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the experiment. The number of prey in the stomach of the predators will increase in the same way until the digestion time T is reached. From this point the ingestion will be
balanced by the egestion, and there will be a steady state with a constant number of prey N present in the stomach of the predators. Thus, if a constant feeding intensity holds
true the digestion time can be easily estimated by using simple geometry:
N T 5 N T →
T 5 N ? T N The digestion rate will then be 1 T . The advantage of the method is that the digestion
estimate is based on predators that are feeding, they can be left completely undisturbed and it is easy to get a good statistical basis for the estimate.
4. Discussion
4.1. Implications for designing digestion experiments From the experiment on individual variability in digestion time there are several
novelty results worthy of comment. First, variability in ingestion time seems to be inherent in each individual and neither related to any short- or long-term rhythm. Thus,
time of the day and time spent in a given food environment do not seem to influence the digestion time of a defined meal. Second, variability between individuals at a given time
also seems to be high, sometimes exceeding a factor of two. Third, average digestion time from multiple measurements over time on single individuals in a stable food
environment seems to be very stable, with no significant differences between in- dividuals. All these facts have implications on how the experimental design should be
defined to get a precise estimate of the digestion time of an organism. Thus, a good estimate of the digestion time of a given meal will be provided from experiments on a
few individuals that are measured several times. Variability caused by difference in meal composition and meal size is not evaluated here, but must certainly be considered in
studies where the food is of mixed or variable composition. Information on this topic is
˚ given for several gelatinous predators by Martinussen and Bamstedt 1999 and in
references provided by these authors. The design used in the experiments on effects of changed feeding intensity has, as far
as we know, never been used before. The absolute control of the feeding rate, concomitant with the repeated measurements of stomach content means that the
digestion rate and its dependence on changed feeding intensity can be estimated from living animals. This is only possible for a transparent animal, like Aurelia aurita, but we
assume that the results for this species are generally valid for aquatic invertebrates. The estimated digestion times of continuously feeding individuals were usually shorter than
digestion times from single-meal experiments, and the former situation probably also reflects the natural situation better, where predators do not starve when food is available.
It is well known that the rate of gut evacuation is different between feeding and non-feeding animals Baars and Helling, 1985; Penry and Frost, 1990. In the gut-
fluorescence method it is therefore common to use only the first 20–30 min of gut
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contents during incubations without food for the calculations Kiørboe and Tisilius, 1987.
Our results from the experiments with constant feeding rate indicate that the digestion time actually can change both up and down over time in such situations, without any
consistent pattern. This is thus in line with the first experiment, where it was shown a considerable random variability. The experimental implication of this then is that, even
when estimating digestion time from continuously feeding animals, it is not sufficient to make single-point estimates.
The most interesting results are found in the experiments where feeding intensity was changed. Switching over from low to high feeding intensity usually gave stomach
contents corresponding to realistic digestion times but the variability increased con- siderably. The opposite change, starting with high and ending with low feeding intensity,
caused in a few cases an accumulation of prey in the stomach that corresponded to dramatically increased digestion times. This might be an effect similar to the one when
measuring gut evacuation from non-feeding animals that have been pre-fed, where the evacuation rate is gradually reduced. The practical implication of this is that digestion
experiments using animals that have decreased their feeding intensity prior to the measurements might cause a considerable positive bias. In the natural environment, food
is usually patchily distributed. This will introduce high variability in the stomach content of collected animals. Using an experimentally determined digestion time from a
homogeneous food environment will therefore generate bias in the ingestion estimates. This will be especially significant in situations where animals have recently left a dense
patch of food cf. Fig. 4, right panels.
4.2. Evaluating the experimental method The present method of estimating digestion rate is synonymous to the one used by
Peterson et al. 1990 and Irigoien et al. 1996, in which total production and gut contents of faecal pellets are used for the variables N and N, respectively. A similar
method was also used by Dagg and Walser 1987 and Pasternak 1994 in which chlorophyll in gut contents and faecal pellets were used for the variables N and N,
respectively. The present method has been used on the euphausiid Meganyctiphanes
˚ norvegica by Bamstedt and Karlson 1998, and they found an average digestion time of
2.15 h n 514, two outliers excluded at 10–128C, a value in agreement with other published data for euphausiids.
Although our results show that there is a considerable variability in digestion time, even if the food composition is kept constant, and both over time and between
individuals, they also show that integration over time and averaged for many individuals will give a rather robust estimate of the digestion time. Since our first experiment Fig.
1 showed that individual differences were eliminated when averaging over time, it will be a matter of choice if the experiment is based on many replicates at a single time or
fewer replicates repeatedly measured over time.
The experimental design offers no possibility of detecting any non-constant feeding intensity, since it is based on end-point measurements. It can be argued that changing
feeding intensity presumably will occur soon after the experimental animals have been
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transferred to the incubation vessels, due to a gradual adaptation to the new environ- ment. If so, a prolonged incubation will reduce this bias by increasing the relative time
under the new feeding intensity. If the feeding intensity is more or less randomly variable, use of a sufficiently high number of predators will be the way of eliminating
this problem.
The number of predators is also important from another point of view. Suspension- feeding animals and predators taking small prey will always have many prey units algal
cells, microzooplankton, etc. in their stomach when actively feeding, and hence, irrespective of the sampling time, stomach content will be non-zero and reflect the
current feeding intensity. Nevertheless, such animals do show a considerable individual
˚ ¨
variability cf. Bamstedt, 1988; Turner et al., 1993; but see also Paffenhofer, 1984, but this probably reflects a true individual variability in feeding intensity that can be
compensated for by a large sample size. In contrast, animals taking larger prey often have a single prey unit or empty stomachs. For example Øresland 1987, 1990 recorded
an average number of prey items in the guts of three chaetognath species in the ranges 0.06–0.28 Sagitta elegans, 0.22–0.25 S
. setosa and 0.10–0.26 Eukrohnia hamata from Antarctic waters. Similarly Falkenhaug 1991 reported a range in mean gut
contents of the Arctic form of S . elegans of 0.05–0.24 prey items. These animals also
have a slow digestion, which at ambient polar temperatures may exceed 10 h Øresland, 1990; Falkenhaug, 1991. Integrated over an extended period these animals may have a
constant feeding intensity, but sampling will give many individuals with empty guts, irrespective of time. The slow digestion rate and low feeding intensity both necessitate a
long incubation T . T and a high number of experimental animals N and N sufficiently high for a good resolution in the equation.
The present method should be useful also for herbivorous zooplankton. A very simple method is then to incubate zooplankton in the algal suspension, analyse chlorophyll a in
the food medium in the start and end of the experiment, and analyse the gut contents of chlorophyll a derivatives of the experimental animals in the end. The scaling of the
experiment is then especially important, since the reduction in food must be significant, making a prolonged incubation or a high abundance of grazers necessary. The equation
can be used to calculate the digestion time, where N is the gut contents, expressed in chlorophyll a units and N is the reduction of food in terms of chlorophyll a, where algal
growth over the incubation period has been compensated for. Alternatively, N may be estimated from the chlorophyll derivatives in the faecal pellets of the predator, as done
by Dagg and Walser 1987 and Pasternak 1994, although this requires that the faecal pellets can be quantitatively recovered. When using the method based on chlorophyll a,
the problem of pigment destruction has to be considered cf. Conover et al., 1986; Head and Harris, 1996; Pasternak, 1994; Dagg et al., 1997.
5. Summary and recommendations
