Factors affecting the respiration/ETS ratio in marine zooplankton

Journal of Plankton Research Vol.18 no.2, pp.239-255,1996

Factors affecting the respiration/ETS ratio in marine zooplankton

Santiago Herndndez-Le6n and May G6mezFacultad de Ciencias del Mar, Universidad de Las Palmas de G. C, PO Box 550,Las Palmas de G. C, Canary Islands, Spain

Abstract. Respiration (/?) and electron transport system (ETS) activity were studied in marine zoo-plankton of different size classes (100-200,200-500 and 500-1000 p.m) in order to determine the factorsaffecting the variability of the relationship between the two parameters. The R/ETS ratio varied withinthe range of values observed in the literature. The variability was more pronounced in the smaller sizefraction. We found this ratio and primary production to vary in parallel during the development of thelate winter bloom in the Canary Island waters. Higher ratios were observed to coincide with highervalues of primary production. When the data from different oceanic environments were pooled, wefound the ratios to be inversely related to temperature. The R/ETS ratio seemed to reflect the scope ofmetabolic activity in the zooplankton. Using a flow-through system, we observed short-term respir-ation rates and ETS activities to be related to the activity level of the organisms. This suggests thatvariability in the relationship between both measurements could be due to the inadequacy of assessingrespiration rates using the classical balance method and the problem of measuring enzymes at VamK.

Introduction

The assessment of respiration rates in planktonic communities is a prerequisite inestimating energy flow in marine ecosystems. Its measurement in zooplanktonprovides their minimum energetic requirements. These animals energize the syn-thesis of new organic compounds by oxidizing the carbohydrates, lipids and pro-teins in their diet. This rate of oxidation can be calculated from respiration data.However, these measurements are tedious, labour intensive and time consuming.As a result, it is difficult to obtain respiration values at the same data acquisitionrate as other modern oceanographic parameters are obtained. This precludes themesoscale assessment (in both time and space) of oceanic respiration. Partially forthis reason, Curl and Sandberg (1961) and Packard (1969) proposed the use ofphysiologically related enzymatic activities in the cell as an index of respiration.This enzymatic approach was designed to serve as a tool in accessing the verticaldistribution of respiration rates, and as a tool for mapping the ocean. In this fash-ion, the biological effects of mesoscale physical structures could be detected. Inaddition, this approach would circumvent the problems of stress, starvation,crowding and bacterial growth that are associated with incubation-based respir-ation measurements.

However, the variability in the relationship between enzymatic activities andrespiration rates has precluded the use of this approach as a standard respirationmethod. In spite of this, the electron transport system (ETS) activity is commonlyused in biological oceanography to assess plankton metabolism. However, in crus-taceans and in other phyla, the respiration (R)/ETS ratios are highly variable.Bamstedt (1979) observed a high variability of both respiration and ETS activitythroughout the course of different seasons. He found maximum respiration ratesand ETS activity in spring, as expected. However, the variations were not always inphase. In general, the respiration and ETS activity were well correlated, but the

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S.Heinindez-Letia and M.Gomez

Table I. Values of the respiration/ETS ratio obtained by different authors for copepods and mixedzooplankton. For comparison, all the ETS values were converted to the method of Kenner and Ahmed(1975) according to the ratios given by Christensen and Packard (1979)

Organisms R/ETS Reference

Calanoides carinatus 0.65 ± 0.19 (n = 13) Packard el al. (1974)Calanus pacificus (NI) 0.59 ± 0.19 (« = 2) King and Packard (1975)Calanus pacificus (Nil) 0.74 ± 0.07 (n = 2) King and Packard (1975)Calanus pacificus (adults) 0.46 ± 0.06 (n = 48) Owens and King (1975)Acartia tonsa 0.47 ± 0.08 (n = 13) Bamstedt (1980)Acartiaaustralis 0.16 ± 0.02 (n = 4) Ikeda and Skjoldal (1980)Calanus helgolandicusCalanus finmarchicus 0.19 ± 0.02 (n = 10) Hirche (1983)Euchaela norvegica 0.77 ± 0.19 (n = 6) Skjoldal el al. (1984)

Antarctic zooplankton 0.57 ± 0.26 (n = 12) Ikeda and Hing Fay (1981)Mixed zooplankton 0.50 ± 0.17 (n = 146) King and Packard (1975)Mixed zooplankton 0.38 ± 0.05 (n = 12) Bidigare et al. (1982)Mixed zooplankton 2.34 ± 0.76in = 5) Alcaraz and Packard (1989)Copepods 0.71 ± 0.40 (n = 6) King and Packard (1975)Copepods 1.39 ± 0.66 (n = 84) Bamstedt (1979)

R/ETS ratio showed considerable variation. This demonstrated the weakness inusing a single universal ratio to calculate respiration rates from ETS activity val-ues. No conclusions, however, were drawn about the causes of the variability in therelationship between both measurements. From the literature, the R/ETS ratio inzooplanktonic organisms is not constant and at least 3- to 4-fold differences arecommonly observed (Table I). Packard (1985) also showed the average values ofthe R/ETS ratio in bacteria, protozoa, phyto- and zooplankton to vary 3- to 4-fold.Because of this variability, there has been considerable resistance in acceptingenzymatic methods for estimating physiological rates.

When the enzymatic activity is measured, we are indirectly measuring theamount of enzyme because the activity is measured under substrate saturatingconditions, therefore it is the V^. For a determined enzyme concentration, thephysiological rate must behave as a Michaelis-Menten function. The physiologicalrate is measured 'in vivo' with cells and mitochondria exposed to substrate limi-tation. If the enzymatic activity is only measured at V,^, a high variability in theR/ETS ratio will be observed. Packard and co-workers expected a R/ETS ratio of—0.5 (see Packard, 1985) because ETS is measured at K^ and the organismswould respire at ~50% of their capacity if they were not under standard or activemetabolism (sensu Brett, 1964). This is the so-called Michaelis-Menten assump-tion, which is to say that the R/ETS ratio could depend on the physiological activityof the organisms.

Excluding temperature and size, the respiration rates are affected by the level offeeding and locomotory activity, and the so-called specific dynamic action as theeffect of the energy cost of protein synthesis (Ki0rboe etal., 1985; Brown and Cam-eron, 1991). Thus, the respiration rates of zooplankton must be influenced by con-ditions of quantity and quality of food in the environment, as was stated byMarshall and Orr (1964) several years ago. In this study, we have used respiration

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Respiration and ETS in zooplankton

and ETS activity measured in different environmental conditions from tropical topolar waters in order to assess the factors governing the variability of the ratiorelating both processes. We asked whether (i) respiration rates and ETS activityare coupled, (ii) chlorophyll, primary production, temperature and the size oforganisms affect the R/ETS ratio and (iii) what is the variability of this ratio inquite different ecosystems, such as those of warm and cold waters. Because ETSactivity is measured at Vmtx, one expects to find a constant level of ETS activity anda variable respiration rate in short-term experiments. In order to evaluate whetherthis feature (the Michaelis-Menten assumption) affected our calculation of respir-ation from ETS activity, we used a flow-through system to measure respirationrates at different food levels. The results obtained allowed us to discuss the physio-logical rates that are really estimated by measuring enzyme activities under sub-strate saturation.

Method

Samples were collected on different cruises from October 1988 to April 1994, fromdifferent oceanic areas (Table II). The 'late winter bloom' refers to the periodbetween October 1988 and June 1989. The development of this small enrichmentwas studied in a coastal station, east of Gran Canaria (Canary Islands). Primaryproduction was measured in situ using the 14C method of Steemann-Nielsen (1952).Zooplankton biomass (as protein content) was measured following the method ofLowry et al. (1951). EMIAC cruises took place leeward of the island of GranCanaria. This area was characterized by the presence of island-induced eddies(Aristegui et al., 1994). The tropical North Atlantic experiments (ATLEX cruise)were performed in an east-west transect at 21°N. Experiments carried out in Ant-arctica refer to samples taken in the Bransfield Strait (Antarctic Peninsula). 'Adlibitum' was an experiment with organisms collected from a coastal area of theisland of Gran Canaria, which were fed at different food levels for measurementsof respiration and ETS activity. In order to compare the R/ETS ratio in relation totemperature, this parameter was normalized, 0% taken as the coldest tempera-ture, 100% the warmest for every environment, and grouped at 20% intervals. Forexample, in the Canary Island waters (late winter bloom experiments) tempera-ture varied between 18.0 and 22.4°C during the period of study. A temperature of18.0°C was taken as 0% and 22.4°C as 100%.

Measurement of oxygen uptake

The animals were gently collected with a WP-2 plankton net (UNESCO, 1968)equipped with 100 u,m mesh and a large cod-end. Animals were screened in orderto obtain the 100-200, 200-500, 500-1000 and >1000 \txn size fractions, and thenplaced in filtered seawater for a short time in order to reduce stress problems.Organisms used in the late winter experiment were obtained with a WP-2 netequipped with a 200 u.m mesh and were not size fractionated. However, the largeand rare zooplankton were eliminated when filling the incubation bottles. In orderto compare with other experiments, we assigned those organisms to a 200-500 u.msize fraction. Experiments carried out in Antarctic waters refer only to large

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Table II. Cruises/experiments from which respiration/ETS values were obtained and results of this ratio (± standard deviations) for the different size fractionsobtained

Cruise/experiment

Late winter bloom

ATLEX

BALTEX

EMIAC9006

EM1AC9103

AD LIBITUM experimem

BIOANTAR

All data

Date

October 1988June 1989September 1989

May 1990

June 1990

March 1991

t October 1992March 1993January 1993

Location

Canary Islands

Tropical Atlantic

Baltic Sea

Canary Islands

Canary Islands

Canary Islands

Antarctica

Size fraction (jun)

100-200

-

0.94 ± 0.47("=13)2.55 ± 0.86

1.59 ±0.08(" = 2)0.95 ± 0.98(n = 10)-

1.93 ± 2.14(" = 31)

200-500

0.77 ± 0.28(n = 60)0.25 ± 0.07(n = 4)0.42 ±0.19(n = 16)0.66 ± 0.26

1.18 ±0.14("=2)0.65 ± 0.39(" = 42)-

0.65 ± 0.32(n = 130)

500-1000

0.35 ± 0.06in = 4)0.40 ± 0.06(rt = 4)0.57 ±0.17

n o ±0.11(« = 4)-

-

0.61 ±0.31(n = 18)

> 1000

-

_

-

0.46 ± 0.05("=3)-

0.19 ± 0.11(n=20)

0.23 ±0.14(" = 23)

n

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copepods and euphausiids (>1000 n-m). In experiments performed with the 100-200 u,m size class in the Baltic Sea, care was taken to wash out phytoplankton cells.Those samples which presented a high proportion of phytoplankton were dis-carded. Carnivorous organisms were always removed and the remaining zoo-plankters were siphoned into 500 and 1000 ml bell jars for incubation in filteredseawater at in situ temperature and in darkness or dim light. After the respirationmeasurements, zooplankton were removed from the bottles and immediately fro-zen in liquid nitrogen (-196°C) for the ETS activity and protein assays. The adlibitum experiments lasted 4 h and the field experiments 18-24 h.

Dissolved oxygen concentrations were measured during the late winter exper-iment according to the Winkler method described in Parsons et al. (1984), using amanual multipipette for titration. We were able to dispense a minimum volume of10 |JL1 of sodium thiosulphate. Later, it was possible to use the method described byBryan et al. (1976) using a photometric end-point detector and a digital micro-burette capable of dispensing 0.1 u,l of sodium thiosulphate. The difference in oxy-gen concentration between control bottles and incubation bottles was assumed tobe the zooplankton respiration (Marshall et al., 1935).

In order to study the effect of food on the respiration rates of copepods, we useda flow-through system (FTS) which consisted of two chambers: one filled withcopepods and the other acting as a control. A peristaltic pump maintained a con-stant flow of 36 ml Ir1 from a well-aired container which could be filled with sea-water containing the desired food concentration. The volume of the experimentalchambers was 73 ml. Respiration rates in the FTS were calculated according toPropp et al. (1982), recording the oxygen concentration with a pulse electrode(Strathkelvin Instruments). Experimental measurements were taken frombetween 3 and 5 h after the copepods were introduced into the experimental cham-ber in order to allow the FTS to stabilize, but also to reduce stress effects. Twoexperiments were performed with the copepod Calanus finmarchicus collected inRaunefjorden (Bergen, Norway). In the first experiment, ETS activity was mea-sured every 3 h in copepods incubated in a series of parallel chambers of the FTS,at the same flow rate as the chamber used to measure respiration. This experimentwas run at a constant food level. In a second experiment, copepods were incubatedin a 5 1 container with oxygen-saturated seawater. Copepods were at the same foodand light levels as organisms in the FTS. Copepods were removed every 2 h andfrozen in liquid nitrogen in order to measure ETS activity. We also measured ETSactivity of C.finmarchicus from Raunefjorden in order to observe values of theenzymatic activity of this copepod in nature during different days.

Determination of ETS activity

The ETS assay was run according to Packard (1969,1971), with the modificationsintroduced by Owens and King (1975) and Kenner and Ahmed (1975). Thehomogenates were obtained by grinding the animals in a Teflon-glass homoge-nizer for 2 min at 0-4°C using a phosphate buffer (0.05 M, pH 8.0) containing Tri-ton X-100. A diluted aliquot of the crude homogenate was incubated for 20 min indarkness and close to in situ temperature, in the presence of NADH, NADPH,

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S.Hernandez-Leon and M.Gomez

Table III. Relationships between respiration and ETS activity obtained in our late winter bloom andflow-through system experiments and all data presented in this work. Regression equations from otherauthors are also presented for comparison

Source

Late winter bloomFlow-through systemFinlaywa/. (1983)

King and Packard (1975)Bamstedt(1979)

All data (present work)

Equation

R = (0.327 x £75)+ 21.26/? = (0.244 x £75)+ 13.38R = (0.250 x ETS) + 0.01

log R = 0.953 log £75 + 0.202log R = 0.859 log ETS + 0.436

log R = 0.835 log £75 + 0.056

r

0.750.790.92

0.990.93

0.71

n

181131

9884

202

P

< 0.001<0.01

-

< 0.001

succinate and the iodo-nitro-tetrazolium salt INT (Biomedical Lab.). The reactionwas halted by the addition of a quench solution made up of 50% formalin and 50%phosphoric acid. After centrifugation for 10 min at 1500 g, the reaction colour wasmeasured at 490 nm with a turbidity baseline at 750 nm with respect to a blankwithout substrates and treated as the samples. In order to ensure a proper compari-son between respiration and ETS, the enzymatic activity was recalculated for thein situ temperature using the Airhenius equation and an activation energy of15 kcal moh1 (Packard et at, 1975). Proteins were assayed following the methodproposed by Lowry et al. (1951) using bovine serum albumin (BSA) as standard.The modification of the Lowry method by Peterson (1977,1983) was used for thesamples with a low protein content.

Results

R/ETS variability in zooplankton in relation to chlorophyll a and primaryproduction

The R/ETS ratio showed a high variability during the development of the late win-ter bloom in the coastal location of the Canary Islands (Figure 1). We observed agood relationship between both parameters (Table III), but we also found arelationship between phytoplankton chlorophyll a and primary production valuesand the zooplankton R/ETS ratio during the period of study. Higher values of theR/ETS ratio and high SDs were observed during the late winter mixing period,when the highest values of primary production were found. When the watercolumn was stratified and primary production decreased, the ratio gave values ofaround 0.5. The average R/ETS ratios (dots in Figure 1) were slightly correlatedwith chlorophyll a, primary production and with the primary production/zoo-plankton biomass ratio (Figure 2), which represents the primary production avail-able to the organisms on a biomass basis. Using the ANOVA I test, we observedthat primary production accounted for 42.8% of the variability of the R/ETS ratio.

R/ETS variability in relation to temperature and size

During the late winter bloom in the Canary Islands area, we observed high zoo-plankton R/ETS ratios coinciding with lower temperatures in the water column(Figure 3). The lower R/ETS ratios were found at the highest temperatures.

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NOV DEC JAN FEB MAR APR MAY JUN

Fig. 1. R/ETS ratio during the development of the late winter bloom in the Canary Islands. Mixing in thewater column started at the end of December and lasted until mid-April. Vertical bars are SDs.

Analysis of variance (ANOVA I) indicated that temperature accounted for 44%of the variability of the R/ETS ratio, while the weight of individuals as protein(estimated by direct counting of the sample, not shown) accounted for 38% (both99% level of significance).

In order to compare the results of different environments, from polar areas tothe tropics, we pooled all the R/ETS estimations (Figure 4) at the different sizefractions (100-200, 200-500, 500-1000 and >1000 u.m). Very little data wereobtained for the >1000 ^m size class, therefore they are not presented in the fig-ure. The three size fractions showed a tendency towards high values at the lowesttemperatures, as observed in the experiment carried out during the late winterbloom in the Canary Islands (Figure 3). This tendency seems to be more pro-nounced in the small size fractions. Average values of the R/ETS ratio for thedifferent size fractions also showed higher values in the lowest size classes (TableII). Using ANOVA I, we observed no temperature influence in the highest sizefraction considered (500-1000 n-m), while for the 100-200 and 200-500 ^m sizeclasses temperature accounted for 48.3 and 28.5% of the variability, respectively,both with a significance level of 99%. The slope of the regression between respir-ation and ETS activity for all data was quite similar to the ones obtained by Kingand Packard (1975) and Bamstedt (1979) (Table III). However, Figure 5 shows thevariability of the R/ETS ratio for all the estimations presented in this study.Although the higher number of observations were in the 0.4-0.6 interval, asexpected, the normal range had an important variability.

ETS activity and respiration rates obtained using a flow-through system

Because the variability of the R/ETS ratio seemed to be related to the presence/absence of food, two respiration experiments using C.finmarchicus were per-formed. ETS activity was measured in organisms incubated at the same food levelsas copepods in the FTS. Parallel variability was observed between ETS activity andrespiration rates in experiment 2 (Figure 6B), where both parameters were well

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S.HemAndez-Letin and M.G6mez2

02 04 08 OB 1

«gCMa/m3

0 10 20 30 40 SO (0 70 80 M

Production

0 1 0 2 0 3 0 4 0 6 0 0 0 7 0 1 8

Primary prodoctlon/ZoopUnktoa MommFig. i Relationship between the average R/ETS ratio and chlorophyll a (r = 0-549, P < 0.02, n = 18),primary production (r = 0.692, P < 0.002, n = 17) and the primary production/zooplankton biomassratio (r = 0.697, P < 0.002, n = 17) during the late winter bloom experiment.

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correlated (Figure 7A). This was not the case for experiment 1 (Figure 6A) wherelow variability in ETS activity was found (Figure 7A). The R/ETS ratio for eachpair of values was also calculated (Figure 7B). As can be observed, this ratio canchange from ~0.5 to 4 as the regression slope is different from unity, due to thewider scope of ETS activity. Parallel to the experiments of Figure 6, we measuredETS activity in samples of C.finmarchicus collected in Raunefjorden (Bergen,Norway). Most of the ETS activity measurements (Figure 8) were observed in therange of values where we found an important correlation between respirationrates and ETS activity (Figure 7A).

Discussion

A general relationship has been observed between respiration rates and ETSactivity from bacteria to metazooplankton (Finlay et al., 1983; Packard, 1985). Inzooplankton, King and Packard (1975) and Bamstedt (1979) found similarrelationships between respiration and ETS activity. The error (mean error of theprediction using each equation) of estimating respiration from ETS activity was34% (King and Packard, 1975), which also coincides with recent estimations of thesame relationship in microplankton from tropical to polar areas by Arfstegui andMontero (1995). For a given species at a given time, the coefficient of variationbetween respiration and ETS activity was 21-22% (King and Packard, 1975; Bam-stedt, 1979), which includes the methodological error, which was calculated to bein the range of 2-10% by Ikeda (1989). However, as pointed out by Bamstedt(1979), regressions between respiration and ETS activity must be viewed with cau-tion because respiration and ETS activity are proportional to body weight (orpopulation or community biomass), and the relationship between both measureswill always exist. This correlation will improve as the difference between lower andhigher biomasses of organisms increases. This would probably be the reason whyFinlay et al. (1983) found bacteria, protists and zooplankton falling in the sameregression line. Proper calibration of respiration versus ETS activity might be car-ried out using specific rates and activities at constant biomass, as observed inFigure 6.

Our results confirm the variability of the R/ETS ratio usually found in the litera-ture. The observed range coincides with previous results from several authors(Table I). The variability of the R/ETS ratio in relation to chlorophyll and primaryproduction suggests that the relationship between enzymatic activity and respir-ation depends on the amount and/or quality of food if we assume that species com-position (which varied seasonally) does not affect the ETS measurement (novariability of Km for the different species). Although primary production is not aclassic parameter used to assess the role of food in respiration experiments, it isknown that phytoplankton removal by zooplankton is greatest where the assimi-lation number is higher in the water column (Roman etal, 1986). These organismsingest the active growing cells, as shown several years ago by Richman and Rogers(1969). However, the variability of the R/ETS ratio in relation to temperatureseems to have an indirect effect, as primary production and mixing in the watercolumn are normally related. Moreover, the relationship between the R/ETS ratio

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S.Hernandez-Leon and M.Gomez

2

CO

UJ

oc

O-1O 3O-4O 80-70 90-100

TEMPERATURE < % Variation)

Fig. 3. Relationship between temperature and the R/ETS ratio during the development of the latewinter bloom in the Canary Islands. Temperature was normalized, 0% taken as the coldest tempera-ture, 100% the warmest and grouped at 20% intervals. Vertical bars are SDs.

and temperature also suggests that the variability of this ratio in relation to primaryproduction is observed in quite different environments, such as those of warm andcold areas. The highest response of smallest size classes to temperature might berelated to the more direct dependency of these organisms on the phytoplanktoncrop. As zooplankters increase in size, they change to omnivorous feeding, becom-ing slightly more independent of autotrophic production. Furthermore, smallerorganisms could display a higher specific respiratory response than larger ones inrelation to an increase in their ingestion rates.

The importance of food in the variability of the R/ETS ratio suggests that respir-ation is the factor which produces the high variability of this ratio. Feeding hasbeen observed to vary during the diel cycle at post-dawn and pre-dusk (e.g. Simardet al, 1985) or in relation to a satiation-starvation effect (Ishii, 1990). This dielfeeding activity produces an increase in respiration, as observed by Duval andGeen (1976) and Gyllenberg (1981), as well as an increase in ammonia excretion(Checkley et al., 1992). The respiration rate will therefore change according to thelevel of feeding activity and the so-called specific dynamic action (Ki0rboe et al,1985). This shift in the metabolic activity reflected the metabolic scope (sensuBrett, 1964) of the organisms and its range of variation (normally 3- to 4-fold) is ofthe same magnitude as the one found for the R/ETS ratio.

A source of uncertainty in experiments to calibrate respiration and ETS activityis related to the procedure used. Respiration rates estimated by the classical bal-ance method measure an average (or integrated) value between the level of respir-ation at the beginning of the experiment and the lower level in starved animals at

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UJ

oc

7-

6-

5-

4-

3-

2-

1-

2-

1-

0-2-

1-

0-

4

1

(

\

A

B

C

0 20 2040 4O6O eoao 80 100

TEMPERATURE

Fig. 4. Relationship between normalized temperature and the R/ETS ratio of pooled data from differ-ent environments (see the text) and different size classes: (A) 100-200 ujn, (B) 200-500 u-m and (C)500-1000 jun. Normalization of temperature for every location was carried out as in Figure 2. Note thedifferent scale in (A). Vertical bars are SDs.

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60

R/ETS

Fig. 5. Frequency histogram (n = 202) of R/ETS ratios obtained in the different oceanic environments.Nine very high values, ranging from 3.29 to 8.19, have been omitted for clarity.

the end of the experiment. Respiratory rates at the beginning of the experimentmainly depend on the previous feeding acclimation of animals, as stated severalyears ago by Marshall and Orr (1964), Conover and Lalli (1974), Ikeda (1976) andKi0rboe et al. (1985), among many others. However, the ETS activity in exper-iments designed to determine the R/ETS ratio is normally measured at the end ofthe respiration experiment on the incubated animals. This is a normal proceduretaken from early studies by Packard and co-workers up to recent estimations suchas those of Thuesen and Childress (1993) using citrate synthase (CS) as an esti-mator of respiration. As the physiological and enzymatic activities could vary inparallel (Figure 6), the ETS activity could reflect a precise metabolic level at theend of the experiment, which is normally low because of starvation. In fact, Bam-stedt (1980), Ikeda and Skjoldal (1980) and Skjoldal et al. (1984) observed thisparallelism between respiration and ETS activity in the decline of both measure-ments as the incubated animals starved. This could explain the higher R/ETS ratiosduring the development of the late winter bloom in the Canaries, as well as thehigher R/ETS as the temperature decreases in the environment. Fed organismshave higher average respiration, but lower ETS activity, at the end of incubation(high R/ETS ratio). In contrast, when food is scarce (or there is a low primaryproduction), the decline in the respiration rate is lower and much more similar tothe final ETS value, and so a lower R/ETS ratio is normally found. Therefore, it issuggested that calibrations of ETS activity using bottle incubations have givenspurious results throughout recent decades. Moreover, the R/ETS ratio determi-nation cannot be regarded as precise because the slope in the relationship betweenETS activity and respiration is different from unity, and ETS activity has a widerange of variation with respect to respiration rates (Figure 7). Furthermore, the

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o

10

g

4*

oflj

(A

Ui

1010

T I M E ( h )

Fig. 6. Electron transport system activity (filled circles) and respiration rates (open circles) obtainedusing a FTS. Experiment 1 (A) was carried out at a constant food level (6.6 ji.gChla.1"1) and samples forETS activity were taken every 3 h. In experiment 2 (B), the food level was changed from low food (LF,0.07 ± 0.06 M.gChla.1-') to high food (HF.6.0 ji.gChla.l-') coinciding with the dark period (black bar).Samples for ETS activity were taken every 2 h.

use of other enzymes to estimate respiration rates seems to add nothing new towhat we already know about the R/ETS ratio. Thuesen and Childress (1993) alsoobserved a rather low correlation between CS and respiration in chaetognaths.This lack of correlation could be related to the use of a similar procedure to thebottle incubation, as well as to the fact that this enzyme is coupled to carbonoxidation and not to oxygen utilization.

The respiration rate varied parallel to ETS activity in experiment 2 of the FTSand the important increase in both parameters developed in only 4 h. There is apossible interpretation for such a behaviour. It is the so-called respiratory control(see e.g. Lehninger, 1976). Respiration in the mitochondria, the velocity of theelectron transfer (and therefore of the activity of its enzymes) and the velocity ofATP production are driven by the relative concentrations of ADP, ATP and phos-phate in the environment of mitochondria, and not by the concentration of therespiratory substrates. When these substrates are not a limiting factor, the

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S.Hernandez-Letin and M.Gomez

10 16 20

ETS activity

26 30

Fig. 7. (A) Relationship between average respiration rate (from half an hour before and after the sam-pling for ETS activity) in the flow-through system. Triangles and dots correspond to experiment 1 and 2,respectively. Point 1 correspond to an uncoupled vaJue (arrow in Figure 6A) and point 2 is the firstsample of experiment 1. For both experiments, it was observed that the flow-through system was notstabilized at the beginning of the experiment. (B) R/ETS ratio for each pair of data of (A). Symbols areas in (A).

maximum velocity of oxygen consumption appears when the ADP and phosphateconcentrations are high, and ATP is low. On the other hand, when the ATP level ishigh, the mitochondria show low respiration rates. Here, the concentration ofADP is critical. In the muscle at rest, the respiration rate is low and ATP concen-tration is high. If the muscle is now in exercise, the ATP is used and disappearsquickly, producing ADP and phosphate, thus giving the signal for an increase inrespiration. This is a regulatory process and controls respiration and the electrontransport for a given enzymatic concentration. Moreover, when an enzyme has a

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Specific ETS activity

Fig. 8. ETS activity in samples of C.finmarchicus from the same Norwegian fjord (Raunefjorden) fromwhich the copepods were collected for experiments using the FTS.

regulatory role, there is variability in Km (see e.g. Lehninger, 1976), and thereforethis parameter cannot be considered as being constant for one species.

We can suggest that ETS activity is measuring respiration when there is no sub-strate limitation (Figure 6). At low food concentration, or when animals are incu-bated in filtered seawater, substrate limitation can be observed 'in vivo' and thenrespiration is not predictable from ETS activity because it is measured at V ^ (stip-pled line in Figure 7A). If organisms in nature are not substrate limited, the abovekind of calibration could be used to predict respiration from ETS activity. Fromthe field samples collected in Raunefjorden, we observed few data falling (Figure8) in the values of ETS activity where animal cells could be substrate limited in the'in vivo' experiment to determine respiration (around 5-7 JJLI O2 mg"1 protein h"1 inFigure 7). It seemed that those copepods have an ETS activity which varied in therange not affected by substrate limitation, where this enzyme has a regulatory role.Needless to say, this feature, as well as the calibration procedure using the FTS,need further research.

Finally, Mayzaud (1986) pointed out some advantages of the enzymaticapproach such as those derived from its slow adaptation to changes in the environ-ment and its use in high-frequency sampling. From our results and those of Bam-stedt (1980), Ikeda and Skjoldal (1980) and Skjoldal et al. (1984), it seemscontroversial to claim that enzymes are medium-term adapting processes display-ing lower responses than physiological rates to changes in the environment.

Acknowledgements

The authors wish to thank Dr J.Arfstegui for providing us with his unpublishedprimary production data and for the oxygen determinations from the Baltic cruise.Thanks are also given to Dr T.T.Packard for his review of an early draft of thismanuscript. Two anonymous reviewers also gave valuable comments to amend thepaper. We also thank Carmen Fraga Saavedra for drawing the figures. This work

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S.Heraandez-Leon and M.Gomez

was partly supported by project 22/01.06.88 of the Canary Islands EducationCouncil, projects PB88-0436 and BioAntar-93 of CICYT (Ministry of Educationof Spain) and project M AST-0031 of the European Union. We are also grateful toDr H.R.Skjoldal and Dr U.Bamstedt for facilitating us in the opportunity to attendthe ICES workshops of zooplankton production held at the University of Bergen(Norway) where flow-through experiments were performed.

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Received on July 15, 1995; accepted on October 20, 1995

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Factors affecting the respiration/ETS ratio in marine zooplankton