Spongilla lacustris. Photosynthesis by symbiotic algae in the freshwater sponge,

Transcription

1 Limnol. Oceanogr., 39(3), 1994, , by the American Society of Limnology and Oceanography, Inc. Photosynthesis by symbiotic algae in the freshwater sponge, Spongilla lacustris Kaj Sand- Jensen and Marten Foldager Pedersen Freshwater Biological Laboratory, Helsingssrgade 5 1, DK 3400 Hillerod, Denmark Abstract Spongilla lacustris is a common freshwater sponge which becomes dark green at high illumination due to the presence of numerous symbiotic zoochlorellae. Oxygen metabolism of Spongilla from a shallow Danish stream was analyzed in relation to concentration of tissue chlorophyll, incident light, and external CO, concentration. Photosynthesis at light saturation increased linearly with chlorophyll content of the sponge. High light intensity was needed to saturate photosynthesis in green Spongilla because of strong light attenuation in the sponge tissue, whereas. isolated zoochlorellae in suspension saturated at low light. High CO, concentration- substantially above air saturation- was also required to saturate photosynthesis of green Spongilla. We found that daily net production of SpongilIa was low based on photosynthesis alone, and filtration of particles was probably needed to support the prolific growth observed in the stream. The estimated net gain from symbiotic algae could, however, substantially increase Spongilla growth in the light. Spongilla lacustris L. is distributed worldwide and is a prolific sponge in temperate ponds, lakes, and streams. The nutrition of Spongilla is based on filtration of organic particles and photosynthesis of endosymbiotic zoochlorellae (Gilbert and Allen 1973; Frost and Williamson 1980). Individuals without zoochlorellae occur in deep shade and can grow well given suitable food particles (Kilian 1964), while green individuals with zoochlorellae are found associated with high illumination (Williamson 1977). Both types have similar clearance rates of food particles in laboratory experiments (Frost and Williamson 1980), but light stimulates growth of Spongilla in field experiments (Frost and Williamson 1980). Spongilla forms a l-lo-mm-thick cover on solid substrates from which erect branches (2-10 mm thick) develop under suitable growth conditions. The internal system of ventilated channels lined with choanoflagellates is essential for particle filtration and gas exchange with the surrounding water. The thick tissue should attenuate light and may limit the supply of O2 and CO2 to algae and host cells within the tissue. In the dark, the symbiotic algae and host use O2 supplied from the surrounding wa- Acknowledgments This work was supported by grants from the Danish Natural Science Research Council (1 l-7795). We thank Ole Tendal for help with taxonomic literature and Patricia Kremer, Niels Peter Revsbech, Marten Sondergaard, and two anonymous referees for comments on the manuscript. 551 ter. CO, limitation of photosynthesis is more likely than O2 limitation of respiration, because light-saturated photosynthesis often exceeds dark respiration (Gilbert and Allen 1973), and air-saturated water contains -20 times more O2 (3 15 PM at 15 C) than CO, (15 PM, Stumm and Morgan 1970). The supply rate of particles and dissolved materials to Spongilla depends on the ventilation rate and the concentrations in the bulk medium. The growth of Spongilla in Denmark is luxurious in streams compared with standing waters (Sand-Jensen pers. obs.), implying that the continuous flow of water bringing food particles, 02, and CO, to the sponge surface is bene ;cial to growth. The objective of this study was to analyze photosynthesis of Spongilla in relation to concentration of chlorophyll from zoochlorellae, incident light, and external CO2 concentration. We evaluated the importance of self-shading and transport limitation within the sponge and compared the results with photosynthesis of isolated algae from Spongilla in suspension. Finally, we estimated the contribution of symbiotic photosynthesis to the growth of Spongilla in the field by applying field measurements of light to relationships between photosynthesis and light measured in the laboratory. Methods Field site-spongilla was sampled between late June and early August 1990 in Mattrup

2 552 Sand- Jensen and Pedersen A, an unshaded stream in Jutland. The stream and illuminated at 120 or 1,000 PEinst rna2 is 5 m wide and -0.5 m deep in summer. s-l during l-2-h incubations or kept in the Water discharge is liters s- l, and mean dark for 2-4 h. Oxygen concentrations were stream velocity is m s-l. The stream determined by duplicate micro-winkler titrais nutrient-rich, alkaline, and supersaturated tion (precision, 0.01 mg 0, liter-l). with CO, (Jacobsen and Sand-Jensen 1992). A photosynthesis chamber was used in de- Water temperature during collections varied tailed kinetic analysis of photosynthesis curves between 13 and 18 C. Light attenuation was as a function of light (P-Z) and CO, concenmeasured at depth with a cosine quantum sen- tration of intact individuals and suspensions sor (LiCor). The same instrument measured of dark green Spongilla. The photosynthesis light intensities in laboratory experiments. chamber was made of Perspex (290-ml vol) Spongilla was dark green and received 50- and supplied with a Clark-type O2 electrode, 60% of surface light at 0.5-m depth; it was a ph electrode, a temperature transducer, and brown or light green under shaded conditions a submersible pump; the chamber was operbelow overhanging vegetation and bridges. The ated as described by Sand-Jensen ( 1983). flat encrusting part of the sponge covering the Spongilla was attached in the chamber and substratum was thicker and erect branches were illuminated from above with halogen projector more common under high illumination. lamps. Light intensity was varied with neutral Encrusting forms of entire sponges, 2-5 mm density filters. The ph was manipulated in thick, were sampled for experiments and kept some experiments by adding HCl or NaOH to in running tapwater (high quality groundwater) alter the proportions of free CO2 in DIC (prinin dim light. Tapwater was used in the exper-$ ciples from Sand-Jensen 198 3, 1987). The CO2 iments because it had about the same ionic concentration was kept high and saturating to composition, alkalinity (2.10 meq liter- ), and photosynthesis in experiments at varying irdissolved inorganic C content (DIC, 2.30 mm) radiances by maintaining a low, constant ph as the stream water and negligible O2 con- (b 7.0) by repeated additions of HCl. sumption, which may otherwise interfere with The slopes of initial linear parts of the phomeasurements of O2 metabolism. Alkalinity tosynthesis curves at low light or CO2 were and DIC were measured by potentiometric obtained by linear regression analysis. The endpoint- titration (Golterman et al. 1978). photosynthetic rates at high light and CO2 were Conductivity and ph in the stream water and fitted visually and saturation points taken as in experiments were measured with Radiom- light and CO2 levels where 95% of maximum eter equipment. The CO1 concentration was rates (P,,,) were attained. We preferred to excalculated from alkalinity, ph, conductivity, press light saturation levels (I,,,) in this way and temperature according to Rebsdorf (1972). instead of using the Zk value (intersection of The mean CO, concentration at the field site, initial linear slope with the P,,, level) because measured at noon on five dates, was 190 Ifi: 40 of the slow progression of photosynthesis to- PM (*SD) or 13-fold above air saturation. ward saturation at increasing light. Spongilla composition - Spongilla used for Net production (PN) of Spongilla is the rate experiments was blotted dry, and subsamples of O2 production in the light, and dark respiwere analyzed for fresh, dry, and organic dry ration (R,) is the rate of O2 consumption in weight (loss on ignition at 55O C), C and N darkness. Gross production (PC) of zoochlo- (Carlo-Erba CHN analyzer), and chlorophyll. rellae is estimated as the sum of PN and RD, Chl a + b was extracted in N,N-dimethyl-form- assuming that algae and host maintain unalamide and measured spectrophotometrically tered respiration rates in the dark and the light according to Inskeep and Bloom (198 $). during short-term experiments. Photosynthesis and respiration-photosynthesis and respiration rates of Spongilla over a range of chlorophyll concentrations were determined from bottle incubations. Bottles (120 ml) with pieces of Spongilla (N 125 mm3) and water-filled blanks were placed on a rotating wheel (60-cm diam; 12 rpm) in an incubator The diel gross production and respiration of green Spongilla in the stream were estimated from the average P-Z curve (based on five experiments with different sponges) and mean hourly values of available light in July at the mean depth in the stream. Net O2 production was calculated for each light hour, and dark

3 Sponge photosynthesis 553 respiration was assumed constant over the diel cycle. This estimate does not account for rhythmicity in photosynthesis and respiration which may occur in some field populations of symbiotic organisms (Kremer et al. 1990). Surface light was measured at a nearby meteorological station, and the proportion of surface light at the mean depth was measured on several occasions in July. Spongilla suspensions - Suspensions were prepared by homogenizing (cutting and squeezing) green Spongilla with a scalpel in tapwater and filtering the homogenate through a loo-pm Nitex screen. Photosynthesis as a function of light intensity and CO2 concentration was measured in the photosynthesis chamber. Subsamples were collected onto glassfiber filters and analyzed for chlorophyll content. Cell diameter was measured with an ocular micrometer. Oxygen, ph, and photosynthesis within Spongilla- Internal 0, concentrations in Spongilla were measured with a minielectrode (0.6-mm tip diam), a calomel reference electrode, and an O2 meter (Diamond, Electro- Tech Inc.). Oxygen readings were calibrated in air and N,-equilibrated water. The O2 electrode gave identical O2 output in both stagnant and stirred water, making measurements in Spongilla reliable. The response rate of the electrode was measured by moving it rapidly from O,-rich water (86%) to O,-free water. The 98.4% of full response lasted 10 s, allowing measurements of photosynthetic rates up to 5.4 pmol O2 cm-3 min-l, grossly above the rates encountered here. The O2 minielectrode was moved through SpongiZZa by a micromanipulator (accuracy, If: 10 pm). SpongiZZa was fastened on an agar surface in a Petri dish under constant temperature (20 C) and illuminated from above by a projector lamp. Free CO2 was kept constant at - 15 PM by air bubbling or elevated to 300 PM by reducing ph. During the latter experiment, the water surface was covered by plastic film to reduce COZ loss to the air. Oxygen concentrations in the sponge were measured at different light intensities and ambient COZ concentrations. Gross photosynthesis was measured at depth by applying the light-dark switch technique of Revsbech and Jorgensen (1983). Internal ph in SpongiZZa was measured with Table 1. Composition of green Spongilla lacustris. Mean SD n Fresh wt : dry wt 9.5 ko.7 16 Organic dry wt : dry wt 0.62 ko.05 8 Carbon content, g C (g DW) ko.03 8 C : N weight ratio 5.97 ko.07 8 a combination minielectrode (Microelectrode Inc.) connected to a millivoltmeter with high internal resistance. The minielectrode has a 0.5-mm-diameter sensing tip and was advanced by the micromanipulator. Light transmission -Light transmission was measured through the 2-3-mm upper surfaces of several specimens of green SpongiZZa positioned above the quantum sensor in a light beam from the projector lamp. Attenuation [ln(l,/z)] (where I, and I represent the quantum flux density incident on and through the sample) was calculated as a function of sponge thickness (d) (accuracy, +O. 1 mm) and chlorophyll concentration [Chl], according to In&/l) = kd.d or kchl [Chl], thus providing dimensions of the attenuation coefficient (k) as mm- l and m2 (mg Chl)- l, respectively. The depth for 1% light penetration was estimated as In 100/k,. Light transmission was also measured by means of Spongilla suspensions kept in a small glass beaker placed above the quantum sensor. Light attenuation was linearly proportional to the light path through the suspension. Chlorophyll concentrations and light attenuation coefficients of suspensions were calculated as dimensions of m2 (mg Chl)- l. Results Composition and conversions-the composition of SpongiZZa (fresh and dry weight, C, and N content) is shown in Table 1. Conversion of zoochlorella cell volume ( pm3) to C (Strathmann 1967) yielded a C : Chl ratio of 25 : 1 in the zoochlorellae. Chlorophyll concentrations in green SpongiZZa were usually between2and3mg(gdw)+.thus, % of the C pool in green specimens is contained in symbiotic algae. Production andpigmentation - The [Chl] was close to zero in SpongiZZa growing in shade (< 2% of surface light) and up to 3.4 mg (g DW)l in high light (70% of surface light) in late June. Gross and net production of SpongiZZa at CO2 saturation increased linearly and

4 554 Sand- Jensen and Pedersen E irradiance (PEinst mm2 s ) Fig. 2. Light saturation curve of net O2 production at high CO, of an entire green sponge (0) [1.93 mg Chl (g Dw>- 1 and a suspension of green Spongilla lacustris (0). -3 I I Chlorophyll content (mg g DW ) Fig. 1. Oxygen production at CO, saturation as a function of chlorophyll content in whole specimens of Spongilla lacustris at high (O- 1,000 PEinst m-2 s-l) and low (0-120 PEinst m-2 s-l) light levels. A. Gross production (high light, r2 = 0.85; low light, y2 = 0.82). B. Net production (high light, r2 = 0.7 1; low light, y2 = 0.59). C. Dark respiration (r2 = 0.41). All regression lines have slopes significantly different from zero (P < 0.001). significantly over this chlorophyll range both at 120 and 1,000 PEinst m-2 s-l (Fig. 1). The relationships of net production with chlorophyll showed that and 1.44 mg Chl (g DW)-1 in SpongiZZa were needed for O2 production to balance host and algal respiration (PN = 0) at 120 and 1,000 PEinst m-2 s-l, respectively. The slopes of gross production vs. chlorophyll density corresponded to mg O2 (mg Chl)- h-l at 120 PEinst m-2 s- 1 and 1.56 at 1,000 PEinst m-2 s-l. Dark respiration of the sponge ranged from 1.O to 3.9 mg O2 (g DW)- 1 h-l and increased with [Chl], implying enhanced respiration associated with increasing algal content. Production vs. light-oxygen production of green SpongiZZa (3-5 mm thick) in the photosynthesis chamber showed saturation curves with increasing light intensity (Fig. 2). The mean saturation light intensity (Isat) in five separate P-Z experiments was PEinst m-2 s-l (&SD, Table 2), and there was no photoinhibition at the highest light intensity applied. The attenuation coefficient with depth (kd) in the sponge increased with [Chl] according to kd (mm-l) = [Chl] (pg mmd3) (r2 = 0.85, n = 10). The mean value was mm-l for chlorophyll concentrations between 1.6 and 3.2 mg (g DW)-l, corresponding to 99% light reduction within 3.68 mm. The compensation light intensity vc, PN = 0) was high in intact green SpongiZZa (Fig. 2, Table 2) because of self-shading and high respiration rates in the sponge. Self-shading was reduced when SpongiZZa was homogenized and kept in suspensions of low cell concentrations. The P-Z relationships of these suspensions (Fig. 2) were characterized by markedly lower Zsat and Zc values (Table 2). The initial slope (CU) of the P-Z curve was more than 10 times higher with the zoochlorellae in suspension than in intact SpongiZZa (Table 2). The quantum efficiency (4, = cuk) of zoochlorellae was estimated at mol

5 Sponge photosynthesis 555 Table 2. Parameters describing 0, metabolism in relation to light of green, intact individuals of Spongilla lacustris [ mg Chl (g DW)- 1 and suspensions of the sponge. Experiments were run between late June and early August. PAmax) and P,(max)-net and gross production at light and CO2 saturation; R,- dark respiration, Imt - light saturation level; I,-light compensation point; cy- initial slope of production vs. light. Mean values &SD are shown. PAmax) RLI P,(max) I sat Ic a mgo,(gdw)-i h-l mg 0, (mg Chl)- h- mg 0, (g DW)-l h- mg 0, (mg Chl)- h-l mg 0, (g DW)- h-l mg 0, (mg Chl)- h-l PEinst m-2 s-~ PEinst m-2 s-~ pg 0, (mg Chl)- h-l (PEinst me2 s-*)-l 1.66kO kO f kO kO kO kO f & kO O2 produced (mol absorbed photon)- l, based on the measured a! values and attenuation values [k, ko m2(mg Chl)- ] of suspensions. The presence of host tissue in the suspensions contributed to respiration and light attenuation. The Zc value of zoochlorellae alone will therefore be smaller and the quantum efficiency higher than the values for suspensions. The initial slope and the saturation light intensity apply to zoochlorellae alone, because host respiration reduces net O2 production by the same magnitude at all light intensities, maintaining the shape of the P-Z curve. The maximum gross production of intact green SpongiZZa in the photosynthesis chamber averaged 2.62kO.32 mg O2 (g DW)-l h-l. Dark respiration was - 37% of maximum gross production (Table 2). Diel gross production of green SpongiZZa in the siream was estimated at 3 1.O mg O2 (g DW)- 1 id- and respiration at 23.3 mg O2 (g DW)-l cl-. Net production of the sponge was 7.7 mg 0, (g DW)-l d-l, corresponding to 7.7 mg C (g sponge C)- 1 d- 1 (PQ assumed to be 1.25 mol O2 mol-1 C). This production would allow a specific growth rate of only d-l if p.hotosynthesis was the only mode of nutrition. Because the host can exploit the symbiotic algae by receiving translocated products (Gi1be:t-t and Allen 1973) and perhaps by digesting th[e algae, it is not necessary to account for percentages of translocated photosynthetic products in the carbon balance of the sponge, provided that photosynthetically fixed C is not exported (dissolved organic C and zoochlorellae) to the surrounding water. The net gain derived from the algae was estimated assuming an algal respiration rate of 11% of maximum gross production (average for eight planktonic green algae, Geider and Osborne 1989). Diel algal respiration was 6.9 mg O2 (g DW)- 1 d-l, and the net gain was 24.1 mg O2 (g DW)-1 d-l [24.1 mg C (g sponge C)- 1 d- 1. Photosynthesis could therefore increase the specific growth rate of the sponge by d- 1 above that achieved based on particle feeding alone under the same assumptions as above. The d-l represents the net gain achievable by photosynthesis of symbiotic algae, whereas the d-l is the possible growth rate if photosynthesis must also support respiration of the host animal. Production vs. CO2 - Oxygen production of intact green SpongiZZa at light saturation as a function of CO2 concentration is shown in Fig. 3. The relationship agrees with photosynthetic utilization of CO2 alone (see Sand-Jensen 1983). As CO2 declines below 10 PM, between ph 8.0 and 9.0, HC03- constitutes most of the DIC pool (> 2 mm), and HC03- use would have been reflected by high gross photosynthetic rates. High CO2 concentrations ( PM) were needed to saturate O2 production. The CO2 compensation point was t 5.0 PM. When transport limitation of the CO, supply to zoochlorellae was mini- mized in suspensions, the saturating CO2 concentration was reduced to PM and the CO2 compensation point to 2+ 1 PM (Fig. 3). Because host tissue respires in the suspension, the CO2 compensation point of zoochlorellae alone will be lower than 2 PM C02, while the

6 556 Sand- Jensen and Pedersen.Y c CO2 concentration (mm), Fig. 3. CO, saturation curve of net 0, production at high light of an entire green sponge (0) [2.25 mg Chl (g DW)- 1 and a suspension of green Spongilla lacustris (@): saturating CO2 concentration will remain unaffected. Profiles of 02, ph, and photosynthesis-oxygen depth profiles in green SpongiZZa tissue differed markedly with location (Fig. 4). The variation is due to the variable distance in the O2 profiles to ventilation channels in SpongiZZa which distribute O,-rich water vertically. Oxygen concentrations were lowest in the dark and increased with surface light and ambient n CO, concentrations. The profiles at 400 PEinst m-2 s-l and 15 PM CO2 (Fig. 4C) suggest a small inward flux of 02. These data agree with results of experiments in which CO, concentrations above 17 PM were needed for net production of 0,. At 300 PM CO, in the water (Fig. 4D), O2 concentrations at 0.1 -mm depth in SpongiZZa were times higher than ambient levels and O2 was released from Spongilla, clearly showing the importance of CO2 limitation of photosynthesis with depth in Spongilla. Profiles of gross photosynthesis and O2 concentration were measured in a 2-mm thick dark-green individual SpongiZZa containing 184 pg Chl cm-3 and attenuating surface light (400 PEinst m-2 s-l) to 6% at 2-mm depth (Fig. 5). With 15 PM CO2 in the water, gross photosynthesis was nm01 O2 cm-3 min-l in the upper parts and close to zero in the lower 1 mm of SpongiZZa. Gross photosynthesis integrated with depth was mg O2 (mg Chl)- h- l. The O2 profile suggests an O2 flux close to zero. Thus, gross photosynthesis should approximate respiration, which averaged 0.47&O. 17 mg O2 (mg Chl)- h-l when measured in the photosynthesis chamber (Table 2). With 300 FM CO, in the ambient water, Oxygen concentration (jm) Fig. 4. Oxygen concentration with depth in green Spongilla lacustris. Each profile is derived from a separate position or individual: A-in the dark; B-at 40 PEinst m-2 s-l and 15 PM CO,; C-at 400 PEinst m- 2 s- and 15 PM C02; D-at 400 PEinst m-2 s-l and 300 PM CO,. Arrows indicate the ambient 0, concentrations at air saturation.

7 Sponge photosynthesis 557 gross photosynthesis was about twice as high and extended to 2-mm depth, demonstrating that the smaller rates and the shallower photosynthetic profile at low CO2 were due to CO2 limitation-also seen in Fig. 4. The integrated gross photosynthesis at 300 PM CO2 [ mg O2 (mg Chl)- h-l] resembled maximum rates measured in the bottle experiments [ 1.56 mg O2 (mg Chl)- h-l] and in the photosynthesis chamber [ 1.26 mg 0, (mg Chl)- h-l, Table 4. Internal ph was only measured at l-mm depth in green Spongilla at air saturation in the ambient water. The average ph at four different positions was in the light (400 PEinst m-2 s-l) and 7.54kO.10 in the dark. Ambient ph during these measurements was close to 8.2. If we assume that internal DIC resembled external DIC, ph measurements suggested that COZ concentrations were - 10 PM (Rebsdorf 1972) at 1 -mm depth and thus markedly below the COZ concentration (-60 PM) needed to saturate photosynthesis of isolated zoochlorellae. This finding supports our conclusion that zoochlorellae within Spongilla are CO, limited when light availability is high. Discussion Production vs. light--the high light saturation levels and compensation points of green Spongilla are comparable to the highest values for marine symbionts (table 4 of Kremer et al. 1990) and exceed the values for other freshwater symbionts, e.g. Hydra viridis (Phipps and Pardy 1982), the colony-forming ciliate Ophrydium versatile (Sand-Jensen et al. 199 l), and the solitary ciliate Paramecium bursaria (Pado 1967; Reisser 1980). The photosynthetic efficiency [avg 5.3 pg O2 (mg Chl)- m-2 s-l)-l] is also much lower than in the marine cnidarians and freshwater symbionts (20-120, Pado 1967; Reisser 1980; Phipps and Pardy 1982; Kremer et al. 1990; Sand-Jensen et al. 1991). This apparent poor efficiency of light utilization of green Spongilla is caused by light attenuation by tissue. Zoochlorellae from Spongilla in suspension had much higher photosynthetic efficiency [avg 69 pg O2 (mg Chl)- h-l (PEinst me2 s-~)-~], similar to that of phytoplankton (avg 64, Langdon 1988). The quantum efficiency for Spongilla sus iz z c 1.0 '; d Oxygen concentration (j.all Gross photosynthesis (nm01 O2 cmb3 min ) Fig. 5. Profiles of 0, (PM) and gross photosynthesis (nmol O2 cm-3 min-l) at two separate positions (open and closed symbols, respectively) in green Spongilla lacustris: A-at 400 PEinst m-2 s-l and 15 PM CO,; B-at 400 PEinst m-2 s-l and 300 PM CO,. Arrows indicate the ambient 0, concentration at air saturation. pensions represents, as emphasized before, an underestimate of the value for zoochlorellae alone, because host tissue contributed to light absorption. If we instead apply the chlorophyll-specific light attenuation coefficients for cultures of Chlorella pyrenoidosa [O.OlOS m2 (mg Chl)-, Bannister to the photosynthetic efficiency of zoochlorellae, we estimate a quantum efficiency of mol O2 (mol photons)- 1 -within the typical range for phytoplankton ( , Langdon 1988). The maximum gross production rates based on chlorophyll of intact Spongilla and suspended zoochlorellae resemble those for freshwater Hydra and ciliates (Pado 1967; Phipps and Pardy 1982; Sand-Jensen et al , 1994) but are clearly at the lower end of the range for marine cnidarians with zooxanthellae (table 4 of Kremer et al. 1990) and 5-lo-fold lower than values for phytoplankton at 15 C [8-14 mg O2 (mg Chl)- h-, Harris Our results suggest that the zoochlorellae of Spongilla are efficient in using the low light intensities that prevail inside green Spongilla. Further, they photosaturate at low light inten-

8 558 Sand- Jensen and Pedersen sities (avg, 40 PEinst m-2 s-l), yielding low ceive more than 1% of light incident on the maximum rates. In this respect, their photo- organism. This distribution was expected besynthesis appears to resemble photosynthesis cause the light compensation point of zoochloof zoochlorellae in the freshwater symbionts rellae in suspensions was - 12 PEinst m-2 s-l mentioned above. or 1% of surface illumination at noon. Photosynthetic variables within and among symbiotic species must be compared cautiously. The photosynthetic characteristics of SpongiZZa depend on tissue thickness, density of zoochlorellae, light attenuation, metabolic activity of the host, and external physicochemical conditions (e.g. temperature and CO, concentration). Also, photoadaptation of individual zoochlorellae may vary with these conditions and their location in Spongilla. Accordingly, it is difficult to ascertain whether different species differ in photoadaptation of their symbiotic algae. This difficulty is accen- tuated by differences in taxonomy and the contribution of accessory pigments relative to chlorophylls of zoochlorellae and zooxanthellae. Thus, zooxanthellae have peridinins which assist in light capture and tend to show higher photosynthetic rates when normalized to chlorophyll compared to those of zoochlorellae, where chlorophylls are the most important light-harvesting pigments (table 4.1 of Raven 1984). Evaluation of light availability to symbiotic algae based solely on measurements of incident illumination provides an insufficient description in thick organisms such as corals (Dubinsky et al. 1990) and S. Zacustris, where areal chlorophyll concentrations are high and most light is absorbed within a few millimeters from the surface. In the stream we studied, the incident illumination on green SpongiZZa varied IO-fold among sites. The same variation in light availability, however, is generated within - 1. g-mm depth in green SpongiZZa, and the light attenuation coefficient is strongly influenced by the chlorophyll concentration in the tissue. In shaded habitats, the thin Spongilla tissue of low zoochlorella density can therefore assure sufficient light exposure to the individual alga. In well-illuminated habitats, SpongiZZa can develop thicker tissue and erect branches of high zoochlorella density and still maintain sufficient light exposure to the symbiotic algae, yet with marked differences depending on location. The zoochlorellae are, however, predominately restricted to the upper 2-3 mm from the sponge surface and re- The minimum light requirement also sets an upper limit to the areal chlorophyll concentration (-60 pg Chl a+ b cm-2) in S. Zacustris and apparently somewhat lower (-20 pg Chl a cm-2) in the coral Stylophora pistillata (Dubinsky et al. 1990), with zooxanthellae rich in accessory pigments. As these high concentrations of symbiotic algae are approached, the algae are bound to use a larger fraction of the photosynthetically assimilated carbon for their own respiratory requirements, leaving less photosynthate available for translocation to the animal host. Carbon supply to Spongilla-The CO, supply to photosynthesis of SpongiZZa is derived from respiration in the sponge and from the surrounding water. We showed that external CO, concentrations had a profound influence on photosynthesis and internal CO, concentration and that high CO, concentrations were needed for saturation. Danish streams are usually supersaturated with CO, (avg, 130 PM: Rebsdorf et al. 199 l), providing an advantage to SpongiZZa photosynthesis compared with ponds and lakes, where CO, concentrations are more commonly close to air saturation. Because internal CO2 depletion and competition for CO2 is enhanced by high density of symbiotic algae, it is likely that external CO2 concentration also influences the maximum chlorophyll concentrations attainable and that the high levels observed here for the stream pop- ulations of SpongiZZa reflect the CO,-rich flowing water. Although photosynthesis by freshwater H. viridis and P. bursaria was C saturated in alkaline waters at 1 mm DIC and ph 7.5, rates were markedly lower at 0.15 mm DIC (Reisser 1980; Phipps and Pardy 1982). Also, photo- synthesis of 0. versatile in air-saturated water was only 70% of maximum CO,-saturated rates (Sand- Jensen et al ). Thus, C limitation of photosynthesis is a common phenomenon in freshwater symbionts, where an ability to exploit external HC03- has never been found. Limitation should be particularly pronounced in bulky organisms, like SpongiZZa that have high densities of algae.

9 Sponge photosynthesis 559 Marine corals presumably use HC03- actively and may provide free protons to elevate CO2 concentrations associated with formation of the CaCO, skeleton (Drew 1973). Zooxanthellae in corals also show high carbonic anhydrase activity, which is believed to enhance carbon acquisition in algae (Weis et al. 1989). Only one investigation has examined photosynthesis dependence of external DIC in corals and that study found a relatively small photosynthetic enhancement at HC03- concentrations above the ambient 2.3 mm (Burris et al. 1983). The experimental material, however, had low photosynthetic capacity, and the role of free CO2 vs. HC03- availability was not evaluated. Dubinsky et al. (1990) found a profound decline in light-saturated photosynthesis per symbiotic alga as algal density increased in S. pistillata. They proposed that this decline is the result of increasing CO, limitation. The influence of inorganic C supply on photosynthesis and density of symbiotic algae warrants further examination in freshwater and marine symbionts. It is often suggested that one advantage of the symbiotic association for the algae is CO2 supply from the host (cf. Kilian 1964). Gross production balances total respiration at the light and CO2 compensation points. The total respiration in SpongiZZa was -37% of maximum gross production. Applying a conventional respiration rate of green algae at 11% of maximum gross production (Geider and Osborne 1989) would therefore indicate that host respiration and the associated CO2 release could support photosynthetic rates of zoochlorellae at -26% of their maximum. This advantage of CO2 supply from the host, however, is more than offset by the increase of transport resistance from the host tissue. Photosynthetic rates of zoochlorellae released from the association could be supported at 55-65% of their maximum by concentrations at 17 PM C02, equal to the compensation point of intact SpongiZZa. The sponge does, however, provide a habitat for zoochlorellae, and respiratory activity of the host, combined with ventilation, contributes to reduce O2 accumulation, reduce ph, and increase CO2 in the tissue during intensive photosynthesis in the light. Thus, internal O2 and ph measured by the minielectrodes in the light were close to those in air-saturated water. Measurements with microsensors in differ- ent microalgal communities on solid surfaces have repeatedly shown massive O2 accumulation (2-5 times air saturation) and ph increase (ph = 9-l 0) because of high densities of algae, high photosynthetic rates per unit volume, and slow gas diffusion through stagnant boundary layers above the algal mats (Revsbech and Jorgensen 1986). In a series of measurements (Jorgensen et al. 1983; Revsbech and Jorgensen 1983; Revsbech et al. 198 l), maximum photosynthetic rates were 0.4-l.2 pmol cme3 min-l in different types of algal mats with estimated chlorophyll densities of 100-l,000 pg Chl cm-3. We measured maximum photosynthetic rates in SpongiZZa at 0.2 pmol crnb3 min-l with chlorophyll densities of 180 pg Chl cmu3, implying that lower photosynthetic rate, respiration of animal cells, and bulk flow by ventilation contributed to reduce O2 and ph in SpongiZZa tissue. Internal O2 and photosynthesis-our minielectrode measurements in SpongiZZa were in close agreement with those expected from photosynthetic O2 exchange in chambers. Internal O2 concentrations in the light were close to ambient levels at air saturation. In the dark, internal O2 concentrations in the surface layers were typically 30-50% of air saturation. Changes between light and dark produced new steady state levels after 2 min (data not shown). Oxygen microelectrode measurements in other. symbiotic organisms are rare, but another example is from the reef-building coral Acropora sp. from the Red Sea (Revsbech and Jorgensen 1986). In normal daylight, the oxygen concentration in Acropora rose to 80% of ambient air saturation when the microelectrode was positioned in a layer of symbiotic zooxanthellae at - 1 -mm depth. In the dark, the animal tissue became nearly anoxic. Measurements in other positions of the coral showed variable O2 concentrations in the light, including substantial supersaturation. The lightdark variation in O2 tension was rapid and fully completed within 3 min. The microenvironment is even more dynamic at the shell surface of the symbiotic foraminifera, Globigerinoides sacculijk (Jorgensen et al. 1985), both in terms of the O2 amplitude observed and the rate of change between light and dark. At the surface of G. sacculijkr, the O2 amplitude is % of air saturation, and changes are completed in -K 1 min upon light-

10 560 Sand- Jensen and Pedersen dark switches. The photosynthetic rates were up to 2.7 pmol O2 cmd3 min-l in G. sacculifer and exceeded the maximum rates in SpongiZZa by 10 times. These few measurements stress the potentially high spatial and temporal variability in concentrations of 0, and presumably other substrates or products (e.g. C02) associated with algal and host metabolism in symbiotic organisms and stress the likelihood of substrate limitation of photosynthesis. The importance of symbiotic algae-the nutrition of SpongiZZa is based on clearance and digestion of particles (mainly bacteria and microalgae) and photosynthesis of symbiotic algae (Gilbert and Allen 1973; Frost and Williamson 1980). Field experiments showed that SpongiZZa in the dark grew to only 20 and 49% of the size of the sponge in the light, while the light stimulus in three other experiments increased the specific growth rate of SpongiZZa by d-l (Frost and Williamson 1980). Our estimates in the stream during July suggested that net photosynthesis of algal symbionts could potentially increase SpongiZZa growth rate by d-l if photosynthetic products were fully utilized within the sponge. This net gain occurred because gross photosynthesis increased more than dark respiration of the sponge with increasing chlorophyll concentration. The advantage to the sponge of symbiotic photosynthesis could be that the animal would not have to respire a large fraction of the prey carbon to cover its energy metabolism. The symbiosis would allow the host to efficiently retain and recycle the nutrients from its prey internally. Net photosynthesis of zoochlorellae in green SpongiZZa could theoretically provide all the organic C required for growth and respiration of the host. However, if photosynthesis was the only source of organic C, the specific growth rate would be only d-l. This calculation suggests that particle filtration is needed to obtain the observed prolific growth of the organism. Microscopy of the collected individuals showed digestion of bacteria and microalgae, and captured prey could also supply other essential compounds not transferred from symbiotic algae. We present no direct evidence here for increased photosynthesis of SpongiZZa leading to increased animal biomass through direct consumption of the algae or translocation of pho- tosynthates to the animal. In theory, photosynthates could be used solely for algal growth and their density increase without affecting growth of the animal. Previous work has shown, however, that inorganic 14C supplied to SpongiZZa in the light can be traced by autoradiography to the animal tissue (Gilbert and Allen 1973), and sponge growth was indeed stimulated in the light (Frost and Williamson 1980). Moreover, if the diel C surplus by photosynthesis of symbiotic algae measured here was used exclusively to support growth of new algae, this growth would take place at a rate of 0.12 d-l -much faster than the registered growth rates of SpongiZZa in the field (Frost and Williamson 1980; Sand-Jensen and Pedersen unpubl.) - and would result in the zoochlorellae outgrowing their host. Therefore, when we also consider the prolific growth of illuminated SpongiZZa, we are confident that photosynthates benefited the host. Likewise, light stimulated the growth of symbiotic P. bursaria (Karakashian 1963; Pado 1965), 0. versatile (Sand-Jensen et al. 1994), and green Hydra (Muscatine and Lenhoff 1965) in experiments where external prey was in limited supply. The light effect was negligible in Hydra (Muscatine and Lenhoff 1965; Muller-Parker and Pardy 1987) and less pronounced in Paramecium (Pado 1965) fed to repletion. Some of the controversy about growth regulation in symbiotic organisms could therefore result from variability in food supply and light intensity among experiments and the fact that these key parameters are often poorly described and light effects are erroneously evaluated at very low light intensities barely sufficient to sustain the respiratory requirements of the symbiotic algae. References BANNISTER, T. R Quantitative description of steady state nutrient-saturated algal growth, including adaption. Limnol. Oceanogr. 24: BURRIS, J. E., J. W. PORTER, AND W. A. LAING Effects of carbon dioxide concentration on coral photosynthesis. Mar. Biol. 95: 113-l 16. DREW, E. A The biology and physiology of algainvertebrate symbiosis. 3. In situ measurements of photosynthesis and calcification in some hermotypic corals. J. Exp. Mar. Biol. Ecol. 13: DUBINSKY, Z., AND OTHERS The effect of external nutrient resources on the optical properties and photosynthetic efficiency of Stylophora pistillata. Proc. R. Sot. Lond. Ser. B 239:

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