Starved copepods were incubated in bottles with each phytoplankton, then gut pigment was analyzed on a microplate reader. Microplate readers are capable of measuring low levels of fluorescence on small volumes of sample, allowing for feeding estimates on individual or relatively small groups of copepods (5–50).

All experiments were conducted at 19 ± 1°C. Copepods were sorted from the culture and allowed to evacuate their guts as above. Copepods were then pipetted individually into clean polystyrene Nalgene® bottles (P. marinus adults: 2 L; O. davisae adults: 1 L; all nauplii: 500 mL) containing fresh GF/F culture water. For P. marinus, four bottles containing 15 adult females each and six bottles containing 40 nauplii each were used. For O. davisae, four bottles containing 50 adult females each and six bottles containing 175 nauplii each were used.

Phytoplankton culture was added to the bottles to attain a final concentration of approximately 500 µg C L-1, and inoculations of each bottle were staggered by 10 min to allow for processing time for each bottle. The same phytoplankton culture was used for both adults and nauplii.

Incubations were conducted as in the GPI experiment except that durations were 30 min for P. marinus and 60 min for O. davisae, based on the results of preliminary experiments (see below). Longer incubation times were chosen for O. davisae since this copepod is smaller than P. marinus, and more feeding time was needed for this species to ingest detectable amounts of pigment. Incubations were terminated as for the GPI experiments. Copepods were processed for analysis individually (P. marinus adults) or in groups (5 P. marinus nauplii; 5 O. davisae adults, 50 nauplii).

Under dim light, copepods were pipetted onto ethanol-rinsed, 1x1 cm, 20 µm pore size nylon mesh filter squares. Excess moisture was wicked away with a Kimwipe™. Filter squares were then put into 1.5 mL microcentrifuge tubes that were immediately placed on dry ice in a dark container and stored at -80°C. Processing times averaged two min per bottle.

A control group was processed with each experiment consisting of copepods that were treated identically to the experimental group, except that the incubation was terminated immediately after the phytoplankton was added. The purpose of this control was to account for any sources of contamination (e.g., phytoplankton cells that may have stuck to the copepod).

One hundred µL of 95% ethanol was dispensed into each microcentrifuge tube and samples were extracted for 24 h. Ethanol was used as the extraction solvent because it does not damage polystyrene microplates. Filters were removed using forceps rinsed with 95% ethanol between samples.

A Tecan Infinite F200 or Biotek Synergy 2 microplate reader was used for each analysis. Each microplate reader contained a 430/20 EX, 680/20 EM filter pair for chlorophyll a. The microplate reader was kept in a dark room maintained at 14ºC to minimize pigment loss and evaporation of solvent during analysis.

A Chl a standard was prepared by dissolving commercially available pure Chl a (source: Anacystis nidulans cyanobacteria, Sigma Chemical: C6144) in 95% ethanol. The concentration of the standard was measured on a Agilent 8453 spectrophotometer (Agilent Technologies) and calculated using published equations (Ritchie 2008).

The Chl a-ethanol stock standards were dispensed in 5 mL aliquots into 5 mL glass ampoules. The headspace was displaced using a commercially available 99.9% argon gas dispenser. The ampoules were flame sealed in dim light and on dry ice in a ventilated fume hood, then placed in a darkened container and kept in a -20 ºC freezer. The concentration of the standards was checked every 1–2 months by measuring the Chl a concentration of the stock standards on a spectrophotometer periodically for one year. No degradation in the standards was detected during that period. A standard dilution series for each experiment was prepared by dispensing known volumes of Chl a standard into 95% ethanol to achieve final concentrations of 0.05, 0.1, 0.3, 1, and 2 ng Chl a (100 µL)-1, the range of pigment concentrations in the copepod samples.

For each assay, standards, sample extracts, and 95% ethanol blanks were dispensed in 60 µL aliquots into white, skirted 96-well microplates (Bio-Rad, model HSP-9601) fitted with Optical Flat 8-cap strips (Bio-Rad, model TCS-0803). White plates provided the highest sensitivity for the assay. All samples and materials were allowed to acclimate to the temperature of the room before analysis. The microplate reader settings were: 25 flashes, optimal gain, center read.

Each microplate was read once, and then all samples were acidified with 1.0 µL of 1 mol L-1 HCl using a 0.1–2 µL multichannel pipettor. The microplate was then re-read with the gain set manually to the value set automatically by the instrument for the initial readings. The plate was read repeatedly for 10 min until readings stabilized, which typically occurred in under three min, but some samples took ~5–7 min to completely stabilize. The last readings in the 10-min cycle were used as the post-acidification fluorescence values to ensure that steady state had been reached.

2_feeding.csv