Photosynthesis and respiration of two species of algal symbionts in the anemone Anthopleura elegantissima (Brandt) (Cnidaria; Anthozoa)

Journal

ELSEVIER

of

Experimental Marine Biology 195 (1996) 187-202

and Ecology

JOURNAL OF EXPERIMENTAL MARINE WOLOGY AND ECOLOGY

Photosynthesis and respiration of two species of algal symbionts in the Anemone Anthopleura elegantissima (Brandt) (Cnidaria; Anthozoa) E. Alan Verde”, L.R. McCloskeyb‘* *Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA hDepartment of Biological Sciences, Walla Walla College. College Place, WA Y9.724, USA

Received 22 August 1994; revision received 20 March 1995; accepted 2 May 1995

Abstract

In parts of its range, the anemone Anthopleura elegantissima (Brandt) is populated by two different symbiotic algae: zoochlorellae and zooxanthellae. Anemones with exclusively one or the other symbiont were compared under identical conditions. A zooxanthella was about twice the volume and carbon content of a zoochlorella, and contained more chlorophyll. Zooxanthellae population density was 24% less than zoochlorellae, consequently zooxanthellae protein biomass per unit animal was only 58% greater than zoochlorellae. The mitotic index (MI) of zoochlorellae was almost 22 times greater than zooxanthellae, and the die1 cell division pattern was asynchronous in both algae. Using duration of cytokinesis times (r,) of 28 and 69 h for zooxanthellae and zoochlorellae, respectively, we estimated that zoochlorellae grew eight times faster than zooxanthellae. with a commensurately shorter population doubling time. Zooxanthella respiration was two and a half times and net photosynthesis twice that of a zoochlorella; this coupled with a comparatively low growth rate allows the zooxanthellae population to potentially translocate almost five times as much carbon to its host as do zoochlorellae. The contribution of carbon to animal respiration (CZAR) was estimated at 48% for the zooxanthellate and only 9% for zoochlorellate anemones. It is suggested that these associations may be similar to ancestral symbioses. Keywords: Anemone; Anthopleura elegantissima; Translocation; Zoochlorellae; Zooxanthellae

* Corresponding

Carbon

author.

0022-0981/96/$15.00 @ 1996 Elsevier SSDI 0022-0981(95)00080-l

Science

B.V. All rights

reserved

budgets;

CZAR;

Symbiosis:

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E.A. Verde, L.R. McCloskey

I J. Exp. Mar. Biol. Ecol. 195 (1996) 187-202

1. Introduction

Two congeneric anemones, Anthopleura elegantissima and A. xanthogrammica, each host two species of algal symbionts: a dinoflagellate, Symbiodinium cafiforgreen unicellular alga nium (Banaszak et al., 1993); and a Chlorophycean (Muscatine, 1971; O’Brien, 1978). These two should not be confused with symbiont species residing in other hosts, however, we subsequently refer to them and “zoochlorellae”, by the conventional, non-specific terms “zooxanthellae” respectively. While mutual exclusiveness of the two species within a single host seems to be the norm, occasional individuals may harbor both algae. Various trophic characteristics have been attributed to zooxanthellae in Anthopleura (Muscatine, 1971; O’Brien, 1980; Fitt et al., 1982; Dykens & Shick, 1984; Shick & Dykens, 1984) but much less is known about zoochlorellae. There has been no systematic comparison of the growth, morphology, or productivity of these two symbionts. This paper compares the biomass and productivity features of zooxanthellae and zoochlorellae and provides estimates of their relative carbon flux and budgets in the anemone A. elegantissima (Brandt). We show that these two symbionts contribute to the trophic state of the animal host in remarkably different ways.

2. Methods 2.1. Collection and maintenance

of anemones

Similar sized specimens of Anthopleura elegantissima containing exclusively zooxanthellae or zoochlorellae were collected during June-August 1985, in the intertidal zone of Swirl Rocks in the Strait of Juan de Fuca south of Lopez Island, Washington. The collection sites for zooxanthellate and zoochlorellate anemones were within 2 m of each other. The animals were maintained for no longer than 48 h prior to experimentation in an outdoor seawater tank at the Walla Walla College Marine Station, Anacortes, Washington. The temperature of the seawater in the flow-through system ranged from 12 to 14°C. At the end of each experiment, each anemone was homogenized in distilled water with a laboratory blender. The homogenate volume was determined and samples taken for various assays. 2.2. Algal biomass Size and density. Average diameter of freshly isolated zooxanthellae (n = 76) and zoochlorellae (n = 76) was determined with an ocular micrometer. Algal density was determined from hemacytometer counts of algal cells in 20 replicate samples taken from each homogenate, and converted to total number of algae per anemone (standing stock, SS). Biomass; carbon and protein. Algal cell-specific carbon values of 124.4 and 64.9

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I J. Exp. Mar. Biol. Ecol. 19-f (1996)

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1x9

pgecelll’, for zooxanthellae and zoochlorellae, respectively, were obtained from Verde (1993). Algal protein was calculated from measures of N mass and algal cell counts. Average N values of 15.21 and 9.68 pg. cell-’ (Verde, 1993) multiplied by 6.25 (Muscatine et al., 1986) yielded protein values of 95.1 and 60.5 pg. cell-’ for zooxanthellae and zoochlorellae, respectively. Cell-specific protein multiplied by SS provided a measure of the total algal protein. Chlorophyll. Chlorophyll was extracted from 12 anemones - six zooxanthellate and six zoochlorellate-collected from the same site as the anemones subsequently used in respirometry experiments. Each anemone was homogenized, algal counts made, and three replicate 3 ml samples of the homogenate were filtered through GF/C filters, each followed by a wash of 1% MgCO, (0.5 ml), and the filters frozen. The filters were macerated in 4 ml of 90% Photrex acetone and the chlorophyll extracted 12 h in the dark at 3°C. The preparation was centrifuged for 10 min to pellet the glass fiber fragments and algal cell debris. The absorbance of the acetone supernatant was determined with a diode array spectrophotometer (Hewlett Packard model 8452A) and were converted to chlorophyll mass by the equations of Jeffrey & Humphrey (1975) as described by Parsons et al. (1984). MI and growth. Die1 mitotic activity (MI) of zooxanthellae and zoochlorellae was determined using a technique similar to that of Wilkerson et al. (1983). At 2-h intervals over a 24-h cycle under natural light, 14 similarly-sized, freshly collected anemones - seven zooxanthellate and seven zoochlorellate - were frozen, indiand the algae in each homogenate counted in a vidually homogenized, hemacytometer. The number of algae undergoing cytokinesis per 1000 cells was noted, and the resultant percentage taken as the MI. MI was also similarly determined for each anemone used for respiration and photosynthesis experiments. The formula of McDuff & Chisholm (1982) and Wilkerson et al. (1983) for asynchronously dividing (non-phased) cells was used to calculate the algal-specific growth rate (,u~) per day K =(24+t,-‘)ln(l

+fl

(1)

where t, is the duration of cytokinesis and f is the average fraction of cells undergoing non-phased division, obtained from MI. We have used t,‘s of 28 and 69 h for zooxanthellae and zoochlorellae, respectively, consistent with the rationale developed in a companion paper (McCloskey et al., 1996). The carbonspecific growth rate (C, in ,cLgC. day-‘) of the algal population was calculated as C, = [(SS)(C. celll’)(r.L,)]. 2.3. Anemone

(2)

(animal) biomass

Animal protein was determined by Bio-Rad assays (Bradford, 1976; Bio-Rad Labs.) on three replicate samples from each whole anemone homogenate, using bovine serum albumin as the protein standard. Proteins determined in this

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I J. Exp. Mar. Biol. Ecol. 195 (1996) 187-202

manner were multiplied by 1.56 to make them comparable to proteins determined with the Lowry method (Cook et al., 1988). Since the Bio-Rad assay does not measure the protein of the intact algal symbionts (unpublished data from our laboratory), the resultant value is protein of the animal fraction alone. Total anemone protein was determined as animal plus algal protein. The animal and algal proteins, expressed as fractions of total protein, are subsequently referred to as j3 and l-p, respectively (Muscatine et al., 1981). 2.4. Photosynthesis and respiration Respirometry. Die1 oxygen production (P”) and respiration (R”) were measured on zooxanthellate and zoochlorellate anemones (n = 9 and 10 respectively). To ensure similarity of conditions, a zoochlorellate anemone was measured at the same time in each experiment. Each anemone was placed in its own chamber in a self-contained underwater respirometer (McCloskey et al., 1985), the respirometer submerged (0.5 m) in an outdoor seawater tank, and the 0, flux of the anemones, as well as incident irradiance, monitored for 24 h. Aerial and aquatic measurements of P” and R” were synchronized to approximately coincide with natural tide cycles. Aerial measurements were made from 0700 to 1300, the average low tide period during June through August at the collection site. 0, flux was measured during this simulated low tide after draining and drying the respirometer chambers, reintroducing the anemones without water, and resubmerging the respirometer to 0.2 m. Submergence maintained constant temperature within the dry chambers. P and R data. Daytime fluxes of photosynthetically produced oxygen, summed with anemone respiration rate values obtained at night, provided daily gross photosynthesis measures (P”) (McCloskey et al., 1978, 1994; Muscatine et al., 1981). Net photosynthesis (!‘y), was computed by subtracting algal respiration from PO. Respn-ation of the algae (RF) and animal (R$) were estimated based on the animal (/3) and algal (1-p) components of total biomass, according to the convention of Muscatine et al. (1981). Nighttime oxygen flux measurements provided average hourly respiration rates (rye) of the whole anemone (where 0 = algal [r”] plus animal [rz] fraction), which when extrapolated to 24 h, &vided an &timate of total daily anemone respiration (Rye). Carbon budgets and CZAR. The oxygen flux measurements of photosynthesis and respiration were used to estimate the distribution of carbon within the association. The percent contribution of algal carbon to animal respiration (CZAR) was determined by the modified formula of McCloskey et al. (1994): CZAR

= u-375

. p~)(pa,~-‘1-

[cl - ~xo.375.

(N(O.375

where RQ,, =

W-kWQ,)-‘>

K~(RQ,,)~ - [c,i . loo (3)

* R?J(RQ,,)

+ (W)(RQ,,)-‘)I-’

and values of 1.1,0.9, and 1.0

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I J. Exp. Mar. Biol. Ecol. 19.5 (1994)

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191

were used for photosynthetic quotient (Pa,), respiratory quotient of the animal (RQ,,), and respiratory quotient of the algae (RQ,), respectively. The numerator of Eq. (3) represents the carbon available for translocation to the host (C,). 2.5. Statistics All data were checked for normality and homogeneity of variance (Cochran’s test). Non-normal or heterogenous data were log transformed, and CZAR and MI were square root-arcsine transformed prior to analysis. Student’s t-test. One-Way ANOVA, and post-hoc Tukey tests were performed using the software program Statistica/ W 4.0@ (StatSoft, Inc.).

3. Results 3.1. Algal biomass and growth parameters Density and biomass. Zooxanthella cell diameter was 1.3 times greater. so its volume was about 2.2 times that of a zoochlorella (Table 1). Although zooxanthellae cells are about twice as large, the density of zooxanthellae in hospite was only about three-fourths that of zoochlorellae. The average algal biomass ratio (1-p) was 0.045 for zooxanthellate, and 0.038 for zoochlorellate anemones. Chlorophyll. The average zooxanthella had 1.6 times as much chlorophyll-a as a zoochlorella (Table 1). Zooxanthellae lacked chlorophyll-h and zoochlorellae Table 1 Algal size, density. eleganrissitna

and

chlorophyll

content

of zooxanthellae

(ZX)

zx * -t SF

and

zoochlorellae

(ZC)

zc:zx

Significance

(n) Cell diameter Cell volume Density

(lm)

12.46 + 0.15

9.57 f 0.12

1.3

***

(pm’)

(76) 104.5.6 + 36.4

(76) 473.9 i 16.5

2.2

:<* *

(76) 0.49 + 0.04

(76) 0.65 ? 0.05

0.8

*

(9) 3.87 2 0.14

(10) 2.38 5 0.14

1.6

*-ad;

1.5

***

(10“ cells. rng-’

protein) Chlorophyll-a

animal

(pg. cell-‘)

<‘hlorophyll-h

(pg. cell

‘)

(6) _

(6) 0.77 ? 0.05

Chlorophyll-c

(pg. cell

‘)

0.92 ? 0.04

(6) _

(6) 4.79 k 0.18

3.15 + 0.19

(6)

(6)

Total Chl-m + b or c (pg. cell

Significance

of differences

‘)

was determined

in A.

by the r-test (* = p i 0.05: *** = p < 0.001).

192

E.A. Verde, L.R. McCloskey

2

I J. Exp. Mar. Biol. Ecol. 195 (1996) 187-202

4

6

6

TIME

10

12 14 16 16 20 22 24

OF

DAY

(h)

Fig. 1. Mitotic indices (MI) of freshly isolated zooxanthellae (ZX) and zoochlorellae (ZC) from Anfhopleura elegantissima (n = 7) as a function of time of day. The horizontal bar denotes the period when the anemones experienced aerial exposure in a simulated low tide. Each point is the mean of seven anemones with the vertical bars indicating standard error.

lacked chlorophyll-c. Total chlorophyll for a zooxanthella was about 1.5 times that of a zoochlorella. MI and growth. The die1 patterns of MI for zooxanthellae and zoochlorellae are shown in Fig. 1. No significant peaks of activity were evident during the 24-h period for either alga; we interpret this as similar to the non-phased (asynchronous) division described by McDuff and Chisholm (1982). The MIS and derived values for the two algae are compared in Table 2. Zoochlorellae MI was nearly 22 times greater than zooxanthellae. Consequently the daily algal-specific growth rate (Pi) of zoochlorellae was commensurately increased, and zoochlorellae population doubling time (D,) decreased. Given these MIS and the t,‘s used, the zoochlorellae population would double about every 28 days, compared to 239 days for zooxanthellae. The carbon-specific growth rate (C,) of zoochlorellae was nearly seven times greater than zoox-

Table 2 Growth measures of zooxanthellae (ZX) and zoochlorellae (ZC) in A. elegantissima, based index (MI) measures (n = 84 anemones for each species of algae)

Measured parameter MI (X% ? SE) Parameters calculated @, (day-‘) D, (day) C, (cLg C.day-‘)

zx

zc

zc:zx

Significance

0.34 -t 0.03

7.34 5 0.23

21.6

***

8.5 0.1 6.8

***

from MI 0.0029 239.0 56.2 + 3.6

0.0246 28.2 380.8 2 16.1

on mitotic

Algal specific growth per day (p,), population doubling time (D, = In 2. pi’), and carbon-specific growth (C&) were calculated using cd’s of 28 and 69 h for zooxanthellae and zoochlorellae, respectively. Significance of difference in MI and C,, between the two algae was determined by the r-test ( *** =p
E.A. Verde. L.R. McCloskey

anthellae, necessitating maintain algal growth.

I J. Exp. Mar. Biol. Ecol. 19.5 (1996)

a corresponding

increase

in carbon

187-202

requirement

1%

to

3.2. Photosynthesis and respiration Photosynthesis and algal respiration. The respiration and photosynthesis of the two algae, in carbon equivalents, are compared in Figs. 2 and 3 respectively. The average daily respiration of the zooxanthellae (Rz . day ’ . lO_ ’ cells; Fig. 2A) was nearly twice that of zoochlorellae. However total algal respiration per whole

1500

z

01 I=

NS g

1353

d oE 1400 E ii

1344

C

-

I m

a& 1300

-

$0 I: za

1200

Fig. 2. Respiration rates (A. B) of zooxanthellae (2X) (n = 8) and B zoochlorellae (ZC) (n = 10) in Anthopleura eleganhsima. (C) Respiration of the animal fraction only. Significance of differences determined by the f-test (*** = p < 0.001; N.S. = not significant). Vertical bars represent standard error. Values above the bars indicate the averages.

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anemone (RF. day-’ * anemone-‘; Fig. 2B), while slightly higher for zooxanthellae, was not significantly different. Net photosynthesis normalized to algal cells (I’:. day-’ * 10e6 cells; Fig. 3A) and whole anemone (Pz. day-’ . anemone ’ ; Fig. 3B), were significantly greater for zooxanthellae (ZX:ZC between 1.4 and 2.2). Animal respiration. Animal respiration (Rz, . day-’ . anemone-‘) was not significantly different in zooxanthellate and zoochlorellate anemones (Fig. 2C). CZAR. The mean CZAR for zooxanthellate anemones, under the conditions of our measurements, was 5.3 times higher than their zoochlorellate counterparts (Fig. 3C).

mzx

Ezlzc

ttt

I

60 *et

60

48.4

C

Fig. 3. Net photosynthesis (A) and (B) and CZAR estimates (C) of zooxanthellae (ZX) (n = 9) and zoochlorellae (ZC) (n = 10) from Anrhopleura elegantissima. Significance of differences determined by the f-test (** = p < 0.01: *** = p < 0.001). Vertical bars represent standard error. Values above the bars indicate the averages.

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195

4. Discussion Algal density biomass and chlorophyll. The decided differences in mass between the two symbionts from the same host (Table 1) coupled with the differences in photosynthetic capacity of the two algae we describe, raise questions about host-symbiont specificity and fidelity, as well as what limits the symbiont population size. In a companion paper (McCloskey et al., 1996), we have presented data on differential expulsion of zooxanthellae and zoochlorellae from Anthopleura elegantissima, and discussed its potential for limiting symbiont populations. While the chlorophyll-a in zooxanthellae from A. efegantissima (3.9 pg. cell ‘: Table 1) was 1.6 to 2.6 times greater than for symbionts in the anemones Aiptasiu palfida and A. pufchella, the chlorophyll ax ratio of the algae in all three anemone species was similar (Muller-Parker. 1984. 1987: Cook et al.. 1988). The chlorophyll-a from zoochlorellae (2.4 pg. cell ‘: Table 1) falls within the midrange of chlorophyll-a values for known cnidarian algal symbionts. Both pigment values are high, probably due to low irradiances reaching individual algae, the consequence of host tissue and algal self-shading (Rees, 1991). Algal MI and growth. As Hoegh-Guldberg & Smith (1989; also Smith & Hoegh-Guldberg, 1987) have pointed out. phased MI appears to be the general rule among most cnidarian symbionts, with notable exceptions reported by Wilkerson et al. (1988). The two algal species in A. elegantissima. exhibiting non-phased division, join the exceptions. Wilkerson et al. (1988) proposed that non-phased algal division may indicate host control of algal population density, although this interpretation has been questioned by Hoegh-Guldberg & Smith (1989). Since A. efegantissima has exhibited a capacity to effectively control its algal population through expulsion as we have shown in a companion paper (McCloskey et al., lYY6), we believe the parsimonious explanation for symbiont control in A. elegantissima is simply expulsion rather than internal nutrient restriction. The average MI for zooxanthellae in A. elegantissima (0.34%: Table 2) was about an order of magnitude less than values reported by Wilkerson et al. (19X3) for zooxanthellae in the same hosts from California (4.69%) and Washington (2.88%), and also lower than MI values of 1.2-1.7% reported by McCloskey et al. (1996). Rather, the MI of this sample is closer to the values of 0.76% and 0.2Y% reported for zooxanthellae in the anemone Aiptasia pufchella (Wilkerson et al.. 1983: Muller-Parker, 1985), and 0.43-0.61% for zooxanthellae in the coral Stylophora pistillatrr (Wilkerson et al.. 1983). Two years after the experiments described here, we re-examined anemones from the same collection site and obtained somewhat higher MIS for zooxanthellae. but the average was still slightly less than 1%. While low MIS have been ascribed to host starvation (Cook et al., lYX8), we do not believe this is the reason for low MIS in zooxanthellae from the anemones in this experiment. The anemones were collected from a site where they are obviously thriving. and appear to have full coelenterons when collected.

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Comparable data for zoochlorellae include MI values up to 20% from the congeneric anemone A. xanthogrammica (O’Brien & Wyttenbach, 1980) and we have determined similar MI values for zoochlorellae from this species in our laboratory (unpublished). McCloskey et al. (1996) reported MI values for zoochlorellae from A. elegantissima from 3.2 to 5.6%. The MI of 7.34% for zoochlorellae from A. elegantissima (Table 2) was consistent and repeatable for the anemones from our collection site. The implications and conclusions we shall subsequently draw regarding carbon budgets for both algae, which are based on MI values measured in this study, should be viewed as single-case comparisons. Photosynthesis and algal respiration. The higher net photosynthesis (PF . cell-‘; Fig. 3) of zooxanthellae compared to zoochlorellae is consistent with observations of McFarland & Muller-Parker (1993) who report greater productivity of zooxanthellae from A. elegantissima, especially at higher irradiances. Anemones symbiotic with zooxanthellae were not light saturated up to 550 pmol . rnp2 * SC’, whereas anemones with zoochlorellae were light saturated around 100 pmol* -2 m * s-’ (Muller-Parker, pers. comm.). Our experiments placed anemone pairs, one zooxanthellate and the other zoochlorellate, under identical light regimes, where maximum midday irradiance usually exceeded 2000 pmol . me2 . SC’. Because we have observed no photoinhibition at these irradiance levels, we have dismissed photoinhibition as a factor suppressing the photosynthesis of either alga in A. elegantissima. The comparatively reduced photosynthesis and low saturation values of zoochlorellae could also be ascribed to algal self-shading, given that zoochlorellae are about 33% denser than zooxanthellae (Table 1). Shick & Dykens (1984) reported Pz values of 1255 lug C. day-’ for high intertidal zooxanthellate anemones in California - about 1.8 times those reported here. Such a discrepancy is difficult to explain. Differences in the respective methods include different respirometry techniques and longer aerial exposure (15 h compared to 6 h) in Shick and Dykens’ work. We cannot evaluate what differences in instrumentation might contribute, but latitudinally related increased daily irradiance could also contribute to the higher values obtained by Shick and Dykens. If the lower latitude anemones were photoadapted, with a higher P,,,,,, Pz should be higher. As well, there could be different strains of zooxanthellae in these two geographically separated localities, with different photophysiological responses. Regardless of the discrepancies between P: for zooxanthellae in this and other comparable studies, our data show remarkable differences between zooxanthellae and zoochlorellae in A. elegantissima under the conditions of our experiment. Algal P:R ratios calculated from the PF and RF values of Figs. 2 and 3 reveal relatively high values of close to 11 for zooxanthellae and 10 for zoochlorellae. Although algal respiration of symbiotic algae in hospite based upon relative algal protein biomass (1-p) (Muscatine et al., 1981) has become a widely used convention, significant uncertainty remains, with in vitro algal respiration perhaps being five to eight times greater than predicted by the biomass ratio method (Muller-Parker, 1984; Smith and Muscatine, 1986; Muscatine, 1990). We suspect

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197

that we have underestimated algal respiration for both algae (which would inflate the algal P:R), but have no way presently to measure Ry in hospite. In spite of the uncertainties of algal respiration measurements, the two algae are sufficiently different to invite comparison. CZAR. The mean CZAR for zooxanthellate anemones of 48.4% (Fig. 3) is almost three times the CZAR of 17.7% reported by Shick and Dykens (1984). They used a translocation factor (T, obtained by the 14C method) of 40% to calculate CZAR. Although the revised formula for CZAR (Eq. 3) does not use T. the de facto T in our study is T=

P; - c, P’,

. 100

and lies close to 90%. When a T of 90% is substituted in the CZAR equation used by Shick & Dykens (1984) their CZAR estimates range from 40 to 65%, bracketing the mean of 48% we obtained in this study (see also Zamer & Shick, 1987). The average CZAR of 9.1% for zoochlorellate anemones, though among the lowest ever reported for a symbiotic cnidarian, is probably a maximum for this association. When t, values less than 69 h are used, CZAR for zoochlorellae drops even lower, and below 54 h, CZAR becomes a negative number (Verde & McCloskey, 1996). Thus, the high MI of zoochlorellae represents a significant carbon sink. and under low irradiance conditions, zoochlorellae may direct more carbon per day into new cells (pY and C,) than is fixed in net photosynthesis. Consequently, C, decreases, and provides an explanation as to why Muscatine (1971) and O’Brien (1980) found minimal translocation products in zoochlorellate host fractions. This reduction in C, results in lower CZAR estimates. Carbon budgets. As we have pointed out (Verde & McCloskey, 1996: McCloskey et al.. 1995) a number of the components of carbon budgets (CL,, CF. and CZAR) are influenced by the duration of cytokinesis (td), which becomes less important in CZAR and carbon budget calculations at longer times. While our choice of t, of 28 and 69 h for zooxanthellae and zoochlorellae, respectively, was not based on empirical measurements, we believe the rationale for t, times longer than the 11 h proposed by Wilkerson et al. (1983) is compelling. Consequently we have chosen to report first-approximation budgets for symbiotic A. elegantissimu rather than wait for the development of the technology to precisely measure t, in hospite. Our estimates of average daily fluxes of carbon for both algal types are given as a flow diagram (Fig. 4). The higher net photosynthesis of zooxanthellate anemones, coupled with much lower algal growth demand, results in much more fixed carbon available for translocation to the host (i.e. C, = 651 for zooxanthellae versus 130 ,ug C. day-’ for zoochlorellae). The respiration of the animal (R:,) can be satisfied by carbon translocated or heterotrophically derived (including transepidermal uptake; Preston, 1993), or both. The carbon flow models in Fig. 4 show that only about 9% of respiratory

198

E.A. Verde, L.R. McCloskey

I J. Exp. Mar. Biol. Ecol. 195 (1996) 187-202 Heterotrophy

L 702

Heterotrophy

2 1214

I-

___-

A. 2X in Anthopleura elegantissima

/

. /,

cc-

N / I I I I

New Animal

I

(Growth) = ?

\ I \ \ \ \

\ \ \

.

. -_

B. ZC in Anthoplewa elegantissima Fig. 4. Average carbon fluxes for (A) zooxanthellae (ZX) and (B) zoochlorellae (ZC) in Anthopleura eleganfissima; values arc given as pg C. day-’ for an average anemone. P,c is gross and Pz is net of algae and animal fractions, respectively; C, is the photosynthesis; Ry and Rz are respiration carbon-specific growth rate (defined in Eq. 2 of text); C, is carbon translocated to the host ([p:] [C,]); K is algae expelled (from McCloskey et al., 1995); and CBAG is carbon translocated from the animal back to the algae.

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demand is potentially satisfied by algal translocate in zoochlorellate anemones, while about 48% is satisfied in zooxanthellate animals. It follows that considerably less carbon assimilation by heterotrophy would therefore be necessary in zooxanthellate animals (2702 versus 21214 pug C. day-’ for zoochlorellate). Heterotrophic consumption clearly has to be greater than this if the animal is to grow, and if any carbon is translocated back to the algae from the animal (indicated as CBAG in Fig. 4). If Rf: is in fact higher than the estimates of Fig. 2 (based on l-/3), then the carbon translocated (C,) would be proportionately reduced. The possibility also exists for carbon to be translocated from the host to the algae, and such heterotrophically derived carbon could subsidize either C,, C,, or both. The idea of translocation from host to symbiont has received support from the work of Douglas & Smith (1980) McAuley (1986, 1987) Steen (1986a,b), Rees (1987) and Cook et al. (1988), and we suggest that zoochlorellae are likely recipients of translocation products from A. elegantissima. Neither of these estimates of minimal heterotrophic carbon consumption appear to be beyond the capabilities of A. elegantissima. Shick (pers. comm.) has estimated that A. elegantissimu is capable of heterotrophic prey capture at a rate of 2550 pug C. day-‘, and Zamer (pers. comm.) has determined that “high” intertidal anemones receive -3150 pg C. day-’ from captured prey. Jensen & Muller-Parker (1994) have also provided evidence for high feeding rates by A. eleguntissimu in tidepools. Clearly, heterotrophic feeding represents a major source of carbon for the association regardless of whether the animal utilizes it for respiration, growth, or CBAG. A more precise accounting of the carbon translocated to the host (C,) is dependent upon the development of new technology to directly measure respiration of the algae (RF) in hospite and refinements in our ability to confirm algal growth (C,L). As well, the magnitude of CBAG, if it occurs in this association. remains unmeasured. We have evidence (unpublished work by Minnick & McCloskey) that not just the quantity, but also the photosynthetic products of zooxanthellae and zoochlorellae are different. While zooxanthellae translocate primarily glycerol, fumarate/succinate, malate, and citrate, as well as small quantities of alanine (Trench, 1971a,b), zoochlorellae translocate several amino acids (isoleucine, alanine, tryptophane, glycine, aspartate, and asparagine) as well as glycerol, succinate. and lactate. While the major intracellular product of zoochlorellae is expected to be sucrose (Muscatine, 1971) we have been unable to detect any sugars in zoochlorellate translocate. If zooxanthellate anemones receive so much more algal translocate than do zoochlorellate hosts, the question arises as to why does the zoochlorellae symbiosis persist? We suggest two possibilities. First, under some conditions, a host obtaining primarily amino acids and lipids from zoochlorellae conceivably could be favored over one with zooxanthellae. This could be especially significant during periods of low food availability. Second, A. eleguntissimu simply may not be dependent on symbiotic algae to any significant degree (the success of

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nonsymbiotic A. elegantissima proliferating in sea caves is indicative), but rather both algae may be exploiting the host - unlike their tropical counterparts in reef corals. A complete accounting of the energy and fluxes of materials in both associations is yet necessary to adequately evaluate the adaptive value of the respective symbionts; the work of Zamer & Shick (Zamer, 1986; Zamer & Shick, 1987, 1990) may be considered as models for such an effort. It is not currently possible to determine the evolutionary time frame for either symbiotic association (cf. Trench, 1993), and therefore one cannot do more than agree with Muscatine (1971) that the relationship between zoochlorellae and Anthopleura, at least, seems to be one where the alga is in some early stage of evolutionary development, and may even be parasitic. McFarland & MullerParker (1993) have similarly proposed that their algal-nudibranch association represents an early phase in the evolution of plant-animal symbiosis. Even if zoochlorellae, and perhaps zooxanthellae as well, in A. elegantissima, are not relatively recently established symbioses, they have been subjected to decidedly different selection forces than their tropical counterparts.

Acknowledgements

This research was supported by National Science Foundation grant OCE8303513 (to LRM), the Department of Biological Sciences and the Marine Station of Walla Walla College. We thank D. Riley for constructing Fig. 4, and G. Muller-Parker for numerous helpful comments on an early draft of the manuscript.

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