Since the initiation of the industrial revolution, human activity has dramatically increased atmospheric carbon dioxide (CO₂) levels (Karl and Trenberth, 2003). Consequently, the ocean has absorbed almost one-third of anthropogenically produced CO₂, resulting in ocean acidification (OA), an increase in the partial pressure of CO₂ (pCO₂), and a subsequent decrease in oceanic pH (Sabine et al., 2004). The predicted increase of oceanic pCO₂ is expected to further decrease sea surface pH by 0.3-0.4 units by the year 2100 (Stocker et al., 2013). This decrease in pH will be particularly pronounced in arctic and sub-arctic climates, such as Alaska’s oceans, due to the increased solubility of CO₂ gas at lower temperatures (Sabine et al., 2004; Orr et al., 2005).

Nearshore marine ecosystems are often characterized as highly dynamic, driven by fluctuations in tidal action, wave activity, freshwater input, and primary production (Carstensen and Duarte, 2019; Miller and Kelley, 2021). Specifically, seasonal booms in primary productivity can drive cyclical, diel variability between photosynthesis and respiration during certain periods of the year, leading to major pH swings over a 12-hour period (Miller and Kelley, 2021). However, OA experimental designs have historically only investigated the effects of static pCO₂ on marine organisms. For marine species, habitats characterized by high carbonate chemistry variability have been theorized to increase the potential for adaptive capacity to OA (Kapsenberg and Cyronak, 2019). By designing OA experiments that consider the historic environmental exposure and natural variability in carbonate chemistry parameters a species experiences, we can begin to make more ecologically relevant predictions of species’ resilience or susceptibility to ocean change. This is vital for the effective management of fisheries species inhabiting nearshore environments.

The Pacific razor clam (Siliqua patula; Dina’ina: qiz’in; Sugpiaq: Cingtaataq) is a narrow-shelled bivalve ranging from the Aleutian Islands to Northern California (Alaska Department of Fish and Game, 2020). Razor clams inhabit sandy beaches and can range in size from 15 to 28 cm in length, depending on the region (Washington Department of Fish and Wildlife, 2010; Alaska Department of Fish and Game, 2020). In Southcentral Alaska, S. patula are prized as a commercial, recreational and subsistence resource. S. patula is harvested as a subsistence resource by numerous Indigenous communities throughout Lower Cook Inlet (LCI) (Alaska Department of Fish and Game, 2022). In LCI, AK, S. patula has been harvested commercially since 1919, and constitutes the most popular recreational shellfish species in the region (Alaska Department of Fish and Game, 2020). Unfortunately, in 2015 all recreational harvest was closed along the east side of the inlet due to record low numbers in the area, the cause of which is still unclear (Olsen, 2015).

While Siliqua patula follows the typical bivalve developmental process, they deviate during their initial shell formation. S. patula produces a concretion in early shell development (Alcantar et al., in review) ¹ , delaying the formation of more organized calcium carbonate (CaCO₃) polymorphs, relative to other bivalves, in favor of a flexible, more elementally diverse shell structure during the early stages of development (Fretter and Pilkington, 1971; Hinzmann et al., 2015). The impact of OA exposure on an organism that produces a concretion during larval development remains unexplored. Given the importance of S. patula as a resource and recent population declines, investigating how S. patula will respond to the effects of OA during its early life-history is critical to understanding how this species will fare in future ocean conditions.

Ocean acidification exerts negative impacts on a variety of marine organisms, including high-latitude, intertidal species (Bacus and Kelley, 2023), and biocalcifiers such as bivalves (Kroeker et al., 2013). While OA results in a decrease in seawater pH, it can also decrease carbonate ion saturation state (Feely et al., 2008), limiting the pool of CaCO₃ necessary for biocalcification (Fabry et al., 2008). The negative effects of OA are generally most pervasive during the early life-history stages of bivalves due to the critical series of events that occur during early shell formation (e.g., Kroeker et al., 2013; Kapsenberg et al., 2018). Previous research examining the effects of OA on various larval bivalve species documented shell malformation, alteration to calcification, delays in development and growth, and alterations to elemental composition (Barton et al., 2012; Frieder et al., 2014). Exposure to elevated pCO₂ during larval bivalve development has also resulted in smaller shell sizes and reduced survival in Mercenaria mercenaria and Argopecten irradians (Talmage and Gobler, 2010).

Additionally, changes in gene expression under OA conditions have been documented in numerous marine bivalves (Cummings et al., 2011; Wang et al., 2016; Peng et al., 2017). Expression of heat shock protein 70 (HSP70), a known biomarker of organismal environmental stress, is significantly increased under OA exposure in bivalves (Cummings et al., 2011; Wang et al., 2016). Given the established impact of OA during early bivalve shell development (Watson et al., 2009; Bylenga et al., 2017), investigating genes, such as Calmodulin (CaM), important for the generation of calcium binding proteins involved in shell formation (Li et al., 2004) and larval settlement (Chen et al., 2012), may provide some insight into the mechanism that larval bivalves utilize to offset OA exposure.

Our goal was to characterize the response of S. patula to future OA conditions, including daily variability, an ecologically relevant dynamism that is seasonally present in LCI during the key spawning and larval periods of July and August (Southcentral Region Division of Sport Fish, 2017). Here, we provide information about the current and future sensitivity of this species under future conditions of OA, characterizing the response of larval S. patula across levels of biological organization. Our research also represents the first study that examines how a concretion-producing bivalve will respond to OA. This latter point is vital to establish a baseline for future OA work examining bivalves, particularly those with varied life-history traits.

While the Raman spectroscopy results were limited, we believe that ACC and vaterite are the CaCO₃ polymorphs present during the biomineralogical transitions of larval S. patula. Firstly, the persistence of ACC and vaterite was evident in the visual assessment of SEM micrographs. Secondly, the diverse elemental constituency of larval S. patula shells is in line with known compositional diversity seen in both ACC and vaterite (Nebel et al., 2008; Soldati et al., 2008). Finally, the Raman spectra presented are consistent with known ACC (Wehrmeister et al., 2011) and vaterite (Jacob et al., 2008), with potential shift of the vateritic dual peak due to alternative mineral inclusion (as seen in the compositional data, Seknazi et al., 2019). ACC was the polymorph present in larval S. patula concretions, while vaterite was evident in fully calcified larval S. patula shells. Aragonite was the final CaCO₃ polymorph present in adult S. patula. This biomineralogical journey, from ACC to vaterite to aragonite, while relatively unique, is not necessarily unprecedented (Hasse et al., 2000). However, it does raise some concerns regarding the sensitivity to dissolution of S. patula under future OA conditions as ACC, vaterite and aragonite represent three of the most soluble polymorphs of biogenic CaCO₃ (Gal et al., 1996).

ACC is a known precursor for other polymorphs of CaCO₃; however, ACC typically only persists for several hours (Raz et al., 2003; Politi et al., 2008), likely due to its relatively unstable structure (Weiner et al., 2003). Unfortunately, there are a dearth of studies examining the direct effects of OA on organisms that possess ACC as a polymorphic precursor to other forms of calcium carbonate. Previous research on Mytilus edulis suggests that ACC production is increased under future OA conditions as a shell repair mechanism (Fitzer et al., 2016); however, the implications of persistent use of ACC by larval bivalves has yet to be investigated. Given established knowledge regarding the thermodynamics of ACC (Gal et al., 1996), it is possible to infer that prolonged use of this form of CaCO₃ increases a biocalcifier’s vulnerability to dissolution under future OA conditions.

While ACC is a known precursor of aragonite (Weiss et al., 2002), and aragonite is a common CaCO₃ polymorph found in marine bivalves (Sonak, 2017), biogenic vaterite is not well understood. Biogenic vaterite has been found in fish otoliths (McConnell et al., 2019), pearls (Jacob et al., 2008), juvenile freshwater snail shells (Hasse et al., 2000), bird egg shells (Portugal et al., 2018), and even in a species of alpine plant (Wightman et al., 2018). However, in light of vaterite’s less organized crystalline structure, there is uncertainty regarding whether vaterite exists as a typical, transitional CaCO₃ polymorph, or as result of stress-induced, deficient mineralization from ACC to calcite (Hasse et al., 2000; Melancon et al., 2005). Moreover, if the presence of biogenic vaterite is not a result of malformation, its benefit over another, more stable polymorph of CaCO₃ is unknown (Portugal et al., 2018), and should be further investigated. Unfortunately, there were no ambient individuals present in the 28 DPF Raman spectroscopy sample from this study, so we are unable to make a direct comparison to the presence of vaterite in S. patula reared under ambient pCO₂ conditions to those exposed to elevated and variable pCO₂. Meaning, we cannot definitively state that the use of vaterite as a precursor phase is common across all LCI S. patula, or if it is present due to maladaptive mineralization.

While the transition from vaterite to aragonite constitutes a reduction of the potential solubility of S. patula shells, it is unclear (based on the scope of this study) at what point during development this transition occurs, and therefore how long this more vulnerable vaterite phase persists. Further, it is important to note that while less soluble than vaterite, aragonite is still a more soluble polymorph of CaCO₃ and is also vulnerable to dissolution under OA (Gal et al., 1996). Exposure to future OA conditions has resulted in external, aragonitic shell dissolution in various marine mollusks (Green et al., 2004; Watson et al., 2009; Busch et al., 2014), as well as increased dissolution of the aragonitic, nacreous layer of Mytilus edulis (Melzner et al., 2011). While S. patula may also prove vulnerable to future OA conditions due to dissolution, future research is needed to establish the response of aragonitic individuals.

ACC and vaterite are the two of the most soluble polymorphs of CaCO₃ (Gal et al., 1996; Weiner et al., 2003) due to disordered crystallography (Rodriguez-Blanco et al., 2011). ACC is composed of disorganized, scattered molecules so that the overall surface area of an ACC aggregate is larger than a more ordered polymorph of CaCO₃, with more interstitial space between molecules (Weiner et al., 2003). Vaterite is similar in nature to ACC in that it has a more disordered molecular structure than other CaCO₃ polymorphs, but the overall structure is tighter (less inter-molecular space) than that of ACC (Christy, 2017). The increased porosity of ACC and vaterite polymorphs permits greater solvent access along the crystalline interface (Ogino et al., 1987), which is one of the mechanism that increases the overall solubility of these polymorphs.

In addition to the increased vulnerability to dissolution, the greater interstitial space present in these polymorphs allows for the intrusion of organic molecules within the CaCO₃ amalgamation (Addadi et al., 2003), leading to the potential for increased elemental diversity within overall CaCO₃ composition in ACC aggregates, which is supported by the compositional data from the concretion stage of S. patula presented here ( Figure 2 ). The tighter lamination of CaCO₃ molecules in vaterite results in a reduction of organic molecule intrusion compared to ACC, and therefore an increase in the relative calcium presence. However, the interstitial space present in vaterite, while less than ACC, still allows for the intrusion of organic molecules within the CaCO₃ crystalline matrix (Fernández-Díaz et al., 2010). This reduction in organic molecule intrusion due to tighter crystal lamination between ACC and vaterite is further supported by the significant difference in the relative calcium contribution to overall composition between concretion-stage individuals (using ACC) and fully calcified individuals in our study (using vaterite, Figure 6 ).

Although we expected alterations of CaM expression due to the observed treatment-related differences in the timing of biomineralogical transition given the prior expression of CaM in shell building and preparation for settlement (Li et al., 2004; Chen et al., 2012), there was no significant effect of elevated or variable pCO₂ exposure on CaM expression. This further contradicts previous studies which have shown upregulation under OA conditions, likely intended to maintain shell calcification under acidic conditions (Dineshram et al., 2013; Dineshram et al., 2015). It is possible that the lack of upregulation of CaM was due to the concretion development technique used by S. patula, and the subsequent delay in overall calcification. A prolonged exposure beyond our experimental duration may have resulted in CaM upregulation as described in Dineshram et al. (2013); Dineshram et al., (2015). There was no significant alteration to HSP70 expression as a result of exposure to elevated or variable pCO₂. This contradicts results described in Cummings et al. (2011) which reported HSP70 upregulation in Laternula elliptica under OA conditions, and Lardies et al. (2014), which identified similar upregulation of HSP70 family genes in juvenile Concholepas concholepas. As with CaM, perhaps HSP70 expression was not significantly upregulated in the present study as stress effects due to exposure to acidic conditions were not observed within this experimental timeframe. It should be noted that individuals were sampled from the variable pCO₂ treatment during the day, when pCO₂ was low. Subsequently, the HSP70 and CaM expression results may not fully represent the spectrum of gene expression experienced by S. patula in the variable treatment, as was documented by Podrabsky and Somero (2004) where gene expression varied with daily, cyclical temperature fluctuations.

While this study was unable to capture any potential dissolution or malformation as a result of ACC and vaterite presence during initial shell formation, or altered gene expression, individuals who were reared under elevated and variable pCO₂ did appear to transition from ACC to vaterite at an accelerated rate. Furthermore, this relationship between shell calcification and exposure to future OA conditions appears to be directly correlated to the time spent to elevated pCO₂. Larvae reared in consistently high pCO₂ conditions first exhibited fully calcified individuals at 21 DPF (although not all individuals had calcified), whereas larvae exposed to intermittently elevated pCO₂ (the variable treatment) demonstrated individuals with complete calcification at 28 DPF. Conversely, not a single individual reared under ambient pCO₂ conditions displayed complete calcification at any time during the experiment.

The elevated calcium presence in transitional shells and fully calcified shells, compared to the calcium levels found in a concretion, also suggests an increase in the developmental rate of S. patula larvae exposed to consistent and intermittently high pCO₂. Calcium contribution to overall shell composition was significantly higher in these two treatments compared to the ambient shells at 28 DPF ( Figure 5 ). While calcification had begun at 21 DPF, the maximum mean percentage of calcium present was only 41% at 28 DPF. An analysis of adult LCI S. patula composition showed a mean calcium contribution of 96.2% ± 2.31% (N = 5, Alcantar & Severin ² , unpublished data), indicating that by 28 DPF, the majority of calcification is yet to occur.

Contrary to the results of this study, previous research looking at the response of larval mollusks under OA conditions has typically produced an overall decrease in developmental rate. A study examining the impact of OA on the early development of abalones Haliotis diversicolor, and Haliotis discus hannai demonstrated that both species experienced a decrease in developmental rate under elevated pCO₂ conditions, and that this was directly correlated to the concentration of pCO₂ experienced (Guo et al., 2015). The abalone, Haliotis tuberculata, presented a similar pattern to Guo et al. (2015), with the timing of different developmental stages being pH dependent (Wessel et al., 2018). Bivalves have also responded to elevated pCO₂ exposure by altering normal developmental timelines. Both Mercenaria mercenaria and Argopecten irradians exhibited decreasing developmental rates with increasing pCO₂ conditions across veliger, pediveliger and metamorphosed transitions (Talmage and Gobler, 2010).

Given the results from previous molluscan studies, the question arises as to what an accelerated developmental timeline means for larval S. patula. Past research on the fish species Lates calcarifer has revealed that exposure to future OA conditions results in an increase in developmental rate (Rossi et al., 2015). However, this acceleration of development led to negative timing mismatches of vital settlement cues in larval individuals (Rossi et al., 2015). In light of the negative consequences associated with accelerated development highlighted by Rossi et al. (2015), it is vital to consider the implications of accelerated shell development in LCI S. patula, as the energy needed to maintain an accelerated development rate is likely to come at a metabolic cost.

Bivalves have displayed varied metabolic responses to future OA conditions (Waldbusser et al., 2015a; Kelley and Lunden, 2017; Bacus and Kelley, 2023), mandating the need for further metabolic examination of S. patula under elevated pCO₂ conditions. However, if larval S. patula are deviating from the normal shell developmental strategy (like that seen in the ambient pCO₂ treatment group), and do not demonstrate an upregulation in overall metabolism, like 58% of other bivalve species (Kelley and Lunden, 2017), larvae may need to divert the energy that would otherwise be devoted to growth, or other important developmental processes. In fact, although it was not statistically significant, the size data of S. patula 28 DPF demonstrates that despite being further along in the calcification process, individuals in the high and variable pCO₂ treatments were smaller overall than individuals from ambient pCO₂ conditions ( Figure 4 ). It is possible that the cost of an earlier mineralogical transition from ACC to vaterite, with a faster increase in overall calcium contribution (possibly at the expense of overall size), may have been more pronounced had the experiment been carried out longer, or had the pH been reduced by more than 0.4 units, as noted in Waldbusser et al. (2015b). Additional research is needed to determine if accelerated calcification comes at a cost to overall size of LCI S. patula, a possibility that has substantial implications for commercial, recreational and subsistence harvest.

There was an expectation that variable pCO₂ treatment larvae would exhibit fewer negative effects than the static, elevated pCO₂ treatment larvae given the variable carbonate chemistry conditions experienced by LCI S. patula, however, our results did not bear this out. With respect to the CaCO₃ polymorphs present and shell development, the variable pCO₂ treatment larvae were somewhat aligned the elevated pCO₂ treatment larvae, although temporally delayed, with both ACC and vaterite present as CaCO₃ polymorphs, and shell development appearing to be accelerated compared to the ambient pCO₂ S. patula. The relationship between variability and growth is not straightforward. Although not statistically significant, S. patula reared under variable pCO₂ conditions were larger overall than both ambient and elevated pCO₂ S. patula at 7 and 14 DPF. However, at both 21 and 28 DPF, variable pCO₂ individuals were smaller overall than both ambient pCO₂ treatment S. patula and elevated pCO₂ treatment S. patula. While these results do not provide a clear understanding of S. patula from LCI, and future research is needed to explore potential population specific responses to variable carbonate chemistry conditions, these results do highlight the importance of including variable conditions for relevant species.

This study suggests that one possible response of LCI S. patula to future OA conditions is an increase in shell developmental rate. These rates are proportional to the amount of time exposed to elevated pCO₂, with high treatment individuals fully calcifying first, and individuals from the variable treatment following. Furthermore, this study has characterized the calcium carbonate polymorphism of S. patula for the first time, identifying a potential OA vulnerability present in the form of vaterite during the initial calcification phase.

While this study did not find any other impacts of elevated pCO₂ on LCI S. patula, the alteration of shell developmental rate was contradictory to that documented in previous mollusca studies (Talmage and Gobler, 2010; Guo et al., 2015; Wessel et al., 2018). This deviation of developmental rate could prove detrimental to overall S. patula fitness if individuals divert energy from other processes to increase their developmental rate. Moreover, alterations to developmental rate could result in detrimental timing mismatches of vital processes during S. patula life-history stages (Rossi et al., 2015).

Given that S. patula’s biomineralogical response to elevated pCO₂ contradicts the current research regarding mollusk development under OA conditions, it is evident that there is still a lot of work to be done to lay the baseline of knowledge about the response of early life-history stage bivalves to future OA conditions. Furthermore, the presence of vaterite as a transitional calcium carbonate polymorph, or as an artifact of mineral malformation remains unresolved. Overall, this study highlights the need for multivariate, species and population specific experimental evaluations to be conducted in order to better inform bivalve management decisions, and mariculture practices in a future acidified ocean.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The requirement of ethical approval was waived by the Institutional Animal Care and Use Committee at the University of Alaska Fairbanks for the studies involving animals because current U.S. regulations do not apply to work with invertebrate species. The studies were conducted in accordance with the local legislation and institutional requirements.

MA: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. JH: Funding acquisition, Methodology, Resources, Supervision, Writing – review & editing. JR: Data curation, Formal analysis, Investigation, Writing – review & editing. AK: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing.