This mortality data was obtained from several ~8 hour small-boat cruises done about every other week from August to October. Vertical tow samples were taken from the mid-bay of the Chesapeake from several stations in a transect, corresponding to the mid-line transect sampled by the two, week-long cruises (stations M1-M3, cruises 1301/1302). More stations along the same transect were included in the collection of this data set.
The plankton net was 0.5m in diameter and made of 64µm mesh, equipped to close with a mechanical messenger trigger, and was deployed from the winch arm of the R/V Parker.
A CTD cast was done at each station prior to sampling with the plankton net. Sampling depths were determined based on the location of the pycnocline; the aim was to capture zooplankton below and above the pycnocline. For sampling below the pycnocline, the net was deployed below the pycnocline, towed up to the pycnocline, triggered to close, and returned to the surface. Sampling above the pycnocline was done by standard vertical tow: deploy net to desired depth and winch to the surface.
Once brought on board, the nets were rinsed down with seawater to collect plankton in the codend. Codends were gently poured into 64µm sieves in a water bath of ambient seawater, then carefully poured into numbered glass jars for incubation; jar number, depth sampled, station, and cast number were entered into a log book.
Neutral red stain was added based on the volume in the jar- the desired concentration was 150μl per 100ml (for a stock solution of 0.1g Neutral Red powder per 10 ml DI water). The jars were returned to the water bath and incubated in the shade for 15-20 minutes (recorded in the log). After incubation, each sample was vacuum filtered onto a 64 μm mesh filter which was then transferred to a small petri dish. The dish was capped and labeled with the jar number of the sample, then wrapped in parafilm to seal. The samples were placed in a Ziploc bag labeled with the date and station and given a burst of Flash Freeze, then stored in a cooler with ice for the duration of the cruise.
After returning from each cruise, samples were immediately transferred to -20°C freezers. Samples were individually processed within a week after collection using the protocol developed by David Elliott:
The petri dish containing the sample was thawed at room temperature, then the mesh was submerged in filtered seawater and gently shaken to resuspend the sample. The sample was then transferred to a counting wheel where it was checked for density and subsampled if necessary.
The sample was then slightly acidified by adding 10% HCl drop-wise until the stained animals distinctly changed from pink to bright red. The sample was then tallied for live or dead status and general stage under dissecting microscope with darkfield illumination. Copepods which were bright red throughout their tissues were considered alive at the time of staining; copepods which had no stain, were cloudy white, or were only light pink were considered dead. Occasionally, photographs were taken of the acidified sample for secondary confirmation since the stain fades after 1 hour after acidification.
Data were entered into Excel spreadsheets and checked for transcription errors, then imported into MatLab for data analysis.
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