Title Phototrophic potential and form II ribulose-1,5-bisphosphate carboxylase/oxygenase expression in five organs of the fluted giant clam, Tridacna squamosa Author(s) Jeslyn S. T. Poo, Celine Y. L. Choo, Kum C. Hiong, Mel V. Boo, Wai P. Wong, Shit F. Chew, and Yuen K. Ip Source Coral Reefs, 39, 361–374 Published by Springer
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This is a post-peer-review, pre-copy/edit version of an article published in Coral Reefs. The final authenticated version is available online at: https://doi.org/10.1007/s00338-020-01898-7
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1 Phototrophic potential and form II ribulose-1,5-bisphosphate
2 carboxylase/oxygenase expression in five organs of the fluted giant clam,
3 Tridacna squamosa
4
5 Jeslyn S. T. Poo1, Celine Y. L. Choo1, Kum C. Hiong1, Mel V. Boo1, Wai P. Wong1, Shit
6 F. Chew2 and Yuen K. Ip1*
7 1Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore
8 117543, Republic of Singapore
9 2Natural Sciences and Science Education, National Institute of Education, Nanyang
10 Technological University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
11 *author for correspondence
12 13 Running head: RuBisCO in T. squamosa
14 ------
15 Correspondence address: 16 Dr. Y. K. Ip 17 Professor 18 Department of Biological Sciences 19 National University of Singapore 20 Kent Ridge 21 Singapore 117543 22 Republic of Singapore 23 24 Phone: (65)-6516-2702 25 Fax: (65)-6779-2486 26 Email: [email protected] 27
28 Keywords: Coral reefs, light-enhanced calcification, photosynthesis, symbiosis, zooxanthellae
29
0 30 Abstract
31 Despite living in oligotrophic tropical waters, giant clams can grow to large sizes because they
32 live in symbiosis with extracellular phototrophic dinoflagellates (zooxanthellae) and receive
33 photosynthates from them. The physical presence of zooxanthellae in five organs (colorful outer
34 mantle, whitish inner mantle, ctenidium, hepatopancreas and foot muscle) of Tridacna
35 squamosa had been confirmed by microscopy, and high densities of zooxanthellae were
36 detected in specific regions of the inner mantle and foot muscle. Symbiotic dinoflagellates use
37 form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) to fix inorganic carbon
38 during C3 photosynthesis. Using qPCR primers that were designed comprehensively against all
39 known zooxanthellal form II RuBisCO gene sequences (rbcII) in existing databases, we
40 demonstrated that the outer mantle of T. squamosa (TS) had the greatest phototrophic potential
41 as reflected by its high zooxanthellal rbcII (TSZrbcII) transcript level, which varied among
42 different regions of the outer mantle. The other four organs also expressed moderate levels of
43 TSZrbcII, despite the lack of iridophores and direct light exposure. Importantly, light exposure
44 led to significant increases in the protein abundance of TSZRBCII in the outer mantle but not
45 the other four organs. Taken together, these results indicate that organs inside the mantle cavity
46 had low phototrophic potentials, but zooxanthellae residing inside these organs might serve
47 some unidentified functions to benefit the host.
1 48 Introduction
49 Dinoflagellates (Phylum: Dinoflagellata, Division: Pyrrhophyta) are a group of unicellular
50 alveolates with unique characteristics (Carty 2003). They can be phototrophic, heterotrophic or
51 mixotrophic, depending on their reliance on photosynthesis or food ingestion, or both, for
52 energy and nutrition needs (Carty and Parrow 2015). Nonetheless, all of them need inorganic
53 carbon (Ci), phosphorus and nitrogen for growth and reproduction. Some dinoflagellates
54 (Symbiodiniaceae) have free-living and symbiotic stages, and can live as intracellular or
55 extracellular symbionts inside host animals (Trench 1987). The symbiotic stage of these
56 dinoflagellates lack flagella, and are known as zooxanthellae. Phototrophic animal-
57 zooxanthellae associations, including giant clams, scleractinian corals and symbiotic anemones,
58 can flourish in nutrient-poor tropical waters where light is adequately available (de Goeij et al.
59 2013).
60 Giant clams belong to Phylum: Mollusca, Family: Cardiidae, Subfamily: Tridacninae,
61 and Genus: Tridacna or Hippopus. The host clam is known to associate with three genera of
62 dinoflagellates, Symbiodinium, Cladocopium, and Durusdinium (LaJeunesse et al. 2018), which
63 were named previously as Symbiodinium clades A, C and D, respectively (LaJeunesse et al.
64 2004; Takabayashi et al. 2004; Hernawan 2008; Lee et al. 2015). The host harbors extracellular
65 zooxanthellae in a branched tubular system surrounded by hemolymph (Norton et al. 1992),
66 and the majority of zooxanthellae reside in the tiny tertiary zooxanthellal tubules located in the
67 fleshy, colorful and extensible outer mantle peculiar to giant clams (Norton et al. 1992; Hiong
68 et al. 2017a; Ip et al. 2017a). During insolation, these symbionts conduct photosynthesis and
69 donate >95% of the photosynthates to the host, satisfying a major portion of the host’s energy
70 requirements (Fisher et al. 1985; Klumpp et al. 1992). This helps to sustain light-enhanced shell
71 formation and a high growth rate in the host clam (Sano et al. 2012; Ip et al. 2017a). In turn,
72 the host must supply essential nutrients to the symbionts that have no access to the ambient
73 seawater.
2 74 In giant clams, the outer mantle possesses host pigments and iridophores, which are
75 aggregations of special iridocytes containing tiny reflective platelets (Griffiths et al. 1992).
76 These platelets scatter light of photosynthetically conducive wavelengths to the zooxanthellae
77 embedded in the tissue while back-reflecting light of other wavelengths (Holt et al. 2014),
78 engendering multiple and variable colors to the outer mantle. Due to its prominent coloration
79 and ability to extend fully during insolation, research on giant clams in the past has focused on
80 the fleshy outer mantle and its zooxanthellae, with the general notion that the primary function
81 of these phototrophic zooxanthellae is to conduct photosynthesis and share the photosynthates
82 with the host clam. By contrast, the inner mantle adjacent to the extrapallial fluid, as demarcated
83 by the pallial line of the shell-valve, is thin and whitish, and is involved principally in shell
84 formation (calcification). It has no direct exposure to light and lacks iridophores, yet the region
85 of the inner mantle adjacent to the hinge of the shell-valves is always brownish in color,
86 indicating the probable presence of zooxanthellae (supplementary material, Fig. S1). Other
87 organs, like the ctenidia, hepatopancreas and foot muscle, also lack direct illumination and have
88 no iridophores, as they are located inside the mantle cavity and shaded from light by the
89 siphonal mantle. However, the foot muscle may possess a substantial quantity of zooxanthellae
90 at the tip that is also brownish in color (supplementary material, Fig. S1). Although the ctenidia
91 can be visible at an angle when the incurrent siphon is open, they are located below the siphonal
92 mantle and not directly under the opening. Hence, the intensity of light reaching the ctenidia
93 could be much lower than that received by the outer mantle. Taken together, zooxanthellae
94 residing in these four tissues/organs shaded from light might not be able to perform
95 photosynthesis effectively. Zooxanthellae conduct C3 photosynthesis (Streamer et al. 1993),
96 and the fixation of Ci in the Calvin cycle is catalyzed by form II ribulose-1,5-bisphosphate
97 carboxylase/oxygenase (RuBisCO) (Rowan et al. 1996; Mayfield et al. 2014). The form II
98 RuBisCO gene (rbcII) has been cloned and characterized from zooxanthellae of Tridacna gigas
99 (Rowan et al. 1996), and reported to be completely different from form I RuBisCO gene (rbcL)
3 100 of eukaryotes, as it bears ~65% similarity with RuBisCO gene (cbbM) of the proteobacterium,
101 Rhodospirillum rubrum (Rowan et al. 1996). Therefore, it is important to ask the foundational
102 question whether these four iridophore-free tissues/organs of giant clams would display any
103 phototrophic potential as reflected by the transcript and protein expression levels of
104 rbcII/RBCII of zooxanthellal origin.
105 Unlike the oligomeric structure of form I RuBisCO with both large and small subunits,
106 form II RuBisCO is devoid of the small subunit, and has the quaternary structure of (L2)n (Tabita
107 et al. 2008). The estimated molecular masses of the monomer and the native protein isolated
108 from the zooxanthellae of Tridacna gigas are 56 kDa and 300–350 kDa, respectively (Whitney
109 and Yellowlees 1995), suggesting a value of 3 for n. Besides structural differences, form II
110 RuBisCO also differs from form I RuBisCO in terms of kinetics and efficiency. Form II
111 RuBisCO cannot differentiate CO2 from O2 as effectively as form I RuBisCO (Morse et al.
112 1995; Tabita et al. 2008). The evolution of form I RuBisCO to associate with its small subunits
113 and assemble into an oligomer with the quaternary structure of L8S8 greatly improves its
114 catalytic efficiency and specificity (Erb and Zarzycki 2018). Consequently, form I RuBisCO
115 can support higher rates of CO2 fixation than form II RuBisCO. Nonetheless, both form I and
116 form II RuBisCO catalyze the same carboxylation and oxygenation reactions. As form I
117 RuBisCO is the main rate-limiting enzyme of CO2 fixation in the Calvin cycle, form II
118 RuBisCO can probably serve the same role (Rowan et al. 1996; Mayfield et al. 2014).
119 Therefore, it is logical to hypothesize that the transcript level of rcbII could probably act as
120 an indicator of the phototrophic potential of zooxanthellae in various organs of giant clams.
121 The first objective of this study was to confirm the presence of zooxanthellae in the five
122 organs (colorful outer mantle, whitish inner mantle, ctenidium, hepatopancreas and foot muscle)
123 of the fluted giant clam, Tridacna squamosa, which were obtained from Vietnam, by ethanol
124 treatment as well as differential interference contrast (DIC) and fluorescence (red channel)
125 microscopy. The second objective was to estimate the phototrophic potential, by determining
4 126 the transcript levels of T. squamosa zooxanthellal rcbII (TSZrbcII), in these five organs using
127 quantitative real-time PCR (qPCR). Efforts were made to design a set of qPCR primers that
128 would react comprehensively with all known sequences of rbcII available in existing databases
129 for Symbiodinium, Cladocopium, and Durusdinium (Bayer et al. 2012; Rosic et al. 2015;
130 Aranda et al. 2016; Levin et al. 2016; Liew et al. 2016; Shoguchi et al. 2018). The third objective
131 was to quantify the transcript levels of TSZrbcII in eight arbitrarily defined regions of the outer
132 mantle of T. squamosa, in order to verify whether the distribution of zooxanthellae in the outer
133 mantle would be uniform. Based on all the deduced amino acid sequences of RBCII from
134 various databases, a comprehensive anti-RBCII antibody was custom-made commercially to
135 achieve the fourth objective of confirming the localization of TSZRBCII in the plastids
136 (chloroplasts) of zooxanthellae in the outer mantle of T. squamosa. Finally, the fifth objective
137 was to test the hypothesis that the gene and protein expression levels of TSZrbcII/TSZRBCII
138 in the outer mantle, but not those in the other organs shaded from light, would be upregulated
139 during light exposure.
140 Due to anthropogenic activities, pollution and overharvesting for food and aquarium
141 trade, giant clam populations have been remarkably reduced in the wild, and they have been
142 listed in CITES Appendix II since 1985 (Wells and Barzdo 1991). In particular, the internal
143 population of zooxanthellae in giant clams can decrease drastically through a bleaching process
144 under certain environmental conditions, especially in relation to climate change, resulting in
145 the disruption of giant clam-zooxanthellae symbiotic associations (Leggat et al. 2003).
146 Traditionally, zooxanthellae have been assumed to be located predominantly in the colorful
147 outer mantle, and discoloration of the outer mantle is regarded as a major facet of the bleaching
148 process. Results obtained from this study were expected to shed light on the presence of
149 zooxanthellae in organs that are shaded from light in T. squamosa and draw attentions to their
150 possible roles, besides phototrophy, in the giant clam-zooxanthellae association.
151
5 152 Materials and Methods
153 Maintenance of animals
154 Institutional approval was not required for research on giant clams (National University of
155 Singapore Institutional Animal Care and Use Committee). Adult T. squamosa individuals (550
156 ± 50 g, mean ± SD; N=38) were purchased from Xanh Tuoi Tropical Fish, Ltd (Vietnam) and
157 housed in an indoor aquarium in three glass tanks (L90 × W62 × H60 cm) with recirculating
158 seawater. The conditions of the seawater maintained as follows: temperature at 26 ± 1°C;
159 salinity of 30–32; pH 8.1–8.3; hardness 143–179 ppm; calcium at 280–400 ppm; phosphate at
160 <0.25 ppm; nitrate at 0 ppm; total ammonia at <0.25 ppm. Giant clams were kept under a 12 h
161 light:12 h dark regime, and the light intensity measured under water at the level of the giant
162 clams was 120 μmol photons m-2 s-1 throughout the 12-h light period.
163 The light intensity used for experimental conditions should mimic the actual light
164 intensity received by the clams in their natural habitats. Although peak irradiances at the water
165 surface at noon on a sunny day can reach 2000 µmol photons m-2 s-1 in the tropical Pacific
166 (McFarland and Munz 1975), such high light intensities last several hours at the most (Marra
167 1978). Furthermore, only 5-8% of the incident light at the surface would reach the sea floor at
168 25 m, because of light attenuation in water (Fine et al. 2013). Tridacna squamosa happens to
169 be a deep water dwelling species, as the depth of occurrence ranges from 11 m to 20 m (Juinio
170 et al. 1989; Weber 2009; Andréfouët et al. 2014) and mature individuals can be found up to 42
171 m deep (Jantzen et al. 2008). As follows, Jantzen et al. (2008) exposed T. squamosa to 128 ±
172 59 μmol photons m-2 s-1, corresponding to the actual light intensity at ~20 m water depth, in
173 their study. Therefore, it is logical to postulate that zooxanthellae in the extensible outer mantle
174 of T. squamosa could display their full phototrophic potential as they would receive a light
175 intensity of 120 µmol photons m-2 s-1 in this study. Using light intensities that are
176 physiologically too high poses the danger of inducing photo-inhibition and even bleaching. In
177 fact, the photochemical efficiencies of corals (Montipora digitate and Stylophora pistillata) are
6 178 the lowest at noon, but increase as light intensity dropped (Jones and Hoegh‐Guldberg 2001).
179 Furthermore, the photosynthetic activity of several free-living dinoflagellates species could be
180 reduced to 20-30% of its maximum when exposed to high light intensities at mid-day (Ryther
181 1956).
182 Exposure of animals to experimental conditions for organ/tissue collection
183 After a month of acclimation to laboratory conditions following procurement, a clam was
184 sacrificed for ethanol treatment as well as DIC and fluorescence microscopy (N=1) to confirm
185 the presence of zooxanthellae in the five organs of T. squamosa, while another three clams were
186 used to determine the localization of TSZRBCII using immunofluorescence microscopy (N=3).
187 Various organ samples were collected from another ten clams for analyses of the zooxanthellae
188 populations by qPCR (N=10). Of these ten clams, three clams were also analyzed for the
189 distribution of zooxanthellae in different regions of the outer mantle, while five clams were
190 used to examine the distribution of zooxanthellae between the brown region and the white
191 region of the inner mantle and the foot muscle. To examine the effects of light on the transcript
192 level and protein abundance of TSZrbcII/TSZRBCII, the remaining 24 clams were maintained
193 in darkness for 12 h to be used as controls or exposed to 3, 6 or 12 h of light (N=6 for each time
194 point) before organ collection. The 12-h light intensity measured at the level of these giant
195 clams was 120 μmol photons m-2 s-1. Clams were anaesthetized with 0.2% phenoxyethanol, and
196 were forced open to cut the adductor muscles. The colorful outer mantle, inner mantle,
197 ctenidium, hepatopancreas and foot muscle were excised, freeze-clamped with pre-cooled
198 aluminium tongs and stored at –80°C until further processing for molecular analyses.
199 Photography, fluorescence microscopy and immunofluorescence microscopy
200 Organs excised from T. squamosa were fixed with 3.7% paraformaldehyde in seawater for 18
201 h, before dehydration in 70% ethanol for another 18 h at 4°C. Images of the five organs before
202 and after dehydration in 70% ethanol were obtained. Dehydration was continued with
203 increasing ethanol concentrations and Histoclear (Sigma-Aldrich Co., St. Louis, MO, USA).
7 204 The organs were embedded in paraplast (Sigma-Aldrich Co.) and sectioned to 5 μm, followed
205 by deparaffinization and rehydration in order to capture DIC and fluorescence microscopy (red
206 channel) images. For immunofluorescence microscopy, antigen retrieval was performed on the
207 deparaffinized sections with 1% sodium dodecyl sulfate in TPBS (0.05% Tween 20 in
-1 -1 -1 208 phosphate-buffered saline: 10 mmol l Na2HPO4, 1.8 mmol l KH2PO4, 137 mmol l NaCl,
209 2.7 mmol l-1 KCl, pH 7.4) for 10 min, and blocked with 1% BSA in TPBS for 30 min at room
210 temperature. The sections were incubated with the custom-made anti-RBCII antibody (2.5 µg
211 ml-1 in TPBS) for 1.5 h followed by Alexa-fluor-488 secondary antibody (2.5 µg ml-1 in TPBS)
212 for 1 h at 37°C. Nuclei were then stained with 6-diamidino-2-phenylindole dihydrochloride
213 (DAPI, Sigma-Aldrich Co.) for 10 min before capturing of images. All microscopy images were
214 captured using the Olympus microscope imaging system with universally adjusted brightness
215 and contrast, and processed by Adobe Photoshop CC (Adobe Systems, CA, USA).
216 A rabbit polyclonal anti-RBCII antibody was custom-made by GenScript (Piscataway,
217 NJ, USA) with the epitope ALDQSSRYADLSLD, which was designed at a conserved region
218 of 13 CbbM/RBCII sequences of various proteobacteria and dinoflagellates that were retrieved
219 from GenBank (supplementary material, Table S1). The epitope sequence had an 85.7-100%
220 identity with all these CbbM/RBCII sequences. Hence, in theory, this anti-RBCII antibody
221 could react comprehensively with the TSZRBCII sequences from all species of Symbiodinium,
222 Cladocopium and Durusdinium in T. squamosa.
223 Extraction of total RNA and cDNA synthesis
224 Total RNA from the various excised organs was extracted using TRI Reagent® (Sigma-Aldrich)
225 and purified with the PureLinkTM RNA Mini Kit (Invitrogen, Thermo Fisher Scientific Inc.,
226 Waltham, MA, USA). The concentration of purified RNA was determined with a Shimadzu
227 BioSpec-nano spectrophotometer (Shimadzu Corporation, Tokyo, Japan), after which RNA
228 integrity was verified with electrophoresis before being reverse-transcribed into cDNA with
229 RevertAidTM first strand cDNA synthesis kit (Thermo Fisher Scientific Inc.).
8 230 Determination of mRNA expression by quantitative real-time PCR (qPCR)
231 The mRNA transcript levels of TSZrbcII in various organs of T. squamosa were quantified
232 through qPCR with a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad,
233 CA, USA), following the methods described in Ip et al. (2017b). A set of gene-specific forward
234 and reverse qPCR primers (forward: 5’-CAGTTCTTGCACTACCACCG-3’; reverse: 5’-
235 CATCTTGCCGAAGCTCATGG-3’) was designed at a conserved region of all available rbcII
236 sequences from various dinoflagellate databases. These included 19 sequences from
237 Symbiodinium databases (Bayer et al. 2012; Aranda et al. 2016), three sequences from
238 Cladocopium databases (Levin et al. 2016; Liew et al. 2016; Shoguchi et al. 2018) and six
239 sequences from Durusdinium database (Rosic et al. 2015). This set of TSZrbcII qPCR primers
240 would theoretically react comprehensively with all known rbcII sequences from Symbiodinium,
241 Cladocopium and Durusdinium (see supplementary material, Table S2 for primer design), and
242 had an overall amplification efficiency of 95.2%. The absolute transcript levels of TSZrbcII
243 were calculated using a plasmid standard curve, and expressed as copies of transcript per ng
244 total RNA.
245 Western blotting
246 Proteins were extracted from various excised organs of T. squamosa according to the protocol
247 of Hiong et al. (2017b). Twenty micrograms (for the outer mantle) or 100 μg (for the other four
248 organs) of protein were heated at 95°C for 5 min before running through 10% SDS-PAGE and
249 transferred onto nitrocellulose membranes. Western blotting was then conducted with Pierce
250 Fast Western Blot kit, SuperSignal® West Pico Substrate (Thermo Fisher Scientific Inc.),
251 adhering to the manufacturer’s instructions. For the outer mantle samples, the membrane was
252 incubated with the custom-made anti-RBCII antibody at a 1:2000 dilution in Fast Western
253 Antibody Diluent, while the same antibody was used at 1:1000 dilution for the other four organ
254 samples. Bands were visualized via chemiluminescence using ChemiDocTM Imaging Systems
255 (Bio-Rad Laboratories, Hercules, CA, USA). The optical density of the bands was calculated
9 256 using ImageJ (version 1.50, NIH) after calibration with a 21-step reflection scanner scale (1″9
257 8″; Stouffer #R3705-1C). The protein abundance of TSZRBCII was expressed as arbitrary
258 densitometric units (a.u.) per 20 μg protein from the outer mantle or per 100 μg protein from
259 each of the other four organs. In this study, the protein abundance of the targeted protein in each
260 of the five organs was not normalized with a reference protein because the assumption of a
261 constant expression of the reference protein across different organs could not be justified. It has
262 been established that commonly used reference proteins such as tubulin and actin do not serve
263 as good reference proteins for studying relative expression in various tissues/organs because of
264 variable expression among different tissues, cell lines or experimental conditions (Thorrez et
265 al. 2008).
266 Statistics
267 Results were presented as means + S.E.M. Data obtained were analyzed with the SPSS Statistics
268 version 20 software (IBM Corporation, Armonk, NY, USA). Levene’s test was used to test the
269 homogeneity of variance. Following the fulfilment of the assumption of equal variance, either
270 independent t-test or one-way analysis of variance (ANOVA) was conducted to analyze the
271 differences between or among the means. After performing one-way ANOVA, the Tukey’s test
272 was conducted to evaluate the differences among the means of multiple experimental groups.
273 When the assumption of equal variance was not fulfilled, the differences among the means of
274 these experimental groups were analyzed using the Dunnett’s T3 test. Differences between
275 means with a P value that is less than 0.05 were regarded as statistically significant.
10 276 Results
277 Zooxanthellae are present in all five organs of T. squamosa
278 The originally brown outer mantle turned green after alcohol treatment, which was indicative
279 of a high chlorophyll content (Fig. 1a). In comparison, the inner mantle appeared to contain less
280 zooxanthellae, which were concentrated in a tiny region at the edge near the hinge, rendering it
281 a brownish color (supplementary material, Fig. S1), or green after alcohol treatment as
282 compared to the whitish middle part of the mantle (Fig. 1b). Similarly, the amount of
283 zooxanthellae present in the ctenidium seemed to be small, as indicated by the light
284 brown/green coloration observed in the middle ctenidial suspensory ligament (Fig. 1c). The
285 hepatopancreas also contained zooxanthellae, but they were not concentrated in a specific
286 region (Fig. 1d). In contrast, the tip of the foot muscle appeared to have high concentrations of
287 zooxanthellae as compared to the whitish region of the same organ (Fig. 1e; supplementary
288 material, Fig. S1).
289 In histological sections, red auto-fluorescence of chlorophyll under blue light made it
290 possible to identify and localize zooxanthellae. The overlay of DIC images with the red channel
291 revealed the localization of auto-fluorescing zooxanthellae inside the tubular networks of the
292 five organs (Fig. 2). In the outer mantle, the tertiary zooxanthellal tubules contained high
293 densities of zooxanthellae, arranged in a columnar-like pattern (Fig. 2a). In the inner mantle,
294 the tertiary tubules and the associated zooxanthellae were located closer to the seawater-facing
295 epithelium than the shell-facing epithelium. The amount of zooxanthellae present in the whitish
296 middle part of the inner mantle (Fig. 2b) was lower than that in the brownish region close to the
297 valve hinge (Fig. 2c). Zooxanthellae were distributed scarcely in the ctenidium, as only a few
298 zooxanthellae were found in the middle ctenidial suspensory ligament (Fig. 2d). In the
299 hepatopancreas, morphologically intact zooxanthellae and degraded zooxanthellae could be
300 found within the macrophage centers (Fig. 2e). No zooxanthella was found in the whitish part
301 of the foot muscle (Fig. 2f), but the tip of the foot muscle harbored high densities of
11 302 zooxanthellae (Fig. 2g). Immunofluorescence microscopy using the anti-RBCII antibody
303 confirmed the green staining of TSZRBCII in the pyrenoids of zooxanthellae (Fig. 2h), which
304 was the correct localization of RBCII in algae and dinoflagellates (with reproducible results
305 obtained from three individuals).
306 TSZrbcII is detected not only in the outer mantle, but also other organs that are shaded
307 from light
308 TSZrbcII transcripts were detected in all five organs of T. squamosa examined, of which, the
309 inner mantle, ctenidium, hepatopancreas and foot muscle have no direct exposure to light (Fig.
310 3). The TSZrbcII transcript level in the outer mantle was significantly higher than those in other
311 organs (P<0.05; Fig. 3a), which was in agreement with the results from fluorescence
312 microscopy. Furthermore, TSZrbcII transcript levels in the inner mantle and foot muscle were
313 comparable, and they were significantly greater than those in the ctenidium and the
314 hepatopancreas. The brown regions of the inner mantle and the foot muscle contained
315 significantly more TSZrbcII transcripts than the corresponding white regions (P<0.05; Fig. 3b).
316 TSZrbcII transcripts are distributed non-uniformly in the colorful outer mantle
317 The transcript levels of TSZrbcII were apparently non-uniform across the length of the colorful
318 outer mantle of all three clams examined (Table 1), with differences ranging between 2- and
319 4.5-fold, indicating that certain regions of the outer mantle might contain more zooxanthellae
320 than others. Consequently, the mean TSZrbcII transcript levels across the eight arbitrarily
321 defined regions of the outer mantle displayed high percentage deviations (26-44%).
322 Effects of 3, 6 or 12 h of light exposure on the transcript level and protein abundance of
323 TSZrbcII/TSZRBCII in the outer mantle
324 The transcript level of TSZrbcII remained unchanged in the outer mantle of T. squamosa after
325 exposure to light for 3, 6 or 12 h as compared with the control kept in darkness for 12 h (Fig.
326 4). Western blotting revealed a band of interest at ~50 kDa, which corresponded to the estimated
327 molecular size of the RBCII monomer (Fig. 5a). The protein abundance of TSZRBCII (in
12 328 arbitrary units) increased significantly by ~4 fold in the outer mantle of T. squamosa after
329 exposure to 12 h of light as compared to that of the control kept in 12 h of darkness (P<0.05;
330 Fig. 5b).
331 Effects of light exposure on the protein abundance of TSZRBCII in the inner mantle,
332 ctenidium, hepatopancreas and foot muscle
333 The protein abundances of TSZRBCII (in arbitrary units) in the inner mantle, ctenidium,
334 hepatopancreas and foot muscle were apparently lower than that in the outer mantle. In contrast
335 with the outer mantle, exposure to light for 3, 6 or 12 h had no significant effect on the protein
336 abundances of TSZRBCII in these four organs as compared with the corresponding control kept
337 in darkness for 12 h (Fig. 6).
13 338 Discussion
339 In giant clams, the zooxanthellal tubular system originates from the stomach as a single primary
340 zooxanthellal tube, which passes through the digestive diverticula and the muscular wall that
341 encloses the digestive mass and reproductive organ, and then divides into two secondary tubes
342 that pass through the kidney and the adductor muscle, followed by the mantle tissues (Norton
343 et al. 1992). The secondary zooxanthellal tubes branch into the upper portion of the fleshy and
344 colorful outer mantle where they terminate in convolutions of tertiary zooxanthellal tubules
345 containing zooxanthellae (Norton et al. 1992). Other secondary zooxanthellal tubes form
346 tertiary zooxanthellal tubules in organs such as the ctenidium (gill), the connective tissue
347 surrounding the adductor muscle, connective tissues of the bulbus arteriosus of the heart and
348 some areas of the inner mantle adjacent to the shell (Norton et al. 1992). Overall, tertiary tubules
349 are located mainly in the surface tissues of the outer mantle, which harbor the highest density
350 of zooxanthellae, and are surrounded by iridocytes (Ip et al. 2018). However, apart from the
351 outer mantle, the structure of the tubular system denotes the presence of zooxanthellae in organs
352 located inside the mantle cavity, which is supported by results obtained from this study.
353 Besides the outer mantle, zooxanthellae are present in organs that have no direct exposure
354 to light
355 Among the five organs of T. squamosa examined, the outer mantle, which contains iridophores
356 and is fully extensible during insolation, had the greatest density of zooxanthellae and the
357 highest transcript level of TSZrbcII. Nevertheless, even with direct light exposure, the amount
358 of light reaching the deeper layers of the outer mantle (which is several millimetres thick) is
359 low, and zooxanthellae therein would receive low irradiance due to shading by the layers of
360 cells on top (Holt et al. 2014). Therefore, the host iridophores peculiar to the outer mantle are
361 crucial for zooxanthellal photosynthesis, as they forward-scatter light to the underlying
362 zooxanthellae. This would enhance the quantum of light reaching the zooxanthellae by five-
363 fold as compared to the absence of iridophores. However, even with the help of iridophores,
14 364 only ~5-10% of the incident light can penetrate into the deeper cell layers of the outer mantle
365 (Holt et al. 2014), and the quantum of light reaching the zooxanthellae without iridophores
366 would presumably be negligible.
367 As the inner mantle, ctenidium, hepatopancreas and foot muscle of T. squamosa are
368 located inside the mantle cavity, they would logically receive low levels of illumination.
369 However, our results demonstrated that they contained considerable amounts of zooxanthellae,
370 with auto-fluorescing zooxanthellae being detected by fluorescence microscopy. Furthermore,
371 specific regions of the inner mantle (near the valve hinge) and the foot muscle (at the tip)
372 contained substantial quantities of zooxanthellae as revealed by the green coloration after
373 ethanol treatment and the relatively high TSZrbcII expression levels. It has been accepted
374 traditionally that the primary function of zooxanthellae is to conduct photosynthesis and share
375 the photosynthates with the host (Klumpp et al. 1992). As follows, the presence of considerable
376 quantities of zooxanthellae in these four organs shaded by the siphonal mantle is intriguing.
377 Although all four organs expressed moderate levels of TSZrbcII, the phototrophic potentials of
378 these organs are unlikely to be high as they lack both iridophores and direct exposure to light.
379 In fact, correlations between insufficient light and reduced carbon fixation by form I RuBisCO,
380 as well as reduced efficiency of photosystem II, have been reported in plants (Sui et al. 2012).
381 Nonetheless, the discovery of substantial quantities of zooxanthellae in special locations of the
382 inner mantle and the foot muscle of T. squamosa suggests that they might serve some special
383 functions, besides photosynthesis, yet to be elucidated in these two organs.
384 TSZrbcII is distributed unevenly across different regions of the outer mantle
385 The distribution of TSZrbcII transcripts across eight arbitrarily defined regions of the colorful
386 outer mantle of T. squamosa was non-uniform, as reflected by the high variability (26-44%) in
387 the mean transcript levels among these eight regions. These results indicate the presence of
388 disparate quantities of zooxanthellae in different regions of the outer mantle. Notably, it has
389 been reported that the two halves of the outer mantle of Tridacna maxima showed different
15 390 susceptibility to bleaching (Rouzé and Hédouin 2018). This supports the proposition that
391 different parts of a giant clam might contain dissimilar zooxanthellae communities, possibly
392 correlated with differences in bleaching tolerances. The high variability in transcript levels of
393 TSZrbcII in the outer mantle of T. squamosa could result from the difference in densities of
394 tertiary zooxanthellal tubules in these eight regions. As the distribution of zooxanthellae in the
395 outer mantle was non-uniform, it should be noted that tissue clipping of the outer mantle may
396 not be a valid method of representative sampling of zooxanthellae from this organ.
397 Light exposure upregulates the protein abundance of TSZRBCII in the outer mantle,
398 indicating an increased rate of CO2 fixation
399 The transcript levels of TSZrbcII in the outer mantle of T. squamosa remained statistically
400 unchanged after 12 h of light exposure. As the baseline transcript level of TSZrbcII in the outer
401 mantle of control clams exposed to 12 h of darkness was very high (x106 copies), any further
402 increase in the transcript level in response to light would not make a substantial difference.
403 Hence, regulation of TSZrbcII expression at the translational level could be more effective, as
404 has been suggested by Mayfield et al. (2014) for symbiotic dinoflagellates. Indeed, the protein
405 abundance of TSZRBCII increased significantly in the outer mantle of T. squamosa after 12 h
406 of light exposure compared to the control kept in darkness for 12 h, denoting that TSZRBCII
407 expression is light-dependent. It can be seen that the increase in expression of TSZRBCII
408 occurred progressively in the outer mantle between hour 6 and hour 12. As TSZRBCII is
409 presumed to be the main rate-limiting enzyme in the Calvin cycle (Rowan et al. 1996), an
410 upregulation of its expression in the outer mantle of T. squamosa would lead to an increase in
411 CO2 fixation and the production of photosynthates during insolation.
412 Although the expression of rbcL in some cyanobacteria and phytoplankton peaks at
413 sunrise (Pichard et al. 1996; Zinser et al. 2009), the expression of rbcII in cultured zooxanthellae
414 isolated from the sea anemone, Aiptasia pulchella, increases with light and peaks after 12 h of
415 light exposure (Mayfield et al. 2014). As suggested by Mayfield et al. (2014), regulation of
16 416 photosynthetic genes and proteins may differ between free-living and symbiotic phototrophs.
417 Free-living dinoflagellates have direct access to inorganic nutrients in the ambient seawater,
418 but zooxanthellae have to depend on their host for a regular supply of inorganic nutrients.
419 Although light may be readily available from morning to noon, photosynthesis in zooxanthellae
420 may be limited by the availability of Ci. In T. squamosa, the host clam conducts light-enhanced
421 absorption of Ci through a light-dependent mechanism consisting of Dual-Domain Carbonic
422 Anhydrase (DDCA; Koh et al. 2018), Vacuolar-type H+-ATPase (VHA; Ip et al. 2018), and
423 Na+/H+ Exchanger 3-like (NHE3-like; Hiong et al. 2017b) in its ctenidia. Evidently, the
424 transcript and protein levels of these enzymes and transporters are only significantly
425 upregulated in the ctenidium of T. squamosa between hour 6 and hour 12 of light exposure to
426 augment Ci absorption and assimilation (Hiong et al. 2017b; Ip et al. 2018; Koh et al. 2018).
427 Hence, even with an upregulation of the host carbon-concentrating mechanism (CCM) to
428 translocate Ci from the hemolymph to the tubular fluid at hour 3 of light exposure (Ip et al.
429 2017b, 2018), it is unlikely that the Ci available in the hemolymph at that time point would
430 support the peak capacity of carbon-fixation. In fact, the pattern of light-dependent expression
431 of TSZRBCII in the outer mantle of T. squamosa aligns well with that of the Ci uptake
432 mechanisms in the ctenidium.
− 433 The absorbed Ci is transferred as HCO3 in the hemolymph to other parts of clam’s
434 body, and it must permeate both the basolateral and apical membranes of the epithelial cells of
435 the tertiary zooxanthellal tubules before it can be absorbed by the symbionts. This is achieved
436 through a host-mediated and light-dependent CCM that increases the translocation of Ci from
437 the hemolymph to the tubular fluid. VHA (Ip et al. 2018) and Carbonic Anhydrase 2-like (CA2-
438 like; Ip et al. 2017b) constitute the host CCM that involves iridocytes and tubular epithelial
439 cells in the outer mantle of T. squamosa. Specifically, the transcript and protein expression
440 levels of VHA subunit A (ATP6V1A/ATP6V1A) increase significantly in the outer mantle of T.
441 squamosa during light exposure. In the outer mantle, the iridocytes, which deflect light of useful
17 442 wavelengths to the zooxanthellae, are strongly ATP6V1A-immunopositive. In light, they can
+ − 443 increase the secretion of H to the hemolymph sinuses to promote the dehydration of HCO3 to
444 CO2, which is then transported across the basolateral membrane of the epithelial cells of the
445 zooxanthellal tubules. Notably, iridocytes are known to be present only in the outer mantle and
− 446 not in any other organ in giant clams. Inside the epithelial cells, CO2 is converted to HCO3
− 447 catalyzed by CA2-like, and HCO3 is then transported across the apical membrane into the
448 tubular fluid of the zooxanthellal tubules. Simultaneously, VHA located in the apical membrane
449 (Armstrong et al. 2018; Ip et al. 2018) secretes H+ into the tubular fluid to promote the
− 450 dehydration of HCO3 to CO2, augmenting the supply of Ci to the zooxanthellae residing in the
451 lumen of the tubule. Overall, the entire host CCM present in the outer mantle can effectively
452 supply photosynthesizing zooxanthellae inside the tertiary tubules with the amount of CO2
453 needed to produce adequate quantities of photosynthates for the host-symbiont association
454 during insolation.
455 The protein abundances of TSZRBCII in the other four organs shaded from light remain
456 unchanged during light exposure
457 TSZRBCII was also detected in the inner mantle, ctenidium, hepatopancreas and foot muscle
458 of T. squamosa, although at levels considerably lower than that in the outer mantle. However,
459 unlike the outer mantle, the protein abundance of TSZRBCII in these four organs remained
460 statistically unchanged during 12 h of light exposure as compared with the control kept in 12 h
461 of darkness. Besides being shaded from light, these four organs do not have iridophores, and
462 therefore lack the mechanism (VHA of iridocytes; Ip et al. 2018) that augments the supply of
463 Ci from the hemolymph into the epithelial cells of zooxanthellal tubules. Even with the presence
464 of VHA in the epithelial cells (Armstrong et al. 2018; Ip et al. 2018), the host CCM is
465 incomplete and the transport of Ci from the hemolymph through the basolateral membrane into
466 the epithelial cells of the zooxanthellal tubules would be inefficient in the absence of iridocytes.
467 This would imply that zooxanthellae residing in these organs would be deprived of an efficient
18 468 supply of Ci absorbed by the host clam to support light-enhanced CO2 fixation. Taken altogether,
469 it can be concluded that these four organs have low phototrophic potentials. It could be that the
470 transport of nutrients through the open circulatory system of giant clams is slow and inefficient,
471 and therefore these organs need to possess some zooxanthellae in order to produce small
472 quantities of photosynthates under low light intensities. Nevertheless, the possibility of these
473 zooxanthellae having some special physiological functions unrelated to photosynthesis in these
474 organs of the giant clam holobiont cannot be ignored.
475 Notably, plastids can perform important functions other than photosynthesis (Füssy et
476 al. 2019). In many non-photosynthetic lineages including stramenopiles and apicomplexans,
477 plastids are retained despite the loss of photosynthetic capabilities. In apicomplexans, plastids
478 (known as apicoplasts) perform important biosynthetic functions including the synthesis of Fe-
479 S clusters, fatty acids, isoprenoids and heme (van Dooren and Hapuarachchi 2017), which are
480 essential for the growth of the parasite in its host (van Dooren and Striepen 2013). Similarly, in
481 non-photosynthetic organs of plants such as the tubers, roots and seeds, plastids may
482 differentiate into colorless leucoplasts that lack photosynthetic pigments (Heldt and Piechulla
483 2011). Instead of photosynthesis, they may be specialized for the storage of starch, lipids or
484 proteins (Zhu et al. 2018; Sadali et al. 2019). Additionally, leucoplasts may possess biosynthetic
485 functions, including syntheses of fatty acids, amino acids and tetrapyrrole compounds such as
486 heme (Zhu et al. 2018). In roots where nitrate assimilation occurs, the reduction of nitrite to
487 ammonia occurs in the leucoplasts (Heldt and Piechulla 2011). Even in phototrophs, the
488 syntheses of fatty acids, terpenoids and tetrapyrroles occur solely in the plastids of chromerids
489 (Füssy et al. 2019). Therefore, it is highly probable that plastids of zooxanthellae with low light
490 exposure might engage in some important physiological functions, such as the biosyntheses of
491 fatty acids and amino acids, to benefit the host. Furthermore, it is logical to deduce that some
492 of these functions could be organ specific, for instance, to support protein synthesis in the foot
493 muscle or light-enhanced calcification in the inner mantle.
19 494 Zooxanthellae of the inner mantle, ctenidium, hepatopancreas and foot muscle naturally
495 live in conditions of very low light intensities. Therefore, they should not be affected too
496 severely during the bleaching of the outer mantle caused by a lack of insolation due to floating
497 sediments resulting from anthropogenic activities. Theoretically, the survival of zooxanthellae
498 outside the outer mantle may act as ‘seeds’ for the build-up of the zooxanthellae population
499 during recovery in subsequently more favourable environmental conditions. Upon migrating to
500 the outer mantle during recovery, plastids of these zooxanthellae may then engage in
501 photosynthesis with illumination. In particular, this phenomenon has been well documented in
502 etioplasts of plants. In the absence of light during leaf development, proplastids differentiate
503 into etioplasts which are chloroplast progenitors (Kowalewska et al. 2016) without chlorophyll
504 pigments (Plöscher et al. 2011). Then, with illumination, etioplasts can differentiate into
505 functioning chloroplasts (Solymosi and Schoefs 2010).
506 Perspective
507 Based on microscopy and TSZrbcII/TSZRBCII expression, we have demonstrated for the first
508 time the presence of zooxanthellae in five different organs of T. squamosa, with the colorful
509 outer mantle having the highest phototrophic potential among them. The discoveries of the
510 presence of zooxanthellae in special locations of the inner mantle and foot muscle, the existence
511 of substantial quantities of intact zooxanthellae in the hepatopancreas, and the apparent non-
512 uniform distribution of zooxanthellae in the colorful outer mantle are novel. As Symbiodinium
513 is known to require high light intensities to flourish (Ikeda et al. 2017) while Cladocopium may
514 be better adapted to low light conditions (Grasso 2015), the abundance of Symbiodinium could
515 be lower than that of Cladocopium in the shaded organs. It is possible that T. squamosa would
516 harbor Symbiodinium, Cladocopium and Durusdinium in unequal proportions in various organs,
517 as defined by their exposure to different light intensities, but future work is needed to confirm
518 this proposition. Importantly, it is logical to deduce that zooxanthellae residing in organs shaded
519 from light serve some kind of physiological functions, besides photosynthesis, to benefit the
20 520 host. Therefore, efforts should be made in the future to elucidate the functional roles of
521 zooxanthellae in the shaded organs of T. squamosa, and to examine their responses to
522 environmental conditions that would induce bleaching.
21 523 Acknowledgements
524 This study was supported by the Singapore Ministry of Education through grants (R-154-000-
525 A37-114 and R‐154‐000‐B69-114) to Ip YK.
526 Ethical approval
527 Institutional approval was not required for research on giant clams (National University of
528 Singapore Institutional Animal Care and Use Committee). Nonetheless, all applicable
529 international, national, and/or institutional guidelines for the care and use of animals were
530 followed.
531 Conflict of interest
532 The authors declare that the research was conducted in the absence of any commercial or
533 financial relationships that could be construed as a potential conflict of interest.
22 534 References
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27 714 Table 1. Transcript levels (x106 copies of transcript per ng total RNA) of Tridacna squamosa
715 zooxanthellal form II ribulose-1,5-bisphosphate carboxylase/oxygenase (TSZrbcII) in
716 eight arbitrarily defined regions of the outer mantle (OM R1-8) of T. squamosa
717 (N=3). The eight regions of the outer mantle are defined in the supplementary
718 material, Fig. S2.
TSZrbcII transcript level (x106)
Clam A Clam B Clam C
OM R1 1.95 1.21 0.78
OM R2 0.62 1.05 0.57
OM R3 1.12 0.99 0.89
OM R4 0.90 1.10 1.35
OM R5 1.06 1.50 1.42
OM R6 0.90 1.53 1.12
OM R7 1.26 2.05 0.30
OM R8 0.91 1.60 0.67
Mean ± SD 1.09 ± 0.39 1.38 ± 0.36 0.89 ± 0.39
719
28 720 Fig. 1. The detection of zooxanthellae in five organs of Tridacna squamosa by alcohol
721 treatment. A visual comparison of the coloration of a colorful outer mantle (OM), b
722 whitish inner mantle (IM), c ctenidium, d hepatopancreas, and e foot muscle of T.
723 squamosa immersed in formaldehyde fixative (left panel) followed with overnight
724 dehydration with alcohol (right panel). Arrowheads show the regions of organs that are
725 concentrated with zooxanthellae. Note that there is a change in color from brown to
726 green (representing zooxanthellae) during pre- and post-alcohol treatments.
727 Fig. 2. Differential interference contrast (DIC) and fluorescence microscopy of the five organs
728 of Tridacna squamosa. Zooxanthellae (ZX) appear in red (autofluorescence) in a the
729 inner fold of outer mantle facing the sun (OM), b the whitish region (WR) of inner
730 mantle (IM, WR), c the zooxanthellae-concentrated region (ZR) of IM (IM, ZR), d the
731 middle ctenidial suspensory ligament (MCL) of ctenidium (CT), e the hepatopancreas
732 (HP), f the whitish region (WR) of foot muscle (FM, WR), and g the zooxanthellae-
733 concentrated region (ZR) of FM (FM, ZR) of T. squamosa. h Composite image of DIC
734 with corresponding red (autofluorescence) and green (immunolabelling of TSZRBCII
735 using the custom-made anti-RBCII antibody) channels of OM. Arrowheads show the
736 presence of whole zooxanthellae in the organs; asterisks show partially degraded
737 zooxanthellae specifically in the HP; and open arrowheads show immunostaining of
738 TSZRBCII in the pyrenoid of ZX. Abbreviations: CF: ctenidial filament, DT: digestive
739 tubules, EP: epithelium; EPF: extrapallial fluid, EBV: efferent branchial vein, HL:
740 hemolymph; MC: macrophage centre, SW: seawater, SWC: secondary water channel,
741 TWC: tertiary water channels, ZT: zooxanthellae tubules. Scale bar: 50 µm for a-g, 20
742 µm for h, 20 µm for all insets.
743 Fig. 3. Transcript levels (copies of transcript per ng total RNA) of Tridacna squamosa
744 zooxanthellal form II ribulose-1,5-bisphosphate carboxylase/oxygenase (TSZrbcII) in
745 various organs of T. squamosa. a Transcript levels (x106 copies of transcript per ng 29 746 total RNA) of TSZrbcII in five organs of T. squamosa. Results represent means +
747 S.E.M. (N=10). One way analysis of variance (ANOVA), followed by Dunnett’s T3
748 test, were conducted to analyze the differences between the means. Significantly
749 different means are indicated with different letters (P<0.05). b Transcript levels (x106
750 copies of transcript per ng total RNA) of TSZrbcII in the inner mantle and the foot
751 muscle of T. squamosa. The black and white bars represent the transcript levels of
752 TSZrbcII obtained from the brown and white regions, respectively, of the organ.
753 Results represent means + S.E.M. (N=5). An independent t-test was conducted to
754 analyze the differences between the means. Significantly different means between the
755 brown regions and the white regions are indicated with asterisks (P<0.05).
756 Fig. 4. Effects of light on the mRNA expression of Tridacna squamosa zooxanthellal form II
757 ribulose-1,5-bisphosphate carboxylase/oxygenase (TSZrbcII) in the outer mantle of T.
758 squamosa. Transcript levels (x106 copies of transcript per ng total RNA) of TSZrbcII
759 in the outer mantle of T. squamosa exposed to 12 h of darkness (control) or 3, 6 or 12 h
760 of light. Results represent means + S.E.M. (N=6). One way analysis of variance
761 (ANOVA), followed by Tukey’s test, were conducted to analyze the differences
762 among the means.
763 Fig. 5. Effects of light on the protein abundance of Tridacna squamosa zooxanthellal form II
764 ribulose-1,5-bisphosphate carboxylase/oxygenase (TSZRBCII) in the outer mantle of
765 T. squamosa exposed to 12 h of darkness (control) or 3, 6 or 12 h of light. a An
766 immunoblot of TSZRBCII. b TSZRBCII protein abundance expressed as arbitrary
767 densitometric units (a.u.) per 20 μg protein from the outer mantle. Results represent
768 means + S.E.M. (N=5). One way analysis of variance (ANOVA), followed by Tukey’s
769 test, were conducted to analyze the differences among the means. Significantly
770 different means are indicated with different letters (P<0.05).
30 771 Fig. 6. Effects of light on the protein abundance of Tridacna squamosa zooxanthellal form II
772 ribulose-1,5-bisphosphate carboxylase/oxygenase (TSZRBCII) in the a inner mantle, b
773 ctenidium, c hepatopancreas, and d foot muscle of T. squamosa exposed to 12 h of
774 darkness (control) or 3, 6 or 12 h of light. An immunoblot of TSZRBCII is presented
775 for each of the four organs. TSZRBCII protein abundance is expressed as arbitrary
776 densitometric units (a.u.) per 100 μg protein. Results represent means + S.E.M. (N=5).
777 One way analysis of variance (ANOVA), followed by Tukey’s test, were conducted to
778 analyze the differences among the means.
31 779 Fig. 1
780
32 781 Fig. 2
782
0 783 Fig. 3
784
0 785 Fig. 4
786
1 787 Fig. 5
788
0 789 Fig. 6
790
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