Mesozooplankton Gut Fluorescence

Methods

Mesozooplankton sampling by Bongo net

Mesozooplankton are sampled with quantitative plankton nets, usually a 0.71-m diameter Bongo net frame, using the CalCOFI sampling protocol (https://cce.lternet.edu/data/methods-manual/augmented-cruises/calbobl-net-depolyment). However, unlike CalCOFI, we use 202-um Nitex mesh nets and hard cod ends with the same size mesh. A calibrated General Oceanics flow meter is mounted in the mouth of one net. Samples are typically double oblique (from surface to 210 m to surface; depth is based on 300 mwo with a 45° wire angle) or vertical (from surface to 100 m to surface; nominal 0° wire angle). A stainless steel weight (ca. 50 kg) is mounted to the bottom of the bongo frame. Samples from one side of the bongo net are preserved in sodium borate-buffered Formalin (final concentration of 5% Formalin = 1.85% formaldehyde). Samples from the other net are used for gut fluorescence and dry mass biomass determination. The latter samples are anesthetized with soda water immediately upon retrieval to inhibit net feeding and regurgitation. Both nets are carefully washed to ensure quantitative recovery of all sampled material.

Size fractionation and flash freezing samples

The anaesthetized zooplankton sample is taken promptly into a shipboad laboratory and split on a Folsom splitter to obtain 3/8 of the sample for gut fluorescence, 3/8 of the sample for dry biomass, and 1/4 of the sample for molecular probes. Samples are kept cool while processing. The gut fluorescence and the biomass aliquots are passed separately through a nested 5-filter series of Nitex sieves resulting in 5 size fractions (0.2-0.5 mm, 0.5-1.0 mm, 1.0-2.0 mm, 2.0-5.0 mm, > 5.0 mm). Each of these samples is concentrated on a 202-um mesh filter, placed in a labelled plastic petri dish, and flash frozen in liquid N2 for analysis ashore.

Sorting and fluorometric analysis in the laboratory.

In the laboratory, frozen filters of zooplankton are placed on a dividing block and sliced into fractions for analysis using a clean razor blade. Typically filters are divided as follows: 5 mm - ½ filter ; 2 mm - ¼ filter ; 2 mm - two ¼ filters; 0.5 mm - two 1/8 filters; 0.2 mm - two 1/8 filters. Each fraction to be analyzed is kept on ice for a short time, in the dark, until sorting. Sorting is done under a dissecting microscope with cool white illumination and no room lights, to avoid photodegrading pigments. Detritus and phytoplankton debris are separated from zooplankton using fine needles. The cleaned zooplankton are transfered to a vial containing 10 mL of 90% acetone in an extraction tube and kept on ice, in the dark, until extraction. Extraction is done by immersing a micro-sonication probe into the acetone solution and applying four 5-s bursts at a sonciator setting of 7, while holding the extraction tube in a beaker in ice water. If organisms remain intact, the sample is sonciated with two additional 5-s bursts. Pigments are extracted in a -20 C freezer for one hour in the dark, then inverted twice to mix, placed in a clincial centrifuge, and spun for 10 min at maximum speed. For analysis: Two 4-mL aliquots of supernatant are pipetted into separate cuvettes. Fluorescence is recorded in a calibrated Turner Designs 10AU fluorometer, before and after acidification with 10% HCl. 90% acetone blanks are subtracted from sample readings.

Calculation of grazing per unit volume and grazing rate per unit volume.

Fluorometric readings are converted to equivalent concentration of Chl-a and Phaeopigments (phaeophorbide plus phaeophytin) using the standard equations from Strickland and Parsons (1972. A practical handbook of seawater analysis, 2nd ed. Fisheries Research Board of Canada. Bull. 167: 328 p). The amount of pigment analyzed is corrected for filter aliquot fraction, for the fraction of the sample split at sea, and for the volume of water filtered by the plankton net, to obtain the mass of phytoplankton pigment ingested by zooplankton per m3 of seawater filtered. Data can also be expressed as the mass of pigment ingested by mesozooplankton per m2 of sea surface by multiplying by the maximum depth of the tow. Concentrations of pigment ingested can be converted a grazing rate (i.e., mass of pigment ingested per m3 or m2 per unit time) by correcting for the temperature-dependent gut turnover rate of pigments. Gut turnover rates have been determined by Dam and Peterson (1988) as updated in Bamstedt et al. (2000. p. 297-399. In R. P. Harriset al [eds.], ICES Zooplankton Methodology Manual. Academic.) For this purpose, zooplankton are assumed to feed primarily at the temperature corresponding to the depth of the chlorophyll-a maximum layer.