Moderate levels of dissolved iron stimulate cellular growth and increase lipid storage in Symbiodinium sp.

Moderate levels of dissolved iron stimulate cellular growth and increase lipid storage in Symbiodinium sp. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Moderate levels of dissolved iron stimulate cellular growth and increase lipid storage in Symbiodinium sp. Walter Dellisanti*, Swathi Murthy*, Elena Bollati, Sara Prehn Sandberg, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5160718/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Dinoflagellates in the family Symbiodiniaceae are fundamental in coral reef ecosystems and facilitate essential processes such as photosynthesis, nutrient cycling, and calcium carbonate production. Iron (Fe) is an essential element for the physiological processes of Symbiodiniaceae, yet its role remains poorly understood in the context of cellular development and metabolic health. Here, we investigated the effect of iron availability (0 to 100 nM Fe(III)) on Symbiodinium sp. ITS2 type A1 cultures and quantified cellular content using flow cytometry and holotomography. Moderate levels of dissolved Fe (50 nM) enhanced growth rates and cellular content development in Symbiodinium sp., including lipids and proteins. We observed distinct growth patterns, pigment concentrations, and cellular morphology under increasing Fe concentrations, indicating the influence of iron availability on cellular physiology. Non-destructive, label-free holotomographic microscopy enabled single cell in vivo revealing higher intracellular lipid accumulation (+57%) in response to 50 nM Fe(III) enrichment. Our findings contribute to a deeper understanding of the relationship between iron availability and Symbiodinium sp. growth and cellular development, with potential implications for coral health and reef resilience in the face of environmental stressors. *Walter Dellisanti and Swathi Murthy contributed equally to this manuscript. Marine and Freshwater Biology symbiodinium cell development in vivo iron enrichment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Symbiotic dinoflagellates belonging to the family Symbiodiniaceae (LaJaunesse et al., 2018) are of crucial significance to coral reef ecosystems. They have a fundamental role in facilitating key processes to corals through photosynthesis, supporting the macro- and micronutrient cycle, and the production of calcium carbonate as the foundation of the reefs (Frommlet et al., 2015; Coffroth and Santos, 2005). However, the current global environmental changes, including ocean warming, nutrient pollution, and deoxygenation, affect the cellular dynamics of the coral-dinoflagellate symbiosis by altering, among other processes, nutrient exchange (Morris et al., 2019; Johnson et al., 2021; Rädecker et al., 2021). Disruption of nutrient exchange can lead to the breakdown of symbiosis, i.e., bleaching, ultimately compromising coral survival (Pernice 2014). Among the essential nutrients, iron (Fe) plays a fundamental role in the physiology of Symbiodiniaceae and other microalgae (Reich et al., 2020 ). Iron is typically present in seawater in nanomolar concentrations, and it is considered a trace metal (Entsch et al., 1983) essential for metabolic processes such as photosynthesis, phagocytosis, and prey digestion in mixotrophic dinoflagellates (Rodriguez et al., 2016; Reich et al., 2020 ). While iron concentration refers to the total amount of iron present, its bioavailability refers to the fraction of iron that is accessible and usable by the organism. The balance between iron concentration and its bioavailability directly influences the homeostasis of photosynthetic organisms, as insufficient iron availability can limit growth, while excess iron can induce toxicity (Romero et al., 2022 ; Reich et al., 2020 ). Iron is an enzyme cofactor in electron transfer and catalysis (Balk & Schaedler, 2014 ; Müller, 2023 ), and its limitation can lead to reduced chlorophyll synthesis resulting in decreased pigment content and reduced photosynthetic efficiency in Symbiodiniaceae (Iglic, 2011 ), phytoplankton (Koch & Trimborn, 2019 ), and freshwater green microalgae (Yadavalli et al., 2012 ). Iron limitation can also induce the expression of iron transporters and siderophore production to enhance iron acquisition from the environment (Sandy & Butler, 2009 ). Recent observations suggest that the photochemical performance of Symbiodiniaceae is enhanced in association with Marinobacter sp. and L. alexandrii bacteria, which may support nutrient exchange and siderophore production to bind Fe into bioavailable forms (Amin et al., 2009 ; Matthews et al., 2023a , b ). On the other hand, excess iron can lead to stressful conditions in Symbiodinium spp. potentially leading to the production of reactive oxygen species (ROS) through Fenton chemistry, and causing oxidative stress (Wietheger et al., 2018 ; Deleja et al., 2022 ). This in turn can disrupt cellular homeostasis, damage cellular components, such as proteins and lipids, and inhibit the photosynthetic electron transport chain (Rai et al., 2021 ; Reich et al., 2023 ). Despite its importance, only a few Symbiodiniaceae species have been extensively studied regarding their iron requirements (Rodriguez et al., 2016; Reich et al., 2020 , 2021). Iron availability can also affect the lipid content and lipid profile of microalgae (Liu et al., 2008 ; Wang et al., 2023 ). Lipids are fundamental components of cells and serve in the cellular metabolism of microalgae, including Symbiodiniaceae (Garrett et al., 2013 ; Kneeland et al., 2013 ; Pasaribu et al., 2024 ). They contribute to energy reserves and form integral components of cellular membranes (Patton & Burris, 1983 ; Tchernov et al., 2004 ). These functions have implications for nutrient stress responses, energy storage, and proliferation rates of Symbiodiniaceae in nitrogen-limited host tissue directly affecting lipid accumulation (Wang et al., 2015 ). Quantification of lipids and other cellular contents (e.g. protein) in relation to inorganic iron concentration is therefore relevant to understand the potential role of this micronutrient in optimizing Symbiodiniaceae cellular conditions, and its role in influencing the health and resilience of Symbiodiniaceae populations, both free-living and in hospite . Recently, optical diffraction tomography (also known as holotomography) techniques have emerged, providing quantitative morphological and biochemical information about individual cells and tissues without the need for exogenous labeling agents (Kim et al., 2024 ). The use of interferometry allows the measurement of complex optical fields of diffracted light from biological samples enabling the mapping of refractive index (RI) (Popescu, 2011 ; Lee et al., 2013 ). RI is an optical property that varies with the composition and density of cellular components, enabling the identification and quantification of intracellular structures. Holotomography measurements of phase-distortions of light from various incident angles enable the construction of 3D refractive index distributions in cells and tissues at sub-micron resolution (Choi et al., 2007 ; Habaza et al., 2017 ), which can then be segmented to identify particular cellular structures with characteristic RI. For example, lipids typically have a higher RI than other cellular components, and this allows their detection and quantification within live cells (Jung et al., 2018 ). Holotomography thus provides a rapid, quantitative method for intracellular lipid detection in live cells (Kim et al., 2016 ; Park et al., 2020 ). It has been used as an effective imaging technique for studying biological samples including microalgae (Jung et al., 2018 ), phytoplankton (Lee et al., 2014 ), bacteria (Kim et al., 2014a ; Bennet et al., 2017 ), yeast (Rappaz et al., 2009 ; Habaza et al., 2015 ), and red blood cells (Kim et al., 2014b , c ). However, potential applications of this technique as a non-invasive, label-free, in vivo approach to quantify cellular content in Symbiodiniaceae have not yet been explored. In this study, we investigate the effect of iron enrichment on Symbiodinium sp. cultures and quantify their cellular content using a range of label-free, in vivo techniques such as flow cytometry and holotomography. The objectives of this study are to i) evaluate the impact of varying concentrations of dissolved Fe(III) on the growth rate and cellular content development of Symbiodinium sp., ii) visualize and quantify cellular structures, including proteins and lipids, under different iron enrichment conditions through holotomography techniques, and iii) to investigate the role of inorganic iron in optimizing cellular growth conditions. We hypothesized that controlled iron enrichment could enhance Symbiodinium sp. growth and cellular content. 2. Materials and Methods 2.1 Symbiodinium cultures Symbiodinium sp. cultures (K-1618) were acquired from the Norwegian Culture Collection of Algae (NORCCA, Norway) and maintained in TL-30 medium in 250 mL polycarbonate flask, at 20°C with a photoperiod of 12:12 at 100 µmol photons m − 2 s − 1 (400–700 nm), as measured with a Universal Light Meter (ULM-500) equipped with a spherical micro quantum sensor (US-SQS/L, Heinz Walz, Effeltrich, Germany). Subsequently, subcultures were acclimated to standard f/2 medium for one month and maintained under the same conditions prior to the start of experiments. All experimental analyses, except for the flow cytometry assay, were performed during the mid-exponential phase, as determined via cell counts identifying optimal growth and cellular activity (Lee et al., 2015 ). At the start of the experimental period, aliquots of Symbiodinium sp. cultures were transferred into each culture flask (20 mL, n = 3 replicates per condition) to reach an initial density of ~ 10,000 cells mL − 1 , and maintained under the same conditions as above until they reached the stationary phase (Fig. 1 ). Symbiodinium sp. cultures were exposed to increasing iron Fe(III) concentrations (0, 10, 50, and 100 nM total dissolved Fe) at a controlled temperature of 20°C. A dissolved Fe(III) solution (FeCl 3 *6H 2 O, Sigma-Aldrich) was used to enrich the standard f/2 medium to different iron concentrations (Rodriguez et al., 2016; Reich et al., 2021). Experimental conditions were prepared as follows: modified f/2 medium with depleted dissolved iron, 0 nM Fe(III); standard f/2 medium, 10 nM Fe(III); f/2 media with a concentrated solution of dissolved iron to reach the initial concentrations of 50 nM Fe(III) and 100 nM Fe(III), respectively. 2.2 Culture identification To verify the taxonomic assignment of the Symbiodinium sp. strain, we performed Sanger sequencing of the ITS2 region of rDNA. Aliquots of the cultures (500,000 cells mL − 1 ) were collected and pelleted by centrifugation during the mid-exponential growth phase and stored at -80°C. DNA was extracted using the DNAEasy PowerBiofilm kit (Qiagen) following manufacturer protocol with slight modifications (all centrifugation steps performed at 8,000G for 90s). The ITS2 region was amplified using primers SYM_VAR_5.8S2 and SYM_VAR_REV (Hume et al. 2018 ) in the following reaction: 0.5 µL of each 10 µM primer, 1 µL of 2 mg mL − 1 BSA, 12.5 µL KAPA HiFi HotStart Ready Mix (Roche), 1 µL template DNA (2.65 ng µL − 1 ) and water to a total reaction volume of 25 µL. The amplification cycle was: 98 °C for 2 min, followed by 35 cycles of 98 °C for 20 s, 61 °C for 30 s, 72 °C for 30 s, followed by 72 °C for 5 min. Successful amplification, amplicon size, and contamination were visually assessed via agarose gel electrophoresis. PCR products were cleaned on AMPure XP beads (Beckman Coulter) and submitted for Sanger sequencing to a commercial provider (Eurofins). Taxonomy was assigned via blastn to the NCBI nt database, as well as local blastn to the SymPortal RefSeq database (github.com/reefgenomics/SymPortal_framework downloaded on 25/03/2024, Hume et al., 2019 ). 2.3 Growth measurements Symbiodinium cell growth was assessed every third day with a CytoFLEX cytometer (Beckman Coulter, USA), which was equipped with a 50-mW laser (excitation = 488 nm, fluorescence channel 690/50 BP). Flow cytometry was performed throughout the light phase of the light: dark cycle until cultures reached the stationary phase. The instrument gain settings were as follows: forward scatter (FSC) = 200, side scatter (SSC) = 83, phycoerythrin-Cy5.5 (PC5.5) = 350 (Galotti et al., 2020 ). Before each analysis, quality control procedures were implemented, as recommended by the CytoFLEX software. All measurements, including culture media blanks, were conducted using an analyzed 1 mL sample at a slow rate (10 µL min − 1 ) with a threshold of 300 seconds or 10,000 events. The gating process relied on FSC and SSC patterns, with only cells displaying a positive signal for photosynthetic pigments (PC5.5) being chosen for downstream analysis, aiming to minimize potential contamination from non-algal particles ( Fig. S1 ). Cell counts, size, and shape were determined in vivo by FSC and SSC on unstained cells using the CytExpert software v2.5. 2.4 Pigment analysis Quantitative determination of chlorophyll and pigment content was carried out using High-Performance Liquid Chromography (HPLC, 1260 Infinity, Agilent Technologies). Aliquots of 1 mL for each culture were collected during the mid-exponential growth phase and transferred to 1.5 mL tubes, centrifuged at 5000g, and the pellet was kept at − 80°C until analysis. The pigments were extracted by adding 0.4 mL acetone:methanol (7:2 vol:vol) to each tube, which was briefly vortexed, and then kept on ice for a 2 min extraction time in darkness. Subsequently, samples were sonicated in an ice-cooled high-power ultrasonicated bath (Misonix 4000; Qsonica LLC., Newtown, CT) in darkness at 80% power for 60 s consisting of 10 pulses of 2 s ON and 4 s OFF (with an amplitude setting of 100%) and then centrifuged at 12,000 g for 1 min in a mini centrifuge (MiniSpin, Eppendorf AG, Hamburg, Germany). The supernatant was filtered through a 0.2 µm pore size syringe filter (Sartorius Minisart SRP 4 filter; Sartorius AG, Goettingen, Germany). 100 µL of the extract was then immediately injected into the HPLC. Pigment extracts were separated and analyzed in the HPLC by a diode array detector (HPLC-DAD and Agilent 1260 Infinity; Agilent Technologies, Santa Clara, CA) fitted with an Ascentis C18 column (25 cm × 4.6 mm, Sigma-Aldrich cat. no. 581325 U), detecting specific absorption wavelengths of compounds. The extracts were run at a constant column temperature of 30°C for 69 min., and a flow rate of 1.0 mL min − 1 in a changing gradient of solvent A (methanol:acetonitrile:water, 42:33:25, vol/vol/vol), and solvent B (methanol:acetonitrile:ethylacetate, 50:20:30, vol/vol/vol), where the mobile phase changed linearly from 30% solvent B at the time of injection to 100% at 52 min, staying at 100% for 15 min before returning to 30% within 2 min. Elution profiles from the absorbance detector signal at 664 nm (chlorophyll a ), 460 nm (carotenoids), and 387 nm (scytonemin) (Lichtenthaler, 1987 ), were used to calculate pigment ratios from the derived integrated peak areas for each of the identified pigments of interest, using the manufacturer’s software (OpenLAB CDS ChemStation Edition; Agilent Technologies). 2.5 Optical diffraction tomography Quantitative 3D imaging of individual Symbiodinium cells was carried out with an optical diffraction tomography (ODT) system (Tomocube HT-2H; Tomocube Inc., South Korea). The system also has an inbuilt ability to perform fluorescence microscopy in parallel with RI mapping. The system is based on Mach-Zehnder interferometry, employing a diode-pumped green solid-state laser (532 nm) as a light source. A spatially modulated hologram of the cell was recorded from the diffracted light by a quantitative phase imaging technique (Popescu, 2011 ). The 3D refractive index (RI) distribution of individual cells was then reconstructed from multiple 2D images measured at various illumination angles (controlled by a digital micromirror device), using the Fourier diffraction theorem (Choi et al., 2007 ). The theoretical spatial resolution of the system was 119 nm and 336 nm for lateral and axial directions, respectively (Kim et al., 2016 ). From the measured 3D RI tomograms, different RI regions were visualized with a unique color and rendered as a 3D image. Maximum intensity projection images of the RI tomograms were also generated to visualize the 3D data set on a 2D plane. Data visualization and rendering were carried out using Tomostudio software (Tomocube Inc., South Korea). 2.6 Wide-field fluorescence imaging. To determine the presence of chlorophyll in the Symbiodinium cells grown at different Fe concentrations, we used the in-built wide-field fluorescence imaging capability of the HT-2H using the green channel with excitation and emission wavelengths centered around 475 nm and 520 nm, respectively. For every cell imaged, fluorescence images were captured immediately after the ODT images. At least 30 cells were imaged for each Fe concentration. The fluorescence image was overlaid on the rendered RI tomograms. While the system is not equipped with a standard Chl a filter set, these settings still enabled clear detection of a Chl a fluorescence signal due to strong coupling in Symbiodiniaceae with Chl c and peridinin (Zigmantas et al., 2002 ). To confirm the identification of the high RI regions (RI > 1.46) in the 3D tomograms as neutral lipids (see below), aliquots of Symbiodinium cell cultures were stained with a Nile Red solution (10 µg mL − 1 ) mixed in 30% ethanol solution according to Storms et al. ( 2014 ). About 20 µL of the cell sample solution was sandwiched between a pair of coverslips. Cells were then imaged on the Tomocube HT-2 using the in-built wide-field fluorescence system using the red channel, with an excitation wavelength centered around 575 nm, and an emission band of 600–800 nm. 2.7 Symbiodinium sp. cellular lipid and protein determination For single-cell imaging, Symbiodinium cell cultures were diluted with culture medium so that the cell density was low enough to avoid having multiple cells within the imaging field. The regions from the RI tomogram with n > 1.46, were identified as lipid droplets, as verified by Nile red staining and from the reported average RI for vegetable oils (Ullmann, 1985; Firestone, 1999 ). Then, cell volume and lipid dry mass were calculated for cells grown under different Fe concentrations. The cell volume was calculated from the cell boundary, as determined in the Tomocube software using Otsu thresholding. For further analysis, only mature cells (indicated by a sphericity value > 0.8) were chosen, while dividing cells were disregarded. The lipid and the non-lipid (mostly protein) regions were segmented out based on the RI, i.e., assigning RI 1.46 as lipid. Here, we assume proteins to be the major non-lipid component inside the cell (Zhou et al., 2022 ). The dry mass of these two cell components was then calculated, as per Eq. 1 , using the parameter RI increment (RII), defined as an increment of RI of the solution per unit increment of the solute concentration (Barer & Joseph, 1954 ). $$n(x,y,z)=nm+\sum\limits_{i}^{{}} {\alpha iCi(x,y,z)}$$ 1 Where n m is the RI of the medium, and \(\alpha i\) and \(Ci\) are the RII and concentration of protein or lipid, respectively. The typical RII for proteins is 0.19 mL/g (Barer & Joseph, 1954 ), and that for lipids is 0.135 mL g − 1 (Mashaghi et al., 2008 ). Integrating the concentration over the volume of the component yields its mass. All data were log-transformed and checked for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene’s test. When data did not meet the assumptions of normality, a Kruskal-Wallis test was used to compare differences in cell counts, size, chlorophyll fluorescence, and morphology. Permutational analysis of variance (PERMANOVA) with 999 permutations was used to compare differences in cell size (FSC) and complexity (SSC) between conditions. A principal component analysis (PCA) was used to compare all cellular morphology data from tomography analysis between conditions. The lipid: protein ratio was calculated by subtracting the log-transformed protein concentration from the log-transformed lipid concentration. To indicate the variability of data around the mean, the coefficient of variation (CV%) was used. All statistical analyses were run in R v4.2.3 (R Core Team, 2016 ) using the dplyr package v1.1.2 (Wickman et al., 2023) and visualized with the ggplot2 package v3.4.4 (Wickman, 2016). 3. Results 3.1 ITS2 identification Sanger sequencing of the ITS2 rDNA region returned two sequence variants separated by a single SNP ( Table S1 ), as detected via manual inspection of the chromatogram. Subsequent blastn taxonomic assignment confirmed the identification of the cultures as Symbiodinium sp.. The most closely related sequence (98.64% identity, query cover 100%, e-value 7x10 − 104 ) available from the NCBI nt database had been previously detected from a symbiont of the coral Montastraea faveolata collected in the Florida Keys, USA (accession: HQ317739; Granados-Cifuentes & Rodriguez-Lanetty, 2011 ), from a free-living strain collected from Hawaii, USA (accession: AF184948), and from a free-living strain of unspecified origin (accession: EU449053). The same sequence was labeled as Symbiodinium A1dh in the SymPortal RefSeq database (Hume et al., 2019 ). 3.2 Cellular growth rate and pigment concentration The effect of Fe(III) on cell abundance and morphology varied with different Fe concentrations in the culture media. Symbiodinium sp. cultures exhibited distinct growth curves when exposed to different iron concentrations (χ 2 = 36.757, p < 0.01, Fig. 2 a, Table S2 ). Under depleted iron conditions (0 nM), no changes in cell abundance were observed, and fluorescence (PC5.5) was not detected after day 18, indicating the absence of viable cells. Under low (10 nM), moderate (50 nM), and replete (100 nM) Fe(III) concentrations, cultures reached their maximum growth on day 26 (Fig. 2 a). The highest abundances were observed in cells growing under 50 nM Fe(III), with a cell density of 1.1 ± 0.04 x 10 6 cells mL − 1 . Cells growing under 100 nM iron exhibited the second highest density of 0.81 ± 0.08 x 10 6 cells mL − 1 , followed by those growing under 10 nM iron with a concentration of 0.6 ± 0.09 x 10 6 cells mL − 1 . Iron concentration also influenced cell size (ANOVA, F = 26.24, p < 0.01), although no differences were observed between 50 and 100 nM (Fig. 2 b). Cells growing at 10 nM Fe were on average 4.6% and 9.3% larger than those in the 100 and 50 nM treatment, respectively. The depleted Fe condition did not support cellular growth as the overall cell size at 0 nM Fe was 12.5% smaller than 10 nM Fe. In the chlorophyll fluorescence signal (Fig. 2 c), no significant differences were observed among the various Fe concentrations tested. However, in terms of cell size (FSC) and complexity (SSC), significant differences were detected under increasing Fe concentrations (PERMANOVA, F = 47.773, p < 0.01, Fig. 2 d, Table S3 ) indicating small and granular cells at 0 nM Fe, large and relatively smooth cells at 10 nM Fe, medium-size and smooth cells at 50 and 100 nM Fe, respectively. Fe(III) concentration affected the concentration of Chl a , carotenoids, and scytonemin in Symbiodinium sp. cultures (Table 1 , S4 ). Pigment concentrations of cells grown under depleted Fe concentration (0 nM Fe) were undetectable. Chlorophyll a concentration increased from 11.09 ± 0.48 µg mL − 1 in cells at 10 nM Fe to 24.87 ± 2.5 µg mL − 1 at 100 nM Fe (ANOVA, F = 113.3, p < 0.01). Similarly, the concentration of carotenoids increased from 0.24 ± 0.01 to 0.54 ± 0.05 µg mL − 1 in cells grown in 10 nM Fe and 100 nM Fe, respectively (ANOVA, F = 121.2, p < 0.01). The concentration of scytonemin ranged from 5.39 ± 0.23 µg mL − 1 in cells at 10 nM Fe, to 12.08 ± 1.23 µg mL − 1 at 100 nM Fe (ANOVA, F = 114.3, p < 0.01). Table 1 Photosynthetic pigments analyzed by HPLC: Chlorophyll a ; carotenoids; scytonemin per each condition. All data are expressed as pigment mass per mL of culture sample. Mean ± standard deviation is shown. Condition Fe conc. nM Chlorophyll a µg mL − 1 Carotenoids µg mL − 1 Scytonemin µg mL − 1 0 Fe 0 0 0 0 10 Fe 10 11.09 (0.48) 0.24 (0.01) 5.39 (0.23) 50 Fe 50 19.69 (2.46) 0.42 (0.05) 9.56 (1.19) 100 Fe 100 24.87 (2.50) 0.54 (0.05) 12.08 (1.21) The availability of Fe(III) also had a significant impact on cellular volume and content, including proteins and lipids, and on refractive index (RI) (ANOVA, F = 16.34, p < 0.01, Fig. S2 ). 3.3 Mapping RI distributions within Symbiodinium cells. Optical diffraction tomography (ODT) was employed to map the 3D refractive index (RI) distribution of Symbiodinium cells. The reconstructed 3D RI distribution ( Fig. 3a-d ) and maximum intensity projection images ( Fig. 3i-l ) of cells grown under different Fe (III) concentrations in the culture medium indicated the presence of high RI (>1.46) regions in many of the cells. Based on previous studies (Kim et al., 2016) these regions were suspected to be lipid droplets. To confirm the identification of the high RI (> 1.46) regions as lipids, the cells were stained with Nile red dye and imaged using the in-built wide-field fluorescence imaging (see methods section). The Nile red fluorescence images (red channel) were compared with the 3D RI tomograms of the same cell (Fig. 4 ). The images confirmed that regions with RI > 1.46 corresponded to lipid droplets. This is also in accordance with values found in the literature for vegetable oils (Ullmann, 1985) and for lipid droplets in microalgal cells (Jung et al., 2018 ). The Nile red and chlorophyll fluorescence signals co-localize in most cells and were difficult to separate, as Nile red could also stain membranes of organelles like chloroplasts (Greenspan et al., 1985 ; An et al., 2021 ). However, in cases of large lipid droplets (as shown in Fig. 4 , of a dividing cell) it was possible to separate the two signals (to some extent) by appropriate thresholding. The chlorophyll fluorescence (green channel), in Fig. 4 , appears as a ring around the lipid droplets. This could again be an artifact due to intense light scattering from intra-cellular components and a possible spillover from Nile red fluorescence. To assess the effect of Fe(III) concentration in the culture medium on Symbiodinium sp. cell growth, morphology, and biochemical content, the 3D RI images were used to quantify (see methods section) cell size, mean RI, protein, and lipid content (Fig. 5 , Table S5 ). Cells grown in 0 nM Fe displayed smaller volume and refractive index (RI), lower lipid concentration and dry mass, as well as reduced protein concentration and dry mass (Fig. 3 and Fig. 5 ). In terms of RI, cells grown in 0 nM Fe had a mean value of 1.37 ± 0.02, while the highest mean RI was measured in cells grown in 50 nM Fe condition at 1.4 ± 0.005, compared to 10 and 100 nM conditions (ANOVA, F = 22.83, p < 0.01, Fig. 5 a, Table S5 ). The cell volume ranged from 262.9 ± 420.6 µm 3 in 0 nM Fe to 649.07 ± 298.6 µm 3 in 10 nM Fe (ANOVA, F = 23.94, p < 0.01, Fig. 5 b, Table S5 ), with no significant differences observed in 50 and 100 nM Fe conditions. The lowest protein dry mass was recorded in 0 nM Fe 61.04 ± 98.55 pg (ANOVA, F = 29.50, p < 0.01), while the highest was measured in 10 nM Fe 171.58 ± 66.55 pg, with no significant difference among the other conditions (Fig. 5 c, Table S5 ). Protein concentration was lowest in cells grown in 0 nM Fe, at 0.17 ± 0.1 10 − 4 pg µm 3 (ANOVA, F = 22.91, p < 0.01), while the highest values were measured in cells grown in 50 nM Fe at 0.3 ± 0.02 10 − 4 pg µm − 3 ( Fig. 5 d, Table S5) . Lipid dry mass and concentration were higher in cells cultured in 50 nM Fe condition than all other conditions and measured at 1.54 ± 2.2 pg (χ 2 = 18.619, p < 0.01) and 31.47 ± 46.61 10 − 4 pg µm − 3 (χ 2 = 17.178, p < 0.01), respectively (Fig. 5 e, f, Table S5 ). Lipid:protein ratio was higher in cells grown in 50 nM condition at 1.66 ± 0.57 (ANOVA, F = 4.82, p < 0.01, Fig. 5 g, Table S5 ), with no significant difference observed in other conditions. 4. Discussion In this study, we investigated the influence of dissolved Fe concentration on the cellular growth and content of Symbiodinium sp. As hypothesized, Fe enrichment enhanced cellular growth and increased cellular contents. However, this was only true up to an intermediate concentration of 50 nM Fe(III), which seemed to provide the most favorable conditions among the investigated concentrations. Iron is recognized for its support of the photosynthetic process in coral endosymbionts and its essential role as a cofactor in numerous enzymatic reactions involved in photosynthesis, electron transport, and antioxidant activities (Raven et al., 1999 ; Reich et al., 2020 ). Thus, iron limitation can limit the growth of Symbiodinium spp. (Rodriguez and Ho, 2018 ) as well as marine phytoplankton (Sunda & Huntsman, 1997 ), as observed in our experiment for cells grown in depleted iron conditions (0 nM). An increase in the volume of Symbiodinium sp. cells was observed at 10 nM Fe condition, which could indicate that lower Fe concentrations may facilitate cellular expansion. However, the large variation in cell volume (46%) suggests that cells were at a different growth stage compared to other conditions. Iron availability can influence the expression and activity of cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases (CDKs), which control the progression of cell cycle phases in phytoplankton (Peter & Herskowitz, 1994 ; Smith et al., 2017 ). The variability of cell volume in the growth stages might be related to differences in the utilization of Fe within the cells, with varying requirements at different stages of growth. The highest cell growth rate was measured at moderate Fe levels (50 nM Fe), resulting in a smaller cell volume compared to 10 nM (-36%) and 100 nM Fe (-13%). Similar patterns of high growth rate and reduced cell volume were observed in S. microadriaticum (ITS2 type A1, Camp et al., 2022 ), indicating a strict correlation between trace metal availability and cellular growth. However, in hospite symbionts are often nitrogen-limited (Falkowski et al., 1993 ; Cui et al., 2022 ), and enriching iron under coral reef-like nutrient conditions may exacerbate nitrogen limitation and further unbalance nutrient exchange, photosynthetic activity, and host-symbiont relationship, which could explain the reduced growth rate we observed at high iron levels (100 nM Fe). At the coral host-endosymbiont level, increasing iron levels may stimulate photosynthetic symbiont proliferation but it could lead to their overgrowth in the coral host tissue, which eventually results in expulsion as a stress-response and bleaching. Moreover, the exposure of corals to high iron levels can in turn lead to decreased rates of photosynthesis and reduced maximum quantum yield of PSII (Brown, 1989; Dellisanti et al., 2024 ). Iron is a limiting nutrient for primary producers, including symbiotic dinoflagellates (Entsch et al., 1983) and eukaryotic marine algae (Greene et al., 1972). However, the concentration of iron in seawater is affected by mechanisms of scavenging, such as complexation by strong ligands (Johnson et al., 1997). Iron in seawater is present in ferrous Fe(II) and ferric Fe(III) forms, and its speciation depends on oxidation states and thermodynamics (Blain & Tagliabue, 2016). Most of the iron present in seawater is in the form of ferric ion Fe(III) and can undergo inorganic speciation to ferrous ion Fe(II) through hydrolysis and organic speciation with ligands, such as EDTA and siderophores produced by marine cyanobacteria (Sandy & Butler, 2009 ; Blain & Tagliabue, 2016). When considering the bioavailable amount of Fe (Westall et al., 1976 ), the Fe levels utilized in this study (0–100 nM Fe) were lower compared to previous studies, which used a range of 0–250 nM Fe, corresponding to 0–1250 pM bioavailable Fe (Rodriguez et al., 2016; Rodriguez & Ho, 2018 ; Reich et al., 2020 , 2021). Moreover, in this study, we did not use EDTA as an inorganic ligand to increase the bioavailability of iron, which might account for a lower bioavailable amount of iron. Despite these methodological differences, previous findings have also highlighted a similar role of Fe(III) in the growth of Symbiodiniaceae, evidenced by higher growth rates when S. microadriaticum is cultured with Fe(III) complexes (Romero et al., 2022 ). This response is, however, species-specific, with other Symbiodiniaceae species responding faster than S. microadriaticum (Romero et al., 2022 ). Iron uptake and intracellular content are also enhanced when Symbiodinium spp. are exposed to Fe(III), although high Fe levels reduce the utilization efficiency of this element in S. microadriaticum , with a shift to the utilization of other trace metals such as zinc, nickel, and copper to maintain the enzymatic activities and cellular functioning (Blay-Haas & Merchant, 2012). In our study, the higher Chl a and carotenoid concentration at 100 nM Fe(III) indicates enhanced pigment production, potentially due to excess Fe stimulating pigment synthesis. Iron is present in almost all the components of the electron transport chain in the chloroplast, including cytochromes, and it is a precursor of chlorophyll synthesis (Pushnik et al., 1984 ) through hemoproteins in the cytochrome b6f complex (Hogle et al., 2014 ). The absence of these pigments at 0 nM Fe(III) indicates severe stress or nutritional limitations leading to impaired cellular growth (Rodriguez and Ho, 2018 ; Romero et al., 2022 ). However, excessive iron (100 nM Fe(III)) may lead to toxic levels negatively impacting Symbiodiniaceae growth and, in turn, the symbiotic relationships within the coral host (Brown, 1989; Leigh-Smith et al., 2018). Moreover, our findings indicate that Symbiodinium sp. can produce higher levels of scytonemin when cells are grown in 100 nM Fe(III). The biosynthesis of scytonemin in Symbiodinium sp. is influenced by osmotic and oxidative stress (Dillon et al., 2002; Liu et al., 2018), potentially serving as an indicator of stress for cells grown under high Fe conditions. This could explain why moderate levels of 50 nM Fe(III) are more favorable, providing sufficient Fe for pigment production, while supporting the development of cellular content without reaching toxic levels (as observed in this study). Non-destructive label-free tomographic imaging represents an advanced tool for investigating the morphology and cellular content of live single cells, such as Symbiodinium sp. Previous studies have demonstrated its capability in visualizing lipids within cells and delineating cell morphologies of microalgal cells, showing comparability with traditional staining techniques. Recent advancements in holotomography have further expanded the potential of this approach by enabling the study of subcellular components, such as lipid droplets without the need for fixation or staining methods (Kim et al., 2024 ). This offers researchers the opportunity to investigate cellular structures and contents in-vivo providing insights into dynamic cellular processes that conventional staining techniques may not cover. Using non-destructive holotomography, we measured higher lipid content (+ 57%), refractive index (+ 0.35%), and protein concentration (+ 9%) when Symbiodinium sp. was exposed to 50 nM Fe(III), indicating a physiological response to moderate iron concentrations by increasing cellular content. We speculate that moderate levels of Fe enhance the metabolic activity of Symbiodinium sp., which leads to increased lipid production to store excess energy as a response mechanism to cellular division. This suggests that there is an optimal Fe(III) concentration in the culture medium, which leads to optimal cellular biochemical content, resulting in the highest growth rates of the Symbiodinium cells. It is important to note that different Symbiodinium species may exhibit distinct strategies for energy storage, reflecting their specific physiological adaptations to their respective ecological niches (Wang et al., 2015 ). The relatively higher lipid content observed in cells grown at 50 nM Fe(III) may represent a cellular response for energy storage, as observed in Symbiodinium cultures and other microalgae (Roessler, 1990 ; Jiang et al., 2014 ; Sun et al., 2018 ). Lipids generate more energy than carbohydrates upon oxidation and can be efficiently packed into the cell, thus providing the best energy reserve for cells to return to homeostatic conditions, particularly in response to stress conditions like temperature fluctuations (Rosset et al., 2019 ) and nutrient deprivation, such as nitrogen limitation (Jiang et al., 2014 ). In conclusion, holotomography allows real-time observation of live Symbiodiniaceae cells, providing insights into their response to environmental stressors. It enables non-invasive monitoring of cellular adaptations, such as lipid droplet formation and protein concentration via tomographic mapping of refractive index. This method can also be applied to compare different Symbiodiniaceae species, potentially revealing strategies for energy storage and environmental adaptation, which might help to explain their evolutionary success in various ecological niches. Importantly, it should be noted that the Fe levels utilized in our study (0-100 nM) are higher than ranges found in natural environments. Coastal waters typically exhibit Fe levels around 14.5 nM (Sarthou & Jandel, 2001), whereas tropical waters often have Fe concentrations below 5 nM (GEOTRACES IDP, 2021). Moreover, our study primarily examined the short-term responses of cultured Symbiodinium sp. to increasing Fe concentrations, which may not fully capture the long-term implications or acclimation potential of these organisms to fluctuating Fe levels in their natural habitats. Additionally, while specific growth rates provide valuable insights, they offer only a partial understanding of overall fitness and ecological performance. Incorporating additional metrics such as specific growth rate, nutrient uptake rates, photophysiological measurements or gene expression profiles could provide a more comprehensive understanding of the physiological responses of Symbiodinium sp. to Fe availability either in culture or in hospite . 5. Conclusions Marine photosynthetic organisms are profoundly influenced by the availability of trace metals, which are fundamental for numerous physiological processes including photosynthesis, cellular homeostasis, and antioxidant enzymatic reactions. Increasing levels of dissolved iron, Fe(III), have been shown to significantly impact the growth of Symbiodinium sp., while also stimulating the production of cellular lipids. However, the observed variation in pigment concentrations versus growth and reproduction at different iron concentrations in Symbiodiniaceae cultures suggests that iron availability and pigment concentration are just one facet of the complex network of factors influencing their physiology. Our study highlights the significance of iron availability as an essential trace metal in regulating the growth of Symbiodinium sp. It emphasizes the necessity of considering potential interactions of trace metals when investigating the response of these organisms to environmental changes. Moreover, it highlights the need for further research to elucidate the specific mechanisms and regulatory pathways involved in Symbiodinium sp. responses to iron availability. For future studies, it would be valuable to incorporate measurements e.g. of cellular scavenging of reactive oxygen species (ROS) levels under environmental stress, such as ocean warming, and under nitrogen and phosphorus limitation. Additionally, exploring the role of inorganic Fe in supporting thermo-tolerance may provide insights into the mechanisms of metal availability in the growth of Symbiodiniaceae. Declarations Acknowledgment We thank Sofie Jakobsen and Jonathan Wolters for excellent technical assistance, and Dr. Stefano Amalfitano at the Water Research Institute (CNR-Italy) for discussing flow cytometry procedure and data analysis. Data availability Data and code for data analysis are available in the Zenodo repository at https://zenodo.org/doi/10.5281/zenodo.13845002. Funding This research was supported by the European Union (Grant Agreement no. 101062810, MedCorP; WD), the Villum Foundation (Grant no. VIL57413; MK), the Carlsberg Foundation (Grant no. CF21-0599; MK) and the Gordon and Betty Moore Foundation (Grant no. GBMF9206; https://doi.org/10.37807/GBMF9206; MK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Amin, S. A., Green, D. H., Hart, M. C., Küpper, F. C., Sunda, W. G., & Carrano, C. J. (2009). Photolysis of iron–siderophore chelates promotes bacterial–algal mutualism. Proceedings of the National Academy of Sciences of the United States of America, 106(40), 17071. https://doi.org/10.1073/PNAS.0905512106. An, J., Miao, X., Wang, L., Li, X., Liu, X., & Gao, H. (2021). Visualizing the integrity of chloroplast envelope by rhodamine and Nile Red staining. Frontiers in Plant Science, 12: 668.414. https://doi.org/10.3389/fpls.2021.668414. Balk, J., & Schaedler, T. A. (2014). Iron cofactor assembly in plants. Annual Review of Plant Biology, 65, 125–153. https://doi.org/10.1146/ANNUREV-ARPLANT-050213-035759. Barer, R. & Joseph, S. (1954) Refractometry of living cells. J. Cell Sci. 95, 399–423. Bennet, M., Gur, D., Yoon, J., Park, Y. K., & Faivre, D. (2017). A Bacteria-Based Remotely Tunable Photonic Device. Advanced Optical Materials, 5(1), 1600617. https://doi.org/10.1002/ADOM.201600617. Blaby-Haas CE, Merchant SS (2012) The ins and outs of algal metal transport. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research 1823:1531–1552. Camp, E. F., Nitschke, M. R., Clases, D., Gonzalez de Vega, R., Reich, H. G., Goyen, S., & Suggett, D. J. (2022). Micronutrient content drives elementome variability amongst the Symbiodiniaceae. BMC Plant Biology, 22(1), 1–14. https://doi.org/10.1186/S12870-022-03512-0. Choi, W., Fang-Yen, C., Badizadegan, K., Oh, S., Lue, N., Dasari, R. R., & Feld, M. S. (2007). Tomographic phase microscopy. Nature Methods, 4(9), 717–719. https://doi.org/10.1038/NMETH1078. Cui, G., Liew, Y.J., Konciute, M.K. et al. Nutritional control regulates symbiont proliferation and life history in coral-dinoflagellate symbiosis. BMC Biol 20, 103 (2022). https://doi.org/10.1186/s12915-022-01306-2. Deleja, M., Paula, J. R., Repolho, T., Franzitta, M., Baptista, M., Lopes, V., Simão, S., Fonseca, V. F., Duarte, B., & Rosa, R. (2022). Effects of Hypoxia on Coral Photobiology and Oxidative Stress. Biology, 11(7), 1068. https://doi.org/10.3390/BIOLOGY11071068. Dellisanti W, Zhang Q, Ferrier-Pagès C, Kühl M. 2024. Contrasting effects of increasing dissolved iron on photosynthesis and O 2 availability in the gastric cavity of two Mediterranean corals. PeerJ 12:e17259 https://doi.org/10.7717/peerj.17259. Falkowski, P. G., Dubinsky, Z., Muscatine, L., & McCloskey, L. (1993). Population Control in Symbiotic Corals. BioScience, 43(9), 606–611. https://doi.org/10.2307/1312147. Firestone D. (1999). Physical and chemical characteristics of oils, fats, and waxes. 2 edn, (AOCS press Champaign, IL). Galotti, A., Jiménez-Gómez, F., & Parra, G. (2020). Flow Cytometry Assessment of Microalgae Physiological Alterations under CO 2 Injection. Cytometry Part A, 97(11), 1136–1144. https://doi.org/10.1002/cyto.a.24028. Garrett, T. A., Schmeitzel, J. L., Klein, J. A., Hwang, J. J., & Schwarz, J. A. (2013). Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont. PLoS ONE, 8(3), 57975. https://doi.org/10.1371/JOURNAL.PONE.0057975. Granados-Cifuentes, C., & Rodriguez-Lanetty, M. (2011). The use of high-resolution melting analysis for genotyping Symbiodinium strains: a sensitive and fast approach. Molecular Ecology Resources, 11(2), 394–399. https://doi.org/10.1111/J.1755-0998.2010.02933.X. Greenspan, P., Mayer, E. P., & Fowler, S. D. (1985). Nile red: a selective fluorescent stain for intracellular lipid droplets. Journal of Cell Biology, 100 (3): 965-973. https://doi.org/10.1083/jcb.100.3.965. Habaza, M., Gilboa, B., Roichman, Y., & Shaked, N. T. (2015). Tomographic phase microscopy with 180 rotation of live cells in suspension by holographic optical tweezers. Optics Letters, Vol. 40, Issue 8, Pp. 1881-1884, 40(8), 1881–1884. https://doi.org/10.1364/OL.40.001881. Habaza, M., Kirschbaum, M., Guernth-Marschner, C., Dardikman, G., Barnea, I., Korenstein, R., Duschl, C., Shaked, N. T. (2017). Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation. Advanced Science, 4(2), 1600205. https://doi.org/10.1002/ADVS.201600205. Hansen, G., & Daugbjerg, N. (2009). Symbiodinium natans sp. Nov.: A “free-living” dinoflagellate from Tenerife (northeast-atlantic ocean). Journal of Phycology, 45(1), 251–263. https://doi.org/10.1111/j.1529-8817.2008.00621.x. Hogle, S. L., Barbeau, K. A., & Gledhill, M. (2014). Heme in the marine environment: from cells to the iron cycle. Metallomics, 6(6), 1107–1120. https://doi.org/10.1039/C4MT00031E. Hume, B. C. C., Ziegler, M., Poulain, J., Pochon, X., Romac, S., Boissin, E., de Vargas, C., Planes, S., Wincker, P., & Voolstra, C. R. (2018). An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region. PeerJ, 2018(5). https://doi.org/10.7717/PEERJ.4816. Hume, B. C. C., Smith, E. G., Ziegler, M., Warrington, H. J. M., Burt, J. A., LaJeunesse, T. C., Wiedenmann, J., & Voolstra, C. R. (2019). SymPortal: A novel analytical framework and platform for coral algal symbiont next-generation sequencing ITS2 profiling. Molecular Ecology Resources, 19(4), 1063–1080. https://doi.org/10.1111/1755-0998.13004. Iglic, Katrina Lynn, "Trace metal limitation and its role in oxidative stress of coral algal symbionts; implications for thermally induced coral bleaching events." (2011). Electronic Thesis and Dissertation Repository. 282. https://ir.lib.uwo.ca/etd/282. Kim, T., Zhou, R., Mir, M., Babacan, S. D., Carney, P. S., Goddard, L. L., & Popescu, G. (2014a). White-light diffraction tomography of unlabelled live cells. Nature Photonics 2014 8:3, 8(3), 256–263. https://doi.org/10.1038/nphoton.2013.350. Kim, K., Yoon, H., Diez-Silva, M., Dao, M., Dasari, R. R., & Park, Y. (2014b). Special Section on Advanced Biomedical Imaging and Sensing: High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography. Journal of Biomedical Optics, 19(1), 1. https://doi.org/10.1117/1.JBO.19.1.011005. Kim, Y., Shim, H., Kim, K., Park, H. J., Jang, S., & Park, Y. K. (2014c). Profiling individual human red blood cells using common-path diffraction optical tomography. Scientific Reports, 4(1), 1–7. https://doi.org/10.1038/srep06659. Kim, K., Lee, S., Yoon, J., Heo, J., Choi, C., & Park, Y. (2016). Three-dimensional label-free imaging and quantification of lipid droplets in live hepatocytes. Scientific Reports 2016 6:1, 6(1), 1–8. https://doi.org/10.1038/srep36815. Kim, H., Oh, S., Lee, S., Lee, K. suk, & Park, Y. (2024). Recent advances in label-free imaging and quantification techniques for the study of lipid droplets in cells. Current Opinion in Cell Biology, 87, 102342. https://doi.org/10.1016/J.CEB.2024.102342. Kneeland, J., Hughen, K., Cervino, J., Hauff, B., & Eglinton, T. (2013). Lipid biomarkers in Symbiodinium dinoflagellates: New indicators of thermal stress. Coral Reefs, 32(4), 923–934. https://doi.org/10.1007/S00338-013-1076-3. Koch, F., & Trimborn, S. (2019). Limitation by fe, zn, co, and b12 results in similar physiological responses in two antarctic phytoplankton species. Frontiers in Marine Science, 6(AUG), 469522. https://doi.org/10.3389/FMARS.2019.00514. Jiang, P. L., Pasaribu, B., & Chen, C. S. (2014). Nitrogen-Deprivation Elevates Lipid Levels in Symbiodinium spp. by Lipid Droplet Accumulation: Morphological and Compositional Analyses. PLOS ONE, 9(1), e87416. https://doi.org/10.1371/JOURNAL.PONE.0087416. Jung, J. H., Hong, S. J., Kim, H. B., Kim, G., Lee, M., Shin, S., Lee, S. Y., Kim, D. J., Lee, C. G., & Park, Y. K. (2018). Label-free non-invasive quantitative measurement of lipid contents in individual microalgal cells using refractive index tomography. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-24393-0. Lee, K. R., Kim, K., Jung, J., Heo, J. H., Cho, S., Lee, S., Chang, G., Jo, Y. J., Park, H., & Park, Y. K. (2013). Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications. Sensors 2013, Vol. 13, Pages 4170-4191, 13(4), 4170–4191. https://doi.org/10.3390/S130404170. Lee, S., Lee, S., Kim, K., Mubarok, A., Park, Y., Panduwirawan, A., Park, H., & Lee, K. (2014). High-Resolution 3-D Refractive Index Tomography and 2-D Synthetic Aperture Imaging of Live Phytoplankton. Journal of the Optical Society of Korea, Vol. 18, Issue 6, Pp. 691-697, 18(6), 691–697. https://doi.org/10.3807/JOSK.2014.18.6.691. Lee, E., Jalalizadeh, M., & Zhang, Q. (2015). Growth kinetic models for microalgae cultivation: A review. Algal Research, 12, 497–512. https://doi.org/10.1016/J.ALGAL.2015.10.004 Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology, Volume 148, 1987, Pages 350-382. Liu, Z. Y., Wang, G. C., & Zhou, B. C. (2008). Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresource Technology, 99(11), 4717–4722. https://doi.org/10.1016/J.BIORTECH.2007.09.073. Mashaghi, A., Swann, M., Popplewell, J., Textor, M., & Reimhult, E. (2008). Optical Anisotropy of Supported Lipid Structures Probed by Waveguide Spectroscopy and Its Application to Study of Supported Lipid Bilayer Formation Kinetics. Analytical Chemistry, 80(13), 5276. https://doi.org/10.1021/AC800988V. Matthews, J. L., Hoch, L., Raina, J. B., Pablo, M., Hughes, D. J., Camp, E. F., Seymour, J. R., Ralph, P. J., Suggett, D. J., & Herdean, A. (2023a). Symbiodiniaceae photophysiology and stress resilience is enhanced by microbial associations. Scientific Reports 2023 13:1, 13(1), 1–13. https://doi.org/10.1038/s41598-023-48020-9. Matthews, J. L., Khalil, A., Siboni, N., Bougoure, J., Guagliardo, P., Kuzhiumparambil, U., DeMaere, M., le Reun, N. M., Seymour, J. R., Suggett, D. J., & Raina, J. B. (2023b). Coral endosymbiont growth is enhanced by metabolic interactions with bacteria. Nature Communications 2023 14:1, 14(1), 1–13. https://doi.org/10.1038/s41467-023-42663-y. Müller, B. (2023). Iron transport mechanisms and their evolution focusing on chloroplasts. Journal of Plant Physiology, 288, 154059. https://doi.org/10.1016/J.JPLPH.2023.154059. Park, S., Ahn, J. W., Jo, Y., Kang, H. Y., Kim, H. J., Cheon, Y., Kim, J. W., Park, Y., Lee, S., & Park, K. (2020). Label-Free Tomographic Imaging of Lipid Droplets in Foam Cells for Machine-Learning-Assisted Therapeutic Evaluation of Targeted Nanodrugs. ACS Nano, 14(2), 1856–1865. https://doi.org/10.1021/ACSNANO.9B07993. Pasaribu, B., Primadona Purba, N., Dewanti, L. P., Pasaribu, D., Muhammad, A., Khan, A., Harahap, S. A., Syamsuddin, M. L., Ihsan, Y. N., Siregar, S. H., Faizal, I., Herawati, T., Irfan, M., Partogi, T., Simorangkir, H., & Kurniawan, T. A. (2024). Lipid Droplets in Endosymbiotic Symbiodiniaceae spp. Associated with Corals. Plants 2024, Vol. 13, Page 949, 13(7), 949. https://doi.org/10.3390/PLANTS13070949. Patton, J. S., & Burris, J. E. (1983). Lipid synthesis and extrusion by freshly isolated zooxanthellae (symbiotic algae). Marine Biology, 75(2–3), 131–136. https://doi.org/10.1007/BF00405995. Peter, M., & Herskowitz, I. (1994). Joining the complex: Cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell, 79, 181–184. Popescu, G. 2011. Quantitative Phase Imaging of Cells and Tissues. 1st ed. New York: McGraw-Hill. https://www.accessengineeringlibrary.com/content/book/9780071663427. Pushnik, J. C., Miller, G. W., & Manwaring, J. H. (1984). The role of iron in higher plant chlorophyll biosynthesis, maintenance and chloroplast biogenesis. Journal of Plant Nutrition, 7(1-5), 733–758. https://doi.org/10.1080/01904168409363238 R Core Team (2016) R: A language and environment for statistical computing [Computer software]. R Foundation for Statistical Computing, Vienna. Rai, S., Singh, P. K., Mankotia, S., Swain, J., & Satbhai, S. B. (2021). Iron homeostasis in plants and its crosstalk with copper, zinc, and manganese. Plant Stress, 1, 100008. https://doi.org/10.1016/J.STRESS.2021.100008. Rappaz, B., Cano, E., Colomb, T., Kuhn, J., Depeursinge, C. D., Simanis, V., Magistretti, P. J., & Marquet, P. P. (2009). Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy. Journal of Biomedical Optics, 14:3, 034049. https://doi.org/10.1117/1.3147385. Raven, J. A., Evans, M. C. W., & Korb, R. E. (1999). The role of trace metals in photosynthetic electron transport in O2-evolving organisms. Photosynthesis Research, 60(2–3), 111–150. https://doi.org/10.1023/A:1006282714942. Reich, H. G., Rodriguez, I. B., LaJeunesse, T. C., & Ho, T. Y. (2020). Endosymbiotic dinoflagellates pump iron: differences in iron and other trace metal needs among the Symbiodiniaceae. Coral Reefs, 39(4), 915–927. https://doi.org/10.1007/s00338-020-01911-z. Reich, H. G., Camp, E. F., Roger, L. M., & Putnam, H. M. (2023). The trace metal economy of the coral holobiont: supplies, demands and exchanges. Biological Reviews, 98(2), 623–642. https://doi.org/10.1111/brv.12922. Rodriguez, I. B., & Ho, T. Y. (2018). Trace metal requirements and interactions in Symbiodinium kawagutii . Frontiers in Microbiology, 310350. https://doi.org/10.3389/FMICB.2018.00142. Romero, J. M. D., Botana, M. T., Elias, A. de C., Nomura, C. S., Saldanha-Corrêa, F., & Espósito, B. P. (2022). Effect of iron speciation on growth and heat resistance of Symbiodiniaceae. Ocean and Coastal Research, 70, e22016. https://doi.org/10.1590/2675-2824070.21103JMDR. Roessler, P. G. (1990). Environmental Control of Glycerolipid Metabolism in Microalgae: Commercial Implications and Future Research Directions. Journal of Phycology, 26(3), 393–399. https://doi.org/10.1111/J.0022-3646.1990.00393.X. Rosset, S., Koster, G., Brandsma, J., Hunt, A. N., Postle, A. D. & D’Angelo, C. (2019). Lipidome analysis of Symbiodiniaceae reveals possible mechanisms of heat stress tolerance in reef coral symbionts. Coral Reefs 38, 1241–1253. https://doi.org/10.1007/s00338-019-01865-x. Sandy, M., & Butler, A. (2009). Microbial Iron Acquisition: Marine and Terrestrial Siderophores. Chemical Reviews, 109(10), 4580. https://doi.org/10.1021/CR9002787. Smith SR, Gillard JTF, Kustka AB, McCrow JP, Badger JH, et al. (2017) Correction: Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation. PLOS Genetics 13(3): e1006688. https://doi.org/10.1371/journal.pgen.1006688. Storms, Z. J., Cameron, E., de la Hoz Siegler, H., & McCaffrey, W. C. (2014). A simple and rapid protocol for measuring neutral lipids in algal cells using fluorescence. Journal of Visualized Experiments, 87. https://doi.org/10.3791/51441. Sun, X. M., Ren, L. J., Zhao, Q. Y., Ji, X. J., & Huang, H. (2018). Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation. Biotechnology for Biofuels 2018 11:1, 11(1), 1–16. https://doi.org/10.1186/S13068-018-1275-9. Sunda, W., Huntsman, S. Interrelated influence of iron, light and cell size on marine phytoplankton growth. Nature 390, 389–392 (1997). https://doi.org/10.1038/37093. Tchernov, D., Gorbunov, M. Y., de Vargas, C., Yadav, S. N., Milligant, A. J., Häggblom, M., & Falkowski, P. G. (2004). Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals. Proceedings of the National Academy of Sciences of the United States of America, 101(37), 13531–13535. https://doi.org/10.1073/PNAS.0402907101. Ullmann, F., Gerhartz, W., Yamamoto, S. Y., Campbell, F. T., Pfefferkorn, R. & Rounsaville, J. F. Ullmann’s encyclopedia of industrial chemistry. Vol. 5 (Vch Weinheim, Germany, 1985). Wang, L. H., Chen, H. K., Jhu, C. S., Cheng, J. O., Fang, L. S., & Chen, C. S. (2015). Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp. Journal of Phycology, 51(6), 1127–1136. https://doi.org/10.1111/JPY.12349. Wang, H., Su, Q., Zhuang, Y., Wu, C., Tong, S., Guan, B., Zhao, Y., & Qiao, H. (2023). Effects of Iron Valence on the Growth, Photosynthesis, and Fatty Acid Composition of Phaeodactylum tricornutum . Journal of Marine Science and Engineering 2023, Vol. 11, Page 316, 11(2), 316. https://doi.org/10.3390/JMSE11020316. Westall, J. C., Zachary, J. L. & Morel, F. M. M. (1976) A computer program for the calculation of chemical equilibrium composition of aqueous systems, Civil Eng. Tech. Note 18. Cambridge: Massachusetts Institute of Technology. Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org. Wickham H, François R, Henry L, Müller K, Vaughan D (2023). dplyr: A Grammar of Data Manipulation. https://dplyr.tidyverse.org, https://github.com/tidyverse/dplyr. Wietheger, A., Starzak, D. E., Gould, K. S., & Davy, S. K. (2018). Differential ROS Generation in Response to Stress in Symbiodinium spp. The Biological Bulletin, 234(1), 11–21. https://doi.org/10.1086/696977. Yadavalli, V., Jolley, C. C., Malleda, C., Thangaraj, B., Fromme, P., & Subramanyam, R. (2012). Alteration of Proteins and Pigments Influence the Function of Photosystem I under Iron Deficiency from Chlamydomonas reinhardtii . PLoS ONE, 7(4). https://doi.org/10.1371/JOURNAL.PONE.0035084. Zhou, Y., Liu, L., Li, M., & Hu, C. (2022). Algal biomass valorisation to high-value chemicals and bioproducts: Recent advances, opportunities and challenges. Bioresource Technology , 344 , 126371. https://doi.org/10.1016/J.BIORTECH.2021.126371. Zigmantas, D., Hiller, R. G., Sundström, V., & Polívka, T. (2002). Carotenoid to chlorophyll energy transfer in the peridinin-chlorophyll-a-protein complex involves an intramolecular charge transfer state. Proceedings of the National Academy of Sciences of the United States of America , 99 (26), 16760–16765. https://doi.org/10.1073/PNAS.262537599. Additional Declarations Walter Dellisanti and Swathi Murthy contributed equally to this manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5160718","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":359497423,"identity":"09d08e93-cf0e-4b6d-bfff-fcdfd06e7229","order_by":0,"name":"Walter Dellisanti*","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1336-4578","institution":"University of Copenhagen","correspondingAuthor":true,"prefix":"","firstName":"Walter","middleName":"","lastName":"Dellisanti*","suffix":""},{"id":359497424,"identity":"20083bea-1167-47ff-8da4-b27f0a00a037","order_by":1,"name":"Swathi Murthy*","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Swathi","middleName":"","lastName":"Murthy*","suffix":""},{"id":359497425,"identity":"4e25bf62-e305-408c-885c-a33b0082fbae","order_by":2,"name":"Elena Bollati","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Bollati","suffix":""},{"id":359497426,"identity":"9897fb4f-98a7-4ade-a291-e6f24363f7ab","order_by":3,"name":"Sara Prehn Sandberg","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"Prehn","lastName":"Sandberg","suffix":""},{"id":359497427,"identity":"b8a70cd4-d17e-4cca-9990-ed9d9471388d","order_by":4,"name":"Michael Kühl","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Kühl","suffix":""}],"badges":[],"createdAt":"2024-09-26 19:16:02","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5160718/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5160718/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65580242,"identity":"1ee003c1-cdf8-4e3a-90f4-b8b310eba90e","added_by":"auto","created_at":"2024-09-30 08:20:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":358652,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental procedure followed in this study. (1) Cultures of \u003cem\u003eSymbiodinium\u003c/em\u003esp. K-1618 were acquired from the Norwegian Culture Collection of Algae (NORCCA). (2a) Aliquots of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures were transferred into treatment flasks at increasing levels of inorganic Fe (0, 10, 50, and 100 nM Fe(III)). (2b) The ITS2 region was sequenced with the Sanger method to genotype the culture. (3) \u003cem\u003eSymbiodinium\u003c/em\u003e cells were sorted and counted using flow cytometry. (4) Pigment analysis of chlorophyll \u003cem\u003ea\u003c/em\u003e, carotenoids, and scytonemin was performed with HPLC. (5) Cell content (protein and lipids) was analyzed with optical diffraction tomography \u003cem\u003ein vivo\u003c/em\u003e. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/f3c037e0af84282da57a422d.png"},{"id":65580241,"identity":"5abda804-1148-4557-aef2-12fa00ea2440","added_by":"auto","created_at":"2024-09-30 08:20:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147387,"visible":true,"origin":"","legend":"\u003cp\u003eResults of flow cytometric analysis of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cells exposed to increasing Fe concentrations (0 to 100 nM Fe). A) cell abundance and growth pattern; B) median values of cell size (FSC-A); C) median values of chlorophyll fluorescence (PC5.5-H); D) relationship of median cellular size (FSC-A) vs median complexity (SSC-A), and ellipses indicating data distribution (95% confidence intervals) in each condition. All values, except for cell abundance, are on a logarithmic scale; the gray area indicates confidence intervals (95%).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/a33fc457365e081f8387f1b9.png"},{"id":65580079,"identity":"3e207364-2309-4725-8be7-9e91c8853691","added_by":"auto","created_at":"2024-09-30 08:12:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1069002,"visible":true,"origin":"","legend":"\u003cp\u003eDigital holotomography images of representative \u003cem\u003eSymbiodinium\u003c/em\u003e cells grown under different Fe concentrations in the culture media. (a-d) 3D rendered image of the reconstructed RI distribution. The legend on the right indicates the RI values of the different regions; (e-h) Overlay of chlorophyll fluorescence images on the 3D RI rendering; (i-l) Maximum intensity projection images of the tomograms of the reconstructed 3D RI distribution.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/4242478889cbbc681c86864f.png"},{"id":65580076,"identity":"2c254ae3-f44b-4778-89fe-6aaef78a890a","added_by":"auto","created_at":"2024-09-30 08:12:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1437843,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of lipid droplets inside \u003cem\u003eSymbiodinium \u003c/em\u003ecells: (a) Maximum intensity projection image of the tomogram of the reconstructed 3D RI distribution; (b) 3D rendered image of the reconstructed RI distribution. The color coding is the same as in the previous image (\u003cstrong\u003eFig. 3\u003c/strong\u003e); (c) Overlay of red channel fluorescence image on the 3D RI rendering; (d) Overlay of green channel fluorescence image on the 3D RI rendering; (e) Overlay of both red and green channel fluorescence images on the 3D RI rendering.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/91cc8590bc8ea349e374ecb9.png"},{"id":65580074,"identity":"3e54b3b5-8d6c-4bcf-bbcb-56908b11e42d","added_by":"auto","created_at":"2024-09-30 08:12:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119388,"visible":true,"origin":"","legend":"\u003cp\u003eCellular parameters calculated from digital holotomography imaging for different growth conditions (0 – 100 nM Fe). A) mean refractive index (RI); B) volume of individual \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cells (mm\u003csup\u003e3\u003c/sup\u003e); C) cellular protein dry mass (pg); D) protein concentration per cell volume (pg mm\u003csup\u003e-3\u003c/sup\u003e); E) cellular lipid dry mass (pg); F) lipid concentration per cell volume (pg mm\u003csup\u003e-3\u003c/sup\u003e); G) lipid to protein ratio on a log10 scale.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/c3c9e5b1ba55b0cc607d3de5.png"},{"id":65791378,"identity":"2190c15b-3716-41ae-8600-29b311e07f4d","added_by":"auto","created_at":"2024-10-02 17:37:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5966379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/8166845e-d52b-4bb1-bf42-a2ecaf795a7a.pdf"},{"id":65580239,"identity":"4168798d-2ad0-4147-ad59-4714f17254d5","added_by":"auto","created_at":"2024-09-30 08:20:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":382965,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5160718/v1/e4539dc8dc4619fed691c1f5.docx"}],"financialInterests":"\u003cp\u003eWalter Dellisanti and Swathi Murthy contributed equally to this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","formattedTitle":"\u003cp\u003e\u003cstrong\u003eModerate levels of dissolved iron stimulate cellular growth and increase lipid storage in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSymbiodinium \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp.\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSymbiotic dinoflagellates belonging to the family Symbiodiniaceae (LaJaunesse et al., 2018) are of crucial significance to coral reef ecosystems. They have a fundamental role in facilitating key processes to corals through photosynthesis, supporting the macro- and micronutrient cycle, and the production of calcium carbonate as the foundation of the reefs (Frommlet et al., 2015; Coffroth and Santos, 2005). However, the current global environmental changes, including ocean warming, nutrient pollution, and deoxygenation, affect the cellular dynamics of the coral-dinoflagellate symbiosis by altering, among other processes, nutrient exchange (Morris et al., 2019; Johnson et al., 2021; R\u0026auml;decker et al., 2021). Disruption of nutrient exchange can lead to the breakdown of symbiosis, i.e., bleaching, ultimately compromising coral survival (Pernice 2014).\u003c/p\u003e \u003cp\u003eAmong the essential nutrients, iron (Fe) plays a fundamental role in the physiology of Symbiodiniaceae and other microalgae (Reich et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Iron is typically present in seawater in nanomolar concentrations, and it is considered a trace metal (Entsch et al., 1983) essential for metabolic processes such as photosynthesis, phagocytosis, and prey digestion in mixotrophic dinoflagellates (Rodriguez et al., 2016; Reich et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While iron concentration refers to the total amount of iron present, its bioavailability refers to the fraction of iron that is accessible and usable by the organism. The balance between iron concentration and its bioavailability directly influences the homeostasis of photosynthetic organisms, as insufficient iron availability can limit growth, while excess iron can induce toxicity (Romero et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reich et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Iron is an enzyme cofactor in electron transfer and catalysis (Balk \u0026amp; Schaedler, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; M\u0026uuml;ller, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and its limitation can lead to reduced chlorophyll synthesis resulting in decreased pigment content and reduced photosynthetic efficiency in Symbiodiniaceae (Iglic, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), phytoplankton (Koch \u0026amp; Trimborn, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and freshwater green microalgae (Yadavalli et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Iron limitation can also induce the expression of iron transporters and siderophore production to enhance iron acquisition from the environment (Sandy \u0026amp; Butler, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Recent observations suggest that the photochemical performance of Symbiodiniaceae is enhanced in association with \u003cem\u003eMarinobacter\u003c/em\u003e sp. and \u003cem\u003eL. alexandrii\u003c/em\u003e bacteria, which may support nutrient exchange and siderophore production to bind Fe into bioavailable forms (Amin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Matthews et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003eb\u003c/span\u003e). On the other hand, excess iron can lead to stressful conditions in \u003cem\u003eSymbiodinium\u003c/em\u003e spp. potentially leading to the production of reactive oxygen species (ROS) through Fenton chemistry, and causing oxidative stress (Wietheger et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Deleja et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This in turn can disrupt cellular homeostasis, damage cellular components, such as proteins and lipids, and inhibit the photosynthetic electron transport chain (Rai et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Reich et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite its importance, only a few Symbiodiniaceae species have been extensively studied regarding their iron requirements (Rodriguez et al., 2016; Reich et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, 2021).\u003c/p\u003e \u003cp\u003eIron availability can also affect the lipid content and lipid profile of microalgae (Liu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Lipids are fundamental components of cells and serve in the cellular metabolism of microalgae, including Symbiodiniaceae (Garrett et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kneeland et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pasaribu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). They contribute to energy reserves and form integral components of cellular membranes (Patton \u0026amp; Burris, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Tchernov et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These functions have implications for nutrient stress responses, energy storage, and proliferation rates of Symbiodiniaceae in nitrogen-limited host tissue directly affecting lipid accumulation (Wang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Quantification of lipids and other cellular contents (e.g. protein) in relation to inorganic iron concentration is therefore relevant to understand the potential role of this micronutrient in optimizing Symbiodiniaceae cellular conditions, and its role in influencing the health and resilience of Symbiodiniaceae populations, both free-living and \u003cem\u003ein hospite\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRecently, optical diffraction tomography (also known as holotomography) techniques have emerged, providing quantitative morphological and biochemical information about individual cells and tissues without the need for exogenous labeling agents (Kim et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The use of interferometry allows the measurement of complex optical fields of diffracted light from biological samples enabling the mapping of refractive index (RI) (Popescu, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). RI is an optical property that varies with the composition and density of cellular components, enabling the identification and quantification of intracellular structures. Holotomography measurements of phase-distortions of light from various incident angles enable the construction of 3D refractive index distributions in cells and tissues at sub-micron resolution (Choi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Habaza et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which can then be segmented to identify particular cellular structures with characteristic RI. For example, lipids typically have a higher RI than other cellular components, and this allows their detection and quantification within live cells (Jung et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Holotomography thus provides a rapid, quantitative method for intracellular lipid detection in live cells (Kim et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has been used as an effective imaging technique for studying biological samples including microalgae (Jung et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), phytoplankton (Lee et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), bacteria (Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e; Bennet et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), yeast (Rappaz et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Habaza et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and red blood cells (Kim et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003ec\u003c/span\u003e). However, potential applications of this technique as a non-invasive, label-free, \u003cem\u003ein vivo\u003c/em\u003e approach to quantify cellular content in Symbiodiniaceae have not yet been explored.\u003c/p\u003e \u003cp\u003eIn this study, we investigate the effect of iron enrichment on \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures and quantify their cellular content using a range of label-free, \u003cem\u003ein vivo\u003c/em\u003e techniques such as flow cytometry and holotomography. The objectives of this study are to \u003cem\u003ei)\u003c/em\u003e evaluate the impact of varying concentrations of dissolved Fe(III) on the growth rate and cellular content development of \u003cem\u003eSymbiodinium\u003c/em\u003e sp., \u003cem\u003eii)\u003c/em\u003e visualize and quantify cellular structures, including proteins and lipids, under different iron enrichment conditions through holotomography techniques, and \u003cem\u003eiii)\u003c/em\u003e to investigate the role of inorganic iron in optimizing cellular growth conditions. We hypothesized that controlled iron enrichment could enhance \u003cem\u003eSymbiodinium\u003c/em\u003e sp. growth and cellular content.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 \u003cem\u003eSymbiodinium\u003c/em\u003e cultures\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures (K-1618) were acquired from the Norwegian Culture Collection of Algae (NORCCA, Norway) and maintained in TL-30 medium in 250 mL polycarbonate flask, at 20\u0026deg;C with a photoperiod of 12:12 at 100 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (400\u0026ndash;700 nm), as measured with a Universal Light Meter (ULM-500) equipped with a spherical micro quantum sensor (US-SQS/L, Heinz Walz, Effeltrich, Germany). Subsequently, subcultures were acclimated to standard f/2 medium for one month and maintained under the same conditions prior to the start of experiments. All experimental analyses, except for the flow cytometry assay, were performed during the mid-exponential phase, as determined via cell counts identifying optimal growth and cellular activity (Lee et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the start of the experimental period, aliquots of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures were transferred into each culture flask (20 mL, n\u0026thinsp;=\u0026thinsp;3 replicates per condition) to reach an initial density of ~\u0026thinsp;10,000 cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and maintained under the same conditions as above until they reached the stationary phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures were exposed to increasing iron Fe(III) concentrations (0, 10, 50, and 100 nM total dissolved Fe) at a controlled temperature of 20\u0026deg;C. A dissolved Fe(III) solution (FeCl\u003csub\u003e3\u003c/sub\u003e*6H\u003csub\u003e2\u003c/sub\u003eO, Sigma-Aldrich) was used to enrich the standard f/2 medium to different iron concentrations (Rodriguez et al., 2016; Reich et al., 2021). Experimental conditions were prepared as follows: modified f/2 medium with depleted dissolved iron, 0 nM Fe(III); standard f/2 medium, 10 nM Fe(III); f/2 media with a concentrated solution of dissolved iron to reach the initial concentrations of 50 nM Fe(III) and 100 nM Fe(III), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Culture identification\u003c/h2\u003e \u003cp\u003eTo verify the taxonomic assignment of the \u003cem\u003eSymbiodinium\u003c/em\u003e sp. strain, we performed Sanger sequencing of the ITS2 region of rDNA. Aliquots of the cultures (500,000 cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were collected and pelleted by centrifugation during the mid-exponential growth phase and stored at -80\u0026deg;C. DNA was extracted using the DNAEasy PowerBiofilm kit (Qiagen) following manufacturer protocol with slight modifications (all centrifugation steps performed at 8,000G for 90s). The ITS2 region was amplified using primers SYM_VAR_5.8S2 and SYM_VAR_REV (Hume et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in the following reaction: 0.5 \u0026micro;L of each 10 \u0026micro;M primer, 1 \u0026micro;L of 2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BSA, 12.5 \u0026micro;L KAPA HiFi HotStart Ready Mix (Roche), 1 \u0026micro;L template DNA (2.65 ng \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and water to a total reaction volume of 25 \u0026micro;L. The amplification cycle was: 98 \u0026deg;C for 2 min, followed by 35 cycles of 98 \u0026deg;C for 20 s, 61 \u0026deg;C for 30 s, 72 \u0026deg;C for 30 s, followed by 72 \u0026deg;C for 5 min. Successful amplification, amplicon size, and contamination were visually assessed via agarose gel electrophoresis. PCR products were cleaned on AMPure XP beads (Beckman Coulter) and submitted for Sanger sequencing to a commercial provider (Eurofins). Taxonomy was assigned via blastn to the NCBI \u003cem\u003ent\u003c/em\u003e database, as well as local blastn to the SymPortal RefSeq database (github.com/reefgenomics/SymPortal_framework downloaded on 25/03/2024, Hume et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Growth measurements\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSymbiodinium\u003c/em\u003e cell growth was assessed every third day with a CytoFLEX cytometer (Beckman Coulter, USA), which was equipped with a 50-mW laser (excitation\u0026thinsp;=\u0026thinsp;488 nm, fluorescence channel 690/50 BP). Flow cytometry was performed throughout the light phase of the light: dark cycle until cultures reached the stationary phase. The instrument gain settings were as follows: forward scatter (FSC)\u0026thinsp;=\u0026thinsp;200, side scatter (SSC)\u0026thinsp;=\u0026thinsp;83, phycoerythrin-Cy5.5 (PC5.5)\u0026thinsp;=\u0026thinsp;350 (Galotti et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Before each analysis, quality control procedures were implemented, as recommended by the CytoFLEX software. All measurements, including culture media blanks, were conducted using an analyzed 1 mL sample at a slow rate (10 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with a threshold of 300 seconds or 10,000 events. The gating process relied on FSC and SSC patterns, with only cells displaying a positive signal for photosynthetic pigments (PC5.5) being chosen for downstream analysis, aiming to minimize potential contamination from non-algal particles (\u003cb\u003eFig. S1\u003c/b\u003e). Cell counts, size, and shape were determined \u003cem\u003ein vivo\u003c/em\u003e by FSC and SSC on unstained cells using the CytExpert software v2.5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Pigment analysis\u003c/h2\u003e \u003cp\u003eQuantitative determination of chlorophyll and pigment content was carried out using High-Performance Liquid Chromography (HPLC, 1260 Infinity, Agilent Technologies). Aliquots of 1 mL for each culture were collected during the mid-exponential growth phase and transferred to 1.5 mL tubes, centrifuged at 5000g, and the pellet was kept at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. The pigments were extracted by adding 0.4 mL acetone:methanol (7:2 vol:vol) to each tube, which was briefly vortexed, and then kept on ice for a 2 min extraction time in darkness. Subsequently, samples were sonicated in an ice-cooled high-power ultrasonicated bath (Misonix 4000; Qsonica LLC., Newtown, CT) in darkness at 80% power for 60 s consisting of 10 pulses of 2 s ON and 4 s OFF (with an amplitude setting of 100%) and then centrifuged at 12,000 g for 1 min in a mini centrifuge (MiniSpin, Eppendorf AG, Hamburg, Germany). The supernatant was filtered through a 0.2 \u0026micro;m pore size syringe filter (Sartorius Minisart SRP 4 filter; Sartorius AG, Goettingen, Germany). 100 \u0026micro;L of the extract was then immediately injected into the HPLC. Pigment extracts were separated and analyzed in the HPLC by a diode array detector (HPLC-DAD and Agilent 1260 Infinity; Agilent Technologies, Santa Clara, CA) fitted with an Ascentis C18 column (25 cm \u0026times; 4.6 mm, Sigma-Aldrich cat. no. 581325 U), detecting specific absorption wavelengths of compounds. The extracts were run at a constant column temperature of 30\u0026deg;C for 69 min., and a flow rate of 1.0 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in a changing gradient of solvent A (methanol:acetonitrile:water, 42:33:25, vol/vol/vol), and solvent B (methanol:acetonitrile:ethylacetate, 50:20:30, vol/vol/vol), where the mobile phase changed linearly from 30% solvent B at the time of injection to 100% at 52 min, staying at 100% for 15 min before returning to 30% within 2 min. Elution profiles from the absorbance detector signal at 664 nm (chlorophyll \u003cem\u003ea\u003c/em\u003e), 460 nm (carotenoids), and 387 nm (scytonemin) (Lichtenthaler, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), were used to calculate pigment ratios from the derived integrated peak areas for each of the identified pigments of interest, using the manufacturer\u0026rsquo;s software (OpenLAB CDS ChemStation Edition; Agilent Technologies).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Optical diffraction tomography\u003c/h2\u003e \u003cp\u003eQuantitative 3D imaging of individual \u003cem\u003eSymbiodinium\u003c/em\u003e cells was carried out with an optical diffraction tomography (ODT) system (Tomocube HT-2H; Tomocube Inc., South Korea). The system also has an inbuilt ability to perform fluorescence microscopy in parallel with RI mapping. The system is based on Mach-Zehnder interferometry, employing a diode-pumped green solid-state laser (532 nm) as a light source. A spatially modulated hologram of the cell was recorded from the diffracted light by a quantitative phase imaging technique (Popescu, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The 3D refractive index (RI) distribution of individual cells was then reconstructed from multiple 2D images measured at various illumination angles (controlled by a digital micromirror device), using the Fourier diffraction theorem (Choi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The theoretical spatial resolution of the system was 119 nm and 336 nm for lateral and axial directions, respectively (Kim et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). From the measured 3D RI tomograms, different RI regions were visualized with a unique color and rendered as a 3D image. Maximum intensity projection images of the RI tomograms were also generated to visualize the 3D data set on a 2D plane. Data visualization and rendering were carried out using Tomostudio software (Tomocube Inc., South Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Wide-field fluorescence imaging.\u003c/h2\u003e \u003cp\u003eTo determine the presence of chlorophyll in the \u003cem\u003eSymbiodinium\u003c/em\u003e cells grown at different Fe concentrations, we used the in-built wide-field fluorescence imaging capability of the HT-2H using the green channel with excitation and emission wavelengths centered around 475 nm and 520 nm, respectively. For every cell imaged, fluorescence images were captured immediately after the ODT images. At least 30 cells were imaged for each Fe concentration. The fluorescence image was overlaid on the rendered RI tomograms. While the system is not equipped with a standard Chl \u003cem\u003ea\u003c/em\u003e filter set, these settings still enabled clear detection of a Chl \u003cem\u003ea\u003c/em\u003e fluorescence signal due to strong coupling in Symbiodiniaceae with Chl \u003cem\u003ec\u003c/em\u003e and peridinin (Zigmantas et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). To confirm the identification of the high RI regions (RI\u0026thinsp;\u0026gt;\u0026thinsp;1.46) in the 3D tomograms as neutral lipids (see below), aliquots of \u003cem\u003eSymbiodinium\u003c/em\u003e cell cultures were stained with a Nile Red solution (10 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) mixed in 30% ethanol solution according to Storms et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). About 20 \u0026micro;L of the cell sample solution was sandwiched between a pair of coverslips. Cells were then imaged on the Tomocube HT-2 using the in-built wide-field fluorescence system using the red channel, with an excitation wavelength centered around 575 nm, and an emission band of 600\u0026ndash;800 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cellular lipid and protein determination\u003c/h2\u003e \u003cp\u003eFor single-cell imaging, \u003cem\u003eSymbiodinium\u003c/em\u003e cell cultures were diluted with culture medium so that the cell density was low enough to avoid having multiple cells within the imaging field. The regions from the RI tomogram with n\u0026thinsp;\u0026gt;\u0026thinsp;1.46, were identified as lipid droplets, as verified by Nile red staining and from the reported average RI for vegetable oils (Ullmann, 1985; Firestone, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Then, cell volume and lipid dry mass were calculated for cells grown under different Fe concentrations. The cell volume was calculated from the cell boundary, as determined in the Tomocube software using Otsu thresholding. For further analysis, only mature cells (indicated by a sphericity value\u0026thinsp;\u0026gt;\u0026thinsp;0.8) were chosen, while dividing cells were disregarded. The lipid and the non-lipid (mostly protein) regions were segmented out based on the RI, i.e., assigning RI\u0026thinsp;\u0026lt;\u0026thinsp;1.46 as protein and RI\u0026thinsp;\u0026gt;\u0026thinsp;1.46 as lipid. Here, we assume proteins to be the major non-lipid component inside the cell (Zhou et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The dry mass of these two cell components was then calculated, as per Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, using the parameter RI increment (RII), defined as an increment of RI of the solution per unit increment of the solute concentration (Barer \u0026amp; Joseph, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1954\u003c/span\u003e).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$n(x,y,z)=nm+\\sum\\limits_{i}^{{}} {\\alpha iCi(x,y,z)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003en\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the RI of the medium, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\alpha i\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(Ci\\)\u003c/span\u003e\u003c/span\u003e are the RII and concentration of protein or lipid, respectively. The typical RII for proteins is 0.19 mL/g (Barer \u0026amp; Joseph, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1954\u003c/span\u003e), and that for lipids is 0.135 mL g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mashaghi et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Integrating the concentration over the volume of the component yields its mass.\u003c/p\u003e \u003cp\u003eAll data were log-transformed and checked for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene\u0026rsquo;s test. When data did not meet the assumptions of normality, a Kruskal-Wallis test was used to compare differences in cell counts, size, chlorophyll fluorescence, and morphology. Permutational analysis of variance (PERMANOVA) with 999 permutations was used to compare differences in cell size (FSC) and complexity (SSC) between conditions. A principal component analysis (PCA) was used to compare all cellular morphology data from tomography analysis between conditions. The lipid: protein ratio was calculated by subtracting the log-transformed protein concentration from the log-transformed lipid concentration. To indicate the variability of data around the mean, the coefficient of variation (CV%) was used. All statistical analyses were run in R v4.2.3 (R Core Team, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) using the \u003cem\u003edplyr\u003c/em\u003e package v1.1.2 (Wickman et al., 2023) and visualized with the \u003cem\u003eggplot2\u003c/em\u003e package v3.4.4 (Wickman, 2016).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 ITS2 identification\u003c/h2\u003e\n \u003cp\u003eSanger sequencing of the ITS2 rDNA region returned two sequence variants separated by a single SNP (\u003cstrong\u003eTable S1\u003c/strong\u003e), as detected via manual inspection of the chromatogram. Subsequent blastn taxonomic assignment confirmed the identification of the cultures as \u003cem\u003eSymbiodinium\u003c/em\u003e sp.. The most closely related sequence (98.64% identity, query cover 100%, e-value 7x10\u003csup\u003e\u0026minus;\u0026thinsp;104\u003c/sup\u003e) available from the NCBI \u003cem\u003ent\u003c/em\u003e database had been previously detected from a symbiont of the coral \u003cem\u003eMontastraea faveolata\u003c/em\u003e collected in the Florida Keys, USA (accession: HQ317739; Granados-Cifuentes \u0026amp; Rodriguez-Lanetty, \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e), from a free-living strain collected from Hawaii, USA (accession: AF184948), and from a free-living strain of unspecified origin (accession: EU449053). The same sequence was labeled as \u003cem\u003eSymbiodinium\u003c/em\u003e A1dh in the SymPortal RefSeq database (Hume et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Cellular growth rate and pigment concentration\u003c/h2\u003e\n \u003cp\u003eThe effect of Fe(III) on cell abundance and morphology varied with different Fe concentrations in the culture media. \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures exhibited distinct growth curves when exposed to different iron concentrations (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;36.757, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cstrong\u003eTable S2\u003c/strong\u003e). Under depleted iron conditions (0 nM), no changes in cell abundance were observed, and fluorescence (PC5.5) was not detected after day 18, indicating the absence of viable cells. Under low (10 nM), moderate (50 nM), and replete (100 nM) Fe(III) concentrations, cultures reached their maximum growth on day 26 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). The highest abundances were observed in cells growing under 50 nM Fe(III), with a cell density of 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Cells growing under 100 nM iron exhibited the second highest density of 0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, followed by those growing under 10 nM iron with a concentration of 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 x 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Iron concentration also influenced cell size (ANOVA, F\u0026thinsp;=\u0026thinsp;26.24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), although no differences were observed between 50 and 100 nM (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Cells growing at 10 nM Fe were on average 4.6% and 9.3% larger than those in the 100 and 50 nM treatment, respectively. The depleted Fe condition did not support cellular growth as the overall cell size at 0 nM Fe was 12.5% smaller than 10 nM Fe. In the chlorophyll fluorescence signal (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), no significant differences were observed among the various Fe concentrations tested. However, in terms of cell size (FSC) and complexity (SSC), significant differences were detected under increasing Fe concentrations (PERMANOVA, F\u0026thinsp;=\u0026thinsp;47.773, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cstrong\u003eTable S3\u003c/strong\u003e) indicating small and granular cells at 0 nM Fe, large and relatively smooth cells at 10 nM Fe, medium-size and smooth cells at 50 and 100 nM Fe, respectively.\u003c/p\u003e\n \u003cp\u003eFe(III) concentration affected the concentration of Chl \u003cem\u003ea\u003c/em\u003e, carotenoids, and scytonemin in \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cultures (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e). Pigment concentrations of cells grown under depleted Fe concentration (0 nM Fe) were undetectable. Chlorophyll \u003cem\u003ea\u003c/em\u003e concentration increased from 11.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in cells at 10 nM Fe to 24.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 100 nM Fe (ANOVA, F\u0026thinsp;=\u0026thinsp;113.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, the concentration of carotenoids increased from 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 to 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in cells grown in 10 nM Fe and 100 nM Fe, respectively (ANOVA, F\u0026thinsp;=\u0026thinsp;121.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The concentration of scytonemin ranged from 5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in cells at 10 nM Fe, to 12.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 100 nM Fe (ANOVA, F\u0026thinsp;=\u0026thinsp;114.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhotosynthetic pigments analyzed by HPLC: Chlorophyll \u003cem\u003ea\u003c/em\u003e; carotenoids; scytonemin per each condition. All data are expressed as pigment mass per mL of culture sample. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation is shown.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCondition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFe conc.\u003c/p\u003e\n \u003cp\u003enM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarotenoids\u003c/p\u003e\n \u003cp\u003e\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScytonemin\u003c/p\u003e\n \u003cp\u003e\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 Fe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 Fe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.09 (0.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24 (0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.39 (0.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 Fe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.69 (2.46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42 (0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.56 (1.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 Fe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.87 (2.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54 (0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.08 (1.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe availability of Fe(III) also had a significant impact on cellular volume and content, including proteins and lipids, and on refractive index (RI) (ANOVA, F\u0026thinsp;=\u0026thinsp;16.34, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cstrong\u003eFig. S2\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cu\u003e3.3 Mapping RI distributions within \u003cem\u003eSymbiodinium\u0026nbsp;\u003c/em\u003ecells.\u003c/u\u003e\u003c/p\u003e\n \u003cp\u003eOptical diffraction tomography (ODT) was employed to map the 3D refractive index (RI) distribution of \u003cem\u003eSymbiodinium\u003c/em\u003e cells. The reconstructed 3D RI distribution (\u003cstrong\u003eFig. 3a-d\u003c/strong\u003e) and maximum intensity projection images (\u003cstrong\u003eFig. 3i-l\u003c/strong\u003e) of cells grown under different Fe (III) concentrations in the culture medium indicated the presence of high RI (\u0026gt;1.46) regions in many of the cells. Based on previous studies (Kim et al., 2016) these regions were suspected to be lipid droplets.\u003c/p\u003e\n \u003cp\u003eTo confirm the identification of the high RI (\u0026gt;\u0026thinsp;1.46) regions as lipids, the cells were stained with Nile red dye and imaged using the in-built wide-field fluorescence imaging (see methods section). The Nile red fluorescence images (red channel) were compared with the 3D RI tomograms of the same cell (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The images confirmed that regions with RI\u0026thinsp;\u0026gt;\u0026thinsp;1.46 corresponded to lipid droplets. This is also in accordance with values found in the literature for vegetable oils (Ullmann, 1985) and for lipid droplets in microalgal cells (Jung et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The Nile red and chlorophyll fluorescence signals co-localize in most cells and were difficult to separate, as Nile red could also stain membranes of organelles like chloroplasts (Greenspan et al., \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e; An et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, in cases of large lipid droplets (as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, of a dividing cell) it was possible to separate the two signals (to some extent) by appropriate thresholding. The chlorophyll fluorescence (green channel), in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, appears as a ring around the lipid droplets. This could again be an artifact due to intense light scattering from intra-cellular components and a possible spillover from Nile red fluorescence.\u003c/p\u003e\n \u003cp\u003eTo assess the effect of Fe(III) concentration in the culture medium on \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cell growth, morphology, and biochemical content, the 3D RI images were used to quantify (see methods section) cell size, mean RI, protein, and lipid content (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cstrong\u003eTable S5\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eCells grown in 0 nM Fe displayed smaller volume and refractive index (RI), lower lipid concentration and dry mass, as well as reduced protein concentration and dry mass (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cstrong\u003eand\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). In terms of RI, cells grown in 0 nM Fe had a mean value of 1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, while the highest mean RI was measured in cells grown in 50 nM Fe condition at 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005, compared to 10 and 100 nM conditions (ANOVA, F\u0026thinsp;=\u0026thinsp;22.83, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cstrong\u003eTable S5\u003c/strong\u003e). The cell volume ranged from 262.9\u0026thinsp;\u0026plusmn;\u0026thinsp;420.6 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e in 0 nM Fe to 649.07\u0026thinsp;\u0026plusmn;\u0026thinsp;298.6 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e in 10 nM Fe (ANOVA, F\u0026thinsp;=\u0026thinsp;23.94, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, \u003cstrong\u003eTable S5\u003c/strong\u003e), with no significant differences observed in 50 and 100 nM Fe conditions. The lowest protein dry mass was recorded in 0 nM Fe 61.04\u0026thinsp;\u0026plusmn;\u0026thinsp;98.55 pg (ANOVA, F\u0026thinsp;=\u0026thinsp;29.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the highest was measured in 10 nM Fe 171.58\u0026thinsp;\u0026plusmn;\u0026thinsp;66.55 pg, with no significant difference among the other conditions (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cstrong\u003eTable S5\u003c/strong\u003e). Protein concentration was lowest in cells grown in 0 nM Fe, at 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e pg \u0026micro;m\u003csup\u003e3\u003c/sup\u003e (ANOVA, F\u0026thinsp;=\u0026thinsp;22.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the highest values were measured in cells grown in 50 nM Fe at 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e pg \u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed, \u003cstrong\u003eTable S5)\u003c/strong\u003e. Lipid dry mass and concentration were higher in cells cultured in 50 nM Fe condition than all other conditions and measured at 1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 pg (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;18.619, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 31.47\u0026thinsp;\u0026plusmn;\u0026thinsp;46.61 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e pg \u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;17.178, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), respectively (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee, f, \u003cstrong\u003eTable S5\u003c/strong\u003e). Lipid:protein ratio was higher in cells grown in 50 nM condition at 1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 (ANOVA, F\u0026thinsp;=\u0026thinsp;4.82, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg, \u003cstrong\u003eTable S5\u003c/strong\u003e), with no significant difference observed in other conditions.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we investigated the influence of dissolved Fe concentration on the cellular growth and content of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. As hypothesized, Fe enrichment enhanced cellular growth and increased cellular contents. However, this was only true up to an intermediate concentration of 50 nM Fe(III), which seemed to provide the most favorable conditions among the investigated concentrations.\u003c/p\u003e \u003cp\u003eIron is recognized for its support of the photosynthetic process in coral endosymbionts and its essential role as a cofactor in numerous enzymatic reactions involved in photosynthesis, electron transport, and antioxidant activities (Raven et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Reich et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, iron limitation can limit the growth of \u003cem\u003eSymbiodinium\u003c/em\u003e spp. (Rodriguez and Ho, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) as well as marine phytoplankton (Sunda \u0026amp; Huntsman, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), as observed in our experiment for cells grown in depleted iron conditions (0 nM). An increase in the volume of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. cells was observed at 10 nM Fe condition, which could indicate that lower Fe concentrations may facilitate cellular expansion. However, the large variation in cell volume (46%) suggests that cells were at a different growth stage compared to other conditions. Iron availability can influence the expression and activity of cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases (CDKs), which control the progression of cell cycle phases in phytoplankton (Peter \u0026amp; Herskowitz, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Smith et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The variability of cell volume in the growth stages might be related to differences in the utilization of Fe within the cells, with varying requirements at different stages of growth. The highest cell growth rate was measured at moderate Fe levels (50 nM Fe), resulting in a smaller cell volume compared to 10 nM (-36%) and 100 nM Fe (-13%). Similar patterns of high growth rate and reduced cell volume were observed in \u003cem\u003eS. microadriaticum\u003c/em\u003e (ITS2 type A1, Camp et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), indicating a strict correlation between trace metal availability and cellular growth. However, \u003cem\u003ein hospite\u003c/em\u003e symbionts are often nitrogen-limited (Falkowski et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Cui et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and enriching iron under coral reef-like nutrient conditions may exacerbate nitrogen limitation and further unbalance nutrient exchange, photosynthetic activity, and host-symbiont relationship, which could explain the reduced growth rate we observed at high iron levels (100 nM Fe). At the coral host-endosymbiont level, increasing iron levels may stimulate photosynthetic symbiont proliferation but it could lead to their overgrowth in the coral host tissue, which eventually results in expulsion as a stress-response and bleaching. Moreover, the exposure of corals to high iron levels can in turn lead to decreased rates of photosynthesis and reduced maximum quantum yield of PSII (Brown, 1989; Dellisanti et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIron is a limiting nutrient for primary producers, including symbiotic dinoflagellates (Entsch et al., 1983) and eukaryotic marine algae (Greene et al., 1972). However, the concentration of iron in seawater is affected by mechanisms of scavenging, such as complexation by strong ligands (Johnson et al., 1997). Iron in seawater is present in ferrous Fe(II) and ferric Fe(III) forms, and its speciation depends on oxidation states and thermodynamics (Blain \u0026amp; Tagliabue, 2016). Most of the iron present in seawater is in the form of ferric ion Fe(III) and can undergo inorganic speciation to ferrous ion Fe(II) through hydrolysis and organic speciation with ligands, such as EDTA and siderophores produced by marine cyanobacteria (Sandy \u0026amp; Butler, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Blain \u0026amp; Tagliabue, 2016). When considering the bioavailable amount of Fe (Westall et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), the Fe levels utilized in this study (0\u0026ndash;100 nM Fe) were lower compared to previous studies, which used a range of 0\u0026ndash;250 nM Fe, corresponding to 0\u0026ndash;1250 pM bioavailable Fe (Rodriguez et al., 2016; Rodriguez \u0026amp; Ho, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Reich et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, 2021). Moreover, in this study, we did not use EDTA as an inorganic ligand to increase the bioavailability of iron, which might account for a lower bioavailable amount of iron. Despite these methodological differences, previous findings have also highlighted a similar role of Fe(III) in the growth of Symbiodiniaceae, evidenced by higher growth rates when \u003cem\u003eS. microadriaticum\u003c/em\u003e is cultured with Fe(III) complexes (Romero et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This response is, however, species-specific, with other Symbiodiniaceae species responding faster than \u003cem\u003eS. microadriaticum\u003c/em\u003e (Romero et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Iron uptake and intracellular content are also enhanced when \u003cem\u003eSymbiodinium\u003c/em\u003e spp. are exposed to Fe(III), although high Fe levels reduce the utilization efficiency of this element in \u003cem\u003eS. microadriaticum\u003c/em\u003e, with a shift to the utilization of other trace metals such as zinc, nickel, and copper to maintain the enzymatic activities and cellular functioning (Blay-Haas \u0026amp; Merchant, 2012).\u003c/p\u003e \u003cp\u003eIn our study, the higher Chl \u003cem\u003ea\u003c/em\u003e and carotenoid concentration at 100 nM Fe(III) indicates enhanced pigment production, potentially due to excess Fe stimulating pigment synthesis. Iron is present in almost all the components of the electron transport chain in the chloroplast, including cytochromes, and it is a precursor of chlorophyll synthesis (Pushnik et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) through hemoproteins in the cytochrome \u003cem\u003eb6f\u003c/em\u003e complex (Hogle et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The absence of these pigments at 0 nM Fe(III) indicates severe stress or nutritional limitations leading to impaired cellular growth (Rodriguez and Ho, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Romero et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, excessive iron (100 nM Fe(III)) may lead to toxic levels negatively impacting Symbiodiniaceae growth and, in turn, the symbiotic relationships within the coral host (Brown, 1989; Leigh-Smith et al., 2018). Moreover, our findings indicate that \u003cem\u003eSymbiodinium\u003c/em\u003e sp. can produce higher levels of scytonemin when cells are grown in 100 nM Fe(III). The biosynthesis of scytonemin in \u003cem\u003eSymbiodinium\u003c/em\u003e sp. is influenced by osmotic and oxidative stress (Dillon et al., 2002; Liu et al., 2018), potentially serving as an indicator of stress for cells grown under high Fe conditions. This could explain why moderate levels of 50 nM Fe(III) are more favorable, providing sufficient Fe for pigment production, while supporting the development of cellular content without reaching toxic levels (as observed in this study).\u003c/p\u003e \u003cp\u003eNon-destructive label-free tomographic imaging represents an advanced tool for investigating the morphology and cellular content of live single cells, such as \u003cem\u003eSymbiodinium\u003c/em\u003e sp. Previous studies have demonstrated its capability in visualizing lipids within cells and delineating cell morphologies of microalgal cells, showing comparability with traditional staining techniques. Recent advancements in holotomography have further expanded the potential of this approach by enabling the study of subcellular components, such as lipid droplets without the need for fixation or staining methods (Kim et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This offers researchers the opportunity to investigate cellular structures and contents \u003cem\u003ein-vivo\u003c/em\u003e providing insights into dynamic cellular processes that conventional staining techniques may not cover. Using non-destructive holotomography, we measured higher lipid content (+\u0026thinsp;57%), refractive index (+\u0026thinsp;0.35%), and protein concentration (+\u0026thinsp;9%) when \u003cem\u003eSymbiodinium sp.\u003c/em\u003e was exposed to 50 nM Fe(III), indicating a physiological response to moderate iron concentrations by increasing cellular content. We speculate that moderate levels of Fe enhance the metabolic activity of \u003cem\u003eSymbiodinium\u003c/em\u003e sp., which leads to increased lipid production to store excess energy as a response mechanism to cellular division. This suggests that there is an optimal Fe(III) concentration in the culture medium, which leads to optimal cellular biochemical content, resulting in the highest growth rates of the \u003cem\u003eSymbiodinium\u003c/em\u003e cells. It is important to note that different \u003cem\u003eSymbiodinium\u003c/em\u003e species may exhibit distinct strategies for energy storage, reflecting their specific physiological adaptations to their respective ecological niches (Wang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The relatively higher lipid content observed in cells grown at 50 nM Fe(III) may represent a cellular response for energy storage, as observed in \u003cem\u003eSymbiodinium\u003c/em\u003e cultures and other microalgae (Roessler, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Lipids generate more energy than carbohydrates upon oxidation and can be efficiently packed into the cell, thus providing the best energy reserve for cells to return to homeostatic conditions, particularly in response to stress conditions like temperature fluctuations (Rosset et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and nutrient deprivation, such as nitrogen limitation (Jiang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In conclusion, holotomography allows real-time observation of live Symbiodiniaceae cells, providing insights into their response to environmental stressors. It enables non-invasive monitoring of cellular adaptations, such as lipid droplet formation and protein concentration via tomographic mapping of refractive index. This method can also be applied to compare different Symbiodiniaceae species, potentially revealing strategies for energy storage and environmental adaptation, which might help to explain their evolutionary success in various ecological niches.\u003c/p\u003e \u003cp\u003eImportantly, it should be noted that the Fe levels utilized in our study (0-100 nM) are higher than ranges found in natural environments. Coastal waters typically exhibit Fe levels around 14.5 nM (Sarthou \u0026amp; Jandel, 2001), whereas tropical waters often have Fe concentrations below 5 nM (GEOTRACES IDP, 2021). Moreover, our study primarily examined the short-term responses of cultured \u003cem\u003eSymbiodinium\u003c/em\u003e sp. to increasing Fe concentrations, which may not fully capture the long-term implications or acclimation potential of these organisms to fluctuating Fe levels in their natural habitats. Additionally, while specific growth rates provide valuable insights, they offer only a partial understanding of overall fitness and ecological performance. Incorporating additional metrics such as specific growth rate, nutrient uptake rates, photophysiological measurements or gene expression profiles could provide a more comprehensive understanding of the physiological responses of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. to Fe availability either in culture or \u003cem\u003ein hospite\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eMarine photosynthetic organisms are profoundly influenced by the availability of trace metals, which are fundamental for numerous physiological processes including photosynthesis, cellular homeostasis, and antioxidant enzymatic reactions. Increasing levels of dissolved iron, Fe(III), have been shown to significantly impact the growth of \u003cem\u003eSymbiodinium\u003c/em\u003e sp., while also stimulating the production of cellular lipids. However, the observed variation in pigment concentrations versus growth and reproduction at different iron concentrations in Symbiodiniaceae cultures suggests that iron availability and pigment concentration are just one facet of the complex network of factors influencing their physiology. Our study highlights the significance of iron availability as an essential trace metal in regulating the growth of \u003cem\u003eSymbiodinium\u003c/em\u003e sp. It emphasizes the necessity of considering potential interactions of trace metals when investigating the response of these organisms to environmental changes. Moreover, it highlights the need for further research to elucidate the specific mechanisms and regulatory pathways involved in \u003cem\u003eSymbiodinium\u003c/em\u003e sp. responses to iron availability. For future studies, it would be valuable to incorporate measurements e.g. of cellular scavenging of reactive oxygen species (ROS) levels under environmental stress, such as ocean warming, and under nitrogen and phosphorus limitation. Additionally, exploring the role of inorganic Fe in supporting thermo-tolerance may provide insights into the mechanisms of metal availability in the growth of Symbiodiniaceae.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Sofie Jakobsen and Jonathan Wolters for excellent technical assistance, and Dr. Stefano Amalfitano at the Water Research Institute (CNR-Italy) for discussing flow cytometry procedure and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and code for data analysis are available in the Zenodo repository at https://zenodo.org/doi/10.5281/zenodo.13845002.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the European Union (Grant Agreement no. 101062810, MedCorP; WD), the Villum Foundation (Grant no. VIL57413; MK), the Carlsberg Foundation (Grant no. CF21-0599; MK) and the Gordon and Betty Moore Foundation (Grant no. GBMF9206; https://doi.org/10.37807/GBMF9206; MK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmin, S. A., Green, D. H., Hart, M. C., K\u0026uuml;pper, F. C., Sunda, W. G., \u0026amp; Carrano, C. J. (2009). Photolysis of iron\u0026ndash;siderophore chelates promotes bacterial\u0026ndash;algal mutualism. Proceedings of the National Academy of Sciences of the United States of America, 106(40), 17071. https://doi.org/10.1073/PNAS.0905512106.\u003c/li\u003e\n\u003cli\u003eAn, J., Miao, X., Wang, L., Li, X., Liu, X., \u0026amp; Gao, H. (2021). Visualizing the integrity of chloroplast envelope by rhodamine and Nile Red staining. Frontiers in Plant Science, 12: 668.414. https://doi.org/10.3389/fpls.2021.668414.\u003c/li\u003e\n\u003cli\u003eBalk, J., \u0026amp; Schaedler, T. A. (2014). Iron cofactor assembly in plants. Annual Review of Plant Biology, 65, 125\u0026ndash;153. https://doi.org/10.1146/ANNUREV-ARPLANT-050213-035759.\u003c/li\u003e\n\u003cli\u003eBarer, R. \u0026amp; Joseph, S. (1954) Refractometry of living cells. J. Cell Sci. 95, 399\u0026ndash;423.\u003c/li\u003e\n\u003cli\u003eBennet, M., Gur, D., Yoon, J., Park, Y. K., \u0026amp; Faivre, D. (2017). A Bacteria-Based Remotely Tunable Photonic Device. Advanced Optical Materials, 5(1), 1600617. https://doi.org/10.1002/ADOM.201600617.\u003c/li\u003e\n\u003cli\u003eBlaby-Haas CE, Merchant SS (2012) The ins and outs of algal metal transport. Biochimica et Biophysica Acta (BBA) \u0026ndash; Molecular Cell Research 1823:1531\u0026ndash;1552.\u003c/li\u003e\n\u003cli\u003eCamp, E. F., Nitschke, M. R., Clases, D., Gonzalez de Vega, R., Reich, H. G., Goyen, S., \u0026amp; Suggett, D. J. (2022). Micronutrient content drives elementome variability amongst the Symbiodiniaceae. BMC Plant Biology, 22(1), 1\u0026ndash;14. https://doi.org/10.1186/S12870-022-03512-0.\u003c/li\u003e\n\u003cli\u003eChoi, W., Fang-Yen, C., Badizadegan, K., Oh, S., Lue, N., Dasari, R. R., \u0026amp; Feld, M. S. (2007). Tomographic phase microscopy. Nature Methods, 4(9), 717\u0026ndash;719. https://doi.org/10.1038/NMETH1078.\u003c/li\u003e\n\u003cli\u003eCui, G., Liew, Y.J., Konciute, M.K. et al. Nutritional control regulates symbiont proliferation and life history in coral-dinoflagellate symbiosis. BMC Biol 20, 103 (2022). https://doi.org/10.1186/s12915-022-01306-2.\u003c/li\u003e\n\u003cli\u003eDeleja, M., Paula, J. R., Repolho, T., Franzitta, M., Baptista, M., Lopes, V., Sim\u0026atilde;o, S., Fonseca, V. F., Duarte, B., \u0026amp; Rosa, R. (2022). Effects of Hypoxia on Coral Photobiology and Oxidative Stress. Biology, 11(7), 1068. https://doi.org/10.3390/BIOLOGY11071068.\u003c/li\u003e\n\u003cli\u003eDellisanti W, Zhang Q, Ferrier-Pag\u0026egrave;s C, K\u0026uuml;hl M. 2024. Contrasting effects of increasing dissolved iron on photosynthesis and O\u003csub\u003e2\u003c/sub\u003e availability in the gastric cavity of two Mediterranean corals. PeerJ 12:e17259 https://doi.org/10.7717/peerj.17259.\u003c/li\u003e\n\u003cli\u003eFalkowski, P. G., Dubinsky, Z., Muscatine, L., \u0026amp; McCloskey, L. (1993). Population Control in Symbiotic Corals. BioScience, 43(9), 606\u0026ndash;611. https://doi.org/10.2307/1312147.\u003c/li\u003e\n\u003cli\u003eFirestone D. (1999). Physical and chemical characteristics of oils, fats, and waxes. 2 edn, (AOCS press Champaign, IL).\u003c/li\u003e\n\u003cli\u003eGalotti, A., Jim\u0026eacute;nez-G\u0026oacute;mez, F., \u0026amp; Parra, G. (2020). Flow Cytometry Assessment of Microalgae Physiological Alterations under CO\u003csub\u003e2\u003c/sub\u003e Injection. Cytometry Part A, 97(11), 1136\u0026ndash;1144. https://doi.org/10.1002/cyto.a.24028.\u003c/li\u003e\n\u003cli\u003eGarrett, T. A., Schmeitzel, J. L., Klein, J. A., Hwang, J. J., \u0026amp; Schwarz, J. A. (2013). Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont. PLoS ONE, 8(3), 57975. https://doi.org/10.1371/JOURNAL.PONE.0057975.\u003c/li\u003e\n\u003cli\u003eGranados-Cifuentes, C., \u0026amp; Rodriguez-Lanetty, M. (2011). The use of high-resolution melting analysis for genotyping Symbiodinium strains: a sensitive and fast approach. Molecular Ecology Resources, 11(2), 394\u0026ndash;399. https://doi.org/10.1111/J.1755-0998.2010.02933.X.\u003c/li\u003e\n\u003cli\u003eGreenspan, P., Mayer, E. P., \u0026amp; Fowler, S. D. (1985). Nile red: a selective fluorescent stain for intracellular lipid droplets. Journal of Cell Biology, 100 (3): 965-973. https://doi.org/10.1083/jcb.100.3.965.\u003c/li\u003e\n\u003cli\u003eHabaza, M., Gilboa, B., Roichman, Y., \u0026amp; Shaked, N. T. (2015). Tomographic phase microscopy with 180 rotation of live cells in suspension by holographic optical tweezers. Optics Letters, Vol. 40, Issue 8, Pp. 1881-1884, 40(8), 1881\u0026ndash;1884. https://doi.org/10.1364/OL.40.001881.\u003c/li\u003e\n\u003cli\u003eHabaza, M., Kirschbaum, M., Guernth-Marschner, C., Dardikman, G., Barnea, I., Korenstein, R., Duschl, C., Shaked, N. T. (2017). Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation. Advanced Science, 4(2), 1600205. https://doi.org/10.1002/ADVS.201600205.\u003c/li\u003e\n\u003cli\u003eHansen, G., \u0026amp; Daugbjerg, N. (2009). \u003cem\u003eSymbiodinium natans\u003c/em\u003e sp. Nov.: A \u0026ldquo;free-living\u0026rdquo; dinoflagellate from Tenerife (northeast-atlantic ocean). Journal of Phycology, 45(1), 251\u0026ndash;263. https://doi.org/10.1111/j.1529-8817.2008.00621.x.\u003c/li\u003e\n\u003cli\u003eHogle, S. L., Barbeau, K. A., \u0026amp; Gledhill, M. (2014). Heme in the marine environment: from cells to the iron cycle. Metallomics, 6(6), 1107\u0026ndash;1120. https://doi.org/10.1039/C4MT00031E.\u003c/li\u003e\n\u003cli\u003eHume, B. C. C., Ziegler, M., Poulain, J., Pochon, X., Romac, S., Boissin, E., de Vargas, C., Planes, S., Wincker, P., \u0026amp; Voolstra, C. R. (2018). An improved primer set and amplification protocol with increased specificity and sensitivity targeting the \u003cem\u003eSymbiodinium ITS2\u003c/em\u003e region. PeerJ, 2018(5). https://doi.org/10.7717/PEERJ.4816.\u003c/li\u003e\n\u003cli\u003eHume, B. C. C., Smith, E. G., Ziegler, M., Warrington, H. J. M., Burt, J. A., LaJeunesse, T. C., Wiedenmann, J., \u0026amp; Voolstra, C. R. (2019). SymPortal: A novel analytical framework and platform for coral algal symbiont next-generation sequencing \u003cem\u003eITS2\u003c/em\u003e profiling. Molecular Ecology Resources, 19(4), 1063\u0026ndash;1080. https://doi.org/10.1111/1755-0998.13004.\u003c/li\u003e\n\u003cli\u003eIglic, Katrina Lynn, \u0026quot;Trace metal limitation and its role in oxidative stress of coral algal symbionts; implications for thermally induced coral bleaching events.\u0026quot; (2011). Electronic Thesis and Dissertation Repository. 282. https://ir.lib.uwo.ca/etd/282.\u003c/li\u003e\n\u003cli\u003eKim, T., Zhou, R., Mir, M., Babacan, S. D., Carney, P. S., Goddard, L. L., \u0026amp; Popescu, G. (2014a). White-light diffraction tomography of unlabelled live cells. Nature Photonics 2014 8:3, 8(3), 256\u0026ndash;263. https://doi.org/10.1038/nphoton.2013.350.\u003c/li\u003e\n\u003cli\u003eKim, K., Yoon, H., Diez-Silva, M., Dao, M., Dasari, R. R., \u0026amp; Park, Y. (2014b). Special Section on Advanced Biomedical Imaging and Sensing: High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography. Journal of Biomedical Optics, 19(1), 1. https://doi.org/10.1117/1.JBO.19.1.011005.\u003c/li\u003e\n\u003cli\u003eKim, Y., Shim, H., Kim, K., Park, H. J., Jang, S., \u0026amp; Park, Y. K. (2014c). Profiling individual human red blood cells using common-path diffraction optical tomography. Scientific Reports, 4(1), 1\u0026ndash;7. https://doi.org/10.1038/srep06659.\u003c/li\u003e\n\u003cli\u003eKim, K., Lee, S., Yoon, J., Heo, J., Choi, C., \u0026amp; Park, Y. (2016). Three-dimensional label-free imaging and quantification of lipid droplets in live hepatocytes. Scientific Reports 2016 6:1, 6(1), 1\u0026ndash;8. https://doi.org/10.1038/srep36815.\u003c/li\u003e\n\u003cli\u003eKim, H., Oh, S., Lee, S., Lee, K. suk, \u0026amp; Park, Y. (2024). Recent advances in label-free imaging and quantification techniques for the study of lipid droplets in cells. Current Opinion in Cell Biology, 87, 102342. https://doi.org/10.1016/J.CEB.2024.102342.\u003c/li\u003e\n\u003cli\u003eKneeland, J., Hughen, K., Cervino, J., Hauff, B., \u0026amp; Eglinton, T. (2013). Lipid biomarkers in Symbiodinium dinoflagellates: New indicators of thermal stress. Coral Reefs, 32(4), 923\u0026ndash;934. https://doi.org/10.1007/S00338-013-1076-3.\u003c/li\u003e\n\u003cli\u003eKoch, F., \u0026amp; Trimborn, S. (2019). Limitation by fe, zn, co, and b12 results in similar physiological responses in two antarctic phytoplankton species. Frontiers in Marine Science, 6(AUG), 469522. https://doi.org/10.3389/FMARS.2019.00514.\u003c/li\u003e\n\u003cli\u003eJiang, P. L., Pasaribu, B., \u0026amp; Chen, C. S. (2014). Nitrogen-Deprivation Elevates Lipid Levels in \u003cem\u003eSymbiodinium\u003c/em\u003e spp. by Lipid Droplet Accumulation: Morphological and Compositional Analyses. PLOS ONE, 9(1), e87416. https://doi.org/10.1371/JOURNAL.PONE.0087416.\u003c/li\u003e\n\u003cli\u003eJung, J. H., Hong, S. J., Kim, H. B., Kim, G., Lee, M., Shin, S., Lee, S. Y., Kim, D. J., Lee, C. G., \u0026amp; Park, Y. K. (2018). Label-free non-invasive quantitative measurement of lipid contents in individual microalgal cells using refractive index tomography. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-24393-0.\u003c/li\u003e\n\u003cli\u003eLee, K. R., Kim, K., Jung, J., Heo, J. H., Cho, S., Lee, S., Chang, G., Jo, Y. J., Park, H., \u0026amp; Park, Y. K. (2013). Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications. Sensors 2013, Vol. 13, Pages 4170-4191, 13(4), 4170\u0026ndash;4191. https://doi.org/10.3390/S130404170.\u003c/li\u003e\n\u003cli\u003eLee, S., Lee, S., Kim, K., Mubarok, A., Park, Y., Panduwirawan, A., Park, H., \u0026amp; Lee, K. (2014). High-Resolution 3-D Refractive Index Tomography and 2-D Synthetic Aperture Imaging of Live Phytoplankton. Journal of the Optical Society of Korea, Vol. 18, Issue 6, Pp. 691-697, 18(6), 691\u0026ndash;697. https://doi.org/10.3807/JOSK.2014.18.6.691.\u003c/li\u003e\n\u003cli\u003eLee, E., Jalalizadeh, M., \u0026amp; Zhang, Q. (2015). Growth kinetic models for microalgae cultivation: A review. Algal Research, 12, 497\u0026ndash;512. https://doi.org/10.1016/J.ALGAL.2015.10.004\u003c/li\u003e\n\u003cli\u003eLichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology, Volume 148, 1987, Pages 350-382.\u003c/li\u003e\n\u003cli\u003eLiu, Z. Y., Wang, G. C., \u0026amp; Zhou, B. C. (2008). Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresource Technology, 99(11), 4717\u0026ndash;4722. https://doi.org/10.1016/J.BIORTECH.2007.09.073.\u003c/li\u003e\n\u003cli\u003eMashaghi, A., Swann, M., Popplewell, J., Textor, M., \u0026amp; Reimhult, E. (2008). Optical Anisotropy of Supported Lipid Structures Probed by Waveguide Spectroscopy and Its Application to Study of Supported Lipid Bilayer Formation Kinetics. Analytical Chemistry, 80(13), 5276. https://doi.org/10.1021/AC800988V.\u003c/li\u003e\n\u003cli\u003eMatthews, J. L., Hoch, L., Raina, J. B., Pablo, M., Hughes, D. J., Camp, E. F., Seymour, J. R., Ralph, P. J., Suggett, D. J., \u0026amp; Herdean, A. (2023a). Symbiodiniaceae photophysiology and stress resilience is enhanced by microbial associations. Scientific Reports 2023 13:1, 13(1), 1\u0026ndash;13. https://doi.org/10.1038/s41598-023-48020-9.\u003c/li\u003e\n\u003cli\u003eMatthews, J. L., Khalil, A., Siboni, N., Bougoure, J., Guagliardo, P., Kuzhiumparambil, U., DeMaere, M., le Reun, N. M., Seymour, J. R., Suggett, D. J., \u0026amp; Raina, J. B. (2023b). Coral endosymbiont growth is enhanced by metabolic interactions with bacteria. Nature Communications 2023 14:1, 14(1), 1\u0026ndash;13. https://doi.org/10.1038/s41467-023-42663-y.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller, B. (2023). Iron transport mechanisms and their evolution focusing on chloroplasts. Journal of Plant Physiology, 288, 154059. https://doi.org/10.1016/J.JPLPH.2023.154059.\u003c/li\u003e\n\u003cli\u003ePark, S., Ahn, J. W., Jo, Y., Kang, H. Y., Kim, H. J., Cheon, Y., Kim, J. W., Park, Y., Lee, S., \u0026amp; Park, K. (2020). Label-Free Tomographic Imaging of Lipid Droplets in Foam Cells for Machine-Learning-Assisted Therapeutic Evaluation of Targeted Nanodrugs. ACS Nano, 14(2), 1856\u0026ndash;1865. https://doi.org/10.1021/ACSNANO.9B07993.\u003c/li\u003e\n\u003cli\u003ePasaribu, B., Primadona Purba, N., Dewanti, L. P., Pasaribu, D., Muhammad, A., Khan, A., Harahap, S. A., Syamsuddin, M. L., Ihsan, Y. N., Siregar, S. H., Faizal, I., Herawati, T., Irfan, M., Partogi, T., Simorangkir, H., \u0026amp; Kurniawan, T. A. (2024). Lipid Droplets in Endosymbiotic Symbiodiniaceae spp. Associated with Corals. Plants 2024, Vol. 13, Page 949, 13(7), 949. https://doi.org/10.3390/PLANTS13070949.\u003c/li\u003e\n\u003cli\u003ePatton, J. S., \u0026amp; Burris, J. E. (1983). Lipid synthesis and extrusion by freshly isolated zooxanthellae (symbiotic algae). Marine Biology, 75(2\u0026ndash;3), 131\u0026ndash;136. https://doi.org/10.1007/BF00405995.\u003c/li\u003e\n\u003cli\u003ePeter, M., \u0026amp; Herskowitz, I. (1994). Joining the complex: Cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell, 79, 181\u0026ndash;184.\u003c/li\u003e\n\u003cli\u003ePopescu, G. 2011. Quantitative Phase Imaging of Cells and Tissues. 1st ed. New York: McGraw-Hill. https://www.accessengineeringlibrary.com/content/book/9780071663427.\u003c/li\u003e\n\u003cli\u003ePushnik, J. C., Miller, G. W., \u0026amp; Manwaring, J. H. (1984). The role of iron in higher plant chlorophyll biosynthesis, maintenance and chloroplast biogenesis. Journal of Plant Nutrition, 7(1-5), 733\u0026ndash;758. https://doi.org/10.1080/01904168409363238\u003c/li\u003e\n\u003cli\u003eR Core Team (2016) R: A language and environment for statistical computing [Computer software]. R Foundation for Statistical Computing, Vienna.\u003c/li\u003e\n\u003cli\u003eRai, S., Singh, P. K., Mankotia, S., Swain, J., \u0026amp; Satbhai, S. B. (2021). Iron homeostasis in plants and its crosstalk with copper, zinc, and manganese. Plant Stress, 1, 100008. https://doi.org/10.1016/J.STRESS.2021.100008.\u003c/li\u003e\n\u003cli\u003eRappaz, B., Cano, E., Colomb, T., Kuhn, J., Depeursinge, C. D., Simanis, V., Magistretti, P. J., \u0026amp; Marquet, P. P. (2009). Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy. Journal of Biomedical Optics, 14:3, 034049. https://doi.org/10.1117/1.3147385.\u003c/li\u003e\n\u003cli\u003eRaven, J. A., Evans, M. C. W., \u0026amp; Korb, R. E. (1999). The role of trace metals in photosynthetic electron transport in O2-evolving organisms. Photosynthesis Research, 60(2\u0026ndash;3), 111\u0026ndash;150. https://doi.org/10.1023/A:1006282714942.\u003c/li\u003e\n\u003cli\u003eReich, H. G., Rodriguez, I. B., LaJeunesse, T. C., \u0026amp; Ho, T. Y. (2020). Endosymbiotic dinoflagellates pump iron: differences in iron and other trace metal needs among the Symbiodiniaceae. Coral Reefs, 39(4), 915\u0026ndash;927. https://doi.org/10.1007/s00338-020-01911-z.\u003c/li\u003e\n\u003cli\u003eReich, H. G., Camp, E. F., Roger, L. M., \u0026amp; Putnam, H. M. (2023). The trace metal economy of the coral holobiont: supplies, demands and exchanges. Biological Reviews, 98(2), 623\u0026ndash;642. https://doi.org/10.1111/brv.12922.\u003c/li\u003e\n\u003cli\u003eRodriguez, I. B., \u0026amp; Ho, T. Y. (2018). Trace metal requirements and interactions in \u003cem\u003eSymbiodinium kawagutii\u003c/em\u003e. Frontiers in Microbiology, 310350. https://doi.org/10.3389/FMICB.2018.00142.\u003c/li\u003e\n\u003cli\u003eRomero, J. M. D., Botana, M. T., Elias, A. de C., Nomura, C. S., Saldanha-Corr\u0026ecirc;a, F., \u0026amp; Esp\u0026oacute;sito, B. P. (2022). Effect of iron speciation on growth and heat resistance of Symbiodiniaceae. Ocean and Coastal Research, 70, e22016. https://doi.org/10.1590/2675-2824070.21103JMDR.\u003c/li\u003e\n\u003cli\u003eRoessler, P. G. (1990). Environmental Control of Glycerolipid Metabolism in Microalgae: Commercial Implications and Future Research Directions. Journal of Phycology, 26(3), 393\u0026ndash;399. https://doi.org/10.1111/J.0022-3646.1990.00393.X.\u003c/li\u003e\n\u003cli\u003eRosset, S., Koster, G., Brandsma, J., Hunt, A. N., Postle, A. D. \u0026amp; D\u0026rsquo;Angelo, C. (2019). Lipidome analysis of Symbiodiniaceae reveals possible mechanisms of heat stress tolerance in reef coral symbionts. Coral Reefs 38, 1241\u0026ndash;1253. https://doi.org/10.1007/s00338-019-01865-x.\u003c/li\u003e\n\u003cli\u003eSandy, M., \u0026amp; Butler, A. (2009). Microbial Iron Acquisition: Marine and Terrestrial Siderophores. Chemical Reviews, 109(10), 4580. https://doi.org/10.1021/CR9002787.\u003c/li\u003e\n\u003cli\u003eSmith SR, Gillard JTF, Kustka AB, McCrow JP, Badger JH, et al. (2017) Correction: Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation. PLOS Genetics 13(3): e1006688. https://doi.org/10.1371/journal.pgen.1006688.\u003c/li\u003e\n\u003cli\u003eStorms, Z. J., Cameron, E., de la Hoz Siegler, H., \u0026amp; McCaffrey, W. C. (2014). A simple and rapid protocol for measuring neutral lipids in algal cells using fluorescence. Journal of Visualized Experiments, 87. https://doi.org/10.3791/51441.\u003c/li\u003e\n\u003cli\u003eSun, X. M., Ren, L. J., Zhao, Q. Y., Ji, X. J., \u0026amp; Huang, H. (2018). Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation. Biotechnology for Biofuels 2018 11:1, 11(1), 1\u0026ndash;16. https://doi.org/10.1186/S13068-018-1275-9.\u003c/li\u003e\n\u003cli\u003eSunda, W., Huntsman, S. Interrelated influence of iron, light and cell size on marine phytoplankton growth. Nature 390, 389\u0026ndash;392 (1997). https://doi.org/10.1038/37093.\u003c/li\u003e\n\u003cli\u003eTchernov, D., Gorbunov, M. Y., de Vargas, C., Yadav, S. N., Milligant, A. J., H\u0026auml;ggblom, M., \u0026amp; Falkowski, P. G. (2004). Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals. Proceedings of the National Academy of Sciences of the United States of America, 101(37), 13531\u0026ndash;13535. https://doi.org/10.1073/PNAS.0402907101.\u003c/li\u003e\n\u003cli\u003eUllmann, F., Gerhartz, W., Yamamoto, S. Y., Campbell, F. T., Pfefferkorn, R. \u0026amp; Rounsaville, J. F. Ullmann\u0026rsquo;s encyclopedia of industrial chemistry. Vol. 5 (Vch Weinheim, Germany, 1985).\u003c/li\u003e\n\u003cli\u003eWang, L. H., Chen, H. K., Jhu, C. S., Cheng, J. O., Fang, L. S., \u0026amp; Chen, C. S. (2015). Different strategies of energy storage in cultured and freshly isolated \u003cem\u003eSymbiodinium\u003c/em\u003e sp. Journal of Phycology, 51(6), 1127\u0026ndash;1136. https://doi.org/10.1111/JPY.12349.\u003c/li\u003e\n\u003cli\u003eWang, H., Su, Q., Zhuang, Y., Wu, C., Tong, S., Guan, B., Zhao, Y., \u0026amp; Qiao, H. (2023). Effects of Iron Valence on the Growth, Photosynthesis, and Fatty Acid Composition of \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e. Journal of Marine Science and Engineering 2023, Vol. 11, Page 316, 11(2), 316. https://doi.org/10.3390/JMSE11020316.\u003c/li\u003e\n\u003cli\u003eWestall, J. C., Zachary, J. L. \u0026amp; Morel, F. M. M. (1976) A computer program for the calculation of chemical equilibrium composition of aqueous systems, Civil Eng. Tech. Note 18. Cambridge: Massachusetts Institute of Technology.\u003c/li\u003e\n\u003cli\u003eWickham H (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org.\u003c/li\u003e\n\u003cli\u003eWickham H, Fran\u0026ccedil;ois R, Henry L, M\u0026uuml;ller K, Vaughan D (2023). dplyr: A Grammar of Data Manipulation. https://dplyr.tidyverse.org, https://github.com/tidyverse/dplyr.\u003c/li\u003e\n\u003cli\u003eWietheger, A., Starzak, D. E., Gould, K. S., \u0026amp; Davy, S. K. (2018). Differential ROS Generation in Response to Stress in \u003cem\u003eSymbiodinium\u003c/em\u003e spp. The Biological Bulletin, 234(1), 11\u0026ndash;21. https://doi.org/10.1086/696977. \u003c/li\u003e\n\u003cli\u003eYadavalli, V., Jolley, C. C., Malleda, C., Thangaraj, B., Fromme, P., \u0026amp; Subramanyam, R. (2012). Alteration of Proteins and Pigments Influence the Function of Photosystem I under Iron Deficiency from \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. PLoS ONE, 7(4). https://doi.org/10.1371/JOURNAL.PONE.0035084.\u003c/li\u003e\n\u003cli\u003eZhou, Y., Liu, L., Li, M., \u0026amp; Hu, C. (2022). Algal biomass valorisation to high-value chemicals and bioproducts: Recent advances, opportunities and challenges. \u003cem\u003eBioresource Technology\u003c/em\u003e, \u003cem\u003e344\u003c/em\u003e, 126371. https://doi.org/10.1016/J.BIORTECH.2021.126371. \u003c/li\u003e\n\u003cli\u003eZigmantas, D., Hiller, R. G., Sundstr\u0026ouml;m, V., \u0026amp; Pol\u0026iacute;vka, T. (2002). Carotenoid to chlorophyll energy transfer in the peridinin-chlorophyll-a-protein complex involves an intramolecular charge transfer state. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e, \u003cem\u003e99\u003c/em\u003e(26), 16760\u0026ndash;16765. https://doi.org/10.1073/PNAS.262537599. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"081cdd96-f084-4a2c-ab84-795cf618b541","identifier":"10.13039/501100000780","name":"European Commission","awardNumber":"101062810","order_by":0},{"identity":"1d978e05-694a-41fc-9e6f-9494308574c5","identifier":"10.13039/100008398","name":"Villum Fonden","awardNumber":"VIL57413","order_by":1},{"identity":"0221f3b6-7fbc-4b2d-ae64-1a43640fbfb9","identifier":"10.13039/501100002808","name":"Carlsbergfondet","awardNumber":"CF21-0599","order_by":2},{"identity":"3fd8a839-8fff-4985-b3a0-673f3715fef1","identifier":"10.13039/100000936","name":"Gordon and Betty Moore Foundation","awardNumber":"GBMF9206","order_by":3}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Copenhagen","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"symbiodinium, cell development, in vivo, iron enrichment","lastPublishedDoi":"10.21203/rs.3.rs-5160718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5160718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDinoflagellates in the family Symbiodiniaceae are fundamental in coral reef ecosystems and facilitate essential processes such as photosynthesis, nutrient cycling, and calcium carbonate production. Iron (Fe) is an essential element for the physiological processes of Symbiodiniaceae, yet its role remains poorly understood in the context of cellular development and metabolic health. Here, we investigated the effect of iron availability (0 to 100 nM Fe(III)) on \u003cem\u003eSymbiodinium\u003c/em\u003esp. ITS2 type A1 cultures and quantified cellular content using flow cytometry and holotomography. Moderate levels of dissolved Fe (50 nM) enhanced growth rates and cellular content development in \u003cem\u003eSymbiodinium\u003c/em\u003e sp., including lipids and proteins. We observed distinct growth patterns, pigment concentrations, and cellular morphology under increasing Fe concentrations, indicating the influence of iron availability on cellular physiology. Non-destructive, label-free holotomographic microscopy enabled single cell \u003cem\u003ein vivo\u003c/em\u003e revealing higher intracellular lipid accumulation (+57%) in response to 50 nM Fe(III) enrichment. Our findings contribute to a deeper understanding of the relationship between iron availability and \u003cem\u003eSymbiodinium\u003c/em\u003esp. growth and cellular development, with potential implications for coral health and reef resilience in the face of environmental stressors.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*Walter Dellisanti and Swathi Murthy contributed equally to this manuscript.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Moderate levels of dissolved iron stimulate cellular growth and increase lipid storage in Symbiodinium sp.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-30 08:12:23","doi":"10.21203/rs.3.rs-5160718/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"78b8bb5d-6b8a-45c1-a953-f667d3bb31c6","owner":[],"postedDate":"September 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38256155,"name":"Marine and Freshwater Biology"}],"tags":[],"updatedAt":"2024-09-30T08:12:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-30 08:12:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5160718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5160718","identity":"rs-5160718","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}