Establishing typical values for hemocyte mortality in individual mussels ( Mytilus californianus ) using fluorescence‐activated cell sorting

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. The ability to serially sample hemocytes in an individual animal would allow one to monitor the animal’s temporal responses to stressors, potentially providing insights into the cellular mechanisms that govern survival under stress. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter‐individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter‐ and intraindividual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus , free from typical abiotic stressors. This animal is a dominant species in Eastern Pacific rocky intertidal habitats, where it encounters wide variation in abiotic stress, e.g., from temperature, due to microhabitat variation and transitions between air and water during the tidal cycle. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence‐activated cell sorting (FACS) of cells stained with propidium iodide (PI), which enters and fluoresces in dead cells. Our study establishes the first FACS gating methods for M. californianus , and demonstrates that FACS can distinguish the two hemocyte subtypes: granulocytes and hyalinocytes. Furthermore, hemolymph can be repeatedly sampled from individual mussels without killing them, however, there is substantial inter‐ and intra‐individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels’ hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%. Pooled‐sample means were similar to individual‐sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels’ temporal responses to various stressors on a cellular level. Support or Funding Information NSF IOS 1655529 to M.W.D. and Myers Oceanographic & Marine Biology Trust to N.E.M.Group hemocyte mortality across samples. The midline of the boxplots indicates the median % of dead hemocytes (assessed by PI using FACS) of the four Groups that were each sampled repeatedly at four timepoints, with either: 24, 48, 72, or 168 h between samples. Diamonds are the mean. *Significant difference between that sample vs. Sample 4. ‡Significant difference from 24 h Group for that sample timepoint. †Significant difference from all other Groups for that sample timepoint (all p <0.05).Mean ± SD Individual variability & reliability of hemocyte mortality across samples.Sample Group Individual Sample SD Individual Sample Range Individual Sample Differences Individual Sample CV24 h 3.7 ± 2.7 8.2 ± 5.8 4.3 ± 3.4 0.77 ± 0.7248 h 3.3 ± 2.7 7.1 ± 5.6 3.5 ± 2.6 0.56 ± 0.3072 h 1.7 ± 1.0 3.7 ± 2.2 2.3 ± 1.6 0.49 ± 0.21168 h 2.0 ± 1.3 4.3 ± 2.8 2.0 ± 1.4 0.52 ± 0.23Overall 2.7 ± 2.2 5.9 ± 4.7 3.0 ± 2.5 0.59 ± 0.43All data presented in the table are percentages (of hemocyte mortality, except for the CV). Each of these metrics were calculated by first taking each individual’s SD, range, between‐sample differences, and CV, and then taking the overall Group mean ± SD. No statistical analyses were completed for these data, as they are simply meant to describe the overall variability and reliability of repeated sampling within the same individuals across four repeated hemolymph samples.