H E L G O I ~ D E R MEERESUNTERSUCHUNGEN
Helgol~nder Meeresunters. 33, 36-46 (1980)
Low-salinity stress in the American lobster, Homarus
americanus, after chronic sublethal e x p o s u r e to
cadmium: Biochemical effects
E. Gould
National Marine Fisheries Service, Northeast Fisheries Center;
Milford Laboratory, Milford, Connecticut 06460, USA
ABSTRACT: Lobsters (Homarus americanus) were exposed to cadmium (6/~g 1-1, 30 days) in flowing
seawater, then held for 7 days in aerated "clean" seawater at either ambient (27 %0) or low (17 %0)
salinity. Cadmium exposure alone (ambient salinity) induced a general elevation of enzyme activity
(heart, antennal gland, and muscle MDH; heart LDH and GPI), despite the probability of some
clearance of cadmium from body tissues during the "clean" seawater holding period. Low-salinity
alone (non-exposed lobsters) caused a decrease of enzyme activity (AAT, LDH, GPI, PK) in most
tissues examined, except for tail muscle IDH, file activity of which was increased, and MDH, which
was significantly elevated above ambient controls in all tissues except heart. Most low-salinity
effects were observed in tail muscle, and most cadmium effects, in heart; low-salinity effects
outnumbered cadmium stress by nine to four. In heart and tail muscle of cadmium-exposed lobsters
held at low salinity, each of the two stresses apparently operated to nullify the other's effects. The
most prominent single biochemical response to these sublethal stresses was the elevation of MDH
activity. The ratio MDH: LDH gave the clearest indication of overall relative stress.
INTRODUCTION
For the b e t t e r part of the past d e c a d e , w e h a v e e x a m i n e d in our laboratory the
p h y s i o l o g i c a l effects of h e a v y m e t a l s on m a r i n e an i m al s (Calabrese et al., in press).
Short-term bioassay w o r k l e d to l o n g - t e r m s u b l e t h a l e x p o s u r e s to w h i c h has b e e n added,
in r e c e n t years, s u b s e q u e n t c h a l l e n g e by a n at u r al variable. Th e p u r p o s e is to discover
h o w s u b l e t h a l m e t a l e x p o s u r e affects an a n i m a l ' s capacity to a d a p t to e n v i r o n m e n t a l
c h a n g e . This report d e s c r ib e s such a n e x p e r i m e n t : a 1-month e x p o s u r e of A m e r i c a n
lobsters, Homarus americanus, to s u b l e t h a l c a d m i u m (6 /~g 1-1), f o l l o w e d by a 7-day
h o l d i n g p e r i o d in s e a w a t e r of e i t h e r a m b i e n t or low salinity. At v e r y l o w concentrations,
c a d m i u m can alter m e t a b o l i c patterns in adult m a r i n e a n i m a l s (Calabrese et al., 1977),
i n c l u d i n g the lobster, a n d also strongly modifies, in Mytilus edulis larvae, life functions
that are s a l i n i t y - d e p e n d e n t ( L e h n b e r g & T h e e d e , 1979).
Th e lobster w a s s e l e c t e d as an o s m o c o n f o r m e r that adapts only slowly to c h a n g i n g
ionic concentrations. It will, h o w e v e r , s u r v iv e an abrupt drop in salinity w i t h i n its
t o l e r a n c e r a n g e (Dall, 1970). Milford H a r b o r water, in w h i c h the test a n i m a l s w e r e
accl i m at ed , h a d an a v e r a g e salinity of 27 %o at the time of the e x p e r i m e n t ; for the lowsalinity c h a l l e n g e , therefore, w e s e l e c t e d 17 %0 as b e i n g stressful b u t w e l l w i t h i n the
l e t h a l limits set for this a n i m a l (McLeese, 1956) in fully o x y g e n a t e d s e a w a t e r at 20 ~
9 Biologische Anstalt Helgoland
0017-9957/80/0033/0036/$ 02.00
L o w - s a l i n i t y stress
37
M e t a b o l i c r e s p o n s e in a n i m a l tissue often d e p e n d s u p o n e n v i r o n m e n t a l salinity. As
M u n d a y & Poat (1971) h a v e p o i n t e d out, " . . . not o n l y do tissues from the s a m e s p e c i e s
r e a c t differently to osmotic variation, b u t . . , effects on one tissue m a y often m a s k the
effect on others". Four different tissues, therefore, w e r e e x a m i n e d for b i o c h e m i c a l
c h a n g e in this study: h e a r t a n d tail muscle, b e c a u s e m u s c l e a m i n o a c i d s d e c r e a s e in
o s m o c o n f o r m e r s at low s a l i n i t y (Prosser, 1973)i the a n t e n n a l g l a n d , b e c a u s e of its
r e g u l a t o r y function in ionic b a l a n c e ; a n d the m a l e gonad, b e c a u s e of the p o s s i b i l i t y that
it m a y b e b u f f e r e d from the m e t a b o l i c d e m a n d s of g e n e r a l stress, as Bayne has o b s e r v e d
to b e the case w i t h Mytilus edu]is (cited in Livingstone, 1975).
E n z y m e s s e l e c t e d for m o n i t o r i n g in t h e s e tissues w e r e almost e n t i r e l y those of
glycolysis, as our p r i m a r y i n t e r e s t w a s in e n e r g y e x p e n d i t u r e . Pyruvate k i n a s e (PK; E.C.
2.7.1.40) a n d g l u c o s e p h o s p h a t e i s o m e r a s e (GPI; E.C. 5.3.1.9) s t a n d at e i t h e r e n d of the
E m b d e n - M e y e r h o f p a t h w a y of a n a e r o b i c glycolysis; g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (G6PDH; E.C. 1.1.1.49) is the m a g n e s i u m - m o d u l a t e d r e g u l a t o r of p e n t o s e shunt
activity; m a l a t e d e h y d r o g e n a s e (MDH; E.C. 1.1.1.37) a n d l a c t a t e d e h y d r o g e n a s e (LDH;
E.C. 1.1.1.27) are closely i n v o l v e d in c e l l u l a r r e d o x r e g u l a t i o n , p r o v i d i n g t h e o x i d i z e d
c o e n z y m e that is e s s e n t i a l for c o n t i n u i n g glycolysis; a n d N A D P - l i n k e d isocitrate d e h y d r o g e n a s e (IDH; E . C . I . i . I . 4 2 ) r e p r e s e n t s the citric a c i d cycle. T r a n s a m i n a t i o n , the most
i m p o r t a n t m e a n s of i n i t i a t i n g a m i n o a c i d c a t a b o l i s m (Banks et al., 1976), w a s m o n i t o r e d
in the l o b s t e r heart, u s i n g a s p a r t a t e a m i n o t r a n s f e r a s e (AAT; E.C. 2.6.1.1.).
MATERIALS A N D M E T H O D S
A n i m a 1 e x p o s u r e. Lobsters w e r e c o l l e c t e d b y trap from the w a t e r s of Long
I s l a n d S o u n d n e a r Milford, C o n n e c t i c u t (USA), a n d a c c l i m a t e d in the l a b o r a t o r y in
flowing, s a n d - f i l t e r e d s e a w a t e r for one to two w e e k s before e x p e r i m e n t a l exposure.
T h e y w e r e fed c h o p p e d c l a m s (Spisula solidissima) d a i l y d u r i n g a c c l i m a t i o n a n d
t h r o u g h o u t the e x p o s u r e period. Test a n i m a l s w e r e e x p o s e d to c a d m i u m (6/~g 1-1) as
c a d m i u m c h l o r i d e in a f l o w - t h r o u g h s y s t e m as d e s c r i b e d b y T h u r b e r g et al. (1977).
S i x t e e n lobsters s e r v e d as controls (4 a n i m a l s p e r 285-1 f i b e r g l a s s tank) a n d the s a m e
n u m b e r for c a d m i u m e x p o s u r e in e a c h of the d u p l i c a t e e x p e r i m e n t a l series. S a l i n i t y a n d
t e m p e r a t u r e r a n g e s d u r i n g the 3 0 - d a y e x p o s u r e p e r i o d s w e r e 25.9-27.5 %0 a n d 11.2-18.2
~ r e s p e c t i v e l y , for the first series, a n d 25.7-28.5 %0 a n d 14.0-23.2 ~ for the second.
T e m p e r a t u r e r a n g e d u r i n g b o t h 7 - d a y h o l d i n g periods, at e i t h e r a m b i e n t or low salinity,
was 18-23 ~.
At the e n d of the first e x p e r i m e n t a l exposure, the control lobsters w e r e r e m o v e d in
t h r e e lots over t h r e e consecutive d a y s (29, 30, a n d 31 days' exposure), c o l o r - c o d e d w i t h
r u b b e r b a n d s to d e s i g n a t e the d a y of removal, a n d p l a c e d in a l a r g e f i b e r g l a s s h o l d i n g
t a n k c o n t a i n i n g a e r a t e d w a t e r at low s a l i n i t y (17 %0). The c a d m i u m - e x p o s e d lobsters
w e r e r e m o v e d in l i k e fashion a n d p l a c e d in a n o t h e r l a r g e t a n k w i t h a e r a t e d , l o w - s a l i n i t y
s e a w a t e r . A n i m a l s from the s e c o n d e x p e r i m e n t a l e x p o s u r e w e r e s i m i l a r l y h a n d l e d , b u t
p l a c e d in a e r a t e d w a t e r of a m b i e n t s a l i n i t y (27 4- 0.5 ~
The w a t e r w a s c h a n g e d daily,
b o t h low s a l i n i t y a n d a m b i e n t , a n d the a n i m a l s w e r e fed c h o p p e d clams e v e r y o t h e r day.
After 7 days, t h e lobsters for e a c h series w e r e r e m o v e d in t h r e e lots over t h r e e consecutive d a y s a n d t h e i r tissues dissected, p a c k a g e d in air-tight p l a s t i c bags, a n d frozens t o r e d at - 8 0 ~ to a w a i t testing.
38
E. G o u l d
T i s s u e p r e p a r a t i o n. Tissues w e r e h o m o g e n i z e d as follows: a n t e n n a l g l a n d ,
1:9, w/v, in cold 0.25 M s u c r o s e c o n t a i n i n g 2.4 m M N a deoxycholate~ heart, 1:19 in cold,
d o u b l y g l a s s - d i s t i l l e d water; tail muscle, 1:4, also in cold water~ a n d m a l e gonads, 1:9 in
cold 1 % Triton, a n o n - i o n i c surfactant (W-1339, R u g e r C h e m i c a l C o m p a n y , Irvingtono n - H u d s o n , N. Y.)*. A l l tissues w e r e p r e p a r e d in s m a l l a l l - g l a s s h o m o g e n i z e r s containi n g a s m a l l a m o u n t of 25-nm glass p o w d e r . C e n t r i f u g a t i o n at 4 ~ w a s at 20,000 g for 45
m i n for a n t e n n a l g l a n d a n d heart, a n d 32,000 g for 60 rain for tail m u s c l e a n d m a l e
gonads. T h e c l e a r s u p e r n a t e s s e r v e d as the crude e n z y m e p r e p a r a t i o n s , a n d p r o t e i n
content w a s d e t e r m i n e d b y t h e b i u r e t m e t h o d (Gornal et al., 1949, cited in Layne, 1957}.
E n z y m e a s s a y s. A l l solutions w e r e m a d e in d o u b l y g l a s s - d i s t i l l e d water.
B i o c h e m i c a l s w e r e o b t a i n e d from S i g m a C h e m i c a l C o m p a n y , St. Louis, Mo. *. Activities
of all e n z y m e s w e r e d e t e r m i n e d b y f i x e d - w a v e l e n g t h s p e c t r o p h o t o m e t r y u s i n g p y r i d i n e
n u c l e o t i d e c o e n z y m e s e i t h e r directly or b y a c o u p l e d - a s s a y procedure~ a s s a y t e m p e r a ture w a s 25 ~ Unit of activity in e a c h case w a s ffmole NAD(P)* * r e d u c e d or N A D H
o x i d i z e d p e r m i n p e r m g b i u r e t protein. A s s a y concentrations (mM} in a 3.00-ml r e a c t i o n
v o l u m e w e r e : MDH, LDH, GPI, a n d PK - as in G o u l d & N i t k o w s k i (1979}, e x c e p t for PK
substrate c o n c e n t r a t i o n s ADP, 0.23, a n d PEP, 0.80; A A T - K p h o s p h a t e buffer p H 7.5,
100~ K aspartate, 200; NADH, 0.30~ ocKG, 6.7~ 9 6 / ~ M u n i t s M D H (Sigma #410-9)~ I D H Tris buffer p H 8.0, 90~ NADP, 0.3~ MnC12, 3.3; NaC1, 5.0; IsoC, 5.0; G 6 P D H - a s in G o u l d
& N i t k o w s k i (1979), e x c e p t substrate concentrations G6P, 0.04 a n d 0.40. D e t e r m i n a t i o n
of statistical s i g n i f i c a n c e w a s m a d e u s i n g S t u d e n t ' s t-test.
RESULTS
T a i l - m u s c l e e n z y m e s e x a m i n e d w e r e g e n e r a l l y i n f l u e n c e d m o r e strongly b y lows a l i n i t y stress t h a n b y s u b l e t h a l c a d m i u m exposure. E l e v a t i o n of M D H activity w a s the
only o b s e r v e d effect that could b e a t t r i b u t e d to c a d m i u m e x p o s u r e alone, an effect also
s e e n in control a n i m a l s at low salinity; a n d together, t h e s e stresses e l e v a t e d the activity
to a g r e a t e r d e g r e e t h a n d i d e i t h e r stress alone, for a n a d d i t i v e effect (Pig. la). M u s c l e
LDH activity w a s d e p r e s s e d b y l o w - s a l i n i t y stress in b o t h control a n d c a d m i u m - e x p o s e d
a n i m a l s (Pig. lb}~ c a d m i u m e x p o s u r e d i d not affect this e n z y m e activity. Low s a l i n i t y
d e p r e s s e d the activity of GPI o n l y in control a n i m a l s (Fig. lc), a n effect a b o l i s h e d b y
c a d m i u m exposure, a l t h o u g h c a d m i u m e x p o s u r e a l o n e d i d not affect GPI activity. T h e r e
was no c a d m i u m effect on the activity of e i t h e r PK or IDH in this tissue at a m b i e n t
s a l i n i t y (Table 1), b u t l o w s a l i n i t y i n h i b i t e d PK b o t h in controls a n d in c a d m i u m - e x p o s e d
lobsters, a n d s t i m u l a t e d IDH in the controls, w i t h w h a t a p p e a r e d to b e a n a d d i t i v e effect
w i t h c a d m i u m , in the m e t a l - e x p o s e d animals.
In the l o b s t e r heart, c a d m i u m e x p o s u r e c a u s e d m o r e m e t a b o l i c c h a n g e s t h a n in the
tail muscle, a n d m o r e t h a n low s a l i n i t y alone. MDH, LDH, a n d GPI activities w e r e all
* Reference to trade names does not constitute endorsement by the NMPS, NOAA
* * Abbreviations used: NAD(P), nicotinamide adenine dinucleotide (phosphate)~ NADH, reduced
NAD~ Tris, tris (hydroxymethyl) aminomethane;. ADP, adenosine 5'-diphosphate; PEP, phospho(enol) pyruvate, tricyclohexylamine salt; P6P, dl-fructose-6-phosphate, potassium salt; QcKG,ocketoglutaric acid, potassium salt~ IsoC, dl-isocitric acid, sodium salt~ G6P, d-glucose-6-phosphate,
monosodium salt~ Km, substrate concentration at one-half maximal velocity, Vmax; M, tail muscle; H,
heart; AG, antennal gland~ MG, male gonad
Low-salinity stress
a
39
r
001
LDH
:,
oF, SNs-
NS
4OOO
i
4c~c
e
i
I
.=_
I
.~ 15o0
~
1o0c
0~)
AMBIENT LOW
SALINITY SALINITy
CONTROLS
_
qBIE
LIN
(~2:
i_
LOW i
SALINITY
!
I (13) I
Oi
(14) l
CONTROLS
CADMIUMEXPOSED
~.1203
CADMIUMEXPOSED
CONTROLS
CADMIUM-
EXPOSED
Fig. 1. Effects of sublethal cadmium exposure and of low salinity, individually and combined, on
enzymes in lobster tail muscle. Bar height represents arithmetic mean, and the ticked vertical lines,
standard error. Numerals in parentheses are sample number. Level of confidence is inserted in each
arrow connecting a pair of bars, representing compared data sets. The same conventions are
observed for Figures 2-4
s t i m u l a t e d b y the s u b l e t h a l m e t a l stress (Figs. 2a, 2b, 2c), w h e r e a s low salinity d e p r e s s e d
heart LDH (Fig. 2b) a n d AAT {Fig. 2d} i n control lobsters. Low salinity a b o l i s h e d the
effect of c a d m i u m on heart LDH a n d GPI b u t did not c h a n g e the c a d m i u m - i n d u c e d
e l e v a t i o n of MDH activity. C a d m i u m exposure did not i n f l u e n c e AAT activity.
In a n t e n n a l glands, either c a d m i u m exposure or low salinity alone p r o d u c e d a n
e l e v a t i o n of MDH activity, b u t this effect was significantly d i m i n i s h e d w h e n both
stresses were c o m b i n e d (Fig. 3a). A n t e n n a l g l a n d LDH, on the other h a n d , was not
affected b y either stress alone, b u t the two together acted to i n h i b i t LDH activity (Fig.
3b}. The result of the c o m b i n e d stress i n lobster a n t e n n a l g l a n d i n each of these cases,
therefore, was repressive synergism. U n d e r optimal assay conditions. G6PDH activity in
the a n t e n n a l g l a n d s h o w e d no significant effects either for c a d m i u m exposure or for low
salinity. Activation b y a tenfold increase i n assay c o n c e n t r a t i o n of substrate (from 0.04 to
Table I. Effects of cadmium exposure (6 ~g l-I) and of low salinity (17 %0) on pyruvate kinase and
isocitrate dehydrogenase in lobster tissues
Tissue
Cadmium effect
(at ambient salinity)
Low-salinity effect
(in controls)
Cadmium effect
at low salinity
PK
Heart
Muscle
Male gonad
None
None
None
Muscle
None
None
~ Depressed activity (< .05)
None
IDH
~ Elevated activity (< .05)
None
~ LS Effect only
None
~ Additive (< .02)
E. Gould
40
~o,--.~
c
" /oo1~.~
MDH
15oo,
op,
/-~o2
,~ ,oooo
r
=
T
~ 9ooo.
1
i3
'
~ "4oo.
-2
~ 1300-
x 8coo-
~
9
•
~ ~2oo-
7~o-
o
looo-
~BPENT LOW
~L~NITY SALINrTY
(13)
(13)
AMBIENT LOW
~LINIT~ ~LINITY
(~2)
(14)
CONTROLS
CONTROLS
b
LDH
d
AAT
CADMIUMEXPOSED
# / , / j ~ NS~
~
600,
-NS~
T
Zi
s
~ 150o
500"
.c
~3
c 400-
10~0
~
I
CADMIUMEXPOSED
5oo
2o0.
AMBtENT
CONTROLS
LC~
AMBIENT
CADMIUM"
EXPOSED
CONTROLS
CADMIUMEXPOSED
Fig. 2. Effects of sublethal cadmium exposure and of low salinity, individually and combined, on
enzymes in lobster heart
0.40 mh4}, h o w e v e r , w a s significantly higher (P < 0.05) in the low-salinity controls
(72.0 %) as compared with ambient-salinity controls (58.4 %), w h e r e a s values for the
c a d m i u m - e x p o s e d animals, at both a m b i e n t (70.0 %) and low-salinity (67.5 ~
did not
differ significantly from either a m b i e n t or low-salinity controls.
In Figures 4a, 4b, and 4c, it is clear that there w a s no c a d m i u m effect at ambient
salinity on three e n z y m e s tested from m a l e gonad (MDH, LDH, and GPI). Low salinity
e l e v a t e d M D H activity in both control and c a d m i u m - e x p o s e d lobsters, and d e p r e s s e d
LDH in control animals, an effect that disappeared in the c a d m i u m - e x p o s e d animals.
Combined, the two stresses e l e v a t e d GPI activity, another synergism.
L o w - s a l i n i t y stress
o. f
41
100- b
ol-~
LDH
._=
!
3~0'
)
.E
@
J=
o~
)
~ 2000"
50.
-g_
:oo1
<
Z
Z
Z
~1000-
o
E
01
I
- t ~ NS ----~
AMBIENT LOW
AMBIENT LOW
SALINITYSALINITY SALINITYSALINITY
(12)
(13)
CONTROLS
.05
CADMIUMEXPOSED
A:B,ENT LO:
A:BIENT L ~
O'~SALINITY
SALN
ITIY SALINITY
SALINITY
CONTROLS
CADMIUMEXPOSED
Fig. 3. Effects of sublethal cadmium exposure and of low salinity, individually and combined, on
enzymes in lobster antennal gland
a
.~
S.s~
!
fNS-
5oo
-{-\~
.E
r
o.,
.=_
§
fNS~
-NS~
§
4OO
~NS~
T I ~
I
~o
i
|
/
300
5. ~
=
ISlENT LOW ~MBENTLOW
..... I. . . . . I . . . . . I. . . . .
c
o
~ooo
AMBIEN3
LOW
S*LINITY SALINITY
(7)
(0)
CONTROLS
AMBIENt
LOW
SALINITY SALINITY
(S)
(12)
/
CONTROLS
CONTROLS
CADMIUMEXPOSED
CADMIUMEXPOSED
CADMIUMEXPO6ED
Fig. 4. Effects of sublethal cadmium exposure and of low salinity, individually and combined, on
enzymes in male lobster gonad
DISCUSSION
T h e g e n e r a l characteristic of l o w - s a l i n i t y stress in o t h e r w i s e u n t r e a t e d lobsters w as
an i n h i b i t i o n of most of the e n z y m e s e x a m i n e d {Table 2), w i t h the e x c e p t i o n o f M D H in
all tissues an d tail m u s c l e IDH. Cripps & Reish (1973) f o u n d that LDH activity d e c r e a s e d
in a m a r i n e p o l y c h a e t e h e l d u n d e r e i t h e r h y p e r o s m o t i c or h y p o s m o t i c conditions, an d
that M D H i s o e n z y m e s v a r i e d in their response: cy t o p l asm i c (s) M D H d e c r e a s e d slightly,
42
E. G o u l d
a n d m i t o c h o n d r i a l (m) M D H i n c r e a s e d u n d e r b o t h forms of osmotic stress. T h e i r o b s e r v a tion p r o m p t s the s p e c u l a t i o n t h a t M D H activity o b s e r v e d in t h e p r e s e n t s t u d y w a s
p r e d o m i n a n t l y the m i t o c h o n d r i a l form, w h i c h is a s s o c i a t e d w i t h the citric a c i d cycle~ this
i n t e r p r e t a t i o n is s u p p o r t e d b y t h e concurrent e l e v a t i o n of a n o t h e r citric a c i d cycle
e n z y m e , IDH. I n c r e a s e d activity of this cycle w i t h d e c r e a s i n g o s m o l a r i t y has b e e n
r e p o r t e d in i s o l a t e d c r a b - g i l l m i t o c h o n d r i a , a c c o m p a n i e d b y i n c r e a s e d o x y g e n c o n s u m p tion (King, 1966). Moreover, b o t h M D H a n d IDH in a q u a t i c a r t h r o p o d s are r e p o r t e d to b e
i n h i b i t e d b y i n c r e a s e d c e l l u l a r ionic concentrations (Schoffeniels 8, Gilles, 1970b);
a n a l o g o u s r e a s o n i n g s u g g e s t s the d o m i n a n c e of s M D H in the m u s c l e tissue of m a r i n e
c r u s t a c e a n s u n d e r h y p e r o s m o t i c conditions and, conversely, of m M D H u n d e r h y p o s m o tic conditions. The d e c r e a s e in h e a r t A A T activity o b s e r v e d in b o t h control a n d c a d m i u m - e x p o s e d lobsters at low s a l i n i t y supports e a r l i e r o b s e r v a t i o n s that t r a n s a m i n a s e
activities (in m a r i n e bivalves) are p r o p o r t i o n a l to the rates of loss of a m i n o a c i d s
(Hammen, 1969), a n d t h a t a m i n o a c i d concentrations (in e u r y h a l i n e invertebrates)
d e c r e a s e d u r i n g h y p o s m o t i c stress (Schoffeniels & Gilles, 1970a; Gilles, 1973).
H y p o x i c stress can e x e r t b i o c h e m i c a l effects that a r e d i s c o n c e r t i n g l y s i m i l a r to those
h e r e a t t r i b u t e d to low salinity: Blackstock (1978) r e p o r t e d a s i g n i f i c a n t l y l o w e r A A T
activity in a m a r i n e p o l y c h a e t e that h a d b e e n e x p e r i m e n t a l l y s u b j e c t e d to h y p o x i c
conditions, a n d a l o w e r PK in the s a m e s p e c i e s f i e l d - c o l l e c t e d from s e d i m e n t s at a
p o l l u t e d , often h y p o x i c site, t h a n w a s s e e n in w o r m s t a k e n from less s t r e s s e d areas.
I n h i b i t i o n of LDH activity, too, is a c o m m o n e v e n t in cells g e a r e d t o w a r d a n a e r o b i o s i s
(Schoffeniels & Gilles, 1970b), as is also the case w i t h PK ( H o c h a c h k a & Somero, 1973).
Yet d u r i n g the 7 - d a y h o l d i n g p e r i o d at the two salinities, the w a t e r w a s c o n t i n u o u s l y
a e r a t e d , as w e l l as c h a n g e d daily. The a m b i e n t - s a l i n i t y tanks, 27.5 %o at a n a v e r a g e 21 ~
h a d a d i s s o l v e d o x y g e n c o n c e n t r a t i o n of 7.57 p p m , a c c o r d i n g to G r e e n & Carritt 0967);
in the l o w - s a l i n i t y tanks, at 17 %~ a n d 20 ~ it w a s 8.24 p p m . The a n i m a l s , therefore,
w e r e not hypoxic; the o b s e r v e d stress c a n r e a s o n a b l y b e a t t r i b u t e d to low salinity.
Tissues of lobsters e x p o s e d to s u b l e t h a l c a d m i u m a n d s u b s e q u e n t l y h e l d for 7 d a y s
(clearing) at a m b i e n t salinity, on the other hand, s h o w e d m a r k e d e l e v a t i o n in the activity
of glycolytic e n z y m e s (Table 2). In e a r l i e r e x p e r i m e n t a t i o n w i t h c a d m i u m - e x p o s e d
lobsters (30 days, 6 ~5 1-1 b u t w i t h no c l e a r a n c e period), a d d i t i o n a l o b s e r v e d c a d m i u m
effects w e r e also stimulatory, on m a l e g o n a d LDH a n d tail m u s c l e PK a n d GPI (Gould,
u n p u b l i s h e d data). The m a j o r overall effect of s u b l e t h a l c a d m i u m stress in all l o b s t e r
tissues e x a m i n e d , then, w a s i n c r e a s e d glycolysis, a n e x p e n d i t u r e of e n e r g y r e s e r v e s
t y p i c a l of m a n y t y p e s of s u b l e t h a l stress, as in t h e c o l d - a c c l i m a t i o n of terrestrial
i n v e r t e b r a t e s (Rao, 1966). This o b s e r v a t i o n accords w i t h T h e e d e ' s report (1980) of a n
i n c r e a s e d gross e n e r g y d e m a n d in c a d m i u m - f e d Mytilus larvae, a n d w i t h that of Price &
U g l o w (1980) of i n c r e a s e d rate of h e a r t b e a t in c a d m i u m - e x p o s e d Crangon.
Jointly, the two stresses (sublethal c a d m i u m a n d low salinity} o p e r a t e d to p r o d u c e a
v a r i e t y of effects in all tissues e x a m i n e d (Table 2). Two e s p e c i a l l y n o t e w o r t h y effects
w e r e o b s e r v e d in the a n t e n n a l g l a n d of c a d m i u m - e x p o s e d lobsters at low salinity.
Firstly, a l t h o u g h s u b l e t h a l c a d m i u m e x p o s u r e a n d low s a l i n i t y i n d i v i d u a l l y c a u s e d
e l e v a t i o n of M D H activity, t o g e t h e r t h e y a c t e d s y n e r g i s t i c a l l y to d e p r e s s it. The activity
w a s not d e p r e s s e d b e l o w a m b i e n t - c o n t r o l levels, a l t h o u g h a l o n g e r p e r i o d of s u b j e c t i o n
to the d o u b l e stress m i g h t h a v e done so; the significant fact here, h o w e v e r , is that the
n o r m a l r e s p o n s e of the l o b s t e r a n t e n n a l g l a n d to e a c h of the two s u b l e t h a l stresses
(H)
(H)
LDH (MG)
LDH
AAT
Depressed activity:
LDH (M)
GPI (M)
PK (M)
Elevated activity:
MDH (M)
IDH (M)
MDH (AG)
MDH (MG)
Low-salinity
effect
(in controls)
M D H (M)
M D H (H)
LDH (H)
GPI (H)
M D H (AG)
C a d m i u m effect
(at a m b i e n t
salinity)
M D H (M)
IDH (M)
Additive effect
M D H (AG)
LDH (AG)
LDH (H)
GPI (MG)
Synergism
Cd > LS
Abolition of
depressed
activity:
GPI (M)
LDH (MG)
Antagonism
Abolition of
elevated
activity:
GPI (H)
LDH (H)
LS < Cd
Table 2. Classification of effects of c a d m i u m exposure a n d low salinity, alone a n d in combination, on e n z y m e activity i n lobster tissues
F
44
E. G o u l d
i n d i v i d u a l l y w a s M D H elevation, a n d that this r e s p o n s e was s i g n i f i c a n t l y b l u n t e d b y the
d o u b l e stress. Moreover, LDH activity w a s also d e p r e s s e d . C o n c u r r e n t i n h i b i t i o n of t h e s e
two r e d o x r e g u l a t o r s i n d i c a t e s a d i m i n i s h e d c a p a c i t y for r e g e n e r a t i n g o x i d i z e d NAD
and, c o n s e q u e n t l y , l i m i t e d glycolytic efficiency (Banks et al., 1976). The s e c o n d effect of
the d o u b l e stress in the a n t e n n a l g l a n d w a s the w e a k e n i n g of a n e n z y m e - l i g a n d affinity.
T h e prior w o r k w i t h c a d m i u m - e x p o s e d lobsters, m e n t i o n e d above, s h o w e d a significant
loss of sensitivity to m a g n e s i u m m o d u l a t i o n (Km, P < 0.01; Vmax, P < 0.001) in a n t e n n a l
g l a n d G 6 P D H (Gould, u n p u b l i s h e d data), s i m i l a r to that f o u n d in the k i d n e y - p r o n e p h r o s
of c a d m i u m - e x p o s e d (60 days, 10 ppb) w i n t e r flounder, Pseudopleuronectes americanus
(Gould & N i t k o w s k i , 1979). P r e p a r a t i o n s of a n t e n n a l g l a n d from lobsters in the p r e s e n t
report, insufficient for p e r f o r m i n g similar k i n e t i c studies, w e r e u s e d i n s t e a d to test
G6PDH s u b s t r a t e activation. The s u b s t r a t e - a c t i v a t i o n figures (see s e c t i o n "Results")
s u g g e s t a p r o t e c t i v e m e c h a n i s m at l o w - s a l i n i t y for e n z y m e - s u b s t r a t e affinity that is
p r e s e n t in control b u t not in c a d m i u m - e x p o s e d lobsters. A n a n a l o g o u s loss of l i g a n d
sensitivity w a s r e p o r t e d for h e a r t A A T in lobsters s i m i l a r l y e x p o s e d to s u b l e t h a l cadm i u m (Thurberg et al., 1977).
P e r h a p s the most p r o m i n e n t s i n g l e b i o c h e m i c a l r e s p o n s e to s u b l e t h a l stress w a s the
e l e v a t i o n of M D H activity, a stress s i g n a l that in this study w a s often a c c o m p a n i e d b y
d e p r e s s i o n of LDH activity. T h e ratio of t h e s e two e n z y m e activities, MDH: LDH (Table
3), constitutes a fairly a c c u r a t e reflection of the d e g r e e of stress: C d - L S > C o n t r o l - L S > C d A m b i e n t > C o n t r o l - A m b i e n t . O x y g e n c o n s u m p t i o n of gill tissue from the lobsters in this
s t u d y confirms this o r d e r of stress: Cd-LS (1323 #1 h -1 g-l), Control-LS (1104), CdA m b i e n t (772), a n d C o n t r o l - A m b i e n t (703) (F. P. T h u r b e r g , p e r s o n a l communication),
a n d the only significant difference in h e m a t o l o g i c a l p a r a m e t e r s was a n i n c r e a s e in N a +
in the c a d m i u m - e x p o s e d lobsters at low s a l i n i t y (M. A. Dawson, p e r s o n a l c o m m u n i cation).
Roberts (1964) h a s c o m m e n t e d that like m e t a b o l i c systems in different tissues in the
s a m e species, a n d in l i k e tissues in different species, m a y o p e r a t e to p r o d u c e d i v e r s e
effects b e c a u s e of the v a r y i n g d e g r e e s of s y s t e m i c control. This is u n d o u b t e d l y true for
the o b s e r v a t i o n s in this study, a n d it w o u l d b e u n w i s e to a t t e m p t a n y firm conclusions.
N e v e r t h e l e s s , one m i g h t p r o f i t a b l y c o n s i d e r the following p a r a d o x : E l e v a t i o n of e n z y m e
activity ( i n c l u d i n g a b o l i t i o n of depression) b y the c o m b i n e d stresses was e v i d e n t only in
the tail m u s c l e a n d m a l e gonad; y e t in this study, such e l e v a t i o n w a s the h a l l m a r k of
s u b l e t h a l c a d m i u m stress, a n d tail m u s c l e w a s the tissue in w h i c h l o w - s a l i n i t y stress was
most p r o m i n e n t . Conversely, i n h i b i t i o n of e n z y m e activity ( i n c l u d i n g a b o l i t i o n of
Table 3. Effects of cadmium exposure and of low salinity on the ratio of MDH : LDH activities in
lobster tissues
Tissue
Control-Ambient
Cd-Ambient
Heart
5.31
5.61
Muscle
0.89
1.01
Antennal gland
35.32
38.00
Male gonad*
7.96
7.52
*No cadmium effect was observed in the male gonad
Control-LS
Cd-LS
7.49
1.75
49.98
11.74
7.77
1.87
53.53
10.53
Low-salinity stress
45
increased activity) was seen only in the heart and antennal gland; yet such inhibition
w a s o b s e r v e d to b e t h e m a j o r e f f e c t of l o w s a l i n i t y , a n d t h e h e a r t w a s w h e r e c a d m i u m
e f f e c t s w e r e t h e m o s t n u m e r o u s . It t h u s a p p e a r s t h a t i n t h e g e n e r a l m u s c l e m e t a b o l i s m of
t h i s d e c a p o d c r u s t a c e a n , t h e e f f e c t s of o n e s u b l e t h a l s t r e s s t e n d to o f f s e t t h o s e of t h e
other.
Acknowledgements. I a m grateful to M. Nitkowski a n d N. A. Rawson for their expert technical
assistance, a n d to Dr. J o h n J. Stegeman, Woods Hole O c e a n o g r a p h i c Institution, for a critical review
of the manuscript.
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