Marine Biology 41, 293-305 (1977)
MARINE BIOLOGY
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 u t i l i z a t i o n of i n o r g a n i c c a r b o n (C) and p h o s p h a t e (P) by p h y t o p l a n k t o n and b a c t e r i a in the R h o d e R i v e r e s t u a r y has b e e n e s t i m a t e d u s i n g l i q u i d - e m u l s i o n a u t o r a d i o g r a p h y d u r i n g four t i m e s of the year. 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 was e s t i m a t e d by c a l c u l a t i n g silver g r a i n s a b o v e i n d i v i d u a l species. F r o m t h e s e counts, the r e l a t i v e C and P u p t a k e rates of i n d i v i d u a l s p e c i e s per u n i t of b i o m a s s and per v o l u m e of w a t e r w e r e estimated. E x a m i n a t i o n of the a u t o r a d i o g r a m s s h o w e d that the m e t a b o l i c a l l y m o s t a c t i v e algae w e r e s m a l l e r than 10 ~m. B o t h C and P w e r e t a k e n up by these s m a l l e r s p e c i e s at a h i g h e r r a t e t h a n their p r o p o r t i o n of the to- tal b i o m a s s per v o l u m e of w a t e r w o u l d indicate. U p t a k e of C and P p e r u n i t of c e l l v o l u m e v a r i e d w i t h i n a s p e c i e s and among the v a r i o u s p h y t o p l a n k t o n at t h e d i f f e r e n t
seasons of the year. M e t a b o l i c a c t i v i t y of p l a n k t o n i c b a c t e r i a in s a f r a n i n - s t a i n e d a u t o r a d i o g r a m s was also e s t i m a t e d by c o u n t i n g b a c t e r i a w i t h a s s o c i a t e d g r a i n s and c e l l s w i t h o u t grains. The r a t i o of 3 3 p - l a b e l e d to u n l a b e l e d b a c t e r i a w a s h i g h e s t in November. T h i s h i g h m e t a b o l i c a c t i v i t y of b a c t e r i a in N o v e m b e r c o r r e s p o n d e d w i t h h i g h P u p t a k e r a t e s of the p h y t o p l a n k t o n at that time. T h r o u g h o u t this study, o n l y 28 to 42% of the total p h y t o p l a n k t o n b i o m a s s was m e t a b o l i c a l l y a c t i v e and 63 to 85%
of the b a c t e r i a .
I ntroduction
Size f r a c t i o n a t i o n of n a t u r a l a q u a t i c p l a n k t o n p o p u l a t i o n s a s s o c i a t e d w i t h 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 n u t r i e n t u p t a k e has b e e n e m p l o y e d p r e v i o u s l y for p h o s p h o r u s (Correll et al., 1975; F a u s t and Correll, 1976) and for c a r b o n uti- l i z a t i o n (Derenbach and Le B. W i l l i a m s , 1974; Berman, 1975). Size f r a c t i o n a t i o n is b a s e d u p o n the a s s u m p t i o n t h a t r a d i o - a c t i v i t y c o l l e c t e d on v a r i o u s p o r e - s i z e f i l t e r s r e p r e s e n t s the total u p t a k e of n u t r i e n t s in the p h o t o s y n t h e t i c (algal;
l a r g e size) and in the h e t e r o t r o p h i c (bacterial; small size) p o p u l a t i o n s . In the above studies, a s i g n i f i c a n t a m o u n t of the r a d i o a c t i v i t y was a s s o c i a t e d w i t h o r g a n i s m s of less t h a n 5 ~m, i n d i c a t i n g b a c t e r i a l i n v o l v e m e n t , and only a small p a r t of the total r a d i o a c t i v i t y was as- s o c i a t e d w i t h a l g a l cells. However, size f r a c t i o n a t i o n d o e s not p e r m i t d e t e r m i n a -
E v e n t h o u g h a s e p a r a t i o n of algae from b a c t e r i a c a n be a c h i e v e d w i t h size f r a c t i o n a t i o n of plankton, c e r t a i n prob- lems h a v e b e e n r e c o g n i z e d w h i c h are in- h e r e n t in the use of this t e c h n i q u e (Sheldon and S c u t c l i f f e , 1969). B a c t e r i a l u p t a k e c a n n o t be fully d e t e r m i n e d by f i l t r a t i o n , b e c a u s e some b a c t e r i a m a y a d h e r e to algal s u r f a c e s (Bell and M i t - chell, 1972; Jones, 1972) or to non- living p a r t i c u l a t e s . O t h e r s are large r o d s and r e m a i n in the large size frac- t i o n (Faust and Correll, 1976). In con- trast, b r o k e n a l g a l cell f r a g m e n t s c a n a l s o p a s s t h r o u g h s m a l l e r p o r e - s i z e fil- ters and a p p e a r in the f r a c t i o n r e p r e - s e n t i n g m o s t l y b a c t e r i a (Berman, 1975;
F a u s t and Correll, 1976).
T h e m e t h o d s w i d e l y u s e d in p r e v i o u s s t u d i e s are i n a d e q u a t e to p r o v i d e esti- m a t i o n s of the n u t r i e n t u p t a k e and m e t a - bolic a c t i v i t y of i n d i v i d u a l species of m i c r o o r g a n i s m s in the p l a n k t o n community.
t i o n of s e l e c t i v e u p t a k e by m e m b e r s of In i n v e s t i g a t i n g the r o l e of s p e c i f i c p l a n k t o n ; it o n l y m e a s u r e s total n u t r i e n t m i c r o o r g a n i s m s in n u t r i e n t uptake, auto- u p t a k e for a s p e c i f i c size class w i t h i n r a d i o g r a p h y o v e r c o m e s some of t h e s e d i f - the p l a n k t o n c o m m u n i t y , f i c u l t i e s (Brock and Brock, 1966; F u h s
294 M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
and C a n e l l i , 1970). T h i s t e c h n i q u e en- a b l e s 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 o n t h e f u n c t i o n of s p e c i f i c m i c r o o r g a n i s m s , s i n c e t h e 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 t h e m i c r o - b i a l cell c a n be a s c e r t a i n e d . It a l s o a l l o w s 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 ac- t i v e s p e c i e s in a g i v e n e n v i r o n m e n t . In t h i s study, we e x a m i n e d the p h o s p h o r u s a n d 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 t h e t e c h n i q u e of l i q u i d - e m u l s i o n a u t o r a d i o g r a p h y . H e r e we d e - scribe: (a) t h e 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 p o p u l a t i o n s ; (b) 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 of p h y t o p l a n k t o n s p e c i e s ; (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 a c t i v e 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 a n d d i f f i c u l t i e s of t h e a u t o r a d i o g r a p h i c a n d s i z e f r a c t i o n a t i o n m e t h o d s .
Materials and Methods
P l a n k t o n Experiments
T h e e x p e r i m e n t s w e r e c a r r i e d o u t w i t h 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 t h e m a i n b a s i n of t h e R h o d e R i v e r e s t u - ary, a n a r m of the C h e s a p e a k e Bay, f r o m M a r c h 1973 t h r o u g h F e b r u a r y 1974 (Faust and C o r r e l l , 1976). A c l e a r g l a s s a n d a black, e p o x y p a i n t m d i p p e d b o t t l e e a c h of I 1 c a p a c i t y w e r e f i l l e d w i t h p l a n k t o n t a k e n f r o m a d e p t h of I m w i t h a p e r i - s t a l t i c p u m p (Correll et al., 1975). T w o 4 ~ C i e a c h of 3 3 p - o r t h o p h o s p h a t e (car- r i e r - f r e e ) or 1 4 C - c a r b o n a t e w e r e a d d e d to e a c h b o t t l e . B o t t l e s w e r e 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. A t 15 and 30 m i n a f t e r t h e s t a r t of the ex- p e r i m e n t , 500 m l a l i q u o t s f r o m t h e b o t - t l e s w e r e r e m o v e d and f i x a t i v e w a s a d d e d
O r t h o p h o s p h a t e in s a m p l e s w h i c h h a d b e e n f i l t e r e d t h r o u g h 0 . 4 5 ~ m p o r e - s i z e m e m b r a n e s w a s a n a l y z e d c o l o r i m e t r i c a l l y b y 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 s t a n n o u s c h l o r i d e ( ~ e r i - c a n P u b l i c H e a l t h A s s o c i a t i o n , 1971).
W a t e r w a s a n a l y z e d at the b e g i n n i n g a n d end of the e x p e r i m e n t . A n a l y s e s of R h o d e R i v e r w a t e r d u r i n g t h i s 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 o r t h o - p h o s p h a t e c o n c e n t r a t i o n s f r o m a h i g h of 82 a n d 80 ~g P / I on J u n e 14 and S e p t e m - b e r 6 to a l o w of 6.5 a n d 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
F i x e d s a m p l e s w e r e t a k e n to the l a b o r a - tory, a n d c e n t r i f u g e d for 30 m i n at 2000 x g. T h e p e l l e t w a s w a s h e d w i t h 12 ml of 0 . 0 0 2 N HCI to r e m o v e a n y r a d i o t r a c e r w h i c h m a y h a v e b e e n b o u n d to the c e l l
s u r f a c e . A f t e r c e n t r i f u g i n g , t h e p e l l e t w a s w a s h e d two m o r e t i m e s w i t h 12 m l d i s t i l l e d w a t e r a n d f i n a l l y r e s u s p e n d e d in 3 m l d i s t i l l e d w a t e r . E a c h s a m p l e w a s t h e n s o n i c a t e d f o r 5 to 6 sec to b r e a k u p a n y c l u m p s of c e l l s (Model W 140 C, H e a t S y s t e m s Co., at 60 W o u t p u t ) , as d e s c r i b e d b y M a g u i r e and N e i l l (I 971).
A c i d - w a s h e d g l a s s m i c r o s c o p e s l i d e s w e r e c o a t e d w i t h O.5% a q u e o u s g e l a t i n and a i r - dried. S l i d e s w e r e p r e p a r e d f r o m sub- s a m p l e s by p l a c i n g 5 d r o p s of cell sus- p e n s i o n on e a c h of 3 m i c r o s c o p e slides.
C e l l s on the s l i d e s w e r e q u i c k l y f r o z e n on a b l o c k of d r y 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 a b o u t 6 h.
In a d a r k room, K o d a k N T B - 2 e m u l s i o n w a s d i l u t e d w i t h an e q u a l v o l u m e of d i s t i l l e d w a t e r a n d l i q u i f i e d at 40~ in a w a t e r bath. T w o d r o p s of e m u l s i o n w e r e p l a c e d on t h e end of e a c h s l i d e and the to g i v e a f i n a l c o n c e n t r a t i o n of 0 . 4 % e m u l s i o n w a s s p r e a d w i t h a s t a i n l e s s g l u t e r a l d e h y d e in O . O 1 M s o d i u m p h o s p h a t e s t e e l " r a c l e t t e " to o b t a i n a c o n s t a n t b u f f e r , 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 t h i c k n e s s t h r o u g h o u t t h e s l i d e (Bogoroch, c o n t r o l s w e r e r u n 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 a c i d (IAA) to c o n t r o l s a m p l e s (Faust a n d C o r r e l l , 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 w e r e i d e n t i - f i e d f r o m w a t e r s a m p l e s in t h e p l a n k t o n e x p e r i m e n t s .
water C h e m i s t r y
W a t e r s a m p l e s 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 u n d e r p l a n k t o n b o t t l e e x p e r i m e n t s . I n o r g a n i c c a r b o n a v a i l a b l e for u p t a k e w a s d e t e r - m i n e d b y a l k a l i n i t y t i t r a t i o n ( A m e r i c a n P u b l i c 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 R i v e r w a t e r d u r i n g t h i s s t u d y y i e l d e d e s t i m a t e s of i n o r g a n i c c a r - b o n c o n c e n t r a t i o n s r a n g i n g f r o m 11.6 to 13.3 mg C/I.
1972). T h e s l i d e s w e r e d r i e d in the d a r k at r o o m t e m p e r a t u r e (ca. 18~ and t h e n t r a n s f e r r e d to a l i g h t - p r o o f b o x a n d s t o r e d in the r e f r i g e r a t o r for 3 to 5 days. A f t e r e x p o s u r e , the e m u l s i o n w a s 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, f o l l o w e d by a 10 sec d i s t i l l e d w a t e r rinse, 5 m i n in a c i d f i x e r a n d 5 to 10 m i n w a s h i n g in r u n n i n g t a p w a t e r . A f t e r d r y i n g , t h e e m u l s i o n w a s 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 c o v e r slip (Watt, 1971). S o m e s l i d e s w e r e d i p p e d , a f t e r d e v e l o p m e n t b u t b e f o r e w a s h i n g , in 0 . 0 5 % a q u e o u s s a f r a n i n for 30 sec.
Grain C o u n t i n g
S i l v e r g r a i n c o u n t s w e r e m a d e of p h y t o - p l a n k t o n on s l i d e s p r e p a r e d b o t h f r o m
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton 295
l i g h t and d a r k b o t t l e s . W h e n e v e r p o s - sible, e n o u g h c e l l s of e a c h s p e c i e s w e r e e x a m i n e d to g i v e 30 n e t c o u n t s p e r spe- cies. S o m e s p e c i e s w e r e t o o i n f r e q u e n t , and for t h e s e s p e c i e s f e w e r t h a n 30 c o u n t s w e r e e n u m e r a t e d . G r a i n c o u n t s w e r e m a d e u n d e r o i l i m m e r s i o n , w i t h t h e m i c r o s c o p e f o c u s e d at t h e b a s e of t h e c e l l s a n d t h o s e g r a i n s in f o c u s w e r e c o u n t e d . T h e n t h e m i c r o s c o p e w a s f o c u s e d up u n t i l t h e n e x t set of g r a i n s w a s in
f o c u s a n d t h e s e c o u n t e d . T h i s p r o c e d u r e w a s r e p e a t e d u n t i l a l l g r a i n s w e r e o u t of focus. G r a i n s w i t h i n a b o u t 3 ~m of the m a r g i n of e a c h cell w e r e a l s o c o u n t e d , s i n c e g r a i n s 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 f r o m t h e c e l l m a y n o t lie di- r e c t l y o v e r the p o i n t of e m i s s i o n .
I A A - t r e a t e d s a m p l e s s h o w e d l i t t l e or no s i l v e r g r a i n s o v e r a n d a r o u n d p h y t o - p l a n k t o n c e l l s . H o w e v e r , d e n s e g r a i n a g - g 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 o v e r d e t r i t a l p a r t i c l e s .
A u t o r a d i o g r a m s s t a i n e d w i t h s a f r a n i n w e r e u s e d to e s t i m a t e m e t a b o l i c a l l y ac- t i v e b a c t e r i a . M i c r o s c o p i c f i e l d s w i t h d i s p e r s e d b a c t e r i a l c e l l s w e r e u s e d to e s t i m a t e t h e g r a i n s o v e r b a c t e r i a . B a c - t e r i a w i t h o u t g r a i n s w e r e a l s o c o u n t e d a n d t h e r a t i o of l a b e l e d to u n l a b e l e d c e l l s 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 a n d 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 w e r e h e a v i l y c o v e r e d w i t h s i l v e r g r a i n s . B e c a u s e of the u n - c e r t a i n n a t u r e of t h e s e a g g r e g a t e s , s u c h f i e l d s w e r e not i n c l u d e d in the c a l c u l a - tions.
Biomass Determination
T h e e s t i m a t i o n of c e l l n u m b e r s of p h y t o - p l a n k t o n and b a c t e r i a by the d i r e c t c o u n t p r o c e d u r e (Rodina, 1972; C a m p b e l l ,
1973) h a s b e e n d e s c r i b e d by F a u s t a n d C o r r e l l (1976). C e l l b i o m a s s of o r g a n i s m s w a s c a l c u l a t e d f r o m t h e m e a n d i a m e t e r , l e n g t h a n d d e p t h of i n d i v i d u a l c e l l s as d e s c r i b e d by R o d i n a (1972). T h e b i o m a s s p e r u n i t of w a t e r v o l u m e w a s d e t e r m i n e d .
Light and Scanning Electron Microscopy
M i c r o s c o p i c f i e l d s f r o m b o t h l i g h t - a n d d a r k - b o t t l e p l a n k t o n e x p e r i m e n t s w e r e e x a m i n e d u s i n g t h e 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 Z e i s s l i g h t m i c r o s c o p e at 1 O O O x m a g n i f i c a t i o n . P i c - t u r e s w e r e r e c o r d e d on H i g h C o n t r a s t C o p y or P a n c h r o m a t i c films.
P l a n k t o n s a m p l e s for s c a n n i n g e l e c - t r o n m i c r o s c o p y w e r e p r e p a r e d as d e - s c r i b e d by F a u s t (1974).
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 r a t e s f r o m a u t o r a d i o g r a m s w e r e 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). T h e i n i t i a l s p e c i f i c a c t i v i t y of C a n d P w a s e s t a b l i s h e d f r o m the i n o r - g a n i c C a n d o r t h o p h o s p h a t e c o n c e n t r a - t i o n s of t h e w a t e r a f t e r f i l t r a t i o n t h r o u g h a 0 . 4 5 ~un p o r e s i z e f i l t e r a n d f r o m t h e c p m / m l of w h o l e w a t e r .
T h e m e a n n u m b e r of g r a i n s per cell w a s c o r r e c t e d for 33p d e c a y to the d a y of e x p e r i m e n t . G r a i n y i e l d for 14C w a s 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 s t r i p p i n g f i l m by P e l c (1972). T h i s v a l u e w a s u s e d in our c a l c u l a t i o n s .
S i n c e 33p has a r a n g e of m a x i m u m e n e r g i e s c l o s e to t h a t of 14C, a 1.8 g r a i n y i e l d w a s a l s o u s e d on K o d a k N T B - 2 e m u l s i o n f o r 33p, w h i c h h a s a s e n s i t i v i t y s i m i l a r to t h a t of K o d a k A R - I O s t r i p p i n g f i l m
(Herz, 1959). A f t e r t h e s e c o r r e c t i o n s , the m e a n n u m b e r of g r a i n s p e r c e l l w a s c o n v e r t e d to c o u n t s p e r m i n u t e p e r l i t e r for e a c h s p e c i e s as u s e d 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 t i m e
= cpm/l.
T h e c h a n g e in c p m / l in a p h y t o p l a n k - t o n s p e c i e s for a 3 0 - m i n t i m e i n t e r v a l w a s d i v i d e d b y t h e a v e r a g e s p e c i f i c ac- t i v i t y f o r i n o r g a n i c c a r b o n or o r t h o - p h o s p h a t e . U p t a k e r a t e s w e r e e s t i m a t e d as ~g C / h 1-I and ~g P / h 1 -I of i n d i v i d - u a l a l g a l s p e c i e s . U p t a k e r a t e s w e r e a l s o e x p r e s s e d as ug P / ~ m 3 and ~g C/~un 3 of a l g a l b i o m a s s of e a c h s p e c i e s .
Results
Association of Microbial Cells
B a c t e r i a a n d 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 R h o d e R i v e r , as i l l u s - t r a t e d in the s c a n n i n g 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 a p p e a r to be j o i n e d w i t h f i b r o u s m a t e r i a l a n d w i t h s m a l l p i e c e s of n a t u r a l d e b r i s in Fig. I: I. S i m i l a r - ly, a f i l a m e n t o u s o r g a n i s m f o r m s a f i r m a t t a c h m e n t on Prorocentrum mariae-lebouriae c e l l s u r f a c e a n d on d e b r i s a d j a c e n t to the c e l l in the l o w e r 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 s a m p l e s v a r i e d in s h a p e a n d size, a n d s o m e w e r e c l o s e l y a s s o c i a t e d w i t h t h e m i c r o o r g a n i s m s , w h i l e o t h e r s w e r e not. T h e a b o v e o b s e r - v 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 a l g a e a n d b a c t e r i a
d i f f i c u l t .
296 M.A. Faust and D.L. Correll: A u t o r a d i o g r a p h y of Estuarine P l a n k t o n
Fig. i. Scanning electron micrographs (SEM) of association of m i c r o o r g a n i s m s from the Rhode River.
1: M i c r o o r g a n i s m s 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, indi- cated b y 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 p e r i p h e r y of cell are debris from natural environment; flagellar pores at anterior end of cell are
indicated by d o u b l e - a r r o w
M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton 297
Some of the d i f f i c u l t i e s w e r e over- come, however, by m i l d s o n i c a t i o n of f i x e d p l a n k t o n s a m p l e s b e f o r e c e l l s w e r e m o u n t e d o n plates. A few seconds of s o n i c a t i o n s e p a r a t e d b o t h b a c t e r i a and a l g a l c e l l s of small a g g r e g a t e s . E x a m - p l e s of b a c t e r i a in an u n s t a i n e d s a m p l e a f t e r b r i e f s o n i c a t i o n , u s i n g p h a s e con- t r a s t o p t i c s is i l l u s t r a t e d in Fig. 2.
A t t e m p t s to s e p a r a t e c e l l s in large ag- g r e g a t e s w e r e not s u c c e s s f u l . A l o n g e r time of s o n i c a t i o n d i s r u p t e d the cells;
thus, s o n i c a t i o n c o u l d not be u s e d to b r e a k u p l a r g e r a g g r e g a t e s .
P h y t o p l a n k t o n of f i x e d p l a n k t o n sam- p l e s w e r e a l s o s e p a r a t e d a f t e r a f e w s e c o n d s of s o n i c a t i o n w i t h o u t d i s r u p t i n g their c e l l 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 a p p e a r e d as s i n g l e c e l l s (Fig.2: 6,7), in p a i r s (Fig. 2: 9) and in g r o u p s of t h r e e (Fig. 2: 8) are i l l u s t r a t e d in t h e s e l i g h t m i c r o g r a p h s . C e l l s d i s p e r s e d in a m i c r o s c o p i c f i e l d c o u l d be u s e d for g r a i n c o u n t s and for t a x o n o m i c a l i d e n t i f i c a t i o n . A t the same time, p h y t o p l a n k t o n r e m a i n i n g in large a g g r e g a t e s w e r e of l i t t l e v a l u e in this study.
Au toradiography
A f e w e x a m p l e s of b a c t e r i a seen in the a u t o r a d i o g r a m s of the R h o d e R i v e r p l a n k - t o n s a m p l e s a r e i l l u s t r a t e d in Fig. 2:
4 , 5 . A n u n s t a i n e d f i l a m e n t o u s o r g a n i s m in an a u t o r a d i o g r a m is c o v e r e d w i t h grains, but the o r g a n i s m c a n n o t be d i s t i n g u i s h e d w i t h the m i c r o s c o p e u n d e r b r i g h t f i e l d o p t i c s (Fig. 2: 4). The m i c r o s c o p e is f o c u s e d o n the grains.
This a u t o r a d i o g r a m is a n u n s t a i n e d p r e p - aration, and the c e l l s are not v i s i b l e d u e to the low c o n t r a s t of the o r g a n i s m u n d e r b r i g h t - f i e l d optics. However, in p r e p a r a t i o n s s t a i n e d w i t h safranin, bac- t e r i a are v i s i b l e and l a b e l e d and un- l a b e l e d b a c t e r i a can be c l e a r l y d i s t i n - g u i s h e d (Fig. 2: 5). T h i s l a t t e r a u t o - r a d i o g r a m is f o c u s e d o n the b a c t e r i a ,
A u t o r a d i o g r a m s of a l g a l c e l l s w i t h silver g r a i n s c o n c e n t r a t e d a r o u n d and on the c e l l s i n d i c a t e a c t i v e l y m e t a b o l i z i n g o r g a n i s m s . G r a i n s w i t h i n a b o u t 3 ~ m of the m a r g i n of e a c h c e l l w e r e c o n s i d e r e d to be d u e to r a d i o a c t i v i t y e m i t t e d f r o m the c e l l (Fig. 2: 7 , 8 ) . A 1 4 C - a u t o r a d i o - g r a m of Gyrodinium dominans w i t h g r a i n s a b o v e and a r o u n d the p e r i p h e r y of the cell is e v i d e n t in Fig. 2: 7. 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 p h y t o - p l a n k t o n s p e c i e s is i l l u s t r a t e d in Fig.
2: 8. N u m e r o u s silver g r a i n s are a b o v e a Peridinium s p e c i e s l o c a t e d in the center, none are f o u n d a b o v e Prorocentrum mariae- lebouriae, and o n l y a f e w g r a i n s are a b o v e a Euglenoid species (arrow). Q u i t e often, p h y t o p l a n k t o n i n c u b a t e d in the d a r k ap- p e a r e d to h a v e 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 s at a g r e a t e r d i s t a n c e t h a n 3 ~m. A n e x a m p l e of a 1 4 C - a u t o r a d i o g r a m of G. estuariale i n c u b a t e d in the d a r k s h o w i n g n u m e r o u s g r a i n s is s h o w n in Fig.
2: 6.
A large n u m b e r of g r a i n s w e r e c o n c e n - t r a t e d o n and a r o u n d Gyrodinium dominans c e l l s w i t h b o t h t r a c e r 14C and 33p in the light and d a r k b o t t l e s (Fig. 2: 9).
The g r a i n s w e r e l o c a t e d w i t h i n an a v e r a g e d i a m e t e r of 25 ~ m a r o u n d cells, and
their a p p e a r a n c e was v e r y d i s t i n c t f r o m the b a c k g r o u n d of the a u t o r a d i o g r a m . This o r g a n i s m w a s the o n l y p h y t o p l a n k t o n ex- h i b i t i n g s u c h a g r a i n pattern. T h e rea- son for this u n u s u a l g r a i n p a t t e r n w a s n o t r e a d i l y a p p a r e n t .
A u t o r a d i o g r a m s w e r e also p r e p a r e d of c o n t r o l p l a n k t o n s a m p l e s w h i c h had b e e n e x p o s e d to the m e t a b o l i c i n h i b i t o r IAA.
T h e n u m b e r of g r a i n s a b o v e the m i c r o - o r g a n i s m s w e r e g r e a t l y r e d u c e d w h e n IAA was added. However, g r a i n s c o v e r i n g nat- u r a l d e b r i s w e r e o f t e n o b s e r v e d in the p r e p a r a t i o n s . U n m e t a b o l i z e d or a d s o r b e d r a d i o t r a c e r s w e r e w a s h e d out w i t h m i l d a c i d and r i n s e d t w i c e w i t h d i s t i l l e d w a - ter from the samples. T h i s p r o d u c e d low b a c k g r o u n d on the r a d i o a u t o g r a m s . It is p o s s i b l e t h a t w a s h i n g was i n a d e q u a t e to and the g r a i n s are s o m e w h a t out of focus, free r a d i o t r a c e r s f r o m n a t u r a l debris.
T h e r e f o r e , s t a i n i n g of the a u t o r a d i o g r a m s T h e r e f o r e , g r a i n s over c l u m p s of n a t u r a l was n e c e s s a r y to e n h a n c e b a c t e r i a l m o r -
p h o l o g y u n d e r the a u t o r a d i o g r a p h i c e m u l - sion. S t a i n e d a u t o r a d i o g r a m s w e r e exam- ined w i t h b r i g h t - f i e l d o p t i c s to o b s e r v e g r a i n - c o n t a i n i n g b a c t e r i a . In s u c h
fields, s i l v e r g r a i n s a p p e a r as b l a c k d o t s and u n d e r n e a t h the g r a i n s b a c t e r i a a r e v i s i b l e .
R e p r e s e n t a t i v e a u t o r a d i o g r a m s of p h y t o p l a n k t o n a r e i l l u s t r a t e d in Fig. 2:
6 - 9 . T h e p h y t o p l a n k t o n s h a p e and o t h e r t a x o n o m i c a l f e a t u r e s a r e r e c o g n i z a b l e u n d e r the e m u l s i o n , h o w e v e r the r e s o l u - t i o n of the c e l l u l a r f e a t u r e s is some- w h a t lowered.
d e b r i s w e r e n o t counted.
Estimation of Carbon and Phosphorus Uptake by Phytoplankton
I n c o r p o r a t i o n of 1 4 C - c a r b o n a t e and 33p_
o r t h o p h o s p h a t e into p h y t o p l a n k t o n was e s t i m a t e d as g r a i n s per cell of m e t a b o l - i c a l l y a c t i v e s p e c i e s d u r i n g the four s e a s o n s of the year. U p t a k e of b o t h t r a c e r s c o m p u t e d as g r a i n s p e r c e l l v a r i e d s e a s o n a l l y and f r o m s p e c i e s to s p e c i e s (Table I). A large n u m b e r of g r a i n s w e r e f o u n d o n l y in the d i n o f l a g e l -
298 M.A. Faust and D.L. Correll: A u t o r a d i o g r a p h y of Estuarine Plankton
M . A . F a u s t a n d D . L . C o r r e l l : A u t o r a d i o g r a p h y o f E s t u a r i n e P l a n k t o n 299
T a b l e i. R e l a t i v e c a r b o n (C) a n d p h o s p h o r u s (P) u p t a k e b y p h y t o p l a n k t o n i n R h o d e R i v e r a t d e p t h o f i m, e s t i m a t e d a s g r a i n s p e r c e l l . -: a b s e n t
O r g a n i s m S i z e D a t e ( 1 9 7 3 - 1 9 7 4 )
(um) J u n e 14 S e p t e m b e r 6 N o v e m b e r 26 F e b r u a r y 2 8
B i o - G r a i n s B i o - G r a i n s B i o - G r a i n s B i o - G r a i n s m a s s ( n o . / c e l l ) m a s s ( n o . / c e l l ) m a s s ( n o . / c e l l ) m a s s ( n o . / c e l l )
(ram3/ C P (ram3~ C P (ram3/ C P (mm3/ C P
i) i) i) i)
Chlamydomonas sp. 1 3 x i 0 . . . . . . . . . 0 . O 1 4 . 3 1 . 8 Chlorella sp. 5 X 5 - - - 0 . 0 2 1 1 . 9 9 . 3 - - - 0 . 0 0 2 -
Cryptomonas s p . l 1 5 x 1 2 - - - 0 . 0 2 . . . . . . 0 . 0 4 1 1 . 6 3 . 0 Cryptomonas S p . 2 1 3 x l O . . . . . . . . 0 . 0 5 7 . 8 7 . 8 Diplosalis sp. 1 5 x 1 5 - - - 0 . 0 4 4.1 3 . 8 . . . . . .
Euglena sp. 5 x 5 5 - - 0 . 0 2 5 . 4 6.1 - - - 0 . 0 5 9 . 9 2 . 7
F l a g e l l a t e 1 5 x 1 2 . . . . . . . . 0 . O 5 9 . 9 2 . 7
Gymnodinium
aurenticum 1 3 x 7 - - - O . O 1 8 . 8 4 . 5 . . . . . Gymnodinium s p . l 1 2 x 8 0 . 0 8 9.2 8 . 2 0 . 1 4 1 4 . 0 1 2 . 5 . . . . .
Gymnodinium s p . 2 1 5 x 1 3 . . . . . . . . 0 . 0 3 9 . 6 11.2 Gyrodinium
dominans 1 5 x 1 2 O . 1 8 3 0 . 6 3 0 . 0 . . . . . . . . .
G. estuariale 1 2 x i O 0 . 0 3 - - 0 . 0 9 7 . 0 6 . 6 0 . 1 0 1 6 . 7 1 5 . 7 - - - Katodinium
rotundatum f O x 6 . . . . . . 0 . 0 5 1 9 . 0 1 7 . 7 - - -
N a n n o p l a n k t o n i O x 5 . . . . . 0 . 0 2 18.1 16.1 - - -
Pavlova gyrans 1 6 x 6 . . . . . . 0 . 0 4 1 4 . 5 12.1 - - - Peridinium sp. 1 7 x 1 3 0 . 0 2 3 0 . 5 1 8 . 2 . . . . . . . . .
Prorocentrum mariae-lebou-
riae 1 6 x 2 0 0 . 3 5 2 5 . 8 2 1 . 8 0 . 0 8 9 . 9 4 . 0 . . . . . . Pseudopedinella
pyriforme 5x3 . . . . . . . . . 0 . 0 5 3 . 0 2.1
Tetraselmis
gracilis 1 5 x 1 2 . . . . . . . . . 0 . 1 0 0 1 3 . 5 11.7
% b i o m a s s o f g r a i n - c o n -
t a i n i n g c e l l s 3 7 . 2 4 1 . 9 2 8 . 0 3 6 . 3
( F i g . 2. L i g h t m i c r o g r a p h s o f b a c t e r i a a n d p h y t o p l a n k t o n f r o m R h o d e R i v e r e x a m i n e d w i t h p h a s e - a n d b r i g h t - f i e l d o p t i c s (all x 1 5 0 0 ) . 3: R o d - s h a p e d b a c t e r i a i n p a i r s ( d o u b l e 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 ( s i n g l e 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 3 3 p a u t o - r a d i o g r a m o f 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 i n 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 i n f o c u s i n p h o t o m i c r o g r a p h o f a 3 3 p - a u t o r a d i o g r a m ( b r i g h t - f i e l d o p t i c s ) ; l a r g e b a c t e r i a l f i l a m e n t ( s i n g l e b l a c k a r r o w ) a n d s e v e r a l s m a l l b a c t e r i a l a g g r e g a t e s ( s i n g l e w h i t e - e d g e d b l a c k a r r o w s ) w i t h o u t g r a i n s a r e s h o w n ; o t h e r b a c - t e r i a i n a g g r e g a t e s ( d o u b l e b l a c k a r r o w ) h a v e g r a i n s a b o v e t h e m , 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 o f f o c u s . N o t e c o n t r a s t i n d e t e c t a b i l i t y o f c e l l 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 o f 1 4 C - a u t o r a d i o g r a m o f p l a n k t o n s a m p l e i n c u b a t e d i n t h e d a r k ; Gyrodi- nium 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 t h e c e l l ( a r r o w s ) . (In i l l u s t r a t i o n s 6 a n d 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 a s a l g a l u p t a k e o f 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 o f p h y t o p l a n k t o n . ) 7: Gyrodinium dominans w i t h s o m e 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 i s e v i d e n t i n t h e 1 4 C - a u t o r a d i o g r a m . G r a i n s a r e w i t h i n 3 ~ m o f c e l l p e r i p h e r y a n d a r e u s e d i n e s t i m a t i n g g r a i n s p e r c e l l . 8: S e l e c t i v e u p t a k e o f 3 3 p - o r t h o p h o s p h a t e b y p h y t o p l a n k t o n ; 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 i n t h e c e n t e r , n o n e a r e 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 ( a r r o w ) . 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 i n 3 3 p - a u t o r a d i o g r a m a r e s h o w n , u s i n g p h a s e - c o n t r a s t o p t i c s ; l a r g e n u m b e r o f g r a i n s i s a c c u m u l a t e d o n 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 h a l o , i n d i s c r i m i n a t e d l y , i n 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 i n l i g h t a n d d a r k b o t t l e s
300 M.A. Faust and D.L. Correll: Autoradiography of Estuarine Plankton
late s p e c i e s Gymnodinium sp. I, Gyrodinium dominans, Peridinium sp. and Prorocentrum mariae-!ebouriae in June. Thus, t h e s e s p e c i e s w e r e c o n s i d e r e d the m o s t m e t a - b o l i c a l l y a c t i v e a l g a e at this time of the year. The b i o m a s s of g r a i n - c o n t a i n i n g a l g a l s p e c i e s was o n l y 37.2% of the to- tal a l g a l b i o m a s s in J u n e (Table I).
G r a i n s per c e l l of the same algal s p e c i e s v a r i e d seasonally. The h i g h num- ber of g r a i n s per Prorocentrum mariae-
lebouriae cell in J u n e d e c l i n e d s e v e r a l fold to 10 g r a i n s per cell of C and 4 g r a i n s per c e l l of P in S e p t e m b e r (Table I). In contrast, i n c o r p o r a t i o n of the t r a c e r s i n c r e a s e d f r o m 9 to 14 g r a i n s p e r cell for C and 8 to 12 g r a i n s per cell for P in Gymnodinium sp. I from J u n e to S e p t e m b e r .
P h y t o p l a n k t o n c a r b o n and p h o s p h o r u s u p t a k e d u r i n g the s u m m e r was due to d i n o f l a g e l l a t e s and to other s p e c i e s d u r i n g spring, fall and w i n t e r seasons
(Table I). In S e p t e m b e r , 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 n u m b e r of g r a i n s w e r e f o u n d in Chlorella sp., Cryptomonas
sp. I , Katodinium rotundatum, n a n n o p l a n k t o n , and Tetraselmis gracilis. All a c t i v e l y m e t a b o l i z i n g c e l l s w e r e less t h a n 20 ~ m
in size and o n l y 28 to 42% of the t o t a l algal b i o m a s s w e r e m e t a b o l i c a l l y a c t i v e at any g i v e n s e a s o n (Table I).
T h e t o t a l b i o m a s s of p h y t o p l a n k t o n
bon u p t a k e was c o m p a r a t i v e l y low (41.2 and 51.1 ~ g C / l h-1 x 10 -2 ) in J u n e and S e p t e m b e r and h i g h (393.4 and 170.2 ugC/l h-1 x 10 -2 ) in N o v e m b e r and F e b r u a r y , r e s p e c t i v e l y . P h o s p h o r u s u p t a k e r a t e s w e r e 60.0 and 50.9 ~ g P / l h -I x 10 -2 in J u n e and S e p t e m b e r and 66.36 and 2.42 u g P / l h -I x 10 -2 in N o v e m b e r and
F e b r u a r y . No p e r s i s t e n t r e l a t i o n s h i p was o b s e r v e d b e t w e e n C and P u p t a k e r a t e s per v o l u m e of w a t e r (Table 3) and t o t a l a l g a l b i o m a s s (Table 2). It m u s t be n o t e d that o n l y a b o u t o n e - t h i r d of the t o t a l b i o m a s s of a l g a e was a c t i v e at a n y g i v e n time.
C a r b o n and p h o s p h o r u s u p t a k e r a t e s per u n i t of cell v o l u m e w e r e h i g h e r for the s m a l l e r t h a n for the larger s p e c i e s
(Table 4). Pseudopedinella pyriforme, the s m a l l e s t f l a g e l l a t e in the samples, had the h i g h e s t u p t a k e r a t e s for b o t h C and P per unit of cell v o l u m e (817 ~ g C / ~ m 3 h -I x 10 -10 and 5.9 ~ g P / ~ m 3 h -I x 10 -10 in F e b r u a r y ) , f o l l o w e d b y a n a n n o p l a n k - ton (366.8 ~ g C / ~ m 3 h-1 x 10-10 a n d 7.5
~gP/~/n 3 h -I x 10 -10 in N o v e m b e r ) ; w h i l e the t h i r d h i g h e s t r a t e was b y a Chlorella sp. (190.7 ~ g C / ~ m 3 h -I x 10 -]0 and 15.2
~ g P / ~ m 3 h-1 x 10-10 in S e p t e m b e r ) . C a r - b o n and P u p t a k e per c e l l v o l u m e was m u c h lower for the l a r g e r 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 / v a r i e d f r o m 0.5 to 1.7 m m 3 / l d u r i n g the ~m3 h-1 x 10-10) and Gymnodinium sp.1 seasons (Table 2). The b i o m a s s of v a r i o u s (11.5 to 24.1 ~ g C / ~ m 3 h-1 x 10-10 and a l g a l c l a s s e s was a l s o e s t i m a t e d . D i n o - O.18 to 0.22 ~ g P / ~ m 3 h-1 x 10-10) in f l a g e l l a t e s r e p r e s e n t e d 69 to 80% of the
total a l g a l b i o m a s s in June, S e p t e m b e r and N o v e m b e r ; w h e r e a s o r g a n i s m s of the C r y p t o p h y c e a e , B a c i l l a r i o p h y c e a e , C r y s o - phyceae, f l a g e l l a t e s , and n a n n o p l a n k t o n e a c h m a d e u p 18 to 24% of the t o t a l al- gal b i o m a s s in F e b r u a r y . In F e b r u a r y the d i n o f l a g e l l a t e b i o m a s s d e c l i n e d to 5% of total a l g a l biomass.
R a t e s of c a r b o n and p h o s p h o r u s u p t a k e for all p h y t o p l a n k t o n s p e c i e s are pre- s e n t e d in T a b l e 3. The c o n t r i b u t i o n of a l g a l s p e c i e s to the u p t a k e of C and P per v o l u m e of w a t e r c h a n g e d seasonally.
U p t a k e of C b y Gymnodinium sp.1 v a r i e d f r o m 8.8 ~ g / l h-1 x 10-2 in J u n e and 33 ~ g / l h -I x 10 -2 in S e p t e m b e r to none in N o v e m b e r and F e b r u a r y . The P u p t a k e b y t h i s a l g a f o l l o w e d a s i m i l a r course.
In c o n t r a s t , Prorooentrum mariae-!ebouriae C and P u p t a k e r a t e s w e r e h i g h e r in J u n e and lower in S e p t e m b e r . The h i g h e s t ob- s e r v e d u p t a k e r a t e s for C and P w e r e
J u n e and S e p t e m b e r , r e s p e c t i v e l y . The b i o m a s s of P. mariae-lebouriae was the
l a r g e s t of a n y s p e c i e s m e a s u r e d d u r i n g the e x p e r i m e n t a l period, yet C and P up- take per u n i t of cell v o l u m e was 160 and 30 t i m e s less t h a n t h a t e s t i m a t e d for Pseudopedinel!a pyriforme. T h e u p t a k e m a y h a v e b e e n g o v e r n e d b y the s u r f a c e : v o l u m e r a t i o in the v a r i o u s p l a n k t o n size
classes.
T h e r e l a t i o n s h i p C and P u p t a k e per u n i t of c e l l v o l u m e v a r i e d b e t w e e n algal
s p e c i e s (Table 4). The r a t i o of C to P u p t a k e per ~m3 r a n g e d f r o m 10: I for Diplo-
salis sp. , Gynmodinium aurenticum and Peri- dinium sp. ; and 50: I for Katodinium rotun- datum, n a n n o p l a n k t o n , and Pavlova gyrans, to a b o v e IOO : I for Gymnodinium sp. 1 and Pseudopedinella pyriforme.
Autoradiography of Bacteria
e x h i b i t e d by Katodinium rotundatum (145 ~ g C / M e t a b o l i c a l l y a c t i v e b a c t e r i a l p o p u l a - 1 h-1 x 10-2 and 31 ~ g P / l h -I x 10 -2 ) tions f r o m light and d a r k b o t t l e s w e r e and b y Pavlova gyrans (79.5 ~ g C / l h-1 x d e t e c t e d in s t a i n e d 3 3 P - a u t o r a d i o g r a m s . 10-2 and O.16 ~ g P / l h-1 x 10-2) in N o v e m - The p e r c e n t a g e s of l a b e l e d and u n l a b e l e d
ber. b a c t e r i a in the a u t o r a d i o g r a m s w e r e
T o t a l C and P u p t a k e per v o l u m e of e s t i m a t e d four times a y e a r (Table 5).
w a t e r w a s a l s o e s t i m a t e d (Table 3). C a r - The d i s t r i b u t i o n of l a b e l e d b a c t e r i a
M . A . F a u s t a n d D . L . C o r r e l l : A u t o r a d i o g r a p h y o f E s t u a r i n e P l a n k t o n 301
T a b l e 2. B i o m a s s o f v a r i o u s a l g a l c l a s s e s a n d t h e i r s e a s o n a l o c c u r - r e n c e i n R h o d e R i v e r a t d e p t h o f 1 m . V a l u e s a s % o f t o t a l b i o m a s s
A l g a l c l a s s e s D a t e ( 1 9 7 3 - 1 9 7 4 )
J u n e 1 4 S e p t e m b e r 6 N o v e m b e r 2 6 F e b r u a r y 2 8
D i n o p h y e e a e 7 9 . 9 7 2 . 0 6 9 . 1 4 . 9
C h r y s o p h y c e a e 2 . 5 5 . 7 3 . 4 1 0 . 4
P r a s i n o p h y c e a e 9 . 7 1 . 4
C h l o r o p h y c e a e i . i 8 . 1 - 1 . 8
B a c i l l a r i o p h y c e a e 1 5 . 9 0 . 7 4 . 7 1 8 . 3
C r y p t o p h y c e a e - 0 . 9 3 . 7 2 3 . 7
E u g l e n o p h y c e a e - 1 . 0 - -
H a p t o p h y c e a e - - 4 . 7 3 . 0
F l a g e l l a t e s - 4 . 7 - 1 8 . 2
N a n n o p l a n k t o n 0. i 6 . 9 4 . 7 1 8 . 3
T o t a l b i o m a s s ( m m 3 / l ) 1 . 7 0 . 9 i . i 0 . 5
T a b l e 3. C a r b o n (C) a n d p h o s p h o r u s (P) u p t a k e r a t e s ( u g l - I h - I x 1 0 - 2 ) o f p h y t o - p l a n k t o n i n R h o d e R i v e r a t d e p t h o f i m
O r g a n i s m S i z e D a t e ( 1 9 7 3 - 1 9 7 4 )
(um) J u n e 1 4
C P
S e p t e m b e r 6 N o v e m b e r 2 6 F e b r u a r y 2 8
C P C P C P
Chlamydomonas s p . 1 3 x l O - - -
Chlorella s p . 5 x 5 - 7 . 2
Cryptomonas s p . l 1 5 x 1 2 - - -
Cryptomonas s p . 2 1 3 x l O - - -
Diplosalis s p . 1 5 x 1 5 - - i . O
Euglena s p . 5 x 5 5 - - 0 . 8
F l a g e l l a t e 1 5 x 1 2 - - -
Gymnodinium s p . l 1 2 x 8 8 . 8 1 4 . O 3 2 . 9 3 0 . 0
Gymnodinium s p . 2 1 5 x 1 3 - - -
Gymnodinium aurenticum 1 3 x 7 - - i . i
Gyrodinium dominans 1 5 x 1 2 8 . 8 1 5 . O -
G. estuariale 1 2 x l O - - 7 . 1
Katodinium rotundatum 1 O x 6 - - -
N a n n o p l a n k t o n i O x 5 - -
Pavlova gyrans i O x 6 - - -
Peridinium s p . 1 7 x 1 3 6 . 4 7 . 0 -
Prorocentrum mariae-
lebouriae 1 6 x 2 0 1 7 . 2 2 4 . 0
Pseudopedinella
pyriforme 5 x 3 - - -
Tetraselmis gracilis 1 5 x 1 2 - - -
- - 9 . 4 O . 0 4
6 . 0 . . . .
- - - 2 5 . 5 0 . 0 7
- - 3 8 . O O . 4 O
0 . 9 . . . .
0 . 9 - - -
- - - 4 4 . 4 O . 1 2
- - 1 2 . 8 1 . 5 0
0 . 8 . . . .
6 . 8 6 8 . 4 1 4 . 8 - -
- 1 4 5 . O 3 1 . 2 1 - -
- 7 4 . 8 1 5 . i O - -
- 7 9 . 5 O . 1 6 - -
1 . O 4 . 6
- - 4 0 . I O . 2 9
2 5 . 7 5 . 1 5 - -
T o t a l u p t a k e
( u g l - I h - I x 1 0 - 2 ) 4 1 . 2 6 0 . 0 5 1 . 1 0 5 0 . 9 3 9 3 . 4 6 6 . 3 6 1 7 0 . 2 2 . 4 2
302 M . A . F a u s t a n d D . L . C o r r e l l : A u t o r a d i o g r a p h y o f E s t u a r i n e P l a n k t o n
T a b l e 4. C a r b o n (C) a n d p h o s p h o r u s (P) u p t a k e (ug u m 3 h -I x 10 - 1 0 ) o f p h y t o p l a n k - t o n p e r u n i t b i o m a s s i n R h o d e R i v e r a t d e p t h o f i m
O r g a n i s m S i z e
(um)
D a t e ( 1 9 7 3 - 1 9 7 4 )
J u n e 14 S e p t e m b e r 6 N o v e m b e r 2 6 F e b r u a r y 28
C P C P C P C P
Chlamydomonas sp. 1 3 x l O . . . . . . 1 6 0 . 8 6 . 9 4
Chlorella sp. 5 x 5 - - 1 9 0 . 7 6 1 5 . 2 0 . . . .
Cryptomonas s p . l 1 5 x 1 2 . . . . . . 6 5 . 4 O . 1 7
Cryptomonas s p . 2 1 3 x i 0 . . . . . . 7 3 . 0 0 . 7 5
Diplosalis sp. 1 5 x 1 5 - - 2 . 4 2 0 . 2 3 . . . .
Euglena sp. 5 x 5 5 - - 5 . 1 8 0 . 0 5 . . . .
F l a g e l l a t e 1 5 x 1 2 . . . . . . 5 5 . 7 1 . 5 0
Gymnodinium aurenticum 1 3 x 7 - - 1 8 . 3 2 O . 1 6 . . . .
Gymnodinium s p . 1 1 2 x 8 1 1 . 5 0 . 1 8 2 4 . 1 0 0 . 2 2 . . . .
Gymnodinium s p . 2 1 5 x 1 3 . . . . . . 4 6 . 1 0 . 5 5
Gyrodinium dominans 1 5 x 1 2 4 . 9 0 . 8 4 . . . . . .
G. estuariale 1 2 x l O - - 0 . 0 7 O . 1 6 7 0 . 4 1 . 5 2 - -
Katodinium rotundatum I 0 x 6 . . . . 1 6 4 . 8 3 . 5 3 - -
N a n n o p l a n k t o n i O x 5 . . . . 3 6 6 . 8 7 . 5 5 - -
Pavlova gyrans 1 6 x 6 . . . . 1 2 7 . 5 2 . 4 2 - -
Peridinium sp. 1 7 x 1 3 1 2 . 7 O . 1 3 . . . . . .
Prorocentrum mariae-
lebouriae 1 6 x 2 0 4 . 9 0 . 2 0 2 . 5 6 0 . 1 5 . . . .
Pseudopedinella
pyriforme 5 x 3 . . . . . . 8 1 7 . 1 5 . 9 1
Tetraselmis gracilis 1 5 x 1 2 . . . . 3 1 . 6 0 . 6 2 - -
T a b l e 5. P r o p o r t i o n s o f 3 3 p - o r t h o p h o s p h a t e m e t a b o l i z i n g b a c t e r i a a t v a r i o u s s e a s o n s i n R h o d e R i v e r a t d e p t h o f i m
D a t e T r e a t m e n t a N o . b a c t e r i a / f i e l d % l a b e l e d L a b e l e d : C e l l s / m l B i o m a s s ( 1 9 7 3 - 1 9 7 4 ) L a b e l e d U n l a b e l e d • S D b U n l a b e l e d x 1 0 6 ( m m 3 / l
J u n e 14 L 3 3 . 3 2 0 . 3 6 3 + 3.7 1 . 6 4 0 . 9 4 . 9
D 3 4 . 1 1 9 . 8 63 + 4 . 5 1 . 7 2
S e p t e m b e r 6 L 3 2 . 7 1 3 . 8 7 0 + 3.5 2 . 3 6 2 . 5 3 . 4
D 3 0 . 1 1 7 . O 63 +- 4.2 1.77
N o v e m b e r 26 L 4 7 . 8 7 . 4 8 5 + 5.1 6 . 4 5 3.1 5.1
D 4 6 . 1 7 . 6 8 4 -+ 5 . 0 6 . 0 6
F e b r u a r y 28 L 6 5 . 8 14.1 81 + 4 . 4 4 . 6 6 2 . 6 1.6
D 5 5 . 4 1 2 . 0 82 -+ 3 . 7 4 . 6 1
a
L: L i g h t b o t t l e ; D: d a r k b o t t l e . b S D : S t a n d a r d d e v i a t i o n .
