Using Transcript Levels of Nitrate Transporter 2 as Molecular Indicators to Estimate the Potentials of Nitrate Transport in Symbiodinium, Cladocopium, and Durusdinium of the Fluted Giant Clam, Tridacna squamosa

Giant clams are important ecosystem engineers of coral reefs because they harbor large quantities of phototrophic Symbiodiniaceae dinoflagellates of mainly genera Symbiodinium, Cladocopium, and Durusdinium. The coccoid dinoflagellates donate photosynthate and amino acids to the clam host, which in return needs to supply inorganic carbon and nitrogen to them. The host can conduct light-enhanced absorption of nitrate (NO3–), which can only be metabolized by the symbionts. This study aimed to clone nitrate transporter 2 (NRT2) from the symbionts of the fluted giant clam, Tridacna squamosa. Here, we report three major sequences of NRT2 derived from Symbiodinium (Symb-NRT2), Cladocopium (Clad-NRT2) and Durusdinium (Duru-NRT2). Phenogramic analysis and molecular characterization confirmed that these three sequences were NRT2s derived from dinoflagellates. Immunofluorescence microscopy localized NRT2 at the plasma membrane and cytoplasmic vesicles of the symbiotic dinoflagellates, indicating that it could partake in the uptake and transport of NO3–. Therefore, the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 could be used as molecular indicators to estimate the potential of NO3– transport in five organs of 13 T. squamosa individuals. The transcript levels of form II ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcII) of Symbiodinium (Symb-rbcII), Cladocopium (Clad-rbcII) and Durusdinium (Duru-rbcII) were also determined in order to calculate the transcript ratios of Symb-NRT2/Symb-rbcII, Clad-NRT2/Clad-rbcII, and Duru-NRT2/Duru-rbcII. These ratios expressed the potentials of NO3– transport with reference to the phototrophic potentials in a certain genus of coccoid dinoflagellate independent of its quantity. Results obtained indicate that Symbiodinium generally had a higher potential of NO3– transport than Cladocopium and Durusdinium at the genus level. Furthermore, some phylotypes (species) of Symbiodinium, particularly those in the colorful outer mantle, had very high Symb-NRT2/Symb-rbcII ratio (7–13), indicating that they specialized in NO3– uptake and nitrogen metabolism. Overall, our results indicate for the first time that different phylotypes of Symbiodiniaceae dinoflagellates could have dissimilar abilities to absorb and assimilate NO3–, alluding to their functional diversity at the genus and species levels.

Giant clams are important ecosystem engineers of coral reefs because they harbor large quantities of phototrophic Symbiodiniaceae dinoflagellates of mainly genera Symbiodinium, Cladocopium, and Durusdinium. The coccoid dinoflagellates donate photosynthate and amino acids to the clam host, which in return needs to supply inorganic carbon and nitrogen to them. The host can conduct light-enhanced absorption of nitrate (NO 3 − ), which can only be metabolized by the symbionts. This study aimed to clone nitrate transporter 2 (NRT2) from the symbionts of the fluted giant clam, Tridacna squamosa. Here, we report three major sequences of NRT2 derived from Symbiodinium (Symb-NRT2), Cladocopium (Clad-NRT2) and Durusdinium (Duru-NRT2). Phenogramic analysis and molecular characterization confirmed that these three sequences were NRT2s derived from dinoflagellates. Immunofluorescence microscopy localized NRT2 at the plasma membrane and cytoplasmic vesicles of the symbiotic dinoflagellates, indicating that it could partake in the uptake and transport of NO 3 − . Therefore, the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 could be used as molecular indicators to estimate the potential of NO 3 − transport in five organs of 13 T. squamosa individuals. The transcript levels of form II ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcII) of Symbiodinium (Symb-rbcII), Cladocopium (Clad-rbcII) and Durusdinium (Duru-rbcII) were also determined in order to calculate the transcript ratios of Symb-NRT2/Symb-rbcII, Clad-NRT2/Clad-rbcII, and Duru-NRT2/Duru-rbcII. These ratios expressed the potentials of NO 3 − transport with reference to the phototrophic potentials in a certain genus of coccoid dinoflagellate independent of its quantity. Results obtained indicate that Symbiodinium generally had a higher potential of NO 3 − transport than Cladocopium and Durusdinium at the genus level. Furthermore, some phylotypes (species) of Symbiodinium, particularly those in the INTRODUCTION Giant clams (genera Tridacna and Hippopus) are members of reef ecosystems in the tropical Indo-Pacific. They generally live in symbiosis with three genera of phototrophic dinoflagellates (Symbiodinium, Cladocopium, and Durusdinium) belonging to family Symbiodiniaceae (LaJeunesse et al., 2004(LaJeunesse et al., , 2018Takabayashi et al., 2004;Hernawan, 2008), although giant clams of the French Polynesia may also contain Gerakladium (Pochon et al., 2019;Guibert et al., 2020). As animal-dinoflagellate associations, giant clams can flourish in nutrient-poor tropical waters where light is adequately available. The life cycle of Symbiodiniaceae dinoflagellates consists of a free-living flagellate stage and a symbiotic coccoid stage. Giant clams harbor the coccoid dinoflagellates (also called zooxanthellae) extracellularly inside a tubular system surrounded by hemolymph. These symbionts are found predominantly in the lumen of tertiary zooxanthellal tubules located in the colorful outer mantle, which can be extended beyond the edge of the shell-valve to receive irradiance needed by the symbionts for photosynthesis (Norton et al., 1992;Ip et al., 2017b). Photosynthesizing symbionts release a large portion of photosynthate to the host to support its energy and nutritional needs (Fisher et al., 1985;Klumpp et al., 1992). As a result, the host can conduct light-enhanced shell formation and grow to large sizes (Ip et al., 2017a;Rossbach et al., 2019; see Ip and Chew, 2021 for a review). In return, the host must supply the symbionts with inorganic carbon, phosphorus and nitrogen as they are separated from the ambient seawater.
Symbiodiniaceae dinoflagellates possess form II ribulose-1,5bisphosphate carboxylase/oxygenase (RuBisCO) (Rowan et al., 1996;Mayfield et al., 2014;Poo et al., 2020Poo et al., , 2021, and fix inorganic carbon through C3 photosynthesis (Streamer et al., 1993). Poo et al. (2020) used the transcript level of zooxanthellae-form II RuBisCO (Zoox-rbcII), which comprised rbcII of Symbiodinium (Symb-rbcII), Cladocopium (Clad-rbcII), and Durusdinium (Duru-rbcII), as a molecular indicator to examine the phototrophic potentials of five organs (colorful outer mantle, whitish inner mantle, foot muscle, hepatopancreas and ctenidium) in the fluted giant clam, Tridacna squamosa. They reported that the outer mantle of T. squamosa had a significantly higher transcript level of Zoox-rbcII, and hence a higher phototrophic potential, than the other four organs that are located inside the mantle cavity and shaded from direct irradiance (Poo et al., 2020). Subsequently, Poo et al. (2021) made a pioneering attempt to design three sets of genus-specific primers that could differentiate Symb-rbcII, Clad-rbcII, and Duru-rbcII with the aim of estimating the relative abundances of Symbiodinium, Cladocopium, and Durusdinium harbored by T. squamosa. They reported that Durusdinium was the dominant genus of Symbiodiniaceae dinoflagellates present in individuals of T. squamosa obtained from Vietnam . They also examined coccoid dinoflagellates freshly isolated from the outer mantle of T. squamosa, and demonstrated that Symb-rbcII, Clad-rbcII, and Duru-rbcII exhibited different responses to light at the transcriptional level . Thus, they suggested that similar methods could be applied to study other genes of physiological importance in order to elucidate the functional diversity of various phylotypes of Symbiodiniaceae dinoflagellates .
While inorganic carbon is needed for the production of carbohydrates through photosynthesis, nitrogen is crucial for the formation of amino acids, proteins and nucleic acids. For instance, nitrogen is a vital element for chlorophyll biosynthesis in phototrophic dinoflagellates (Bernhard, 2010). For coccoid dinoflagellates, they have to synthesize amino acids not only for themselves but also for the host, which requires a large supply of amino acids for the production of muscle proteins. The clam host needs muscles to retract the extended colorful outer mantle, to close the shell valves (adductor muscle) and to generate lateral movements (foot muscle). It has been reported that the muscle of T. squamosa contains essential amino acids (Liu et al., 2019) that cannot be synthesized by the clam host (Wang and Douglas, 1999). Yet, T. squamosa can live and grow in Millipore-filtered seawater with light as the sole energy source for more than 10 months (Fitt and Trench, 1981), implying that the host can obtain all their nutrients, including essential amino acids, from its phototrophic symbionts (Klumpp and Griffiths, 1994). However, coccoid dinoflagellates are nitrogen-deficient (Wilkerson and Trench, 1986) and do not have access to the ambient seawater. Therefore, the host must absorb exogenous ammonia (Fitt et al., 1993a), urea (Chan et al., 2018(Chan et al., , 2019, and NO 3 − (Ip et al., 2020) to support nitrogen metabolism in the symbionts. This is unique among aquatic animals, which generally excrete ammonia as the major nitrogenous waste, often together with a small quantity of urea (Ip and Chew, 2010;Chew and Ip, 2014).
In seawater, dissolved inorganic nitrogen is present as NO 3 − and NH 4 + , while dissolved organic nitrogen is available as urea and amino acids. The concentration of NO 3 − in seawater (<500 µmol N 1 −1 ) is much higher than those of NH 4 + (2 µmol N 1 −1 ) and urea (25 µmol N 1 −1 ) (Collos and Berges, 2003). Although the environmental toxicity of NO 3 − is relatively low (Westin, 1974;Tomasso and Carmichael, 1986;Jensen, 1996), nitrite (NO 2 − ) is toxic to most aquatic animals. Hence, unlike algae and plants, aquatic animals generally absorb little NO 3 − from the environment because the reduction of NO 3 − to NO 2 − inside their bodies can lead to NO 2 − poisoning (Camargo and Alonso, 2006). Instead, aquatic animals excrete small quantities of endogenous NO 3 − in order to avoid NO 3 − reduction and the resulting NO 2 − toxicity. By contrast, T. squamosa absorbs NO 3 − from the external seawater, and the rate of NO 3 − absorption is augmented by illumination (Ip et al., 2020). The ctenidium (gill) of T. squamosa expresses a homolog of SIALIN, which functions as an electrogenic H + :2NO 3 − cotransporter. SIALIN is localized at the apical membrane of the epithelial cells near the tips of ctenidial filaments to absorb exogenous NO 3 − . Furthermore, illumination leads to significant increases in the transcript level of SIALIN and the protein abundance of SIALIN in the ctenidium, indicating that it can play a role in light-enhanced NO 3 − absorption. As the clam host cannot assimilate NO 3 − , the NO 3 − absorbed through the ctenidium must be dedicated to the symbionts. Indeed, the addition of NO 3 − to the ambient seawater can increase the growth rate of the host and the density of its symbionts (Fitt et al., 1993b). Hence, coccoid dinoflagellates must be able to absorb NO 3 − from the luminal fluid of the zooxanthellal tubules through the plasma membrane. As NO 3 − is an anion that cannot permeate the hydrophobic phospholipid bilayer freely, its transport through the symbiont's plasma membrane must involve certain types of membrane transporters or channels.
As the NO 3 − concentration in the extracellular fluid of the clam host needs to be low to avoid NO 3 − reduction leading to NO 2 − toxicity, it is logical to hypothesize that coccoid Symbiodiniaceae dinoflagellates would possess some types of high-affinity type NO 3 − transporter. Therefore, this study was undertaken to clone and sequence NRT2 from the symbionts residing in the outer mantle of T. squamosa. Due to the presence of various phylotypes (species) of Symbiodinium, Cladocopium, and Durusdinium, we had obtained multiple NRT2 sequences. However, only one major cDNA coding sequence of NRT2 for each genus of dinoflagellate was presented in this report. These three major sequences were named Symbiodinium-NRT2 (Symb-NRT2), Cladocopium-NRT2 (Clad-NRT2), and Durusdinium-NRT2 (Duru-NRT2). Their identities as NRT2 and origins from dinoflagellates were confirmed through molecular characterization and phenogramic analysis. An antibody that could bind comprehensively with NRT2 derived from all three genera of dinoflagellates, named zooxanthellae-NRT2 (Zoox-NRT2), was custom-made to confirm the localization of Zoox-NRT2 at the plasma membrane by immunofluorescence microscopy. In addition, we made a pioneering attempt to use the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 as molecular indicators to estimate the potential of NO 3 − transport in phylotypes of Symbiodinium, Cladocopium, and Durusdinium. Three sets of genus-specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to determine the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 in five organs (colorful outer mantle, whitish inner mantle, foot muscle, ctenidium, and hepatopancreas) of T. squamosa. However, the transcript levels of these three NRT2s could vary considerably among individuals of T. squamosa as they naturally harbor different quantities and proportions of dinoflagellate phylotypes in various organs. To resolve this problem, we also determined the transcript levels of Symb-rbcII, Clad-rbcII, and Duru-rbcII based on the genus-specific qPCR primers designed by Poo et al. (2021). The aim was to calculate the ratios of Symb-NRT2/Symb-rbcII, Clad-NRT2/Clad-rbcII, and Duru-NRT2/Duru-rbcII for a specific organ of each T. squamosa individual, as these ratios could provide information on the potential of NO 3 − transport with reference to the phototrophic potential for each genus of dinoflagellate independent of its quantity in the tissue sample.
Giant clams are important ecosystem engineers of coral reefs because they harbor large quantities of coccoid Symbiodiniaceae dinoflagellates. They can expel intact and viable dinoflagellates that can repopulate bleached Symbiodiniaceae-bearing hosts including scleractinian corals (Morishima et al., 2019;Umeki et al., 2020). While giant clams are known to harbor multiple phylotypes of Symbiodiniaceae dinoflagellates, the physiological reasons behind it remain enigmatic. Results obtained from this study were expected to furnish novel information on whether different phylotypes of Symbiodinium, Cladocopium, and Durusdinium would have disparate potentials of NO 3 − transport, and hence different abilities to use NO 3 − as a substrate to produce essential nitrogenous compounds for themselves and the host. Such information may shed light on the divergent physiological roles of Symbiodiniaceae dinoflagellates at the genus or even the phylotype (species) level, and provide insights into their distinct contributions to the physiological needs of the giant clam-dinoflagellate holobiont.

Giant Clam and Maintenance
Sixteen T. squamosa weighing 550 ± 150 g were imported directly from Vietnam through Xanh Tuoi Tropical Fish Co. Ltd. On arrival, the specimens were distributed into three tanks, each with a dimension of 92 cm (L) by 62 cm (W) by 62 cm (H) containing approximately 320 l of seawater at Salinity 30-32 and 26 • C. Artificial seawater was prepared with Red Sea salt (Red Sea, Houston, TX, United States). The salinity and temperature of the seawater were monitored using a Pro30 conductivity meter (YSI Incorporated, Yellow Springs, OH, United States). The pH of the seawater was maintained at 8. 2-8.4; the hardness at 143-179 ppm; the calcium content at 380-420 ppm; the phosphate content at <0.28 ppm; the total ammonia and NO 3 − contents at 0 ppm. Each tank was illuminated with two sets of four feet Aquazonic T5 lighting systems, and each system consisted of two white light tubes and two actinic blue light tubes. The underwater light intensity (photosynthetic photon flux density; PPFD) reaching the clams was ∼115 − 125 µmol photons m −2 s −1 (400−700 nm) as determined by a SKP 215 PAR Quantum sensor connected to the SKP 200 display meter (Skye Instruments Ltd, United Kingdom). This level of irradiance mimicked the light intensity received by T. squamosa in its natural habitat at a depth of ∼20 m (Jantzen et al., 2008). No food was supplied to the giant clams during the 1 month of acclimatization under a 12 h: 12 h dark: light regimen. Approval on the use of giant clams in this study was exempted by the Nanyang Technological University Institutional Animal Care and Use Committee.

Exposure to Light Conditions and Collection of Tissues
After 1 month of acclimatization, individuals of T. squamosa (n = 13) that had been exposed to light for 3 h were sampled randomly from the three tanks. Three hours of light exposure is chosen as giant clams are known to display light-enhanced phenomena, and the transcript levels of many transporters and enzymes could be enhanced after 3 -6 h of illumination (see Ip and Chew, 2021 for a review). To minimize stress on the clams, they were anesthetized in 0.2% phenoxyethanol prior to tissue sampling. The shell valves were forced open to sever the adductor muscle. Samples of the outer mantle, inner mantle, foot muscle, hepatopancreas, and ctenidium (gill) were excised. Excised tissue samples were blotted dry, freezeclamped in liquid nitrogen, and stored at −80 • C until further processing. Separately, tissue samples of the outer mantle were collected from three other individuals of T. squamosa for immunofluorescence microscopy (n = 3). Excised outer mantle tissues were fixed in 3.7% paraformaldehyde prepared with seawater for 18 h at 4 • C.

Total RNA Extraction and cDNA Synthesis
The total RNA of a tissue sample was extracted using TRI Reagent TM (Sigma-Aldrich Co., St Louis, MO, United States), and purified with a PureLink TM RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, United States). The concentration of the purified RNA was determined using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, United States), and the RNA integrity was checked by agarose gel electrophoresis. A RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific) was used to convert the purified RNA into cDNA.

Amino Acid Sequences and Phenogramic Analysis
The ExPASy Proteomic server 1 was used to deduce the amino acid sequences of Symb-NRT2, Clad-NRT2, and Duru-NRT2 from their respective nucleotide sequences. TMpred provided by Expasy 2 , was used to identify the transmembrane regions and pore lining amino acid residues. The identities of Symb-NRT2, Clad-NRT2 and Duru-NRT2 were confirmed by conducting a phenogramic analysis together with NRT2 sequences obtained from various databases. The phenogram was generated using Maximum Likelihood analysis using the program RaxML 8.2.5 (Stamatakis, 2014) with 2000 bootstraps. Using ModelGenerator v0.85 (Keane et al., 2006), the bestfitting evolutionary model for NRT2 was determined to be WAG + G + F (Whelan and Goldman, 2001).

Determination of the Transcript Levels by qPCR
Three sets of genus-specific qPCR primers, one each for Symb-NRT2 (forward: 5 -TGAAGACAGGTCTGGAGTA-3 ; reverse: 5 -CGCATATGGGCTCTTCT-3 ), Clad-NRT2 (forward: 5 -AGAATGATGATACCAATCCCAC-3 ; reverse: 5 -CAAA CACAGTCCGCCAG-3 ), and Duru-NRT2 (forward: 5 -GAAGTACAAGAACATTTCCACGAC-3 ; reverse: 5 -AAAC GCACTTGGACAGCAC-3 ) were designed by aligning Symb-NRT2, Clad-NRT2, and Duru-NRT2 with nine NRT2 sequences selected from various dinoflagellate databases (Supplementary Table S2). In order to verify the specificity of the designed Symb-NRT2 primer, Clad-NRT2 primer, and Duru-NRT2 primer, efforts were made to generate three different plasmid clones, each of which contained the insert of the amplicon region of Symb-NRT2, Clad-NRT2, or Duru-NRT2 following the method of Hiong et al. (2017). Then, qPCR was performed using these three plasmid clones as substrates to confirm that each set of genusspecific primers would only react with the plasmid containing the corresponding insert. The amplification efficiencies of the qPCR primers for Symb-NRT2, Clad-NRT2, and Duru-NRT2 were 102.4, 92.9, and 101.9%, respectively. Genus-specific qPCR primers designed by Poo et al. (2021) were adopted to quantify the transcript levels of Symb-rbcII, Clad-rbcII, and Duru-rbcII from T. squamosa. The amplification efficiencies of the primer set for Symb-rbcII, Clad-rbcII, and Duru-rbcII were 95.1, 95.1, and 112.0%, respectively. qPCR was performed using a 96-well StepOnePlus TM Real-Time PCR System (Thermo Fisher Scientific). Each reaction, in a total volume of 10 µl, consisted of 5 µl of qPCRBIO SyGreen Mix Hi-ROX (PCR Biosystems Inc., Wayne, PA, United States), 0.3 µl of forward primer (10 µmol l −1 ), 0.3 µl of reverse primer (10 µmol l −1 ), and an appropriate amount of cDNA. The qPCR cycling conditions included a 20 s denaturation and enzyme activation at 95 • C, followed with 40 cycles of 95 • C for 3 s and a specific temperature for a certain gene for 30 s. The specific temperature that lasted 30 s for Symb-rbcII, Clad-rbcII, and Duru-rbcII were 58, 56, and 55 • C, respectively. For Symb-NRT2, Clad-NRT2, and Duru-NRT2, the respective temperature used in the 40 cycles were 57, 60, and 60 • C. The dissociation curve obtained after each run was analyzed to verify the homogeneity of the PCR product and the specificity of the PCR reaction. Three standard curves were constructed using the three different plasmid clones as standards for Symb-rbcII, Clad-rbcII, and Duru-rbcII. The transcript levels Symb-rbcII, Clad-rbcII, and Duru-rbcII in a sample were calculated based on these three standard curves and expressed as copies of transcripts per ng of total RNA.

Immunofluorescence Microscopy
The fixed outer mantle sample was dehydrated in ethanol and cleared using HistoChoice Clearing Agent (Sigma-Aldrich Co.) before embedding in Paraplast Plus (Sigma-Aldrich Co.). Sections of 5 µm was prepared using a Leica RM2125 RTS microtome (Leica, Wetzlar, Germany) and mounted on Menzel Gläser SuperFrost Plus Adhesion slides (Thermo Fisher Scientific). The deparaffinized section was treated with citraconic anhydrase (Nacalai Tesque, Kyoto, Japan) at 95 • C for 5 min, followed with 1% SDS solution at 25 • C for 10 min for the retrieval of antigen. The section was then washed with TPBS containing 0.2% Triton-X, 10 mmol l −1 Na 2 HPO 4 , 1.8 mmol l −1 KH 2 PO 4 , 137 mmol l −1 NaCl, and 1.8 mmol l −1 KCl at pH 7.4. To reduce autofluorescence, the section was treated with 0.1% Sudan Black B (Sigma-Aldrich Co.) in 70% ethanol for 10 min and washed three times with TPBS. Blocking was performed with 1% bovine serum albumin in TPBS at 25 • C for 1 h. Thereafter, the section was incubated with the anti-Zoox-NRT2 antibody (2.5 µg ml −1 diluted with Signal Enhancer HIKARI Solution A obtained from Nacalai Tesque) at 25 • C for 1 h and rinsed three times with TPBS. The section was incubated with 2.5 µg ml −1 of fluorochrome-coupled goat anti-rabbit gamma globulin diluted with Signal Enhancer HIKARI Solution A (Alexa Fluor 488; Thermo Fisher Scientific) for 1 h at 25 • C. Finally, they were mounted in Prolong Gold antifade reagent (Thermo Fisher Scientific Inc.) for microscopy.
The mounted section was examined under a fluorescence microscope (Olympus BX43F; Olympus Corporation, Tokyo, Japan) and the images were acquired using an Olympus DP80 camera and the cellSens Imaging software (Olympus). Differential interference contrast microscopy (DIC) was applied to examine tissue structures and orientation. The red autofluorescence of the plastids of dinoflagellates was examined using the U-MWIG Interference Green Fluorescence Filter with an excitation wavelength of 520-550 nm. The green fluorescence resulting from the staining by the primary antibody and Alexa Fluor 488 was acquired using the Olympus U-WNIBA Blue Fluorescence Filter with an excitation wavelength of 470-490 nm. Overlaying of the images and adjustment of brightness were performed using Adobe Photoshop CC (Adobe Systems, CA, United States).

Data Analysis
Values were reported as means ± SEM unless otherwise stated. The non-parametric Friedman test of differences followed by the Wilcoxon Signed-Rank Test were applied for data expressed as percentages or ratios in Tables 4-8 and Figure 4. Differences obtained among means were considered statistically significant with p-values < 0.017 after Bonferroni adjustment. For Table 3, One-way Analysis of Variance (ANOVA) was used for comparison among the means of the transcript levels of the genus-specific NRT2 or those of genus-specific rbcII in a particular organ while Levene's test was used to assess the homogeneity of the variance. This was followed by Dunnett's T3 test as the variance was assumed not to be equal. On the other hand, the paired t-test was used to compare the means between the genus-specific NRT2 and the corresponding genusspecific rbcII in a particular organ in Table 3. The differences between the two means were regarded as significant when the p-value was <0.05. All comparisons were performed with the use of SPSS Statistics software v26 (IBM Corporation, Armonk, NY, United States).

Localization of NRT2 by Immunofluorescence Microscopy
NRT2 immuno-labeling was detected at the plasma membrane of the coccoid dinoflagellates in the outer mantle of T. squamosa, in support of a possible role in transporting NO 3 − across the plasma membrane (Figure 3). NRT2 immunofluorescence was also detected in certain intracellular vesicles inside the cytoplasm of the coccoid dinoflagellates.

Genus-Specificity of the Three Sets of qPCR Primers
The genus-specificity of the primer sets designed for Symb-NRT2, Clad-NRT2 and Duru-NRT2 were validated with three plasmid clones, each containing the insert of one of the three targeted amplicons. Indeed, a specific set of primer reacted positively only with its related plasmid clone ( Table 2). Poo et al. (2021) had verified the genus-specificity of the primer sets designed for Symb-rbcII, Clad-rbcII, and Duru-rbcII previously.

The Proportions of Genus-Specific NRT2 and rbcII Transcripts in Various Organs
A comparison of genus-specific NRT2 and rbcII in T. squamosa indicates that the mean transcript levels of Symb-NRT2 were significantly higher than those of Symb-rbcII in the outer mantle, foot muscle and hepatopancreas, but they were comparable in the inner mantle and ctenidium (n = 13; Table 3). Of note, the most prominent difference in transcript level of Symb-NRT2 and Symb-rbcII was observed in the outer mantle. By contrast, the mean transcript levels of Clad-NRT2 were either significantly lower than (as in the outer mantle and ctenidium) or comparable to (as in the inner mantle, foot muscle and hepatopancreas) the transcript levels of Clad-rbcII. The transcript levels of Duru-NRT2 were significantly lower than those of Duru-rbcII in these five organs (n = 13; Table 3).
For rbcII, the mean transcript level of Duru-rbcII was significantly higher than those of Symb-rbcII and Clad-rbcII (n = 13; Table 3) in all the five organs studied, indicating that Durusdinium was the major dinoflagellate genus in the individuals of T. squamosa obtained from Vietnam (Figure 4). Based on ANOVA, the transcript levels of Clad-rbcII were not significantly different from those of Symb-rbcII in the five organs because equal variance could not be assumed (n = 13; Table 3). Overall, the percentage proportions of transcript levels of rbcII derived from the three genera of Symbiodiniaceae dinoflagellates in the five organs of these 13 individuals of T. squamosa were Duru-rbcII > > Clad-rbcII = Symb-rbcII (Figure 4).
For NRT2, the mean transcript level of Duru-NRT2 was also significantly higher than those of Symb-NRT2 and Clad-NRT2 in four of the organs studied, except the hepatopancreas (n = 13; Table 3), corroborating the proposition that Durusdinium was the major dinoflagellate genus in these T. squamosa individuals (Figure 4). Furthermore, the mean transcript levels of Clad-NRT2 were statistically comparable to those of Symb-NRT2 in the outer mantle, inner mantle, foot muscle, and ctenidium (Table 3) due to the high variation of transcript levels among different individuals (n = 13). By contrast, the mean transcript level of Symb-NRT2 was significantly higher than that of Clad-NRT2 in the hepatopancreas (n = 13; Table 3). Hence, unlike rbcII, the percentage proportions of transcript levels of NRT2 derived from the three genera of Symbiodiniaceae dinoflagellates varied among the five organs of T. squamosa (Figure 4). For instance, the estimated proportions of Symb-rbcII transcripts in the outer mantle, hepatopancreas and ctenidium were 0.8, 3.0, and 1.9%, respectively, which were considerably lower than the estimated proportions of Symb-NRT2 transcripts in these three organs (outer mantle, 10.5%; hepatopancreas, 28.3%; ctenidium, 4.4%; Figure 4).

Symb-NRT2 clone
Clad-NRT2 clone Results are presented as average cycle threshold (Ct) values obtained from reactions between each of the three pairs of primers and the three clones generated, whereby a low Ct value represents a favorable reaction. UND indicates undetermined or no reaction between the specific set of primer and the clone.
Out of the 13 individuals examined, only clams 1 and 13 had Symb-NRT2/Symb-rbcII values > 1.0 ( Table 8). As a result, the mean transcript ratio of Symb-NRT2/Symb-rbcII for the ctenidium (0.77; Table 8) was the lowest among the five organs studied (Tables 4-7). The transcript ratios of Clad-NRT2/Clad-rbcII in the ctenidium of all 13 individuals were <1.0, with five of them having particularly low values (<0.10). In comparison, the transcript ratios of Duru-NRT2/Duru-rbcII in the hepatopancreas were relatively constant among the 13 individuals.

DISCUSSION
Giant clams harbor multiple phylotypes of Symbiodiniaceae dinoflagellates in variable proportions. The phylotype composition of dinoflagellates can influence the host's growth rate (Hernawan, 2008;DeBoer et al., 2012) and affect the host's ability to withstand environmental changes (Ikeda et al., 2017). Hence, it is logical to deduce that different phylotypes of Symbiodiniaceae dinoflagellates could play distinct physiological roles in the giant clam-dinoflagellate holobiont. One important role of coccoid dinoflagellates is to synthesize essential amino acids and share them with the host, but the host must supply them with nitrogen. Uniquely, the 3 | Transcript levels (copies of transcript per ng of total RNA) of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcII) and nitrate transporter 2 (NRT2) derived from Symbiodinium (Symb-rbcII and Symb-NRT2, respectively), Cladocopium (Clad-rbcII and Clad-NRT2, respectively) and Durusdinium (Duru-rbcII and Duru-NRT2, respectively) in the outer mantle, inner mantle, foot muscle, hepatopancreas, and ctenidium (n = 13) of Tridacna squamosa.

Organ
Transcript levels (copies of transcript per ng of total RNA) clam host conducts light-enhanced NO 3 − absorption through the ctenidium to benefit its symbionts. Indeed, our results confirm that Symbiodinium, Cladocopium, and Durusdinium of T. squamosa express NRT2 in the plasma membrane. They also indicate for the first time that different phylotypes of Symbiodiniaceae dinoflagellates in the coccoid stage could have different potentials of NO 3 − transport and hence different ability to assimilate NO 3 − into nitrogenous compounds such as amino acids.

NRT2 Is Localized at the Plasma Membrane of Coccoid Dinoflagellates
NRT2 is localized at the plasma membrane of the coccoid dinoflagellates of T. squamosa, indicating that it can transport NO 3 − from the luminal fluid of the zooxanthellal tubule into the symbionts. The intracellular vesicles that display NRT2 immunofluorescence could be involved in the transfer of NRT2 from the internal membranes to the plasma membrane as suggested previously for Arabidopsis thaliana (Wirth et al., 2007). Hence, the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 can be appropriately used as molecular indicators to estimate the potential of NO 3 − transport (or uptake) in Symbiodinium, Cladocopium, and Durusdinium, respectively.
Molecular characterization indicates that Symb-NRT2, Clad-NRT2, and Duru-NRT2 probably rely on an inwardly directed [H + ] gradient to drive the active absorption of NO 3 − . Recently, Mani et al. (2021) reported that coccoid dinoflagellates of T. squamosa could excrete H + into the luminal fluid of zooxanthellal tubules through the merging of intracellular vesicles containing vacuolar-type H + -ATPase (VHA) subunit B (VHA-B) with the plasma membrane. The excreted H + could augment the dehydration of luminal HCO 3 − and promote the absorption of CO 2 by the photosynthesizing symbionts. The inwardly directed [H + ] gradient generated by VHA across the plasma membrane could also drive the transport of NO 3 − through NRT2 into the symbiont. The absorbed NO 3 − could be reduced to NH 4 + . Then, a portion of the carbon fixed during photosynthesis could be utilized to assimilate NH 4 + into amino acids catalyzed by glutamate dehydrogenase, glutamine synthetase and glutamine oxoglutarate aminotransferase (Padgett and Leonard, 1996). Some of the amino acids synthesized could be donated to the host in support of its growth and development. Of note, glutamine synthetase and glutamine oxoglutarate aminotransferase have been cloned from dinoflagellates of T. squamosa (Fam et al., 2018;Teh et al., 2021).

Obtained From Vietnam
Cladocopium has been reported as the major genus of dinoflagellate found in T. squamosa from Japan (Ikeda et al., 2017), which is temperate and subtropical. However, based on the transcript levels of Symb-rbcII,  4 | Ratios of the transcript levels of nitrate transporter 2 (NRT2) to that of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcII) from Symbiodinium (Symb-NRT2/Symb-rbcII), Cladocopium (Clad-NRT2/Clad-rbcII), and Durusdinium (Duru-NRT2/Duru-rbcII) obtained from the outer mantle of Tridacna squamosa (n = 13).  Clad-rbcII, and Duru-rbcII, Poo et al. (2021) reported that T. squamosa (n = 4) obtained from Vietnam contained mainly Durusdinium (85-95%). Although we also worked with T. squamosa from Vietnam, the individuals involved were different from those examined by Poo et al. (2021). In general, the transcript levels of Symb-rbcII, Clad-rbcII, and Duru-rbcII reported by Poo et al. (2021) were higher than the corresponding results obtained in this study, which could be due to differences in the environmental conditions in the natural habitats of these two batches of T. squamosa. Nonetheless, in agreement with Poo et al. (2021), results of this study (n = 13) also denoted Durusdinium as the dominant genus of dinoflagellates in our experimental animals based on the transcript levels of NRT2 (78-92%) and rbcII (88-94%). This could be related to the tropical environmental conditions of and the availability of Symbiodiniaceae dinoflagellate phylotypes in Vietnam waters. Durusdinium is generally tolerant of a variety of environmental stressors (Brown et al., 2002;van Oppen et al., 2009), including high light intensity that would result in bleaching (Kemp et al., 2014) and turbid reef environments (Ulstrup and Van Oppen, 2003;LaJeunesse et al., 2010;Tonk et al., 2013). Thus, the high abundance of Durusdinium in the outer mantle of T. squamosa from Vietnam could have provided these clams with an advantage under stressful conditions, particularly at elevated temperature in the tropics. Based on the transcript level of Symb-rbcII, individuals of T. squamosa obtained from Vietnam contained relatively small populations of Symbiodinium (1-13%, Poo et al., 2021;0.1-3%, this study), which could be due to the rarity of Symbiodinium in Indo-Pacific waters as compared to the Red Sea and the Caribbean waters (LaJeunesse, 2002;Baker, 2003).

The Implications of Different Percentage
Proportions of Symb-rbcII and Symb-NRT2 Transcripts in Certain Organs of Tridacna squamosa There are discrepancies in the percentage proportions of Symb-rbcII and Symb-NRT2 transcripts in three organs of the 13 individuals of T. squamosa examined in this study. The percentage proportions of Symb-rbcII in the outer mantle, ctenidium and hepatopancreas (0.8, 1.9, and 3.0%, respectively) were apparently lower than the percentage proportions of Symb-NRT2 in the same organ (10.5, 4.4, and 28.3%, respectively). A logical explanation is that some phylotypes of Symbiodinium in these three organs expressed more transcripts of NRT2 per dinoflagellate cell than phylotypes of Cladocopium and Durusdinium. Hence, our results indicate the presence of different phylotypes of Symbiodinium with different expression levels of NRT2 in the five organs T. squamosa. They also signify that some Symbiodinium phylotypes had a higher potential of NO 3 − transport than the phototrophic potential. Thus, it was crucial to analyze the transcript ratios of NRT2 to rbcII.
Symbiodinium Generally Have Higher Potential of NO 3 − Transport Than Cladocopium and Durusdinium The transcript ratio of NRT2/rbcII estimated for each genus of dinoflagellates (e.g., Symb-NRT2/Symb-rbcII) could indicate whether that genus would have a high or low potential of 6 | Ratios of the transcript levels of nitrate transporter 2 (NRT2) to that of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcII) from Symbiodinium (Symb-NRT2/Symb-rbcII), Cladocopium (Clad-NRT2/Clad-rbcII), and Durusdinium (Duru-NRT2/Duru-rbcII) obtained from the foot muscle of Tridacna squamosa (n = 13).  NO 3 − transport relative to the potential of phototrophy. Any difference in the transcript ratios among the three genera of dinoflagellates would suggest different potentials of NO 3 − transport and therefore different abilities to assimilate NO 3 − for the production of amino acids and other nitrogenous compounds. The mean transcript ratios of Symb-NRT2/Symb-rbcII were significantly higher than those of Clad-NRT2/Clad-rbcII and Duru-NRT2/Duru-rbcII in the outer mantle, inner mantle, foot muscle and hepatopancreas of T. squamosa, and the greatest differences were observed in the outer mantle. Hence, it can be concluded that the phylotypes of Symbiodinium generally had higher potential of NO 3 − transport than the phylotypes of Cladocopium and Durusdinium in these four organs. For the ctenidium, Symb-NRT2/Symb-rbcII was also significantly higher than Clad-NRT2/Clad-rbcII but comparable to Duru-NRT2/Duru-rbcII. These results imply that the composition of Symbiodinium phylotypes present in the ctenidium of T. squamosa were dissimilar to those present in the other four organs, and particularly distinct from those in the outer mantle.

Foot muscle of individuals
Some Phylotypes (Species) of Symbiodinium Might Have Higher Potential of NO 3 − Transport Than the Phototrophic Potential Symbiodiniaceae dinoflagellates are phototrophic and express high transcript levels of rbcII (Poo et al., 2020). Yet, the mean transcript levels of Symb-NRT2 in the outer mantle, inner mantle, foot muscle and hepatopancreas of 13 T. squamosa individuals were substantially higher than the corresponding mean transcript levels of Symb-rbcII, resulting in the mean Symb-NRT2/Symb-rbcII ratios of 4.0-8.4 in these four organs. In the outer mantle, the mean Symb-NRT2/Symb-rbcII ratio of 8.4 was much greater than the mean Clad-NRT2/Clad-rbcII ratio of 0.39 and the mean Duru-NRT2/Duru-rbcII ratio of 0.63. These results are unusual as they indicate for the first time that certain phylotypes of Symbiodinium could specialize in NO 3 − absorption, and hence NO 3 − assimilation, more so than photosynthesis. They also indicate that the potentials of NO 3 − transport in these Symbiodinium phylotypes were greater than those in phylotypes of Cladocopium and Durusdinium in general.

Two Physiologically Distinct Phylotypes (Species) of Symbiodinium Based on Symb-NRT2/Symb-rbcII
Of the 13 individuals examined, 11 had ratios of Symb-NRT2/Symb-rbcII that ranged between 7.2 and 13.1 in the outer mantle of T. squamosa, but the ratios for clam 3 (0.41) and clam 10 (1.34) were distinctly < 7. These results indicate that the phylotypes of Symbiodinium in clam 3 and clam 10 had strikingly lower potential of NO 3 − transport than those in the other 11 clams. Hence, there could be two physiologically distinct phylotypes (species) of Symbiodinium as defined by high or low potential of NO 3 − transport. To analyze our results, a Symb-NRT2/Symb-rbcII ratio of 1.0 (i.e., equal transcript levels of Symb-NRT2 and Symb-rbcII) was set arbitrarily to differentiate the Symbiodinium phylotypes with high potential of NO 3 − transport from those with low potential of NO 3 − . In the 13 individuals examined in this study, the  numbers of clams that displayed a ratio of >1.0 for the outer mantle, inner mantle, foot muscle, hepatopancreas and ctenidium were 12, 10, 11, 11, and 2, respectively. These results indicate that the proportion of phylotypes of Symbiodinium with high potential of NO 3 − transport varied among the five organs, and that the ctenidium contained mainly Symbiodinium phylotypes with low potential of NO 3 − transport. It is noteworthy that while muscle tissues can be found in the other four organs, the ctenidium comprises mainly epithelial tissues dedicated for gases exchange, membrane transport and acid-base balance. It could be that the demand for using NO 3 − to form amino acid is low in the symbionts of the ctenidium in comparison with the symbionts of the other four organs. It could also imply that the symbionts of the ctenidium prefer to use other source of nitrogen, e.g., ammonia, for nitrogen metabolism. On the other hand, the predominant presence of Symbiodinium phylotypes with high potential of NO 3 − transport in the outer mantle facilitates the effective coupling of NO 3 − transport and assimilation with photosynthesis. Hence, it can be concluded that the phylotype compositions of Symbiodiniaceae dinoflagellates could vary among organs of T. squamosa based on the host's physiological needs. Nonetheless, how the relevant phylotypes of Symbiodinium find the way to and establish in different organs of T. squamosa needs to be elucidated in the future.

Ctenidium of individuals
Cladocopium Phylotypes (Species) Also Display Different Potentials of NO 3 − Transport Based on Clad-NRT2/Clad-rbcII, the outer mantle of T. squamosa contained two physiologically distinct phylotypes of Cladocopium. Of the 13 individuals examined, eight had ratios of Clad-NRT2/Clad-rbcII ranging between 0.39 and 0.88. However, clams 2, 7, 9, 11, and 13 had distinctly lower Clad-NRT2/Clad-rbcII ratios (<0.1) and hence lower potential of NO 3 − transport than the other eight individuals. An examination of Clad-NRT2/Clad-rbcII ratios in the inner mantle, foot muscle, and hepatopancreas indicates the presence of some phylotypes of Cladocopium with Clad-NRT2/Clad-rbcII slightly > 1. The inner mantle, foot muscle, hepatopancreas, and ctenidium apparently contained dissimilar proportions of Cladocopium phylotypes with either high or low potentials of NO 3 − transport. Overall, Cladocopium phylotypes in the ctenidium, with a mean Clad-NRT2/Clad-rbcII ratio of 0.21 (n = 13), appeared to have lower potentials of NO 3 − transport than those in the other four organs (0.32-0.57). Again, this might indicate the relative low demand for using NO 3 − to form amino acids in the ctenidium as compared with the other four organs.

Implications and Perspective
Using NRT2 and rbcII as molecular indicators, our results indicate for the first time that Symbiodiniaceae dinoflagellates harbored by T. squamosa could have different potentials of NO 3 − transport, and offer insights into the functional diversity among coccoid dinoflagellates at the genus level (Symbiodinium, Cladocopium, and Durusdinium). A corollary of some phylotypes of Symbiodinium having a preference of NO 3 − uptake and assimilation is that different members of Symbiodiniaceae could be specialized in absorbing and utilizing distinct types of nitrogenous compounds (e.g., ammonia, urea and glutamine) furnished by the host. An important implication is that it would not be possible for the clam host to regulate its general symbiont population by simply reducing the availability of endogenous ammonia to them through the recycling of metabolic ammonia into non-essential amino acids, as has been suggested for scleractinian corals (Falkowski et al., 1993;Cui et al., 2019;Xiang et al., 2020). This is particularly the case for those Symbiodinium phylotypes that can effectively absorb and assimilate NO 3 − . Rather, the clam host might be able to regulate the population of a specific Symbiodiniaceae phylotype in a certain organ by controlling the translocation of a particular nitrogen-containing compound from the hemolymph through the tubular epithelial cells into the luminal fluid of the zooxanthellal tubules.
Importantly, our results signify that, in the coccoid stage, certain phylotypes (species) of Symbiodinium might specialize in nitrogen metabolism more so than photosynthesis as reflected by their high potential of NO 3 − transport with transcript ratios of Symb-NRT2/Symb-rbcII much greater than one. This implies that different phylotypes of Symbiodiniaceae dinoflagellates could have different abilities to donate amino acids and carbohydrates to the host. As the host requires ∼20 different types of amino acid for growth and reproduction, it is highly unlikely that these amino acids are donated equally by all the phylotypes of dinoflagellates. Rather, there could be a division of labor among different phylotypes of coccoid dinoflagellates in the supply of nutrients (e.g., specific types of carbohydrate or amino acid) to the host. Therefore, efforts should be made in the future to develop a large variety of molecular indicators (e.g., transporters of ammonia, urea and glutamine, as well as enzymes involved in the syntheses of various amino acids) in order to elucidate the distinct physiological roles of various Symbiodiniaceae phylotypes in the giant clam-dinoflagellate holobiont.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI (accession: MZ014639, MZ014640, and MZ014641).

AUTHOR CONTRIBUTIONS
SC and YI designed the experiments, wrote the manuscript, and involved in the analysis of data and approval of the manuscript. CP performed the experiments and analyzed the data. SC and CP participated in animal subjection and sample collection. All the authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by the Singapore Ministry of Education through grants to SC through National Institute of Education (NIE) Academic Research Fund RI 3/19 CSF and the NIE Research support for Senior Academic Administrator Grant RS 1/21 CSF.