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
haddoni
mertensii
Family Thessianthidea
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
β
1-β
μaf
μzx
Caf
Ch
Cμ
Ct
CBAG
CZAR
DOM
Dt
f
fo
Fo
Ik
Iopt
MI
P
Pcg
Pmax
Pnomax
Pczx net
PQzx
R
roal
Rcal
roan
Roan
rozx
Rczx
RQ
RQal
RQan
RQzx
SS
td
t
Measure of photosynthetic efficiency
Animal:intact anemone biomass ratio
Algal:intact anemone biomass ratio
Algae ingested by anemonefish
Algal population growth
Carbon from anemonefish
Carbon from heterotrophy
Carbon specific growth rate
Carbon available for translocation to the host (equation 10)
Carbon translocated from animal back to algae
Percent contribution of algal translocated carbon to daily animal respiratory
requirements
Dissolved organic matter
Algal doubling time
Average fraction of zooxanthellae cells undergoing non-phased division
Hourly net oxygen flux
Net oxygen flux by the anemone (all respiration values are considered to have a
positive sign. Fo is typically positive over normal diel cycle. However f o may be
negative at night or positive during the day (Figure 1)
Intersection of α and Pnomax tangents
Optimum irradiance during Pnomax
Mitotic index
Photosynthesis
Daily gross photosynthesis (in carbon equivalents)
Maximum (light-saturated) rate of photosynthesis seen in a P-I curve
Maximum net photosynthesis
Net daily carbon fixation by the zooxanthellae
Photosynthetic quotient for zooxanthellae, the molar ratio of O2 produced to CO2 fixed
Daily respiration, expressed in units of energy or carbon
Hourly respiration of the animal fraction per unit protein biomass (equation 6)
Daily respiration by the animal (daily animal respiratory requirement)
Hourly respiration of a symbiotic anemone (intact association; ral + rzx) per unit protein
biomass (equation 6)
Daily respiration by intact anemone
Hourly respiration of the algal fraction (zooxanthellae) per unit protein biomass
(equation 6)
Daily carbon respiration by zooxanthellae
Respiratory quotient, the molar ratio of CO2 produced to O2 consumed
Respiratory quotient for only the animal portion of the anemone
Respiratory quotient for the intact anemone
Respiratory quotient for zooxanthellae
Standing Stock (total number of algae per anemone)
Duration of cytokinesis
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 (μzx) per day:
μzx =ln[(1 + fmax)(1 + fmin)-1]
(1)
where fmax and fmin are the maximum and minimum daily fraction of dividing cells over a
24 hour period. The carbon-specific growth rate (Cμ in μg C • day-1) of the algal
population:
Cμ = [(SS)(C • cell-1)(μzx )]
(2)
and algal doubling time (Dt, in days) were calculated using the equations of Wilkerson et
al. (1983):
Dt = [(ln 2)(μzx)-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 (Fo, mg O2 • 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 ddH2O 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 O2 flux (fo) versus time of
10
day, derived from continuous pO2 recordings. The daytime fluxes of photosynthetically
produced oxygen, summed with anemone respiration rate values obtained at night,
provided daily gross photosynthesis measures (Pog) (McCloskey et al. 1978; Muscatine et
al. 1981; McCloskey et al. 1994; Verde and McCloskey 1996b). Net photosynthesis
(Pozxnet) was calculated by subtracting algal respiration from Pog.
P-I analysis consisted of plotting hourly net photosynthesis normalized to algal
density (μg O2 • 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 (Iopt), maximum net photosynthesis
(Pnomax), and the intersection of α and Pnomax tangents (Ik) determined. Nighttime
oxygen flux measurements provided average hourly respiration rates (roan) of the whole
anemone where
roan = rozx + roal
(6)
which when extrapolated to 24 h, provided an estimate of the total daily anemone
respiration (Roan). Respiration of the algae (Rozx) and animal (Roal) 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 O2 flux for Entacmaea quadricolor, along
with the average daily integrated irradiance regime.
12
0.8
f o (mg O2 • min-1)
0.6
0.5
0.4
irradiance
0.3
0.2
fo
0.1
-0.0
-0.1
-0.2
0
2
4
6
8
10
12
14
16
18
Experiment running time (h)
13
20
22
average integrated irradiance
(104 E • m -2 • min-1)
0.7
14
12
10
8
6
4
2
0
-2
-4
-6
-8
-10
-12
-14
24
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:
mg C assimilated by zooxanthellae photosynthesis
= (mg O2 produced • 0.375)(PQzx) -1
(7a)
mg C lost by anemone respiration
= (mg O2 consumed • 0.375)(RQan).
(7b)
and
The percent contribution of algal carbon to animal respiration (CZAR) was
determined by the formula of McCloskey et al. (1994):
CZAR ={[(0.375•Pog)(PQzx)-1]-[(1-β)(0.375•Roan)(RQan)]-[Cμ] / [β)(0.375•Roan)(RQan)]} •100
(8)
where the photosynthetic quotient for the zooxanthellae (PQzx) is the ratio of algal O2
produced to CO2 fixed. The respiratory quotient for the anemone (RQan) is the ratio of CO2
produced to O2 consumed by the whole anemone where
RQan = [((1-β)(RQzx)-1)+((β)(RQal)-1)]-1.
(9)
The respiratory quotient of the animal (RQal) is the ratio of CO2 produced to O2 consumed
by only the animal portion of the anemone, whereas the respiratory quotient of the
zooxanthellae (RQzx) is the ratio of CO2 produced to O2 consumed by only the algal portion
of the association. Values of 1.1, 0.9, and 1.0 were used for PQzx , RQal , and RQzx
respectively (cf. Muscatine et al.1981; Porter et al. 1984; and Kremer et al. 1990).
14
The carbon available for translocation to the host (Ct) is:
Ct = [(0.375 · Pog)(PQzx)-1]-[(1-β)(0.375 · Roan)(RQan)]-[Cμ]
(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 μm3 ± 238.7, 743 and 261.5 μm3 ± 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
solitary
n=8
± SD
n = 20
± SD
clonal : solitary
Significance
Cell diameter (μm)
7.21 ± 2.4
7.08 ± 2.45
1.02
n. s.
Cell volume (μm3)
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
300
Number of cells
250
225
200
175
150
5.2 ± 1.2, 578
9.5 ± 1.2, 465
Median (7.3 μ m)
275
125
100
75
50
25
0
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.
MI (%)
large cells
small cells
Algal cell
diameter
Tentacles
Oral Disk
& Gullet
Column
Wall
Mesentary
Foot
0.34
0.00
0.00
0.66
0.60
0.00
1.21
0.44
0.00
0.00
11.8 ± 3.0
6.8 ± 2.0
10.7 ± 1.7
9.4 ± 3.6
9.5 ± 3.6
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
( μm ± SD)
% of total
% large cells
% small cells
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;
r2= 0.49, Chl(a+c) = 1.99 (Biomass) + 3.77. Density; for solitary r2= 0.44,
Chl(a+c) = 0.029 (Density) + 1.49, for clonal r2= 0.53, Chl(a+c) = 0.007
(Density) + 3.48.
22
A
45
r2 = 0.49*
Total Chlorophyll
(mg a + c)
40
35
30
25
20
15
10
solitary
clonal
5
0
0
2
4
6
8
10
12
14
16
18
20
Anemone Biomass
(g protein)
B
Total Chlorophyll
(mg a + c)
45
40
solitary, r2 = 0.44*
35
clonal, r2 = 0.53*
30
25
20
15
10
5
0
0
500
1000
1500
2000
2500
Algal Cell Density
(106 cells)
23
3000
3500
Total chlorophyll and algal cell density (106 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 fmax and fmin were 2.31% and
0.31%, respectively. The algal-specific growth rate (μzx) was 0.0197 d-1 and the resultant
algal doubling time (Dt) was 35.1 days.
Photosynthesis. Clonal and solitary daily gross photosynthesis (Pg) 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
106 cells • mg-1 protein
0.7
0.36
*
0.6
0.5
0.4
0.3
0.12
0.2
0.1
0.0
Solitary
Clonal
Anemone morph
B
6
Algal Density
(10 cells • mg -1 protein)
1.00
solitary, r 2 = 0.13
clonal, r 2 = 0.10
0.75
0.50
0.25
0.00
0
2
4
6
8
10
12
Anemone Biomass
(g protein)
26
14
16
18
20
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 Pg is directly related to total chlorophyll (Fig. 6A). Pg 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 Pg, when normalized to total chlorophyll,
were not significantly different (ANCOVA, p>0.05). However the Pg 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, Ik, and Pnmax. In contrast, both solitary and
clonal Iopt were not significantly different.
Carbon Budgets
Solitary and clonal daily net photosynthesis (Pzxnet), carbon-specific growth rate
(Cμ), translocated carbon (Ct), and carbon available for host growth (Cavail) are presented
as a function of anemone biomass in Figure 7. Solitary and clonal Pzxnet and Ct (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 Cμ (Fig. 7C) was
significantly higher than clonal Cμ (ANCOVA, p<0.05). Solitary and clonal Cavail (Fig.
7D) were significantly different (ANCOVA, p<0.05) with clonal Cavail increasing with
greater anemone biomass while solitary anemones had a negative slope, therefore
showing the opposite relationship.
29
Figure 6. Daily gross photosynthesis (Pg) of zooxanthellae in Entacmaea quadricolor
as a function of total chlorophyll (Chla+c). Best-fit curves for the
relationship between Pg and Chla+c are linear regression. (A) Pg = 4.6
(Chla+c) -6.3, r2 = 0.78. (B) normalized to algal chlorophyll: solitary Pg =
0.07 (Chla+c) + 3.51, r2 = 0.00; clonal Pg = 0.01 (Chla+c) + 3.51, r2 = 0.61.
(C) normalized to algal protein: solitary Pg = 0.02 (Chla+c) - 11.5, r2 =
0.82; clonal Pg = 0.01 (Chla+c) - 4.16, r2 = 0.41. (D) normalized to algal
density: solitary Pg = 0.002 (Chla+c) - 10.8, r2 = 0.43; clonal Pg = 0.008
(Chla+c) - 4.92, r2 = 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-Ia
clonal
n=8
± SD
solitary
n = 17
± SD
clonal: solitary
Significance
α
0.202 ± 0.198
0.066 ± 0.044
3.1
*
Ik
595.1 ± 251.9
301.9 ± 158.2
2.0
**
Iopt
1968 ± 147.1
1544 ± 58.5
1.3
n. s.
Pnmax
140.3 ± 211.6
17.31 ± 10.66
8.1
***
Significance of differences was determined by the two-sample t-test or MannWhitney 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 O2· h-1· 10-6 cells)
(μE· m-2· s-1)-1]
Ik is the intersection of α and Pnomax tangents (μE· m-2· s-1)
Iopt is the optimum irradiance during Pnomax (μE· m-2· s-1)
Pnomax is the maximum net photosynthesis (μg O2· 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 r2 value indicates a linear regression slope
significantly different from zero (p<0.05). (A) Net photosynthesis
(Pzxnet): Pzxnet = 10.8 (Biomass) + 3.94, r2= 0.53. (B) Potentially
translocated carbon (Ct): Ct = 10.5 (Biomass) + 3.39, r2= 0.52. (C) Algal
carbon-specific growth rate (Cμ): solitary Cμ = 0.25 (Biomass) + 0.95, r2=
0.43; clonal Cμ = 0.14 (Biomass) + 0.09, r2= 0.56. (D) Available
translocated carbon in excess of that utilized for animal respiration
(Cavail): solitary Cavail = -7.78 (Biomass) + 21.8, r2= 0.63; clonal Cavail =
12.2 (Biomass) - 18.5, r2= 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 Pzxnet and Ct (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 Cμ and Cavail 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 r2 value indicates a linear regression
slope significantly different from zero (p<0.05). (A) Net photosynthesis
(Pzxnet): Pzxnet = 3.60 (Chlorophyll) - 22.8, r2= 0.78. (B) Potentially
translocated carbon (Ct): Ct = 3.52 (Chlorophyll) - 23.5, r2= 0.77. (C)
Algal carbon-specific growth rate (Cμ): solitary Cμ = 0.1 (Chlorophyll) +
0.86, r2= 0.44; clonal Cμ = 0.03 (Chlorophyll) + 0.21, r2= 0.53. (D)
Available translocated carbon in excess of that utilized for animal
respiration (Cavail): solitary Cavail = -1.33 (Chlorophyll) + 1.65, r2= 0.12;
clonal Cavail = 3.30 (Biomass) - 15.4, r2= 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 (Chla+c), and morph. Best-fit curves for the relationship
between CZAR, anemone biomass, and Chla+b are linear regression. (A)
solitary CZAR = 3.05 (Biomass) + 48.8, r2 = 0.01; clonal CZAR = 26.5
(Biomass) + 20.9, r2 = 0.73. (B) solitary CZAR = 3.00 (Chla+b) + 24.4, r2 =
0.07; clonal CZAR = 6.63 (Chla+b) + 33.2, r2 = 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
solitary, r 2 = 0.01
-300
clonal, r 2 = 0.73*
-400
0
2
4
6
8
10 12 14 16 18 20
Anemone Biomass
(g protein)
B
250
200
100
150
0
-100
100
solitary, r2 = 0.07
50
clonal, r2 = 0.93*
0
0
5
10
15
20
25
30
Total Chlorophyll
(mg a + c)
39
35
40
-200
-300
-400
45
Solitary CZAR (%)
(mg C • day-1)
Clonal CZAR (%)
(mg C • day-1)
200
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
(Ban) which is in g protein · anemone-1 and total chlorophyll (Chla+c)
which is in mg a+c. Pg and Pn: gross and net photosynthesis,
respectively; Rzx and Ral: algal and animal respiration, respectively; Cμ:
carbon specific growth rate; Ct: carbon translocated to the host; μx:
expelled algae; μaf: algae ingested by anemonefish; Cavail: carbon
available to the animal for growth, storage products, mucus production,
and reproduction; CBAG: carbon back-translocated from the animal to
the algae; Ch: carbon from heterotrophy; Caf: 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 (Ran) 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; r2 =
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 O2 d-1 mg-1 protein)
200
r 2 = 0.82
150
100
50
0
0
2
4
6
8
10
12
Anemone Biomass
(g protein)
44
14
16
18
20
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
(Pnmax), 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 multi46
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 · 106 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 NH4-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, Ik, and Pnmax (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, Pn, Ct, and Rzx 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 Pn, Ct, and Rzx 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 (Ch; 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
than 1.0, which suggests that even under well-illuminated conditions, a heterotrophic
source of carbon is required for E. quadricolor to survive. Even though the solitary and
clonal P:R ratios were not statistically different, notably the ratio ( ± SD, n) for clonal
anemones of 1.04 ± 0.83, 8 was higher than that for solitary anemones (0.63 ± 1.20, 20),
suggesting that solitary anemones are more dependent upon heterotrophic carbon.
CZAR. The large variation in CZAR from Entacmaea quadricolor (especially
the solitary morph) has never before been observed from single taxa. This increased
variability from solitary anemones support the findings of Shick and Dowse (1985)
where variance in several physiological measures (including aquatic oxygen
consumption) were significantly less in monoclonal groups of anemones than in
genotypically diverse conspecifics or closely related species.
As Figure 9A illustrates, Entacmaea quadricolor does not support the large
polyp, lower CZAR hypothesis. Surprisingly clonal E. quadricolor shows an exact
opposite trend, while solitary anemones seem to have the capacity to utilize algal derived
carbon regardless of size. As a clonal anemone increases in size, I speculate the relative
concentration of anemonefish fecal organic carbon and nitrogenous waste products
decreases, thereby possibly resulting in an increased dependence by the anemone on
CZAR, or a decrease in the host’s metabolism. If so, this selective pressure on clonal
anemones to remain small may explain the observed smaller size of clonal anemones
compared to solitary morphs. Solitary anemones seem to observe the description of
allometric growth described by Sebens (1981) where feeding surfaces are amplified to
compensate for a decreasing surface-to-volume ratio. Solitary anemones are unique in
that they have the capacity to utilize algal produced carbon, but do not seem to be
53
trophically limited to this ecophysiological strategy. Conversely if an entire clone is
considered energetically, clonal aggregations also increase their feeding-surface-tobiomass ratios (Sebens 1981), but in the case of clonal E. quadricolor the larger a single
individual becomes, the greater its trophic dependence is upon algal derived carbon.
CZAR for Entacmaea quadricolor, however, is more consistent with the “junk
food” hypothesis of Falkowski et al. (1984), where the zooxanthellae symbionts can
provide a rich carbon source for the anemone host. Falkowski et al. (1993) hypothesize
this “junk food” as important because although it is nitrogen poor (Davies 1984), it may
supply some essential amino acids to the host animal. The positive selective pressure for
the presence of zooxanthellae due to the possible translocation of essential amino acids
may be one reason that natural populations of E. quadricolor do not remain aposymbiotic
for extended periods of time (pers.obs.).
Entacmaea quadricolor likely has a higher metabolism when small (concurrent
with greater relative concentrations of anemonefish contributed fecal organic carbon and
nitrogen, and algal contributed photosynthetate). If so, small (young) E. quadricolor
could grow quickly until the compensation point of Cavail from these sources is reached
and significant anemone growth ceases. In contrast, very large anemones would then
likely have a slower metabolism, slow growth rate, and achieve long lifespans.
Clonal and solitary Entacmaea quadricolor do not closely model other symbiotic
cnidarians from oligotrophic tropical waters, which are generally thought to be more
dependent upon their algae as a carbon source and, consequently to exhibit higher
CZARs than symbiotic cnidarians from eutrophic waters (McCloskey et al. 1994).
CZAR values in excess of 100% have been measured in Anemonia viridis (Tytler &
54
Davies 1986), several species of tropical scleratinian and soft corals (Schlichter et al.
1983; Muscatine et al. 1984; Davies 1984; Edmunds & Davies 1986, 1989; Davies
1991), the tropical sea anemone Bunodeopsis antilliensis (Day 1994), and the tropical
jellyfish Mastigias sp. (McCloskey et al. 1994) and Linuche unguiculata (Kremer et al.
1990) (reviewed by Davy et al. 1996). Verde & McCloskey (1998) also report CZAR
values in excess of 100% from the jellyfish Cassiopea xamachana. Two tropical
symbiotic cnidarians that have CZARs less than 100% are Zoanthus sociatus and
Palythoa variabilis (Steen and Muscatine 1984).
Based on the results of this study, I hypothesize that the ecophysiological strategy
of Entacmaea quadricolor involves a variable trophic plasticity approach to maintaining
themselves in the oligotrophic waters of the tropics. The wide ranging photosynthetic
rates, metabolic rates, and resulting carbon budget values of solitary and clonal E.
quadricolor indicate that these anemones have the capacity to obtain carbon using an
equally wide spectrum of trophic strategies. With the appropriate combination of light,
these individuals presumably utilize excess algal derived carbon when available. The
data suggests E. quadricolor’s trophic strategy is highly flexible, with the ability to
change strategies depending upon algal density, chlorophyll content, algal translocation,
heterotrophic contribution, and presumably algal diversity. This unique adaptation seems
fitting for organisms which are both solitary and clonal, presumably achieve long
lifespans, and have the ability to associate with both zooxanthellae and anemonefish
simultaneously.
55
ACKNOWLEDGEMENTS
I wish to extend my appreciation and gratitude toward the many individuals who
made this investigation possible. I thank Dr. Larry Aamodt for his invaluable time spent
updating the Respirometry programs and for providing “technical support” from half way
around the world and also the experts of WWC Technical Support Services for your work
on the Mark V Respirometer. I am indebted to the Christensen Research Institute, Dr.
Larry Orsak, Ann, Martin, John and the CRI Binnatang Lab for there camaraderie, taste
in music, and “help” with the local land owners. They made my stay in Papua New
Guinea a successful learning experience. Stephen Southern and Rose Ledinao of
Astrolabe Analytical, I appreciate the generous use of their lab and spec--the only one
found in a 500 mile radius. To my guidance committee members, Dr. Joe Galusha, and
Dr. Jim Nestler, my external observer and graduate studies representative Dr. Jon Cole,
and Dr. Scott Ligman our link to WWC while in New Guinea. My colleague Andrew
Rice provided an invaluable discussion partner, with whom many ideas first took shape,
and problems became manageable. This study would have been at a standstill without
Kevin McCloskey’s unique and efficient collecting abilities. The Habenenicht family for
home cooked meals, shelter for my fiancée and for always making my time at Rosario
exciting; even while counting algae for two months. I am grateful to my parents for
financial support, encouragement, and for giving me diverse opportunities to explore and
observe nature. My wife Jen, “When are you going to finish that thing?” for her help,
self-sacrifice, and patience. Especially to my friends and mentors Dr. Larry McCloskey,
for maintaining your commitment to me, even while working at a University 2000 miles
away and Dr. Alan Verde for your instrumental and invaluable help in all aspects of this
project. Finally, I am grateful for the funding assistance provided by the following
organizations:
Department of Biological Sciences, Walla Walla College
Project AWARE Foundation
Christensen Research Institute
Walla Walla College Marine Station
La Sierra University Department of Biology
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62
Appendix 1. Anemonefish diel respiration experiments and gut content analysis.
INTRODUCTION
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 trophic contribution of anemonefish to the
anemone host can be addressed by quantifying its nutrient contribution to the host in the
forms of nitrogenous waste and fecal organic carbon. Nutrient contribution by the
anemonefish in the form of nitrogenous waste should result in a more eutrophic
environment for the anemone and zooxanthellae, likely resulting in enhanced algal
growth. While we collected some relevant samples, this hypothesis was not fully
addressed in this thesis.
METHODS AND MATERIALS
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. Anemonefish
respiration (described in main text) was monitored simultaneously with anemone
respiration.
To observe what Amphiprion melanopus with Premnas biaculeatus were eating
and ingesting, as well as information to compare and contrast these anemonefish, the
contents of the stomach and intestine from representative fish (collected and sacrificed
for this purpose) were examined. Observations made on four areas, the gullet and
stomach, foregut, midgut, and hindgut. Fish length, gut length, and sex were also noted.
To correct values of measurements for allometric relations the intestinal weight length
(IWL) or Zihler Index is used (Zihler 1982) where
IWL=(Intestinal length)(10 · weight1/3)-1.
(1)
Other freshly collected anemonefish were also isolated in clean aquaria for 24 h
and their feces collected throughout the day and at dawn. Fecal samples were frozen in
seawater, and the feces prepared for C:N analysis by rinsing three times with distilled
water and drying at 40° C overnight.
RESULTS
Fidelity. The symbiotic fish Amphiprion melanopus was only observed with
clonal Entacmaea quadricolor, and Premnas biaculeatus was only observed with solitary
E. quadricolor.
Wet Weight. The mean (± SD) wet weights of Amphiprion melanopus (n=8) and
Premnas biaculeatus (n=19) were 9.4 ± 7.6 and 10.9 ± 8.5 grams, respectively, with no
significant difference between the mean wet weights of either species (t-test, p>0.05).
63
Length. The mean (± SD) lengths of Amphiprion melanopus (n=8) and Premnas
biaculeatus (n=19) were 53.8 ± 18.6 and 58.7 ± 19.3 millimeters, respectively, with no
significant difference between the mean lengths of either species (t-test, p>0.05).
Displacement Volume. The mean (± SD) displacement volumes of Amphiprion
melanopus (n=8) and Premnas biaculeatus (n=19) were 9.8 ± 8.3 and 13.7 ± 11.4
milliliters, respectively, with no significant difference between the mean volumes of
either species (t-test, p>0.05).
Zihler Index. The ( ± SD, n) Zihler Index for Amphiprion melanopus and
Premnas biaculeatus was 2.6 ± 0.4, 3 and 2.4 ± 0.85, 2 respectively, with no significant
difference between the Zihler Index for these two species (t-test, p>0.05).
Respiration. There was a direct relationship between anemonefish respiration
(mg O2 · day-1) and anemonefish wet weight (grams), (Fig 1A). This relationship was
also observed for anemonefish length (mm), (Fig. 1B) and displacement volume (ml),
(Fig 1C).
The ( ± SD) respiration rates of Amphiprion melanopus (n=8) and Premnas
biaculeatus (n=19) were 19.0 ± 11.9 and 21.5 ± 20.2 mg O2 • day-1 • gram-1 anemonefish,
respectively, with no significant difference between respiration rates of these two species
(t-test, p>0.05). When fish respiration (independently measured) was summed with
anemone respiration (independently measured), the sum was not significantly different (ttest, p>0.05) than measured respiration of the symbiosis (anemone + fish).
Light respiration rates (μg O2 · min-1) of Amphiprion melanopus and Premnas
biaculeatus were significantly higher than their corresponding dark respiration rates (ttest, p<0.01). There was a decided two-phase exponential-decay relationship between
anemonefish respiration rate normalized to anemonefish wet weight (μg O2 · min-1 ·
gram-1 anemonefish) as a function of anemonefish wet weight. The weight-specific
respiration of smaller fish was higher than those of larger fish (Fig. 2).
Gut Examination. Gut content analysis of the anemonefish is summarized in
Table 1. Zooxanthellae, nematocysts, and mucus were observed in all samples but one.
This particular fish happened to be associated with an azooxanthellate anemone. Other
types of food items included filamentous red algae, copepods, ostracods, amphipods, and
sponge spicules.
DISCUSSION
Zihler Index. The mean Zihler Index for Amphiprion melanopus (2.6) is similar
with the values reported for Amphiprion ocellaris (2.2), Amphiprion frenatus (1.7), and
Amphiprion sp. (2.0), (Zihler 1982). No previous sources describing the Zihler Index
from Premnas biaculeatus were found.
Fish Respiration. No sources describing respiration in other Perciformes were
found. However, the anemonefish respiration per wet weight given here was over twice
as high as that recorded for five species of freshwater fishes (Beamish 1964).
Benefits to Fish Symbionts. Zooxanthellae were present among the gut contents
of the anemonefish (Table 1), however the amount of zooxanthellae assimilated by the
fish was not quantified. As zooxanthellae pass though the gut, they begin to appear more
64
and more featureless (i.e. no chloroplasts or nuclei). Zooxanthellae found in the
anemonefish gut also seemed to be associated with amorphous mucus (most likely from
the anemone). Zooxanthellae in the hindgut appear clear, while the golden brown color
of mucus increases, possibly as pigments from the zooxanthellae are leached. It seems
plausible that both Amphiprion melanopus and Premnas biaculeatus can obtain nutrition
from ingested zooxanthellae since Augustine and Muller-Parker (1998) showed a 40%
and 60% respective reduction in chlorophyll a and c from zooxanthellae in the feces of
the mosshead sculpin (Clinocottus gobiceps). The concept of algal fixed carbon directly
contributing to the heterotrophic requirements of the anemonefish is supported and
deserves further investigation.
Benefits to Anemone and Algal Symbionts. Extrinsic organic carbon and
nitrogenous waste products from anemonefish feces is suggested to contribute to the
anemone’s carbon budget. Contribution from this source was not quantified or estimated.
However, there are documented cases of extrinsic organic carbon from the feces of
associated schooling fish transported to reef communities (Meyer & Schultz 1985a;
Meyer & Schultz 1985b; Meyer et al. 1983; Bray et al. 1981; Ogden & Ehrlich 1977),
and feeding (although infrequent) of anemones by associated fish (summarized by Fautin
1991).
Spotte (1996) has found that the spotted anemone shrimp Periclimenes
yucatanicus (symbiotic with Condylactis gigantea) excretes ammonia at 0.0393 μ mol
total NH4-N (g of shrimp · min-1) which enriches the nitrogen concentration among the
anemone’s tentacles. If the fish symbionts of Entacmaea quadricolor excrete NH4,
which is then absorbed by the host anemone, this elevated N enrichment could be what is
driving phased division in the algal symbionts. Further investigation is required to test
this hypothesis.
65
Figure 1. Anemonefish respiration from both Amphiprion melanopus and Premnas
biaculeatus as functions of anemonefish wet weight (A), length (B), and
displacement volume (C). Best-fit curve for these relationships is linear
regression; resp. = 10.3 (wet weight) + 36, resp. = 4.1 (length) - 93.4, resp.
= 7.2 (displacement volume) + 52.7. Asterisk following r2 value indicates
a linear regression slope significantly different from zero (p < 0.05).
Amphiprion melanopus and Premnas biaculeatus sample sizes were 8 and
19, respectively.
66
A
Anemonefish Respiration
(mg O2 • day-1 )
A. melanopus
P. biaculeatus
350
r2 = 0.90*
300
250
200
150
100
50
0
0
5
10
15
20
25
30
B
Anemonefish Respiration
(mg O2 • day-1 )
Anemonefish Wet Weight
(grams)
350
r2 = 0.79*
300
250
200
150
100
50
0
0
10
20
30
40
50
60
70
80
90
100 110
C
Anemonefish Respiration
(mg O2 • day-1 )
Anemonefish length
(mm)
350
r2 = 0.76*
300
250
200
150
100
50
0
0
5
10
15
20
25
30
35
40
Anemonefish Displacement Volume
(ml)
67
45
Figure 2. Light and dark respiration rates of both Amphiprion melanopus and
Premnas biaculeatus normalized to anemonefish wet weight as a function
of anemonefish wet weight. Best-fit curve for the relationship between
respiration and weight is a two-phase exponential-decay; light resp. =
44.2(-1.4 · wet weight) - 18.0(0.008 · wet weight) + 24.3, dark resp. = 51.4(-5.4 · wet weight) +
8.4(-0.15 · wet weight) + 5.2. Asterisk following r2 value indicates a non-linear
regression slope significantly different from zero (p < 0.05). Amphiprion
melanopus and Premnas biaculeatus sample sizes were 8 and 19,
respectively.
68
Anemonefish
Respiration Rate
(μg O2 min-1 g -1 )
50
45
light, r2 = 0.90*
40
dark, r2 = 0.87*
35
30
25
20
15
10
5
0
0
5
10
15
20
Anemonefish Wet Weight
(grams)
69
25
30
Table 1
Summary of gut content analysis from Premnas biaculeatus and Amphiprion
melanopus. Samples were dissected from freshly collected anemonefish.
Anemonefish
Sex
Fish
length
(cm)
Host Anemone
Total gut
Characteristics length (cm)
P. biaculeatus
F
NMa
Solitary
NMa
zxb, mucus,
copepods
P. biaculeatus
F
6.8
Solitary
4.0
zx, mucus,
copepods
P. biaculeatus
M
4.5
Solitary,
Azooxanthellate
3.0
mucus, nematocysts
arthopods,
polychaetes
A. melanopus
F
5.5
Clonal
7.5
very few zx, mucus,
arthopods, ostrocod,
algaec, sponge
spicules
A. melanopus
F
5.8
Clonal
9.0
zx, algae, arthopods,
amphipods,
copepods, sponge
spicules, diatoms
A. melanopus
M
5.6
Clonal
6.3
zx, algae, copepods,
arthopods, tentacle
tip from E.
quadricolor
a
Not Measured
refers to zooxanthellae
c
refers to filamentous red algae
b
70
Gut
Contents