Increased Apical Sodium-Dependent Glucose Transporter Abundance in the Ctenidium of the Giant Clam Tridacna Squamosa Upon Illumination Christabel Y
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© 2019. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2019) 222, jeb195644. doi:10.1242/jeb.195644 RESEARCH ARTICLE Increased apical sodium-dependent glucose transporter abundance in the ctenidium of the giant clam Tridacna squamosa upon illumination Christabel Y. L. Chan1, Kum C. Hiong1, Celine Y. L. Choo1, Mel V. Boo1, Wai P. Wong1, Shit F. Chew2 and Yuen K. Ip1,3,* ABSTRACT relationships with certain dinoflagellates (zooxanthellae) (Trench, Giant clams contain phototrophic zooxanthellae, and live in nutrient- 1987). Giant clams are common members of coral reefs throughout deficient tropical waters where light is available. We obtained the the tropical Indo-Pacific (Neo et al., 2017). They can harbor three – complete cDNA coding sequence of a homolog of mammalian sodium/ genera of dinoflagellates of the family Symbiodiniaceae glucose cotransporter 1 (SGLT1) – SGLT1-like – from the ctenidium of Symbiodinium (formerly Symbiodinium clade A), Cladocopium the fluted giant clam, Tridacna squamosa. SGLT1-like had a host origin (formerly Symbiodinium clade C) and Durusdinium (formerly and was expressed predominantly in the ctenidium. Molecular Symbiodinium clade D) (Takabayashi et al., 2004; Hernawan, – characterizations reveal that SGLT1-like of T. squamosa could 2008; LaJeunesse et al., 2018) extracellularly in a branched transport urea, in addition to glucose, as other SGLT1s do. It has an tubular system. These symbionts live inside the zooxanthellal tubules apical localization in the epithelium of ctenidial filaments and water located mainly in the extensible and colorful outer mantle (Norton channels, and the apical anti-SGLT1-like immunofluorescence was et al., 1992), where they conduct photosynthesis during insolation. stronger in individuals exposed to light than to darkness. Furthermore, They transfer >95% of photosynthates to the host, which can ’ the protein abundance of SGLT1-like increased significantly in the generally satisfy the host s energy requirements (Fitt, 1993; Griffiths ctenidium of individuals exposed to light for 12 h, although the SGLT1- and Klumpp, 1996). With that, giant clams can perform light- like transcript level remained unchanged. As expected, T. squamosa enhanced shell formation (calcification) and maintain a high growth could perform light-enhanced glucose absorption, which was impeded rate in nutrient-deficient tropical waters where light is available by exogenous urea. These results denote the close relationships (Lucas et al., 1989). As the photosynthesizing zooxanthellae require a between light-enhanced glucose absorption and light-enhanced supply of inorganic carbon, research in the past has focused on SGLT1-like expression in the ctenidium of T. squamosa. Although inorganic carbon uptake and metabolism in giant clams (Rees et al., glucose absorption could be trivial compared with the donation of 1993b; Baillie and Yellowlees, 1998; Leggat et al., 2002, 2005; photosynthates from zooxanthellae in symbiotic adults, SGLT1-like Yellowlees et al., 2008). Nonetheless, giant clams can also obtain might be essential for the survival of aposymbiotic larvae, leading to some nutrients through filter feeding (Fankboner and Reid, 1986), its retention in the symbiotic stage. Apriori, glucose uptake through possible digestion of zooxanthellae in the digestive tract (Reid et al., SGLT1-like might be augmented by the surface microbiome through 1984), and uptake of dissolved organic molecules from the external nutrient cycling, and the absorbed glucose could partially fulfill the medium (Fitt, 1993). metabolic needs of the ctenidial cells. Additionally, SGLT1-like could More than a century ago, Putter (1909) suggested that marine partake in urea absorption, as T. squamosa is known to conduct light- organisms might be able to obtain nutrients by directly absorbing enhanced urea uptake to benefit the nitrogen-deficient zooxanthellae. certain molecules from the ambient seawater. Since then, evidence has been gathered to substantiate the absorption of isotopically labelled KEY WORDS: Carbohydrate, Calcification, Nitrogen, Symbiodinium, sugars and amino acids against their concentration gradients through Urea, Zooxanthellae epidermal tissues of annelids, echinoderms and pogonophores (Stephens and Schinske, 1961; Ferguson, 1967; Ahearn and INTRODUCTION Gomme, 1975; Davis and Stephens, 1984; Manahan, 1989; Pajor Tropical waters are poor in nutrients owing to a lack of overturn, and et al., 1989). Péquignat (1973) provided categorical evidence to therefore referred to as ‘deserts’ of the sea. To compensate for nutrient support the presence of a similar epidermal route of absorption for shortage in tropical waters, some marine invertebrates, including glucose and amino acids in the ctenidium (gill) and mantle of the giant clams and scleractinian corals, acquire and maintain symbiotic filibranch bivalve Mytilus edulis. Subsequently, by measuring D-glucose transport in brush-border membrane vesicles derived from the ctenidium of M. edulis, the functional presence of a sodium- 1Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore 117543, Republic of Singapore. 2Natural Sciences and Science dependent glucose transporter (SGLT) in its labial palps has been Education, National Institute of Education, Nanyang Technological University, confirmed (Pajor et al., 1989). Furthermore, in vitro incubation of the 1 Nanyang Walk, Singapore 637616, Republic of Singapore. 3The Tropical Marine ctenidium of the oyster Crassostrea gigas in artificial seawater reveals Science Institute, National University of Singapore, Kent Ridge, Singapore 119227, Republic of Singapore. that it can absorb D-glucose and D-galactose, but not 3-O-methyl-D- glucose, via an active carrier-mediated system (Bamford and Gingles, *Author for correspondence ([email protected]) 1974). In the absence of exogenous Na+, glucose uptake occurs by + Y.K.I., 0000-0001-9124-7911 simple diffusion, but an Na -dependent carrier-mediated process sensitive to phlorizin [a potent inhibitor of sodium/glucose Received 1 November 2018; Accepted 5 March 2019 cotransporter 1 (SGLT1); Panayotova-Heiermann et al., 1996] Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb195644. doi:10.1242/jeb.195644 inhibition is operating to absorb glucose in the ctenidium and the glucose (and urea) uptake in giant clams, which may offer insights mantle of C. gigas. A kinetic analysis of glucose uptake confirms the into ways to enhance their growth and survivorship in this rapidly presence of a saturable component at low substrate concentrations, and changing climate. a diffusive component at high substrate concentrations. Subsequently, asequenceofSGLT-like has been obtained from the oyster C. gigas MATERIALS AND METHODS (Huvet et al., 2004) that has a high mRNA expression level in the Animals ctenidium and the mantle edge but low expression in other tissues/ Adult Tridacna squamosa Lamarck 1819 (mass=500±180 g, N=42) organs (Hanquet et al., 2011). were procured from Xanh Tuoi Tropical Fish (Ho Chi Minh City, SGLTs cotransport Na+ and glucose down the electrochemical Vietnam). The giant clams were maintained in tanks as described by potential gradient of Na+ but up against the concentration gradient of Ip et al. (2015) but with slight modifications. The water temperature glucose across cell membranes. Six isoforms of the SGLT gene was maintained at 26±1°C, the salinity was 30–32 and the belonging to the SLC5 gene family have been identified in humans pH ranged between 8.1 and 8.3. The carbonate hardness was (Wright et al., 2011). All SGLTs have 14 transmembrane regions 143–179 ppm and the calcium concentration was 280–400 ppm. (TMs) in topology (Wright et al., 2011). In humans, SGLT1 is Each tank was illuminated from the top by two sets of Aquazonic T5 expressed mainly in the intestine and kidney, where it functions lighting systems (Yi Hu Fish Farm Trading, Singapore), and each as a glucose/galactose transporter. In mouse intestine, SGLT1 is system consisted of two sun and two actinic blue fluorescence tubes expressed in the brush-border membranes, and glucose absorption (39 W each). Using a Skye SKP 200 display meter connected with a across these membranes disappears in SGLT1-deficient mouse, SKP 215 PAR Quantum sensor (Skye Instruments, UK), the light indicating that intestinal glucose absorption is mediated intensity at the level of the giant clams was determined as predominantly by SGLT1 (Gorboulev et al., 2012). ∼100 μmol m−2 s−1. Institutional approval was not necessary for Until now, it was unknown whether giant clams express a homolog research on giant clams (National University of Singapore of SGLT1 (SGLT1-like) in their ctenidia, and whether they can Institutional Animal Care and Use Committee). absorb glucose from the ambient seawater. Nonetheless, giant clams are known to display light-dependent physiological properties, Exposure of animals to experimental conditions for tissue including light-enhanced shell formation (Sano et al., 2012; Ip collection et al., 2017a) and light-enhanced nitrogen uptake (Wilkerson and At the end of the 12 h dark period of the 12 h:12 h light:dark regime, Trench, 1986; Chan et al., 2018). In addition, the gene and/or protein one batch of giant clam (N=5; control) was killed and sampled. The expression levels of some of their enzymes and transporters are also other 15 individuals were separated into three batches (N=5 for light dependent