Hochnea 29(3): 241-247, I tab.. 6 fig., 2002

Release ofextracellular carbohydrate by Peridinium willei (Dinophyceae)under different irradiances

Armando Augusto Henriques Vieira 1,2, Vanessa Colombo I and Odete Rocha I

Received: 15 January. 2002; accepted: 13 August. 2002

ABSTRACT - (Release of extracellular carbohydrate by Peridillil//Il lVillei (Dinophyceae) under different irradiances).Peridillilllll lVillei forms heavy blooms in an oligotrophic reservoir (Lagoa Dourada), with high incident irradiance and aneuphotic column usually reaching the bottom. The high value ofEk (600 ~mol photons m-2S-I ), K

m(270 ~mol photons m-2S-I),

the low relative excretion rates «4.5%) and the fact that total carbon fixation is not affected by photoinhibition, even whenexposed to irradiances as high as 2000 ~mol photons m-2

S-I in cultures of three different ages, show a high adaptation of thisspecies to Lagoa Dourada environmental conditions. Another clue for this high adaptation to the environment from wherethe species was isolated is the release of a rhamnose rich heteropolysaccharide proportional to the total carbon fixation incells in the exponential growth phase and a decrease in the aging cells at intermediate irradiances but with slight increase athigh irradiances.Key words: PeridilliulI1 willei, irradiance, extracellular polysaccharide, rhamnose, excretion, heteropolysaccharide

RESUMO - (Libera~ao de carboidrato extracelular por Peridillilllll lVi/lei (Dinophyceae) em diferentes intensidades de luz).Peridillillll1 wi/lei ocorre em grandes popula~6es em uma represa 01 igotr6fica, com alta irradiancia incidente e com colunaf6tica que freqiientemente alcan~a 0 fundo. 0 alto valor de E k (600 ~mol photons m,2 S-I), de K

m(270 ~mol photons m-2S-I),

as baixas taxas relativas de excre~ao «4,5%) eo fato da fixa~ao de carbono total nao ser afetada por fen6menos defotoinibi~ao, mesmo em exposi~ao a irradiancias tao altas quanto 2000 ~mol photons m-2

S-I em tres idades diferentes,mostram a boa adapta~ao da especie as caracterfsticas ambientais da Lagoa Dourada. Outro indfcio da boa adapta~ao daespecie ao ambiente de onde foi isolada foi a libera~ao de urn heteropolissacarfdeo rico em ramnose, proporcional a fixa~ao

de carbono total em celulas na fase exponencial de crescimento e uma diminui~ao em celulas mais velhas sob irradianciasintermediarias, com urn pequeno aumento em altas irradiancias.Palavras-chave: Peridilli1111l wilLei, irradiancia, polissacarfdeo extracelular, excre~ao, heteropolissacarfdeo, ramnose

Introduction

The autotrophic dinoflagellate Peridiniu/Il willei

is a phytoplanktonic species frequently forming heavy

blooms in some tropical and subtropical oligotrophic

small shallow lakes and reservoirs, dominating the

autotrophic plankton at certain times of the year (Dias

1990, Rocha & Sampaiol991, 1995. Melao 1995,

Talamoni 1995).

High light ilTadiances, together with oligotrophic

nitrogen and phosphorus limiting conditions, constitute

environmental conditions propitious to trigger

physiological stress in phytoplankton populations. Inan environment where high irradiances occurthroughout the year, and along almost the entire

euphotic column, species not adapted to high irradiance

display a reduction of photosynthesis due to

photoinhibition processes and a decrease in cytokinesis

rates, and can even disappear, at least during the

periods when such conditions predominate. However,in species adapted to high light irradiance, the

photoinhibition phenomenon is attenuated, ifeventually

occurring, and therefore the rates of organic matter

production depend on the nutritional conditions,

physiological state, and species characteristics, such

as cell size, which in turn influence cytokinesis rates(Vieira et al. 1998). Under these environmental

conditions, high light intensity acts as a stress factor

during growth of the organisms but does not causephotoinhibition. These conditions can trigger an

I. Univer idade Federal de Sao Carlos. Departamento de Botiiniea. Caixa Postal 676. 13565-905 Sao Carlos, SP. Brasil.2. Corresponding author: ahvieira@poweLuf car.br

242 Hoehnea 29(3), 2002

increase in carbon losses via processes involved inphoto-protection, such as the increase in mitochondrialrespiration (Grande et at. 1989) or the excretion oforganic matter, mainly carbohydrates (Guillard &Wangersky 1958, Zlotnik & Dubinsky 1989). Theincreased excretion rates in these conditions and therelease of extracellular carbohydrates can provide anadditional substrate and energy source to heterotrophicpopulations, mainly bacteria, that can contribute tonutrient cycling (Wood & Van Valen 1990).

Stressing conditions that reduce cell division ratesbut do not completely interrupt photosynthesis lead tothe increase in C/N ratios by directing photosynthesistoward nitrogen-poor compounds, for example lipids,such as triacylglycerols, (Mayzaud et at. 1989, Choen1990) and carbohydrates (Fogg 1983, Vieira &Myklestad 1986). The effect of nitrogen andphosphorus limitation in establishing stress conditionsthat raise the production of total carbohydrates and ofextracellular carbohydrates in particular, have alreadybeen observed for several taxonomic groups ofmicroalgae (Myklestad 1974, Vieira & Myklestad1986, Vieira et at. 1998, Alcoverro et at. 2000).

The present study was designed to verify theoccurrence of photosynthesis inhibition, increase ofthe excretion rates and the release of extracellularcarbohydrates due to high irradiance in a populationcoming from a shallow and oligotrophic reservoircharacterized by high irradiances throughout the watercolumn.

Material and methods

Sampling locality and culture conditions: thedinoflagellate Peridinium willei (Huitfeld-Kaas 1900)(Dinophyceae) was isolated under light microscopein 1991 from a sample collected by a phytoplanktonnet with 20 ,.UTI porous at surface water from LagoaDourada, a small reservoir with a mean depth of 2.6 m,located in the district of Brotas, SP (22°01' Sand4r53'W). This species is part of the MicroalgalCulture Collection of the Botany Department of theFederal University of Sao Carlos (strain 00103PY),being kept in axenic culture. Experimental cultureswere prepared with cells maintained in WC medium(Guillard & Lorenzen 1972) under a photoperiod of12112 h light/dark. Illumination was provided by 40Wday light type fluorescent tubes (300 Jlmolphotons m·2 S·I ) and kept under a temperature of 23°C.

The experimental cultures were grown in 10 L pyrex

glass carboys containing 8 L of culture medium, in

duplicate.

Experiments with light: to perform light experiments2.3 L were collected from each of the carboys at thebeginning of exponential growth (six days), at the endof this phase (37 days) and during the stationarygrowth phase (72 days), each of which wasdetermined from the growth curve obtained bycounting the cells on a Palmer-Maloney chamber.These aliquots were inoculated with NaH I4C03(0.054 JlCi m L· t

) and subdivided into 250 mL lots innine pyrex glass bottles. These were submerged inflowing water, maintained at 23°C and exposed tovarious light iO'adiances (0, 33, 66, 166,430,830 1460,and 2000 J-Lffiol photons m·2 S·I) produced by combiningdifferent neutral aluminized glass filters. Halogenicbulbs of 500W were used as light source. After fourhours of exposure, aliquots of 10 mL were taken intriplicate from each flask, and filtered under lowvacuum pressure through cellulose acetate membrane(Schleicher & Schuell) of0.45 JllTI pore size and 47 mmdiameter to determine the incorporation of 14C in theparticulate fraction. The remaining contents of theflasks were also filtered through the same type of

. d h' 14Cmembrane and use to measure t e organicexcreted and for analysis of the dissolvedcarbohydrates released in the culture medium by thecells. From the filtrate of each flask, 40 mL aliquotswere taken, adjusted to pH 3.0 and bubbled withfiltered air for 40 minutes to eliminate any inorganicI-IC not assimilated by cells, which was captured in2M KOH solution. After bubbling, the aliquots hadtheir pH adjusted to 7.0 and from each flask four subsamples of 8 mL were placed in scintillation vials towhich 10 mL of the scintillation solution 7T:6R (7 paftsof toluene + 6 parts of Renex-95™) + 3 g L· 1 ofPPO(2,5 diphenyloxazole) + 100 mg L ·1 of dimethyl­POPOP (I 4-bis [2-(4-methyl-5-phenyloxazole)]­benzene}were added. The filters containing themarked cells were placed in scintillation vials to which10 mL of the scintillation solution 2T: lR (solution ofaforementioned compounds in two parts of tolueneand one paft of Renex-95™) were added. Renex-95™is a nonyl-phenol with an average number of nineethylene oxide units per molecule. Sample radioactivityin disintegrations per minute (DPM) was determinedin a Packard Tricarb mod. 1550 liquid scintillationcounter. The rest of the filtrate from each flask wasused to quantify carbohydrates released by the cells

exposed to each light intensity by the method of Dubois

A.A.H. Vieira, V. Colombo & O. Rocha: Release of extracellular polysaccharide by Peridillil/lll willei 243

100

2000

80

500 1000 1500

Irradiance (flmol m'zs·,)

20

1.5 +-----.----.---,-------,---~

o 40 60

Time (days)

Figure I. Growth of Peridillilllll willei in we medium, 300 J-Imphotons m'z S·I, photoperiod of 12/12 hand 23°e. Values shownare means ± SE (n=4).

3.5

4

14000

12000

".g~10000"'._ Vl

'2 ~"Vi u 8000Vl ~",0

}l; 6000

-"-.. 0 4000~~

2000

2

:..J-= E 3~ Vl0=~ "o u 2.5

00..9

of 13 days that might be considered too low, ifcompared with other planktonic species, but is notuncommon for other species of dinoflagellates(Chau 1978).

Figure 2 shows that in the cells of P willei,pre-adapted to ilTadiances of 300 fJInol photons m·2 S·I,the assimilation of carbon was not inhibited by theirradiance of 2000 fJmol photons m·2 S·I. Thephenomenon of photoinhibition did not occur even incells at the age of 72 days in the stationary phase. Thefact that chlorophyll-a concentrations (figure 3) didnot significantly vary over all tested irradiances(according to Anova) is also a proof of that. TheE

k= 600 fJInol photons m·2 S·I obtained for the species

is quite high, as is Km

(270 /-lmol photons m·2 S·I).

Besides the lack of photoinhibition at high irradiances,

et al. (1956) and PAD-HPLC (Gremm & Kaplan1997). Chlorophyll-a concentrations in the culturesafter exposure to light were determined by the methoddescribed by Tailing & Driver (1963).

Qualitative analysis of extracellular polysaccharide:the extracellular polysaccharide (EPS) was obtainedfrom a 4 L culture, after 70 days growing under theconditions previously described. After collecting thecells by tangential flow filtration in hollow fibercartridge of pore size 0.65 mrn, the medium withoutcells was concentrated five times in a rotaryevaporator and dialyzed in a dialysis membrane(12,000-14,000 Da) for 24 hours in flowing tap waterand an additional 24 hours in distilled water. Somedrops of toluene were added to the medium in orderto avoid contamination during the period of dialysis.After dialysis the concentrated medium wasfreeze-dried and kept in the freezer until the momentof the analysis. In order to check the purity ofEPS, a5 mg sample of the freeze-dried material was dissolvedin 5 mL of distilled water and injected on a column ofgel Sepharose gel CL-4B (Pharmacia) with an

I . 4 6exc uSlon range of 3.10 -3.10 Da. The sample wasthen eluted at a flow rate of 1.66 mL min· l

, with 2%butanol in water. Fractions were collected in a GradiFrac 200 (Pharmacia) collector and assayed forcarbohydrates by the phenol-sulfuric method (Duboiset al. 1956) and for proteins by the method of Lowryet al. (1951). Identification and quantification ofEPScomponent monosaccharides were performed by gaschromatography according to the method describedby Chaplin (1982) and modified by Paulsen & Vieira(1994). Protein content of EPS fraction wasdetermined according to the method described byLowry et al. (1951).

The medium without cells was also assayed fordissolved free monosaccharides by HPLC-PAD(Gremm & Kaplan 1997).

Nomenclature and definitions of photosyntheticparameters are according to Sakshaug et al. (1997).Hence, the former Ik , Iight saturation parameter[I

k= Pm / a = 1(8pSH 1')], here is represented by E k

and the former PFD, photon flux density, by E =irradiance.

Results

The growth curve of P willei (figure I) undercomplete nutrient conditions (WC medium) andE = 300 fJmol photons m·2 S·I, shows a doubling time

Figure 2. Total 14e assimilation vel' us irradiance by cells ofPeridillilllll willei of di fferent ages. Ek = 600 J-Im photons m-z s";Km = 270 pm photons m· 2 s·'. Values shown are means ± SE(n=8). -II- 6 days, --37 days, -A- 72 days.

244 Hoehnea 29(3), 2002

200015001000500

o~----.--------.-------,------,

o

50

350

300

-7 250'"c=

.~ ~ 200EoU - 150~2

0.. 100e.

0.5

0.45

0,4

~-7 0,35'"~=..c: 0)

Q.U 0,3OM~O

0- 0,25..c: 00U 2;

0,2

0.15

0,1

0 500 1000 1500 2000

Irradiance (pmol m,2 S,I)

Figure 3. Chlorophyll-a in cells of Peridinilll/1 willei of diffcrcntages after four hours of exposure to different ilTadiances. Valuesshown are means ± SE (n=3). -- 6 days, -e- 37 days,-"'-72 days.

Figure 4. Labeled dissolved organic carbon excreted by cells ofPeridiniulIl willei at differcnt ages after exposurc to differentirradiances. Values shown are means ± SE (n=8). -.- 6 days,-e-- 37 days, -"'-72 days.

there was no great increase in the rates of excretionof total organic 14C as a consequence of the stresscaused by those ilTadiances, even in cells in transitionand stationary growth phases, as can be observed infigure 4. The highest relative excretion rates occurredin cells 72 days old under in'adiances of66 and 166 ~olphotons m-2

S-l, 3.6 and 4.3%, respectively, whereasat 2000 ~mol photons m-2

S-I the rates were 2.0, 1.0and 1.3 % for cells 6, 37 and 72 days old respectivelyas can be seen in figure 5.

In figure 6 it can be observed that the totalcarbohydrate excretion, measured by the method ofDubois et al. (1956), for the cells in the exponential

growth phase is a process approximately parallel tophotosynthesis whereas for the cells in the transitionand stationary phases the excretion decreases atintermediate irradiances but increases at highirradiances. The amounts of carbohydrates releasedby the cells at different ages were 1.32, 2.72 and3.38 mg L- I at the exponential, transition, andstationary ages, respectively. The carbohydratereleased was an extracellular polysaccharide (EPS)and only trace amounts of dissolved free glucose andrhamnose were detected in the medium.

The identification and concentration of EPSmonosaccharide components is shown in table 1. The

65,5 I

5 0,9

~4.5 ~ 0.8

4 ~~ 0,7-0 •c

3,5:>.~

0 ..c: - 0.6o "~ 3 ~ U

~M 0,5U "0>< 2,5 u_0)

'E 2 0.4'- 20 "'0-0) 1,5 20 0.3c; ~~

c:: I c:: 0.2

0,5 0,1

0 0

0 500 1000 1500 2000 0 500 1000 1500 2000

Irradiance (pmolm'2 S,I)

Figure 5. Relative rates (%) of excretion oforganic carbon relea cdby cells of PeridinillJll willei at different ages after exposure to

different irradiances. Values shown are means ± SE (n=8).6 days, -e-- 37 days, -"'-72 days.

Figure 6. Carbohydratc rcleased by cells of Peridinilll/1 willei ofdifferent ages after exposurc to diffcrent ilTadiances. Values shown

are means ± SE (n:8). -1- 6 day, -.- 37 days, -.&72 days.

A.A.H. Vieira. V. Colombo & O. Rocha: Release of extracellular polysaccharide by Peridilli/llll willei 245

Discussion

Table I. Rclative monosaccharide composition of extracellularpolysaccharide produccd by Peridilli/{II/ Ivillei at stationarygrowth phase and determined by gas chromatography as theTMS derivatives of the corresponding methyl-glycosides.

EPS is a rhamnose-rich (52%) heteropolysaccharidewith small quantities of acidic sugars (l % glucuronicacid and 3% galacturonic acid) and neutral sugars (14%man nose, 13% galactose, 10% glucose and 2%

fucose). Gel filtration of the EPS in Sepharose CL-4B(Pharmacia) resulted in a single fraction which waseluted together with the Blue Dextran standard ofmolecular weight of 2 .106 Da.

Peridiniales occur throughout the year in LagoaDourada, a shallow, highly transparent and oligotrophiclake (Rocha & Sampaio 1991). They were mostabundant in the summer of 198911990, numericallyrepresenting 47.4% and 53.7% in October andNovember, 1989, respectively, and 81.1 % in January,1990 (Talamoni 1995). In 1995 the dominance ofPeridiniales was extremely high during January. Theyrepresented 50 to 94% of the total phytoplanktondensity and when biovolumes were considered, thedominance was even higher (Melao 1995). AlthoughPeridiniopsis and Gymnodinium are also present inthe reservoir, P willei was the domina~t species ofPeridiniales and although P willei reaches itsmaximum cell density in summer, it can be found allyear round, even in winter, although the densities arelow in this season (Talamoni 1995, Melao 1995).

Lagoa Dourada reservoir is highly transparent dueto low concentrations of suspended matter andchlorophyll-a with a mean depth of2.5 m, Secchi Diskreadings of2.5 m around the year. The light reachingthe bottom is sufficient for the maintenance of densepopulations of MoyacaJ711viatilis (Mayacaceae) andUtricularia gibba (Lentibulariaceae) (Melao 1995).

In this environment, algal cells are exposed to

high light ilTadiances, both in the summer, when theirradiance reaching the water surface is extremelyhigh, and in the winter when there are bright skies,without any clouds, for many consecutive weeks. Lightintensity under these conditions can easily reacharound 2000 flmol photon m-2 s-t on the surface at

noon. The high values of standard Ek

and of Km

forthis species, even in the transition and stationary growthphases, coupled with the low doubling rates, indicate

that P willei is a species adapted to this type ofenvironment, which is also characterized by lowconcentrations of nutrients which varied from 7.8 to16.2 pg L- 1 of nitrate, and from 0.3 to 1.9 pg L- I forphosphate, during 1995 (Melao & Rocha 1999).

Prezelin & Matlick (1980) reported the occurrenceof a type of photoadaptive response in a species ofdinoflagellate which was closely linked to the lightregime to which the species was adapted. In fact ahigh Km value indicates that even being tolerant to

high irradiances P willei presents a slow reaction to

light, a response already expected considering that ahigh value of E k often means inefficiency in the useof low ilTadiances and greater efficiency in usinghigher irradiances (Henley 1993). This probablyexplains the highest excretion rates at ilTadiancesbelow E k. Under irradiances below Ek the highestexcretion rates occurred in cells with 36 and 76 daysof age, indicating the decrease in photosyntheticefficiency determined by the cell aging and nutrientdepletion. At ilTadiances above E

k, nevertheless, the

highest excretion occurred with cells of six days, atthe exponential growth phase, and in this case the

amount of organic dissolved carbon excreted wasproportional to the total carbon assimi lated.Nevettheless in the present work the relative excretionrates (up to 4.3%) at all irradiances and at various

cell ages can be considered low. In fact, rates around5% of total carbon assimilation are reported as typicalrates for healthy phytoplankton populations (Fogg1983).

The absence of photoinhibition of the synthesisof the organic matter and the maintenance ofconstantchlorophyll-a concentration per cell, besides the lowrelative organic carbon excretion rates at all irradiancestested, including 2000 )lITlol m-2 S-I, are indicators thathigh irradiances do not promote severe stress effectsin P willei cells. However. the low doubling rates ofthe species makes the growth cycle of P willeirelatively long, with a stationary growth phaseextending for a hundred days, as observed in the

521413105321

Mol%Monosaccharide

RhamnoseMannoscGalactoscglucoscXyloseGalaclUronic acidFucoseGlucuronic acid

246 Hoehnea 29(3), 2002

cultures. Reduced growth rates are found In large,voluminous cells (Chau 1978, Brook 1981), as is thecase of P. willei, which has a biovolume of 4.104 ~m3

and is also a characteristic of many dominant speciesin oligotrophic environments (Brook 1981, Reynolds

1988). Such low growth rates imply that, in a population

of P. wilLei, cells differing greatly in age can be found.It is therefore expected that in a phytoplanktonassemblage dominated by P. willei, cells of differentphysiological states are found and, therefore, differentphysiological responses to high irradiances. Thus,despite the low excretion rates, the concentration ofcarbohydrate in the total organic carbon excreted bycells in the exponential phase is reasonably constantat all irradiances, slightly increasing as irradianceincreases in a process approximately parallel tophotosynthesis. Nevertheless, cells in the transition andstationary growth phases, when cytokinesis processesare well reduced or stopped and the concentrationsof nitrate and phosphate are already depleted as aconsequence of the culture age, excreted higherconcentrations of carbohydrates than the cells in theexponential phase, exposed to high irradiances. Suchconcentrations of carbohydrates released mayhowever be considered low and do not represent astrong stress effect caused by high in-adiances (Giroldo& Vieira 1998). In fact, the excreted carbohydratespresents monosaccharide composition which differsfrom intracellular reserve polymer, a starch, a cluethat suggests that the integrity of the cells wasmaintained. On the other hand, despite the smallamounts of excreted carbohydrates, the fact that theyare constantly produced make them a reasonablesubstrate for microbial heterotrophic populations (Bellet al. 1974), an important role in oligotrophicenvironments.

Acknowledgements

The authors thank FAPESP (Grants 97/05449,99/07766-0) and CNPq (PRONEX Program) forfinancial support.

Literature cited

Alcoverro, T., Contre, E. & Mazzellla, L. 2000. Productionof mucilage by the Adriatic epipelic diatomCylindrotheca closteriulIl (Bacillariophyceae) undernutrient limitation. Journal ofPhycology 36: 1087-1095.

Bell, W.H., Lang, j.M. & Mitchell, R. 1974. Selectivestimulation of marine bacteria by algal extracellularproducts. Limnology and Oceanography 19: 833-839.

Brook, A.J. 1981. The Biology of Desmids. Blackwell,Oxford, 286 p.

Chaplin, M.F. 1982. A rapid and sensitive method for theanalysis of carbohydrate components in glycoproteinsusing gas-liquid chromatography. Analytical Chemistry123: 336-341.

Chau, A. 1978. Comparative physiological study of marinediatoms and dinoflagellates in relation to irradiance andcell size. I. Growth under continuous light. Journal ofPhycology 14: 396-402.

Choen, Z. 1990. The production potential ofeicosapentaenoic and arachidonic acids by the red algaPorphyridillIl/ Criel/tllll/. Journal of American OilChemistry Society 67: 916-920.

Dias, C. 1990. Cicio anual do fitoplancton e algumasvariaveis ambientais na Lagoa do Infernao (SP).Disserta<;:iio de Mestrado, U ni versidade Federal de SaoCarlos, Sao Carlos, 108 p.

Dubois, M., Gilles, K.A., Hamilton, j.K., Rebers, P.A. &Smith, F. 1956. Colorimetric method for determinationof sugars and related substances. Analytical Chemistry28: 350-356.

Fogg, G.E. 1983. Ecological significance of extracellularproducts of phytoplankton photosynthesis. BotanicaMarina 26: 3-14.

Giroldo, D. & Vieira, A.A.H. 1998. Release of dissolvedorganic matter (DOM) in a tropical strain ofCryptolllonas obovata exposed to several irradiances.Journal of Plankton Research 21: 1911-1921.

Grande, K.D., Marra, j., Langdon, c., Heinemann, K. &Bender, M.L. 1989. Rates of respiration in the lightmeasured in marine phytoplankton using an 180isotope-labeling technique. Journal of ExperimentalMarine Biology and Ecology 129: 95-120.

Gremm, T.j. & Kaplan L.A. 1997. Dissolved carbohydratesin the stream water determined by HPLC and pulsedamperometric detection. Limnology and Oceanography42: 385-393.

Guillard, RRL. & Lorenzen, C.j. 1972. Yellow-green algaewith chlorophyllid-c. Journal ofPhycology 8: 10-14.

Guillard, R.RL. & Wangersky, P.j. 1958. The productionof extracellular carbohydrates by some marineflagellates. Limnology and Oceanography 3: 449-454.

Henley, W.J. 1993. Measurement and interpretation ofphotosynthetic light-response curves in algae in thecontext of photoinhibition and diel changes. Journal ofPhycology 29: 729-739.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. & RandaU, R.J.1951. Protein measurement with the folin phenol reagent.Journal of Biological Chemistry 193: 265-75.

Mayzaud, P., Chanut, J.P. & Ackman, R.G. 1989. Seasonalchanges of the biochemical composition of marineparticulate matter with special reference to fatty acidsand sterols. Marine Ecological Programme Serie56: 189-204.

A.A.H. Vieira. V. Colombo & O. Rocha: Release of extracellular polysaccharide by Peridinilllll lI'ilIei 247

Melao, M.G.M. 1995. The plankton community(phytoplankton and zooplankton) and zooplanktonsecondary productivity of a oligotrophic reservoir. Te ede Doutorado, UniversidadeFederal de Sao Carlos, SaoCarlos, 380 p.

Melao, M.G.M. & Rocha, 0.1999. Biomass and productivityof a freshwater sponge Melal/ia spinara (Metaniidae)in a Brazilian reservoir. Hydrobiologia 390: 1-10.

Myklestad, S. 1974. Production of carbohydrates byplanktonic diatoms. I. Comparison of nine differentspecies in culture. Journal of Experimental MarineBiology and Ecology 15: 261-274.

Paulsen, B.S. & Vieira, A.A.H. 1994. Structural studies onextracellular dissolved and capsular polysaccharidesproduced by Spol/dylosilllll pandllrijonlle. Journal ofPhycology 30: 638-641.

Prezelin, B.B. & Matlick, H.A. 1980. Time course ofphotoadaptation in the photosynthesis-irradiancerelationship of a dinoflagellate exhibitingphotosynthetic periodicity. Marine Biology 58: 85-96.

Reynolds, C.S. 1988. Functional morphology and theadapti ve strategies of freshwater phytoplankton. In:C.D. Sandgren (ed.). Growth and reproductive strategiesof freshwater phytoplankton. Cambridge UniversityPress, Cambridge, pp. 388-433.

Rocha, O. & Sampaio, A. V. 1991 Composi~ao,

caracteriza~ao e varia~ao sazonal da comunidadezooplanctonica da Lagoa Dourada, Bacia Hidrogriificado Lobo. Anais do 6° Semimlrio Regional deEcologia,Sao Carlos, pp. 23-46.

Sakshaug, E., Bricaud, A., Dandonneau,Y., Falkoski, E.G.,Kiefer, D.A., Legendre, L., Morel, A., Parslow, j. &Takahashi, M. 1997. Parameters of photosynthesis:definitions, theory and interpretation of results. Journalof Plankton Research 19: 1637-1670.

Talamoni, j.L.B. 1995. A comparative study on theplankton communities of lakes of different trophicdegrees and the analysis of the effect of Microcystisaertlgil/osa (Cyanophyceae) upon somemicrocrustaceans. Tese de Doutoramento. Uni versidadeFederal de Sao Carlos, Sao Carlos, 305 p.

Tailing, j.F. & Driver, D. 1963. Some problems in theestimation of chlorophyll-a in phytoplankton. In: M.S.Doty (ed.). Proceedings of the Conference of PrimaryProductivity Measurements, Marine and Freshwater.University of Hawaii, UA Atomic Energy Commission.TID-7633,Hawaii, pp. 142-146.

Vieira, A.A.H. & Myklestad, S. 1986. Production ofex tracellu lar carbohydrates of AI/kistrodesl/llIs del/slls

Kors. (Chlorophyta). Journal of Plankton Research8: 985-994.

Vieira, A.A.H., Lombardi, A.T. & Sartori, A.L. 1998.Release of dissolved organic matter in a tropical strainof SYI/Ilra petersel/ii (Chry ophyceae) grown underhigh irradiances. Phycologia 37: 57-362.

Wood, A. M. & Van Valen, L.M. 1990. Paradox lost? On therelease of energy-rich compounds by phytoplankton.Marine Microbial Food Web 4: 103-116.

Zlotnik, I. & Dubinsky, Z. 1989. The effect of light andtemperature on DOC excretion by phytoplankton.Limnology and Oceanography 34: 831-839.