Carbon Budgets of the Bulb-Tentacle Sea Anemone (Entacmaea quadricolor) Symbiotic with Zooxanthellae and Anemonefish Nathan T. Schwarck A Thesis in partial fulfillment of the requirements for the degree of Master of Science Department of Biological Sciences Walla Walla College Major Professor: Dr. Lawrence R. McCloskey 9 August, 1999 1
INTRODUCTION All 10 species of sea anemones known to associate with anemonefish (Table 1), and 9 of the 28 known species of anemonefish, can be found near Madang on the north coast of Papua New Guinea (Fautin 1988; Fautin & Allan 1992). These anemones also host endosymbiotic unicellular dinoflagellate algae (zooxanthellae). Fautin (1991) states that the intimacy of this three-way symbiosis invites investigation of the degree to which the actinians depend on their algae for fixed carbon and their fish for nitrogen and possibly other nutrients. Entacmaea quadricolor (Rueppell & Leuckart 1828) is one of the 10 species of anemones known to associate with both anemonefish and zooxanthellae, and also exhibit two generally distinct forms (solitary and clonal) described as ecophenotypes (Dunn 1981). The solitary and clonal ecophenotypes (morphs) of Entacmaea quadricolor are often associated with the commensal anemonefish Premnas biaculeatus and Amphiprion melanopus, respectively (Fautin & Allan 1992). The flux and distribution of carbon in anemones symbiotic with anemonefish, including Entacmaea quadricolor have never been estimated. Consequently, the primary focus of this study is the carbon budget of E. quadricolor symbiotic with zooxanthellae and anemonefish. The components used to estimate carbon flux between cnidarian hosts and their algal symbionts include photosynthesis and respiration, algal reproduction (cytokinesis), and algal translocation rates (Verde & McCloskey 1996b, Hinde 1989). From diel photosynthesis and respiration measurements, the amount of translocated carbon from zooxanthellae which contributes to the anemone s daily carbon requirements, can be estimated (CZAR; Muscatine et al. 1981; McCloskey et al. 1994; 2
Table 1. The ten species of anemones symbiotic with anemonefishes in Papua New Guinea (Fautin and Allen 1992). Phylum Cnidaria Class Anthozoa Order Actiniaria Family Actiniidae Genus Entacmaea Species quadricolor* Genus Macrodactyla Species doreensis Family Stichodactylidae Genus Heteractis Species aurora crispa magnifica malu Genus Stichodactyla Species gigantea Family Thessianthidea haddoni mertensii Genus Cryptudendium Species adhaesivum *Occurs in two separate ecophenotypes, one solitary and the other clonal (Dunn, 1981). 3
Verde & McCloskey 1996b). Organisms with CZARs approaching zero obtain little or no carbon from their symbionts whereas those with CZARs approaching 100% potentially obtain all of their carbon for respiration from their symbiotic algae. The parameters used to estimate CZAR are defined in Table 2. Coral species with large polyps have been predicted to show lower CZARs than those with small polyps (McCloskey and Muscatine 1984; Porter et al. 1984), because their adaptation for capturing commensurately larger and greater quantities of prey should render them less dependent upon their algal symbionts for sustenance. If this hypotheses holds, solitary Entacmaea quadricolor, which is among the largest cnidarian polyps, should show the lowest CZAR when compared to smaller-sized clonal anemone morphs. 4
Table 2 Terms used throughout this thesis. Consistent with published conventions for the abbreviation of respiration estimates, uppercase letters denote daily values while lower case letters refer to hourly rates. Superscripts indicate units of measure (i.e. o = oxygen, c = carbon), and subscripts refer to the particular fraction of the symbiotic association for which the respiration is attributed (an = whole anemone equaling the animal and algal fractions of the symbiosis; al = animal fraction of symbiosis; zx = zooxanthellae, or algal fraction of symbiosis; μ = growth; t = time; d = duration). α Measure of photosynthetic efficiency β Animal:intact anemone biomass ratio 1-β Algal:intact anemone biomass ratio μ af Algae ingested by anemonefish μ zx Algal population growth C af Carbon from anemonefish C h Carbon from heterotrophy C μ Carbon specific growth rate C t Carbon available for translocation to the host (equation 10) CBAG Carbon translocated from animal back to algae CZAR Percent contribution of algal translocated carbon to daily animal respiratory requirements DOM Dissolved organic matter D t Algal doubling time f Average fraction of zooxanthellae cells undergoing non-phased division f o Hourly net oxygen flux F o Net oxygen flux by the anemone (all respiration values are considered to have a positive sign. F o is typically positive over normal diel cycle. However f o may be negative at night or positive during the day (Figure 1) I k Intersection of α and P o n max tangents I opt Optimum irradiance during P o n max MI Mitotic index P Photosynthesis P c g Daily gross photosynthesis (in carbon equivalents) P max Maximum (light-saturated) rate of photosynthesis seen in a P-I curve P o n max Maximum net photosynthesis P c zx net Net daily carbon fixation by the zooxanthellae PQ zx Photosynthetic quotient for zooxanthellae, the molar ratio of O 2 produced to CO 2 fixed R Daily respiration, expressed in units of energy or carbon r o al Hourly respiration of the animal fraction per unit protein biomass (equation 6) R c al Daily respiration by the animal (daily animal respiratory requirement) r o an Hourly respiration of a symbiotic anemone (intact association; r al + r zx ) per unit protein biomass (equation 6) R o an Daily respiration by intact anemone r o zx Hourly respiration of the algal fraction (zooxanthellae) per unit protein biomass (equation 6) R c zx Daily carbon respiration by zooxanthellae RQ Respiratory quotient, the molar ratio of CO 2 produced to O 2 consumed RQ al Respiratory quotient for only the animal portion of the anemone RQ an Respiratory quotient for the intact anemone RQ zx Respiratory quotient for zooxanthellae SS Standing Stock (total number of algae per anemone) t d Duration of cytokinesis t Time 5
METHODS AND MATERIALS Location, Identification, and Collection This research was conducted at the Christensen Research Institute in Madang, Papua New Guinea between 28 April and 15 June 1996. The anemones and fish were identified in the field based upon appearance and habitat, using Fautin & Allen (1992) as a reference. Both clonal and solitary morphs of the anemone Entacmaea quadricolor and their associated commensal fish were used for experimentation. The anemones and their anemonefish were collected using SCUBA from sites within Madang Lagoon (the natural history of which has been described by Jebb & Lowry 1995). Prior to collecting the anemones, the fish symbionts were captured with modified dipnets according to the method of Allen (1972, 1980) and Fautin (1986). Anemones were removed from their original attachment site by carefully freeing the foot of the anemone from the substrate, transporting the anemone to a holding tank supplied with fresh running saltwater, and carefully removing any remaining substrate fragments from the anemone s foot. Anemones and fish were held in running seawater tanks for several hours before transporting them back to their corresponding depth at the study location on the reef. Sample Size The sample sizes used in this study were out of concern for conservation and preservation of these organisms. Non-lethal methods of determining anemone carbon budgets were not available; therefore, sample sizes were kept at minimal levels so that impact on the local populations was minor yet statistically valid results could still be obtained. 6
Commensal Fish The anemonefish Amphiprion melanopus is most often associated with the clonal form of Entacmaea quadricolor, and Premnas biaculeatus with the solitary form of E. quadricolor (Fautin & Allen 1992). The social structure of both anemonefish species usually involves a hierarchy where one large female fish is dominant (Fautin & Allen 1992). Since this individual most likely has the greatest trophic contribution to the anemone, this larger dominant anemonefish was the only one used in the respiration experiments. Algal Density, Diameter, and Biomass Algal density (cells ml -1 ) was determined from averaged hemacytometer counts (n = 20) from a minimum of 1,000 algal cells counted from homogenate samples of each anemone. Algal density multiplied by total homogenate volume (ml), provided an estimate of the total standing stock number of algal cells per anemone (SS). The mean diameter of the zooxanthellae (n = 40) from each anemone was determined from measurements of individual algae using an ocular micrometer. Algal cell-specific protein biomass (pg N cell -1 ) was determined from C:N analysis using the method outlined by Verde (1993). The nitrogen value obtained was converted to protein biomass by multiplying by 6.25 pg (Muscatine et al. 1986). Total algal protein (mg) was estimated by multiplying the algal cell-specific biomass by the algal standing stock (SS). Mitotic Index and Growth Mitotic index (MI) was determined using a technique modeled after that of Wilkerson et al. (1983). The number of algal cells undergoing cytokinesis was noted, 7
then divided by the total number of cells counted from the sample. The resultant percentage was taken as the MI. The phased-division formula of Vaulot (1992) was used to calculate the algal-specific growth rate (μ zx ) per day: μ zx =ln[(1 + f max )(1 + f min ) -1 ] (1) where f max and f min are the maximum and minimum daily fraction of dividing cells over a 24 hour period. The carbon-specific growth rate (C μ in μg C day -1 ) of the algal population: C μ = [(SS)(C cell -1 )(μ zx )] (2) and algal doubling time (D t, in days) were calculated using the equations of Wilkerson et al. (1983): D t = [(ln 2)(μ zx ) -1 ]. (3) Diel Mitotic Activity Diel zooxanthellae mitotic activity was investigated in two experiments on groups of freshly collected and individually marked anemones. The first experiment included six clonal and two solitary anemones, while the second experiment (one month later) consisted of four clonal and three solitary anemones. From each anemone, tentacle snips were collected every hour for 24 h and frozen. Diel mitotic index data was determined later from frozen tentacle snips at the Walla Walla College Marine Station, Washington. Each sample was homogenized and the dividing algal cells counted in a hemacytometer. The MI was calculated as described by Wilkerson et al. (1983). 8
Algal Chlorophyll Zooxanthellae chlorophyll was extracted using the technique of Verde & McCloskey (1996b). Chlorophyll mass (pg cell -1 ) was calculated according to the equations of Jeffrey & Humphrey (1975) as described in Parsons et al. (1984). Animal Biomass The Pierce BCA colorimetric assay was used for animal protein measurements with bovine serum albumin as the protein standard (Pierce 1991). Three replicate 0.1 ml samples of each anemone homogenate were assayed for animal protein per volume (mg protein ml -1 ). The mean of the three replicates, multiplied by total homogenate volume (ml), was used to estimate total animal biomass (mg protein anemone -1 ). These biomass values were only for the animal fraction because the Pierce BCA assay does not detect the protein of the intact algal symbionts (Verde 1987). Total anemone protein was determined as the sum of animal plus algal protein. The equations for calculating relative biomass, used to normalize respiration measures, utilize the conventions beta (β), which is the animal: intact anemone biomass ratio, and 1-β which is the algal: intact anemone biomass ratio (Muscatine et al. 1981): β = (animal biomass)(anemone biomass) -1 (4a) 1-β = (algal biomass)(anemone biomass) -1. (4b) 9
Diel Photosynthesis and Respiration Anemone and resident anemonefish diel net oxygen flux (F o, mg O 2 liter -1 min -1 ) and incident irradiance (μe m -2 s -1 ) were monitored in situ using a microcomputer-controlled, self-contained submersible respirometers (described in McCloskey et al. 1978; McCloskey et al. 1985). All oxygen-flux measurements were corrected for background photosynthesis and respiration measured in a control (empty) chamber. Respirometers were placed at depths between 1.4 and 7.0 m corresponding to the anemone s original collection depth. Diel photosynthesis and respiration incubation experiments were done simultaneously on two separate anemone and anemonefish associations; therefore, each complete experiment was 48 h. During one diel cycle both the fish and anemone were enclosed together within the same bell jar; for the next diel cycle, the fish and the anemone were separated with the order of this sequence alternated. At the end of each 48 h experiment, the organisms were brought into the laboratory. The displacement volume of each anemone was recorded, and the anemone was homogenized in filtered rainwater (because ddh 2 O was not available) in a blender. Total anemone homogenate volume was recorded, and the homogenate filtered through a mesh strainer to remove any mesoglea chunks. Two 5 ml aliquots from the homogenate were immediately frozen for protein determination. Displacement volume, standard length, and wet weight of the associated anemonefish were recorded before they were returned to the reef. Diel oxygen flux and light measurements were used to generate photosynthesis and irradiance (P-I) response curves according to the method of Verde & McCloskey (1998). Figure 1 presents an example of a plot of anemone O 2 flux (f o ) versus time of 10
day, derived from continuous po 2 recordings. The daytime fluxes of photosynthetically produced oxygen, summed with anemone respiration rate values obtained at night, provided daily gross photosynthesis measures (P o g) (McCloskey et al. 1978; Muscatine et al. 1981; McCloskey et al. 1994; Verde and McCloskey 1996b). Net photosynthesis (P o zxnet) was calculated by subtracting algal respiration from P o g. P-I analysis consisted of plotting hourly net photosynthesis normalized to algal density (μg O 2 h -1 10-6 cells) versus incident irradiance (μe m -2 s -1 ). These curves were then iteratively fit to a hyperbolic tangent function (Jassby & Platt 1976) and the photosynthetic efficiency (α), optimum irradiance (I opt ), maximum net photosynthesis o (P n max), and the intersection of α and o Pn max tangents (Ik ) determined. Nighttime oxygen flux measurements provided average hourly respiration rates (r o an) of the whole anemone where r o an = r o zx + r o al (6) which when extrapolated to 24 h, provided an estimate of the total daily anemone respiration (R o an). Respiration of the algae (R o zx) and animal (R o al) was estimated based on the animal (β) and algal (1-β) components of total biomass (Muscatine et al. 1981). 11
Figure 1. Representative diel curve of net O 2 flux for Entacmaea quadricolor, along with the average daily integrated irradiance regime. 12
f o (mg O 2 min -1 ) 0.8 14 0.7 12 10 0.6 8 0.5 6 4 0.4 2 irradiance 0.3 0 0.2-2 0.1 f o -4-6 -0.0-8 -0.1-10 -12-0.2 0 2 4 6 8 10 12 14 16 18 20 22-14 24 Experiment running time (h) average integrated irradiance (10 4 E m -2 min -1 ) 13
Carbon Budgets and CZAR Diel oxygen flux measurements were converted to carbon equivalents using the carbon to oxygen ratio 12:32 = 0.375 (McCloskey et al. 1978). The equations for the conversion of mg oxygen to mg carbon (McCloskey et al. 1978; and Muscatine et al. 1981) are: and mg C assimilated by zooxanthellae photosynthesis = (mg O 2 produced 0.375)(PQ zx ) -1 (7a) mg C lost by anemone respiration = (mg O 2 consumed 0.375)(RQ an ). (7b) The percent contribution of algal carbon to animal respiration (CZAR) was determined by the formula of McCloskey et al. (1994): CZAR ={[(0.375 P o g)(pq zx ) -1 ]-[(1-β)(0.375 R o an)(rq an )]-[C μ ] / [β)(0.375 R o an)(rq an )]} 100 (8) where the photosynthetic quotient for the zooxanthellae (PQ zx ) is the ratio of algal O 2 produced to CO 2 fixed. The respiratory quotient for the anemone (RQ an ) is the ratio of CO 2 produced to O 2 consumed by the whole anemone where RQ an = [((1-β)(RQ zx ) -1 )+((β)(rq al ) -1 )] -1. (9) The respiratory quotient of the animal (RQ al ) is the ratio of CO 2 produced to O 2 consumed by only the animal portion of the anemone, whereas the respiratory quotient of the zooxanthellae (RQ zx ) is the ratio of CO 2 produced to O 2 consumed by only the algal portion of the association. Values of 1.1, 0.9, and 1.0 were used for PQ zx, RQ al, and RQ zx respectively (cf. Muscatine et al.1981; Porter et al. 1984; and Kremer et al. 1990). 14
The carbon available for translocation to the host (C t ) is: C t = [(0.375 P o g)(pq zx ) -1 ]-[(1-β)(0.375 R o an)(rq an )]-[C μ ] (10) which is the numerator of equation 8. Data and Statistical Analysis The coral data acquisition program Hcoral (ver 3.11) was used for all respirometry data. All data were checked for normality and homogeneity of variance. Non-normal or heterogeneous data were log transformed and retested, or non-parametric tests were utilized. Mitotic index and CZAR percentage data were square root-arcsine transformed prior to analysis (Sokal & Rohlf 1995, Zar 1996). Two-sample unpaired t-tests were used to determine significance of differences (p<0.05) between biomass parameters of clonal and solitary anemone morphs. The average diameter of the zooxanthellae and chlorophyll mass between anemone morphs were similarly compared. One-way analysis of covariance (ANCOVA, post hoc Tukey HSD Spjotvoll/Stoline test, α = 0.05 for both tests; Zar 1996) was used to determine significant differences between anemone morph data as a function of anemone biomass or total chlorophyll. Statistical and graphical analysis were done using the software packages GraphPad Prism 2.01 (GraphPad Software, Inc.), Systat 7.0.1 (SPSS, Inc.), Statistica\W (Statsoft, Inc.), and MS Excel 7.00. Carbon budget figures were created using CorelDRAW! 3.00. 15
RESULTS Environmental Parameters Light and Thermal Regime. The mean (SD, n) daily integrated irradiance experienced by clonal anemones (30.7 ± 4.7 E m -2 d -1, 8) was significantly higher than for solitary anemones (20.2 ± 8.1 E m -2 d -1, 20), (t-test, p<0.05). There was no significant difference in the water temperatures (19.1 C ± 0.3, 20) experienced by the clonal and solitary anemones (t-test, p>0.05). Depth. The experimental depths in meters ( ± SD, n) of the solitary and clonal anemones was (3.3 ± 1.5, 20) and (2.7 ± 0.0, 8), respectively. Algal Parameters Algal Biomass: Nitrogen, Carbon, and Protein. The nitrogen and carbon contents ( pg cell -1 ± SD) of freshly isolated zooxanthellae from solitary anemones (n = 5) were 11.2 ± 2.5 and 82.6 ± 68.6, respectively. The calculated protein value ( pg cell -1 ± SD) was 70.0 ± 15.3. Cell Size. The average (± SD, n) algal cell diameter from clonal anemones (7.21 μm ± 2.40, 300) was not significantly different (t-test, p>0.05) than for algae from solitary anemones (7.08μm ± 2.45, 743) (Table 3). It follows that the average cell volume for algae in the two morphs also was not significantly different (t-test, p>0.05; 254.4 μm 3 ± 238.7, 743 and 261.5 μm 3 ± 218.3, 300 for solitary and clonal, respectively). A bimodal distribution of algal diameters (t-test, p<0.001) was observed (Fig. 2). When the median (7.3μm) was used to divide the data set, means (± SD, n) of 16
Table 3 Algal size and chlorophyll (Chl) measures of zooxanthellae from solitary and clonal morphs of Entacmaea quadricolor. clonal n = 8 ± SD solitary n = 20 ± SD clonal : solitary Significance Cell diameter (μm) 7.21 ± 2.4 7.08 ± 2.45 1.02 n. s. Cell volume (μm 3 ) 261.5 ± 218.3 254.4 ± 238.7 1.03 n. s. Chl-a (pg cell -1 ) 20.02 ± 12.4 5.97 ± 2.9 3.35 * Chl-c (pg cell -1 ) 11.60 ± 7.3 7.11 ± 11.1 1.63 n. s. Chl-a+c (pg cell -1 ) 31.62 ± 21.4 13.09 ± 13.6 2.42 * Chl a:c Ratio 1.74 ± 0.1 1.77 ± 1.5 0.98 n. s. Significance of differences was determined by the two-sample t-test with Welch s correction (n. s. = not significant; * = p < 0.05). Confidence interval at 95%. 17
Figure 2. Algal cell diameters of zooxanthellae from both clonal and solitary Entacmaea quadricolor (n = 28). Numbers are from measured (± 0.5 μm) cell diameters (n = 1069). The bimodal distribution ( ± SD, n) was significantly different (t-test, p<0.001) when divided at the median (7.3 μm). 18
Number of cells 300 275 250 225 200 175 150 125 100 75 50 25 0 5.2 ± 1.2, 578 9.5 ± 1.2, 465 Median (7.3μ m) 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 13.5 Zooxanthellae diameter (μm) 19
5.2 μm (± 1.2, 578) and 9.5 μm (± 1.2, 465) were calculated for each, which better describes the average diameters of the two populations. Mean zooxanthellae cell size (μm) and concentration (% of total algae observed) varied depending upon location in the one anemone where this was examined (Table 4). The majority of the zooxanthellae (61.1%) was located evenly between the tentacles and the column wall. A binomial distribution of algal diameters ( ± SD, n) was again observed in this anemone, with the concentrations of large (11.8 μm ± 1.7, 36) and small (5.8 μm ± 1.1, 64) zooxanthellae varying with the body region of the anemone. The tentacles and column wall contained almost exclusively large algae, while the oral disk, gullet, and foot contained more small algae than large. Chlorophyll. Zooxanthellae chlorophyll-a from clonal anemones was significantly higher (Welch s corrected t-test, p<0.05) than chlorophyll-a from algae in solitary anemones (Table 3). Similarly, chlorophyll-a + c from clonal anemones was significantly higher (Welch s corrected t-test, p<0.05) than chlorophyll-a + c from solitary anemones. However chlorophyll-c and the chlorophyll-a:c ratio were not significantly different between clonal and solitary morphs. The average clonal anemone had 3.4 times as much chlorophyll-a and 1.6 times as much chlorophyll-c as the average solitary anemone. Total algal chlorophyll (mg chl a + c) and host anemone biomass (g protein) were directly related, with larger anemones containing significantly higher (linear regression, p<0.001) total chlorophyll than smaller anemones (Fig. 3A). Data were combined, because solitary and clonal values were not significantly different (ANCOVA, p > 0.05). 20
Table 4 Summary of position-dependent algal parameters from a single solitary anemone (Entacmaea quadricolor). Zooxanthellae for mitotic index (MI) calculation are divided into two categories: large (> 7μm) and small ( 7 μm). A total of 4722 cells were observed for MI from five separate sections; 20 cell diameters were measured per section. Tentacles Oral Disk & Gullet Column Wall Mesentary Foot MI (%) large cells small cells 0.34 0.00 0.00 0.66 0.60 0.00 1.21 0.44 0.00 0.00 Algal cell diameter 11.8 ± 3.0 6.8 ± 2.0 10.7 ± 1.7 9.4 ± 3.6 9.5 ± 3.6 ( μm ± SD) % of total % large cells % small cells 31.3 98.8 1.1 4.8 29.0 71.0 29.8 95.4 4.6 15.6 67.4 32.6 18.4 41.3 58.7 21
Figure 3. Total chlorophyll (Chl (a+c) ; mg Chl a + c organism -1 ) as a function of both anemone size (A) and algal cell density (B). Asterisk indicates linear regression slopes significantly different from zero (p<0.001). Biomass; r 2 = 0.49, Chl (a+c) = 1.99 (Biomass) + 3.77. Density; for solitary r 2 = 0.44, Chl (a+c) = 0.029 (Density) + 1.49, for clonal r 2 = 0.53, Chl (a+c) = 0.007 (Density) + 3.48. 22
A 45 Total Chlorophyll (mg a + c) 40 35 30 25 20 15 10 5 0 r 2 = 0.49* solitary clonal 0 2 4 6 8 10 12 14 16 18 20 Anemone Biomass (g protein) B Total Chlorophyll (mg a + c) 45 40 35 30 25 20 15 10 5 solitary, r 2 = 0.44* clonal, r 2 = 0.53* 0 0 500 1000 1500 2000 2500 3000 3500 Algal Cell Density (10 6 cells) 23
Total chlorophyll and algal cell density (10 6 cells) were also directly related, with anemones containing higher algal cell densities having significantly more (linear regression, p < 0.001) total chlorophyll (Fig. 3B). Density. Algal densities from clonal and solitary anemones were significantly different (t-test, p < 0.05), (Fig. 4A). Host anemone biomass and algal density were directly related, with large anemones containing significantly higher (linear regression, p < 0.05) algal cell densities than small anemones (Fig. 4B); and the regression slopes between clonal and solitary algal densities as a function of anemone size also were significantly different (ANCOVA, p<0.05). However, there is no significant relationship when algal cell density as a function of anemone biomass is normalized to animal protein (Fig. 4B). Diel Mitotic Activity and Growth. There was no significant difference between anemone morphs in algal diel mitotic activity (Mann-Whitney, p>0.05), therefore data from both morphs were combined and treated as one group (Fig. 5). A nocturnal peak of activity at 0200 was significantly higher than the rest of the diel period (Tukey, p<0.05), therefore the mitotic activity of Entacmaea quadricolor is considered phased. The grand mean (± SD, n) of MI was 0.97% (± 1.07, 360), while f max and f min were 2.31% and 0.31%, respectively. The algal-specific growth rate (μ zx ) was 0.0197 d -1 and the resultant algal doubling time (D t ) was 35.1 days. Photosynthesis. Clonal and solitary daily gross photosynthesis (P g ) of zooxanthellae in Entacmaea quadricolor as a function of total chlorophyll (mg chl a + c) were not significantly different (ANCOVA, p>0.05). Data was therefore combined to 24
Figure 4. Algal densities of zooxanthellae in solitary and clonal Entacmaea quadricolor (A). Asterisk indicates significant differences between morphs (t-test, p<0.05). Values above bars are means. Vertical lines represent standard deviation. Algal density of zooxanthellae within Entacmaea quadricolor normalized to animal protein as a function of anemone size and morph (B). Linear regression was not significant (p > 0.05). Clonal and solitary sample sizes were 8 and 20, respectively. 25
A Mean Algal Density 10 6 cells mg -1 protein 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.36 * 0.12 0.0 Solitary Clonal Anemone morph B Algal Density (10 6 cells mg -1 protein) 1.00 0.75 0.50 0.25 solitary, r 2 = 0.13 clonal, r 2 = 0.10 0.00 0 2 4 6 8 10 12 14 16 18 20 Anemone Biomass (g protein) 26
Figure 5. Mitotic index (MI) of zooxanthellae in Entacmaea quadricolor as a function of time of day. Each point is the mean MI from fifteen anemones of both morphs. Vertical lines indicate standard deviation. Horizontal lines denote darkness. 27
Mitotic Index (%) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0-0.5 0 2 4 6 8 10 12 14 16 18 20 22 24 Time of day (h) 28
show P g is directly related to total chlorophyll (Fig. 6A). P g normalized to total chlorophyll (Fig. B), algal protein (Fig. C), and cell density (Fig. D), was also directly related to total chlorophyll. Solitary and clonal P g, when normalized to total chlorophyll, were not significantly different (ANCOVA, p>0.05). However the P g linear regression slopes for clonal anemones, when normalized to both algal protein and density, were significantly greater (ANCOVA, p<0.01) than those of solitary anemones (Figs. 6C & D). Photosynthetic Parameters. Photosynthesis versus irradiance (P-I) parameters of solitary and clonal Entacmaea quadricolor are summarized in Table 5. Clonal anemones displayed significantly higher α, I k, and P n max. In contrast, both solitary and clonal I opt were not significantly different. Carbon Budgets Solitary and clonal daily net photosynthesis (P zx net), carbon-specific growth rate (C μ ), translocated carbon (C t ), and carbon available for host growth (C avail ) are presented as a function of anemone biomass in Figure 7. Solitary and clonal P zx net and C t (Figs. 7A, B) showed a significant correlation with anemone biomass, but were not significantly different from each other (ANCOVA, p>0.05), therefore the linear regressions were calculated from the combined data of both morphs. Solitary C μ (Fig. 7C) was significantly higher than clonal C μ (ANCOVA, p<0.05). Solitary and clonal C avail (Fig. 7D) were significantly different (ANCOVA, p<0.05) with clonal C avail increasing with greater anemone biomass while solitary anemones had a negative slope, therefore showing the opposite relationship. 29
Figure 6. Daily gross photosynthesis (P g ) of zooxanthellae in Entacmaea quadricolor as a function of total chlorophyll (Chl a+c ). Best-fit curves for the relationship between P g and Chl a+c are linear regression. (A) P g = 4.6 (Chl a+c ) -6.3, r 2 = 0.78. (B) normalized to algal chlorophyll: solitary P g = 0.07 (Chl a+c ) + 3.51, r 2 = 0.00; clonal P g = 0.01 (Chl a+c ) + 3.51, r 2 = 0.61. (C) normalized to algal protein: solitary P g = 0.02 (Chl a+c ) - 11.5, r 2 = 0.82; clonal P g = 0.01 (Chl a+c ) - 4.16, r 2 = 0.41. (D) normalized to algal density: solitary P g = 0.002 (Chl a+c ) - 10.8, r 2 = 0.43; clonal P g = 0.008 (Chl a+c ) - 4.92, r 2 = 0.80. For all figures, asterisk indicates linear regression slopes significantly different from zero. Clonal and solitary sample sizes were 7 and 20, respectively. 30
31
Table 5 Photosynthesis versus irradiance (P-I) parameters of zooxanthellae from solitary and clonal Entacmaea quadricolor. P-I a clonal n = 8 ± SD solitary n = 17 ± SD clonal: solitary Significance α 0.202 ± 0.198 0.066 ± 0.044 3.1 * I k 595.1 ± 251.9 301.9 ± 158.2 2.0 ** I opt 1968 ± 147.1 1544 ± 58.5 1.3 n. s. P n max 140.3 ± 211.6 17.31 ± 10.66 8.1 *** Significance of differences was determined by the two-sample t-test or Mann- Whitney U-test, (n. s. = p > 0.05, * = p < 0.05; ** = p < 0.01; *** = p < 0.001). Confidence interval at 95%. a P-I parameters: α is a measure of photosynthetic efficiency [(μg O 2 h -1 10-6 cells) (μe m -2 s -1 ) -1 ] I k is the intersection of α and P n o max tangents (μe m -2 s -1 ) I opt is the optimum irradiance during P n o max (μe m -2 s -1 ) P n o max is the maximum net photosynthesis (μg O 2 h -1 10-6 cells) 32
Figure 7. Daily carbon budget parameters of Entacmaea quadricolor as a function of anemone biomass and morph. Best-fit curves for the relationship between carbon budget parameter and anemone biomass are linear regression. Asterisk following r 2 value indicates a linear regression slope significantly different from zero (p<0.05). (A) Net photosynthesis (P zx net): P zx net = 10.8 (Biomass) + 3.94, r 2 = 0.53. (B) Potentially translocated carbon (C t ): C t = 10.5 (Biomass) + 3.39, r 2 = 0.52. (C) Algal carbon-specific growth rate (C μ ): solitary C μ = 0.25 (Biomass) + 0.95, r 2 = 0.43; clonal C μ = 0.14 (Biomass) + 0.09, r 2 = 0.56. (D) Available translocated carbon in excess of that utilized for animal respiration (C avail ): solitary C avail = -7.78 (Biomass) + 21.8, r 2 = 0.63; clonal C avail = 12.2 (Biomass) - 18.5, r 2 = 0.64. Clonal and solitary sample sizes were 8 and 20, respectively. 33
34
These same parameters normalized to total chlorophyll are also presented (Fig. 8). Solitary and clonal P zx net and C t (Figs. 8 A, B) showed a significant correlation with total chlorophyll, but were not significantly different from each other (ANCOVA, p>0.05), therefore the linear regressions were calculated from the combined data of both morphs. The C μ and C avail from solitary anemones were significantly higher than clonal anemones (ANCOVA, p<0.05). The zooxanthellae carbon contribution toward animal respiration (CZAR) exhibited wide ranging values (7.9% to 244.3% for clonal; -363.5% to 201.6% for solitary). Mean CZAR (± SD, n) for solitary and clonal anemones was 63.47% (± 120.9, 20) and 105.6% (± 84.92, 8) respectively. When data from both morphs were combined, CZAR was 75.5% (± 111.9, 28). However, CZAR of clonal anemones showed a direct correlation with anemone biomass (Fig. 9A; linear regression, p<0.01), and total chlorophyll (Fig. 9B; linear regression, p<0.01). In contrast, CZAR of solitary anemones showed no correlation with anemone biomass or total chlorophyll. First-approximation carbon budgets for symbiotic Entacmaea quadricolor are given as flow diagrams (Fig. 10) using estimates of average daily fluxes of carbon. The carbon budgets are presented for both small and large anemones of each morph because there were significant size-dependent changes in all parameters (except solitary CZAR). Anemones were determined to be either small or large by using median biomass as the dividing point. As a result both small and large clonal carbon budgets (Figs. 10A, B) are calculated means (± SD) of 4 anemones, and both small and large solitary carbon budgets (Figs. 10C-D) are calculated means (± SD) of 10 anemones. 35
Figure 8. The daily carbon budget parameters of Entacmaea quadricolor as a function of total chlorophyll and morph. Best-fit curves for the relationship between carbon budget parameter and total chlorophyll are linear regression. Asterisk following r 2 value indicates a linear regression slope significantly different from zero (p<0.05). (A) Net photosynthesis (P zx net): P zx net = 3.60 (Chlorophyll) - 22.8, r 2 = 0.78. (B) Potentially translocated carbon (C t ): C t = 3.52 (Chlorophyll) - 23.5, r 2 = 0.77. (C) Algal carbon-specific growth rate (C μ ): solitary C μ = 0.1 (Chlorophyll) + 0.86, r 2 = 0.44; clonal C μ = 0.03 (Chlorophyll) + 0.21, r 2 = 0.53. (D) Available translocated carbon in excess of that utilized for animal respiration (C avail ): solitary C avail = -1.33 (Chlorophyll) + 1.65, r 2 = 0.12; clonal C avail = 3.30 (Biomass) - 15.4, r 2 = 0.94. Clonal and solitary sample sizes were 8 and 20, respectively. 36
37
Figure 9. The percent contribution of algal translocated carbon to daily animal respiratory requirements (CZAR) as a function of anemone biomass, total chlorophyll (Chl a+c ), and morph. Best-fit curves for the relationship between CZAR, anemone biomass, and Chl a+b are linear regression. (A) solitary CZAR = 3.05 (Biomass) + 48.8, r 2 = 0.01; clonal CZAR = 26.5 (Biomass) + 20.9, r 2 = 0.73. (B) solitary CZAR = 3.00 (Chl a+b ) + 24.4, r 2 = 0.07; clonal CZAR = 6.63 (Chl a+b ) + 33.2, r 2 = 0.93. Asterisk indicates linear regression slopes significantly different from zero, (p<0.01). Clonal and solitary sample sizes were 8 and 20, respectively. 38
A CZAR (%) (mg C day -1 ) 200 100 0-100 -200-300 -400 solitary, r 2 = 0.01 clonal, r 2 = 0.73* 0 2 4 6 8 10 12 14 16 18 20 Anemone Biomass (g protein) B Clonal CZAR (%) (mg C day -1 ) 250 200 150 100 50 solitary, r 2 = 0.07 clonal, r 2 = 0.93* 200 100-100 -200-300 0-400 0 5 10 15 20 25 30 35 40 45 Total Chlorophyll (mg a + c) 0 Solitary CZAR (%) (mg C day -1 ) 39
Figure 10. Distribution of carbon ( ±SD) within a small (A) and large (B) clonal anemone and a small (C) and large (D) solitary anemone. All values are in mg C d -1 except for the combined algal and animal biomass (B an ) which is in g protein anemone -1 and total chlorophyll (Chl a+c ) which is in mg a+c. P g and P n : gross and net photosynthesis, respectively; R zx and R al : algal and animal respiration, respectively; C μ : carbon specific growth rate; C t : carbon translocated to the host; μ x : expelled algae; μ af : algae ingested by anemonefish; C avail : carbon available to the animal for growth, storage products, mucus production, and reproduction; CBAG: carbon back-translocated from the animal to the algae; C h : carbon from heterotrophy; C af : carbon from anemonefish. Sample sizes for small and large clonal anemones were 4 each, while sample sizes for small and large solitary anemones were 10 each. 40
41
Anemone Respiration Respiration rates normalized to anemone protein of clonal anemones were not significantly different from solitary rates (t-test, p>0.05) and therefore were combined (Fig. 11). There was a decided two-phase exponential-decay relationship between respiration and anemone size, with protein-specific respiration of smaller anemones being higher than for larger anemones. 42
Figure 11. Respiration rate (R an ) of Entacmaea quadricolor normalized to total anemone protein as a function of anemone biomass and morph. There was no significant difference between clonal and solitary respiration rates (ANCOVA, p<0.05), therefore the best-fit curve (two-phase exponential-decay, Resp. = 92.3 (0.01 biomass) + 232.3 (-4.2 biomass) -65.2; r 2 = 0.82) is for solitary and clonal data combined. Clonal and solitary sample sizes were 8 and 20, respectively. 43
Clonal Solitary Anemone Respiration Rate (μ g O 2 d -1 mg -1 protein) 200 150 100 50 0 r 2 = 0.82 0 2 4 6 8 10 12 14 16 18 20 Anemone Biomass (g protein) 44
DISCUSSION The ecophysiological strategy of Entacmaea quadricolor involves a trophic plasticity suggesting E. quadricolor s trophic strategy is highly flexible, with the ability to change strategies depending upon anemone size, algal density, chlorophyll content, algal translocation, heterotrophic contribution, and presumably algal diversity. With the appropriate combination of algal densities, light, and prey availability, these individuals presumably utilize excess algal derived carbon when available. The wide ranging algal densities, photosynthetic rates, metabolic rates, and resulting carbon budget values of solitary and clonal E. quadricolor indicate this. Furthermore, the carbon budgets of solitary and clonal E. quadricolor indicate these anemone morphs use entirely different trophic strategies. Physical and Environmental Parameters Depth. Solitary anemones came from a variety of locations and depths within the same small lagoon, whereas clonal anemones were collected from two separate colonies at similar depths. This increased range in solitary experimental depth likely contributed to the greater variation among individuals. Light Regime. Observed differences in average daily-integrated irradiance during experiments (although minimal due to the tropical latitude of 5 00 S) could contribute to the individual photosynthetic variation. However, irradiance was virtually always sufficient to achieve the algal maximum light-saturated rate of photosynthesis (P n max), reducing the likelihood of light influenced variations in the carbon budget estimates. 45
Algal Parameters Diameters. The bimodal distribution of algal diameters (Fig. 2), and the difference in mitotic index between small and large cells (Table 4) suggest the anemones in this study host more than one zooxanthellae species. Muller-Parker et al. (1996) reports no significant differences in mean cell diameter of zooxanthellae between well fed and several starved groups of the anemone Aiptasia pallida. Therefore it is unlikely that the variability in algal diameters from Entacmaea quadricolor is due to differences in nutrient availability. Wilkerson et al. (1988) report some evidence of bimodality in zooxanthellae cell size frequency distribution from MI and zooxanthellae size experiments in some scleractinian coral species. The zooxanthellae cell diameters reported by Wilkerson et al. (1988) ranged from 4.6 μm in Madracis mirabilis to 19 μm in Porites astreoides. Interspecific zooxanthellae diameters showed almost a two-fold difference with means ranging from 6.4 to 12.6 μm. In this study, the cell diameters of zooxanthellae from E. quadricolor ranged in size from 2.7 μm to 13.6 μm. If E. quadricolor has but one species of symbiont, it exhibits the greatest reported zooxanthellae diameter range of any cnidarian. Algal Diversity. The symbiotic dinoflagellates associated with the anemones of this study most likely belong to the genus Symbiodinium (Trench, 1993). Species diversity among these microalgae can be recognized in some instances by restriction fragment length polymorphism (RFLP) analysis in small ribosomal subunit RNA genes (Rowan & Powers 1991a; 1991b). In some corals, a single colony may be simultaneously associated with multiple species (three distantly related taxa) of zooxanthellae (Rowan & Knowlton 1995). Rowan et al. (1997) found dynamic multi- 46
species communities of Symbiodinium within the Caribbean corals Montastraea annularis and M. faveolata. For Entacmaea quadricolor diverse zooxanthellae populations seems equally likely, and this possible increased variation in algal dynamics (i.e. photosynthetate translocation) could effect the resulting carbon budgets. A possible additional variable might be depth-related differences in zooxanthellae species zonation. Recent studies (Rowan & Knowlton 1995 and Rowan et al. 1997) on zooxanthellae using RFLP provides evidence for depth-dependent zooxanthellae species zonation. Rowan et al. (1997) also report that the composition of communities of Symbiodinium follow gradients of environmental irradiance. The scope of the present study did not permit analysis for different species of zooxanthellae, but it is evident that intraspecific variation in carbon budget parameters for Entacmaea quadricolor resulting from multiple zooxanthellae species is a reasonable possibility and should be investigated. Chlorophyll. Clonal anemones had significantly more total chlorophyll (chl a + c) than solitary anemones, while both solitary and clonal algal chlorophyll-a and chlorophyll-c content (Table 3) was higher than for most reported algal-invertebrate associations (Rees, 1991). Even though clonal anemones experienced higher average irradiance, it is unlikely any photo-acclimation took place (i.e. short or long term increases in portions of the photosynthetic machinery and various Chl-protein complexes, as discussed in Trench 1993). The higher chlorophyll-a and chlorophyll-c observed in clonal anemones likely contributed to the observed higher clonal photosynthetic efficiencies (Table 5). The 47
significant variability in chlorophyll content among these anemones is surprising, especially for the clonal anemones which were sampled relatively close to each other. Density. Anemones with highly variable zooxanthellae densities were commonly found in the field, which surprisingly conflicted with the impressively constant and fairly predictable population densities ranges for zooxanthellae associations of 0.6 to 8.5 10 6 discussed in Muscatine et al. (1985). In one clonal collecting area (containing 50+ individuals), the anemones (previously dark brown with zooxanthellae) were found to appear all white, apparently having released their algae sometime within the 4-week period since the previous collection at this site. These observations of large fluctuations occurring within relatively short periods provide further evidence of the dynamic nature of algal densities in these anemones. On several occasions aposymbiotic or white anemones without zooxanthellae were observed in the field. Aposymbiotic Entacmaea quadricolor has also been observed at multiple locations off Silliman University Marine Lab in the Sulu Sea, Philippines (pers. obs.). Zooxanthellae bleaching in E. quadricolor appear to be a basic physiological attribute as discussed in Buddemeier and Fautin (1993), both in a response to a variety of stresses and in the absence of obvious stress. Population density of zooxanthellae is controlled by systematic nitrogen limitation of algal growth which (when coupled with excess photosynthetic capacity and host factors that promote the leakage of low-molecular-weight products from the algae) subsequently keep zooxanthellae far from balanced growth and simultaneously ensures a supply of photosynthetically derived carbon from the host metabolism (c.f. Falkowski et al 1993). Since zooxanthellate associations are essentially closed systems with regard to dissolved inorganic nitrogen (Lewis 1989; c.f. Falkowski et al 1993) variable 48
zooxanthellae densities in Entacmaea quadricolor may be the result of increased nutrient concentrations (including inorganic nitrogen) from the environment. Studies on the effect of external nutrient resources on zooxanthellae population dynamics and photosynthetic efficiency have shown that cnidarians supplemented with nitrogen and ammonium support greater zooxanthellae densities (Muscatine et al. 1989; Muller-Parker et al. 1994a; Muller-Parker et al. 1994b; Cook et al. 1994). Biomass: Nitrogen, Carbon, and Protein. The nitrogen content of zooxanthellae from solitary Entacmaea quadricolor (11.2 pg N cell -1 ) was similar to values reported for field collected Aiptasia pallida (10.7 pg N cell -1 ) (Cook et al. 1988). In laboratory studies using A. pallida, C:N ratios increased for starved anemones (Cook et al. 1988). The wide carbon content range (and therefore wide C:N ratio range) for zooxanthellae from E. quadricolor (10.5-193.3 pg C cell -1 ) may indicate varied feeding strategies for E. quadricolor. If so, anemones showing low C:N ratios presumably have recently fed, while those with high C:N ratios may have experienced significant time since last feeding. The mean zooxanthellae protein content of 70.0 pg cell -1 was lower than that reported for Cassiopea xamachana (95.2 pg cell -1, Verde & McCloskey 1998). The reported carbon and nitrogen values were obtained using empirical C:N measurements of isolated algae. It is possible to derive zooxanthellae biomass using the Strathmann (1967) equation for dinoflagellates; however, the errors introduced by this method (see Davy et al. 1996) make C:N the preferred approach. Since C:N analyses was performed on algae from solitary anemones, C:N data from algae in clonal anemones is not known. Values from algae from solitary anemones were used for all carbon budget 49
estimates, recognizing that these numbers may be refined when the appropriate data become available. Diel Mitotic Activity. Like the corals Seriatopora hystrix (Hoegh-Guldberg & Smith 1989), Stylophora pistillata, Fungia repanda, and Pocillopora damiconis (Smith & Hoegh-Guldberg 1987), the jellyfish Mastigias sp. (Wilkerson et al. 1983) and Cassiopea xamachana (Verde & McCloskey 1998), Entacmaea quadricolor exhibit phased symbiont division. Zooxanthellae in the hydroid Myrionema ambionense exhibit high rates of phased division that apparently correlate with feeding, addition of dissolved inorganic nutrients, and nutrient fluxes (Cook & Fitt 1989; Fitt & Cook 1989). Fitt & Cook (1989) also report a peak in MI 34 hours following feeding in the marine hydroid M. ambionense. The phased, diel cell division profile of Mastigias sp. is also attributed to the algal symbionts being exposed to a pulse of ammonium during the night (Wilkerson et al. 1983). Mastigias sp. exhibits a peak at 0445 hours (Wilkerson et al. 1983), approximately 10 hours after the jellyfish s first visit to the ammonium rich chemocline. Spotte (1996) has found that the spotted anemone shrimp Periclimenes yucatanicus (symbiotic with Condylactis gigantea) excretes ammonia at 0.0393 μ mol total NH 4 -N (g of shrimp min -1 ) which enriches the nitrogen concentration among the anemone s tentacles. Consequently, the division peak for zooxanthellae at 0200 hours may be heavily influenced by an increased input of anemonefish-derived dissolved organic and inorganic nutrients. This flux would most likely occur during the presence of the anemonefish in the anemone s oral cavity, where the fish hides during the night (pers. obs.). 50
Photosynthesis. Even though the algal density of solitary anemones was greater (Fig. 4A & 4B), photosynthesis of the clonal anemones was always significantly higher. Higher α, I k, and P n max (Table 6) in clonal Entacmaea quadricolor suggest these anemones have adapted for light capture over a wide range of light regimes, whereas the P-I parameters of solitary E. quadricolor are more characteristic of low light adapted organisms. This is not surprising considering solitary anemones generally occur at greater depths than clonal anemones (Dunn, 1981) and usually are attached deep within the shaded interstices of the reef. Carbon Budgets The large standard deviations (Figs. 10A-D) and inclusion of apparent outliers (Fig. 9A) in the carbon budget data set may raise concern about the integrity of the data. However there is ample evidence that both data and procedures are accurate. One indication of data integrity is the inherent internal consistency of the data. All carbon budget experiments were forty-eight hours long, while only twenty-four hours of data is reported. It is of key importance that data from the first and second twenty-four hour periods are inherently consistent and these two data separate sets are not statistically different. The only element that changed in the method of collecting these two consecutive data sets was the presence or absence of the anemonefish. A second possible concern is the inability to directly measure daytime respiration, however Verde and McCloskey (1996a) showed that calculations or interpretations of carbon budgets for Anthopleura elegantissima were not compromised and that nighttime respiration adequately reflected daytime respiration. A third point of consideration is the likelihood of any suspect data resulting from a mechanical failure or miscalibration of the 51
respirometery equipment. Since two separate organisms were simultaneously undergoing respirometery experiments, any problems with the equipment would likely yield two suspect data sets with outlying carbon budget parameters and not only one. A final factor contributing to greater standard deviations for the carbon budget parameters is that the mean of calculated numbers from multiple organisms are used, therefore increasing the variance of the final values presented in figures 10A-D. In figures 10A-D, P n, C t, and R zx are influenced by several factors, including water temperature, light intensity, and daylength. Clonal anemones experienced significantly higher average daily-integrated irradiance; however, no significant difference in water temperature or daylength was observed. Clonal P n, C t, and R zx are possibly influenced by this higher irradiance regime. Verde & McCloskey (1998) discuss the possibility of jellyfish produced mucus and released DOM as significant carbon sinks. Carbon lost by these methods was not quantified in this study. Entacmaea quadricolor produces a significant amount of mucus (pers. obs.) and carbon lost by this form likely has a significant effect on the carbon budget from these organisms. Heterotrophic contribution (C h ; Figure 10) through tentacular capture of zooplankton was also not measured but is expected to be in excess than that previously observed (1.82 mg C d -1 ; McCloskey et al. 1994). Assuming algal to anemone photosynthetate translocation is occurring, support for heterotrophic contribution can be taken from the daily photosynthesis:respiration (P:R) ratios. Like the P:R ratios (<1.0 for both) reported for the temperate symbiotic anemones Cereus pedunculatus and Anthopleura ballii (Davy et al. 1996), the P:R ratio for Entacmaea quadricolor was less 52