The effect of food on the respiration rates of Daphnia magna using a flow-through system *

Respiration in aquatic organisms gives a base line of consumption in the marine environment. Their measurement is of paramount importance in order to understand the flow of energy and matter in the ocean. Excluding temperature and size, the respiration rates are mostly affected by the level of feeding and animal activity, and the so-called specific dynamic action as the effect of the energy cost of protein synthesis (Jobling, 1983; Kiørboe et al., 1985; Brown and Cameron, 1991). Thus, the respiration rates of zooplankton must be influenced by conditions of quantity and quality of food in the environment, as was stated long ago by Marshall and Orr (1955). Respiration is frequently measured in aquatic organisms but in most cases food is omitted during the experiments. The effect of food on the respiratory rate has not yet been studied very extensively SCI. MAR., 67 (3): 361-365 SCIENTIA MARINA 2003

Respiration in aquatic organisms gives a base h e of consumption in the marine environment.Their measurement is of paramount irnportance in order to understand the flow of energy and matter in the ocean.Excluding temperature and size, the respiration rates are mostly affected by the leve1 of feeding and animal activity, and the so-called specific dynarnic action as the effect of the energy cost *Received Febmary 27,2002. Accepted December 12, 2002. of protein synthesis (Jobling, 1983;Kiorboe et al., 1985;Brown and Cameron, 1991).Thus, the respiration rates of zooplankton must be infiuenced by conditions of quantity and quality of food in the environment, as was stated long ago by Marshall  and Orr (1955).
Respiration is frequently measured in aquatic organisms but in rnost cases food is omitted during the expenments.The effect of food on the respiratory rate has not yet been studied very extensively EFFECT OF FOOD ON RESPIRATION 361 because of technical reasons related to the balance (closed bottle) method.Omori and Ikeda (1984) suggested that measurements of respiratory rates by the classical bottle method can be taken as somewhere between routine and active metabolism and this value depends on the previous feeding history of the animals.However, short-tenn variability of physiological rates remains unknown and this could cause underestimation of respiration.Classical measurements are normally performed in filtered seawater where suppression of feeding takes place and a decline in oxygen demand presumably occurs as organisms enter in starvation (Skjoldal et al., 1984).Organisms can only reach high rates of respiration for short periods which should not be representative of the physiological condition.In such a procedure, the range of animal activity cannot be determined accurately because the rate measured is an average (or integrated) value of respiration during the experiment.There meth~d~!ngicd prnhlemc: recl&n th_e assessment of metabolic rates in relation to ambient food (and therefore physical processes) in biological oceanography.
Thus, to study the factors affecting respiration rates in manne zooplankton it is necessq to know the scope of animal response in order to obtain a m e picture of their metabolic budgets.The scope of activity can be assessed by the use of a flow-through system (FTS), which allows one to monitor physiological rates continuously, as well as to change the food environment of animals.Using this methodology, Kigrboe et al. (1985) observed that respiration rate was related to food concentration in the copepod Acartia clausi.Larnpert (1986) and Thor (2000) were also able to measure the increase in metaboiic rate at increasing food levels in an FTS.The method shows the scope of respiratory activity of animals, which in the cited studies ranged from two to more than four times the standard respiration rate.Mackas and Burns (1986) were able to synchronise the feeding response of starved organisms of

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in food.The copepods responded to food by increasing the ingestion rates as observed from the increase in gut fluorescence.They observed that gut fullness and swimming activity at the time of sampling were strongly correlated.We wondered whether metabolic rates of zooplankton respond to such increases in feeding.The aim of this work was therefore to study the variability of the metabolic response in zooplankton induced by the increase in ingestion due to different pulses of food.Short-term respiration rates of the cladoceran Daphnia magna were measured, looking for the response of metabolic activity to the ingested food (measured by the gut fluorescence method) using a fiow-through system.Organisms were forced into extreme situations of low and high levels of food in order to reflect the metabolic range that animals can experience in nature, Daphnia magna was cultured in the laboratory and allowed to grow in saturating food concentrations in large containers.Food consisted of the freshwater phytoplankton Selenastrum capricomu- tum (Chlorophyta).In order to study the effect of food on the respiration rates we built a flow-through system (ITS) which consisted of two chambers (120 ml), one filled with organisms (10-15 individuals) acting as the experiment and the other acting as a control (Fig. 1).A peristaltic pump maintained a constant flow (36 and 60 rnl-h-') from a well-aerated reservoir, which could be filled with filtered water mc! tn which the f ~n d c ~d d be iddec!zt t !~ se!ected time intervals.
Respiration rates in the FTS were measured at 19°C and calculated by multiplying the difference in oxygen concentration between the control and the experimental bottles by the flow rate instead of measuring it at the inflow and outfiow.Oxygen values were recorded every 20 seconds using a Strathkelvin Instruments (SI) pulse electrode.Experimental measurements were taken 24 hours after the organisms were introduced into the experimental chamber in order to allow the FTS to stabilise, but also to minirnise stress effects and, in some experiments, to be s u e that animals were in starvation.After this time, we replaced filtered water with 300 ml of saturating FIG. 1. -Schematic representation of the flow-through system used in our experiments.R is the reservoir containing the focd source or well-aired water.Freshwater and food could be changed in this reservoir without allowing bubbles to enter into the system.Ti3 stands for thermostatic bath, C and E are the control and expenmental chambers, EL are the oxygen electrodes, V is a three-way valve which allows one to inject oxygen-free or saturated sea-water in order to calibrate the oxymeter, OX is the oxymeter, CO is the computer and PP the penstaltic pump.The solid lines indicate flow of water.levels of Selenastrurn capricomuturn and measured their concentration (cells-rnl-') at the out-flow in the control and experimental chambers.When the reservoir was empty, filtered water was replaced and the experiment continued.Water containing food was allowed to reach the temperature of incubation prior to the food treatment.The smaii Merences in oxygen content due to water with and without food were prevented by the use of the control chamber.
Organisrns used for gut fluorescence measure-  was always observed despite the previous metabolic level and was followed by a decrease in respiration rates after 10 to 15 hours; except in experim&t 4 in which respiration rates remained at high levels for more than 14 hours.In these experiments, gut fluorescence was tightly coupled with the corresponding respiration rates (Fig. 3) and a significant relationship was observed between the index of feeding and the respiration rates obtained (Fig. 4).
Severe variability in respiration rates were observed in our experiments with the FTS.As the temperature in the system and the body size of the organisms did not change, respiration rates increased as a consequence of food availability.This effect agree with previous studies in which food level was the main cause of respiration rate variability (Conover and Lalli, 1974;Ikeda, 1976;Kigrboe et al., 1985;Larnpert, 1986;Thor, 2000).In our study, two pulses of food at saturating levels gave rise to two reproducible increases in respiration rates (Q.2).
a"erage "f iespiraíion was 2.-)7 plO,.ind-l.h-'with maximum values of about 4 p10,aind-'.h-l.Assuming that standard metabolic rates are in the range 18-22% of maximum rates (Kigrboe et al,, 1985; Macy ct o!,, 19991, o1.r Standard metabolic rate would be in the range 0.72-0.88p10,-ind-'.h-',which in fact is similar to the lower values obtained in the present work (see Fig. 2) and the range found in the literature of 0.70-0.92plO,.ind-'.h-lfor Daphnia nzagna by Schindler (1968), Kersting and Van der Leeuw-Leegwater (1976), Goss and Bunting (1980) and Lampert (1986), among others.The similarity between our estimated standard respiration rates and the results of the bottle method is not surprising as no differences have been observed between food and non-food treatments using this methodology (Christou  and Moraitou-Apostolopoulou. 1995).The more than four-fold increase in respiration rates found in our work agree with the observations by Kigrboe et al. (1985) for the copepod Acar-tia toma and reflect the metabolic scope of zooplanktonic organisms.The observed increase in respiration rates suggests that feeding and the related activity (see Mackas and Burns, 1986) were responsible for the observed metabolic variability.
Our results suggest that the closed bottle method does not reflect the true metabolic rate of an animal during the diel cycle due to inherent variability in feeding (see Duval and Geen, 1976;Gyllenberg, 1981;Simard et al., 1985;Durbin et al., 1990, Atkinson et al., 1992;Cervetto et al., 1993).However, Lampert (1986) compared his FTS measurements in Daphnia nzagna with previous estimations of oxygen consumption rates using the closed bottle method and found no differences between the methods.The metabolic scope observed using the FTS was two-fold, and thus lower than that observed in , the present work, which is comparable to that a observed by other authors (Duval and Geen, 1976; a Kicirboe et al., 1985;Cervetto et al., 1993;Thor, 2000).Our average value of respiration rates in E experiment 1 (Fig. 2), in which the two pulses of food were piven every 24 hours, was 2.07 p102.
ind-l-h-l.This value is more than twice the standard !metabolism of those organisms.Therefore, metabolic rates in aquatic organisms should be estimated by taking into account their average feeding level in $ nature.
The close correlation between respiration rates and gut fluorescence observed in the present work suggests a coupling between ingestion rates and rneiaboiism anci couici heip [o correcc for [he effect of feeding in the assessment of metabolism in the field.Unfortunately, the gut fluorescence method suffers from the uncertainty of pigment destruction anc! the use of ?heC!GX re!zti~nship f ~u ~c !
In the present work between respiration and gut tluorescence to predict the metabolic level of animals in the field is debatable.First, the rate at which pisments disappear in zooplankton guts is not constant and therefore is difficult to evaluate (see McLeroy-Etheridge and McManus, 1999).Secondly, zooplankton are omnivorous and the proportion of nonpigmented food in the gut varies from 10 to more than 80%, depending on the availability of phytoplankton and other non-pigmented protista (Dam et  al., 1995; Hernández-León et al., 2002).The knowl-edge of the daily integrated or average gut fluorescence that an organisrn can achieve in nature and its r ~l a t i n n c h i n w i t h respiratiyfi rUtey c i i n n ~c t c t h n t it ..d*US,L".A"&A* Y " '"' YU66YYLY C*A-L will provide a more realistic daily integrated or average estimate of metabolic requirements of zooplankton compared with the classical bottle rnethod.This rate would stili, however, be an underestimate of the true metabolic leve1 because of pigment destruction and the arnount of non-pigmented food in the gut.
Finally, further research on this relationship in e.g.different species of copepods of different size is required.The use of a flow-through system with a low chamber volume or higher flow rates in order to measure very short-term changes in respiration rates would increase its sensitivity and improve monitoring to follow the parallel changes in oxygen consumption and gut content of zooplankton.Nevertheless, the significant rclationskip obtained betwen both measurements suggests that feeding and the related swimrning activity were responsible for the VUXX vcu IIIGLCIUULIC; VaLlauuLLy.
RESUMIEN: EL EFECTO DEL ALIMENTO SOBRE LAS TASAS DE RESPIRACIbN DE DAPHNIA MAGNA MEDIANTE EL USO DE UN SISTE-MA ABIERTO.
ments were incubated in parailel to oxygen consumption measurements.A batch of 60-70 organisms were placed in the same conditions of temperature, flow and food as the others in the FTS.The volume of the chamber was 250 m1 and, after introduction of phytoplankton, 3-4 anirnals were pickedup every hour for 20-24 hours.These individuals were immediately placed in an Eppendorf tube and frozen at -80°C.For the measurement of gut fluor m r p n r p c a r n p ] ~~ ~yere h ~m p g i ~y y bz --"--AA--, "%.& pestle at 04°C and an aliquot of the homogenate was placed i n a test tube with 10 ml of 90% acetone and stored at -20°C (24 hours).Fluorescence of the samples was measured before and after acidification in a Tumer Design fluorometer, previously calibrated with pure chlorophyll (Yentsch and Menzel, 1963).Pigments were calculated with the equations given by Stnckland and Parsons (1972) slightly modified to Chlorophyll = k.(Fo-Fa)-individual4Pheopigments = k.(R.F,-Fo).individual-lwherek is the instrument calibration constant, F, and Fa are the fluorescence readings before and after acidification and R is the acidification coefficient.Gut pigment concentration in this study refers to the addition of chlorophyll and pheopigments.Experiments perforrned with Daphnia magna showed the effect of changes in food concentration m:-3\ D ~~. .: -~c : ---1.h-1depending on the presence or not of food.The increase in respiration rates lasted for about 20 hours after the introduction of food.The increase in respiration rates after the introduction of food was reproducible in our experiment (Fig.2).Additional experiments were made at flushing times (chamber volume/flow rate) of 2 (Figs.3a,b) and 3.3 hours (Fig.3c).Total removing of food was observed after 4-5 times the flushing time.After the introduction of food, an increase in respiration rates .2. -Respiration rates (flO2.ind-I-h-l) of Daphnia magna versus time using an flow-through system (experiment 1).The arrows indicate the start of a pulse of phytoplankton.See text for explanation.
FIG. 3. -Respiration rates (flO,.ind-l-h-l)and gut fluorescence (ng pigments4nd-l) of Daphnia magna versus time using a flow-through systern at flushing times of 2 hours (a and b) and 3.3 hours (c).Cell density are given in relative units (right scale).