Analysis methodology referenced in:
Roman, M.R. et al, Zooplankton variability on the equator at 140W during the JGOFS EqPac Study. Deep Sea Research II, 42(2-3) : 673-693, 1995

Mesozooplankton Grazing - Isotope Method

Measurements of grazing rates by the radioisotope uptake technique were done day and night, immediately following the MOCNESS tows, in short term, in situ incubations at 1, 10, 30, 50, 70 and 90 m (Roman and Rublee, 1981). Five-liter Plexiglas chambers (General Oceanics) with 64 µm-mesh covering the bottom were lowered 10 m past the desired depths and then raised to concentrate zooplankton in the chambers. A messenger triggered the close of the bottles and released the radioisotope tracers (25 µCi l of methylH-thymidine, > 75 Ci mmol and 50 µCi l Na CO, 55.0 Ci mmol ) into the chambers. After a 45-minute incubation on the hydrowire, the chambers were retrieved, zooplankton collected on nested 200 and 64 µm sieves. The zooplankton were rinsed (filtered seawater, 10% HCl and deionized water) onto preweighed 12 µm pore-size Nuclepore filters and dried. Using a dissecting microscope, visible detritus and phytoplankton were removed with a sable brush. The filters were weighed and the weight-specific dpm of the isotopes were measured. Zooplankton carbon was assumed to be 40% of their dry weight to calculate the dpm mg C of the zooplankton. The labelled particulate matter (< 64 µm) was collected on 0.2 µm and 2.0 µm pore-size Nuclepore filters to determine the specific activity of the particulate matter. We derived weight-specific corrections for the absorption and adsorption of the isotopes in shipboard experiments using both filtered seawater and time-0 controls. These corrections were generally less than 10% of experimental values. The isotope activity of the labelled particulate matter (> 2 µm) and zooplankton were used to calculate zooplankton filtration rates, F = liters filtered. mg zooplankton C h, after Daro (1978). The grazing impact of the zooplankton community, expressed as liters filtered m h, was calculated as the product of the weight-specific filtration rate determined from the in situ incubations and the zooplankton biomass in the same depth interval determined immediately prior to the grazing incubation. We used both C bicarbonate and [methyl] H thymidine to estimate the filtration rates of mesozooplankton upon autotrophs (phytoplankton and protozoa that consumed phytoplankton) and heterotrophs (free-living and attached bacteria and protozoa that have consumed bacteria).

Mesozooplankton Grazing - Gut Fluorescence Method

Zooplankton were collected day and night, immediately preceding MOCNESS tows, from vertical tows with opening-closing, 3/4 meter-Puget Sound nets equipped with 200 µm mesh and solid cod ends. Nets were towed to sample depth strata similar to those sampled by the MOCNESS in the upper 120 m. Three depth strata sampled were: 0-20 m, 20-60 m and 60-120 m. The contents of each net was immediately sieved under dim light red light to yield three size fractions: 2.0-1.0 mm; 1.0-0.5 mm and 0.5-0.2 mm. Each fraction was immediately filtered onto a glass fiber filter (GF/A) and frozen at -20 C. This procedure took about 5 min. Samples were analyzed in the laboratory ashore. We employed a modification of the procedure described by Dam and Peterson (1988). Thawed samples were placed under a dissecting scope, animals picked individually with jeweler's forceps without regard to species, gently rinsed with 0.2 µm-filtered seawater and placed in centrifuge tubes containing 90% high-grade, cold acetone solution. The whole procedure was performed under dim, red-light illumination. We typically took triplicate subsamples of 30-100 animals, with the number of individuals depending on the size fraction considered. A similar number of animals per sample was also picked for weight (C,N) determination. The tubes containing the acetone and animals were kept frozen at -20 C for at least 24 h for pigment extraction. The amount of pigment (chlorophyll and phaeopigments) per individual was measured fluorometrically and expressed in terms of chlorophyll (body carbon) (Dam et al., 1993).

To convert gut pigment values to pigment ingestion rates, knowledge of the gut passage time is necessary. This is typically estimated from the reciprocal of the gut clearance rate constant (GCRC, units of time) of animals placed in filtered seawater (review in Dam and Peterson, 1988). GCRC estimated over the first 30-40 min of gut evacuation is not statistically different from the gut evacuation rate constant of feeding animals (Kiorboe and Tiselius, 1987; Ellis and Small, 1990). We estimated GCRC every time collections for gut pigment were made. To collect animals for gut evacuation experiments, one tow (upper 60 m) was done immediately after the ones for gut fluorescence. We estimated GCRC of animals in the different size fractions (0.2-0.5 mm; 0.5-1.0 mm; 1.0-2-0 mm) by employing the procedure described by Small et al. (1989); i.e., animals were quickly sorted into separate containers, kept in the dark in a temperature-controlled room. We monitored the decline in gut pigment, at 5-min. intervals, over the first 25 min of gut evacuation. Seventy-five per cent of the variance of GCRC is explained by temperature, with a Q of 2.2 (Dam and Peterson, 1988). Therefore, we corrected GCRC for differences in temperature from the depth at which animals were collected. Weight-specific ingestion rates for each depth layer were estimated from knowledge of gut pigment and GCRC. The grazing impact of zooplankton was estimated from the product of weight-specific ingestion rates and biomass for each size class.

Literature Cited

Dam, H.G., C.A. Miller and S.H. Jonasdottir (1993). The trophic role of mesozooplankton at 47 N, 20 W during the North Atlantic Bloom Experiment. Deep-Sea Research, 40: 197-212.

Dam, H.G. and W.T. Peterson (1988). The effect of temperature on the gut clearance rate constant of planktonic copepods. Journal of Experimental Marine Biology and Ecology, 123: 1-14.

Daro, M.H. (1978). A simplified C method for grazing measurements on natural planktonic populations. Helgolander wiss. Meeresunters, 31: 241-248.

Ellis, S.G. and L.F. Small (1989). A comparison of gut evacuation rates of feeding and non-feeding Calanus marshallae. Marine Biology, 103: 175-181.

Kiorboe, T. and P. Tiselius (1987). Gut clearance and pigment destruction in a herbivorous copepod, Acartia tonsa, and the determination of in situ grazing rates. Journal of Plankton Research, 9: 525-534.

Roman, M.R. and P.A. Rublee (1981). A method to determine in situ grazing rates on natural particle assemblages. Marine Biology, 65: 303-309.

Small, L.F., M.R. Landry, R.W. Eppley, F. Azam and A.F. Carlucci (1989). Role of plankton in the carbon and nitrogen budgets of the Santa Monica Basin, California. Marine Ecology Progress Series, 56: 57-74.

Wiebe, P.H., A.W. Morton, A.M. Bradley, R.H. Backus, J.E. Craddock T.J. Cowles, V.A. Barber and G.R. Flierl (1985). New developments in the MOCNESS, an apparatus for sampling zooplankton and micronekton. Marine Biology, 87: 313-323.

Analysis methodology referenced in:
Roman, M.R. et al, Zooplankton variability on the equator at 140W during the JGOFS EqPac Study. Deep Sea Research II, 42(2-3) : 673-693, 1995

Mesozooplankton Grazing - Isotope Method

Measurements of grazing rates by the radioisotope uptake technique were done day and night, immediately following the MOCNESS tows, in short term, in situ incubations at 1, 10, 30, 50, 70 and 90 m (Roman and Rublee, 1981). Five-liter Plexiglas chambers (General Oceanics) with 64 µm-mesh covering the bottom were lowered 10 m past the desired depths and then raised to concentrate zooplankton in the chambers. A messenger triggered the close of the bottles and released the radioisotope tracers (25 µCi l of methylH-thymidine, > 75 Ci mmol and 50 µCi l Na CO, 55.0 Ci mmol ) into the chambers. After a 45-minute incubation on the hydrowire, the chambers were retrieved, zooplankton collected on nested 200 and 64 µm sieves. The zooplankton were rinsed (filtered seawater, 10% HCl and deionized water) onto preweighed 12 µm pore-size Nuclepore filters and dried. Using a dissecting microscope, visible detritus and phytoplankton were removed with a sable brush. The filters were weighed and the weight-specific dpm of the isotopes were measured. Zooplankton carbon was assumed to be 40% of their dry weight to calculate the dpm mg C of the zooplankton. The labelled particulate matter (< 64 µm) was collected on 0.2 µm and 2.0 µm pore-size Nuclepore filters to determine the specific activity of the particulate matter. We derived weight-specific corrections for the absorption and adsorption of the isotopes in shipboard experiments using both filtered seawater and time-0 controls. These corrections were generally less than 10% of experimental values. The isotope activity of the labelled particulate matter (> 2 µm) and zooplankton were used to calculate zooplankton filtration rates, F = liters filtered. mg zooplankton C h, after Daro (1978). The grazing impact of the zooplankton community, expressed as liters filtered m h, was calculated as the product of the weight-specific filtration rate determined from the in situ incubations and the zooplankton biomass in the same depth interval determined immediately prior to the grazing incubation. We used both C bicarbonate and [methyl] H thymidine to estimate the filtration rates of mesozooplankton upon autotrophs (phytoplankton and protozoa that consumed phytoplankton) and heterotrophs (free-living and attached bacteria and protozoa that have consumed bacteria).

Mesozooplankton Grazing - Gut Fluorescence Method

Zooplankton were collected day and night, immediately preceding MOCNESS tows, from vertical tows with opening-closing, 3/4 meter-Puget Sound nets equipped with 200 µm mesh and solid cod ends. Nets were towed to sample depth strata similar to those sampled by the MOCNESS in the upper 120 m. Three depth strata sampled were: 0-20 m, 20-60 m and 60-120 m. The contents of each net was immediately sieved under dim light red light to yield three size fractions: 2.0-1.0 mm; 1.0-0.5 mm and 0.5-0.2 mm. Each fraction was immediately filtered onto a glass fiber filter (GF/A) and frozen at -20 C. This procedure took about 5 min. Samples were analyzed in the laboratory ashore. We employed a modification of the procedure described by Dam and Peterson (1988). Thawed samples were placed under a dissecting scope, animals picked individually with jeweler's forceps without regard to species, gently rinsed with 0.2 µm-filtered seawater and placed in centrifuge tubes containing 90% high-grade, cold acetone solution. The whole procedure was performed under dim, red-light illumination. We typically took triplicate subsamples of 30-100 animals, with the number of individuals depending on the size fraction considered. A similar number of animals per sample was also picked for weight (C,N) determination. The tubes containing the acetone and animals were kept frozen at -20 C for at least 24 h for pigment extraction. The amount of pigment (chlorophyll and phaeopigments) per individual was measured fluorometrically and expressed in terms of chlorophyll (body carbon) (Dam et al., 1993).

To convert gut pigment values to pigment ingestion rates, knowledge of the gut passage time is necessary. This is typically estimated from the reciprocal of the gut clearance rate constant (GCRC, units of time) of animals placed in filtered seawater (review in Dam and Peterson, 1988). GCRC estimated over the first 30-40 min of gut evacuation is not statistically different from the gut evacuation rate constant of feeding animals (Kiorboe and Tiselius, 1987; Ellis and Small, 1990). We estimated GCRC every time collections for gut pigment were made. To collect animals for gut evacuation experiments, one tow (upper 60 m) was done immediately after the ones for gut fluorescence. We estimated GCRC of animals in the different size fractions (0.2-0.5 mm; 0.5-1.0 mm; 1.0-2-0 mm) by employing the procedure described by Small et al. (1989); i.e., animals were quickly sorted into separate containers, kept in the dark in a temperature-controlled room. We monitored the decline in gut pigment, at 5-min. intervals, over the first 25 min of gut evacuation. Seventy-five per cent of the variance of GCRC is explained by temperature, with a Q of 2.2 (Dam and Peterson, 1988). Therefore, we corrected GCRC for differences in temperature from the depth at which animals were collected. Weight-specific ingestion rates for each depth layer were estimated from knowledge of gut pigment and GCRC. The grazing impact of zooplankton was estimated from the product of weight-specific ingestion rates and biomass for each size class.

Literature Cited

Dam, H.G., C.A. Miller and S.H. Jonasdottir (1993). The trophic role of mesozooplankton at 47 N, 20 W during the North Atlantic Bloom Experiment. Deep-Sea Research, 40: 197-212.

Dam, H.G. and W.T. Peterson (1988). The effect of temperature on the gut clearance rate constant of planktonic copepods. Journal of Experimental Marine Biology and Ecology, 123: 1-14.

Daro, M.H. (1978). A simplified C method for grazing measurements on natural planktonic populations. Helgolander wiss. Meeresunters, 31: 241-248.

Ellis, S.G. and L.F. Small (1989). A comparison of gut evacuation rates of feeding and non-feeding Calanus marshallae. Marine Biology, 103: 175-181.

Kiorboe, T. and P. Tiselius (1987). Gut clearance and pigment destruction in a herbivorous copepod, Acartia tonsa, and the determination of in situ grazing rates. Journal of Plankton Research, 9: 525-534.

Roman, M.R. and P.A. Rublee (1981). A method to determine in situ grazing rates on natural particle assemblages. Marine Biology, 65: 303-309.

Small, L.F., M.R. Landry, R.W. Eppley, F. Azam and A.F. Carlucci (1989). Role of plankton in the carbon and nitrogen budgets of the Santa Monica Basin, California. Marine Ecology Progress Series, 56: 57-74.

Wiebe, P.H., A.W. Morton, A.M. Bradley, R.H. Backus, J.E. Craddock T.J. Cowles, V.A. Barber and G.R. Flierl (1985). New developments in the MOCNESS, an apparatus for sampling zooplankton and micronekton. Marine Biology, 87: 313-323.