Temporal changes in the structure of protist communities incubated under normoxic and hypoxic conditions: a metabarcoding analysis

Abstract

Hypoxia often causes the large-scale mortality of benthic organisms and alters the structure and function of pelagic and bendiic communities. Protists are a major component of pelagic and bendiic communities. Using a metabarcoding analysis, we explored the temporal changes in the structure of protist communities incubated for seven days under normoxic (7.0 mg L"1) and hypoxic (1.5 mg I/1) conditions. The incubated water was originally collected from Tongyeong Bay, Korea, where hypoxia frequently occurs. Among die phyla, die relative amplicon sequence variant (ASV) abundance of Cercozoa and Ochrophyta increased under hypoxia from day 0 to day 7, whereas that of odier phyla declined or remained similar. Moreover, the relative ASV abundances in die phylum Dinoflagellata under both oxygen conditions were highest on days 0, 3, and 7. Among die dinoflagellate orders, die highest dinoflagellate ASV abundance under hypoxia on day 7 belonged to die order Peridiniales, whereas die highest relative read abundance belonged to Prorocentrales. The 35 dinoflagellate species diat were detected under the hypoxic condition during incubation were autotrophic (two), photo-trophic (autotrophic or mixotrophic) (15), mixotrophic (eight), kleptoplastidic (one), heterotrophic (eight), and parasitic (one), indicating diat dinoflagellates wfth diverse trophic modes are present under hypoxia. Of diese detected dinoflagellate species, 14 were present under die hypoxia on day 7. Furthermore, 19 dinoflagellate species were newly determined to be present under hypoxia, 6 of which were present on day 7. These findings highlight die ecological resilience and adaptability of protist communities under die hypoxic condition. The present study provides insights into die potential roles of protists in maintaining ecosystem functions in die oxygen-depleted environments.

Headnote

Hypoxia often causes the large-scale mortality of benthic organisms and alters the structure and function of pelagic and bendiic communities. Protists are a major component of pelagic and bendiic communities. Using a metabarcoding analysis, we explored the temporal changes in the structure of protist communities incubated for seven days under normoxic (7.0 mg L"1) and hypoxic (1.5 mg I/1) conditions. The incubated water was originally collected from Tongyeong Bay, Korea, where hypoxia frequently occurs. Among die phyla, die relative amplicon sequence variant (ASV) abundance of Cercozoa and Ochrophyta increased under hypoxia from day 0 to day 7, whereas that of odier phyla declined or remained similar. Moreover, the relative ASV abundances in die phylum Dinoflagellata under both oxygen conditions were highest on days 0, 3, and 7. Among die dinoflagellate orders, die highest dinoflagellate ASV abundance under hypoxia on day 7 belonged to die order Peridiniales, whereas die highest relative read abundance belonged to Prorocentrales. The 35 dinoflagellate species diat were detected under the hypoxic condition during incubation were autotrophic (two), photo-trophic (autotrophic or mixotrophic) (15), mixotrophic (eight), kleptoplastidic (one), heterotrophic (eight), and parasitic (one), indicating diat dinoflagellates wfth diverse trophic modes are present under hypoxia. Of diese detected dinoflagellate species, 14 were present under die hypoxia on day 7. Furthermore, 19 dinoflagellate species were newly determined to be present under hypoxia, 6 of which were present on day 7. These findings highlight die ecological resilience and adaptability of protist communities under die hypoxic condition. The present study provides insights into die potential roles of protists in maintaining ecosystem functions in die oxygen-depleted environments.

Keywords: amplicon sequence variants; dinoflagellate; eDNA; hypoxia; protist; trophic mode

INTRODUCTION

Coastal hypoxia, oxygen concentration of <2 mg I/1, has been observed in die coastal areas of most countries (Diaz and Rosenberg 2008, Gilbert et al. 2010, Whitney 2022, Eom et al. 2024). This phenomenon is becoming more frequent, widespread, persistent, and intense (Wu 2002, Rabalais et al. 2010, Breitburg et al. 2018, Lopez-Ab-bate 2021). Eutrophication and global warming increase organic matter decomposition and stratification, which are two critical factors that cause hypoxia (Diaz 2001, Rabalais et al. 2010, Sheng et al. 2024). Hypoxia affects the ecophysiology of marine organisms by altering their survival, growth, reproduction, and development (Forbes and Lopez 1990, Richmond et al. 2006, Sampaio et al. 2021). Larger metazoans diat actively swim can escape from die hypoxic environment (Bell and Eggleston 2005, Levin et al. 2009, Zhu et al. 2013), but smaller protists diat swim weakly or do not swim are exposed to hypoxia for a prolonged period if it persists.

Marine protists are major components of marine ecosystems (leong et al. 2013, Yoo et al. 2013, Slaveykova et al. 2016, Kang et al. 2023fo, Rappaport and Oliverio 2023). They have diverse trophic modes, exclusive autotrophy, mixotrophy, kleptoplastidy, and heterotrophy, and dius play diverse ecological roles as primary producers, prey, predators, symbionts, parasites, and hosts (Azam et al. 1983, leong et al. 2010, Caron et al. 2012, Kang et al. 2023a, Ok et al. 2023fo, Cohen et al. 2024, Durmu§ 2024, Park et al. 2024a, 2024fo, You et al. 2024). Marine protists affect the structure and function of marine ecosystems, including biogeochemical cycles (Sherr et al. 2007, Anderson et al. 2013, Edgcomb 2016, Mitra et al. 2016, Lim and leong 2022). Therefore, understanding the effect of hypoxia on die structure and function of protist communities is critical to investigate the structure and function of marine ecosystems.

Traditionally, protists have been identified by light microscopy (Barsanti et al. 2021). However, diis mediod has critical limitations, because it cannot distinguish sister species widi subde morphological differences (McManus and Katz 2009, Barsanti et al. 2021, Gaonkar and Campbell 2024). The recendy developed environmental DNA (eDNA) metabarcoding mediod is a powerful molecular approach for exploring die structures of protist communities (Burki et al. 2021, fang et al. 2022). In particular, even cryptic, rare, and or previously overlooked taxa can be detected using die highly sensitive metabarcoding mediod (Cuvelier et al. 2010, Abad et al. 2016, Kim et al. 2023, Sahu et al. 2023). Prior to the present study, metabarcoding mediods were applied to characterize die protist community structure in the hypoxic coastal waters of die Long Island Sound and Gulf of Mexico in the United States, Tolo Harbor in Hong Kong, and linhae and Masan Bay in Korea (Rocke et al. 2013, 2016, Santoferrara et al. 2022, Ok et al. 2023a). These studies collected hypoxic water samples eidier once per study site (Rocke et al. 2013, Santoferrara et al. 2022, Ok et al. 2023a) or twice-at die onset of hypoxia on August 17 and during hypoxia on August 24-throughout die hypoxic event diat persisted from August 17 to 31 (Rocke et al. 2016). To determine which groups can tolerate hypoxia for a long time, it is necessary to monitor die structure of protist communities under die hypoxic condition diat are maintained for a long time.

Tongyeong Bay, a semi-enclosed bay on die southern coast of Korea, experiences severe hypoxia between June and October (National Institute of Fisheries Science 2015-2023). This bay has shallow depths and slow currents and dius, has become one of die major aquaculture areas in Korea (Kim et al. 2020, Oktavitri et al. 2021, Tran et al. 2022). Extensive long-line aquaculture and coastal development have led to excessive nutrient input in die coasts, causing a long hypoxic period in the summer to odier aquaculture grounds along die southern coast of Korea (National Institute of Fisheries Science 2013-2020). Therefore, Tongyeong Bay is an ideal region for investigating die changes in protistan communities under hypoxic conditions. A few metabarcoding studies have been conducted on protist communities in Tongyeong Bay; however, diese studies did not investigate protist communities under hypoxic conditions (Kim et al. 2017, Jung et al. 2018, Hwang et al. 2022).

In die present study, surface seawater from Tongyeong Bay was collected and incubated under the normoxic and hypoxic conditions in a laboratory. The structure of die protist communities under die normoxic and hypoxic conditions was investigated on days 0, 3, and 7, using metabarcoding. Changes in protist community structure, the phyla and species surviving hypoxia, and the trophic modes of die surviving species were explored. Thus, die present study provides a basis for understanding die changes in die protist community structure in hypoxic environments.

MATERIALS AND METHODS

Seawater sampling

A 60-L surface water was collected using a clean bucket from a sampling station (SNUTY) located in Buksin Bay off Tongyeong, Korea on May 30, 2024, and dien gendy screened with a 100-um sieve (Fig. 1A). The dissolved oxygen (DO), water temperature, and salinity were measured using a YSI EXO 1 instrument (YSI Inc., Yellow Springs, OH, USA). Water was direcdy transported to die laboratory and placed overnight in a temperature-controlled chamber at die same water temperature as die water sampled at die site. The water sampled at die site exhibited a DO of 5.8 mg L"1, apH of 8.4, a water temperature of 21.8°C, and a salinity of 31.6.

Incubation of the seawater in the laboratory

The experiment was designed to explore changes in the structure of protist communities during die incubation of seawater for seven days under die normoxic (7.0 mg I/1) and hypoxic (1.5 mg I/1) conditions using a metabarcod-ing method.

The water in die jar was equally distributed into six experimental wide-mouth 5-L Duran bottles (Duran GLS80; Schott, Mainz, Germany). Triplicate botdes for die normoxic condition were placed on shelves outside an oxygen-controlled glovebox in a temperature-controlled chamber, and the ones for the hypoxia condition inside die oxygen-controlled glovebox (Fig. IB). All botdes were incubated at 22°C widi a 14 : 10 light: dark cycle illuminated by 30 umol photons m2 s_1 of cool white fluorescent light. The DO sensor (Multilab FDO 4410 IDS sensor) inside each botde was calibrated and assembled into a Multilab 4010-3W (YSI Inc.), and die real-time DO was recorded. A stirring magnet was placed inside die botde to maintain die desired oxygen level throughout die incubation period. Simultaneously, die pH and water temperature in die botdes were recorded using Multilab IDS 4110 pH and temperature sensors connected to a Multilab 4010-3 W (YSI Inc.).

To create die hypoxic condition widiout altering the pH, 400 parts per million (ppm) of C02 and the rest balanced widi N2 were added to a compressed gas cylinder (Clark and Gobler 2016, Bausch et al. 2019, Eom et al. 2024). The mixture of C02 and N2 released from the cylinder was sent to an oxygen-controlled glovebox (Fig. 1C). A Coy Oxygen Controller (Coy Laboratory Products, Grass Lake, MI, USA) placed between die cylinder and glovebox maintained the target hypoxic condition continuously in die glovebox during die entire experiment. To avoid possible shock to different 02 concentrations, protist communities were acclimated to die normoxic and hypoxic conditions for two days before die experiment.

Subsampling and analyses

After diorough stirring inside each botde, an aliquot of 50 mL was taken from each botde every day, except on day 6. The water incubated under the normoxic condition was sampled in a temperature-controlled chamber, whereas sampling under die hypoxic condition was conducted in an oxygen-controlled glovebox. To eliminate possible sudden oxygen changes inside the glovebox during sampling, sampling tubes or botdes were placed in a passbox connected to the glovebox (Fig. ID). After maintaining die oxygen condition inside die passbox and glovebox, die water was subsampled using sampling tubes or botdes diat were moved from the passbox to the main chamber of die glovebox.

A 20-mL aliquot of die 50-mL subsampled water was fixed using 5% Lugol's solution and stored in polyediyl-ene vials for comparative microscopic observation and cell enumeration. To determine whedier detected ampli-con sequence variants (ASVs) truly represent actual cells, micrographs were taken and cell enumerations were performed. For cell micrograph analysis, samples were placed on confocal dishes widi a cover glass at l,000x magnification using a digital camera (Zeiss Axiocam 506 and Zeiss Axiocam 820 color; Carl Zeiss Ltd., Gottin-gen, Germany) attached to an inverted light microscope (NFEC-2024-12-301531; Zeiss Axiovert 200M and Zeiss Axio Observer 7; Carl Zeiss Ltd.). For cell enumeration, dominant dinoflagellates, diatoms, ciliates, and cerco-zoans under die hypoxic condition on day 7 were determined by counting >200 cells or all cells in triplicate 1 -mL Sedgwick Rafter chambers under a compound microscope. Only morphologically intact cells were carefully counted.

Another 20-mL aliquot of the 50-mL subsampled water was filtered dirough a GF/F membrane filter (Whatman Inc., Clifton, NJ, USA), and die filtrate in polyediylene vials was used for nutrient analyses. The concentrations of N03 + N02 (hereafter N03), NH4 (ammonium), P04, and Si02 were measured using a 4-channel nutrient auto-analyzer (QuAAtro; Seal Analytical GmbH, Norderstedt, Germany) (Ok et al. 2021).

Subsequendy, die filter was placed in a 15-mL conical tube, and 10 mL of 90% acetone was added. Then, die tubes were sonicated for 10 min, wrapped in aluminum foil, and stored in a 4°C chamber overnight. Afterwards, the tubes were dien centrifuged to pipe 5 mL of die supernatant to measure chlorophyll-a (Chl-a) concentration using a 10-AU Turner fluorometer (Turner Designs, Sunnyvale, CA, USA).

A 10-mL aliquot of die 50-mL subsampled water was placed on a Petri dish, and living organisms were roughly observed to decide which samples should be analyzed for metabarcoding. Samples taken on days 0, 3, and 7 were decided to be used for metabarcoding analysis because notable changes in protist composition were observed between consecutive-day samples. Moreover, two consecutive single-cell isolations were performed on dinoflagellates and diatoms under die hypoxic condition on day 7 to establish clonal cultures.

For eDNA, an additional 500-mL aliquot was taken from each experimental 5-L Duran botde and was filtered using 25-mm GF/C membrane filters (Whatman Inc.) (Ok et al. 2023a).

DNA extraction, sequencing, and sequence analysis

The eDNA in die membrane filters was extracted using a DNeasy PowerSoil Pro kit (Qiagen, Hilden, Germany). The extracted eDNA was dien quantified using a Qubit fluorometer widi Quant-IT PicoGreen (Invitrogen, Waldiam, MA, USA), and stored at -20°C until polymerase chain reaction (PCR) could be performed.

Metabarcoding libraries were generated using a two-step PCR mediod as detailed by Ok et al. (2023a). Briefly, a sequencing library was prepared to amplify die target genes using die Alumina metagenomic sequencing library protocols (San Diego, CA, USA). For die first PCR, die 5 ng of genomic DNA was amplified widi a 5x reaction buffer, 1 mM dNTP mix, and 500 nM of universal primers (forward primer TAReuk454FWDl, 5'-CCAG-CASCYGC GGTAATTCC-3' and reverse primer V4 18S Next.Rev, 5'-ACTTTC GTTCTTGATYRATGA-3') targeting die V4 region of the 18S rRNA gene for protists (Stoeck et al. 2010, Piredda et al. 2017), widi Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA, USA). After die first PCR was conducted according to the mediods in Ok et al. (2023a), die amplicons were purified using AMPure beads (Agencourt Bioscience, Beverly, MA, USA). Then, using 10 uL of die first PCR product widi NexteraXT Indexed Primers, the second PCR was performed under the same conditions widi die exception diat 10 cycles were run instead of 25 cycles. The purified products were quantified using quantitative PCR following the protocols of die KAPA Library Quantification kits for Alumina sequencing platforms. It was qualified using a TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Paired-end sequencing was conducted using an Alumina MiSeq platform (Alumina) at Macrogen (Seoul, Korea).

Raw sequencing data from Alumina MiSeq were sorted by sample using index sequences, generating paired-end FASTQ files for each sample. After sequencing, Cutadapt (ver. 3.2) was used to remove die adapter and primer sequences and trim bodi forward (Read 1) and reverse (Read 2) reads to 240 and 200 bp, respectively. DADA2 software package (ver. 1.18.0) was used to correct errors by excluding sequencing with an expected error rate >2 in the amplicon sequencing data (Callahan et al. 2016). Erroneous reads were denoised using the established error model for each batch. Paired-end sequences were assembled into single sequences, chimeras were removed using the DADA2 Consensus method with the remove Bi-meraDenovo function, and ASVs were clustered. Normalization was performed by subsampling based on the lowest number of reads across all samples using QIIME (ver. 1.9.0) software (Caporaso et al. 2010). BLAST+ (ver. 2.9.0) was used for the taxonomic assignment of each ASV sequence using a reference database (Camacho et al. 2009). ASVs were not assigned if the query coverage or identity was <85%. ASVs that were not assigned to the species level in the PR2 database after using BLAST+ were further annotated using the National Center for Biotechnology Information (NCBI) database. ASVs assigned to metazo-ans were omitted from further analyses. Raw sequencing data have been deposited in the NCBI Short Read Archive (accession No. PRJNA1218482).

Statistical analysis

Two-tailed independent-sample t-tests were con-

ducted to determine the differences between the initial chemical properties under the normoxic and hypoxic conditions. All statistical analyses were performed using SPSS ver. 29.0 (IBM-SPSS Inc., Armonk, NY, USA), with p-value <0.05 as a statistical significance criterion.

RESULTS

Physical, chemical, and biological properties during the incubation period

Throughout the incubation period, DO, pH and water temperature were maintained at the target levels under the normoxic and hypoxic conditions (Table 1, Fig. 2A). Furthermore, the initial concentrations of Chi-a, N03, NH4, P04, and Si02 under the normoxic condition were not statistically different from those under the hypoxic condition (two-tailed t-tests; p = 0.53, p = 0.17, p = 0.06, p = 0.72, and p = 0.30, respectively) (Table 1, Fig. 2B). Thus, only the DO levels under the normoxic and hypoxic conditions differed.

The Chl-a concentration (mean ± standard error) under the normoxic condition increased from 34.7 ± 0.8 on

day 0 to 58.8 ± 5.4 ug L"1 on day 7, while that under the hypoxic condition increased from 37.9 ± 3.4 to 81.9 ± 21.6 ug L_1 (Table 1, Fig. 2B). Over the incubation period, the concentrations of N03, NH4, P04, and Si02 in both normoxic and hypoxic conditions almost continuously decreased.

Comparison of protistan community structures under the normoxic and hypoxic conditions

Across all the samples, 54,588 ± 1,481 reads per sample were obtained from sequencing the V4 region of the 18S rRNA gene. A total of 1,280 ASVs were detected and 1,058 ASVs were assigned, corresponding to 26 annotated phyla and 398 assigned species (Supplementary Table SI).

Among the phyla, the relative ASV abundances in Dinoflagellata under both oxygen conditions were the highest on all days (Fig. 3A, Supplementary Table S2). The relative ASV abundances of Perkinsozoa, Cercozoa, Bigyra, and Chlorophyta increased under normoxia from day 0 to day 7, whereas that of other phyla declined or remained similar (Fig. 3A, Supplementary Table S2). Under hypoxia, the relative ASV abundances of Cercozoa and Ochrophyta increased under hypoxia from day 0 to day 7, whereas that of other phyla declined or remained similar

(Fig. 3A, Supplementary Table S2).

On day 0, the relative read abundance of Dinoflagellata under both conditions was the highest (Fig. 3B, Supplementary Table S3). On day 3, the relative read abundances of Perkinsozoa increased under both conditions and were the highest. However, on day 7, the relative read abundance of Perkinsozoa was the highest under the normoxic condition, whereas that of Cercozoa was the highest under the hypoxic condition.

Comparison of Dinoflagellata community structures under the normoxic and hypoxic conditions

Among the dinoflagellate orders, the relative ASV abundances of the order Syndiniales under both oxygen conditions were the highest on days 0 and 3, whereas that of the order Peridiniales were the highest on day 7 (Fig. 4A, Supplementary Table S4). The relative ASV abundance of Prorocentrales increased under hypoxia but decreased under normoxia from day 0 to day 7 (Fig. 4A, Supplementary Table S4).

On day 0, without unassigned taxa, the relative read abundances of the orders Gymnodiniales and Prorocentrales under the normoxic condition were the highest and second highest, respectively, but they were very similar (Fig. 4B, Supplementary Table S5). However, on day 7, the relative read abundance of die order Suessiales was die highest under die normoxic condition, whereas that of the order Prorocentrales was die highest under the hypoxic condition. The survival of species belonging to the order Prorocentrales under the hypoxic condition was confirmed by microscopic observations (Supplementary Fig. SI).

Dinoflagellate species detected and their trophic modes under the hypoxic condition

In total, 272 dinoflagellate ASVs were assigned to the phylum Dinoflagellata in die present study. In all die samples, 46 species were taxonomically identified at die species level (Tables 2 & 3). Among the 46 identified species, 31 were detected under bodi die normoxic and hypoxic conditions, 11 under the normoxic condition only, and four under the hypoxic condition only on days 0, 3, and 7 (Tables 2 & 3). Prior to die experiment, species detected on day 0 had been exposed to dieir respective oxygen conditions dirough a two-day preliminary incubation. Thus, under die normoxic condition, 42 species were taxonomically identified, whereas 35 species were identified under the hypoxic condition on days 0,3, and 7.

In terms of the relative ASV abundance of dinoflagel-lates, widiout unassigned order, die proportion of parasitic dinoflagellates was the highest under bodi oxygen conditions on days 0, 3, and 7 (Fig. 5A). Under die normoxic condition, die proportion of photo trophic dinoflagellates (autotrophic or mixotrophic, not yet identified) was die second highest on days 0, 3, and 7. Under die hypoxic condition, die proportion of mixotrophic dinoflagellates was the second highest on days 0 and 7.

In die relative read abundance of dinoflagellates under die normoxic condition, die proportion of mixotrophic dinoflagellates was die highest on day 0, whereas that of phototrophic dinoflagellates was die highest on days 3 and 7 (Fig. 5B). In the relative read abundance of dinoflagellates under die hypoxic condition, die proportion of mixotrophic dinoflagellates was die highest on days 0, 3, and 7 (Fig. 5B).

The number of taxonomically identified dinoflagellate species diat were detected on day 7 was 16 under the normoxic condition and 14 under the hypoxic condition (Tables 2 & 3). Under the normoxic condition, the trophic modes of the identified dinoflagellate species on day 7 were one exclusively autotrophic, nine phototrophic, four mixotrophic, one kleptoplastidic taxon, and one heterotrophic. However, under die hypoxic condition, die trophic modes of die identified dinoflagellate species on day 7 were one exclusively autotrophic, six phototrophic, three mixotrophic, one kleptoplastidic taxon, and diree heterotrophic.

Enumeration of cell density and establishment of clonal cultures of dominant protistan taxa under hypoxia on day 7

As die relative ASV abundances cannot directly confirm die presence or abundance of actual cells, die dominant protistan taxa under the hypoxic condition on day 7 were furdier quantified under a compound microscope. Among dinoflagellates, Prorocentrum triestinum exhibited the highest cell density, followed by Biecheleria-like spp. (Table 4). Odier dinoflagellates such as Gyrodinium spp. and Scrippsiella sp. were also observed under die compound microscope. Among diatoms, Skeletonema spp. showed die highest density, followed by Psammodic-fyore-like species. Odier diatoms, including Cylindrotheca closterium, Thalassiosira spp., Pseudo-nitzschia spp., and Chaetoceros spp., were also present. Ciliates, including the loricate ciliate Eutintinnus sp. and naked ciliates of various sizes, and cercozoans, including Vampyrella-Wke sp., were also observed. In addition, cells of two dinoflagellate species {Prorocentrum triestinum and Scrippsiella acuminata) and diree diatom species {Chaetoceros cur-visetus, Cylindrotheca closterium, and Skeletonema ja-ponicum) were isolated from die water incubated under the hypoxic condition on day 7 and successfully established as clonal cultures (Supplementary Fig. S2). Each species was identified based on die internal transcribed spacer and large subunit ribosomal DNA sequences.

DISCUSSION

The present study is the first to explore temporal changes in die structure of protist communities in field-collected water incubated for one week under both the normoxic and hypoxic conditions using a metabarcoding mediod. Previous studies typically collected water samples from different depths, usually normoxic water near die surface and hypoxic water near die bottom, or on different dates, with normoxic water collected on one day and hypoxic water on anodier (Rocke et al. 2013, 2016, Santoferrara et al. 2022, Ok et al. 2023a). Therefore, it has been difficult to determine which groups or species are genuinely affected by hypoxia because protist communities in waters collected from different depths or times are not identical. Moreover, previous studies could not establish a clear trend in die changes in die structure of protist communities because the communities observed under die hypoxic conditions in die field were not die same as diose observed under die normoxic conditions. In contrast, die incubation of die collected water in the present study allowed us to explore trends in changes to die structure of protist communities because no species could enter or escape from the experimental botdes. Fur-diermore, incubating collected water samples containing nearly identical protist communities under bodi the normoxic and hypoxic conditions enabled us to understand die effects of hypoxia on community structure under die same physicochemical conditions. This approach effectively excludes die influence of factors odier than hypoxia. Moreover, the mediod used in die present study allowed us to determine which species or groups widiin die protist community were affected by hypoxia.

The present study investigated die temporal changes in die community structure of marine protists under die oxygen-depleted condition over a period of 7 days. However, on day 7, die concentrations of P04 and Si02 declined to low levels, potentially affecting die protist community dynamics. This low nutrient concentration may have limited die growdi and survival of specific protist groups such as diatoms, particularly dependent on P04 and Si02. Thus, shifts in community composition observed on day 7 could be influenced not only by oxygen depletion but also by nutrient limitation. Therefore, careful consideration of this experimental condition on day 7 is needed when interpreting die findings.

Comparison of protist groups and species under the normoxic and hypoxic conditions

On day 7, under die hypoxic condition, Dinoflagellata, Cercozoa, and Ochrophyta accounted for die majority of the relative ASV abundance. Bodi relative ASV abundance and read abundance of Cercozoa and Ochrophyta under die hypoxic condition increased during seven-day incubation. Cercozoa has been found in die hypoxic waters and sediments of die world's oceans (Table 5) (Stock et al. 2009, Kalu et al. 2023, Ok et al. 2023a). In particular, Cercozoa has been reported to be abundant in marine bendiic and interstitial communities (Pawlowski et al. 2011, Harder et al. 2016), suggesting that diey may be resilient to low-oxygen environments. The present study showed that die read abundance of die cercozoan Pseu-dopirsonia mucosa was die highest among cercozoans under the hypoxic condition on day 7 (Table 5). P. mucosa is known to be a parasitic protist diat infects various diatoms (Kuhn et al. 2004). Thus, its highest proportion in hypoxic waters on day 7 may be partially related to die increase in die relative read abundance of Ochrophyta, die majority of which were diatoms (Supplementary Table SI). Moreover, the presence of diis species under die hypoxic condition has not been previously reported. Thus, the present study added P. mucosa to the list of species that are dominant under die hypoxic condition.

In terms of relative read abundance, die top five species in Ochrophyta under the hypoxic condition belonged to diatoms according to the present and previous study (Table 5) (Ok et al. 2023a). They can tolerate hypoxia by producing oxygen. Few studies have been conducted on die effects of hypoxia on diatom growdi (Wu et al. 2012, Chen et al. 2025). The growdi rate of the diatom Thalassiosira weissflogii increased under hypoxia widi an increased net photosyndietic rate (Sun et al. 2022). The growdi rates of diatom Thalassiosira pseudonana and Skeletonema costatum were affected by hypoxia; however, die rates were still positive at die DO concentration of 1.3 mg L ' for T. pseudonana, and 0.5 and 2 mg I/1 for S. costatum, respectively (Wu et al. 2012). The results of die present study provide information on diatoms which can tolerate and even show higher growdi rates under hypoxia, and further studies on the growth rate of die odier dominant diatoms Thalassiosira tenera, Psammodictyon panduri-forme, and Minidiscus variabilis are needed.

In Perkinsozoa, die relative read abundance of die dominant species Parvilucifera infectans increased under bodi oxygen conditions on day 3, but decreased on day 7. Parvilucifera infectans is an endoparasitoid diat infects a wide range of dinoflagellates, including diose from Dino-physiales, Gonyaulacales, Gymnodiniales, Peridiniales, and Suessiales (Garces et al. 2013, Alacid et al. 2015). The decline in die relative read abundance of Dinoflagel-lata under bodi die normoxic and hypoxic conditions on day 3 co-occurred widi die increase in die relative read abundance of Perkinsozoa. This result suggests diat P. infectans might have been actively infecting and potentially reducing dinoflagellate populations. However, on day 7, the population of die parasitic P. infectans declined under bodi die oxygen conditions, possibly due to a lack of available dinoflagellate hosts. Furthermore, die relative read abundance of Perkinsozoa on day 7 declined more under the hypoxic condition than under die normoxic condition. P. infectans has not been reported to infect Proro-centrales, including die dominant species in the present study, Prorocentrum triestinum (Garces et al. 2013). Thus, the high relative read abundance of Prorocentrales on day 7 under hypoxia may have partially contributed to a lower relative read abundance of Perkinsozoa compared to normoxia. To verify this hypodiesis, furdier investigation is needed to determine whedier die increase in the Prorocentrum triestinum population affected die decline of Parvilucifera infectans under the hypoxic condition.

Based on relative read abundance on day 0, die dino-flagellate composition differed between die hypoxic and normoxic conditions in the present study. For example, Prorocentrales was die dominant dinoflagellate order under hypoxia, whereas Gymnodiniales dominated under normoxia. This discrepancy may be attributed to die preliminary incubation, which allowed protists to acclimate to dieir respective oxygen conditions before die experiment. Prorocentrales possess thick diecal plates, which may provide protection against environmental stressors such as the sudden oxygen reduction (lanouskovec et al. 2017). In contrast, Gymnodiniales have relatively thin cell walls, making diem less adapted to the sudden transition to hypoxic environments (lanouskovec et al. 2017). To verify diis hypodiesis, furdier investigation into the mechanisms diat enable Prorocentrales to dominate under hypoxia is needed.

In Dinoflagellata, die relative ASV abundance of the order Peridiniales was die highest under die hypoxic condition on day 7, whereas the relative read abundance of the order Prorocentrales was die highest. Prorocentrum. triestinum belonging to Prorocentrales, Scrippsiella pre-caria and Scrippsiella acuminata belonging to Peridiniales, and Biecheleria brevisulcata and Biecheleria tirezensis belonging to Suessiales ranked in die top five dinoflagellate species in read abundance under die hypoxic condition (Table 5, Supplementary Fig. SI). Prorocentrum triestinum, B. brevisulcata, S. acuminata, and S. precaria have been found in hypoxic seawater (Table 5) (Ok et al. 2023a). Moreover, clonal cultures of P. triestinum and S. acuminata from single-cell isolation were established from day 7 under die hypoxic condition (Supplementary Fig. S2). It is highly possible diat B. tirezensis is present in the hypoxic waters of natural environments.

In die present study, 35 dinoflagellate species were detected under die hypoxic condition throughout die entire study period including days 0, 3, and 7. Of diese detected species during die entire study period, 16 species were previously known to be present under hypoxia, but 19 species were newly determined (Table 6). These results extend the number of die dinoflagellate species are present under hypoxia. Among die newly discovered dinoflagellates that were present under hypoxia, autotrophic species were two, phototrophic species 12, mixotro-phic species one, and heterotrophic species four (Table 6). Furthermore, of these 35 detected dinoflagellate species, 14 were present on day 7, and 6 were newly identified to be detected under die hypoxic condition: one autotrophic, four phototrophic, and one heterotrophic dinoflagellates (Table 6). Thus, some dinoflagellate species remained detectable longer than die odiers, suggesting differential tolerances to hypoxia. Therefore, careful consideration of the presence of dinoflagellate species in relation to the duration of hypoxia is needed when interpreting the findings. Autotrophic, mixotrophic, phototrophic, and kleptoplastidic dinoflagellates can produce oxygen and may release it into ambient water, which may alleviate hypoxia. Moreover, mixotrophic dinoflagellates are known to survive die hypoxic condition by feeding on dieir prey. Under die hypoxic condition, die growdi rate of die mixotrophic dinoflagellate Alexandrium po-hangense satiated with prey is positive, whereas diat of A. pohangense starved is negative (Eom et al. 2024). Fur-diermore, Rocke et al. (2016) observed diat mixotrophic dinoflagellates became more dominant as die hypoxia intensified, suggesting a shift from autotrophy to phagot-rophy. Heterotrophic dinoflagellates can also survive the hypoxic condition by growing on dieir prey. That is, if the growdi rate of a heterotrophic dinoflagellate species feeding on prey exceeds its mortality rate due to hypoxia, the heterotrophic dinoflagellate species can survive. Under hypoxic conditions, the growth rate of die heterotrophic dinoflagellate Gyrodinium dominans satiated widi prey was positive, whereas that of G. dominans starved was negative (Eom et al. 2024). Therefore, feeding is a critical survival strategy for mixotrophic and heterotrophic dinoflagellates under die hypoxic condition.

In previous metabarcoding studies, relative ASV abundances have been used to assess marine protist communities (Vasselon et al. 2018, Santoferrara et al. 2022, Ok et al. 2023a). However, die choice of barcoding region, the efficiency and specificity of PCR primers, and die varying completeness of reference databases across protist taxa can result in underestimation or overestimation of ASVs in different protist groups (Pawlowski et al. 2016, Martin et al. 2022). Although the V4 region of the 18S rRNA gene is commonly employed in protist metabarcoding (Kezlya et al. 2023), it may lead to an overestimation of dinoflagellate ASVs, as die high variability in die rDNA regions widi-in cells can result in die detection of multiple ASVs (Stuart et al. 2024). Furthermore, die incompleteness of a diatom barcoding database in the V4 region can lead to an underestimation of diatom ASVs (Pawlowski et al. 2016). In die present study, a higher number of diatom species under the microscopy were enumerated compared to diat of dinoflagellate species, aldiough a higher number of dinoflagellate ASVs were detected compared to die diatoms on day 7. This may have been caused by die underestimation and overestimation of ASVs in diatoms and dinoflagellates, respectively. Thus, potential taxonomic biases in metabarcoding results need to be carefully considered. Furdiermore, in the present study, die quantitative cell enumeration, microscopic observations, and clonal culture establishment from single-cell isolation and identification of species confirmed that die dominant taxa were composed of morphologically intact cells or viable cells and not cellular debris (Table 4, Supplementary Figs SI & S2). Therefore, integrating microscopic observation, cell counting, and live cell cultivation with metabarcoding analyses is essential for accurately assessing the protist community structure and distinguishing actual cells from degraded material.

Under die closed condition of the present study, die absence of protist introduction and die progressive accumulation of cellular debris may have affected die detected ASV profile, making it different from natural environments. Therefore, it is important to consider diat die present study was conducted in a closed system when interpreting its results in die context of natural environments. Furdiermore, in ocean, low pH conditions are frequently associated widi hypoxia (Howarth et al. 2011, Gobler and Baumann 2016, Guo et al. 2022). However, in die present study, die low DO condition was established by using N2 + 400 ppm C02 gas to maintain stable pH levels. This provides an ideal condition for investigating die single effects of hypoxia on protistan community structures. However, die combined effects of hypoxia and acidification resulted in different physiological responses (e.g., survival, growdi, photosyndiesis, and dark respiration) of a marine protist, die dinoflagellate Amphidinium carterae, compared to single hypoxic effect (Bausch et al. 2019). Therefore, when applying the findings of die present study to natural environments, die potential impact of C02 accumulation should be considered.

The present study provides valuable insights into die dynamics of protist communities under the hypoxic condition. The results of the present study suggest diat hypoxia can alter protist community composition. Future research should explore die physiological mechanisms of protist survival under die low-oxygen condition.

ACKNOWLEDGEMENTS

This research was supported by the National Research Foundation (NRF) funded by die Ministry of Science and ICT (RS-2021-NR058847; RS-2021-NR057869; RS-2023-00291696) award to Hll and Korea Basic Science Institute (National Research Facilities and Equipment Center) funded by the Ministry of Science and ICT (RS-2024-00399598) award to IHO.

CONFLICTS OF INTEREST

The audiors declare diat diey have no potential conflicts of interest.

SUPPLEMENTARY MATERIALS

Supplementary Table SI. Detailed taxonomic information on die protist communities analyzed by sequencing the V4 region of die 18S rRNA gene amplicon during die study period (https://www.e-algae.org).

Supplementary Table S2. Relative amplicon sequence variants abundance (mean and standard error, %) based on different phyla for each incubation period under the normoxic and hypoxic conditions (https://www.e-algae. org).

Supplementary Table S3. Relative read abundance (mean and standard error, %) based on different phyla for each incubation period under the normoxic and hypoxic conditions (https://www.e-algae.org).

Supplementary Table S4. Relative amplicon sequence variants abundance (mean and standard error, %) based on different orders in Dinoflagellata for each incubation period under the normoxic and hypoxic conditions (https://www.e-algae.org).

Supplementary Table S5. Relative read abundance (mean and standard error, %) based on different orders in Dinoflagellata for each incubation period under the normoxic and hypoxic conditions (https://www.e-algae. org).

Supplementary Fig. SI. Micrographs of actual cells fixed in Lugol present under the hypoxic condition on day 7 (https://www.e-algae.org).

Supplementary Fig. S2. Micrographs of live cells established as single-cell isolated cultures under the hypoxic condition on day 7 (https://www.e-algae.org).

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Research Article

Algae 2025,40(2): 147-161 https://doi.org/10.4490/algae.2025.40.424