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. Bamstedt J. Exp. Mar. Biol. Ecol. 251 2000 239 –263
respiration rates of aquatic organisms in diverse environments e.g., Packard, 1971;
˚ Kenner and Ahmed, 1975; King and Packard, 1975; Bamstedt, 1979, 1980; Martinez,
1991, 1992, 1997; Martinez and Estrada, 1992; Hernandez-Leon and Gomez, 1996; Ikeda, 1996; Madon et al., 1998. The modified method Owens and King, 1975 gives
an estimate of the potential respiration rate, because the analytical protocol includes saturation conditions in substrate level NADH, NADPH which makes the reactions
work at the maximum rate V
. This means that the amount of available enzymes will
max
be the rate-limiting step in the electron transport and the result will then be a reflection of the enzyme level in the sample. However, it is unlikely that this is the major
regulating factor in response to short-term changes in energy expenditure. A general conversion factor from ETS activity to respiration rate of 0.5 was suggested by Packard
et al. 1974 based on empirical results. The rather weak theoretical basis for this constant is that organisms would respire at around 50 of their capacity in normal
situations Packard, 1985. However, in vivo ETS activity is more likely controlled through typical enzyme kinetics, according to a Michaelis–Menten function, where the
rate is regulated by the present substrate level. This, in turn, implies that the ratio between respiration rate and ETS activity R ETS will vary with the physiological
activity of the organism. The rather wide R ETS ratios reported in the literature support
˚ this explanation see, e.g., Bamstedt, 1979; Hernandez-Leon and Gomez, 1996, for
zooplankton, although other sources of variability are certainly possible. By excluding the addition of substrates used in the original method, the measured reduction of INT
will be a result of the naturally occurring amounts of enzymes and substrates in the homogenate. Without the intact cell system that can regenerate the substrate there will be
an exponential decrease of the substrate level, causing a gradual decrease in the rate of reduction of INT as described by a Michaelis–Menten function. Theoretically, the initial
rate of reduction should give the actual ETS activity but, in practice, this is very difficult to measure, since substrate is being used up immediately when homogenisation starts,
and a considerable time is needed before a spectrophotometric reading can be taken. The alternative is to continue the reaction until total consumption of the substrates has taken
place. The spectrophotometric reading would then give a result that should be directly related to the total amount of substrates available during the incubation. Since the total
amount of substrates available will be the controlling factor for the initial activity of the ETS as defined by the Michaelis–Menten equation, the result should be a good index
of the respiration rate. In the present paper, I report on methodological tests of such a method and compare results for different marine invertebrates with this method, the
original ETS activity method as modified by Owens and King, 1975 and direct measurements of the respiration rate in incubation experiments.
2. Material and methods
2.1. Chemical reagents 1. ETS reagent. A buffer of 0.05 M Tris Tris hydroxymethyl aminomethan and 0.01
21
M phosphate with 0.075 mM MgSO , 1.5 mg ml PVP polyvinylpyrrolidone, 2 ml
4
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. Bamstedt J. Exp. Mar. Biol. Ecol. 251 2000 239 –263 241
21 21
l Triton X-100 and 2 mg ml
INT p-iodonitrotetrazolium violet was adjusted to pH 8.5 with HCl and stored frozen.
2. Dilution medium. The same as under point 1 but without INT. 3. Quench. A mixture of concentrated formaldehyde and 1 M phosphoric acid, 1:1 by
volume. 4. Extraction medium. Chloroform methanol, 2:1 by volume.
5. Methanol.
21
6. Reducing agent. Ascorbic acid, 70 mg ml dissolved in distilled water. Kept in a
refrigerator for up to 1 month and stored frozen for a longer period. 2.2. Analytical procedure
A number of modifications of the original analytical method Owens and King, 1975 were adopted:
• No substrate added to the homogenate sample.
• Only one reagent is used, and the material is homogenised in this reagent.
• Incubation time and temperature are set to levels that ensure that the enzymatic
reduction of INT is close to complete. •
After stopping the reaction, the samples are mixed with chloroform methanol 2:1 by volume in order to dissolve lipids from the material. This is important for lipid-
storing organisms, since the reduced INT is dissolved and concentrated in any lipid droplets that are present. It also dissolves particle-bound dye and thereby represents a
general improvement of the method.
• The extraction procedure can be used both to concentrate and dilute the samples,
thereby increasing the range of sample size biomass analysed considerably. •
A procedure for preparing a standard curve is defined, making it possible to test the quality of each specific batch of ETS reagent prepared.
• Omitting the addition of substrates also saves the main costs of the analytical
procedure, since NADH and NADPH are very expensive components. With a final reagent volume of 3 ml the amount of living biological material used
should be between 3 and 30 mg, corresponding to 0.6–6 mg dry weight or 0.3–3 mg protein. By adjusting the final volume up or down this range can be expanded.
The material is homogenised in an appropriate volume of ETS reagent usually 1–3 ml, transferred to a graduated centrifuge tube and incubated for 1 h at 408C. A blank of
ETS reagent without biological material is run in the same way. The reaction is stopped by adding 0.2 ml quench per ml of homogenate. One millilitre extraction medium is
added, mixed and the sample centrifuged at ca. 3000 rpm for a few minutes. The clear upper phase is discarded. The lower phase with its remaining inter-phase is made up to
3.0 ml by adding methanol. If the homogenised sample is dark red or hardly red at all, the final volume may be adjusted appropriately. The tube is mixed well and centrifuged
at ca. 3000 rpm for a few minutes. The absorbency of the biological sample is read against the blank in a spectrophotometer at 475 nm. The reduced INT in the medium
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. Bamstedt J. Exp. Mar. Biol. Ecol. 251 2000 239 –263
used here has a well defined peak at ca. 475 nm Fig. 1A and natural pigments from, for example, crustaceans do not seem to interfere.
2.2.1. Standardisation of the ETS reagent It is recommended to perform a calibration procedure, described below, for each new
batch of ETS reagent. As a control for the ETS reagent at each time of analysis it is recommended to use a duplicate standard sample with known concentration, treated as
described below. If there is a significant change in absorbance, the ETS reagent should be discarded and a new one prepared.
One millilitre of ETS reagent is diluted to 50.0 ml with dilution medium. This then has a concentration of 79.1 mM INT. A series of samples is made up from this by taking
0, 0.3, 0.6, 0.9, 1.2 and 1.5 ml and making up to 1.5 ml with dilution medium. A few
Fig. 1. A Absorbance spectrum of an analytical sample for ETS activity prepared with the new method, with the broken line indicating the wavelength of peak absorbance. B Standard curve for formazan reduced INT
read at 475 nm.
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. Bamstedt J. Exp. Mar. Biol. Ecol. 251 2000 239 –263 243
drops of reducing agent are added and mixed and the samples are incubated at 408C for 10 min. The samples are added 0.3 ml quench and 1 ml chloroform methanol and
centrifuged for a few minutes at ca. 3000 rpm. The upper phase is discarded and the remaining made up to 3 ml with methanol. The samples will now contain the red
formazan reduced INT with a concentration of 0, 7.91, 15.82, 23.73, 31.64 and 39.55 mM. The samples are read at 475 nm in a spectrophotometer and formazan concentration
is plotted against absorbance at 475 nm. A linear regression equation is calculated and used for the calculations of the subsequent biological samples. INT from Sigma grade I,
I 8377 gave a very close linear relationship Fig. 1B with a coefficient of de- termination of 0.9999. The equation used to convert from absorbance to formazan is
mM formazan 5 49.704A 2 0.225
475
Since 1 mol of formazan corresponds to 1 mol of oxygen or 0.5 mol O 516 g O the
2 2
formula for calculating the corresponding oxygen utilization from absorbance measure- ments is
ETS activity mg O used 5 16 3 49.704A 2 0.225 3V 1000 3 1 L
2 475
5 0.795A 2 0.0036 3VL
475
where A is the absorbance of the sample, corrected for the blank absorbance, V is the
475
final reagent volume in ml, and L is the cell width of the cuvette used for the samples. This calculated value has no meaning without converting it to the actual respiration rate
by using a ratio factor between respiration rate and ETS activity R ETS, i.e.
Respiration rate 5 ETS ? R ETS The present paper gives some examples of R ETS for different zooplankton organisms.
2.3. Test of the analytical procedures 2.3.1. Time schedule of biological reduction of INT
Freshly collected Praunus flexuosus Mysidacea and eggs of the lumpsucker Cyclopterus lumpus Teleostei were used in this test. One homogenate with high and
one with low concentration was used for the mysid, whereas a single homogenate was prepared with the fish eggs. The homogenates were prepared as described above and
incubated at 408C. The reduction of INT was stopped by adding quench at different time intervals between 0.1 and 270 min after the start. The following analytical steps were the
same as described above and the samples were read in a spectrophotometer at 475 nm against a blank prepared without biological material.
2.3.2. Homogenate preparation Single individuals in triplicate of similar sized Praunus flexuosus were used for this
test. Each animal was weighed wet weight and then homogenised either in buffer three samples or in buffer containing INT three samples. From each homogenate was
taken two subsamples, one was centrifuged and the clear supernatant used for the incubation, whereas the other one was used as crude homogenate. Four treatment groups
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were then produced by a combination of 1INT 2INT during homogenisation and
21
supernatant crude homogenate during incubation. ETS was expressed as mg O mg
2
wet weight. 2.3.3. Incubation temperature
Homogenates were prepared according to the standard procedure see above using temperatures between 10 and 508C in intervals of 108 both for preparation and
incubation. Three different biological materials were used: fresh mysids Praunus flexuosus; fresh soft tissue of the blue mussel Mytilus edulis; formalin preserved P
. flexuosus. In addition, a blank without biological material was included.
2.3.4. Linearity and concentration and dilution of the analytical sample Fresh mysids Praunus flexuosus were used in these experiments. The linearity when
using various volumes of homogenate was tested by preparing a stock homogenate and withdrawing between 0.25 and 2.0 ml homogenate for further processing. Dilution
medium see above was added to samples smaller than 2 ml, making the final volume 2.0 ml, and the standard procedure was followed see above.
In order to test the possibility of diluting concentrating the formazan produced in the analytical samples, a 25 ml homogenate of Praunus flexuosus was prepared and
incubated according to the standard procedure see above and the reaction was stopped by adding 5 ml quench. From this was taken 2, 4, 6 and 8 ml samples to which was
added 2.0 ml extraction medium. An additional 4 ml sample was added to 1.0 ml extraction medium. After mixing, centrifugation and discarding the upper phase, the
volume was made up to 4.0 ml by methanol 2.0 ml for the additional 4 ml sample. In this way a concentration of up to four times the lowest concentration was obtained.
2.3.5. Storage of processed samples ETS activity should be analysed on freshly collected material in order to obtain
realistic measurements. Therefore, it might be a problem to perform all the analytical steps and the final spectrophotometric reading in field applications onboard a small boat.
The effect of storing the analytical samples incubated and quenched at different temperatures all in darkness was therefore investigated using homogenate of freshly
collected mysids Praunus flexuosus. A batch homogenate was prepared, incubated and stopped, and thereafter divided into 30 subsamples. These were divided randomly into
three series, which were stored frozen, refrigerated and at room temperature, corre- sponding to 222, 15 and 1228C, respectively. One sample from each series was taken
out at different times, up to 103 days, and the samples prepared in the general way and read at 475 nm on a spectrophotometer.
2.3.6. Storage of live zooplankton A mixture of zooplankton collected in September and dominated by copepods was
kept refrigerated at 168C and subsamples were taken out regularly over a 3 day period by dipping a small jar in the well mixed container. Each time, three subsamples were
taken and filtered through a 20 mm plankton gauze and prepared as described for ETS analysis. Duplicate subsamples for respiration measurements were taken in the same way
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. Bamstedt J. Exp. Mar. Biol. Ecol. 251 2000 239 –263 245
on three occasions, incubated for 4–21 h and analysed for oxygen content by Winkler titration.
2.3.7. Effects of changes in environmental variables The mysid Praunus flexuosus was used in some manipulation experiments. Freshly
collected individuals were held at in situ temperature in June 148C, August 178C and September 138C for 3 days before being analysed. In September, one group was also
held at 58C and another one at 168C for 3 days. All animals were first used for respiration measurements, thereafter for ETS activity. Respiration measurements were
made in small glass bottles with seawater with known oxygen content. Together with appropriate blanks these were sealed and incubated in the dark at predefined tempera-
tures. Subsamples of water were taken by siphoning to a smaller bottle, letting water overflow with at least the volume of the bottle. The oxygen content was then determined
by the Winkler technique or using a Clarke-type polarographic electrode. In a second series of experiments a range of combinations of feeding starving, light dark and
different temperatures and acclimation periods were used Table 1. The experimental mysids were given natural zooplankton and small pieces of mussel meat as food. After
the treatment the animals were blotted on a paper towel, weighed and then analysed for ETS activity according to the standard procedure see above.
2.3.8. Respiration and ETS activity of organisms from natural environments Newly collected animals from different habitats were used. All were sampled with as
little stress as possible using non-filtering cod-ends of nets and trawls and direct catching with a bucket from surface water. Results are presented for the mysid prawn Praunus
flexuosus and the decapod shrimp Palaemon adspersus living in the sublittoral. These ¨
¨ were collected from the pier at Tjarno laboratory
Ten species of macrozooplankton from Kosterfjorden, western Sweden, were sorted from gently sampled material taken in winter by a conical net, 200 mm mesh size,
equipped with a non-filtering cod-end. After sorting, animals were incubated for respiration measurements as described above. The incubation temperature was close to
the in situ temperature of 5–68C and incubation was performed in darkness. After
Table 1 Treatments of Praunus flexuosus used to study the effects of changes in environmental variables on ETS
activity Treatment group
Duration Temp.
Food Light dark
days 8C
I 16
17 Yes
Dark II
7 3
Yes Dark
III 16
17 Yes
12 12 L D IV
16 17
No 12 12 L D
a 16
14 Yes
Dark b
16 4
Yes Dark
c 16
11.5 Yes
Dark d
4 4
Yes Dark
e 4
14 Yes
Dark
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respiration measurement the average individual wet weight was measured by blotting the collected material with a paper towel and weighing on a micro-balance. The ETS
activity was then measured using both the new and the traditional method of Owens and King 1975 using an incubation temperature of 218C for the latter method. A
conversion was made to the in situ temperature using the Arrhenius equation and a value
21
˚ for the energy of activation of 15 kcal mol
see Bamstedt, 1980. ETS activity, using the new method, was also measured on macrozooplankton from
the Barents Sea, collected during summer. Gentle collection with a conical net with a non-filtering cod-end was used. Single individuals of separate species were each placed
into a plastic capsule with 0.3 ml ETS reagent and frozen at 2228C. The material was thawed in the laboratory after 3.5 months and the animals picked up with forceps. The
animals were blotted on a paper towel and weighed see procedure above before being homogenised for ETS analysis. The weight of the animals might be negatively biased
due to loss of water during freezing. This might apply particularly to the hydromedusa, which has the highest water content.
Finally, ETS activity was also measured on POM particulate organic material at different depths in Kosterfjorden, western Sweden. Duplicate water samples of 2 l
volume were filtered through Whatman GF C filters, one was used for POM, the other one for ETS activity. POM was estimated as the weight loss from a dried 608C for 24 h
to an ashed sample 5008C for 24 h. ETS activity was analysed according to the standard procedure see above.
3. Results
