Banded dye murex, Hexaplex trunculus measurements of total weight, soft tissue weight and net calcification rate in males and females over the 310 day ocean acidification experiment

Physiological traits of banded dye murex were measured during the exposure to a range of pH over the course of 310 days. Gastropods were collected in Bistrina Bay, part of the Mali Ston Bay in the Southeastern Adriatic Sea (42°52'19.1 N 17°42'02.3 E). Experiment was performed in Laboratory for Mariculture, University of Dubrovnik. Banded dye murexes were maintained in nine treatment tanks (volume 130L) with a flow-through water system with filtered, UV-sterilized, and aerated ambient seawater pumped directly from the Bistrina Bay adjacent to the laboratory facilities. Nine pH treatments were assigned to tanks, ranging from pHT 7.95 to 7.22. pH was manipulated by bubling pure CO2 gas, controlled with pH computers (Milwaukee MC122).Temperature, salinity, dissolved oxygen concentration were measured using multiparametric probes (YSI Pro 30 & Oxygen Handy Polaris, respectively). pH was measured on a total scale (pHT) by the potentiometric method with TRIS (2-amino-2-hydroxy-1,3-propanediol) buffer. Total alkalinity (TA, µmol kg-1) was determined with the potentiometric two-point open-cell 0.1 M HCl titration. Other seawater carbonate chemistry parameters (pCO2, ΩCa, ΩAr) were calculated based on known TA and pHT for a given salinity using CO2SYS software. Measurments of total weight were performed eight times during the experiment: day 28, 59, 91, 133, 172, 196, 236 & 310. Total wet weight was measured to the nearest 0.01 g on the top-load scale (Mettler toledo JL602-G/L) for the same individuals nine times over the course of experiment. Prior to weighing, snails were placed on absorbent blotting paper for approximately 20 minutes to remove excess water. The procedure was repeated eight times during the experiment (day 28, 59, 91, 133, 172, 196, 236, 310). Total weight growth rate (TWGR in g day-1) was calculated for each individual as the change in the shell weight between successive observation points divided by time. Net calcification rate was measured with a buoyant weight technique where the weight of the whole snails in seawater (buoyant weight) was determined by placing them on an improvised holder made of copper wire attached directly to the hook at the bottom of scale (Mettler toledo JL602-G/L). The scale was placed on a stand with a hole in the centre, under which was the container of seawater. Immediately before weighing, the snails were carefully forced to close the operculum to clear their mantle cavities of possible air. The balance was tared to compensate for the weight of the improvised holder, and the weight of the submerged snails was recorded to the nearest 0.01 g. The procedure was repeated seven times during the experiment (day 59, 91, 133, 172, 196, 236, 310). To obtain estimates of shell weight from immersed weight, a regression between shell weight and buoyant weight was performed. Net calcification rate (NCR in g day-1) was calculated for each individual as the change in shell weight between successive observation points divided by time. The soft body weight was calculated for each gastropod as the difference between the total weight and the shell weight. Soft body growth rate (STWGR in g day-1) was calculated for each individual as the difference in soft body weight between successive observation points divided by time. At the end of the experiment, specimens from all treatments were sampled and frozen until further analysis. For the sex determination, shells were cracked with a vice to expose the soft tissue. Males were identified by the presence of a penis behind the right tentacle, females by the vaginal opening. As temperature was varying naturally over the course of the experiment, statistical analysis was divided into periods to account for the modulating effect of the temperature. Carbonate Chemistry data were downloaded from the PANGAEA dataset (see Source). In order to allow full comparability with other ocean acidification data sets, the R package seacarb (Gattuso et al, 2024) was used to compute a complete and consistent set of carbonate system variables, as described by Nisumaa et al. (2010). In this dataset the original values were archived in addition with the recalculated parameters (see related PI). The date of carbonate chemistry calculation by seacarb is 2024-02-26.

本数据集针对带纹染料骨螺（banded dye murex）开展了为期310天的实验，测定了其在不同pH梯度暴露条件下的生理性状。实验所用腹足类（gastropods）采集于亚得里亚海东南部马里什通湾（Mali Ston Bay）的比斯特里纳湾（Bistrina Bay），坐标为42°52'19.1 N 17°42'02.3 E，实验在杜布罗夫尼克大学海水养殖实验室（Laboratory for Mariculture, University of Dubrovnik）完成。

将带纹染料骨螺饲养于9个容积为130 L的试验养殖缸中，采用流水系统，水源为直接从实验室邻近的比斯特里纳湾抽取的天然海水，经过滤、紫外灭菌与充气处理。为9个养殖缸设置9组pH处理梯度，范围覆盖总尺度pH（pHT）7.95至7.22，通过通入纯二氧化碳（CO₂）气体调控pH，由Milwaukee MC122型pH控制器自动执行调控。

水温、盐度与溶解氧浓度分别采用多参数水质检测仪（YSI Pro 30）与溶解氧手持测定仪（Oxygen Handy Polaris）进行测定。总尺度pH（pHT）采用电位滴定法，以TRIS（2-氨基-2-羟基-1,3-丙二醇）缓冲液为校准物质完成测定。总碱度（total alkalinity, TA，单位为µmol kg⁻¹）采用两点开放式电位滴定法，以0.1 mol/L盐酸（HCl）为滴定液测定。其余海水碳酸盐化学参数，包括二氧化碳分压（pCO₂）、方解石饱和度（ΩCa）与文石饱和度（ΩAr），均基于已知的总碱度、总尺度pH与盐度，通过CO2SYS软件计算得到。

实验期间共开展8次总重量测定，时间点分别为第28、59、91、133、172、196、236与310天。针对同一批个体共进行9次总湿重测定，测定精度可达0.01 g，使用梅特勒托利多JL602-G/L型顶载天平。称重前，将螺类置于吸水纸上静置约20分钟以去除体表多余水分，该操作在实验期间共重复8次，对应上述8个时间点。个体总重量生长率（total weight growth rate, TWGR，单位为g day⁻¹）通过相邻两次观测点的壳重差值除以间隔时间计算得到。

净钙化速率采用浮力重量法测定：将螺类置于铜线自制的固定架上，直接悬挂于梅特勒托利多JL602-G/L型天平底部挂钩处，测定其在海水中的浮力重量。将天平放置于带有中心开孔的支架上，支架下方放置海水容器。称重前需小心按压螺类使其厣闭合，以排出外套腔中可能残留的空气。先对天平进行去皮校准以抵消自制固定架的重量，随后记录浸没于海水中的螺类重量，精度可达0.01 g。该操作在实验期间共重复7次，对应时间点为第59、91、133、172、196、236与310天。为通过浮力重量估算壳重，本研究建立了壳重与浮力重量之间的回归关系。个体净钙化速率（net calcification rate, NCR，单位为g day⁻¹）通过相邻两次观测点的壳重差值除以间隔时间计算得到。

腹足类的软体组织重量通过总重量与壳重的差值计算得到。个体软体组织生长率（soft body growth rate, STWGR，单位为g day⁻¹）通过相邻两次观测点的软体组织重量差值除以间隔时间计算得到。实验结束后，采集所有处理组的标本并冷冻保存，以待后续分析。为鉴定性别，使用台虎钳夹碎螺壳以暴露软体组织：雄性可在右触手后方发现阴茎，雌性则可见阴道开口。

由于实验期间水温存在自然波动，为消除温度的调控效应，统计分析按实验阶段进行划分。本数据集的碳酸盐化学数据下载自PANGAEA数据库（详见数据源）。为确保与其他海洋酸化数据集具有完全可比性，本研究参照Nisumaa等（2010）的方法，使用R语言包seacarb（Gattuso等，2024）计算得到一套完整且一致的碳酸盐系统变量。本数据集同时存档了原始测定值与重新计算得到的参数（详见相关首席研究员，PI）。使用seacarb完成碳酸盐化学参数计算的日期为2024年2月26日。