Soluble extracellular products of algae symbiotic with a ciliate, a sponge and a mutant hydra

Comp. Biochem. Physiol., 1967, Vol. 20, pp. 1 to 12. PergamonPressLtd. Printedin Great Britain

SOLUBLE EXTRACELLULAR PRODUCTS OF ALGAE SYMBIOTIC W I T H A CILIATE, A SPONGE AND A M U T A N T HYDRA* LEONARD MUSCATINE, S T ~ E N J. KARAKASHIAN and MARLENE W " ~ K A S I : I T K N Department of Zoology, University of California, Los Angeles Department of Biology, Reed College, Portland, Oregon, U.S.A. (Received 14 June 1966)

Abstract--1. Soluble extracellular products of symbiotic algae (zoochlorellae) from Paramecium bursaria, Spongilla sp. and a mutant Chlorohydra viridissima were identified after photosynthesis in the presence of NaHCtaO 3 and compared to the products of ten strains of free-living algae. 2. All of the symbiotic strains liberated soluble maltose principally or glucose to the extent of 5"4-86"7 per cent of their total photosynthate. All of the freeliving strains liberated 0"4-7'6 per cent of their total photosynthate principally as glycolic acid. 3. Liberation of soluble carbohydrate by symbiotic algae from fresh-water representatives of three animal phyla. ~md by liehe~ algae is viewed as an adaptation to symbiosis. INTRODUCTION QUANTITATIVE experimental studies show that growth and survival of the ciliate Paramecium bursaria, the sponge SpongiUa sp. and the hydra Chlorohydra viridissima are enhanced by the presence of endosymbiotic algae (Karakashian, 1963 ; Miller, 1964; Muscatine & Lenhoff, 1965; Stiven, 1965). The mechanism of enhancement in each case has not yet been conclusively demonstrated. However, the hypdthesis that organic carbon from the algae is the primary growth-augmenting factor in the hydra association is consistent with two experimental observations: (1) 10-12 per cent of the C1402 initially incorporated by the algae in the intact C. viridissima association subsequently appears incorporated into animal cell proteins, nucleic acids and other organic compounds (Muscatine & Lenhoff, 1963). (2) Algae isolated from C. viridissima and incubated for 30 rain with NaHCt4Oa liberate 85 per cent of their total photosynthate to the extemal medium. The principal extracellular product is maltose. In contrast, free-living algae serving as controls liber~dce only traces of glycolic acid (Muscatine, 1965). In view of the striking difference in the quality and quantity of metabolites released by symbiotic vs. free-living algae, and the possibility that production of * Supported by research grants from the National Science Foundation (GB-1540, GB-3720) and National Institutes of Health (GM-10730). 1

1

2

LEONARDMUSCATINE, STEPHEN J. KARAKASHIAN AND MARLENE W. KARAKASHIAN

unique extracellular products by symbiotic algae is an adaptation to hereditary endosymbiosis, we have attempted to determine if excretion of soluble carbohydrate in relatively large amounts is characteristic of symbiotic algae from a variety of fresh-water invertebrate hosts or unique to hydra symbionts alone. We have examined algae from the three major phyla (Protozoa, Porifera, Coelenterata) whose fresh-water representatives exhibit hereditary endocellular symbiosis. Our selection of organisms within these groups has been restricted somewhat by the availability of simple methods for culturing the isolated algal symbionts or the hosts themselves, from which the algae can be obtained en masse. However, the survey includes those symbionts which have been shown to influence favorably growth or survival of their respective hosts. This paper describes soluble metabolites liberated by algae symbiotic with P. bursaria, Spongilla sp. and a mutant strain of C. viridissima not available for a previous study of hydra symbionts (Muscatine, 1965). The results are compared with those obtained from a variety of free-living strains of fresh-water urficeUular algae. The difference between the behavior of the symbiotic and free-living strains is considered highly significant.

METHODS 1. Maintenance and culture of organisms Algal stocks employed in this study and their sources are listed in Table 1. All stocks, with the exception of C. viridis~ima symbionts, were maintained on 1.5 per cent agar slants of a medium (LMM) modified (cf. Karakashian, 1963) from that described by Loefer (1936) for axenic culture of P. bursaria. Each liter of medium (LMM) contained 3.0 mg NaCl, 1.5 mg CaSO4, 4-5 mg MgSO4, 1.3 mg KNO3, 0.2 mg FeC12, 5-0 mg KH2PO4, 0.28 mg H3BO,, 0.011 mg MnC12, 0.012 mg ZnSO4, 0.00017 mg Na,MoO4, 2.2 mg ethylenediaminetetraacetic acid, 7.25 g proteose peptone, 5.0 g glucose, 0.5 g yeast extract and an aqueous extract of 1-0 g Cerophyl. (Cerophyl is a preparation of dried cereal grasses m~de by the Cerophyl Laboratories, Inc., Kansas City, Missouri.) Several weeks prior to experimentation the algae were transferred to 250 ml Erlenmeyer flasks containing 50 ml of liquid LMM. These were illuminated continuously by two 40 W fluorescent lights situated 5 cm below the flasks. Temperature ranged from 24 to 26°C. All strains grew well under this rdgime but invariably the free-living strains grew faster than symbiotic strains. Subcultures of these liquid stocks, after 3-5 days of growth, yielded at least 0.1 ml of wet-packed algae for experimentation. Symbionts from C. viridissima were isolated from the host and assayed immediately, without intervening maintenance or culture, as described by Muscatine (1965). All samples of algae were washed by centrifugation (2 rain, 3000 rev/min, International Clinical Centrifuge, Model CL) in an inorganic salt solution consisting of (rag/l): Ca(NO3)2.4H20 , 250; MgSO,.7H20 , 50; K,HPO4, 75, diluted to 1 part solution to 3 parts deionized water.

SOLUBLE EXTRACELLULAR PRODUCTS OF ALGAE

3

T h e experiments, all of short duration, were carried out in this dilute inorganic m e d i u m because the complex organic L M M m e d i u m interfered in various ways with chemical and chromatographic assay of the m e d i u m for extracellular products. T A B L E 1 - - S U M M A R Y OF ALO.M~ EMPLOYED IN THIS STUDY

Strain* 1. Symbiotic algae

Host

3H

P. bursaria syngen 1

32B1

P. bursaria syngen 1

34A

P. bursaria syngen 1

NC64A P. bursaria syngen 1

2. Free-living algae

41K

P. bursaria syngen 1

130C

P. bursaria syngen unknown

838

SpongiUa sp.

C61m

C. viridissimat

GrB1

C. vulgaris

490 20 250 261 262 263 326 397 398

Chlorella miniata C. ellipsoldea C. protothecoides C. vulgaris C. vulgaris C. vulgaris Selenastrum minutum C. vulgaris C. vulgaris

Remarks Isolated and cultured by Karakashian (1963) Isolated and cultured by Karakashian (1963) Isolated and cultured by Karakashian (1963) Isolated and cultured by Karakashian (1963) Isolated and cultured by Karakashian (1963) Isolated and cultured by Loefer (1936). Obtained from IUCCA Isolated and cultured by R. A. Lewin. Obtained from IUCCA Isolated en masse from cultures of the host but not grown in culture (cf. Muscatine, 1965) Obtained from Prof. D. Appleman, Univ. Calif., Los Angeles Obtained from IUCCA Obtained from IUCCA Obtained from IUCCA Obtained from lUCCA Obtained from IUCCA Obtained from IUCCA Obtained from IUCCA Obtained from IUCCA Obtained from IUCCA

* Numbers of strains from the Indiana University Culture Collection of Algae (IUCCA) are those of the collection. Other strain numbers are our arbitrary designations. t Some properties of this mutant hydra are described by Lenhoff (1965). 2. Experimental procedures

O n e - t e n t h of a milliliter of wet-packed algae f r o m vigorously growing subcultures was suspended in a mixture of 1-5 ml of dilute inorganic m e d i u m and 1-5 ml M c I l v a i n e citrate-phosphate buffer at the desired p H in a 12 ml conical

4

LEONARDMUSCATINE,STEPHENJ. KARAKASHIANANDMARLENEW. KARAKASHIAN

centrifuge tube and illuminated from the side (1500 ft-candles). After 1 hr of equilibration 20/zc of NaHC140 s in 20/zl of solution was added to the suspension. The tube was stoppered and incubated for 30 rain at 24-26°C, 1500 ft-candles. Tubes were agitated briefly by hand from time to time to offset the tendency of the cells to settle. The suspensions were then centrifuged (2 rain, 3000 rev/min) and the clear supernatant drawn off with a pipette and saved. The cells were washed twice with 0.5 ml aliquots of buffered inorganic medium and the supernatants drawn off and combined with the original medium and then evaporated to 0-5 ml in vacuo with warming to 40°C. A 20/zl aliquot of supernatant medium was then placed on a planchet within a prescribed area, acidified with 0.1 N HC1, dried under an infra-red heat lamp and assayed for radioactivity with a thin window Geiger tube (Lionel-Anton, model 1007TA). The C14-1abeled algal pellet was resuspended in 0.5 ml 80% ethanol and a 20 ~l sample of the suspension was removed and counted. Ca~-labcled extracellular organic matter was expressed as a percentage of the total (medium plus cells) labeled photosynthate. 3. Analytical procedures The medium and alcoholic extracts of the algae were analyzed for labeled organic material by paper chromatography in two dimensions on Whatman No. 4 paper (46 × 57 era) using phenol-water (100 : 39, w/v) for the first dimension and n-butanol-propionic acid-water (142 : 71 : 100 v/v/v) for the second dimension. A third solvent consisted of n-butanol-ethyl alcohol-water (5 : 1 : 4, v/v/v, organic phase) used in one dimension. Paper electrophoresis of carbohydrates was carried out at 500-600 V (Model E-800-2, Research Specialties Co., Richmond, California), using a conductant solution of sodium borate (pH 9.9). Radioactive compounds were located on chromatography papers by autoradiography with Kodak singlecoated blue-sensitive medical X-ray film. Levels of activity were determined from the sum of square centimeter areas counted separately, and for a given compound are expressed as a percentage of the total activity on the paper after developing the chromatogram. Unknowns were eluted with water for further analysis, and provisionally identified by co-chromatography in three different solvents. Maltose was identified by co-crystallization of the 3-octa-acetate derivative with authentic maltose 3-octa-acetate to constant specific activity. Full details of all analytical procedures and criteria for identification have been published previously (cf. Muscatine, 1965). Maltase activity in P. bursaria was assayed by suspending lyophilized algae-free P. bursaria in 0"5 ml of Mcllvaine citrate-phosphate buffer at pH 5-0. To this solution was added 10 tA of a solution containing 10 mg/ml maltose in distilled water. Maltose, glucose and the crude P. bursaria lyophilisate, each in buffer solution at pH 5-0, served as the necessary controls. After a 3 hr incubation at 24-26°C enzymatic activity was stopped by immersing the tubes in boiling water for 3 min. Tubes with precipitates were centrifuged and from each tube an aliquot of supernatant was removed for paper chromatography in one dimension (descending), using butanol-ethanol-water ( 5 : 1 : 4 , v/v/v) on Whatman No. 4 paper.

SOLUBLE EXTRACELLULARPRODUCTS OF ALGAE

Reducing substances, including sugars, were detected with the silver nitrate reagent (Trevelyan et al., 1950). Prior to lyophilization the algae-free P. bursaria were washed twice by centrifugation in a sterile salt solution to reduce contamination with the bacteria (Aerobacter cloacae) normally provided as particulate food. The tendency of the ciliates to lyse precluded further washing, and it proved necessary to estimate the extent to which residual bacteria may have contributed to maltase activity of the dried P. bursaria. As a control for this purpose, lyophilized A. cloacae were also assayed for maltase activity, RESULTS

1. Liberation of organic material (a) P. bursaria symbionts. Figure 1 is a radioehromatogram of an alcoholic extract of the cells of strain NC64A showing labeled photosynthetic intermediates after 30 rain photosynthesis. The major intracellular products have been provisionally identified as sucrose, glutamic acid and alanine. Figure 2 is a radiochromatogram of the medium from this strain showing a major radioactive spot identified as maltose. Glucose, glycolic acid and several unidentified substances are present in trace amounts. Since the water-soluble intracellular products, especially sucrose, are absent from the medium, we conclude that cell lysis during the experiment was negligible, and that the labeled organic material in the medium represents a selective liberation of products formed during photosynthesis. Table 2 shows that these results are representative of all of the strains of P. bursaria symbionts. CX4-1abeledorganic material was liberated into the medium in amounts ranging from 5-4-86 per cent of the total C aa fixed. In all cases, maltose comprised at least 95 per cent of the products liberated. (b) Spongilla sp. symbionts. The amounts of C 14 fixed photosynthetically by the 0.1 ml aliquots of sponge symbionts were among the highest encountered in these experiments, yet the yield to the medium was the lowest among the symbiotic algae; a maximum of 4"4 per cent as indicated in Table 2. Radiochromatographic analysis of the medium (Fig. 3) disclosed one major radioactive compound identified as glucose after comparative electrophoretic studies and other tests. Other materials (glycolic acid, unknowns) comprised less than 1 per cent of the total organic matter liberated. Maltose was not detected in the medium. As with P. bursaria symbionts, the principal water-soluble intracellular product was sucrose. In addition, an unusually large proportion of the label was present in lipid or lipid-like material as judged from its chromatographic behavior (Fig. 4). (c) C. viridissima (mutant) symbionts. Algae from the mutant hydra produced relatively large amounts of extracellular material (Table 2; Fig. 5). The major extracellular product was glucose. In addition, we occasionally detected traces of maltose. This led us to suspect that the material actually liberated was maltose and that maltase (~-glueosidase) in the medium, known from prior studies to be carried over with the algae when they are separated from the host, was mediating hydrolysis to glucose. In a previous study on wild-type C. viridissima it was found

6

LEONAm)MUSCATINE,STEPHENJ. KARAgaSmANaND MARLENEW. KARAKASHIAN TABLE 2 ~ D I S T R I B U T I O N OF C a* AFTER PHOTOSYNTHESIS FOR 3 0

Strain

1. P. bursaria

NC64A 32B1 130C 3H 34A 42 41K

Total C 14 fixed Per cent (103 cpm/ in 0.1 ml cells) medium 1214"4 577.0 664.9

86"0 64"5 29.1

1581.6 922.4 1114.2 1423.0 2155.8 1305.1 1344-6 192-0 1253-6

15-3 5"4 84"5 41.1 46.2 29"1 86"7 74"5 86"5

rain, 1500 ft-candles

As maltose (%)

As As glycolic glucose acid and (%) unknown

(%)

97"1

1.2

1"7

99'1

0.3

0'6

97.0

0"0

3.0

95.2

4.8

0'0

99"4

0'5

0"1

99"2

0.7

0.1

99.1

0"4

0.5

2. Spongilla sp.

838

2207.8 3238"4

4.4 3.0

0.0

99.9

0'1

3. C. viridissima

C61m

1013.8 3969.0

48"8 37"0

0.0 2"2

90"8 94"2

9.2 3.6

4. Free-living algae 20 GrB1 250 490 262 263 326 397 398

1206"1 610"4 875"5 1456.2 1093"8 1192"3 104"3 1230"7 787"5

6"5 1"1 0"9 7"6 0"4 0-5 3"8 1"0 0"6

None None None None None None None None None

None None None None None None None None None

100 100 100 100 100 100 100 100 100

Replicate values for the per cent excretion by symbiotic algae are uniformly lower than initial values because the initial experiment (including free-living strains) was carried out at pH 4"5 and the replicate at pH 5"0. As shown in Fig. 6, an increase in pH in the acid range tends to lower the per cent of photosynthate excreted. that exhaustive washing eliminated maltase activity from algal suspensions. I n the present study, exhaustive washing did not change the end-products, which may indicate that washing does not completely eliminate enzymatic activity in this instance. Since liberation of carbohydrate by the symbionts from mutant C. viridissima varied with pH, we believe that the carbohydrate may be liberated as maltose, since, in other symbiont strains (as will be shown below), liberation of maltose but not glucose is influenced by pH. It is possible that maltose is then

I~7~;. 1. R a d i o c h r o m a t o g r a m of an 80c!'~ ethanol extract of cells of strain N C 6 4 A showing photosynthetic intermediates (3() rain, 1500 ft-candles). In this and subsequent chromatograms, origin at lower left, the first dimension in p h e n o l - w a t e r is horizontal, the second dimension in b u t a n o l - p r o p i o n i c acid-water is vertical.

["i(;. 2. R a d i o c h r o m a t o g r a m of the aqueous m e d i u m in which N C 6 4 A cells were incubated, showing soluble extracellular products of photosynthesis. Conditions as in Fig. 1.

FJt;. 3. Radiochromatogram of the aqueous medium in which Spongilla symbionts (838) were incubated, showing soluble extraceIlular products. Conditions as in Fig. 1.

FIG. 4. Radiochromatogram of 80'~ ethanol extract of

Npongilla algae.

Fro. 5. R a d i o c h r o m a t o g r a m of t h e a q u e o u s m e d i u m in w h i c h algae f r o m m u t a n t C. z,iridissima were i n c u b a t e d . C o n d i t i o n s as before. R a d i o c h r o m a t o g r a m s of the extracts of these cells are n e a r l y identical to t h a t d e p i c t e d in Fig. 1.

SOLUBLE EXTRACELLULARPRODUCTS OF ALGAE

immediately hydrolyzed by enzymes bound to the algal cell surface. In addition to these carbohydrates and traces of glycolic acid, there also appeared in the medium an unknown product (R t 0.77, 0.56 in PW, BPAW) as yet unidentified, which did not appear in the medium of symbionts from wild-type C. viridissima. The significance of the excretion of this product is under investigation. (d) Free-living algae. In striking contrast to the symbiotic algae, all strains of free-living algae, including several capable of infecting P. bursaria (cf. Karakashian & Karakashian, 1965, and Discussion) liberated relatively small amounts (0.4-6.5 per cent) of the total photosynthate entirely as glycolic acid and trace unknown (Table 2). Neither maltose nor glucose nor any other carbohydrate was detected in the medium. In all strains sucrose was the major intracellular product.

2. lnfluence of hydrogen-ion concentration on liberation of extracellular organic material In previous studies on symbionts from wild-type C. viridissima it was noted that the pH optimum for liberation of maltose was in the acid range. As one might expect, the same pH optimum was noted in the present study among symbionts which liberated maltose. Figure 6 shows the effect of pH on excretion by selected algal strains. The effect is more pronounced on some symbiotic strains than others (compare NC64A vs. 34A). Excretion of glucose by sponge symbionts appears to be independent of pH except for a slight increase at pH 8.1. Again, in contrast to symbiotic algae, the excretion of none of the strains was pH-dependent. I00

80

~E 2 6o o i.iJ z -~ 4o

20

5

6 pH

7

S

FIG. 6. Influence of pH on excretion by symbiotic and free-living algae. Symbiotic algae: NC64A (O), C61 (A), C61m ( I ) , 34A (O), 838 (A). Free-living algae: C. pyrenoidosa Emerson strain (O).

8

LEONARDMUSCATINE, STEPI-IEN J. KARAKASHIANAND MARLENE W. KARAKASHIAN

3. Maltase activity in P. bursaria

The capability for enzymatic hydrolysis of maltose by P. bursaria was investigated by qualitative assay of maltase (~-glucosidase) activity in algae-free host cells. This was a necessary inquiry since any hypothesis involving host utilization of maltose from symbionts would require evidence that the host at least possesses the enzyme required for hydrolysis of this substrate. The results are shown in Fig. 7.

0

GLUCOSE

MALTOSE

P. 8URSARIA P. BURSARIA A.CLOACAE A. CLOACAE t ONLY + ONLY MALTOSE MALTOSE

.I

.2

.3 Rf .4

.5

0 FIG. 7. One-dimensional descending chromatogram (butanol-ethanol-water) showing maltase activity in P. bursaria and A. cloacae. Incubation with P. bursaria lyophilisates resulted in extensive hydrolysis of maltose as evidenced by the appearance of glucose in the experimental reaction mixture and the concomitant disappearance of part of the original maltose substrate. Maltose in the absence of the lyophilisate was not hydrolyzed. Substances reducing silver nitrate were present in the lyophilisates but these were easily distinguishable from glucose and maltose. Figure 7 also shows relatively weak maltase activity in the .4. cloacae preparation. The number of bacteria in the lyophilized preparation of A. cloacae was approximately 6 x 109 cells. This is two orders of magnitude more than the number of bacteria (ca. 4 x 107) actually encountered in the washed ciliate suspension just prior to lyophilization as determined by plate counts. Therefore, we tentatively conclude that the maltase activity in the ciliate preparation was predominantly native to P. bursaria, while recognizing that part of the activity may have originated from contaminating bacteria.

S O L U B I ~ E X T R A C E L L U L A R P R O D U C T S O F ALGAE

DISCUSSION

The results of the present study together with information from a previous study (Muscatine, 1965) show that symbiotic algae from three major animal phyla, whose fresh-water representatives exhibit stable endosymbiosis with algae, liberate soluble carbohydrate to the external medium. The phenomenon of carbohydrate liberation is now known for lichen algae as well. The blue-green algal constituent (Nostoc sp.) of the lichen Peltigera polydactyla liberates substantial amounts of extracellular glucose (Drew & Smith, 1966), while green algae (Trebouxia sp.) in lichens liberate ribitol and other carbohydrates (Richardson & Smith, 1966). As shown in Table 2, there are consistent differences between the types and amounts of compounds excreted by symbiotic and free-living algae. The symbiotic algae excrete primarily soluble carbohydrate (maltose or glucose) in amounts ranging from 3.0 to 86.7 per cent of their total photosynthate. The rate of liberation of maltose is high, easily detectable in experiments of 1-30 min duration and profoundly influenced by pH of the medium. In contrast, the ten to twelve freeliving strains which have been studied excrete primarily glycolic acid along with traces of other organic materials. Levels of excretion are relatively low and unaffected by changes in pH of the medium, at least in our short-term experiments. These latter observations are consistent with other recent investigations on excretion of organic material by free-living fresh-water unicellular algae under laboratory conditions. Excretion levels of 0.5-5.5 per cent have been reported most frequently with glycolic acid as the major extracellular product. Higher values for excretion of Chlorella sp. in the laboratory have been observed, especially under conditions of alkaline pH and limited availability of CO S. Excretion by natural populations of phytoplankton from lakes has been studied in some detail. In short-term experiments ( < 3 hr) under the most favorable conditions, excretion by the several dozen or so species encountered seems not to exceed 2 per cent of the total carbon fixed. In longer experiments (3-24 hr) values for excretion are higher (7-50 per cent). In most cases, the higher values for per cent excretion are only apparent values. This is encountered when photosynthesis is inhibited to a greater extent than excretion, as in darkness or intense light, or when accumulation of radioactive material in the medium represents an exchange of labeled and unlabeled substances between cells and medium rather than a net loss by the cells. (For details of the foregoing discussion see Tolbert & Zill, 1956; Nalewajko et al., 1963 ; Fogg & Nalewajko, 1964; Fogg et al., 1965 ; Nalewajko, 1966 ; Watt & Fogg, 1966). The biochemical pathways involved in the liberation of soluble carbohydrate by symbiotic algae remain obscure. Similarly, the effect of pH and other environmental variables on excretion awaits further inquiry. On the other hand, aspects of the effect of environmental and other variables on excretion by free-living Chlorella have been investigated and are discussed elsewhere (Kearney & Tolbert, 1962; Pritchard et al., 1962; Miller et al., 1963; Nalewajko, 1966; Watt & Fogg, 1966).

10

LEONARDMUSCATINE, STEPHEN J. KARAKASHIANAND MARLENEW. KARAKASHIAN

The evidence accumulated to date is consistent with the hypothesis that liberation of soluble, low-molecular weight carbohydrate by symbiotic algae is a primary factor in enhancing the growth and survival capabilities of their host. Karakashian & Karakashian (1965) infected aposymbiotic P. bursaria with (1) symbiotic algae from different strains of P. bursaria, (2) symbiotic algae from different host species and (3) free-living species. Ability to enter the host cells, to persist, to be transmitted from one host generation to the next, and to promote growth and survival of the host were the parameters compared. Although some free-living strains of algae were ingested by the ciliates, persisted within them for at least several days and reproduced slowly within the host in light, none survived or reproduced in the host in darkness. Moreover, free-living algae were virtually unable to enhance the growth of the host. On the other hand, symbiotic algae characteristically persisted in darkness and in all cases supported growth of the host when food was limited (cf. also Karakashian, 1953). Symbiotic algae from other host species, for example, C. viridissima, supported growth of P. bursaria at least as well as did the host's native symbionts. Finally, the probable occurrence of maltase activity in P. bursaria indicates that maltose can be used by the host. For the C. viridissima association it is known that (1) the presence of algae favorably influences growth and survival of the host; (2) carbon-14 labeled photosynthate is transferred from the algae to the host. The labeled material accumulated by the animal is incorporated into proteins and other substances; (3) the animal exhibits maltase activity; and (4) when unlabeled green hydra heads are grafted onto S35-1abeled albino bodies, the rate of loss of S 35 from the albino bodies via protein catabolism is significantly lowered. The implication drawn from these experiments is that the symbiotic algae, which grow relatively slowly in situ but photosynthesize at rates which are comparable with a variety of free-living algae, release 'excess' photosynthate which is then accumulated by the animal host. Since C. viridissima and P. bursaria exhibit maltase activity, and glucose but not maltose is demonstrable in C. viridissima animal tissues, it is likely that maltose from the algae is hydrolyzed to glucose residues upon entering the host carbon pool. The net increase of usable organic carbon in the host cells would ultimately result in conservation of existing host substrates (Muscatine & Lenhoff, 1963, 1965; Muscatine, 1965). Finally, we have the conclusions of Slobodkin (1964) and Stiven (1965), drawn from quantitative experimental studies, which show that symbiotic algae contribute quantitatively to host growth in the light, and that when food intake is reduced, growth efficiency of green hydra increases 40-60 per cent above that measured in albino C. viridissima. T h e algae from Spongilla differ in several interesting ways from the other symbionts we have studied. First, as we have shown, they excrete glucose rather than maltose, and in these experiments only a small proportion of the total carbon fixed was excreted. In addition, these algae are ellipsoidal in shape and smaller than the rounded, larger symbionts of C. viridissima or P. bursaria. Finally, although the sponge algae do not as readily infect aposymbiotic strains of P. bursaria as do algae from P. bursaria or C. viridissima (Karakashian & Karakashian,

SOLUBLE EXTRACELLULAR PRODUCTS OF ALGAE

11

unpublished observations), the influence of the sponge algae on their own host is similar to that which has been described for C. viridissima and P. bursaria. Miller (1964) has demonstrated experimentally that the presence of symbiotic algae reduces the rate of protein catabolism in Spongilla gemmules. It is noteworthy that the P. bursaria symbionts have been in culture for more than 3 years and still display high levels of excretion. On the other hand, strain 130C, which has been in culture for some 35 years, also exhibits the lowest level of excretion among the P. bursaria symbionts. The Spongilla symbionts have been in culture for about 12 years and display the lowest level of excretion of all symbionts tested. Since Richardson & Drew (1966) have observed that excretion of ribitol by lichen algae in culture diminishes over a period of several days, perhaps some effect of prolonged culture could explain the low excretion levels of Spongilla algae. We are endeavouring to obtain fresh material to test this hypothesis. SUMMARY 1. Soluble extracellular products of algae symbiotic with P. bursaria, Spongilla sp. and a mutant C. viridissima were isolated by radiochromatography and identified after a short period of photosynthesis in the presence of NaHCI40 a. 2. Algae from six strains of P. bursaria liberated 5.4-86.7 per cent of their photosynthate. The principal product of each strain was maltose. Aposymbiotic P. bursaria exhibit some maltase activity. 3. Algae from a mutant strain of C. viridissima liberated approximately 40-50 per cent of their total photosynthate to the medium. Their principal soluble extracellular product was glucose. A second minor product was not identified. Previous work (Muscatine, 1965) has shown that algae isolated from wild-type C. viridissima excrete maltose and do not produce detectable quantities of the unidentified compound. 4. Algae from Spongilla sp. liberated a maximum of 4.4 per cent of their total photosynthate, principally as glucose. 5. Liberation of maltose by symbiotic algae was enhanced by acid pH of the medium. Liberation of glucose by Spongilla algae did not exhibit a pH-dependence. 6. Ten strains of free-living algae liberated 0.4-7.6 per cent of their photosynthate. The principal product of each strain was glycolic acid. Excretion was not influenced by pH of the medium. 7. The implications of the liberation of relatively large amounts of soluble carbohydrate by symbiotic algae, as contrasted with the liberation of small amounts of glycolic acid by free-living algae, are discussed in terms of adaptation to endocellular symbiosis. Acknowledgements--We thank Drs. A. A. Benson and F. T. Haxo for their interest and for providing facilities at Scripps Institution of Oceanography where this investigation was initiated. T h e skilful technical assistance of Mrs. Elsa Cernichiari is gratefully acknowledged.

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LEONARDMUSCATINE, STEPHENJ. KARAKASHIANAND MARL~-NEW. KARAKASHIAN

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