Mar Biol (2012) 159:939–950

DOI 10.1007/s00227-011-1871-8

ORIGINAL PAPER

The eVects of symbiotic state on heterotrophic feeding in the temperate sea anemone Anthopleura elegantissima

Terra C. Hiebert · Brian L. Bingham

Received: 14 September 2011 / Accepted: 23 December 2011 / Published online: 6 January 2012© Springer-Verlag 2012

Abstract The temperate sea anemone Anthopleura ele-gantissima is facultatively symbiotic with unicellular algae.Symbiotic A. elegantissima can supplement heterotrophicfeeding with excess photosynthate from their algal partners,while asymbiotic individuals must rely solely on heterotro-phy. A. elegantissima individuals were collected from SwirlRocks, Washington (48°25�6� N, 122°50�58� W) in July2010, and prey capture and feeding characteristics weremeasured to determine whether asymbiotic individuals aremore eYcient predators. Feeding abilities were then mea-sured again after a 3-week exposure to full sunlight orshaded conditions. Freshly collected asymbiotic anemoneshad larger nematocysts, but symbiotic individuals showedgreater nematocyte sensitivity. Sunlight enhanced digestionand reduced cnida density in all anemones regardless ofsymbiotic state. Results suggest that the phototropicpotential of A. elegantissima, as inXuenced by symbioticcondition, has little eVect on heterotrophic capacity. Theanemones appear to maximize heterotrophic energy inputindependent of the presence or identity of their algalsymbionts.

Introduction

Cnidarian–algal symbioses are generally considered mutu-alistic associations with photosynthetic products from thealgae being exchanged for respiratory waste products fromthe cnidarian host (Yellowless et al. 2008). The metabolicneeds of the host are met by some combination of auto-trophic energy from the symbionts and host heterotrophicfeeding (Verde and McCloskey 1996; Grottoli et al. 2006;Palardy et al. 2008; Hoogenboom et al. 2010). The relation-ship between the animal and the symbiont can be obligateor facultative (Dean 1983; Bronstein 1994). Obligate asso-ciations are typically seen in tropical environments wherethe water is clear, sunlight irradiances are consistently highand plankton biomass is relatively low (Muscatine andPorter 1977). Temperate cnidarians, however, exist in moreproductive waters where plankton and suspended particu-late matter decrease light penetration, but increase hetero-trophic feeding opportunities (Muller-Parker and Davy2001). Therefore, temperate symbiotic cnidarians rely moreon heterotrophic feeding than do their tropical counterparts,and facultative symbioses are more common (Muller-Parker and Davy 2001; Piniak 2002).

Anthopleura elegantissima, a common temperate seaanemone, facultatively hosts both dinoXagellates of thegenus Symbiodinium (LaJeunesse and Trench 2000) and thechlorophyte Elliptochloris marina (Letsch et al. 2009)(commonly called zooxanthellae and zoochlorellae, respec-tively). The nutritional relationship between A. elegantiss-ima and its symbionts is not clearly understood, butresearch suggests that A. elegantissima receives greaternutritional beneWt from hosting zooxanthellae than zoo-chlorellae and that both zooxanthellate and zoochlorellateanemones have a nutritional advantage over anemones thatlack symbionts altogether (Verde and McCloskey 1996;

Communicated by J. Purcell.

T. C. Hiebert (&) · B. L. BinghamDepartment of Environmental Sciences, Western Washington University, Bellingham, WA 98225, USAe-mail: terrah@uoregon.edu

Present Address:T. C. HiebertOregon Institute of Marine Biology, P.O. Box 5389, Charleston, OR 97420, USA

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Bergschneider and Muller-Parker 2008). While asymbioticanemones feed exclusively heterotrophically, symbiotic(zooxanthellate and zoochlorellate) individuals may acquireenergy both heterotrophically and autotrophically. If theautotrophic contribution is substantial for symbiotic anem-ones, asymbiotic individuals may compensate for the lackof autotrophic energy by being more eYcient heterotrophicfeeders.

Heterotrophic feeding in A. elegantissima is aVected by atleast three features. As suspension feeders, anemones relyon prey contact with their tentacles and oral disk and contactprobability increases with surface area (Sebens 1981; Zamer1986). Following contact, the eVectiveness of the tentaclecnidae inXuences capture success. Cnida sizes may be adap-tive and dependent on anemone size, habitat and prey type(Francis 2004; Kramer and Francis 2004) and could also bevariable by symbiotic state. Finally, the processing of prey(ingestion, digestion and absorption) is potentially aVectedby nutritional need. Zamer (1986) found that A. elegantiss-ima individuals living in the upper intertidal zone hadheightened receptivity to prey compared to low-shore indi-viduals and speculated that one might also Wnd diVeringingestion rates among symbiotic states of A. elegantissimadue to diVerential autotrophic contributions from algal sym-bionts, but that possibility has not been tested.

To investigate variation in heterotrophic feeding as inXu-enced by symbiotic state, the prey capture and processingabilities of zooxanthellate, zoochlorellate and asymbioticA. elegantissima were compared. In particular, anemonetentacle number, prey-capture surface area and cnida size,density, sensitivity and strength were measured. We alsocompared ingestion rate, digestion rate and absorptioneYciency in anemones of each symbiotic state. These mea-surements were performed with freshly collected anemonesand were subsequently repeated under natural sunlight andshade treatments to assess the eVect of symbiont productiv-ity on these feeding features. Our goal was to determinewhether autotrophic contribution from symbiotic algaeaVects how the host anemones feed heterotrophically.

Methods

Anemone collection

Anthopleura elegantissima individuals were collected fromSwirl Rocks, Washington (48°25�6� N, 122°50�58� W) on14 July 2010. This location was chosen because A. elegan-tissima was present in zooxanthellate, zoochlorellate andasymbiotic forms all at approximately equal tidal heights(+0.3 m above mean lower low water) within 2 m of eachother. Asymbiotic anemones were collected from a shadedoverhang and zooxanthellate, and zoochlorellate anemones

were collected from opposing sides of a shallow crevice.Nine similar-sized anemones of each symbiotic state weretransported to the Shannon Point Marine Center in Anacor-tes, Washington, where they were placed on numbered5 £ 6.5 mm slate tiles and acclimated in a Xow-throughseawater table for 1 week before experiments began. Theseanemones were used in all experiments; their attachment tothe tiles permitted easy transfer during experiments.

Initial measurements

Photographs were taken of the expanded anemones 5 daysafter collection, and Image J software (National Institutesof Health) was used to determine oral disk and tentaclecrown diameters as the mean of three measurementsextending between the bases or tips of tentacles and directlyacross the oral disk (Fig. 1a, b). All visible tentacles werethen counted, and length and midpoint width were mea-sured for one arbitrarily chosen, fully visible tentacle ineach of three quadrants of the oral disk (Fig. 1c). Oral disksurface area was calculated as the area of a circle circum-scribed within the oral disk diameters (Fig. 1a), and thetentacle surface area was calculated as if the tentacles wereperfect cylinders (Sebens 1981, Fig. 1c, d). The totalprey-capture surface area (PCSA) of each anemone wascalculated as the sum of the total tentacle surface area (theaverage tentacle surface area of three tentacles multipliedby the total number of tentacles) and the oral disk surfacearea (Zamer 1986). One-way ANCOVAs were used to testfor diVerences in tentacle number, tentacle crown diameter orPCSA between anemones of each symbiotic state. Anemoneprotein mass (determined at conclusion of all experiments)was used as the covariate.

To determine whether symbiotic state aVects cnida sizeand density, one arbitrarily chosen tentacle from eachanemone was removed and frozen at ¡70°C. These tenta-cles were later homogenized in 20 �l of 5 �m Wltered sea-water using a TeXon tissue grinder and motorized stirrer.The homogenates were examined on a hemocytometer, andcnida density was determined with separate counts for basi-trichs (penetrating cnidae with spines) and spirocysts (cni-dae with coiled, adhesive tubules). These densities werenormalized to tentacle protein content, which was deter-mined later from the homogenates. Length and width of theWrst three unWred cnidae seen in each homogenate weremeasured using ImagePro Plus image analysis software.Basitrich and spirocyst densities were compared for zoo-xanthellate, zoochlorellate and asymbiotic anemones withseparate one-way ANOVAs. Cnida length and width datawere analyzed with nested ANCOVA with individualnested in symbiotic state and anemone protein biomass asa covariate. The nesting was necessary because multiplemeasurements were taken from each anemone.

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To determine tentacle adhesive force and test thehypothesis that adhesive force varies by symbiotic state, weused a method modiWed from Thorington and Hessinger(1988), Giebel et al. (1988) and Francis (1991). Anemoneswere placed in an 11.5-l clear Plexiglas tank Wlled with5 �m Wltered seawater. A 0.2-mm-diameter steel wire(Ernie Ball) with a 1-mm-diameter glass bead coated in30% (w/v) Knox gelatin at its tip was lowered with a micro-manipulator (1 cm s¡1) until it just touched the tip of a testanemone’s tentacle. Upon contact, the wire was immedi-ately raised at 1 cm s¡1 until the upward force exceeded thetentacle’s adhesive force, and the test probe broke free.This process was digitally recorded in side view, and themaximum deXection of the wire was later determined fromthe recording. A new probe was used for each measure-ment, and two tentacles were tested on each of the 27anemones (9 per symbiotic state). A calibration curve relat-ing deXection of the wire to mass was determined using themicromanipulator to press the wire on the surface of ananalytical balance. The force required to bend the cantile-vered test probe was approximated by mass and convertedto �N (Thorington and Hessinger 1988, 1996).

Because adhesive force is related to the number of cni-dae discharged, it was necessary to count discharged cnidaefor every trial. After an adhesion measurement was made,the bead from the tip of the wire was soaked for 4 h in 10 �lof 2% Trizyme (Amway). This dissolved the gelatin, leav-ing behind the Wred cnidae (Thorington and Hessinger1988). Discharged basitrich capsules were clearly visibleand could be counted with a microscope under 1,000£magniWcation. Each force measurement was normalized tothe number of basitrichs Wred (i.e., �N basitrich¡1) andadhesive force per basitrich, and the total number of Wredbasitrichs were analyzed with one-way ANOVAs to deter-mine whether anemones in diVerent symbiotic statesexerted diVerent amounts of contact force. Spirocysts werealso present, but the Wred adhesive tubules could not bediVerentiated in the samples and had to be omitted from theanalysis.

To determine whether symbiotic state aVects ingestiontime, digestion time or absorption eYciency, anemones ontheir tiles were cleared of debris and placed in 8-cm-diame-ter glass Wnger bowls resting in a Xow-through seawaterbath that maintained water temperature between 13 and

Fig. 1 Anemone morphological data generated from three measurements of a oral disk diameter and oral disk surface area, b tentacle crown diam-eter, c tentacle length and width and d tentacle surface area

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15°C yet isolated each anemone and, ultimately, the egestait produced. A pellet of squid mantle tissue was placed oneach anemone’s outer tentacle tips. The pellet sizes wereapproximately 5% of each anemone’s body weight as esti-mated from oral disk diameter. A. elegantissima is anopportunistic feeder, and little is known about its feedingactivities in the Weld. The ration size, therefore, was chosento allow comparison with previous work on A. elegantiss-ima feeding (e.g., Sebens 1980; Zamer 1986).

Each anemone was examined at 5-min intervals for theWrst 30 min after placement of the pellet then at 1-h inter-vals thereafter. Ingestion time was measured as the timeafter the pellet was placed on the tentacles until it was com-pletely consumed and no longer visible. Digestion time wasfrom complete ingestion of the squid to the Wrst appearanceof egested material. Egesta produced over the following24 h was collected and placed in labeled microfuge tubes.These samples were then rinsed with 2 ml of Nanopurewater onto pre-dried and weighed 25-mm GF/C Wlters withvacuum Wltration. The Wlters were dried at 60°C for 24 hand weighed on a microbalance to determine absorptioneYciency as (mg ingested—mg egested)/mg ingested(Zamer 1986). Ingestion time, digestion time and absorp-tion eYciency were analyzed individually with one-wayANOVAs with symbiotic state as the independent factor.Anemones that failed to ingest the squid ration within 4 hof contact with the tentacle were not used in the analyses.

Light experiment

To test the eVect of light level on heterotrophic feeding ofthe anemones, we imposed light treatments immediatelyfollowing the experiments described above. The same nineanemones from each symbiotic state were randomly splitbetween shade (n = 4) and full sunlight (n = 5) treatmentsin an outdoor Xow-through seawater tank. The anemonesexperienced low tides that corresponded to daily low tidesat their collection site. All anemones were held in the treat-ments from 5 to 26 Aug 2010 and were fed a single squidpellet, weekly. Seawater temperatures were monitored withHobo data loggers, and irradiances were measured with aQSL-101 Light Meter (Biospherical Instruments, Inc.). Fol-lowing the three-week test period, all measurements ofcnida size, density and strength as well as prey processingrates were repeated.

Cnida length and width were again measured from tenta-cle homogenates and individually analyzed with two-waynested ANCOVAs with symbiotic state and light treatmentas the main eVects and protein biomass as the covariate.Cnida density data were analyzed with a two-way ANOVA.Prey ingestion, digestion and absorption eYciency weremeasured as described above with three consecutive 24-hfeeding trials separated by 48-h intervals to investigate

individual variability over time. Anemones remained intheir respective light treatments during the experiments.Separate ANOVARs were used to test the eVects of light(sunlight or shade), symbiotic state (zooxanthellate, zoo-chlorellate or asymbiotic) and trial (1–3) on anemoneingestion, digestion and absorption eYciency. Seventy-twohours after the Wnal feeding experiment, an adhesive forceexperiment was done as previously described, and datawere analyzed as a two-way ANOVA with light treatmentand symbiotic state as main eVects.

Algal density and protein analysis

To conWrm symbiotic state and to measure anemone size atthe conclusion of all experiments, the anemones werehomogenized in 5 �m Wltered seawater and frozen at¡70°C until they could be processed. The homogenateswere later examined with a hemocytometer at 100£ todetermine symbiont complement and density. Four subsam-ples of homogenate were examined with at least 100 cellscounted in each. Anemone soluble protein content wasdetermined using the method of Lowry et al. (1951), withbovine serum albumin (BSA) as a standard. Algal cell den-sity was normalized to anemone protein content. Anemoneprotein content was used as a covariate in statistical analy-ses where appropriate to determine whether individual sizeaVected the heterotrophic feeding features.

Statistical analysis

All data were analyzed using SPSS v 18, and Levene’s testswere used to test the homogeneous variance assumption.For covariate analyses, the interaction between anemonesize and treatment was tested to conWrm the assumption ofparallel covariate lines. If homogeneous variance or parallelcovariate assumptions were violated (P < 0.05), data weretransformed. Count data were square root transformed, sizedata were log transformed and percentage values were arc-sine transformed where appropriate. If variances of trans-formed data remained heterogeneous, a more conservative� was adopted (� = 0.025, Underwood 1981). If a covariateinteraction persisted despite transformation, the covariatewas not included in the analysis. Pairwise comparisons(Tukey’s HSD) were used to compare treatment means orcovariate-adjusted treatment means where appropriate.

Results

Anemone size and symbiont density

Protein analysis of anemone homogenates at the conclusionof the experiments showed that biomass of the anemones in

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the three symbiotic states was similar (Table 1). Cell countsveriWed that anemones initially identiWed as zooxanthellateor zoochlorellate had at least 95% Symbiodinium muscatineior Elliptochloris marina, respectively. Zooxanthellate anem-ones also hosted small numbers of E. marina (<1%, Table 1),and zoochlorellate anemones hosted some S. muscatinei(<5%, Table 1). One anemone, initially assigned to the zoo-chlorellate group, was removed from all analyses because ithosted a mixed algal complement by number of symbionts(55% zooxanthellae, 45% zoochlorellae). Symbiont densi-ties (cells mg¡1 protein) were nearly twice as high in zoo-chlorellate anemones as in zooxanthellate individuals.However, the volume of an S. muscatinei cell (9–10 �mdiameters) is 2–3 times greater than that of an E. marinacell (11–13 �m diameter, Bergschneider and Muller-Parker2008), so the total volume of symbionts within the zooxan-thellate anemones probably exceeds that of the zoochlorellateindividuals (Table 1). Anemones designated asymbioticactually hosted low densities of S. muscatinei (averagingone sixth of the density hosted by zooxanthellate individu-als, Table 1).

Conditions in the light experiment

Anemones held in the sunlight treatment experienced awider range of irradiances than did anemones in the shaded

treatment. Irradiances in the shade were about 2% of that inthe sunlight. Average temperatures in the sunlight andshade treatments diVered by 0.3°C or less (Table 2). Maxi-mum temperature diVerences between the treatmentsoccurred during trial 1 when temperatures in the sunlighttreatment exceeded those in the shade treatment by 1.1°Cfor nearly 3 h (Table 2). One zoochlorellate anemone diedwhile being held in the sunlight treatment and was removedfrom the experiment. One asymbiotic anemone in the shadetreatment divided, and both individuals were included inthe remaining experiments. Sample sizes were, therefore, 4,5 and 5 for zoochlorellate, zooxanthellate and asymbioticanemones in the sunlight and 4, 4 and 5 in the shaded treat-ment.

Anemone morphology

Total number of tentacles was signiWcantly related to thesymbiotic state of the anemone (F2,22 = 4.7, P = 0.02) whenanemone size was removed as a signiWcant covariate(F1,22 = 7.4, P = 0.013, Table 1). Asymbiotic anemones, onaverage, had 32% fewer tentacles than zoochlorellate indi-viduals; zooxanthellate individuals were intermediate intentacle number (Table 1). Though they were fewer innumber, the tentacles of the asymbiotic anemones were sig-niWcantly wider (F2,22 = 4.0, P = 0.03, Table 1). Tentacle

Table 1 Morphological features of A. elegantissima used in all experiments

Protein content, algal cell density and algal species composition were measured at the end of all experiments. The remaining morphological fea-tures were measured immediately upon collection. Errors are standard errors. Uppercase letters indicate statistically signiWcant diVerences betweensymbiotic states in algal cell density, number of tentacles and tentacle widthsa Values are adjusted for the protein biomass covariateb PCSA prey-capture surface area

Symbiotic state

Protein content (mg)

Algal cell density (cells mg¡1 protein)

Algal species composition

Number of tentaclesa

Tentacle widtha (mm)

Tentacle crown diametera (mm)

PCSAa,b (mm2)

Zooxanthellate 27.9 § 3.1 52,468 § 6,846 B >99% S. muscatinei 77.5 § 7.0 A,B 1.09 § 0.11 B 27.8 § 1.7 71.0 § 16.1

Zoochlorellate 24.1 § 4.6 100,111 § 24,505 A >95% E. marina 91.9 § 7.4 A 0.91 § 0.11 B 24.9 § 1.8 76.4 § 16.9

Asymbiotic 21.6 § 2.9 8,334 § 2,478 C >99% S. muscatinei 62.9 § 6.9 B 1.34 § 0.11 A 25.3 § 1.7 110.0 § 15.8

Table 2 Maximum and average daily irradiance and temperature for three feeding trial days in both sunlight and shaded treatments

Irradiance (�mol photons m¡2 s¡1) Temperature (°C)

Sunlight Shade Sunlight Shade

Trial #1: 26–27 Aug

Average 195 § 24 4 § 0.6 12.6 § 0.03 12.4 § 0.02

Maximum 1,743 30 14.5 13.4

Trial #2: 29–30 Aug

Average 205 § 15 4 § 0.3 12.7 § 0.03 12.7 § 0.03

Maximum 349 17 13.2 13.0

Trial #3: 01–02 Sep

Average 426 § 40 9 § 0.9 12.8 § 0.03 12.5 § 0.02

Maximum 1,826 30 14.9 14.0Standard errors are shown

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crown diameter (F1,22 = 24.9, P < 0.001) and prey-capturesurface area (F1,22 = 22.3, P < 0.001) both increased withanemone size, but neither feature was related to symbioticstate (F2,22 = 0.8, P = 0.47 for crown diameter andF2,22 = 2.5, P = 0.11 for PCSA, Table 1).

Cnida characteristics

Among freshly collected individuals, mean basitrich widthswere similar for all anemones (2.70 § 0.05 �m, Table 3),but the basitrichs were signiWcantly longer in asymbioticthan in zoochlorellate individuals (P = 0.0015, Fig. 2a).Neither spirocyst width (2.75 § 0.07 �m) nor length(Fig. 2c) was signiWcantly diVerent among symbiotic states,though there was again a trend toward longer cnidae in theasymbiotic individuals.

Following 2 weeks in the light treatments, basitrichlengths were no longer aVected by symbiotic state(Table 3), nor was there a signiWcant light eVect or a symbi-otic state by light interaction (Fig. 2b). Symbiotic stateagain had no signiWcant eVect on spirocyst length, but spi-rocyst sizes were aVected by light with longer spirocysts inthe shaded anemones (Fig. 2d), but no signiWcant symbioticstate by light interaction (Table 3). Cnida widths were notaVected by light, symbiotic state or the interaction of thetwo (Table 3). Average widths were similar to those mea-sured before the light experiment (2.8 § 0.1 �m, for basi-trichs and 2.9 § 0.1�m, for spirocysts).

Densities of basitrichs and spirocysts were not signiW-cantly diVerent between symbiotic states in freshly col-lected A. elegantissima (Table 3; Fig. 3a, c). In thesubsequent light experiment, both basitrich and spirocystdensity nearly quadrupled over what we had measured3 weeks earlier in the same anemones (Fig. 3b, d), and den-sities were signiWcantly higher for anemones in the shade.All symbiotic states were aVected similarly by the light

experiment with respect to basitrich and spirocyst densities(Table 3).

Tentacle adhesive force

There were no statistically signiWcant diVerences in thenumber of basitrichs discharged or the adhesive force perbasitrich among freshly collected anemones in diVerentsymbiotic states (Tables 3, 4).

All anemones Wred many more basitrichs following thelight experiment than they had immediately after collection.Zooxanthellate anemones Wred signiWcantly more basitrichsthan did anemones in the other symbiotic states (Table 3).However, there were no signiWcant diVerences in the forceexerted per basitrich between the three symbiotic states.The number of basitrichs discharged and the resulting adhe-sive forces per basitrich were not signiWcantly diVerentbetween anemones in the sunlight or the shaded treatment,and there were no signiWcant symbiotic state by light inter-actions for either parameter (Table 3).

Prey processing

Neither ingestion time (F2,21 = 1.17, P = 0.33), digestiontime (F2,21 = 1.20, P = 0.32), nor absorption eYciency(F2,21 = 0.05, P = 0.94) were aVected by symbiotic state infreshly collected anemones. Ingestion times were widelyvariable (ranging from 5 to 60 min), while digestion timeswere remarkable consistent (averaging 8–9 h in all treat-ments).

In the light experiment, all symbiotic anemones ingestedtheir squid ration, but only 73% of the asymbiotic anemo-nes maintained in the sunlight did. Anemones that failed toingest their ration were removed from the analysis, reduc-ing the sample size for asymbiotic anemones in the sunlighttreatment to 2, and there was no eVect of symbiotic state on

Table 3 Summary of ANOVA results for cnida characteristics of freshly collected A. elegan-tissima and for the same individ-uals subsequently held under natural sunlight or shaded conditions for 3 weeks

Freshly collected After 3-week light experiment

Symbiont Symbiont Light Symbiont * light

Measure F2,21 (p) F2,20 (p) F1,20 (p) F2,20 (p)

Basitrich width 0.70 (0.51) 2.38 (0.12) 0.04 (0.85) 0.05 (0.95)

Basitrich length 3.52 (0.03) 1.70 (0.21) 0.17 (0.69) 0.32 (0.73)

Spirocyst width 0.46 (0.64) 0.80 (0.47) 0.01 (0.98) 0.19 (0.83)

Spirocyst length 0.90 (0.42) 2.89 (0.06) 6.31 (0.01) 0.57 (0.57)

F2,22 (p) F2,20 (p) F1,20 (p) F2,20 (p)

Basitrich density 0.14 (0.87) 1.52 (0.24) 5.03 (0.04) 0.72 (0.50)

Spirocyst density 0.19 (0.83) 2.24 (0.13) 10.3 (0.004) 0.08 (0.93)

F2,20 (p) F2,17 (p) F1,17 (p) F2,17 (p)

Basitrichs discharged 1.81 (0.19) 5.44 (0.015) 2.42 (0.14) 1.44 (0.26)

Force basitrich¡1 0.24 (0.79) 1.59 (0.23) 0.39 (0.54) 0.41 (0.67)SigniWcant eVects are highlighted in bold

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the ingestion time of the remaining anemones (Table 5;Fig. 4a–c). Ingestion times were also not statistically diVer-ent between anemones in the sunlight and those in theshade (Table 5). Digestion time was the only prey process-ing factor signiWcantly aVected by light exposure. Allanemones digested faster in the sunlight than in the shadetreatment (Fig. 4d–f), but there was no signiWcant eVect ofsymbiotic state on digestion time (Table 5). Ingestion timesand digestion times were signiWcantly diVerent between tri-als, with the faster times seen in successive trials.

Absorption eYciencies exceeded 70% in all anemonesand were not aVected by light treatment or symbiotic state(Table 5; Fig. 4g–i). Consistent with other prey processingparameters, absorption eYciency was signiWcantly diVerenton each trial day, and the highest absorption eYciencieswere measured on the 3rd trial day (Fig. 4i). There were nostatistically signiWcant interactions in any of the ANOVARanalyses (Table 5).

Discussion

The productivity of symbiotic algae can aVect the heterotro-phic feeding of their tropical cnidarian hosts (Anthony andFabricius 2000; Grottoli et al. 2006; Houlbrèque and Ferrier-Pagès 2009). Our goal was to determine whether such inXu-ences occur in the temperate anemone, A. elegantissima. Wespeculated that the identity and productivity of algal symbi-onts would inXuence the host anemones’ heterotrophic nutri-tional need, resulting in diVerences in anemone morphologyor feeding behavior. However, we found only subtle diVer-ences in heterotrophic feeding among anemones in diVerentsymbiotic states, suggesting that autotrophic energy derivedfrom the algal symbionts had little direct eVect on heterotro-phy. Because the asymbiotic individuals we collected fromthe Weld were not entirely symbiont free (Table 1), this resultshould be interpreted with some caution. Future work couldfocus on A. elegantissima individuals from which all

Fig. 2 Cnida lengths for A. elegantissima in three symbiotic states: a,c freshly collected from Swirl Rocks and b, d after the 3-week lightexperiment. The three symbiotic states include zooxanthellate (ZX),zoochlorellate (ZC) and asymbiotic (AS). Symbiotic individuals areindicated by shaded bars (a and c) with uppercase letters above the

bars indicating statistically signiWcant diVerences. Black bars repre-sent individuals in the shaded treatment, and white bars represent thosein the sunlight treatment (b and d). SigniWcant diVerences betweenlight treatments across all symbiotic states are indicated by uppercaseletters above the Wrst set of bars. Standard errors are shown

a b

dc

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symbionts had been artiWcially removed prior to experimen-tation (e.g., Fitt and Pardy 1981).

Anthopleura elegantissima feeds on planktonic or ben-thic prey that contacts its tentacles or oral disk, and prey

capture increases with feeding surface area (Sebens 1981).Since asymbiotic individuals must rely solely on heterotro-phic feeding, they might be expected to produce a largerprey-capture surface area with larger or more numerous

Fig. 3 Cnida density for A. ele-gantissima in three symbiotic states: a, c freshly collected from Swirl Rocks and b, d after the 3-week light experiment. Symbiotic individuals are indi-cated by shaded bars (a and c). Black bars represent individuals in the shaded treatment, and white bars represent those in the sunlight treatment (b and d). SigniWcant diVerences between light treatments across all symbiotic states are indicated by uppercase letters above the Wrst set of bars. Standard errors are shown

a b

dc

Table 4 Adhesive force and basitrichs discharged in three symbioticstates of A. elegantissima

Treatment Basitrichs discharged

Adhesive force (�N basitrich¡1)

Zooxanthellate Freshly collected 134 § 23 1.35 § 0.39

Light experiment

Sunlight 661 § 222 0.45 § 0.20

Shade 401 § 118 1.59 § 0.69

Zoochlorellate Freshly collected 128 § 35 1.36 § 0.78

Light experiment

Sunlight 197 § 69 2.36 § 0.98

Shade 261 § 118 1.89 § 1.37

Asymbiotic Freshly collected 74 § 16 1.85 § 0.59

Light experiment

Sunlight 337 § 64 0.40 § 0.20

Shade 173 § 43 1.06 § 0.88

Table 5 Summary of ANOVAR results for the eVects of symbioticstate, light and trial on ingestion time, digestion time and absorptioneYciency of Anthopleura elegantissima

SigniWcant eVects are highlighted in bold

Source df Ingestion time

Digestion time

Absorption eYciency

F (p) F (p) F (p)

Between subjects

Symbiont 2,16 3.08 (0.07) 0.01 (0.99) 0.32 (0.73)

Light 1,16 0.10 (0.75) 24.09 (<0.001) 4.31 (0.05)

Symbiont * light 2,16 0.22 (0.81) 0.24 (0.79) 0.22 (0.80)

Within subjects

Trial 2,32 3.30 (0.04) 19.0 (<0.001) 3.69 (0.04)

Trial * light 2,32 2.20 (0.13) 0.67 (0.52) 1.70 (0.20)

Trial * symbiont 4,32 1.10 (0.36) 0.21 (0.93) 0.82 (0.52)

Trial * light * symbiont

4,32 0.20 (0.92) 0.90 (0.48) 0.46 (0.77)

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cnidae for more eYcient prey capture. Instead, we foundthat A. elegantissima had similar prey-capture surface areasand cnida densities, regardless of symbiotic state. However,the tentacles of freshly collected asymbiotic anemones didhave longer basitrichs with a trend toward greater adhesiveforces per basitrich. Purcell (1984) found that siphono-phores with larger nematocysts could capture larger preythan those with smaller nematocysts. Our results suggestthat asymbiotic anemones in the Weld may be able to cap-ture larger prey while discharging fewer basitrichs.

Light aVected the heterotrophic feeding of all A. elegan-tissima individuals, regardless of symbiotic state. Cnidadensity increased in anemones held in sunlight and shadedtreatments during a three-week acclimation period, suggest-ing that our feeding regime, prey type or tank conditionschanged the feeding capacity of the anemones. The magni-

tude of the increase depended on the light treatment. Bothspirocyst and basitrich densities increased 3–6£ in anemo-nes held in the shade, but the increase was smaller amonganemones in the sunlight. We predicted that shading anem-ones with symbionts would increase their nutritional needcausing them to produce additional cnidae, while cnidadensities in asymbiotic anemones would remain relativelyunchanged. In fact, under shaded conditions, asymbioticindividuals showed the greatest increase in basitrich densi-ties. We believe that sunlight had direct eVects on cnidaproduction in these asymbiotic individuals. Anemonesexposed to sunlight invest energy in pigments and enzy-matic repair mechanisms as a defense against light (Shickand Dykens 1984; Dykens and Shick 1984) and tempera-ture (Snyder and Rossi 2004) damage. Symbiotic anemoneswe collected from a high-light Weld location, therefore,

Fig. 4 a–c Ingestion time, d–f digestion time and g–i absorption eY-ciency over three trial days for zooxanthellate (ZX), zoochlorellate(ZC) and asymbiotic (AS) A. elegantissima. SigniWcant diVerences

between light treatments across all symbiotic states are indicated byuppercase letters above the Wrst set of bars. Standard errors are shown

a b c

fed

g h i

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948 Mar Biol (2012) 159:939–950

were accustomed to the high irradiance they subsequentlyexperienced in the experimental tank. In contrast, asymbi-otic individuals came from a deeply shaded microhabitat.Transplanting them into full sunlight may have producedsigniWcant costs as the anemones responded by investingenergy in photoprotective pigments and enzymatic repairmechanisms. This may have reduced energy available forother activities (e.g., production of cnidae).

Light stress was also evident in our feeding experiments.Twenty-seven percent of asymbiotic anemones held in sun-light dropped and failed to ingest their food pellets, a behav-ior that was not seen among symbiotic anemones. Webelieve that our sunlight treatment stressed the asymbioticA. elegantissima and negatively aVected their prey-captureability by reducing cnida production or function. Althoughsunlight reduced the eVectiveness of tentacle cnidae, itdecreased digestion time in all anemones. This may haveresulted from slightly warmer seawater temperatures in thesunlight treatment; warmer water temperatures decreasedigestion times in the jellyWsh Aurelia spp. (Purcell 2009)and in the gorgonian, Leptogorgia sarmentosa (Rossi et al.2004). Anemones in our sunlight treatment experiencedshort periods of slightly warmer water temperatures than didanemones in the shade. However, the maximum temperaturediVerence between the treatments was only 1°C. This islower than the temperature diVerence required to see eVectsin either cnidarian mentioned above. Strom (2001) showedthat sunlight exposure enhances digestion in the protozoanNoctiluca scintillans, presumably through partial breakdownof ingested food by reactive oxygen species produced byirradiant energy. Photodynamic reduction of oxygen (form-ing superoxide and hydroxyl radicals and hydrogen perox-ide) also occurs in A. elegantissima tissues (Dykens andShick 1984), which may enhance digestive processes ofindividuals exposed to sunlight. Algal productivity increasesthe formation of reactive oxygen species, so symbioticanemones might be expected to digest faster than asymbioticanemones in the sunlight. However, to mitigate oxygen tox-icity, symbiotic anemones produce enzymes that suppressthe formation of reactive oxygen (Dykens and Shick 1984).These competing processes may explain why digestion wasfaster in the sunlight for all anemones, but was not furtherenhanced by the presence of symbiotic algae.

If a signiWcant portion of daily nutrition derives fromalgal photosynthesis, anemones lacking symbionts mightcompensate with more eVective heterotrophic feeding.However, we saw no clear diVerences in heterotrophyamong the three symbiotic states of A. elegantissima. Itshould be noted that none of the anemones used in ourstudy were truly asymbiotic; even those we categorized asasymbiotic hosted small numbers of zooxanthellae(Table 1). However, densities were suYciently diVerent inour treatments that eVects of the symbionts, if important,

should have been evident. There are at least 3 possibleexplanations for the lack of clear eVects.

First, it is possible that the cost of maintaining the sym-biotic algae just balanced their nutritional contribution.Algal symbionts may acquire nutrients directly from theirhosts (Steen 1986) and, under some conditions, symbioticanemones may actually have to feed more eVectively tomeet the needs of the symbionts (Hoogenboom et al. 2010).In addition, symbiotic anemones must experience suYcientsunlight to support the photosynthetic activities of theirsymbionts. The sunlight that allows the symbionts to sur-vive, however, may produce costs for the anemones, whichmust protect themselves from sunlight damage (Dykensand Shick 1984; Snyder and Rossi 2004). These costs couldoVset energetic contributions from the symbiotic algae.While these trade-oVs are theoretically possible, measure-ments have repeatedly shown that, at least for zooxanthel-late individuals, the energetic beneWts of algal symbiosisexceed the costs in A. elegantissima, (e.g., Sebens 1980;Fitt and Pardy 1981; Tsuchida and Potts 1994), which canexperience high irradiance and temperature conditions(Secord and Muller-Parker 2005).

Another possible explanation for our results is that thesymbiotic algae contributed less than expected to their hostanemones, which would have made diVerences in feedingstrategies diYcult to detect. Davy et al. (1997) studied auto-trophy and heterotrophy in three temperate sea anemones anda temperate zoanthid symbiotic with Symbiodinium. Theirresults suggested that heterotrophy largely supplied theenergy necessary to meet metabolic needs and that these tem-perate symbiotic cnidarians were not dependent on energyfrom the symbionts. A common way to quantify the potentialbeneWt of algal symbionts is as the percent contribution of thesymbiont to anemone respiration (CZAR, Muscatine et al.1981). The inXuence of host environment and symbiont con-dition and density result in widely variable literature esti-mates for CZAR in A. elegantissima, ranging from 34 to126% for zooxanthellate anemones and 9–82% for zoochlo-rellate anemones (Muller-Parker and Davy 2001). When esti-mating CZAR, it is assumed that the host receives allphotosynthetic carbon not used by the algae for growth orrespiration (Engebretson and Muller-Parker 1999). However,this may not be the case. Verde and McCloskey (1996) cal-culated the amount of heterotrophically derived carbonrequired for respiration in zooxanthellate and zoochlorellateanemones based on their respective CZAR estimates. Theysuggest that zooxanthellate anemones require only half theheterotrophic carbon per day that zoochlorellate anemonesdo (702 and 1,214 �g C day¡1, respectively). Despite thispotential diVerence in the need to feed heterotrophically, wesaw little variation in heterotrophic feeding abilities of anem-ones in any symbiotic state, suggesting that the actual algalcontribution may be lower than expected.

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Finally, it has been estimated that, in the highly produc-tive intertidal zones of the PaciWc Northwest, A. elegantiss-ima can heterotrophically acquire almost twice the dailycarbon they require (3,000 �g C potentially acquired day¡1

and only 1,300 �g C required day¡1, Verde and McCloskey1996). Therefore, A. elegantissima may meet daily nutri-tional requirements heterotrophically, and autotrophicenergy from their symbionts is directed to other processes.Davy et al. (1996) suggested that obligate symbiotic cnidar-ians depend on autotrophy for their daily nutritionalrequirement, and excess heterotrophic feeding contributesto non-essential functions. For example, heterotrophy hasbeen shown to increase skeletal growth in the coral Cladoc-ora caespitosa (Hoogenboom et al. 2010) and resiliency tobleaching in Montipora capitata (Grottoli et al. 2006; Pal-ardy et al. 2008). Facultatively symbiotic cnidarians, suchas A. elegantissima, may show the reverse pattern, depend-ing on heterotrophic feeding to meet daily nutritionalrequirements, and using excess autotrophic energy forreproduction, growth (Tsuchida and Potts 1994) or storage(Fitt and Pardy 1981). Energy provided by the symbiontsmay also permit A. elegantissima to live in areas withhigher temperature and irradiance (Secord and Muller-Parker 2005), expanding their intertidal range.

Temperate cnidarians are less reliant on their algal sym-bionts for nutrition than are their tropical counterparts(Muller-Parker and Davy 2001). Results from our studyemphasize this fact for A. elegantissima. Productivity of thesymbionts does not appear to inXuence the heterotrophicfeeding of the host anemone. Piniak (2002) found that prey-capture eYciency was similarly unaVected by the presenceof algal symbionts in the facultatively symbiotic, temperatecoral, Oculina arbuscula. The ability to shift betweenmodes of nutrient acquisition (heterotrophic and auto-trophic) may vary with latitude. Heterotrophic plasticity maybe common in tropical symbioses (Anthony and Fabricius2000; Grottoli et al. 2006; Houlbrèque and Ferrier-Pagès2009), while autotrophic plasticity is the more commonnutritional pattern in temperate ones.

Acknowledgments We thank G. Muller-Parker, J. Dimond, L. Fran-cis, G. McKeen, N. Schwarck and M. Ponce-McDermott for laboratoryassistance. Comments from D. Donovan, S. Strom, G. Ayres and twoanonymous reviewers signiWcantly improved the work. Funding wasprovided by Western Washington University and by National ScienceFoundation grant IOS-0935820.

References

Anthony KRN, Fabricius KE (2000) Shifting roles of heterotrophy andautotrophy in coral energetics under varying turbidity. J Exp MarBiol Ecol 252:221–253

Bergschneider H, Muller-Parker G (2008) Nutritional role of two algalsymbionts in the temperate sea anemone Anthopleura elegantiss-ima Brandt. Biol Bull 215:73–88

Bronstein JL (1994) Our current understanding of mutualism. Quar-terly Rev Biol 69:31–51

Davy SK, Lucas IAN, Turner JR (1996) Carbon budgets in temperateanthozoan-dinoXagellate symbioses. Mar Biol 126:773–783

Davy SK, Turner JR, Lucas IAN (1997) The nature of temperate antho-zoan-dinoXagellate symbioses. Proceedings of 8th internationalcoral reef symposium, vol 2, pp 1307–1312

Dean AM (1983) A simple model of mutualism. Am Nat 121:409–417Dykens JA, Shick JM (1984) Photobiology of the symbiotic sea anem-

one, Anthopleura elegantissima: defenses against photodynamiceVects, and seasonal photoacclimatization. Biol Bull 167:683–697

Engebretson HP, Muller-Parker G (1999) Translocation of photosyn-thetic carbon from two algal symbionts to the sea anemone Antho-pleura elegantissima. Biol Bull 197:72–81

Fitt WK, Pardy RL (1981) EVects of starvation, and light and dark onthe energy metabolism of symbiont and aposymbiotic sea anem-ones, Anthopleura elegantissima. Mar Biol 61:199–205

Francis L (1991) Sailing downwind: aerodynamic performance of theVelella sail. J Exp Biol 158:117–132

Francis L (2004) Microscaling: why larger anemones have longer cni-dae. Biol Bull 207:116–129

Giebel GEM, Thorington GU, Lim RY, Hessinger DA (1988) Controlof cnida discharge: II. Microbasic p-mastigophore nematocystsare regulated by two classes of chemoreceptors. Biol Bull175:132–136

Grottoli AG, Rodrigues LJ, Palardy JE (2006) Heterotrophic plasticityand resilience in bleached corals. Nat Lett 440:1186–1189

Hoogenboom M, Rodolfo-Metalpa R, Ferrier-Pagès C (2010) Co-var-iation between autotrophy and heterotrophy in the Mediterraneancoral Cladocora caespitosa. J Exp Biol 213:2399–2409

Houlbrèque F, Ferrier-Pagès C (2009) Heterotrophy in tropical scle-ractinian corals. Biol Rev 84:1–17

Kramer A, Francis L (2004) Predation resistance and nematocyst scal-ing for Metridium senile and M. farcimen. Biol Bull 207:130–140

LaJeunesse TC, Trench RK (2000) Biogeography of two species ofSymbiodinium (Freudenthal) inhabiting the intertidal sea anem-one Anthopleura elegantissima (Brandt). Biol Bull 199:126–134

Letsch MR, Muller-Parker G, Friedl T, Lewis LA (2009) Elliptochlorismarina sp. nov. (Trebouxiophyceae, Chlorophyta), symbioticgreen alga of the temperate PaciWc sea anemones Anthopleuraxanthogrammica and A. elegantissima (Anthozoa, Cnidaria).J Phycol 45:1127–1135

Lowry OH, Rosenbrough NJ, Farr HL, Randall RJ (1951) Protein mea-surement with the Folin phenol reagent. J Biol Chem 193:265–275

Muller-Parker G, Davy SK (2001) Temperate and tropical algal-seaanemone symbioses. Invert Biol 120:104–123

Muscatine L, Porter JW (1977) Reef corals: mutualistic symbiosesadapted to nutrient-poor environments. Biosci 27:454–460

Muscatine L, McCloskey LR, Marian RE (1981) Estimating the dailycontribution of carbon from zooxanthellate to coral animal respi-ration. Limnol Oceanogr 26:601–611

Palardy JE, Rodrigues LJ, Grottoli AG (2008) The importance of zoo-plankton to the daily metabolic carbon requirements of healthy andbleached corals at two depths. J Exp Mar Biol Ecol 367:180–188

Piniak GA (2002) EVects of symbiotic status, Xow speed, and prey typeon prey capture by the facultatively symbiotic temperate coralOculina arbuscula. Mar Biol 141:449–455

Purcell JE (1984) The functions of nematocysts in prey capture byepipelagic siphonophores (Coelenterata, Hydrozoa). Biol Bull166:310–327

Purcell JE (2009) Extension of methods for jellyWsh and ctenophoretrophic ecology to large-scale research. Hydrobiol 616:23–50

Rossi MR, Coma R, Gili J-M (2004) Temporal variability in zooplank-ton prey capture rate of the passive suspension feeder Leptogorgiasarmentosa (Cnidaria: Octocorallia). Mar Biol 144:89–99

123

950 Mar Biol (2012) 159:939–950

Sebens KP (1980) The regulation of asexual reproduction and indeter-minate body size in the sea anemone Anthopleura elegantissima(Brandt). Biol Bull 158:370–382

Sebens KP (1981) The allometry of feeding, energetics, and body sizein three sea anemone species. Biol Bull 161:152–171

Secord D, Muller-Parker G (2005) Symbiont distribution along a lightgradient within an intertidal cave. Limnol Oceanogr 50:272–278

Shick JM, Dykens JA (1984) Photobiology of the symbiotic sea anem-one Anthopleura elegantissima: photosynthesis, respiration, andbehavior under intertidal conditions. Biol Bull 166:608–619

Snyder MJ, Rossi S (2004) Stress protein (HSP70 family) expressionin intertidal benthic organisms: the example of Anthopleura ele-gantissima (Cnidaria: Anthozoa). Sci Mar 68:155–162

Steen RG (1986) Evidence for heterotrophy by zooxanthellate in sym-biosis with Aiptasia pulchella. Biol Bull 170:267–278

Strom SL (2001) Light-aided digestion, grazing and growth in herbiv-orous protists. Aquat Microb Ecol 23:253–261

Thorington GU, Hessinger DA (1988) Control of Cnida discharge: I.Evidence for two classes of chemoreceptor. Biol Bull 174:163–171

Thorington GU, Hessinger DA (1996) EVerent mechanisms of dis-charging cnidae: I. Measurements of intrinsic adherence of cnidae

discharged from tentacles of the sea anemone, Aiptasia pallida.Biol Bull 190:125–138

Tsuchida CB, Potts DC (1994) The eVects of illumination, food andsymbionts on growth of the sea anemone Anthopleura elegantiss-ima (Brandt, 1835). I. Ramet growth. J Exp Mar Biol Ecol183:227–242

Underwood AJ (1981) Techniques of analysis of variance in experi-mental marine biology and ecology. Oceanogr Mar Biol AnnuRev 19:513–605

Verde EA, McCloskey LR (1996) Photosynthesis and respiration oftwo species of algal symbionts in the anemone Anthopleura ele-gantissima (Brandt) (Cnidaria; Anthozoa). J Exp Mar Biol Ecol195:187–202

Yellowless D, Rees TAV, Leggat W (2008) Metabolic interactionsbetween algal symbionts and invertebrate hosts. Plant Cell Envi-ron 31:679–694

Zamer WE (1986) Physiological energetics of the intertidal sea anem-one Anthopleura elegantissima. I. Prey capture, absorption eY-ciency and growth. Mar Biol 92:231–299

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