Dear Anonymous Referee #1,

We really appreciate the very constructive, helpful and overall encouraging comments on our manuscript and agree that addressing the issues raised in the specific comments section would improve the manuscript. We aim to improve the clarity of Fig. 4, distinguish between true replicates grown in the same water and replicates that just grew under the same experimental conditions, add detail to the feeding procedure and add an error estimate to the alkalinity measurements. To add a comparison of the presented El/Ca data from cultured specimens to those from naturally-grown Tridacna is a good idea, which we will aim to include in the discussion. We are also grateful for the technical corrections and will try to shorten sentences that seem too long and complicated. A decision on final edits, however, can only be made once all reviews have been received.

Best regards,

Iris Arndt on behalf of all co-authors

Dear Nils de Winter, dear Anonymous Referee #1, dear Daniel Killam,

We are very grateful for the positive feedback as well as the helpful suggestions. All comments are very constructive, and we aim to implement them to improve our manuscript. Main changes would include:

• Updating the details of the feeding regime to the methods section
• Adding assigned species for all specimen
• Providing an error estimate for the alkalinity measurements and the resulting calcification rates
• Improving the clarity of Fig. 3.
• Expanding the discussion to include a comparison of elemental ratio data of cultured to naturally-grown specimen
• Strengthening the link to paleoenvironmental applications
• Going into more detail on the measured oxygen concentrations
• Discussing the potential response of the volume of the extrapallial fluid (EPF) with valve closure

In the following we provide answers to the referee comments (which are highlighted in italic fond) individually.

RC1: 'Comment on egusphere-2025-3479', Anonymous Referee #1, 02 Sep 2025

General Comments

The manuscript “Culturing experiments reveal mechanisms of daily trace element incorporation into Tridacna shells” presents novel and well-designed culturing experiments that address important knowledge gaps in our understanding of biomineralization processes in Tridacna and their application as high-resolution paleoclimate archives. The use of ¹³⁵Ba as a tracer to unequivocally differentiate between day- and night-time shell growth represents a novel methodological advancement, providing robust constraints on the mechanisms of trace element (El/Ca) incorporation. The link made between EPF dynamics, light availability, and elemental ratios is compelling and will be of interest to both geochemists and marine biologists. The work is highly relevant to Biogeosciences, as it improves the mechanistic understanding of high resolution geochemical proxy development in marine bivalves, with implications for interpreting sub-daily climate signals.

The manuscript is clearly structured, generally well-written (although I would recommend making many sentences shorter where possible), and supported by a thorough literature review. The experimental setup is carefully designed and technologically sharp and the analytical work by the novel LA-ICPMS configuration is impressive - clearly the author's expertise. The conclusions are both significant and well supported by the data.

Overall, the study provides important insights for the Tridacna sclerochronology community (myself included) and for paleoclimatology. With some very minor clarifications, improvements in figure presentation, and minor language editing, the paper will be an excellent contribution.

We appreciate the supportive comments on our manuscript.

Specific Comments

-             Figures 3 and 4 are central to the paper. In Figure 3, I would suggest expanding it width-wise. I know Fig. 4 provides an expanded version but the legibility in Fig. 3 is not great and should be improved. This would make the timing of the spiking vs the periodicity in the El/Ca values (which is key) a bit clearer. In Fig. 4, explain the green bars (as you do in Fig. 3) in the figure caption or in the figure.

We agree that Fig. 3 is quite full of information and will do our best to improve legibility. We will include the explanation of the green bars in the caption of Fig. 4, thank you for noticing the missing description.

 In terms of experimental, given that two clams share each jar, and therefore the same water, can they be considered as true replicates? Isn’t the carbonate chemistry in the water an important part of the experimental unit?

The carbonate chemistry is indeed an important part of the experiment. For the two clams grown in the same jar only one water measurement is available. The resulting elemental ratios (El/Ca) data could be expected to be the same, given water chemistry and treatment was identical, if one assumes that only environmental factors impact El/Ca. The observed variability is likely caused by the individual’s physiological performance and behaviour. Since the El/Ca data are different between some clams grown in the same culturing jar, we deduce that the individual response of each clam places a limit on overall signal reproducibility. However, most features, like the daily cycles and trends from changing the water source they grew in, are reproducible. The reproducibility between two clams given the same treatment in two different jars is just as good (or as bad) as between two clams grown in the same jar. Nevertheless, we agree that a distinction between replicates grown in the same water and replicates that just grew under the same experimental conditions would add clarity to the experimental setup and we will provide this clarification in the revised document.

-             Line 109: A few more details about the feeding regime would be good. Quantity? Timing? DOM vs particulate matter? This is important for understanding food vs light contributions.

The food is from the German brand “fauna marine”, and the used food powder is called “coral sprint”. It is a fine powder with 85 % protein, 11 % fat, 3% fibre and 1 % ash. It contains additives per 1 kg: 600 i.u./kg Vitamin D3 (E671), 50 mg iron-sulphate-monohydrate, 2.2 mg calcium-iodate, 6 mg copper-sulphate, 17 mg manganese-monohydrate, 120 mg zinc-monohydrate, 57 mg antioxidants. The recommended dosage is one measurement cup every two days for 500 l of water. We scaled the dosage down to be appropriate for the 11 l water reservoirs attached to the culturing jars. The clams grew in the prepared reservoir water for the initial spike (experiment days 1-3) and in a new set of reservoir water prepared in the same way for the three days of day and night-spiking (experiment days 4-7). The reservoir water was pumped through the culturing jars continuously, with approximately 2 of the 11 l flowing through the culturing jar every 12 h. There were no feeding events that could have introduce spiked nutrient availability, but the constant pumping of new reservoir water into the jars provided constant nutrient inflow. Through reintegrating the 2 l flown through the culturing jars to the respective 11 l reservoirs every 12 h the nutrient availability within the reservoir water was slightly diluted over the 2 to 3 days of usage. Following the feeding instructions, the food within the reservoir water was sufficient throughout the culturing. Before and after the experiment, as well as overnight from experiment day 3 to 4 the clams stayed in a 400 l aquarium with corals, anemones, hermit crabs, fish, sea urchins and other molluscs. In this aquarium 10 ml of “Reef Energy Plus” from the brand “Red Sea” are added daily during the week. We will add this information in the revised manuscript.

-             Consider uncertainties in using 12h ΔALK to estimate calcification.

This is a good point, thank you.

-             How does the data from this study compare with daily-resolved trace element/Ca data from clams that grew in natural reef settings (e.g. in some of your other publications)? E.g. amplitude, relative phasings. If similar, this would strengthen any arguments you had that your observations apply to Tridacna more generally, not just those with very specific culture conditions. (Juvenile, small individuals in 28 °C, 37 psu, constant light regime (12:12 h) under lab flow are quite different from heterogeneous reef conditions. Whether the 5× daytime calcification and the precise phasing of maxima/minima are similar across sizes, species, and natural diel PAR/temperature cycles remains to be tested – worth acknowledging perhaps?)

Regarding daily cycle amplitudes and absolute El/Ca values, data from the cultured Tridacna of this study compare well to other data, namely those from the late Miocene Tridacna of Arndt et al. (2025) as well as the recent and fossil clams from Water and Müller (2017), which were all measured using a very similar method. We do not see a way to compare, in required detail, the relative phasing of the daily cycles without a tracer experiment under laboratory conditions that provides the timing of sub-daily growth. We will provide a comparison to natural cycles and will point out that this particular experiment resulted in this calcification pattern which might not be uniformly translated to all species, sizes and temperature regimes. Having said this, the choice of experimental conditions was based on the findings in Warter et al. (2018) regarding temperature, light and food availability and optimized for high calcification rates through several pre-experiments.

Arndt, I., Bernecker, M., Erhardt, T., Evans, D., Fiebig, J., Fursman, M., Kniest, J., Renema, W., Schlidt, V., Staudigel, P., Voigt, S., Müller, W., 2025. 20,000 days in the life of a giant clam reveal late Miocene tropical climate variability. Palaeogeography, Palaeoclimatology, Palaeoecology 112711. https://doi.org/10.1016/j.palaeo.2024.112711

Warter, V., Erez, J., Müller, W., 2018. Environmental and physiological controls on daily trace element incorporation in Tridacna crocea from combined laboratory culturing and ultra-high resolution LA-ICP-MS analysis. Palaeogeography, Palaeoclimatology, Palaeoecology 496, 32–47. https://doi.org/10.1016/j.palaeo.2017.12.038

Warter, V., Müller, W., 2017. Daily growth and tidal rhythms in Miocene and modern giant clams revealed via ultra-high resolution LA-ICPMS analysis — A novel methodological approach towards improved sclerochemistry. Palaeogeography, Palaeoclimatology, Palaeoecology 465, 362–375. https://doi.org/10.1016/j.palaeo.2016.03.019

-             The discussion could more explicitly link findings to potential paleoenvironmental applications (ENSO, storm reconstructions, etc.), strengthening the broader impact.

Thank you for pointing this out. We aim to strengthen the link to paleoenvironmental applications in the discussion chapter.

Technical Corrections

-             Language generally fluent, but some sentences are long and could be simplified for clarity.

We will give the text a critical reread for sentences that are too long.

-             Don’t capitalise ‘tridacnid’

-             Minor typos: Hyphen needed in ‘non spiked’ on p121. Centre data in table.

Thank you for noticing, we will change these points accordingly.

RC2: 'Comment on egusphere-2025-3479', Daniel Killam, 14 Sep 2025

The work of Arndt et al represents a valuable expansion of the previous investigation of Warter et al., 2018, with many novel interpretations that make it a valuable contribution to the advancement of giant clam biology. The effort to quantify the daily calcification of these animals in particular is quite important to other efforts to understand how they will fare during a time of environmental change. The methodology of LA-ICPMS sampling at 2 micron resolution, and mention of the variation of different under-studied elemental ratios such as Na/Ca and B/Ca is also an important advancement. This approach shows how shell records are valuable to investigate the modern-day physiology of these living animals, and I endorse its publication once the following line-by-line comments are addressed.

Daniel Killam

Thank you for the positive comments on our manuscript.

22: I'd advocate for the authors noting here that they are estimating the EPF composition based on the composition of external seawater in a culture setup. This is totally a valid approach, but the abstract made it sound like direct sampling of the EPF was conducted, which does not seem to be the case.

We will state more clearly that we estimate the composition of the EPF from that of the seawater, thanks.

86: It is crucial to know the species of these clams, for reproducibility purposes. I would be happy to personally identify the species if the authors have pictures of the clams. Feel free to email pictures.

Thank you very much for this kind offer. We will give the identification a try and reach out to you when we need an expert opinion.



109: Do you know the brand/composition of coral food? The diet could be important for other workers to reproduce the results. I think it was useful that the clams were fed (that is, of course different than knowing if they actually ate the food), since other studies often do not feed the clams, which leads to changes in behavior, particularly for juvenile clams of this size, which are generally more reliant on filter-feeding. Did you observe them feeding during the experiment? Production of feces/pseudofeces, valve clapping, etc.

Please find the details to the food composition and feeding procedure in the comment for Referee #1. I did not directly observe feeding behaviour during the experiment.

140: The measurement of alkalinity differences at night and day is a great approach and one of those things that makes me slap my forehead, and wonder why someone hadn't thought to do that before, especially since in the aquarium trade, giant clams are known as infamous alkalinity sinks. Were any other environmental parameters measured, even intermittently? I'd be particularly interested in nutrient measurements like nitrate or ammonium (more on that below)

Sadly, we did not conduct nutrient measurements on the water. We agree that nutrient measurements of the culturing water would be an interesting dataset for future culturing setups.

204: What direct influence would the calcification have on DO? I would have thought O2 is more related to photosynthesis. In daylight hours, the clams can be net sources of O2 (Fisher et al., 1985), while at night, they conduct respiration, analogous to similar processes seen in plants. Were there any algae in the tanks with them adding to these processes, or were they the only significant biomass in the observation tanks?

While the giant clams should consume more oxygen when they are active during the day through respiration, the photosymbionts also produce oxygen during daytime. We clearly see an increase in the oxygen concentration during the day (high calcification rates) and a reduction in oxygen during the night (low calcification rates). That would strengthen the hypothesis that the photosymbiont activity is strongly coupled to the calcification rate. The clams (with their symbionts) were the only significant biomass in the tank. Some clams had small amounts of algae still stuck on their shells but that was very little (we did not brush them before transfer into the culturing jars). These algae will have contributed little to the overall oxygen production at daytime.

336: One source of potential error in estimations of day-night differences in EPF residence time: did the clams partially close at night? They tend to close partially at night (see Killam et al., 2023) in a defensive posture (this is not always the case, such as some aquarium settings where predation is not an issue, so yours might not have). But if they close, their overall internal volume would be smaller, and as such their extrapallial space would be smaller in volume.

They were not fully closed when the lights were off, but the mantle may have been more extended outward over the ventral shell rim during daytime (light exposure). As we did not monitor valve movement it is difficult to say how this affected the EPF volume. They can retreat their mantles and close quite quickly and that rapid response should not change the EPF volume, although it is logical that if they have less space for several hours, they might reduce the EPF volume as well. Either way it is a point worth adding to the discussion.

359: What nutrients are higher in daytime? Generally, N-bearing nutrients would be expected to be lower in the day on average due to assimilation and higher at night due to remineralization/respiration being dominant. 

You are absolutely right; nutrient availability should be higher at nighttime. Thanks a lot for noticing this mistake, we will change the statement accordingly.

Section 4.4.1: I suspect that in your tanks, this was not a major factor, but the reason I asked about nutrient measurements is that in other experimental setups where other biota might be present in the aquarium, I believe nitrification would represent a significant source of error trying to replicate this approach. In closed-system aquarium setups, ammonium waste from the inhabitants is converted to nitrate, a process which can destroy alkalinity. In your constrained single-species tanks, I bet that the clams were quickly re-absorbing all ammonium produced to feed their symbionts, making it not a factor of concern in your calculations. However, I'd advise adding a mention here of that process to aid in reproducibility, since this hidden sink of alkalinity could make replication difficult in some research aquaria. Also for that reason, if you have any observations of nutrients like NH4/NO3/NO2/etc during the experiment, please include them.

As stated above we did unfortunately not measure nutrient contents in the water. We will include the point that isolated culturing experiments are important for reliable alkalinity measurements.

376: It is better to refer to them as Tridacninae or tridacnines, since they are in the family Cardiidae

Thanks for pointing this out. We will change it.

Kind regards,

Iris Arndt on behalf of all coauthors