Effects of food concentration and diet on chromophoric dissolved organic matter accumulation and fluorescent composition during grazing experiments with the copepod Calanus finmarchicus

Abstract

Laboratory experiments were conducted with Calanus finmarchicus copepodites IV–V to examine the effects of food concentration, food type, and life stage on chromophoric dissolved organic matter (CDOM) release from copepods. Changes in CDOM absorption and fluorescence were monitored by incubating copepods in rotating bottles for 24 h in the dark with controlled diets. Copepods were fed a range of food concentrations from 800 to 15 000 cells l ⁻¹ of either Thalassiosira weissflogii and Skeletonema costatum or Prorocentrum minimum in stationary and exponential growth phases. Results from these studies indicate that diet does have an effect on the amount and type of CDOM released by C. finmarchicus . A diet of either diatoms or dinoflagellates in exponential growth will result in a release of humic-like material, while feeding on senescent cells leads to a net input of protein-like material. Only at very high senescent cell concentrations (>5000 cells l ⁻¹ ) can humic-like material accumulate. This could lead to seasonal cycles in the amount and type of CDOM released by copepods, such that humic-like material will be released in the spring and at the height of phytoplankton blooms, while protein-like material will be added to the water as the bloom dies. The humic-like material found in these studies is blue-shifted compared to the standard humic material (Peaks A, M, and C) measured in seawater. The role of this humic-like material in the environment is currently unknown, but it could be important in absorbing UV radiation.

Introduction

Zooplankton have been known for some time to contribute to dissolved organic matter (DOM) through sloppy feeding, excretion, and faecal pellet dissolution ( Copping and Lorenzen, 1980 ; Urban-Rich, 1999 ; Steinberg et al ., 2000 and 2001; Nagata, 2000 ). In fact, previous studies have found that 10–40% of the ingested carbon can be lost to dissolved organic carbon (DOC; Copping and Lorenzen, 1980 ; Strom et al ., 1997 ), indicating that a substantial portion of the carbon that is grazed by zooplankton can end up in the DOM pool. This suggests that these animals could be an important source of DOM in the marine environment. Indeed, recent studies have found that 1–13% of the annual DOC flux at the Bermuda Atlantic Time Series station (BATS) could be from vertically migrating zooplankton ( Steinberg et al ., 2000 ). In addition, 50% of the carbon within faecal pellets can be in the dissolved form and is released over several days as the pellets sink through the water providing labile DOC to the upper 500 m ( Urban-Rich, 1999 ).

Little is known about what factors regulate the amount or type of DOM released by grazers. Food concentration and cell size can be important in determining DOM release from grazers ( Jumars et al ., 1989 ; Møller and Nielsen, 2000 ). In addition, food concentration, quality in terms of carbon and nitrogen ratios and food type, e.g. diatom, dinoflagellate, or ciliate, are known to affect numerous metabolic processes, e.g. copepod grazing rates, fecundity, growth rates, faecal pellet production rates, and pellet carbon content and ammonia excretion rates ( Klein Breteler et al ., 1999 ; Lincoln et al ., 2001 ; Turner et al ., 2001 ; Besiktepe and Dam, 2002 ; Liu and Wang, 2002 ). Thus, it is logical to conclude that the type and amount of food ingested by a zooplankter will affect the amount and type of DOM released by the grazer.

DOM is a complex pool made up of many known and unknown compounds ( Benner, 2002 ). One important fraction of the DOM pool is chromophoric dissolved organic matter (CDOM). CDOM is a material that contains aromatic rings that can absorb visible and ultraviolet light. This property of CDOM means it can be important in the biogeochemistry of natural waters. Since CDOM absorbs strongly in the UV-A and UV-B regions, it is the primary factor of controlling the depth to which potentially harmful UV penetrates ( Blough and Del Vecchio, 2002 ). In addition, it can also be the dominant material absorbing the blue portion of the visible light spectrum, thereby influencing the amount and quality of photosynthetically available radiation (PAR) that reaches the phytoplankton ( DeGrandpre et al ., 1996 ). Thus, by absorbing light, CDOM can reduce primary production and affect ecosystem structure. However, absorption of light also causes photochemical reactions in CDOM that lead to the destruction or bleaching of its absorption and fluorescence ( Moran and Zepp, 1997 ; Zepp et al ., 1998 ; Blough, 2001 ). This serves as feedback, which can allow greater penetration of damaging UV radiation and beneficial PAR. In addition, absorption of light by CDOM can lead to the photo-oxidation of organic material and the production of reactive oxygen species, e.g. superoxide (O ₂ ) and hydrogen peroxide (H ₂ O ₂ ), which can react with trace metals and change their bioavailability. Since CDOM is also a complex mixture of many different aromatic compounds (e.g. humic and fulvic acids, tannins, proteins, etc.), knowledge about the sources, composition, and turnover of different components of CDOM is needed before we can fully assess its ecological significance.

Like DOM, knowledge about the chemical composition of CDOM is difficult to obtain; however, a subset of CDOM fluoresce. Since fluorescence is a highly sensitive method, this characteristic of CDOM allows us to examine the production of different CDOM compounds and trace the origin of a portion of the CDOM pool. Monitoring changes in the fluorescent characteristics of the CDOM can give information on the sources of the material, its oxidative state and the biological processing of the material ( Coble, 1996 ; McKnight et al ., 2001 ; Klapper et al ., 2002 ).

Recent studies conducted in our laboratory have found that copepods and larvaceans can excrete CDOM (Urban-Rich unpublished data). The object of this project was to use fluorescent excitation–emission matrix spectroscopy (EEMS) along with CDOM absorption and spectral slopes to examine the effect of food concentration and food type (diatom vs. dinoflagellate) on CDOM accumulation and composition during laboratory grazing studies with the copepod Calanus finmarchicus . The aim of the experiments was to determine if changes in the copepod's diet would be reflected in the CDOM. Three hypotheses were tested in these experiments:

• CDOM absorption and fluorescence would increase in the water with increasing food concentration during the grazing studies.

• CDOM fluorescence would be dominated by protein fluorescence during grazing experiments with exponentially growing cells and humic material would dominate with diets of senescent cells.

• CDOM fluorescence will be different for diets of diatoms vs. dinoflagellates.

Materials and methods

Laboratory grazing experiments were conducted with freshly caught CIV–CV C. finmarchicus during June, July, and August 2002. Copepods were collected from vertical net tows in the upper 15 m, over Stellwagen Bank in Massachusetts Bay, diluted in 20 l of surface seawater and then brought back to the laboratory. Sixty healthy CIV–CV, C. finmarchicus were sorted and placed in 6-l beakers filled with 0.2 μm filtered seawater and the desired diet ( Table 1 ). The copepods were caught, diluted, sorted, and placed in beakers for preconditioning within 4 h, thereby minimizing the stress to the animals. The copepods were allowed to acclimate to the diets for 24 h in a walk-in incubator at 15°C with a 12 h light/dark cycle, before being used in the experiments. This temperature and light field approximated that experienced by the copepods in Massachusetts Bay in the summer. The food mixtures were made by combining 6 l of 0.2 μm filtered photo-oxidized seawater with predetermined concentrations of either Thalassiosira weissflogii (10×14×18 μm) and Skeletonema costatum (8×6×14μm) or Prorocentrum minimum (10×14μm) in either stationary or exponential growth ( Table 1 ). The phytoplankton cultures were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton in Maine and were grown in the laboratory on F/2 medium at 15°C under a 12 h light/dark cycle. Prior to the start of an experiment, an aliquot of the cell culture was counted using a haemacytometer to determine the actual cell concentration. The required volume was then gently filtered through an 8.0-μm polycarbonate filter and rinsed in filtered seawater. The cells were immediately backwashed into the experimental seawater and gently stirred to obtain uniform distribution. The culture was recounted and there was always a ≤2% loss in cells due to the filtration and rinsing.

Table 1

Laboratory grazing experiments were conducted with CIV–CV, Calanus finmarchicus fed cultured phytoplankton, Thallassiosira weissflogii (T.w.), Skeletonema costatum (S.c.), or Procentrum minimum (P.m.) at various concentrations in either exponential or stationary growth for 24 h incubations. Description of the fluorescent material that accumulated during the incubation in terms of excitation/emission ranges and the type of material are recorded. n.s = not sampled.

Experiment	Food concentration (cells/l)/Chl (μg/l)	Food type	Growth stage	Exc max (nm)	Em (nm)	Description
1	800/0.54	T.w., S.c.	Stationary	250	304–320	Protein-like (Peak B)
2	2 000/1.22	T.w., S.c.	Stationary	250	300–320	Protein-like (Peak B)
3	8 000/2.86	T.w., S.c.	Stationary	275	330–350	Protein-like (Peak T)
				285	330–345	Unknown
4	8 000/3.1	T.w., S.c.	Stationary	250	315–320	Protein-like (Peak B)
5	8 500/3.88	T.w., S.c.	Stationary	275	290–305	Protein-like (Peak B)
				275	330–350	Protein-like (Peak T)
6	8 000/4.66	T.w., S.c.	Stationary	275	290–305	Protein-like (Peak B)
				275	340–355	Protein-like (Peak T)
7	10 000/7.68	T.w., S.c.	Stationary	250	304–320	Protein-like (Peak B)
				250	345–350	Protein-like (Peak T)
				276	350–400	Humic-like 1
				300	350–380	Humic-like 2
8	15 000/14.45	T.w., S.c.	Stationary	270	345–355	Protein-like (Peak T)
				295	350–390	Humic-like 2
9	5 000/4.12	T.w.	Stationary	250	290–315	Protein-like (Peak B)
				275–280	330–345	Protein-like (Peak T)
10	5 000/4.3	T.w.	Exponential	260	290–305	Protein-like (Peak B)
				260	350–490	Humic-like 3
				285	360–390	Humic-like 1
				315	380–400	Humic-like (Peak M)
11	8 000/4.56	P.m.	Exponential	285	405–460	Humic-like 1
12	8 000/2.63	P.m.	Stationary	265–270	315–320	Protein-like (Peak B)
13	8 000/n.s.	P.m.	Stationary	270	300–320	Protein-like (Peak B)
14	10 000/9.75	T.w., S.c., P.m.	Stationary	270	290–304	Protein-like (Peak B)
15	8 000/6.26	T.w., S.c., P.m.	Exponential	285	410–460	Humic-like 1
16	10 000/10.25	T.w., S.c., P.m.	Exponential	275	345–355	Protein-like (Peak T)
				300	350–400	Humic-like 2
				280	410–420	Humic-like 1

After the 24 h acclimation, 25 copepods were picked and rinsed with filtered seawater and five were transferred into each of 5, 0.5-l acid-washed, glass treatment bottles with Teflon lined tops. Each experiment was conducted with the same design, which consisted of five treatment (with copepods) and five control (without copepods) bottles filled with the desired food mixture. The bottles were placed on a plankton wheel and rotated at 1 rpm in the dark at 15°C for 24 h. After 24 h, the bottles were removed from the plankton wheel and subsampled for CDOM absorption and fluorescence, chlorophyll, and bacterial counts. Inherent to incubations are possible artifacts due to bottle effects; we tried to minimize these artifacts by keeping the incubation to only 24 h and using only five copepods per 0.5 l, while obtaining enough material for significant inputs to be detected. The concentration of C. finmarchicus used in these experiments, can be found in Massachusetts Bay.

Water analysis

Samples for CDOM absorption and fluorescence were collected by filtering 200 ml of water through a 0.2-μm Nucleopore filter. The filtrate was stored frozen in amber vials for analysis in the laboratory. Samples were analysed for absorption within one week and fluorescence within one month of each experiment. Previous work has shown us that there is less than a 2% change in the fluorescence intensities when the samples are stored for this time period (Urban-Rich and McCarty, unpublished data). CDOM absorption was measured on a Cary 50 spectrophotometer using a 10-cm cuvette and corrected with a Milli-Q blank. Absorption coefficients at 290 and 355 nm were calculated as described by Green and Blough (1994) . These absorptions were chosen, as 290 nm is in the region of greater sensitivity and in the region detected with the fluorescence measurements, while 355 nm is in the visible light region and represents that seen with remote sensing. Spectral slopes (S) were determined using a linear, least squares regression of absorption vs. wavelength from 290–400 nm ( Blough and Del Vecchio, 2002 ). S describes the rate of decrease of CDOM absorption with increasing wavelength. S can vary with the source of CDOM and changes in S can reflect biological or chemical modifications of CDOM.

Three-dimensional, CDOM fluorescent excitation–emission matrices were measured on a SPEX FluoroMax-3. Excitation scans from 250 to 550 nm at 5-nm intervals and emission scans from 265 to 710 nm at 2-nm intervals and 2-s integration created 61 individual, excitation–emission scans. Slit widths were 5.0 nm for excitation and 2.0 nm for emission. The instrument was corrected in accordance with the manufacturer's instructions. Data were normalized to the water Raman Peak at ex/em = 275/303 nm and converted into normalized quinine sulphate units (NQSU) using a correction curve generated with a quinine sulphate standard in 0.05 M sulphuric acid ( Coble et al ., 1993 ; Hoge et al ., 1993 ). The EEMs were corrected for Raman and Rayleigh scatter peaks with MATLAB, using an algorithm developed by Zepp et al . (2004) to excise the scatter peaks and replace them with values developed using a three-dimensional interpolation. The net input of FDOM was calculated for each experiment by:  

Fluorescent excitation–emission matrix scans (EEMS) can give information on different humic and protein components of the CDOM pool. Regions within EEMS have been defined by Coble (1996) as humic-like or protein-like ( Table 2 ). In order to compare our data with other published data we used these same notations for the peak regions.

Table 2

The major seawater fluorescent peak regions. FDOM peaks with their maximum excitation and emission ranges as reported by Coble (1996) .

Peak	Excitation (nm)/emission (nm)	Description
B	275/310	Tyrosine, protein-like
T	275/340	Tryptophan, protein-like
A	260/380–460	Humic-like
M	312/380–420	Marine humic-like
C	350/420–480	Humic-like

Water from each sample and control bottle (25 ml) was preserved with 0.1% gluteraldehyde for bacteria counts. The water was gently filtered onto a black Poretics 0.2-μm membrane filter and stained with acridine orange ( Hobbie et al ., 1977 ). Slides were frozen until they were counted in the laboratory with epifluorescence microscopy.

Chlorophyll concentrations were monitored before and after the incubation by filtering 25 ml through a GF/F filter. The filter was placed in 90% acetone for 24 h in a freezer to extract the chlorophyll. Chlorophyll was analysed on a Turner Designs Model AU-10 fluorometer using the methods of Strickland and Parsons (1968) .

Results

Food concentration

The effects of food concentration on C. finmarchicus inputs to CDOM were examined by feeding the copepods a diatom diet ( T. weissflogii and S. costatum ) in stationary and exponential growth. Copepod ingestion rates increased with chlorophyll concentration to 10 μg Chl l ⁻¹ and then decreased slightly at the highest food concentration ( Figure 1 ). CDOM absorption at 290 nm and 355 nm responded in a similar manner, so only the results from 355 nm are shown. CDOM absorption in both the treatment and control bottles did not appear to be related to food concentration for either stationary or exponential diets, excluding the highest food concentration in exponential growth ( Figure 2 ). There were significant changes in the spectral slopes between the treatment and control bottles for experiments with higher chlorophyll and higher ingestion rates ( Table 3 ).

Figure 1

Ingestion of chlorophyll in the laboratory grazing experiments with diatoms and dinoflagellates in stationary growth. Ingestion rates were based on the clearance of chlorophyll using the equations of Frost (1972) . Ingestion rates increase to 8 μg Chl l ⁻¹ and then appear to decrease.

Figure 2

Diatom mixtures (1/1) of Thalassiosira weissflogii and Skeletonema costatum in either stationary or exponential growth phase were fed to Calanus finmarchicus at various food concentrations for 24 h incubation in the dark at 15°C. CDOM absorption at 355 nm was measured in the initial water and then in the treatment and control bottles water after the incubation. The treatment and control bottles are corrected for the CDOM absorbance initially present in the water. A. Diatoms were in stationary growth; CDOM absorbance was consistently above the initial water values once the food concentration was ≥4 μg Chl l ⁻¹ . B. Diatoms were in exponential growth only at the highest food concentration >10 μg Chl l ⁻¹ . CDOM absorbance was greater than initial values.

Table 3

The spectral slopes of the treatment (T), control (C), and initial (I) water were determined from 260–400 nm for the experiments where there was a net input of CDOM. The spectral slopes are reported for experiments 4,7, 9, and 14 as listed in Table 1 . For all analysis n=141 and p was always less than 0.001.

Experiments	S±s.e×10 −nm	R 2	CDOM net input
4	T=15.1±0.26	0.961	0.200
	C=20.1±0.32	0.965
	I=17.6±0.29	0.963
7	T=18.72±0.25	0.976	0.090
	C=20.82±0.28	0.975
	I=15.98±0.24	0.970
9	T=17.4±0.21	0.980	0.110
	C=18.3±0.23	0.978
	I=24.6±0.34	0.973
14	T=12.7±0.27	0.942	0.110
	C=13.4±0.29	0.938
	I=14.4±0.29	0.948

There was a positive relationship between ingestion rates and net CDOM inputs that led to measurable net inputs of CDOM at ingestion rates >0.02 μg Chl copepod ⁻¹ h ⁻¹ (r ² =0.84, p<0.01). However, at the highest food concentration, net inputs of CDOM absorbance were negative, suggesting that at extreme chlorophyll concentrations exudation from the phytoplankton cells exceeds that added by grazers ( Figure 3A ). There was an apparent positive relationship between food concentration and the fluorescent fraction of CDOM ( Figure 4 ); protein fluorescence increased with food concentration and at high food concentrations humic-like fluorescence was detected. There was a positive correlation between the net inputs of fluorescent protein-like material with ingestion rates ( Figure 3B , r ² =0.68, p<0.05).

Figure 3

Net input = (treatment−initial)−(control−initial) of CDOM during the grazing experiments with a diet of diatoms in stationary growth was positively correlated with copepod ingestion rates. A. Net inputs of CDOM absorption, measured at 355 nm (r ² =0.84, p<0.01). The circled point corresponds to the highest food concentration and slight lower ingestion rate. This point does not fit with remaining data points and was dropped from the regression analysis. B. Net inputs of protein-like fluorescence (peaks B (ex 275/em 305) + T(ex 275/345)) were measured (r ² =0.68, p<0.05).

Figure 4

Excitation–emission matrix scans from three grazing experiments with the diatom cells in stationary growth and at different food concentrations. The protein-like fluorescent peaks (B and T) increase with increasing food concentrations. Humic-like peaks 1 and 2 were detected at the highest food concentration. A. Treatment consisted of a 1:1 Thalassiosira weissflogii and Skeletonema costatum diet at 800 cells l ⁻¹ (0.54 μg Chl l ⁻¹ ). Protein-like peak B (tyrosine) is present. B. Treatment diet was the same food source, but 2000 cells l ⁻¹ (1.22 μg Chl l ⁻¹ ), protein-like peaks B (tyrosine) and T (tryptophan) were present. C. Treatment diet was the same food source, but at 10 000 cells l ⁻¹ (7.68 μg Chl l ⁻¹ ) protein-like peaks B (tyrosine) and T (tryptophan) and humic-like peaks 1 and 2 were abundant. Note the fluorescence scale changes with each graph.

Phytoplankton diet

The effects of diet type were examined by feeding C. finmarchicus either diatoms ( T. weissflogii ) or dinoflagellates ( P. minimum ) ( Table 1 : experiments 4, 5, 9, and 12) in either stationary or exponential growth phase. Food type had a limited effect on the type of CDOM released. Diatom diets led to both protein-like peaks B and T, while a dinoflagellate diet resulted in only peak B material accumulating in the water. The humic-like material that accumulated during both diatom and dinoflagellate diets had a similar excitation maximum (280–285 nm), but the diatom diet resulted in a material that had a shorter wavelength emission maximum compared to the material from the dinoflagellate diet, 365–400 nm vs. 400–450 nm.

Phytoplankton growth stage had a major effect on the type of CDOM released. For a diet of either diatoms or dinoflagellates in exponential growth there was a net input of humic-like material, while a diet consisting of senescent cells resulted in a net input of protein-like material ( Figure 5 , Table 1 ). Only at high cell concentrations could net inputs of humic-like material be seen with stationary phase diatom diets ( Figure 4 ). While growth stage of the phytoplankton affected the bulk pools that accumulated during grazing, food type affected the chemical composition of the material within these bulk pools.

Figure 5

Fluorescent excitation–emission matrix scans from grazing experiments conducted with diatom diets ( Thalassiosira weissflogii , Plots A and B) and dinoflagellate diets ( Prorocentrum minimum , Plots C and D) in stationary and exponential growth. These graphs represent net inputs and thus the amount of material added in the treatment bottles due to the grazers. For both the diatom and dinoflagellate diet in stationary growth (Plots A and C), protein-like fluorescence (Peak B) was detected. Exponential growth (Plots B and D) had measurable inputs of humic-like material.

Overall, the humic-like material that accumulated in these experiments had a lower excitation maximum (280–290 nm) than that reported for marine humic material (Peak M ex/em 300–320/380–420 nm: Table 2 ) ( Coble, 1996 ; Blough and Del Vecchio, 2002 ). In many ways the material appears intermediate between humic-like peaks A and M in that the excitation maxima is around 285 nm compared to 312 nm for peak M and 260 nm for peak A, and the emission range is from 365–460 nm compared to 380–420 and 380–460 nm for peaks M and A, respectively. The dinoflagellate diet tended to release humic-like material that had a longer wavelength emission than that found with the diatom diet ( Figure 5 ), possibly representing a different arrangement of the fluorophore. Within the diatom treatments, longer emission wavelengths were generally observed for cells in exponential growth compared to stationary growth.

Bacteria were absent or extremely few (100–500 cell ml ⁻¹ ) in all the experiments. No changes in bacterial numbers were seen between treatment or control bottles. Thus changes in fluorescence were most likely due to grazing by C. finmarchicus and not to bacterial modification of non-coloured DOM.

Discussion

Food quality, quantity, and type are known to affect many aspects of zooplankton ecology from the vertical distribution of the animals within the water column ( Huntley and Brooks 1982 ; Fischer and Visbeck, 1993 ) to population dynamics (growth rates and reproduction; Kleppel and Burkart, 1995 ; Guisande et al ., 2000 ; Lincoln et al ., 2001 ; Turner et al ., 2002 ) to carbon and nitrogen cycling ( Kiørboe et al ., 1982 ; Feinberg and Dam, 1998 ; Urban-Rich, 2001 ) to toxicity and trophic cascade effects ( Tester et al ., 2000 ; Turner and Tester, 1997 ; Turner et al ., 2000, 2002 ). Since DOM can be produced via sloppy feeding, excretion, and faecal pellet dissolution and studies have shown that feeding rates increase with food concentrations ( Kiørboe et al ., 1982 ; Ayukai 1987 ), it would seem that higher food levels would lead to increased sloppy feeding. Also, there are increased faecal production rates with increases in food, so again it seems there would be more DOM released via excretion and faecal pellet dissolution with increased food. Thus, finding that net inputs of CDOM and fluorescent protein-like material increased with ingestion rates and food concentration ( Figure 3 ) supported our first hypothesis, i.e. that CDOM concentrations would increase with food concentration.

The small net inputs of CDOM ( Figure 3 ) combined with the significant changes in the spectral slope ( Table 3 ) suggest changes in the chemical composition of the material between the treatment and control bottles. Little is known about the type of material released by grazing copepods, but it appears the copepods release humics that absorb in the short wavelength regions from 285 to 300 nm. This humic-like material will be primarily involved in UV absorption opposed to visible light absorption. It is highly likely that this material is less aromatic, which would decrease its absorption and make changes in CDOM harder to detect. The ecological significance of this material is not known, but it could be important in regulating UV penetration in the water column.

The type of fluorescent CDOM that accumulated with each diet (exponential vs. stationary) was the opposite of that in our second hypothesis. As phytoplankton grow, their biochemical composition reflects their life stage ( Myklestad et al ., 1989 ; Belkoura et al ., 1997 ). During exponential growth, the cells generally have higher protein content and a lower C/N ratio ( Poulet and Martin-Jezequel, 1983 ). We therefore expected that there would be a large input of fluorescent protein material with this type of food due to sloppy feeding and to a greater excretion of protein material by the copepods. However, there was little to no net input of fluorescent protein material with a diet of exponentially growing diatoms or dinoflagellates. Instead, there was a net input of humic-like material ( Table 3 ). In the diet of stationary diatoms or dinoflagellates there was a net input of fluorescent protein material. Generally, during senescence phytoplankton cells contain little protein and larger amounts of carbohydrates and carbon-based compounds ( Poulet and Martin-Jezequel, 1983 ). While we did not measure the amino acid composition of the cells used in our experiments, the results from our study suggest that sloppy feeding is not the primary avenue by which CDOM is released into the water during copepod grazing, since there was no net input of protein-like material on a diet of exponentially growing cells. Instead, these results suggest that excretion or faecal pellet dissolution is the means by which copepods produce CDOM. This is in agreement with other studies conducted in our laboratory, where it has been found that copepods and larvaceans can excrete CDOM (Urban-Rich unpublished data). The consistency of the results in terms of protein vs. humic net inputs for the diatom and dinoflagellate diet suggests that the same digestive or mechanistic methods are responsible for the type of CDOM present rather than the actual food type. Whether this is due to digestive processes in the copepod or physiological and biochemical properties in the phytoplankton cannot be determined, yet the lack of CDOM accumulation in the controls would suggest that it is from the copepod digestive processes.

Variations in the fluorescent protein composition were seen between the diatom and the dinoflagellate diet ( Table 3 ). The diatom diet resulted in a net input of both the tyrosine peak (275/305 nm) and tryptophan peak (275/340 nm). Previous work has shown that both of these protein signatures occur within diatoms ( Determann et al ., 1998 ). In comparison, the dinoflagellate diet had a net input of only the tyrosine peak ( Table 3 ). While we do not know the amino acid composition of the food used in our study, the CDOM results could be due to variations in the biochemical composition of the phytoplankton species used in these experiments. Previous work has shown variations in the amino acid composition of different phytoplankton classes and variations with the life stage of the phytoplankton cells ( Wheeler, 1983 ; Brown and Jeffrey, 1992 ).

Previous work on the fluorescence of CDOM has found three major humic-like regions (A, C, and M) ( Table 2 ) that can reflect sources of humic material ( Coble, 1996 ). Generally, marine material (Peak M) is blue-shifted compared to terrestrial or riverine humic material (Peak C), owing to the decreased aromaticity of the material. The humic-like material that accumulated during the grazing studies had a lower excitation maxima than most reported studies ( Table 3 ). The shift to UV dominated or shorter visible wavelengths is in agreement with the general finding for marine waters. Since bacterial numbers remained constant and low throughout these experiments it would suggest that the grazing C. finmarchicus is the source of the humic-like material. It may be possible to use this humic-like material to trace zooplankton grazing in situ , though additional experiments would be needed to determine the effect of bacteria on this material.

C. finmarchicus is a dominant large copepod in the North Atlantic and occurs in regions that are dominated by spring diatom blooms followed by a low mixture of different phytoplankton in the summer and a frequent fall phytoplankton (diatom or dinoflagellate) bloom. Based on the results from these laboratory studies, there should be a seasonal cycle to the amount and type of CDOM released by C. finmarchicus . There would be a spring input of humic-like material that would be followed in early summer with an input of protein-like fluorescence and then in the fall there would be another pulse of humic-like fluorescence. Thus the impact of copepods on CDOM will be seasonal. Before we can totally assess the role of copepods in CDOM production we need to understand the significance and turnover of the low wavelength, UV-absorbing humics released by the copepods.

We thank Dr Bernie Gardner and Captain Peter Edwards for use of the RV “Neretic” in collecting C. finmarchicus . We also acknowledge the help provided by Dr Bob Chen, University of Massachusetts, Boston in lending us his Cary 50 Spectrophotometer and Prassede Vella for her help with data analysis. The Office of Naval Research supported the study through a grant (N000014-01-1-0247) to J. Urban-Rich.