ELSEVIER
Marine Micropaleontology
28 ( 1996) 5- I7
Survival, ATP pool, and ultrastructural characterization of benthic
foraminifera from Drammensfjord (Norway) : response to anoxia
Joan M. Bernhard n, Elisabeth Alve b
” Wadsworth Center, New York State Department of Health,P.O. Box 509, Albany, NY 12201-0509, USA
’ Department of Geology,University ofoslo, P.O. Box 1047, Blindem, N-0316 Oslo 3, Norway
Received 13 March 1995; accepted
17 May 1995
Abstract
While much evidence indicates that certain benthic foraminifera are facultative anaerobes, little is known regarding the
physiologic response of foraminifera to anoxia. In order to assess their response, specimens of four foraminiferal species,
collected from a typically dysoxic area of Drammensfjord, Norway (45 m water depth), were incubated in seawater purged with
nitrogen. Over a time course of > 3 weeks, the specimens were extracted for adenosine triphosphate (ATT’) in a nitrogen-flushed
glove bag to assess their survival and ATP reserve under such conditions. For comparative purposes, similar extractions were
done on conspecifics one week after their collection from the seafloor, as well as on other conspecifics, obtained from the same
site, incubated in aeratedconditions.
The survival rates of nitrogen-treatedAdercotryma
glomeratum,
Psammosphueru
bowmunni.
and Stuinfi,rtlzia,f~s~~~rmis were not significantly lower than those of the control specimens. However, the ATP concentrations
of nitrogen-incubated A. glomerutum and S. fisiformis
were significantly lower than those of their aerated conspecifics, while
there was no significant difference between the [Al?]
of P. bowmnnni from the two treatments. Both the survival rate and the
ATP concentrations of nitrogen-incubated
Bulimina marginatu were significantly lower than those of control specimens. The
ultrastructure
of B. murginatu and S.fis$“otmis
incubated in N2 for 18 days were compared with those of specimens fixed within
I5 minutes of collection. For both species, the specimens that survived the experimental treatment had ultrastructures indistinguishable from those fixed just after field collection. However, the ultrastructure of B. murginatu differed from that of S.
.fir.sifi~rmi.s in that it lacked the numerous peroxisome-endoplasmic
reticulum (ER) complexes and what appeared to be algal
chloroplasts observed in S. .fusiformis. Copious arrays of paracrystals were observed in both species from the experimental
treatment as well as the shipboard-fixed specimens, suggesting that neither population had extensive pseudopodial networks.
When considered in combination, our results indicate that the four species respond to and survive anoxia differently, with
responses including dormancy and, as yet unidentified, anaerobic metabolic pathways.
1. Introduction
Dissolved
oxygen
groups to successfully
is depleted
in the lower parts of the biological
sediments.
Benthic
foraminifera,
to undetectable
values
zone of most marine
the principal
mei-
ofaunal group in many marine sediments (e.g., Snider
et al.. 1984; Gooday, 1986), are one of few eukaryotic
0377.8398/96/$15,00
0 1996 Elsevier Science B.V. All rights reserved
SSn10377.839X(95)00036-4
inhabit
dysoxic
and anoxic’
’ There is no consensus on the definitions of the terms “dysoxic”
and “anoxic” (see, e.g., Tyson, 1987; Fenchel and Finlay, 1995).
In this paper, the term “dysoxic” refers to dissolved oxygen concentrations of 0. l-l .O ml O2 I- ’ while the term “anoxic” refers to
<O. I ml O2 I-‘. There is considerable analytical uncertainty in
measuring oxygen concentrations of < 0.1 ml O2 I ’ , prohibiting
confident distinction between the presence of a trace amount versus
absence of oxygen.
6
J.M. Bernhard. E. Alse /Murk
environments (e.g., Bernhard, 1989; Bernhard and Reimers, 1991; Sen Gupta and Machain-Castillo,
1993;
Alve, 1994; Sen Gupta and Aharon, 1994). While
experimental studies support the conclusion that some
benthic foraminifera are facultative anaerobes (Moodley and Hess, 1992; Bernhard, 1993)) nearly nothing
is known regarding the physiological response of foraminifera to anoxia. In a previous study where adenosine triphosphate (ATP) analysis was used to establish
foraminiferal survival under anoxia (Bernhard, 1993))
the specimens were reaerated prior to ATP extraction
and, therefore, only survival could be ascertained.
In the present study, we extracted ATP from specimens in a nitrogen-flushed
glove bag, which enabled
us to gain insight into the physiologic response (i.e.,
[ ATP] ) , as well as survival, of foraminifera exposed
to conditions of undetectable oxygen. In addition, the
ultrastructure of specimens incubated in nitrogen for
18 days was compared with that of specimens fixed
within 15 minutes after they were obtained from the
collection site.
2. Materials and methods
All material was collected on 21 October 1992 from
45 m water depth in Drammensfjord,
Norway
(59”40.77’N; 10”25.75’E), a typically dysoxic environment (Alve, 1990, 1991). One gravity core (6.7
cm inner diameter; - 20 cm long) and one box core
(0.25 m’) were obtained from the same site. Just after
the gravity core was retrieved on deck, a bottom-water
sample was collected from - 10 cm above the sediinterface
dissolved
oxygen
ment-water
for
determinations
by Winkler titration. The gravity core
was extruded and sliced into 0.5 cm intervals to a depth
of 1.5 cm. These sediment intervals were placed and
maintained in ambient temperature (7°C) seawater.
After returning to the laboratory, the sediment intervals
were sieved with ambient seawater over a 63-pm
screen within 24 hours of collection, From the > 63pm fraction, foraminifera were removed via mouth
pipette with the aid of a Zeiss stereomicroscope. One
hundred specimens were removed for ATP extraction
in the superficial 0.5 cm interval, and 50 wererecovered
from each of the two remaining intervals. A week later,
the length of each specimen was recorded just prior to
its extraction for ATP following the method of Bern-
Micrr,l,aleontol(~~.v 2X (I 996) 5-I 7
hard (1992). Extracts were frozen ( - 30°C) until
luciferin/luciferase
analysis using an LKB model 1250
luminometer. The data were analyzed according to
Bernhard ( 1992) and Alve and Bernhard ( 1995) ?
where specimens with > 145 ng ATP mm-’ of test
volume were considered alive at the time of extraction.
The surface 1.5 cm of sediments from the box core
were collected in bulk, sieved over a 63-pm screen,
and the > 63-pm fraction was maintained in ambient
temperature
seawater. Specimens
of Adercotryma
glomeratum, Bulimina marginata, Psammosphaeru
bowmanni, and Stainforthia fusiformis were removed
from the sediments as described above. Since live specimens were needed for the experiment, individuals
were selected only if they either had material collected
around their apertures (e.g., Linke, 1992; Moodley and
Hess, 1992) or if the cytoplasm of calcareous species
appeared green (e.g., Haynes, 1965; Goldstein and
Corliss, 1994). The foraminifera were divided into 7
groups, each containing 5 specimens of A glomeratum,
10 of P. bowmanni, and 15 each of B. marginata and
S. fusifonnis. One group was placed in each well of two
six-well tissue-culture plates. The remaining group was
extracted for ATP, as describe above, at the beginning
of the experiment ( 1 November). One culture plate
was incubated in a nitrogen-flushed glove bag located
in a darkened environmental room maintained at 8°C
while the other (the control), was incubated on an
adjacent bench top in the same environmental
room
(i.e., under aerated conditions). A hole was fashioned
into the glove bag to allow the inclusion of a stereomicroscope body, while the oculars remained outside
the bag. The edge of the hole was securely fastened to
the stereomicroscope
body with electrical tape. All
materials and reagents required for Winkler sampling,
ATP extractions and ultrastructural fixations (e.g., buffered glutaraldehyde)
were also placed in the glove
bag. Two 800-ml
beakers containing
seawater
remained uncovered in the glove bag. The water in
these beakers served as the source for Winkler samples
as well as to humidify the glove bag so that the salinity
of seawater bathing the specimens did not increase due
to evaporation. A Jenway portable dissolved-oxygen
meter (model 9070; Essex, England) was used to monitor the oxygen concentration inside the bag. Once all
equipment, supplies, and specimens were in place, the
glove bag was sealed and flushed with nitrogen. Positive pressure was maintained in the nitrogen bag at all
J.M. Bernhard. E. Abe/Marine
times. Over a time course of 3.5 weeks from 1 to 25
November,
all specimens in a given group were
extracted for ATP in the glove bag. The specimens were
manipulated within the glove bag using a pipette operated with a Pasteur pipette bulb. After each sampling
event, the glove bag was opened for a brief period of
time ( <30 seconds) to remove ATP extracts for
proper handling. Just prior to opening the glove bag,
the tissue-culture plate containing the specimens was
securely fastened inside two sealed ziplock bags to
prevent oxygen contamination. The tissue culture plate
was not removed from these bags until just prior to the
next sampling event. During each sampling event, all
specimens from one group in the aerated (control)
tissue culture plate were similarly extracted for ATP.
The environmental room received light from a 60 Watt
bulb only for - 2 hours during each set of extractions
and for < 5 minutes daily while the nitrogen flow was
monitored.
Some additional specimens of Bulimina marginata
and Stainforthia fusiformis,
which were incubated in
the N,-tilled glove bag for 18 days, were fixed for highvoltage electron microscopy (HVEM) using standard
procedures (Bernhard, 1993). Specimens were fixed
in 6% glutaraldehyde/O.l
M cacodylate buffer (PH
7.2) for 5 hours, rinsed three times in buffer, decalcified
in 0.1 A4EDTA (PH 7.0), postfixed in Os04, and poststained in uranyl acetate. After dehydration in a graded
series of ethanol, the specimens were cleared in propylene oxide, embedded in Epon 812 (Polysciences,
Warrington, PA), and sectioned into 0.25 pm-thick
sections. To serve as controls, some specimens were
similarly fixed - 15 minutes after collection from the
ambient dysoxic environment.
3. Results
The ambient dissolved oxygen concentration at the
collectionsitewas0.95m1021-’
(i.e.,41.5 flkg-‘).
Temperature and salinity data were not obtained at the
sample site on the day of collection, but values for
September and November, 1992 were 7.2”-7.5”C and
30.2-30.4%0,
respectively
( Alve
and Bernhard,
unpubl. data). Some spionid polychaete tubes extended
above the sediment-water
interface in both the gravity
and box cores. The sediments were brown in the surface
centimeter, but were black below that depth.
Micropaleontology 28 (1996) 5-l 7
7
Throughout the experiment, the oxygen concentrations in the nitrogen-flushed glove bag were below the
detection limit of the Winkler titration method (i.e.,
< 0.1 ml 0, I- ‘), and, therefore, considered anoxic by
definition. In fact, trace levels of hydrogen sulfide
( < 0.1 ml H?S 1 ’ ) were detected in the glove bag on
4 November.
3. I. Community response
The numerical densities of live foraminifera (i.e.,
> 145 ng ATP mrnp3 test volume) in the gravity core
are presented in Table 1. The ATP concentrations of
the live specimens from both treatments and those
extracted one week after field collection were highly
variable (Fig. 1A). The [ ATP] of the live population
extracted 7 days after collection (i.e., 28 October) were
significantly higher than those of the live specimens in
the aerated culture plate pooled over the course of the
experiment (rank sum, p < 0.05, Zar, 1984). While the
foraminiferal populations in the gravity core and box
core were similar in species composition, there were
no live Adercotryma glomeratum or Psammosphaera
bowmanni in the populations removed from the gravity
core for ATP determinations.
However, rose-Bengal
stained specimens of both species were recorded in the
samples ( Alve, unpubl. data).
The nitrogen treatment did not have a significant
effect on survival rate of the populations (Fig. 1B; rank
sum, p > 0.05). Conversely, the ATP concentrations of
live specimens from the N, treatment were significantly
lower than those of the controls
(rank sum,
p < 0.0001) The average [ ATP] for all N,-incubated
live specimens was depressed - 53% compared with
the average concentration of aerated specimens. On 4
November, only three days after the initiation of the
experiment, the average [ATP] of specimens in the
Table 1
Numerical densities (number cm-‘) of live fomminiferain the gravity core (extracted 28 October 1992), presented by depth interval
Sediment
interval
All species
Bulimincl
marginata
14.7
5.2
5.1
93
3.3
2.6
(cm)
o-o.5
0.5-l .o
1.0-1.5
3.1
I .9
1.9
8
J.M. Bernhard.
E. Alve /Murine
Microl~aleontolofiy
28 (1996) 5-17
glove bag was substantially depressed, compared with
control values. While [ ATP] in Nz-treated specimens
remained lower than the controls over the remainder of
the experiment, the average values increased compared
with the values of 4 November.
3.2. Species response
Survival rates of Adercotryma glomeratum, Psammosphaera bowmanni, and Stainforthia fustformis
were not significantly influenced by the nitrogen treatment (Fig. 2A-C; rank sum, p > 0.05). However, the
survival rate of nitrogen-incubatedBulimina
marginata
was significantly
lower than that of the controls
(Fig. 2D; one-tailed rank sum, p < 0.05).
The ATP concentrations
of Adercotryma glomeraturn, Bulimina marginata and Stainforthia fustformis
incubated in the glove bag were significantly lower than
their aerated conspecifics
(Fig. 3A,C,D; rank sum,
each p < 0.005). However, the [ ATP] of N,-incubated
Psammosphaera bowmanni were not statistically different from aerated conspecifics (Fig. 3B; rank sum,
p > 0.05). For the N2 treatment, all four species had
their lowest average [ ATP] on 4 November (Fig. 3).
The average ATP levels of Adercotryma glomeratum
and Bulimina marginata incubated in nitrogen were
depressed 67% and 68%, respectively, compared with
ATP concentrations in aerated specimens. The average
[ ATP] in Stainforthia fusiformis incubated in N, was
only depleted by 26% compared to the average of aerated controls. The average ATP concentration of nitrogen-incubated
Psammosphaera
bowmanni
(590;
S.D. = 548) was comparable to values of aerated conspecifics (530; S.D. = 414) when one outlier with an
[ ATP] of approximately an order of magnitude higher
than other live conspecifics was omitted from the analyses (from the 11 November N2 treatment).
3.3. Ultrastructural
observations
ultrastructural observations were made on 0.25ym thick sections, rather than conventional
70-nm
ultrathin sections. There are numerous advantages in
sectioning and viewing thick sections (see Rieder et
al., 1985), which are particularly pertinent for investigations of foraminiferal ultrastructure. In brief, specimens can be sectioned using a relatively inexpensive
synthetic diamond knife and more material can be
Our
Extraction
date -, 28 Oct.
1 Nov.
4 Nov.
11 Nov.
25 Nov.
lo
24
11 Nov.
25 Nov.
No. days in N2 ---t
P
3
Begin Nz
incubation of
appropriate groups
B
28 Oct.
1 Nov.
4Nov.
Fig. 1.(A) Bar graphshowing the average ( + 1 standard deviation)
ATP concentration of all live foraminifera from the three gravity
core samples (28 October), initial control sample (box core), and
each of both control and experimental treatments over time. (B) Bar
graph showing the proportion of specimens determined to be alive
at the time of extraction.
examined per unit time compared with viewing ultrathin sections.
The ultrastructure of Bulimina marginata and Stainforthia fustjormis incubated in the glove bag for 18
days did not differ from that of specimens fixed just
after recovery from the collection site (Fig. 4 and
Fig. 5). The nucleus was clearly visible in one nitrogen-incubated B. marginata (Fig. 4A,B). The mito-
J.M. Bernhard, E. Alve / Marine Micropaleontc~logy28 (1996)
A
A. glomeratum
100
80
80
$60
.3
el
g 40
60
20
20
40
0
0
28Oct.
1 Nov.
4Nov.
11 Nov.
25 Nov.
28 Oct.
Extraction date
28 Oct.
1 Nov.
4 Nov.
11 Nov.
25 Nov.
28 Oct.
Fig. 2. Bar graphs showing the proportion of live Adercottyna glomeratum (A),
11 Nov.
25 Nov.
1 Nov.
4 Nov.
11 Nov.
25 Nov.
Aerated controls
q N$ncubated
groups
Psammosphaeru howmanni (B), Slainfi,rthitrfirsvhnnis (C),
at the time of extraction.
and Golgi complexes of N,-incubated
B.
were similar in appearance and abundance
to those found in conspecifics fixed just after field collection (Fig. 4C,D). Paracrystals, a storage form of
tubulin (Rupp et al., 1986)) were concentrated near the
nucleus in nitrogen-incubatedIS.
marginata
(Fig. 4B).
Tubulin paracrystals were also observed in B. marginato fixed just after field collection. The cytoplasm of
all specimens examined with HVEM was highly vacuolated (e.g., Fig. 4A); some vacuoles contained partially degraded food (e.g., Fig. 4C).
In Stainforthiafisiformis,
the mitochondria of nitrogen-incubated
specimens were similar in abundance
marginata
4 Nov.
Extraction date
q
and Bulirnimr mrrrginaru (D)
1 Nov.
Extraction date
Extraction date
chondria
9
P. bowmanni
B
100
S-l7
and appearance to those of conspecifics fixed just after
field collection. The mitochondria are most abundant
in the cytoplasm nearest the aperture (i.e., extrathalamous cytoplasm sensu Alexander and Banner, 1984;
Fig. 5A). However, the mitochondria also appear concentrated at the periphery of chambers but not clearly
associated with pore plugs (Fig. 5B). This pattern is
observed not only in areas where the pores of that
region open to the external environment, but in chamber peripheries where two chambers abut (Fig. 5B).
Numerous organelles similar in appearance to peroxisomes are associated with endoplasmic reticulum (ER)
in N,-incubated S. fusiformis
(Fig. 5C). These com-
28 Oct.
1 Nov.
4 Nov.
11 Nov.
28 Oct.
25 Nov.
1 Nov.
C
4 Nov.
11 Nov.
25 Nov.
Extraction date
Extraction date
D
S. fusformis
B. murginata
6ooo-
sooo4ooQ.
3om
2ooo.
1fJw
0.
28 Oct.
1 Nov.
4 Nov.
11 Nov.
25 Nov.
28 Oct.
1 Nov.
4 Nov.
I1 Nov.
25 Nov.
Extraction date
Extraction date
q Aerated
controls H
Nz-incubated groups
Fig. 3. Bar graphs showing the average ( i 1 st~d~d
deviation) ATP concen~tion
of all live Aderc~t~y~a ~I~~~r~tu~ (A), ~~li~jnu
f~~t~~j~~ft# (8 f, St~in~~~t~iafusifnris
(C), and ~~~~~~.~~~er~ ~~~~u~ff~ (D) from each treatment over time. Note the different scales on
the y-axes.
plexes were also abundant in S. fusifotmis fixed just
after field cohection (Fig. 5D). Tubulin paracrystals
were also observed in S. fiaiformis fixed just after field
collection (Fig. 5D) as well as in ~*-incubated specimens. The nucleus was observed in a Na-incubated S.
fusiformis (not shown). Structures that appeared similar to chloroplasts were aumerous in both the fieldfixed and N,-incubated
S. fisiformis
(Fig. 5B,D).
Some appeared intact, while others exhibited various
degrees of degradation. Both N,-incubated and fieldfixed specimens had numerous vacuoles, some of
which were food vacuoles (Fig. SC,D).
4. Discussion
In the four species studied, there were three different
observed physiological responses to nitrogen incubation: f I ) both survival and [ ATP] were significantly
decreased (e.g., Bulimina marginata),
(2) survival
was not affected but [ ATP] was significantly depleted
(e.g., Stainforthia fusiformis and Adercottyma glomeraturn), and ( 3) no significant decrease in either survival or [ ATP] (e.g., Psammosphaera
bowmanni).
The ul~astructure of the species examined provides
some possible explanations for our results. ~~~irnina
Fig 4. High-voltage electron micrographs ofBuliGza
murginuta (250.nm sections). (A-C) Specimen incubated in Nz for 18 days. (,A) Lowmagnification view showing a complete chamber between two adjacent chambers. Note highly vacuolated (V) nature of cytoplasm. N = nucleus.
E- region that was the calcareous test wall or, beyond that, the external environment prior to fixation. Scale bar 20 pm. (8) View of pnracrystals
(F) near nucleus NU = nucfeolus. SC& bar 1 Fm. (C) High-~gn~fication
view of numerous mitochondria (84) at chamber periphery. Also
note Golgi ( G). food vacuole f FV). Scale bar = 1 pm. (D) Specimen fixed just after field collection. from 0.5-I .O cm sediment depth. Note
the mitochondt-ia and Golgi. Scale bar I pm.
Fig. 5. High-voltage efectron mi~mgrap~s of Strrdnforthiu~ic;if;?rmis (2%nm sections). (A. C) Specimen incubate in nitrogen for 18days.
(B. D) Specimen fixed just after field collection, fromO.~-Jam cm sediment depth. (A) View ofm~tochond~a (M) in aperturai ~y~oplasrl~.CN )
View of mitochondria (M) St periphery of chamber abutting anather chamber. (C, B) View of peroxisomes ( * ) and ~ndo~~asn~~~
reti~uJurn
(arrowheads). Also note the chloroplast-like structures (C), paracrystals (F)? and food vacuoles (FV). Scale bars = I pm.
J.M. Bernhard.
E. Abe / Murine
marginatu, the species most drastically affected by our
N1 treatment in terms of survival and ATP depletion,
did not possess any ultrastructural
characteristics
exhibited by many anaerobes (e.g., endosymbionts).
Distributional data support our inference that B. marginatu is not well adapted to anoxia. For instance, it
has only recently colonized the Drammensfjord
sampling area, because in 1984, B. marginata was not
recorded at any water depths in this part of the fjord
(Alve. 1995). In 1988, scattered populations of this
species were present, but never comprised over 2% of
the rose Bengal-stained
assemblages (Alve, 1995).
However. when we sampled in 1992, B. marginata
comprised 45% of the stained assemblage
(Alve,
unpubl. data). with numerical densities of nearly 10
live specimens per cm.’ (Table 1). This substantial
increase in numerical density and dominance can be
attributed to the return of aerated conditions at this
water depth. a result of decreased organic-matter pollution pressure from the areas surrounding the fjord
(Alve. I995).
On the other hand, Stainforthiafusiformis,
a species
that was not affected in terms of survival during nitrogcn incubation but did have significantly decreased
[ ATP 1, had LWOultrastructural components that were
rarely observed in Bulimina marginatu. First, S. fusi,fijrrk
possessed
numerous
peroxisome-ER
complexes. A similar difference in the occurrence of these
complexes was noted in two other foraminiferal species
from severely oxygen-depleted
sediments ( <O.l ml
O7 I ‘: Bernhard and Rcimers, 1991). Distributional
observations suggest that the two species with numer-ous peroxisomes-ER
complexes (i.e., S. fusiformis
I’rom this study and Nonionella stella from the Santa
Barbara Basin) are more tolerant to anoxia than other
I’oraminifera. We know that S. fusiformis is the most
abundant species in areas closest to the redox boundary
and was the first species 10 recolonize the deeper parts
of the Fjord after return of dysoxic conditions (Alve,
I995 ) In the case of N. stellu, distributional data suggest that it survived anoxia longer than other foraminiferal species -just prior to mass mortality (Bernhard
and Reimers. 199 1) Peroxisome-ER complexes have
been noted in the cytoplasm of additional benthic foraminifera from dysoxic fjord environments
(Nyholm
and Nyholm, 1975 ) Widespread in nature (see Miiller,
1975; Van den Bosch et al., 1992 for reviews), peroxisomcs arc organelles where oxidative reactions pro-
Microl,uleontolog?,
28 (I 996) 5-I 7
I7
duce hydrogen peroxide, which is then metabolized by
catalase to water and oxygen. A variety of additional
enzymes exist in peroxisomes, whose distributions
depend on culturing conditions and organism type. Little is known regarding the identity of enzymes in foraminiferal
peroxisomes
although
Anderson
and
Tuntivate-Choy
( 1984) showed that the peroxisomes
of planktonic foraminifera at least have catalase. Since
our identification of peroxisomes was made on a structural, rather than biochemical, basis, we do not know
what enzymes are active in the peroxisomes of S.,fi~siformis. Some peroxisomes are known to have the
enzymes necessary for the production of glucose,
which can be metabolized anaerobically by glycolysis
in the cytoplasm. We agree with the previous suggestion (Nyholm and Nyholm, 1975; Bernhard and Reimers, 199 I ) that glycolysis may play an important role
in the metabolismofforaminiferain
dysoxic and anoxic
environments.
Another ultrastructural difference between Stainftirthia fusiformis and Bulimina marginata is that S. ,fusiformis had what appeared, on a morphological basis,
to be chloroplasts. While these chloroplast-like
stuctures, which were numerous, appeared in various stages
of digestion, many of them appeared intact. Cedhagen
( 1991) noted that this species retained chloroplasts in
its cytoplasm. While the light levels reaching 45-m
water depth in Drammensfjord
are undoubtedly
extremely low, it is possible that the chloropl,ast-like
structures in S. ,fusiformis received enough light to
remain active. A decade before our collections were
made, the depth of the euphotic zone was - IO m in
the Drammensfjord (Magnusson and NZS, 1986), hut
comparable measurements were not made during OUIcollections. However, since we know that pollution in
the fjord has decreased over recent years ( Alve. I99 I.
1995), the euphotic zone undoubtedly extends deepet
than observed in the early 1980s. Even if the depth ot
the euphotic zone, as conventionally
calculated (i.c.,
I % light level), is shallower than our sample site, there
are instances where significant amounts of photosynthesis have been recorded from depths deeper than the
1% light level (e.g., Venrick et al., 1973).
Cases of chloroplast husbandry in foraminifera arc
documented (e.g., Lopez, 1979; Leutenegger,
1984;
Lee et al., 1988; Cedhagen,
199 I and rcl‘erences
therein) ; some instances being in specimens from areas
with possible or extended oxygen depletion (c.g..
fjords, Cedhagen, 1991; mudflats, Lee et al., 1988).
We do not know what benefit the foraminifera gain
from these chloroplast-like structures, but it is possible
that they play a significant role in foraminiferal metabolism. This role may range from merely being a food
source to providing a site for crucial metabolic activities that enable the foraminifer to survive anoxia. For
instance, at least one case is known where oxygen produced by an algal endosymbiont is utilized by its ciliate
host (e.g., Reisser and Wiessner, 1984; Lee et al.,
198.5). Since an additional foraminiferal species that is
often found in dysoxic to anoxic environments also has
retained ~hloroplasts and peroxisome-ER
complexes
(i.e., ~~~~~~~ell~ stella, Leutenegger, 1984; Bernhard
and Reimers, 199 1) , it is possible that the peroxisomes
are actively involved in a metabolic interplay involving
the chloroplasts and mitochondria, as observed in certain plants (Tolbert, 1971). However, it should be
noted that our inferences about the biochemical interactions between foraminifera1 host and chloroplasts, as
well as the metabolic pathways of foraminifer~
peroxisomes, must be considered to be highly speculative
since we have not done the biochemical analyses
required to support such hypotheses.
An additional explanation for the observed pattern
of depleted [ATP] without decreased survival in
response to nitrogen incubation is that the specimens
became dormant. The presence of tubulin paracrystals
in foraminiferal
cytoplasm suggests that extensive
pseudopodial networks were not extended (Bowser et
al., 1984; Rupp et al., 1986), which further suggests
that specimens were not actively feeding. While this
may be expected for the N,-incubated
specimens
because they were not fed, the field-fixed specimens
certainly had available food since total organic carbon
values in that areaof Drammens~jord~eapproximately
2% ( Alve, 1990). However, food vacuoles in the N,incubated specimens appeared similar in abundance
and content as those in field-fixed specimens, indicating
that the foraminifera had not digested all food reserves.
Although possibly dormant while in situ, the specimens
may have exhibited a physiological reawakening during sanlpling ( i.e., increase in [ ATP] ), similar to that
caused by organic enrichment, as observed by Linke
( 1992). Alternatively,
it is possible that the tubulin
paracrystals
were formed because the specimens
retracted their pseudopods during box coring, but paracrystals have not been observed in other foraminiferal
species collected in a similar manner (Bernhard and
Reimers, 1991). Dormancy, where specimens do not
exert energy by actively feeding, may be an energetically advantageous approach to surviving periods of
anoxia.
The third observed response to nitrogen incubation
(i.e., no effect on either [ ATP] or survival as observed
in Psammosphaera bowmanni) is difficult to interpret
since distributional data show that this species does not
proliferate nor consistently
occur in the severely
dysoxic areas of Drammensfjord ( Alve, 1990; unpubl.
data). This species has been found in another dysoxic
fjord ( < 0.35 ml O2 1~ ‘, Byfjorden, western Sweden,
1. Olsson, pers. commun., 1989) and a congener was
found in oxic as well as anoxic sediments (P. parun,
Bernhard, 1989). However, neither of these two occurrences was in abundance, suggesting that reproduction
of P. bowmanni in anoxic and dysoxic environments is
inhibited, even though it maintains high ATP conccntrations. Thus, in a certain sense, this species may also
be considered to respond to anoxia by becoming dormant. It is unfortunate that P. bowmanni was not investigated ultrastructurally because it may have bacterial
associates, as observed for at least two other benthic
foraminiferal species obtained from dysoxic to anoxic
environments
(Bernhard and Reimers, 1991; Bernhard, 1993), mat may enable the foraminifer to survive
extended exposure to anoxia without depleted ]ATP].
A previous investigation suggested that the mitochondria of foraminifera collected from dysoxic environments are associated with test pores (Leutenegger
and Hansen, 1979). The conventional
interpretation
regarding such distributions is that the mitochondria
are strategically located in order to efficiently acquire
oxygen (Corliss, 1985; Moodley and Hess, 1992; Sen
Gupta and Machain-CastiIlo,
1993). While our study
was not quantitative, we found that the mitochondria
appeared concentrated at chamber peripheries, but they
were not clearly associated with pores. Furthermore,
mitochondria
were not just observed at chamber
peripheries adjacent to the environment, but also at the
peripheries of two abutting chambers. Thus, our observations do not appear to support the idea that mitochondria congregate at pores; distributions may be an
artifact resultant from cytoplasmic movements during
fixation (e.g., Lister, 1895; Jepps, 1942). In our specimens, it appeared that the higbest mitochondrial densities were in apertural cytoplasm, suggesting that
pseudopodia serve as a site for mitochondrial activity,
as first proposed by Doyle ( 1935), who observed mitochondrial movement through foraminiferal pseudopodia. While it is generally thought that mitochondria
possess only the aerobic respiratory chain, an instance
has been documented where mitochondria are known
to activate an anaerobic
respiratory
chain when
required (Finlay et al., 1983). Alternatively, organelles
with anaerobic respiratory capabilities, which are indistinguishable from mitochondria on an ultrastructural
basis. may co-occur with “normal”
mitochondria
(Takamiya et al., 1994). Rigorous, quantitative studies
are required to determine whether either of these possibilities occurs in foraminifera.
The highly vacuolated nature of the observed cytoplasm is typical of foraminifera (e.g., see Anderson
and Lee, 199 1 for review). Two types of vacuoles have
been noted in foraminiferal cytoplasm (Bowser et al.,
1985): digestive food vacuoles (i.e., lysosomes) and
those with an unidentified function. Determining this
role may be crucial for better understanding
the cell
biology of foraminifera from both aerated and oxygendepleted environments.
Because all four species in our experiment survived
nnoxia for at least 3.5 weeks, our results support the
assertion that benthic foraminifera
are facultative
anacrobes. However, even though Oz levels were undetectable, it is possible that enough oxygen was present
to permit continued respiration. Regardless of whether
the foraminifera were respiring aerobically or anaerobically, the significant decrease in the ATP concentrations of N,-incubated
specimens in three of the four
species studied indicate that most species are affected
by extended exposure to severe oxygen depletion. Furthermore, prelimin~y
studies of the [ATP] in reaerated N?-incubated
specimens
indicate
that ATP
concentrations
remained depressed even after the
return of 0, (Bernhard and Alve, unpubl. data), suggesting that foraminifera are permanently affected by
extended exposure to anoxia. Decreased ATP coneentrations in foraminifera have been attributed to a lack
of food (Graf and Linke, 1992; Linke, 1992) since
concentrations
in some deep-sea foraminifera
were
drastically increased after an input of organic material
to the seafloor. In our experiment, it is likely that the
significantly lower [ATP] in the aerated specimens
compared with that of specimens extracted one week
after collection was also due to a lack of food. However,
the significantly decreased [ ATP] in the N,-incubated
specimens compared with the controls must be attributed to an additional factor since neither treatment was
fed.
It is unfortunate that ATP turnover rates could not
be determined in this study, since that data could be
used in conjunction with [ ATP] data to infer possible
metabolic pathways employed by the nitrogen-incubated foraminifera. Because the [ ATP] in Stuinforthia
fusiformis was depleted less than in BuEimina murginara or Adercotryma glomeratum, it is likely that S.
fus~furmis uses a different, more efficient pathway or
has a higher metabolic rate than the other two species.
The presence of high numbers of peroxisome-ER complexes and/or chloroplast-like structures may in some
way account for this inferred greater efficiency. Elxperiments are presently underway to identity the alternative metabolic pathways employed by facultative
anaerobic foraminifera.
Acknowledgements
We thank the crew of the R/V Trygoe Braarud, the
staff of the University of Oslo Biology Department’s
TEM facility, Hans Grav, Sam Bowser, and Grisel Osorio for their assistance in various aspects of this study.
This work was partially supported by Biotechnological
Resource grant PHS RR 01219 to support the Wadsworth Center’s Biological Microscopy
and Image
Reconstruction facility and a National Biotechnological Resource and primarily supported by a Norwegian
Research Council for Science and the Humanities grant
to EA, the Fulbright Foundation
(research grant to
JMB), Norge-Ame~ka
Foreningen, and NSF grants
OCE-92 11166 and OCE-94 1’7097 to JMB.
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