Gut Microbiome Alteration of Cold-adapated Antarctic copepod Tigriopus kingsejongensis with Temperature Changes and Developmental Stages

Tigriopus kingsejongensis, a copepod species, reported from the King Sejong Station, Antarctica, serves as a valuable food resource in ecosystems. Some copepods were temperature-sensitive in growth and post-embryonic development. We cultured T. kingsejongensis at three different temperatures (2°C, 8°C, and 15°C) in a laboratory to observe the alterations in the stool microbiome of copepods depending on the cultivation temperature and developmental stages. We observed copepod gut microbiome changes by increasing temperatures: a lower microbial diversity, a higher abundance of aquatic microbes, Vibrio, and a lower abundance of the psychrophilic microbes, Colwellia. Also, the copepod gut microbiome, according to the developmental stage, was changed: a lower microbial diversity in egg-attached copepods than nauplius at 8°C. We further analyzed three shotgun metagenomes from T. kingsejongensis stool samples at different temperatures and obtained 44 metagenome-assembled genomes (MAGs). We noted that MAGs of V. splendidus D contained glycosyl hydrolase (GHs) encoding chitinases and virulence factors with higher relative abundance at 15°C than at lower temperatures. These results that temperature and developmental stages affect the gut microbiome of copepods are helpful to understand the changes in the low-temperature adapted copepod with climate change.

bacteria belonging to the Vibrionaceae family are commonly found in plankton [13]. Bacteria colonizing copepods' gut and body surfaces utilize chitin as the carbon and nitrogen sources and attract organic and inorganic nutrients and biomasses produced by copepods' fecal pellet and feeding [14]. For e cient chitin utilization, marine bacteria, including Vibrio species, require chitinase [15,16]. The exoskeletons of copepods are composed of chitin, and bacterial chitinase may be involved in host pathogenesis and growth [17]. Besides, bacterial virulence factors are required to survive bacteria and are often involved in direct interactions with the host's defense mechanisms and growth. However, most studies have used cultivation-dependent approaches and molecular cloning methods and showed limited bacterial community structure and genes in copepods' bacterial colonization areas [18,19].
In this study, we conduct the metagenome sequencing and bioinformatics analysis for examining copepods' gut microbiome. According to environmental and regional distribution, we intend to understand their role and function and observe gut microbial community composition changes and their genetic information by copepods' growing temperatures and development stages. The key objective is to see how cold-adapted Antarctic copepods and their gut microbiome change by experimentally applying temperature rise caused by global climate change.

Sample collection
Tigriopus kingsejongensis was isolated from tidal pools on the coast of the Barton Peninsula in Maxwell Bay (628140S, 588460W), King George Island, Antarctica [20]. Seawater collected with T. kingsejongensis from the same area in the tidal pool was used to cultivate and wash the copepods after ltration and sterilization. T. kingsejongensis was cultivated in a 2 l ask containing natural seawater ( ltered using 0.2 µm lters) with moderate shaking at a 12:12 h light:dark cycle. The copepods were maintained at 8°C and 15°C before the experiment and were fed frozen Chlorella (purchased from Eleven Street Co.,Ltd). After 2-48 h of incubation, their stool was collected and used as samples for analyzing the stool microbial community. Seawater was used in the experiment after ltration and sterilization following the methods of Lee et al. [21]. Adult copepods were transferred to three different temperatures, namely 2°C, 8°C, and 15°C, after washing with sterilized seawater. To determine the temperature at which T. kingsejongensis can grow and propagate well, the standard temperature for arti cial culture was set at 8°C, and copepods with eggs were selected to grow at this temperature. Nauplii hatched from the eggs of copepods were isolated. To compare the stool microbial community of different Tigriopus species, adult T. japonicus isolated from the tidal pool of Seonnyeo Rock Beach, Korea, was cultured at 15°C. DNA extraction and 16S rRNA gene amplicon sequencing DNA from the stool samples of copepods was extracted using pooled (n = approximately 100) 180-220 mg stool samples at each temperature and developmental stage using the QIAGEN QIAamp Fast DNA Stool Mini Kit. The concentration and purity of the extracted DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scienti c Inc., Waltham, MA, USA). For each stool sample, the V4-V5 region of the 16S rRNA gene was ampli ed by amplicon PCR using the barcoded fusion primers 518F-926R and Nextera XT Index II primer 2. In brief, the forward primer 5′-TCG TCG GCA GCG TCA GAT GTG  TAT AAG AGA CAG CCA GCA GCY GCG GTA AN-3′ contained the Illumina sequencing primer. The reverse   primer 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GCC GTC AAT TCN TTT RAG T-3′  contained the Illumina pre-adapter. The ampli cation condition consisted of an initial denaturation at  95°C for 3 min; 25 cycles of ampli cation involving denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s; and a nal extension at 72°C for 5 min. The ampli ed PCR products were puri ed using AMPure XP beads. Index PCR was performed under the same conditions as the ampli cation procedure, with the exception that eight ampli cation cycles were used. The obtained product was pooled for the Illumina Miseq sequencing library after quanti cation using PicoGreen and Nanodrop. The nal product was quanti ed by qPCR according to the qPCR Quanti cation Protocol Guide (using KAPA Library Quanti cation Kits for Illumina sequencing platforms), and the quality was assessed using the LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Massachusetts, USA). Pairedend (2 × 300 bp) sequencing was performed by Macrogen using the MiSeq™ platform (Illumina, San Diego, USA).

Bacterial community analysis
The obtained sequences were processed using meren/illumina-utils [22] and the Quantitative Insights into Microbial Ecology (QIIME) pipeline (v.1.9.1) [23]. Paired-end sequence reads were merged with qualitybased ltering using default parameters (the P-value was 0.3, Q-score was 10, and quality lter was Q30). Sequences that did not contain the primer sequence or contained an uncorrectable primer sequence were removed from the analysis. The merged sequences were clustered into operational taxonomic units (OTUs) with a sequence similarity threshold of 99% using the de novo UCLUST method [24].
Representative sequences of OTUs were used to identify the taxonomy using the Silva 132 reference database through the RDP classi er with a 0.5 minimum support threshold [25,26]. Then, OTUs assigned to chloroplast and mitochondria were removed from our datasets. To assess the alpha diversity of the copepod's stool microbiome, the richness index (Chao1) was calculated using the QIIME script. Principal coordinate analysis (PCoA) was performed to evaluate the differences in the composition of microbial communities among the samples on the Bray-Curtis distance matrices.
Whole genome ampli cation (WGA) and metagenome sequencing WGA was performed using the QIAGEN REPLI-g Mini Kit because of an insu cient quantity of DNA for metagenome sequencing. The stool samples of adult T. kingsejongensis at 2°C, 8°C, and 15°C were ampli ed. In total, 2.5 µl of the template DNA was added to a 1.5 ml microcentrifuge tube, following which 2.5 µl of the buffer D1 was added to the DNA. The DNA-buffer mixture was centrifuged and incubated at room temperature for 3 min. After the addition of 5 µl of the buffer N1, 40 µl of the master mix (10 µl of nuclease-free water, 29 µl of REPLI-g Mini Reaction Buffer, and 1 µl of REPLI-g Mini DNA Polymerase) was added to the sample and incubated at 30°C for 10-16 h. The sample was heated for 3 min at 65°C with REPLI-g Mini DNA Polymerase. Finally, the ampli ed DNA was quanti ed and quali ed using PicoGreen and agarose gel electrophoresis. To prepare the sequencing library, 100 ng of genomic DNA for a 350 bp insert size was fragmented using Covaris. The fragmented DNA was blunt-ended and phosphorylated. After end repair, the appropriate library size was selected using different ratios of sample puri cation beads. A single "A" was ligated to the 3′ end, and Illumina adapters were ligated to the fragments. The nal ligated product was quanti ed by qPCR according to the qPCR Quanti cation Protocol Guide, and the quality was assessed using the Agilent Technologies 2200 TapeStation (Agilent Technologies, Palo Alto CA, USA). The nal samples were sequenced on the Illumina HiSeq™ 4000 platform (Illumina, San Diego, USA) as paired-end (2 × 100 bp) reads.

Metagenomic data analysis: Preprocessing and assembly
Shotgun metagenomic sequencing of the stool samples collected from adult T. kingsejongensis (after wash) at three different temperature conditions yielded 100,227,276 pair-end reads. Preprocessing of the metagenomic data was performed for the quality check and duplicate removal steps. The sequence quality was checked using FaQCs included in the Empowering the Development of Genomics Expertise (EDGE) pipeline, with a Q-score of 20 being the quality lter [27]. Duplicate reads were then removed using FastUniq with quality-ltered reads [28]. After the preprocessing steps, a total of 92,081,431 pair-end reads were used for metagenomic data analysis. Co-assembly of the metagenomic data was performed using MEGAHIT v1.1.3 with the option "--meta-sensitive presets." Contigs less than 500 bp were discarded [29]. Finally, 112,246 contigs were generated with 325,730,737 bp, a maximum of 881,242 bases, and 9,035 N50 bases.
Metagenomic phylogenetic analysis: Taxonomic classi cation and genome reconstruction CONCOCT, which uses a Gaussian mixture model and a dimension reduction algorithm with PCA based on the coverage, was used to reconstruct metagenome-assembled genomes (MAGs) from the metagenomic data [30]. To perform CONCOCT, original contigs longer than 20 kb were cut into fragmented contigs of 10 kb to enable more accurate coverage and reduce the impact of chimeric assemblies. Then, reads were mapped to fragmented contigs using BWA-MEM with default parameters [31]. CONCOCT was performed using fragmented coverage information with default parameters.
Fragmented contigs were merged with the original contig, and a total of 138 genomes were reconstructed using a co-assembly contig.

Metagenomic data analysis: Pruning of MAGs
For genome quality, pruning of the genomes was performed employing Additional Clustering Re ner (ACR; https://github.com/hoonjeseong/acr). Overall, prediction of the open reading frame of contigs and annotation of single-copy proteins was performed using prodigal and HMMER, respectively. K-means clustering using scikit-learn v0.20.2 was performed based on the coverage of mapped reads. The 120 ubiquitous single-copy proteins were used for calculating completeness and redundancy of each MAG, for which we obtained more than MAG-Quality-score (Completeness-5×Redundancy) was 50. MAGs which were MAG-Quality-score < 50 were removed after the pruning step, and pruned MAGs are denoted by '-' (e.g. Flavobacteriales_C.68.Bac to Flavobacteriales_C.68 − 0.Bac and Flavobacteriales_C.68 − 1.Bac). In the end, we obtained 44 high-MAG-Quality-score MAGs. For 44 pruned MAGs, we assigned taxonomy using Genome Taxonomy Database Toolkit (GTDB-Tk) [32].
CAZymes were annotated using run-dbcan v2.0.6 [33]. Three tools were used for detecting CAZomes: HMMER search, DIAMOND search, and Hotpop search. CAZymes detected in more than two tools were selected, yielding a total of 1,991 predicted CAZymes. To compare the genes found in MAGs from three T. kingsejongensis stool samples, the relative abundance of CAZymes was calculated as RPKM using featureCounts. Moreover, BLASTp was performed against the Virulence Factor Database (VFDB) to assess the virulence factors in these MAGs [34]. Only genes with ≥ 60% identity and ≥ 0.5 sequence coverage were considered associated with virulence factors and used for further analyses. To compare the distribution of virulence factors found at different cultivation temperatures, RPKM of virulence factors, such as CAZymes, was calculated.

Characteristics of the copepod stool microbiome
To identify the differences in the stool microbiome of Antarctic copepods depending on temperatures, we collected the pooled (approximately 100 copepods) stool microbiome of T. kingsejongensis at three cultivation temperatures (2°C, 8°C, and 15°C; T.2, T.8, and T.15 groups, respectively) during different developmental stages (nauplii, egg-attached, and adult stages; the adult group was separated to before (BW) and after (AW) wash of seawater) (Fig. 1a). We observed that one-fth to one-third of female egg sacs detached early and often failed to hatch when T. kingsejongensis was cultivated at 20°C. Through repeated experiments, we considered 15°C to be a signi cant boundary temperature for optimal growth. We thus set the cultivation temperatures at 2°C, 8°C, and 15°C. On the other hand, T. japonicas showed slow growth and reproductive retardation at 8°C.
We also analyzed a total of 13 samples, including T. japonicas as control using 16S rRNA gene sequencing, and generated a total of 1,405,607 reads. The 949,990 merged sequences were generated and clustered into 25,305 OTUs based on 99% sequence identity. PCoA with Bray-Curtis distance matrices revealed a clear separation among copepod microbiomes, especially on cultivation temperatures (Fig. 1b). The stool microbiome composition differed with the cultivation temperature and with the developmental status of copepods. Regarding the alpha diversity of the stool microbiome, an increase in the cultivation temperature decreased the Shannon diversity index (Fig. 1c). Moreover, the microbial richness of nauplii was higher than that of egg-attached copepods. When comparing two different copepod species, we found that the Shannon diversity index of T. kingsejongensis was lower than that of T. japonicas.

Recovery of MAGs from copepod gut microbiome
To examine the relationship of the microbiome with slow copepod growth and the reduced population of egg-attached copepods at 15℃, metagenome sequencing was performed using three pooled samples (AW adult of T.2, T.8, and T.15 stools) obtained from T. kingsejongensis. The DNA extracted from the three stool samples was sequenced. A total of 92,081,431 paired-end reads were co-assembled using MEGAHIT. Following genomic binning by CONCOCT, 44 high-quality MAGs with MAG quality scores > 50 were obtained; after pruning ( Fig. 3a), taxonomic assignment of each MAG was performed using GTDB.
The most abundant phylum was Bacteroidota, followed by Proteobacteria, Patescibacteria, Campylobacterota, and Verrucomicrobiota (Fig. 3b). Flavobacteriaceae was the most frequently assigned taxon at the family level in the T. kingsejongensis stool microbiome. Of the 44 MAGs, only three were assigned to the species level: V. splendidus D, Croceibacter atlanticus, and Pseudomonas E GCF_001050345.1. The most predominant MAGs were Arcobacter, 1G12 within the order Flavobacteriales, and V. splendidus D in the AW T.2, T.8, and T.15 groups, respectively. Interestingly, V. splendidus D, known as a dominant culturable Vibrio found in seawater, had a higher relative and absolute abundance in the AW T.15 group. In addition, MAGs were distributed in three samples; however, their abundance depended on the cultivation temperature. The abundances of MAGs belonging to Proteobacteria (V. splendidus D, Loktanella, and Pseudoalteromonas) and to Bacteroidota (Maribacter, C. atlanticus, and Polaribacter) were higher with an increase in temperature. In contrast, Bizionia, LS-SOB, and UBA8649, belonging to Bacteroidota, decreased with an increase in temperature. However, some MAGs were evenly distributed in all the samples regardless of the cultivation temperature; these included those belonging to Maribacter, Winogradskyella, and LS-SOB.
Features and distribution of virulence factor-related genes in MAGs from T. kingsejongensis stool samples To identify the virulence factors in the T. kingsejongensis stool microbiome, 44 MAGs were identi ed based on VFDB using BLASTp. A total of 351 virulence factors were found in all MAGs, except for SW10 (Bin0) and UBA1006 (Bin99). Thirty-two kinds of virulence factor genes were identi ed. Most virulence factors were more frequently observed in the AW T.15 group than in the AW T.2 and T.8 groups (Fig. 4a). The salicylate biosynthesis protein PchB (pchB), sugar nucleotidyltransferase (Cj1416c), and dTDP-4dehydrohamnose 3,5-epimerase (rfbC) were the top three virulence factors in the copepod stool microbiome. The relative abundances of nine genes, including dTDP-4-dehydrorhamnose 3,5-epimerase (rfbC), the type III secretion system inner membrane export apparatus protein AscS (ascS), the agellar basal body rod modi cation protein FlgD ( gD), the alginate regulatory protein AlgQ (algQ), and urease accessory protein (ureG), were higher in high temperature. On the other hand, the relative abundance of only one gene was higher in low temperature: N − acetylglucosaminyltransferase involved in polysaccharide intercellular adhesin (PIA) synthesis (icaA).
Comparison of the number of virulence factors in each MAG and the size of the MAG revealed that most MAGs did not contain a high number of virulence factors (Fig. 4b). Of these, MAGs belonging to V. splendidus D (Bin95), GCF_001050345.1 (Bin82), Pseudoalteromonas (Bin136), Colwellia (Bin64_0), and Amphritea (Bin115) contained many virulence factors. The rst three MAGs had a 130-2359-fold higher abundance in the AW T.15 group, while the latter two MAGs had a 1044-3558-fold higher abundance in the AW T.2 group.
We then compared the distribution of virulence factors present in each MAG depending on the cultivation temperature. For most MAGs, there was a proportional relationship between the log ratio of abundance of MAGs in the AW T.15 group/abundance of MAGs in the AW T.2 group and the log ratio of RPKM of the virulence factors in the AW T.15 group/RPKM of the virulence factors in the AW T.2 group (Fig. 4c). MAGs with a log-ratio greater than 1 for RPKM of the virulence factors, i.e., MAGs with more virulence factors in the AW T.15 group than in the AW T.2 group, belonged to 1G12 (Bin54), V. splendidus D (Bin95), and Pseudomonas E GCF_0010345.1 (Bin82). On the other hand, MAGs with a log-ratio lesser than 1 for RPKM of the virulence factors belonged to Arcobacter (Bin44_0), Nonlabens (Bin122), and LS-SOB (Bin23). To determine which virulence factor genes were frequently found in the T.2 and T.15 groups, we con rmed the virulence factors present in six MAGs: three MAGs with the highest log ratio of AW T.15/AW T.2 for RPKM of the virulence factors and three with the lowest (Table 1). A sugar nucleotidyltransferase (Cj1416c) was found in all six MAGs. Moreover, a two-component response regulator ( eR), the agellar basal body rod modi cation protein FlgD ( gD), and dTDP-4-dehydrohamnose 3,5-epimerase (rfbC) were detected in all three MAGs with the highest log ratio of AW T.15/AW T.2. , and GH20 (Fig. 5a). Most GHs were more frequently found in MAGs in the AW T.15 group than in the AW T.8 and AW T.2 groups.
Then, MAGs were screened for the presence of GHs annotated to chitinase, which bacteria use to mineralize the exoskeleton and fecal pellets of copepods. Three GHs (GH18, GH19, and GH23) encoding chitinase were detected (Fig. 5b). The relative abundance of these GHs increased with increasing cultivation temperature; GH23 was the most abundant GH annotated to chitinase. Moreover, the number of CAZymes increased as the MAG size increased (Fig. 5c)

Discussion
The present study aimed to identify the effects of increased seawater temperature due to climate changes in the changes in Antarctic copepods' stool microbiome and the population reduction of this cold-adapted copepod. Changes in the seawater temperature can affect the abundance of copepods, which are the primary producers and consumers of seawater. In addition, changes in the copepod population can alter the entire seawater ecosystem. There are two explanations of how copepods are affected by temperature increases: (1) changes in the temperature of the host environment directly affect the physiology of copepods and/or (2) increased temperature alters the gut microbiome, which then changes the host physiology and other aspects. In general, the gut microbiome affects the host physiology, growth, immune system, and health. We used this approach to characterize the copepod stool microbiome depending on cultivation temperatures and developmental stages in the present study.
16S rRNA gene sequencing revealed different stool microbial communities at different temperatures and a decreasing alpha diversity pattern with increasing temperature. We identi ed Colwellia as a strict psychrophilic microbe found in cold marine environments, including deep-sea and Antarctic sea ice, primarily observed in T. kingsejongensis in the T.2 group [35]. On the other hand, in the T. kingsejongensis at 15°C, the genus Vibrio was higher than in low temperatures. These results could be explained in two possibilities. One is that a higher temperature is suitable for the tness of Vibrio and its relative abundance increased with increasing temperatures. The second is that there is no control in the microbial community, and a new microbe cannot enter the bacterial pool in the closed experimental condition, the T. kingsejongensis gut. Microbes may need to adapt to changes in cold-adapted copepods with temperature changes. Therefore, in bacterial communities that lose their diversity, the population of speci c bacteria rapidly increases, eventually leading to the loss of intrinsic functions, such as digestion, nutrient uptake, reproduction, immune response, and other host defenses.
The abundance of copepod populations shows a clear seasonal and horizontal distribution pattern [36,37]. Copepod species show the highest abundance during spring, and their abundance is low in the warm water environment (> 10-12°C) during later summer and fall in the Gulf of Maine region [38]. Moreover, temperature affects to reproduction and development of some copepod. Different copepod species had distinct temperature ranges for reproduction and development and showed different reproduction patterns in each incubation temperature [39]. The egg production of female calanoid copepod Pseudodiaptomus annandalei was signi cantly lower compared to females at higher temperatures [40]. It is clear that the growth, development, and reproduction of copepods are in uenced by water temperature changes [39,41]. Here, we observed the differences of microbial diversity and the gut microbial community of nauplius and egg-attached at the same temperature, 8°C. These results that change of copepod's gut microbiome depending on their developmental stage suggested that the gut microbiome indirectly affects the changes in copepod populations.
In the present study, we recovered 44 MAGs from T. kingsejongensis stool samples. We found that both Proteobacteria and Bacteroidetes were dominant in the copepod stool microbiome at three different temperatures (2°C, 8°C, and 15°C). Proteobacteria and Bacteroidetes are commonly observed in the intestines of aquatic animals. These two phyla have been found to be distributed in the stools of marine animals, including Litopenaeus vannamei [42], Penaeus monodon [43], the mitten crab Eriocheir sinensis [44], and the sea lamprey Petromyzon marinus [45]. At the genus level, the relative abundance of Colwellia and Arcobacter lowered, while Vibrio, Pseudomonas, and Pseudoalteromonas increased with an increase in cultivation temperature. These results are similar to those reported Moisander et al. [46]. They revealed that Proteobacteria and Bacteroidetes were the predominant phyla, and Vibrionaceae and Pseudoalteromonas were abundant in copepod stools at 16°C.
Vibrio, belonging to Vibrionaceae, showed higher abundance at higher temperatures. A number of Vibrio species have a mechanism to degrade chitin, a component of the copepod surface [17]. We assumed that an increase in the abundance of Vibrio species having certain genes, including the gene encoding chitinase, affects copepod development at higher temperatures. Through CAZyome annotation, we identi ed GHs encoding chitinase, including GH18, GH19, and GH23, were more abundant at higher temperatures. These GHs were mainly found in the MAG of V. splendidus D, and this MAG was abundant at 15°C. More chitinase is found at a higher temperature than at a lower temperature, implying that higher temperature is more suitable for bacteria to mineralize the exoskeleton and fecal pellets of copepods. In addition, MAG annotation using VFDB revealed the virulence factors present at a higher temperature (15°C). Virulence factors can be divided into three groups according to their function. We revealed that virulence factors belonging to all three groups were present in MAGs from T. kingsejongensis [47]: membrane proteins playing a role in adhesion, colonization, and invasion (pchB, gD, ureG, algQ, and algU); polysaccharide capsules for surrounding the bacterial cell (rfbC); and secretory proteins (ascS and virD4). An increase in virulence factors suggests that speci c Vibrio strains are likely to affect copepod development directly. The gene pchB (encoding salicylate biosynthesis protein) and ureG and ureB (encoding urease-related protein) have been studied with regard to antibiotic resistance or the colonization of bacteria [48,49], whereas rfbC (encoding dTDP-4-dehydrorhamnose 3,5-epimerase) is known for increasing bacterial adherence and immune invasion [50].
As the current temperature of Antarctic seawater has not increased to 8°C or 15°C, it is not possible to know the effects in the real environment. However, this study emphasizes the need to prepare for climate change based on the experimental results that an increasing temperature affects the population and growth of cold-adapted copepods and their stool microbiome. In the present study, we con rmed a lower microbial diversity and a lower number of egg-attached copepods at a higher temperature, 15°C. Therefore, failure to regulate the growth of copepods with an increase in temperature will affect the population of copepods, which play an essential role in the seawater ecosystem, thereby disrupting the seawater ecosystem as a whole.  Sample collection method and characteristics of the copepod microbiome. a Sample information used in the present study. b Principal coordinate analysis was performed on the basis of Bray-Curtis distance matrices. c Alpha diversity of the stool microbiome of copepods using the Shannon diversity index.

Figure 2
Bar graph of the copepod stool microbiome at the phylum level (a), genus level (b), and species level (c).
The X-axis represents each sample, and the y-axis represents the relative abundance of bacteria. Thirty-four MAGs were high-quality MAGs qualitatively ltered with the criterion of MAG quality score (calculated as completeness -5 × redundancy) > 50, and 10 MAGs were obtained after pruning. The bubble represents one MAG taxonomically assigned to the order level using the GTDB database, and the size of the bubble is based on the relative abundance. b Heat map for the relative abundance of MAGs of T. kingsejongensis at different cultivation temperatures. The color of the circle above the heat map represents a MAG assigned to the phylum level.