APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1986, p. 1340-1347 Vol. 52, No. 60099-2240/86/121340-08$02.00/0Copyright © 1986, American Society for Microbiology

Effect of Temperature on Growth, Respiration, and NutrientRegeneration by an Omnivorous Microflagellatet

DAVID A. CARON,* JOEL C. GOLDMAN, AND MARK R. DENNETTWoods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543

Received 2 May 1986/Accepted 5 September 1986

The effect of temperature on the rates and extent of carbon and nitrogen cycling by the heterotrophicmicroflagellate Paraphysomonas imperforata (diameter, 7 to 12 ,um) fed with the diatom Phaeodactylumtricornutum was investigated over an ecologically pertinent temperature range (14 to 26°C). All physiologicalrates investigated increased with increasing temperature. Qlo values were similar for all rate changes and werecomparable to those which have been reported for other protozoa. In contrast to all rates, microflagellate grossgrowth efficiency and cell volume were unaffected by temperature. Decreases in the concentrations ofparticulate carbon and particulate nitrogen from grazed diatom cultures also were similar when summed overthe entire growth phase of the microflagellate population. Therefore, the proportions of ingested carbon andnitrogen which were incorporated or remineralized by the microflagellate were independent of temperaturebetween 14 and 26°C. At temperatures above 18°C, growth rates of P. imperforata were greater than themaximum growth rates reported for most phytoplankton. We conclude that the impact of P. imperforata onnatural phytoplankton communities is not controlled by temperature above 18°C but may be affected by therate at which zooplankton or microzooplankton prey on the microflagellate, as well as the inability of themicroflagellate to graze efficiently when phytoplankton are present at low cell densities.

It is now widely recognized that protozoa play a vital rolein regulating energy flow and nutrient cycling in marineplankton communities. It has been demonstrated, for exam-ple, that ciliates are important consumers of phytoplankton(4, 5, 25), and heterotrophic microflagellates recently havebeen identified as the primary predators of bacterioplankton(2, 9, 14, 15). Indeed, we now have evidence that somemicroflagellates are omnivorous and thus may be importantconsumers of small (<20 ,um) phytoplankton as well asbacteria (18). Clearly, a firmer understanding of the trophicrelationships of aquatic protozoa is beginning to emerge.However, we still know very little about how physicalvariables such as temperature influence growth and survivalof these protozoan populations.

Intuitively, we would expect temperature to have a directeffect on the rates of numerous physiological processes ofprotozoa including rates of grazing, growth, respiration, andnutrient regeneration. In general, this expectation has beenrealized in experimental studies with cultured protozoa, butthe direction and magnitude of the physiological response ofindividual species to temperature changes has not beenconsistent. Temperature dependence of growth rate hasbeen most commonly examined in this regard. Protozoangrowth rates in a number of studies increased with increasingtemperatures (3, 12, 16, 28-30, 36, 37, 40, 41), although somerates decreased at the higher temperatures studied (whichpresumably were near the upper limit for growth of thespecies). For several species, however, there was no changein growth rate over temperature ranges of 5 or 10°C (28, 32,39, 43). Similarly, ingestion rates have been shown to bepositively correlated with temperature (33, 39, 41, 46) orindependent of water temperature over ranges of 5 or 15°C(27, 28, 30, 44). The ability of some protozoan species tomaintain constant physiological rates over significant tem-

* Corresponding author.t Contribution 6320 of the Woods Hole Oceanographic Institute.

perature ranges makes it difficult to generalize about temper-ature dependence in these organisms.Temperature also may have an effect on other growth

processes of protozoa, but as with physiological rates, thiseffect is difficult to predict. Protozoan cell volume, forexample, often decreases with increasing water temperaturewithin the temperature limits of the species (17, 26, 32, 41),but this trend is not without exceptions (3, 31). Likewise, ithas been observed that gross growth efficiency for protozoanspecies may increase (30, 46), decrease (39, 43), or remainunchanged (41, 43) in response to increasing water temper-ature.Because of the far-reaching, and often unpredictable,

consequences of temperature on the rates and efficiencies ofcarbon and nutrient utilization by protozoa, it is imperativeto establish empirically these relationships for ecologicallyimportant species. These effects have ecological implica-tions because changes in the rates of these processes mayaffect the outcome of competition between two species, ashas been demonstrated with algae competing for a growth-limiting substance (21, 23), or influence the populationdynamics of predator and prey species (1). In addition,temperature dependence of growth parameters such as grossgrowth efficiency would have a direct effect on the amount ofenergy transferred to higher trophic levels and on nutrientregeneration efficiency by protozoa.We previously described the energetics of growth, graz-

ing, respiration, and nutrient regeneration by the omnivo-rous microflagellate Paraphysomonas imperforata in a nar-row temperature range of 20 to 24°C (6, 18, 19). On the basisof these results, we hypothesized that this microflagellate (orecologically analogous species), which is ubiquitous in themarine environment, may be an important consumer of algaein the marine plankton. In support of this hypothesis,apochlorotic chrysomonads have been shown to numericallydominate the cultivable protozoa from samples collectedthroughout the western North Atlantic (D. A. Caron, P. G.Davis, L. P. Madin, and J. M. Sieburth, J. Mar. Res., in

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TEMPERATURE EFFECTS ON P. IMPERFORATA 1341

press) and to be the major grazers of phytoplankton inoutdoor mass algal cultures (10, 23).Even though there was only a 4°C temperature range in

our previous experiments (6, 18, 19), a significant effect oftemperature on the physiological processes of this protozoanwas observed. To expand these earlier findings, we investi-gated the effect of temperature on the rates and extent ofgrazing, growth, nitrogen cycling, and carbon cycling by P.imperforata over an ecologically pertinent temperaturerange (14 to 26°C). In this study, we distinguished betweenphysiological rates (processes carried out during the expo-nential growth of the microflagellate with units of matterprocessed per flagellate per unit time) and carbon or nitrogenassimilation-remineralization summed over the growth cycleof the microflagellate population (units of percent diatomcarbon or nitrogen assimilated or remineralized).

All physiological rates investigated were significantly af-fected by temperature. The magnitude of the Qlo values forall rates averaged 2.4 over the 12°C temperature range. Incontrast, temperature had no discernable impact onmicroflagellate cell volume or gross growth efficiency. Whensummed over the entire growth cycle (early exponentialgrowth to stationary phase), temperature had no effect onthe total amount of particulate carbon (PC) removed fromthe cultures by grazing, the total amount of NH4' regener-ated, or the amount of dissolved organic carbon (DOC)produced. We conclude that, although the rates of materialand energy processing by P. imperforata are highly temper-ature dependent, the overall assimilation efficiencies of thismicroflagellate for carbon and nitrogen are independent oftemperature.

MATERIALS AND METHODS

P. imperforata was obtained from a tidal channel connect-ing Vineyard Sound, Mass., with an estuarine pond near theWoods Hole Oceanographic Institution. Seawater from thislocation was filtered through 35-,im-pore screening, gentlycentrifuged, and added to the second stage of a two-stagecontinuous culture system. The marine diatom Phaeo-dactylum tricornutum (clone TFX-1) was cultured in the firststage of this system on a synthetic seawater medium (22).The microflagellate was isolated, made free of bacteria bymicropipetting herbivorous protozoa growing in the secondstage into axenic batch cultures of Phaeodactylumtricornutum, and maintained by approximate weekly trans-fers to fresh cultures of axenic Phaeodactylum tricornutum.The bacterium-free status of the cultures in this study waschecked by epifluorescence microscopy (47).A single culture of Phaeodactylum tricornutum was used

as food for P. imperforata in all experimental trials. An8-liter axenic culture of the diatom was grown in continuouslight at 20°C on synthetic seawater medium containing 100,ug-atoms of NH4' and 10 ,ug-atoms of P04 3 liter-' (N/Pratio = 10). The carboy was continuously stirred by meansof a magnetic mixer and aerated with filter-sterilized air. Thediatom culture was grown to late exponential phase, andthen 800-ml portions were transferred aseptically into eachof eight sterile 1-liter wide-mouthed flasks which allowedgood air exchange with the cultures. Four of the sampleswere inoculated with a culture of P. imperforata previouslygrown to late exponential growth phase on bacteria-freePhaeodactylum tricornutum. Two cultures of Phaeo-dactylum tricornutum, one with a microflagellate inoculumand one without, were placed in each of four water baths at14, 18, 22, and 26°C. The cultures were not stirred during the

grazing phase of the experiment and were kept in continuousdarkness except for 3- to 5-min periods during sampling.Time course sampling for various biological and chemical

measurements was performed at approximately 8-h intervalsover a 4-day period, but not all cultures were sampled everytime. Samples (75 ml) were withdrawn aseptically at eachsampling. Cell counts were performed by epifluorescencemicroscopy with acridine orange (8, 47). Volume estimatesof the microflagellates during exponential and early station-ary growth were obtained by microscopical observation ofthe first 100 cells encountered in acridine orange prepara-tions. PC and particulate nitrogen (PN) were measured witha no. 240 elemental analyzer (The Perkin-Elmer Corp.) on25-ml samples retained on precombusted GF/F glass fiberfilters (Whatman, Inc.). Filtrates from the PC-PN sampleswere used to measure NH4' and DOC by the methods ofMcCarthy and Kamykowski (34) and Menzel and Vaccaro(35) as modified by Sharp (42), respectively.The respiration rate of P. imperforata was determined

with a Strathkelvin radiometer-type oxygen electrode duringthe late exponential and early stationary phases ofmicroflagellate growth. Respiration rates (nanoliters of 02milliliter-' hour-') in Phaeodactylum tricornutum cultureswith and without microflagellates were measured at eachtemperature after equilibration of the oxygen electrode tothe appropriate temperature. Microflagellate respiration wascalculated by subtracting the contribution of diatom respira-tion in the two-member cultures. Total diatom respiration inthe cultures of diatoms with microflagellates was determinedby measuring diatom respiration rate (nanoliters of 02 cell-1hour-') in the cultures without microflagellates, multiplyingthe resulting rate by the density of diatoms in the cultureswith microflagellates, and subtracting this value (nanolitersof 02 milliliter-1 hour-) from the total respiration rate of thetwo-member culture. The remaining respiration rate wasdivided by the density of microflagellates in the sample toyield the microflagellate respiration rate (nanoliters of 02cell-' hour-'). The weight-specific respiration rate of themicroflagellate was calculated by using the cell volumeestimates derived from microscopical observation.

Specific growth rates (day-') of the microflagellate werecalculated from the slope of the linear portion of the curvesof In cell number versus time. Gross growth efficiency (cellyield equals the conversion of prey biomass into micro-flagellate biomass) was calculated in two ways at the sam-pling period for which maximum microflagellate abundancewas observed. Gross growth efficiency based on biovolumewas estimated by comparing the biovolume of diatomsconsumed with the biovolume of microflagellates produced.The biovolume of the ingested prey was calculated bymultiplying Phaeodactylum tricornutum cell volume (deter-mined by microscopical observations) by the decrease in thedensity of diatoms. The biovolume of microflagellates wascalculated as the product of microflagellate density andaverage cell volume (also determined microscopically).Gross growth efficiency based on the percent PC remainingin the cultures was the ratio of the PC concentration in aculture at the time of peak density of microflagellates to thePC concentration initially. The amount of unconsumed dia-tom PC at the time of maximum microflagellate density wascalculated to be an insignificant contribution to the total PC.

Ingestion rates (diatoms consumed flagellate-' day-1) andclearance rates (milliliters filtered flagellate-' day-1) for P.imperforata were calculated from microscopical populationcounts by the equations of Heinbokel (24). Rates werecalculated for each two consecutive samples, beginning with

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1342 CARON ET AL.

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Phaeodactylum tricornutum inoculated with the microflagellate P. imperforata and placed in continuous darkness at four temperatures.Changes in the cell number of P. imperforata (A) and Phaeodactylum tricornutum (B) are shown, as are concomitant changes in theconcentrations of NH4' (C) and DOC (D) and the percentages of PN (E) and PC (F) in the cultures. Symbols: 0, 14°C; 0, 18°C; A, 22°C;A, 260C.

the sample which showed the first significant decrease inprey density (coefficient of variation of counts is 10 to 15%).The highest rates observed during this period were assumedto be the maximum ingestion and clearance rates.

Rates of NH4' excretion of P. imperforata (picograms ofN cell-' day-') were calculated during exponential, earlystationary, and late stationary growth of the microflagellateaccording to the equation EN = (N2 - N1) x (F x At)-',where EN is the nitrogen excretion rate, N1 and N2 are theconcentrations of NH4' at the beginning and end of the timeinterval At, and F is the ln average concentration ofmicroflagellates during each time interval (19).Changes in the rate of growth, respiration, ingestion,

clearance, and nitrogen excretion of P. imperforata as aresult of changes in temperature were used to calculate Qlovalues for these processes over the temperature range inves-tigated (14 to 260C) according to the equation Qlo = (R1 xR2-1)[10 X (tl - t2) 1, where R1 and R2 are the magnitudes of theparticular rate process at the temperatures t1 and t2, respec-

tively (38).

RESULTS

Growth rate and cell density. All measured physiologicalrates of P. imperforata increased with increasing tempera-

ture between 14 and 26°C (Fig. 1; Table 1). Microflagellategrowth rates (depicted by the linear portion of the growthcurves in Fig. 1A) varied by a factor of 2.9 over the 12°Ctemperature range from 1.37 day-' at 14°C to 4.04 day-1 at26°C. A plot of the natural logarithm of growth rate as afunction of the reciprocal of absolute temperature (an Ar-rhenius-type plot) yielded a straight line corresponding to a

Qlo of 2.48 (r = 0.98). Although time course experimentswere not replicated at all temperatures, a consistent andsignificant effect of temperature is indicated by the highcorrelation coefficient of this line. Comparison of our data onthe relationship between growth rate and temperature for P.imperforata with published data for freshwater and marinemicroalgae showed that the microflagellate growth rate wasgreater than most published maximum specific growth ratesof microalgae over the temperature range examined in thisstudy (Fig. 2).

Differences in the growth rate due to temperature resultedin temporally offset peaks in microflagellate abundance (Fig.1A). Maximum microflagellate cell density was, however,relatively similar for all four temperatures (.x = 7.2 x 104ml-,; range = 6.0 x 104 to 8.5 x 104 ml-'). Decreases in thedensity of Phaeodactylum tricornutum were concomitantwith increases in microflagellate density. Minimum densities

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TEMPERATURE EFFECTS ON P. IMPERFORATA 1343

TABLE 1. Physiological rate processes of the omnivorous microflagellate P. imperforata grazing on the diatom Phaeodactylumtricornutum as a function of temperature

Temp (TC) Growth rate Respiration ratea NH4+ regeneration rateb Ingestion rate Clearance rateb,d(day-') (nl of 02 cell-' h-') (nl of 0° p.m3 h-') (pg of N cell-' day-')

14 1.37 2.00 x 10-3 1.42 x 10' 8.0 43 1.80 x 10-418 1.92 1.92 x 10-3 1.62 x 10-5 11.5 54 2.19 x 10-422 2.52 2.34 x 10-3 1.91 X l0-5 11.8 65 2.01 x l0-426 4.04 5.27 x 10-3 3.59 x 10-5 13.8 209 4.05 x 10-4

a Determined at the time of maximum flagellate density.b Highest rate calculated between consecutive sampling periods.c Measured in cells flagellate-' day-'.d Measured in milliliters flagellate-' day-'.

of the diatom ranged from 6.1 x 103 to 1.0 x 104 ml-1 for allculture vessels (x = 8.5 x 103 ml-').

Respiration and ammonium regeneration rates. The effectof temperature on the respiration rate of P. imperforataduring the late exponential growth phase was similarwhether calculated on a per cell basis or on a weight-specificbasis. Respiration increased by a factor of 2.5 (on a per cellbasis) or 2.6 (on a weight-specific basis) between 14 and26'C, but most of the increase occurred between 22 and 26'C(Table 1). Respiration rate on a per cell basis decreased in allfour cultures as the microflagellate population entered thestationary growth phase. These decreases were concomitant

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FIG. 2. Effect of temperature on the maximum specific growthrate of marine and freshwater algae and the omnivorous microfla-gellate P. imperforata feeding on Phaeodactylum tricornutum. Theregression lines for algal growth rate were taken from Eppley (11)for batch culture experiments and from Goldman and Carpenter (20)for continuous culture experiments. Data on P. imperforata havebeen summarized from Table 1 (solid symbols) and from Goldman etal. (19). Symbols: 0, nonbacterized cultures; F1, bacterizedcultures.

with decreases in microflagellate cell volume and, therefore,the weight-specific respiration rate remained relatively con-stant at each temperature. This relationship has been previ-ously described (6).

Regeneration of NH4+ was due primarily to the growth ofmicroflagellates in the cultures (Fig. 1A and C). However, asmall amount of NH4+ was produced in the axenic diatomcultures at 22 and 26'C after 4 days of incubation, equalling3.8 and 6.8%, respectively, of the amount produced indiatom cultures inoculated with microflagellates at the sametemperatures. No measurable NH4' was produced in theaxenic diatom cultures at 14 and 18°C. Rates of NH4'regeneration by P. imperforata (Table 1), like respirationrates, were temperature dependent, but the overall increasein regeneration rate (1.7-fold increase over the temperaturerange examined) was less than that observed for respirationrate. Regeneration rates were maximal for the microflagel-late during the late exponential growth phase and decreasedas the population entered stationary growth.

Ingestion and clearance rates. Ingestion rates (diatomsconsumed flagellate-' day-1) and clearance rates (milliliterscleared flagellate-' day-') of P. imperforata were calculatedfor each two consecutive sampling periods throughout lateexponential and early stationary growth at the four experi-mental temperatures. These rates also increased between 14and 26'C by a factor of 4.8 for ingestion rate and a factor of2.2 for clearance rate (Table 1). The highest ingestion rates ateach temperature always were observed at the first samplingperiod which showed a significant decrease in diatom density(i.e., the first sample for which ingestion rate could becalculated) and then decreased as prey density decreased.Clearance rates at each temperature were lowest when preywere abundant and reached a maximum as diatom densityapproached its threshold density (Fig. 1B). As with respira-tion and growth rate, ingestion and clearance rates wereparticularly sensitive to the temperature change between 22and 26°C.

Qlo values. Qlo values were calculated for changes in allphysiological rates for each 4°C temperature increment andfor the entire 12'C range (Table 2). With one exception (the

TABLE 2. Qlo values for the growth of the omnivorousmicroflagellate P. imperforata fed the diatom Phaeodactylum

tricornutum over the temperature range 14 to 26'C

NH., +Temp Growth Wt-specific regeneration Ingestion Filtration

range ('C) rate respiration rate rate rate rate

14-18 2.5 1.4 2.5 1.8 1.618-22 2.0 1.5 1.1 1.6 0.822-26 3.2 4.8 1.5 18.5 5.814-26 2.5 2.2 1.6 3.7 2.0

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APPL. ENVIRON. MICROBIOL.

Qlo of <1.0), these values all demonstrated an expectedpositive effect of temperature on metabolic processes of themicroflagellate. The average Qlo value for all five rates over

the 12°C range was 2.4, with the highest Qlo value for eachrate generally occurring for the temperature interval from 22to 26°C (with one exception). The Qlo values for ingestionand filtration rates were unusually high for the temperatureinterval between 22 and 26°C, and were due to a singlepopulation count that was exceptionally high.Changes in PC and PN. Losses of PC from the

Phaeodactylum tricornutum cultures were mediated largelyby microflagellate grazing (Fig. IF; Table 3). Losses of PC inthe axenic Phaeodactylum tricornutum cultures ranged from10 to 19% of the initial PC plus DOC. Although the rate of PCloss was temperature dependent, the overall effect ofmicroflagellate grazing was similar at all temperatures. PCand DOC constituted an average of 91 and 9%, respectively,of the organic carbon initially present in the cultures at thetime of inoculation with microflagellates. Over the course ofthe experiment, PC concentrations were reduced by 39 to61%. The largest and smallest losses of PC occurred incultures at 26 and 14°C, respectively, and presumably were

due to the fact that microflagellates at warmer temperatureswere in more advanced stages of the stationary growthphase.

Approximately 13% of the organic carbon initially presentas PC reappeared as DOC, presumably due to release bymicroflagellates during grazing and growth (Table 3). Weobserved no relationship between temperature and the finalDOC concentration. On the basis of changes in PC and DOCconcentration, respiration of the microflagellate and diatomtogether accounted for a loss of 23 to 48% of the total amountof organic carbon initially present.More than 95% of the NH4' initially present in the growth

medium was converted to diatom PN at the start of grazing(Table 4). There was an average loss of 3% of the PN in theaxenic cultures of Phaeodactylum tricornutum over thecourse of the 4-day experiment. An equivalent increase inthe concentration of NH4+ was not observed, and the PNwas presumably released as dissolved organic nitrogen. Incontrast, losses of PN in diatom cultures inoculated withmicroflagellates were 40 to 73% over the course of theexperiment. Nitrogen appearing as NH4' accounted for 64 to78% of the loss of PN (x = 70%). Concomitant increases ofNH4' in the cultures represented 27 to 51% of the total PNplus NH4' initially present in the cultures. The total amountof nitrogen which was not accounted for by PN or NH4' atthe end of the experiment ranged from 13 to 26% of the

TABLE 3. Changes in the concentration of PC and DOC incultures of the omnivorous microflagellate P. imperforata grazing

on Phaeodactylum tricornutum in the dark as a function oftemperature

Initial concn" Final concn"

Temp (mg of C liter-)' (mg of C liter-'l)(OC)

PC DOC PC DOC

14 8.02 (90) 0.87 (10) 4.50 (51) 2.27 (25)18 7.98 (89) 0.97 (11) 3.47 (39) 1.59 (18)22 8.12 (92) 0.68 (8) 3.55 (40) 2.08 (24)26 8.41 (94) 0.58 (6) 3.00 (33) 1.84 (21)

" Samples were taken at the time of inoculation of the microflagellate(initial) and after 3.2 (26'C) or 3.9 days of incubation (final).

" Numbers in parentheses are PC or DOC concentrations as a percentage ofthe initial PC plus DOC concentrations.

TABLE 4. Changes in the concentration of NH4' and PN incultures of the omnivorous microflagellate P. imperforata grazing

on Phaeodactylum tricornutum in the dark as a function oftemperature

TempInitial concn' (piM)Y Final concn" (,uM)"

(OC) PN NH4+ PN NH4+

14 88.1 (95) 4.3 (5) 51.0 (55) 29.7 (32)18 87.1 (97) 2.8 (3) 31.9 (35) 41.8 (47)22 88.4 (99) 0.7 (1) 30.4 (34) 46.0 (52)26 96.5 (99) 0.5 (1) 25.6 (26) 46.6 (48)

As in Table 3, footnote a.Numbers in parentheses are PN or NH4+ concentrations as a percentage

of the initial PN plus NH4+ concentrations.

nitrogen initially present and presumably was released asdissolved organic nitrogen during microflagellate grazing.This range is in agreement with our previous work in whichwe demonstrated that urea constituted 15% of the totalexcreted nitrogen (19). We observed no relationship betweentemperature and the amount of nitrogen released as dis-solved organic nitrogen. As was the case with carbon, lessremineralization of nitrogen took place over the course ofthe experiment at 14°C than at the other three temperatures.

Cell volume and gross growth efficiency. Gross growthefficiency of P. imperforata feeding on Phaeodactylumtricornutum showed no relationship with temperature (Table5). Efficiency calculated on the basis of cell volume averaged42% for the four culture temperatures atid was virtuallyindistinguishable from the cell yield obtained for thismicroflagellate grown at 20 and 24°C in a previous study (6).Efficiency based on the change in PC averaged 63% for thefour treatments and also showed no relationship to temper-ature. Assuming that cell volume is directly related to carboncontent (43, 45), we observed a 20% difference in grossgrowth efficiency between these two methods.

Microflagellate cell volume also showed no significantdifference as a result of growth at different temperatures(Table 5). Microflagellates sampled during exponentialgrowth before a significant decrease in cell volume due tostarvation (6) averaged 220 pLm3 cell-1 when examined byepifluorescence microscopy. A relatively large error (stan-dard deviation, "40%) was associated with the cell volumemeasurements because the microflagellate population con-tained individuals in all stages of the cell cycle.

TABLE 5. Cell volume and gross growth efficiency of theomnivorous microflagellate P. imperforata fed on the diatomPhaeodactylum tricornutum and grown at four temperatures

Gross growthTemp Initial PC concn efficiency" (%) Flagellate cell(OC) (mg of C liter-') volumeb (,um3)

Vol PC

14 8.02 43 62 226 9518 7.89 41 65 224 9022 8.12 41 65 228 7626 8.41 45 60 210 96

x = 42 x = 63 x = 220

" Calculated based on cell volumes (determined by microscopical counts)and by changes in total PC in the cultures during exponential growth of themicroflagellate.

I Based on 100 organisms for each trial and determined by microscopicalmeasurement of flagellates in mid-exponential growth before significant de-crease in diatom density.

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TEMPERATURE EFFECTS ON P. IMPERFORATA 1345

DISCUSSION

Most investigations dealing with temperature effect onprotozoan physiology have been performed with ciliates oramoebae, and relatively few data are available for flagellatedprotozoa. Sherr et al. (43) examined grazing, growth, andammonium excretion in the bacterivorous microflagellateMonas sp. at four temperatures ranging from 3 to 30°C. Thegrowth rate of Monas sp. in that study was reduced at theupper and lower temperature extremes compared with thetwo intermediate temperatures (18 and 23.5°C). The Qlovalues calculated from their data for the growth rate andammonium excretion rate of Monas sp. between 18 and23.5°C (1.1 and 1.2, respectively) were less than the Qlovalues observed for P. imperforata in the present study. Inaddition, Monas sp. displayed higher rates of NH4' excre-tion and lower gross growth efficiencies at 3 and 30°C than at18 and 23.5°C. In contrast, there was no relationship be-tween the gross growth efficiency of P. imperforata andtemperature in the present study, and NH4' excretion rateincreased with increasing temperature over the entire rangefrom 14 to 26°C. Differences in the response of thesephysiological processes to temperature by these twomicroflagellates exemplify the species-specific effect of tem-perature on protozoa.Although Qio values for the physiological processes ex-

amined in this study varied somewhat, the differences ap-pear not to be significant. For example, the overall Qlo valuefor ingestion rate increased more rapidly than growth ratewith increases in temperature. If this difference were signif-icant, we would expect that the respiration rate and theNH4' regeneration rate would have Qlo values of approxi-mately 3.7 because incorporation of ingested prey (grossgrowth efficiency) did not change with temperature. How-ever, Qio values for respiration rate and regeneration ratewere 2.2 and 1.6, respectively. A more likely explanation forthe Qlo data is that the Qlo values are essentially the same,and the variability associated with the measurement of thephysiological rates, and the calculations used to generate therates, are largely responsible for the observed differences.We conclude that caution must be exercised when compar-ing Qlo values based on different rate processes.Although physiological rates of P. imperforata were af-

fected by temperature, we found no evidence to suggest thatthe basic pattern of carbon and energy flow was affected bytemperature between 14 and 26°C when summed over thewhole population growth cycle from exponential to station-ary phase. The overall changes in the concentration of PC,PN, NH4', and DOC in diatom cultures inoculated with P.imperforata were relatively unaffected by temperature, al-though these changes occurred at different rates at the fourtemperatures (Fig. 1C to F). For example, the microflagel-late populations in the three cultures at 18 to 26°C had allreached the late stationary growth phase by the terminationof the experiment, and the concentrations of PC, PN, DOC,and NH4' in these cultures were all quite similar at this time(ranges were 33 to 40% of the initial PC concentration, 26 to35% of the initial PN concentration, 1.59 to 2.08 mg of DOCliter-1, 41.8 to 46.6 pg-atoms of NH4' liter-'). Therefore,despite the fact that the incubations were conducted at threedifferent temperatures, the overall effect of microflagellategrazing in the diatom cultures was unchanged. The concen-trations of these constituents in the culture at 14°C weredifferent because the microflagellate population had onlyreached the early stationary growth phase at the time thatthe experiment was terminated (Fig. 1A).

We observed no significant change in the average cellvolume of the microflagellate as a result of temperature. Thisobservation is contrary to the results of studies in which adecrease in average cell size with increasing temperature hasbeen found (16, 17, 26, 32, 41). In previous work, wedemonstrated that the cell volume of P. imperforata is highlydependent on both the size of the prey (18) and the growthstage of the microflagellate (6). Average cell volume in thosestudies was largest during exponential growth of themicroflagellate and decreased by a factor of 4 as the popu-lation entered stationary growth. The average cell volumesof microflagellate populations growing exponentially weredirectly related to the size of the prey and varied by a factorof 10 for the prey items investigated. Therefore, changes inthe average cell volume of a population of P. imperforatadue to its stage of growth and the prey type were muchgreater than any effect of temperature.The gross growth efficiency of P. imperforata also showed

no relationship to temperature over the range investigated. Atemperature-independent gross growth efficiency has beenobserved for Amoeba proteus between 10 and 20°C (41) andfor the bacterivorous microflagellate Monas sp. between 18and 23.5°C (43). In the latter study, however, efficiency ofMonas sp. was significantly lower at 3 and 30°C than at thetwo intermediate temperatures. From these data, it seemsprobable that gross growth efficiency is relatively constantfor a protozoan species within the temperature range foroptimal growth but decreases as the temperature approachesthe species growth limits. If this is true, then the constantgross growth efficiency observed for P. imperforata over thetemperature range of 14 to 26°C would indicate that themicroflagellate is capable of optimal growth (i.e., energeti-cally) over a wide temperature range.As previously stated (6), gross growth efficiency may be

overestimated if calculated on the basis of changes in the PCconcentration because it is assumed that microflagellates andunconsumed diatoms constitute all of the PC in the cultures(i.e., egested PC is insignificant). Since efficiency based onchanges in PC concentration averaged 21% higher thanefficiency based on cell volume in the present study, egestedPC may have constituted approximately 20% of the total PCat the time of maximum microflagellate density. Alterna-tively, some shrinkage of the microflagellates may haveoccurred during fixation and preparation for microscopy,resulting in an underestimation of gross growth efficiencybased on volume and an overestimation of the amount of PCpresent as egested PC.

It is clear from results described above that, over thetemperature range examined in this study, temperature had adirect effect on the rates of all physiological processes of P.imperforata which we investigated but did not affect theoverall magnitude of carbon or nutrient cycling by themicroflagellate when summed over its population growthcycle. We conclude that, within this temperature range innature, the actual proportion of the ingested carbon andnitrogen which will be incorporated or remineralized isdependent not on temperature, but on the growth stage ofthe microflagellate population.We have previously speculated on the potential impor-

tance of herbivory by microflagellate populations in planktoncommunities (6, 18). We concluded, on the basis of acomparison of phytoplankton maximum specific growthrates and the growth rate of P. imperforata, that the rela-tively rapid growth rate of this microflagellate would allow itto keep pace with, or exceed, the growth of most phyto-plankton species at 20 and 24°C. In this investigation we

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established that the Qlo value for growth of the microflagel-late (Table 2) exceeds the Qio value predicted previously forphytoplankton, 1.88 from Eppley (11) and 2.08 from Gold-man and Carpenter (20), and that the growth rate of P.imperforata exceeds the maximum expected phytoplanktongrowth rates over the temperature range 18 to 26°C (Fig. 2).Therefore, the growth rate of P. imperforata is intrinsicallygreater than that of most phytoplankton over the tempera-ture range of 14 to 26°C, and the difference between theirgrowth rates increases with temperature over this tempera-ture range. Indeed, reported maximum growth rates offlagellated and ciliated protozoa typically are greater thangrowth rates reported for phytoplankton species (12, 13).These results support our hypothesis that P. imperforata

(or ecologically analogous species of microflagellates) maybe an important consumer of small phytoplankton in somemarine plankton communities since the microflagellate pos-sesses the ability to keep pace with rapid phytoplanktongrowth rates over a relatively wide temperature range. Ourhypothesis also is supported by studies in which it has beennoted that large-scale mass cultures of marine diatoms oftenare severely grazed by flagellated protozoa when the watertemperature exceeds 20°C (10, 23). We conclude that theomnivorous microflagellate P. imperforata is capable ofoutgrowing its phytoplankton prey under optimal growthconditions and should, therefore, be a major consumer ofsmall phytoplankton in marine plankton communities. Wespeculate, on the basis of this and our previous studies (6,18, 19), that the effect of grazing by P. imperforata in natureis not limited by temperature between 18 and 26°C, but bythe rate at which zooplankton or microzooplankton prey onthe microflagellate, its inability to efficiently graze lowphytoplankton densities, and the nutritional quality of theavailable prey.

ACKNOWLEDGMENTS

We gratefully acknowledge Judith Capuzzo for the loan of theoxygen electrode and Bonnie Woodward for performing the CHNanalysis.

This study was supported by National Science Foundation grantsOCE-858578 and OCE-8511283, and a Woods Hole OceanographicInstitution Mellon Study Award.

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