Watson, S. W., T. J. Novitsky, H. L. Quinby, and F. W. Valois. 1977. Determination of bacterial number and biomass in the marine environment. Appl. Environ.
Vol. 54, No. 8
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1988, p. 2149-2151 0099-2240/88/082149-03$02.00/0 Copyright © 1988, American Society for Microbiology
Improved Method for Determining Bacterial Filtration Rates in Zooplankton Laboratoire de Zoologie
et
OLIVIER MARVALIN* AND STANISLAW LAZAREK Protistologie, Univ'ersite Blaise Pascal de Clerinont-Ferrand, 63177 Aubicre Cedex, France
Received 23 November 1987/Accepted 14 May 1988
Filtration rates were determined for a natural population of zooplankton grazers (Bosmina longirostris [Mull.], Cyclops vicinus vicinus [Ulianine], Acanthodiaptomus denticornis [Wierz.], and Daphnia longispina [Mull.]) by using 3H-labeled bacteria as food for these organisms. There was a relationship between filtration rates of the major zooplankton grazers and the prevailing algal and bacterial composition in the lake water. Low filtration rates were obtained in the presence of colonial and filamentous cyanobacteria. The rapid process of bacterial adhesion to the external organs of grazers can result in an overestimation of filtration rates. By using the simple method presented here, filtration rates, with simultaneous correction for bacterial adhesion, can be quickly determined.
The epidermis and external organs of grazers feeding on and among planktonic algae provide habitats for attached bacteria. Although it has been recognized that attached bacteria are an important component of lake ecosystems (1, 4, 5), the process of bacterial adhesion relative to zooplankton grazing and its possible implications on calculated filtration rates have been largely ignored. This paper presents a simple methodological investigation with the following aims: (i) to evaluate the magnitude of passive bacterial adhesion to grazers in a short-term experiment, (ii) to facilitate routine determinations of zooplankton filtration rates, and (iii) to provide a means for rapid discrimination between the activity of radiolabeled bacteria ingested by grazers and the activity of the bacteria adhering to the external organs of grazers. A composite sample of 20 liters of water was collected with a Van Dorn sampler from a depth of 0 to 4 m in a eutrophic lake, Lake Aydat, in France. The water was filtered through a net (mesh size, 63 p.m) to remove the grazers. A round water tank (50 cm in diameter; volume, 15 liters) was filled with 6 liters of homogenized lake water without grazers. The tank (Fig. 1) consisted of two compartments. The bottom of the removable internal compartment A was made of netting (mesh size, 180 [Lm). Another 6 liters of lake water was concentrated for 10 min in a pump (Amicon Corp., Lexington, Mass.) by filtration through a column with 0.2-,um pores. Bacterial concentrate (100 ml) was obtained, and the resultant 5.9 liters of ultrafiltrate was added to the tank. Zooplankton was collected from a depth of 0 to 14 m by using a net (mesh size, 180 [Lm). The samples were kept for a maximum of 1 h at 4°C. The dominant zooplankton grazers in the lake which were used in the experiment were Bosmina longirostris (Mull.), which was most abundant in June; Cyclops vicinuts vicinuis (Ulianine), which was most abundant in May; Acanthodiaptomius denticornis (Wierz.), which was most abundant in September; and Daphnia longispina (Mull.), which was most abundant in July. The grazers represented four size classes (Table 1). The bacterial concentrate was incubated for 12 h in
darkness at 20°C with [3H]thymidine (Amersham, France). The final concentration of [3H]thymidine was 1 V.Ci ml-' (4 nM). Gentle agitation (30 rpm) was provided. The 3H-labeled bacteria were centrifuged (3 x 104 rpm), and the pellet was suspended in the ultrafiltrate (specific activity, 7.5 105 dpm ml-'). This bacterial suspension was then added to the water tank, thus simulating the natural composition of microorganisms in the lake water as established by comparative epifluorescence analysis (3). Zooplankton, in the density and composition found in Lake Aydat, was acclimated for 12 h at in situ temperatures ranging from 11 to 22°C. Ten minutes after the addition of FIELD
SAMPLING 20 LAKE WATER
ZOOPLAN KTON
VAN DORN SAMPLER pm MESH SIZE NE -180
60
pm
LABORATORY 6.0
6.0
r
ULTRAFILTRATION
IN THE AMICON PUMP (0.2pnaI 100 ml
5.9
BACTERIOPLANKTON CONCENTRATE
IH-THYMIDINE LABELLING (12 hr)
_____________ CENTRIFUGATION
GRAZING (10 miii) RINSING SEVERAL TIMES
IN ULTRAFILTERED LAKE
CO, ANAESTHETIZING
OF GRAZERS
WATER
(5 min)
FREEZING IN LIQUID N2
FREEZE STORAGE
*
FIG. 1. Schematic diagram of the generalized procedures for determining bacterial filtration rates in zooplankton.
Corresponding author. 2149
2150
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Filtration rates, corrected for passive adhesion of bacteria, for the dominant zooplankton grazers in Lake Aydat in 1986 (average values per individual and hour; n = 3)
Body size Organismsize Organism
Vol MI) Of (ml)of water
filtered
B. longirostris C. vicinus vicinus A. denticornis D. longispina
0.2-0.4 0.3-1.2 0.3-1.3 0.5-1.5
0.17 0.30 0.42 0.25
June May Amt Vol Amt (106 Crates(ponig (MI Of)16 Corresponding) (ml)gof (p.g of filtered rates of filtered Corresponding water of carbon flow of carbon flow filtered bacteria bacteria
0.77 1.35 1.90 1.13
1.82 3.22 4.51 2.68
3H-labeled bacteria, the experiment was terminated. Compartment A, containing the grazers, was lifted and transferred for 5 min into an identical tank containing carbonated water. The initial activity (at time zero) of the bacterial suspension in the tank was checked. No dissolved [3H]thymidine was present in the tank. To distinguish between the bacteria ingested and those passively adhering to grazers, an identical experiment was run with animals which were first anesthetized by transferring them in compartment A into the tank with carbonated water. The animals were rinsed thoroughly with ultrafiltered water and frozen in liquid N2. With a stereoscopic microscope, they were then sorted according to species and size. One hundred animals were placed in 3-ml vials to which 250 Rl of Protosol (Sigma Chemical Co., St. Louis, Mo.), a few drops of acetic acid, and Ready-SolvEP (Beckman Instruments, Inc., Fullerton, Calif.) were added prior to activity counting in a Beckman liquid scintillation counter. The numbers of bacteria in the lake water and in the experimental tank were determined by epifluorescence microscopy. A corresponding carbon biomass was calculated by multiplying an average cell volume by a carbon content of 1.21 x 10-13 g of C um-3 (6). Filtering rates (F) were calculated in volume (ml) of water filtered by an individual grazer and hour according to the formula: F = [(Ra -RRaa) X h]/(Ri x 10) where Ra is radioactivity (in dpm) in grazing animals, R,a is radioactivity in anesthetized animals, Ri is the initial activity of bacteria per milliliter, and 10 is the grazing time in minutes. The filtration rates, which were corrected for passively adhering bacteria (Table 1), showed no relation to the mean sizes of the four species of grazers tested. The filtration rates differed significantly within species (Friedman two-way analysis of variance, P = 0.194) for the three experimental dates. This might have resulted from the different algal and bacterial compositions of the water on these dates. Consistently high filtration rates were obtained in June, which corresponded to the lack of filamentous and colonial forms of algae and the highest (2-m) annual water transparency. The low filtration rates in May coincided with the highest annual density of filamentous cyanobacteria (Anabaena flos-aquae [Breb. ex Born. et Flah.] and A. spiroides [Kleb.]), whereas in September the low filtration rates for all tested grazers might have resulted from a dominance of the colonial cyanobacterium Gomphosphaeria lacustris (Chod.) as examined by the epifluorescence analysis (O. Marvalin, unpublished results). This agrees with earlier observations (7) that the entanglement of cyanobacterial filaments reduces filtering rates of cladocerans.
0.36 0.51 0.86
1.30 1.85 3.15
Amt
(Ml) Of
of Cells) (106
Corsndg
Crates(ponig 1itee 10te)
(102cells)
(106cells)
July Vol
1.63 2.33 3.96
filtered
of filtered bacteria
of carbon flow
0.28 0.28 0.36
1.08 1.05 1.68
1.69 1.72 2.10
water
These short-term experiments demonstrated that the process of adhesion of free-living bacterial cells to grazers is rapid. Despite the short incubation time (10 min), thorough rinsing of the animals only partly removed bacterial contamination. The 3H activity recovered from preexperimentally anesthetized animals showed little variance (coefficient of variation = 4 to 5%) on the three experimental dates. Consistently high activity (30 to 32% of the total activity in living animals) was encountered for anesthetized C. vicinus vicinus (Ulianine) and A. denticornis (Wierz.). Adhering bacteria constituted up to 13% of the total activity in live B. longirostris (Mull.) and only 9% of the activity in live D. longispina (Mull.). The pattern of bacterial adhesion seems to be related to the total surface area of the external organs of the grazers and not to their actual body size. The low activity found in B. longirostris (Mull.) and D. longispina (Mull.) may, therefore, be explained by the fact that these cladocerans close their chambers when anesthetized. The low variance in 3H activity of passively adhering bacteria recovered after 10 min of incubation on the three different dates suggests that adhesion was a purely physical process independent of the bacterial forms prevailing in the water. It is likely, however, that in natural conditions, the amount of adhering bacteria equilibrates with that of bacteria detaching in the process of filtration. The method presented here describes a quick and easy way to determine filtration rates in natural populations of grazers by using natural populations of bacteria as food for the grazers. The method allows a minimum prehandling of the living components and eliminates the risk that concentrating zooplankton will lead to reduced grazing rates (e.g., see reference 2). Because of the magnitude of passive adhesion by bacteria, we strongly suggest that filtration rates be corrected for passive adhesion in experimental work on
zooplankton grazing. This work was financially supported by the Centre National de la Recherche Scientifique and the Swedish Institute. We thank Lars Bern and Hans J. Hartmann for their comments. Thora M. Press corrected the grammar. LITERATURE CITED 1. Bern, L. 1985. Autoradiographic studies of [methyl-3H]thymidine incorporation in a cyanobacterium (Microcystis wesenbergii)bacterium association and in selected algae and bacteria. Appl. Environ. Microbiol. 49:232-233. 2. Bj$rnsen, P. K., J. B. Larsen, G. Hansen, and M. Olesen. 1986. A field technique for the determination of zooplankton grazing on natural bacterioplankton. Freshwater Biol. 16:245-253. 3. Hobbie, J. E., R. J. Daley, and S. Jasper. 1977. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:1225-1228.
VOL. 54, 1988 4. Kirchman, D. 1983. The production of bacteria attached to particles suspended in a freshwater pond. Limnol. Oceanogr. 28:
858-872. 5. Pedros Alio, C., and T. D. Brock. 1983. The importance of attachment to particles for planktonic bacteria. Arch. Hydrobiol. 98:354-379.
NOTES
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6. Watson, S. W., T. J. Novitsky, H. L. Quinby, and F. W. Valois. 1977. Determination of bacterial number and biomass in the marine environment. Appl. Environ. Microbiol. 33:94(-946. 7. Webster, K. E., and R. H. Peters. 1978. Some size-dependent inhibitions of larger cladoceran filterers in filamentous suspensions. Limnol. Oceanogr. 23:1238-1245.
