Isolation, Fatty Acid Profiles and Cryopreservation of Marine Thraustochytrids from Mangrove Habitats in Thailand

Botanica Marina 2017; 60(4): 363–379

Panida Unagul*, Satinee Suetrong, Sita Preedanon, Anupong Klaysuban, Wunna Gundool, Chanwit Suriyachadkun and Jariya Sakayaroj* Isolation, fatty acid profiles and cryopreservation of marine thraustochytrids from mangrove habitats in Thailand

DOI 10.1515/bot-2016-0111 Received 11 October, 2016; accepted 14 June, 2017; online first 14 Introduction July, 2017 Thraustochytrids, marine protists belonging to Class Lab- Abstract: Thraustochytrids, marine protists, have attracted yrinthulomycetes (Kingdom Straminipila, Super-group attention as a proven alternative source of polyunsatu- Chromalveolata), serve many biological roles as saprobes, rated fatty acids (PUFAs). We isolated a high diversity of parasites and commensals in marine ecosystems (Adl thraustochytrids from Thailand and tested their potential et al. 2005, Tsui et al. 2009). These microbes are predomi- use in the production of high-value fatty acids. The iso- nantly associated with detrital materials, including decay- lated thraustochytrids can be categorized into seven major ing mangrove leaves and sediments in marine habitats groups based on unique morphological features, molecular (Barclay et al. 1994, Raghukumar 2008). Thraustochytrids phylogeny and fatty acid profiles. Two of the seven isolated are capable of producing many enzymes, such as protease, thraustochytrid groups could potentially be new lineages in lipase, esterase, cellulase and xylanase (Raghukumar et al. the Labyrinthulomycetes. The production of total fatty acid 1994, Bremer and Talbot 1995, Bongiorni et al. 2005b). In (TFA) from these thraustochytrids ranged from 2.4 to 35.6% addition, thraustochytrids have recently attracted atten- (w/w), with biomass varying from 0.3 to 8.9 g l−1. The pro- tion as an alternative source of polyunsaturated fatty acids duction of arachidonic acid (C20:4) ranged from 0.3 to 8.2% (PUFAs), such as arachidonic acid (ARA; 20:4), eicosap- of TFA, eicosapentaenoic acid (C20:5) from 1.5 to 12.4%, entaenoic acid (EPA; 20:5) and particularly docosahexae- docosapentaenoic acid (C22:5) from 9.7 to 27.3%, and doco- noic acid (DHA; 22:6; Ward and Singh 2005, Raghukumar sahexaenoic acid (C22:6) from 14.4 to 51.7%. In addition, the 2008). Notably, DHA has been shown to be involved in cryopreservation of selected thraustochytrids at −80°C with the development of brain and retina during fetal life and 10% glycerol, the combination of 5% trehalose and 10% infancy and in the maintenance of brain functions in glycerol, and freezing in vapor-phase liquid nitrogen were adults (Muskiet et al. 2004, Singh 2005, Ward and Singh found to maintain significant cell viability. The information 2005). Intake of DHA shows positive effects in the preven- provided in this study could be applied to cryopreservation tion of cardiovascular and neurological diseases (Kang and biotechnological applications of the economically val- and Leaf 1996, Simopoulous 1999, Kris-Etherton et al. uable marine Labyrinthulomycetes from Thailand. 2002). These benefits have led to the addition of DHA to most infant food products, drugs and animal feeds (Innis Keywords: cryopreservation; diversity; fatty acid profile; 2008, Raghukumar 2008). DHA has also been shown to mangroves; thraustochytrids. promote growth and development of the marine crusta- cean Artemia and this has implications for designing feeds *Corresponding authors: Panida Unagul and Jariya Sakayaroj, for commercial fish and prawns (Jaritkhuan 2002). For National Center for Genetic Engineering and Biotechnology these reasons, a number of novel thraustochytrid strains (BIOTEC), National Science and Technology Development Agency showing substantial yields of PUFAs have been explored (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang, Pathum Thani 12120, from different marine habitats (Yang et al. 2010, Hong Thailand, e-mail: [email protected] (P. Unagul); et al. 2011, Chang et al. 2012, Leaño and Damare 2012, Li [email protected] (J. Sakayaroj) et al. 2013, Gupta et al. 2013, 2016). Given the increasing Satinee Suetrong, Sita Preedanon, Anupong Klaysuban, Wunna evidence for the economic significance of these microor- Gundool and Chanwit Suriyachadkun: National Center for Genetic ganisms, it is worth investigating their existence in a wider Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science range of unexplored marine habitats. Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe The mangrove ecosystem serves important roles Khlong Luang, Pathum Thani 12120, Thailand in natural ecosystems by protecting shorelines from 364 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation storms, reducing coastal erosion and serving as valu- Materials and methods able nursery areas for marine animals (Mwangi 2001, Nagelkerken et al. 2008). Mangrove sediments and Sample collection and isolation detritus are involved in nutrient recycling in the food web (Kristensen et al. 2008). The mangrove-derived of microorganisms organic matter in mangrove ecosystems also promotes Decaying mangrove leaves, seagrasses and seawa- a rich microbial community (Kathiresan and Bingham ter samples were collected from 22 mangrove habitats 2001). Thailand supports a rich diversity of marine located in 12 provinces from central, eastern and south- habitats, including offshore islands, healthy coral reefs, ern Thailand (Figure 1). The leaf samples were washed seagrass beds and estuaries, with a significant number with sterile natural seawater (NSW) containing penicillin of mangrove stands (Jones and Hyde 2004). In 2009, G and streptomycin sulfate (500 mg l−1 each, Bio Basic the estimated mangrove area in Thailand comprised Inc., Canada). These substrata were cut into circular discs 240,000 ha, mainly distributed along the coastline of (0.5 cm in diameter) and plated directly on the isolation the Gulf of Thailand and the Andaman Sea. There are agar medium, Glucose Peptone Yeast Extract (GPY) con- 81 species of mangrove trees recorded for Thailand taining 1 g peptone (Difco Laboratories, Detroit, USA), 2 g (DMCR 2017). Currently, Thailand’s coastline faces most yeast extract (Difco), 4 g glucose (Difco) and antibiotics of the usual disturbances, such as coastal erosion, sedi- (Yang et al. 2010), prepared in 1 l of half-strength NSW mentation, habitat degradation and declining mangrove forests due to natural and human activities (DMCR 2017). The recent decline in the mangrove population will affect the ecosystems and cause a decline in biodiversity (Mwangi 2001, Stone 2006). Little attention has been paid to the occurrence of thraustochytrids in Thailand. Studies that have recorded them include those of Jones et al. (2006) and Jarit- khuan (2002), whereas others focused on the effects of culture conditions on DHA production and the application of certain thraustochytrid species for aquaculture (Unagul et al. 2005, 2006, 2007). These findings represent only limited information on the diversity of Thai thraustochytrids, which are known to be ubiquitous microorganisms. Additionally, long-term preservation of these micro organisms is of importance for reducing contamination and preventing genetic change (Snell 1991). Among the limited literature concerning cryopreservation of marine thraustochytrids, the only recent documented cryopreservation technique for marine thraustochytrids was developed by Cox et al. (2009). The use of a combination of 30% horse serum and 10% dimethyl sulphoxide (DMSO) was proven to be the most effective cryoprotective agent (CPA) for liquid nitrogen cryopreservation of thraustochytrids isolated from New Zealand. To complement the sporadic knowledge of marine Labyrinthulomycetes in Thailand, the present study reports on novel PUFA-producing thraustochytrids from various mangrove locations. In addition, we evaluate the fatty acid profiles of these microorganisms, iden- tify them based on morphological and molecular characteristics and determine a suitable method for their cryopreservation. Figure 1: Map of collection sites in 12 provinces of Thailand. P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 365

(1:1 NSW/distilled water). To obtain thraustochytrids Amplification using the primer pair Thr404f/Thr1017r from seawater, pine pollen was used as a bait and added was performed as follows: initial denaturation (4 min at to the seawater samples (Porter 1990). After 72 h incu- 95°C), followed by 30 cycles of denaturation (30 s at 94°C), bation, the pine pollen grains were transferred to new annealing (40 s at 50°C), extension (90 s at 72°C) and a GPY agar plates and subsequently incubated at 25°C. final extension step (20 min at 72°C; Harel et al. 2008). The The thraustochytrid-like colonies were aseptically trans- PCR products were then directly sequenced by Macrogen ferred to new agar plates and repeatedly sub-cultured Inc. (Korea). on freshly prepared GPY medium until axenic cultures were obtained. The isolated thraustochytrids were char- acterized based on morphological features and molecu- Sequence alignment and phylogenetic lar identification. All axenic cultures were kept in the analyses BIOTEC Culture Collection (BCC), Thailand. Pure cultures were grown in Glucose Yeast Extract (GY) medium The nuclear SSU rDNA sequences of the thraustochytrids containing (per liter of half-strength NSW) 10 g yeast obtained in this study (Table 1) were aligned with nucleo- extract and 30 g glucose, and shaken at 200 rpm at 25°C tide sequences retrieved from GenBank, with Bacillaria for 7 days. The cultured cells were harvested by centrifu- paxillifera (O. F. Müll.) Hendey M87325 chosen as the out- gation at 10,640 g, washed twice with sterilized 0.85% group, using the multiple alignment program CLUSTAL NaCl (Carlo Erba, Lombardy, Italy), and lyophilized to W 1.6 (Thompson et al. 1994). The alignment was refined determine cell dry weight. visually in BioEdit 6.0.7 (Hall 2004) and incorporated into PAUP 4.0b10 (Swofford 2003) for phylogenetic analyses through a maximum parsimony (MP) criterion. Morphological observation All characters were equally weighted, followed by heuristic searches with a stepwise starting tree, a random Colonies of 26 thraustochytrid strains were sub-cultured stepwise addition of 10 replicates and tree-bisection- using a sterilized inoculating loop, streaked across seawa- reconnection (TBR) branch-swapping algorithm. The ter GPY agar and incubated at 25°C for 3–7 days. Colonies most parsimonious trees (MPT) were tested for the best appearing on the plates were transferred to 3 ml seawa- topology with the Kishino-Hasegawa (K-H) maximum ter GPY broth in six-well plates and incubated at 25°C. likelihood test (Kishino and Hasegawa 1989) to find the Aliquots of the cell suspension (100 μl) were transferred most likely tree for the dataset. Tree length, consist- into a slide chamber for microscopic observation. Mor- ency indices (CI), retention indices (RI) and rescaled phological characteristics of the thraustochytrid strains consistency indices (RC) were calculated for each tree during cell cycle development, such as presence of ecto- generated. Finally, 1000 replicates of MP bootstrapping plasmic net, formation of zoospores, amoeboid cells, veg- analysis (Felsenstein 1985) were performed through full etative cell, zoospores and zoosporangia were observed heuristic searches, stepwise addition of sequence, 10 (Yokoyama et al. 2007). replicates of random addition of taxa and TBR branch- swapping algorithm. The Bayesian inference was calculated with Genomic DNA extraction, PCR amplification Mr. Bayes 3.0b4 with the general time reversible (GTR) and DNA sequencing model of DNA substitution and a gamma distribution rate variation across sites (Huelsenbeck and Ronquist Genomic DNA of selected strains was extracted by using 2001). Four Markov chains were run from random DNeasy Plant Mini kit (Qiagen, USA). Partial nuclear small starting trees for 3,000,000 generations and sampled subunit (SSU) rDNA was amplified using primer sets: NS1/ every 100 generations. The first 300,000 generations NS4 and Thr404f/Thr1017r (White et al. 1990, Honda et al. were discarded as burn-in of the analysis. A majority 1999, Harel et al. 2008). All the PCR primer pair amplifica- rule consensus tree of all remaining trees, as well as tions were carried out using BIORAD T100 Thermal Cycler the posterior probabilities (PP), was calculated. The (USA). Amplification using the primer pair NS1/NS4 was maximum-likelihood (ML) analyses were conducted in performed as follows: initial denaturation (2 min at 94°C), the CIPRES web portal (Miller et al. 2010) using RAxML followed by 35 cycles of denaturation (1 min at 94°C), 8.2.4 (Stamatakis 2014) with the Broyden-Fletcher- annealing (1 min at 55°C), extension (2 min at 72°C) and Goldfarb-Shanno (BFGS) method to optimize GTR rate a final extension step (10 min at 72°C; White et al. 1990). parameters. The maximum-likelihood bootstraps were 366 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation KX688798 KX688821 KX688823 KX688810 KX688813 KX688814 KX688811 KX688812 KX688802 KX688801 KX688800 KX688809 KX688815 KX688806 KX688822 KX688805 KX688816 KX688808 KX688820 KX688803 KX688819 KX688804 KX688817 KX688807 KX688818 GenBank accession accession GenBank rDNA) (SSU nos. KX688799 E E E E ′ ′ ′ ′ 42.0 ′ 42.0 ′ 42.0 ′ 42.0 ′ 51 ′ 51 ′ 51 ′ 51 ′ N 99 ° N 99 ° N 99 ° N 99 ° ′ ′ ′ ′ 18.0 ′ 18.0 ′ 18.0 ′ 18.0 ′ ′ 12 12 ′ 12 ′ 12 ′ ° Si Thammarat/9 Nakhon ° 25 ′ 39.4 N 99 46 24.6 E Khan/12 Khiri Prachuap ° 23 ′ 31.2 N 99 16 42.1 E Chumphon/10 ° Si Thammarat/9 Nakhon Phang-nga/ 8 ° 23 ′ 16.8 N 98 27 40.1 E Phang-nga/ Krabi/ 8 ° 04 ′ 30.0 N 98 55 03.1 E Krabi/ Trang/ 7 ° 21 ′ 43.3 N 99 30 31.2 E Trang/ Trang/ 7 ° 21 ′ 43.3 N 99 30 31.2 E Trang/ Ranong/ 9 ° 58 ′ 00.0 N 98 38 E Ranong/ Trang/ 7 ° 21 ′ 43.3 N 99 30 31.2 E Trang/ Trang/ 7 ° 21 ′ 43.3 N 99 30 31.2 E Trang/ Trang/ 7 ° 21 ′ 43.3 N 99 30 31.2 E Trang/ ° 22 ′ 13.6 N 100 01 44.8 E Songkhram/13 Samut ° 12 ′ 04.1 ″ N 99 28 29.1 E Thani/9 Surat ° 22 ′ 13.6 N 100 01 44.8 E Songkhram/13 Samut ° 25 ′ 39.4 N 99 46 24.6 E Khan/12 Khiri Prachuap ° 31 ′ 31.5 N 100 16 11.6 E Sakhon/13 Samut ° 21 ′ 42.0 N 100 58 48.0 E Buri/13 Chon ° 31 ′ 31.5 N 100 16 11.6 E Sakhon/13 Samut ° 21 ′ 42.0 ″ N 100 58 48.0 E Buri/13 Chon Ranong/ 9 ° 58 ′ 00.0 N 98 38 E Ranong/ ° 21 ′ 42.0 N 100 58 48.0 E Buri/13 Chon ° 30 ′ 18.0 N 101 00 09.6 E Chachoengsao/13 ° Si Thammarat/9 Nakhon Samut Sakhon/ 13 ° 31 ′ 31.5 N 100 16 11.6 E Sakhon/ Samut ° Si Thammarat/9 Nakhon Collecting site (Province)/GPS site Collecting ) hemprichii Thalassia Submerged mangrove leaf mangrove Submerged Seawater Submerged mangrove leaf leaf mangrove Submerged Submerged mangrove leaf mangrove Submerged Seawater sp.) ( Rhizophora leaf Submerged Seagrass ( ) parviflora ( Bruguiera leaf Submerged Seawater Submerged mangrove leaf leaf mangrove Submerged Submerged mangrove leaf leaf mangrove Submerged Submerged mangrove leaf leaf mangrove Submerged Submerged mangrove leaf mangrove Submerged Seawater Submerged mangrove leaf leaf mangrove Submerged Seawater sp.) ( Rhizophora leaf Submerged Seawater Seawater Seawater Seawater Seawater ) mucronata ( Rhizophora leaf Submerged Seawater Submerged mangrove leaf leaf mangrove Submerged Seawater Substrata Parietichytrium sakarianum Parietichytrium Parietichytrium sakarianum Parietichytrium sp. Thraustochytrium sp. Thraustochytrium sp. Thraustochytrium sp. Thraustochytrium sp. Thraustochytrium sp. Thraustochytrium sp. Thraustochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium Aurantiochytrium limacinum Aurantiochytrium sp. Thraustochytrium Aurantiochytrium limacinum Aurantiochytrium sp. Schizochytrium Aurantiochytrium limacinum Aurantiochytrium sp. Schizochytrium Aurantiochytrium limacinum Aurantiochytrium Unidentified thraustochytrids Unidentified Aurantiochytrium limacinum Aurantiochytrium Unidentified thraustochytrids Unidentified Aurantiochytrium limacinum Aurantiochytrium Unidentified thraustochytrids Unidentified Aurantiochytrium limacinum Aurantiochytrium Unidentified thraustochytrids Unidentified Taxa 75567 52209 77550 72477 60508 60491 55445 55166 52189 56601 55449 55421 60533 60518 60532 77563 60447 75569 60446 75583 52190 75573 51316 54862 51304 54861 BCC code BCC JS1087 JS510 JS1115 JS1085 JS964 JS947 JS727 JS679 JS489 JS736 JS732 JS702 JS989 JS974 JS988 JS1128 JS828 JS1089 JS827 JS1103 JS490 JS1093 JS446 JS660 JS434 JS659 Original Original code IV III II VII VI I V Newly isolated thraustochytrids from the present study and used in the phylogenetic analyses and their GenBank accession numbers. accession their GenBank and analyses in the phylogenetic used and study the present from thraustochytrids isolated 1: Newly Table Group P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 367 estimated with the substitution matrix as GTR model (iii) 17% skim milk (Difco) combined with 20% glycerol and a discrete gamma distribution. (SG), (iv) 30% horse serum (GIBCO, USA) combined with 10% DMSO (HD) Fatty acid extraction and analysis (v) 5% trehalose (H + B Lifescience, Japan) combined with 10% glycerol (TG). Strains cultivated in GY medium at 25°C and 200 rpm for 7 days were harvested by centrifugation and lyo- Cryotubes were placed into a controlled-rate freez- philization. Freeze-dried cells were then directly trans- ing container (Mr. Frosty, Nalgene Cat. No. 5100) and esterified with 4% sulphuric acid in methanol and then frozen at 1°C min−1 before storage in a −80°C freezer for heated in a 90°C water bath for 1 h. Nonadecanoic acid 6 months. To test the survival of thraustochytrids, the (C19:0) was used as an internal standard (Sigma-Aldrich, cryotubes retrieved from the −80°C freezer were directly St. Louis, MO, USA). The esterified samples were applied thawed by immersion in a 30°C water bath for 3 min to a gas chromatograph (GC17A, Shimadzu), equipped (Nishii and Nakagiri 1991). One micro liter of the con- with a 30 m × 0.25 mm Omegawax™ 250 fused silica tents was serially diluted, and 0.01 ml was then directly capillary column (Supelco, USA), an automatic sampler spread onto GPY agar plates. The colonies formed on the and flame ionization detector (FID). The injector and plate were counted to calculate the proportion of viable detector temperature were kept at 250°C and 260°C, cells. The survival rates were calculated relative to the respectively. Helium was used as a carrier gas at a linear cell density before freezing. velocity of 30 cm s−1. The initial column temperature of The survival rates of thraustochytrids preserved in 200°C was held for 10 min and then increased at 20°C liquid nitrogen were subsequently determined by placing min−1 to 230°C where it was held for 17 min. Peaks were the cryotubes containing the concentrated cells in the identified based on the retention times relative to fatty best CPA obtained from the previous experiment in a acid methyl ester standards (Supelco 18919-1 AMP; all vapor-phase of nitrogen for 12 months. The survival of the from Sigma-Aldrich, USA). thraustochytrids was tested in the same way. Statistical significance was established with one-way ANOVA using SPSS 11.5 software for Windows. Cryopreservation of selected thraustochytrids

Four marine thraustochytrids (JS510, JS702, JS974 and Results JS1085) were selected, based on differences in morphological features and phylogenetic position, for cryo- Isolation, morphological observation preservation at −80°C and vapor-phase liquid nitrogen. and molecular phylogeny The thraustochytrid strains were grown in 50 ml GPY medium at 25°C with shaking at 80 rpm (Cox et al. 2009). More than 300 strains of thraustochytrids were isolated in Cells were harvested during the log phase growth period this study. The majority of the isolated strains (approxi- (at day 3–5, depending on the species) by centrifugation mately 90%) were obtained from decaying mangrove (10,640 g) for 5 min. The resulting pellet was re-suspended leaves by the direct plating technique, while only 10% was in an equal volume of fresh GPY medium, and the total obtained from seawater by the pine pollen baiting tech- number of cells was enumerated through serial dilution nique (data not shown). Twenty-six selected strains of dif- and a total plate count on GY agar plates. The number of ferent colony morphology were examined for microscopic colonies (colony forming unit, CFU) formed per milliliter morphological features and molecular phylogeny based (CFU ml−1) was calculated for each thraustochytrid strain on SSU rDNA sequences (Table 1). before freezing. The selected thraustochytrids can be categorized into To determine the most suitable CPA for cryopreserva- seven major groups based on morphological features and tion of selected thraustochytrids in −80°C, an aliquot of molecular evidence (Table 2, Figures 2 and 3). The analy- the concentrated cells was added to cryotubes containing ses of SSU rDNA sequences resulted in nine MPTs [tree five different CPA treatments: length = 2427 steps, CI = 0.3976, RI = 0.776, and RC = 0.413], (i) 5% DMSO (D, Bio Basic Inc., Canada), and the best tree topology as determined by the K-H test is (ii) 10% glycerol (G, Bio Basic Inc., Canada), presented in Figure 3. Of the 868 total characters, 360 were 368 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation Absent Present Absent Absent Absent Absent Absent Absent Present Present Present Binary cell division Present Present Present Present Present Present Present Present Present Present Present Amoeboid Amoeboid cell Thick- walled Thin- walled Thick- walled Thick- walled Thin- walled Thin- walled Thin- walled Thin- walled Thin- walled Thin- walled Thin- walled Cell Cell wall m diam., with a large a large with 10–90 μ m diam., Globose, (1–6 bodies Multiproliferous vesicle. contain Zoosporangia produced. are cells) retracted protoplast with vesicles large wall zoosporangium mature from Globose to subglobose, 21.2–92.5 × subglobose, to Globose 10–80 um Globose, 10–45 μ m diameter Globose, Globose, subglobose 20–177 × 20–82.5 subglobose Globose, vesicle a large μ m, with Globose, subglobose to pyriform, pyriform, to subglobose Globose, 8.7–57.5 × 8.7–50 μ m Globose, 26.2–51.2 μ m diameter Globose, Globose, 20–50 μ m diameter Globose, Globose, 16.2–55 μ m diameter Globose, m diameter 12–30 μ m diameter Globose, Globose, 15–31.2 μ m diameter Globose, m diameter 12.5–47.5 μ m diameter Globose, Shape and size of a zoosporangium a of size and Shape Globose, 10–57 μ m Globose, diam Globose to to Globose 10– subglobose, 38.7 × 10–27.5 μ m Globose, 10–40 μ m Globose, diam Globose, 22.5–60 μ m Globose, diam Globose, 10–40 μ m Globose, diam Globose, 7.5–15 μ m Globose, diam Globose, 10–22.5 μ m Globose, diam Spherical, 16.2– Spherical, 47.5 μ m diam Spherical, 5–20 μ m Spherical, cells diam.Vegetative 12.5– limaciform, 30 × 5–11.25 μ m wide pseudopodia with Spherical, 7.5– Spherical, 16.2 μ m Spherical, 5–20 μ m Spherical, cells Vegetative diam. 10– limaciform, 45 × 3.75–16.25 μ m pseudopodia with Shape and size of of size and Shape cells vegetative Well- developed Well- developed Un-developed Un-developed N/O Un-developed Un-developed Un-developed Well- developed Well- developed Well- developed Ectoplasmic Ectoplasmic network Colonies are circular in shape, in shape, circular are Colonies with raised yellowish, pale to cream Numerous margin. an undulate produced zoosporangium Colonies are circular in shape, in shape, circular are Colonies an with raised yellowish, to cream margin undulate Colonies are circular in shape, in shape, circular are Colonies an entire with raised yellowish, margin Colonies are circular in shape, in shape, circular are Colonies an entire with raised yellowish, margin Colonies are circular in shape, in shape, circular are Colonies margin. an entire with raised white, wall persistentCell after releasing protoplast N/O N/O Colonies are circular in shape, in shape, circular are Colonies an entire with raised cream, a into developing Thallus margin. zoosporangium single Colonies are larger than group I, group than larger are Colonies in shape, irregular growing, slow margin an undulate with flat white, Colonies are irregular in shape, in shape, irregular are Colonies margin. an undulate with flat white, Colonies are irregular in shape, in shape, irregular are Colonies margin an undulate with flat white, Colony morphology Colony JS974 JS1089, JS1128 JS1093, JS1103 JS659, JS660 JS510, JS1087 JS1085 JS1115 JS489, JS679, JS964, JS727, JS947 JS702, JS732, JS736 JS490 JS434, JS446, JS828, JS988, JS989, JS827 Original code Original N/O, not observed. not N/O, VII- Thraustochytrium sp. VI- Schizochytrium sp. V- Unidentified Unidentified V- thraustochytrids IV- IV- Parietichytrium sarkarianum III- Thraustochytrium sp. II- Aurantiochytrium sp. I – Aurantiochytrium limacinum Morphological features of the examined thraustochytrid strains. thraustochytrid the examined of features 2: Morphological Table Group P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 369

A B C D

E F G H

I J K L

M N O P

Q R S T

U V W X

Y Z AA AB

AC AD AE AF

AG AH AI AJ

Figure 2: Thraustochytrid colonies and cells. (A–D) Aurantiochytrium limacinum (Group I, JS434). (E–H) Aurantiochytrium sp. (Group II, JS736). (I–L) Thraustochytrium sp. (Group III, JS489). (M–P) Parietichytrium sarkarianum (Group IV, JS510). (Q–X) Unidentified thraustochytrids (Group V, JS660). (Y–AB) Schizochytrium sp. (Group VI, JS1089). (AC–AJ) Thraustochytrium sp. (Group VII, JS974). 370 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation

Figure 3: One of the nine most parsimonious trees resulting from maximum parsimony analyses obtained from nuclear SSU rDNA sequence analyses of thraustochytrids. Maximum parsimony (left) and maximum likelihood (right) bootstrap values greater than 50% are shown above each branch. Bayesian posterior probabilities greater than 0.95 are given below each branch. Scale bar indicates 10 character state changes. P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 371 constant, 94 were parsimony-uninformative and 414 were JS1103 possessed smaller vegetative cells and zoospo- parsimony-informative. rangia. Isolates JS1089 and JS1128 (Figure 2Y–AB) lie Sequences of the isolated thraustochytrids cluster within Group VI comprising Schizochytrium aggregatum in the Thraustochytridae, Labyrinthulomycetes S. Goldst. et Belsky AB022106 and Schizochytrium sp. (Figure 3). Group I comprises seven thraustochytrid AB290576 with nucleotide similarity ranging from 92.9 to strains (JS446, JS989, JS828, JS988, JS434, JS827, JS490) 97.2%. One thraustochytrid strain (JS974) forms a clade possessing similar morphological features, except for with T. gaertnerium R. Jain, Raghuk., Bongiorni et Aggar- JS490, which had smaller zoosporangia (Figure 2A–D). wal AY705753 with 88% sequence similarity (Group VII). They are closely related to Aurantiochytrium limaci- Strain JS974 is distinct in morphological characteriza- num (D. Honda et Yokochi) R. Yokoyama et D. Honda tion. It had irregularly shaped colonies, thalli with thick sequences retrieved from GenBank. Group II contains hyaline walls, a globose sporangium and cytoplasm that three strains (JS702, JS732, JS736) forming a strongly sup- cleaved into zoospores. Multiproliferous bodies (1–6 ported monophyletic group without any closely related cells) were produced. Zoosporangia contained large taxa. They share similar morphological characteristics vesicles with the protoplast retracted from the mature with the genus Aurantiochytrium R. Yokoyama et D. zoosporangium wall. Numerous cigar-shaped, limax Honda, but differ from A. limacinum in colony morphol- amoebae were observed (Figure 2AC–AJ). ogy and growth rate (Figure 2E–H). Group III comprises seven strains (JS964, JS679, JS489, JS727, JS947, JS1085, JS1115), clustering with species of Thraustochytriidae Polyunsaturated fatty acid composition and Thraustochytrium striatum Joa. Schneid., although JS1085 nestled separately from the others. The vegeta- Mean values of cell biomass, TFA and PUFA composi- tive cell and zoosporangium measurement varied among tion of the isolated thraustochytrids are shown in Table 3 strains within this group (Figure 2I–L). Moreover, two and Figure 4. Profiles of the fatty acids, particularly isolates (JS1087, JS510) nestled within Group IV and have arachidonic acid (ARA, C20:4), eicosapentaenoic acid a close affinity with strains of Parietichytrium sarkari- (EPA, C20:5), docosapentaenoic acid (DPA, C22:5) and anum (A. Gaertn.) R. Yokoyama, Salleh et D. Honda, with docosahexaenoic acid (DHA, C22:6), were determined sequence similarities of 98.8–99.7%. Our strains share in each group. The production of C22 acids, particularly morphological features with P. sarkarianum in possess- DHA (C22:6), ranged from 7 to 221 mg l−1, whereas the C20 ing vegetative globose cells, with thin walls. Amoeboid (C20:4 and C20:5) content ranged from 0.3 to 16.8 mg l−1. cells were actively motile and the cell wall was persis- The highest contents of DPA, DHA and EPA were obtained tent after releasing the protoplast (Figure 2M–P). Four from Group II strains and the highest content of ARA was isolates (JS660, JS659, JS1093 and JS1103) are well placed found in Group IV (Table 3). in Group V without any closely related reference strains. The fatty acid composition (as% of TFA) of individual The morphological characteristics for this group are strains is shown in Table 4. DHA was the predominant unique (Figure 2Q–X). JS659 and JS660 possessed large fatty acid present in all groups, as well as palmitic acid vesicles inside the zoosporangia, whereas JS1093 and (C16:0) for Groups I and IV and pentadecanoic acid (C15:0)

Table 3: Mean biomass and polyunsaturated fatty acid contents, total fatty acids (± standard deviation) of thraustochytrids in each group.

Group Biomass Total fatty Polyunsaturated fatty acids (mg l−1) (g l−1) acid (% w/w) C20:4 (ARA) C20:5 (EPA) C22:5 (DPA) C22:6 (DHA)

I 1.0 ± 0.1 10.2 ± 2.3 0.31 1.53 9.89 52.73 II 3.1 ± 0.9 19.4 ± 3.0 6.01 16.84 73.37 221.92 III 1.3 ± 0.6 7.9 ± 4.0 2.36 11.56 24.34 54.15 IV 2.6 ± 1.3 8.3 ± 1.8 14.24 10.14 42.73 31.08 V 0.6 ± 0.1 4.9 ± 0.8 2.41 3.65 6.29 9.53 VI 0.9 ± 0.4 3.5 ± 1.6 3.68 1.92 2.74 7.08 VII 0.59 5.4 2.07 2.52 8.70 10.86

ARA, arachidonic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. 372 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation

Group JS490 C20:4 preservation for 6 months (p < 0.05). Aurantiochytrium JS827 C20:5 sp. JS702 had a cell density before freezing of 3.6 × 106 JS434 C22:5 CFU ml−1 and retained concentrations of 1.1 × 105–6.0 × 106 I JS988 −1 JS828 C22:6 CFU ml (3–29% viability) after 6 months of storage for JS989 all CPAs tested. Of these CPAs: D, HD and SG gave the JS446 JS736 lowest cell viability (p < 0.05), while TG and G had the II JS732 highest survival rates (29% and 16% viability, respec- JS702 JS1085 tively) (p < 0.05). JS1115 Thraustochytrium sp. JS1085 had a cell density JS489 × 8 −1 III JS727 before freezing of 2.6 10 CFU ml , and it retained 7 7 −1 JS964 concentrations of 1.7 × 10 − 5.6 × 10 CFU ml (6–21% JS947 viability) after 6 months of storage for all CPAs tested. JS679 JS510 All five CPAs yielded a similar range of survival rates, IV JS1087 with no significant differences (p > 0.05). Finally, JS1103 JS1093 Thraustochytrium sp. JS1089 had an initial cell density V JS659 of 7.0 × 106 CFU ml−1 and retained concentrations of JS660 3.2 × 105 − 5.8 × 106 CFU ml−1 (4–60% viability) after JS1128 VI JS1089 6 months of storage for all CPAs tested. The viability VIII JS0974 for JS1089 preserved with TG was the highest (60%) fol- 0 20 40 60 80 Fatty acids (as % total fatty acid) lowed by G (17%).

Figure 4: Polyunsaturated fatty acid profiles of the seven phylogenetic thraustochytrid groups isolated from the present study (as% of Cryopreservation of selected total fatty acids). thraustochytrids in different freezing conditions for Groups II and III. All groups studied contained high The survival rates of thraustochytrids preserved at levels of C22 PUFAs, ranging from 34.2 to 61.4% of TFA, −80°C and the vapor-phase of nitrogen were determined with lower levels of C20 PUFAs, varying from 1.8 to 20.5% using the best CPAs for each thraustochytrid tested over of TFA. Group I contained the highest level of DHA (51.7% 6 months. The best CPA used for cryopreservation at of TFA). Group V had the highest levels of ARA and EPA −80°C and liquid nitrogen for JS510 was 10% glycerol (G), (8.2 and 12.4% of TFA, respectively). In addition, Group while for JS702, JS1085 and JS1089, 5% trehalose com- VII contained relatively high levels of DPA (27.3% of TFA; bined with 10% glycerol (TG) was the best. The survival Table 4). rates of all strains tested at −80°C and in liquid nitrogen were similar during the first month of preservation (results not shown), but were significantly greater after Cryopreservation of thraustochytrids freezing in liquid nitrogen for 12 months than those kept at −80°C (Figure 5B). Parietichytrium sarkarianum JS510 Effect of cryoprotective agents for cryopreservation retained a cell concentration of 2.0 × 107 CFU ml−1 (20% at 80°C viability) after freezing at −80°C, whereas the cell density after freezing in liquid nitrogen was 3.5 × 107 CFU ml−1 The survival rates of thraustochytrids for all tested CPAs (35% viability). Aurantiochytrium sp. JS702 had a cell con- varied with species, whereas no viability of thraus- centration of 7.0 × 105 CFU ml−1 (20% viability) after freez- tochytrid cells was observed for the control experiment ing at −80°C, but 1.5 × 106 CFU ml−1 (43% viability) after (no CPA added). Figure 5A shows the survival rates of freezing in liquid nitrogen. Similarly, Thraustochytrium each species after freezing at −80°C. Parietichytrium sp. JS1085 showed 11% viability (3.1 × 107 CFU ml−1) after sarkarianum JS510 had a cell density before freez- freezing at −80°C, but 24% viability (6.5 × 107 CFU ml−1) ing of 7.3 × 107 CFU ml−1 and retained concentrations after freezing in liquid nitrogen. Finally, Thraustochytrium of 1.4 × 107 − 6.0 × 107 CFU ml−1 (20–61% viability) after sp. JS1089 showed 12% viability (8.7 × 105 CFU ml−1) after 6 months storage for all CPAs tested. Three CPAs (D, G freezing at −80°C, but 21% viability (1.5 × 106 CFU ml−1) and SG) provided the highest survival rates for JS510 after after freezing in liquid nitrogen. P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 373

Table 4: Fatty acid composition of the isolated thraustochytrids (as% of total fatty acids).

Group JS code BCC C14:0 C15:0 C16:0 C17:0 C18:0 C18:1 C18:2 C20:0 C20:2 C20:3n6 C22:0 C20:4 C20:5 C22:5 C22:6 code

I JS446 51316 0.15 1.23 32.68 1.73 0.42 0.25 0.29 – – – – – – 9.61 53.64 JS989 60533 0.36 1.61 35.10 1.47 0.94 – – – – – – – – 9.36 51.16 JS828 60447 0.58 1.99 27.03 1.43 0.45 – – – – – – – – 12.97 55.53 JS988 60532 0.49 1.64 32.89 1.54 0.62 – – – – – – – – 10.03 51.26 JS434 51304 0.25 1.67 31.78 1.97 0.49 – – – – – – 0.41 2.13 8.92 52.37 JS827 51305 0.26 1.18 32.58 1.81 0.40 0.18 0.07 – – – – – 2.22 8.81 52.48 JS490 52190 1.79 6.67 34.05 1.36 0.76 0.08 – 0.18 – – 0.06 0.30 0.28 8.57 45.91 Mean 0.55 2.28 32.30 1.62 0.58 0.07 0.05 0.04 – – 0.01 0.10 0.66 9.75 51.76 II JS702 55421 0.57 19.57 11.13 5.57 0.23 0.08 – 0.48 – 0.18 – 1.29 3.47 14.63 42.62 JS732 55449 0.81 25.29 14.40 8.09 0.31 0.12 – 0.29 – 0.19 – 0.76 2.22 11.16 36.36 JS736 56601 1.00 29.34 16.75 5.63 0.44 0.10 – 0.00 0.38 – – 1.00 2.74 10.86 31.76 Mean 0.79 24.73 14.09 6.43 0.33 0.10 – 0.26 0.13 0.12 – 1.02 2.81 12.22 36.91 III JS679 55166 0.19 20.19 5.23 10.33 0.79 – – – – – – 2.40 6.40 19.46 35.01 JS947 60491 0.29 23.52 3.69 8.61 0.12 – – – – – – 2.18 7.66 17.10 36.83 JS964 60508 – 16.47 5.20 6.87 – – – – – – – 2.72 10.18 19.72 38.84 JS727 55445 1.09 18.46 13.13 5.77 – – – 0.15 – 0.11 – 2.65 8.24 15.97 34.43 JS489 52189 0.45 18.34 13.75 7.82 0.16 – – – – – – 2.29 8.62 13.60 34.95 JS1115 77550 0.61 12.30 8.33 10.22 – – – – – – – 3.60 10.07 16.80 38.08 JS1085 72477 0.80 40.20 5.90 10.00 – – – – – – – 0.00 2.50 9.70 31.00 Mean 0.49 21.35 7.89 8.52 0.15 0.00 – 0.02 0.00 0.02 – 2.26 7.67 16.05 35.59 IV JS1087 75567 0.83 7.23 20.39 7.30 3.34 13.01 – – – – – 6.47 5.89 19.75 15.80 JS510 52209 1.48 2.05 24.49 2.48 7.58 15.79 – – – 2.94 – 6.78 3.51 19.88 13.01 Mean 1.15 4.64 22.44 4.89 5.46 14.40 – – – 1.47 – 6.62 4.70 19.81 14.40 V JS660 54862 0.00 0.00 17.58 5.55 0.63 0.26 – – 1.15 – – 6.17 13.24 19.16 36.25 JS659 54861 0.37 7.46 15.71 6.34 0.79 2.41 – – 0.00 – – 3.63 11.60 12.98 38.70 JS1093 75573 0.00 4.20 13.90 5.50 0.00 0.00 – – 0.00 – – 11.10 11.70 26.90 26.70 JS1103 75583 0.00 3.20 13.20 4.00 0.00 0.00 – – 0.00 – – 12.10 13.00 26.50 27.90 Mean 0.09 3.72 15.10 5.35 0.36 0.67 – – 0.29 – – 8.25 12.39 21.39 32.39 VI JS1089 75569 1.74 19.15 20.29 7.48 0.00 0.00 – – – – – 11.29 5.16 12.56 22.33 JS1128 77563 1.31 9.31 23.99 9.90 4.08 4.60 – – – – – 12.05 7.10 4.93 22.71 Mean 1.53 14.23 22.14 8.69 2.04 2.30 – – – – – 11.67 6.13 8.75 22.52 VII JS974 60518 1.06 2.92 16.73 1.33 1.55 0.59 – – – – – 6.52 7.93 27.30 34.08

well as thraustochytrids, as they were found to be a rich Discussion source of sugars, starch, protein, amino acids, lipid, sterol and other trace vitamins (Hayakawa et al. 1991, Phuphu- Isolation, diversity and fatty acid production mirat et al. 2011, Gupta et al. 2013). In this study, pine of marine thraustochytrids from Thailand pollen baiting was found to be a suitable technique for obtaining slow-growing thraustochytrids from seawater, In this study, thraustochytrids isolated from decay- especially those that potentially represent new lineages. ing mangrove leaves represented the dominant group, Seven major lineages of the isolated thraustochytrids whereas a small proportion of thraustochytrids was iso- can be distinguished based on morphological features lated from seawater using the pine pollen baiting tech- and molecular phylogeny. Groups I and IV can be identi- nique (Porter 1990). Most of the strains isolated from pine fied as Aurantiochytrium limacinum and Parietichytrium pollen baiting were placed in clades II, V and VII in the sarkarianum, respectively, based on their morphological phylogenetic tree (Figure 3). The direct plating of fallen features. Groups II, III, and VI can be identified to generic mangrove leaves poses a higher risk of contamination by level as Aurantiochytrium sp., Schizochytrium sp. and rapidly growing fungi, yeasts and antibiotic-resistant bac- Thraustochytrium sp., respectively. Finally, observations teria (Gupta et al. 2013). Pollen grains have been widely of the remaining taxa in Groups V and VII revealed that used as a bait for zoosporic fungi and actinobacteria, as they could potentially represent new lineages within the 374 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation

A 100 This study assessed the diversity of newly isolated marine thraustochytrids from various mangrove locations DG HD SG TG 80 in Thailand. The most common species was identified as A. limacinum, with other strains identified as species of 60 the genera Schizochytrium, Parietichytrium and Thraus- 40 tochytrium. This is in agreement with the observations of Viability (%) Yang et al. (2010), in which newly isolated thraustochytrids 20 from Taiwan belonged to Aurantiochytrium and Thraus- tochytrium, although Aplanochytrium and Oblongichytrium 0 Parietichytrium Aurantiochytrium Thraustochytrium Schizochytrium sp. R. Yokoyama et D. Honda were also found. Chang et al. sarkarianum JS510 sp. JS702 sp. JS1085 JS1089 (2012) identified four genera, namely Aurantiochytrium, B 60 Schizochytrium, Thraustochytrium and Ulkenia A. Gaertn. from Australian marine environments. Moreover, Gupta 50 –80°C LN et al. (2013) identified thraustochytrid strains from Aus- 40 tralia as species of Schizochytrium, Thraustochytrium and

30 Ulkenia using a pine pollen baiting technique. In addition, our observation is in agreement with a recent study by Ou

Viability (%) 20 et al. (2016), in which Aurantiochytrium was dominant in 10 Malaysian mangroves. Differences in species diversity and composition may be attributed to different substrata and 0 Parietichytrium Aurantiochytrium Thraustochytrium Schizochytrium sarkarianum sp. JS702 (TG) sp. JS1085 (TG) sp. JS1089 (TG) environmental factors, such as water temperature, salin- JS510 (G) ity and nutrient sources (Raghukumar 2002, Ueda et al. Figure 5: Results of cryopreservation studies on Thai 2015). Identification of thraustochytrids has been found thraustochytrids. to be difficult. No single morphological character can (A) Mean viability (±standard deviation, n = 15) of four strains of be used to classify these organisms (Honda et al. 1999). marine thraustochytrids after cryopreservation for 6 months at Major limitations for the identification of marine thraus- ° = −80 C using five different cryoprotective agents (D 5% DMSO; tochytrids include their morphological complexity and G = 10% glycerol; HD = 30% horse serum combined with 10% DMSO; SG = 17% skim milk combined with 20% glycerol; TG = 5% trehalose the limited number of named sequences in the public combined with 10% glycerol). (B) Mean viability (±standard devia- databases for comparison. tion, n = 15) of four strains of marine thraustochytrids after cryo- In our study, all the thraustochytrids isolated from preservation for 12 months at −80°C and in vapor-phase nitrogen Thailand produced PUFAs, such as ARA, EPA, DPA and (LN) using the optimal cryoprotective agent for each strain (shown DHA. This result is similar to earlier studies by Chang et al. in brackets). (2012) and Gupta et al. (2016), which showed the presence of PUFAs in thraustochytrids isolated from various habitats in Australia and India. They noted that DHA consti- Labyrinthulomycetes based on morphological and mole- tuted 20–50% of TFA, and was the major PUFA found in cular evidence. Morphologically, all members nestled in their strains. The others, ranging from 1 to 12% of TFA, Group V possessed hyaline thick-walled thalli and unde- were DPA, ARA and EPA. In the present study, the highest veloped ectoplasmic net elements. Strains JS659 and yielding isolates of DHA appeared in Group I belonging to JS660 had large zoosporangia (20–177 μm), with large vesi- A. limacinum (51% of TFA), which is similar to the strains cles appearing inside the zoosporangia (Figure 2Q–X). In BL8 and BL10 (51% and 47%, respectively) reported by addition, strain JS974 (clade VII, Figure 2AC–AJ) was dis- Chang et al. (2012). Since the biomass and TFA production tinct in its irregularly shaped colonies with an undulating of Group I isolates (1.0 g l−1, 10.2% w/w) were lower than margin, sometimes producing elongated amorphous cells those of Group II isolates (3.1 g l−1, 19.4% w/w), however, at the margin of the colony. The zoosporangia were unique the DHA production of Group I isolates was lower than with large vesicles and ectoplasmic net elements. Numer- Group II per unit volume (52.7 and 221.9 mg l−1, respec- ous cigar-shaped and limax amoebae were also observed. tively). Thus, the strains in Groups I and II should be Comparison of Thraustochytrium sp. JS974 with T. gaertne- examined for further manipulation in order to maximize rium, the most closely similar taxon, revealed that JS974 DHA production and evaluate their potential for com- differs by possessing larger zoosporangia and a greater mercial application. Thraustochytrids from other groups number of proliferation bodies (Bongiorni et al. 2005a). produced other C20–22 PUFAs including ARA, EPA and P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 375

DPA ranging from 0.3 to 27.3% of TFA. Interestingly, EPA and trehalose have been widely used as CPAs in preserv- is an important fatty acid for commercial applications. ing various microorganisms. Glycerol has the ability to The present study indicates that the level of EPA produced penetrate both cell wall and cell membrane, whereas tre- by the strains in this study (12.3% of TFA) is comparable halose is a non-permeable CPA (Hubálek 2003). The use of with that of Australian strains (12.6%; Gupta et al. 2016). combined CPA types, which have synergistic effects, may The results also showed that the highest amount of DPA enhance the viability of cells or tissues after cryopreserva- (27.3% of TFA) was produced by a potentially new thraus- tion (Crowe et al. 1984). tochytrid lineage JS974 (Table 4). In the present study, thraustochytrids stored in vapor- PUFA composition is a key chemotaxonomic character phase nitrogen showed higher viability than preservation for thraustochytrids, in addition to saturated fatty acids in −80°C (Figure 5B). After 12-month storage in liquid nitro- and the presence of odd chain PUFA (OC-PUFA; Huang gen, the survival rates of thraustochytrids ranged from et al. 2003, Yokoyama et al. 2007, Chang et al. 2014). The 21 to 43% in different species. The method for long-term present study revealed that the profile and proportion of preservation of thraustochytrid cultures in liquid nitrogen PUFAs (particularly C20–22) of the strains clustered in the was originally developed by Bremer (2000). The cultures same phylogenetic clade were similar (Figure 4, Tables 3 were preserved under liquid nitrogen together with pollen and 4). However, the fatty acid profile might be affected grains, without the use of a cryoprotective agent. Some by culture conditions (Chang et al. 2014). Yokoyama et al. cultures survived after many years of storage, but not all (2007) distinguished the genus-level phylogenetic groups (Bremer 2000). The only recent documented cryopreserva- in the Labyrinthulomycetes by a combination of mor- tion technique for marine thraustochytrids was developed phological and chemotaxonomic features such as PUFA by Cox et al. (2009). They noted that the use of a combi- profiles and carotenoid pigments. Thus, it is necessary to nation of 30% horse serum and 10% DMSO (“HD” in this establish taxonomic criteria for thraustochytrids based study) was the most effective CPA for liquid nitrogen cryo- on a combination of phenotypic, chemical and molecular preservation (1 month of storage) of thraustochytrids from characteristics. New Zealand. Storage in liquid or vapor-phase nitrogen is the most universally applicable preservation method for various microbes, such as fungi, bacteria, viruses and pro- Cryopreservation of selected marine tozoa, as well as animal, algal and plant cells (Snell 1991). thraustochytrids However, there are some limitations to using liquid nitrogen for long-term preservation of microbes. It requires The survival rates of thraustochytrid strains isolated specialized equipment, operating and maintenance from Thailand after preservation in a −80°C freezer and systems and a regular supply of liquid nitrogen (Day and vapor-phase nitrogen (−187°C) varied between species. Brand 2005). Our observations revealed that the thraus- Aurantiochytrium sp. JS702 and Thraustochytrium sp. tochytrid strains tested can be frozen in vapor-phase liquid JS1085 exhibited relatively poor survival rates after being nitrogen and retain 26–50% viability for 6 months (data frozen at −80°C. Parietichytrium sarkarianum JS510 and not shown). Nevertheless, the effectiveness of cryopreser- Schizochytrium sp. JS1089, with larger vegetative cells and vation in microorganisms will be influenced by multiple zoosporangia, appeared to be more tolerant to cryopreser- factors, including species or strain, cell structure, growth vation. This is in agreement with a study suggesting that phase and rate, growth medium, incubation, tempera- algae with a large cell size are more tolerant to cryopreser- ture, cell density before freezing, type and concentration vation (Day and Brand 2005). Prolonged storage at −80°C of CPAs, cooling rate, warming rate, preserved time and as well as in liquid nitrogen was found to reduce cell via- recovery medium (Hubálek 2003). bility. The loss of viability is related to cell damage due In conclusion, our study has investigated the diversity to the formation of ice and osmotic pressures (Snell 1991, of newly isolated marine thraustochytrids from Thailand. Miyamoto-Shinohara et al. 2000). All of the strains isolated produced commercially useful Our results have established suitable CPAs and cryo- high-value fatty acids. Two of the seven thraustochytrid preservation conditions for the economically valuable groups isolated could represent potential new lineages marine thraustochytrids recently discovered in Thailand. in the Labyrinthulomycetes based on their unique mor- The most effective CPAs after cryopreservation at −80°C phological features, molecular phylogeny and fatty acid were 10% glycerol (G) and the combination of 5% treha- profiles. The thraustochytrids were successfully pre- lose and 10% glycerol (TG), which maintained good viabil- served at −80°C or in liquid nitrogen. Glycerol, alone and ity for most of the thraustochytrid strains tested. Glycerol in combination with trehalose, yielded relatively high 376 P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation survival rates for the thraustochytrid strains. The thraus- Cox, S.L., D. Hulston and E.W. Maas. 2009. Cryopreservation of tochytrids tested were well-preserved for at least 1 year in marine thraustochytrids (Labyrinthulomycetes). Cryobiology 59: 363–365. vapor-phase liquid nitrogen. The information provided in Crowe, J.H., L.M. Crowe and D. Chapman. 1984. Prevention of this study could be applied to long term preservation of membranes in anhydrobiotic organisms: the role of trehalose. the economically valuable marine Labyrinthulomycetes. Science 223: 701–703. Further investigation of media optimization for higher Day, J.G. and J.J. Brand. 2005. Cryopreservation methods for PUFA yields and production is necessary. maintaining microalgal cultures. In: (R.A. Andersen, ed.) Algal Culturing Techniques, Academic Press, New York, USA. pp. 165–187. Acknowledgments: This work was supported by National DMCR (Department of Marine and Coastal Resources). 2017. Central Science and Technology Development Agency (grant nos. Database System and Data Standard for Marine and Coastal P-11-00363 and P-13-50213). The authors acknowledge Prof. Resources. http://marinegiscenter.dmcr.go.th/km/mangroves_ Morakot Tanticharoen, Dr. Kanyawim Kirtikara, Dr. Lily doc01/#.WIhJH1N97IW. Eurwilaichitr, and Dr. Janet Jennifer Luangsa-ard for con- Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. tinued support. We are grateful to Department of Marine Gupta, A., S. Wilkens, J.L. Adcock, M. Puri and C.J. Barrow. 2013. and Coastal Resources staff for field support. Thanks go to Pollen baiting facilitates the isolation of marine thraus- BIOTEC Culture Collection for the laboratory facility. tochytrids with potential in omega-3 and biodiesel production. J. Ind. Microbiol. Biotechnol. 40: 1231–1240. Gupta, A., A. Singh, A.R. Byreddy, T. Thyagarajan, S.P. Sonkar, A.S. Mahtur, D.K. Tuli, C.J. Barrow and M. Puri. 2016. 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Yokoyama, R., B. Salleh and D. Honda. 2007. Taxonomic rearrange- research interests include marine mycology and molecular phyloge- ment of the genus Ulkenia sensu lato based on morphology, netics. Recently, she has worked on a morphological and molecular chemotaxonomical characteristics, and 18S rRNA gene phylog- phylogeny study of marine fungi. Other research work includes eny (Thraustochytriaceae, Labyrinthulomycetes): emensation crude lipid and fatty acid extraction from marine microbes. for Ulkenia and erection of Botryochytrium, Parietichytrium and Sicyoidochytrium gen. nov. Mycoscience 48: 329–341. Anupong Klaysuban National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency Bionotes (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Panida Unagul Amphoe Khlong Luang, Pathum Thani 12120, National Center for Genetic Engineering and Thailand Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Anupong Klaysuban is a lab technician at National Center for Phahonyothin, Tambon Khlong Nueng, Genetic Engineering and Biotechnology (BIOTEC), Thailand. He Amphoe Khlong Luang, Pathum Thani 12120, holds a Bachelor’s degree in Applied Biology from Chandrakasem Thailand Rajabhat University. His research interests focus on marine, endo- [email protected] phytic fungi and polyunsaturated fatty acid producing microbes.

Panida Unagul is a researcher at National Center for Genetic Engi- Wunna Gundool neering and Biotechnology (BIOTEC), Thailand. She was awarded National Center for Genetic Engineering and a PhD in Fermentation Technology by King Mongkut’s University of Biotechnology (BIOTEC), National Science Technology Thonburi for working on production of docosahexaenoic and Technology Development Agency acid (DHA) by Schizochytrium. Her current research is focused on (NSTDA), 113 Thailand Science Park, Thanon microbial production of a variety of bio-products such as fatty acids, Phahonyothin, Tambon Khlong Nueng, bioactive compounds and enzymes. Amphoe Khlong Luang, Pathum Thani 12120, Thailand Satinee Suetrong National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science Wunna Gundool is a researcher assistant at Fungal Biodiversity and Technology Development Agency Laboratory, National Center for Genetic Engineering and Biotechnol- (NSTDA), 113 Thailand Science Park, Thanon ogy (BIOTEC), Thailand. She obtained her BSc from Mahasarakham Phahonyothin, Tambon Khlong Nueng, University. She currently works on the diversity of marine fungi in Amphoe Khlong Luang, Pathum Thani 12120, Thailand. Thailand Chanwit Suriyachadkun National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science Satinee Suetrong is a researcher at Fungal Biodiversity Labora- and Technology Development Agency tory, Biodiversity and Biotechnological Resource Research Unit (NSTDA), 113 Thailand Science Park, Thanon (BBR), National Center for Genetic Engineering and Biotechnology Phahonyothin, Tambon Khlong Nueng, (BIOTEC), Thailand. She earned her PhD in Microbiology from Prince Amphoe Khlong Luang, Pathum Thani 12120, of Songkla University, Thailand. Her research interests include Thailand marine Dothideomycetous fungi, rock inhabiting and deteriorating fungi in Thailand − isolation, characterization, and their natural products and polyunsaturated fatty acid producing microbes. Chanwit Suriyachadkun is a researcher at BIOTEC Culture Collection Sita Preedanon Laboratory, National Center for Genetic Engineering and Biotech- National Center for Genetic Engineering and nology (BIOTEC), Thailand. He was awarded a PhD in Bioscience by Biotechnology (BIOTEC), National Science Kasetsart University, Thailand for working on selection and taxo- and Technology Development Agency nomic characterizations of novel species belonging to the family (NSTDA), 113 Thailand Science Park, Thanon Streptosporangiaceae isolated from Thailand and evaluation of Phahonyothin, Tambon Khlong Nueng, selected strains for biological control of rice pathogens. His current Amphoe Khlong Luang, Pathum Thani 12120, research is focused on diversity of Actinomycetes and microbial Thailand preservation.

Sita Preedanon works as a research assistant at Fungal Biodiversity Laboratory, Bioresources Technology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Her P. Unagul et al.: Thai marine thraustochytrids and their cryopreservation 379

Jariya Sakayaroj National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang, Pathum Thani 12120, Thailand [email protected]

Jariya Sakayaroj works as a researcher and Lab Head of Fungal Biodiversity Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. She has published extensively on diversity, molecular phylogenetics and chemical constituents of various fungal groups. Her research interests include diversity and phylogenetic study of marine and endophytic fungi. Other areas of current research include study and application of fungal-like microbes producing high value fatty acids.