Dataset: 4b_min fluorescence thresholds

The copepod Pseudodiaptomus marinus was cultured at RTC and used in the experiments. The evening before an experiment the copepods were size fractioned to separate the adults from the nauplii (larval stage) and were placed in 0.45 µm filtered San Francisco Bay water with the alga Rhodomonas salina, to allow for feeding. We handpicked 25 adults and 50 nauplii (Naupliar stage 3 and 4) which were maintained in 3 L of previously described water. The following morning (0900) the copepods were rinsed into freshly filtered 0.45 µm San Francisco Bay water and held for three hours to allow the individuals to clear the contents of their guts. While the copepods cleared their guts, the ciliates (Codonellopsis sp. or Favella sp.) were stained with the fluorescent stain, CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate) (Li et al. 1996) for one hour at 1 µmolar solution. After staining was complete, the ciliates were rinsed from the stain by being rinsed over a 35µm mesh and gently dunked into clean 0.45 µm filtered San Francisco Bay water. The ciliates were then resuspended and introduced to the copepods, which were allowed to feed for 1 hour.

To obtain the best images of the copepod digestive track the individuals needed to be alive. After 1 hour of feeding the copepods were rinsed over a 100 µm mesh which allowed for the separation of the ciliates and copepods. Using a dissecting microscope, individuals were transferred to a microscope slide in a small amount of water, which held each individual on its side for the best image of the gut. Once in place the microscope slide was transferred to the Olympus IX83 Epifluorescence microscope, and each individual was imaged under two settings. The first setting was bright-field and the second setting was the GFP filter (395 nm excitation, 500 nm emission) which allowed for the detection of the stained ciliate in the digestive track. This experiment was repeated twice.

Our next approach to identify the grazing of copepods on ciliates focused on offering the copepods a natural assemblage of food that was spiked with a known amount of stained ciliates. Three liters of surface seawater was collected from the seawall at RTC, size fractioned by reverse filtration into a 20-50 µm cohort, which removed other large grazers yet maintained other species of ciliates and phytoplankton. Cultured Favella sp. was stained as previously described and then introduced to the natural assemblage. Two containers (1500 ml each) were made and the copepods were allowed to feed for 1 hour. At the end of the grazing period 3 replicate samples of 50 ml each were preserved with 0.1% Acid Lugol and 3 replicate samples of 50 ml each were preserved in 50% Glutaraldehyde.