Validity of Eucaryote Inhibitors for Assessing Production

Vol. 53, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1987, p. 119-1280099-2240/87/010119-10$02.00/0Copyright © 1987, American Society for Microbiology

Validity of Eucaryote Inhibitors for Assessing Production andGrazing Mortality of Marine Bacterioplanktont

GORDON T. TAYLOR* AND MICHAEL L. PACEt

Hawaii Institute of Geophysics and Department of Oceanography, University of Hawaii, Honolulu, Hawaii 96822

Received 14 May 1986/Accepted 15 October 1986

Application of eucaryote inhibitors to the estimation of production and grazing mortality of bacterioplanktonwas evaluated. Exposure to a range of concentrations of thiram, cycloheximide, and neutral red (0.4 to 210, 36to 1,777, 4 to 346 ,uM, respectively) was 98 to 100% effective at inhibiting growth of a chrysomonad in culture.Exposure to colchicine and griseofulvin (50 to 1,000 ,uM for both) yielded only 24 to 94 and 53 to 79%inhibition, respectively. Exposures to thiram, neutral red, and griseofulvin were 90 to 100% effective atinhibiting growth in culture of a ciliate, Cyclidium sp., and the responses to colchicine and cycloheximide were

variable (64 to 100 and 0 to 100% inhibition, respectively). Thiram and neutral red inhibited field populationsof nanozooplankton more effectively than cycloheximide and colchicine. Direct effects of eucaryote inhibitorson growing cultures of bacterioplankton varied with parameters measured and duration of exposure. After3-day exposures, specific growth rates and "instantaneous" heterotrophic potential ([14C]glucose uptake) were

not consistently affected, but biosynthetic activity (RNA and DNA syntheses) was depressed. The degree ofinhibition of isolates and field populations of phytoplankton depended upon type of inhibitor and phytoplank-ton species. In field experiments, it was possible to calculate rates of bacterioplankton production and grazingmortality for only 16 of 29 inhibitor experiments and for 4 of 10 size fractionation experiments.Bacterioplankton production and mortality estimates varied greatly with the eucaryote inhibitor used, andthose derived from inhibition techniques were substantially different from those derived from fractionationtechniques. The poor performances of both techniques are attributed to the following: (i) effects of inhibitorson phytoplankton, (ii) indirect effects of the inhibitors on bacterioplankton, and (iii) insufficient separation ofgrazers from prey by filtration techniques. Because of the inconsistent results obtained in this investigation, westrongly recommend exercising caution in the application of inhibitor techniques to ecological problems,especially in phototrophically dominated systems.

Selective inhibitors have been used in aquatic microbiol-ogy as a method to separate the trophic activity ofprocaryotes and eucaryotes in planktonic associations (6, 9,11). Recently, selective eucaryote and procaryote inhibitorshave been used to estimate bacterioplankton productionrates, to assess the impact of grazing by phagotrophic nano-and microplankton on bacterioplankton production, and toexamine bacterial-protistan interactions on sedimentingbiogenic particles in marine systems (2, 12, 13, 16; B. F.Sherr, E. B. Sherr, T. L. Andrew, R. D. Fallon, and S. Y.Newell, Mar. Ecol. Prog. Ser., in press).The use of selective inhibitors has provided a novel

approach to the examination of bacterial-protistan interac-tions. This approach, however, has not been carefully eval-uated for marine pelagic systems. Newell et al. (12) incu-bated coastal surface water samples (collected 0.25 to 15 kmfrom the beaches of Sapelo Island, Ga.) with the eucaryoteinhibitors thiram and cycloheximide and compared bacterialnumbers in these treatments with uninhibited controls. Bytheir estimates, the proportion of bacterial productiongrazed by protists was variable, ranging from 0 to 100%.Fuhrman and McManus (2) performed similar experiments,with some refinements, using beach samples from Crane

* Corresponding author.t Hawaii Institute of Geophysics contribution no. 1799.t Present address: Institute of Ecosystem Studies, New York

Botanical Garden, Mary Flagler Cary Arboretum, Millbrook, NY12545.

Neck, N.Y., which were incubated with cycloheximide,colchicine, or benzylpenicillin (procaryote inhibitor of celldivision). Nongrazing bacterial mortality caused by autolysisand viral or bacterial endoparasites, or both, and grazingmortality were uncoupled from bacterial growth by treat-ments with benzylpenicillin and eucaryote inhibitors to-gether (nongrazing mortality), treatments with benzylpeni-cillin alone (nonspecific mortality), and control treatmentswithout inhibitors (growth and mortality). Most of the in-ferred bacterivory was attributable to small or very flexibleorganisms (passing through 0.6- and 1.0-,um membranes),and inferred grazing rates for unfiltered samples ranged from22 to 336% of estimated bacterial production rates (x =

123%; standard deviation = 102; growth based on [3H]-thymidine incorporation). Taylor et al. (16) deployed repli-cate particle interceptor traps at seven depths (50 to 2,000 m)and three stations in the eastern North Pacific. The trapswere filled with a nontoxic density gradient solution and halfof them were precharged with thiram. Comparisons ofconcentrations of particulate organic carbon/nitrogen,-aplastidic mastigotes, bacteria, and ATP were made amonglive control traps, live thiram traps, and traps prechargedwith preservatives. Aplastidic mastigotes were observed tobe the numerically dominant phagotrophic organism; com-parisons of inhibited and control traps indicated thataplastidic mastigotes accelerated decomposition of sedi-menting organic particles and nutrient regeneration.

Theoretically, in the experiments described above, theselective inhibitor debilitates or kills the eucaryotic grazers,

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primarily protists, and thereby uncouples the presumedmajor agent of bacterial mortality from bacterial production.Inhibitor techniques assume that bacterial growth is notaffected by (i) direct chemical inhibition, (ii) use of theinhibitor as a growth substrate, or (iii) indirect communityresponses such as release of intracellular organic pools byphytoplankton. If any of these assumptions are invalid, theapplicability of this technique is seriously compromised.The present study examines the effects of the eucaryote

inhibitors thiram (inhibits protein synthesis), cycloheximide(inhibits 80S ribosomal operation), neutral red (stimulatesautocytosis and inhibits endocytosis), colchicine (inhibitsmicrotubule polymerization), and griseofulvin (inhibitsmicrotubule polymerization) on the growth and metabolismof isolated cultures of marine microorganisms. On the basisof laboratory findings, field experiments were conductedwith samples collected from meso- and oligotrophic watersat six different geographical sites, using the most reliableinhibitors at their lowest effect doses. For the sake of clarity,the following text utilizes a more precise terminology pro-posed in reference 14, in which the terms protist, mastigote,plastidic, aplastidic, Apico, and Anano supplant protozoa,flagellate, phototrophic, heterotrophic, Hpico, and Hnano,respectively.

MATERIALS AND METHODS

Culture maintenance. An unidentified aplastidic chryso-monad species (mastigote) and a Scuticociliatida species,Cyclidium sp. (ciliate), isolated from oligotrophic waters(-900 miles [1,448 km] northeast of Hawaii), were main-tained in a two-stage continuous culture system. In the firststage, a mixed bacterial assemblage obtained from the<0.8-,um filtrate (Nuclepore membrane) of an offshore watersample was grown in f/2 medium (3), which was made fromfiltered oligotrophic seawater and amended with 3.0 and 1.6mg of Casamino Acids and sucrose, respectively, liter-'.Outflow from the first stage was split equally to supply twoprotist culturing vessels (one for Cyclidium sp. and one forthe chrysomonad). Prior to experiments with protists, aseries of culture vessels containing filter-sterilized seawateramended with one rice grain (-20 mg) and 1 ,uM glucosewere inoculated with either log-phase bacteria from the firststage of the chemostat or log-phase protists and attendantbacteria (specific growth rate [pu] = 1 to 3 day-'); thesesamples were incubated for 24 h at 22°C without light oragitation (= preincubation).Phytoplankton stock cultures (Thalassiosira pseudonana

clone 3H, Synechococcus sp. clone DC2, Dunaliellatertolectica, Phaeodactylum tricornutum, Chaetocerosgracile, and an unidentified chrysophyte) were maintained inf/2 medium at 22°C under a light bank (67.9 microeinsteinsm-2 S-1)

Inhibitor preparation. Stock solutions of eucaryote inhib-itors were prepared in distilled water in the following con-centrations (millimolar): thiram, 2.08; cycloheximide, 17.79;neutral red, 3.46; colchicine, 10.00; and griseofulvin, 10.00;they were stored at -20°C. Prior to use, stock solutions werewarmed to 60°C to increase solubility of the least solubleinhibitors (thiram, cycloheximide, and griseofulvin), and therequired volumes were filter sterilized through disposable0.2-p.m Acrodisc filters (Gelman Sciences, Inc., Ann Arbor,Mich.). Concentrations of thiram, cycloheximide, andgriseofulvin reported below are slightly higher than actualconcentrations because these inhibitors have low solubilitiesin water, and some insoluble material was undoubtedly lost

during filtration. In preliminary experiments, all inhibitorswere supplied by Sigma Chemical Co., St. Louis, Mo. (asreported in reference 2), but at low concentrations the thiramand cycloheximide from Sigma Chemical Co. were found tobe 93 to 100% less effective at inhibiting mastigote and ciliategrowth than those supplied by Aldrich Chemical Co., Mil-waukee, Wis. Therefore, thiram, cycloheximide, andcolchicine used in experiments reported below were sup-plied by Aldrich Chemical Co.

Inhibitor effects on cultured protists. Following the stan-dard preincubation (outlined above), log-phase cultures ofthe chrysomonad or Cyclidium sp. were transferred to sixseries of four flasks each (five inhibitors and one controlseries), a range of four concentrations of inhibitors (1.5 to 2.5orders of magnitude) was introduced, and the series wasincubated for 24 (chrysomonad) or 48 (Cyclidium sp.) h.Incubations for all experiments, except phytoplanktongrowth experiments, were conducted in darkness at 22°Cwithout agitation or aeration, unless otherwise noted.Protistan cell concentrations at the beginning and end ofincubations with inhibitors were determined from preservedsubsamples (2% [vol/vol] borate-buffered formaldehyde,final concentration). Chrysomonads were enumerated (20fields per sample) by epifluorescence microscopy at x400magnification, using acridine orange-stained preparations(5). Cyclidium cells were enumerated by assessing 5 to 10random transects on a Sedgewick-Rafter counting chamber(1.0-ml capacity).

Inhibitor effects on bacterial growth and metabolism. Short-term effects were examined by inoculating bacterioplanktoninto filter-sterilized seawater amended with 1.0 p.M glucose,0.005% (wt/vol) yeast extract (Difco Laboratories, Detroit,Mich.), and 0.1 ,uCi of [2-3H]adenine (specific activity, 20 Cimmol-1; New England Nuclear Corp., Boston, Mass.) ml-'and incubating for 1 h on a shaker table. Thiram, cyclohex-imide, or neutral red was then added at its lowest effectiveconcentration (-95% inhibition of protistan growth; 8, 178,or 4 FiM, respectively), and incubations were continued untilthe 3H20 yield was equal to approximately 30% of totaladded [3H]adenine activity. Biosynthetic activity ([3H]ATP,[3H]RNA, [3H]DNA, and 3H20 production) and total ATPconcentrations were measured with material captured onWhatman GF/F filters as described in references 7 and 8.Radioactive samples were suspended in Aquasol-2 (NewEngland Nuclear) and assayed on a Tri-Carb 4640 (PackardInstrument Co., Inc., Rockville, Md.), using a channels ratioquench correction program.Long-term inhibitor effects on growth and biosynthetic

activity of bacterioplankton were measured after a 3-dayincubation in two subsequent experiments. In the first ex-periment, bacterioplankton from the continuous culture sys-tem was inoculated into four flasks containing 100 ml offilter-sterilized seawater amended with 1.0 puM glucose and asingle sterile grain of rice. After a 4-h incubation, thecultures were amended with inhibitors and incubated on ashaker table at 150 rpm. After 3 days, 0.1 ,uCi of [3H]adenineml-1 was added, and total ATP, [3H]ATP, [3H]RNA,[3H]DNA, and 3H20 production were measured after 2.5 h(7, 8). In the second experiment, a mixture of senescentphytoplankton cultures (C. gracile, T. pseudonana, and anunidentified coccolithophore) replaced rice to simulate nat-ural nutrient sources more closely. Bacteria were enumer-ated by epifluorescent microscopy from preserved sampleswhich were stained with acridine orange and captured on0.2-p.m Nuclepore membranes (5). Growth rates were calcu-lated assuming exponential growth and using the formula, p.

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= (In CQ - In Co)It,, where p. = divisions per day, Co and C,= bacterial concentrations at beginning and end of incuba-tion, and t, = length of incubation.Long-term effects of the inhibitors on the heterotrophic

potentials of bacterioplankton were examined by using thesame protocol described for the first long-term experiment,except that on day 3 each flask was amended with 2.28 p.Ciof D-[U-'4C]glucose (NEC 042X; specific activity, 346 mCimmol-1; New England Nuclear). [14C]glucose incorporationat 0 and 4 h was determined by the radioactivity of materialfrom 1-ml subsamples captured on a Whatman GF/F filter(16). Respired '4CO2 was evolved by syringe injection of 0.1N HCl into sealed serum bottles containing 15-mlsubsamples and was captured on a suspended filter paperwick soaked with ,B-phenethylamine (4). Samples wereradioassayed as described above.

Effects of inhibitors on phytoplankton. Inocula (0.1 ml)from monocultures of marine phytoplankton (listed above)were introduced into triplicate culture tubes containing 10 mlof f/2 medium with or without inhibitors. Cultures wereincubated for 7 days as described above, and then in vivofluorescences were measured on a Turner Designfluorometer. Fluorescences of inhibited treatments werecompared with those of controls to calculate inhibition.

Field experiments. During the VERTEX 5 cruise (4 June to3 July 1984), 30-liter samples were collected with Niskinbottles from the chlorophyll a maximum (100 to 135 m) atthree stations (A, B, C) and from 10 m at station X on awest-southwest transect from Point Sur, Calif. (northeasternPacific transect experiment). Stations were 102 to 1,448 kmoffshore (see reference 16). Macrozooplankton were elimi-nated in all field experiments by prescreening the samplesthrough a 200-p.m Nitex mesh. The sample was then sub-jected to a concentration-fractionation protocol designed toenhance the influence of nano- and microplankton grazers (2to 20 and 20 to 200 p.m in diameter, respectively). Themicroplankton fraction (MICRO) was concentrated from 30to 1 or 2 liters across a 20-p.m Nitex mesh by gravity-drivenreverse-flow tangential filtration. The nanoplankton fraction(NANO) was concentrated two- to threefold from the<20-p.m filtrate across a 2.0-p.m Nuclepore membrane (142mm in diameter). The <2.0-p.m filtrate was not concentratedand represented the picoplankton fraction (PICO).Subsamples (100 ml) from each fraction (MICRO, NANO,and PICO) were incubated in acid-washed polyethylenebottles for 24 h in darkness at in situ temperatures afteraddition of inhibitors. Subsamples from each fraction werepreserved in 2% borate-buffered formaldehyde at 0 and 24 hfor subsequent epifluorescent microscopic analysis andgrowth rate calculations (as described above).

In the Hilo coast experiment, a 24-liter sample was col-lected at a station 6.7 km off Hilo, Hawaii (19 February1985), with Niskin bottles from 120 and 125 m (chlorophyll amaximum). A 4-liter subsample was passed through a1.0-p.m Nitex (nominal pore size) mesh-covered cylindricalframe by reverse-flow filtration as described above andrepresented a reduced grazer treatment. Duplicate 1-literaliquots for each treatment were placed in sterile 1.5-literNasco Whirl-Pak bags in an effort to minimize containmenteffects and were treated as follows: (i) <200 p.m (MICRO);(ii) <1.0 p.m (PICO); (iii) MICRO, 4 p.M thiram; (iv)MICRO, 178 p.M cycloheximide; (v) MICRO, 4 p.M neutralred; (vi) MICRO, 50 p.M colchicine. All incubations wereconducted under a simulated in situ light and temperature(23.5°C) regime in an on-deck incubator equipped withneutral density filters and flowthrough cooling water. Enu-

meration of picoplankton and mastigotes was performed onpreserved subsamples from 0- and 24-h time points asdescribed above. Mastigotes (30- to 50-ml subsamples) werecollected on Irgalan Black-stained 0.8-p.m Nuclepore mem-branes. Chlorophyll a was extracted in 90% acetone fromreplicate GF/F filters that had collected material from 200-mlsubsamples taken at 0 and 24 h. Chlorophyll a samples werestored at -20°C and subsequently analyzed on a TurnerDesign fluorometer (15).

Detailed examination of the effects of thiram on themicroplankton community was performed on a 25-liter sam-ple collected from the middle of an oligotrophic embayment(Kaneohe Bay, Oahu, Hawaii, 30 October 1984) at 1-mdepth. A 12-liter subsample was fractionated through a2.0-p.m Nuclepore membrane by reverse-flow filtration tominimize predator concentrations. Duplicate 2.3-liter ali-quots were placed in acid-washed clear polycarbonate bot-tles to further reduce containment effects. Experimentaltreatments were as follows: (i) <200 p.m (MICRO), To; (ii)MICRO, T24; (iii) MICRO, T24, plus 4 p.M thiram; (iv) <2.0p.m (PICO), To; (v) PICO, T24; (vi) PICO, T24, plus 4 p.Mthiram; where To and T24 are time zero and 24 h, respec-tively. Incubation bottles were tethered on a weighted lineand submerged at a depth of 1 to 2 m off the pier at theHawaii Institute of Marine Biology, Coconut Island, for 24h. In addition to quantification of bacterioplankton (Apico),aplastidic mastigotes (Anano), and chlorophyll a, enumera-tions of plastidic picoplankton (Ppico) and plastidicnanoplankton (Pnano) and measurements of microbial ATPwere performed. Enumerations of Ppico and Pnano wereperformed on autofluorescing cells from 30-ml subsamplescaptured on 0.2-p.m darkened Nuclepore membranes. Mi-crobial ATP from duplicate 350-ml subsamples captured onGF/F filters were extracted and analyzed as described inreference 8.

RESULTS

Laboratory studies. (i) Inhibition of protistan growth.Growth of the chrysomonad was 0 to 2% of the controls inthe presence of thiram, neutral red, and cycloheximide overthe ranges examined (Fig. 1A). Colchicine and griseofulvin,which both inhibit microtubule polymerization, were lesseffective; growth of the chrysomonad ranged from 24 to 94%of that of the control (Fig. 1A). Growth of the ciliateCyclidium sp. was 0 to 10% of the control in the presence ofthiram, neutral red, and griseofulvin over the ranges exam-ined (Fig. 1B). Growth responses of Cyclidium sp. to cyclo-heximide and colchicine were variable and these compoundswere less effective at inhibiting growth (Fig. 1B). Thiram andneutral red were, therefore, most effective at inhibitinggrowth of both types of protists, and these compounds wereeffective at very low concentrations (4 p.M for both).

Cultures of both the mastigote and the ciliate were ob-served microscopically to determine whether these protistswere viable after incubation with the inhibitors.Chrysomonads were observed swimming up to 3 days afteraddition of thiram at the lowest concentration (4 p.M), andCyclidium sp. remained active in all concentrations of cy-cloheximide and in all but the highest concentration ofthiram (208 p.M) up to 6 days after inhibitor addition.

(ii) Effects of inhibitors on bacterial metabolism and growth.Over short-term incubations, production of [3H]RNA and[3H]DNA by bacterioplankton cultures was impeded by thepresence of thiram, cycloheximide, and neutral red to 28, 68,and 38% of the control, respectively (Fig. 2). 3H20 produc-

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122 TAYLOR AND PACE

control values for the inhibitors thiram, cycloheximide, and[A neutral red (Fig. 4).

(iii) Effects of inhibitors on phytoplankton. Growth re-sponses of six species of phytoplankton to inhibitors werehighly variable among species and between inhibitors (Table1). The three diatom species were uniformly sensitive tothiram, cycloheximide, and neutral red. Two of these spe-

O4- =° -----9cies (P. tricornutum and C. gracile) were less affected by-'\ colchicine; growth rates were 50 and 58% of the control,_`<-b respectively. The procaryote Synechococcus sp. was rela-

tively unaffected by cycloheximide and neutral red but_ 8 severely inhibited by thiram (,u= 5% of control). The

chlorophyte D. tertolectica was slightly inhibited by thiramand neutral red and was severely inhibited by cycloheximide(,u = 4% of control). The unidentified chrysophyte wassensitive to thiram (,u = 19% of control) and cycloheximide([L = 2% of control) and less sensitive to neutral red andcolchicine.

\B Field experiments. (i) Northeastern Pacific transect experi-ment. For comparative purposes, size fractionation and

_ -Ax inhibitor techniques were used concurrently in all fieldexperiments. The PICO fraction in this experiment repre-sented the picoplankton assemblage released from grazing

7/ \ / \, pressure. In the absence of substrate limitation, we expected-<t/ \ / \\ that positive and similar growth rates would occur in all

treatments. Apparent specific growth rates of aplastidicpicoplankton (Apico) were positive in 13 of 16 samples (Fig.

A \1 \ 5A), and these positive values varied from 0.02 to 1.37_~@--sZ,.-__-s>*- )J day-'. There was, however, no consistent effect of the

inhibitors; i.e., growth in the inhibited treatments varied0 1 2 3 4 inconsistently with respect to one another and to the con-

trols (Fig. 5A).Inhibitor Concentration (log10 I.LM) The NANO fraction represented picoplankton in the pres-Dose responses of cultured protistan production to ence of both nanoplankton predators (Anano; concentratedinhibitors as percentage of control (mean of 5 to 20 two to three times) and plastidic nanoplankton (Pnano). We

'A) Responses of a phagotrophic chrysomonad to a 24-h predicted that, if the eucaryote inhibitors were effective,(B)espnsesof-he ciliate Cyclidium sp. to a 48-h;(B) responses of th clitCyldusptoa4- apparent growth rates of Apico would be lower in the

Symbols: *, neutral red; A, thiram; O, cycloheximide; aAO, griseofulvin.' ' ' controls relative to the inhibited treatments. This was not the

case. Apparent growth rates of Apico in the presence ofinhibitors exceeded those of controls in only 7 of 12 samples(Fig. SB). Apparent growth rates were consistently higher

treatments with inhibitors was similar to control than controls only in treatments with the inhibitor cyclohex-)n the basis of a close correlation between 3H20 imide. Overall, the apparent growth rates of Apico in thison and adenine uptake (8), these results suggest that size fraction were higher than in PICO and MICRO frac-mine was taken up at comparable rates in all treat- tions, ranging from 0.07 to 1.51 day-'.at that biosynthesis (as measured by [3HJRNA and The MICRO fraction represented pico- and nanoplankton

[3H]DNA production) was less efficient in the inhibitedtreatments relative to the control (Fig. 2). Growth rates overthe entire incubation were estimated by using changes inATP and assuming exponential increase. Growth ratesranged from 0.073 to 0.202 h-', and the inhibitors impededgrowth in proportions similar to those of the biosynthesismeasurements (Fig. 2).

Since most published inhibitor studies are based on -1-day incubations, we were interested in the effects of inhibi-tors in long-term experiments, such as in particle trapdeployments (16). The specific production rates (,u; per day)of ATP and cells for control, thiram, cycloheximide, neutralred, and colchicine treatments after 3 days were similar forbacterioplankton grown on rice detritus (Fig. 3A) and onsenescent phytoplankton (Fig. 3B). On the other hand,production rates of [3H]RNA, [3H]DNA, and 3H20 in theinhibited treatments were generally lower in the short incu-bations (<2.5 h) at the end of the 3-day period (Fig. 3A andB). The rates of incorporation and respiration of ['4C]glucoseat the end of a 3-day incubation period were comparable to

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FIG. 2. Short-term (6-h) effects of inhibitors on production ofATP, [3H]RNA, [3H]DNA, and 3H20 by laboratory cultures ofbacterioplankton: 8 ,uM thiram; 178 ,uM cycloheximide (Cyclo.); 4,uM neutral red (N.R.). Bars represent means of two replicates;ranges of most were too narrow to present.

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ParameterFIG. 3. Long-term (3-day) effects of inhibitors on production rates of ATP, cells, [3H]RNA, [3H]DNA, and 3H20 by laboratory cultures

of bacterioplankton. Addition of [3H]adenine and radioassay were performed in the last 2.5 (A) or 1 (B) h of incubation. (A) Marinebacterioplankton incubated for 3 days with rice grain, 1 ,uM glucose, and inhibitors. (B) Same protocol as (A) but senescent phytoplanktonused as a nutrient source. Concentrations: 8 ,uM thiram; 178 puM cycloheximide (Cyclo.); 4 p.M neutral red (N.R.); 50 ,uM colchicine (Colch.).Error bars represent ranges of duplicate samples.

in the presence of enhanced microplankton concentrations(15 to 20 times). Apparent growth rates of Apico were

predicted to be higher in the inhibited treatments than in thecontrols but, as in the NANO size fraction, they were higherin only 7 of 12 inhibited samples (Fig. 5C). Apparent growth

rates in the presence of cycloheximide at three stations wereagain greater than controls, which indicated that either graz-ing was blocked or bacterial growth was stimulated. The ap-parent growth rates of Apico in all three size fractions in thepresence of the three inhibitors did not match expectations.

TABLE 1. Effects of eucaryote inhibitors on chlorophyll a production of phytoplankton isolatesa% of control'

Species Thiram Cycloheximide Neutral Red Colchicine

SD x SD x SD SD

Synechococcus sp. (clone DC2) 5.03 0.22 97.35C 15.89 83.77c 18.33 NTd NTD. tertolectica 91.07c 102.63 4.21 11.55 81.60c 141.89 NT NTUnidentified chrysophyte 18.59 3.22 1.73 1.11 94.58c 5.77 74.73C 31.43T. pseudonana (clone 3H) 0.17 0.20 0.04 0.04 24.77 16.32 NT NTP. tricornutum 1.45 0.61 1.03 0.31 2.65 1.01 50.00 28.28C. gracile 0.88 1.97 6.38 8.18 1.80 0.15 58.16 68.24

a In vivo fluorescence measured after 1 week of incubation of 1% inocula in triplicate culture tubes (100% = 0% inhibition).b 4 ,uM thiram; 178 ,uM cycloheximide; 2 ,uM neutral red; 50 pFM colchicine.c Not significantly different from control (Student's t test; P < 0.05)d NT, Not tested.

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Thiram Cyclo. N.R.

14 C-incorporation 14C-respiration

in the PICO fraction but was inhibited in the MICRO fraction(6% of control). At the initiation of the experiment, bothfractions contained the same chlorophyll a concentrations(0.196 + 0.029 ,ug liter-'). Ppico accounted for a substantialportion of the chlorophyll a in Kaneohe Bay, and Pnano cellsdid pass through the 2-p.m filter. Initial concentrations ofPnano were 6.96 ± 3.23 x 106 and 10.42 ± 5.22 x 106 liter-'in the PICO and MICRO fractions, respectively, indicatingthat only 40% of these organisms were effectively excludedby our reverse-flow fractionation. Specific chlorophyll aproduction was inhibited in the presence of thiram relative tothe controls in both size fractions (Table 2).

ParameterFIG. 4. Long-term effects of inhibitors on potential for incorpo-

ration and respiration of [U-14C]glucose by laboratory cultures ofmixed assemblages of bacterioplankton. Parallel incubation to Fig.3A, except samples were incubated last 4 h of day 3 with [U-"4C]glucose. Concentrations: 8 ,uM thiram; 178 ,uM cycloheximide(Cyclo.); 4 F.M neutral red (N.R.). Bars represent means of dupli-cates; ranges were too narrow to present.

(ii) Hilo coast experiment. At the initiation of the experi-ment, the PICO (<1.0 m) and MICRO (<200 [.m) fractionscontained nearly the same Apico (mean + 95% confidenceinterval, n = 40 counts; 5.0 ± 2.4 x 108 and 4.2 ± 0.9 x 108liter-) and chlorophyll a (0.163 t 0.110 and 0.142 ± 0.096pg liter-) concentrations. The reverse-flow filtration proce-dure also did not totally eliminate mastigotes from the PICOfraction which contained 2.14 ± 0.71 x 105 versus 6.22 +

1.19 x 105 cells liter-' in the MICRO fraction. Hence,grazing pressure on picoplankton was lowered but noteliminated in the PICO fraction. As in the previous fieldexperiment, apparent growth rates of Apico were not en-hanced in the PICO fraction or in the MICRO fractionsamended with inhibitors relative to the controls (Fig. 6A).Apparent Apico specific growth rates were positive in alltreatments, and mean cell volumes increased two- to three-fold over the 24-h incubation. The increase in cell volumesuggests that containment effects, such as organics leachingfrom Whirl-Pak bags, may have influenced the results of theexperiment.Growth of mastigotes was positive in the control treat-

ments, and growth was effectively blocked by thiram, cyclo-heximide, neutral red, and colchicine (Fig. 6B). Apparentspecific growth rates of phototrophs (primarily chroococcoidcyanobacteria, Chlorella-like chlorophytes, and plastidicmastigotes) as measured by chlorophyll a production werenegative in all treatments except thiram (Fig. 6C). Chloro-phyll a concentrations could not be determined in the neutralred treatment because of interference resulting from thestain. The nearly uniform decrease in chlorophyll a againsuggests that containment effects may have influenced theresults of the experiment.

(iii) Kaneohe Bay experiment: community effects of thiram.ATP concentrations increased in all treatments during the24-h incubation; ILATP varied from 0.03 to 1.374 day-' (Table2), indicating net growth of the community or some compo-

nent thereof. Although apparent growth rates in both PICOtreatments were greater than in the MICRO treatments,initial ATP concentrations in the PICO fraction were 22% ofthe MICRO fraction (82 versus 380 ng liter-'), and final ATPconcentrations were 46 to 84% of the MICRO treatments.The net rate ofATP accumulation was not affected by thiram

Control m Thiram * Cyclo. E N.R.

2.0 .

1.5 .

1.0

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0)

40)-c

3

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ci

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0-0.0 9_ _"

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1.5-

1.0

0.5-

0.0 I

-0.5

2.0 MICRO1.5 -

1.0

0.0

-0.5A B x C

StationFIG. 5. North Pacific transect experiment. Effects of inhibitors

on production of aplastidic picoplankton (Apico) in three sizefractions of samples collected from four stations. (A) PICO fraction= 0.2 to 2.0 p.m. (B) NANO = 2.0 to 20 p.m (concentrated two- tothreefold plus PICO fraction). (C) MICRO = 20 to 200 p.m (concen-trated 15- to 30-fold) plus NANO and PICO fractions (see text fordescription). Station locations, sample depth, and date: A-35055'N, 122°34'W, z = 100 m, 16 June 1984; B-34°45'N, 129054'W,Z = 120 m, 25 June 1984; X-34011'N, 133°15'W, z = 10 m, 9 June1984; C-33006'N, 139°34'W, z = 125 m, 30 June 1984. Samplesincubated for 24 h with inhibitors (4, 178, or 2 p.M thiram, cyclo-heximide [Cyclo.], or neutral red [N.R.]) in darkness at ambienttemperature. Error bars represent 95% confidence intervals aroundmeans (n = 20 fields). d, Day.

0

L.

c0

0

125

100

75

50

25

0



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TABLE 2. Estimated net production rates for components of the microplanktonic community based on ATP, chlorophyll a,and cell concentrationSa

Mean net production rate per day (range)Parameterb PICO (0.2-2.0 pm) MICRO (0.2-200 ,um)

Control +Thiram Control +Thiram

ATP 1.328 (0.073) 1.374 (0.210) 0.538 (0.265) 0.030 (0.033)Chlorophyll a 0.668 (0.491) -0.807 (0.017) 0.862 (0.030) 0.159 (0.043)Apico volc 0.545 (0.149) 0.818 (0.025) 0.482 (0.062) 0.771 (0.009)Ppico 0.102 (0.071) -0.400 (0.065) 0.086 (0.087) -0.206 (0.051)Anano 3.113 (0.046) -1.972 (0.752) 1.171 (0.146) -3.190 (0.247)Pnano 0.523 (0.102) 0.040 (0.083) 0.360 (0.057) -0.009 (0.000)

a Sample was collected from Kaneohe Bay, Oahu, Hawaii (9 November 1984), size fractionated, and incubated for 24 h with or without thiram (4.2 ,uM). Meanand ranges for two replicate bottles are reported.bApico, Aplastidic picoplankton (0.2 to 2.0 pum in diameter); Ppico, plastidic picoplankton (autofluorescent, 0.2 to 2.0 ,um in diameter); Anano, aplastidic

nanoplankton (2 to 20 ,um in diameter); Pnano, plastidic nanoplankton (autofluorescent, 2 to 20 p.m in diameter).c Apico volume was used in calculation because mean cell volumes varied between treatments for bacteria, but did not for other organisms.

3.5 [

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0.5

0.3

0.1

-0.1

-0.3

-0.5

Il

Micro Pico Thiram Cyclo. N.

TreatmentFIG. 6. Hilo Coast experiment. Effects of inh

fractionation on apparent production ratesnanomastigotes (B), and chlorophyll a (C). Samplo120 to 125 m at a station 6.7 km northeast of HilcFebruary 1985. Error bars represent 95% confaround means (n = 20, 40, and 4 in A, B, and (Cyclo., Cycloheximide; N.R., neutral red; ColUND, Not detectable.

Apico cell concentrations and mean cell volumes in thePICO and MICRO fractions were indistinguishable at the

A initiation of the experiment (1.20 x 109 and 1.22 x 109Apico liter-', respectively, and 0.147 p.m3 cell -1 for both). After 24

h, Apico cell concentrations increased uniformly in alltreatments and fractions (2.00 x 109 to 2.26 x 109 liter-').Mean cell volumes, however, in the thiram treatments were1.4-fold greater than in the controls (0.194 and 0.147 p.m3cell-', respectively). On the basis of total biovolume pro-duced, specific growth rates of Apico in thiram treatments ofPICO and MICRO fractions were 1.5- to 1.6-fold greaterthan those of the controls. Net growth rates in the PICOfractions were greater than in comparable MICRO fractions.At the initiation of the experiment, concentrations of Ppico,

B composed mostly of chroococcoid cyanobacteria and, to aNano lesser extent, Chlorella-like cells, were the same in PICO

and MICRO fractions, 9.59 + 1.66 x 107 and 8.58 + 1.91 x107 liter-', respectively. Growth rates of Ppico were positivein the control treatments and approximately equal in thePICO and MICRO fractions (Table 2). Apparent growthrates of Ppico in the thiram treatments were negative in bothsize fractions. A similar negative impact of thiram on theprocaryote Synechococcus sp. was observed in the labora-tory experiments.

Prefractionation excluded 82% of the Anano from thePICO fraction; initial concentrations were 0.61 + 0.14 x 105

C and 2.43 + 0.30 x 105 liter-' in the PICO and MICROrophyll fractions, respectively. Unlike Pnano cells, the Anano cells

did not tend to pass through the filter. This difference can beattributed to the observed overall larger size of Anano cells

z relative to Pnano (many Anano cells were in the 2- to 5-p.mD

size range) and possibly to a greater abundance of individu-als with rigid cell walls among the Anano. Thiram effectivelyblocked growth of Anano and Pnano in both fractions (Table2). Anano were especially sensitive to the inhibitor. It is notclear why there was an apparent difference in the sensitivity

R. Coich. of Anano and Pnano. Since thiram inhibits protein synthesis,its effect should be independent of trophic mode. In thecontrol treatments, net growth rates of both Anano andPnano were high (Table 2). Anano growth rates were espe-

ibitors and size cially high in the PICO relative to the MICRO fraction (Tableof Apico (A), 2), reflecting both the rapid increase in their prey and,e collected from probably, release from predation.n,Hawaii, on 16 In this experiment the effect of thiram on Apico growth,drespectively), appeared to conform with expectation. Apparent growth in thech., colchicine- inhibited treatment in both size fractions exceeded growth in

the control, and the difference in growth rates might have been

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126 TAYLOR AND PACE

due to grazing. It is also clear, however, that thiram had a

negative effect on the phototrophs and that Apico cells in thethiram treatment were larger at the end of the experiment. Weattribute this difference in part to stimulation of the Apico cellsas a result of dying phytoplankton.

DISCUSSION

On the basis of our laboratory results, the requirementsfor an inhibitor that blocks protistan activity completely and

selectively were not entirely met by any of the inhibitorstested. At the concentrations required to block protistangrowth, the inhibitors also affected bacterial metabolism andphytoplankton growth. Sherr et al. (in press) also observedthat several eucaryote inhibitors, including thiram, cyclo-heximide, demicolchicine, and colchicine, had mixed effectson the growth of a ciliate, Uronema sp., an aplastidicmastigote, natural assemblages of bacterioplankton, andaplastidic nanoplankton. They found that colchicine andcycloheximide, when used in combination, blocked growthof lab and field populations of protists to varying degrees anddid not affect the growth rates of bacterioplankton in theDuplin River estuary. Similarly, Sanders and Porter (13)report that use of cycloheximide and penicillin to assess

grazing mortality and production of bacterioplankton inLake Oglethorpe, Ga., yielded inconsistent results. Further-more, they demonstrated that three freshwater species ofciliates continued swimming and feeding in the presence ofcycloheximide (13). Our own observations demonstrate that,although protistan cell concentrations remained constant ordeclined during incubations with inhibitors, growth-incompetent protists remained viable for long periods oftime. The possibility cannot be excluded that these protistscontinue to feed, resulting in underestimates of bacterialgrowth and grazing mortality.The inhibitors had a strong negative effect on instanta-

neous measurements of bacterial biosynthesis in both theshort- and long-term experiments, whereas substrate utiliza-tion and growth rates calculated from ATP and cell yield inthe inhibited and control cultures were similar. The effect ofinhibitors was, therefore, to alter bacterial growth patterns.Although all cultures eventually reached the same endpointin 3 days, the rate at which they reached the endpoint mayhave varied, which violates an important assumption thatthese inhibitors have no effect on procaryotes. This effect islikely to be most critical in shorter incubations (c24 h) inwhich the rate of bacterial growth in inhibited treatments isthe key variable for determining grazing and productionrates. Also, the inconsistent impact of these inhibitors on

phytoplankton isolates suggests that field experiments thatuse inhibitors to uncouple bacteria-protistan interactions are

likely to be influenced indirectly by inhibitor effects on thephytoplankton.Our field experiments illustrate the interpretational prob-

lems associated with inhibitor experiments in planktonicsystems. If we assume that the production rates (k) of Apicoare approximated by production rates calculated for inhib-ited treatments and that production rates calculated for thecontrol represent realized production with grazing mortality(r), then we can calculate grazing rates (d) from the differ-ence: d = k - r (equation 1). For the fractionation studies(PICO, NANO, MICRO treatments), the same approach canbe applied where k is the production rate in the PICOfraction and r is the realized production rate in the NANO orMICRO fraction. These rates can be derived in each type ofexperiment by solving the equation: P, = Po e(k - d)t (equation

2), where P is the concentration of the organisms of interest(i.e., Apico) at the beginning (to) and end (t,) of the experi-ment (10). These rates are calculated for our field experi-ments for cases in which k was greater than r (Table 3). Inthe northeastern Pacific transect experiment, k was less thanr in 9 of 35 inhibited treatments and in 5 of 8 fractionations.In the experiment off the Hilo coast, k was less than or verynearly equal to r in every case. Calculation of d in thesecases would lead to the conclusion that production rateswere greater in the presence of grazing mortality than in itsabsence, which cannot be demonstrated in these experi-ments. Where calculation of the rates was possible, thevalues for production and grazing mortality within a singlesize fraction vary inconsistently among treatments. Forexample, at station C in the NANO size fraction there is atwo-fold difference in the values of k measured with thiramrelative to cycloheximide and neutral red and a 20-folddifference in the values of d. The good agreement in the rates

TABLE 3. Estimated Apico production (k) and mortality (d) ratesdetermined by inhibitor and fractionation techniques

for field samplesGrazer impact

Samplea Treatmentb k (day -1) d (day -) (% of dailyproduction)

N. Pacific transectStation APICO T 0.350 0.156 45

C 0.313 0.119 38NANO C 1.00 0.184 18MICRO N 1.03 0.198 19

Station BPICO C 0.796 0.379 48NANO T 0.571 0.296 52

C 0.948 0.673 71F 0.417 0.142 34

MICRO T 0.288 0.388 135C 0.677 0.777 115F 0.417 0.517 124

Station XPICO T 0.421 0.521 124

N 0.085 0.185 218NANO T 0.647 0.353 55

C 0.717 0.423 59MICRO T 0.096 0.176 183

C 0.188 0.268 143

Station CPICO C 1.370 0.760 55

N 0.652 0.042 6NANO T 0.874 0.032 4

C 1.500 0.658 44N 1.510 0.668 44

MICRO C 0.884 0.347 39N 1.010 0.473 47F 0.610 0.073 12

Hilo Coast Cannot be calculated

Kaneohe BayPICO T 0.818 0.273 33MICRO T 0.771 0.289 37

F 0.545 0.063 12

a Prefractionated samples: PICO = 0.2 to 2.0 pLm; NANO = 0.2 to 20.0 ,um;MICRO = 0.2 to 20 ,um.bT, Thiram; C, cycloheximide; N, neutral red; F, fractionation (apparent

growth from PICO fraction used as k).



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calculated for the inhibitors neutral red and cycloheximide inthis case is the exception rather than the rule (Table 3). Inthe Kaneohe Bay experiment, there is a rough agreementbetween the production rates (k) calculated with the frac-tionation procedure and the inhibition method (Table 3, Tand F treatments). Grazing mortality (d) calculated by thetwo methods differs by a factor of 5. The inconsistency ofthese rates argues that these estimates are largely spuriousand that the techniques are not reliable.

It is possible to criticize our field experiments and suggestthat the disparities observed were the result of artifacts. Forexample, in the northeastern Pacific transect experiment,two potential artifacts may have biased our results. First, the24-h incubations were carried out in 100-ml polyethylenebottles, which may have resulted in containment effects (1).In addition, our reverse-flow fractionation may have in-creased nutrient concentrations by disrupting cells or byleaching primary amines from Nuclepore membranes (1). Inthe latter cases, the final fraction (PICO) should have con-tained the highest concentrations of extracellular metabo-lites and primary amines and thereby exhibited the highestgrowth rates. We consistently observed lower Apico growthrates in the PICO fraction and believe that any disruption ofcells or leaching resulting from our procedure was minimal.In the other two field experiments, we attempted to minimizecontainment effects by using sterile 1.5-liter Whirl-Pak bagsand acid-washed 2.5-liter polycarbonate bottles, and we stillobtained inconsistent results.

Despite possible experimental artifacts, we believe theinhibitor method failed largely because of effects on thephytoplankton, which confounded the experiments. Thissupposition is supported by the observed sensitivity oflaboratory cultures of phytoplankton to inhibitors and therepeated observations of net mortality of phytoplankton andstimulation of bacteria in terms of production and increasedaverage size in the presence of inhibitors. Findings reportedin reference 11 also support this hypothesis. They report avariable response of picoplankton from the Sargasso Sea andthe Canadian Arctic to a 6-h exposure to cycloheximide (100,uM); 3H-labeled amino acid and [3H]glucose incorporationswere only inhibited slightly, whereas 33P04 incorporationwas markedly depressed. Some inhibition of 33P04 incorpo-ration may be attributable to altered Ppico activity, but itseems unlikely because incorporation of [14C]bicarbonate bypicoplankton was unaffected by cycloheximide. Incorpora-tion of [14C]bicarbonate into Pmicro, however, was totallyinhibited by cycloheximide. These observations are consis-tent with our findings that the cyanobacterium Synechococ-cus sp. (a numerically significant component of Ppico) wasnot inhibited by cycloheximide, whereas all eucaryotic phy-toplankton species tested (some of which may have beenincluded in Li and Dickie's microplankton fraction [11])were sensitive to cycloheximide.Use of an additional procaryote inhibitor, such as benzyl-

penicillin (2, 13; Sherr et al., in press), to block bacterial celldivision may circumvent the problem of stimulated bacterialgrowth but is more likely to introduce a new set of artifacts.Penicillin has been shown to inhibit ingestion of latex beadsby three freshwater species of ciliates relative to controls(13). Furthermore, Iturriaga and Zsolnay (6) demonstratedthat the procaryote inhibitor gentamicin, which inhibitsbacterial metabolism, also inhibits ['4C]-bicarbonate incor-poration by phytoplankton at concentrations of >214,uM.The problems associated with phytoplankton inhibition po-tentially compromise results from epipelagic systems but areprobably avoided in systems in which phototrophic pro-

cesses do not dominate, such as below the photic zone, insediments, and in turbid detritus-based estuaries (16; Sherret al., in press).

Conclusions. We conclude that the application of inhibitortechniques to epipelagic systems does not produce reliableresults and that careful use of these techniques shouldprobably be restricted to situations in which phototrophicprocesses are not important, such as subeuphotic zonesamples and sediments. Even with these restrictions, ratemeasurements must be interpreted cautiously, because ofthe presence of nontarget organisms and because of thedifferential effects of inhibitors on target organisms.

ACKNOWLEDGMENTSWe are grateful to the crews of the RIV Wecoma and the

Hokkaido University training ship Hokusei Maru and to the staff ofthe Hawaii Institute of Marine Biology for assistance in fieldsampling, to S. Taguchi and U. Magaard for technical assistance,and to C. Hironaka, P. Sexton, D. Haneda, and D. Henderson formanuscript preparation. We thank D. M. Karl, C. D. Winn, and M.Bailiff for their comments on and criticisms of this manuscript.

This study was supported, in part, by National Science Founda-tion grant OCE 82-16673 (awarded to D. M. Karl) and by anintramural grant from the Office of Research Administration, Uni-versity of Hawaii (awarded to G. T. Taylor).

LITERATURE CITED1. Ferguson, R. L., E. N. Buckley, and A. V. Palumbo. 1984.

Response of marine bacterioplankton to differential filtrationand confinement. Appl. Environ. Microbiol. 47:49-55.

2. Fuhrman, J. A., and G. B. McManus. 1984. Do bacteria-sizedeukaryotes consume significant bacterial production? Science224:1257-1260.

3. Guillard, R. R. L., and F. H. Ryther. 1962. Studies on marineplanktonic diatoms. I. Cyclotella nana Hustedt and DetonulaConferiacea (Cleve) Gran. Can. J. Microbiol. 8:229-239.

4. Hobbie, J. E., and C. C. Crawford. 1969. Respiration correc-tions for bacterial uptake of dissolved organic compounds innatural waters. Limnol. Oceanogr. 14:528-536.

5. Hobbie, J. E., R. J. Daley, and S. Jasper. 1977. Use ofNuclepore filters for counting bacteria by fluorescence micros-copy. Appl. Environ. Microbiol. 33:1225-1232.

6. Iturriaga, R., and A. Zsolnay. 1981. Differentiation betweenauto- and heterotrophic activity: problems in the use of sizefractionation and antibiotics. Bot. Mar. 24:399-404.

7. Karl, D. M. 1981. Simultaneous rates of ribonucleic acid anddeoxyribonucleic acid syntheses for estimating growth and celldivision of aquatic microbial communities. Appl. Environ. Mi-crobiol. 42:802-810.

8. Karl, D. M., C. D. Winn, and D. C. L. Wong. 1981. RNAsynthesis as a measure of microbial growth in aquatic environ-ments. I. Evaluation, verification, and optimization of methods.Mar. Biol. 64:1-12.

9. Krempin, D. W., S. M. McGrath, J. Beeler SooHoo, and C. W.Sullivan. 1981. Orthophosphate uptake by phytoplankton andbacterioplankton from the Los Angeles Harbor and SouthernCalifornia coastal waters. Mar. Biol. 64:23-33.

10. Landry, M. R., L. W. Haas, and V. L. Fagerness. 1984.Dynamics of microbial plankton communities: experiments inKaneohe Bay, Hawaii. Mar. Ecol. Prog. Ser. 16:127-133.

11. Li, W. K. W., and P. M. Dickie. 1985. Metabolic inhibition ofsize-fractionated marine plankton radiolabeled with amino ac-

ids, glucose, bicarbonate, and phosphate in the light and dark.Microb. Ecol. 11:11-24.

12. Newell, S. Y., B. F. Sherr, E. B. Sherr, and R. D. Fallon. 1983.Bacterial response to presence of eucaryote inhibitors from a

coastal marine environment. Mar. Environ. Res. 10:147-157.13. Sanders, R. W., and K. G. Porter. 1986. Use of metabolic

inhibitors to estimate protozooplankton grazing and bacterialproduction in a monomictic eutrophic lake with anaerobichypolimnion. Appl. Environ. Microbiol. 52:101-107.

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14. Sieburth, J. M., and K. W. Estep. 1985. Precise and meanin$fulterminology in marirqe microbial ecology. Mar. Microb. FoodWebs'l:f-16.

15. Strickland, J. D. H., and T. R. Parsons. 1972. A practicalhandbook of seawater analysis. Fisheries Research Board ofCanada? Ottawa.

16. Taylor, G. T., D. M. Karl, and M. L. Pace. 1986. Impact ofbacteria -and zooflagellates on the composition of sinking par-ticles: an in situ experiment. Mar. Ecol. Prog. Ser: 29:141-155.

17. Wright, R. T., and J. E. Iobbie. 1966. Use of glucose andacetate by bacteria in aquatic ecosystems. Ecology 47:447-464.

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Validity of Eucaryote Inhibitors for Assessing Production