Interactive comment on “ Light-dependent calcification in Red Sea giant clam Tridacna maxima ” by Susann Rossbach et al

Rossbach et al. demonstrated depth-dependent abundances of Tridacna maxima in natural reefs and experimentally examined short term net calcification rates of T. maxima in different light conditions. Tridacna is abundant bivalves in coral reefs and has demand as fishery resources and environmental proxies. However, the knowledge about their calcification rates are scarce. While calcification rates of tridacna shells seem to be also strongly related to temperature conditions (Warter et al., 2018), this study provide new insight of the relationship between their calcification and light. I recommend this paper published in “Biogeosciences” after some revisions.


Introduction
Giant clams (Family Cardiidae, Subfamily Tridacninae) are among the largest and fastest growing bivalves on earth, reaching up to one meter 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 Southeast Asia, giant clams are 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 of restocking 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 under a 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 as corals and sea anemones.They are generally described as being mixotrophic (Klumpp et al., 1992), obtaining their energy both from 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 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 it has 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(Ip et al., , 2015(Ip et al., , 2017;;Hiong et al., 2017aHiong et al., , 2017b;;Chan et al., 2018;Chew et al., 2019).There is also evidence for the light enhanced expression of genes encoding for those transporters / enzymes needed for calcification within the inner mantle and ctenidium of Tridacna squamosa (Hiong et al., 2017a;Hiong et al., 2017b;Ip et al., 2017;Chew et al., 2019).As both tissues are lacking 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 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) effect 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 is the calcification rate 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 -14m) and in the dark, as well as photosynthetic rates at three experimental light level corresponding to the high light conditions in shallow waters (0 -4m).Further, we assessed in situ abundances of T. maxima in different depth zones (0.5 -11m) at a sheltered 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 with their abundances in the field.

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,39.048278E), a small inshore reef, were abundances were examined at the sheltered, leeward side (Southeast) of the reef, which are relatively protected from wave action and currents (Khalil et al., 2013).Additionally, abundances were assessed at a second station (20.753764N, 39.442561 E), a fringing reef close to Almojermah, were we conducted transects at the exposed, windward side (Northwest) of the reef.At both stations, belt transects were conducted in six different depths (0.5, 1.5, 3, 5, 8 and 11 m).At the sheltered reef, a total area of 1,000 m 2 was covered and we conducted six transects at each depth.At the exposed reef 560 m 2 were covered, with three transects at each depth.Transect lines of 25 m were deployed and all T. maxima specimen within 2 meters of the transect where counted (e.g., 50m 2 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 cm.

Clam incubations to obtain net calcification rates
We determined net calcification (see section 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 level, mimicking light intensities at different water depths ranging from 4 to 14 m and during a dark incubation.
In November 2016, 20 specimen of T. maxima (shell length of 17 ± 2 cm; mean ± SD) were collected in 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 ten flow-through independent LDPE (low density polyethylene) outdoor aquaria (30 L).Each aquaria contained two 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 m 3 h -1 , leading to a complete water exchange in each single aquaria every 80 minutes.To maintain ambient Red Sea surface water temperatures, all aquaria were immerged to the last top cm in a large flow-through pool of 12 m 3 , 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 hours (from approx.09:30 to 15:30 mean solar time) under four different shadings and one dark incubation (at night) (n = 10), allowing 3 days 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 of 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 depth.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 level, addressing light effects encountered in very shallow waters (between 0 and 4 m).We collected eight specimen of T. maxima (shell length of 17 ± 1 cm; mean ± SD) in a water depth of about 4 m at an exposed, fringing reef close to Almojermah (Station 2) (Fig. 2).The incubation experiment was conducted on board of R/V Thuwal in a setup consisting of two big PVC flow-through tanks (350 L each), containing 9 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 m 3 h -1 ), assuring a constant water exchange and movement in the individual tanks.Temperature and salinity were checked four times a day using a handheld CTD 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 airtight 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 hours (from approx.11:00 to 14:00 mean solar time) under three different shadings and one dark incubation (at night), allowing one day 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 m, 0.5 m and at 4 m.No additional food was provided, as raw unfiltered seawater was used.

Carbonate chemistry
At the start, after three and after six hours of incubation, seawater was sampled from each experimental aquaria in gas tight 100 mL borosilicate bottles (Schott Duran, Germany) and poisoned with mercury chloride, following (Dickson et al., 2011).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 the 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 HSO4 - (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 ΩAra 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 (Supplementary Material_S1).

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): Where ΔTA is the variation of TA during the time (t) of the incubations and the factor 2 accounts for a decrease of TA by 2 equivalents per CaCO3 precipitated (Zeebe and Wolf-Gladrow, 2001).Calcification rates were expressed relative to either mantle surface area (cm 2 ) or tissue dry-mass (g).For mantle surface area, the power relationship between standard length in cm (L) and mantle area (cm 2 ) (Jantzen et al., 2008) was used to calculate mantle surface in cm 2 .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, the mantle and other tissues were separated from the shells and dried at 60 °C for 24 to 48 hours 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 level (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.

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 minute intervals over the three-hour 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):

Statistical analyses
For assessing the comparisons of clam abundance at the six survey depths, an analysis of variance (ANOVA) and pairwise posthoc 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 net calcification rates and clam tissue mass.This model was selected against other concurrent models by using Akaike information criterion (AIC) (Burnham and Anderson 2003).
Statistical analyses were performed using R (Foundation for Statistical Computing, Vienna, Austria, Version 3.4.2) and Statistica (Dell Software).

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 in a water depth of 3 m (ANOVA, p < 0.001, F = 35.6;Post-hoc Tukey test p < 0.001; Supplementary Material_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 with 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).In 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 140.4 ± 90.4 cm 2 (mean ± SD, n = 422) respectively.
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.

Depth-dependent abundances
In the Red Sea, T. maxima shows a significant dependence of net calcification rates with 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 to 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 peak values of 0.80 clams m -2 (Roa-Quiaoit, 2005) and 0.42 clams m -2 in Kiribati (Chambers, 2007).
In 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 and sheltered reef, respectively.Abundances at the sheltered reef are ranking amongst the highest abundances reported world-wide, 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) character of the examined sites.As reviewed by Van Wynsberge ( 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.Similar to Roa-Quiaoit (2005), we found that T. maxima abundances in the Red Sea seems 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) in water depths between 0.5 and 11 m.Explanations for the observed contrasts in numbers of clams per m 2 at both reefs could lay in the probable differences in abiotic environmental conditions at the surveyed sites.For instances, 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, report similar patterns for T. crocea, opposite patterns were observed for abundances of T. maxima in that region.In the reefs surveyed by Militz and collegues (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 for the observed differences by considering exposure alone.For example, T. maxima specimens from our study, which were located at the more southern reef, could be possibly also exposed to higher surface water temperatures due to location of this reef at lower latitudes.Mean sea surface annual temperature 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 in water depth of maximum 2 m, while we assessed abundances of T. maxima in six different depths (0.5, 1.5, 3, 5, 8 and 11 m).Previous studies have shown that the depth of abundance surveys significantly impact the estimates (Van Wynsberge et al., 2016), even though generally, 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 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 m, 1.5 m and 3 m depth level 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 few specimens of T. maxima in 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 preventing 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).

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), 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 at the air-water interface in the reef of 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 μmol quanta m -2 s -1 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 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 level received in 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 highest densities in 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.
The mechanisms of light-enhanced calcification (LEC) have been intensely studied in zooxanthellate scleractinian corals, leading  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), report 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.Yet, 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 be also supported by the pronounced decrease in gross primary production rates at this light treatment.High incident light level, especially high level 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 level 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).

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 system, 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 for 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 giant clams seem to decrease with age once they reached 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 of produced egg numbers with increasing shell size (Jameson, 1976).

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, normalisation of calcification rates per mantle surface area would be also appropriate, as Symbiodinium cells in giant clams are mostly found in the upper 5 mm of the mantle (Ishikura et al. 1997).

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.5 Albeit 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 to 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 10 overall understanding of light enhanced calcification in Red Sea Tridacninae and their relationship with the symbiotic algae.

Figure 1 :
Figure 1: (a) Map of the Red Sea.Abundance surveys and sampling of clams for incubation experiments where 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 sheltered reef (Station 1), (c) satellite image of exposed, fringing reef (Station 2).

Figure 5 :
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.

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