Light-dependent calcification in Red Sea giant clam Tridacna maxima

Tropical giant clams of the subfamily Tridacninae, including the species Tridacna maxima, are unique among bivalves as they live in a symbiotic relationship with unicellular algae and generally function as net photoautotrophs. Light is therefore crucial for these species to thrive. Here we examine the light dependency of calcification rates of T. maxima in the central Red Sea as well as the patterns of its abundance with depth in the field. Red Sea T. maxima show the highest densities at a depth of 3 m with 0.82±0.21 and 0.11±0.03 individuals m⁻² (mean ± SE) at sheltered and exposed sites, respectively. Experimental assessment of net calcification (µmol CaCO₃ cm⁻² h⁻¹) and gross primary production (µmol O₂ cm⁻² h⁻¹) under seven light levels (1061, 959, 561, 530, 358, 244, and 197 µmol quanta m⁻² s⁻¹) showed net calcification rates to be significantly enhanced under light intensities corresponding to a water depth of 4 m (0.65±0.03 µmol CaCO₃ cm⁻² h⁻¹; mean ± SE), while gross primary production was 2.06±0.24 µmol O₂ cm⁻² h⁻¹ (mean ± SE). We found a quadratic relationship between net calcification and tissue dry mass (DM in gram), with clams of an intermediate size (about 15 g DM) showing the highest calcification. Our results show that the Red Sea giant clam T. maxima stands out among bivalves as a remarkable calcifier, displaying calcification rates comparable to other tropical photosymbiotic reef organisms such as corals.

1 Introduction

Giant clams (family Cardiidae, subfamily Tridacninae) are among the largest and fastest growing bivalves on earth, reaching up to 1 m in size (Rosewater, 1965) and growth rates of up to 8–12 cm yr⁻¹ in the largest species, Tridacna gigas (Beckvar, 1981). In the Indo-Pacific, giant clams are considered ecosystem-engineering species (Neo et al., 2015), playing multiple roles in the framework of coral reef communities, such as providing food for a number of predators and scavengers (Alcazar, 1986), shelter for commensal organisms (De Grave, 1999), and substrate for epibionts (Vicentuan-Cabaitan et al., 2014). By producing calcium carbonate shell material they can occasionally even form reef-like structures (Andréfouët et al., 2005). However, due to their specific habitat preference (Yonge, 1975; Hart et al., 1998) and their presumed longevity (Chambers, 2007), Tridacninae are exceedingly vulnerable to exploitation and environmental degradation (Ashworth et al., 2004; Van Wynsberge et al., 2016). In South-East Asia, giant clams have been harvested for human consumption (adductor muscle and mantle meat) and for their shells (Lucas, 1994) already since pre-historic times (Hviding, 1993). Giant clams are also reared in aquaculture farms for the fishkeeping market (Bell et al., 1997) and in an effort to restock the natural population (Gomez and Mingoa-Licuanan, 2006). Currently, all giant clam species are listed in the IUCN Red List of Threatened Species (IUCN, 2016) and protected under Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Most of them are considered to be lower risk/conservation-dependent status; however, the IUCN status of tridacnine species is in need of updating according to Neo et al. (2017). Besides the pressure of fishing on natural stocks, giant clams are also predicted to be vulnerable to the effects of climate change, including heat waves which have been associated with mass die-off events of Tridacninae in French Polynesia (Andréfouët et al., 2013).

Giant clams are one of the few molluscan groups living in symbiotic relationship with dinoflagellates of the genus Symbiodinium (Yonge, 1936; Taylor, 1969; LaJeunesse et al., 2018), likewise to corals and sea anemones. They are generally described as being mixotrophic (Klumpp et al., 1992), obtaining their energy from both filter feeding and photosynthesis; however, some species appear to be even functionally autotrophic (Beckvar, 1981; Jantzen et al., 2008). This dual capacity is assumed to support their fast calcification and growth rates, exceeding those of most other bivalves (Klumpp and Griffiths, 1994). Thus, the availability of light seems to be a critical factor affecting the growth and overall performance of giant clams (Lucas et al., 1989). To date, several studies have examined long-term growth rates of giant clams in response to different environmental factors, such as nutrient enrichment (Hastie et al., 1992; Hoegh-Guldberg, 1997; Belda-Baillie et al., 1998), water temperature (Hart et al., 1998; Schwartzmann et al., 2011), and wave exposure (Hart et al., 1998). Only a few studies assessed net calcification of Tridacninae as a short-term process and how environmental factors, especially light, are influencing the calcification, physiology, and general metabolic rates of Tridacninae.

A positive correlation between light and calcification has been observed in several photosynthetic calcifying organisms, symbiotic (e.g. scleractinian corals) or not (e.g. coccolithophorids and calcifying algae) (Allemand et al., 2011). For corals, the term light-enhanced calcification (LEC) has been coined (Yonge, 1931); however, the underlying mechanisms remain poorly understood and various hypotheses have been proposed. (1) The photosynthetic uptake of carbon dioxide by the symbionts lowers CO₂ levels while increasing pH and the concentration of carbonate ions at the calcification site, which eventually could favour calcium carbonate precipitation (McConnaughey and Whelan, 1997); (2) the removal of inhibiting substances (such as phosphates) by the symbionts during photosynthesis (Simkiss, 1964) or (3) the light-induced production of signalling molecules by the symbionts could lead to an increase in enzymatic activity, essential for the calcification of the host (Ip et al., 2015). Only within the last years has it been possible to investigate LEC mechanisms at the molecular level (Moya et al., 2008; Bertucci et al., 2015), leading to an increasing number of publications reporting light-enhanced expression of enzymes, such as carbonic anhydrase, supporting shell formation in giant clams (Ip et al., 2006, 2015, 2017; Hiong et al., 2017a, b; Chan et al., 2018; Chew et al., 2019). There is also evidence of the light-enhanced expression of gene encoding for those transporters/enzymes needed for calcification within the inner mantle and ctenidium of Tridacna squamosa (Hiong et al., 2017a, b; Ip et al., 2017; Chew et al., 2019). As both tissues lack the presence of symbiotic algae, it has been supposed that light could also directly affect the giant clam host. Despite recent progress in understanding LEC processes in Tridacninae, much remains unknown to date. Previous studies mostly focussed on molecular processes or long-term (several months) effects of light on growth rates, assessed either as an increase in shell length (Lucas et al., 1989; Adams et al., 2013) or total weight (Adams et al., 2013), and did not differentiate between different light intensities. Only a small number of studies actually reported short-term (hours to few days) effects of light on calcification. They either focused on the development of proxies (strontium ∕ calcium ratio) for parameters of the daily light cycle (Sano et al., 2012) through tracer (strontium) incorporation or aimed to understand environmental and physiological parameters controlling daily trace element incorporation, using the total alkalinity (TA) anomaly technique (Warter et al., 2018). As growth and calcification rates in calcifying organisms are considered to be controlled by the corresponding light intensities (Barnes and Taylor, 1973) and as the penetration of light decreases with depth, so the calcification rate is expected to decrease (Goreau, 1963). Tridacna maxima, the most abundant giant clam species in the Red Sea, can be found on shallow reef flats and edges, usually shallower than 10 m, where light intensity is high due to these transparent waters of tropical, oligotrophic oceans (Van Wynsberge et al., 2016). Although tridacnid clams are one of the most dominant and charismatic molluscan taxa in the Red Sea (Zuschin et al., 2000), little is known about their ecology in this area. In addition, the majority of studies on Tridacninae in the region exclusively focused on the Gulf of Aqaba in the northern Red Sea (Roa-Quiaoit, 2005; Jantzen et al., 2008; Richter et al., 2008), which represents less than 2 % of the entire basin of the Red Sea (Berumen et al., 2013).

In the present study, we assessed the net calcification rates (as µmol calcium carbonate per hour) of T. maxima in two short incubation experiments under seven different incident light levels (corresponding to a water depth of 0–14 m) and in the dark, as well as photosynthetic rates at three experimental light levels corresponding to the high-light conditions in shallow waters (0–4 m). Further, we assessed in situ abundances of T. maxima in different depth zones (0.5–11 m) at a sheltered reef and an exposed reef in the central Red Sea. To our knowledge, this is the first study quantifying the light dependence of short-term net calcification rates of tridacnid clams of the Red Sea, interrelating these rates to their abundances in the field.

2 Material and methods

2.1 Clam abundance surveys

Abundance surveys on T. maxima were conducted either via snorkelling or SCUBA diving at two reefs in the eastern central Red Sea (Fig. 1). The first station was Abu Shosha (22.303833^∘ N, 39.048278^∘ E), a small inshore reef, where abundances were examined at the sheltered, leeward side (south-east) of the reef which are relatively protected from wave action and currents (Khalil et al., 2013). Additionally, abundances were assessed at a second station (20.753764^∘ N, 39.442561^∘ E), a fringing reef close to Almojermah, where we conducted transects at the exposed, windward side (north-west) of the reef. At both stations, belt transects were conducted at six different depths (0.5, 1.5, 3, 5, 8, and 11 m). At the sheltered reef, a total area of 1000 m² was covered, and we conducted six transects at each depth. At the exposed reef 560 m² were covered, with three transects at each depth. Transect lines of 25 m were deployed and all T. maxima specimens within 2 m of the transect were counted (e.g. 50 m² area was covered on each transect). In addition, their length (maximum anterior to posterior distance) was recorded at the sheltered reef, using a measuring tape to the nearest centimetre.

Figure 1 (a) Map of the Red Sea. Abundance surveys and sampling of clams for incubation experiments were conducted at both a sheltered reef (Station 1; 22.303833^∘ N, 39.048278^∘ E) and an exposed reef (Station 2; 20.753764^∘ N, 39.442561^∘ E). (b) Satellite image of the sheltered reef (Station 1). (c) Satellite image of the exposed, fringing reef (Station 2).

2.2 Clam incubations to obtain net calcification rates

We determined net calcification (see Sect. 2.4 below) in T. maxima during two consecutive incubation experiments. During the first incubations, conducted in December 2016, we assessed net calcification of T. maxima under four different, moderate experimental light levels, mimicking light intensities at different water depths ranging from 4 to 14 m and during a dark incubation. In November 2016, 20 specimens of T. maxima (shell length of 17±2 cm; mean ± SD) were collected at a water depth of about 4 m at a sheltered reef site (Station 1) (Fig. 2). As T. maxima is often embedded in the substrate, specimens were removed by carefully cutting their byssus with a knife. The incubations took place in December 2016 at the Coastal and Marine Resources Core Lab (CMOR) of King Abdullah University of Science and Technology (KAUST) in Thuwal, Saudi Arabia. The experimental setup consisted of 10 flow-through independent LDPE (low-density polyethylene) outdoor aquaria (30 L). Each aquarium contained 2 clams (in total 20), cleaned with a brush from epibionts prior to the experiment. Aquaria were supplied with water by gravity through an intermediate PVC (polyvinylchloride) tank of 77 L, itself receiving water pumped from the adjacent Red Sea surface water at a flow of 0.22 m³ h⁻¹, leading to a complete water exchange in each single aquarium every 80 min. To maintain ambient Red Sea surface water temperatures, all aquaria were immerged to the last top centimetre in a large flow-through pool of 12 m³, receiving the overflowing water from the intermediate tank and the 10 experimental tanks. An Exo1 probe (YSI Incorporated, Yellow Springs, USA) was used to log water temperature and salinity at 30 min frequency. Both remained constant during the experimental period, with an average temperature of 27.2±0.8 ^∘C (mean ± SD, n=672) and salinity of 38.4±0.8 (mean ± SD, n=672). Experimental aquaria were shaded with nets to reproduce light levels that mimicked natural conditions at different depths on the reef. We conducted short-term incubations of 6 h (from approximately 09:30 to 15:30 mean solar time) under four different shadings and one dark incubation (at night) (n=10), allowing a 3 d acclimatization period to the clams prior to each incubation. During the incubations, the flow-through system was turned off in order to determine changes in seawater carbon chemistry over time as a measure for calcification processes. Photosynthetically active radiation (PAR) was recorded with a light logger (Odyssey Logger, Dataflow Systems Ltd., New Zealand) as µmol quanta m⁻² s⁻¹ and averaged over the incubation period, as natural light conditions fluctuated over the course of the day. Experimental light levels comprised 530, 358, 244, and 197 µmol quanta m⁻² s⁻¹. Using data on depth-dependent decrease in light levels (Dishon et al., 2012), we calculated the extinction of light with water depth. The experimental irradiation levels therefore correspond to incident light conditions at about 4, 8, 12, and 14 m water depths. No additional food was provided, as natural and unfiltered seawater was flowing into the tanks. During the subsequent incubation, conducted in April 2018, we examined net calcification and primary production of T. maxima under three additional experimental high-light levels, addressing light effects encountered in very shallow waters (between 0 and 4 m). We collected eight specimens of T. maxima (shell length of 17±1 cm; mean ± SD) at a water depth of about 4 m at an exposed, fringing reef close to Almojermah (Station 2) (Fig. 2). The incubation experiment was conducted onboard R/V Thuwal in a setup consisting of two big PVC flow-through tanks (350 L each) containing nine individual PVC tanks (10 L), eight of them containing one clam each (cleaned from epibionts) and one serving as a control tank. To maintain ambient Red Sea surface water temperatures, all aquaria were immerged into the flow-through pool and water was constantly pumped (0.36 m³ h⁻¹), ensuring a constant water exchange and movement in the individual tanks. Temperature and salinity were checked four times a day using a handheld CTD (conductivity, temperature, depth) probe (CastAway-CTD, SonTek, USA). Both remained constant during the experimental period, with an average temperature of 31.5±0.3 ^∘C (mean ± SD, n=16) and salinity of 38.2±0.1 (mean ± SD, n=16). During the incubations, the individual tanks were closed air-tight with see-through PVC lids and water movement was generated with battery-driven motors (Underwater motor, Playmobil, Germany). Nets were used for shading and therefore to reproduce light levels that mimicked natural conditions at different depths on the reef. We conducted closed short-term incubations of 3 h (from approx. 11:00 to 14:00 mean solar time) under three different shadings and one dark incubation (at night), allowing 1 d acclimatization to the clams prior to each incubation. Measurements of PAR intensities were identical to the first round of incubations. Experimental light levels comprised 561, 959, and 1061 µmol quanta m⁻² s⁻¹. The amount of light received by the highest experimental light level was identical to light received directly at the water surface in the reef of collection at the same time of the day. Experimental irradiation levels correspond to incident light conditions at 0, 0.5, and 4 m. No additional food was provided, as raw unfiltered seawater was used.

Figure 2 Changes in the abundance (individuals m⁻² ± SD) of T. maxima with depth at a sheltered reef (a) and an exposed reef (b) in the central Red Sea. Different capital letters describe statistically significant differences in abundance between survey depths.

2.3 Carbonate chemistry

At the start, after 3 h and after 6 h of incubation, seawater was sampled from each experimental aquarium in gas-tight 100 mL borosilicate bottles (Schott Duran, Germany) and poisoned with mercury chloride, following Dickson et al. (2007). Each sample was analysed for TA by open-cell titration with an AS-ALK2 titrator (Apollo SciTech,USA) using certified seawater reference material (CRM) (Andrew Dickson, Scripps Institution of Oceanography). During the incubations at moderate light levels (530, 358, 244, and 197 µmol quanta m⁻² s⁻¹), additional samples for dissolved inorganic carbon (DIC) were analysed using an AS-C3 infrared DIC analyser (Apollo SciTech, USA). Further components of the carbonate system were calculated with R package Seacarb (Lavigne and Gattuso, 2013) using first and second carbonate system dissociation constants of Millero (2010) as well as the dissociations of HF and ${\mathrm{HSO}}_{\mathrm{4}}^{-}$ (Dickson, 1990; Dickson and Goyet, 1994), respectively. Carbonate chemistry at the beginning of each incubation and in all experimental aquaria were comparable with mean (± SD) TA of 2324±83 and Ω_Ara of 3.44±0.33 (n=50) during the moderate light incubations and a TA of 2489±38 (n=4) during the high-light incubations (Sect. S1 in the Supplement).

2.4 Net calcification

Net calcification (G in µmol CaCO₃ h⁻¹) was estimated from changes in total alkalinity (TA) using the alkalinity anomaly technique (Smith and Key, 1975) using the following equation (Eq. 1):

$$\begin{array}{}\text{(1)}& G=-{\displaystyle \frac{\mathrm{\Delta }\mathrm{TA}}{\mathrm{2}}}\times {\displaystyle \frac{\mathrm{1}}{\mathrm{\Delta }t}},\end{array}$$

where ΔTA is the variation of TA during the time (t) of the incubations and the factor 2 accounts for a decrease in TA by two equivalents per CaCO₃ precipitated (Zeebe and Wolf-Gladrow, 2001). Calcification rates were expressed relative to either mantle surface area (cm²) or tissue dry mass (g). For mantle surface area, the power relationship between standard length in centimetres (L) and mantle area (cm²) (Jantzen et al., 2008) was used to calculate the mantle surface in cm². For tissue dry mass (DM in gram) of clams, all clams were dissected and their biomass was determined subsequently to the incubation experiment. Clams were opened by cutting the adductor muscle with a scalpel, and the mantle and other tissues were separated from the shells and dried at 60 ^∘C for 24 to 48 h to determine tissue DM to the nearest 0.01 g. Experimentally determined net calcification rates (µmol CaCO₃ h⁻¹) of T. maxima for different DM under four moderate light levels (197, 244, 358, and 530 µmol quanta m⁻² s⁻¹) were used to create a multiple regression modelling net calcification for any given light level and DM.

2.5 Primary production

Primary production was assessed during the high-light incubations (561, 959, and 1061 µmol quanta m⁻² s⁻¹) only. Therefore, oxygen (µmol L⁻¹) content in the incubation chambers was automatically logged (miniDOT, Precision Measurement Engineering, Inc., USA) in 15 min intervals over the 3 h incubation period. Net photosynthesis (NPP) was calculated from the variation of oxygen concentration over time and normalized for clam mantle surface area (µmol O₂ cm⁻² h⁻¹). Dark respiration rates (R), also given in µmol O₂ cm⁻² h⁻¹, were used to calculate gross primary production (GPP) as Eq. (2):

$$\begin{array}{}\text{(2)}& \mathrm{GPP}=\mathrm{NPP}+R.\end{array}$$

2.6 Statistical analyses

To assess the comparisons of clam abundance at the six survey depths, an analysis of variance (ANOVA) and pairwise post hoc Tukey analysis (Tukey HSC) were performed. A statistical model was built to explain calcification rates from the combination of PAR and clam tissue dry mass. The model chosen was a multiple non-linear relationship built as the combination of a linear dependency between PAR and calcification rates and a quadratic dependency of net calcification rates and clam tissue mass. This model was selected against other concurrent models by using the Akaike information criterion (AIC) (Anderson and Burnham, 2004). Statistical analyses were performed using R (Foundation for Statistical Computing, Vienna, Austria, Version 3.4.2) and Statistica (Dell Software).

3 Results

3.1 Depth-dependent abundances

At the sheltered reef site, significantly highest abundances of T. maxima (0.82±0.21 individuals m⁻²; mean ± SE) were observed at a water depth of 3 m (ANOVA, p<0.001, F=35.6; post hoc Tukey test p<0.001; Sect. S2.1), being twice as high as in shallower waters (between 0.41±0.02 and 0.44±0.01 individuals m⁻² mean ± SE; at 0.5 and 1.5 m, respectively) (Fig. 2). No clams were found at the deepest survey depth of 11 m and abundances at 8 m were low, at 0.04±0.01 individual m⁻² (mean ± SE). Giant clams were significantly less abundant in deeper water when compared to shallow reef areas (p<0.001 for both 0.5 and 1.5 m when compared to 8 and 11 m). On average, the density of T. maxima at the sheltered reef (0.5–11 m depth) was 0.32±0.05 individuals m⁻²; mean ± SE). The average size of clams was 16.6±5.1 cm (mean ± SD, n=422) and their calculated mantle surface area was 140.4±90.4 cm² (mean ± SD, n=422), respectively. At the exposed reef, abundances of T. maxima were overall lower, at 0.04±0.01 individuals m⁻² (mean ± SE); however, we also found highest densities of clams at a water depth of 3 m (0.11±0.03 individuals m⁻²; mean ± SE) (ANOVA, p=0.027, F=3.813; Sect. S2.2). However, they were only significantly higher than those found at 8 and 11 m (with mean ± SE of 0.02±0.01 and 0.01±0.01, respectively) (post hoc Tukey test; Sect. S2.2) (Fig. 2).

3.2 Net calcification and primary production

We combined observed net calcification (as the balance between calcification and dissolution) at all seven experimental incident light levels and the dark incubation and identified a polynomial relationship (R²=0.77) between net calcification (NC, µmol CaCO₃ cm⁻² h⁻¹) and incident light (I, µmol quanta m⁻² s⁻¹) (Eq. 3) (Fig. 3):

$$\begin{array}{}\text{(3)}& \mathrm{NC}=-\mathrm{2}\times {\mathrm{10}}^{-\mathrm{6}}\times I^{\mathrm{2}}+\mathrm{0.0019}\times I+\mathrm{0.1643}.\end{array}$$

Among all light incubations, net calcification rates of T. maxima were highest (mean ± SE 0.65±0.03 µmol CaCO₃ cm⁻² h⁻¹) at experimental incident light levels of 530 to 561 µmol quanta m⁻² s⁻¹ (Fig. 3). T. maxima still showed positive but low net calcification during the night (0.18±0.02 µmol CaCO₃ cm⁻² h⁻¹; mean ± SE). The lowest NC rates (mean ± SE of 0.01±0.01 µmol CaCO₃ cm⁻² h⁻¹) were observed at the highest incident irradiance of 1061 µmol quanta m⁻² s⁻¹. Overall, we observed a decline in net calcification with both decreasing and increasing light intensities (Table 1), with polynomial regression indicating the maximum calcification (NC_max) to be reached at an incident light level of 475 µmol quanta m⁻² s⁻¹. From an incident light level of 1033 µmol quanta m⁻² s⁻¹ on, we expect to see dissolution processes outweighing calcification (NC_min, −0.01 µmol CaCO₃ cm⁻² h⁻¹). Gross primary production (GPP) under the high-light incubations (561, 959, and 1061 µmol quanta m⁻² s⁻¹) showed an identical decreasing trend with increase in incident light as observed for net calcification. At 561 µmol quanta m⁻² s⁻¹, GPP was highest (2.06±0.24 µmol O₂ cm⁻² h⁻¹; mean ± SE) and production rates were significantly lower (ANOVA, p=0.039, F=4.982; Sect. S3) during the incubations at 959 and 1061 µmol quanta m⁻² s⁻¹ (Table 1, Fig. 3), with mean ± SE of 1.76±0.28 and 0.87±0.37 µmol O₂ cm⁻² h⁻¹, respectively. Two specimens died after the second highest light treatment of 959 µmol quanta m⁻² s⁻¹.

Table 1 Net calcification (µmol CaCO₃ cm⁻² h⁻¹; ± SE) and gross primary production (µmol O₂ cm⁻² h⁻¹; ± SE) under the seven experimental incident light levels (µmol quanta cm⁻² h⁻¹) and in the dark.

NA – not available

Figure 3 Boxplots showing net calcification rates (µmol CaCO₃ cm⁻² h⁻¹) of T. maxima under seven different light regimes (197, 244, 358, 530, 561, 959, and 1061 µmol quanta m⁻² s⁻¹) (n=10 in December 2016 and n=8 in April 2018) and in the dark, as well as gross primary production (µmol O₂ cm⁻² h⁻¹) (n=8) as dots (± SE), under three high-light regimes (561, 959, and 1061 µmol quanta m⁻² s⁻¹). Calculated maximum net calcification (NC_max) at 475 µmol quanta m⁻² s⁻¹ and incident light level where dissolution outweighs calcification processes (NC_min) are symbolized by a cross (×). Net calcification rates obtained during incubations under moderate light conditions are symbolized by light grey boxplots and those from the high-light incubations by dark grey boxplots, the central line represents the median, the boxes encompass the central 50 % of the data, and the lines extend to the 95 % quartiles.

We identified a quadratic relationship between net calcification and tissue dry mass, with clams of an intermediate size (DM of about 15 g), showing the highest calcification rates at the four incubations at moderate light level (197, 244, 358, 530 µmol quanta m⁻² s⁻¹) (Fig. 4). Therefore, we combined the influence of light and dry mass into a statistical model, explaining 77 % of the variance in observed calcification rates (all parameters p<0.05, Table 2, Fig. 5). Based on this model, we identify maximum rates on clams of an intermediate size (DM of about 15 g), showing the highest calcification rates at the four light-level incubations.

Table 2 Description of the statistical model parameters (Fig. 5) combining the influence of irradiance (E, µmol m⁻² s⁻¹) and clam dry mass (DM; g) on calcification (G; µmol CaCO₃ ind⁻¹ h⁻¹), where d is the fitted intercept.

R²: 0.77

Figure 4 Net calcification (µmol CaCO₃ ind⁻¹ h⁻¹) (n=10) in T. maxima at four different incident light levels (197, 244, 358, and 531 µmol photons cm⁻² h⁻¹) and in the dark, plotted against tissue dry mass (g). Data are shown with polynomial trend lines.

Figure 5 Model considering a linear relationship between light and calcification and a first-order polynomial relationship between dry mass (DM) and net calcification (NC) explaining 77 % of the variance.

4 Discussion

4.1 Depth-dependent abundances

In the Red Sea, T. maxima shows a significant dependence of net calcification rates on incident light. This light dependency is consistent with significantly higher abundances of this species in shallow, sunlit reef flats. Globally, densities of T. maxima range between 0.1 and 0.0001 individuals m⁻² (Van Wynsberge et al., 2016), with some exceptions such as at the Ningaloo Marine Park in Western Australia with 0.86 clams m⁻² (Black et al., 2011), the Egyptian Sinai Peninsula with a peak value of 0.80 clams m⁻² (Roa-Quiaoit, 2005), and 0.42 clams m⁻² in Kiribati (Chambers, 2007).

At water depths between 0.5 and 11 m, we found averaged (± SD) abundances of T. maxima of 0.04±0.01 individuals m⁻² and 0.32±0.05 individual per m⁻² (mean ± SE) at an exposed reef and a sheltered reef, respectively. Abundances at the sheltered reef rank amongst the highest abundances reported worldwide, representing a 50 % higher abundance than previously reported for a local reef (Bodoy, 1984) with 0.22 clams m⁻². This difference in average abundances between the two reefs observed in this study could be explained by the leeward and windward (sheltered or exposed, respectively) characters of the examined sites. As reviewed by Van Wynsberge et al. (2016), the “reef type” can influence Tridacna abundances, as it potentially affects the water exchange (and thus water temperature and nutrient availability) as well as the exposure to waves. Similarly to Roa-Quiaoit (2005), we found that T. maxima abundances in the Red Sea seem to display great differences between locations, as we found significant lower numbers of giant clams at the exposed reef, with an average of 0.04±0.01 individuals m⁻²; (mean ± SE) at water depths between 0.5 and 11 m. Explanations for the observed contrasts in numbers of clams per m² at both reefs could lie in the probable differences in abiotic environmental conditions at the surveyed sites. For instance, giant clams at the exposed reef are potentially more at risk from high wave action than at the sheltered reef site, which could impact the initial settlement (Jameson, 1976) as well as the survival of juveniles (Foyle et al., 1997), as both have been shown to be influenced by geographical factors (Foyle et al., 1997). While a previous study (Militz et al., 2015), in which abundances of giant clam species in French Polynesia were examined, reports similar patterns for T. crocea, opposite patterns were observed for abundances of T. maxima in that region. In the reefs surveyed by Militz et al. (2015), T. maxima showed higher abundances at reef sites with a high exposure, in comparison to those with low exposure levels. However, additional factors such as temperature and local geomorphology might also have an influence on giant clam densities. Therefore, it is not possible to confidently identify the underlying causes of the observed differences by considering exposure alone. For example, T. maxima specimens from our study, which were located at the more southern reef, could possibly also be exposed to higher surface water temperatures due to the location of this reef at lower latitudes. Mean sea surface annual temperatures of the Red Sea have been shown to increase towards lower latitudes and can be as high as 33 ^∘C in the central and southern Red Sea (Chaidez et al., 2017). Further, the local geomorphological features of each reef could influence the light availability of benthic habitats. Consequently, differences in the local topography could have led to different angles of incident light and shading conditions, which would then result in differences between reefs even though the examined depths are identical.

Contrasting findings to previously reported Tridacninae abundances in the central Red Sea could be further a result of differences in sampling depths in the respective studies, as e.g. Bodoy et al. (1984) only accounted for clams at water depths of a maximum of 2 m, while we assessed abundances of T. maxima at six different depths (0.5, 1.5, 3, 5, 8, and 11 m). Previous studies have shown that the depth of abundance surveys significantly impacts the estimates (Van Wynsberge et al., 2016), even though generally, the highest densities of T. maxima are always reported for shallow reefs (0–5 m) (Jantzen et al., 2008; Andréfouët et al., 2009). This is also reflected in the results of previous studies in the Red Sea (Roa-Quiaoit, 2005) showing the highest abundances of T. maxima in shallow water (<3 m). However, Roa-Quiaoit (2005) accumulated abundances at all depths less than 3 m, while we differentiated even between the 0.5, 1.5, and 3 m depth levels and thereby found that although T. maxima shows the highest density at 3 m, abundances in shallower depths are significantly reduced. Furthermore, we found only a few specimens of T. maxima at water depths between 5 and 11 m. This finding is similar to previous studies describing T. maxima as being mostly restricted to reefs shallower than 10 m, principally reef flats and edges (Van Wynsberge et al., 2016). This depth distribution is most likely a result from a trade-off between maximizing light-dependent photosynthesis while minimizing temperature stress, UV irradiation, wave exposure, and/or emersion stress. All these stressors have been previously reported to lead to massive bleaching and mass die-off events in T. maxima (Addessi, 2001) and prevent settlement and recruitment in the shallow waters of the reef flat (Watson et al., 2012). The average size of T. maxima specimens at the sheltered reef was 16.6±5.1 cm (± SD), similar to previous studies on this species in the Red Sea (Roa-Quiaoit, 2005), corresponding, according to the size classification by Manu and Sone (1995), to broodstock (i.e. sexually mature individuals) hermaphrodites. However, the number of small, juvenile specimens (<4 cm) is potentially underestimated, as they are extremely cryptic (Munro and Heslinga, 1983).

4.2 Light-dependent calcification and production in Red Sea giant clam T. maxima

Overall, we found significantly enhanced net calcification rates in Red Sea T. maxima during light incubations compared to the dark incubation. Net calcification rates also significantly increased with light intensity up to 475 µmol photons m⁻² s⁻¹ (incident light level corresponding to a water depth of approximately 5 m, at the same time of the day and season, when the incubations were conducted), and thenceforward decrease until an eventual dominance of dissolution over calcification at approximately 1033 µmol photons m⁻² s⁻¹ (corresponding to light conditions received directly at the air–water interface in the reef of the collection). Likewise to net calcification in T. maxima, we observed gross primary production (GPP) to be highest at intermediate light levels of around 560 µmol photons m⁻² s⁻¹ (corresponding to a water depth of about 4 m) and to decrease with increasing light intensities (at 959 and 1061 µmol quanta m⁻² s⁻¹, corresponding to 1.5 and 0.5 m water depth, respectively). We conclude that net calcification in the Red Sea giant clam T. maxima is not only enhanced by light, but is also likely coupled to the photosynthetic activity of their algal symbionts. Further, our results show that both net calcification and primary production in Red Sea T. maxima are highest at incident light levels received at water depths between 5 and 3 m at Red Sea reefs. This is especially noteworthy as these findings correlate with the observed depth-related abundances of T. maxima, displaying the highest densities at intermediate water depths around 3 m in the central Red Sea. The observed irradiance optima for both net calcification and primary production of T. maxima could therefore provide an explanation for the maximum in abundances in intermediate waters (3–5 m) and the decreasing numbers of observed clams at both shallower and deeper reef sites.

Table 3 Comparison of net calcification rates in relation to light conditions in different marine phototrophic and mixotrophic calcifiers. Values are given as average value mean (± SE) or ^a (± SD). Experimental light incubation levels are given in µmol photons m⁻² s⁻¹. Net calcification values were converted to µmol CaCO₃ cm⁻² h⁻¹ from ^bmg CaCO₃ cm⁻² d⁻¹.

Overall, our findings of enhanced calcification rates under light are consistent with reports on the related species Tridacna gigas (Lucas et al., 1989), Tridacna derasa (Sano et al., 2012), and Tridacna squamosa (Adams et al., 2013). The mechanisms of LEC have been intensely studied in zooxanthellate scleractinian corals, leading to several hypotheses proposed to explain LEC (Tambutté et al., 2011). The majority of these refer to mechanisms that are influenced by the symbiotic relationship of host and Symbiodinium, with the most supported hypothesis relating photosynthetic CO₂ uptake by the algal symbionts to increased pH and the concentration of carbonate ions, thereby favouring calcification through the corresponding elevated saturation state for carbonate minerals (McConnaughey and Whelan, 1997).

Table 4 Comparison of net calcification rates of different marine heterotrophic calcifiers. Values are given as average value mean (± SE) or ^a (± SD). All net calcification values were normalized for gram dry mass (DM); rates given in fresh weight were converted to DM ^b following Ricciardi and Bourget (1998) or ^c Dame (1972). Net calcification values were converted to µmol CaCO₃ g DM⁻¹ h⁻¹ from ^d µg CaCO₃ g DM⁻¹ d⁻¹.

Giant clams, including T. maxima, can potentially harbour multiple genera of Symbiodiniaceae simultaneously (DeBoer et al., 2012; Ikeda et al., 2017), including Symbiodinium, Cladocopium, and Durusdinium (previously referred to as clades A, C, and D; LaJeunesse et al., 2018; DeBoer et al., 2012). The composition of these associated algal symbionts might therefore also impact the susceptibility to (high) light levels, as different genera of Symbiodiniaceae (in symbiosis) exhibit different physiological and ecological patterns, including sensitivity to light and temperature (Rowan et al., 1997; Berkelmans and Van Oppen, 2006). However, a previous study on Red Sea giant clams and their associated Symbiodiniaceae (Pappas et al., 2017) reports that T. maxima in the region exclusively associated with Symbiodinium spp. (previously clade A), which was thus assumed to represent an optimal group for the local environmental conditions. However, the reliance of calcification of host organisms (e.g. T. maxima) on their relationship with symbiotic algae could provide an explanation for the significant decrease in net calcification rates at the highest light treatment (1061 µmol photons m⁻² s⁻¹). These diminished rates could be the result of photoinhibition and even photodamage of the associated unicellular algae when exposed to these high incident light levels. This would also be supported by the pronounced decrease in gross primary production rates at this light treatment. High incident light levels, especially high levels of UV radiation in shallow waters, have been previously shown to be correlated with decreased calcification rates in other marine calcifiers such as stony corals, e.g. Porites compressa (Kuffner, 2001). However, recent findings for hermatypic corals also report that the contribution by the symbionts might not be the primary or sole driver for LEC, but the blue light spectrum could trigger the light sensors of the host itself, leading to higher calcification rates (Cohen et al., 2016). It is suggested that blue light photoreceptors in coral tissues of Porites lutea and Acropora variabilis could potentially sense the light which is ultimately activating a cascade of processes involved in blue light-enhanced calcification (Cohen et al., 2016). However, our experimental light level, produced by different layers of neutral screen shading, only differed in light intensities but not in the wavelength that T. maxima would receive in the respective water depth. In a previous study on Tridacna crocea, short-term calcification rates were also reported to be strongly light dependent (Warter et al., 2018). However, in this their experiment, Warter et al. (2018) exposed the clams not only to artificial light, but also light levels that were not comparable to actual conditions in the environment, as the average treatment comprised only 162±7 µmol quanta m⁻² s⁻² (corresponding to a water depth of approximately 16 m in an oligotrophic ocean such as the Red Sea).

4.2.1 Allometric relationship between calcification and biomass

We determined a non-linear relationship between net calcification and biomass (as tissue DM) in T. maxima. Clams of an intermediate DM of approximately 15 g showed the highest net calcification throughout the four incubations as moderate light levels (530, 358, 244, and 197 µmol quanta m⁻² s⁻¹). Specimens of a smaller or higher biomass calcified less during the incubations. A similar allometric relationship has been previously described for the photosynthetic metabolic performance of the zooxanthellae in T. maxima (Yau and Fan, 2012). This allometric pattern is most likely due to an optimal ratio of symbionts to clam body mass at intermediate sizes. As the clam grows, its mantle tissue increases in thickness and thus the three dimensional tubular systems bearing the utmost of symbionts (Fisher et al., 1985). However, as the mantle thickens, impinging light must penetrate through more tissue before reaching the stacked zooxanthellae (Trench et al., 1981), and there is evidence of increased shading of the symbionts in the mantles of bigger clams (Fisher et al., 1985). With further increasing size, the number of symbionts per unit clam biomass also decreases (Fisher et al., 1985; Fitt et al., 1993; Griffiths and Klumpp, 1996). In general, growth rates of giant clams seem to decrease with age once they reach the threshold for maturity and become broodstock hermaphrodites (Van Wynsberge et al., 2016). Past this age, a growing portion of their energy is invested in reproduction (Romanek and Grossman, 1989; Van Wynsberge et al., 2016), especially since there is an exponentially increase in produced egg numbers with increasing shell size (Jameson, 1976).

4.2.2 Comparison with other calcifiers

We compared net calcification rates of T. maxima with those of other benthic phototrophic and mixotrophic calcifiers (Table 3). In most calcifying organisms that live in symbiotic relationship with zooxanthellae (such as corals), metabolic rates and calcification are normalized by surface area. In contrast to corals, however, which host their symbiotic algae intracellularly in their endodermal cell layer, the symbionts of Tridacninae are located in delicately branching and specialized channels within the mantle, which extend from the stomach (Trench et al., 1981; Norton et al., 1992). Although this difference makes the comparison to other calcifiers conceptually difficult, normalization of calcification rates per mantle surface area would also be appropriate, as Symbiodinium cells in giant clams are mostly found in the upper 5 mm of the mantle (Ishikura et al., 1997). The Red Sea giant clam T. maxima shows averaged net calcification rates of 0.47±0.03 µmol CaCO₃ cm⁻² h⁻¹ (mean ± SE), which are comparable to those reported for hermatypic corals (0.42±0.08 µmol CaCO₃ cm⁻² h⁻¹; mean ± SE) and those for calcifying macroalgae (0.43±0.38 µmol CaCO₃ cm⁻² h⁻¹; mean ± SE). However, in comparison with averaged rates of other heterotrophic, temperate bivalve species, such as Mytilus edulis, Argopecten purpuratus, and Crassostrea gigas with 0.08±0.07 µmol CaCO₃ g DM⁻¹ h⁻¹ (mean ± SD), calcification in T. maxima is about 70 times higher (5.38±0.42 µmol CaCO₃ g DM⁻¹ h⁻¹; mean ± SE) (Table 4). When compared to heterotrophic cold-water coral species, such as Lophelia pertusa and Madrepora oculata, net calcification rates of T. maxima are more than 7 times higher (0.80±0.70 µmol CaCO₃ g DM⁻¹ h⁻¹; mean ± SD); Table 4). Our comparative assessment of the net calcification rates of giant clams with temperate/azooanthellate species show that rates in the Red Sea T. maxima tested here are comparable to other photosymbiotic organisms (such as corals) and calcifying algae.

5 Conclusion

The present study shows that net calcification and photosynthetic rates of Red Sea T. maxima are light dependent but show a maximum at intermediate irradiance, suggesting strong inhibition at the highest incident light levels received in very shallow (0–1.5 m) waters. This is consistent with the depth-related distribution of this species in the Red Sea, and elsewhere, which showed maximum abundances in shallow (3 m), sunlit coral reefs but a decrease in abundance from 3 m towards the surface and below. Although enhanced calcification is consequently beneficial for T. maxima, the light dependency of both calcification and production restricts them to shallow waters, which also makes them more vulnerable to potentially harmful environmental changes, such as predicted increasing water temperatures associated with global warming (Hughes et al., 2003) as well as high levels of incident light, including high levels of UV radiation (Shick et al., 1995). The present study provides an important baseline for future studies examining the impact of wavelength-specific responses of calcification and metabolic rates on giant clams as well as for a better overall understanding of light-enhanced calcification in Red Sea Tridacninae and their relationship with the symbiotic algae.