Culture Characteristics and Orthophosphate Excretion of a Marine Oligotrich Ciliate, Strombidium Sulcatum, Fed Heat-Killed Bacteria

MARINE ECOLOGY PROGRESS SERIES Vol. 105: 159-165.1994 Published February 17 Mar. Ecol. Prog. Ser.

Culture characteristics and orthophosphate excretion of a marine oligotrich ciliate, Strombidium sulcatum, fed heat-killed bacteria

K. Allali, J. Dolan*,F. Rassoulzadegan

Observatoire des Sciences de I'Univers, Universitk Paris V1 IINSUI CNRS, URA 716, Station Zoologique, BP 28, F-06230 Villefranche-Sur-Mer, France

ABSTRACT: Ciliate microzooplankton have long been hypothesized as probably being important in nutrient regeneration. However, few estimates of excretion rates exist, especially with regard to phosphorus, identified as the limiting nutrient in many marine and estuarine systems. The utility of using heat-killed prey, which avoids prey uptake of excretory products, to measure excretion in feeding ciliates was examined. The growth characteristics of Strombidium sulcaturn fed heat-killed vs live bacteria were investigated and orthophosphate excretion rates were estimated. Organismal and orthophosphate concentrations were monitored over an 8 d incubation period in triplicate solutions of live bacteria, live bacteria plus ciliates, heat-killed bactena, and heat-killed bacteria plus ciliates. Ciliate cultures fed heat-lulled or live bacteria were indistinguishable in terms of the duration of different growth phases, maximum growth rates, maximum concentrations and average cell dry weights based on volume estimates. Calculated orthophosphate excretion rates of S. sulcatum, based on changes in concentrations of orthophosphate in cultures fed heat-killed bacteria, were highest for lag-phase cells, 13 X IO-~pg-at. ciliate-' h-' (24.6 lgP mg-' dry wt h-') and decreased to zero for late stationary-phase cells.

KEY WORDS: Phosphorus . Microzooplankton . Culture

INTRODUCTION nids (Verity 1985) and an oligotrich (Ferrier & Rassoul- zadegan 1994), phosphorus excretion has only been Nearly 30 yr ago Johannes (1964, 1965) suggested estimated in terms of turnover time for a fresh- that marine ciliates, even if only a minor fraction of water mixotrophic oligotrich, the chloroplast-retaining total planktonic biomass, could be responsible for a Strombidium viride (Taylor & Lean 1981). To our large portion of nutrient recycling in plankton com- knowledge, data on phosphorus excretion rates for munities. The suggestion was based on evidence that marine ciliates are limited to a single study of a scutic- weight-specific excretion rates increase with dimin- ociliate (Berman et al. 1987);there are no phosphorus ishing body size, a relationship which is now well excretion estimates for marine oligotrichs, generally established (Caron & Goldman 1990). Johannes' the dominant group of planktonic ciliates in marine hypothesis is also supported by findings that, rather systems. than being minor constituents, ciliates are often a The lack of data on phosphorus excretion may be major component of marine plankton communities partly due to a lack of interest because nitrogen rather (Beers & Stewart 1969). than phosphorus is commonly thought to limit primary Despite the possible importance of ciliates, very few production in marine systems (Hecky & Kilham 1988). estimates of excretion rates are available for typical However, some oceanic systems such as the Mediter- marine forms, i.e. tintinnids and oligotrichs. While ranean and the tropical Atlantic may be phosphorus- nitrogen excretion has been investigated in both tintin- limited (Berland et al. 1980, Krom et al. 1991, Raim- bault & Pujo-Pay 1993). Recent studies of coastal 'Addressee for correspondence systems have concluded that phosphorus can be limit-

O Inter-Research 1994 Mar. Ecol. Prog. Ser. 105: 159-165, 1994

ing, depending on the season, for phytoplankton or incubation period in solutions of live bacteria, live bac- bacteria in systems as diverse as the Chesapeake teria plus ciliates, heat-killed bacteria, and heat-killed (Fisher et al. 1992) and Florida Bays, USA (Fourqurean bacteria plus ciliates. Ciliate cultures were compared et al. 1993), Norwegian fjords (Sakshaug & Olsen 1986, in terms of the duration of different growth phases, Thingstad et al. 1993), the Baltic Sea (Lignell et al. maximum growth rates, maximum concentrations and 1992, Zweifel et al. 1993) and the NW Mediterranean average cellular dry weights based on volume esti- (Zweifel et al. 1993). Another possible explanation for mates. We calculated orthophosphate excretion rates the paucity of data may be the difficulties involved of S. sulcatum based on changes in concentrations of with measuring ciliate excretion rates. orthophosphate in cultures fed heat-killed bacteria. Estimates based on excretion of isotopes involve long incubation times with labeled food to insure equi- librium of all cellular P pools followed by removal of METHODS AND MATERIALS ciliates from their prey (Taylor & Lean 1981, Taylor 1986), generally, a labor-intensive protocol. Simply fol- Cultures. Inocula for cultures of bacteria were lowing changes in concentration of excretion products prepared by filtering Villefranche Bay (NW Mediter- in batch cultures neglects absorption of predator ranean, France) water twice through 0.4 pm Nuclepore excretion by prey organisms. This latter problem has filters. Double filtration greatly reduces micro- been successfully circumvented in studies using heat- flagellate concentrations. A small volume of this fil- killed prey; the use of inactivated prey allows accumu- trate, 2 or 3 drops, was used to inoculate l l volumes of lation of excretion products (Caron et al. 1990, Ferrier 0.2 pm filtered seawater which had been autoclaved & Rassoulzadegan 1994). The use of heat-killed prey after adding 4 wheat grains and 0.1 g of yeast extract has become an increasingly popular means of investi- (Difco). Bacterial cultures were incubated at 18°C in gating protistan feeding (Gonzalez et al. 1993, Landry darkness until cell concentrations were approximately 1994). While growth of ciliates fed heat-killed prey has 5 X 107cells ml-' (7 d). Cultures, which were predorni- been recorded (Sherr et al. 1987, Ferrier & Rassoul- nantly large rods, were periodically checked for zadegan 1994), to our knowledge the culture charac- microflagellate contamination. teristics of ciliates fed heat-killed prey have not been Stock cultures of Strombidium sulcatum were main- compared to cultures fed live prey. tained on bacterized wheat-grain media as previously Culture characteristics of protists, in terms of cell described (Rivier et al. 1985). For experiments, a ciliate sizes and growth rates, can vary with the quality of culture was pre-grown on heat-killed bacteria. Ciliates prey (Goldman & Caron 1985). There is evidence that from a stock culture were individually brought through marine ciliates discriminate against heat-killed prey 10 washes of 0.2 pm filtered seawater and inoculated (Stoecker 1988, Putt 1991). Using heat to kill bacteria into a solution of heat-killed bacteria (1 X 107 bacteria does not appear to change the composition of bacteria ml-') prepared following the protocol outlined below. as regards carbon, nitrogen and phosphorus contents The ciliate culture was incubated for 7 d at 18OC in (Caron et al. 1990, Ferner & Rassoulzadegan 1994) but darkness. it is not unreasonable to assume that many macromol- Experimental protocol. Bacterial cultures were fil- ecules and vitamins are altered. In addition, prey tered through 5 pm Nuclepore filters to remove ag- motility is clearly altered and might influence inges- gregates and detrital material and then pooled in an tion rates and perhaps growth efficlencies in an unpre- autoclaved container to yield a single 10 1 culture. dictable manner. A previous study of ammonium Aliquots, 750 m1 volumes, were then dispensed into excretion in the oligotrich Strombidium sulcatum fed twelve 1 1 containers. Containers of bacterial cultures heat-Idled bacteria showed relatively low cell yields were assigned one of 4 treatments: (1) no manipula- with heat-killed bacteria (Ferrier & Rassoulzadegan tion, (2) addition of ciliates, (3) heat-killing, (4) heat- 1994). Hence, we were interested in comparing cul- killing followed by addition of ciliates. Thus, there tures fed live and heat-killed prey in a detailed manner were 3 replicates for each treatment. Bacterial cul- to establish the validity of using heat-killed prey as a tures were heat-killed by placing containers in a 60 "C food source for ciliate microzooplankton and making water bath for 2 h; ciliate additions represented inocu- estimates of orthophosphate excretion, generally lations from the Strombidium sulcatum culture grown thought to account for most phosphorus excretion in on heat-killed bacteria added to give a final ciliate marine protlsts (Caron & Goldman 1990). concentration of 10 ml-l. The containers were incu- Here we report the results of an experiment in which bated in darkness at 18OC. Samples for determina- we examined the growth of Strombidium sulcatum fed tions of orthophosphate and organismal concentra- heat-killed and live bacteria. Orthophosphate and tions were removed, after gentle mixing, at time zero organismal concentrations were monitored over an 8 d and every 24 h for 8 d. Allali et al.: Orthophosphate excretion by Strornbjdlurn sulcatunl 161

Sample processing. Orthophosphate concentrations where C, and C2 are concentrations of cells (ml-') at tl were determined on unfixed samples using a Techni- and t2 (h),respectively. Excretion rates for ciliates feed- con AutoAnalyzer (model 11). Aliquots removed for ing on heat-killed bacteria (Excil,pg-at. P ciliate-' h-') bacterial enumerations were preserved with formalde- were determined using the equation: hyde and processed following the DAPI protocol of Porter & Feig (1980).Samples for ciliate data were preserved with Lugol's solution and 10 m1 (Days 1, 2 & 3) where C,,,,, is the average concentration of ciliates or 2 m1 (Days 4 to 8) subsamples settled and examined (cells ml-l), calculated as in Eq. (2). Ingestion rates using an inverted microscope. Entire surfaces of the (I, bacteria ingested ciliate-' h-') of Strombidium sulca- sedimentation chambers were scanned for estimates of tum feeding on heat-killed bacteria were estimated as: ciliate concentrations. Ciliate cell volume estimates were made based on measurements of 30 individuals for samples from Day 4 to Day 8, when ciliates were where B, and B2 are bacterial concentrations (cells sufficiently abundant. For these estimates, the first 30 ml-l) at tl and t2,respectively, and C,,,., is the average similarly oriented cells in a sample were measured and concentration of ciliates for the corresponding interval volumes calculated following the protocol of Ohman & calculated according to Eq. (3). Snyder (1991) for oligotrichs. Cell volumes, corrected Data analysis. Analysis of variance (ANOVA) was for shrinkage due to fixation in 2% Lugol's - 65 % live used to test for differences between average biomasses volume (Ohman & Snyder 1991), were converted to dry of ciliates fed heat-killed or live bacteria. Correlation weights assuming a specific gravity of 1 and a wet-to- analysis was used to examine the relationships dry weight ratio of 0.2 (Caron & Goldman 1990). between measured variables (organismal and phos- Rate calculations. Growth rates were calculated phate concentrations) as well as calculated rates assuming exponential growth. For live bacteria, phos- (excretion and ingestion). Unless otherwise noted, phorous absorption rates (A&, in pg-at. P bacterium-' means and SD of the 3 replicates are reported. h-') were calculated using the relationship: RESULTS There was little difference between cultures of cili- where [P1]and [P,] represent P concentrations (,ug-at. ates grown on heat-lulled bacteria compared to cells ml-l) at times t, and t2 in h, respectively, and (cells ml-') is the average concentration of bacteria feeding on live bacteria in terms of the duration of dif- during the interval t1 to t2 calculated using the Hein- ferent growth phases, maximum growth rates, maxi- bokel (1978) equation for an exponentially growing mum concentrations or calculated dry weights. Both population: treatments yielded growth curves showing a lag phase of 2 d and an exponential phase of 4 d followed (2) by a stationary phase (Figs. 1A & 2A). Maximum

mrep 1

.-@-A = CIL rep 1,2, 3 U-- -0- -A = BACT rep 1,2, 3 - I dr;iaeQ DAYS DAYS Fig. 1. Strombidiurn sulcaturn.1 Temporal changes in the concentrations of (A) ciliates and bacteria and (B) orthophosphate in ciliate cultures feeding on heat-killed bacteria. Lines join averages of 3 replicates Mar. Ecol. Prog. Ser. 105: 159-165, 1994

35 F- 30-

?l,5 25- W l- / X' a 20- X L rep 1 0 15- 0 cl I / 3' rep 3 10P~Ek-d - A g E X m C 5- V) LT W - U-- -0- -A = BACT rep 1,2,3 k A 0 B 9 1 0 I I I I I I I iOi345678 012345678 DAYS DAYS

Fig. 2. Strombidiurn sulcatum. Temporal changes in the concentrations of (A) ciliates and bactena and (B) orthophosphate in ciliate cultures feeding on live bacteria. Lines join averages of 3 replicates growth rates were 0.09 f 0.01 and 0.09 +0.02 h-' for declined with time on both a per ciliate and a per unit ciliates feeding on heat-killed and live bacteria, re- dry weight basis (Fig. 4). spectively. The peak ciliate density recorded for cili- Phosphorus concentrations increased regularly in ate~feeding on heat-killed bacteria was 818 f 52.2 solutions of heat-killed bacteria with ciliates from a compared to 880 k 24.0 cells ml-' for cells fed live beginning value of 10.57 +0.137 to 32.75 f 0.23 pg-at. bacteria. For both treatments the average cell biomass P 1-l at the end of 8 d (Fig. 1B).Calculated excretion increased from Day 2 to Day 5 and then decreased as rates were highest at the beginning of the experiment, prey concentrations declined (Fig. 3). There was no 13 +12 X IO-~pg-at, ciliate-' h-' and decreased to zero significant difference between the calculated dry for late stationary phase cells (Fig. 5). Excretion varied weights of ciliates fed heat-killed or live bacteria. In- inversely with the concentrations of P as well as ciliates gestion rates of ciliates feeding on heat-killed bacteria and were positively related to concentrations of bacteria and calculated ingestion rates (Table 1). Overall, a similar pattern was apparent in solutions of ciliates feeding on live bacteria in terms of increases in P and

10000,

d - 100: a B W 10 0 -1 U U 0 A rep l, 2, 3 per Cil m - W A rep I.2. 3 per mg DW 2000,I 2000,I 1 IIIIIII 1 23456789 012345678 DAY S DAYS

Fig. 3. Strornbidiurn sulcatum. Temporal changes in the aver- Fig. 4. Strombidium sulcatum. Temporal changes in calcu- age per cell biomass In mg dry weight (mg DW) of ciliates lated ingest~onrates of c~liatesfeedng on heat-Wed bacteria feed~ngon heat-killed (HK) and live (L) bacteria. Lines joln on a per ciliate (per cil) and per mg dry weight (mg DW) averages of the 3 replicates basis. Lines join averages of the 3 replicates Allali et al.: Orthophosphate excretion by Slrombidium sulcatum 163

lxloO 7

C? 1x10-'7 - HK rep2 A - HK rep 3 X - L rep 1 7 1x10-~: 0 - L rep 2 F A-Lrep3 W + 1~10-~: 5 W d I A = BACT rep l, 2, 3 O 0 A=Prep1,2,3 a0 1~10-~: m c. A l I I I I I I g 1x10-~: A 012345678 zi DAYS

1 xl o-~ I I I I I I I 012345678 DAYS Fig. 5. Strombidium sulcatum Temporal changes in calculated phosphorus excretion rates based on increases in orthophosphate concentratlons in replicate (rep) cultures of ciliates feeding on heat-k~lled(HK) and live (L) bacteria. Llnes join averages of the 3 replicates. Zero and negative values not plotted

Table 1. Results of linear correlation analysis.No data trans- formation was used and n = 24 for all correlations

Treatment YI yz r value DAYS Fig. 6. Strombidium sulcatum. Temporal changes in the con- Live bacteria Bacteria P conc. -0.890 centrations of bacteria and orthophosphate in solutions of & no cil~ates (A)heat-killed and (B) live bacteria. Lines join averages of the 3 replicates Heat-killed bacteria Bacteria lngestion 0.903 & ciliates lngestion Excretion 0.992 DISCUSSION ciliates coupled with decreases in bacterial concentra- We found little difference between Strombidiumsul- tions (Figs. 2 & 5). However, in the beginning of the catum feeding on heat-killed bacteria and live bacte- incubations, the solutions of ciliates with live bacteria ria. Duration of growth phases of the cultures, cell sizes showed an increase in bacterial densities coupled with and cell yields were indistinguishable. Hence, using a decrease in P and ciliate concentrations (Fig. 2). heat to kill bacteria did not appear to affect their nutn- In solutions of heat-killed bacteria without ciliates, tional quality for the ciliate. This finding is of some bacterial and phosphate concentrations varied little importance. Heat-killed prey (stained or unstained) (Fig. 6A). Bacterial numbers at the beginning of the are often discriminated against, relative to live prey, by experiment were 2.73 f 0.11 X 107 cells ml-' vs 2.68 f microflagellates (Landry et al. 1991, Monger & Landry 0.289 X 107 cells ml-l. Phosphate varied between 1991) as well as ciliates (Stoecker 1988, Putt 1991), 12.92 f 0.14 and 12.23 f 0.03 yg-at. I-'. In contrast, in leading some investigators to question the validity of the bottles of live bacteria without ciliates, bacterial using heat-killed prey as tracers in grazing studies concentrations increased and phosphate decreased (Gonzalez et al. 1993, Landry 1994). A previous study, (Fig. 6B). Bacterial density increased from 2.7 to 9.50 k using heat-killed prey and focusing on ammonium 0.026 X 107 cells 1111-' and phosphate declined from an excretion of S. sulcatum (Ferrier & Rassoulzadegan initial value of 11.43 +0.28 to 5.1 It 0.1 yg-at. I-'. Cal- 1994), indicated that heat-killed prey supported culated rates of phosphorus absorption by live bacteria growth but gave smaller cell yields relative to live bac- varied from 4.43 k 0.13 to 2.16 k 0.62 X 10-l2 yg-at, bacteria. Our finding of similar culture characteristics with terium-' h-' at the beginning and end of the experi- live or heat-killed prey were likely due to the high con- ment, respectively. centrations of prey employed and the nutrient-replete 164 Mar. Ecol. Prog. Ser

nature of the prey, especially with regard to phosphorus (orthophosphate concentrations of 10 to 11 pg-at. P 1-' at time 0). It is possible that heat-killing of nutrient- limited prey could change nutritional quality. The similarity between the 2 sets of cultures also extended to final concentrations of orthophosphate (Figs. 1B & 2B) and estimates of excretion rates for late- exponential and stationary-phase cells (Fig. 5). This may seem surprising given that in the control cultures orthophosphate did not change in solutions of heat- killed bacteria but did diminish in solutions of live bacteria. Rather than indicating that live bacteria in the presence of ciliates did not absorb orthophosphate, this 4x1o'~ finding is probably due to the relative concentrations 012345676 and activities of bacteria and ciliates in the cultures. DAYS Rough calculations can be made using absorption rates Fig. 7. Strombidium sulcatum. Temporal changes in the ratio of bacteria from cultures of live bacteria alone. Beyond of excretion to ingestion of ciliates [P excreted (pg-at. P cili- Day 3 of incubation, bacterial concentrations ranged ate-' h-') per bacterium ingested] feeding on heat-killed bacteria. Lines join averages of the 3 replicates from 107 to 106 ml-' in cultures of ciliates feeding on live bacteria (Fig. 1A). Based on the maximum rates estimated from control cultures (4 X 10-12 fig-at. bac- time (Fig. 7). Perhaps, with phosphorus-replete prey, terium-' h-'), living bacteria absorbed approximately excretion could be a simple function of ingestion, 1 to 0.1 pg-at. P 1-l d-', an insignificant amount rela- regardless of the physiological state of the grazer. This tive to ciliate excretory activity when ciliate concentra- hypothesis should be examined by estimating excre- tions ranged from 100 to 800 cells ml-'. tion and ingestion rates in short-term experiments Other data on phosphorus excretion rates of ciliates using ciliates in different growth phases or physiologi- are sparse and difficult to compare directly. Rate esti- cal states feeding on a homogeneous prey population. mates have been made only for a marine scuticociliate, Such experiments have been conducted with Colpid- Cyclidium sp. (Berrnan et al. 1987), and a freshwater ium sp. for excretion but not ingestion rates (Taylor colpodid, Colpidium sp. (Taylor 1986); both studies 1986). In our experiment the ciliates could have been examined excretion rates with nutrient-limited prey. feeding on different sub-populations of heat-killed For Cyclidium sp., excretion of soluble reactive phos- bacteria in lag vs stationary phases complicating calcu- phorus was reported as 0.5 to 15.1 pg P rng-' dry wt lations of excretion as a function of ingestion. h-'; the comparable values for Strombidium sulcatum Heat-killing bacteria does not appear to alter the are 0 to 24.6 pg P mg-' dry wt h-', measured as nutritional value of phosphorus-replete bacteria and orthophosphate. For Colpidium sp. phosphorous re- allows measurement of excretion rates which are not lease was reported as 7.8 pg P cell-' h-' for stationary- confounded by prey absorption of excretory products. phase cells and 12.3 pg P cell-' h-' for exponential- Future studies will address ciliate excretion when phase cells compared to 0 pg P cell-' h-' and 416 pg P feeding on nutrient-limited prey and address questions cell-' h-' for stationary and exponential-phase cells, of variability with the physiological state of the grazer. respectively, for S. sulcatum. Our maximum rate estimates for S. sulcatum are higher likely due to the nutri- Acknowledgements. The research reported is based on a ent-replete nature of the prey used. 'Rapport du Stage' submitted by K.A. to the Univers~teP. &M. Excretion rates estimated from ciliates feeding on Curie (Paris VI) in partial fulfillrnent of the requirements for a heat-killed bacteria covaried with estimates of inges- D.E.A, degree in Biological Oceanography. Financial support was provlded by the Commission of the European Communl- tion rates and prey concentration (Table 1). While the ties, MAST I1 grant MAS 2 - CT93-0063 'Medipelagos' and association of high excretion with high ingestion and the Centre National de Recherche Scientifique, URA 716 low excretion w~thlow ingestion could be expected, a (France). Comments by anonymous reviewers led to signifi- strong correlation between excretion and ingestion cant improvements in the manuscnpt. rates throughout different growth phases was surprising. We expected to find somewhat lower rates of LITERATURE CITED excretion, relative to ingest~on,in rapidly growing cells Beers, J. R.,Stewart, G L. (1969).Mlcro-zooplankton and its which synthesize large amounts of membrane and abundance relative to the larger zooplankton and other nucleic acids. There is little evidence of such a pattern seston components. Mar. Biol. 4: 182-189 when excretion divided by ingestion is plotted against Berland, B., Bonin, D. J., Maestrini, S. Y (1980). Azote ou Allall et a1 : Orthophosphate excretion by Strombidium sulcatum

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I his arD.cle was submitted to the editor Manuscript first received: August 17, 1993 Revised version accepted: December 16, 1993