Introduction
Calcification is a biomineralization process where many marine organisms,
such as corals, mollusks, polychaetes and echinoderms, deposit carbonate
minerals and form their calcareous shells or skeletons. This process is
highly associated with the fitness and survival of calcifying organisms
because shell growth not only allows continuous somatic growth, but also
strengthens protection against physical and chemical damages. The protective
role of shells is particularly important under life-threatening conditions
(e.g., following non-lethal shell damage), where many calcifying organisms
are able to produce stronger shells at a higher rate to increase physical
protection (Cheung et al., 2004; Brookes and Rochette, 2007; Hirsch et al.,
2014). Indeed, such inducible defence response via enhanced calcification
plays an important role in the survival of calcifying organisms (Harvell,
1990).
In view of the accelerated anthropogenic emission of carbon dioxide,
the calcification and hence defence response of calcifying organisms may be
dampened by climate change stressors, such as ocean acidification and hypoxia
(Bijma et al., 2013). While ocean acidification was expected to retard
calcification (Orr et al., 2005), it is now realized that calcification is
not primarily driven by the pH and carbonate saturation state of seawater
(Roleda et al., 2012), meaning that the impact of ocean acidification on
calcifying organisms through the changes in seawater carbonate chemistry is
less deleterious than previously thought (e.g., Garilli et al., 2015; Ramajo
et al., 2016; Leung et al., 2017a, b). Indeed, calcification is an
energy-dependent physiological process actively regulated by calcifying
organisms (Roleda et al., 2012); therefore, this process is likely determined
by the energetics of calcifying organisms. As such, hypoxia (i.e., dissolved
oxygen concentration in seawater ≤ 2.8 mg O2 L-1 or
≤ 87.5 µmol L-1, Wu, 2002) can probably compromise
calcification through its direct, adverse effect on aerobic metabolism and
hence production of metabolic energy (Wu, 2002; Leung et al., 2013a). Since
calcification is an energy-demanding process (Palmer, 1992), the impaired
aerobic metabolism under hypoxia could be the underlying mechanism causing
the reduced calcification as previously observed (e.g., Cheung et al., 2008;
Wijgerde et al., 2014). As the occurrence of hypoxia is predicted to become
more prevalent in future marine ecosystems owing to ocean warming and
human-induced eutrophication (Diaz and Rosenberg, 2008; Keeling et al., 2010;
Bijma et al., 2013), the impact of hypoxia on calcifying organisms would be
continuously escalated.
However, few previous studies showed that some calcifying organisms are able
to maintain calcification under hypoxia (Mukherjee et al., 2013; Frieder et
al., 2014; Keppel et al., 2016) and even anoxia (Nardelli et al., 2014).
These unexpected results suggest potential mechanisms which can help
compensate for the reduced metabolic energy under hypoxia in order to
sustain calcification. This could be mediated by phenotypic plasticity,
which involves trade-offs between phenotypic traits in response to altered
conditions (Malausa et al., 2005). For example, shell growth may be
maintained under hypoxia at the expense of shell quality or other
physiological processes (e.g., soft tissue growth, reproduction and somatic
maintenance) via energy trade-offs (Nisbet et al., 2012; Sokolova et al.,
2012). Alternatively, energy demand for calcification may be reduced by
changing geochemical properties of shells, which in turn favors shell
growth when metabolic energy is reduced (Ramajo et al., 2015; Leung et al.,
2017b). For instance, bimineralic calcifying organisms (i.e., organisms which
can produce both calcite and aragonite) may precipitate a greater proportion
of calcite to promote shell growth under metabolic stress conditions (e.g.,
ocean acidification, Chan et al., 2012; Leung et al., 2017b) because calcite
has a lower packing density and its production requires less metabolic
energy than aragonite (Weiner and Addadi, 1997; Hautmann, 2006). For
calcite-producing organisms, a small quantity of magnesium ions is
incorporated into the calcite lattice and impacts the quality of shells
(e.g., solubility). While Mg incorporation could be physiologically regulated
by calcifying organisms per se (Bentov and Erez, 2006), this energy-consuming
regulation may be reduced under hypoxia so that more energy can be allocated
to shell growth. To form crystalline calcium carbonate, metabolic energy is
required for stabilization of amorphous calcium carbonate (ACC) because it
involves some matrix proteins and transport of carbonate ions (Addadi et
al., 2006; Bentov, 2010; Weiner and Addadi, 2011). In order to conserve
energy for shell growth, therefore, less crystalline shells may be produced
under hypoxia as the trade-off. Whether calcifying organisms can exhibit
these plastic responses to alleviate the impact of hypoxia-induced metabolic
depression on calcification and defence response remains largely unknown and
deserves a comprehensive investigation.
In this study, we examined how hypoxia affects the calcification and defence
response of a common calcifying polychaete (Hydroides diramphus), which is tolerant to hypoxia
(Leung et al., 2013b). Calcification was indicated by shell growth, while
defence response was indicated by both shell growth and mechanical strength. We analyzed
the mineralogical properties of shells (organic matter content, calcite to
aragonite ratio, magnesium to calcium ratio in calcite and relative
amorphous calcium carbonate content) to indicate the possible changes in
calcifying mechanism in response to hypoxia. Respiration rate and feeding
rate were measured to represent aerobic metabolism and energy gain,
respectively. Given the possible impact of hypoxia on aerobic metabolism, we
hypothesized that (1) the mineralogical properties of newly produced shells
would be modified to reduce the energy demand for calcification so that
shell growth can be sustained and (2) the defence response would be undermined as
the reduced metabolic energy is possibly insufficient to enhance both shell
growth and mechanical strength. If changing mineralogical properties of
shells can help alleviate the impact of hypoxia on calcification and even
defence response without causing significant adverse effects by trade-offs,
this suggests that calcifying organisms may be more robust to
metabolic stress conditions than previously thought.
Materials and methods
Collection and maintenance of specimens
A calcifying polychaete Hydroides diramphus was selected as the
study species, which lives on hard substrate and is widely distributed within
circumtropical regions (Çinar, 2006). Adult polychaetes (tube length:
35–45 mm) were collected from a fish farm at Yung Shue O
(22∘25′ N, 114∘16′ E), Hong Kong, in summer when
hypoxia was commonly observed (Leung et al., 2013a). Other fouling organisms
on the calcareous tube of H. diramphus, such as mussels and
tunicates, were carefully removed. Then, the polychaetes were temporarily
reared in plastic tanks (50 × 40 × 30 cm) filled with
natural seawater under laboratory conditions (dissolved oxygen concentration:
6.00 ± 0.10 mg O2 L-1, pH: 8.10 ± 0.05,
temperature: 28.0 ± 1.0 ∘C and salinity:
33.0 ± 0.5 psu). Algal suspension containing live Isochrysis galbana and Dunaliella tertiolecta (1 : 1, v/v) was daily
provided as food. The polychaetes were allowed to acclimate under these
laboratory conditions for 1 week before experimentation.
Experimental design and rearing method
The impact of hypoxia on the calcification and defence response of adult
H. diramphus was examined using a full factorial experimental
design, involving two dissolved oxygen levels (normoxia vs. hypoxia) and two
contexts (unthreatened vs. threatened). Thus, there were four treatment
conditions based on their crossed combinations: (1) normoxia and
unthreatened, (2) normoxia and threatened, (3) hypoxia and unthreatened, and
(4) hypoxia and threatened. Normoxia
(∼ 6.0 mg O2 L-1, i.e., control) and hypoxia
(∼ 2.0 mg O2 L-1) were achieved by continuously
aerating seawater with air and a mixture of nitrogen and air, respectively
(Leung et al., 2013b). Digital flow meters (Vögtlin Instruments,
Switzerland) were used to adjust the flow rate of each gas (i.e., nitrogen and
air) so that the desired dissolved oxygen concentration for hypoxia was
maintained. To induce a life-threatening condition on the polychaetes,
non-lethal shell damage was done by carefully trimming the calcareous tube
until the radioles were exposed, while the body was still fully covered. The
polychaetes with “intact” (tube length: ∼ 40 mm; body length:
∼ 20 mm) and “damaged” (tube length: ∼ 20 mm; body length:
∼ 20 mm) tubes were then allowed to acclimate under either normoxia or
hypoxia for another week before experimentation, which can particularly help
the “damaged” polychaetes to recover from the stress induced by tube
trimming (i.e., fight-or-flight response) so that they were only subject to
the stress induced by non-lethal shell damage in the following experiments.
A total of 120 adult polychaetes were evenly and randomly assigned to each of
the four treatment conditions (i.e., n=30 polychaetes per treatment). The
rearing method for the polychaetes was previously described (Leung and
Cheung, 2017). Briefly, polychaetes with their initial tube length measured
(see Sect. 2.3) were individually transferred into 2 mL labeled
microcentrifuge tubes with the radioles pointing upward. A small hole
(∼ 2 mm) was drilled at the bottom of each microcentrifuge tube to
allow water exchange. The microcentrifuge tubes were glued together by
hot-melt adhesives (3M, USA) to maintain an upright position and then put
into a lidded glass bottle (10 polychaetes per bottle, 3 bottles per
treatment) containing 180 mL filtered seawater (FSW) (pore size:
0.45 µm). Bottles assigned to the same dissolved oxygen level
(i.e., normoxia or hypoxia) were connected to the same gas inlet and had the target
dissolved oxygen concentration manipulated as described above. Stable
equilibrium between gases in seawater was achieved rapidly by the constant
aeration (< 5 min) and thus the target dissolved oxygen
concentration in seawater, which was daily recorded using an optical
dissolved oxygen probe (SOO-100, TauTheta Instruments, USA), was very stable
over time (Fig. S1 in the Supplement). To simulate the summer seawater
temperature at the collection site, the whole setup was incubated in a water
bath with temperature maintained at 28 ∘C using a heating bath
circulator. The polychaetes were reared under a day/night cycle of
14 : 10 h. Algal suspension (20 mL) containing live I. galbana
and D. tertiolecta (1 : 1, v/v) at ∼1×106 cells mL-1 was provided daily as food to ensure adequate food
supply for normal shell growth. The microcentrifuge tubes were cleaned and
the seawater was renewed once every 3 days to prevent accumulation of
excreted waste. The exposure lasted for 3 weeks, excluding the initial
acclimation period (see Table S1 in the Supplement for the seawater
chemistry). After the 3-week exposure period, only 4 out of 120 polychaetes
died across treatments (2 from “intact, normoxia” and 2 from “damaged,
hypoxia”), meaning that the treatment conditions per se did not
cause fatality.
A micrograph showing the newly produced shell and original shell of
H. diramphus, where the former is easily distinguished from the
latter by the white color. The original shell appears slightly colored due
to the biofilm (e.g., bacteria and algae) growing on the surface in the
field.
Shell growth
Shell growth was indicated by the increase in tube length over time, where
the newly produced shells can be easily identified by the difference in color
from the original shells (Fig. 1). The tube length of all individuals was
measured on day 1, 11 and 21 to estimate
shell growth (n=30 polychaetes per treatment). During the tube length
measurement, a polychaete was temporarily placed in a Petri dish (diameter:
90 mm) filled with seawater at its corresponding dissolved oxygen
concentration to avoid potential desiccation. Tube length was measured under
a dissecting microscope with a scale to the nearest 0.1 mm, followed by
putting the polychaete back to the respective glass bottle immediately
(< 30 s for each measurement). Since tube growth can be measured
with sufficient accuracy and precision under the dissecting microscope, the
tube growth of each individual was analyzed as a replicate.
Cumulative change in the tube length of H. diramphus in
different treatments across the 3-week exposure period (mean ± S.E.; n=30 for “intact, hypoxia” and “damaged, normoxia”; n=28 for
“intact, normoxia” and “damaged, hypoxia” due to the mortality).
Physiological performance
Following the 3-week exposure period, the respiration rate and feeding rate
of polychaetes were measured using the method described in Leung et
al. (2013a) with minor modifications. Briefly, 25 individuals from the same
treatment were randomly sampled and evenly transferred into five airtight
polypropylene syringes (Terumo® hypodermic
syringe without needle, Terumo Corporation, Japan) each containing
∼ 35 mL FSW with dissolved oxygen concentration adjusted to the
corresponding treatment level (n=5 replicate syringes per treatment).
They were allowed to rest in the syringe for 15 min. Then, the initial
dissolved oxygen concentration of FSW was measured using an optical dissolved
oxygen probe (SOO-100, TauTheta Instruments, USA), calibrated according to
the manual of the manufacturer. The atmospheric air inside the syringe, which
helps buffer the change in dissolved oxygen concentration during the resting
period, was then fully expelled and the tip of the syringe was sealed by Blu
Tack to ensure an airtight condition. After 1 h, the final dissolved
oxygen concentration of FSW was recorded when it became steady by gently
stirring the FSW to ensure uniform dissolved oxygen concentration inside the
syringe. Blank samples without individuals were prepared to correct the
background change in dissolved oxygen concentration, which fluctuated less
than 1 %. Respiration rate was expressed as
µg O2 ind-1 h-1.
To measure feeding rate, we determined the decrease in concentration of
microalgae in a given period of time (i.e., clearance rate), as previously
described (Riisgård, 2001; Leung et al., 2013a; Leung and Cheung, 2017).
For each treatment, 25 randomly selected individuals, which had been starved
for 1 day to standardize their hunger level, were put into five glass vials
(i.e., n=5 replicate glass vials per treatment) each containing
80 mL FSW with an initial concentration of ∼1×106 cell mL-1 live D. tertiolecta. After feeding for 1 h under light conditions, 1 mL seawater was taken from the bottle and the
microalgae were enumerated using a hemocytometer (six trials per bottle).
Prior to counting, 1 % Lugol's solution was used to fix the microalgae.
Clearance rate was calculated using the following formula to represent
the feeding rate (Coughlan, 1969):
CR=Vnt×lnCoCt,
where CR is the clearance rate (mL ind-1 h-1); V is the volume
of seawater; n is the number of individuals; t is the feeding time; and
Co and Ct are the initial and final concentrations of
microalgae, respectively.
Shell properties
After measuring respiration rate and feeding rate, the newly produced shells
for the analyses of mechanical and geochemical properties were carefully
removed using a pair of forceps and then rinsed with deionized water to
remove the microalgae and other debris on the shell surface.
Hardness and elastic modulus were measured using a micro-hardness tester (Fischerscope
HM2000, Fischer, Germany) to estimate mechanical strength. For each
treatment, five shell fragments from five randomly selected individuals were
mounted firmly onto a metal disc with the inner shell surface facing upwards
using cyanoacrylate adhesives (n=5 fragments per treatment). Then, the
fragment was indented by a Vickers four-sided diamond pyramid indenter for 10 s
in the loading phase (Peak load: 300 mN; Creep: 2 s). In the unloading
phase, the load decreased at the same rate as the loading phase until the
loading force became zero. At least five random locations on each fragment
were indented. Vickers hardness (H) and elastic modulus (E) were
calculated based on the load-displacement curve using software WIN-HCU
(Fischer, Germany). In this study, the Vickers hardness to elastic modulus ratio (H/E),
which represents the resistance of a material to deformation (Leyland and Matthews, 2000), was
calculated as a proxy for the mechanical strength of shells.
The organic matter content of the newly produced shells collected from
another five individuals was determined by mass loss upon ignition at
550 ∘C in a muffle furnace for 6 h (n=5 replicates
per treatment).
Vickers hardness to elastic modulus ratio (H/E), indicating
mechanical strength, of H. diramphus shells produced in different
treatments (mean + S.E.; n=5).
Given the limited amount of newly produced shells, shells from three to five
individuals from the same treatment were powdered to make one composite shell
powder sample as a replicate for the analyses of the following geochemical
properties. Shell powder was prepared by removing the newly produced shells
using a pair of forceps, rinsing them with deionized water to remove the
microalgae and other debris, drying them at room temperature, and finally
grinding them into powder (particle size: ∼ 5 µm) using a mortar
and pestle. Carbonate polymorphs were analyzed using an X-ray diffractometer
(D4 ENDEAVOR, Bruker, Germany). A small quantity of shell powder was
transferred onto a tailor-made sample holder and then scanned by Co Kα radiation (35 kV and 30 mA) from 20 to 70∘ 2θ with
step size of 0.018∘ and step time of 1 s (n=3 replicates
per treatment). Carbonate polymorphs were identified based on the X-ray
diffraction spectrum using the EVA XRD analysis software (Bruker, Germany).
The calcite to aragonite ratio was calculated using the following equation
(Kontoyannis and Vagenas, 2000):
IC104IA221=3.157×XCXA,
where IC104 and IA221 are the intensity of calcite 104 peak
(34.4∘ 2θ) and aragonite 221 peak (54.0∘ 2θ),
respectively; XC/XA is the calcite to aragonite ratio.
The magnesium to calcium ratio was determined by energy dispersive X-ray
spectroscopy under the Philips XL30 field emission scanning electron
microscope (Ries, 2004; Leung et al., 2017a). A small quantity of shell
powder was transferred onto a stub and coated by carbon (n=3 replicates
per treatment; 3 trials per replicate). The shell powder was irradiated by an
electron beam with an accelerating voltage of 12 kV to obtain the energy
spectrum with background correction. Elements were identified and the magnesium
to calcium ratio was calculated using software Genesis Spectrum SEM Quant
ZAF (EDAX, USA). To determine relative amorphous calcium carbonate content,
1 mg shell powder was mixed with 10 mg potassium bromide, followed by
compressing the mixture into a disc (diameter: 13 mm) using a manual
hydraulic press (n=3 replicates per treatment) (Chan et al., 2012). An
infrared absorption spectrum ranging from 600 to 1800 cm-1 with
background calibration for the baseline was obtained using a
Fourier-transform infrared spectrometer (Avatar 370 DTGS, Nicolet, USA). The relative
ACC content was estimated as the intensity ratio of the peak at 856 to that
at 713 cm-1 (Beniash et al., 1997).
Geochemical properties of H. diramphus shells, including
(a) organic matter content, (b) calcite to aragonite ratio,
(c) magnesium to calcium ratio in calcite and (d) relative
amorphous calcium carbonate content, in different treatments
(mean + S.E.; n=3, except n=5 for organic matter content).
Statistical analysis
Two-way permutational analysis of variance (PERMANOVA) was applied (number
of permutations: 999; Euclidean distance calculated) to test the effects of
hypoxia and non-lethal shell damage on the shell growth, mechanical strength,
organic matter content, calcite to aragonite ratio, magnesium to calcium
ratio, relative ACC content, respiration rate and clearance rate using
software PRIMER 6 with the PERMANOVA+ add-on (Anderson, 2001).
Discussion
Hypoxia is expected to diminish the fitness and survival of marine
organisms, probably leading to serious ramifications on marine ecosystems,
such as changes in species populations, community structure and ecosystem
functioning (Wu, 2002; Diaz and Rosenberg, 2008). Nevertheless, many less
mobile marine organisms (e.g., mollusks, polychaetes and echinoderms) are
generally tolerant to hypoxia in the short term (Vaquer-Sunyer and Duarte,
2008), suggesting their potential capacity to accommodate its impacts.
Despite the substantial reduction in respiration rate and feeding rate under
hypoxia, we found that the calcification and defence response of a calcifying
polychaete were generally maintained, which might be associated with
mineralogical plasticity, such as increased calcite to aragonite ratio and
magnesium to calcium ratio.
SEM images of the inner surface of H. diramphus shells
produced in different treatments, indicating the shell integrity. The
carbonate crystals of newly produced shells appear to be thicker and more
compact following non-lethal shell damage, regardless of the dissolved oxygen
level. Scale bar: 20 µm.
Since energy demand for calcification is enormous mainly due to the
production of the organic matrix (Palmer, 1983, 1992), the reduction in
energy gain by feeding and energy production by aerobic respiration under
hypoxia would undermine both the quality and quantity of shells produced by
calcifying organisms (Cheung et al., 2008; Wijgerde et al., 2014). Under
unthreatened conditions (i.e., without non-lethal shell damage), we found
that hypoxia slightly hinders the shell growth of H. diramphus, but
did not affect the mechanical strength of newly produced shells. The retarded
shell growth under hypoxia could be pertinent to the reduced feeding rate,
and hence energy reserves for calcification. While energy gain by feeding is
suggested to be fundamental for shell growth (Melzner et al., 2011; Thomsen
et al., 2013; Leung et al., 2017b), aerobic respiration is necessary to
efficiently convert energy reserves into metabolic energy for various
biological processes, including calcification. As such, the retarded shell
growth is more likely ascribed to the hypoxia-induced metabolic depression,
which reduces the amount of metabolic energy allocated to calcification. The
quantity of organic matter (e.g., matrix proteins) occluded in the shell is a
key factor affecting mechanical strength (Marin et al., 2008). Since the
organic matter content of newly produced shells was not affected by hypoxia,
mechanical strength can be maintained. Our results imply that a similar
amount of metabolic energy is allocated to the production of organic matter
for shell strength, while less is allocated to inorganic components (i.e.,
calcium carbonate) for shell growth under hypoxia. This strategy (i.e., shell
quality over shell quantity) is favorable under energy-limiting conditions
because there is no exigency to expedite shell growth when risk is not
imminent and the shell can already offer sufficient protection.
Under life-threatening conditions (i.e., following non-lethal shell damage),
H. diramphus exhibited a defence response, indicated by the production
of stronger shells at a higher rate. As H. diramphus is sessile,
enhancing the protective function of shells is probably the most effective
defence response. Therefore, more organic matter was produced and occluded in
the newly produced shell to augment mechanical strength. Additionally, the
carbonate crystals in the shell appeared to be more compacted (Fig. 6), which
could also strengthen the shell. Such inducible defence response is commonly
exhibited by calcifying organisms because shell repair should be prioritized
to restore and enhance protection (Cheung et al., 2004; Hirsch et al., 2014;
Brom et al., 2015). However, trade-offs are involved to activate the defence
response, such as reduction in the less essential biological processes or
activities (Trussell and Nicklin, 2002; Hoverman and Relyea, 2009; Babarro et
al., 2016). For example, Brookes and Rochette (2007) showed that the
calcification rate of a grazing gastropod is promoted under predation risk at
the expense of grazing activity and somatic growth. Here, similar trade-offs
were observed in H. diramphus (i.e., enhanced shell growth against
reduced feeding rate). Indeed, when animals are under life-threatening
conditions and the chance of survival becomes very low, they have to
prioritize defence response (e.g., production of stronger shells for
calcifying organisms) as the last resort to maximize survival rate. This
proposition is corroborated by our results showing that H. diramphus
allocated more metabolic energy not only to enhance shell growth, but also to
synthesize a greater quantity of organic matrix to augment the mechanical
strength of shells following non-lethal shell damage.
We expected that defence response would deteriorate under hypoxia in view of
the substantial energy demand for shell production. Contrary to this
prediction, H. diramphus can still produce stronger shells at a
higher rate (cf. “intact”), meaning that the effect of hypoxia on defence
response is mild in view of the slight impact on shell growth. This
unexpected finding not only reveals the strong tolerance of H. diramphus to hypoxia, but also suggests potential mechanisms that enable
efficient calcification under hypoxia despite the reduced metabolic energy.
We propose that changing mineralogical properties could help compensate for
the reduced metabolic energy in order to sustain the defence response. In fact,
the mineralogical properties of H. diramphus were altered
consistently in response to hypoxia, irrespective of context. We found that
hypoxia resulted in a greater proportion of calcite in the shell. When
metabolic energy is reduced, precipitation of calcite is favorable because
it requires less metabolic energy and allows faster shell growth than that of
aragonite (Weiner and Addadi, 1997; Hautman, 2006; Ries, 2011). For instance,
Ramajo et al. (2015) showed that gastropod Concholepas concholepas
increases calcite precipitation under metabolic depression; Chan et
al. (2012) found that the calcite to aragonite ratio in the shell of
polychaete Hydroides elegans is elevated at pH 7.4, which incurs
metabolic cost for acid–base regulation. Apart from changing carbonate
minerals, we found that more magnesium ions were incorporated into the
newly produced shell under hypoxia. It is evident that the incorporation of
magnesium ions into calcite is actively regulated through various biological
mechanisms, such as active extrusion of excess magnesium ions at the
calcification site (Bentov and Erez, 2006). The elevated Mg / Ca in
calcite under hypoxia may suggest that the energy-requiring regulation of
magnesium ions is reduced to conserve energy, which warrants further
investigation. Furthermore, crystallization of amorphous calcium carbonate
was slightly reduced by hypoxia, indicated by the higher relative ACC
content. Since crystallization requires metabolic energy for the transport of
carbonate ions (Addadi et al., 2006; Weiner and Addadi, 2011), our results
suggest that metabolic energy allocated to crystallographic control also
decreased. Given the aforementioned changes in mineralogical properties, the
energy cost for sustaining shell growth could be lessened. Such plastic
response, also shown in some calcifying organisms under metabolic stress
conditions (Ramajo et al., 2015; Leung et al., 2017b), may explain why the
defence response of H. diramphus can generally be maintained under
mild hypoxia in the short term. Interestingly, we found that such maintenance
can last for at least 3 weeks, even though the energy intake by feeding
was markedly reduced by hypoxia. While the change in somatic tissue was not
examined in this study, it is likely that H. diramphus consumes its
energy reserves to enable the boosted shell growth (Palmer, 1983; Leung et
al., 2013b).
Despite the benefit of changing mineralogical properties as the plastic
response, trade-offs against other phenotypic traits are inevitably incurred
(Malausa et al., 2005; Leung et al., 2013b). For instance, shell solubility
increases due to the higher relative ACC content and Mg / Ca in calcite
(Ries, 2011). In other words, while the changes in mineralogical properties
may allow sustained shell growth and mechanical strength under hypoxia, the
chemical stability of shells may be weakened. Nevertheless, our results
suggest that the benefit of defence response probably outweighs the cost of
this trade-off under life-threatening conditions.
Based on the present findings, we support the paradigm that calcification is
mainly driven by the physiology of calcifying organisms rather than the
seawater carbonate chemistry (Pörtner, 2008; Roleda et al., 2012). For
example, the shell growth of H. diramphus decreased when the carbonate saturation state
slightly increased under hypoxia. This is contradictory to the paradigm that
calcification generally increases with carbonate saturation state and vice versa (Orr et
al., 2005). Indeed, most calcifying organisms do not directly utilize
carbonate ions, but bicarbonate ions, as the substrate for calcification,
meaning that formation of calcareous shells is not a chemical reaction
between calcium and carbonate ions (Pörtner, 2008; Roleda et al., 2012;
Bach, 2015). This concept based on physiology explains why many calcifying
organisms can maintain or even enhance calcification when carbonate
saturation state is reduced (e.g., Ries et al., 2009; Garilli et al., 2015;
Ramajo et al., 2016; Leung et al., 2017b).
Hypoxia can last for a long period of time (e.g., month) as observed in many
coastal and marine open waters worldwide (Helly and Levin, 2004; Diaz and
Rosenberg, 2008) and is predicted to be more prevalent in the future due to
ocean warming and human-induced eutrophication (Bijma et al., 2013). In
order to maintain populations under hypoxia, calcifying organisms have to
counter its impact on calcification. Despite the impaired aerobic
metabolism, this study revealed that hypoxia only mildly hampers the shell
growth of a calcifying polychaete, whereas its defence response (i.e., harder
shells produced at a higher rate) can be sustained in the short term. This
is likely mediated by modifying mineralogical properties of shells to reduce
the energy demand for calcification. While some potential trade-offs are
incurred, such plastic response could be the cornerstone of calcifying
organisms to acclimate to metabolic stress conditions and hence sustain
their populations and ecological functions in coastal and marine ecosystems.