INHIBITION OF CLADOCERAN FEEDING BY STAINING
WITH ACRIDINE ORANGE1
JOHN A. DOWNING
Department of Biology, McGill University,
Montreal, Quebec H3A 1B1, Canada
Downing, J. A. 1980. Inhibition of cladoceran feeding by staining with acridine
orange. Trans. Amer. Micros. Soc, 99: 398-403. Acridine orange has been proposed
recently as a marker for living cladocerans used in predation experiments in situ. Tests
of the effect of one brand of acridine orange on the filtering rates of cladocerans and
observations on survival of stained individuals show that staining is detrimental to both
laboratory and field animals. These findings suggest that the staining procedure may be
responsible for high predation and mortality rates obtained in predation experiments in
situ.
Collection of data on community interactions in situ is an important goal
for ecologists. This is especially important when one is investigating such
fundamental characteristics as predatory interactions. Lane et al. (1976) have
recently published an interesting technique for the assessment of the pre
dation rates of naturally occurring copepods on cladocerans in situ. Lane
(1979) has applied this technique to natural zooplankton communities to im
ply that predation of cladocerans by invertebrate predators is more intense
than predation by pelagic fishes. Using this technique, prey organisms (e.g.,
Bosmina longirostris) are stained with the fluorescent dye acridine orange
(AO) and released inside a grazing chamber (Haney, 1971) in situ to be
preyed upon by natural predators. The predators are allowed to feed for 1 h,
and then the chamber is removed from the lake. The animals are collected
from its interior, and predation rates are calculated from the number of pred
ators with fluorescent material (AO) in their guts. As a reference check, mor
tality rates of prey are calculated from the difference between the number of
fluorescent prey added and the number of fluorescent prey recovered at the
end of the experiment. In order for this technique to produce realistic pre
dation rates, the stained prey in the enclosure must be ingested or killed at
the same rate as natural prey organisms.
I calculated the total rate of B. longirostris predation by the three main
predator species used by Lane et al. (1976) (Cyclops bicuspidatus, Tropocyclops prasinus, and Diaptomus sp.), and found that in their experiments,
more prey organisms were killed each day than existed in the experimental
chamber (Table I). This is disturbing since prey populations remained quite
stable over the 12-h period investigated. Even if we assume that more than
one predator could feed on each prey organism, the mortality rate of stained
B. longirostris in the chamber shows that more prey died each day than were
initially available (Table II). This would be possible if B. longirostris pro1 A contribution to the Lake Memphremagog Project, Limnology Research Group, McGill
University. This work was supported by the Inland Waters Directorate of Fisheries and Envi
ronment, Canada, the Department of Education of the Province of Quebec, and the National
Research Council of Canada through the Lake Memphremagog Project. I thank R. Lamarche for
help with fluorescence microscopy, and R. H. Peters and E. L. Schmidt for helpful discussions.
Trans. Amer. Micros. Soc, 99(4): 398^03. 1980.
399
DOWNING—INHIBITION OF CLADOCERAN FEEDING
TABLE I1
Comparison of specific and total predation rates with prey concentrations in in situ experiments
performed by Lane et al. (1976)
5m, 1200h
5m, 2400h
20m, 1200h
20m, 2400h
Specific daily predation rate (PCl)
Tropocyclops
Cyclops
Diaptomus
23
91
—
123
164
82
Total daily predation rate (Pc)
114
367
187
421
Bosmina (prey) concentration
19
23
11
17
Turnover time for stable
Bosmina population (h)
4.0
157
23
7
259
162
1.5
—
1.4
1.0
1 The specific daily predation rates on Bosmina longirostris (PC1) weTe obtained by multiplying the daily predation rate per
predator by the predator densities (D, + Dn) in table II and V of Lane et al. (1976). Pc is a summation of the Pt|. The concentrations
of experimental prey were taken from table I of Lane etal. (1976). All densities and rates are expressed per 8.91. This table shows
that if the predation rates are correct, then the population of B. longirostris would be required to reproduce its own biomass
once every 1-4 h in order to maintain a stable population.
duction rates were extremely high, but is unlikely because biomass turnover
time should range from 0.10 to 0.25 days in order to maintain stable prey
biomass. Biomass turnover times for unstained filter-feeding cladocerans
range from 6.7 to 58.9 days in nature (Wetzel, 1975). It is clear then, that
mortality rates of AO-stained B. longirostris in the experiments of Lane et al.
(1976) were at least 25 times greater than those that could have been main
tained by production rates.
There are two possible circumstances that could lead to unnaturally high
apparent mortality rates. The first of these arises from increased prey density
in experimental chambers. The added prey were 2-5 times more abundant
than the natural prey. It is well known that ingestion rates can increase mark
edly with prey concentrations (Holling, 1965). The second possible cause of
predation overestimation is that AO-labeled prey might have been captured
more easily than other prey animals because of the staining procedure. The
first circumstance seems unlikely since a 2-5-fold increase in prey concen
tration would result in a relatively small increase in predation rate, unless
the initial prey concentrations were very low. The second possibility was not
tested by Lane et al. (1976), and could have resulted in considerable over-
TABLE IP
Comparison of the mortality rate of stained Bosmina longirostris with its population from in situ
experiments performed by Lane et al. (1976).
5m, 1200h
Number stained prey added
Number stained prey remaining
Stained prey killed/h
Turnover time for stable
Bosmina population (h)
20
16
4
5.0
5m, 2400h
20m, 1200h
20
20
12
8
8
2.5
12
1.7
20m, 2400h
20
9
11
1.8
1 The number of Buorescent prey remaining at the end of the experiment was obtained from table II of Lane et al. (1976). AH
concentrations and rates are expressed per 8.91. This table shows that if the measured mortality rates mimic natural mortality rates,
then B. longirostris must reproduce its population from 4 to 14 times per day to maintain a stable population.
400
TRANS. AMER. MICROS. SOC, VOL. 99, NO. 4, OCTOBER 1980
SIMOCEPHALUS
VETULUS, LABORATORY
60• MEASURED
IN
STAIN
o MEASURED AFTER RINSE
0
40
30
AO CONCENTRATION
Fig. 1.
50
(ing/1)
The effect of acridine orange stain (AO) on the filtering rate (v>, ml/animal/day ± 2
SE) of Simocephalus vetulus in laboratory experiments. AO decreased the filtering rate and the
effect of AO remained after 30 min recovery time.
estimation of predation rates if AO staining induced mortality or slowed prey
movement.
In developing a technique for measuring the uptake rate of periphyton by
littoral cladocerans (Downing, 1981), I used AO as a marker for fresh animals
added to chambers containing macrophytes labeled with 32P. I performed a
series of experiments to assess the effect of AO staining on the behavior and
filtering rate of a few cladoceran species.
Materials and Methods
Because I found that some Cladocera (Daphnia pulex, and Simocephalus
vetulus) died after 30 min of exposure to low concentrations (14 mg/1) of AO,
I performed laboratory experiments to determine the effects of short-term
staining on their suspension feeding behavior. Ten large (—1.5 mm) S. vet
ulus were transferred by Pasteur pipette from laboratory cultures to each of
five 250-ml beakers of yeast suspension (Rhodotorula sp., 8 x 104 cells/ml in
filtered dechlorinated tap water). Animals were allowed to feed normally for
about 30 min following which time 103 cells/ml of radioactive (32P) Rhodo
torula were added (Downing & Peters, 1980) and the concentration of AO
(Fisher Scientific Co., A-971) in the beaker was raised to between 5 and 50
mg/1. Animals were allowed to feed in the radioactive stain solution for 5 min
before they were anaesthetized with CO2 (Burns & Rigler, 1967), concen
trated on a 102 /im mesh, and preserved with buffered sucrose-formalin so
lution (Haney & Hall, 1973). Animals were transferred immediately to indi-
DOWNING—INHIBITION OF CLADOCERAN FEEDING
401
SIDA CRYSTALLINA, IN SITU
100-
• NATURAL
o
o STAINED
Ld
or
«
50-
o
•
•
•
•/• V/#o c
LU
o° o
>
0 0.5
o
o 41
BODY
1
1.5
LO
LENGTH
2.0
(mm)
FIG. 2. The effect of acridine orange (AO) on the filtering rate (VK, ml/animal/day) of Sida
crystallina of various body lengths (anterior margin of head to posterior margin of carapace) in
situ. AO staining, even at low concentrations, inhibits feeding.
vidual planchettes and covered with Parafilm®. The ingested radioactivity
was measured on a Geiger-Miiller counter under gas-flow. Filtering rates (VF),
in ml/animal/day, were calculated (Rigler, 1971) as
VF = R x 24/Rf x t
where R is the radioactivity of one animal, Rf is the radioactivity of the food
cells in 1 ml of the suspension, and t is the feeding time in hours.
Another experiment was performed to determine if S. vetulus recovered
from stain effects after removal from the stain solution. Ten animals were
added to a yeast suspension as described above, but were stained (5 mg/1
AO), rinsed, and returned to a fresh yeast suspension before the filtering rate
was measured with no further AO addition.
Since I felt that natural and laboratory animals might respond differently
to AO staining, I also tested the effect of staining on the filtering rates of
cladocerans in situ. Littoral cladocerans on the surfaces of macrophytes were
enclosed in a Plexiglas® chamber (Downing & Peters, 1980) in Lake Memphremagog (Ross & Kalff, 1975). Additional specimens were collected from
an adjacent macrophyte, stained for 3 min with a low concentration of AO
(0.83 mg/1), rinsed with fresh lakewater, and added to the Plexiglas® chamber.
Radioactive yeast cells (Rhodotorula, <103 cells/ml) were added to the cham
ber and the animals were allowed to feed for 13 min (a period less than gut
clearance time). All animals were anaesthetized and removed from the cham
ber and preserved as above. Stained animals were separated from the un
stained animals using a Zeiss binocular dissecting microscope equipped with
402
TRANS. AMER. MICROS. SOC, VOL. 99, NO. 4, OCTOBER 1980
No. 44 barrier filters in the oculars. Illumination was supplied by a Zeiss
HBO incandescent illuminator with a BG12 excitation filter. AO-stained
animals could be distinguished from unstained animals by their bright yellowgreen fluorescence. Individuals of the most abundant species (Sida crystallina) were placed on planchettes, and filtering rates were measured and
calculated as above. In earlier experiments (Downing, 1981), I found that han
dling of the stained specimens had no effect on the feeding behavior of S.
crystallina; thus, any difference in filtering rate observed between stained
and unstained animals was a result of staining.
Results and Discussion
In the laboratory experiments, addition of AO decreased the filtering rate
10-fold in Simocephalus vetulus (Fig. 1). The effect of AO was somewhat
less severe if the animals were removed from the AO solution, and then
rinsed before the filtering rate was measured (open circle, Fig. 1). The swim
ming behavior of S. vetulus also became erratic in high AO concentrations
where the animals swam rapidly, often spinning in tight circles. In another
laboratory experiment, I was able to obtain only slightly depressed filtering
rates by staining for 30 min at 0.05 mg/1 AO. However, animals stained in this
manner sometimes did not appear to be fluorescent.
My field experiment shows that even brief staining at low AO concentration
depressed the filtering rate of Sida crystallina significantly (Fig. 2). The an
imals in this experiment were unable to feed normally, even though visual
observations did not suggest altered behavior. They swam normally and had
partially filled guts.
If the Bosmina longirostris in Lane et al.'s (1976) experiments were af
fected by AO in a similar manner, then the high mortality rates and high
apparent predation rates could have been a result of decreased viability of
the prey animals. Lane et al. (1976) stained their prey cladocerans for 3 min
at 50 mg/1, a concentration high enough to inhibit feeding in Simocephalus
vetulus and Sida crystallina (Figs. 1, 2). In fact, I observed that S. crystallina
were killed after staining for 1 min at just 10 mg/1. If B. longirostris are
influenced by AO, then they may be less able to avoid predators. They also
might die from starvation or AO toxicity, and fall to the bottom of the feeding
chamber becoming more readily ingestible (Lane et al., 1976). Thus, AO
staining in these kinds of experiments might cause overestimation of preda
tion rates, and also erroneous classification of detritus-feeding and scavenging
copepods as predators.
I believe that the in situ technique developed by Lane et al. (1976) should
be applied with caution because of the toxicity of AO to cladocerans. When
such a new technique is presented, it is important to demonstrate that pro
cedures do not induce errors. Such a step is requisite to confidence in a
measured variable as an accurate representation of the characteristic being
studied. I did not use AO to stain cladocerans in subsequent experiments.
Accordingly, I do not recommend that others use it, unless it can be dem
onstrated that AO does not affect the behavior of the stained animals.
Literature Cited
BURNS, C. W. & Rigler, F. H. 1967. Comparison of filtering rates of Daphnia in lakewater and
suspension of yeast. Limnol. Oceanogr., 12: 492-502.
DOWNING—INHIBITION OF CLADOCERAN FEEDING
403
Downing, J. A. 1981. In situ foraging responses of three species of littoral Cladocera. Ecol.
Monogr. (in press)
Downing, J. A. & Peters, R. H. 1980. The effect of body size and food concentration on the
in situ filtering rate of Sida crystallina. Limnol. Oceanogr., 25: 883-895.
Haney, J. 1971. An in situ method for the measurement of zooplankton grazing rates. Limnol.
Oceanogr., 16: 970-977.
Haney, J. & Hall, D. J. 1973. Sugar-coated Daphnia: a preservation technique for Cladocera.
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HoLLING, C. S. 1965. The functional response of predators to prey density and its role in
mimicry and population regulation. Mem. Entomol. Soc. Can., 45: 1-60.
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communities. Nature, 280: 391-393.
Lane, P. A., Klug, M. J. & Louden, L. 1976. Measuring invertebrate predation in situ on
zooplankton assemblages. Trans. Amer. Micros. Soc, 95: 143-155.
RlGLER, F. H. 1971. Feeding rates: zooplankton. In W. T. Edmondson & G. G. VVinberg, eds.,
Secondary productivity in fresh waters. IBP Handbook No. 17, Blackwell, Oxford, pp.
228-255.
Ross, P. R. & Kalff, J.
1975. Phytoplankton production in Lake Memphremagog, Quebec
(Canada)-Vermont (USA). Verb. Int. Verein. Limnol., 19: 760-769.
Wetzel, R. G. 1975. Limnology. Saunders, Philadelphia. 743 pp.