M.A. Faust and D.L. Correll. Chesapeake Bay Center for Environmental Studies, Smithsonian Institution; F~ter, Maryland, USA. Abstract. The rate of utilization of ...
MARINE BIOLOGY
Marine Biology 41, 293-305 (1977)
9 by Springer-Verlag 1977
Autoradiographic Study to Detect Metabolically Active Phytoplankton and Bacteria in the Rhode River Estuary M.A. Faust and D.L. Correll Chesapeake Bay Center for Environmental Studies, Smithsonian Institution; F ~ t e r ,
Maryland, USA
Abstract
The rate of utilization of inorganic carbon (C) and phosphate (P) by p h y t o p l a n k t o n and bacteria in the Rhode River estuary has been estimated using liquid-emulsion autoradiography during four times of the year. Metabolic activity of phytoplankton was estimated by calculating silver grains above individual species. From these counts, the relative C and P uptake rates of individual species per unit of biomass and per volume of water were estimated. Examination of the autoradiograms showed that the m e t a b o l i c a l l y most active algae were smaller than 10 ~m. Both C and P were taken up by these smaller species at a higher rate than their proportion of the total biomass per volume of water would indicate. Uptake of C and P per unit of cell volume varied w i t h i n a species and among the various phytoplankton at the different seasons of the year. Metabolic activity of planktonic bacteria in safranin-stained autoradiograms was also estimated by counting bacteria with associated grains and cells without grains. The ratio of 33p-labeled to unlabeled bacteria was highest in November. This high metabolic activity of bacteria in November corresponded with high P uptake rates of the phytoplankton at that time. Throughout this study, only 28 to 42% of the total phytoplankton biomass was m e t a b o l i c a l l y active and 63 to 85% of the bacteria.
I ntroduction
Size fractionation of natural aquatic plankton populations associated with autotrophic and heterotrophic nutrient uptake has been employed previously for phosphorus (Correll et al., 1975; Faust and Correll, 1976) and for carbon utilization (Derenbach and Le B. Williams, 1974; Berman, 1975). Size fractionation is based upon the assumption that radioactivity collected on various pore-size filters represents the total uptake of nutrients in the photosynthetic (algal; large size) and in the heterotrophic (bacterial; small size) populations. In the above studies, a significant amount of the radioactivity was associated with organisms of less than 5 ~m, indicating bacterial involvement, and only a small part of the total radioactivity was associated with algal cells. However, size fractionation does not permit determination of selective uptake by members of plankton; it only measures total nutrient uptake for a specific size class within the plankton community,
Even though a separation of algae from bacteria can be achieved with size fractionation of plankton, certain problems have been recognized which are inherent in the use of this technique (Sheldon and Scutcliffe, 1969). Bacterial uptake cannot be fully determined by filtration, because some bacteria may adhere to algal surfaces (Bell and Mitchell, 1972; Jones, 1972) or to nonliving particulates. Others are large rods and remain in the large size fraction (Faust and Correll, 1976). In contrast, broken algal cell fragments can also pass through smaller pore-size filters and appear in the fraction representing m o s t l y bacteria (Berman, 1975; Faust and Correll, 1976). The methods widely used in previous studies are inadequate to provide estimations of the nutrient uptake and metabolic activity of individual species of microorganisms in the plankton community. In investigating the role of specific m i c r o o r g a n i s m s in nutrient uptake, autoradiography overcomes some of these difficulties (Brock and Brock, 1966; Fuhs
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M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
and Canelli, 1970). This t e c h n i q u e enables the i n v e s t i g a t o r to o b t a i n d i r e c t i n f o r m a t i o n on the f u n c t i o n of specific microorganisms, since the p r e s e n c e of a r a d i o a c t i v e s u b s t a n c e w i t h i n the m i c r o bial cell can be a s c e r t a i n e d . It also allows r e c o g n i t i o n of m e t a b o l i c a l l y active species in a g i v e n environment. In this study, we e x a m i n e d the p h o s p h o r u s and c a r b o n u p t a k e of a n a t u r a l p l a n k t o n p o p u l a t i o n by the t e c h n i q u e of liquide m u l s i o n a u t o r a d i o g r a p h y . Here we describe: (a) the m e t a b o l i c a c t i v i t y of a s s o c i a t e d p h y t o p l a n k t o n and b a c t e r i a l populations; (b) r e l a t i v e n u t r i e n t uptake rates of p h y t o p l a n k t o n species; (c) c o n t r i b u t i o n of m e t a b o l i c a l l y active b a c t e r i a to the n u t r i e n t u t i l i z a t i o n ; (d) a d v a n t a g e s and d i f f i c u l t i e s of the a u t o r a d i o g r a p h i c and size f r a c t i o n a t i o n methods.
Materials and Methods
Plankton Experiments
The e x p e r i m e n t s were carried out with n a t u r a l p l a n k t o n p o p u l a t i o n s o b t a i n e d in the m a i n b a s i n of the Rhode River estuary, an arm of the C h e s a p e a k e Bay, from M a r c h 1973 t h r o u g h F e b r u a r y 1974 (Faust and Correll, 1976). A clear glass and a black, epoxy p a i n t m d i p p e d b o t t l e each of I 1 c a p a c i t y were filled w i t h p l a n k t o n taken from a d e p t h of I m w i t h a peristaltic pump (Correll et al., 1975). Two 4 ~Ci each of 3 3 p - o r t h o p h o s p h a t e (carrier-free) or 1 4 C - c a r b o n a t e were added to each bottle. B o t t l e s were s u s p e n d e d for i n c u b a t i o n at a d e p t h of I m. At 15 and 30 m i n after the start of the experiment, 500 ml a l i q u o t s from the bottles were r e m o v e d and f i x a t i v e was added to give a final c o n c e n t r a t i o n of 0.4% g l u t e r a l d e h y d e in O . O 1 M sodium p h o s p h a t e buffer, pH 7.0. M e t a b o l i c a l l y i n h i b i t e d c o n t r o l s were run as d e s c r i b e d b e f o r e by a d d i n g i o d o a c e t i c acid (IAA) to control samples (Faust and Correll, 1976). P h y t o p l a n k t o n p o p u l a t i o n s were identified from water samples in the p l a n k t o n experiments.
water Chemistry
Water samples for c a r b o n and p h o s p h o r u s a n a l y s i s w e r e t a k e n as d e s c r i b e d under p l a n k t o n b o t t l e experiments. Inorganic c a r b o n a v a i l a b l e for u p t a k e was determ i n e d by a l k a l i n i t y t i t r a t i o n (American Public H e a l t h A s s o c i a t i o n , 1971). A n a l y ses of R h o d e River water d u r i n g this study y i e l d e d e s t i m a t e s of inorganic carbon c o n c e n t r a t i o n s r a n g i n g from 11.6 to 13.3 mg C/I.
O r t h o p h o s p h a t e in samples w h i c h had been f i l t e r e d t h r o u g h 0.45 ~m p o r e - s i z e m e m b r a n e s was a n a l y z e d c o l o r i m e t r i c a l l y by r e a c t i o n w i t h a m m o n i u m m o l y b d a t e and r e d u c t i o n w i t h stannous c h l o r i d e ( ~ e r i can Public H e a l t h A s s o c i a t i o n , 1971). Water was a n a l y z e d at the b e g i n n i n g and end of the experiment. A n a l y s e s of Rhode River water d u r i n g this i n v e s t i g a t i o n y i e l d e d e s t i m a t e s of d i s s o l v e d orthop h o s p h a t e c o n c e n t r a t i o n s from a high of 82 and 80 ~g P/I on J u n e 14 and September 6 to a low of 6.5 and 1.5 ~g P/I on N o v e m b e r 26 and F e b r u a r y 2 8 , r e s p e c t i v e l y . A u toradiograph y
Fixed samples w e r e taken to the laboratory, and c e n t r i f u g e d for 30 m i n at 2000 x g. The p e l l e t was w a s h e d with 12 ml of 0.002 N HCI to r e m o v e any r a d i o t r a c e r w h i c h m a y have been bound to the cell surface. A f t e r c e n t r i f u g i n g , the pellet was w a s h e d two m o r e times w i t h 12 ml d i s t i l l e d water and f i n a l l y r e s u s p e n d e d in 3 ml d i s t i l l e d water. E a c h sample was then s o n i c a t e d for 5 to 6 sec to break up any clumps of cells (Model W 140 C, Heat S y s t e m s Co., at 60 W output), as d e s c r i b e d by M a g u i r e and Neill (I 971). A c i d - w a s h e d glass m i c r o s c o p e slides were coated w i t h O.5% aqueous g e l a t i n and airdried. Slides w e r e p r e p a r e d from subsamples by p l a c i n g 5 drops of cell susp e n s i o n on each of 3 m i c r o s c o p e slides. Cells on the slides were q u i c k l y frozen on a block of dry ice and d r i e d in a v a c u u m d e s i c c a t o r for about 6 h. In a d a r k room, Kodak NTB-2 e m u l s i o n was d i l u t e d with an equal v o l u m e of d i s t i l l e d water and l i q u i f i e d at 40~ in a w a t e r bath. Two drops of e m u l s i o n were placed on the end of each slide and the e m u l s i o n was spread w i t h a stainless steel "raclette" to o b t a i n a c o n s t a n t t h i c k n e s s t h r o u g h o u t the slide (Bogoroch, 1972). The slides were dried in the dark at r o o m t e m p e r a t u r e (ca. 18~ and then t r a n s f e r r e d to a l i g h t - p r o o f box and stored in the r e f r i g e r a t o r for 3 to 5 days. A f t e r exposure, the e m u l s i o n was d e v e l o p e d in D e k t o l d e v e l o p e r at 15~ for 2 min, followed by a 10 sec d i s t i l l e d water rinse, 5 m i n in acid fixer and 5 to 10 m i n w a s h i n g in r u n n i n g tap water. A f t e r drying, the e m u l s i o n was c l e a r e d for I h in m e t h y l s a l i c y l a t e and m o u n t e d in P e r m o u n t w i t h a cover slip (Watt, 1971). Some slides were dipped, after d e v e l o p m e n t but b e f o r e washing, in 0.05% a q u e o u s s a f r a n i n for 30 sec. Grain Counting
Silver g r a i n counts w e r e m a d e of phytop l a n k t o n on slides p r e p a r e d both from
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
light and d a r k bottles. W h e n e v e r possible, e n o u g h cells of each species w e r e e x a m i n e d to give 30 net counts per species. Some species w e r e too infrequent, and for these species fewer than 30 counts w e r e enumerated. G r a i n counts w e r e m a d e under oil immersion, w i t h the m i c r o s c o p e f o c u s e d at the base of the cells and those grains in focus w e r e counted. T h e n the m i c r o s c o p e was focused up u n t i l the next set of grains was in focus and these counted. This p r o c e d u r e was r e p e a t e d until all g r a i n s w e r e out of focus. G r a i n s w i t h i n about 3 ~m of the m a r g i n of each cell w e r e also counted, since grains a c t i v a t e d by r a d i o a c t i v i t y e m i t t e d from the cell m a y not lie directly over the p o i n t of emission. I A A - t r e a t e d samples showed little or no silver g r a i n s over and a r o u n d phytop l a n k t o n cells. However, d e n s e g r a i n agg r e g a t e s w e r e o c c a s i o n a l l y p r e s e n t over d e t r i t a l particles. A u t o r a d i o g r a m s stained w i t h s a f r a n i n w e r e used to e s t i m a t e m e t a b o l i c a l l y active bacteria. M i c r o s c o p i c fields w i t h d i s p e r s e d b a c t e r i a l cells w e r e used to e s t i m a t e the grains over bacteria. Bacteria w i t h o u t g r a i n s w e r e also c o u n t e d and the ratio of labeled to u n l a b e l e d cells c o m p u t e d for 40 to 60 m i c r o s c o p i c fields. B a c t e r i a w e r e f r e q u e n t l y o b s e r v e d in a g g r e g a t e s and a t t a c h e d to d e t r i t a l p a r t i c l e s w h i c h were h e a v i l y c o v e r e d w i t h silver grains. B e c a u s e of the unc e r t a i n n a t u r e of these aggregates, such fields were not included in the c a l c u l a tions.
295
Uptake Calculations
C a r b o n (C) and p h o s p h o r u s (P) u p t a k e rates from a u t o r a d i o g r a m s were e s t i m a t e d as p r e v i o u s l y d e s c r i b e d by F r i e b e l e (1975). The initial specific a c t i v i t y of C and P was e s t a b l i s h e d from the inorganic C and o r t h o p h o s p h a t e c o n c e n t r a tions of the water after f i l t r a t i o n t h r o u g h a 0.45 ~un pore size filter and from the c p m / m l of whole water. The m e a n number of grains per cell was c o r r e c t e d for 33p d e c a y to the day of experiment. G r a i n y i e l d for 14C was e s t i m a t e d to be 1.8 to 2.0 on K o d a k A R - I O stripping film by Pelc (1972). This v a l u e was used in our c a l c u l a t i o n s . Since 33p has a range of m a x i m u m e n e r g i e s close to that of 14C, a 1.8 g r a i n yield was also used on K o d a k NTB-2 e m u l s i o n for 33p, w h i c h has a s e n s i t i v i t y similar to that of Kodak A R - I O s t r i p p i n g film (Herz, 1959). After these c o r r e c t i o n s , the m e a n number of g r a i n s per cell was c o n v e r t e d to counts per m i n u t e per liter for each species as used by F r i e b e l e (1975): M e a n g r a i n s / c e l l x no. of c e l l s / l 1.8 g r a i n s / c o u n t x 4320 m i n e x p o s u r e
time
= cpm/l. The c h a n g e in cpm/l in a p h y t o p l a n k ton species for a 30-min time interval was d i v i d e d by the a v e r a g e specific act i v i t y for inorganic c a r b o n or orthophosphate. U p t a k e rates were e s t i m a t e d as ~g C/h 1-I and ~g P/h 1 -I of individual algal species. U p t a k e rates were also e x p r e s s e d as ug P / ~ m 3 and ~g C/~un 3 of algal b i o m a s s of each species.
Biomass Determination
The e s t i m a t i o n of cell n u m b e r s of phytop l a n k t o n and b a c t e r i a by the d i r e c t count p r o c e d u r e (Rodina, 1972; Campbell, 1973) has b e e n d e s c r i b e d by F a u s t and C o r r e l l (1976). Cell b i o m a s s of o r g a n i s m s was c a l c u l a t e d from the m e a n diameter, length and d e p t h of i n d i v i d u a l cells as d e s c r i b e d by R o d i n a (1972). The b i o m a s s per unit of water v o l u m e was determined.
Light and Scanning Electron Microscopy
M i c r o s c o p i c fields from both light- and dark-bottle plankton experiments were e x a m i n e d using the p h a s e c o n t r a s t or b r i g h t - f i e l d o p t i c s of a Zeiss light m i c r o s c o p e at 1OOO x m a g n i f i c a t i o n . Pictures w e r e r e c o r d e d on High C o n t r a s t Copy or P a n c h r o m a t i c films. P l a n k t o n samples for scanning electron m i c r o s c o p y w e r e p r e p a r e d as des c r i b e d by F a u s t (1974).
Results
Association of Microbial Cells
B a c t e r i a and p h y t o p l a n k t o n w e r e o b s e r v e d singly, in a g g r e g a t e s or a t t a c h e d to d e t r i t u s in the Rhode River, as illustrated in the scanning e l e c t r o n m i c r o g r a p h s (Fig. I). C o c c o i d b a c t e r i a in an a g g r e g a t e appear to be joined w i t h f i b r o u s m a t e r i a l and w i t h small p i e c e s of n a t u r a l d e b r i s in Fig. I: I. Similarly, a f i l a m e n t o u s o r g a n i s m forms a firm a t t a c h m e n t on Prorocentrum mariae-lebouriae cell surface and on d e b r i s a d j a c e n t to the cell in the lower p o r t i o n of Fig. I: 2. D e b r i s of p l a n k t o n samples v a r i e d in shape and size, and some were c l o s e l y a s s o c i a t e d w i t h the m i c r o o r g a n i s m s , w h i l e others w e r e not. The above observ a t i o n m a k e s m e a s u r e m e n t of in situ m e t a b o l i c a c t i v i t y of algae and b a c t e r i a difficult.
296
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
Fig. i. Scanning electron micrographs (SEM) of association of microorganisms from the Rhode River. 1: Microorganisms often found in aggregates or attached to surfaces; aggregate of coccoid bacteria is illustrated (x 6300); bacterial cells within aggregate appear to be connected by fibers, indicated by arrows; background delineates surface of SEM stub. 2: Filamentous organism coherent to strongly compressed and flattened surface of P r o r o c e n t r u m m a r i a e - l e b o u r i a e (x 3100); cell surface is covered with tiny spines and trichocyst pores (single arrows); amorphous materials adhering to periphery of cell are debris from natural environment; flagellar pores at anterior end of cell are indicated by double-arrow
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
Some of the difficulties were overcome, however, by mild sonication of fixed plankton samples before cells were mounted on plates. A few seconds of sonication separated both bacteria and algal cells of small aggregates. Examples of b a c t e r i a in an unstained sample after brief sonication, using phase contrast optics is illustrated in Fig. 2. Attempts to separate cells in large aggregates were not successful. A longer time of sonication disrupted the cells; thus, sonication could not be used to break up larger aggregates. P h y t o p l a n k t o n of fixed plankton samples were also separated after a few seconds of sonication without disrupting their cell m o r p h o l o g y (Fig. 2: 6-9). P h y t o p l a n k t o n that appeared as single cells (Fig.2: 6,7), in pairs (Fig. 2: 9) and in groups of three (Fig. 2: 8) are illustrated in these light micrographs. Cells dispersed in a microscopic field could be used for grain counts and for taxonomical identification. At the same time, p h y t o p l a n k t o n remaining in large aggregates were of little value in this study. Au toradiography
A few examples of bacteria seen in the autoradiograms of the Rhode River plankton samples are illustrated in Fig. 2: 4,5. An unstained filamentous organism in an a u t o r a d i o g r a m is covered with grains, but the organism cannot be distinguished with the microscope under bright field optics (Fig. 2: 4). The m i c r o s c o p e is focused on the grains. This a u t o r a d i o g r a m is an unstained preparation, and the cells are not visible due to the low contrast of the organism under bright-field optics. However, in preparations stained with safranin, bacteria are visible and labeled and unlabeled bacteria can be clearly distinguished (Fig. 2: 5). This latter autor a d i o g r a m is focused on the bacteria, and the grains are somewhat out of focus, Therefore, staining of the autoradiograms was necessary to enhance bacterial morp h o l o g y under the autoradiographic emulsion. Stained autoradiograms w e r e examined w i t h bright-field optics to observe g r a i n - c o n t a i n i n g bacteria. In such fields, silver grains appear as black dots and underneath the grains bacteria are visible. R e p r e s e n t a t i v e autoradiograms of p h y t o p l a n k t o n are illustrated in Fig. 2: 6-9. The p h y t o p l a n k t o n shape and other taxonomical features are recognizable under the emulsion, however the resolution of the cellular features is somewhat lowered.
297
A u t o r a d i o g r a m s of algal cells with silver grains concentrated around and on the cells indicate actively metabolizing organisms. Grains within about 3 ~m of the m a r g i n of each cell were considered to be due to radioactivity emitted from the cell (Fig. 2: 7,8). A 14C-autoradiogram of Gyrodinium dominans with grains above and around the p e r i p h e r y of the cell is evident in Fig. 2: 7. Selective uptake of 33p-orthophosphate by phytop l a n k t o n species is illustrated in Fig. 2: 8. Numerous silver grains are above a Peridinium species located in the center, none are found above Prorocentrum mariaelebouriae, and only a few grains are above a Euglenoid species (arrow). Quite often, p h y t o p l a n k t o n incubated in the dark appeared to have numerous grains around the cells at a greater d i s t a n c e than 3 ~m. An example of a 14C-autoradiogram of G. estuariale incubated in the dark showing numerous grains is shown in Fig. 2: 6. A large number of grains were concentrated on and around Gyrodinium dominans cells with both tracer 14C and 33p in the light and dark bottles (Fig. 2: 9). The grains were located within an average diameter of 25 ~m around cells, and their appearance was very distinct from the background of the autoradiogram. This organism was the only p h y t o p l a n k t o n exhibiting such a grain pattern. The reason for this unusual grain pattern was not readily apparent. A u t o r a d i o g r a m s were also prepared of control plankton samples which had been exposed to the metabolic inhibitor IAA. The number of grains above the microorganisms were greatly reduced when IAA was added. However, grains covering natural debris were often observed in the preparations. U n m e t a b o l i z e d or adsorbed radiotracers were washed out with mild acid and rinsed twice with distilled water from the samples. This produced low background on the radioautograms. It is possible that washing was inadequate to free radiotracers from natural debris. Therefore, grains over clumps of natural debris were not counted.
Estimation of Carbon and Phosphorus Uptake by Phytoplankton
Incorporation of 14C-carbonate and 33p_ orthophosphate into p h y t o p l a n k t o n was estimated as grains per cell of metabolically active species during the four seasons of the year. Uptake of both tracers computed as grains per cell varied seasonally and from species to species (Table I). A large number of grains were found only in the dinoflagel-
298
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
M.A.
Faust
a n d D.L.
Correll:
T a b l e i. R e l a t i v e c a r b o n i m, e s t i m a t e d a s g r a i n s Organism
Chlamydomonas sp. Chlorella sp. Cryptomonas sp.l Cryptomonas Sp.2 Diplosalis sp. Euglena sp. Flagellate
Gymnodinium aurenticum Gymnodinium sp.l Gymnodinium sp.2 Gyrodinium dominans G. estuariale Katodinium rotundatum Nannoplankton
Pavlova gyrans Peridinium sp. Prorocentrum mariae-lebouriae Pseudopedinella pyriforme Tetraselmis gracilis
% b i o m a s s of grain-containing cells
Size (um)
Autoradiography
of E s t u a r i n e
(C) a n d p h o s p h o r u s (P) u p t a k e p e r cell. -: a b s e n t
Plankton
299
by phytoplankton
in R h o d e
River
at d e p t h
of
D a t e (1973-1974) J u n e 14 BioGrains mass (no./cell) (ram3/ C P
September 6 BioGrains mass (no./cell) (ram3~ C P
N o v e m b e r 26 BioGrains mass (no./cell) (ram3/ C P
F e b r u a r y 28 BioGrains mass (no./cell) (mm3/ C P
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-
-
3.0
2.1
13.5
11.7
.
.
.
36.3
(Fig. 2. L i g h t m i c r o g r a p h s of b a c t e r i a a n d p h y t o p l a n k t o n from Rhode River examined with phase- and bright-field o p t i c s (all x 1500). 3: R o d - s h a p e d b a c t e r i a in p a i r s (double a r r o w ) , c o c c o i d b a c t e r i a in s m a l l c l u s t e r s (single a r r o w ) , a n d s i n g l e c e l l s ( p h a s e - c o n t r a s t o p t i c s ) . 4: U n s t a i n e d 33p a u t o r a d i o g r a m of a f i l a m e n t o u s o r g a n i s m c o v e r e d w i t h m a n y s i l v e r g r a i n s ( b r i g h t , f i e l d o p t i c s , w i t h t h e s i l v e r g r a i n s in f o c u s ) . 5: S a f r a n i n - s t a i n e d b a c t e r i a a r e in f o c u s in p h o t o m i c r o g r a p h of a 33pautoradiogram (bright-field optics); large bacterial filament (single b l a c k arrow) a n d s e v e r a l small b a c t e r i a l a g g r e g a t e s (single w h i t e - e d g e d b l a c k arrows) w i t h o u t g r a i n s a r e shown; o t h e r b a c t e r i a in a g g r e g a t e s (double b l a c k arrow) h a v e g r a i n s a b o v e them, h o w e v e r s i l v e r g r a i n s a r e o u t of focus. N o t e c o n t r a s t in d e t e c t a b i l i t y of cell m o r p h o l o g y b e t w e e n u n s t a i n e d (4) a n d s a f r a n i n - s t a i n e d (5) p r e p a r a t i o n s . 6: E x a m p l e of 1 4 C - a u t o r a d i o g r a m of p l a n k t o n s a m p l e i n c u b a t e d in the dark; Gyrodinium estuariale c e l l is s h o w n w i t h n u m e r o u s g r a i n s a r o u n d the c e l l (arrows). (In i l l u s t r a t i o n s 6 and 9, g r a i n s a r e t o o f a r f r o m c e l l a n d a r e n o t c o n s i d e r e d as a l g a l u p t a k e of t h e t r a c e r in e s t i m a t i n g m e t a b o l i c a c t i v i t y of p h y t o p l a n k t o n . ) 7: Gyrodinium dominans w i t h some g r a i n s a b o v e a n d a r o u n d t h e c e l l is e v i d e n t in the 1 4 C - a u t o r a d i o g r a m . G r a i n s are w i t h i n 3 ~ m of c e l l p e r i p h e r y a n d a r e u s e d in e s t i m a t i n g g r a i n s p e r cell. 8: S e l e c t i v e u p t a k e of 3 3 p - o r t h o p h o s p h a t e by phytoplankton; n u m e r o u s s i l v e r g r a i n s a r e p o s i t i o n e d a b o v e a Peridinium s p e c i e s in t h e c e n t e r , n o n e are f o u n d a b o v e Prorocentrum mariae-lebouriae, a n d a f e w g r a i n s a r e a b o v e a Euglenoid s p e c i e s (arrow). 9: T w o c e l l s o f Gyrodinium dominans s u r r o u n d e d b y m a n y s i l v e r g r a i n s in 3 3 p - a u t o r a d i o g r a m are shown, u s i n g phase-contrast o p t i c s ; l a r g e n u m b e r of g r a i n s is a c c u m u l a t e d on a n d a r o u n d m o s t c e l l s , f o r m i n g a d e n s e g r a i n halo, i n d i s c r i m i n a t e d l y , in 14C a n d 3 3 p - a u t o r a d i o g r a m s i n c u b a t e d in l i g h t a n d d a r k bottles
300
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
late species Gymnodinium sp. I, Gyrodinium dominans, Peridinium sp. and Prorocentrum mariae-!ebouriae in June. Thus, these species w e r e considered the m o s t metabolically active algae at this time of the year. The biomass of grain-containing algal species was only 37.2% of the total algal biomass in June (Table I). Grains per cell of the same algal species varied seasonally. The high number of grains per Prorocentrum mariaelebouriae cell in June declined several fold to 10 grains per cell of C and 4 grains per cell of P in September (Table I). In contrast, incorporation of the tracers increased from 9 to 14 grains per cell for C and 8 to 12 grains per cell for P in Gymnodinium sp. I from June to September. P h y t o p l a n k t o n carbon and phosphorus uptake during the summer was due to dinoflagellates and to other species during spring, fall and winter seasons (Table I). In September, November and F e b r u a r y the largest number of grains were found in Chlorella sp., Cryptomonas sp. I , Katodinium rotundatum, nannoplankton, and Tetraselmis gracilis. All actively m e t a b o l i z i n g cells were less than 20 ~m in size and only 28 to 42% of the total algal biomass were m e t a b o l i c a l l y active at any given season (Table I). The total biomass of p h y t o p l a n k t o n varied from 0.5 to 1.7 mm3/l during the seasons (Table 2). The biomass of various algal classes was also estimated. Dinoflagellates represented 69 to 80% of the total algal biomass in June, September and November; whereas organisms of the Cryptophyceae, Bacillariophyceae, Crysophyceae, flagellates, and nannoplankton each made up 18 to 24% of the total algal biomass in February. In February the dinoflagellate biomass declined to 5% of total algal biomass. Rates of carbon and phosphorus uptake for all phytoplankton species are presented in Table 3. The contribution of algal species to the uptake of C and P per volume of water changed seasonally. Uptake of C by Gymnodinium sp.1 varied from 8.8 ~g/l h-1 x 10-2 in June and 33 ~g/l h -I x 10 -2 in September to none in November and February. The P uptake by this alga followed a similar course. In contrast, Prorooentrum mariae-!ebouriae C and P uptake rates were higher in June and lower in September. The highest observed uptake rates for C and P were exhibited by Katodinium rotundatum (145 ~ g C / 1 h-1 x 10-2 and 31 ~gP/l h -I x 10 -2 ) and by Pavlova gyrans (79.5 ~gC/l h-1 x 10-2 and O.16 ~gP/l h-1 x 10-2) in November. Total C and P uptake per volume of water was also estimated (Table 3). Car-
bon uptake was comparatively low (41.2 and 51.1 ~ g C / l h-1 x 10 -2 ) in June and September and high (393.4 and 170.2 ugC/l h-1 x 10 -2 ) in November and February, respectively. Phosphorus uptake rates were 60.0 and 50.9 ~gP/l h -I x 10 -2 in June and September and 66.36 and 2.42 ugP/l h -I x 10 -2 in November and February. No persistent relationship was observed between C and P uptake rates per volume of water (Table 3) and total algal biomass (Table 2). It m u s t be noted that only about one-third of the total biomass of algae was active at any given time. Carbon and phosphorus uptake rates per unit of cell volume were higher for the smaller than for the larger species (Table 4). Pseudopedinella pyriforme, the smallest flagellate in the samples, had the highest uptake rates for both C and P per unit of cell volume (817 ~gC/~m3 h -I x 10 -10 and 5.9 ~gP/~m 3 h -I x 10 -10 in February), followed by a nannoplankton (366.8 ~ g C / ~ m 3 h-1 x 10-10 and 7.5 ~gP/~/n 3 h -I x 10 -10 in November); while the third highest rate was by a Chlorella sp. (190.7 ~gC/~m 3 h -I x 10 -]0 and 15.2 ~ g P / ~ m 3 h-1 x 10-10 in September). Carbon and P uptake per cell volume was m u c h lower for the larger species, e.g. Prorocentrum mariae-lebouriae (4.9 to 2.5 ~ g C / ~ m 3 h -I x 10-10 and 0.2 to O.15 U g P / ~m3 h-1 x 10-10) and Gymnodinium sp.1 (11.5 to 24.1 ~gC/~m 3 h-1 x 10-10 and O.18 to 0.22 ~gP/~m3 h-1 x 10-10) in June and September, respectively. The biomass of P. mariae-lebouriae was the largest of any species measured during the experimental period, yet C and P uptake per unit of cell volume was 160 and 30 times less than that estimated for Pseudopedinel!a pyriforme. The uptake may have been governed by the surface:volume ratio in the various plankton size classes. The relationship C and P uptake per unit of cell volume varied between algal species (Table 4). The ratio of C to P uptake per ~m3 ranged from 10: I for Diplosalis sp. , Gynmodinium aurenticum and Peridinium sp. ; and 50: I for Katodinium rotundatum, nannoplankton, and Pavlova gyrans, to above IOO : I for Gymnodinium sp. 1 and Pseudopedinella pyriforme.
Autoradiography of Bacteria
M e t a b o l i c a l l y active bacterial populations from light and dark bottles were detected in stained 33P-autoradiograms. The percentages of labeled and unlabeled bacteria in the autoradiograms were estimated four times a year (Table 5). The distribution of labeled bacteria
M.A.
Faust
and
D.L.
Correll:
Autoradiography
Table
2.
Biomass
rence
in
Rhode
Algal
classes
of
various
River
at
Dinophyeeae
of
Estuarine
algal
depth
classes
of
1 m.
Plankton
and
Values
Date
(1973-1974)
June
14
September
their as
6
total
November
26
February
72.0
69.1
4.9
5.7
3.4
10.4
9.7
1.4
i.i
8.1
-
15.9
0.7
4.7
18.3 23.7
Chlorophyceae
-
0.9
3.7
Euglenophyceae
-
1.0
-
Haptophyceae
-
-
4.7
Flagellates
-
4.7
-
18.2
Nannoplankton
0. i
6.9
4.7
18.3
1.7
0.9
i.i
0.5
3.
plankton
biomass
Carbon in
(C)
Rhode
(mm3/l)
and
River
Organism
phosphorus at
(P)
depth
of
Flagellate
Gymnodinium s p . l Gymnodinium s p . 2 Gymnodinium aurenticum Gyrodinium dominans G. estuariale Katodinium rotundatum
rates
Date
(1973-1974)
(um)
June
14
-
5x5
-
(ug
September
P
13xlO
3.0
l-I
h -I
x
10 -2 ) of
phyto-
i m
Size
C
Chlamydomonas s p . Chlorella s p . Cryptomonas s p . l Cryptomonas s p . 2 Diplosalis sp. Euglena sp.
uptake
28
1.8
Cryptophyceae
Total
C -
P
6
November C
-
-
7.2
6.0
.
15x12
-
-
-
-
-
13xlO
-
-
-
-
-
15x15
-
-
i.O
0.9
.
5x55
-
-
0.8
0.9
-
15x12
-
-
-
-
-
14.O
12x8
8.8
32.9
30.0
15x13
-
-
-
-
-
13x7
-
-
i.i
0.8
.
15.O
26 P
February C
.
.
.
.
.
15x12
8.8 -
-
7.1
6.8
1Ox6
-
-
-
-
145.O
Nannoplankton
iOx5
-
-
-
74.8
Pavlova gyrans Peridinium s p . Prorocentrum mariaelebouriae Pseudopedinella pyriforme Tetraselmis gracilis
iOx6
-
-
-
-
79.5
O.
17x13
6.4
7.0
-
16x20
17.2
24.0
9.4
O.04
25.5
0.07
38.O
O.4O
.
-
.
28 P
.
-
12xlO
Total
occurbiomass
2.5
Prasinophyceae
Table
seasonal
% of
79.9
Chrysophyceae
Bacillariophyceae
301
-
-
44.4
O.12
12.8
1.50
.
-
1 .O
68.4
14.8
-
-
31.21
-
-
15.
iO
-
-
16
-
-
4.6
5x3
-
-
-
-
-
15x12
-
-
-
25.7
5.15
41.2
60.0
393.4
66.36
40. I
O.29
-
-
170.2
2.42
uptake
(ugl -I
h -I
x
10 -2 )
51.10
50.9
302
M.A.
Table
4. C a r b o n
ton per
unit
Faust
(C) a n d p h o s p h o r u s
biomass
in R h o d e
Organism
Flagellate
Gymnodinium aurenticum Gymnodinium sp.1 Gymnodinium sp.2 Gyrodinium dominans G. estuariale Katodinium rotundatum Nannoplankton
Pavlova gyrans Peridinium sp. Prorocentrum mariaelebouriae Pseudopedinella pyriforme Tetraselmis gracilis
at depth
Date June C
13xlO
.
5x5 15x12 13xi0 15x15 5x55 15x12 13x7 12x8 15x13 15x12 12xlO I0x6 iOx5 16x6 17x13
.
4.9
5x3 15x12
. .
. .
.
.
. .
.
.
x 10 - 1 0 ) of p h y t o p l a n k -
.
N o v e m b e r 26 C P .
15.20 . . . . 0.23 0.05 . . O.16 0.22 . . . O.16
.
2.56 .
.
Plankton
i m
190.76 . . 2.42 5.18 . 18.32 24.10 . . . 0.07 . . . . .
0.20
of E s t u a r i n e
160.8 .
.
6.94
.
. .
. .
. .
. .
. .
. .
. .
. .
. 70.4 1.52 164.8 3.53 366.8 7.55 127.5 2.42 . . .
65.4 73.0
O.17 0.75
55.7
1.50
46.1
0.55
.
.
.
.
.
.
metabolizing
.
.
0.15 .
F e b r u a r y 28 C P
31.6
bacteria
0.62
-
-
. 817.1 -
at v a r i o u s
5.91 -
seasons
No. b a c t e r i a / f i e l d Labeled Unlabeled
% labeled • SD b
Labeled: Unlabeled
Cells/ml x 106
Biomass (mm3/l
L D
33.3 34.1
20.3 19.8
63 + 3.7 63 + 4.5
1.64 1.72
0.9
4.9
6
L D
32.7 30.1
13.8 17.O
70 + 3.5 63 +- 4.2
2.36 1.77
2.5
3.4
November
26
L D
47.8 46.1
7.4 7.6
85 + 5.1 84 -+ 5 . 0
6.45 6.06
3.1
5.1
February
28
L D
65.8 55.4
14.1 12.0
81 + 4.4 82 -+ 3.7
4.66 4.61
2.6
1.6
June
Treatment a
(ug u m 3 h -I of
.
. . . . . . 11.5 0.18 . . 4.9 0.84 . . . . . . . . . 12.7 O.13
16x20
Autoradiography
(1973-1974) 14 September 6 P C P
T a b l e 5. P r o p o r t i o n s of 3 3 p - o r t h o p h o s p h a t e in R h o d e R i v e r at d e p t h of i m Date (1973-1974)
Correll:
(P) u p t a k e
River
(um)
Size
Chlamydomonas sp. Chlorella sp. Cryptomonas sp.l Cryptomonas s p . 2 Diplosalis sp. Euglena sp.
a n d D.L.
14
September
a
L:
Light
bottle;
bSD : Standard
D:
dark
deviation.
bottle.
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
within each sample was relatively uniform. In November and February, 81 to 85% of the bacteria were labeled, whereas in June and September 63 to 70% of bacteria had grains. Thus, a greater proportion of planktonic bacteria (Table 5) than p h y t o p l a n k t o n (Table I) had grains above them at the same selected time of the year. The ratio of 33p-labeled to unlabeled bacteria was the highest (6.4) in November, lower (4.6) in February and the lowest (1.6 to 2.3) in June and September (Table 5). The high metabolic activity of bacteria corresponded with high P uptake rates per volume of water of the phytoplankton in November (Table 3). Similarly the number of bacteria was highest, 3.1 x 106 per ml, in November, 2.5 and 2.6 x 106 per ml in September and February, and lowest, 0.9 x 106 per ml, in June. In contrast, the biomass per volume of water was highest, 4.9 and 5.1 mm3/l in November and June, 3 . 4 m m 3 / i in September, and lowest 1.6 mm3/1, in February.
303
in an equivalent area containing no phytoplankton. No correction for the adhering or aggregating bacteria could be made. This is a source of error, and could cause an o v e r - e s t i m a t i o n of phosphorus uptake by individual phytoplankton species. Bacteria are small and difficult to detect under the emulsion of the autoradiograms. Staining enhanced the visibility of these cells. Comparison of counts of labeled and unlabeled bacteria from stained autoradiograms suggested that a large proportion of bacteria w e r e clumped; thus, counts obtained from the autoradiograms might have underestimated unlabeled organisms. The proportion of labeled bacterial cells could also be underestimated in the June experiment when a large number of grains were observed as a dense grain halo around Ggrodiniumdominans. In this halo, bacteria were not visible and thus this activity was not counted or tabulated as bacterial since activity of bacteria was assessed using grains only above recognizable bacterial cells. Several investigators (Fuhs and Canelli, 1970) experienced similar problems and suspected that Discussion clumps of bacteria were the cause of Relative grain counts of phytoplankton such halos, but were unable to provide species as a measure of their metabolic proof of the presence of bacterial cells. activity have been used previously (Brock Nevertheless, the data available suggest and Brock, 1968; Fuhs and Canelli, 1970; that valid counts of labeled bacteria Maguire and Neill, 1971; Watt, 1971). from a m i x e d population could be obHowever, quantitative measurements from tained from autoradiograms provided the autoradiograms are difficult to obtain. organisms are not clumped and the autoRelative grain densities around organisms radiograms are stained. depend not only on the geometry and denResults from the autoradiograms show sity of the specimen, but also on the that phytoplankton smaller than 10 ~m in energy of the tracer and the thickness size were m e t a b o l i c a l l y most active in and sensitivity of the photographic the Rhode River. M c C a r t h y et al. (1974) emulsion (Perry, 1964). measured primary production of phytoEven though autoradiography is speplankton in the Chesapeake Bay. They recific, we recognize some of the diffiported that 94% of the primary producculties in estimating the metabolic activity was by "algae" passing through a tivity of algae and bacteria in an estu10 ~m pore-size net. Taft et al. (1975) ary. Some p h y t o p l a n k t o n and bacteria showed also that "ultra-plankton" (which form m u c i l a g i n o u s capsular material; could include bacteria and small algae) others adhere to algal cell surfaces and were responsible for the majority of the to sediment particles (Bell and Mitchell, P uptake in the Chesapeake Bay. Our pre1972; Jones, 1972; Faust and Correll, vious results have also indicated that 1976). Bacteria are also opportunists, 95% of phosphorus was assimilated by utilizing photosynthetic products of bacteria throughout the year and that P phytoplankton efficiently (Watt, 1971). uptake by algae was only a significant Also, algal h e t e r o t r o p h y may exist in percentage of the total during the sumnature (Ketchum, 1939; Faust and Gantt, mer months in the Rhode River (Faust and 1973; Pollinger and Berman, 1976). DeCorrell, 1976). spite all these d i f f i c u l t i e s , a u t o r a d i o g We have reported earlier (Correll et raphy is still one of the more sensitive a!., 1975) and in this report that P uptechniques for estimation of bacterial take by nannoplankton was greater than and phytoplankton m e t a b o l i s m at the spethe proportion of their biomass would cies level. suggest. Watt (1971) also reported that Grain counts of phytoplankton cells this was true for primary productivity were corrected for 33P-radioactive decay of nannoplankton in the northwest Atlanand for background grains occurring with- tic Ocean. A l t h o u g h nutrient uptake by
304
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
algal c e l l s m a y differ, some s p e c i e s m a y have the a d v a n t a g e in n u t r i e n t u p t a k e over o t h e r s due to h i g h e r s u r f a c e - t o v o l u m e ratios. N a n n o p l a n k t o n c e r t a i n l y h a v e a large s u r f a c e - t o - v o l u m e r a t i o (Fuhs et al., 1972). M u n k and R i l e y (1952) c a l c u l a t e d n u t r i e n t a b s o r p t i o n rates from a t h e o r e t i c a l e x p r e s s i o n c o n t a i n i n g h y d r o d y n a m i c v a r i a b l e s for algal c e l l s and found that a b s o r p t i o n i n c r e a s e s w i t h d e c r e a s i n g cell d i m e n s i o n . F r i e b e l e (1975) and our r e s u l t s w i t h 33p autor a d i o g r a p h y of a n a t u r a l p l a n k t o n p o p u l a t i o n c o n f i r m this theory. P h o s p h o r u s u p t a k e r a t e s per v o l u m e of w a t e r d e t e r m i n e d by a u t o r a d i o g r a p h y w e r e h i g h e s t in N o v e m b e r (Table 3), w h e n the r a t i o of labeled and u n l a b e l e d b a c t e r i a l cells was also h i g h e s t (Table 5). Thus, a close relationship existed between a u t o t r o p h i c and h e t e r o t r o p h i c m e t a b o l i c a c t i v i t y of the p l a n k t o n p o p u l a t i o n , and b a c t e r i a did c o n t r i b u t e s u b s t a n t i a l l y in this process. This r e s u l t is similar to r e p o r t s of b a c t e r i a l p a r t i c i p a t i o n in the p r i m a r y p r o d u c t i o n in the sea (Derenb a c h and W i l l i a m s , 1974) and of P u p t a k e in the R h o d e R i v e r (Faust and Correll, 1976) e s t i m a t e d p r e v i o u s l y by u s i n g size f r a c t i o n a t i o n of the plankton. The g e n e r a l i z e d r e s u l t s show summer p r i m a r y p r o d u c t i v i t y and P u p t a k e in R h o d e R i v e r p h y t o p l a n k t o n to be p r e d o m i n a n t l y a t t r i b u t a b l e to d i n o f l a g e l l a t e s (70 to 80% from J u n e to November) and to other small s p e c i e s (30 and 95% of total b i o m a s s in N o v e m b e r and F e b r u a r y ) . The l a r g e s t d i n o f l a g e l l a t e , Prorocentrum mariaelebouriae, c o n t r i b u t e d s i g n i f i c a n t l y to C and P u p t a k e in June, w h e r e a s the s m a l l e r d i n o f l a g e l l a t e K a t o d i n i u m r o t u n d a t u m and o t h e r n a n n o p l a n k t o n w e r e the p r i n c i p a l p r i m a r y p r o d u c e r s d u r i n g the r e s t of the year (Table 3). Thus, the r a t e s of prim a r y p r o d u c t i o n and P u p t a k e per u n i t of cell v o l u m e (Table 4) d i s p l a y a w i d e r a n g e of values. The n u m b e r of o b s e r v a tions are too small to d e f i n e p r o d u c t i v i ty r a n g e s of the s p e c i e s involved. The metabolic rates encountered within a s p e c i e s such as P. mariae-lebouriae in J u n e and S e p t e m b e r or a m o n g the d i n o f l a g e l late s p e c i e s K. rotundatum and Gyrodinium estuariale at the R h o d e River e s t u a r y and in the o c e a n (Watt, 1971) are e x t r e m e l y variable. The m e t a b o l i c a c t i v i t y per u n i t of cell v o l u m e of small o r g a n i s m s such as Pseudopedinella pyriforme (volume = 35 ~m 3) was h i g h e r than the larger n a n n o p l a n k t o n (volume = 196 ~m3) or K a t o d i n i u m r o t u n d a t u m (volume = 458 ~m3). The C:P u p t a k e r a t i o s f o l l o w e d the p r e v i o u s order (e.g. C:P for P. pyriforme was 138:1, n a n n o p l a n k t o n 49:1 and K. rotundatum 46:1. However, w h e n c o m p a r i n g the u p t a k e r a t e s per
v o l u m e of water, w h i c h r e f l e c t the total p r o d u c t i v i t y of the p l a n k t o n p o p u l a t i o n , K. rotundatum was the m o s t p r o d u c t i v e , followed by the n a n n o p l a n k t o n and by P. pyriforme. P e r h a p s there is no u n i v e r s a l r e l a t i o n s h i p that e x i s t s b e t w e e n a species b i o m a s s (volume) and its c o n t r i b u tion to the total p r o d u c t i v i t y of the c o m m u n i t y as s u g g e s t e d by Watt (1971). The r e a s o n s for c h a n g i n g p a t t e r n s of p r o d u c t i v i t y c o u l d be such f a c t o r s as life history, age, size, p h o t o s y n t h e t i c c a p a c i t y , n u t r i e n t c o n c e n t r a t i o n s and d i u r n a l r h y t h m s of p h y t o p l a n k t o n s p e c i e s (Maguire and Neill, 1971; S t r o s s and Pemrick, 1974). In v i e w of the above, we r e c o g n i z e the l i m i t a t i o n of this a u t o r a d i o g r a p h y study, w h i c h only a t t e m p t e d to e s t i m a t e r e l a t i v e n u t r i e n t u p t a k e r a t e s by p h y t o p l a n k t o n s p e c i e s and to supply i n f o r m a tion on the p r o p o r t i o n of m e t a b o l i c a l l y active heterotrophic bacterial populations. However, a u t o r a d i o g r a p h y and size-fractionation techniques combined may provide a considerable advancement in our u n d e r s t a n d i n g of the m e c h a n i s m s which determine plankton community structure. E s t i m a t i n g s h o r t - t i m e in situ nut r i e n t u p t a k e by p l a n k t o n m a y also be of v a l u e to studies of in situ algal p h y s i o l o g y and the r o l e of a l g a l h e t e r o t r o p h y in the a q u a t i c e n v i r o n m e n t . Acknowledgement. This research was supported, in part, by a grant from the Program for Research Applied to National Needs of the National Science Foundation, and b y t h e Smithsonian Institution's Environmental Sciences Program.
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Date of final manuscript acceptance: February Ii, 1977. Communicated by M.R. Tripp, Newark
