Abstract
Symbiodiniaceae are a diverse group of dinoflagellates, the majority of which are free-living and/or associated with a variety of protists and other invertebrate hosts. Maintenance of isolated cultures is labour-intensive and expensive, and cryopreservation provides an excellent avenue for their long-term storage. We aimed to cryopreserve 15 cultured isolates from six Symbiodiniaceae genera using dimethyl sulfoxide (DMSO) as the cryoprotectant agent (CPA). Under 15% DMSO, 10 isolates were successfully cryopreserved using either rapid freezing or controlled-rate freezing. Cultures that failed or had low survival, were subjected to (1) a reduction of CPA to 10%, or (2) increased salinity treatment before freezing. At 10% DMSO, three further isolates were successfully cryopreserved. At 15% DMSO there were high cell viabilities in Symbiodinium pilosum treated with 44 parts per thousand (ppt) and 54 ppt culture medium. An isolate of Fugacium sp. successfully cryopreserved after salinity treatments of 54 ppt and 64 ppt. Fatty acid (FA) analyses of S. pilosum after 54 ppt salinity treatment showed increased saturated FA levels, whereas Fugacium sp. had low poly-unsaturated FAs compared to normal salinity (34 ppt). Understanding the effects of salinity and roles of FAs in cryopreservation will help in developing protocols for these ecologically important taxa.
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Introduction
Dinoflagellate species belonging to the family Symbiodiniaceae often exist in endosymbiotic relationships with invertebrates including corals, sea anemones, jellyfish, giant clams, flatworms and benthic foraminifera1. Some species also exist as free-living phytoplankton in coastal environments2. Endosymbiotic Symbiodiniaceae are crucial for supporting the growth, survival and metabolic processes of their hosts by providing photosynthetically fixed carbon in the form of carbohydrates, sugars, starches, and amino acids3. Their symbiosis with stony corals is fundamental to the formation and existence of healthy coral reef ecosystems4. Symbiodiniaceae are genetically diverse and have historically been grouped into a number of phylogenetic lineages, i.e. clades A–J5,6 and numerous sub-clade types or species7. However, the recent taxonomic revision of the family Symbiodiniaceae has enabled the formal description of eleven genera8 each containing a variety of Symbiodiniaceae species with distinct ecological attributes.
Many dinoflagellate species are maintained in living culture collections. This requires continuous sub-culturing that is costly and risks strain loss, contamination, and changes in genetic integrity9,10. Cryopreservation, the process of preserving viable cells, tissues or microorganisms in a frozen state over extended periods of time, provides an opportunity to overcome these challenges11. Successful cryopreservation depends on the application of appropriate cryoprotectant agents (CPAs), freezing rates, osmotic balance and ice nucleation processes12. In marine microalgae, CPAs such as glycerol, dimethyl sulfoxide (DMSO) and methanol are added to avoid cellular damage due to ice formation during freezing9,13. The optimal CPA, equilibration time, type of freezing rates used, and thawing temperature varies between species10,14,15.
Successful cryopreservation of symbiotic dinoflagellates will provide a resource that can be used in research and the conservation of coral reefs16. Use of different cryopreservation techniques and appropriate amount of cryoprotectants have led to high cell survival of the Breviolum sp. 10. For Symbiodiniaceae species, successful cryopreservation has been reported in these genera: Durusdinium17,18,19, Breviolum10,14,19,20, Gerakladium15,19, Fugacium and Cladocopium19. However, there is a need to develop robust cryopreservation protocols for various species or sub-clades and other Symbiodiniaceae genera.
The impact of increased salinity treatment in microalgal cryopreservation is an avenue that has not been explored in sufficient detail. High salinity of the culture medium causes water to move out of the cells by osmosis facilitating dehydration during the freezing process21. In high salinity media, microalgae can accumulate lipids as the cells switch from active cell division to the storage of energy and these changes in production of lipids differ among species22. Manipulating salinity conditions prior to cryopreservation has been shown to assist in the successful freezing of some microalgal species21.
Many marine microalgal species can tolerate great variations of salinity, and their chemical and fatty acid (FA) composition can vary in response to such fluctuations23. The main roles of FAs in algae are in the maintenance of cell membrane functions, structure and metabolic processes24. Poly-unsaturated fatty acids (PUFAs) play important roles as structural components of membrane phospholipids24,25. Living organisms maintain their membranes in a semi-fluid state at the growth temperature by regulating the degree of unsaturation of the membrane lipids26. The plasma membrane is the primary site where considerable damage occurs as a result of chilling injury during freezing27. The saturated fatty acids (SFAs) help to reduce the fluidity and permeability of the cell membranes which regulates the flow of substances in and out of the cells24. This is due to the physical properties of these FAs compared to other FA classes28. However, depending on culture conditions, increasing salinity in some microalgal species outside their optimal salinity range can lead to a reduction in biomass productivity that off-sets the high lipid productivity29. However, changes in FA profiles amongst distinct Symbiodiniaceae genera grown in both normal and increased salinity media before cryopreservation have not been investigated.
The aim of the present study was to explore whether a method originally optimized during the cryopreservation of a Breviolum sp.10 could be applied to a range of other Symbiodiniaceae genera and whether increased salinity treatment enhances cryopreservation success in the recalcitrant cultures. It was hypothesized that: [a] Most Symbiodiniaceae cultured isolates will be successfully cryopreserved using the previously optimized protocol10 with DMSO as the CPA, and [b] Survival will be enhanced in selected culture isolates when grown in a high salinity medium before treatment with DMSO due to the increased dehydration of cells that results in changes in the amount and types of FA produced. Investigations of different approaches that lead to high cell viabilities during cryopreservation will help in establishing a robust freezing protocol for multiple Symbiodiniaceae species. The long-term preservation of these species will facilitate future research on their genetic capacities, life cycles, secondary metabolite production, and enhance knowledge on their endosymbiotic relationships.
Materials and methods
Cultivation conditions of the culture isolates
Normal salinity
All culture isolates from the family Symbiodiniaceae utilized in this study were provided by the Marine Symbiosis and Coral Reef Biology Laboratory at Victoria University of Wellington (New Zealand) and Cawthron Institute (CICCM, New Zealand)30. Twelve culture isolates were cultured in f/2 growth medium31 and the other three culture isolates in f/2/L1 growth medium31,32 as shown in Table 1. For all the initial experiments, the salinity of both f/2 and f/2/L1 growth media was maintained at normal salinity (34 ppt). All the culture isolates were grown and maintained in sterile plastic flatbottomed vessels (70 mL Labserv, Thermofisher scientific NZ) with a 12:12 light: dark cycle under 100 µmol m−2 s−1 photosynthetically active radiation light at 25 °C. All culture isolates were harvested during their late exponential phase prior to DNA extraction, cryopreservation, and FA extraction experiments.
High salinity treatments
From our study cultures, five isolates from the genus Symbiodinium; S. microadriaticum [ITS-2 type A1], S. pilosum [A2], S. tridacnidorum [A3], S. tridacnidorum [A6] and S. necroappetens [A13] and one isolate from the genus Fugacium (Fugacium sp. [F5.2]), were subjected to higher salinity treatments. All of these Symbiodinium isolates had failed to survive after thawing during the initial cryopreservation experiments using 15% DMSO while Fugacium sp. [F5.2] had very low cell viability after thawing. The salinity of the f/2 and f/2/L1 media was increased by adding appropriate amounts of NaCl (Emsure, Denmark) to reach either 44 parts per thousand (ppt), 54 ppt or 64 ppt as described by van der Merwe et al.33. These culture isolates were then harvested during their late exponential phase for cryopreservation experiments.
DNA extraction, Polymerase chain reaction and DNA sequencing
Due to the complex diversity of the family Symbiodiniaceae, a phylogenetic analysis of the nuclear ribosomal large subunit (28S) rRNA and the nuclear ribosomal Internal Transcribed Spacer region 2 (ITS-2) rRNA was undertaken to identify all cultures isolates investigated. Molecular analyses (DNA extraction, Polymerase Chain Reaction [PCR] set-up, template addition and PCR amplification) were conducted under sterile conditions with sequential workflow to ensure there was no contamination. DNA extraction, PCR set-up and template addition was carried out in separate rooms equipped with ultra-violet sterilisation.
For each Symbiodiniaceae strain, a subsample (20 mL) was taken from a densely grown culture and transferred to a 50 mL centrifuge tube (Corning CentriStar, China) and centrifuged (3000 × g, 10 min). The supernatant was carefully discarded, and 1 mL of the dense culture isolate was transferred into a sterile 1.7 mL microtube (Axygen, Wujiang, China). The concentrated culture isolate was then centrifuged (3000 × g, 10 min) and all the supernatant discarded. The isolated pellet was stored in − 20 °C before DNA extraction. The pellet was lysed manually using a bead beater machine (1600 MiniG Spex SamplePrep, New Jersey, United States) and DNA was extracted using the DNeasy PowerSoil Kit (QIAGEN, Hilden, Germany) following the protocol from the manufacturer.
Two markers were selected for PCR and sequencing analyses: the 28S rRNA gene using forward LSU1F primer 5′-GCG GAG GAA AAG RAA CTA A-3′34 and reverse LO primer 5′-GCT ATC CTG AGR GAA ACT TCG-3′35 and the ITS-2 rRNA region using forward primer 5′-GTG AAT TGC AGA ACT CCG TG-3′36 and ITS-2-rev2 reverse primer 5′-GCC TCC GCT TAC TTA TAT GCT T-3′37. Polymerase chain reactions were performed in 50 µL reaction volume for each sample with the reaction mixture containing 25 µL of 2× PCR MyFi Mix (Bioline, London, UK), 21 µL of nuclease free water (AM9937; Ambion, CA, USA), 1 µL (10 µM) of each primer, 1 µL of bovine serum albumin (BSA, 20 mg mL−1, Sigma-Aldrich, Auckland, New Zealand) and 1 µL of template DNA. The PCR cycling conditions were held at 94 °C for 3 min, followed by 35 cycles of 94 °C for 20 s, 52 °C for 20 s, 72 °C for 30 s and finally, an extension step of 5 min at 72 °C. During the PCR reaction, negative PCR controls containing nuclease free water as a template were run alongside the samples. All the amplified products were then purified using NucleoSpin Gel and PCR clean up kits (Macherey–Nagel, Düren, Germany) and sent to the Genetics Analysis Services (Dunedin, New Zealand) for direct bi-directional Sanger sequencing.
Phylogenetic analyses of the culture isolates
Forward and reverse sequences were inspected and aligned to generate consensus sequences using Geneious Prime v11.0.11 (Biomatters, Auckland, New Zealand). Consensus 28S rDNA sequences (n = 15) were manually aligned to an existing alignment dataset38 before performing a ClustalW alignment on the final dataset using BioEdit v7.2.0.39. Three additional 28S sequences from the recently described Symbiodiniaceae clade J6, were also incorporated into the alignment. To estimate the best-fit model of evolution and Maximum-likelihood (ML), all the analyses were performed with the use of Mega X v10.1.840. The initial trees for the heuristic search were obtained automatically by applying both Neighbor-Join and BioNJ algorithms to a matrix of pairwise distance estimated using Tamura-Nei model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories; + G parameter = 0.3631). The reliability of internal branches was assessed using 100 bootstraps method41.
Consensus ITS-2 sequences (n = 15) enabled identification of Symbiodiniaceae culture isolates at finer taxonomic level (type or species) using BLASTn in GenBank. Accession numbers of all sequences produced in this study are shown in Table 1.
Cryoprotectant agent
The CPA used during freezing experiments was DMSO (≥ 99.9% pure, Sigma-Aldrich, France). CPA solutions were prepared at double the target concentration in sterilized appropriate growth medium for each culture and added to the cultures in a 1:1 ratio to give final concentrations of 10% and 15% (v/v) of the CPA.
Cryopreservation experiments
Two different concentrations of DMSO and two freezing methods were used for 15 cultured isolates from the family Symbiodiniaceae. The cultures that did not cryopreserve or had a low post-thaw viability were subsequently grown in media with increased salinity to explore its effect during their cryopreservation. The FA profiles of all the culture isolates grown in normal salinity and the isolates that had high cell viabilities post-thaw after being treated with high salinity were analysed and compared to explore whether increased salinity contributes to cryopreservation success.
Three experiments were done: Part A: Cryopreservation experiments with a final concentration of 15% DMSO with normal salinity culture isolates, Part B: Cryopreservation experiments using a final concentration of 10% DMSO for the culture isolates that failed to survive and had low cell viabilities post-thawing (< 15%) in part A and lastly Part C: Cryopreservation experiments using a final concentration of 15% DMSO and high salinity treated culture isolates that failed to survive and had low cell viabilities post-thawing (< 15%) in part A.
During these experiments, aliquots (1 mL) of each culture in their late exponential phase were pipetted into separate 5 mL sterile glass test tubes in triplicates in a laminar flow cabinet. For each culture, 100 µL aliquots of appropriately diluted at double the desired final concentration of DMSO was added to the tubes every 1 min with gentle agitation after each addition up to the 10th min (RT, 30 µmol m−2 s−1 PAR) until a final 1:1 dilution of culture medium to CPA was obtained10. After the last addition, the glass tubes were stoppered and incubated in the dark (30 min). These cultures were aspirated into cryopreservation straws (0.5 cc, IMV, France), plugged with coloured polyvinyl chloride powder, and placed in water (20 °C) to set the powder. The straws were then wiped dry before the freezing procedure.
Two main cryopreservation methods were applied as detailed in Kihika et al. 10.
-
(a)
Rapid freezing method: The straws containing the DMSO treated cultures isolates were arranged horizontally on a metal rack fitted onto polystyrene floats measuring (41 × 14 × 4 cm, l × w × h)10. Each rack was placed over the liquid nitrogen bath (45 × 30 × 6 cm, l × w × h) for 10 min before the straws were plunged into liquid nitrogen10.
-
(b)
Controlled-rate freezing method: The straws were placed in a controlled-rate freezer (Cryologic Pty, Mt Waverley, Australia) which cooled the culture isolates from 20 to − 40 °C at a rate of 1 °C min−1. The straws were maintained at − 40 °C for 10 min before plunging into liquid nitrogen10.
In both cryopreservation methods, the cooling rate difference was 10 min for rapid freezing and 1 h 30 min for the controlled rate freezer. All the frozen straws were stored in a dewar containing liquid nitrogen for one week.
Thawing and culture isolates recovery
After storage, triplicate straws of each culture isolate were retrieved and thawed by rapidly transferring into a water bath (20 °C) for 2 min until all visible ice melted. A tissue moistened with 70% ethanol was used to wipe the straws dry10. The straw contents were transferred to a sterile plastic flatbottomed vessel and individually diluted by stepwise addition of 500 µL of their growth medium at normal salinity (34 ppt), each minute for 10 min (total volume 5 mL)10. The culture isolates that had been treated with higher salinities were grown in normal salinity growth media after thawing. All culture isolates were incubated in the dark (30 min) to equilibrate before adding a final volume of 5 mL of their respective growth medium. The culture isolates were transferred to dark conditions at ⁓ 20 °C for 24 h, followed by a further 48 h under 27 µmol m−2 s−1 red light (OSRAM L18W/60, Germany) to facilitate the recovery of the dinoflagellate cells10. Finally, growth media (~ 40 mL) was added to the recovering culture isolates, which were then incubated in their normal standard light growing conditions.
Cell viability assessment tests
A viability test was conducted for each cultured strain after the recovery phase on the third day to determine cell survival after freezing. The cells were carefully resuspended in the plastic flatbottomed vessel before cell counting was done10. A subsample (1 mL) of the cell culture was taken and an appropriate serial dilution was made following the method used by Kihika et al. 10. Lugol’s iodine (10 µL) was added to the diluted culture to fix the living cells for easier counting under a microscope. The diluted subsample (100 µL) was placed on a glass slide in triplicate and the fixed cells allowed to settle for 30 min10. An inverted microscope was used to enumerate viable fixed cells (stained dark brown), and dead cells (colorless)10.
Fatty acids (FAs) extraction and identification
The FAs were extracted and analysed for all the culture isolates grown in normal salinity. A further two culture isolates grown at specific high salinities were also tested; S. pilosum [A2] was grown in two different salinity treatments of both 44 ppt and 54 ppt, and Fugacium sp. [F5.2] was grown in 54 ppt salinity. These culture isolates and treatments were selected for FA analysis because cells had successfully cryopreserved with viabilities of (> 50%). No culture isolate growing at 64 ppt salinity was selected for FA analysis due to low viability or lack of survival post-thawing. All the FAs were extracted from these culture isolates before cryopreservation experiments. All the culture isolates were grown in Erlenmeyer flask (500 mL) before harvesting for FAs extraction. The culture isolates were then transferred to 50 mL centrifuge tubes (Corning CentriStar, China) and centrifuged (3000 × g, 10 min). The supernatant was then discarded, and the dense culture isolate was resuspended and transferred into a 1.7 mL microtubes (Axygen, Mexico). The culture isolates were centrifuged (3000 × g, 10 min) and the supernatant discarded. The final isolated pellets were weighed from (60 to 120 mg) and stored in − 20 °C before FAs extraction.
The pellet samples (wet weight 60–120 mg) were directly added to 3 mL of methylating solution (methanol:dichloromethane:concentrated hydrochloric acid 10:1:1 v/v/v) made within 7 days. The tube was capped under Teflon, vortexed, and placed in a heating block (100 °C, 2 h). When the solution had cooled, 1 mL of Milli-Q water was added. The solution was extracted with 2 mL of hexane:dichloromethane (4:1 v/v) by shaking and then vortexing, followed by centrifugation (2000 × g, 5 min). The fatty acid methyl esters (FAME) occupying the upper organic layer was blown down and transferred to a vial. The 2 mL extractions were repeated twice42. After adding internal standard solution (19:0 FAME)42, samples were injected from this solution into a gas chromatograph (GC, Agilent Technologies Australia, Victoria, Australia).
FAME samples were run in accordance with AOAC official methods 963.22 “Methyl Esters of fatty acids in oils and fats”43. In brief, FAME was analysed by gas chromatography (GC, Agilent Technologies, Victoria, Australia) performed using an Agilent 6890 with an Agilent SP-2560 silica capillary column (DKSH New Zealand Limited, Auckland, New Zealand) (100 m × 0.25 mm i.d., 0.2 µm film thickness) and peak area determined by flame ionized detection (FID). The samples each (1 µL) were then injected via a split injector at 260 °C. The column temperature program was: 220 °C at 17 min, then raised by 2.8 °C min−1 to 240 °C and held for 5 min. FAs were linked to an external commercial FA standard (Supelco 37 Component FAME Mix, Merck, Auckland, NZ) with the use of ChemStation software (Version A10.02, Agilent, Auckland, NZ). Nitrogen was the carrier gas. To determine the FAs composition, the following formula with minor modifications was used: Cfa = (Afa/Ais) × (Cis/Vs)/RRF, where Cfa was the amounts of individual FAs, Afa is the chromatographic area units of the FA being investigated. Ais is the chromatographic area units for internal standard, Cis is the concentration of the internal standard used in µg/mL and Vs is the volume in mL of the Symbiodiniaceae sample used. The relative response factor (RRF) for each peak was determined from a commercial, equal-weight standard44. The mass of FAME was represented as mg 100 g−1 wet weight.
Descriptive analyses
All of our study data were analysed using R software (4.0.3). To determine if there was a significant difference between the cell viabilities of the culture isolates from the two freezing techniques, unpaired t-tests were undertaken (p < 0.05) and a boxplot used to show the median distribution of the cell survival after freezing. P-values of less than 0.05 was considered to be statistically significant. For the FAs, only descriptive analyses were done. All differences in the production of mean FA classes between normal and increased salinity treatments were represented in bar graphs. Due to the large number of culture isolates and the high cost of FA analysis, there was no replication in this part of the study and only descriptive analysis was possible. All FAs from individual samples were pooled by FA class and compared by salinity (Fig. 4). The changes in FA profiles in the high salinity culture isolates when compared to normal salinity were displayed in bar graphs and the Hierarchical clustering of the FA profiles was undertaken using Bray–Curtis similarity and results presented as a CLUSTER dendrogram. The number of cluster groups (k-means) for the dendrogram was selected using both the elbow method and the gap statistic45,46. SIMPER analysis on PAST software (4.03) was used to identify the species that best explained the differences between the FA classes, and the dominant FA profiles.
Results
Phylogenetic characterisation of Symbiodiniaceae cultures
Phylogenetic analysis of the 721 base-pair 28S rRNA gene fragment showed that the fifteen cultured isolates belonged to six Symbiodiniaceae genera (Fig. 1) including Symbiodinium (n = 5), Breviolum (n = 4), Fugacium (n = 3), Effrenium (n = 1), Durusdinium (n = 1) and Cladocopium (n = 1). Finer scale identification using the ITS-2 marker indicated that the cultures belonged to 13 putative distinct Symbiodiniaceae species (Table 1).
Best Maximum likelihood (ML) topology for the family Symbiodiniaceae based on nuclear large subunit ribosomal 28S rRNA gene, showing the phylogenetic placement of the 15 putative culture isolates (Cultures 1–15 in bold) together with their GenBank accession numbers as provided in Table 1 among the Symbiodiniaceae genera. Numbers at nodes represent the ML bootstrap pseudoreplicate (BP) values, excluding BP values lower than 70%. The phylogram was rooted using the dinoflagellate Gymnodinium simplex.
Cryopreservation results
Part A: Freezing experiments using 15% DMSO
Rapid freezing technique
Nine Symbiodiniaceae culture isolates were successfully cryopreserved using the rapid freezing method (Table 2). Breviolum psygmophilum [B2] had the best survival rate post-thawing (cell viability 87.8 ± 4.0%), followed by E. voratum [E1] (74.0 ± 7.8%). Low cell viabilities were observed in D. trenchii [D1a] and Breviolum sp. [B1] (16.0 ± 1.3% and 10.3 ± 1.7% respectively; Table 2). After thawing, no viable cells were observed for the six remaining culture isolates (Table 2).
Controlled-rate freezing technique
Nine Symbiodiniaceae culture isolates were successfully cryopreserved using the controlled-rate freezer method (Table 2). One isolate of B. psygmophilum [B2] had the best survival rate after thawing (62.1 ± 6.5%). Breviolum minutum [B1] and Fugacium sp. [F5.2] recorded very low cell viabilities (15.6 ± 0.4% and 15.0 ± 0.9% respectively; Table 2). After thawing, no viable cells were observed from the six remaining culture isolates (Table 2).
Part B: Freezing experiments using 10% DMSO
Five Symbiodiniaceae culture isolates from the genus Symbiodinium that failed to cryopreserve at 15% DMSO with both freezing methods and one culture from genus Fugacium (Fugacium sp. [F5.2]) that only cryopreserved using the controlled-rate freezer with a low cell viability with 15% DMSO were all cryopreserved with 10% DMSO.
Rapid freezing technique
One culture isolate from the genus Fugacium and three culture isolates from the genus Symbiodinium were successfully cryopreserved (Table 3). Fugacium sp. [F5.2] had the best survival rate (76.0 ± 4.7%), followed by S. necroappetens [A13], S. microadriaticum [A1] and S. pilosum [A2] survival rates of 62.2 ± 4.7, 25.0 ± 1.4 and 23.1 ± 3.9, respectively (Table 3).
Controlled-rate freezing technique
Fugacium sp. [F5.2] was successfully cryopreserved with a survival rate of 65.1 ± 6.3% (Table 3).
Part C: Freezing experiments using 15% DMSO and high salinity treatment
Rapid freezing technique
Symbiodiniaceae cultures grown in 44 ppt salinity media
Only S. pilosum [A2] was successfully cryopreserved with a post-thaw viability (64.9 ± 5.3%; Table 4).
Symbiodiniaceae cultures grown in 54 ppt salinity treatment
Symbiodinium pilosum [A2] and Fugacium sp. [F5.2] were successfully cryopreserved with high post-thaw viabilities (75.5 ± 7.2% and 57.4 ± 2.7%, respectively; Table 4).
Symbiodiniaceae cultures grown in 64 ppt salinity treatment
Only Fugacium sp. [F5.2] was successfully cryopreserved with a post-thaw cell viability of 22.5 ± 4.9% (Table 4).
Controlled-rate freezer method
Symbiodiniaceae cultures grown in 44 ppt salinity media
Symbiodinium pilosum [A2] and Fugacium sp. [F5.2] were successfully cryopreserved (37.8 ± 2.8% and 33.1 ± 8.2% respectively; Table 5).
Symbiodiniaceae cultures grown in 54 ppt salinity treatment
Only S. pilosum [A2] was successfully cryopreserved (44.1 ± 6.9%; Table 5).
Symbiodiniaceae cultures grown in 64 ppt salinity treatment
When the selected Symbiodiniaceae culture isolates were grown in f/2 media set at 64 ppt salinity, none of the treated strains survived (Table 5).
Cell viabilities between rapid freezing and controlled-rate freezing
The cell survival of all the successfully cryopreserved Symbiodiniaceae culture isolates grown in normal salinity media after both rapid freezing and the controlled-rate freezing techniques was investigated. The cell viabilities of the culture isolates cryopreserved with rapid-freezing were significantly higher when compared to those cryopreserved with the controlled-rate freezer (unpaired t-test, p < 0.05; cell survival after rapid freezing: 50.0 ± 24.4%, cell survival after controlled-rate freezing: 38.0 ± 16.1%).
Fatty acid (FA) profiles
Three classes of FAs were observed: saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). Full FA profiles are given in Supplementary Information excel Table S1. A cluster dendrogram (based on their FA profiles) was produced for all the culture isolates grown in normal salinity and for the two culture isolates Fugacium sp. [F5.2] and S. pilosum [A2] that survived cryopreservation after increased salinity treatments.
Two major clusters were observed that were further grouped into six subclusters that were calculated using a combination of the elbow method and gap statistic (Supplementary Information Fig. S1). The S. pilosum [A2] culture isolates grown in normal salinity and in 44 ppt salinity expressed similar FA profiles that were different to the culture isolate grown in 54 ppt salinity. For Fugacium sp. [F5.2], increased salinity treatment of 54 ppt resulted in the culture expressing different FA profiles that placed the isolate in a different cluster from the other cultures in the same genus Fugacium (Fig. 2). Three cultures of genus Breviolum were found in a similar FA cluster apart from Breviolum sp. [B1], which expressed a similar FA to that of the genus Fugacium (Fig. 2). Symbiodinium tridacnidorum [A6] and S. necroappetens [A13] were found in a FA cluster that was different from other five culture isolates in the genus Symbiodinium. Durusdinium trenchii did not cluster with any of the other culture isolates (Fig. 2).
Cluster analysis dendrogram based on fatty acid (FA) profiles using the hierarchical relationship among 15 culture isolates grown at normal salinity (black font) and three selected culture isolates grown at high salinities. The six different colours of the dendrogram branches represents the six clusters for the FAs similarities among the culture isolates. Culture isolates in blue font = Fugacium sp. [F5.2] and Symbiodinium pilosum [A2] were grown in 54 ppt salinity respectively. The culture isolate in green font = S. pilosum [A2] was grown in 44 ppt salinity. The two culture isolates in red font = S. tridacnidorum [A6] and S. tridacnidorum [A3] did not survive freezing while all the other culture isolates successfully cryopreserved.
Fatty acid composition in all culture isolates at normal and high salinities
Nine different SFAs were identified from C12:0 to C24:0 (Supplementary Information excel Table S1). The mass of the SFAs made up between 26.5 and 40.6% of the total mass of the FAs produced among the culture isolates (Fig. 3). The culture isolate with the highest contribution percentage (14.6%) of SFAs was D. trenchii growing at 34 ppt (normal salinity). The highest percentage of all the total SFAs was in Cladocopium sp. [C1] (40.6%; Supplementary Information Table S1) and the SFA that was highly expressed was Palmitic acid (C16.0) in D. trenchii (Supplementary Information Table S1).
A histogram showing the percentage of the three different fatty acids classes among the culture isolates at 34 ppt (normal salinity) and increased salinities of 44 ppt and 54 ppt for the best performing isolates during cryopreservation. MUFA monounsaturated fatty acids, SFA saturated fatty acids, PUFA polyunsaturated fatty acids, Ppt parts per thousand.
Five different MUFAs were identified from C14:1 to C22:1n9 (Supplementary Information excel Table S1). These MUFAs made up between 11.0 and 19.9% of the total mass of FAs produced among the culture isolates (Fig. 3). The culture isolate with the highest contribution percentage (12.2%) of MUFAs was D. trenchii growing at 34ppt (normal salinity). The highest percentage of all the total MUFAs was in Fugacium sp. (F5.1; 19.9%) (Supplementary Information Table S1, Fig. 3) and the MUFA that was highly expressed was Cetoleic/erucic acid (C22:1n9) in D. trenchii (Supplementary Information Table S1).
Ten different PUFAs from C16:2n4 to C22:6n3 were identified. The PUFAs can also be differentiated by omega 3 and omega 6 FAs (Supplementary Information excel Table S1). These PUFAs made up between 45.4 and 59.3% of the total mass of all the FAs produced among the culture isolates (Fig. 3). The culture isolate with the highest contribution percentage (13.0%) of MUFAs was D. trenchii growing at 34 ppt (normal salinity). The highest percentage of all the total PUFAs was Fugacium sp. at normal (34 ppt) salinity (F5.2; 59.3%) (Supplementary Information Table S1, Fig. 3) and the PUFA that was highly expressed was C18:4n3 stearidonic acid (SDA) in D. trenchii (Supplementary Information Table S1).
Changes in mass of the main classes of fatty acids after high salinity
Symbiodinium pilosum [A2] in 44 ppt and 54 ppt salinities
For the high salinity treatments, our descriptive analysis of the FA analyses after growth in the two increased salinity treatments of 44 ppt and 54 ppt showed major differences in the three main classes of FA when compared to normal salinity. There was higher production of SFAs, MUFAs and PUFAs in 54 ppt salinity compared to normal salinity (Fig. 4a). Conversely, under 44 ppt salinity, these three fatty acid classes were produced in lower amounts compared to normal salinity (Fig. 4a).
Changes in fatty acid (FA) concentration (mg 100 g−1 of FAME production) at different salinity treatments grouped by FA class for all individual FA profiles (MUFA = Monounsaturated fatty acids, SFA = Saturated fatty acids, PUFA = Polyunsaturated fatty acids); (a) Symbiodinium pilosum [A2] culture isolate after treatment with f/2 media at 44 ppt and 54 ppt salinities in comparison to f/2 media at 34 ppt (normal salinity; zero line). (b) Fugacium sp. [F5.2] culture isolate after treatment with f/2 media at 54 ppt salinity only in comparison to f/2 media at 34 ppt (normal salinity, zero line).
Fugacium sp. [F5.2] in 54 ppt salinity
For the FAs expressed by this culture isolate, descriptively there was a low production of all the three FA classes when the isolate was grown in 54 ppt salinity as compared to normal salinity (Fig. 4b).
Changes in the mass of individual fatty acids after high salinity
The initial descriptive analyses of the individual FA profiles of the two culture isolates under high salinity treatment showed some changes in the mass of major FA profiles of S. pilosum [A2] after increased salinity treatments of 44 ppt and 54 ppt as well as Fugacium sp. [F5.2] after 54 ppt salinity were compared to those grown in normal salinity. A further experimentation with appropriate replication with strengthen these findings. Changes of less than 2.0 mg 100 g−1 between increased and normal salinity were not considered in our comparison because many minor FA detected in our isolates had ranges of 0–2.0 mg 100 g−1. Our initial descriptive findings on changes in the FA profiles were visualized in comparative bar graphs (Fig. 5).
The changes in different fatty acid (FA) profiles of two Symbiodiniaceae culture isolates: (a) Symbiodinium pilosum [A2] after treatment with f/2 media at 44 ppt and 54 ppt salinities compared to f/2 media at 34 ppt (normal salinity; zero line); (b) Fugacium sp. [F5.2] after treatment with f/2 media at 54 ppt salinity in comparison to f/2 media at 34 ppt salinity (normal salinity). The values are in mg 100 g−1 change of fatty acid methyl esters.
Symbiodinium pilosum [A2] in 44 ppt and 54 ppt salinities
The initial descriptive findings of the FAs profiles of this culture isolate under high salinity treatments, For the major SFAs produced, myristic acid increased by 6.7 mg 100 g−1 in 54 ppt salinity compared to normal salinity treatment (Fig. 5a). Palmitic acid (C16:0) decreased by 9.1 mg 100 g−1 in 44 ppt salinity but had a large increase of 43.4 mg 100 g−1 in 54 ppt salinity compared to normal salinity. Stearic acid (C18:0), increased by 4.6 mg 100 g−1 in 54 ppt salinity compared to normal salinity (Fig. 5a). There were changes in production of three MUFAs; palmitoleic acid (C16:1) had an increase of 6.1 mg 100 g−1 in 54 ppt salinity treatment as compared to normal salinity (Fig. 5a). The production of oleic acid (C18:1n9c) increased in both salinity treatments by 2.4 mg 100 g−1 in 44 ppt and 6.0 mg 100 g−1 in 54 ppt salinity treatments respectively (Fig. 5a). Cetoleic/erucic acid (C22:1n9), decreased by 10.0 mg 100 g−1 in 44 ppt salinity but in 54 ppt salinity, there was an increase of 15.3 mg 100 g−1 compared to normal salinity (Fig. 5a). Higher changes occurred in the mass of some PUFAs, such as stearidonic acid (SDA) (C18:4n3) that decreased sharply by 30.5 mg 100 g−1 in 44 ppt salinity treatment, but it increased by 18.4 mg 100 g−1 in 54 ppt salinity compared to normal salinity. Docosahexaenoic acid (DHA) (C22:6n3) produced decreased by 10.0 mg 100 g−1 in 44 ppt salinity but it increased by 18.3 mg 100 g−1 in 54 ppt salinity when these two treatments were compared to normal salinity (Fig. 5a).
Fugacium sp. (F5.2) in 54 ppt salinity
For this culture isolate, descriptive analyses of the FA profiles showed that in the SFAs, there was a decrease of 4.3 mg 100 g−1 in lauric acid (C12:0) in 54 ppt salinity compared to normal salinity treatments. Palmitic acid (C16:0) experienced a decrease of 16.4 mg 100 g−1 when this culture was grown in 54 ppt salinity compared to normal salinity (Fig. 5b). Production of the MUFAs; Oleic acid (C18:1n9c), increased by 3.6 mg 100 g−1 under this high salinity treatment. In contrast, cetoleic/erucic acid (C22:1n9) production decreased by 12.4 mg 100 g−1 in 54 ppt salinity as compared to normal salinity (Fig. 5b). Lastly in PUFAs, stearidonic acid (SDA) (C18:4n3) production decreased sharply by 95.7 mg 100 g−1 in 54 ppt salinity treatment as compared to normal salinity. Docosahexaenoic acid (DHA) (C22:6n3) decreased by 29.6 mg 100 g−1 in the increased salinity of 54 ppt as compared to normal salinity (Fig. 5b).
Discussion
The successful cryopreservation of different microalgal species offers an alternative maintenance technique to the conventional serial sub-culturing and is the best method for their long-term storage47. In this study, a cryopreservation protocol optimized by Kihika et al. 10 using DMSO as the CPA and two freezing techniques, was applied to fifteen culture isolates from the family Symbiodiniaceae. DMSO was the preferred CPA because it can readily pass through the cell membrane into the intracellular space, can be removed easily from the cells, does not cause bacterial contaminations13,48 and it has no detrimental effects on the cell health of cryopreserved Breviolum sp.10. In microalgae, optimum conditions for a successful cryopreservation technique should be examined for each species separately47. To successfully cryopreserve the many Symbiodiniaceae cultured isolates with high post thaw survival rates, two main freezing techniques were applied: rapid freezing and controlled-rate freezing.
Before freezing the dinoflagellates, the penetrating CPA, (DMSO) was added in small aliquots to minimize osmotic stress that might affect the cells and their membranes9,10. In the first part of this study, all the culture isolates were treated with 15% DMSO before using the rapid freezing and controlled-rate freezer methods. Cultured isolates from Breviolum, Cladocopium, Durusdinium, Effrenium and Fugacium genera were successfully cryopreserved while all the isolates from the genus Symbiodinium failed to cryopreserve. All successfully cryopreserved isolates had different cell viabilities after thawing, and this illustrates that the optimum conditions for cryopreservation vary from one species to another13.
During cryopreservation, adequate dehydration of the microalgal cells has to be achieved using a suitable concentration of a cryoprotectant for each species10,13,49. Lowering of the DMSO concentration from 15 to 10% before freezing led to higher cell viabilities in Fugacium sp. [F5.2] in both rapid freezing and the controlled-rate freezer method which suggests that a higher concentration of DMSO might be toxic to this species. Surprisingly, Symbiodinium microadriaticum [A1], S. pilosum [A2] and S. necroappetens [A13] successfully cryopreserved only in the rapid freezing technique after using a lower concentration of 10% DMSO. These differences in cell survival among the Symbiodiniaceae culture isolates from both 15% and 10% DMSO treatments shows that the best type of CPA and its concentration has to be determined for each species empirically10.
High salinity treatments were applied to five Symbiodinium culture isolates that failed to cryopreserve with the initial experiments and to Fugacium sp. [F5.2] that had very low post thaw viability after using 15% DMSO. These culture isolates were subjected to salinity treatments of 44 ppt, 54 ppt and 64 ppt and then prepared for cryopreservation by being treated with 15% DMSO to facilitate cell dehydration before freezing10,21. Both S. pilosum [A2] and Fugacium sp. [F5.2] cryopreserved with high cell viabilities post thaw after the high salinity treatments. The high survival rates in these two culture isolates after salinity treatments indicates that ions from the dissolved salts may have acted as a dehydrating agent and the treatment with DMSO protects the cells membranes and proteins before freezing21. In the rapid freezing method, S. pilosum [A2] successfully cryopreserved after both 44 ppt and 54 ppt salinity treatments with very high cell viabilities, while Fugacium sp. [F5.2] cryopreserved at both 54 ppt and 64 ppt salinities. The differences in survival rates after cryopreservation in the salinity treated culture isolates show that the effects of salinity will depend on the sensitivity of the marine microalgae to high salt stress21. Two cultures S. tridacnidorum [A3] and S. tridacnidorum [A6] did not cryopreserve after any of the increased salinity treatments, which may have been caused by a variety of reasons such as the salinity levels applied were not high enough, the DMSO treatment under high salinity may have been toxic to the cells, or the cells underwent excessive dehydration before freezing.
The best cryopreservation technique for S. pilosum [A2] was rapid freezing. Under rapid freezing, the cell viability was very low at normal salinity with 10% DMSO but the viability was very high after increased salinity treatments of both 44 ppt and 54 ppt with 15% DMSO. In Fugacium sp. [F5.2], higher cell viabilities occurred in normal salinity treatments with 10% DMSO under both rapid and controlled rate freezing techniques. However, after 54 ppt salinity treatment with 15% DMSO, this culture isolate had a high cell viability after rapid freezing. In high salinity, permeable CPAs like DMSO reduce the osmotic stress in the cells by binding together the solute molecules21.
The phylogeny of Symbiodiniaceae has undergone many taxonomic revisions as knowledge on their genetics, morphology and ecology has improved8,50,51. The fifteen culture isolates in this study were classified into six out of the eleven known Symbiodiniaceae genera based on the 28S rRNA sequence analysis. The FA profiles of the culture isolates shows an interesting pattern in the genus Symbiodinium where five out of seven isolates cluster together while in other genera the isolates are mixed in different clusters. These results suggest that FAMEs analysis could serve as a chemotaxonomic marker for validation of phylogenetic classification in some species52. The Symbiodiniaceae culture isolates had higher proportions of PUFAs in both normal and high salinities, The most important FA profiles that were highly expressed and are specific to dinoflagellates were stearidonic acid (SDA) (C18:4n3) and docosahexaenoic acid (DHA) (C22:6n3)53. Increased salinity treatment induced S. pilosum [A2] and Fugacium sp. [F5.2] to express FA profiles that were different from those treated with normal salinity. Therefore, increasing salinity modified the type and amount of FA produced. Compared to the FAs produced in normal salinity, both S. pilosum [A2] and Fugacium sp. [F5.2] culture isolates recorded a sharp decrease in stearidonic acid (SDA) (C18:4n3) at 44 ppt and 54 ppt salinities, which is a glycolipid constituent synthesized within the chloroplast54. The reason for this decrease is not clear but we believe it is related to the desaturation of SDA that is an important precursor in the biosynthesis of Eicosapentaenoic acid (EPA) (20:5ω3)55. The decreased level of PUFAs in the two culture isolates under those high salinity treatments might have been caused by the cells trying to regulate the lipid phase to prevent disruption of membrane integrity56. However, the exact role of fatty acid composition in the osmoregulatory function of microalgal cells is not clear57.
There was a large amount of SFAs in S. pilosum [A2] after 54 ppt salinity treatment. The increase in SFAs may prevent leakage of compatible solutes out of the cell and diffusion of excess ions into the cell58. Alternatively, increasing unsaturated FAs in the membrane may stabilize the photosynthetic machinery of the microalgae under salinity stress conditions59,60. The initial investigation of the FA classes showed that the PUFAs were produced in low amounts in both S. pilosum [A2] and Fugacium sp. [5.2] under 54 ppt salinity. This might have been caused by the cells using these FAs to maintain the fluidity of the cell membrane under high salinity. To confirm these findings and results in the future, an increased number of replicates should be used to conduct appropriate statistical analyses. Overall changes in the sum of the number of double bonds and rings in FAs are important in maintaining the fluidity of the microalgal cell membranes and in providing the appropriate environment for membrane functions24. However, the variability in the production of FA classes under increased salinity in the two cultures demonstrates that individual species respond differently to salinity stress levels, and may express their FA classes differently, perhaps as a result of distinct means of acclimation, stress response or changes in the lipid composition of the cells24. Many marine microalgal species are able to tolerate great variations of salinity, but their chemical and fatty acid composition varies greatly with respect to increased salt stress23.
The changes in the FAs composition of cell membranes can affect their fluidity24 and this may therefore have an impact on the ability to cryopreserve different species. The physical properties of fatty acids differ due the length of the carbon chain and the number of double bonds. In general, the longer the FA chain or the more the double bonds, the lower the melting point. Therefore, increases in PUFAs in the FA profiles of these Symbiodiniaceae isolates will theoretically maintain their membrane’s structure24,25 and improve their ability to survive during cryopreservation.
Conclusion
Symbiodiniaceae species are highly diverse, and their existence in natural habitats as well as that of their associated hosts are threatened by the current global climatic crisis. Cryopreservation will help protect these valuable dinoflagellates for future scientific research and establish a seed bank for the conservation of threatened coral reef habitats prioritizing their recovery plans. In this study, we showed that different approaches were required for the Symbiodiniaceae culture isolates including varying the concentrations of DMSO, increasing nutrient medium salinity and application of two different freezing techniques. Our results indicate that, the best cryopreservation technique with the highest survival rates among the Symbiodiniaceae culture isolates was rapid freezing. However, the differential survival rates of some Symbiodiniaceae cultures among the two freezing methods demonstrate that it is unlikely that a common universal cryopreservation protocol for all microalgae can be developed. High salinity treatments of some Symbiodiniaceae isolates show that dehydration of the microalgae cells and the changes in their fatty acid profiles before cryopreservation leads to higher cell viabilities. This may be an important treatment step that should be considered when developing future protocols for Symbiodiniaceae cryopreservation. Application of new approaches to Symbiodiniaceae cryopreservation, such as investigation of their FA profiles and increasing salinity treatments, will greatly improve the current cryopreservation protocols to include more species from other genera and increase their survival rates.
Data availability
All FA data generated or analysed during this study are included in this published article and its Supplementary Information excel file. All DNA sequences generated in this study are available in the GenBank database (National Center for Biotechnology Information) under the following accession numbers: ITS-2 sequences: ON259675–ON259689. 28S rRNA sequences: ON263271–ON263285.
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Acknowledgements
This research was supported by funding from the New Zealand Ministry of Business, Innovation and Employment, Contract number: CAWX0902 and a Cawthron Institute Internal Capability Investment Fund scholarship. We would like to thank Todd C. LaJeunesse for valuable discussion and advice on Symbiodiniaceae molecular taxonomy and Ravneet Kaur for her input in the FA extractions.
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J.K.K., S.A.W., L.R., K.F.S., and K.G.R. designed the research. C.O. provided the culture isolates in the study. J.K.K., L.T. and J.B. conducted the experiments. M.R.M. assisted in the FA extractions and interpretation. X.P. assisted in ITS-2 genotyping identification and phylogenetic analyses. J.K.K. and J.S. analysed all the data and prepared all the figures and tables. S.A.W., L.R., K.F.S., and K.G.R. interpreted the data and formatted the tables. All authors wrote and reviewed the manuscript.
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Kihika, J.K., Wood, S.A., Rhodes, L. et al. Cryopreservation of six Symbiodiniaceae genera and assessment of fatty acid profiles in response to increased salinity treatments. Sci Rep 12, 12408 (2022). https://doi.org/10.1038/s41598-022-16735-w
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DOI: https://doi.org/10.1038/s41598-022-16735-w
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