| 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.
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.