Individuals of T. squamosa specimens (520 ± 180 g with shells; n = 19) were purchased from Xanh Tuoi Tropical Fish Ltd., in Vietnam. They were acclimated in three glass tanks (L90 cm × W62 cm × H60 cm) under a 12 h light:12 h dark regime for one month before experimentation. Each tank contained 350 L of recirculating seawater and 6 or 7 clams. The water conditions were as follows: salinity, 30–32; temperature, ∼26°C; pH, 8.1–8.3; hardness, 143–179 ppm; calcium, 380–420 ppm; phosphate, <0.25 ppm; nitrate, 0 ppm; nitrite, 0 ppm; total ammonia, <0.25 ppm. The underwater light intensity (PPFD) at the level of the clam was ∼120 μmol m^–2 s^–1.

Four individuals (controls; n = 4) were sampled directly and randomly from all the three glass tanks, and killed for tissue sampling after 12 h of darkness in the normal 12 h light:12 h dark regime. Other individuals were then exposed to light for 3, 6, or 12 h. At each specific time points, four individuals (n = 4) were randomly selected and killed for tissue sampling. Individual clams were anesthetized with 0.2% phenoxyethanol before being forced open to cut the adductor muscles. Collected tissue samples were stored at −80°C until analyses.

After being anesthetized in 0.2% phenoxyethanol, three individuals exposed to darkness for 12 h (n = 3) were killed for the sampling of tissues for immunofluorescence microscopy. Samples were immersion-fixed in 3% paraformaldehyde in seawater at 4°C overnight and processed according to the method of Hiong et al. (2017a).

The total RNA from tissue samples was isolated using TRI Reagent^TM (Sigma-Aldrich Co. St. Louis, MO, United States). Samples were homogenized with silica beads (425–600 μm; Sigma-Aldrich) in TRI Reagent^® using a Mini-G^TM tissue homogenizer (SPEX^® Sample Prep, Metuchen, NJ, United States). Remnant of DNA was removed by PureLink^TM RNA Mini Kit (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, United States), and the purified RNA was quantified by a Shimadzu BioSpec-nano spectrophotometer (Shimadzu Corporation, Tokyo, Japan). After verification of the integrity of RNA by electrophoresis, cDNA was synthesized from the purified total RNA using the RevertAid^TM first-strand cDNA synthesis kit (Thermo Fisher Scientific Inc.).

A partial PiT1-like sequence was isolated using a pair of PCR primers (forward: 5′-TGATCGTTGGTTCCTGGTT-3′; reverse: 5′-TCGCCAATCGTATTGATGAC-3′) designed from the conserved regions of the sodium-dependent phosphate transporter 1 of Octopus vulgaris (XM_029794590.1), Crassostrea gigas (XM_020063507.2), Crassostrea virginica (XM_022469514.1), Mizuhopecten yessoensis (XM_021500463.1), and Priapulus caudatus (XM_014826122.1). The PCR was conducted using a 9902 Veriti 96-well thermal cycler (Applied Biosystems, Carlsbad, CA, United States) and DreamTaq^TM polymerase (Thermo Fisher Scientific Inc.). The thermal cycling conditions included an initial denaturation at 95°C held for 3 min, followed by 40 cycles of denaturation, annealing and extension at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, respectively, and a final extension for 10 min at 72°C. Agarose gel electrophoresis was used to separate the PCR products, and the Wizard^® SV Gel and PCR clean-up system (Promega Corporation, Madison, WI, United States) was used to extract the targeted bands.

Following the method of Chan et al. (2020), the pGEM-T Easy vector system (Promega Corporation) was used to clone and transform the extracted PCR product into JM109 Escherichia coli competent cells. Multiple clones of PiT1-like were sequenced bi-directionally. BlastN¹ was used to confirm the identity of the sequences obtained. Analysis of multiple sequences revealed the absence of isoforms. The full coding sequence of PiT1-like was obtained by conducting 5′ and 3′ RACE-PCR (SMARTer^TM RACE cDNA amplification kit; Clontech Laboratories, Mountain View, CA, United States) with specific RACE primers (forward: 5′-TTCCTGCAAATCCTTACTGCTGTGTT-3′, reverse: 5′-CAA TTCGGATCCTTTGTAGAAGACAG-3′).

BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc.) was used to prepare samples for sequencing, and sequencing was performed using a 3130XL Genetic Analyzer (Thermo Fisher Scientific Inc.). Sequences obtained were assembled and analyzed using BioEdit version 7.2.5. The GenBank Accession number of the full coding sequence of PiT1-like obtained from T. squamosa is MN256447.

The nucleotide sequence was translated into PiT1-like amino acid sequence using the ExPASy Proteomic server². The deduced amino acid sequence was aligned with selected PiT1-like sequences from various species of mollusks and vertebrates to confirm its identity. The TOPCONS server was used to predict the transmembrane regions (TMs).

The gene expression of PiT1-like in the outer mantle, inner mantle, ctenidium, kidney, heart, hepatopancreas, adductor muscle, byssal retractor muscle, and foot muscle of T. squamosa was examined quantitatively by qPCR with gene-specific qPCR primers (forward: 5′-CAATCCTGGTTCGTCTGCTG-3′; reverse: 5′-TCTCAACAAGAACTCCCTCCTC-3′). qPCR was carried out with a StepOnePlus^TM Real-Time PCR System (Thermo Fisher Scientific) and each reaction contained various quantities of cDNA of sample, 0.3 μmol l^–1 each of specific forward and reverse primers and 5 μl of qPCRBIO SyGreen Mix (PCR Biosystems Ltd., London, United Kingdom). The cycling conditions consisted of an initial 20 s denaturation and enzyme activation at 95°C, 40 cycles of 95°C for 3 s and 60°C for 30 s. Absolute quantification of transcript level of PiT1-like from the kidney, ctenidum, and outer mantle were performed in triplicates. The specific qPCR primers had a amplification efficiency of 93%. The transcript level in the sample was estimated using a standard curve constructed according to the method of Hiong et al. (2017a).

For immunofluorescence microscopy and western blotting, a commercial firm (GenScript, Piscataway, NJ, United States) was engaged to produce a custom-made rabbit polyclonal anti-PiT1-like antibody against the epitope TPSSGFSIEVGSAA (residue 468–481) of PiT1-like of T. squamosa. For western blotting, an anti-α-tubulin antibody, 12G10 (Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa, Iowa City, IA, United States), was used to detect the reference protein, α-tubulin.

Proteins were extracted from samples of kidney, ctenidium, and outer mantle following the method of Hiong et al. (2017a). SDS-PAGE (10%) electrophoresis was applied to separate proteins of kidney (20 μg), ctenidium (50 μg) or outer mantle (100 μg) samples. Separated proteins were blotted onto a nitrocellulose membrane, which was then incubated for 1 h at 25°C with anti-PiT1-like (2 μg ml^–1) or anti-α-tubulin (12G10, 0.05 μg ml^–1) antibody. The membrane was processed using the Pierce Fast Western Blot kit, SuperSignal^® West Pico Substrate (Thermo Fisher Scientific Inc.). Visualization of protein bands was achieved using a ChemiDoc^TM MP Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, Untied States). Optical density of the protein bands were determined using ImageJ (version 1.50i, NIH) and a 21-step Tiffen (The Tiffen Company, Rochester, NY, Untied States) transmission photographic step tablet (EK1523406T). Protein abundance of PiT1-like was expressed as the optical density of the PiT1-like band normalized with that of the α-tubulin band. As the protein expression level of PiT1-like was the highest in kidney, a peptide competition assay (PCA) was performed to validate the specificity of the custom-made anti PiT1-like antibody on kidney exposed to light for 12 h (Supplementary Figure 1). The anti-PiT1-like antibody was incubated with neutralizing peptide (Genscript) in a ratio of 1:5 for 1 h prior to western blotting.

Samples of the kidney, ctenidium, and outer mantle were excised and immersion-fixed overnight in 3% paraformaldehyde in seawater at 4°C, and processed according to the method of Hiong et al. (2017b). The paraffin-embedded samples were sectioned (3 μm) using a Leitz 1512 rotary microtome (Leica Biosystems, Germany) and collected on slides. Antigen retrieval was performed by treating deparaffinized sections with heated solution of 0.05% citraconic anhydride (Nacalai Tesque, Japan), pH 7.4, for 5 min with a further 10 min incubation in 1% sodium dodecyl sulfate (SDS) solution at room temperature. The section was blocked in 1% BSA in TPBS (0.05% Tween-20 in phosphate-buffered saline: 10 mmol l^–1 Na₂HPO₄, 1.8 mmol l^–1 KH₂PO₄, 137 mmol l^–1 NaCl, 2.7 mmol l^–1 KCl, pH 7.4) for 20 min. The blocked section was incubated with the anti-PiT1-like antibody, which had been diluted to 2.5 μg ml^–1 in Signal Enhancer HIKARI Solution A (Nacalai Tesque), for 1 h in a humid chamber. It was then incubated for another 1 h at 37°C with the fluorochrome-coupled goat anti-rabbit gamma globulin (Alexa Fluor 488; Life Technologies Corporation) diluted to 2.5 μg ml^–1 in Signal Enhancer HIKARI Solution B (Nacalai Tesque). The slide was washed again with TPBS, counter-stained with fluorescing DNA stain, 4′ 6-diamidino-2-phenylindole (DAPI) and mounted in Prolong Gold antifade reagent (Thermo Fisher Scientific Inc.).

Digital photos were taken using an Olympus DP73 camera (Olympus Corporation, Tokyo, Japan) fitted to an Olympus BX60 epifluorescence microscope equipped with a differential interference contrast (DIC) slider (Olympus U-DICT), as well as corresponding filter sets (Olympus U-MWU, U-MWIG, and U-MNIBA) and various objectives. DIC images were taken to visualize tissue structure. For the ctenidium and the outer mantle, nuclei were stained with DAPI (in blue) and visualized using the U-MWU filter (emission at 420 nm). Autofluorescence (in red) of the plastid of symbiotic dinoflagellates in the outer mantle was captured using the U-MWIG band pass filter (emission at 580 nm). PiT1-like immunostaining (in green) was visualized using the U-MNIBA band pass filter (emission at 515–550 nm). All images were processed using the cellSens Standard v1.15 software (Olympus Corporation) and prepared using the Adobe Photoshop CS6 (Adobe Systems, CA, United States). The overlaid of images was performed using Adobe Photoshop CC.

Results were presented as mean + SEM. Statistical analysis was performed using SPSS Statistics version 19 software (IBM Corporation, Armonk, NY, United States). Levene’s test was used to examine the homogeneity of variance among the means. Depending on the homogeneity of variances, one-way analysis of variance (ANOVA), followed by Dunnett T3 post hoc test or Tukey post hoc test, was used to evaluate differences among means. Differences among means with a p-value less than 0.05 were regarded as statistically significant.

The complete cDNA coding sequence of PiT1-like obtained from the kidney of T. squamosa (GenBank accession number MN256447) comprised 1,665 bp, and the deduced amino acid sequence had 553 amino acids with an estimated molecular mass of 59.3 kDa.

An analysis of sequence similarities among PiT1-like of T. squamosa and PiT1 proteins of various species obtained from GenBank (Table 1) indicated that it had the highest sequence similarity with those of mollusks (50.8–54.4%), followed by those of cnidarians, brachiopods and echinoderms (38.4–48.6%), and it had the lowest sequence similarity with those of plants and chlorophytes (8.8–14.3%). The host origin of PiT1-like of T. squamosa was validated by phenogramic analysis (Figure 1). The low similarity of PiT1-like of T. squamosa with various NPT2s confirmed that it was indeed a PiT1 transport protein (Table 1). In addition, the N and C terminals of PiT1-like of T. squamosa contained the ProDom domain (Bottger and Pedersen, 2011) and the 12 amino acid-long signature sequence (Bottger and Pedersen, 2005) characteristic of the PiT family. Typical of PiT proteins, there were 11 predicted TMs in PiT1-like of T. squamosa. An alignment with other PiT1s (Homo sapiens, Xenopus laevis, and Exaiptasia diaphana) revealed the conservation of several important residues that are critical for P_i transport (D39, E66, H403, D407, E476, and S494, numbered according to PiT1-like of T. squamosa in Figure 2). Other conserved residues included those that constituted part of the substrate binding site (V77, I86, and V88) and those responsible for substrate selectivity (S78 and R82) of P_i binding (S125).

In T. squamosa, PiT1-like was expressed strongly in the kidney and ctenidium, and moderately in the outer mantle, inner mantle, heart, and adductor muscle. It was only weakly expressed in the hepatopancreas, byssal retractor muscle, and foot muscle (Figure 3). Hence, efforts were focused subsequently on elucidating the possible functions of PiT1-like in the kidney, ctenidium, and outer mantle of T. squamosa.

The kidneys of giant clams are known to consist of nephrocytes, which are large globular cells with indistinct boundaries and little visible cytoplasmic contents (Norton and Jones, 1992). In the kidney of T. squamosa, PiT1-like was localized in the membrane surrounding a type of nephrocytes (Figure 4). In the ctenidium of T. squamosa, PiT1-like had a basolateral localization in the filaments and tertiary water channels (Figure 5). In addition, PiT1-like was detected in the epithelial cells of the tertiary zooxanthellal tubules in the outer mantle (Figure 6). In order to identify the location of PiT1-like in the tubular epithelium, it was essential to capture, albeit with some difficulties, the nuclei of the tubular epithelial cells. Nonetheless, based on three separate locations of the nuclei of the tubular epithelial cells in the image presented in Figure 6 and many similar observations in other slide preparations, PiT1-like appeared to have a basolateral localization in the tubular epithelium.

In T. squamosa, the transcript level of PiT1-like in the kidney was approximately 10-fold higher than those in the ctenidium and outer mantle (Figure 7). There were significant increases in the transcript levels of PiT1-like in the kidney at hour 12 of light exposure (Figure 7A), in the ctenidium at hour 6 or 12 of light exposure (Figure 7B), and in the outer mantle at hour 3 of light exposure (Figure 7C), when compared with the corresponding values of the control clams kept in darkness for 12 h.

As the kidney and ctenidium had apparently higher protein abundance of PiT1-like than the outer mantle, the protein loads of samples of kidney, ctenidium, and outer mantle for electrophoresis were 20, 50, and 100 μg (Figure 8), respectively. The protein abundance of PiT1-like increased significantly in the kidney of T. squamosa after 6 or 12 h of exposure to light, as compared to the control (Figure 8A). In comparison, the protein abundance of PiT1-like increased significantly in the ctenidium and outer mantle only after 12 h of exposure to light as compared to the control (Figures 8B,C). The specificity of anti-PiT1-like antibody was validated by PCA, whereby the PiT1-like band of the kidney sample was abolished using the peptide-neutralized antibody (Supplementary Figure 1).

PiT1-like of T. squamosa consists of two homology ProDom domains (PD001131), one at the N terminal region and another at the C terminal region (Salaün et al., 2004; Bottger and Pedersen, 2011), which are characteristic of PiT proteins. Moreover, two related sequence motifs (of 12 amino acids long), which are designated as signature sequences of the PiT family and involved in cation translocation (Bottger and Pedersen, 2005), can be found in the PD001131 domains of PiT1-like of T. squamosa. All conserved amino acids known to be critical for P_i transport are located inside the two PD001131 domains of PiT1-like. Based on the characterization of human PiT2 (Bottger and Pedersen, 2011), residues D39, E66, H403, D407, E476, and S494 in PiT1-like of T. squamosa could be involved in the translocation of H₂PO₄^– (Bottger and Pedersen, 2002, 2005, 2011), although the exact mechanism remains elusive (Beck et al., 2009). The two conserved glutamate residues situated in the TMs could act to couple Na⁺ transport with P_i uptake (Bottger et al., 2006). Similar to PiT1 of H. sapiens and X. laevis, PiT1-like of T. squamosa comprises several conserved residues, such as V77, S78, R82, I86, V88, and S125, which constitute the substrate binding site(s) (Ravera et al., 2013; Bon et al., 2017). In particular, S125 may be responsible for binding of H₂PO₄^– (Bon et al., 2017) while S78 and R82 are responsible for substrate selectivity and coordination (Ravera et al., 2013). As V77, S78, R82, I86, and V88 are located in the extracellular loop region 2, they may be involved in the formation of a hydrophilic pore through which substrates might interact with the binding sites during the transport cycle (Ravera et al., 2013). With conserved substrate binding sites, PiT1-like of T. squamosa can co-transport Na⁺ and H₂PO₄^– against the uphill H₂PO₄^– electrochemical gradient at the expense of the downhill electrochemical gradient of Na⁺ through the plasma membrane into the cell.

PiT1-like expression was detected in many organs of T. squamosa, indicating a possible house-keeping role in P_i homeostasis. However, PiT1-like was expressed strongly in the kidney of T. squamosa, which is known to contain much more phosphate than other organs (Belda and Yellowlees, 1995), denoting a probable role in the uptake and accumulation of P_i. The strong expression of PiT1-like in the ctenidium denotes an important physiological function, but whether it could be involved in exogenous P_i absorption depends on its subcellular localization. Furthermore, the moderate gene expression level of PiT1-like in the colorful outer mantle suggests an important role in the supply of P_i to the dinoflagellates that are found in high density therein. In order to elucidate the functions of PiT1-like, it was essential to determine its cellular and subcellular localization in these three organs by immunofluorescence microscopy.

Extracellular P_i can act as a signaling molecule that directly alters gene expression and cellular physiology. Cells can detect changes in extracellular P_i levels through a P_i-sensing mechanism (Camalier et al., 2013; Michigami, 2013) that may involve the MAPK ERK1/2 pathway (Khoshniat et al., 2011). In human skeletal cells, PiT1 and PiT2 heterodimerization can mediate P_i signaling independent of P_i uptake (Bon et al., 2017). The deletion of PiT1 or PiT2 impedes phosphorylation mediated by the P_i-dependent MAPK ERK1/2 signaling (Bon et al., 2017). If P_iT1-like has an apical localization in epithelial cells in T. squamosa, it could not be involved in sensing the concentration of P_i in the hemolymph. However, if it has a basolateral localization in epithelial cells and direct contact with the hemolymph, it could possibly function as a transporter to translocate H₂PO₄^– from the hemolymph into the cell, as well as a sensing mechanism to detect hemolymph H₂PO₄^– but without transporting it. Although it would be difficult to decipher these two functions of P_iT1-like in T. squamosa, attempts were made to interpret our results based on the physiological functions of the three organs studied.

Symbiotic animals absorb P_i from the ambient seawater not only for their own metabolism, but also for the needs of the symbionts (Jackson and Yellowlees, 1990). P_i uptake in corals (Jackson and Yellowlees, 1990; Godinot et al., 2009) and giant clams (Chan et al., 2020) are light-dependent. For giant clams, it has been postulated that the host could maintain low concentrations of P_i (<0.1 μM) in the hemolymph to control the internal population of symbiotic dinoflagellates (Belda and Yellowlees, 1995). Without knowledge of the mechanism involved, Belda and Yellowlees (1995) proposed that the kidney of giant clams could rapidly take up P_i circulating in the hemolymph to limit the availability of P_i to the symbionts. Our results demonstrate for the first time that the plasma membrane of the globular nephrocytes in the kidney of T. squamosa displayed a strong immunolabeling of PiT1-like. Nephrocytes are located adjacent to the hemolymph sinuses. Hence, they can probably sense the extracellular P_i concentration and actively absorb H₂PO₄^– from the hemolymph through PiT1-like, driven by the electrochemical potential gradient of Na⁺ (Virkki et al., 2007). Furthermore, the gene and protein expression levels of PiT1-like increased significantly in the kidney of T. squamosa during illumination, indicating its involvement in the increased removal of P_i from the hemolymph during light-enhanced P_i uptake to maintain a low hemolymph P_i concentration. As P_i is scarce in seawater, the host probably accumulates P_i in nephrocytes to cater for its long-term growth and development, but whether the accumulation of P_i in nephrocytes is meant to control the populations of symbionts (Belda and Yellowlees, 1995) is debatable. Results obtained from the outer mantle of T. squamosa indicate that the host clam could probably increase the supply of P_i to the symbionts through PiT1-like during illumination, as they would logically have a higher demand for P_i in light than in darkness.

The ctenidium of T. squamosa expresses NPT2a-like in the apical membrane of its epithelial cells (Chan et al., 2020). The inwardly directed Na⁺ electrochemical gradient would drive HPO₄^2– from the ambient seawater into the epithelial cells through the apical NPT2a-like. Then, P_i can be translocated through some unknown basolateral transporter to the hemolymph. Our results indicate that the ctenidial epithelial cells of T. squamosa also expressed PiT1-like in the basolateral membrane. The basolateral localization prescribes that PiT1-like would transport H₂PO₄^– from the hemolymph into the epithelial cells. Enterocytes of human intestine also express an apical NPT2b as well as basolateral PiT1 and PiT2 (Kiela and Ghishan, 2018), whereby NPT2b absorbs P_i from the intestinal lumen under normal circumstances, and PiT1/PiT2 function to import P_i from the blood for cellular P_i homeostasis during periods of dietary insufficiency (Kiela and Ghishan, 2018). Hence, the possibility of the basolateral P_iT1-like in the ctenidial epithelial cells of T. squamosa absorbing P_i from the hemolymph during P_i-limiting situation cannot be ignored. However, the light-enhanced expression of P_iT1-like in the ctenidial epithelial cells might suggest that the capacity for P_i-sensing in these cells is enhanced by light. As P_i binding rather than P_i uptake is the key factor in mediating P_i signaling through the PiT protein, it is possible that the basolateral P_iT1-like in the ctenidial epithelial cells could act as part of a P_i-sensing mechanism, and that the physiological activities of these epithelial cells, including P_i uptake, can be regulated by the P_i concentration in the hemolymph, although future works are needed to verify these propositions.

The outer mantle of T. squamosa can be regarded as a crucial site of light-enhanced P_i absorption through NPT2a-like (Chan et al., 2020), and the P_i absorbed can be delivered directly to the dinoflagellates therein. As PiT1-like had a basolateral localization in the epithelial cells that formed the tertiary tubules in the outer mantle of T. squamosa, it could act as a mechanism for P_i sensing and signaling. It could also actively transport H₂PO₄^– from the hemolymph into the tubular epithelial cells against its concentration gradient, and then P_i could be transported through some unknown apical transporter into the luminal fluid of the zooxanthellal tubules. It has been postulated previously that the growth and reproduction of symbionts are determined by the low concentration of P_i in the hemolymph (Belda and Yellowlees, 1995). However, symbiotic dinoflagellates are surrounded by the luminal fluid of the zooxanthellal tubules, and the ionic concentrations in the luminal fluid are most likely different from those in the hemolymph. In fact, PiT1-like could logically facilitate and regulate the translocation of P_i from the hemolymph into the luminal fluid surrounding the symbionts.

Importantly, the transcript level and protein abundance of PiT1-like/PiT1-like increased significantly in the outer mantle at hour 3 and hour 12 of light exposure, respectively. The increase in the protein abundance of PiT1-like in the outer mantle occurred between hour 6 and hour 12, which aligned well with the light–enhanced expression of RuBisCO protein therein (Poo et al., 2020), and with the light-enhanced host’s carbon concentration mechanism (Ip et al., 2017, 2018). In light, photosynthesizing dinoflagellates need P_i to increase the synthesis of ATP during the light-dependent process of photosynthesis. The localization and light-dependency of PiT1-like expression in the outer mantle provide novel insights into the interaction between the host clam and the symbionts.