nutritional role of two algal symbionts in the temperate sea anemone anthopleura elegantissima...

Nutritional Role of Two Algal Symbionts in the Temperate Sea Anemone Anthopleuraelegantissima BrandtAuthor(s): Heather Bergschneider and Gisèle Muller-ParkerSource: Biological Bulletin, Vol. 215, No. 1 (Aug., 2008), pp. 73-88Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/25470685 .

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Reference: Biol. Bull. 215: 73-88. (August 2008) ? 2008 Marine Biological Laboratory

Nutritional Role of Two Algal Symbionts in the Temperate Sea Anemone

Anthopleura elegantissima Brandt

HEATHER BERGSCHNEIDER AND GIS?LE MULLER-PARKER*

Shannon Point Marine Center and Department of Biology, Western Washington University,

Bellingham, Washington 98225-9160

Abstract. The intertidal sea anemone Anthopleura el

egantissima in the Pacific Northwest may host a single type of algal symbiont or two different algal symbionts simulta

neously: zooxanthellae (Symbiodinium muscatinei) and zoo

chlorellae (green algae; Trebouxiophyceae, Chlorophyta). A

seasonal comparison of zooxanthellate and zoochlorellate anemones showed stable symbiont population densities in summer and winter, with densities of zoochlorellae about 4

times those of zooxanthellae. Photosynthesis-irradiance curves of freshly isolated symbionts show that the produc

tivity (Pmax cell) of freshly isolated zooxanthellae was about

2.5 times that of zoochlorellae during July; comparable rates were obtained in other months. Models of algal carbon flux

show that zoochlorellae may supply the host with more

photosynthetic carbon per unit anemone biomass than zoo

xanthellae supply. Zooxanthellate anemone tissue was 2%c

(13C) and 5%c (15N) enriched and zoochlorellate anemone tissue was 6%c (,3C) and 8%o (15N) enriched over their

respective symbionts, suggesting that zoochlorellate anem

ones receive less nutrition from their symbionts than do zooxanthellate individuals. The disparity between predicted contributions from the algal carbon budgets and the stable

isotopic composition suggests that short-term measures of

algal contributions may not reflect actual nutritional inputs to the host. Isotopic data support the hypothesis of substan tial reliance on external food sources. This additional nutri tion may allow both algae to persist in this temperate intertidal anemone in spite of differences in seasonal pho tosynthetic carbon contributions.

Received 25 June 2007; accepted 25 March 2008. * To whom correspondence should be addressed. E-mail: Gis?le.

[email protected]

Introduction

The intertidal sea anemone Anthopleura elegantissima (Brandt, 1835) hosts two known algal symbionts in the

Pacific Northwest: Symbiodinium muscatinei (LaJeunesse and Trench, 2000) and green algae recently placed in the class Trebouxiophyceae (Lewis and Muller-Parker, 2004).

Dinoflagellate symbionts are commonly called zooxanthel

lae, and symbiotic green algae are called zoochlorellae. A.

elegantissima and its congener A. xanthogrammica may

host one of these symbionts (= zooxanthellate or zoochlo

rellate anemone) or both (= mixed anemone) simulta

neously. In the Pacific Northwest, these anemones are ex

posed to large seasonal fluctuations in irradiance and

temperature, the parameters most likely to affect the pro

ductivity of the algal symbionts. The average daily inte

grated fluxes of sea-surface irradiance in summer are 6.5

times those of winter averages (Muller-Parker and Davy,

2001). Because of seasonal differences in the timing of the lower low tide in the San Juan Islands, during aerial expo sure these anemones may also experience internal body

temperatures as high as 28 ?C in the summer and as low as 5 ?C in the winter (Dingman, 1998).

Given these environmental extremes in irradiance and

temperature, it is of interest to know how these two algal symbionts differ in their productivity and contributions to the host anemone during the summer and winter seasons. Studies have shown that zoochlorellae are maintained at

higher densities and have higher maximum photosynthetic rates under conditions of low light and low temperature (Saunders and Muller-Parker, 1997; Engebretson and Mul

ler-Parker, 1999), and that zooxanthellate individuals of A.

elegantissima sustain higher photosynthetic rates than zoo

73



74 H. BERGSCHNEIDER AND G. MULLER-PARKER

chlorellate individuals under summertime conditions of

high light and temperature (Verde and McCloskey, 2001,

2002, 2007). Zooxanthellae and zoochlorellae may respond

differently to seasonal fluctuations in environmental param eters because they differ in their physiological tolerances to

temperature and irradiance (O'Brien, 1980; Saunders and

Muller-Parker, 1997; Engebretson and Muller-Parker,

1999). Verde and McCloskey (2007) recently determined

that in the spring and summer the net productivity of zoo

xanthellate A. elegantissima was greater than that of zoo

chlorellate A. elegantissima, and they calculated a higher

potential contribution of photosynthetic carbon from zoo

xanthellae than from zoochlorellae in all seasons. It is also

possible that during low light conditions in winter, the

nutritional relationship of both algae with their host may shift from mutualistic to parasitic with respect to carbon.

The purpose of this study was to examine how symbiont

populations and the productivity of zooxanthellae and zoo

chlorellae isolated from A. elegantissima vary with seasonal

changes in environmental parameters, and to compare the

nutritional contributions of both algae by estimating the

amount of photosynthetic carbon available for translocation

to the host and by using stable isotopes to examine the

relative importance of allocthonous versus translocated car

bon to the anemone diet. Since the delta13C and delta15N

signatures of an organism are related to those of its diet, the

relative contribution of photosynthetic carbon and hetero

trophically derived food to the diet of A. elegantissima may be deduced by comparing the isotopic signatures of symbi otic anemones with those of nonsymbiotic counterparts, because the latter are exclusive heterotrophs.

Materials and Methods

Anemone collection and isolation of algal symbionts

Zooxanthellate (brown) and zoochlorellate (green) indi

viduals of Anthopleura elegantissima were collected from

Shannon Point Beach (48?30\ 122?41'), Fidalgo Island,

Anacortes, Washington. Collections were made on 9 July, 16 October, 10 December (2004), and 3 February and 29

April (2005). The same cluster of boulders was sampled

repeatedly, and anemones of a similar size (^0.2 g wet

weight) were collected.

At Shannon Point Marine Center, anemones were sepa rated by color, cleaned of rock and shell debris, and placed in a flow-through natural seawater table until analysis. The

conditions in the seawater table approximated those of the

submerged habitat. The seawater temperature was the same

(supplied from the same body of water), and anemones were

exposed to diffuse natural light supplied by large windows

next to the seawater table. Light or olive-colored anemones

(with low or mixed algal symbiont populations) were re

moved, and the remaining green and brown anemones were

selected haphazardly for analysis. For each collection, anemones were processed in six sets of four individuals at a

time (two zooxanthellate and two zoochlorellate anemones). Three sets of anemones were homogenized on the first and on the second day after collection, for a total of 24 anem

ones. All anemones were processed within 48 h of collec

tion.

To isolate the algae from animal tissue, anemones were

homogenized in 5-jLtm filtered seawater, using a 60-ml glass tissue homogenizer and a motorized Teflon pestle. Sub

samples were frozen for later determination of algal density and mitotic index (MI; the number of cells with complete

cleavage furrows), and for protein analysis. The remaining

homogenate was centrifuged in a tabletop swinging bucket

centrifuge at about 1600 X g; the animal supernatant was

discarded and the algal pellet resuspended in filtered sea

water by vortexing. This process of centrifugation and re

suspension was repeated three times. The final algal sus

pension was filtered through a 25-jtmi Nitex screen to

remove residual animal tissue. Of the final algal suspension, 5 to 10 ml was filtered onto a 25-mm GF/C filter under

gentle vacuum pressure (^10 mm Hg) for later analysis of

chlorophyll pigments, and 3 ml was used for immediate

measurements of algal 14C-fixation rates. A sample was also

taken and frozen for later cell counts.

Photosynthesis-Irradiance (P-I) measurements

The protocol of Bachman and Muller-Parker (2007) was

followed, with slight modifications. 14C bicarbonate (2 jllC?) was added to freshly isolated algae suspended in 3 ml of

filtered seawater. The algal-14C mixture was allocated as

0.2-ml subsamples into 12 glass mini-scintillation vials

(7-ml volume) and placed in a temperature-controlled pho

tosynthetron (CHPT Industries). The temperature of the

photosynthetron was set to the 2004-2005 average temper ature for the month of collection (see Fig. 1). Vials were

exposed to a gradient of irradiances ranging from 0 to about

1200 jLtmol photons m~2 s _1, as measured by a Bio

spherical Instruments QSL-101 quantum scalar sensor im

mersed in water in a glass vial placed into each position of

the photosynthetron. After 0.5 h, incubations were termi

nated by acidification of each sample with 1 mol 1_1 HC1 in

a fume hood overnight, followed by neutralization with 1

mol 1_1 NaOH. Disintegrations per minute (DPM) of each

sample was counted in a Packard 1900 TR scintillation

counter, and the photosynthetic rate of the algae at each

irradiance was calculated from the DPM data and the total

inorganic carbon content of the seawater. The total inor

ganic carbon content of seawater at each incubation tem

perature was determined from a linear curve relating total

inorganic carbon content with seawater temperature. This

curve was derived from pH and alkalinity values of a series



ALGAL SYMBIOSIS IN A TEMPERATE ANEMONE 75

of seawater samples (practical salinity =

30) ranging in

temperature from 5 to 30 ?C, using the methods and tables

in Parsons et al (1984). The maximum photosynthetic rate

(Pm3LX cell), and photosynthetic efficiency (a) were derived

from the P-I data of algae from each anemone by using a

hyperbolic tangent function in Sigma Plot 9.0.

Density, size, mitotic index, and chlorophyll content of

algae

A Reichert Bright-line hemacytometer slide was used to

perform cell counts on anemone homogenate samples and

on the final algal suspensions. Cells were viewed at 400X

magnification and at least 100 cells were counted per sub

sample, with a minimum of four subsamples per anemone

sample. No lysed cells were detected in the samples. Anem

one protein content was determined with the method of

Lowry et al. (1951), using bovine serum albumin as a

protein standard (Sigma Co.). Algal densities in the homog enate were normalized to anemone protein content.

To gauge algal division rates, MI was tallied per sub

sample of 1000 cells and reported as percent cells dividing. For the biomass and carbon content of the algae, the diam

eter of 100-150 cells (about 10 per anemone sampled) was

measured with a calibrated ocular micrometer at 400 X

magnification. Zooxanthellae and zoochlorellae from sum

mer (July 2004) and winter (December 2004 and February 2005) were measured, and cell volumes calculated assum

ing spherical algae. The equations of Muscatine et al

(1983) and Menden-Deuer and Lessard (2000; equation for

protist plankton excluding diatoms) were used, respectively, to derive animal and algal biomass ratios and the carbon

content of zooxanthellae and zoochlorellae.

For chlorophyll analysis, filters containing zooxanthellae or zoochlorellae were ground to a pulp in a 10-ml glass tissue homogenizer with a motorized Teflon pestle, using the appropriate solvent (100% HPLC-grade acetone and

100% HPLC-grade methanol for zooxanthellae and zoo

chlorellae, respectively). Samples were analyzed as de

scribed in Seavy and Muller-Parker (2002), using the equa tions of Jeffrey and Humphrey (1975) for zooxanthellae and

Holden (1976) for zoochlorellae.

Carbon translocation and photosynthetic carbon budgets

The percentage of photosynthetically fixed 14C trans ferred from algae to anemone host during the winter months

was determined from carbon translocation experiments; the

incubation protocols of Engebretson and Muller-Parker

(1999) were used to allow direct comparison of our winter values with their summer values. Zooxanthellate and zoo

chlorellate anemones (0.13 + 0.02 g; n = 8) were collected

from Shannon Point Beach on 26 December 2004, and the

experiment was conducted the next day. Anemones (n = 3

of each type) were incubated with 14C bicarbonate (2-6

j?lC?) for 1.5 h under light-saturating irradiance (?400 juimol

photons m"2 s_1) at 9 ?C. A fourth anemone of each type served as a dark control. After incubation, anemones were

rinsed thoroughly and resubmerged in 5 ml of filtered sea

water for a 1.75-h period of dark phase before the algal and

animal fractions were homogenized and isolated, as de

scribed above. Three 0.5-ml subsamples were taken from

the homogenate (total symbiosis), combined supernatant (animal fraction), and resuspended pellet (algal fraction) from each anemone and pipetted into 7-ml plastic scintilla

tion vials. Immediately after collection, samples were acid

ified with 0.3 ml of 6 mol F1 HC1 and placed on a shaker

table overnight to remove un-incorporated 14C. Samples were then neutralized with the addition of 0.3 ml of 6 mol

1_1 NaOH; 4 ml of EcoScint scintillation fluid was added; and the DPM of the homogenate, animal, and algal fractions

was counted by the liquid scintillation counter.

Percent translocation of 14C from algae to anemone host was calculated by dividing the DPM of the animal fraction

by the total homogenate DPM. Heterotrophic 14C fixation was accounted for by subtracting the calculated DPM of the

dark control anemone samples from the corresponding anemone samples incubated in the light.

To calculate carbon budgets, summer and winter daily

productivities of zooxanthellae and zoochlorellae were de

rived from the equation P = Pmax tanh (a-1' PmSiX~l) using

data from the average P-I curves of isolated algae for each season (summer: July 2004; winter: Dec. 2004 and Feb.

2005) and data from the average hourly irradiance levels (I)

experienced during an average summer or winter day for

2004-2005 in Anacortes, Washington. Local values of sea

surface irradiance were obtained from data collected con

tinuously by Shannon Point Marine Center using a LiCor

1400 datalogger and LI-190S A quantum sensor. Daily pro ductivities, expressed on the basis of anemone protein bio

mass, were calculated for the average hourly sea-surface

irradiance (100% of available light) and for 50%, 25%, 15%, 10%, 5%, 3%, and 1% of the average hourly sea

surface irradiance. This range of light levels was selected to

encompass the possible ranges experienced by the algae in

hospite because of light attenuation by seawater and anem one tissues, and also to account for possible differences in

photosynthesis of symbionts in hospite and freshly isolated.

Stable isotopic signatures of C and N

Zooxanthellate, zoochlorellate, and nonsymbiotic anem ones were collected from Shannon Point Beach on the collection dates listed previously, and also on 17 August 2004 and 19 July 2005. The day following collection, six

zooxanthellate, six zoochlorellate, and five nonsymbiotic A.

elegantissima were selected haphazardly for analysis. These




anemones averaged 0.15 ? 0.03 g wet weight, with an oral

disk diameter of 8.20 ? 0.70 mm (n = 100 ? SE). Each

anemone was prodded to contract, forcing expulsion of

undigested food particles, and was frozen at -70 ?C until

processing.

Frozen anemones were cut in half from oral to aboral end; one half serving as a sample of anemone tissue (nonsym biotic anemones) or anemone tissue plus algal symbionts (zooxanthellate and zoochlorellate anemones). To obtain

algae, the other half of each symbiotic anemone was ground in a 10-ml glass tissue homogenizer with a motorized Teflon

pestle, and the algae were isolated using repeated centrifu

gation as described above. The final algal pellets were

resuspended in less than 1 ml of deionized water in mi

crofuge tubes and frozen at -70 ?C before lyophilization.

Lyophilized samples were ground to a fine powder and sent

to the Washington State University stable isotope laboratory where they were analyzed for 13C and 15N isotope signa

tures by flow-through mass spectrometry. Values are re

ported in parts per thousand (%c) relative to the Peedee

belemnite (PDB) limestone and nitrogen in the atmosphere.

Statistical analyses

A two-way analysis of variance (ANOVA) was used to

examine algal density, MI, and productivity. The factors

examined were month of year (5 levels: July, Oct., Dec,

Feb., Apr.) and algae (2 levels: zooxanthellae and zoochlor

ellae). One-way multivariate analysis of variance

(MANOVA) was used to analyze changes in chlorophylls a

and b for zoochlorellae and chlorophylls a and c for zoo

xanthellae over time. A one-way ANOVA was used to

determine whether percent carbon translocation differed for

zooxanthellate and zoochlorellate anemones.

All data were checked for the assumptions of equal variance and a normal distribution. Data that failed to meet

the assumption of equal variance were natural-log or

square-root transformed. When transformed data still failed

to meet the assumption of equal variance or equality of

covariance matrices, the severity of the violation was de

termined (Hartley's Fmax) and alpha adjusted to 0.01 (Un

derwood, 1981). Otherwise all data were analyzed at the

significance level of 5%.

The statistical software package SPSS was used for all

analyses. When a significant effect was found for month, a

priori contrasts were used to determine if summer (July) values were significantly different from winter (Dec. and

Feb.) values. If a significant month X alga interaction was

found, simple main effect contrasts were used to determine

the reason for the significant interaction. All other contrasts

were done using Scheff?'s S Test. The critical difference,

ty(S), that contrast means must exceed to be declared sig nificant was calculated manually.

60

50

40

30

20 ^

10

- 0 12 13

Temperature (?C)

Figure 1. Monthly averages for seawater temperature (?C) and aver

age daily irradiance for the 2004-2005 sampling period (dotted line) and

the long-term 2000-2005 seawater temperatures and 2002-2005 irradiance

values (solid line). Error bars for the long-term data represent ? 1 standard

error of the mean. Temperature and irradiance data from the Shannon Point

Marine Center Water Quality Data Base.

Results

Figure 1 shows the annual distribution of seawater tem

peratures and daily irradiance fluxes for the 2004-2005

sampling period, and the 5-year (temperature) and 3-year

(irradiance) averages for Anacortes, Washington. For both

parameters, the lowest values occur in January (8 ?C, 6 mol

m~2 d_1); the highest are obtained in July for irradiance

(46 mol m~2 d~ l) and in July and August for temperature

(13 ?C). Compared to the long-term averages, seawater

temperatures in this study were slightly cooler in January and slightly warmer in August; irradiance levels were com

parable in January-February and in July, and reduced in the

spring and late summer-early fall.

Algal biomass parameters, pigments, and photo synthetic

parameters

Overall, anemones collected for the productivity and al

gal biomass measurements averaged 0.17 ? 0.02 g wet

weight, with an oral disk diameter of 7.9 ? 0.49 mm (n ?

120, ? SE). There was no significant difference in the

protein biomass of zooxanthellate and zoochlorellate anem

ones, which averaged 9.56 ? 0.95 mg protein and 8.72 ?

0.81 mg protein, respectively (n = 60, ? SE).

Zoochlorellate anemones had significantly higher algal densities than zooxanthellate individuals (Fig. 2; P < 0.001;

sq. rt. transformed; Fmax= 11.06; a adj. 0.01). Although month of collection had a significant effect on algal density

(P < 0.001), a contrast comparing the pooled densities of

zooxanthellae and zoochlorellae in the summer (July) and

the winter (Dec. and Feb.) did not yield a significant differ

ence. The significant effect of month was probably the result

of the elevated density of zoochlorellae in December (Fig.

2); however, due to low power, an a posteriori contrast did




Zoochlorellae Density ? ?Zooxanthellae Density

- - -o- - Zoochlorellae MI - D - Zooxanthellae MI 2.0 -, T 25

0.0 J-,-,-,- -,-.- -,-.-,- -^0 J-04 J-04 A-04 S-04 O-04 N-04 D-04 J-05 F-05 M-05 A-05 M-05 J-05

Date (M-YY)

Figure 2. Density and mitotic index (MI) of zooxanthellae and zoo

chlorellae in Anthopleura elegantissima sampled from 6 July 2004 to 29

April 2005. Error bars represent ? 1 standard error of the mean (n = 9-12

anemones).

not find this density significantly different from that of

zoochlorellae in anemones collected in all other months

(\p(S) =

27.57). Winter densities of zooxanthellae (Dec. and Feb.) also were not significantly different from April densities 0P(S)

= 16.06).

Regardless of month, the mitotic index (MI) of zoochlor

ellae, almost 20%, was significantly higher than that of

zooxanthellae, 3% (P < 0.001; two-way ANO VA; In trans

formed). There was also a significant month X alga inter

action (P =

0.001; two-way ANOVA). Although monthly MI trends appear similar (Fig. 2), zooxanthellae exhibit a

significant seasonal variation (P < 0.001) and zoochlorellae

do not (P =

0.867). A contrast comparing summer versus

winter found that the MI of zooxanthellae was significantly

higher in July 2004 (3.9% ? 0.63; ? SE) than during December 2004 and February 2005 (2.3% ? 0.26; ? SE; P =

0.001). The size and carbon content of zoochlorellae

remained fairly constant between summer and winter, while

zooxanthellae exhibited a decrease of almost 30% in both

parameters during winter (Table 1).

Chlorophyll concentrations of both algae are shown in

Figure 3. Analysis of individual chlorophyll pigments showed that chlorophylls a and c of zooxanthellae vary

during the year (P < 0.001, Pillai's trace; In transformed; a

adj. 0.01). While chlorophyll c exhibited smaller seasonal

changes than chlorophyll a (Fig. 3a), neither pigment showed a significant difference between summer and winter

concentrations (chl a, P = 0.087; chl c, P =

0.511). Month

of year also had a significant effect on chlorophyll a and b

concentrations in zoochlorellae (P < 0.001, Pillai's trace; In

transformed; a adj. 0.01). Both pigments followed the same

seasonal trend, with highest concentrations of chlorophyll a

and b during fall through winter (Oct., Dec, and Feb.; Fig.

Table 1

Seasonal comparison of zooxanthellae and zoochlorellae biomass, productivity (P), growth parameters, and P -

available for translocation to the anemone) in Anthopleura elegantissima

Cj?L (excess fixed C potentially

Parameter Summer

Zooxanthellae

Winter Summer

Zoochlorellae

Winter

Diameter (/im) Volume (jutm3)

pg C alga-1* MI (%) B$ 1-B?

13.0

1204 168

3.86

0.95

0.05

11.7

865 123

2.30

0.95

0.05

9.4

441 65 20.45

0.91

0.09

9.2

418 62 18.41

0.91

0.09

P @ 50% irradiance?

pgC fixed alga-1 day-1

jLtgC fixed mg protein-1 day

28.0

8.9

4.6

1.9

12.3

15.6

5.0

7.0

td (h) (McCloskey et al, 1996)

^z(day-1)t

Dt (day)t C)Lt(pgC-alga-1-day-L)t

CjLt (/?gC mg protein-1 day-1)t

P - Cfji (jtgC mg protein-1 day-1)

28

0.03

21.4

5.5

1.7

7.2

28

0.02

35.6

2.4

1.0

0.9

69

0.07

9.9

4.3

5.4

10.2

69

0.06

11.6

3.7

5.2

1.8

Calculations derived from data obtained during summer (July 2004) and winter (Dec. 2004, Feb. 2005). *

Algal C content (equation for protist plankton, excluding diatoms; Menden-Deuer and Lessard, 2000). ? Animal (B) and algal (1-B) biomass ratios (Muscatine et al., 1983).

? P was calculated using the summer and winter average P-I curves (Fig. 4) and the average hourly irradiances at 50% (Fig. 6). t Algal growth parameters: specific growth rate (/xz), population doubling time (Dt), and carbon-specific growth (C/x), are based on published values for

duration of cytokinesis (td) (equations from Verde and McCloskey, 1996).




a) Zooxanthellae

J-04 J-04 A-04 S-04 O-04 N-04 D-04 J-05 F-05 M-05 A-05 M-05 J-05

Date (M-YY)

b) Zoochlorellae

J-04 J-04 A-04 S-04 O-04 N-04 D-04 J-05 F-05 M-05 A-05 M-05 J-05

Date (M-YY)

Figure 3. The concentration of photosynthetic pigments (a) chloro

phyll a and c for zooxanthellae and (b) chlorophyll a and b for zoochlor

ellae from Anthopleura elegantissima sampled from 6 July 2004 to 29

April 2005. Error bars represent ? 1 standard error of the mean (n = 9-12

anemones).

3b). The concentrations of chlorophyll a and b were signif

icantly higher in winter (Dec. and Feb.) than during July

(P < 0.001; P = 0.002, respectively).

The productivity of freshly isolated zooxanthellae and

zoochlorellae was similar during all months, with the ex

ception of the elevated productivity of zooxanthellae in July

(Fig. 4). The irradiance level at which light-saturated rates

were obtained (/k) ranged from 50 to 100 /xmol photons m-2 s~l for both algae throughout the year. Analysis of the

initial slopes of the P-I curves, the light utilization effi

ciency of the algae (a), revealed that zooxanthellae and

zoochlorellae exhibit similar photosynthetic efficiencies

(P =

0.226; two-way ANOVA) with month of year having a significant effect (P < 0.001). The photosynthetic effi

ciency of both algae declined gradually from July 2004 to

April 2005 and was lowest during April (Fig. 5a). The

photosynthetic efficiency of both algae was about 2-fold

higher in July than during December and February (P =

0.020). The maximum light-saturated photosynthetic capacity de

rived from the P-l curves (Pmax cell), had a significant month X algae interaction (two-way ANOVA P < 0.001).

Although freshly isolated zooxanthellae achieved a maxi

mum photosynthetic rate almost 2.5 times greater than that

of freshly isolated zoochlorellae in July (P < 0.001), the

two algae had comparable and greatly reduced rates during all other months (Fig. 5b). There was a significant effect of

month (P < 0.001), with both zooxanthellae and zoochlor

ellae having higher photosynthetic capacities in July than

during December and February (P < 0.001 for each alga). A posteriori examination revealed that the photosynthetic

capacity of zooxanthellae during October was not signifi

cantly different from Pmax obtained during winter (^P (S) =

1.00).

Figure 6 compares the daily sea-surface irradiance levels

(and 75%, 50%, 25%, 15%, 10%, and 5% of the average

daily distributions of sea-surface irradiance) at the collec

tion site during July 2004 and winter (Dec. 2004, Feb. 2005) with the 4 for zooxanthellae and zoochlorellae obtained

from the P-l curves for these months. In the summer,

light-saturated photosynthetic rates (at irradiance levels >

/k) may occur for more than 6 h each day at irradiance levels

as low as 10% of ambient sea-surface levels (Fig. 6a).

During winter, the comparison shows that Pmax for both

algae may occur at irradiances 15% of ambient sea-surface

for 4 h.

Percent carbon translocation to host and photosynthetic carbon budgets

Results of the 14C anemone incubation method indicate

that during the winter zooxanthellae and zoochlorellae

translocated 55% and 42% of the carbon fixed to the anem

one host, respectively (P =

0.565; one-way ANO VA). A comparison of the potential benefits of hosting photo

synthetic zooxanthellae and zoochlorellae must account for

the carbon required for algal growth and respiration, with

any extra fixed carbon potentially available to the anemone

or to the alga for storage. Since zooxanthellae are larger and

therefore contain 3 (summer) and 2 (winter) times the

carbon content of zoochlorellae (Table 1), a dividing zoo

xanthella requires more carbon for growth. The amount of

carbon (C) utilized for growth (C/x) depends on algal MI

and the duration of cytokinesis (td). While MI is measured

easily and is 6 times higher for zoochlorellae (Table 1), td in

hospite has not been measured directly. Using algal density and expulsion rates of A. elegantissima collected from the

neighboring San Juan Islands in Washington State, McClos

key et al (1996) estimated the td of zooxanthellae and

zoochlorellae to be 28 and 69 h, respectively. Use of these




July 2004

H^-i

g-? S? -5?5- -5-S

0 200 400 600 800 1000 1200 1400

_Zoochlorellae

_ Zooxanthellae

October 2004

j i U

0 200 400 600 800 1000 1200 1400

1.5

0.0 t

February 2005

s-t-v^i-v

200 400 600 800 1000 1200 1400

December 2004

t 0 200 400 600 800 1000 12QQ 1400

1.5

1.0

0.5

0.0

April 2005

itT"??!*!^

0 200 400 600 800 1000 1200 1400

Irradiance (jumol photons m"2- s"1)

Figure 4. Photosynthesis-irradiance (P-I) curves for zooxanthellae and zoochlorellae from Anthopleura

elegantissima sampled from 6 July 2004 to 29 April 2005. Error bars represent ? 1 standard error of the mean.

For each curve, n = 7-10 (zooxanthellate anemones) or n = 11-12 (zoochlorellate anemones).

estimates, with a much longer td (69 h) for zoochlorellae, results in similar C-specific growth (C/ll) for both symbionts during winter and summer (Table 1). When the Cjh is normalized for the respective density of zooxanthellae and

zoochlorellae within the host anemone, the carbon required for algal growth in a zoochlorellate anemone is 4 times

greater than that required in a zooxanthellate anemone (Ta ble 1).

Although the daily productivity (P; pg C fixed alga-1 d_1) of a zooxanthella at 50% of the average hourly irradi

anee is over twice that of a zoochlorella during summer, the two algae exhibit comparable daily productivities during

winter (Table 1). When daily productivity is expressed on

the basis of the density of the alga in the anemone, the high biomass of zoochlorellae means that the productivity of

zoochlorellate anemones is about 2 and 4 times that of zooxanthellate anemones during summer and winter, re

spectively (Table 1; Fig. 7). The calculated daily productivity of zooxanthellate and

zoochlorellate anemones is greater than the carbon re




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V

i S44 CM*'M*/IH*: Jfcfl*

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b) Photosynthetk capadty <PM ̂ )

2.0

t.5

i 0.5 H

0.0 J-04 ?04 M4 S44 O?* *? |>?f ^^ F-0***? A4* M46 J-06

Figure 5. (a) Photosynthetic efficiency (a) and (b) the light-saturated

photosynthetic capacity (Pmax cell) of zooxanthellae and zoochlorellae

from Anthopleura elegantissima sampled from 6 July 2004 to 29 April 2005. Average values were calculated from a and Pmax values obtained

from the P-I curve for each alga. Error bars represent ? 1 standard error

of the mean. Sample sizes as in Fig. 4.

quired for algal growth for daily photon flux densi

ties ^ 3% of sea-surface levels during summer, and for

daily photon flux densities ^ 15% of sea-surface levels

during winter (Fig. 7a, b). When Cjll is subtracted from

the daily productivity calculated for 50% of the average

hourly sea-surface irradiance (P - Cjll), 7.2 (summer) and

0.9 (winter) micrograms of the daily C fixed per milli

gram protein of zooxanthellate anemone and 10.2 (sum

mer) and 1.8 (winter) micrograms of the daily C fixed per

milligram protein of zoochlorellate anemone is provided in excess of algal growth requirements (Table 1). Some

of this carbon is used for algal respiration, and the rest is

potentially available for translocation to the anemone.

These calculations, based on short-term photosynthetic rates of freshly isolated symbionts, show that zoochlor

ellate anemones may be supplied with 30%-50% more

photosynthetic carbon than zooxanthellate anemones at

50% sea-surface irradiance levels.

Symbiont contribution to anemone diet estimated by stable isotopes, delta13C and delta15N

Figure 8 shows the annual distributions of delta 13C and

delta15N values of nonsymbiotic anemone tissue, zooxan

thellate and zoochlorellate anemone tissue, and isolated

algal symbionts. Zooxanthellae and zoochlorellae show dis

tinctly different seasonal patterns in their delta 13C and

delta15N values (Fig. 8a). This difference between symbi onts is reflected in the delta values of symbiotic anemones, which differ from nonsymbiotic individuals. The delta13C and delta 15N values of zooxanthellate and zoochlorellate anemones are intermediate between those of their respective

symbionts and those of nonsymbiotic anemones.

The delta 13C and delta 15N values of the three anemone

types did not vary seasonally. There was little variation in

delta 13C of anemones, with annual ranges of 0.5%o in non

symbiotic anemones and 0.95%c in symbiotic anemones.

Nonsymbiotic anemone tissue had the highest delta 13C val

ft) Summer day

b) Winter day

Figure 6. Average sea-surface irradiance (100%) and 75%, 50%, 25%,

15%, 10%, and 5% of these levels during a typical day experienced by intertidal Anthopleura elegantissima during (a) summer (July 2004) and (b)

winter (Dec. 2004, Feb. 2005). Lines labeled zx and zc represent the Ik

(light saturation irradiance) for zooxanthellae (zx) and zoochlorellae (zc).

Light data were obtained from the Shannon Point Marine Center Water

Quality Data Base.




a) Zooxanthellate

18 ] 15 H

12 -i i

9-1

il

Excess Carbon

dCm

nyy??nnngggg

10o! 50 I 25 I 15 I 10 I 5 ! 3 I 1 I 0 100 50 | 25 | 15 | 10 | 5 ' 3 j 1 | 0 I Summer Winter

Percent Irradiance

b) Zoochlorellate

Excess Carbon

DCu

I

n TTff 100! 50 I 25 I 15 | 10 ! 5 3 | 1 I 0

I Summer 1001 50 I 25 I 15 | 10 I 5 j 3 | 1 | 0

Winter

Percent Irradiance

Figure 7. Derived summer and winter daily productivity (P) per mil

ligram of anemone protein, represented by each bar, as a function of the

percentage of daily photon flux density. The daily productivity is divided

into two portions: (1) the C-specific growth (C/x) per milligram of anemone

protein using td (duration of cytokinesis) of 28 h for (a) zooxanthellate and

69 h for (b) zoochlorellate anemones (white bar), and (2) the excess carbon

that is potentially available for translocation (black bar). Negative values

occur when C-specific growth exceeds the daily productivity of the algae. Data from the average P-I curves for each season (summer: July 2004; winter: Dec. 2004 and Feb. 2005; Fig. 3) and data from the average irradiance levels (I) experienced during a typical summer or winter day

(Fig. 6) were used to calculate daily productivity (P) using the equation P =

Pmax tanh (a I Pmax_1). C?x was calculated using the equations of

Verde and McCloskey (1996).

ues, followed by zooxanthellate anemone tissue, and then

zoochlorellate anemone tissue (? !6.21%o, ?

17.41%o, and

-18.77%c, respectively; Table 2). Compared to the anem

ones, the zooxanthellae and zoochlorellae exhibited low

delta13C and delta15N values (Fig. 8a; Table 2). The

delta13C of zoochlorellae (-24.93%o) was substantially lower than that of zooxanthellae (? 19.67%o) throughout the

year (Table 2). Zooxanthellae did not exhibit a strong sea

sonal signal, whereas the delta13C values of zoochlorellae were more depleted during the winter and spring (Fig. 8a).

Nonsymbiotic anemone tissues consistently had the high est delta15N values, followed by zoochlorellate, and by zooxanthellate anemone tissues (12.50%c, 11.34%o, and

10.30%o, respectively; Fig. 8; Table 2). The delta15N values

of the isolated algae are much lower than values obtained

from the symbiotic anemones (Fig. 8a). Although the mean

delta15N of zooxanthellae (5.15%o) is greater than that of

zoochlorellae (3.35%c) (Table 2), during most of the year the two algae had similar values (Fig. 8a).

DISCUSSION

This study explored seasonal variation in the population

density, mitotic index, chlorophyll content, productivity of

zooxanthellae and zoochlorellae, and assessed algal contri

butions to the host Anthopleura elegantissima by comparing percent carbon translocation, algal carbon budgets, and sta

ble isotope composition of anemones.

The density of zoochlorellae in the anemone host was

almost 4 times that of zooxanthellae. Although slightly greater, this difference in symbiont densities is consistent

with that observed in previous studies with anemones col

lected from the same region, with densities of zoochlorellae

from 1.3, 3, and 2 times higher than densities of zooxan

thellae (studies by, respectively, McCloskey et al, 1996;

Engebretson and Muller-Parker, 1999; Verde and McClos

key, 2002). However, because zoochlorellae are smaller, the

proportional biomass of symbionts in a host anemone may be similar (Verde and McCloskey, 2002) or lower than that

of zooxanthellae (Table 1). Although some seasonal fluctu

ation in algal density was observed, population densities of

both algal symbionts in A. elegantissima were similar be

tween summer and winter, in spite of large differences in

temperature and irradiance. Although Verde and McClos

key (2007) found a significant seasonal change in the den

sities of zooxanthellae and zoochlorellae in A. elegan tissima, two other field studies (Dingman, 1998; Farrant et

al, 1987) found that densities of zooxanthellae in A. el

egantissima and Capnella gaboensis, a temperate soft coral,

are constant and do not differ significantly between winter

and summer. The demonstration of similar symbiont popu lations in summer and winter for zoochlorellae in addition

to zooxanthellae supports the hypothesis of Muller-Parker

and Davy (2001) that temperate algal symbioses exhibit less

fluctuation in algal densities than tropical ones.

Consistent population densities of symbionts may result

from balanced growth of the algae and of the host during summer and winter, or may result from seasonal adjust

ments in algal expulsion rates, since A. elegantissima con

trols symbiont density primarily by expelling excess algae

(McCloskey et al, 1996). Algal expulsion is related to the

percentage of algae dividing (MI). Although the MI of

zooxanthellae and zoochlorellae show similar seasonal pat

terns, only zooxanthellae exhibited a significant seasonal

difference, with a higher percentage of cells dividing in July than in December and February. Verde and McCloskey




$

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12.00

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-17.00 -16.00 -15.00

Figure 8. Delta 13C and delta 15N values of (a) nonsymbiotic and symbiotic Anthopleura elegantissima

compared with values obtained for isolated zooxanthellae and zoochlorellae and (b) nonsymbiotic and symbiotic A. elegantissima (enlargement from Fig. 8a). Error bars represent ? 1 standard error of the mean (n

= 6 for most

samples; n was 2 for zooxanthellae in Aug. 2004).

(2007) obtained similar results. The elevated MI of zooxan

thellae during the summer months may result from a greater

supply of photosynthetic carbon available for algal growth

during this season, as well as from the high thermal and

light tolerances of zooxanthellae (Saunders and Muller

Parker, 1997; Engebretson and Muller-Parker, 1999).




Table 2

Average (? SE) and minimum and maximum delta13C and delta15N (%c) values for nonsymbiotic Anthopleura elegantissima, A. elegantissima with

algal symbionts, and isolated algae

delta13C (%c) delta 15N (%o)

Average Range Average Range

Anemone

Nonsymbiotic -16.27 ? 0.16 -16.54 to -16.04 12.50 ? 0.14 12.32 to 12.65 29

Zooxanthellate -17.52 ?0.17 -17.94 to -16.99 10.04 ? 0.25 9.53 to 10.58 35

Zooxanthellate tissue w/out

algal symbionts -17.41* NA 10.30* NA NA

Zoochlorellate -19.32 ? 0.29 -19.98 to -19.03 10.62 ? 0.18 10.01 to 10.91 36

Zoochlorellate tissue w/out algal

symbionts -18.77* NA 11.34* NA NA

Algae Zooxanthellae -19.67 ? 0.42 -20.17 to -19.10 5.15 ? 0.57 3.43 to 6.63 31

Zoochlorellae_-24.93 ?

0.38_-26.92 to-22.42_3.35 ?

0.68_0.82 to

6.50_33

Samples were obtained between 17 August 2004 and 19 July 2005 (n =

sample size). *

Average delta values for zooxanthellate and zoochlorellate anemone tissue without symbiotic algae were calculated by deriving the fraction of animal

(B) and algal (1- B) protein present in zooxanthellate and zoochlorellate anemones (Muscatine et al, 1983; Table 1); range and sample size (n) are not

applicable (NA) since values were calculated from the isotopic signatures of zooxanthellate and zoochlorellate anemones with symbionts.

During all months, the percentage of zoochlorellae divid

ing was about 6 times greater than that of zooxanthellae

(20% and 3%). The higher MI for zoochlorellae has been

reported previously, but the values of MI for zooxanthellae

and zoochlorellae differ among studies. McCloskey et al

(1996) report MI values of 5.64% and 1.68%, and Verde

and McCloskey (1996) report values of 7.34% and 0.34%, for zoochlorellae and zooxanthellae respectively. Values

comparable to those observed in this study were obtained

for zooxanthellae isolated from A. elegantissima in Califor

nia (4.69%) and Washington (2.88%; Wilkerson et al,

1983), and for zoochlorellae isolated from A. xanthogram mica in British Columbia, Canada (20%; O'Brien and Wyt tenbach, 1980). The difference between the MI of algae in

the anemones collected from the same area by Verde and

McCloskey and this study may be related to the size and

developmental stage of the host. Smith (1986) found that the

photosynthetic capacity and MI of zooxanthellae in the

tropical juvenile anemone Aulactinia stelloides were double those of zooxanthellae in adult anemones, and that algal densities were higher in juveniles than in adults. Anemones in the study by McCloskey et al (1996) were 1.5 to 1.0 g

wet weight compared to about 0.2 g wet weight in this

study. Since younger and smaller invertebrates have higher nutrient excretion rates (Smith, 1986), the elevated MI and

population densities observed in this study might be due to the greater availability of inorganic nutrients for the sym bionts in the small anemones.

The increase in chlorophyll a and b concentrations of zoochlorellae during winter is consistent with photoaccli

matization to the seasonal reduction in irradiance and with

the results obtained by Verde and McCloskey (2007). Lack

of seasonal variation in the chlorophyll content of zooxan

thellae is supported by observations of constant chlorophyll a concentrations in zooxanthellae isolated from the temper ate soft coral Capnella gaboensis (Farrant et al, 1987), and

by two laboratory studies with A. elegantissima that found

zooxanthellae maintained constant chlorophyll content

whereas zoochlorellae increased with decreasing irradiance

(Verde and McCloskey, 2001, 2002). These findings are in

sharp contrast with those for tropical associations, in which anemones and corals exhibit distinct seasonal fluctuations in

the density, photosynthetic capacity, and pigment concen

trations of zooxanthellae (Muller-Parker, 1987; Fagoonee et

al, 1999; Fitt et al, 2000; Warner et al, 2002). The light-saturated productivity (Pmax cell) and photosyn

thetic efficiency (a) of both zooxanthellae and zoochlorellae are significantly higher in July than during December and

February. Verde and McCloskey (2007) observed similar

trends, with zooxanthellate and zoochlorellate anemones

exhibiting significantly higher productivity during the

spring and summer months. The smaller seasonal decline in

the Pmax of zoochlorellae from summer to winter might result from depressed photosynthesis in the summer that is alleviated by a return to cool, low light conditions favoring photosynthesis by this alga (Verde and McCloskey, 2001, 2002). Although the trend of decreasing photosynthetic efficiency with seasonal reduction in light appears counter

intuitive, the actual amount of light received by the symbi onts is unknown and may be affected by anemone behaviors that may change seasonally, including orientation and pho totactic behavior (Pearse, 1974), attachment of gravel/shell




debris, and expansion/retraction of tentacles (Shick and

Dykens, 1984), as well as differences in the distribution of

symbionts within different body regions (Dingman, 1998).

Additionally, a lack of seasonal adjustment in photosyn thetic efficiency might indicate that the symbionts utilize host carbon to satisfy their metabolic requirements during the winter.

During the high-light and high-temperature conditions of

July, the productivity of zooxanthellae was considerably elevated over that of zoochlorellae, with Pmax values of 2

and 0.8 pg C cell-1 h~\ respectively. This trend of

elevated productivity of isolated zooxanthellae is consistent

with that observed in other summer studies (2 and 3 pg C

zooxanthella-1 h_1 and 0.5 and 1.25 pg C fixed zoo

chlorella-1 h_1; McFarland and Muller-Parker, 1993; Au

gustine and Muller-Parker, 1998, respectively). Similarly, the productivity of zooxanthellate A. elegantissima during the summer is much higher than that of zoochlorellate

individuals, especially at irradiances exceeding 100 /?mol

photons m~2 s_1 (Verde and McCloskey, 2002; Secord

and Muller-Parker, 2005). While high light-saturated pro

ductivity during the summer implies that a greater amount

of photosynthate may be available for zooxanthellate anem

ones during this season, the Pmax (cell) of zooxanthellae and

zoochlorellae was greatly reduced and similar during the

rest of the year (fall, winter, spring), suggesting that any

advantage is limited to summer conditions.

Algal contribution to host anemone diet

Carbon translocation rates, carbon budgets, and seasonal

fluctuations in C and N stable isotopes were examined and

compared to determine the possible nutritional benefits to

the anemone of hosting one symbiont over the other. The

short-term measures based on the photosynthetic contribu

tions of the two algae do not correlate with the stable

isotopic composition of the host. Results of the 14C method

indicate that during the winter the percentage of carbon

translocated from symbiont to host tissues was similar for

zooxanthellate and zoochlorellate anemones. The average

value of 48% translocated C is consistent with that obtained

in other 14C studies of carbon translocation in A. elegan tissima in California (Trench, 1971a). Although the percent

age of carbon translocated during winter is higher than that

observed during summer (30%; Engebretson and Muller

Parker, 1999), the actual amount of carbon translocated

during the summer will be much greater since both algae are

more productive during the summer.

Davy et al (1996) modeled carbon budgets for temperate subtidal symbiotic anemones during summer and concluded

that most temperate host anemones and zoanthids need to

feed heterotrophically to obtain their carbon requirements at

other times of the year. In this study we compared the

seasonal productivity of isolated algal symbionts and con

structed carbon budgets for them on an anemone biomass basis for winter and summer light conditions and for a series of light-reduction scenarios in each season because the actual light levels received by the algae in the host are

unknown. We used 50% light reduction as the basis for

comparing carbon budgets of zooxanthellae and zoochlor

ellae, since productivity was measured using isolated algae and Davies (1991) showed that daylight variations (e.g., 50% light reduction) have a significant effect on the energy

budgets of shallow-water corals. Comparing the daily pro

ductivity and C-specific growth, C budgets show that the

pools of C available for translocation are slightly larger for

zoochlorellate anemones than for zooxanthellate anemones

during both summer and winter, and that fixed C exceeds

C/x for most light levels (Fig. 7). The C budgets suggest a low potential for the algae to be

carbon parasites on their host, and the greater potential

availability of fixed C for zoochlorellate anemones contra

dicts the overall conclusions from C budgets constructed for

A. elegantissima by Verde and McCloskey (1996, 2001,

2002, 2007) that zooxanthellae provide a significantly

greater amount of fixed C to the host. Although we used the

same estimates of td as Verde and McCloskey, we deter

mined the productivity of isolated zooxanthellae and zoo

chlorellae in the laboratory, constructing P-l curves for

freshly isolated symbionts and using the P-l curves, daily irradiance levels, and algal density to derive productivity on

an anemone basis. Whereas Verde and McCloskey mea

sured the productivity of substantially larger (^175 mg

protein) whole anemones exposed to natural levels of sea

surface irradiance, our small anemones (^10 mg protein), had a 4-fold higher density of zoochlorellae than of zoo

xanthellae. This large difference in algal density resulted in a much greater contribution of photosynthetic carbon to the

host calculated for zoochlorellae than for zooxanthellae.

Additionally, possible differences in the distribution of zoo

xanthellae and zoochlorellae in different body regions or

changes in anemone posture affecting light levels received

in hospite may also contribute to the differences between

our results and those of Verde and McCloskey (20?7). Stable isotopic comparisons of delta13C and delta15N

signatures of symbionts and hosts were used as a proxy to

examine the potential carbon contributions of zooxanthellae

and zoochlorellae to their hosts. In contrast with the carbon

budgets derived from short-term measures of photosynthetic rates and estimated growth rates, stable isotopes serve as a

long-term index of carbon assimilation (Muscatine et al,

1989) and allow comparisons with nonsymbiotic counter

parts. Zooxanthellae and zoochlorellae exhibit distinctly different patterns in delta13C and delta15N values (Fig. 8a). This difference in the signatures of symbionts is repeated in

the delta values of symbiotic anemones, which are different




from those of nonsymbiotic individuals. The delta13C and

delta15N values of zooxanthellate and zoochlorellate anem

ones are intermediate between those of nonsymbiotic anem

ones and their respective symbionts, indicating that both

external food sources and translocated products contribute

to the diet of the host anemone.

Nonsymbiotic individuals of A. elegantissima were most

enriched in delta13C and delta15N. The delta13C values were

similar to values obtained for Puget Sound estuarine and

marine littoral epibenthic crustaceans (-\6.2%c ? 2.5%o; Simenstad and Wissmar, 1985), which are a likely food

source (Sebens, 1981a). The carbon and nitrogen isotopic

signatures of nonsymbiotic, zooxanthellate, and zoochlorel

late anemones did not vary seasonally (Fig. 8). Compared to

the anemones, both zooxanthellae and zoochlorellae exhib

ited low delta13C and delta15N values, which varied season

ally. The intermediate delta values of symbiotic anemo

nes?between those of their respective symbionts and

nonsymbiotic anemones?combined with a lack of a sea

sonal signal in anemone tissues, may indicate that feeding rates increase in proportion to possible increases in carbon

translocation during the summer, so that the relative contri

bution of autotrophic inputs from the algae remains the same year-round.

Although zooxanthellae and zoochlorellae are both intra

cellular photosynthetic symbionts, the delta 13C values of

zooxanthellae are more similar to those of Puget Sound

phytoplankton (-20.3%o ? 1.4%c; Carpenter and Peterson,

1989) while the delta13C values of zoochlorellae are on

average 5%c lower. The large difference in delta13C values

of zooxanthellae and zoochlorellae may result from differ ences in carbon fixation and photosynthetic pathways

(Wong and Sackett, 1978). Zooxanthellae possess Form II

Rubisco (Whitney et al, 1995); discrimination against

13C02 relative to 12C02 by Form II Rubisco is less than that

observed in plants and green algae with Form I Rubisco.

Alternatively, the two symbionts may primarily utilize dif ferent carbon sources. Due to the 4-fold higher density and

higher growth rate of zoochlorellae in the host, this symbi ont may be forced to rely to a greater extent on host-derived carbon sources (e.g., depleted C02 from animal respiration or depleted host organic carbon via heterotrophic uptake), whereas zooxanthellae may utilize enriched C02 diffusing into the animal host. In a survey of tropical corals sampled at various depths ranging from 1 m to 50 m in Jamaica and

Eilat, Muscatine et al (1989) obtained delta 13C values for zooxanthellae ranging from -9.63%o to -I9.2l%c. The zoo

xanthellae in A. elegantissima have delta13C values similar to the lowest values, obtained for the algal symbionts from

large-polyp corals sampled from deep waters.

Zoochlorellae show a larger seasonal variation than zoo xanthellae in delta13C, with the most depleted values occur

ring during spring and late winter. Similar seasonal fluctu

ations have been observed in maple leaves, marine eelgrass,

macroalgae, and phytoplankton (Lowdon and Dyck, 1974;

Stephenson et al, 1984; Simenstad and Wissmar, 1985;

Goering et al, 1990). Possible causes of seasonal fluctua

tions in 13C include seasonal shifts in the isotopic compo sition of carbon sources, differential storage of isotopically different biochemical components (i.e., depleted lipids vs.

enriched amino acids; Stephenson et al, 1984), and changes in isotopic fractionation due to temperature (Sackett et al,

1965). The observed seasonal trend in delta13C of zoochlor

ellae may be due to their greater C requirements for growth

during winter, resulting in utilization of the internal C02

pool faster than it can be replenished and thus lower frac

tionation (Swart et al, 2005). The seasonal variation observed in the delta15N signa

tures of symbionts may be explained by a shift in nitrogen source or a change in the alga's nitrogen requirements. The

very low delta values in zoochlorellae during February (similar to that of atmospheric nitrogen, 0.00%o) suggest that zoochlorellae utilize more of the isotopically light N

waste metabolites (Adams and Sterner, 2000) excreted by A.

elegantissima during the winter, while zooxanthellae may utilize an enriched external source such as nitrate.

The intermediate delta13C and delta15N values of zoo

xanthellate and zoochlorellate anemones indicate that sym biotic anemones receive nutrition from both external (het

erotrophic) and internal (translocated algal products) sources; however, the delta values of the symbiotic anem

ones are more similar to those of the nonsymbiotic anem

ones than they are to those of their respective symbionts. A

combination of the enriched state of zooxanthellate and

zoochlorellate anemones and the lack of seasonal variation in these and nonsymbiotic anemones suggest that A. elegan tissima relies primarily on external food sources, regardless of symbiotic condition.

Although A. elegantissima derives most of its nutrition from heterotrophic sources, stable isotope data suggest that in terms of carbon, zooxanthellae may be the more favor

able symbionts for the anemone to host. Zooxanthellate anemone tissue was only 2%c (13C) and 5%c (15N) enriched over zooxanthellae, while zoochlorellate anemone tissue

was 6%c (13C) and 8%o (15N) enriched over zoochlorellae. This greater disparity in the delta13C and delta15N values of zoochlorellate anemones and zoochlorellae, combined with the elevated delta15N signature of zoochlorellate anemones

(^\%c higher than that of zooxanthellate anemones), sug gests that more of the carbon fixed by zooxanthellae is translocated to the host in zooxanthellate anemones; zoo

chlorellate anemones receive less nutrition from their sym bionts and rely to a greater extent on external food sources.

Although stable isotope data indicate that zooxanthellae

may be "better" symbionts for nutritional purposes?sup porting the results of Verde and McCloskey (1996, 2001,




2002, 2007) that zooxanthellate anemones receive more

translocated carbon than zoochlorellate individuals?this

conclusion contradicts that based on the C budgets, which

showed that zoochlorellate anemones have slightly larger

pools of C available for translocation during both summer

and winter, and also contradicts the similar percent values

for carbon translocation obtained for both zooxanthellate

and zoochlorellate anemones (Engebretson and Muller

Parker, 1999). There are several possible reasons for this

discrepancy. The productivity measurements were short

term, using isolated symbionts. Since zooxanthellae may

undergo changes after removal from symbiosis (Trench,

1971b) and are also exposed directly to seawater, it is

possible that the physiological performance of zooxanthel

lae and zoochlorellae measured in vitro does not reflect

carbon fixation rates in hospite. Our aim was to directly

compare the photosynthetic performance of these two algae on an individual cell basis, under the same conditions.

Therefore, we used dilute (unshaded) suspensions of algae,

comparing their photosynthetic rates under equivalent con

ditions of light and carbon dioxide supply to construct the

P-l curves. Since the symbionts were isolated from the host

using the same procedures, any effects on isolation from the

host should apply to both zooxanthellae and zoochlorellae.

Another possible reason for the discrepancy in carbon bud

gets constructed for intact symbioses and from photosyn thetic measurements with isolated algae is that the distribu

tion of the two symbionts in the animal host is not the same;

zoochlorellae may be more abundant in the body column, and zooxanthellae more abundant in the tentacles (Ding

man, 1998). Because these distributions may change sea

sonally and with anemone size, the photosynthetic contri

butions of the algae in vivo may differ accordingly. Since

the duration of cytokinesis (td) has not been measured

directly and has a substantial influence on the outcome of

the carbon calculations, actual td measurements for zooxan

thellae and zoochlorellae in A. elegantissima during sum

mer and winter are also needed to resolve carbon budgets and any conclusions concerning the possible nutritional

benefits of the two symbionts. For tropical corals, stable isotopes are good predictors of

the relative contributions of zooxanthellae to the host (Mus catine et al, 1989; Reynaud et al, 2002). For the temperate

Anthopleura elegantissima, either the photosynthetic con

tributions are not retained by the anemones (lost as gametes, dissolved and particulate organic carbon), or translocated

carbon composes a relatively small portion of the host

anemone's diet, with the host deriving most of its nutrition

via heterotrophy. Perhaps the abundant supply and utiliza

tion of external food sources allow the anemone host the

flexibility of associating with both zoochlorellae and zoo

xanthellae, and enable the symbiosis to persist during un

favorable conditions when one or both algae may become

carbon parasites. This flexibility would also allow the alga best able to grow under a particular set of temperature and

light conditions to dominate in an anemone host. Alterna

tively, since anemones may receive the most translocated C

during the summer months when planktonic food is also

plentiful, the additional carbon could be allocated to asexual

reproduction or gonad production and increased sexual out

put of symbiotic individuals (Muller-Parker and Davy, 2001). It is also possible that symbionts translocate some

product of value to the host other than carbon. Although zooxanthellae translocate primarily glycerol, a carbon-rich

compound (Trench, 1971a), zoochlorellae may provide the

host anemone with N-rich amino acids (Minnick, 1984, cited in Verde and McCloskey, 2007).

The long-term advantages (fitness consequences) for the

host of associating with different taxa of symbionts are

unknown, and await studies that measure the relative con

tributions of each algal symbiont to the growth and repro ductive condition of the host sea anemone. Field studies, such as those conducted by Sebens (1981a, b, 1982) for

zooxanthellate A. elegantissima, are needed to compare the

growth, survival, and sexual and asexual reproduction of

natural populations of zoochlorellate, nonsymbiotic, and

zooxanthellate A. elegantissima to determine whether there

is an ecological advantage for these temperate anemones to

be symbiotic?with zooxanthellae, with zoochlorellae, or

with both.

Acknowledgments

We thank K. Kegel, J. Augustyn, M. Towle, J. Berg schneider, and A. Bergschneider for field and laboratory

assistance. We are grateful to R. Lee for the analysis of

stable isotope samples. S. Strom, B. Bingham, and A.

Singh-Cundy provided valuable advice and guidance. This

research was conducted in partial fulfillment of the senior

author's requirements for the M.S. degree at Western Wash

ington University (WWU). Preparation of the manuscript was partially completed while one of us (GMP) served in a

position at the National Science Foundation (NSF). Any

opinion, findings, and conclusions or recommendations ex

pressed in this material are those of the authors and do not

necessarily reflect the view of the NSF. Funding was pro

vided by a Sigma Xi Grant-in-Aid and by the Office of

Research and Sponsored Programs, WWU. The authors

thank two anonymous reviewers and Editor Malcolm Shick,

who provided stimulating and thoughtful questions which

helped to improve the manuscript. GMP dedicates her con

tributions to this study to the memory of Len Muscatine (d.

2007).




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