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extracted text
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Biological Responses of Juvenile Tridacna maxima
(Mollusca:Bivalvia) to Increased pCO2 and Ocean Acidification
By
Charley G. Waters
A Thesis submitted in partial fulfillment
of the requirements for the degree of
Master in Environmental Studies
The Evergreen State College
October 29, 2008
© by Charley G. Waters. All rights Reserved.
ii
This Thesis for the Master of Environmental Studies Degree
By
Charley G. Waters
Has been approved for
The Evergreen State College
By
_________________________________
Erik V. Thuesen, Ph. D.
Member of the Faculty, Zoology
________________________________
Timothy Quinn, Ph. D.
Member of the Faculty, Conservation Biology
_______________________________
Grace Sparks, Ph. D.
Professor, Seattle Central Community College
__________________________
Date
iii
Table of Contents
List of Figures........................................................................................................... vi
List of Tables ...........................................................................................................viii
Acknowledgements .................................................................................................. ix
Abstract ..................................................................................................................... x
1.0
Introduction .................................................................................................... 1
1.1.
Biology of Tridacna maxima Clams .............................................................2
1.2.
Symbiotic Association Between Tridacna spp. and Symbiodinium
microadriaticum...........................................................................................4
1.3.
Overview of Oceanic Carbonate Chemistry and Ocean Acidification.........12
1.4.
Ecological, Economic, Social and Political Implications of Ocean
Acidification...............................................................................................20
1.5.
Biological Responses of Marine Organisms to Ocean Acidification ...........23
1.6.
Biological Responses of Tridacna maxima to Ocean Acidification.............25
2.0
Materials and Methods................................................................................. 25
2.1.
Clam Specimens.......................................................................................25
2.2.
Aquaria .....................................................................................................26
2.3.
Carbonate Chemistry ................................................................................27
2.4.
Data Collection..........................................................................................28
2.5.
Scanning Electron Microscopy Imaging Protocol.......................................30
2.6.
Statistical Analysis ....................................................................................30
3.0
Results......................................................................................................... 30
3.1.
Chemical and Physical Conditions ............................................................30
3.2.
Calcification Response: Shell Length and Height ......................................35
3.3.
Calcification Response: Calcium (Ca2+) Concentrations in Soft Tissue......39
3.4.
Calcification Response: Aragonite Shell Crystal Conditions ......................41
3.5.
Calcification and Soft Tissue Response: Growth Ratios and Weights .......43
iv
3.6.
Symbiotic Association: Zooxanthellae Population Density.........................47
3.7.
Symbiotic Association: Chlorophyll a Density per Algal Cell ......................49
4.0
Discussion ................................................................................................... 51
4.1.
Materials and Methods..............................................................................51
4.2.
Chemical and Physical Experimental Conditions.......................................52
4.3.
Calcification Responses (Shell Growth) ....................................................55
4.4.
Calcification / Soft-Tissue Response.........................................................58
4.5.
Symbiotic Association ...............................................................................58
4.6.
Conclusions ..............................................................................................59
Bibliography ............................................................................................................ 62
v
List of Figures
Figure 1: Examples of variable pigmentation and iridophore regimens in juvenile
Tridacna maxima clams ~4 cm in length. Source: C. Waters. ......................................... 3
Figure 2: Medial view of basic Tridacna maxima anatomy shows epithelial tubes
branching from the stomach to primary and secondary zooxanthellal tubes leading to
tertiary clusters in the hypertrophied tissue. Source: C. Waters, redrawn from Norton et
al. (1992). ....................................................................................................................... 6
Figure 3: Elements and nutrients exchanged between Symbiodinium microadriaticum
symbionts and Tridacna maxima hosts. Source: C. Waters. ........................................... 8
Figure 4: Calcium carbonate concentration curves for aragonite and calcite by depth at a
station in the Atlantic. Source: Marine Biogeochemical Cycles, Elsevier, The Open
University, 2005, modified by C. Waters. .......................................................................19
Figure 5: Mean differences in calcification response rates in terms of (a) shell length and
(b) shell height of juvenile Tridacna maxima clams within populations by pCO2 treatment
over a 13-week period.. .................................................................................................36
Figure 6: Population percentages of Tridacna maxima specimens that gained in both
length and height (BG), lost in both length and height (BL), and gained in one dimension
but lost in the other (G/L) by pCO2 treatment over a 13-week period. ............................38
Figure 7: Mean differences in Ca2+ ion concentrations per gram of dry tissue weight of
Tridacna maxima mantle tissue by pCO2 treatment at the end of a 13-week incubation
period were not significantly different (ANOVA, P > 0.05). ............................................40
Figure 8: Qualitative differences in the condition of T. maxima shell aragonite crystals in
pCO2 180 ppmv (a) and pCO2 840 ppmv (b). Images were taken from leading mantle
shell locations where most recent shell extrusion is expected. Source: R. Peroutka......42
vi
Figure 9: Differences in mean net weight (g) response of Tridacna maxima specimens to
increased pCO2 at introduction and at 13 weeks were significant within treatment
populations (Student’s paired t-test, P < 0.05).. .............................................................44
Figure 10: Mean dry soft-tissue to shell weight response ratios of Tridacna maxima to
decreased pH conditions over a 13-week period were not significantly different (ANOVA,
P > 0.05)........................................................................................................................45
Figure 11: Differences in Condition Index (CI) responses of Tridacna maxima specimens
to different pCO2 conditions over a 13-week period were not statistically significant (P >
0.05).. ............................................................................................................................46
Figure 12: Mean counts of zooxanthellal population density per gram of Tridacna
maxima mantle tissue by pCO2 treatment after a 13-week incubation period.................48
Figure 13: Chlorophyll a content in mantle tissue of Tridacna maxima expressed as pg
chl a per zooxanthella by pCO2 treatment over a 13-week period..................................50
vii
List of Tables
Table 1: Past, present and projected atmospheric and oceanic parameters for chemical
and physical factors based on 2001 IPCC “business as usual” scenarios. Source: IPCC
as modified by Feely et al. (2001) and Kleypas et al. (2005)..........................................14
Table 2: Chemical and physical parameters for each tank of experimental artificial
seawater (ASW) used to incubate Tridacna maxima specimens for a 13-week period. .32
Table 3: Comparison between present oceanic carbonate chemistry parameters per
IPCC estimates before the end of this century, this experiment, and other studies of
bivalve responses to increasing pCO2 ...........................................................................53
viii
Acknowledgements
Thanks are due to E. V. Thuesen, Ph. D., T. Quinn, Ph. D., G. Sparks,
Ph. D., C. Barlow, Ph. D., R. Peroutka for her unselfish dedication to the project
and demonstrated competence in scanning electron microscopy, J. Freeman, J.
Warmack, J. Williford, F. Goetz, P. Robinson and all of the Scientific Instruction
Technicians, LabStores and Lab Receiving personnel at The Evergreen State
College for invaluable support along the way, M. M. Bachtold and L. D’Andrea,
Ph. D., for unwavering encouragement over the years, M. Gutowska for critical
research design input, and T. Rose for extensive editorial support. A generous
Foundation Activity Grant from The Evergreen State College Foundation
contributed to this study.
ix
Abstract
Biological Responses of Juvenile Tridacna maxima
(Mollusca:Bivalvia) to Increased pCO2 and Ocean Acidification
Charley G. Waters
Anthropogenic acidification of oceanic surface waters is expected to increase
~0.4 units from current pH levels of ~8.1 during this century. The effects of increased
ocean acidification on biological processes vital to marine organisms are not well
understood. This paper describes a controlled experiment designed to measure
biological responses of juvenile Tridacna maxima clams to increased levels of
atmospheric CO2 partial pressure (pCO2) and concomitant increases in ocean
acidification. Over a 13-week period, four T. maxima populations (n = 30 each) were
incubated in carbonate chemistry conditions manipulated by pCO2 concentrations
approximating glacial (180 ppmv), present (380 ppmv) and projected (560 ppmv and
840 ppmv) levels of atmospheric CO2 per International Panel on Climate Change
(IPCC) IS92a scenario. Net differences in mean shell lengths and heights
significantly declined between pCO2 180 ppmv and pCO2 840 ppmv (P < 0.05).
Approximately 11% of individual T. maxima specimens exhibited negative shell
growth (dissolution) in pCO2 180 ppmv conditions compared to > 60% in pCO2
conditions of 840 ppmv. The mean net weight of all specimens varied significantly
within populations (P < 0.05), but not between populations (P > 0.05), indicating an
uncoupling of processes that contribute to shell precipitation or dissolution from
normal mantle growth as pCO2 increases. Cumulative results indicate differences in
biological responses of T. maxima occurred at pCO2 levels well below thresholds
previously considered detrimental to other marine organisms in similar conditions.
Results further indicate that tridacnid clams may be more susceptible to ocean
x
acidification than corals. Monitoring the health of Tridacna spp. may prove useful in
helping predict the effects of increasing pCO2 on coral reef ecosystems.
xi
1.0
Introduction
The oceanic carbonate chemistry system mediates a two-way distribution of
carbon species between two of Earth’s great reservoirs of carbon; the atmosphere
and deep ocean sediments. Since the industrial revolution, anthropogenic behaviors
such as fossil fuel emissions, deforestation and cement manufacturing are raising
levels of atmospheric carbon dioxide (CO2) at exponential rates, thereby interfering
with the ocean’s capacity to mediate a gradual transition of carbon from one
reservoir to another (The Royal Society, 2005). The scope of anthropogenic impacts
on atmospheric conditions is numbing. Current accumulation rates of atmospheric
CO2 exceed rates dating back 650,000 years (IPCC, 2007), and even at 2005 levels
of atmospheric CO2, it is anticipated that returning ocean chemistry to what it was
barely 200 years ago may take tens of thousands of years (The Royal Society,
2005).
Marine scientists are closely monitoring levels of atmospheric CO2 as part of
a well understood chain of chemical reactions between CO2 partial pressure (pCO2)
and seawater. These reactions predictably alter oceanic carbonate chemistry by
increasing hydrogen ion (H+) concentrations, thereby reducing pH in a process
colloquially termed ocean acidification (Doney, 2008). Research initiatives directed at
assessing specific effects of ocean acidification on marine organisms as a result of
elevated pCO2 are scarce. This paper describes a controlled experiment designed to
measure biological responses of juvenile Tridacna maxima clams to levels of
acidification that correspond to past, present and expected levels of atmospheric
pCO2. Study results may advance our understanding of how atmospheric conditions
might affect marine organisms with characteristics similar to T. maxima. This study
1
begins with a summary description of biological attributes of T. maxima and their
autotrophic symbionts.
1.1.
Biology of Tridacna maxima Clams
Tridacna clams are a genus of Tridacnidae (Mollusca:Bivalvia) that thrive in
photic zones of equatorial waters ranging from Eastern Africa to the central Pacific.
Of all ten species, Tridacna maxima are the most geographically wide-ranging
(Heslinga et al. 1990; Ellis, 1998). Optimal water conditions for Tridacna clams
include a temperature range of 25ºC to 30ºC, salinity from 32 to 35 parts per
thousand (‰), and pH levels from 8.1 to pH 8.5 (Heslinga et al. 1990; Ellis, 1998).
Commonly referred to as “giant clams”, Tridacna clams range in size from
approximately 15 cm to 137 cm in length and can weigh up to 250 kg (Rosewater,
1965; Heslinga et al. 1990; Knop, 1996; Ellis, 1998). Shell sizes for T. maxima
typically range from 30 cm to 40 cm (Knop, 1996). The iridescent pigmentation of T.
maxima (Figure 1) may serve as protection from potentially damaging ultraviolet light
or perhaps too much light in general (Knop, 1996).
2
Figure 1: Examples of variable pigmentation and iridophore regimens in juvenile
Tridacna maxima clams ~4 cm in length. Source: C. Waters.
3
As protandric hermaphrodites, Tridacna maxima rely on pheromonal
communication to initiate broadcast spawning of fecundate zygotes during different
breeding seasons. Environmental triggers to spawning include tide, lunar cycle and
temperature (Fitt and Trench, 1981; Heslinga et al. 1990; Ellis, 1998). Natural
predators of Tridacna spp. consist of crabs, lobsters, fish, various boring snails,
octopi, eagle rays and humans (Heslinga and Fitt, 1987; Heslinga et al. 1990; Ellis,
1998). Tridacna maxima have three sources of nutrient uptake; filter feeding of
phytoplankton, principally Isochrysis galbana and its congeners (Heslinga and Fitt,
1987), soft-tissue (mantle) absorption of dissolved nutrients from the water column
(Fankboner, 1971), and photosynthates from a symbiotic association with
Symbiodinium microadriaticum, autotrophic dinoflagellates (Heslinga and Fitt, 1981,
Ellis, 1998). Tridacna spp. are the only known species of Bivalvia to engage in
symbiotic associations solely with S. microadriaticum (Taylor, 1969), commonly
referred to as zooxanthellae (Fitt and Trench, 1981). Life expectancy among
Tridacna clams is not well studied although Heslinga et al. (1990) suggest ranges
between several decades for most tridacnid species and perhaps > 100 years for the
largest of the species, Tridacna gigas.
1.2.
Symbiotic Association Between Tridacna spp. and Symbiodinium
microadriaticum
Symbiodinium microadriaticum zooxanthellae are not inherited by larval
clams (Belda-Baille et al. 1999; Hirose et al. 2005). Instead, within 2 to 3 days of
fertilization, veliger larvae begin ingesting (but not digesting) and storing
zooxanthellae in their stomach (Fitt and Trench, 1981). Following larval
metamorphoses, zooxanthellal packets are relocated via a complex network of
epithelial tubes and haemal fluids to extra-cellular tertiary locations of the
4
hypertrophied tissue (Figure 2) where they undergo mitosis (Trench et al. 1981;
Heslinga and Fitt, 1987; Heslinga, 1990; Norton et al. 1992).
5
Figure 2: Medial view of basic Tridacna maxima anatomy shows epithelial
tubes branching from the stomach to primary and secondary zooxanthellal
tubes leading to tertiary clusters in the hypertrophied tissue. Source: C.
Waters, redrawn from Norton et al. (1992).
6
Specific photosynthates generated by zooxanthellae for Tridacna sp. hosts
include varying degrees of respiratory carbon, complex sugars such as glycerol and
glucose, amino acids such as alanine, and small amounts of oxygen (O2)
(Fankboner, 1971; Trench et al. 1981; Heslinga and Fitt, 1987; Paracer and
Ahmadjian, 2000; Hirose et al. 2005). In exchange, tridacnid hosts provide
zooxanthellae safe haven from prey, a homeostatic environmental (Cowen, 1988),
nutrient salts, CO2, and nitrogenous wastes that fuel zooxanthellal photosynthetic
productivity (Fankboner, 1971). The overall exchange of elements and nutrients is
summarized in Figure 3.
7
Figure 3: Elements and nutrients exchanged between Symbiodinium microadriaticum
symbionts and Tridacna maxima hosts. The precise volumes, rates, frequency and
conditions of exchange of nutrients and elements between organisms are not fully
understood. Source: C. Waters.
8
The relationship between zooxanthellal photosynthetic output and host
metabolic activities of Tridacna maxima and other marine symbionts is elegant yet
extremely complex. Cumulative photosynthates generated by zooxanthellae support
multiple metabolic, anabolic and catabolic activities of the host, including mucus
production, byssal development, nutrient digestion and CaCO3 precipitation (shell
formation) (Goreau et al. 1973; Yonge, 1980; Trench et al. 1981). For example,
under favorable conditions, zooxanthellae may provide up to 100% of the respiratory
carbon required by the host (Fisher, 1985; Heslinga and Fitt, 1987), and > 50% of
the host’s nutritional requirements (Hirose et al. 2005). Experimental evidence
suggests that growth rates for juvenile Tridacna spp. increase when zooxanthellae
begin migrating across the alimentary tract of developing siphonal tissue, and that
hosts void of zooxanthellae do not survive beyond three weeks (Fitt and Trench,
1981).
The exact volumes and rates of zooxanthellal contributions of photosynthates
to Tridacna maxima are difficult to quantify because multiple factors have been
shown to influence zooxanthellal photosynthetic activity. Such factors include nutrient
saturation levels of nitrogen (N) and phosphorus (P), ammonia (NH3), community
metabolic output, light and seasonal conditions, availability and source of carbon
species, and salinity (Amariyanto and Hoegh-Guldberg, 1997; McConnaughey et al.
2000; Medakovic et al. 1997; Legget and Yellowlees, 1999; Ringwood and Keppler,
2002). Steady rates and volumes of zooxanthellal photosynthetic activity are
particularly significant to T. maxima because absent or inconsistent sources of
photosynthetic output may jeopardize CaCO3 precipitation.
Calcium carbonate precipitation describes a mechanism organisms use to
create minerals from surrounding reservoirs of elements. Calcification refers to the
9
creation of hard tissue structures from elements in the calcite group of carbonates.
Calcifying marine organisms precipitate CaCO3 structures for skeletal support,
protection, and as a reserve source of carbonate (CO32-) to offset naturally occurring
spikes in carbonic acid (H2CO3) (Lindinger et al. 1984; Lowenstam and Weiner,
1989). Calcite species used to precipitate CaCO3 structures include calcite,
aragonite, High-magnesium calcite and others, or some combination thereof. The
chemistry between carbonate species is similar but differences in structure and
symmetry tend to make aragonite more soluble than calcite (The Royal Society,
2005). Calcification can only occur when surrounding waters are supersaturated (≥
100%) with the required carbonate and trace elements such as strontium (Sr) and
molybdenum (Mo) (Knop, 1996); undersaturated levels result in dissolution (Gattuso
et al. 1998). Tridacna maxima shells primarily consist of aragonite with trace
amounts of calcite (Lowenstam and Weiner, 1989).
Evidence supporting the importance of CaCO3 precipitation in the life history
of Tridacna spp. is compelling. Approximately 12 hours after fertilization, Tridacna
zygotes pass through a gastrula stage of embryonic development to hatch as
trochofore larvae. A shell gland at the base of the trochofore begins precipitating
CaCO3 shells (Heslinga et al. 1990). The soft tissue of a hatched trochofore is
completely surrounded by shell within two days of fertilization, thereby signifying the
veliger stage of development (Heslinga et al. 1990; Ellis, 1998).
The relationship between zooxanthellal photosynthesis and calcification of
marine organisms remains elusive and subject to much debate. Early research
suggested that zooxanthellae per se may not be clearly linked to CaCO3 precipitation
in corals, yet precipitation rates declined in relation to zooxanthellal loss (Goreau,
1959). More recently, it has been suggested that symbiotic photosynthetic output
may account for ~90% of the primary productivity in reef environments that
10
contribute to CaCO3 precipitation (Cowen, 1988). A rigorous study of the relationship
between photosynthesis and calcification by McConnaughey et al. (2000) concluded
that photosynthetic rates and calcification of reef corals generally correlated over the
course of the day, and virtually ceased at night even when levels of aragonite were
supersaturated. Research findings by Legget et al. (1999) reinforce the potential role
of light in the calcification process. This team found Symbiodinium microadriaticum in
Tridacna sp. to exhibit light-activated intra-cellular carbonic anhydrase activity,
suggesting a carbon concentrating mechanism that may facilitate calcification. In
contrast, Woolridge (2008) suggests that reduced pH rather than sunlight interferes
with urease enzyme activity, the enzyme known to facilitate CaCO3 precipitation.
Other research findings indicate an inverse relationship between
photosynthesis and hermatypic scleractinia corals (Hoegh-Guldberg and Smith,
1989; Langdon and Atkinson, 2005). An inverse relationship between photosynthesis
and calcification is corroborated by Marubini and Atkinson (1999), who found that a
combination of light and nutrient availability affected carbon partitioning between
photosynthesis and calcification in corals. Marubini and Davies (1996) suggest that
inverse relationships between photosynthesis and calcification may be explained by
rapid, large-scale absorption of diffusion-limited CO2 by zooxanthellal populations for
photosynthesis, thereby reducing reserves of inorganic carbon ordinarily available for
calcification.
An exhaustive discussion of variables affecting calcification is beyond the
scope of this study. The temptation to transfer knowledge gained from coral
calcification responses to photosynthesis to Tridacna maxima should be mitigated by
the fact that Tridacna spp. select different species of inorganic carbon from
seawater. For example, symbionts in Tridacna gigas specimens have been shown to
select CO2 as a primary source of carbon as opposed to symbionts in corals that
11
select HCO3- (Legget et al. 2000). Yet to be fully explained is whether either species
of inorganic carbon is used for host calcification, symbiont photosynthesis, or
perhaps a combination of the two species depending on prevailing pH conditions
(Legget et al. 2000).
In summary, the relationship between zooxanthellal photosynthesis and
CaCO3 precipitation depends on the hosting organism, life history stage,
zooxanthellal population density, and multiple environmental variables including the
availability of inorganic carbon. The extent to which increasing pCO2 and declining
oceanic pH influence calcification and photosynthesis in complex marine organisms
with symbiotic associations is unknown. Exploring the relationship between elevated
levels of pCO2 and biogeochemical processes such as calcification and
photosynthesis begins with a review of basic oceanic carbonate chemistry.
1.3.
Overview of Oceanic Carbonate Chemistry and Ocean Acidification
Anthropogenic contributions to ocean acidification begin with elevated levels
of atmospheric CO2 as a result of fossil fuel emissions, deforestation and, more
recently, cement manufacturing (Kleypas et al. 2005). It is impossible to categorically
attribute current conditions of atmospheric CO2 to anthropogenic activities, but the
evidence is compelling (Houghton, 2005). For example, current concentrations of
atmospheric CO2 are > 380 ppmv (Doney, 2008), a 38% increase from ~280 ppmv
levels at the beginning of the Industrial Revolution (IPCC, 2007; Kleypas, 2008). The
last time-frame for an increase of 100 ppmv (from inter-glacial ages of ~180 ppmv to
~280 ppmv) is estimated to be between 640,000 and 800,000 years (IPCC, 2007;
Doney, 2008). If current rates of increase continue under IPCC’s “business as usual”
scenario IS92a, atmospheric concentrations of CO2 are expected to reach ~560
ppmv sometime in the middle of this century and ~840 ppmv by the close of the
12
millennium (Orr, 2005; IPCC, 2007) (Table 1). The result may be a decline of ~0.4
units in oceanic surface pH from current averages of ~8.1 to ~7.7 (Caldeira and
Wicket, 2003), lower than it has been in more than 20 million years (Feely et al.
2004).
13
Table 1: Past, present and projected atmospheric and oceanic parameters for chemical
and physical factors based on 2001 IPCC IS92a “business as usual” scenarios. Source:
IPCC as modified by Feely et al. (2001) and Kleypas et al. (2005).
UNIT
GLACIAL
PRE- IND.
PRESENT
2 X CO2
3 X CO2
Temperature
ºC
15.7
19
19.7
20.7
22.7
Salinity
‰
35.5
34.5
34.5
34.5
34.5
2356
2287
2287
2287
2287
180
280
380
560
840
PARAMETER
-
Total
µequiv kg
Alkalinity (TA)
CO2
1
µatm
(ppmv)
Carbonic Acid
µmol kg
-1
7
9
13
18
25
µmol kg
-1
1666
1739
1827
1925
2004
µmol kg
-1
279
222
186
146
115
µmol kg
-1
(H2CO3)
Bicarbonate
-
(HCO3 )
Carbonate
2-
(CO3 )
+
Hydrogen (H )
4.79 x 10
-03
6.92 x 10
-03
8.92 x 10
-03
1.23 x 10
-2
1.74 x 10
Aragonite
Ωarag
4.26
3.44
2.9
2.29
1.81
Calcite
Ωcalc
6.63
5.32
4.46
3.52
2.77
1952
1970
2026
2090
2144
8.32
8.16
8.05
7.91
7.76
Dissolved
µmol kg
-1
-02
Organic
Carbon (DIC)
Total pH
pHT
14
The relationship between atmospheric CO2 and seawater is partially
explained by four primary reactions:
CO2(aq) + H2O(l) ↔ H2CO3(aq)
(1)
Carbonic Acid
H2CO3(aq) ↔ H+(aq) + HCO3-(aq)
(2)
Bicarbonate
HCO3- ↔ H+ (aq) + CO32-(aq)
(3)
Carbonate
pCO2(aq) + H2O(l) + CO32-(aq) ↔ 2HCO3-(aq)
(4)
Atmospheric CO2 follows Henry’s Law by mixing with seawater (pCO2) across
the air-sea interface to form carbonic acid (H2CO3) (1). H2CO3 rapidly dissociates to
form bicarbonate (HCO3-) (2), and dissociates again at a slower reaction rate to form
carbonate (CO32-) (3), thereby increasing concentrations of H+ ions on a logarithmic
scale and reducing pH. In addition to reduced pH, reactions between pCO2 and
seawater increase concentrations of HCO3- and decreases concentrations of CO32(Open University, 1994; Kleypas et al. 2005; Herfort et al. 2008). For example,
atmospheric conditions of 560 ppmv are expected to reduce CO32- concentrations by
30%, and increase H+ ion concentrations ranging from ~60% (Haugan, 2004) to
between 100% and 150% (Orr, 2005). A further reduction in CO32- ions occurs as
pCO2 reacts with H2O and CO32 to form 2HCO3- molecules, thereby compounding
already reduced concentrations of CO32- (4) (Haugan, 2004).
Two additional equations that contribute to understanding ocean acidification
in a larger framework of carbonate chemistry are equilibrium constants for HCO3and CO32- in terms of H+ ion concentrations in solution, and alkalinity of seawater.
15
Reaction rates between equations (2) and (3) contribute to a relatively stable
oceanic pH of ~8.1. The reactions are said to be at equilibrium when equation (3) is
complete as represented by equation (5), where K is the equilibrium constant and
represents the ratio between HCO3- and CO32- to “buffer” H+ ion concentrations; as
the ratio (K) increases, pH declines.
[HCO3-]
(H+) = K
(5)
[CO32-]
In addition to elevated levels of atmospheric CO2, alkalinity is known to affect
levels of pH differently depending on chemical conditions and measurement methods
(Kelypas et al. 2005; Dickson et al. 2007). Alkalinity generally describes the
concentration of negative charge in a solution that reacts with H+ ions.
Concentrations of HCO3- and CO32- in seawater are closely related to alkalinity since
these ions are orders of magnitude more abundant than conjugate bases and weak
acids capable of accepting protons. Carbonate alkalinity (CA) is expressed as:
CA = 2[CO32-] + [HCO3-]
(6)
The carbonate ion in equation (6) is multiplied by 2 because there are 2 units
of negative charge.
Technically, elements other than carbonate and bicarbonate contribute to the
total alkalinity (TA) of the Earth’s oceans. Equation (7) describes total alkalinity,
rigorously defined by Dickson (1981) as “the number of moles of hydrogen ions
equivalent to the excess of proton acceptors…over proton donors,” where ellipses
16
represent such minor acid or base species that they can be omitted, and [H+]F
represents the free concentration of hydrogen (Dickson et al. 2007).
TA = [HCO3-] + 2[CO32-] + [B(OH)4-] + [OH-] + [HPO42-]
(7)
+ 2[PO43-] + [Si(OH)3-] + [NH3] + [HS-]+…
– [H+]F – [HSO4-] – [HF] – [H3PO4]-…
As stated, the precise relationship between photosynthesis and calcification
on the ocean’s carbonate chemistry system remains elusive. Equation (8) shows
how carbon dioxide and water react to fuel photosynthetic activity (light energy), and
how carbon is oxidized through metabolic respiration.
Photosynthesis
CO2(g) + H2O
(CH2O) + O2(g)
(8)
Respiration
CaCO3 Precipitation
(9)
2HCO3- + Ca2+ ↔ CaCO3 + CO2 + H2O
CaCO3 Dissolution
Photosynthesis and respiration are pathways for carbon to enter and exit
organisms. These processes, referred to as organic carbon metabolism, influence
the rate at which CaCO3 is formed (9).
Calcium carbonate precipitation and dissolution is termed inorganic carbon
metabolism; precipitation occurs as reactions move to the right, and dissolution
occurs as reactions move to the left (9). Ocean acidification can negatively affect this
17
reaction regardless of direction. Under normal seawater conditions, Ca2+ is not
considered limiting to CaCO3 precipitation in corals, strongly suggesting that
precipitation rates are influenced primarily by changes in concentrations of CO32(Kleypas et al. 2005; Gazeau et al. 2007). As reaction (3) demonstrates, CaCO3
precipitation is predicted to decline in proportion to expected reductions in CO32- ion
concentrations as levels of pCO2 continue rising.
In terms of dissolution, saturation horizons describe depth boundaries
(lysocline) at which CaCO3 begins dissolving because surrounding waters lack
sufficient concentrations of CO32- ions at equilibrium with Ca2+ ions. The current
saturation horizon for aragonite, the predominant element of Tridacna shells, is
between 0.5 and 2.5 km from the surface, while the saturation horizon for calcite is
between 1.5 and 5 km (The Royal Society, 2005). Figure 4 illustrates CO32- curves
by depth for aragonite and calcite for a station in the Atlantic. Dots represent CO32concentrations; CO32- concentrations to the right of the curve support calcification,
while concentrations left of the curve cause CaCO3 to dissolve.
18
Figure 4: Calcium carbonate concentration curves for aragonite and calcite by
depth at a station in the Atlantic. Source: Marine Biogeochemical Cycles,
Elsevier, The Open University, 2005, modified by C. Waters.
19
Differences in horizon depths occur because aragonite is ~50% more soluble
in seawater than calcite (Mucci, 1993). Rising saturation horizons may have two
significant effects on ocean biogeochemical processes. First, all marine calcifiers
must reside above respective saturation horizons for CaCO3 precipitation to occur
(The Royal Society, 2005). As saturation horizons rise, marine calcifiers will only
survive in waters closer to the surface if they can rapidly adapt to rising saturation
horizons. Second, premature dissolution of CaCO3 structures from dead organisms
is likely to interfere with normal sequestration of carbon to deep ocean sediments,
thereby altering the ocean’s role in the global carbon cycle.
Understanding how atmospheric conditions affect ionic equilibria of seawater
is fundamental to investigating the biological responses of marine organisms to
ocean acidification. The following section briefly investigates the potential
ramifications of ocean acidification on ecological processes, economic systems, and
social and political activities.
1.4.
Ecological, Economic, Social and Political Implications of Ocean
Acidification
Previous sections summarized the relationship between increased oceanic
acidification and biogeochemical processes such as photosynthesis and calcification
that are critical to marine calcifiers engaged in symbiotic associations. This section
describes how ocean acidification may be the first in a series of cascading events
that influence ecological systems, monetary and resource economies, and social and
political activities.
Ocean acidification may affect multiple biological levels from molecules to
populations that combine to support ecological systems. For example, many
organisms beyond the realm of marine environments may be vulnerable to the
20
effects of elevated CO2 in terms of disturbed internal acid-base balance, oxygen
transport and metabolic processes that influence species growth, survival and
reproduction (Seibel and Fabry, 2003). In addition, ocean acidification coupled with
stressors such as water column stratification and decreased upwelling in response to
warmer temperatures and climate change may adversely affect species diversity and
abundance (Kleypas et al. 2005).
The impact of ocean acidification on the growth and survival of planktonic
organisms is particularly salient because planktonic organisms form the base of the
human food web (Kleypas et al. 2005; The Royal Society, 2005). Pteropods are an
example of planktonic organisms known to be particularly vulnerable to reduced pH
conditions (Orr, et al. 2005). This is important because pteropods are at the base of
a food web involving salmon, herring, mackerel, cod and baleen whales (Knutzen,
1981; Fabry, 2005; Orr, et al. 2005). If ocean acidification decreases the fitness of
marine calcifiers such as pteropods, corals, molluscs and oysters, only species
capable of shifting latitudinal distributions or depth ranges may be favored for
survival (Seibel and Fabry, 2003).
By default, community economies that rely on the well-being of marine
environments may be adversely affected by ocean acidification. For example,
increased ocean acidification has been shown to affect certain species of fish
(Pörtner et al. 2004). Although there is insufficient information to conclude ocean
acidification will affect commercial fish stocks, with global revenues of US $78 billion,
any reductions to existing fish stocks may be economically significant. Coral reef
degradation as a result of ocean acidification may have adverse effects on
commercial fish species (The Royal Society, 2005), further exacerbating direct
threats to fish stocks such as over-harvesting or Allee effects. Coral reefs also have
21
indirect economic benefits since they support world-wide subsistence food gathering
by many millions of people (The Royal Society, 2005).
Coastal economies supported by tourism may be economically challenged if
coral reef ecosystems become compromised as a result of ocean acidification. ‘Reef
interested’ tourism accounts for 68% of Queensland Australia’s total gross regional
product, or AUS$1.4 billion dollars (Hoegh-Guldberg and Hoegh-Guldberg, 2004). In
2000, the World Resources Institute estimated that benefits from Caribbean coral
reefs in terms of fisheries, dive tourism and shoreline protection were between
US$3.1 billion and US$4.6 billion. These values do not account for indirect monetary
contributions coral reefs make to stabilizing coastlines or creating favorable
conditions for other ecosystems such as mangrove forests and sea grass beds
(Hoegh-Guldberg and Hoegh-Guldberg, 2004; The Royal Society, 2005).
Social and political implications of ocean acidification are likely to parallel
social and political implications of global warming. In general, society appears more
focused on removing excess CO2 from the atmosphere than curtailing its production
(Libes, 2005). The scientific community is justifiably frustrated with the rapid rate of
information diffusion but slow social acceptance of scientific fact. Yet, the scientific
community has had immeasurable effects in positively influencing the world’s social
values with respect to the environment. For example, an overwhelming percentage
of today’s “reduce, reuse and recycle” consumer behavior is a result of rigorous
science, cogent explanations and persistent attempts to inform social and political
policy. Continual streams of consistently accurate educational material from
scientists may continue to positively influence social values and political decisions as
much as economic interests. In time, ocean acidification will become a priority item
on the world’s enviro-political agenda. For example, on July 9, 2008, the United
States House Senate Subcommittee on Energy and Environment of the Committee
22
on Science and Technology passed H.R. 4174: The Federal Ocean Acidification
Research and Monitoring Act (FOARAM), a topic completely foreign to politicians just
a few years ago.
1.5.
Biological Responses of Marine Organisms to Ocean Acidification
Research pioneers investigating the effects of oceanic acidification on marine
life controlled seawater pH using acid-base solutions such as hydrochloric acid
(HCl), sulfuric acid (H2SO4) and sodium hydroxide (NaOH). Published results were
compelling; a broad spectrum of marine organisms incubated in pH conditions
exhibited adverse biological responses. Experimental organisms included corals
(Marubini and Atkinson, 1999; Langdon et al. 2003; Langdon and Atkinson, 2005),
pteropods (Orr et al. 2005), foraminifera (Wolf-Gladrow et al. 1999),
coccolithophorids (Riebesell et al. 2000; Zondervan et al. 2001), algae (Gao et al.
1993; Kuffner, 2007), fish and squid (Pörtner et al. 2004), copepods and sea urchins
(Kurihara and Shirayama, 2005), bacteria and nematodes (Takeuchi et al. 1997) and
mollusks (Chalebrese, 1966; Bamber, 1990)
An alternative method for controlling seawater pH involves manipulating
seawater carbonate chemistry using CO2 partial pressure (pCO2) (Burnett 1997). The
differences in chemistry between acid-base control of seawater pH and pCO2 have
been explained by Langdon and Atkinson (2005), and the benefits of using pCO2 to
manipulate pH in studies investigating the effects of ocean acidification on marine
organisms have been explained by Fabry et al. (2008). A series of research
initiatives aimed at quantifying calcification responses of marine organisms exposed
to oceanic pH levels controlled by pCO2 concentrations has recently emerged. Shell
length growth rates of Mytilus edulis mussels incubated for 44 days in pH conditions
ranging from pH 6.7 to pH 8.1 were shown to decline as pCO2 increased (Berge et
23
al. 2006). Calcification rates of M. edulis mussels and Crassostrea gigas oysters
exposed to multiple pCO2 treatments in 2 hour incubation periods declined linearly
as pH declined (Gazeau et al. 2007).
Other research initiatives have examined the effects of ocean acidification on
calcification and other processes. Leclercq et al. (2002) has reported on the effects
of elevated pCO2 on primary productivity, respiration and calcification of a coral reef
community. Iglesias-Rodriguez et al. (2008) recently reported that calcification and
respiration rates in coccolithophores increased as levels of pCO2 increased. Intraand extra-cellular acid-base parameters, metabolic rates and overall specimen
growth of Mytilus galloprovincialis mussels have been observed in normocapnia and
hypercapnia conditions (Michaelidis et al. 2005). Results following 3 months of
incubation revealed a slower rate of shell growth and a corresponding slower rate of
soft-tissue growth of M. galloprovincialis in hypercapnic conditions. Bibby et al.
(2008) suggest that Mytilus edulis mussel responses to ocean acidification may
include impaired haemocytes and associated cellular signaling pathways. Their
results indicate that signaling pathways dependent on specific calcium thresholds
may be compromised as CaCO3 dissolution increases in response to reduced
oceanic pH.
Research initiatives aimed at quantifying biological responses of marine
calcifiers such as corals engaged in symbiotic associations exposed to levels of
acidification controlled by increased pCO2 remain rare (Leclerq, 2002). To date,
biological responses of molluscs engaged in symbiotic associations exposed to
ocean acidification controlled by increased pCO2 are unknown.
24
1.6.
Biological Responses of Tridacna maxima to Ocean Acidification
This project examines the biological responses on Tridacna maxima to
increased pCO2 conditions per IPCC IS92a projections outlined in Table 1: 1)
calcification rates measured as shell length, height and percentages of populations
exhibiting change in shell precipitation will decline as pCO2 increases; 2) calcium
[Ca2+] ion concentrations will increase in mantle tissue as pCO2 increases; 3)
qualitative differences in aragonite shell crystals will be visible as pCO2 increases; 4)
mantle and shell growth rates measured by Condition Index (CI) ratios and net
weight change will differ as pCO2 increases; 5) population density of zooxanthellae
will respond to increases in pCO2; 6) concentrations of chlorophyll a [Chl a] per
zooxanthellae will respond to increases in pCO2.
2.0
Materials and Methods
2.1.
Clam Specimens
One hundred twenty Tridacna maxima clams cultured in the Republic of
Vanuatu were shipped via overnight express to the research laboratory at The
Evergreen State College in Olympia, Washington, USA. Shell lengths ranging from
~18 mm to ~49 mm indicated an estimated age range of 6 to 12 months (Heslinga et
al. 1990). Specimens arrived in individual plastic bags ~8 cm3 filled with source
seawater. Individual bags were set afloat in aquaria described below for >1 hour to
facilitate temperature acclimation of specimens. After manually removing visible
commensal or potentially parasitic organisms, clam specimens were randomly
assigned to four indoor 110-l glass aquaria. Each specimen was fastened by epoxy
to individual pebbles marked with unique location identifiers and placed in permanent
25
positions at the interstices of 1-cm plastic mesh no less than 2 cm away from their
nearest neighbor in each aquarium.
2.2.
Aquaria
The chemical and physical water parameters in each aquarium were
designed to match typical seawater of Oceania over a 13-week incubation period.
Artificial seawater (ASW) in all aquaria consisted of Tropic Marin® Pro-Reef sea salt
mixed with deionized water to an optimal salinity of 34.5 psu. Stock ASW was diluted
with a Marine Biological Laboratory (MBL) seawater recipe (Bidwell & Spotte 1985)
but excluded sodium bicarbonate (NaHCO3-) and was mixed to 34.5 psu. This
carbonate-free artificial seawater (CFASW) was used to offset artificially high total
alkalinity (TA) levels in the commercial ASW mix that could skew pCO2 values. Over
the course of the experiment, elevated TA levels were adjusted downward by
exchanging tank water with CFASW, while low TA levels were adjusted upward by
infusions of commercial ASW.
Each aquarium was fitted with a dedicated Rena® Filstar xP2 water filtration
canister containing mechanical and biological media to circulate aquarium water at
~280 L/h. A Rio® 180 circulation power head augmented surface water circulation.
Finnex® (HMT-150) electronic heaters maintained aquaria temperatures at 27.5º C
(± 1.0). Lighting for each tank consisted of 250 Watt (19000 lumen) MINIHQI250
Pendent/Retrofit Krystal Star metal halide lights with a 11000K spectral composition
for an 11:11 light and dark photoperiod punctuated by 1 h of indirect exposure to 60
Watt “dawn” and “dusk” compact fluorescent lighting provided by Aqualight™. White
foam core light barriers separated each tank to eliminate light spill-over from
adjacent aquaria lights.
26
Every evening, 5 ml of DT’s® Premium Reef blend live marine phytoplankton
was added to each tank, and an additional 5 ml of PhycoPure™ phytoplanktonzooxanthellae mix was added on alternate evenings. Once per week, each tank
received 5 ml of Seachem™ Reef Complete to help maintain optimal calcium (Ca2+),
magnesium (Mg) and strontium (Sr) levels.
2.3.
Carbonate Chemistry
Each tank was fitted with a CO2 bubbling system consisting of 5 lb. gas
bottles of commercially available CO2, Milwaukee MA957 CO2 gas regulators
connected to submerged membrane CO2 reactors, check valves and Aqua Medic
(GmbH) pH controllers. The pH probes dedicated to each controller were
recalibrated every other day using Markson LabSales buffers (pH 4.00, pH 7.00 and
pH 10.00 NIST). The pH controllers were programmed to control tank-specific pH set
points reflecting projected levels of pCO2. Hysteresis ranges maintained pH levels at
± 0.05 margins. Chemical and physical conditions in each tank are shown in Table 2.
For this experiment, all pH values were rounded to the nearest 0.1 per IPCC
estimates with the exception of pH condition 7.76, which was rounded to pH 7.70 to
create even intervals between all pH treatments. A mixture of N2 (79%) and oxygen
O2 (21%) gases were bubbled into the tank with the lowest pCO2 to reach a pH level
of 8.30 (± 0.05) since ambient pCO2 tended to reduce pH well below the targeted
8.30 (± 0.05) control threshold. When the N2/O2 mixture failed to raise pH to desired
levels, 0.4 M sodium hydroxide (NaOH) was added, typically in 0.5 ml increments
that never exceeded 40.0 ml/d. Final chemical and physical conditions in each
aquarium are shown in Figure 1.
27
2.4.
Data Collection
Values for pH, temperature and salinity were recorded at least twice daily for
the duration of the experiment. Observed pH values were collected from Aqua Medic
pH micro-processors. An Oakton® Ion 6 pH/Ion/ºC meter was used to collect
temperature values, and a YSI 3100 conductivity meter (Yellow Springs Instrument
Co., Inc.) was used to measure salinity psu. Values for dissolved oxygen (DO %),
nitrate (NO3-), ammonia (NH3), Ca2+ and TA were collected on a weekly basis. DO
was measured using a YSI 85 meter. Nitrate and ammonia levels were determined
with over-the-counter dip test kits. Qualitative data (color matching) test strips were
used to ensure experimental seawater calcium (Ca2+) ranged from 400 mg l-1 to 450
mg l-1. Frequent ASW exchanges in significant volumes precluded the significance
of measuring Ca2+ using atomic absorption spectrometry.
Total alkalinity values were collected weekly by titrating tank-specific water
with standardized hydrochloric acid (HCl). Acidimetric titration data were copied to
the United States Geological Survey (USGS) on-line Alkalinity Calculator (Rev 2.20).
The fixed endpoint method was used to determine total hydroxide (OH- mg l-1), CO32mg l-1, and HCO3- mg l-1 concentrations. USGS calculations were input to a custom
Excel spreadsheet that calculated 2[CO32-] + [HCO3-]. Total alkalinity output values,
daily mean salinity, temperature and pH (seawater scale) values were entered in the
CO2sys_macro_PC.xls program (Lewis and Wallace, 1998) distributed by the
Carbon Dioxide Information and Analysis Center (CDIAC) to calculate tank-specific
pCO2 levels and other elemental data. Output values were used in determining
whether high-carbonate ASW, CFASW (MBL), or a 325:75 mixture (TA = 2200.8
µmol/kg) of the two was added or exchanged in tanks to attain or maintain alkalinity
thresholds needed to achieve targeted pH values at the chosen pCO2.
28
After a 3-week acclimation period at pCO2 180 ppmv in all tanks, pCO2 levels
in 3 tanks were adjusted upward over a 3-day period to achieve chosen experimental
pCO2 levels for each tank. On a weekly basis, all T. maxima specimens were
removed from respective aquaria, cleaned of algal and parasitic growth, pat-dried,
and measured for shell length, height and weight. Cen-tech electronic digital calipers
(accuracy: 0.02 mm) were used to collect shell length and height values. Shell length
measurements were taken at the maximum lateral distance that touched both caliper
arms. Shell height followed the same protocol except in a vertical direction from the
umbo to the farthest (highest) mantle edge.
A combination of methods for calculating condition indices introduced by
Lawrence and Scott (1982) and Rebelo et al. (2005), was used to determine the
condition index (CI) of each specimen based on dry tissue weight (24 h/60ºC) and
shell volume (CI = dry weight (g) x 100/internal cavity volume ml). Dry tissue and
shell weights were measured using a Mettler Toledo AB204-S balance. Tissue [Ca2+]
of whole animals was measured using a Perkin Elmer Elan, model DRC-e Atomic
Absorption spectrophotometer.
To measure zooxanthellal population density per specimen, the mantle tissue
of each specimen was separated from other organs, blotted dry and weighed. Mantle
tissue was placed in a 5 ml homogenizer containing 3 ml of artificial seawater (ASW)
and ground in a pestle mortar 50 times. Homogenate was centrifuged in a test tube
at 1150 rpm for 5 minutes. Supernatant was drained leaving only zooxanthellal
pellets. Pellets were immersed in 3 ml of ASW and shaken vigorously. Sample
solution was immediately transferred via pipette to a 0.1 mm hemocytometer for cell
counts. Cell counts were multiplied by sample volume to determine zooxanthellal
density per ml. Four replicate counts of zooxanthellae were made for each specimen.
29
Chlorophyll a was measured at 664 nm and 647 nm with a Hewlett Packard 3458
diode array spectrophotometer.
2.5.
Scanning Electron Microscopy Imaging Protocol
Tridacna maxima shells were mechanically cleaned of all soft tissue. Leading
shell edges were fractured and labeled. Every effort was made to collect images
from the same relative location of a cross-section of leading shell edges where
precipitation is expected to be recent. Each shell was placed in 35% hydrogen
peroxide solution for 40 minutes to digest organic material on the surface of the shell.
Shell edges were air dried, sputter coated with gold palladium, mounted in 12 mm
outside diameter Pelco mounting tabs and placed in a Jeol JSM-6480LV Scanning
Electron Microscope with a Control User Interface, Version 7.06 ©2004 Jeol
Techniques, Ltd., for image collection.
2.6.
Statistical Analysis
Statistical significance for all tests was determined at P < 0.05. Intra-
treatment differences were evaluated using Student's t-test, paired two samples for
means. Differences across treatments were evaluated using ANOVA, single factor
analysis, followed by Tukey’s HSD. Reported error (±) represents Standard Error of
the Mean (SEM). Linear regression was used to correlate morphometric parameters.
3.0
Results
3.1.
Chemical and Physical Conditions
Tridacna maxima were successfully incubated for 13 weeks in laboratory
aquaria containing experimental seawater with chemical and physical characteristics
30
listed in Table 2. Mean target values for pCO2 deviated less than 1% from target
values for pCO2 in 3 aquaria. Mean pCO2 values for the fourth aquarium were 340
ppmv versus the target value of 380 ppmv. However, the mean seawater pH level for
the tank with 340 ppmv pCO2 was 8.09, very close to the pH target value of 8.10 (±
0.05) for the duration of the experiment. As pCO2 increased, pH levels declined.
Total alkalinity (TA) gradually declined as pCO2 increased and, as expected,
concentrations of HCO3 increased and CO32- declined as pCO2 increased. All
aquaria were supersaturated with aragonite although Ωar declined to 1.43 in highest
pCO2 conditions. All other chemical and physical parameters across experimental
aquaria were similar for the duration of the experiment. Overall, T. maxima mortality
over the 13-week period was < 4%. One specimen died in pCO2 conditions of 180
ppmv, 3 in pCO2 380 ppmv, 1 in pCO2 560 ppmv and zero in pCO2 840 ppmv.
31
Table 2: Chemical and physical parameters for each tank of experimental artificial seawater (ASW) used to incubate Tridacna
maxima specimens for a 13-week period.
The first set of numbers represents mean values for each control parameter. The second set of numbers represent standard
error (±) values for each parameter, and the third set of numbers represent sample size. 1) Input parameters for CO2SYS.xls
program (K1, K2 from Mehrbach et al. 1973, refit by Dickson & Millero, 1987) based on weekly data collection inputs. 2) TA
values from CO2SYS output converted to meq/kg. 3) CO2SYS output based on inputs (1) above. 4). Weekly Ca2+ ion
concentration values are based on Atomic Absorption analysis of ASW. 5) Mean values based on daily a.m., mid-day and
p.m. collections. 6) Values based on data collected on a weekly basis.
CONDITION
PARAMETERS
(UNITS)
GLACIAL
180 PPMV
(PH 8.30)
CURRENT
380 PPMV
(PH 8.10)
2 X PRE-IND.
560 PPMV
(PH 7.90)
3 X PRE-IND.
840 PPMV
(PH 7.70)
34.5, ± 0.1, 16
34.7, ± 0.2, 14
34.8, ± 0.1, 14
34.2, ± 0.2, 13
Temperature (ºC)
27.5, ± 0.1, 16
27.6, ± 0.1, 14
27.6, ± 0.2, 14
27.7, ± 0.3, 13
1
2180, ± 41, 16
2155, ± 20, 14
2108, ± 45, 14
1983, ± 47, 13
8.3, ± 0.0, 16
8.1, ± 0.0, 14
7.9, ± 0.0, 14
7.7, ± 0.0, 13
2.23, ± 0.04, 16
2.20, ± 0.02, 14
2.16, ± 0.04, 14
2.03, ± 0.04, 13
1
Salinity (‰)
1
TA (µmol/kgSW)
1
pH (Seawater)
2
TA (meq/kg)
32
CONDITION
PARAMETERS
(UNITS)
GLACIAL
180 PPMV
(PH 8.30)
CURRENT
380 PPMV
(PH 8.10)
2 X PRE-IND.
560 PPMV
(PH 7.90)
3 X PRE-IND.
840 PPMV
(PH 7.70)
180, ± 5, 16
340, ± 5, 14
564, ± 13, 14
849, ± 23, 13
3
1391, ± 28, 16
1598, ± 18, 14
1719, ± 38, 14
1720, ± 38, 13
3
317, ± 7, 16
223, ± 2, 14
156, ± 4, 14
104, ± 5, 13
4.6, ± 0.1, 16
3.6, ± 0.0, 14
2.2, ± 0.1, 14
1.4, ± 0.1, 13
411, ± 7, 15
438, ± 5, 14
444, ± 2, 15
435, ± 4, 15
8.3, ± 0.0, 210
8.1, ± 0.0, 210
7.9, ± 0.0, 210
7.7, ± 0.1, 210
34.4, ± 0.1, 210
34.4, ± 0.4, 210
34.9, ± 0.4, 210
34.3, ± 0.3, 210
27.5, ± 0.1, 210
27.6, ± 0.1, 210
27.4, ± 0.5, 10
27.6, ± 0.3, 210
71.4, ± 1.6, 12
77.9, ± 1.3, 14
77.7, ± 3.3, 13
76.2, ± 5.5, 13
448, ± 3, 12
447, ± 3, 12
453, ± 10, 12
450, ± 7, 12
0.0, ± N/A, 12
0.0, ± N/A, 12
0.0, ± N/A, 12
0.0, ± N/A, 12
3
pCO2 (µatm/ppmv)
-
HCO3 (mmol/kgSW)
2-
CO3 ( mmol/kgSW)
3
Ω Ar
Ca
2+
-1
4
(mg l ) (AA)
5
pH
5
Salinity (‰)
5
Temperature (ºC)
6
DO (%)
Ca
2+
-1 6
(mg l )
- 6
Nitrate (NO3 )
33
CONDITION
PARAMETERS
(UNITS)
6
Ammonia (NH3) (ppmv)
GLACIAL
180 PPMV
(PH 8.30)
CURRENT
380 PPMV
(PH 8.10)
2 X PRE-IND.
560 PPMV
(PH 7.90)
3 X PRE-IND.
840 PPMV
(PH 7.70)
0.0, ± N/A, 12
0.0, ± N/A, 12
0.0, ± N/A, 12
0.0, ± N/A, 12
34
3.2.
Calcification Response: Shell Length and Height
Differences in shell length and height of Tridacna maxima specimens between
experimental aquaria at the start of the experiment were not statistically significant
(ANOVA, P > 0.05). However this changed by the end of the experiment. Mean
differences in shell length remained unchanged in aquaria with low pCO2 conditions
(Student’s paired t-test, P < 0.05) (Fig. 5a). However, shell lengths declined significantly
between pCO2 conditions of 180 ppmv and pCO2 840 ppmv over the 13-week period
(ANOVA, P < 0.05, Tukey’s post-hoc analysis).
Differences in shell height within each experimental condition over the 13-week
period were more pronounced (Student’s paired t-test, P < 0.05) (Fig. 5b). Mean
specimen shell heights in the three lowest pCO2 conditions increased, while mean shell
heights of specimens in pCO2 conditions of 840 ppmv decreased. Mean differences in
shell height between populations incubated in the three lowest pCO2 conditions were not
significantly different, but were significantly different in pCO2 conditions of 840 ppmv
(ANOVA, P < 0.05, Tukey’s post hoc analysis).
35
(b)
(a)
0.8
0.4
0.6
0.3
0.28
0.48
∆ Shell Height (mm)
∆ Shell Length (mm)
0.4
0.4
0.2
0.1
0.06
0
-0.07
-0.1
-0.2
-0.23
-0.3
0.23
0.2
0
-0.2
-0.4
-0.43
-0.6
-0.4
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
Treatment (p CO2 )
840 ppm
(pH 7.7)
-0.8
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 5: Mean differences in calcification response rates in terms of (a) shell length
and (b) shell height of juvenile Tridacna maxima clams within populations by pCO2
treatment over a 13-week period. Differences within populations were not statistically
significant (Student’s paired t-test, P > 0.05, ) but were significant between
populations (ANOVA, P < 0.05, Tukey’s post hoc analysis). Y-error bars represent
standard error values (±).
36
Shell length and height revealed striking differences in individual shell growth
patterns between pCO2 conditions. Over half of the specimens incubated in the
lowest pCO2 condition of 180 ppmv exhibited shell growth in terms of both shell
length and height (Fig. 6). In contrast, at the highest pCO2 conditions of 840 ppmv,
over 60% of individual specimens exhibited decreases in both shell length and
height. Individual specimens growing in either one or both dimensions by pCO2
condition were 88.9% in 180 ppmv, 92.3% in 380 ppmv and 86.2% in 560 ppmv.
These growth patterns contrasted sharply with those specimens in pCO2 conditions
of 840 ppmv where only 39.3% of specimens exhibited shell growth in either
dimension. There was no significant correlation between shell length and shell height
at the start of the experiment and end of the experiment.
There was substantial individual variation of growth patterns in response to
pCO2 conditions. All tanks had individuals that increased and decreased in size.
However, the patterns between the tanks at the highest and lowest pCO2 are
striking. In the tank with the lowest pCO2, over half of the individuals increased in
both height and length. In the tank with the highest pCO2, over 60% of the individuals
decreased in both height and length. At pCO2 of 180 ppmv, 340 ppmv and 560
ppmv, 88.9%, 92.3% and 86.2% of the individuals, respectively, exhibited growth in
either or both of the parameters. This contrasted greatly with clams at a pCO2 of 840
ppmv, where only 39.3% of the clams exhibited shell growth in one or both
dimensions.
37
90
76.9
80
70
Population %
60.7
58.6
60
51.9
% BG
50
% BL
40
37.0
% G/L
27.6
30
20
15.4
13.8
11.1
25.0
14.3
7.7
10
0
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 6: Population percentages of Tridacna maxima specimens that gained in
both length and height (BG), lost in both length and height (BL), and gained in
one dimension but lost in the other (G/L) by pCO2 treatment over a 13-week
period.
38
3.3.
Calcification Response: Calcium (Ca2+) Concentrations in Soft
Tissue
Concentrations of Ca2+ ions in dry T. maxima soft tissue after a 13 -week
incubation period in pCO2 conditions of 180 ppmv, 380 ppmv and 540 ppmv were
63.8 mg/g (± 12.3, n = 15), 57.8 mg/g (± 7.7, n = 15) and 61.1 mg/g (± 12.5, n = 15),
respectively, averaging 62.2 mg/g (Figure 7). Differences in pCO2 concentrations
between these populations were not statistically significant (ANOVA, P > 0.05). The
mean concentration of Ca2+ ions in the highest pCO2 condition of 840 ppmv was
~35% higher at 82.4 mg/g (± 8.0, n = 15) dry tissue weight, but this was not
statistically different from the lower pCO2 conditions (ANOVA, P > 0.05).
39
90.00
82.4
80.00
70.00
63.8
61.1
60.00
57.8
50.00
Ca
2+
(mg) per gram of Dry Tissue Weight
100.00
40.00
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 7: Mean differences in Ca2+ ion concentrations per gram of dry tissue
weight of Tridacna maxima mantle tissue by pCO2 treatment at the end of a 13week incubation period were not significantly different (ANOVA, P > 0.05). Yerror bars represent standard error values (±).
40
3.4.
Calcification Response: Aragonite Shell Crystal Conditions
Qualitative comparisons between scanning electron microscope images
suggest conditions of individual aragonite crystals differ as pCO2 increases (Figure
8). Individual shell crystals in pCO2 treatments of 180 ppmv appear well defined by
crisp edges, while individual crystals from shells incubated in higher pCO2 conditions
appear rounded and corroded.
41
(a)
(b)
Figure 8: Qualitative differences in the condition of T. maxima shell aragonite crystals
in pCO2 180 ppmv (a) and pCO2 840 ppmv (b). Images were taken from leading
mantle shell locations where most recent shell extrusion is expected. Source: R.
Peroutka
42
3.5.
Calcification and Soft Tissue Response: Growth Ratios and
Weights
Within each aquarium, mean differences in net weight were significantly
different from introduction through week 13 (Student’s paired t-test, P < 0.05) (Figure
9). The mean net differences in weight gain of specimens in pCO2 380 ppmv
conditions were significantly different from specimens incubated in pCO2 180 ppmv
and 840 ppmv (ANOVA, P < 0.05, Tukey’s post hoc analysis). All specimens in the
lowest and highest pCO2 conditions exhibited net weight gain over the 13-week
incubation period. Five of the 27 clams in pCO2 conditions of 380 ppmv did not
increase in net weight, and 1 of the 29 clams in pCO2 conditions of 560 ppmv did not
increase in weight. Ratios between dry tissue weight and shell weight between all
pCO2 conditions were not significant (ANOVA, P > 0.05) (Figure 10). After a 13-week
incubation period, the differences in ratios between dry tissue weight and shell
volume (Condition Index) values between all pCO2 treatments were not significant
(ANOVA, P > 0.05) (Figure 11).
43
10.00
Net Wet Weight (g)
9.00
8.00
7.89
7.69
7.46
7.16
7.00
7.66
7.49
7.45
7.29
6.00
5.00
4.00
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 9: Differences in mean net weight (g) response of Tridacna maxima
specimens to increased pCO2 at introduction () and at 13 weeks () were
significant within treatment populations (Student’s paired t-test, P < 0.05).
Differences in mean net weight gain in pCO2 380 ppmv were statistically
significant between pCO2 treatments of 180 ppmv and 840 ppmv (ANOVA, P <
0.05, Tukey’s post hoc analysis). Y-error bars represent standard error values
(±).
44
∆ Dry Tissue Weight (g) : Valve Weight (g)
3.4
3.3
3.20
3.2
3.1
3.03
3.01
3
2.90
2.9
2.8
2.7
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 10: Mean dry soft-tissue to shell weight response ratios of Tridacna
maxima to decreased pH conditions over a 13-week period were not significantly
different (ANOVA, P > 0.05). Y-error bars represent standard error values (±).
45
Condition Index
Dry Tissue Weight (g x 100) : Internal Volume (cc)
6
5.5
5.38
5.37
5.17
5.10
5
4.5
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 11: Differences in Condition Index (CI) responses of Tridacna maxima
specimens to different pCO2 conditions over a 13-week period were not
statistically significant (P > 0.05). Index values were derived from dry tissue
weight (g) and shell volume (cc3). Y-error bars represent standard error values
(±).
46
3.6.
Symbiotic Association: Zooxanthellae Population Density
Mean net differences in zooxanthellal population density per gram of
wet mantle tissue over a 13-week period were not statistically significant
(ANOVA, P > 0.05, Tukey’s ad hoc analysis). The highest concentrations of
zooxanthellae were 17.48 x 106 and 18.00 x 106 in pCO2 conditions of 180
ppmv and 560 ppmv, respectively (Figure 12).
47
19
18
18.00
17.48
17
16
6
Zooxanthellae x 10 Wet Weight (g)
20
15
14.58
14
13
12
11.38
11
10
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 12: Mean counts of zooxanthellal population density per gram of Tridacna
maxima mantle tissue by pCO2 treatment after a 13-week incubation period.
Differences in population densities across pCO2 treatments were not statistically
significant. Y-error bars represent standard error values (±).
48
3.7.
Symbiotic Association: Chlorophyll a Density per Algal Cell
The concentration of chlorophyll a [chl a] per zooxanthella cell in the pCO2
treatment of 180 ppmv was 22.84 X 1012 (± 2.75 x 1012), significantly different from
concentrations zooxanthella cells incubated in pCO2 conditions of 840 ppmv 11.5212
(± 1.27 x 1012) (ANOVA, P < 0.05, Tukey’s post hoc analysis) (Figure 13).
Concentrations of chl a per zooxanthella cell in pCO2 conditions of 380 ppmv and
560 ppmv were 16.63 x 1012 (± 1.30 x 1012) and 16.43 x 1012 (± 2.59 x 1012).
Differences in the mean concentration of chl a per zooxanthella cell were statistically
significant between pCO2 180 ppmv and all other treatments (ANOVA, P < 0.05,
Tukey’s post hoc analysis).
49
pg Chlorophyll a per Zooxanthellal Cell
30.00
25.00
22.84
20.00
16.63
16.43
15.00
11.52
10.00
5.00
180 ppm
(pH 8.3)
380 ppm
(pH 8.1)
560 ppm
(pH 7.9)
840 ppm
(pH 7.7)
Treatment (p CO2)
Figure 13: Chlorophyll a content in mantle tissue of Tridacna maxima expressed
as pg chl a per zooxanthella by pCO2 treatment over a 13-week period.
Concentrations of chl a in pCO2 conditions of 180 ppmv were significantly
different from all other treatments (ANOVA, P < 0.05, Tukey’s post hoc analysis).
Y-error bars represent standard error values (±).
50
4.0
Discussion
Results of this experiment indicate that juvenile Tridacna maxima specimens
incubated in pCO2 conditions of 840 ppmv over a 13-week period exhibited negative
biological responses in terms of shell growth, population percentages exhibiting net
shell growth, soft-tissue accumulation of un-precipitated Ca2+ and the condition of
aragonite crystals in their shells. Experimental results also indicate that soft-tissue
development continued even though shell precipitation declined. Although it appears
that elevated pCO2 did not effect the symbiotic association between T. maxima and
zooxanthellae population densities, there was a significant decline in the
concentration of chlorophyll a per zooxanthellal cell as pCO2 increased. In general,
we may tentatively predict that T. maxima clams will begin exhibiting negative
biological responses when oceanic levels of pCO2 exceed ~563 ppmv (pH ~7.90).
4.1.
Materials and Methods
Biological responses of Tridacna maxima specimens incubated in pCO2
conditions of 840 ppmv were particularly acute. From a methodology perspective,
this is significant for two reasons. First, the initial experimental design specified a
control of pCO2 180 ppmv and variable treatments of pCO2 380 ppmv and pCO2 pH
560 ppmv; without adding a pCO2 treatment of pCO2 840 ppmv, little would be
known about extended pCO2 tolerance thresholds of T. maxima. Second, observing
results from a broader spectrum of pCO2 intervals and concomitant pH values helped
identify more specific response tolerance thresholds (cf Gazeau et al. 2007).
Inherent to identifying more specific tolerance thresholds is the ability to begin
classifying organisms as bio-indicators of certain environmental conditions.
Several datasets from T. maxima specimens incubated in pCO2 conditions of
380 ppmv appear anomalous. For example, a comparison of datasets for shell length
51
and height reveals a parallel pattern; calcification rates fluctuate between pCO2 180
ppmv and pCO2 560 ppmv before dropping sharply between pCO2 560 ppmv and
pCO2 840 ppmv. The initial decrease in differences from pCO2 180 ppmv to pCO2
380 ppmv in both metrics remains a mystery. One possible explanation may be the
inadvertent introduction of error in my initial size frequency distribution calculations
for specimens in pCO2 380 ppmv conditions.
4.2.
Chemical and Physical Experimental Conditions
Variation in experimental carbonate chemistry parameters used in other
studies of bivalves make comparisons between this experiment and other ocean
acidification research initiatives challenging. Table 3 shows carbonate chemistry
parameters based in the IPCC IS92a “business as usual” scenario compared to
parameters used for this experiment and parameters used in other investigations of
bivalves to increasing levels of pCO2.
52
Table 3: Comparison between present oceanic carbonate chemistry parameters per IPCC estimates before the end of this
century, this experiment, and other studies of bivalve responses to increasing pCO2, where N/A = Not Applicable, and (*) =
not reported in published parameters but calculated by entering three of four reported parameters into the
CO2sys_macro_PC.xls program (Lewis and Wallace, 1998). In each table cell, upper and lower values reflect upper and
lower boundaries of pCO2 concentrations, respectively.
-
2-
pCO2
(ppmv)
pH
HCO3
-1
(µmol kg )
CO3
-1
(µmol kg )
TA
-1
(µEqiv kg )
Ωarag
Summary Results
180
840
8.32
7.76
1666
2004
279
115
2356
2287
4.26
1.81
N/A
180 ± 5
849 ± 5
8.29 ± 0.05
7.72 ± 0.05
1391 ± 28
317 ± 7
2180 ± 41
4.6 ± 0.1
1720 ± 38
104 ± 5
1983 ± 47
1.4 ± 0.1
Shell dissolution, steady mantle
development, increased soft-tissue
2+
concentrations of Ca , inconclusive
change to zooxanthellal population
density, and decreased chl a per
algal cell were observed at pCO2
between 563 and 840 ppmv.
Crassostrea gigas
(Kurihara et al. 2007)
348
2268
8.21 ± 0.08
7.42 ± 0.02
1506
1825
161.4
26.4
1964 ± 0
1964 ± 0
3.00
0.68
Impaired larval morphology and
inhibited shell mineralization were
observed at pCO2 2268 ppmv.
Mytilus galloprovincialis)
(Michaelidis et al. 2005)
1079
5026
8.05 ± 0.02
7.3 ± 0.0
2890 ± 380
2340 ± 130
254*
373*
6427*
4950*
3.85*
0.57*
Reduced shell length, changed acidbase variables in haemolymph
(HCO3 accumulation), and reduced
O2 consumption were observed at
pCO2 between 1079 and 5026
ppmv.
IPCC
Tridacna maxima
53
-
2-
pCO2
(ppmv)
pH
HCO3
-1
(µmol kg )
CO3
-1
(µmol kg )
TA
-1
(µEqiv kg )
Ωarag
Mytilus edulis
Crassostrea gigas
(Gazeau et al. 2007)
421
2351
8.13
7.46
1916*
2015*
189
53
2421
2546
3.4
1.0
Reduced calcification rates were
observed at 740 ppmv.
Crassostrea gigas
(Gazeau et al. 2007)
698
2774
8.07
7.55
2237*
2562*
170
60
2740
2736
3.1
1.1
Shell dissolution was observed at
1800 ppmv.
Summary Results
54
Explaining the disparity between experimental conditions shown in Table 3 is
difficult because oceanic pH values as a result of increased pCO2 values are
influenced by multiple biotic and abiotic factors (Seibel and Fabry, 2003). In addition,
not all research initiatives report conditions known to affect observed pCO2 values.
Any effort to establish experimental standards for parameters in ocean acidification
investigations is mitigated by known variations in responses of species and species
habitats. However, reporting all measured parameters affecting experimental
carbonate chemistry conditions would aid in evaluating similarities and differences in
variables and responses between research initiatives.
Carbonate chemistry for this experiment over a 13 week period closely
mimicked oceanic conditions predicted by IPCC’s “business as usual” IS92a
scenario with the exception of IPCC estimates for current levels of pCO2. The pCO2
levels for this experiment 340.5 ppmv were ~10% below levels estimated by the
IPCC assuming normal air-sea fluctuations (Gloor, 2003), yet well within the
expected oceanic pH value of 8.10 (± 0.05) at 8.09.
4.3.
Calcification Responses (Shell Growth)
Tridacna maxima calcification responses to increased pCO2 differed from
results of other research initiatives measuring responses of molluscs incubated in
high pCO2 conditions. For example, Michaelidis et al. (2005) observed that growth
rates of Mytilus galloprovincialis decline between normocapnic (pH 8.05 ± 0.02) and
hypercapnic (pH 7.3 ± 0.03) treatments. Conversely, T. maxima shell lengths in this
experiment exhibited negative growth (CaCO3 dissolution) between pH 7.9 and pH
7.72. T. maxima responses also differed from experimental observations of Mytilus
edulis mussels incubated in elevated pCO2 conditions (Berge et al. 2006). M. edulis
mussels incubated in pH treatments ranging from pH 6.7 to pH 8.1 exhibited virtually
55
no growth of shell length at pH 6.7, and no significant difference in growth between
pH 7.4 and pH 7.6. In contrast, the mean change in shell lengths of T. maxima was
~70% lower at pCO2 840 ppmv than pCO2 380. Results of this experiment indicate
that differences in T. maxima growth in terms of shell length occurred at pCO2 levels
well below thresholds considered detrimental to M. galloprovincialis and M. edulis
specimens in similar conditions. This result may indicate that bivalves with more
pronounced periostracum such as M. galloprovinicalis and M. edulis living in
naturally fluctuating pCO2 conditions may be more tolerant to ocean acidification
than T. maxima.
The significance of dissolution responses in terms of Tridacna maxima shell
length and height is reinforced by a sharp increase in the number of individuals
incubated in high pCO2 conditions that exhibited shell size reductions in both length
and height. Studies conducted on Mytilus edulis by Berge et al. (2006) revealed
adverse effects on shell growth between pH 7.4 and pH 7.1. In contrast, Tridacna
maxima specimens in this experiment exhibited negative effects between pH 7.7 and
pH 7.9, well above pH levels that affected M. edulis specimens.
In addition to serving as skeletal support or protection from predators,
calcium carbonate (CaCO3) structures are also a source of carbonate (CO32-) that
can be used to offset naturally occurring spikes in carbonic acid (H2CO3) (Lindinger
et al. 1984; Lowenstam and Weiner, 1989). As a result, concentrations of Ca2+ in
specimen tissue would be expected to increase as pCO2 increases. Concentrations
of Ca2+ ions in the mantle tissue of Tridacna maxima varied slightly between pCO2
180 ppmv and pCO2 560 ppmv before rising to their highest levels at pCO2 > 560
ppmv, perhaps indicative of CaCO3 dissolution in response to hypercapnic
acidification of the seawater.
56
Shell growth responses of T. maxima to increased ocean acidification may be
attributable to one or more mechanisms known to affect calcification. For example, a
decline in CaCO3 precipitation – or increase in CaCO3 dissolution – is expected as
levels of CO32- decline. Calcium carbonate (CaCO3) precipitation rates in corals
decline as pCO2 increases (Kleypas et al. 2005), and CaCO3 precipitation of marine
calcifiers is CO32- limited, not Ca2+ limited (Gattuso et al. 1998; Gazeau et al. 2007).
Alternative mechanisms known to mediate the calcification process include
zooxanthellal enzyme proteins such as carbonic anhydrase (CA) and urease
(Yellowlees et al. 1993; Woolridge, 2008). In addition, competition between host and
symbionts for inorganic carbon also mediates calcification rates (Marubini and
Davies, 1996).
Scanning electron microscopy has been used to view structural changes to
shell crystallization patterns of Tridacna gigas incubated in elevated levels of
ammonium and phosphate (Belda et al. 1993). Precise delineation between recent
and prior shell growth for this experiment was not established, rendering the existing
methodology non-repeatable. However, captured images strongly hint at the
feasibility of investigating changes in aragonite shell conditions in response to
elevated pCO2 similar to changes in conditions in response to elevated nutrients
(Belda et al. 1993). Aragonite crystal conditions in high and low pCO2 for this
experiment suggest at least two areas of future research. First, crystalline lattice
uniformity may contribute to shell density and rate of development, which may be
reduced in high pCO2 conditions. Because Tridacna spp. and other molluscs are
prey to boring organisms, compromised shell density or calcification rates may
adversely affect species fitness (Langdon and Atkinson, 2005). Second, several
types of protein are embedded within crystalline lattice-work of shells (Lowenstam
and Weiner, 1989). Identifying shell protein types and volumes may be used to
57
measure differences in protein synthesis rates as pCO2 increases. The similarity of
images between T. Gigas and T. maxima indicate that introducing x-ray
diffractometry to the methodology and refining techniques for distinguishing
crystallization patterns at topical shell locations before and during treatments may
yield similar results.
4.4.
Calcification / Soft-Tissue Response
Condition Index (CI) comparisons in this study indicate that the soft-tissue of
Tridacna maxima continued growing or stayed the same at higher pCO2, even
though shells either stopped growing or dissolved. This result contrasts with results
observed by Michaelidis et al. (2005), who found that shell growth goes hand in hand
with decreased soft tissue weight for M. galloprovincialis in elevated pCO2
conditions. The potential significance of this result relates to the observed
relationship between increased mean net specimen weights and declining shell size
as pCO2 increased. Mean net increases in weight, in conjunction with declines in
mean net shell size and weight, indicate an uncoupling between photosynthetic
activity and shell growth, not uncommon among photosymbionts in larger temporal
scales (Woolridge, 2008). However, the value of these data is mitigated by the fact
that ratios between tissue and shell growth often fluctuate during different life-history
phases (Green et al. 2004) making definite conclusions hard to reach.
4.5.
Symbiotic Association
The densities of zooxanthellae in this study are within the range of those
observed previously for Tridacna gigas (Fitt et al. 1995). Results of this study
indicate there is no detectable effect of increasing pCO2 on zooxanthellae density
between T. maxima populations as pCO2 increased. One explanation for the initial
58
decline is that as levels of pCO2 increased, fewer zooxanthellae were required to
generate the same volumes of photosynthates. Tridacna maxima hosts may regulate
zooxanthellal density in optimal cost-benefit ratios in terms of energy expenditure
versus photosynthate infusion. Energy conserved by the host may be used to
support other activities such as protein synthesis (Hand 1991), respiration,
reproduction and calcification (Seibel and Fabry, 2004; Hoegh-Guldberg, 2007).
Chlorophyll a per cell in the present study was similar, but ~3-7 times higher
than previously reported for T. maxima (Jantzen et al. 2008). T. gigas incubated in
high nutrient conditions showed increased concentrations of zooxanthellae, but chl a
concentration per zooxanthella cell declined (Belda et al. 1993). These results are
similar to observations of chl a concentrations in zooxanthellae of Tridacna maxima
reported here. Higher pCO2 values resulted in reduced concentrations of chl a,
perhaps indicating that T. maxima can not fully regulate the delivery of CO2 to
zooxanthellal symbionts. Zooxanthellae may regulate concentrations of chl a based
on net photosynthetic productivity; the higher the pCO2, the less chl a per algal cell is
needed to maintain net photosynthetic productivity.
4.6.
Conclusions
The presence of zooxanthellae symbionts in Tridacna maxima complicates
direct comparisons between biological responses of Mytilus galloprovinciallis, Mytilus
edulis, Crassostrea gigas and T. maxima in elevated pCO2 conditions. For example,
results of this experiment indicate that negative shell growth of T. maxima occurred
at pCO2 levels well below thresholds known to negatively affect previously studied
bivalves. This response may be attributable to zooxanthellal carbonic anhydrase
enzyme activity that mediates shell precipitation in normal pCO2 conditions, but may
become inhibited as pCO2 increases. In addition, the presence of photosymbionts
59
may explain the continued development of soft-tissue as pCO2 levels increase, a
response not observed in other mollusc bivalves in similar experimental conditions.
Comparing calcium carbonate precipitation responses of corals in reduced
pH conditions to shell growth responses of tridacnid clams is also challenging for
several reasons. First, T. gigas hosts have been shown to select CO2 as a primary
source of carbon in contrast to coral symbionts that select HCO3- (Legget, et al.
2000). Second, photosynthetic organism preference for HCO3 (Leclerq, 2002)
implies that hosts may prefer CO2 while symbionts favor HCO3. Yet to be fully
explained is whether either species of selected inorganic carbon is used for host
calcification, symbiont photosynthesis, or perhaps a combination of the two carbon
species depending on prevailing pH conditions (Legget et al. 2000). Third, while
corals have been shown to share symbiotic associations with multiple zooxanthellal
species, Tridacnid clams are the only known species of Bivalvia to engage in
symbiotic associations solely with S. microadriaticum (Taylor, 1969). As a result,
unlike corals, Tridacnid clams are not capable of selecting or managing zooxanthellal
clades as a way of temporarily adapting to fluctuating environmental conditions.
Fourth, while coral symbionts maintain intra-cellular positions, S. microadriaticum in
Tridacna maxima are inter-cellular and perhaps more vulnerable to internal acidbase conditions of the host. Fifth, comparing biological responses between Tridacna
spp. and corals is particularly challenging because no research investigating the
effects of ocean acidification as a result of elevated pCO2 on individual species of
hermatypic scleractinian corals has been conducted.
The degree to which increased pCO2 and ocean acidification affects
biological processes vital to marine calcifiers appears critical for inclusion in nearand long-term research objectives. Biological responses of marine calcifiers to ocean
acidification may adversely affect the oceanic carbonate chemistry system, deep
60
ocean carbon sequestration, primary productivity, the global food web and healthy
marine ecosystems. The unique physical and biological attributes of juvenile
Tridacna maxima and their symbiotic partners can offer comparably unique insights
into the biological responses of other marine calcifiers to ocean acidification. Results
of this study appear to indicate that tridacnid clams may be more susceptible to
ocean acidification than corals, and monitoring the health of Tridacnidae may prove
useful in helping predict the effects of increasing pCO2 on coral reef ecosystems.
61
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