Establishing typical values for hemocyte mortality in individual California mussels, Mytilus californianus

The purposes of this study were to: (1) establish FACS as a method to analyze hemocytes in M. californianus, (2) evaluate whether hemolymph can be repeatedly drawn from an individual mussel without adverse effects, and if so, whether the time interval between repeated samples affects hemocyte mortality or organism survival, and (3) describe the typical intra- and inter-individual variability in hemocyte mortality across repeated samples in mussels not exposed to typical abiotic stressors. To assess whether individuals’ hemolymph could be repeatedly sampled (without killing the animal), and to establish baseline hemocyte mortality values in unperturbed mussels, hemolymph was sampled from the same individuals at time zero and three subsequent timepoints, with varying intervals between subsequent samples: 24, 48, 72 or 168 h. Each of these four different sampling intervals defined a separate experimental Group (n = 14-15 mussels per Group). We evaluated each individual's hemocyte mortality (using FACS), and then compared these individual-group samples to pooled-group samples that we created (of four individuals each). We assessed total hemocyte mortality, and mortality of the granulocyte and hyalinocyte populations as well. The attached excel file contains columns for the Mussel ID, the specific Group that the mussel was a part of, and the Sampling Timepoint (#1-4). There are two columns for each the hyalinocyte and granulocyte data: 1) the overall percentage of hyalinocytes or granulocytes relative to the total hemocyte population, and 2) the percentage of dead hyalinocytes or granulocytes relative to their respective cell type. There is one column for the overall percentage of dead hemocytes. The pooled samples that were obtained for each Group's sampling timepoint are indicated in MusselID column as "pooled_..." with an identifying number linked to that Group. There were 2 pooled samples for each Group's sample timepoint (i.e., 8 pooled samples per Group). The mussel morphometric data (shell height, width, length, and body mass) are also included for each individual.本研究的目的如下：(1) 建立荧光激活细胞分选术（FACS）作为分析加州贻贝（Mytilus californianus）血细胞的方法；(2) 评估能否从单只加州贻贝体内反复采集血淋巴且不会对其造成不良影响，若可行，则进一步明确反复采样的时间间隔是否会影响血细胞死亡率或生物体存活率；(3) 描述未暴露于典型非生物胁迫的加州贻贝中，重复采样的血细胞死亡率在个体内及个体间的典型变异特征。为评估能否反复采集个体血淋巴（不致死实验动物）并建立未受扰动加州贻贝的基线血细胞死亡率值，我们在零时及后续三个时间点从同一批个体采集血淋巴，后续采样的间隔时间分别设置为24、48、72或168小时。上述四种不同采样间隔分别对应独立实验组，每组包含14~15只加州贻贝。我们通过FACS检测每只个体的血细胞死亡率，并将这些个体样本与我们制备的混合组样本（每组由4只个体组成）进行对比分析。本次研究同时评估了总血细胞死亡率，以及粒细胞和透明细胞两个细胞群体的死亡率。附带的Excel文件包含以下列：贻贝ID（Mussel ID）、该贻贝所属实验组、采样时间点（Sampling Timepoint，1~4号）。透明细胞与粒细胞数据各设两列：1）透明细胞或粒细胞占总血细胞群体的总体百分比；2）死亡透明细胞或死亡粒细胞占对应细胞类型的百分比。另有一列代表总死亡血细胞的百分比。每个实验组的每个采样时间点对应的混合样本在Mussel ID列中以"pooled_..."格式标识，并附带与该实验组关联的识别编号。每个实验组的每个采样时间点设有2个混合样本，即每个实验组共包含8个混合样本。此外，文件还收录了每只个体的贻贝形态测量数据：壳高、壳宽、壳长及体质量。