Algal chloroplasts in the protoplasm of three species of benthic foraminifera: taxonomic affinity, viability and persistence

Marine Biology 53, 201-211 (1979) MARINE BIOLOGY �9 by Springer-Verlag 1979

Algal Chloroplasts in the Protoplasm of Three Species of Benthic Foraminifera: Taxonomic Affinity, Viability and Persistence

E, Lopez*

Institute of Ecology and Genetics; Ny Munkegade, DK-8000 Aarhus C, Denmark

Abstract

The ultrastructure and pigment content of algal chloroplasts (derived from Bacil- lariophyceae or Chrysophyceae) are described from 3 benthic species of brackish- water foraminiferans. Elphidiumwilliamsoni Haynes contains 4 x 106 chloroplasts mg -I , whereas the contents of Nonion germanicum (Ehrenberg) and E. excavatum (Terquem) are about 10% of this value. The two former contain chlorophylls a and c and fucoxanthin, but these pigments were not detectable in the latter. E. williamsoni and N. germanicum had a net uptake of 14C-HCO~, proportional to their content of chlorophyll and number of chloroplasts, increasing linearly up to approximately 10 Klux. At light saturation the former assimilates 2.3 x 10-3 mg C mg -I h -I and the latter only about 20% of this value. Dark uptake was insignificant in all cases. Uptake could not be demonstrated in E. excavatum. The photosynthesis effected by these species is trivial in terms of the total benthic carbon fixation effected by the mi- croflora. The chloroplasts survived longer in foraminiferans kept in the dark than in light/dark adapted individuals. To keep a steady state population of chloroplasts under light/dark conditions, E. williamsoni must eat at least 65 chloroplasts individual-1 h-l, whereas the minimum consumption rate in N. germanicum is 20.

I ntroduction

Associations with algae are common amongst foraminiferans, since the latter often exhibit delayed digestion ("food farming"); the algae survive in the protoplasm of the foraminiferans according to their resistance to digestion (Lee, 1974; Lee et al., 1974). In this respect, different types of associations have been demonstrated in experiments with algae-bearing members of the Nummuliti- dae and Soritidae. Thus, Heterostegina depressa lives solely on its symbiotic diatoms (R~ttger, 1972; R~ttger and Berger, 1972; Schmalljohann and R~ttger, 1978), whereas Archaias angulatus and Sorites marginalis obtain less than 10% of their total carbon budget (Lee and Zucker, 1969; Lee and Bock, 1976) from their symbiotic green algae (Lee et al., 1974) and dino- flagellates (MHller-Merz and Lee, 1976). These differences may be correlated with

*Present address: Curriculum in Marine Sci- ences, University of North Carolina, 12-5 Venable Hall O45-A, Chapel Hill, North Caro- lina 27514, USA.

the survival of the algae in the foraminiferans, since H. depressa needs no exter- nal food source, whereas primary production in A. angulatus is increased when fed isolated symbionts. This study describes a new type of "food farming" in foraminiferans, of isolated algal chloroplasts in the protoplasm of 3 benthic, brackish-water species, Elphidium williamsoni Haynes, Nonion germanicum (Ehrenberg) and E. excavatum (Terquem) (cf. Hansen and Lykke-Andersen, 1976). The study has been concerned with the taxonomic affinity of the chloroplasts, and their photosynthetic ability and persistence in the foraminiferans, in order to investi- gate whether this could constitute a "chloroplast symbiosis", a phenomenon hitherto described only for sacoglossan opisthobranchs (Trench, 1969, 1975; Tay- lor, 1970, 1974; Trench et al., 1973; Hinde and Smith, 1974).

MaWrialsandMcthods

The taxonomic affinity of the chloroplasts was determined by means of their

0025-3162/79/0053/0201/S02.20

202 E. Lopez: Algal Chloroplasts in Foraminifera

ultrastructure, pigment composition and size. Photosynthetic ability was tested in fresh individuals of Eiphidium williamsoni, Nonion germanicum and E. excavatum, as light-induced uptake of 14C-HC03; the persistence of the plastids was analyzed in starved foraminiferans adapted to alternating light and dark or to complete- ly dark conditions, as a decrease in photosynthetic carbon uptake, content of chlorophyll, and the number of chloroplasts.

Collection and Isolation

The foraminiferans were isolated from surface sediments from two stations (depth O to 0.5 m and 6 m) in a Danish brackish water area, Limfjorden. Elphidium williamsoni and Nonion germanicum OCCUr together at the shallowest station, whereas ~. germanicum and E. excavatum coexist at the deeper station, the density of each species being approximately 105 individuals m -2 .

The sediment was wet-sieved, and individuals for the electron microscopic analysis were picked out from the 125 to 250 ~m sediment fraction and fixed imme- diately; in the remaining analyses and experiments, foraminiferans from 250 to 375 Lua sediment fraction were used. These individuals were washed several times in membrane-filtered sea water (HA-Millipore) to remove adhering algae. During this procedure, they were kept in the dark at temperatures below 15~ Samples of washed foraminiferans were examined under the fluorescence microscope; contamination was on an average I algae per 10 foraminiferans, and hence no corrections were made for adhering algae. All analyses and experiments were started within I week of collection, and less than 3 days after isolation of foraminiferans from sediment.

Ultras tructure

The method applied in the preparation of the foraminiferans for ultrastructural investigations with transmission electron microscope was developed by Dr. C. F. Bardele, University of T~bingen, FRG (unpublished method).

The foraminiferans were fixed at room temperature (ca. 20~ and pH 6.6 in 2% glutaraldehyde (I h) and then in osmium tetroxide (2%, lh); they were decalci- fied at all stages of fixation with EGTA (Serva) incorporated into the cacodylate buffer. They were embedded in Epon 812, sectioned with a diamond knife on an LKB Ultrotome, stained with lead citrate and

uranyl acetate, and examined at 60 kV in a Philips 301 microscope.

Pigment composition and Chlorophyll Content

Two-dimensional thin-layer chromatography on sucrose plates (icing sugar with 10% potato starch) was used in the study of single pigments, in accordance with methods described by Jeffrey (1968). Ex- tractions were made with 90% acetone on samples of 500 to 1OOO individuals; the separated pigments were eluted in 90% acetone, and their absorption spectra were measured with a high-sensitivity spectrophotometer (Aminco DW 2a, UV-Vis). The pigments were identified by means of their absorption spectra, Rfvalues, and color.

Crude analyses of pigment content were made with extracts in 80 or 90% acetone on 300 to 500 specimens, and the absorption spectra of the extracts were measured with the Aminco spectrophotometer or a Unicam SP.800. The total amount of chlorophyll was calculated as described by Strickland and Parsons (1972).

Abundance and Size

Abundance and size of the chloroplasts were determined with an epifluorescence microscope, using the autofluorescence of pheophytin and chlorophyll (Fenchel and Straarup, 1971). Counts and measurements were made on samples of 100 crushed individuals on irgalan black- stained 0.2 ~m Nuclepore filters (Hobbie et al., 1977).

Photosynthetic Carbon Fixation

The photosynthetic ability of the chloroplasts was measured in samples containing 52 to 68 ~Ci 14C-HCO~ mmol-1, corresponding to a total concentration of 12.4 ~Ci per 1OO ml sea water (25 to 30~ S). The total CO 2 content was determined by pH and alkalinity measurements (Strickland and Parsons, 1972). Each sample consisted of 20 individuals that were incubated in centrifuge tubes (dark controls were wrapped in aluminium foil), to which a small amount of autoclaved sediment (grain size 10 to 20 ~un) was added to provide a suitable physical substrate. Following incubation, the foraminiferans were collected on a plankton gauze (mesh size 63 ~m) in a Millipore filtering apparatus, and rinsed several times with filtered sea water to remove inorganic label and ad-

E. Lopez: Algal Chloroplasts in Foraminifera 203

hering sediment. They were then fixed in 5% neutral formaldehyde in filtered sea water, crushed, and counted in a liquid scintillation spectrometer, with Insta- gel as scintillation liquid. In all experiments, light intensity was measured with a Gossen luxmeter (I Klux =approximately I. 11 J m -2 sec -I), and a Beckman Microbalance (LM-5OO) was used for de- termination of the ash-free dry weight (2.4 to 2.5 ~g individual-l), for which samples of 50 foraminiferans were dried at 80~ to constant weight and ashed for 4 h.

A series of experiments were con- ducted to determine the effect of light intensity on primary production. All experiments were made with incubations of foraminiferans in artificial light (At- las Super-Gro, 20 W) at 15~ for 4 or 8 h.

Primary production under natural conditions was measured in foraminiferans incubated in sequential 2 h periods for 24 h.

The rate of loss of 14C-photosynthate from Elphidium williamsoni and Nonion germanicum was determined in control experiments at 7,000 lux and 15~ in which the foraminiferans were incubated in sea water containing 14C-HC03 for 8 h, then rinsed, and transferred to non-labelled medium. The loss rates were measured in samples taken O, 4, 8 and 12 h after transfer, giving an average loss rate of 0 to 3% uptake rate in both species.

survival of Chloroplasts

The persistence of chloroplasts in Elphi- diumwilliamsoni and Nonion germanicum was measured in individuals starved either in alternating light (3,500 lux) and dark (16 h light:8 h dark) or in complete darkness, at 15~ and 22~ S. Each day, adapted foraminiferans were incubated with 14C-HCO3 (10.3 ~Ci per 7OO ml sea water) for 8 h at 3,500 lux or as dark controls. The decrease in content of chlorophyll and number of chloroplasts was measured every second day in similarly adapted individuals.

Results

Several specimens of Eiphidiumwilliamsoni, Nonion germanicum and E. excavatum were examined in the transmission electron microscope, and the only recognizable algal structures found were isolated algal chloroplasts, apparently of only one type. This type is illustrated in Fig. I for E. williamsoni. Fig. I: i shows the protoplasm in the proximal part of cham-

bers in the outer whorl of the foraminiferan. Chloroplasts are found throughout the protoplasm of all 3 species, but tend to be concentrated in the outermost parts.

No entire organelles have been observed in association with the chloroplasts, but quite often tubular structures, that could be part of algal organelles, were observed in the lumen between the chloroplast and the vacuolar membrane. These tubules are sometimes aggregated (Fig. I: 2, T).

Fig. I: 2 and 3 illustrate the chloroplasts in detail, and in the following they will be described in accordance with the chloroplast nomenclature of Dodge (1973).

The chloroplasts consist of a central, relatively fine-grained body, the pyrenoid, which is surrounded by a coarser region containing the lamellae of thylakoids, in which the chlorophylls are concentrated.

The pyrenoid is crossed by I lamella, consisting of 3 thylakoids (Fig. I: 2, PL). Sometines a faint membrane-like structure can be distinguished in the distal part of the pyrenoid (Fig. I: 3, M).

The lamellae in the coarse-grained part of the chloroplast consist of 3 thylakoids (L in Fig. I: 2 and 3). Thy- lakoids crossing from one lamella to an- other can be seen relatively frequently (Fig. I: 3, C). No grana (stacks of thylakoids) have been observed.

The entire chloroplast is surrounded by a girdle lamella, mostly consisting of 3 thylakoids (G in Fig. I: 2 and 3). At each end of the chloroplast, close to the girdle lamella, I or 2 electron- translucent areas (Fig. I: 2, D) often occur. These are probably the areas where the chloroplast deoxyribonucleic acid (DNA) is concentrated, a feature closely connected with the presence of a girdle lamella (Dodge, 1973).

The chloroplast is bounded by a double membrane, the chloroplast envelope (Fig. 1: 2, E); sometimes, situated towards the vacuolar lumen, fragments of a second double membrane can be seen (Fig. I: 2, S, between the arrows). This is probably part of the endoplasmic reticu- lum sheath which surrounds the chloroplast in entire algae.

Occasionally, decaying chloroplasts are found in Eiphidium williamsoni (Fig. I: 4).

In Nonion germanicum and Elphidium excavatum, the chloroplasts are of the same type as or very similar to the chloroplasts in E. williamsoni, since they have a girdle lamella and the same type of pyrenoid. However, no detailed study of

Fig. i. Elphidium williamsoni; ultrastructure. 1: Chloroplasts in chambers in outermost whorl ix 3,000). 2, 3: Details of chloroplasts; C: interconnecting thylakoids between lamellae; D: DNA areas; E: chloroplast envelope; G: girdle lamella; L: lamella consisting of 3 thylakoids; M: membrane-like structure bounding pyrenoid; PL: lamella running through pyrenoid, consisting of 3 thylakoids; S: (presumably) part of endoplasmic reticulu~n sheath; T: tubules in vacuolar lumen (2 x 22,600; 3 x 27,500). 4: Decaying chloroplast in outermost part of chamber (x 12,OOO)


the lamellae was possible in these two species, because many of these chloroplasts were decaying at the time of fixation and because of problems in fixation.

The absorption values of the total pigment content in the foraminiferans (Fig. 2) are expressed in units per mg ash-free dry weight of the foraminiferans. Elphi~um williamsoni and Nonion germanicum have very similar absorption curves, that of the former being about 4 times greater than that of the latter (E. williamsoni: 5.6 ~g chlorophyll rag-l; N. germanicum: 1.6 ~g chlorophyll mg-1). E. excavatum achieves maximum absorption at a shorter wavelength than the other two species.

The two-dimensional thin-layer chromatography separated 6 distinct pigments in Elphidiumwilliamsoni and Nonion germanicum, but only 4 in E. excavatum (Fig. 3). Pigment Spot I had an absorption maximum at 425 nm in E. excavatum and was identified as pheophytin c. In E. williamsoni and N. germanicum the same spot had maximum absorption at 444 nm and was predom- inantly chlorophyll c (Smith and Beni- tez, 1955). Pigment Spot 2 has been identified as fucoxanthin (Jeffrey, 1968; Hallegraeff, 1977) (maximum absorption 446 to 450 nm). Pigment Spot 3 was chlorophyll a (maximum absorption 412 to 420 nm; peak at 656 nm) and, like fucoxanthin, it was only found in E. williamsoni and N. germanicum. The rest of the pigments (Spots 4 to 6) occurred in all 3 species; Pigment 4 was a xanthophyll with maximum absorption at 451 nm and a shoulder at 470 to 475 nm; this pigment is probably diatoxanthin or a mixture of diatoxanthin and diadinoxanthin. Pigment 5 was pheophytin a (maximum absorption

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409 nm; peak 662 nm), and Pigment Spot 6 contained I or more carotenes (maximum absorption 450 nm; small peaks at 425 and 475 nm).

Examination with the fluorescence microscope revealed that the diameter of the chloroplasts in all three species is very similar (Elphidium williamsoni: 3. I +- I. 8 ~m; Nonion germanicum: 2.9 + I. 4 ~m; E. excavatum: 2.8 + I .O ~/n).

Elphidium williamsoni contains the largest number of chloroplasts (4.0 _+ 2.0) x 106 mg-1~ corresponding to (9.7 _+ 4.9) x 10 3 individual -I. This is about 8 times the average number in the other two species (Nonion germanicum: (5.2 _+ 1.6) x 105 mg-1 (1.3 _+ 0.4) x 10 3 individual-l; E. excavatum: (5.0 -+ 2.9) x 105 tug-1 (1.2 + 0.7) x 10 3 individual-l). The number of chloroplasts in E. williamsoni and N. germanicum was the same in individuals collected in summer and in winter, and therefore a steady state population of chloroplasts independent upon season is assumed to exist in these foraminiferans.

In the study of photosynthetic carbon fixation at different light intensities, the rates of light fixation in Elphidium williamsoni always differed significantly from the corresponding dark controls -- (9.4 + 3.4) x 10-5 mg C mg -I ash-free dry weight h -I, which is on an average

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per mg ash-free dry weight (80% acetone extracts) green; 6: carotenes, yellow


4% of the total light uptake at saturation level. The light values for Nonion germanicum were also significantly different from dark values, except at the lowest light intensities -- (1.6 • 0.6) x 10 -4 mg C mg -I ash-free dry weight h -I, i.e., 25% of total light fixation at saturation level. There was no difference between light and dark uptake in E. excavatum -- (5.3 • 2.3) x 10-5 mg C mg -I ash-free dry weight h-1.

In Fig. 4 the results of these experiments are given as net light fixation. The lines have been calculated from the mean values at light intensities less than 8,000 lux. The slopes of the lines indicate that Elphidiumwilliam- soni on an average takes up inorganic carbon at a rate about 5 times that in Nonion germanicum, and that the chloroplasts in E. excavatum are inactive with respect to primary production.

The variation in primary production during 24 h was measured in Eiphidium wil-

liamsoni and Nonion germanicum in natural light. The production rates are plotted in Fig. 5, together with the variation in light intensity and temperature. The experiments were made on a cloudy day with relatively small variation in temperature. Both species reached light saturation level a few hours after dawn

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Fig. 4. Elphidium williamsoni (circles), Nonion germanicum (triangles) and E. excavatum (squares). Primary production with artificial light source as a function of light intensity, in mg carbon per mg ash-free dry weight of foraminiferans per hour; dark controls subtracted. Values are means and ranges; for each species, 2 to 4 measurements were made at each light intensity investigated

and remained at this level for most of the remaining day.

In Fig. 6 the mean values of primary production from these experiments in natural light are given as a function of light intensity. This figure also shows the constructed light saturation curves for the two species. The saturation levels of these curves were calculated from the results of samples taken around mid- day. Thus, primary production rates at light saturation are 2.3 x 10-3 mg C mg -I ash-free dry weight h -I for Elphidium williamsoni and 5.0 x 10-4 mg C mg-1 ash- free dry weight h-1 for Nonion germanicum; the latter rate is about 20% of the production rate of E. williamsoni. The under- saturated parts of these curves are the regression lines calculated from the results of the experiments in artificial light. The data suggest that the chloroplasts in the two species have the same

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Fig. 5. El~idi~ willi~soni (circles) and No- nion germanicum (triangles). Variation in primary production throu~out a 24 h period in natural light; values are means and ranges of primary production in mg carbon per mg ash-free dry weight of foraminiferans per hour; dark controls s~tracted. For both species, i to 2 measurements were made d~ing each period. Variations in mean values of light intensity (squares) and temperature (crosses) are also shown


Fig. 6. Elphidium williamsoni (circles) and No- nion germanicum (triangles). Primary production in natural light (data from Fig. 5) as a function of light intensity (mean values), and constructed light-saturation curves. Saturation levels of light-saturation curves were calculated from primary production rates around mid- day in natural light (cf. Fig. 5); undersatu- rated part of the curves (light intensities up

to ca. i0,OOO lux) represents regression lines from Fig. 4. Values are in mg carbon per mg ash- free dry weight of foraminiferans per hour; dark

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Fig. 7. Elphidium williamsoni and Nonion germanicum. Survival of chloroplasts in starved individuals of E. williamsoni (dark-adapted = filled circles; light/dark adapted = open circles) and N. germanicum (dark-adapted = filled triangles; light/dark adapted = open triangles) as a function of adaptation time. (A) Photosynthetic carbon fixation (mean and range, duplicate measurements) as counts min -I 20

individuals -i h -I. (B) Content of chlorophyll, 90% acetone extracts, as ug total chlorophyll per individual. (C) Number of chloroplasts per individual. Equations and correlation coefficients (r 2) are indicated for both adaptation series of each species in all 3 types of measurements


Table i. Elphidium williamsoni and Nonion germanicum. Turnover time (TT, in hours) and initial loss rate (ILR, in chloroplasts individual -i h -l) of chloroplasts in foraminiferans starved either in alternating light and dark (L/D) or in complete darkness (D). Calcula- tions are based on measurements (cf. Fig. 7) of photosynthetic carbon fixation (A), chlorophyll content (B) and number of chloroplasts (C), using the exponent of the equations and the assumed steady state population of chloroplasts in the foraminiferans

Species and Turnover time and initial light condition loss rate

A B C TT ILR TT ILR TT ILR

E. williamsoni

D 830 iO 13OO iO L/D 45 230 155 65

N. germanicum

D IO5 15 150 iO L/D 25 60 6O 20

380 30 105 95

i00 15 55 25

saturation light intensity (ca. 10,0OO lux) .

Chloroplasts in Elphidium williamsoni and Nonion germanicum (Fig. 7) survive for a longer time in individuals adapted to continuous darkness than those kept in alternating light and dark. The regression lines were calculated assuming an exponential loss (least-squares fit) of chloroplasts. The photosynthetic carbon- fixation experiments were terminated be- fore light-fixation values equalled dark controls. In each adaptation series, photosynthetic ability (measured in counts per minute), chlorophyll content, and number of chloroplasts decrease at comparable rates, indicating that the photosynthetic efficiency of the chloroplasts, measured in carbon fixed per mg chlorophyll and per chloroplast, does not differ between fresh and starved individuals. Using the average number of chloroplasts given in the previous sec- tion, the average turnover times and initial loss rates of chloroplasts were calculated (Table I).

Discussion and Conclusions

The isolated chloroplasts in Eiphidium williamsoni, Nonion germanicum and E. excavatum

the pyrenoid and the presence of a girdle lamella, the latter being present only in 5 classes of Chromophyta: Chlo- romonadophyceae, Xanthophyceae, Chryso- phyceae, Bacillariophyceae and Phaeophy- ceae (Dodge, 1973). The 3 thylakoids per lamella, the absence of grana, and the interconnecting thylakoids between lamellae are also characters common to these classes. The pyrenoid is of the simple internal type with one lamella running through it (Dodge, 1973), a type known from several species of Bacillario- phyceae. In many of these species there is a reduction in the number of thylakoids in the lamella of the pyrenoid (Drum and Pankratz, 1964), but in Nitz- schia sp. (Drum and Pankratz, 1964), Nitz- schia palea (Drum et al., 1966), and Melo- sira varians (Crawford, 1973) this lamella consists of 3 thylakoids. In a member of Chrysophyceae, Ankylonoton luteum (Van der Veer, 1970), the same type of pyrenoid, without reduction in number of thylakoids, is present. The investigated members of the remaining 3 classes either do not contain pyrenolds, or these are of a different type than that described here (Dodge, 1973).

The chlorophylls a and c are typical for the algal classes Chrysophyceae, Bacillariophyceae, Phaeophyceae and Di- nophyceae, whereas the most abundant xanthophyll, which was identified as fucoxanthin, is not present in the latter (Egle, 1960).

The ultrastructure and pigment composition therefore suggest that the chloroplasts originate from Chrysophyceae or Bacillariophyceae. However, at this level it is impossible to know how many algal species are donors of chloroplasts to the foraminiferans or to determine whether the chloroplasts of the three species of Foraminifera studied are derived from the same algae. All three species are deposit-feeders, feeding on sediment particles that they collect from their surroundings with their pseu- dopods. Because they have not been able to catch phytoflagellates, motile diatoms and ciliates, when observed under dissection microscope, they are probably unable to capture motile food organisms. Therefore it is assumed that they obtain their supply of chloroplasts from ses- sile algae living on the surface of sediment particles.

Symbiotic algae belonging to either Chrysophyceae or Bacillariophyceae have been described in association with other

are typical eukaryotic organelles (Dodge, species of foraminiferans: Amphistegina Io- 1973) of algal origin, since they pos- bifera, "Cycloclypeus" tuberculatus, Operculina sess a pyrenoid (Griffiths, 1970). The ammonoides and Heterostegina depressa (Leu- most important characteristics for fur- tenegger, 1977; Schmalljohann and R~tt- ther identification are the structure of ger, 1978), but none of these algae con-


tain chloroplasts identical to those in Elphidium williamsoni, Nonion germanicum and E. excava rum.

Thin-layer chromatography revealed that the observed difference in absorption spectrum on crude pigment extracts between Elphidium excavatum and the other two species is caused by a lack in the former of the photosynthetically essential pigments, chlorophylls a, c and fucoxanthin. In accordance with this, no light-induced uptake of 14C-HCO~ by the chloroplasts in E. excavatum could be de- tected, which means that they are probably merely food items being digested by the foraminiferan. As opposed to this, E. williamsoni and Nonion germanicum pos- sessed a photosynthetic ability proportional to their content of chlorophyll and number of chloroplasts. Therefore, the association between the algal chloroplasts and these two species might be "chloroplast symbiosis", a term defined by Hinde and Smith (1974) as "the situ- ation where an animal takes up and re- tains free, undamaged chloroplasts under natural conditions, shows active photosynthesis for at least several days after removal from its host plant, and utilizes products of photosynthesis released from the chloroplasts", although in this study there is no evidence pre- sented for transfer of photosynthetic products from chloroplasts to hosts (see also Trench, 1975).

Total daily production by foraminiferans at the shallow station can be estimated using the results of the primary production experiments in natural light as approximately 3 x 10 -2 mg C mg-1 ash-free dry weight day -I in Elphi- dium williamsoni and 6.5 x 10-3 in Nonion germanicum. Assuming that the thickness of the photic zone in the sediment (Fenchel and Straarup, 1971) is 2 mm (the density of the 2 species being 4.2 x 104 and 1.6 x 104 individuals m -2, respectively) then, at this station, the total primary production by the chloroplasts in the foraminiferans would be 3.26 mg C m -2 day -I. At this station, the total primary production of algae at the same time of the year has been estimated as approximately 680 mg C m -2 day-1 (unpublished results), which means that about 0.5% of this production is achieved by the chloroplasts in the foraminiferans. This is in contrast to estimates in a tropical environment, where symbiotic algae in foraminiferans accounted for most of the primary production (Sournia, 1976).

The turnover times of chloroplasts estimated for Elphidium williamsoni and No- nion germanicum are within the range of previous estimates of survival of symbi-

otic chloroplasts in sacoglossan opisthobranchs (Trench et al., 1969; Greene, 1970; Hinde and Smith, 1972, 1974, 1975), that can vary from a few days to several months, the longevity of the association probably reflecting the overall importance of these chloroplasts in the energy budget of their hosts (Hinde and Smith, 1975). The higher loss rates (ILR in Table I) of chloroplasts in light/dark than in dark are in accordance with measurements of survival in sacoglossans, and are probably caused by a faster depletion in the light of cer- tain photosynthetically essential com- pounds (Hinde and Smith, 1975), whose synthesis is controlled by the algal nuclei (Trench et al., 1973). Furthermore, the different loss rates indicate that the foraminiferans require a different input of chloroplasts to maintain a steady state in light and dark. Under natural conditions, the loss rates in the uppermost part of the illuminated zone of the sediment are probably higher than those measured in these experiments, since light intensity is usually much higher than 3,500 lux. This means that the consumption rates of the chloroplast donors in the foraminiferans are presumably higher at the sediment surface than the estimated loss rates.

The results of the primary production experiments with the foraminiferans rep- resent the total light-induced uptake of 14C-HCO~, assuming that the respiratory loss during the uptake period was negli- gible (control experiment gave an average total loss rate of O to 3% of uptake rate).

The presence of symbiotic dinoflagel- lates in reef-building corals have been demonstrated to accelerate calcification in the light (Goreau, 1959, 1963; Goreau and Goreau, 1959; Vandermeulen and Mus- catine, 1974), and in the reef-coral Po- cillopora damicornis the 14C built into the skeleton was 9 to 12% of total uptake over a 24 h period (Muscatine and Cerni- chiari, 1969). In accordance with this, light-enhancement of calcification has been demonstrated in laboratory experiments with the symbiont-bearing foraminiferans Archaias angulatus (Lee and Zucker, 1969) and Amphistegina lessoni (Mul- ler, 1978); in the latter, the deposition of carbon into CaCO 3 was estimated to approximate 10% of the total uptake. However, in field experiments with Archa- ias angulatus and Sorites marginalis, Lee and Bock (1976) found no evidence of light- enhanced calcification; this may have been due to inadequate incubation time.

From literature values on respiration of other foraminifer species -- Ammonia beccarii tepida (Bradshaw, 1961), Allogromia


laticollaris, Rosalina leei and Spiroloculina hyalina (Lee and Muller, 1973) -- and other types of meiofauna of similar size (Lasserre, 1976), tentative estimates of the respiration of the foraminiferans can be calculated as (0.3 to 1.0) x 10 -3 mg C mg -I ash-free dry weight h -I. As- suming that 10% of the carbon uptake is

deposited as CaC03 in the shells, then the carbon gain through photosynthesis at maximum respiratory rate at light saturation level will be approximately 200% of the respiratory loss in Elphidium williamsoni and 45% of that in Nonion germanicum. However, the actual importance for the foraminiferans of the chloroplasts' photosynthetic activity depends upon the amount of photosynthate released to the hosts. In chloroplast-bearing sacoglossans, 20 to 50% of the photosynthetically fixed carbon is transferred from

chloroplasts to hosts (Trench, 1975). If similar proportions of photosynthate are transferred to the foraminiferans then the photosynthetic activity of the chloroplasts could account for 40 to 100% of respiration in E. williamsoni and 10 to 20% in N. germanicum.

Acknowledgements. My gratitude is due to Drs. C.F. Bardele and J. Theilade for their kind ad- vice and help in connection with the ultrastructural study. I am grateful to Dr. B. Deutch for help in pigment analyses and for providing the energy conversion data for the light measurements. I am also grateful to Ms. T. Kelstrup, Ms. A. SElling and Ms. K. Andersen for assis- tance in preparations. Dr. H.J. Hansen kindly read the manuscript. I am especially thankful to Professor T. Fenchel and Dr. G. Lopez for valu- able discussions and encouragement during this study. The spectrophotometers were provided by the Institute of Medical Biochemistry, Aarhus, and Department of Plant Physiology, Aarhus. The study was supported by a grant from the Danish Natural Science Research Council.

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Algal chloroplasts in the protoplasm of three species of benthic foraminifera: taxonomic affinity, viability and persistence