plasticity, distinct from 81 \nacclimation, we exposed s tickleback embryos to an environmentally -relevant concentration of 82 \ncopper during early development, returned them to control conditions for nine months  (until 83 \nmaturity), and then compared response to copper exposure in primed and naïve adults. 84 \n 85 \n 86 \nResults 87 \n 88 \nEarly-life exposure promotes continued copper accumulation in the gill 89 \n 90 \nStickleback embryos were pre-exposed to an environmentally relevant concentration of copper 91 \n(nominal: 10 µg/L, measured 11.4 ±0.3 µg/L) during early development (one-cell stage to hatched 92 \nlarvae; 1-217 hpf), alongside a synthetic freshwater control group (measured 0.2 ±0.004 µg/L Cu). 93 \nSurvival was high in both groups, but copper exposure caused a small increase in embryo/larval 94 \nmortality rate (Naïve: 0.69%, Pre-exposed: 1.24%; P=0.0384). Pre-exposure also increased larval 95 \nwhole-body copper concentration (t =-9.20, df = 5.58, P<0.001; Figure 1a).  96 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nAfter nine months depuration in clean water, both naïve and primed adults were (re-)exposed to 97 \ntwo concentrations of copper (nominal: 10 µg/L, measured: 13.5 ±0.3 µg/L; and nominal: 20 µg/L, 98 \nmeasured: 21.8 ±0.05 µg/L) for 96-h alongside a control (measured: 5.0 ±0.05 µg/L). Pre-exposed 99 \nadult stickleback had accumulated higher concentrations of copper in their gills compared to 100 \nthe naïve fish, while adult exposure also increased gill copper concentration in both groups (Pre-101 \nexposure: F1,48=8.55, P=0.005, Adult-exposure: F2,48=21.18, P<0.001, Interaction: F2,48 =0.36, 102 \nP=0.69; Figure 1b). In contrast, there was no discernible effect of either pre -exposure or adult 103 \nexposure on the concentration of copper measured in muscle or liver  tissue. No mortalities or 104 \nbehavioural changes were observed during the adult copper exposure, and neither pre-exposure 105 \nnor adult exposure to copper affected fish size.  106 \n 107 \n 108 \n 109 \nFigure 1. Copper measured in A) whole larvae after copper exposure during embryonic development (n= 110 \n12 pools of 5 larvae/group) and B) in the gills of stickleback from each group later exposed to copper for 111 \n96-h as adults after 9 months depuration in clean water (n= 10/group ).   112 \n 113 \nPre-exposure substantially reduces and modifies transcriptional stress response to copper   114 \nWe focused the molecular analyses on the gills of adult fish, given their role in metal uptake and 115 \nthe measured differential accumulation of copper  in this tissue. We conducted transcriptomic 116 \nprofiling in both the naïve and primed groups following (re-)exposure to 0 and 10 µg/L copper.  117 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nPre-exposure to copper during embryonic development had minimal lasting effect s on baseline 118 \ntranscription in adult fish, with only two differentially expressed genes (DEGs) identified between 119 \nprimed and naïve fish. Gene Set Enrichment Analysis (GSEA), identified a limited number (seven) 120 \nof negatively enriched GO terms (Table S2); which were all related to cytoskeleton structure and 121 \nfunction (actin, myosin, troponin, calcium binding).  122 \nWe then compared the transcriptomic response to copper  exposure of primed (pre-exposed) 123 \nadults with that of naïve fish (adult fish exposed to copper for the first time ). The magnitude of 124 \ntranscriptional response was far greater in naïve  fish than in primed fish  (1575 and 45 DEGs, 125 \nrespectively; Figure 2, Table S1). Of these, 18 DEGs were common between groups, including 126 \nthose encoding seven heat shock proteins (HSPs; subtypes 90,70 and 30). These molecular 127 \nchaperones, critical in cellular stress response, were the most significantly up -regulated genes 128 \nin response to copper in both groups, but the magnitude of this up -regulation was markedly 129 \nhigher in naïve fish (ranging 7 -805 fold increase) than in pre -exposed fish (ranging 3 -170 fold 130 \nincrease). Metallothionein B, a metal -sequestering protein, was also strongly up -regulated in 131 \nboth groups, although, in this case, by a greater magnitude in the pre -exposed fish (5.9 fold 132 \nincrease) compared to naïve fish (3.3 fold increase). 133 \nGSEA revealed a greater magnitude of response to copper in naïve fish (63 enriched terms) than 134 \nin pre-exposed fish (34 terms). In naïve fish there was strong enrichment of ‘DNA replication’, 135 \n‘Cell-cycle’ and associated terms, as well as terms related to protein refolding and synthesis. 136 \nSimilar terms were enriched in pre -exposed fish, but to a far lesser extent. Processes regulated 137 \nexclusively in naïve fish included strong enrichment of those associated with DNA repair and the 138 \nproteosome, while terms associated with ion homeostasis were supressed, reflecting down -139 \nregulation of >30 genes encoding potassium, sodium, calcium, magnesium, ammonium and 140 \nbicarbonate channels and cotransporters. A further marked distinction between the response of 141 \neach group was that terms associated with cytoskeleton (including actin, myosin, troponin and 142 \ncalcium ion binding) and extracellular matrix (ECM) interactions were supressed in naïve fish but 143 \nenhanced in pre-exposed fish. 144 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\n 145 \nFigure 2. A) Number of significantly differentially expressed genes (DEGs) identified in response to pre -146 \nexposure alone (baseline) and in naïve or pre-exposed groups exposed to 10 µg/L copper as adults. B) 147 \nHeatmap visualising the expression of selected DEGs, based on their primary function (N= 135 DEGs out 148 \nof a total of 1608 identified in response to copper exposure across both groups (see Fig S1). C) Number and 149 \nthe genomic context of differentially methylated regions (DMRs) identified in response to pre -exposure 150 \nalone (baseline) and in both naïve and pre-exposed groups exposed to 10 µg/L copper. D) Shared enriched 151 \nGO terms associated with both DMRs and DEGs. 152 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\n 153 \nCopper exposure induced marked and long-lasting changes in the gill methylome  154 \nWe conducted genome -wide DNA methylation profiling (RRBS) in the gills of naïve and p rimed 155 \nfish after (re-)exposure, on the same samples used for transcriptomic analysis. In contrast to that 156 \nobserved for transcription, we measured considerable, lasting changes in the gill methylome of 157 \nadult fish following developmental pre-exposure to copper and in the absence of any subsequent 158 \nexposures. A total of 615 differentially methylated regions (DMRs) were identified between the 159 \npre-exposed and naïve groups (328 hyper -methylated, 287 hypo-methylated). Of these, 13.5 % 160 \noverlapped putative promoters (pps), while 20.4%  and 42.2% were associated with exons  and 161 \nintrons, respectively (Figure 2c, Table S3). Among the genes associated with these DMRs 162 \n(overlapping pps, exons, introns), notable examples included those involved in ion homeostasis 163 \n(particularly sodium, potassium and calcium transport), metal transport and binding (including 164 \nthose encoding copper -uptake protein 2, ceruloplasmin and ferritin), those with immune 165 \nfunction, and a number of lncRNAs. GSEA, performed separately for different genomic contexts, 166 \nidentified 41, 50 and 45 enriched terms associated with DMRs located within p ps, exons and 167 \nintrons, respectively (Table S 4, Fig ure S3). Among the most enriched terms were those 168 \nassociated with membrane transport and ion homeostasis, including copper -ion transport. 169 \nRegulation of immune response (interleukin production) and many terms associated with 170 \ncellular growth and division, cell adhesion and signalling, were also evident.  171 \nAdult copper exposure also induced considerable changes in the methylome, and these were 172 \nmore extensive in naïve fish. A total of 571 DMRs (287 hyper-methylated, 284 hypo-methylated) 173 \nwere identified in naïve fish exposed to copper, compared to 385 DMRs (202 hyper -methylated, 174 \n183 hypo-methylated) in pre -exposed fish (Figure 3, Table S 3). Functional enrichment analysis 175 \nidentified a greater number of terms associated with DMRs in the naïve fish (Table S4, Figure S3). 176 \nThese included, most strongly, ‘immune system’, several terms related to chaperone-mediated 177 \nprotein refolding and, more broadly, many terms related to protein, nucleic acid and cellular 178 \nrepair and turnover. In pre -exposed fish, there were some broad similarities in the function of 179 \nenriched terms  to those in the naïve fish , including those related to nucleosome, immune 180 \nresponse and cellular turnover but, notably, ‘ ion transport ’ was only enriched in pre -exposed 181 \nfish.  182 \n 183 \n 184 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nGenes & functional pathways with both epigenetic and transcriptional modifications 185 \nDMRs were identified within the gene body or putative promoter of 13 DEGs in naïve fish exposed 186 \nto copper as adults, and one DEG in pre-exposed fish exposed to copper (Table S5). Functions of 187 \nthese genes included protein degradation, synthesis, folding and damage -repair, as well as 188 \ncalcium signalling, cytoskeleton, and the regulation of cell cycle and cell movement. There were 189 \nalso six shared enriched GO terms associated with both altered methylation and transcription in 190 \nnaïve fish (relating to protein synthesis, folding and calcium signalling) and one shared term (DNA 191 \nreplication) in pre-exposed fish (Figure 2D). 192 \nWe hypothesised that persistent methylation differences following pre-exposure influenced the 193 \ntranscriptional responses of adult stickleback (re -)exposed to copper. We identified 13 genes 194 \nwith differential baseline methylation  following pre-exposure that showed a different 195 \ntranscriptional response to copper between primed and naïve adult fish; all of these genes were 196 \nonly transcriptionally responsive to copper in the naïve fish (Table S5). The functions of these  197 \ngenes were related to protein degradation and synthesis, DNA repair, regulation of cell cycle and 198 \ncell movement, as well as cytoskeleton and regulation of ion channels. Eight shared enriched GO 199 \nterms, related to cytoskeleton and potassium ion transport, were also identified (Figure 2d). 200 \n 201 \nEarly-life priming increases gill microbiota copper-tolerance  202 \n 203 \nWe hypothesised that priming during the early stages of microbiome establishment would 204 \npromote enrichment of gill-associated microbiota better able to withstand copper exposure. To 205 \ntest this, we characterised the gill microbiomes of primed and naïve adult fish exposed to 0, 10 206 \nand 20 µg/L. Exposure to the higher concentration of copper disrupted microbiome community 207 \nstructure (Bray-Curtis dissimilarity) in all fish, regardless of  priming, but the effects of the lower 208 \ncopper concentration differed between groups ( Adult exposure:  F1,56 =5.938, P <0.001, Pre-209 \nexposure: F1,56 =1.221, P=0.189, Interaction: F1,56 =1.55, P=0.0493; Figure 3). While fish from the 210 \nnaïve group exposed to 10 µg/L showed microbiome disruption similar to those exposed to 20 211 \nµg/L copper, the microbiomes of primed fish were more resistant to change and remained similar 212 \nto those of the fish unexposed to copper as adults.  213 \nWe examined the differences in community composition contributing to these structural 214 \nchanges. We identified two amplicon sequence variants (ASVs)  with baseline differential 215 \nabundance between naïve and pre -exposed fish (Vibriomonas and Candidatus Bacilloplasma). 216 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nIn adults exposed to copper, p re-exposure reduced the number of differentially abundant ASVs 217 \nidentified (14 and 29 following exposure to 10 and 20 µg/L, respectively ; Table S6) compared to 218 \nin naïve fish (32 and 40 following exposure to 10 µg/L and 20 µg/L ; Table S6). Notably, the most 219 \nabundant ASV overall, Comamonas sp., was strongly inhibited by copper in naïve fish (reduced 220 \nby 13 - and 62 -fold following exposure to 10 and 20 µg/L ), but not in pre-exposed fish  (2-fold 221 \nreduction in response to 20 µg/L only) . At the same time, there was a marked increase in the 222 \nabundance of ASVs from the genera Deinococcus, Enhydrobacter, Acinetobacter, 223 \nFlavobacterium and Brevundimonas in both groups, but generally the magnitude of increase was 224 \nhigher in naïve fish. 225 \nThere were no detectable effects of pre -exposure or adult exposure on overall richness or 226 \ndiversity of ASVs present (Chao1 richness - Pre-exposure: F1,56=2.05, P=0.158, Adult-exposure: 227 \nF1,56=0.49, P=0. 485; Shannon diversity - Pre-exposure: F1,56=0.02, P=0.880, Adult exposure:  228 \nF1,56=0.09, P=0.768, Interaction: F1,56=1.00, P=0.32). 229 \n 230 \n 231 \nFigure 3. A) Gill microbial community structure in adult sticklebacks (re -)exposed to 0, 10 or 20 ug/L 232 \ncopper, visualised using Bray -Curtis dissimilarity values, and B) Relative abundance of the top 20 most 233 \nabundant bacterial genera identified across all samples.  234 \n 235 \n 236 \n 237 \n 238 \n 239 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nDiscussion 240 \n 241 \nWe examined whether early -life priming induced developmental plasticity in stickleback  using 242 \ncopper as a model toxicant , hypothesis ing that epigenetic and microbiome -mediated 243 \nmechanisms contribute to this phenomenon. We found that priming substantially ameliorated 244 \ncopper toxicity in adult fish, an effect characterised by reduced stress response  at the 245 \ntranscriptional level. In parallel, pre-exposure induced considerable, and persistent, changes in 246 \nthe gill methylome, with notable similarities between differentially methylated and expressed 247 \ngene pathways suggesting a priming effect on cellular stress response pathways . Furthermore, 248 \nearly-life priming markedly increased copper-tolerance in the gill microbiome, likely also 249 \nreducing the toxic effects of copper exposure on the stickleback host. 250 \n 251 \nEarly-life priming increased tolerance to copper toxicity in adult fish 252 \n 253 \nTranscriptional response to copper was markedly reduced in adult fish that had been primed in 254 \nearly life. In both naïve and pre-exposed fish, we identified transcriptional changes dominated by 255 \ngenes and pathways associated with the cellular stress response (CSR), but the magnitude of 256 \ntranscriptional changes and enrichment scores were  far greater in naïve fish. The CSR is highly 257 \nand broadly inducible by many stressors, but its nature varies depending on the severity and 258 \nduration of the stressor 43,44. In both groups, although with a greater magnitude in naïve fish,  we 259 \ncharacterised an extensive compensatory CSR, reflecting the repair of cellular components. As 260 \npart of this, we identified a marked increase in the transcription of heat shock proteins and other 261 \nmolecular chaperones responsible for refolding of damaged proteins, as well as genes involved 262 \nin DNA repair pathways. There was also strong up -regulation of DNA replication, protein 263 \nsynthesis and cell division pathways, consistent with an increase in cellular turnover. In naïve 264 \nfish only, there was a distinct enrichment of the proteosome, responsible for degradation of 265 \nirreversibly damaged proteins . This supports the induction of a more severe CSR in naïve fish, 266 \ncharacterised by a switch from repair to autophagic pathways , indicating that greater copper -267 \ninduced cellular damage occurred in these fish. 268 \n 269 \nFurther evidence that priming reduc ed copper toxicity included markedly different 270 \ntranscriptional effects associated with ion transport and cytoskeleton dynamics. In naïve fish 271 \nonly, we found down -regulation of a suite of ion transporters, particularly potassium channels, 272 \nindicating broadscale disruption of ion homeostasis, a well -known mechanism of copper 273 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\ntoxicity, especially in the gills 45. Metals can induce cytoskeleton toxicity via interference with 274 \ncalcium signalling and its regulation of troponin -tropomyosin-actin dynamics 46–48.  Consistent 275 \nwith this, in naïve fish only, we found inhibition of these pathways, indicative of substantive 276 \ntoxicity. However, in pre -exposed fish, there was instead a consistent stimulatory effect, likely 277 \nassociated with a compensatory CSR, encompassing transcripts associated with enhanced 278 \ncellular signalling, reorganisation and stability43,44.  279 \n 280 \nIn contrast to the reduced magnitude of CSR, we found that metallothionein, a key metal-binding 281 \nprotein that sequesters and reduces the toxicity of free metal ions, was more strongly up -282 \nregulated in pre-exposed fish in response to copper exposure. A similar priming effect, increasing 283 \ninducibility of this protein, has been previously associated with enhanced tolerance to  toxic 284 \nmetals in populations with different exposure histories 49 and, more widely, the increased 285 \ninducibility of genes with protective functions contribute to  developmental plasticity and 286 \nincreased stressor tolerance 50. Importantly, the minimal differences in baseline transcription 287 \nidentified between the naïve and pre-exposed fish are consistent with persistent developmental 288 \nplasticity, rather than acclimatory effects (i.e. frontloading transcription)50. 289 \n 290 \nImportantly, we found no effects on long-term survival or growth in primed fish compared to their 291 \nnaïve counterparts, suggesting that developmental plasticity in copper tolerance was not 292 \nassociated with overt energetic costs. Surprisingly, early-life exposure caused fish to continue to 293 \naccumulate more copper in their gills during the nine -month depuration phase, indicative of 294 \nchanges in metal-homeostasis physiology. Following pre -exposure, w e identified persistent 295 \nmethylation differences in slc31a2 (copper-uptake protein) , ceruloplasmin (copper -transport 296 \nprotein) as well > 20 sodium and calcium transporters and channels, which are also responsible 297 \nfor a substantial amount of copper uptake in fish gills 51, although there were no differences in 298 \ntheir baseline transcription . Increased  copper accumulation was only evident in the gills  299 \nsuggesting that primed fish may have an increased tendency to sequester copper in this tissue, 300 \nlikely in a less -toxic and/or bioavailable form, and consistent with the higher inducibility of 301 \nmetallothionein proteins identified.  302 \n 303 \nEpigenetic mechanisms contribute to developmental plasticity in copper-tolerance 304 \n 305 \nPre-exposure to copper caused extensive and persistent changes in the gill methylome, evident 306 \neven after nine months in control conditions. This has not been reported before for metals but is 307 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nconsistent with  existing evidence that developmental thermal stress  induces long-lasting 308 \nchanges i n DNA methylation  in fish 25,52, and, more broadly,  with there being heightened 309 \nepigenetic sensitivity in early life which, when combined with environmental fluctuation, drives 310 \nphenotypic plasticity 24. There were also extensive changes in the adult gill methylome 311 \nimmediately following copper exposure, including within many of the same gene pathways 312 \nmodulated by pre -exposure. This suggests that DNA methylation plays a similar role in acute  313 \nstress responses as in developmental priming, an effect previously identified in stickleback 314 \nchallenged with thermal stress25. 315 \n 316 \nWe identified a considerable functional overlap in epigenomic and transcriptomic responses to 317 \ncopper, including in many gene pathways broadly associated with the CSR  (especially protein 318 \nturnover, cytoskeleton regulation and cell cycle)  and ion transport . Thirteen genes were both 319 \ndifferentially methylated and expressed in acute response to copper exposure in adults , while 320 \nanother 13 genes , that  were persistently differentially methylated following pre -exposure, 321 \nsubsequently showed different transcriptional responses to copper in primed and naïve fish. In 322 \naddition, we identified  similarities in modulated gene pathways , which were predominantly 323 \nassociated with the CSR and ion homeostasis . Together, our results suggest that  epigenetic 324 \nregulation has a priming effect on CSR and ion homeostasis pathways  and subsequently 325 \ncontributes to the reduction in transcriptional stress response identified in pre -exposed fish. 326 \nPriming could, for example, facilitate a more efficient and/or readily inducible CSR, similar to that 327 \nwhich occurs in plants, where developmental stress priming  induces diverse methylation 328 \nchanges within CSR, defence and s ignalling pathways  that are associated with improved 329 \ntolerance to many stressors, including metals53. 330 \n 331 \nPriming increased copper tolerance of the gill microbiota 332 \n 333 \nCopper exposure caused a dose -dependent, disruptive effect on gill microbial community 334 \nstructure, that was more severe in naïve fish . Early-life priming reduced the  number of 335 \ndifferentially abundant ASVs identified in response to both concentrat ions of copper  and, in 336 \nparticular, protected against significant community disruption by the lower (10 µg/L) 337 \nconcentration. The disruptive effects of copper were characterised by a substantial decline in 338 \nthe presence of the otherwise most abundant community member, Comamonas sp. In parallel, 339 \nthere was an increased prevalence of genera including Deinococcus, known for its high abiotic 340 \nstressor tolerance54, as well as Acinetobacter and Flavobacterium, both of which are genera that 341 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\ninclude opportunistic fish pathogens and have previously been associated with microbiome 342 \nstressor-disruption42,55. Our results  support the hypothesis that priming enabled commensal 343 \nmembers of the core microbiome, including Comamonas, to develop enhanced copper 344 \ntolerance, likely through mechanisms such as  genetic adaptation, gene transfer or plasticity 41. 345 \nThis may have been further reinforced by elevated copper accumulation occurring in the gills of 346 \nprimed fish, providing a sustained selective environment for tolerant strains. 347 \n 348 \nWe propose that the extensive changes in community structure observed were associated with 349 \ndisrupted gill microbiome function, thereby increasing the toxic effects of copper to the host. 350 \nConsidering the increase in opportunistic pathogens  observed, a dverse effects on host 351 \nphysiology could include a disruption of normal microbial contribution to pathogen defence and 352 \nprovision of beneficial metabolites, and exacerbation of  host inflammatory stress responses . 353 \nHaving a more tolerant, less -disrupted microbiome, is likely to be beneficial to the host . In the 354 \ncontext of the holobiont concept, our results support the hypothesis that microbiome tolerance 355 \nalso contributes to increased stickleback copper-tolerance. Further research should establish 356 \nwhether microbiota additionally provide specific copper-adaptive benefits to the host , such as 357 \nsequestration of metals, as reported in plants41.  358 \n 359 \nConclusions 360 \nDevelopmental plasticity, together with acclimation, transgenerational plasticity and  genetic 361 \nadaptation, contribute to  variations in the sensitivity of natural populations to environmental 362 \nstressors, ultimately influencing their ability to survive50. Establishing the capacity for organisms 363 \nto acquire tolerance, and elucidating underlying molecular mechanisms, is therefore essential 364 \nto understanding and predicting the risks posed by pollution and other stressors  in the natural 365 \nenvironment. Here, we provide some of the first evidence for developmental plasticity in 366 \nchemical tolerance in animals and demonstrate that both epigenetic and microbiome-mediated 367 \nmechanisms are associated with this effect.  368 \n 369 \nMethods Summary 370 \n 371 \nEthics approval 372 \nAll experiments were approved by the University of Exeter Ethics committee and conducted 373 \nunder licence from the UK Home Office according to ASPA.  374 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\n 375 \nEmbryo copper experiment  376 \nPools of 50 e mbryos were exposed to either a water control (0 µg/L copper) or  10 µg/L copper 377 \n(added as CuSO4) from 1- 217 hours post fertilisation (covering the period of embryogenesis and 378 \nhatching, and including the period of epigenetic reprogramming  and microbiome colonisation). 379 \nExposures were  conducted in 500 ml acid -washed glass dishes containing aerated synthetic 380 \nfreshwater56, with four replicates per treatment, repeated three times with different parental fish 381 \n(see SI). Embryo mortalities and hatching were recorded daily.  Water samples were collected at 382 \n120 and 217h , and 20 larvae from each replicate (n=12) were collected at 217h  for copper 383 \nmeasurement (see SI). Embryo survival and copper uptake were analysed using a student’s t-test 384 \nin R (v 4.3.3; 57). 200 larvae from each of the control and copper -exposed groups were then 385 \nmaintained in dechlorinated tap water (in duplicate tanks per group) for nine months.  386 \n 387 \nAdult copper exposure experiments 388 \nAdult male stickleback from both the naïve and pre-exposed groups were exposed to either 0, 10 389 \nor 20 µg/L copper for 96 hours. Each treatment was performed in duplicate 40 L tanks, with nine 390 \nfish per tank , supplied with flow-through dechlorinated tap  water. Fish were not fed for the 391 \nduration of the exposure. Water samples were collected at 24 and 72h for copper measurements 392 \n(see SI). After exposure all fish were humanely sacrificed by lethal dose of benzocaine (0.5 g/L; 393 \nSigma-Aldrich), followed by destruction of the brain. Gill, liver and muscle tissue w ere snap 394 \nfrozen and stored at -80°C. All left-side gill arches were used for copper-content analysis (see 395 \nSI), while all right-side gill arches were used for molecular analysis.  The effect of both pre -396 \nexposure and adult exposure on fish weight and tissue copper concentration  was examined 397 \nusing ANOVA in R.  398 \n 399 \nSequencing & Bioinformatics 400 \nTranscriptome, methylome and microbiome analys es were conducted on gill tissue from adult 401 \nfish, with full details in SI. Briefly, for transcriptome and methylome analyses, gill RNA and DNA 402 \nwere co-extracted using Qiagen AllPrep DNA/RNA Mini kits from fish from four treatment groups 403 \n(naïve and pre-exposed fish exposed to 0 and 10 µg/L Cu; n=6 per group). RNA-seq libraries were 404 \nprepared using an Illumina TruSeq Stranded RNA Sample Preparation kit  and sequenced using 405 \nan Illumina HiSeq 2500 ( 100 bp paired end). RRBS libraries were prepared using Ovation RRBS 406 \nMethyl-Seq kit (Tecan Systems) and sequenced using an Illumina NovaSeq (100 bp paired end). 407 \nFor microbiome analysis, DNA was extracted from gill tissue s using the Qiagen PowerSoil  DNA 408 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nIsolation Kit (n= 10 from all six treatment groups). Libraries were prepared, amplifying t he 16S 409 \nrRNA V4 region using primers 515F, 806R 58, based on the Illumina 16S Sequencing Library 410 \nPreparation protocol59 and sequenced using an Illumina MiSeq (300 bp paired end).  411 \n 412 \nRNA-seq reads were quality filtered using Fastp (v0.23.1.3;60) and aligned to the Gasterosteus 413 \naculeatus reference genome61 using STAR (v2.7.9a;62). Mapped reads were quantified with RSEM 414 \n(v1.3.1;63) and d ifferential gene expression analysis was performed using DESeq2 (v1.38.3; 64). 415 \nGene set enrichment analysis (GSEA) was conducted using Cluster Profiler (v4.7.1; 65), 416 \nincorporating customised GO term annotations generated using InterProScan (v5.55.88.0;66) and 417 \nBlast2Go (v1.4.12;67). 418 \n 419 \nRRBS reads were quality filtered using TrimGalore 68 and aligned to the  reference genome with 420 \nBismark (v0.23.1; 69. Differentially-methylated regions (DMRs) were identified using DSS 421 \n(v2.48.0;70). Genomic location of DMRs (classified as putative promoters (within 1000 bp of the 422 \ntranscription start site ), exons, introns, or intergenic regions), and gene annotation s were 423 \ndetermined using Genomation (v3.17;71). GSEA was performed using g:Profiler72 using input gene 424 \nlists ranked by methylation fold change. 425 \n 426 \n16S rDNA data were processed using DADA2 73 within Qiime2 (v2024.2,74). Reads were quality-427 \nfiltered, merged, de-noised, assigned to amplicon sequence variants (ASVs) and taxonomically 428 \nclassified using the Silva reference database (v132;75). Alpha and beta diversity metrics  were 429 \ncalculated using the Vegan package76. The effects of both early-life priming and adult copper 430 \nexposure on Chao1 richness and Shannon diversity were assessed using ANOVA. Bray-Curtis 431 \ndissimilarity was analysed using PERMANOVA and differential ASV abundance w as evaluated 432 \nusing DESeq2 64.  433 \n 434 \nAuthor contributions: 435 \nLL, TUW & ES conceived & designed the study . LL conducted the stickleback experiments with 436 \nhelp from JF, JP and AL. LL, HL, RMF, AL & TUW conducted molecular work and AF, KM and MH 437 \nconducted the Illumina sequencing. NB and LL conducted the metal analysis. TUW (RRBS & 16S), 438 \nJO (RNA-seq) and LL (metal content) led the data analyses with contributions from RvA, JP, NB & 439 \nES. ES supervised the study and, together with LL & TUW, led funding acquisition. TUW wrote the 440 \noriginal draft. All authors contributed to review and editing of the final manuscript. 441 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 29, 2025. ; https://doi.org/10.1101/2025.11.28.691212doi: bioRxiv preprint \n\nAcknowledgements:  442 \nWe thank staff from the University of Exeter Aquatic Resources Centre for assistance with fish 443 \nhusbandry. Funding was received from the Fisheries Society of the British Isles (FSBI), the BBSRC 444 \n(BB/S004300/1), NERC PhD studentships, the Exeter-Cefas strategic alliance, Cefas Seedcorn, 445 \nand the Swansea University College of Science Research Fund. 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