Journal of Plankton Research Vol.8 no.3 pp.505-517. 1986
Some characteristics of the carbon compounds released byDaphnia
Yngvar Olsen*, Kjell Morten Varum and Arne Jensen
Institute of Marine Biochemistry, N-7034 Trondheim-NTH, NorwayPresent address: Division of Applied Chemistry, SINTEF, N7034 Trondheim-NTH,Norway
Abstract. The Daphnia species studied released 1 8 - 100% of the algal carbon ingested as dissolved andpaniculate carbon compounds, presumably mainly as feces. The paniculate fraction constituted on average79 ± 5% of the total released compounds, leaving 21% as dissolved compounds. The panicles releasedwere very small and transparent, not visible by light microscopy Moreover, they contained significant amountsof chlorophyll derivatives. The dissolved compounds consisted mainly of small molecules (mol. wt < 103
daltons), and were shown to be utilized by planktonic bacteria. Our results show that paniculate organiccarbon and chlorophyll a should not be used as measures for algal carbon in grazing experiments with Daphnia.Both these parameters were influenced by the animals' fecal particles, yielding lowered clearance rates com-pared with those obtained by using cell numbers as a measure for algal carbon.
Introduction
Since Johannes and Webb (1970) suggested that the release of dissolved organic carbonfrom marine animals was an important source of reduced carbon in aquatic ecosystems,considerable efforts have been made to establish the composition, production and fateof fecal pellets from copepods (Johannes and Satomi, 1966; Paffenhofer and Knowles,1979; Tanoue etal., 1982; Small etal., 1983). Few authors have, however, been con-cerned with the carbon wastes produced by Daphnia (cf. Lampert, 1978), althoughthese organisms represent a very successful group in limnetic systems.
The organic carbon released by Daphnia comes from the processes of feeding, ex-cretion and defecation. Lampert (1978) quantified the total dissolved organic carbonreleased by Daphnia pulex and found that this fraction amounted to ~ 12 % (range 4.5 —17.5%) of the carbon ingested. On the other hand, the assimilation efficiency of Daphniafeeding upon algae has been reported to be in the range of 45-100% (Lampert, 1977;Porter et al., 1982), dependent on the food algae and on the method applied (cf. Porteret al., 1982). A significant fraction of the carbon wastes produced by Daphnia musttherefore be of paniculate nature, released as feces from the gut or as wastes fromthe process of feeding ('sloppy feeding').
Here we present a rough characterization of the organic carbon released by somespecies of Daphnia grazing on algae. We have not attempted to quantify the variousmodes of release involved. The losses involved in the feeding processes were minimized,however, by using small food algae (i.e. Scenedesmus acutus and Rhodomonas lacustris).
Materials and methods
Incubation experiments
The feeding cultures of the laboratory experiments were prepared from algal cultures(S. acutus or R. lacustris) grown in and diluted to - 1 mg C I"1 by the WC-medium
Table I. Biomass of algae and zooplankton and paniculate carbon in the enclosures at start of the experiments.
Enclosure
Zooplankton biomass, mg dry weight/1D. pulex, % of totalB. longispina, % of totalOthers, % of total
Algal biomass, mg C/lR. lacustns, % of totalC. marconii, % of totalC. erosa, % of total
Paniculate carbon, mg C/l
El
0.3786104
1.22724
24
1.64
± 0.04
± 0.04
± 0.05
E2
1.1094
51
1.14706
24
1.56
± 0.10
± 0.03
± 0.03
The temperature was 15-17°C during the experimental period.
(Guillard and Lorentzen, 1972). In the field experiments, water was taken from twoplastic enclosures (5 m deep and 1.5 m in diameter) mounted in the eutrophic lakeNesj0vatn in central Norway (69 8'N, 11 50'E, 12 m.a.s.). The initial concentrationof algae and paniculate carbon are given in Table I, showing that cryptophytes totallydominated the algal community.
The animals used for the laboratory experiments were either taken from stock cultures(Daphnia magna and D. pulex) or from natural populations (D. longispina or D. pulex).In the field experiments, zooplankton sampled from the enclosures was used. The resultsof Table I show that D. pulex made up most of the zooplankton of the enclosure com-munities, and its fraction of the total zooplankton was even higher in the incubations(>96%) because the Bosmina species originally present were partly lost during therinsing and washing procedure.
The incubation experiments involved addition of known numbers of Daphnia to feedingcultures or water dispersed into four to nine glass bottles (1.2 1). Care was taken tokeep the concentration of food above the incipient limiting concentration of Daphnia(i.e. -0.25 mg C I"1) during incubation which lasted for 2—4 h. Each series of exper-iments involved a zero time control and a control with alga only, and the incubationswere run in dim light to prevent algal growth. In the field experiments each incubationwas run using animals and water (30 (im filtered) sampled from the same enclosure,and incubations were performed on days 1, 2, 4, 6 and 8 after mounting the enclosures.
The incubation experiments were terminated by removal of the animals with a 120 /tmplankton filter. In the laboratory experiments the animals were preserved with Lugol'ssolution. They were counted and measured and the dry weight was computed usinglength—weight regressions established for the actual species. In the field experiments,the dry weight of the animals was determined directly by collecting all animals in theincubation bottle on a dried pre-weighed Whatman GF-C filter and drying it at 60°Cuntil constant weight of the material was obtained (2—3 days). The dry weight of theanimals was then determined as the difference between the filter weight with and withoutanimals.
Cell numbers of the algae were determined by counting under the microscope, andconverted to units of carbon on the basis of the cellular carbon content which wasobtained from the control cultures free from fecal carbon. In the field experiments,
conversion to carbon was based on estimates of cell volumes determined in the micro-scope and the carbon/cell volume relationship established for cultured R. lacustris, whichmade up >70% of the algal biomass in the field. Chlorophyll a was measured fluoro-metrically according to the method of Holm-Hansen et al. (1965) using a Turner 111fluorometer (RCA F475 lamp and standard filters). Paniculate organic carbon wasestimated in samples of the feeding cultures and the water collected on pre-ignited What-man GF-F filters by elementary analysis in a Carlo Erba Elemental Analyer, model1104, after treatment of the samples with acetic fumes to remove any inorganic carbon.Dissolved organic carbon was estimated in an Astro 1850 carbon analyzer.
Preparation and isolation of liC-labeled fecal carbon
Uniform uC-labeling of the algae was accomplished by growing them for at least fivegenerations in a closed system at constant UC/12C ratio. Light stoppered glass bottles(120 ml) were used. To each bottle were added 50 ml sterile WC-medium, 3 ml 83 mMNaHCO3 to supply inorganic carbon for growth, 5 or 10 /iC NaHwCO3 and a diluteinoculum of the actual alga (< 107 cells I"1). The alga was allowed to develop in thisclosed system under constant environmental conditions (18°C, 60 fiE m~2 s"1).
For the feeding experiments with labeled cells, rapidly growing labeled cultures werewashed twice using centrifugation and finally diluted in normal WC-medium to give1 — 2 mg C I"1. The animals (100-300 individuals) were washed with sterile mediumand added to the labeled culture. After 1 h of incubation the animals were collected,washed thoroughly and transferred to an unlabeled (cold) culture of the alga of the sameconcentration as the labeled one. Approximately 10 mg I"1 of sterile yeast extract hadbeen added to the cold culture immediately prior to the introduction of the zooplanktonto reduce bacterial uptake of the released labeled compounds. The cold culture wassampled immediately after addition of the animals and again 1 h later. The total 14C-activity and the dissolved 14C-activity (GF-F filtrate) of the culture was measured onboth occasions. The paniculate 14C-activity was taken to be the difference between totaland dissolved 14C-activity, and the net release of dissolved and paniculate 14C-activitywas defined as the difference between the activities obtained after 1 h and at the startof the incubation.
Size separation of dissolved carbon compounds
Samples (50—100 ml) were taken from the unlabeled culture after incubation of labeledanimals for 1 h (cf. above). They were immediately filtered (GF-F) and frozen. Allequipment used was pre-treated with 1 % aqueous solution of bovine serum albumin(BSA) to reduce adsorption of active material to glass surfaces (Norde, 1980).
For the gel filtration the frozen samples were thawed and 3 - 5 ml applied directlyto a Sephadex G-50 column (20 x. 250 mm). The column was eluted with 0.1 M NaClat a constant flow rate of 15 ml h" 1 (2.8 ml cm"2 h"1). Fractions (1 - 5 ml) were col-lected directly into plastic scintillation vials. The void volume (VQ) of the column wasdetermined with BSA (mol. wt 67 000 daltons). The dissolved organic matter wasgrouped in three size classes: (i) small-sized molecules (eluted in the total volume ofthe column, mol. wt < 1000 daltons); (ii) medium-sized molecules (1000 > mol. wt> 10 000 daltons); (iii) large-sized molecules (excluded from the column, mol. wt >10 000 daltons).
food concentration of control cultures, mg C/lfood concentration of experimental cultures, mg C/ldissolved organic carbon of control cultures, mg C/ldissolved organic carbon of experimental cultures, mg C/lzooplankton biomass, mg dry weight/Iincubation time, hclearance rate, ml/(h mg dry weight)grazing rate, /ig C/(h mg dry weight)ingestion rate, /ig C/(h mg dry weight)release rate of paniculate carbon, /jg C/(h mg dry weight)release rate of dissolved carbon, /tg C/(h mg dry weight)release rate of total carbon, fig C/(h mg dry weight)assimilation efficiency
Radioassay of samples
Prior to the assay all samples were adjusted to pH 2 with HC1 to remove any inorganiccarbon. The radioactivity was determined in a Packard Liquid Scintillation Spectro-meter, model 3375. Optifluor or Instagel was used a scintillation cocktails. The datawere corrected for differences in the counting efficiency between different species andcocktails.
Calculations
In all experiments the animals fed at food concentrations above the incipient limitingconcentration, meaning that the ingestion of food was constant with time. The specificgrazing rate (G) is then given by:
" * (i)ZT
(symbols in Table II). In cases where the ingestion rate is not constant with time, i.e.when starved animals are incubated, the equation expresses an average grazing ratethrough the period of incubation.
The specific clearance rate (CR) of the animals is given by the grazing rate dividedby the food concentration. The average food concentration during incubation is approxi-mated by (Nc + Ne)/2 and hence the specific clearance rate is given by the followingequation:
a? = ()Z 1\NC + Ne)
(symbols in Table II). Under different experimental conditions where the animals fedat food concentrations below their incipient limiting level, exponential rather than lineardecay of food concentration will occur with time, and other equations must be applied(e.g. the Gauld equation, Edmondson and Winberg, 1971).
In the case where N is estimated by direct counting of cells, we assume the grazingand the ingestion rate of carbon (/c) to be equal. This implies no losses through thefeeding process and that no cells pass the gut of the animal unharmed. On the other
(range) mg C/lNumber of incubationsTemperature, °C (range)
1
D. magruf(culture)S. acutus0.54 - 1.44
515 ± 0.5
2
D. pulex"(culture)S. acutus0.41 - 0.88
415 ± 0.5
3
D. longispinab
(natural)R. lacustris0.47 - 1.13
416 ± 1
4
D. pulex'(natural)R. lacustris0.16 - 0.55
516 ± 1
"The animals were adapted to the actual food source for several days.bStarved, non-adapted animals containing a green gelatinous alga in the gut.The alga was diluted in filtered lake water. The animals were adapted to food type and concentration.
oi
l
1,2-
-
0,8-
0,4-
0 -rr i
-—.
i?n
o• — ~ _ _
o
T
h
"i
_
i i40
T
•
60
O
>
Ian
-150
O
-100
- 5 0
ZT, mg dry wthliter"1
Fig. 1. D. magna feeding upon 5. acutus (Experiment 1). Time course development of algal carbon ( • ) ,paniculate organic carbon (T) , chlorophyll a ( o ), dissolved organic carbon ( • ) and paniculate dead organiccarbon (O). Bars indicate 1 SE of the measurements.
hand, the apparent grazing rate obtained using paniculate organic carbon directly inEquation 1 (Gc) is different from the ingestion rate since it is affected by the paniculatewastes released by the animals. We therefore define the specific release rate of par-ticulate carbon (R , mainly feces) as:
where /c is the specific ingestion rate of carbon and Gc is the apparent grazing rateof paniculate carbon, as defined here. The specific release rate of dissolved carbon(Rd) is estimated as:
» = DOCe - DOC, ( 4 )
ZT
and the total specific release rate of carbon (R) as:
R = Rp + Rd (5)
The assimilation efficiency (AE) of the animals has been calculated according toEquation 6:
AE = 7c ~ R (6)
Results
The release of carbon from Daphnia was measured in four grazing experiments(Table III). The results of Experiment 1 are presented in detail in Figure 1, showingthat algal carbon (AC, i.e. algal cells) was removed from the water faster than the par-ticulate organic carbon (POC) (P <0.05, Table IV). This indicate that paniculate carbon,probably mainly feces, accumulated in the water as the incubation proceeded. Theclearance rate of chlorophyll a was slower than that of AC (P <0.05, Table IV), sug-gesting that the released particles contained chlorophyll a derivatives. A small but signifi-cant (P <0.05) increase in dissolved organic carbon (DOC) was detected as theexperiment proceeded. This was also the case for the paniculate carbon from non-livingsources (stippled curve), although this was significant only at the 90% level. This variablewas defined as the difference between POC and AC (see Materials and methods).
Specific clearance rates, grazing rates, release rates and the assimilation efficiencies
Fig. 2. Specific clearance rate of D. pulex feeding in water sampled from the enclosures as a function offood concentration (i.e. POC). Clearance rate of algal cells ( • O), POC (VA) and chlorophyll a ( • o )Solid symbols obtained in enclosure 1 and open in enclosure 2. Bars indicate 1 SE.
for Experiment 1, as well as for the other three experiments, are shown in Table IV.A common trend was found for all experiments. The specific clearance rate of POCand of chlorophyll a were always lower than those of AC {P <0.05 for all cases).The estimated specific release rates of carbon (R) were quite large compared with theactual ingestion rates of carbon (18-106%), as is also indicated by the assimilationefficiencies of the animls. The non-adapted animals of Experiment 3, having gelatinousgreen algae in their gut, did not apparently assimilate any carbon during the first 3 hafter addition to the Rhodomonas culture.
The relative amounts of dissolved and paniculate carbon released by the animals couldonly be estimated in three of the experiments. In Experiment 4 the tests were run infiltered lake water containing ~ 8 mg DOC I"1. Any increase in DOC caused by thedaphnids could not therefore be detected. In Experiments 1 —3 the DOC fraction consti-tuted 21—50% of the total released carbon (i.e. Rd/R), suggesting the two componentswere of about equal size.
The results obtained in the field experiments (see Table I) are given in Figure 2,showing the specific clearance rates for algae, chlorophyll a and POC at various foodconcentrations. Again the specific clearance rate of algal cells (mainly R. lacustris)was found to exceed that of both chlorophyll a and POC. The clearance rates obtainedfor POC at low food concentrations were somewhat uncertain and the apparent increaseis not regarded as significant. The results demonstrate the presence of paniculate wastes
Table VI. The chlorophyll content, expressed as /ig chlorophyll a/mg C ( ± 1 SE) of the food alga andthe panicles released by Daphnia.
Case Species involved Food alga Fecal panicles
1234
S.5.R.R.
acutus -acutus —lacustrislacustris
D. magnaD. pulex— D. longispina— D. pulex
1135.8
12.410.2
±
±±
6.00.10.20.4
45314.04.43.6
±±±±
3397.52.20.8
of carbon in the natural population.A more detailed examination of the relative amounts of dissolved and paniculate carbon
was achieved in experiments with uniformly labeled algae as food source for the animals.The results, given in Table V, are not directly comparable with those given in TableIV because sloppy feeding is no longer involved. The relative importance of the dissolvedand paniculate components did vary between experiments. The paniculate componentwas the most important throughout, constituting 79 ± 5% of total released carbon.The results of Experiment 1 (Table IV) have also been included to illustrate that sloppyfeeding did not significantly change the distribution pattern.
The particles released by the animals were always very small, transparent and noteasily visible in the light microscope. In a case 1 situation (Table III), the amount ofdissolved carbon increased by a factor of two when GF-C filters were used insteadof GF-F filters (pore sizes 1.2 pm and 0.7 /xm, respectively) to separate the dissolvedfrom the paniculate fractions. Most of the particles must therefore be in this size range.The particles were also resistant to mechanical treatment. Homogenization of harvestedparticles for several minutes with a tissue homogenizer did not bring more than 4%of their carbon into solution.
The particles released contained significant amounts of chlorophyll a and/or its degrad-ation products (chlorophyllide a, phaeophytin a and phaeophorbide a). The apparentchlorophyll a content of the particles, and that of the food algae obtained in the experi-ments referred to in Table III, are shown in Table VI. When S. acutus was used asfood alga the apparent chlorophyll a content of the released particles was higher orequal to that of the alga. This was demonstrated for chlorophyll-rich algal cells (light-limited, Experiment 1) as well as for chlorophyll-poor cells (P-limited, Experiment2). On the other hand, when R. lacustris formed the food source (Experiments 3 and
Fig. 4. Uptake of dissolved released carbon compounds in bacteria. A: Time course of the uptake. B: El-ution profiles obtained at start (day 0) and after 2 days (day 2) of growth.
4), the apparent chlorophyll a content of the relased particles was significantly (P <0.05)lower than that of the alga.
Fractionation of the dissolved carbon (i.e. labeled released dissolved carbon) gaveelution profiles as shown in Figure 3, which also shows the elution profile of the solubleexudates of 5. acutus. Compared with the exudates (S0ndergaard and Schierup, 1982),a considerable fraction of the released carbon was found in larger-sized molecules (mol.wt > 10 000). Most of the material (on average 64%, Table VII) was, however, small-sized molecules (mol. wt < 1000). The results of four separations are shown in TableVII, and the relative importance of the different size groups of molecules was moreor less the same from one experiment to another. The losses of activity during hand-ling and gel filtration did vary, but were normally rather small ( < 10%), suggestingthat the numbers of particles passing the GF-F filters were also small.
The uptake of the dissolved labeled compounds into bacteria was examined in a sep-arate experiment. The results are presented in Figure 4, and show that the materialwas removed from the water as the incubation proceeded (left column). The bacteriadid apparently utilize all the different size groups of labeled compounds. From day0 to day 2, 76% of the smaller molecules and 82% of the larger ones were removedfrom the water. It should be emphasized that the inoculated bacteria were acclimatizedto substrate present in the filtrate.
Discussion
The estimation of the specific release rate of paniculate carbon was based upon theassumption that the carbon ingestion rate of the animals was equal to their grazing rateof AC (i.e. algal cells). This requires that breakage of algal cells during the feedingprocess is negligible, that few cells pass the gut unharmed and that the carbon contentof the algal cells must be directly determined. Here we used algal species which areeaten whole by the animals (Lampert, 1978) and which are believed to be efficientlydigested. Sloppy feeding and release of intact cells should therefore be of minor import-ance. The carbon content of the algae was measured directly in each experiment and
systematic errors introduced through, for example, the use of algal volumes were there-fore avoided. As a result we regard our estimates of the specific ingestion rate andthe release rate of the paniculate carbon to be reasonable.
Our results did show, as expected, that both particles and dissolved carbon compoundswere released from the grazers. Fecal carbon was presumably the main carbon com-ponent in the released material, at least in the paniculate fraction, but the importanceof other excretion processes are expected to increase with the efficiency of digestionin the animals.
The particles released were small and more or less transparent, and are presumablyfragments of cell walls or organelles. Their small size indicates that their sinking ratesare very low (SooHoo and Kiefer, 1982), suggesting they are disintegrated by pelagicheterotrophic organisms in nature rather than degraded in the sediments. They mayalso serve as food for grazers, but this has not been established (cf. Poulet, 1983).
The particles released contained significant amounts of chlorophyll a and/or its deriva-tives. When S. acutus formed the feed, the pigment concentration of the wastes wasapparently higher than that of the alga. This can be explained by assuming that mostof the particles were of fecal origin and that the pigment or the chloroplasts themselveswere badly assimilated. What must, however, be pointed out is that neither chlorophylla nor POC, measured according to normal procedures, should be used as a measureof algal numbers or biomass in short-term grazing experiments. A simple acid cor-rection may improve the chlorophyll a estimates in some cases (Riemann, 1982), butis misleading in others (Jeffrey and Hallegraeff, 1980).
The DOC compounds in the wastes made up ~20% of the total wastes, which isa significant proportion. At least 50% of the dissolved carbon was located in small-and medium-sized molecules (mol. wt < 10 000). The distribution of the dissolved com-ounds differed from that found for the exudates of 5. acutus which had a larger propor-tion of low mol. wt compounds ( < 10 000).
Absorption of labeled dissolved organic molecules to the surface of the containersor suspended particles of different nature is a potential source of error in the experimentsand would result in an underestimate of the dissolved fraction. Although care was takento reduce such losses — treatment of all equipment with BSA and addition of yeastextract (10 mg/1) to the feeding culture — some losses may have occurred.
The fate of the dissolved released compounds in nature seems obvious. Johannes andWebb (1970) have suggested that all soluble organic compounds occurring in the foodorganisms could be present in the wastes of the animals, including amino acids, sugars,fatty acids and other more complex compounds. All of these are presumably utilizedmore or less efficiently by aquatic bacteria, as demonstrated in one of our experiments.
The assimilation efficiencies obtained for the animals in the present experiments variedwidely from practically zero for non-adapted D. longispina feeding upon R. lacustristo ~82% for adapted D. magna feeding upon S. acutus. If we assume that the pro-portion of released particles to dissolved compounds in terms of carbon is 1:4 (see TableV), the corresponding absolute amounts of released compounds may be estimated forthe extreme situations of digestibility given above. The amount of dissolved compoundsreleased was then 4 - 2 0 % of the carbon ingested, whereas the amount of paniculatecompounds released made up 12—80%. This estimate is an approximation, and it should
be noted that the higher values were obtained with non-adapted animals (case 3, TableIII). The values do, however, demonstrate that a considerable fraction of the food in-gested by the animals is released back to the water, representing a significant sourceof reduced carbon. In cases where most of the primary production is consumed by thezooplankton, this source of carbon is obviously providing rich substrates for pelagicbacteria (Azam et ai, 1983).
Acknowledgements
We thank Marte Morkved for technical assistance during this study. This study formspart of the research program on eutrophication of inland waters financed by The RoyalNorwegian Council for Scientific and Industrial Research (NTNF), and is also partlysupported by the Norwegian Research Council for Science and Humanities (NAVF).
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