Effects of Different Intensities and Wavelengths of Light on the Growth of Juvenile Tridacna noae

ABSTRACT Giant clams play a vital role in the preservation of coral reef ecosystems; however, the population of juvenile Tridacna sp. has experienced a decline primarily attributed to low survival rates, overfishing, marine pollution and the effects of climate change. Light intensity reportedly influences the growth and survival of giant clams. They possess the ability to thrive in oligotrophic tropical marine environments by utilising photosynthates from symbiotic zooxanthellae (Symbiodiniaeae). This study assessed the impact of different light intensities and wavelengths on growth rate (GR) and survival rate (SR) of juvenile T. noae. GRs of juvenile T. noae cultivated under 12,500 (518 ± 69%) and 20,000 lx (541 ± 40%) were significantly higher compared to those grown in 5,000 lx (271 ± 17%) (P < 0.05). Additionally, juvenile T. noae cultivated under white (662 ± 142%) or red light (554 ± 135%) exhibited lower growth rate than blue light (BL, 727 ± 146%). However, no significant change in SR was observed among experimental groups (P > 0.05). This study emphasised the significance of light intensity and wavelength in captive rearing of giant clams, contributing to the sustainable production of cultured organisms for research and conservation purposes.


Introduction
Tridacninae, a subfamily of bivalve molluscs, inhabits shallow-water coral reef systems throughout the tropical Indo-Pacific region, ranging from the East African coast to the Pitcairn Islands in the eastern Pacific, and from Okinawa to the southern area of the Great Barrier Reef (GBR) in Australia (Copland and Lucas 1988;Lucas 1994).Giant clams have traditionally been regarded as a valuable fishery species.Owing to their colourful mantles, they are often sold in the wildlife ornamental trade; their adductor muscle and mantle have culinary and ornamental uses, respectively (Wabnitz 2003).Overfishing and habitat loss have resulted in a marked decline in the population of giant clams (Van Wynsberge et al. 2016;Neo et al. 2017).One of these giant clams is Tridacna noae (Röding, 1798), also known as Noah's giant clam.Trade in this species is regulated by Appendix II of CITES (United Nations Convention on International Trade in Endangered Species of Wild Fauna and Flora).In the aquarium trade, T. noae was thought to be Tridacna maxima (Röding, 1798), and often sold under a common name 'Tear-drop Maxima' before it was reinstated as a species (Wabnitz and Fauvelot 2014).After a rigorous morphological and genetic analysis, Tridacna noae is now considered a distinct and separate species from T. maxima (Su et al. 2014;Borsa et al. 2015).Morphologically, T. maxima typically exhibits 4-5 ribs with round projections on the upper margins, while T. noae displays 6-7 ribs with sharp projections.Genetically, the 16S rRNA gene reveals a genetic distance of 0.042-0.048between T. maxima and T. noae, while the intraspecific differences within the clades of T. maxima and T. noae are 0-0.003and 0-0.005, respectively.Since the rediscovery of T. noae, mariculture methods for this species have undergone rapid development (Militz et al. 2017;Braley et al. 2018;Su et al. 2021).For example, Su et al. (2021) demonstrated that higher water temperatures (20.3-27.0°C, Linbian, Taiwan) led to increased shell length in T. noae compared to lower temperatures (16.9-23.9°C, Penghu, Taiwan) (Su et al. 2021).Consequently, it is imperative to assess the impact of environmental factors, such as water temperature, water quality and light, on the growth of T. noae.
Giant clams predominantly depend on the energy produced through photosynthesis by their symbiotic zooxanthellae (Symbiodiniaceae) and utilise filterfeeding as a secondary energy source (Watson and Neo 2021).While Tridacna clams cannot survive in an environment abundant in plankton but lacking sufficient light, as filter-feeding alone cannot sustain their basic metabolism, they thrive in oligotrophic tropical coral reef ecosystems.The clam shells are highly calcified and rapidly grow in the presence of light (Sparsis et al. 2001;Wijgerde et al. 2014;Liu et al. 2020;Chan et al. 2021;Boo et al. 2022).In a relevant study, a light intensity of 3000 lx was utilised to establish the symbiotic relationship between zooxanthellae and the pediveliger larvae of Tridacna squamosa (Lamarck, 1819;Zhang et al. 2016).This suggests that, in normal depths and light conditions, phototrophy alone may provide sufficient carbon requirements for giant clam species, and T. squamosa juveniles can survive for over 10 months with light as their sole energy source.Another giant clam, T. crocea Lamarck, 1819, appears to thrive under a light intensity of approximately 15,000 lx (Liu et al. 2018).However, light intensity rapidly diminishes with an increase in seawater depth, impacting light colour and intensity due to varying light penetration.The absorption of visible light from seawater is optimal at wavelengths of red light (RL, 620-750 nm); the relatively short wavelengths of blue light (BL, 450-495 nm) penetrate deep water layers.Notably, BL could facilitate coral growth, improve zooxanthellae density, and increase chlorophyll concentration and photosynthesis rate (Kinzie et al. 1984;D'Angelo et al. 2008).The spectrum of BL increases the rate of calcification in the tissues of Porites lutea Milne Edwards & Haime, 1851 and Acropora variabilis (Klunzinger, 1879) (Cohen et al. 2016).This suggests that BL might have the potential to be used in giant clam mariculture.Therefore, in the present study, we investigated the effects of various light wavelengths and intensities on the growth rate and survival rate (SR) of juvenile T. noae by using artificial seawater and various light-emitting diodes (LEDs; light source).

Animal source, transportation and acclimation
A single batch of juvenile T. noae was cultivated at Penghu Marine Biology Research Center, Taiwan.A total of 500 juveniles with a mean initial shell length of 1.60 ± 0.67 mm (age: 2 months with endosymbionts) was packed in double-layer plastic bags with oxygen supply and transported to Yilan, Taiwan, via air transport.The juveniles were reared in tanks after arrival.Throughout the experimental period, the tank bottom was outfitted with square-shaped bricks (3 cm × 3 cm each) in a grid arrangement to form a large surface area of 21 cm × 21 cm (total of 49 bricks).Each brick was labelled with a number to facilitate individual tracking and prevent duplication in the assessment of specimens.The survival status of all individuals was confirmed upon arrival at the Yilan greenhouse, and they were acclimated for two weeks.

Light sources
We used light sources with different light intensities, including 5000 lx (GCF-626601-B2; Fishlive Aquatic Biotech Co., Ltd., Taiwan), 12,500 lx (KW-GS150W; Shenzhen HXWY Lighting Technology Co., Ltd., China), and 20,000 lx (KW-GS100W; Shenzhen HXWY Lighting Technology Co., Ltd.).We used WL (30 W; GCF-626601-B2; Fishlive Aquatic Biotech Co., Ltd.), BL (GCF-626601-A2; Fishlive Aquatic Biotech Co., Ltd.), and RL (30 W; GCF-616412, Fishlive Aquatic Biotech Co., Ltd.) in this study.The details of the illuminance and wavelength of light sources in the current study are shown in Table 1.The spectra of white, red, and blue lights with different wavelengths are shown in Figure 1.The schematic shown in Figure 2 presents the experimental light emission setup.The intensities and wavelength of light sources were measured by illuminometer (TES-1330A, CHUNG TSANG TECHNOL-OGY LTD.Taiwan) and spectroscope (SRI-2000; Optimum Optoelectronics, Ltd., Taiwan), whereas different wavelengths of light sources were customised and provided by the company.
The transmission of light through air and water is characterised by distinct levels of transparency, resulting in divergent light intensities.Prior to the commencement of the experiment, an empty tank was subjected to illumination from an overhead light source, yielding an illumination reading of 12,800 lx at a 30 cm distance from the tank bottom (with no intervening water).However, when the light source was positioned at the water's surface, the measured illumination at the same 30 cm distance from the tank bottom (accounting for 30 cm of water) decreased to 5000 lx.In addressing this issue, we employed an adaptive approach, calibrating the light intensity in accordance with the distance between the light source and the clam.A chandelier was placed 20 cm above the water surface without alteration of the water temperature.The clams were placed 20 cm below the water surface to ensure that they received 95% of the light from the water surface.This calibration process was rigorously confirmed through the use of an illuminometer, ensuring a consistent and controlled light intensity throughout the experimental conditions.

Experimental water and environment
The clams were maintained in 55 L tanks (length × width × height, 60 × 35 × 30 cm) within an indoor recirculating water system containing pathogen-free artificial seawater, which was prepared by mixing tap water with commercial sea salt (Prime Sea Salt, Fishlive Aquatic Biotech Co., Ltd., Taiwan) containing calcium (400 ± 50 ppm), magnesium (1200 ± 100 ppm), and potassium (380 ± 20 ppm).The pathogen-free artificial seawater was freshly prepared before its use; the following parameters were ensured by aquarium water tests (JBL, Germany): salinity, 34 ± 1‰; pH, 8.2 ± 0.2; dissolved oxygen level, 6 ± 1 mg/L; and alkalinity, 8 ± 1.The macro (Cl, Na, Ca, Mg, K, S) and trace elements (Br, B, Sr, F, Li, I, Ba, Mo, Zn, Al) depleted due to biological absorption were replenished.The concentration of replenished macro and trace elements was confirmed by commercial tests (PROAQUATEST Mg-Ca and PROAQUATEST K 0-15PPM, JBL, Germany) and conductivity ratios.A separate filter was used for each tank (filter pore diameter: 0.01 cm, EHEIM 2213, Germany).Every week, 20% of the water was replaced to eliminate algae, and the water quality was checked before being replenished into the tank.The holding ambient temperatures in the experimental tanks were 27 °C.

Growth parameters
Juvenile T. noae clams (initial SL, 1.37 ± 0.38 mm, mean ± SD) were continuously cultivated for 18 weeks under the following light intensities: 5000 (control), 12,500, and 20,000 lx.A total of 90 healthy juveniles (mean initial SL, 1.37 ± 0.38 mm) was divided into three experimental groups.Three tanks were used for each experimental group (10 juveniles/per tank).In the comparison of different light sources, juvenile T. noae clams (initial SL, 1.10 ± 0.34 mm, mean ± SD) were continuously cultivated for 18 weeks under WL, RL, and BL.A total of 135 healthy juveniles (mean initial SL, 1.10 ± 0.34 mm) was divided into three experimental groups.Each experimental group had three tanks (15 juveniles/per tank) for replication.

Growth rate
The rate of growth in shell length (SL) was measured in terms of the maximum front and rear dimensions of the shell placed on a plane by digital caliper (precision: 0.01 mm; Mitutoyo, Japan).Growth rates were calculated fortnightly using the following formula: (1) Rate of SL growth (mm month −1 )

Survival rate
The fortnightly survival rate (SR) was calculated using the formula below.: Final number of clams Initial number of clams × 100%

Specific growth rate
The wet weight of clams was measured fortnightly by portable balance (220 G X 0.001 G, SPX223, OHAUS, USA).The specific growth rate (SGR) was calculated using the following formula:

Statistical analysis
Statistical analyses were performed using one-way analysis of variance of SAS (Statistical Analysis System, SAS/PC version; SAS Institute, Cary, CA, USA) version 9.0.The one-way ANOVA was carried out for every fortnightly measurement of shell length data of all juveniles within each water tank.An analysis of variance test was performed, and significance (P < 0.05) was analysed using Duncan's new multiple range test.All data were shown as mean ± SD (standard deviation).

Effects of different light sources on the growth rate of juvenile T. noae
As presented in Table 3, the final SLs of clams cultivated under WL, RL, and BL were 8.59 ± 0.39, 7.68 ± 0.13, and 9.75 ± 0.50 mm (mean ± SD), respectively.The experimental groups did not vary significantly in terms of SL (P > 0.05).As shown in Table 3, the corresponding length gains were 662% ± 141% (WL), 553% ± 134% (RL), and 727% ± 146% (BL) (mean ± SD).The experimental groups did not differ significantly in terms of length gain (P > 0.05) or SGR (P > 0.05; Table 3; Figure 5).At the eighth week of cultivation, the SRs of clams cultivated under WL, RL, and BL were 93% ± 13%, 86% ± 13%, and 93% ± 13% (mean ± SD, Figure 6), respectively.As shown in Figure 5, the growth rates in all groups showed no significant difference during the experimental period.In this period, SR stabilised (80% ± 15%), and no deaths occurred.At the end of the experiment, the SRs of clams cultivated under WL, RL, and BL were 66% ± 0%, 73% ± 3%, and 73% ± 7% (mean ± SD), respectively.No significant differences were noted among the groups in terms of SR (P > 0.05; Table 3; Figure 6).Thus, irradiation using various light sources with different wavelengths induced no effects on the SRs of juvenile giant clams.

Discussion
Giant clam distribution on tropical coral reefs is strongly correlated with light intensity (Copland and Lucas 1988).Higher levels of transparency are observed in tropical seas, whose waters are generally low in nutrients (Van Wynsberge et al. 2016).Regions in natural seas, particularly at depths exceeding 1-4 m, are exposed to 10,000 to 25,000 lx of sunlight at midday (Hamner and Jones 1976).However, the global consequences of greenhouse effects and   ocean pollution, which include phenomena such as ocean acidification and microplastic contamination, result in reduced photosynthetic efficiency and increased turbidity in seawater (Fabina et al. 2013;Comeau et al. 2017;Watson and Neo 2021;Li et al. 2023).To illustrate an example, microplastic pollution in seawater could lead to turbidity, which interferes with light penetration and impairs the utilisation of filter-feeding of giant clams (Arossa et al. 2019).Moreover, the limited light penetration in seawater could potentially impact the photosynthesis of the symbiotic zooxanthellae (Symbiodiniaceae) (Klueter et al. 2017).
Additionally, ocean acidification has been associated with a reduction in symbiont photosynthetic yield and zooxanthellae density (Li et al. 2022).Hence, the motivation of this study is to investigate the effects of light intensity and wavelength on growth performance and survival rate for T. noae.
In the present study, the highest rate of SL growth was noted in clams (SL, approximately 1.51 mm) cultivated under 20,000 lx (1.95 mm month −1 ), followed by that in those cultivated under 12,500 lx (1.84 mm month −1 ) and 5000 lx (0.95 mm month −1 ) light intensities.In an indoor study conducted by Liu et al. (Liu et al. 2020), small (SL, approximately 50 mm) and large (SL, approximately 85 mm) Tridacna crocea clams were cultivated under 15,000 lx light intensity; their SLs increased by 1.40 and 1.10 mm month −1 , respectively, during the 16-week growth experiment.
In the present study, Tridacna crocea cultivated under 20,000 lx exhibited growth rates higher than and similar to that of those reported by Hean and Cacho (1.39 mm month −1 ) and .02mm month −1 ), respectively (Hean and Cacho 2003;Yamamoto et al. 2012).Interestingly, Zhang et al. (Zhang et al. 2020) reported a growth rate of 35.66-47.32mm (3.96-5.27mm month −1 ) for T. crocea by outdoor trial, which was considerably higher than that noted in our study.A conspicuous disparity in the growth rates of giant clams between outdoor and indoor environments is apparent.This differential growth suggests that the different intensities and wavelengths inherent to outdoor sunlight have the potential to increase the effectiveness of various photosynthetic processes involving the symbiotic zooxanthellae (Symbiodiniaceae).Consequently, this increase results in a greater availability of nutrients for the giant clams.However, it is essential to note that direct empirical evidence defining the specific repercussions of varying light intensities and wavelengths on the photosynthetic activity of Symbiodiniaceae, and the subsequent influence on the growth performance of giant clams, remains incomplete.This knowledge gap underscores the need for further research to comprehensively clarify the intricacies of these relationships.
When sunlight penetrates clear seawater, the longer wavelengths (620-750 nm) of RL possess low levels of energy and are gradually absorbed across a certain depth gradient (Chiang et al. 2011).A depth of approximately 20 m prevents RL penetration.By contrast, high-energy BL (450-495 nm) can penetrate deep water layers (approximately 100-150 m) (Godwin 2021).This phenomenon is essential for coral growth and colour change (Wijgerde et al. 2014).Almost all members of the Tridacninae subfamily live at depths of ≤ 15 m, which have high levels of BL exposure.Light regulates the cell cycle and photosynthesis, and RL and BL are the primary spectral   ranges for photosynthesis (Kühl et al. 1995;Salih et al. 2000).The proliferation of symbiotic algae depends on light wavelength and intensities (Brunelle et al. 2007;Yang et al. 2020;Gong et al. 2023).In most symbiotic algae, the cell cycle operates only in the presence of WL or BL; the proliferation under RL is considerably lower in these algae (Wang et al. 2008).As an example, Brunelle et al. (2007) conducted a study revealing the presence of the cryptochrome bluelight receptor in the dinoflagellate Karenia brevis (Davis) Daugbjerg et al. 2000.This receptor was identified as having a circadian role in controlling the cell cycle.Moreover, Yang et al. (2020) found that highlight intensity (600 µE .m −2 .s −1 , ∼140,000 cells .mL −1 ) increased the cell proliferation of Fugacium kawagutii (Clade F1) compared to low-light (20 µE .m −2 .s −1 , ∼ 95,000 cells .mL −1 ).In our results, there was no significant difference across the wavelengths tested.Based on our current understanding, there are no studies that have isolated symbiotic Symbiodiniaceae from giant clam species in order to investigate how light-induced cell proliferation of these organisms contributes to the regulation of giant clam growth.
The pivotal role of photosynthetic pigments in influencing the growth and physiological processes of giant clams has garnered substantial attention in related studies.The iridocytes of giant clams transfer incident photons into mantle tissue (Griffiths et al. 1992;Sweeney et al. 2012;Ghoshal et al. 2016).Iridocytes promote the back reflection of nonproductive wavelengths and the lateral and forward scattering of photosynthetically active radiation (PAR) in clam tissues (Rossbach et al. 2020).Tridacna maxima exhibits strong ultraviolet radiation absorption (200-400 nm) at superficial (100 μm) and deep (approximately 500 μm) tissue layers; this absorption is within the range of photosynthetic activity of the superficial layer (PAR, 400-700 nm).As primary constituents within the symbiotic zooxanthellae (Symbiodiniaceae), these pigments play a crucial role in the growth and metabolic processes of the clams.Their capacity to absorb light energy is instrumental, with their absorption spectra exhibiting peaks between 400 and 560 nm, signifying a robust capacity for energy absorption within this range.Conversely, their absorption is lowest within the 600-675 nm range, indicating reduced efficacy in energy absorption within this wavelength range (Rossbach et al. 2020).These spectral variations hold significant implications for the efficiency of clam photosynthesis and overall growth dynamics.Of particular significance is the observation that the blue-light (BL) wavelength range triggers high absorption by the clam's iridophores, a phenomenon frequently harnessed in experiments.Research underscores that BL stimulation significantly fosters clam growth and the deposition of carbonates (Bonham 1965;Ip et al. 2018;Chew et al. 2019;Rossbach et al. 2019).This, in turn, amplifies the efficiency of photosynthesis, a process heightened by the saturation of internal pH and carbonate minerals, thereby augmenting the metabolism of calciuma vital factor in the clams' shell formation (McConnaughey et al. 1997).In this study, we observed that blue light (BL) demonstrated superior growth stimulation compared to white light (WL) and red light (RL).This observation implies an enhanced absorption of iridophores in the clams.
Nonetheless, it is noteworthy that our current understanding of the intricate relationship between clam photosynthesis and growth remains limited.While extant studies have probed the beneficial role of photosynthesis in driving clam growth (Liu et al. 2020;Ip and Chew 2021;Ip et al. 2022;Pang et al. 2022), a comprehensive understanding of the molecular mechanisms governing the interaction between light and clam growth, as well as the specific influencing factors, such as marine microplastic pollution that can alter light reflectance and transmittance (Goddijn-Murphy and Dufaur 2018; Song et al. 2023), necessitates further thorough investigation.Subsequent research endeavours might encompass investigations into the impacts of light on the metabolic pathways within the giant clam's symbiotic consortium, as well as the mechanisms governing cellular proliferation and differentiation under light regulation.This multifaceted approach could unveil a more comprehensive understanding of the intricate mechanisms through which light contributes to the growth and physiological processes of giant clams.Therefore, the role of photosynthetic pigments in the growth and metabolic processes of giant clams is integral.Their influence on photosynthetic efficiency and the regulatory mechanisms by which light governs growth are progressively being unravelled through ongoing research.
In summary, this study highlights the significant impact of light intensity and wavelength on the growth of juvenile T. noae clams.Elevated light intensities of 12,500 lx and 20,000 lx substantially improved growth rates, with blue light (BL) outperforming white (WL) and red light (RL) in stimulating growth.Although survival rates remained consistent among experimental groups, these findings underscore the intricate relationship between light conditions and clam growth.These insights have broad implications, extending beyond coral reef ecosystems.Our studies provide the potential to inform and optimise mariculture practices, particularly in the context of giant clam aquaculture and other marine species with similar light-dependent growth requirements.Improved understanding of the underlying mechanisms can lead to more sustainable and efficient aquaculture operations, ensuring a stable supply of giant clams and contributing to the broader field of sustainable seafood production.Furthermore, this research underscores the importance of considering light-related factors in the development of strategies for sustainable aquaculture and the conservation of marine biodiversity.By applying the knowledge gained from this study, mariculture practitioners and conservationists can work collaboratively to protect and enhance the resilience of giant clam populations and their associated ecosystems.

Figure 1 .
Figure 1.The spectra of white, red, and blue lights with different wavelengths (A: white light, B: red light, C: blue light) were measured for intensities and wavelengths using a spectroscope.

Figure 2 .
Figure 2. Schematic showing the experimental light emission setup.

Table 2 .
Effects of three light intensities on the shell length gain, specific rate gain and survival rates of juvenile Tridacna noae.Data are presented in terms of mean ± standard deviation values.Mean values in the same column denoted by different letters (a, b) indicate significant differences.n = 10.LG: length gain (%); SGR: specific growth rate.

Figure 3 .
Figure 3. Effects of three light intensities on the growth rate of juvenile Tridacna noae during the 18-week experimental period.Data are presented in terms of mean ± standard deviation values.(C): control.Bars with different letters within the set of three for a week are significantly different.

Figure 4 .
Figure 4. Effects of three light intensities on the survival rate of juvenile Tridacna noae during the 18-week experimental period.Data are presented in terms of mean ± standard deviation value.(C): control.

Figure 5 .
Figure 5. Effects of different light sources on the growth rate of juvenile Tridacna noae during the 18-week experimental period.Data are presented in terms of mean ± standard deviation values.

Figure 6 .
Figure 6.Effects of different light sources on the survival rate of juvenile Tridacna noae during the 18-week experimental period.Data are presented in terms of mean ± standard deviation.

Table 1 .
The illuminance and wavelength of light sources in current study.

Table 3 .
Effects of different light sources on the shell length gain, specific rate gain and survival rates of juvenile Tridacna noae.Data are presented in terms of mean ± standard deviation values.Mean values in a column denoted by the same letter (a) indicate non-significant differences.n= 15.LG: length gain (%); SGR: specific growth rate.