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Title
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Eng
Effects of CO2-Induced Acidification on the Anemone
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Date
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2008
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Creator
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Eng
Towanda, Trisha
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Subject
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Eng
Environmental Studies
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extracted text
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Effects of CO2-induced acidification on the intertidal
sea anemone Anthopleura elegantissima (Cnidaria: Anthozoa) and
its algal symbiont Symbiodinium muscatinei (Dinomastigota: Dinophyceae)
by
Trisha Towanda
A thesis
submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
Olympia, Washington
December, 2008
ABSTRACT
Effects of CO2-induced acidification on the intertidal
sea anemone Anthopleura elegantissima (Cnidaria: Anthozoa) and
its algal symbiont Symbiodinium muscatinei (Dinomastigota: Dinophyceae)
Trisha Towanda
Oceanic absorption of anthropogenic CO2 decreases the pH of the ocean and shifts the
carbonate equilibrium of seawater, a process known as hypercapnic acidification.
Acidification can cause physiological stress and decrease the availability of carbonate
ions for the secretion of calcium carbonate; however increased CO2 may benefit some
photosynthetic organisms through increased rates of carbon fixation. This study
investigated the impact of hypercapnic acidification on the photosynthetic symbiosis of
the non-calcifying anemone, Anthopleura elegantissima (Brandt) and it’s symbiotic alga
Symbiodinium muscatinei (zooxanthella). Anemone specimens were maintained in the
laboratory for 1 week under current levels of PCO2 (369 ppmv) and pH (8.1).
Clonal pairs of these specimens were then divided into two groups; each group was
exposed to one of two hypercapnic conditions for 6 weeks: moderate (PCO2 = 450 ppmv,
pH = 8.1) or high (PCO2 = 2340 ppmv, pH = 7.3). After 6 weeks, individuals were
compared for differences in respiratory rate, photosynthetic rate, and the contribution of
zooxanthellae to the animal's respiration (CZAR). Density of algal cells in anemones, algal
cell size, mitotic index and chlorophyll content were measured to compare zooxanthellal
characteristics. After 6 weeks of exposure, A. elegantissima exhibited higher rates of
photosynthesis among anemones at higher PCO2s (moderate- 3.30, high- 4.20 µmol O2 g1 -1
h ) than in anemones at normocapnic levels (1.53 µmol O2 g-1 h-1). Respiration rates
were also higher at moderate and high PCO2 (1.34 and 1.27 µmol O2 g-1 h-1 respectively)
than at current conditions (0.94 µmol O2 g-1 h-1). Anemones at moderate PCO2 received
more of their respiratory carbon and O2 (CZAR = 137.3%) from zooxanthellae than those
at current conditions (CZAR = 66.6%) or at high PCO2 (CZAR = 78.2%). Mitotic index and
zooxanthellal cell diameter were greater among zooxanthellae in hypercapnic conditions.
The response of Anthopleura elegantissima to hypercapnic acidification reveals the
adaptability of an organism that has evolved a tolerance for high PCO2.
Table of Contents
Introduction
1
Materials & Methods
4
Experimental design
4
Collection and maintenance
4
Photosynthesis, respiration and CZAR
7
Zooxanthellal measurements
10
Statistical analyses
11
Results
11
Photosynthesis and respiration
12
Zooxanthellal characteristics
13
Discussion
15
Literature Cited
22
iv
List of Figures
Figure 1. Schematic of experimental set-up
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Figure 2. Mass-specific rates of
photosynthesis and respiration.
30
Figure 3. The ratio of photosynthesis to respiration.
31
Figure 4. The potential contribution of carbon from
zooxanthellae to the animal's respiratory
requirements (CZAR).
32
Figure 5. Mass-specific rate of photosynthesis
in relation to body wet weight.
33
Figure 6. Mass-specific rate of respiration
in relation to body wet weight.
34
Figure 7. Zooxanthellal (Zx) cell diameter
35
Figure 8. Zooxanthellal (Zx) density
36
Figure. 9. Mitotic index of zooxanthellae
37
Figure 10. Chlorophyll a concentrations of zooxanthellae
38
List of Tables
Table 1. Carbonate parameters of aquaria
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Table 2. Oxygen and carbon flux in Anthopleura elegantissima 40
Table 3. Biomass and growth parameters of
Symbiodinium muscatinei
41
Appendix
Abbreviations
42
v
Acknowledgements
I give thanks to my advisor, Dr. Erik Thuesen for his thoughtful support of this research
as well as my thesis committee members, Dr. Gerardo Chin-Leo and Dr. Frank Melzner,
for their reviews and comments. Daniel Cox provided invaluable assistance in the field
and the lab. Magda Gutowska and Charley Waters contributed stimulating feedback
during the experimental design of this project. Drs. Gerardo Chin-Leo, Clyde Barlow and
Steve Haddock graciously offered their expertise and instruments. Staff members of
Evergreen Lab Stores and the Computer Applications Lab were invaluable in the
completion of this project. My thanks also go to the Quinault Tribe for allowing
collections from Quinault Tribal Lands. Funding was provided in part by an Evergreen
Foundation Grant.
vi
Introduction
It is well established that anthropogenic emissions of CO2 are warming the
climate and decreasing the pH of the ocean (Vitousek et al. 1997, Caldeira &
Wickett 2005, Feely et al. 2004, Orr et al. 2005). For 800,000 years prior to
the industrial revolution, Earth’s atmospheric CO2 ranged from 170-300
parts per million volume (ppmv) (Siegenthaler et al. 2005. Lüthi et al. 2008).
At 386 ppmv, the present CO2 concentration is nearly 40% higher than preindustrial levels (Houghton 2003) and is expected to increase by
approximately 2 ppmv y-1 for many decades (IPCC 2007). The projected rate
of atmospheric CO2 increase is approximately 100 times faster than has
occurred in the past 650,000 years (Raven et al. 2005, Siegenthaler et al.
2005). Climate models project that this will result in CO2 concentrations
over 500 ppmv by the middle of this century, over 800 ppmv by the turn of
the next century (Feely et al. 2004), and as high as 2000 ppmv by the year
2340 (Caldeira & Wickett 2003).
The oceans have absorbed more than a third of the anthropogenic CO2
released in the last 200 years (Sabine et al. 2004). This increase in the partial
pressure of CO2 gas (PCO2) is a condition known as hypercapnia.
Hypercapnia shifts the carbonate equilibrium of seawater: [H+] and
bicarbonate ions [HCO-3] increase, pH and carbonate ions [CO-23] decrease
(Millero 2007) and hypercapnic acidification (HA) results. The current
average pH in the ocean is ~ 8.1 units, a decline of 0.1 pH units from preindustrial values (Caldeira & Wickett 2003). By the end of this century, pH
is predicted to decline by approximately 0.4 pH units (Feely et al. 2001,
Caldeira & Wickett 2005), which is equivalent to a 150% increase in H+.
Although the uptake of CO2 by the oceans has ameliorated the climatic
effects of anthropogenic CO2, HA may adversely affect many marine
1
organisms and ecosystems (Leclercq et al. 2002, Raven et al. 2005, HoeghGuldberg et al. 2007, Doney 2009).
Hypercapnic acidification can suppress marine organisms both by
inducing physiological stress and by decreasing the availability of carbonate
ions for calcification. Increasing the concentration of protons perturbs acidbase balance by interfering with diffusion processes, ion transport pumps
and intracellular buffering capacities (Walsh & Milligan 1989, Seibel &
Walsh 2003). Organisms may generate a new steady state to compensate for
short-term acidosis but long term effects can include reduced protein
synthesis (Hand 1996) and therefore reduced growth and reproduction
(Kurihara et al. 2007). Although the physiological impacts of HA have not
been well studied at ecologically relevant temporal and pH scales, HA has
been demonstrated physiologically harmful to both calcifying and noncalcifying organisms (Grainger et al. 1979, Pörtner et al. 2005, Kurihara et
al. 2007, Kurihara 2008, Waters &Thuesen, submitted).
The effects of HA on calcification processes have been more
thoroughly researched. Numerous studies have demonstrated the correlation
of biogenic calcification on the saturation state of calcium carbonate (Ω
CaCO3) (Gattuso et al. 1998, Langdon et al. 2000, Leclercq et al. 2000,
Riebesell et al. 2000, Michaelidis et al. 2005, Shirayama & Thornton 2005).
Decreasing the concentration of carbonate ions reduces the Ω CaCO3,
thereby inhibiting the ability of many calcifying organisms such as
calcareous plankton, molluscs and corals to secrete exoskeletons (Kleypas et
al. 1999, Caldeira & Wickett 2003, Orr et al. 2005, Schneider & Erez 2006,
Gazeau et al. 2007, Fabry et al. 2008). Calcification rates of hermatypic
corals and coralline algae are predicted to decrease by 10-50% over the next
2
century as Ω CaCO3 decreases (Gattuso et al. 1998, Leclercq et al. 2002,
Reynaud et al. 2003) and temperatures rise (Marubini et al. 2008).
Because calcification is intertwined with other physiological
processes (Furla et al. 2000b, Allemand et al. 2004), it is informative to
isolate physiological effects of HA on actinians and their photosynthetic
symbionts from the calcification process. The mutualist symbiosis between
actinians and photosynthetic dinoflagellates, commonly referred to as
zooxanthellae (ZX), is characterized by an exchange of nutritional and
metabolic goods. ZX produce carbohydrates and O2 through photosynthesis
that contribute to the respiration of the host; ZX receive nitrogen compounds
and CO2 from the host in exchange (Weis 1993, Furla et al. 2005). The
interactions of these energetic systems in hypercapnic conditions are
difficult to predict. . While the overall metabolism of the whole animal may
be suppressed (Seibel and Walsh, 2003), photosynthesis may be enhanced as
seen in other CO2-limited organisms such as eelgrass (Palacios &
Zimmerman 2007).
To understand how HA affects metabolic processes in an intertidal,
photosynthetic actinian, this study examined the energetic effects of
increased PCO2 (moderate- 450 ppmv and high- 2340 ppmv) on the
anemone Anthopleura elegantissima Brandt. In the eastern North Pacific
Ocean, A. elegantissima harbors the dinoflagellate Symbiodinium muscatinei
(Dinomastigota) (LaJeunesse & Trench 2000) that is congeneric with the
symbiotic dinoflagellates found in hermatypic corals. Rates of metabolism,
photosynthesis and the contribution of carbon to the needs of animal
respiration (CZAR) were compared between A. elegantissima at current,
moderate and high PCO2 conditions. In addition, the impacts of HA on
3
zooxanthellae were studied by comparing cell density, cell size, mitotic
index and concentration of chlorophyll a within ZX cells.
Materials and Methods
Experimental Design
Anthopleura elegantissima forms aggregations of genetically identical
clones through bilateral fission (Ayer & Grosberg 1995). Experiments were
designed to examine the effects of hypercapnic acidification through paired
comparisons of genetically identical but separated clonal couplets. After the
initial acclimation period, clonemates were separated so that each individual
(n = 24) was maintained in its own chamber in either a moderate or high
PCO2 condition (Fig. 1). Several respiration chambers without anemones
were kept in each experimental aquarium for measurements of background
O2 consumption. Respiratory (MO2) and photosynthetic (Pg) rates of each
specimen were measured after one week in normocapnic conditions and
after 6 weeks in experimental conditions. After the initial metabolic rate
measurements and after 3 weeks in experimental conditions, 4 tentacles were
clipped from each anemone and frozen in liquid nitrogen for zooxanthellal
measurements at the mid-point of the experimental course (Saunders &
Muller-Parker 1997). At the conclusion of the six-week experiments,
anemones were blotted dry and weighed. Oral disks and tentacles of each
specimen were weighed and frozen in liquid nitrogen for later measurements
of zooxanthellae.
Collection and Maintenance
Zooxanthellate Anthopleura elegantissima specimens were collected
in April 2008 from Point Grenville, Washington, USA (47° 18.2’ N, 124°
16.2’ W). This anemone harbors two different types of photosynthetic
symbiont: the dinoflagellate Symbiodinium muscatinei and a
4
trebouxiophycean, unicellular green alga (Lewis & Muller-Parker 2004).
The intertidal distribution of the symbionts is largely determined by
irradiance and temperature (Secord & Muller-Parker 2005, Muller-Parker et
al. 2007). The green alga is restricted from depths that are brightly
illuminated and subject to warmer temperatures. Because A. elegantissima
was collected from colonies at ~1.5 - 2.0 m above mean low low water
(Secord & Augustine 2000), anemones with the green algal symbiont were
excluded. This assumption was verified later by the absence of the green
alga during algal cell counts. The largest anemones from each colony were
chosen to minimize free space in the respiration chambers. Genetically
identical pairs of A. elegantissima (hereafter referred to as clonemates) were
selected from contiguous colonies within the spatial boundaries that separate
genetically distinct clones (Ayre & Grosberg 2005). No clonemate displayed
acrorhagial aggression toward its respective clonemate, which indicates that
they are genetically identical (Ayre and Grosberg, 1995). Clonemates and
individual specimens were collected and transported to the lab at The
Evergreen State College, Olympia, Washington in separate plastic bags
filled with seawater. In the lab, oral disks and tentacles of individual
anemones (n = 12) were weighed and individually frozen in liquid nitrogen
for zooxanthellal measurements. Clonemates were cleaned of debris before
they were blotted dry and weighed. Each individual clonemate was settled
into a labeled, 130-ml glass chamber (with a stir-bar cage adhered to the
bottom using silicone aquarium sealer) for the duration of the experimental
period to minimize disturbance and the risk of damage prior to respiration
measurements. Chambers were covered with a 1-cm mesh screen for the first
two weeks to prevent anemones from escaping the chambers. Individuals
5
obviously damaged and those that failed to adhere to the chamber were not
used for experiments.
Anemones were acclimated for 7 days in a 120-L, recirculating
aquarium with natural seawater adjusted up to 30 psu with Instant Ocean®
synthetic sea salt at 12°C, pH = 8.1, PCO2 = 368 ppmv. Mean irradiance was
adjusted to ~660 µmole m-2 s-1 and followed a natural spring-summer daily
photoperiod (14 h light: 10 h dark) for the acclimation and the two
experimental aquaria to maximize potential zooxanthellal photosynthesis
without risk of photoinhibition (Fitt et al. 1982, Verde & McCloskey 2002).
Every day, each chamber was moved within the aquarium to a different
position to ensure that all anemones received equivalent irradiance
throughout the experimental period. Each specimen was hand fed twice
every week by alternating shrimp and salmon each weighing 5% of the
specimen's initial wet weight (Zamer & Shick 1989). Chambers were
cleaned 24 h after feeding. Aquaria were cleaned of algal growth and water
exchanged twice per week to minimize accumulation of ammonia, nitrate
and extracellular proteins.
Experimental aquaria (120 L) were designed to maintain two levels of
hypercapnic PCO2: moderate HA (PCO2 = 450 ppmv) and high HA (PCO2 =
2340
ppmv) with pH levels of 8.0 units and 7.3 units, respectively (Table 1).
Natural seawater was collected from southern Puget Sound and adjusted up
to 30 psu with a combination of Instant Ocean® synthetic seawater and a
carbonate-free synthetic seawater (Bidwell & Spotte 1985) to maintain the
targeted pH, PCO2 and alkalinity levels. Carbonate alkalinity (acid
neutralizing capacity in mg carbonates L-1 seawater) was estimated by
6
titration with 0.2 N HCl using a Gilmont microburet and Gran Plot analysis
with the USGS web-based Alkalinity Calculator, version 2.20
(http://or.water.usgs.gov/alk/). PCO2 was calculated with the CO2SYS Excel
macro (Lewis & Wallace 1998). CO2 was used to manipulate pH, rather than
mineral acids such as HCL or H2SO4, due to its ecological relevance and its
roles in carbonate chemistry and cellular function (Ishimatsu et al. 2004,
Schneider & Erez 2006, Fabry et al. 2008, Marubini et al. 2008).
Hypercapnic acidification was generated by bubbling CO2 through a reactor.
The reactor was constructed of PVC pipe and contained 6, pegged plastic
balls (Bio-Balls™) to generate turbulence and therefore facilitate dissolution
of the gas into the seawater before entering the aquarium. CO2 was delivered
through a solenoid valve with a Milwaukee SMS122 pH controller; pH of
each aquaria was monitored daily with an Orion Research 601A digital
ionalyzer that was calibrated daily with Markson LabSales National Bureau
of Standards (NBS) buffers.
Photosynthesis, Respiration and CZAR
Respiratory and photosynthetic rates were measured following the
methods of Thuesen et al. (2005). To minimize microbial O2 consumption,
chambers were cleaned 24 h before rate measurements and 100 mg L-1 each
of streptomycin and ampicillin were added to the test chambers. Anemones
were sealed into their chambers with seawater at their respective level of
PCO2 with a stir-bar to ensure adequate mixing. Chambers were submerged
in a circulating water bath at 12°C on stir-plates at 200 rpm. After a 30minute, lighted acclimation period, O2 saturation was measured for 30
minutes in the light followed by 30 minutes of measurement in the dark.
Oxygen saturation was measured with Microx TX3 temperaturecompensated O2 meters fitted with Type B2 NTH fiber-optic, O2 micro7
optodes (Precision Sensing). Meters were calibrated to 0% O2 with a 5%
solution of Na2SO3 and to 100% O2 using oxygen-saturated seawater.
Optodes were inserted into the respiration chambers through gas-tight septa.
Control chambers without anemones were run simultaneously with the same
mixtures of antibiotics and seawater at 450 ppmv and 2340 ppmv PCO2.
Background rates of microbial O2 consumption within chambers at each
PCO2 condition in light and dark were subtracted from corresponding
anemone rates.
Ratios of photosynthesis (Pg) to respiration (MO2) were calculated
from the daily gross photosynthetic rate (based on a 14-hour lighted period)
relative to the daily respiratory rate. The percent contribution of
zooxanthellal carbon to animal respiration (CZAR) was based on the ratio of
animal (ß) and algal (1- ß) biomass components (Muscatine et al. 1981)
assuming the mean algal biomass ratio of 0.09 = (1- ß) calculated for A.
elegantissima (McKinney 1978, as reported in Fitt et al. 1982). Because
zooxanthellate respiration cannot be measured in the animal in the light, the
daytime algal respiratory rate is estimated from the total dark respiration rate
as a ratio of biomass. CZAR was estimated with the formula defined by
Muscatine et al. (1981) and modified by Verde & McCloskey (1996b and
2001):
CZAR = [[(0.375*PgO)(PQZ)-1]- [(1-ß)(RaeO)(RQae)] - [Cµ]· 100] / (ß)(0.375*
RaeO)( RQae)
where daily gross photosynthetic rate (PgO) is equal to the sum of O2
production rate in the light and the O2 consumption rate in the dark for the
number of lighted hours per day. The conversion ratio of C to O2 equivalents
8
(12:32) equals 0.375 (Verde & McCloskey 2001). Daily MO2 of the
anemone (RaeO) was calculated from the rate of O2 consumption in the dark
and extrapolated to 24 h. The photosnthetic quotient (PQz), animal
respiratory quotient (RQal) and zooxanthellal respiratory quotient (RQz) were
assumed to be 1.1, 0.9 and 1.0, respectively (Kremer et al. 1990, McCloskey
et al. 1994, Verde & McCloskey 1996b, 2001). The respiratory quotient of
the anemone (RQae) was determined by:
RQae = [(1- ß)(RQz)-1 +((ß)(RQal)-1)]-1.
Algal-specific growth rates (µz) were calculated as described in Verde and
McCloskey (1996b):
µz = (24 · td-1)ln(l +f)
with the duration of cytokinesis td equal to 28 (Verde & McCloskey et al.
1996a) and f equal to the fraction of cells in the division phase as determined
from mitotic index. The zooxanthellal carbon-specific growth rates (Cµ)
were determined with the formula supplied by Verde & McCloskey (1996a):
Cµ = [(SS)(C·cell-1)(µz)].
Standing stock (SS) was estimated from ZX cell densities, assuming that
~90% of ZX are harbored in the oral disk and tentacles (Shick 1991). Carbon
per ZX cell was calculated as reported by Menden-Deuer & Lessard (2000):
9
pg C·cell-1 = 0.760(cell volume0.819).
Zooxanthellal Measurements
Symbiodinium muscatinei characteristics were measured to gauge the
effects of HA on the photosynthetic symbionts. Previously frozen tentacles
and oral disks were thawed on ice and individually ground in hand-held,
glass tissue-homogenizers with filtered seawater (0.22 µm, 30 psu) at a ratio
of approximately 1 tissue: 10 water. Homogenates were separated into 3
aliquots for protein, chlorophyll and cellular measurements.
Zooxanthellal density was normalized to µg anemone protein. Algal
cell counts were performed with a hemocytometer in 10 replicate grid counts
per anemone and the number of cells per ml homogenate was converted to
the number of cells per µg protein. Algal cell diameters were measured with
an ocular micrometer in replicates of 10 per MI anemone. Mitotic index (MI)
was measured as an indicator of zooxanthellal growth. was calculated as a
percentage from the number of doublets with a complete cleavage furrow
observed per 1000 cells.
To prevent saline interference with the protein assay, aliquots of
anemone homogenates were desalinated with Millipore Microcon®
centrifugal filter units and then diluted to the original concentration with DI
water before digestion of the homogenate protein in 5% NaOH. Protein
concentrations (mg protein/ml homogenate) were measured with a Thermo
Scientific NanoDrop 1000® spectrophotometer against bovine serum
albumen standard, and protein density was determined by a modified Lowry
Assay (Lowry et al. 1951) according to the NanoDrop protocol on three
samples from each anemone.
10
To measure the concentration of chlorophyll a, 3 replicate
homogenates per anemone were centrifuged and resuspended 4 times to
remove animal fractions (Muller-Parker et al. 2007). Resuspended
zooxanthellae were filtered through GF/C Whatman® filters followed by 0.5
mL of 5% MgCO3 (Verde & McCloskey 1996b) to prevent acidification of
the samples. Filters were folded and wrapped in foil to freeze, then later
submerged in 10 mL 90% acetone and stored for 24 h at 4o C (Augustine &
Muller-Parker 1998). Acetone extracts were read for chlorophyll-a
concentration (mg of chlorophyll/ml acetone) with a Turner Designs® 10 AU
Fluorometer and converted to pg per ZX cell-1.
Statistical analyses
Data were analyzed with JMP Statistical Discovery Software, version 7.0.
Paired t-tests were used to determine if values measured following
hypercapnic experimental treatments differed significantly from the values
under the initial normocapnic conditions. Linear regression was used to
determine if there was a significant relationship between mass-specific
metabolism and body mass. ANOVA analyses were performed to identify ZX
differences between oral disks because initial OD parameters were measured
from individuals that were not clonemates. ANOVA tests were followed with
Fisher’s LSD post hoc test.
Results
All anemones survived the experimental period. Three individuals in each
experimental condition reproduced through bilateral fission during this time
and were treated as single individuals. There were no differences in mass of
the anemones after 6 weeks of exposure to experimental conditions. The two
experimental tanks were maintained within 1% of target PCO2 and pH levels
(448 ppmv and 2342 ppmv, pH 8.08 and 7.35, respectively; Table 1).
11
Although there was considerable variation of PCO2 within the moderate
tank, t-test analyses were highly significant between the conditions
(p<0.001). Hereafter, the tank at PCO2 448 ppmv is referred to as moderate
(450 ppmv) and the tank at 2342 ppmv as high (2340 ppmv). Rates of
photosynthesis and respiration were measured on 24 individual specimens of
A. elegantissima; these were 12 pairs of clonemates. Data for one couplet at
the current condition (PCO2 368 ppmv) were discarded due to measurement
errors; however the couplet was included for analyses between the two HA
conditions. Data from initial measurements at current conditions are
combined in the figures as there were no differences between couplet
individuals in any parameter at current conditions.
Photosynthesis and respiration
Anthopleura elegantissima exhibited higher rates of mass-specific
gross photosynthesis (_g) after 6 weeks of exposure to moderate and high
PCO2 than at current (normocapnic) conditions (Table 2). Anemones at
moderate PCO2 (450 ppmv) had a higher mean _g than those at the high
(2340 ppmv) PCO2 (paired t-test, p=0.03, Fig 2). The _g of anemones at both
moderate PCO2 (3.30 ± 0.35 µmol O2 g-1 h-1) and high PCO2 (4.20 ± 0.40
µmol O2 g-1 h-1) were higher than the _g at current levels (1.53 ± 0.11 µmol
O2 g-1 h-1; paired t-test, p < 0.001, Fig. 2).
Respiration rates were also higher in the experimental treatments
relative to the initial condition. Mean mass-specific respiration (_O2) of
anemones at high (1.27 ± 0.15 µmol O2 g-1 h-1) and moderate (1.34 ± 0.13
µmol O2 g-1 h-1) PCO2 were higher than the rates at current conditions (0.94
± 0.05 µmol O2 g-1 h-1, Paired t-test, p < 0.03, Fig. 2). There was no
difference in _O2 between the high and moderate PCO2 groups (paired t-test,
12
Fig 4). Mean _g:_O2 ratios were significantly higher among anemones
exposed to HA than among anemones prior to exposure (p < 0.001, Fig. 3).
Mean _g:_O2 ratios were greater at moderate PCO2 than at high PCO2,
however the difference was not significant (paired t-test, p = 0.1).
The contribution of oxidizable carbon and O2 by zooxanthellae to
animal respiration (CZAR, Fig. 4) was greatest in the moderate PCO2
treatment (CZAR = 137.3%) in comparison to the high PCO2 (CZAR = 78.2%,
paired t-test, p < 0.01) and at current PCO2 (CZAR = 66.6%, paired t-test, p <
0.001). Over the small size range of specimens used in this study, there was
no significant effect of body mass (linear regression analysis) on either _g
(Fig. 5) or _O2 (Fig. 6).
Zooxanthellal characteristics
Zooxanthellae (ZX) from combined oral disks and tentacles (OD) were
compared between Anthopleura elegantissima at current PCO2 (386 ppmv)
and between clonemates held in moderate (450 ppmv) or high (2340 ppmv)
PCO2 seawater for 6 weeks. Comparisons were also made between the ZX of
anemone tentacles prior to exposure to moderate and high PCO2 and after 3
weeks of exposure. The characteristics of ZX (cell diameter, cell density,
mitotic index, and chlorophyll a per cell) were significantly different in
tentacles than in OD in all categories (paired t-test, p ≤ 0.01, Figs. 7-10).
ZX cell diameters were larger in the tentacles than in the OD within
each PCO2 treatment (Fig. 7). Mean ZX cell diameter was larger in the
tentacles of anemones at high (12.56 ± 0.09 µm, p = 0.06) and moderate
PCO2 (12.31 ± 0.20 µm,) than in the tentacles at current conditions (11.79 ±
0.11 µm, Paired t-test, p<0.001). In the OD, there were no significant
differences in ZX cell diameters (11.16 ± 0.08 µm) among any of the PCO2
13
conditions (ANOVA, p > 0.05, Fig. 7). There were no differences in the algal
density relative to animal protein between anemones in current, moderate or
high PCO2, (tentacles- 0.48 ± 0.03 and OD- 0.26 ± 0.02 106 cells mg protein1
, ANOVA, p > 0.05, Fig. 8).
All anemones at moderate and high PCO2 had increased mitotic index
(MI) relative to those from the field (ANOVA, p < 0.05, Fig. 8). MI was
greater in the OD of anemones at high PCO2 (0.61 ± 0.08 %) than in those at
moderate PCO2 (0.40 ± 0.04 %) (Paired t-test, p = 0.03, Fig. 9). MI was also
much higher in tentacles from anemones after 3 weeks of exposure to high
PCO2 (1.20 ± 0.27 %) compared to those in moderate PCO2 (0.48 ± 0.04 %)
(Paired t-test, p < 0.01, Fig. 9). Anemones in the high PCO2 tank released a
large amount of mucus-bound zooxanthellea that contained a very high ratio
of doublets when examined under a microsocpe (data unreported). Although
the concentrations of chlorophyll-a per algal cell were higher in tentacles
(1.70 ± 0.13 pg per ZX cell) than in OD (3.80 ± 0.25 pg per ZX cell, paired ttest, p < 0.001), there were no differences in the concentration of
chlorophyll-a per algal cell between anemones in current, moderate or high
PCO2 (ANOVA, p = 0.15, Fig. 10).
Discussion
This study demonstrated that the non-calcifying actinian Anthopleura
elegantissima and its photosynthetic dinoflagellate Symbiodinium muscatinei
can thrive in hypercapnic acidified seawater for a duration of 6 weeks. In
photosynthesizing symbiotic organisms, the the percent contribution of
zooxanthellae (ZX) to animal respiration (CZAR) is largely determined by the
ratio of photosynthetic to respiratory rates (Verde & McCloskey 1998). In
this study, higher rates of _g at moderate (450 ppmv, pH=8.1) and high
14
(2340 ppmv, pH=7.3) PCO2 levels offset small, corresponding increases in
MO2. Thus the differences in _g and MO2 were reflected in the CZAR.
Assumptions made in the calculation of CZAR such as the ratio of
algal protein to animal protein could affect the outcome of this research (Fitt
& Cook 2001). Although the actual ratio of algal protein to animal protein is
unknown for this study, the lack of differences in the density of ZX cells and
in the concentration of chlorophyll a suggests that the ratio is the same in all
the of the conditions. Zx growth rates (Verde & McCloskey 2007) are more
likely to impact these results due to the differences in MI and may be a
source of error in the CZAR comparisons. The differences between the
animals at increased PCO2 and those fresh from the field may also have been
affected by the consistent temperature and lighting that were provided in the
lab as well as the absence of dessication, UV exposure and wave stress
(Shick 1991, Verde & McCloskey 1996b). Abundant food in the lab may
have contributed to the rise in _O2 as increasing heterothophic feeding can
increase the respiratory rate of A. elegantissima (Fitt et al. 1982). In their
study, _O2 in fed A. elegantissima was twice the rate of starved animals and
CZAR averaged 13% for fed anemones compared to 45% for starved or
newly collected anemones. This would suggest that the CZAR reported in the
current study may under-estimate the potential increase in CZAR that would
occur in the field as PCO2 increases. However Jensen & Muller-Parker
(1994) found high ammonium concentrations in tidepools with high
densities of anemones, which suggests that these organisms also have high
feeding rates in the field. Taking these factors into consideration, there were
still significant increases in metabolic activities that varied with PCO2 and
pH as well as associated changes in ZX cell size and growth rates as
indicated by mitotic index (MI). Because the density of ZX cells and
15
chlorophyll a was consistent between conditions, it is likely that the
increased availability of CO2 boosted the capacity of ZX to photosynthesize.
This is consistent with the findings of Verde & McCloskey (2007) who posit
that ZX-bearing anemones are carbon limited.
The effect of carbon limitation was also exhibited in a recent study by
Anthony et al. (2008). This group researched the effects of HA and
temperature increase on photosynthetic productivity in three types of
calcifying actinians: a crustose coralline alga, a branching coral and
amassive coral. As in the current study, they found increased rates of
productivity (at temperatures increased by 3o C) at moderately increased
levels of CO2 (520-700 ppmv) in the branching coral Acropora intermedia
but productivity was diminished in the massive coral Porites lobata.
However, at the same temperatures, higher concentrations (1000-1300
ppmv) decreased productivity to near zero in both corals. Both moderate and
large increases in PCO2 reduced productivity in the crustose alga Porolithon
onkodes to the point that respiration rates outpaced photosynthesis (45%160% reduction in productivity). The authors speculate that the increase in
productivity in P. lobata may have resulted directly from an increase in CO2
supply but that at higher concentrations, the effects of a greater CO2 supply
are offset by physiological disruption from HA.
The observed excretion of actively replicating ZX in this study was
confirmed by the dramatic increase in the MI of the anemones at 2340 ppmv.
McCloskey et al. (1996) found that MI was higher in expelled pellets of ZX
than within the host (but suggested that algal division may accelerate after
expulsion when released from host restrictions). Several studies have found
that the population density of ZX in other actinians is maintained under host
control through chemically-signalled arrest of the algal reproduction and
16
active expulsion of symbionts (Trench 1987, McCloskey et al. 1996,
Baghdasarian & Muscatine 2000). If the ZX in A. elegantissima are carbon
limited, the ready supply of dissolved inorganic carbon (DIC) in a HA
environment would necessitate an increase in the rate of expulsion to
maintain normal densities of the rapidly reproducing alga in order to avoid
toxicity from excess oxidative products (Furla et al. 2005).
ZX depend on the actinian host to transport the majority of DIC
needed for photosynthesis (Furla et al. 1998), a process facilitated by the
enzyme carbonic anhydrase (Furla et al. 2000a). Carbonic anhydrase (CA) is
commonly employed by autotrophic symbiontic animals to increase the
passage of DIC through host tissues by accelerating the conversion between
HCO-3 and CO2 for the consumption by the symbiont (Weis 1993, Goffredi
et al. 1999, Furla et al. 2005). In the anemone Aiptasia pulchella,
photosynthesis increased with the concentration of DIC and also was
enhanced by CA (Weis 1993). Furthermore, the expression of CA by the
host is induced by the symbiont (Weis & Reynolds 1999). The role of
carbonic anhydrase at increased PCO2 is worthy of additional investigation
as increasing the PCO2 in the environment could reduce the signal for
expression of CA by the symbionts and therefore of CA activitiy in the
anemone. An increase in the concentration of DIC in the seawater
environment also could reduce the energy required from the animal to
concentrate CO2 and result in the increase in CZAR demonstrated by A.
elegantissima in this study.
In corals, the benefit from increased CO2 to photosynthesis is eclipsed
by the impacts of HA on calcification. The above-mentioned research by
Anthony et al. (2008) illustrated that calcification in a corraline crustose alga
was extremely sensitive to the effects of HA and that two corals (one
17
branching, one massive) reduced calcification by 25-40% at PCO2 of ~1200
ppmv. Another recent study found that the scleractinian coral Stylophora
pistillata increased _g when seawater was enriched with HCO-3 even at
decreased pH; however _g was not responsive to decreases in pH nor
increases in PCO2 and calcification rates decreased (Marubini et al. 2008).
In contrast, increasing PCO2 (411ppmv-918 ppmv) increased MO2 but not _g
in a coral community in the Mediterranean (Leclercq et al. 2002) but again
calcification decreased. In a third study, Schneider & Erez (2006)
independently varied pH, CO-23, CO2, total alkalinity and total dissolved
carbon. They found no correlation between any of these carbonate chemistry
variables and _g or MO2 but that calcification diminished in Acropora
eurystoma with a decrease in the concentration of CO-23. The response of _g
to carbonate shifts varies in these studies, perhaps due to differences in
acclimation and exposure durations, however the effect of acidification on
calcification is consistent. Although calcification is tightly linked to
photosynthesis (Gattuso et al. 2000), it is clear that the effect of [CO-23] on
CaCO2 saturation state has a much larger influence on calcification than can
be compensated by the increase in photosynthesis as seen in this report.
Reports of reduced calcification and suppressed metabolism are
countered by others that found enhanced calcification and metabolic activity
with HA. Among photosynthetic organisms, one study of the
coccolithophore Emiliania huxleyi demonstrated increased primary
production and calcification in the presence of high PCO2, both by
experimentation and in geological record with a 40% increase in test mass
over the last 220 years (Iglesias-Rodriguez et al. 2008). Gutowska et al.
(2008) discovered that the cephalopod Sepia officinalis at very high PCO2
(4000 and 6000 ppmv) had the same capacities for growth and for
18
calcification of its internal aragonite shell as control animals at ~675 ppmv.
Even some corals have demonstrated unexpected resilience to acidification
as discovered by Fine & Tchernov (2007) who exposed two Mediterranean
coral species to decreased pH for 12 months. Although their aragonite
skeletons dissolved, the polyps adopted an anemone-like existence and clung
to the rocky substrate until they reformed skeletons after they were returned
to normocapnic conditions.
Non-calcifying organisms also exhibit a variety of responses to HA.
Studied fishes have shown an elastic response to PCO2 and display
adaptability to HA conditions ranging from acute exposure to CO2
concentrations that fall within IPCC projections (Pörtner et al 2004,
Ishimatsu et al. 2005) to long-term exposure at PCO2 up to nearly 5800
ppmv (Melzner et al. 2009). Invertebrates tend to be more sensitive to HA;
however some species appear able to compensate. The swimming crab
Necora puber was found to be resistant to acid-base disruption down to a pH
of 7.5 (Spicer et al. 2007), as was the shallow-water species Cancer magister
at pH 7.1 (Pane & Barry 2007). However the deep-sea Tanner crab revealed
a limited ability to regulate acid-base ions critical for responding to
acidification (Pane et al. 2008). A sipunculid worm likewise was unable to
compensate for a decrease in extracellular pH and its metabolic rate
decreased with exposure to HA (Pörtner et al. 1998). However, in a study of
the effects of HA on burrow structure and sediment nutrient flux of a nereid
worm, there were no observed metabolic or behavioral effects of HA on the
worm at pH 7.5 (Widdicombe & Needham 2007). The brittle star Amphiura
filiformis increased metabolic rate when kept in HA; however this was at the
expense of muscle wasting in the arms (Wood et al. 2008). The variability
19
among these investigations points to a need for additional examination of the
effects of high PCO2 on marine organisms, particularly in non-calcifiers.
Organisms such as A. elegantissima that are pre-adapted to
fluctuations of high CO2 conditions may be better suited to respond to the
challenges of persistent HA. As a rocky intertidal inhabitant, A.
elegantissima is regularly exposed to fluctuations of PCO2 and pH due to
fresh water input, tidal exchanges and localized, organismal production of
metabolic CO2. Fluctuating conditions are typical in estuaries, where PCO2
is much more dynamic than in coastal and open ocean areas and may reach
concentrations high enough to become a source of atmospheric CO2 through
out-gassing (Frankignoulle et al. 1998). Estuarine pH typically lies between
7.5 to 8.2 units but may be punctuated by occurrences above 9.0 and below
7.0 pH units (Hinga 2002), which is lower than the pH predicted from
anthropogenic climate change. Although estuarine PCO2 usually remains
below 2,500 ppmv (Kempe 1982 as reported in Borges 2001), Frankignoulle
et al. (1996) found PCO2 as high as 5700 ppmv. Similar fluctuations in the
carbonate system were found in the Palau coral reef lagoon that resulted
from nightly respiration and calcification processes (Watanabe et al. 2006).
Although estuarine and intertidal inhabitants may be adapted to
periodic acidification, medium to long-term exposure has been shown to be
detrimental to both
non-calcifying (Langenbuch & Pörtner 2004) and calcifying organisms, such
as bivalves (Michaelidis et al. 2005, Shirayama & Thornton 2005, Berge et
al. 2006, Gazeau et al. 2007, Bibby et al. 2008) that inhabit regions of
fluctuating pH and PCO2. Persistent acidification can result in dramatic
shifts in marine communities. Ecological studies in a high PCO2 volcanic
vent community off Ischia, Italy have shown that seagrass shoot density was
20
at its highest at 1827 ppmv PCO2 and pH 7.6 (along a natural gradient from
8.2 to 7.4) and productivity ~30% greater than in surrounding areas (HallSpencer et al. 2008). However epiphytic coralline algae, gastropods and
urchins were diminished or completely absent in areas below pH 7.7.
Although several species of scleractinian corals are common to the region,
photosynthetic anemones were the only cnidarians found in the zones with
reduced Ω CaCO3 (Hall-Spencer et al. 2008).
The response of Anthopleura elegantissima to hypercapnic
acidification reveals the adaptability of organisms that have evolved a
tolerance for high internal or external PCO2. There is much more to be
understood about the effects of HA on organisms adapted to high PCO2,
either from the demands of photosynthetic symbioses or in environments
such as estuaries, intertidal zones and O2 minimum zones where high PCO2
and reduced pH are commonplace. As an intertidal resident, A. elegantissima
possesses the physiological and behavioral means to thrive in a highly
variable environment. The results of this study suggest that A. elegantissima
can tolerate and even benefit from moderate levels of hypercapnic
acidification and indicates the adaptation of A. elegantissima to the broad
range of PCO2 and pH characteristic of its intertidal habitat.
21
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28
Figure 1. Schematic of experimental set-up. Clonemates were collected
from twelve different populations of Anthopleura elegantissima from the
intertidal zone of the eastern North Pacific Ocean at Point Grenville,
Washington. Upon return to the laboratory, specimens were maintained in
the same aquarium at PCO2 conditions of 368 ppmv. After one week, rates
of photosynthesis and respiration were measured and clonemates were
separated into individual respiration chambers and maintained in aquaria
with moderate and high PCO2 conditions (450 and 2340 ppmv, respectively)
for six weeks.
29
5
*
Specific oxygen flux
-1 -1
(µmol O2 g h )
4
3
2
***
1
0
-1
*
-2
368
450
2340
PCO2 (ppmv)
Figure 2. Mean mass-specific rates of gross photosynthesis (positive
oxygen flux) and respiration (negative oxygen flux) in Anthopleura
elegantissima at PCO2 of 368 ppmv (n = 22), 450 ppmv (n = 12) and
2340 ppmv (n = 12). Error bars represent ± one standard error. Significant
differences in the photosynthetic rate between 450 and 2340 ppmv
(paired t-test; *: p < 0.05). Highly significant differences between the
photosynthetic rate at 368 ppmv and the other 2 conditions (paired
t-test; ***: p < 0.001). Significant differences between the respiratory rate
at 368 ppmv and the other 2 conditions (paired t-test; *: p < 0.05).
30
2.5
P:R ratio
2
1.5
***
1
0.5
368
450
2340
PCO2 (ppmv)
Figure 3. The ratio of photosynthesis to respiration of Anthopleura
elegantissima at PCO2 of 368 ppmv (n = 22), 450 ppmv (n = 12), and
2340 ppmv (n = 12). Error bars represent ± one standard error. Highly
significant differenes between ratios at PCO2 368 and the other two
conditions (paired t-test; ***: p<0.001).
31
160
***
CZAR (%)
140
120
100
80
60
368
450
2340
P CO2 (ppmv)
Figure 4. The potential contribution of carbon by zooxanthellae to the
animal's respiratory carbon requirements (CZAR) in the Anthopleura
elegantissima symbiosis at PCO2 of 368 ppmv (n = 22), 450 ppmv
(n = 12) and 2340 ppmv (n = 12). Error bars represent ± one standard
error. Highly significant differences between CZAR at PCO2 450 ppmv
and the other two conditions (paired t-test ***: p<0.001).
32
7
5
-1 -1
(µmol O2 g h )
Specific photosynthetic rate
6
4
3
2
1
0
2
4
6
8
10
12
14
Mass (g)
Figure 5. Mass-specific rate of photosynthesis of Anthopleura elegantissima
at PCO2 of 368 ppmv ( , n = 22), 450 ppmv ( , n = 12) and 2340 ppmv (+,
n = 12) in relation to body wet weight. There was no significant effect of body
weight on photosynthetic rates (linear regression analysis, p > 0.05).
33
2
(µmol O2 g -1 h -1 )
Specific respiratory rate
2.5
1.5
1
0.5
0
2
4
6
8
10
12
14
Mass (g)
Figure 6. Mass-specific rate of respiration of Anthopleura elegantissima
at PCO2 of 368 ppmv ( , n = 22), 450 ppmv ( , n = 12) and 2340 ppmv (+,
n = 12) in relation to body wet weight. There was no significant effect of
body weight on photosynthetic rates (linear regression analysis; p > 0.05).
34
Cell diameter of zooxanthellae
(µm)
13
12.5
12
11.5
11
10.5
368
450
2340
PCO2 (ppmv)
Fig. 7. Zooxanthellal (ZX) cell diameter of Anthopleura elegantissima at
PCO2 of 368 ppmv, 450 ppmv, and 2192 ppmv in tentacles ( , n = 11) and
oral disk with intact tentacles ( , n = 11). Error bars represent ± one
standard error. Highly significant differences between cell diameter of
tentacles at PCO2 368 ppmv and the other two conditions (paired t-test;
***: p < 0.001). Cell diameter of tentacle samples are significantly different
from oral disk and tentacle samples in all cases (paired t-test, p<0.01).
35
Density of zooxanthellae
6
-1
(10 cells mg animal protein)
0.6
0.5
0.4
0.3
0.2
0.1
368
450
2340
PCO2 (ppmv)
Fig. 8. Zooxanthellal (ZX) density of Anthopleura elegantissima at PCO2
of 368 ppmv, 450 ppmv, and 2340 ppmv in tentacles ( , n = 11) and oral disk
with intact tentacles ( , n = 11). No significant difference between any
conditions (ANOVA ≥ 0.27). Error bars represent ± one standard error;
ZX density in tentacle samples are significantly different from oral disk
and tentacle samples in all cases (paired t-test, p ≤ 0.01).
36
***
*
0.7
0.6
Mitotic index (%)
0.5
*
0.4
0.3
***
*
0.2
0.1
0
368
450
2340
PCO2 (ppmv)
Figure 9. Mitotic index of zooxanthellae in Anthopleura elegantissima
oral disk with tentacles at PCO2 concentrations of 368 ppmv (n=11),
450 ppmv (n=11) and 2340 ppmv (n=12). Error bars represent ± one
standard error. Significant differences between MI at PCO2 450 ppmv
and the other two conditions (ANOVA; *: p<0.05) and highly significant
differences between PCO2 368 and 2340 (ANOVA ; ***: p<0.001).
37
5
-1
[Chl a] (pg cell )
4.5
4
3.5
3
2.5
2
1.5
1
368
450
2340
PCO2 (ppmv)
Figure 10. Chlorophyll a concentrations of zooxanthellae in Anthopleura
elegantissima at PCO2 concentrations of 368 ppmv (n=12), 450 ppmv (n=11)
and 2340 ppmv (n=11) in tentacles ( , n = 11) and oral disk with intact
tentacles ( , n = 11). Error bars represent ± one standard error. No significant
difference between any conditions (ANOVA, p ≥ 0.10). Chl a in tentacle
samples are significantly different from oral disk and tentacle samples in all
cases (ANOVA, p<0.01).
38
Table 1. Carbonate parameters of aquaria to investigate effects of hypercapnic acidification on Anthopleura elegantissima. NBS:
National Bureau of Standards, ANC: acid neutralizing capacity, PCO2: partial pressure of carbon dioxide. Values are mean ± SD.
pHNBS ± SE
Current PCO2 (n=2)
8.10 ± 0.02
ANC ± SE
(µmol/kg SW)
2403
Moderate PCO2 (n=14)
8.08 ± 0.04
2566 ± 120
448 ± 48
High PCO2 (n=14)
7.35 ± 0.02
2119 ± 43
2342 ± 151
39
PCO2 ± SE
(ppmv)
368
Table 2. Oxygen and carbon flux in Anthopleura elegantissima at PCO2 of 368, 450 and 2340 ppmv. CZAR: percent of carbon
provided by zooxanthellae to animal respiration.
Parameter: Mean ± SE
Photosynthetic rate- Gross
µmol O2 g wet weight-1 h-1
Photosynthetic rate- Net
µmol O2 g wet weight-1 h-1
Respiratory rate
µmol O2 g wet weight-1 h-1
Gross Photosynthesis: Respiration
Czar (%)
PCO2 (ppmv)
Current - 386
n=23
1.53 ± 0.11
Moderate - 450
n=11
3.30 ± 0.35
High - 2340
n=12
4.20 ± 0.40
0.69 ± 0.08
2.86 ± 0.31
2.03 ± 0.23
0.94 ± 0.05
1.34 ± 0.13
1.27 ± 0.15
1.02 ± 0.08
1.92 ± 0.16
1.53 ± 0.11
66.6 ± 6.6
137.3 ± 13.0
78.2 ± 8.2
40
Table 3. Biomass and growth parameters of zooxanthellae Symbiodinium muscatenei (Zx) exposured to PCO2 368, 450 and 2340
ppmv. OD (oral disk with intact tentacles) values were after 6 weeks of exposure to experimental conditions. Tentacles were measured
after 3 weeks of exposure. Values are pooled where there are no significant differences in mean.
Parameter: Mean ± SE (n)
Mitotic Index- % Zx cells dividing
OD
Tentacles
Zx cell diameter- µm
OD
Tentacles
Zx cell density-106 cells mg protein-1
OD
Tentacles
Chlorophyll a- pg ZX cell-1
OD
Tentacles
PCO2 (ppmv)
Current- 386
Moderate- 450
High- 2340
0.24 ± 0.05 (11)
no data
0.40 ± 0.04 (11)
0.48 ± 0.04 (11)
0.61 ± 0.08 (12)
1.20 ± 0.27 (12)
11.33 ± 0.16 (11)
11.79 ± 0.11 (22)
11.17 ± 0.11 (12)
12.31 ± 0.20 (12)
0.22 ± 0.03 (12)
0.48 ± 0.06 (22)
0.29 ± 0.05 (12)
0.50 ± 0.04 (12)
0.27 ± 0.03 (11)
0.46 ± 0.05 (12)
0.26 ± 0.02 (35)
0.48 ± 0.03 (46)
3.26 ± 0.58 (11)
1.85 ± 0.25 (23)
4.32 ± 0.39 (11)
1.46 ± 0.24 (12)
3.83 ± 0.27 (12)
1.65 ± 0.29 (12)
3.80 ± 0.25 (34)
1.70 ± 0.13 (47)
41
11.01 ± 0.43 (12)
12.56 ± 0.09 (12)
Pooled
11.16 ± 0.08 (35)
12.43 ± 0.11 (46)
Appendix I. Abbreviations
C
CaCO2
CO-23
CO2
CZAR
Cµ
DI
f
h
H2SO4
HA
HCl
HCO-3
IPCC
L
mg
MgCO3
MI
mL
ṀO2
N
NaOH
O2
OD
PCO2
pg
Ṗg
P gO
ppmv
PQZ
Ṗg: MO2
PS
psu
R
RaeO
RQae
RQal
RQz
SD
SE
SS
ß and 1- ß
td
USGS
ZX
µz
Ω CaCO3
carbon
calcium carbonate
carbonate
carbon dioxide
contribution of zooxanthellae to animal respiration as % carbon
carbon-specific growth rate
de-ionized water
fraction of cells in the division phase
hour
sulfuric acid
hypercapnic acidification
hydrochloric acid
bicarbonate
Intergovernmental Panel on climate Change
liter
milligram
magnesium carbonate
mitotic index
milliliter
hourly mass-specific respiration rate
normal (one gram equivalent of a solute per liter of solution)
sodium hydroxide
oxygen
oral disk and tentacles
partial pressure of carbon dioxide
picogram
hourly mass-specific photosynthetic rate (O2)
hourly gross PS rate (O2)
parts per million by volume
photosynthetic quotient of zooxanthellae
ratio of daily gross photosynthetic rate to respiration rate
photosynthesis, photosynthetic
practical salinity units
respiration, respiratory
daily respiratory rate of the anemone (combined animal and zooxanthellae)
respiratory quotient of the anemone (combined animal and zooxanthellae)
respiratory quotient of the animal
respiratory quotient of zooxanthellae
standard deviation
standard error
standing stock
animal and algal proteins expressed as fractions of total protein, respectively
duration of cytokinesis
United States Geological Service
zooxanthellae, zooxanthellal
specific growth rate of zooxanthellae
saturation state of calcium carbonate
42
