the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 22 Jul 2026
Biological modulation of shell δ13C in Mytilus galloprovincialis and Phorcus sp.: Divergent responses between CO2 vents and laboratory mesocosms
Abstract. Stable isotopes are fundamental proxies for deciphering past environmental conditions and carbon cycle perturbations. However, applying these geochemical tools with a forward-looking perspective can also help predict the resilience of marine biota in future acidified oceans. This study investigates carbon uptake and biomineralization pathways under reduced pH in two ecologically keystone calcifying species: the bivalve Mytilus galloprovincialis and the gastropod Phorcus sp. To unravel these mechanisms, we adopted a dual approach, comparing isotopic trajectories (δ13C) under strictly controlled laboratory mesocosms with observations from natural CO2 vent systems in Ischia, Italy. Our results reveal a striking discrepancy between settings. Under laboratory conditions, both species exhibited a significant depletion in shell δ13C as pH decreased, directly tracking seawater carbonate chemistry. Conversely, specimens from the volcanic vents displayed a paradoxical δ13C enrichment at low-pH sites (pH ~7.4). These divergent trajectories indicate that in complex natural environments, the geochemical signal of ocean acidification can be overridden by metabolic overprints and trophically driven vital processes. Consequently, while laboratory experiments are essential to isolate kinetic fractionation, natural analogues remain crucial to capture the biological resilience and complex ecological feedback of future oceans. Future research combining these geochemical trends with direct, in vivo physiological assessments will be essential to precisely constrain the underlying metabolic kinetics and refine predictive metabolic models.
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Status: open (until 15 Sep 2026)
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RC1: 'Comment on egusphere-2026-4016', Anonymous Referee #1, 10 Aug 2026
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AC1: 'Reply on RC1', Annalisa Capasso, 02 Sep 2026
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We thank the referee #1 for the rigorous and constructive evaluation of our manuscript. The issues raised identify substantive concerns that we have taken very seriously. In response, we have carefully re-examined our raw data and analysis scripts, discussed the overall structure of the study with all co-authors, and reviewed and clarified the information concerning sampling permits for the Marine Protected Area.
We address the reviewer's comments individually below.
Structural problems in the experimental design and in the comparability of the field and laboratory arms:
We accept and agree with the reviewer's criticism regarding the differences between the laboratory and field arms of the study, such as source populations, life stage, exposure duration and history, and diet. We recognise these as important limitations of the original framing and we worked to address them throughout the revision. Rather than presenting the laboratory and field datasets as a direct, mechanistically interpretable comparison, we now present them as separate case studies, each examining whether and how shell δ¹³C in marine calcifiers tracks seawater carbonate chemistry under ocean acidification, under two different environmental contexts (controlled laboratory conditions and natural CO₂ vent systems). We believe this reframing more accurately reflects what the data can support. We have revised the Title, Abstract, Introduction, and Discussion accordingly.
We propose revising the title to: “Shell δ¹³C responses to ocean acidification across contrasting biological and ecological contexts”
R) Regarding the pH data, following the reviewer’s comment, we were able to retrieve the continuous pH records collected at the deployment stations throughout the 2024 field experiment. We have therefore incorporated these data into the revised manuscript and added a new Supplementary Table reporting the mean ± SD and range of pH recorded at each station over the deployment period. This information provides a more robust characterization of the actual pH conditions experienced by the organisms during the field exposure.
Regarding the absence of TA, DIC, pCO₂, or Ω measurements: the manuscript, as submitted, characterises the acidification gradient only in terms of pH classes and we rely on biogeochemical characterization available for each provided in the previous studies.
For Castello Aragonese vent system, we draw on as reference at the well-established biogeochemical characterization, derived from continuous in-situ monitoring reported in previous studies (Hall-Spencer et al., 2008 - dataset: PANGAEA, https://doi.org/10.1594/PANGAEA.728719); Hofmann et al., 2011 [https://doi.org/10.1371/journal.pone.0028983]; Kroeker et al., 2011 [https://doi.org/10.1073/pnas.1107789108]; Foo et al., 2018; Gambi et al., 2020 [Art7]; Teixidò et al., 2018, 2024 [https://doi.org/10.1111/gcb.17105]) some of these already cited in the manuscript. These confirm the pattern already assumed in the manuscript (L104–106): total alkalinity is essentially stable across the gradient, while pH decline is driven by DIC/pCO₂ increase.
For Vullatura, published characterization is more limited than for Castello Aragonese, but not entirely absent (Gambi et al., 2019/2020; Mecca et al., 2020 [https://doi.org/10.12681/mms.20795]; Lee et al., 2022 [https://doi.org/10.1111/gcb.16265]), and a three-zone classification consistent with our own (Ambient 8.2–8.0; Low 7.8 ± 0.3; Extremely low 7.2 ± 0.5 - Low and Extremely low categories correspond respectively to our own Medium and Low classes; Barruffo et al., 2021 [https://doi.org/10.3390/d13070311]). We have added these citations and values at the paragraph “2.1.1 Study Sites and Sample collection” and to the relevant supplementary table.
These are historical characterization data for the same vent systems, not measurements taken during our own 2024 deployment, and vent chemistry at these sites is known to fluctuate both seasonally and sub-daily. They therefore can’t substitute for site- and time-specific measurements, and we no longer imply that our nominal pH classes are equivalent to a full carbonate system characterization of the 2024 field experiment. We agree that full, deployment-specific carbonate system characterization, for both sites, especially for Vullatura, remains a priority for future work, and we now state this explicitly as a limitation in the Discussion: in the absence of a directly measured characterization of the surrounding carbonate chemistry, the interpretation of our results based on carbonate system dynamics should be considered accordingly constrained.
R) Regarding the taxonomic indeterminacy of Phorcus sp., we thank the reviewer for raising this important point. We agree that genus-level identification does not allow us to unequivocally assign all sampled individuals to a single Phorcus species.
Photographic documentation of the collected individuals is available; however, after re-examination this material, we concluded that species-level identification based exclusively on shell morphology would not be sufficiently reliable, particularly considering the morphological plasticity of the genus and the degree of shell erosion observed at the vent sites. Unfortunately, no soft tissue samples were preserved for subsequent molecular identification. We have therefore revised the manuscript to explicitly acknowledge this taxonomic uncertainty and to avoid species-specific interpretations of the observed patterns. In particular, the inter-individual variability in shell δ¹³C can no longer be attributed exclusively to individual physiological plasticity or distinct calcification strategies, as taxonomic heterogeneity may also contribute to this variability. We have revised Section 4.2 to explicitly flag this alternative explanation.
Similarly, we have revised our interpretation of the lower abundance and smaller size of individuals observed at the most acidified station. Rather than interpreting this pattern as evidence of a demographic response to acidification, we now acknowledge that differences in the occurrence and distribution of Phorcus taxa along the environmental gradient may also contribute to the observed pattern. Throughout the revised manuscript we therefore treat the field material conservatively as Phorcus sp. and restrict our conclusions to patterns observed at the genus-level assemblage represented by our samples. We do not attempt to attribute these patterns to the response of any particular Phorcus species. This taxonomic uncertainty is now explicitly acknowledged as a limitation when interpreting the biological mechanisms underlying the observed isotopic variability.
R) Regarding the pseudoreplication point: we have re-analyzed both the mussel and Phorcus laboratory experiment datasets accordingly, as detailed below.
Mussel laboratory experiment. We re-fitted the model as nested design, with Tank as a random effect (linear mixed-effects model: δ¹³C ~ Condition + (1|Tank), fitted via "lme4/lmerTest" packages), in which each tank belongs to a single condition and is therefore not crossed with it. We additionally cross-checked this against the simplest possible correction, aggregating each tank to its mean δ¹³C and analyzing the resulting n = 3 vs. n = 3 tank means as experimental unit, following the recommendation of Harrison et al. (2018) [https://doi.org/10.7717/peerj.4794] for designs with very few clusters, since mixed-model variance-component estimates can be unstable when the number of random-effect levels is this small. Both approaches confirm that the treatment effect remains robust once pseudoreplication is corrected: the mixed model yields F₁,₃.₉ = 160.3, p = 0.0002 (Satterthwaite-approximated degrees of freedom), and the tank-aggregated analysis yields F₁,₄ = 175.2, p = 0.0002 (control mean = −2.51‰, treatment mean = −4.11‰, both expressed as means of tank means), compared to the original pseudoreplicated result of F₁,₇₃ = 169.7. We have replaced the original one-way ANOVA in the Methods and Table 1 with this tank-corrected analysis.
Phorcus laboratory experiment. We have re-analyzed the Phorcus laboratory dataset accordingly, following the same tank-corrected approach applied to the mussel laboratory experiment (mixed-effects model with Tank as a random effect, δ¹³C ~ Condition + (1|Tank), cross-checked against a tank-aggregated t-test on the n = 3 vs. n = 3 tank means). Unlike the mussel dataset, where both corrected approaches converged on the same conclusion, here the two approaches disagree, and we report this transparently rather than selecting the more favourable result. The mixed model returns a non-significant effect (F₁,₁₅ = 3.01, p = 0.103) and converges to a singular fit, with the between-tank variance component estimated at zero; the tank-aggregated t-test, in contrast, returns a significant effect (t₄ = 7.42, p = 0.0018, control mean = −1.60‰, treatment mean = −2.14‰, both as means of tank means).
We investigated the source of this disagreement and traced it to the small and uneven number of individuals per tank in this experiment (2–4 individuals, compared to 20 in the mussel experiment), and in particular to one tank (Treatment replicate 1, n = 2) with very high internal dispersion between its two individuals. With only three tanks per condition, the mixed model cannot reliably distinguish genuine between-tank variability from the noise contributed by this single, sparsely sampled tank. The tank-aggregated test, by construction, does not attempt to estimate this variance component and is therefore more stable, but it is correspondingly more sensitive to the fact that tank means themselves are based on as few as two individuals. We have revised the manuscript to present the Phorcus laboratory result descriptively, reporting both corrected analyses and explicitly stating that the tank-level replication in this experiment is insufficient to draw a statistically robust conclusion about the treatment effect. We have added this as an explicit limitation in the Discussion.
R) We thank the reviewer for highlighting this point and we agree that the role of San Pietro in the study required clearer description. San Pietro has already been used as an external ambient-pH reference site in several previous studies conducted at the Castello Aragonese vent system, based on its comparable hydrodynamic conditions, temperature and salinity (Ricevuto et al., 2015a [http://dx.doi.org/10.1016/j.marenvres.2015.09.005]; Migliaccio et al., 2019 [https://doi.org/10.1016/j.scitotenv.2019.04.005]; Palombo et al., 2023 [https://doi.org/10.1016/j.marpolbul.2023.114700]; Signorini et al., 2025 [https://doi.org/10.1016/j.envres.2025.121874];). We have added these references at and revised the corresponding text to make clear that the environmental comparability of San Pietro is supported by previous studies rather than by the shell δ¹³C measurements obtained here. We nevertheless acknowledge the reviewer’s point that site-specific carbonate-system measurements, including δ¹³C_DIC, are not available for San Pietro. We have therefore revised the manuscript to avoid implying that San Pietro is independently validated as equivalent to the Castello Ambient station in terms of carbonate chemistry. Instead, we identify it explicitly as an external ambient-pH reference site for the Phorcus dataset and acknowledge the absence of contemporaneous carbonate-system and δ¹³C_DIC measurements as a limitation when interpreting the isotopic patterns.
Finally, regarding the statistical test cited in the manuscript (F₁,₁₂ = 0.45, p = 0.514, San Pietro vs. Castello Ambient shell δ¹³C): we agree that the absence of a significant difference in shell δ¹³C between San Pietro and Castello Ambient cannot independently demonstrate environmental equivalence between the two sites. We have therefore removed this interpretation from the revised manuscript and now present San Pietro strictly as an external site with prior-documented hydrodynamic/physical comparability.
San Pietro was used exclusively as an external reference site for the Phorcus field dataset and was not part of the mussel transplantation experiment, which instead included the Castello Aragonese and Vullatura vent systems. We have clarified which sites pertain to which dataset in the Methods, and revised Fig. 1 and its caption to describe San Pietro explicitly and to assign it a distinct symbol, differentiating it visually from the ambient stations of the vent gradient. The revised figure is included in the Supplementary Material.
R) Regarding the shell mineralogy: we acknowledge that it was not directly characterised in this study. Shells of M. galloprovincialis and Phorcus sp. were consistently collected from the growing shell margin and the shell aperture, respectively, following the same powder-drilling procedure described in the Methods. The resulting powders therefore represent an integrated signal from the mineral phases present in these shell regions (a mixture of calcite and aragonite for the bimineralic growing margin of M. galloprovincialis, and aragonite alone for the exclusively nacreous shell aperture of Phorcus sp.). The standardised sampling protocol should have minimised differences in mineralogical composition among samples; if the relative proportions of the two mineral phases remained constant, their different carbon-isotope fractionation factors would primarily produce a systematic offset rather than affect the observed δ¹³C trends.
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AC1: 'Reply on RC1', Annalisa Capasso, 02 Sep 2026
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General comments
This manuscript addresses a relevant and interesting question: whether shell δ¹³C in marine calcifiers tracks seawater carbonate chemistry under acidification, and whether laboratory and natural CO₂ vent systems yield consistent isotopic responses. The paired vents/mesocosm comparison on two taxa with contrasting life histories, complemented by an ontogenetic component in Phorcus, is in principle a valuable approach, and the underlying dataset has genuine documentary value for the Ischia vent system. I therefore consider the scientific significance of the topic to be good.
However, I regret to conclude that the manuscript in its current form suffers from major deficiencies in scientific quality that cannot be remedied through revision. My concerns fall into two categories: 1: structural problems in the experimental design and in the comparability of the field and laboratory arms, and 2: serious issues of internal consistency and traceability of the reported results.
Regarding (1), the central claim of the paper, that biological/trophic processes override the geochemical signal in complex natural environments, requires that the field and laboratory arms differ only in ecological complexity. Instead, the two approaches differ simultaneously in: source populations (Adriatic vs. Tyrrhenian; the origin of the transplanted mussels is never stated), life stage (juvenile mussels in the lab vs. adults in the field; lifetime-exposed native Phorcus vs. 8-week exposed lab adults), exposure duration and history, diet (cultured microalgae of unmeasured δ¹³C for lab mussels vs. natural seston; field-collected epiphytes for lab gastropods), thermal regime (constant 28.5 °C vs. autumn cooling in situ), and pH regime (stable setpoint vs. the highly fluctuating exposure characteristic of vent systems). In addition the pH logger data are mentioned but never shown. The observed lab/field divergence is thus overdetermined: it is compatible with at least half a dozen explanations besides the trophic/metabolic override favoured by the authors. Critically, the interpretation invokes precisely the variables that were not measured in either system (δ¹³C of DIC, POM, and diet), as the authors themselves acknowledge (L308–311). A descriptive juxtaposition of the two datasets would be defensible; the mechanistic conclusions drawn in the Abstract and Discussion are not supported by this design.
In addition, three structural problems are not easily fixable in revision:
1) Absence of carbonate system characterization. No measurements of TA, DIC, pCO₂, or Ω are reported for either the laboratory treatments or the field sites; only nominal pH values are given, and the deployment logger data are not presented. For an ocean acidification study whose entire interpretive framework rests on DIC pool dynamics, this falls short of established best practices, and the field chemistry of the 2024 deployment cannot be reconstructed retroactively.
2) Taxonomic indeterminacy of Phorcus sp. The Mediterranean hosts four Phorcus, at least three of which (P. turbinatus, P. richardi, P. mutabilis) co-occur in the Tyrrhenian and segregate primarily by shore height. The sampling protocol ("from the lower intertidal to the upper subtidal") targets exactly the zone of maximum species overlap. Consequently: (a) the pronounced inter-individual variability interpreted as physiological plasticity and "distinct calcification strategies" (Section 4.2) is indistinguishable from interspecific variance; (b) since species composition may co-vary with the pH gradient (microhabitat and depth change along it), the treatment effect is confounded with a possible taxon effect; (c) even the reported scarcity and reduced size at the Low pH station (L137–139) could reflect species turnover rather than a demographic response. Shell morphology in this genus is notoriously plastic and vent-induced corrosion further degrades its diagnostic value. Unless voucher tissues or photographic documentation of live animals are available for identification or DNA barcoding, this confound cannot be resolved. I ask the authors to state explicitly whether such material exists.
3) Pseudoreplication. In the mussel laboratory experiment, three treatment and three control tanks held 20 individuals each, yet individuals are treated as independent replicates (df ≈ 70) with no tank effect in the models. For the Phorcus lab experiment, the number of tanks is never stated, and the Welch test df (7.35) imply a very small effective sample. A mixed-model reanalysis with tank as a random effect would be the minimum requirement, but the design itself (effectively n = 3 tanks per treatment, and possibly fewer for gastropods) remains weak.
Regarding (2), traceability: the manuscript reports two irreconcilable statistics for the same analysis (one-way ANOVA on lab mussels: F₁,₆₉ = 34.79 in the text at L227 vs. F = 169.7, df = 1, 73 in Table 1), inconsistent pH values between Methods and Discussion (8.2/7.7 vs. ~8.0/~7.5), and three mutually incompatible statements of experimental duration (August–October ≈ 60 days; "3 months"; "90 days"). Furthermore, the Abstract states that "both species exhibited a significant depletion" in the laboratory, whereas the Phorcus result is explicitly non-significant (p = 0.144, L265–268), and the "paradoxical enrichment at low-pH sites (pH ~7.4)" was statistically significant only at Vullatura for mussels, while Phorcus was never sampled at Low pH at all. The Abstract thus presents a substantially cleaner pattern than the data support. Individually, some of these could be typographical; collectively, they undermine confidence in the analysis pipeline. I encourage the editor and authors to make the raw data and R scripts available for verification, as offered in the Code and Data Availability statement.
In addition, San Pietro reference site (L107–109): The site is stated to "share comparable biogeochemical parameters with the Castello's ambient zones", but no comparative data are shown or cited to support this claim. No TA, DIC, pCO₂, Ω, temperature, or salinity measurements for San Pietro are reported anywhere in the manuscript, nor, critically for this study, any δ¹³C DIC characterization. The validity of San Pietro as an external control for the Phorcus dataset rests entirely on this undocumented assertion. Note also that the non-significant difference in shell δ¹³C between San Pietro and Castello Ambient (F₁,₁₂ = 0.45, p = 0.514; L245–246) cannot by itself validate the site as a control, since with n as low as 3 per site the test is severely underpowered, and shell δ¹³C is the response variable, not an independent verification of water chemistry. Please provide the environmental data supporting the comparability claim, or cite the specific source from which it derives. Relatedly, in Fig. 1 the westernmost marker (San Pietro) is shown in the map and legend but is never described in the caption, which mentions only Castello Aragonese and Vullatura; please describe San Pietro in the caption, clarify which sites pertain to which dataset (mussel transplants: Castello and Vullatura; Phorcus collections: Castello and San Pietro), and consider a distinct symbol/color for San Pietro, as it is an external reference site rather than an ambient station of the vent gradient.
Finally, The Castello Aragonese vent system falls within the 'Regno di Nettuno' Marine Protected Area, where the collection of marine fauna requires authorization from the managing body. The manuscript does not mention any collection or sampling permits for the field activities (mussel cage deployment at Castello Aragonese and Vullatura; Phorcus collection at Castello Aragonese and San Pietro). Please state the permits obtained and the issuing authority.
Specific comments
Technical corrections
Concluding remark. Beyond the individual issues listed above, I believe this manuscript effectively contains two studies that are not mutually comparable: a field study at the Ischia vent systems (mussel transplants and native Phorcus populations, including the ontogenetic component) and a laboratory mesocosm study. As detailed above, the two arms differ simultaneously in source populations, life stage, exposure duration and history, diet, thermal regime, pH stability and range, and habitat, that is, in essentially every variable that is isotopically relevant. The lab–field 'divergence' that frames the title and Abstract is therefore not an interpretable result but an artefact of comparing systems that were never designed to be compared. I would encourage the authors to consider developing the two datasets as separate manuscripts: a descriptive field study contributing to the well-established Ischia vent literature (contingent on resolving the taxonomic identity of Phorcus and providing site-level carbonate chemistry and δ¹³C DIC data), and a laboratory study of isotopic responses to controlled acidification (contingent on a tank-level reanalysis and characterization of dietary and seawater δ¹³C). Each dataset has merit within its own scope; neither is served by the current framing, which asks the juxtaposition to support mechanistic conclusions that the design cannot deliver.