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Get Information clear JSmol Viewer clear first_page Download PDF settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing: Column Width: Background: Open AccessArticle Comparison of Bacterial Community Composition in Gut of Chinese Mitten Crabs from Three Distinct Rivers in Korea by Hyung-Eun AnHyung-Eun An SciProfiles Scilit Preprints.org Google Scholar 1, Adeel MalikAdeel Malik SciProfiles Scilit Preprints.org Google Scholar 2,*, Jeongho LeeJeongho Lee SciProfiles Scilit Preprints.org Google Scholar 1, Min-Ho MunMin-Ho Mun SciProfiles Scilit Preprints.org Google Scholar 1, Kang Hyun LeeKang Hyun Lee SciProfiles Scilit Preprints.org Google Scholar 1,3, Hah Young YooHah Young Yoo SciProfiles Scilit Preprints.org Google Scholar 1 and Chang-Bae KimChang-Bae Kim SciProfiles Scilit Preprints.org Google Scholar 1,* 1 Department of Biotechnology, Sangmyung University, Seoul 03016, Republic of Korea 2 Institute of Intelligence Informatics Technology, Sangmyung University, Seoul 03016, Republic of Korea 3 Department of Bio-Convergence Engineering, Dongyang Mirae University, Seoul 08221, Republic of Korea * Authors to whom correspondence should be addressed. Fishes 2024, 9(4), 144; https://doi.org/10.3390/fishes9040144 Submission received: 20 March 2024 / Revised: 17 April 2024 / Accepted: 17 April 2024 / Published: 20 April 2024 (This article belongs to the Special Issue Aquaculture Ecology and the Environmental Microbiome) Download keyboard_arrow_down Download PDF Download PDF with Cover Download XML Download Epub Download Supplementary Material Browse Figures Versions NotesAbstract: The Chinese mitten crab (CMC) also known as Eriocheir sinensis has great significance in the aquaculture industry. The bacterial communities inhabiting the CMC’s gut may differ depending on the host habitat and can aid in their normal biological functioning. These microbes are also known to have certain effects on their flavor. In this study, we utilized MiSeq high-throughput sequencing technology to explore the diversity of bacterial communities in the gut of CMCs from three different geographical locations in Korea: the Geum (GD), Han (HD), and Tamjin (TD) rivers. Although most of the environmental parameters were similar at the three sites, significant differences in conductivity (CDS), dissolved oxygen (DO), and salinity were observed. The results show that CMCs sampled from these locations exhibited distinct microbial composition and abundance. For example, the genus Candidatus Hepatoplasma displayed significantly higher abundance in CMCs from HD than those in the other locations, suggesting nutritional stress. Similarly, the crabs collected from TD showed a higher abundance of pathogenic Helicobacter than those from HD and GD sites. We also observed differences in the amino acid, nucleotide, and lactic acid concentrations between different tissues such as the muscle, hepatopancreas, and testis of CMCs. However, only small differences were observed when these characteristics were compared in CMCs from different locations. Our results offer important insights into the intestinal bacterial composition in CMCs which in turn may help in designing better culturing strategies for these important species of crabs. Keywords: Eriocheir sinensis; Geum River; Han River; Tamjin River; gut microbiota Key Contribution: The present study describes the differences in the gut microbiome of Chinese mitten crabs collected from three distinct rivers in Korea. The findings of this study might be useful for future studies on improving CMC aquaculture. 1. IntroductionEriocheir sinensis, commonly known as the Chinese mitten crab (CMC), is a freshwater crustacean of huge economic significance [1]. The adult CMC has the unique characteristics of spending its entire life in freshwater and then migrating to saline (brackish) water to undergo reproduction [2]. It is native to China and Korea, and in Korea, it is distributed along the west coast, including the Geum and Han rivers. The CMC is also found as an invasive crab in the United States of America (USA) and Europe [3,4]. CMCs rank among one of the most important aquaculture organisms in East Asia and are highly sought after by consumers for their unique flavor, characterized by umami and distinct sweetness [5,6]. Based on the recent report of The State of World Fisheries and Aquaculture (SOFIA), the annual worldwide production of CMCs comes close to 800 thousand tons and is among one of the top three crustaceans after whiteleg shrimp and red swamp crawfish (https://www.fao.org/home/en/, accessed on 6 April 2024). In China, the yield of CMCs has swiftly increased in the last three decades and reached around 800,000 tons in 2023, making it the top ranking aquacultural crab species [7]. According to 2009 records from Korea, about 2000 tons of mitten crabs are consumed by Korean people each year, of which 1500 tons are imported from China [8]. However, with the recent expansion of CMC farming, it was reported that the Korean government purchased 12,057,751 domestically farmed CMCs for KRW 2,177,873,000 (approximately USD 1,611,730) and released them in nature [9]. These trends have further expanded the economic scale of the aquacultured crab market in Korea and increased the focus on the quantity and quality of aquacultured CMCs.In the aquaculture of CMCs, factors that have been shown to influence taste and quality include the geographical environment of the habitat, gut microbiota, and diet [10]. Among them, the environmental parameters of the habitat play crucial roles in the aquatic ecosystem, and any unfavorable environmental condition may create a stressful situation for CMCs and their capability for homeostasis may be disrupted [11]. These environmental parameters can regulate the survivability of CMCs by influencing different physiological processes in animals such as osmoregulation, growth, development, and reproduction [12,13,14]. Besides their impact on growth, development, and other physico-chemical processes, environmental parameters such as salinity have an impact on the flavor and meat quality as well. It has been reported that the environment of CMCs has a significant impact on their flavor as well as market price [10].The digestive tract harbors trillions of microbial cells known as gut microbiota, which plays essential roles in maintaining the host’s health, by performing many beneficial functions for the host such as balancing the immune response, absorbing nutrients, and maintaining homeostasis [14,15]. The host environment is also one of the most crucial factors that can also regulate the composition of gut microbial communities [16,17]. Environmental factors such as salinity can lead to stress (hyposaline or hypersaline) to CMCs, which can influence the growth condition of the host [18] and can also affect the host microbiota. For example, depending on the salinity tolerance, strict freshwater bacteria may be destroyed while saline tolerant bacteria may live, and marine bacteria could immigrate [19] in the gastrointestinal trails of a gastropod living in freshwaters and mesohaline waters. In spite of these facts, a lot of information is available regarding the mechanisms of the intestinal immune regulation of the host and its relationship with the symbiotic microorganisms on the gut mucosa surface [20]. However, limited information regarding the gut immune mechanisms of invertebrates at the barrier epithelia [21] is available especially for CMCs. Additionally, the relationship between the salinity, intestinal microbiota, and growth condition of the CMCs remains unclear. Therefore, maintaining a functional and stable gut microbiota is essential not only for the health of CMCs but it can also have a significant impact on their flavor and market price [10].To the best of our knowledge, no studies have been conducted to identify any potential relationship between the geographical environment of the habitat and the flavor characteristics of cultivated CMCs in Korea. To date, research on the flavor properties of CMCs has mainly focused on analyzing taste-related characteristics such as fatty acids, amino acids, and nucleotides and their association with diet [6]. Recently, although some studies have attempted to identify the differences in the intestinal microbiome and flavor substances in CMCs from freshwater lakes and rivers [10], these studies represent geographically distant locations from three main rivers (Geum, Han, and Tamjin rivers) in Korea that represent the CMC habitat. In this work, an attempt was made to determine the influence of different geographical locations on the gut microbiota of CMCs and its potential implications on crab physiology. To investigate the differences in the gut bacterial communities of CMCs inhabiting different environments, we exploited MiSeq technology to sequence the bacterial 16S rRNA sequences. Additionally, to understand the potential associations between different geographical environments of the CMCs’ habitat and flavor characteristics, we also analyzed the taste-determining metabolites through HPLC analysis. Therefore, this is the first study to investigate the influence of geographical location on the gut microbiome of CMCs in Korea, which in turn may affect their culturing and flavor. Our results may suggest what environmental conditions may be a good solution for culturing CMCs within Korea and may have consequences for their flavor and quality, cultivation, and adaptation. 2. Materials and Methods 2.1. Sample CollectionA total of 54 healthy adult male crabs were collected from three different locations in Korea, namely 18 specimens from the Geum River (GD), Chungcheongnam-do, 18 specimens from the Han River (HD), Seoul, and 18 specimens from the Tamjin River (TD), Jeollanam-do (Figure 1, Table S1). The environmental factors, including water temperature (WT), salinity, pH, and dissolved oxygen (DO), were measured using a portable meter (YK-2001PHA, LUTRON Co., Taipei City, Taiwan) during sample collection (Table S2). The crabs were transported to the laboratory while alive and stabilized for an hour. To extract DNA, the carapace of twenty-seven adult male crabs was irrigated thoroughly using sterile water and disinfected with 70% ethanol for three minutes in the laboratory. The crabs were dissected immediately after washing. For the DNA extraction and bacterial 16S rRNA gene sequencing of the gut microbiome, the digestive tracts of CMCs were aseptically removed and stored at −80 °C for subsequent analysis. For HPLC analysis, the muscle, hepatopancreas, and testis were extracted from twenty-seven healthy adult male crabs and immediately stored at −80 °C for subsequent analysis. The remaining tissues were stored in 99% ethanol at room temperature as voucher specimens. 2.2. Species IdentificationSpecies identification was performed using tissue from the pereopods in each specimen. DNA extraction was performed using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. A Maestro Nano spectrophotometer (Maestrogen, Hsinchu, Taiwan) was used to evaluate the purity and concentration of the extracted DNA. To amplify the mitochondrial cytochrome c oxidase subunit I (COI) gene, polymerase chain reaction (PCR) was performed using the LCO1490/HCO2198 primer set [22]. The PCR conditions included an initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 45 s, and extension at 72 °C for 1 min, with a final extension step at 72 °C for 5 min. After amplification, the PCR products were observed by electrophoresis on 1% agarose gels in tris-acetate buffer. All specimens were identified as E. sinensis (accession numbers: OR842741-OR842794) by both morphological characteristics and molecular phylogenetic analysis based on the COI sequence. The preserved E. sinensis specimens, stored in 99% absolute ethanol, were deposited in the Department of Biotechnology at Sangmyung University, assigned voucher numbers from SMU00218 to SMU00271. 2.3. DNA Extraction and Bacterial 16S rDNA Gene Sequencing of CMC GutAbout 0.5 g of the intestinal tissue samples was placed in 1.5 mL microcentrifuge tubes, and the DNA was isolated using Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The bacterial 16S rDNA V3-V4 regions were utilized to analyze and compare the gut microbiota among CMCs originating from various habitats [23]. The 16S rDNA was sequenced using high-throughput technology with the MiSeq platform (Illumina, San Diego, CA, USA). The paired-end sequencing data of CMCs from three different locations were deposited in NCBI’s Sequence Read Archive (SRA) SRR26926093~SRR26926095 under BioProject PRJNA1044169. 2.4. Bioinformatic AnalysesThe paired-end sequencing data of CMCs from three different locations were first imported into the QIIME2 ver 2023.2 [24] pipeline. Next, the DADA2 plugin within the QIIME2 pipeline was used to improve the joining of forward and reverse reads by filtering the poorest quality ends of the reads and keeping the read length enough for what is required for overlap. The output of this step comprises the amplicon sequencing variant (ASV) table and representative sequences. The sequencing depth was estimated using rarefaction analysis. To estimate the genera richness and α-diversity, the Simpson, Chao 1, and Shannon index and Good’s coverage in each sample were calculated by exporting the QIIME2 output to the R package Phyloseq ver 1.32.0 [25]. In general, microbiome datasets are sparse; therefore, it is indispensable to filter the dataset by excluding the low quality or superfluous variables (ASVs) for better downstream analyses [26]. In this work, we retained only those ASVs which have at least two counts in at least 11% of the samples and normalized the data by the rarefying method. To assign a species-level taxonomy to the unclassified species, we performed a BLAST-based search against the NCBI non-redundant nucleotide database. The R vegan (https://github.com/vegandevs/vegan. accessed on 14 February 2024) package was used to perform the statistical analyses. 2.5. Determination of Free Amino Acid ContentAnalyses of taste-determining metabolites through HPLC analysis were performed to understand the potential associations between different geographical environments of the CMCs’ habitat and flavor characteristics. To determine the free amino acid content in hepatopancreas, muscle, and testis tissues, equal amounts of tissue samples were taken from nine crabs and mixed to a total of 1 g, and the mixture was quickly ground using a mortar and pestle. Then samples were pretreated according to Tao et al. [10]. The concentration of free amino acid was determined using the high-performance liquid chromatography–fluorescence detector (HPLC-FLD) system (Shimadzu HPLC 40 Series-FLD; Kyoto, Japan) by Korea Quality Testing Institute (KQT, Suwon, Korea). Pre-column derivatization with FMOC-Cl (9-fluorenylmethyl chloroformate) and OPA (o-Phthalaldehyde) was carried out. The column used was a C18 Column (250 × 4.6 mm, 5 μm). The gradient elution for the HPLC analysis was conducted using 20 mM KH2PO4 solution with 225 μL TEA (triethylamine) and 5 mL THF (tetrahydrofuran) per liter (mobile phase A) and the mixture of deionized water, acetonitrile, and methanol in a volume ratio of 15:45:40 (mobile phase B). The linear gradient conditions are shown in Table S3. The operating conditions were as follows: a flow rate of 0.8 mL/minute; column temperature, 35 °C; injection volume, 2 μL; excitation wavelength, 350 nm and 266 nm; and emission wavelength, 450 nm and 305 nm. 2.6. Determination of Free Nucleotide ContentTo determine the free nucleotide content in hepatopancreas, muscle, and testis tissues, samples were pretreated according to modified methods of Tao et al. [10]. Equal amounts of tissue samples were taken from nine crabs and mixed to a total of 1 g, and the mixture was quickly ground using a mortar and pestle. Then, 5 mL of 0.6 M perchloric acid was added, homogenized for 2 min, and shaken for 20 min. After that, the mixture was centrifuged at 13,500× g for 10 min, and the pH of the supernatant was adjusted to 7.0 using 1.0 M potassium hydroxide and subsequently centrifuged at 13,500× g for 10 min. The supernatant was collected, adjusted to a total volume of 10 mL, filtered through a 0.45 μm membrane filter, and finally analyzed by the HPLC–diode array detector system (HPLC-DAD, Hitachi, Tokyo, Japan). The analysis was carried out under the following conditions: column, INNO column C18 (250 × 4.6 mm, 5 μm); detector, DAD at 260 nm; and mobile phase, 13.6 g of KH2PO4 dissolved in 1 L water (pH 5.6) (A) and 13.6 g of KH2PO4 dissolved in 750 mL water and 150 mL methanol (pH 5.6) (B). The gradient analysis was set as follows: start, 100% A; 30 min, 100% A; 65 min, 100% B; 90 min, 100% B; 95 min, 100% A; and 130 min, 100% A at a flow rate of 0.5 mL/minute. 2.7. Determination of Lactic Acid ContentTo determine the lactic acid content in hepatopancreas, muscle, and testis tissues, equal amounts of tissue samples were taken from nine crabs and mixed to a total of 1 g, and the mixture was quickly ground using a mortar and pestle. Then, samples were pretreated according to Tao et al. [10]. The concentration of lactic acid was determined using the HPLC-RID (refractive index detector) system (Shimadzu HPLC-RID-10A; Kyoto, Japan). The column used was a Shodex SUGAR SH1011 H+ ion exclusion column (8 mm × 300 mm). The isocratic elution was performed with 0.005 N H2SO4 at a flow rate of 0.8 mL/minute. The temperature of the column oven was set to 50 °C, and the sample injection volume was 20 μL. 3. Results 3.1. Differences in the Environmental ParametersThe environmental parameters for each sampling site are provided in Table S2. Since Korea is a country with a narrow range of latitudes, the climate is generally similar across the region; the WT in the geographical locations was similar. Overall, the WT ranged between 16.3 °C (HD) and 19.2 °C (TD). The WT in GD was found to be 17.3 °C. However, we observed significant differences in a few parameters such as CD (conductivity), DO, and salinity. Specifically, DO levels were the highest (21.6 mg/L) in HD, followed by TD (12.7 mg/L) and GD (10.2 mg/L), respectively. In contrast, GD showed the highest concentration of CD and the lowest salinity. 3.2. A 16S rDNA Metabarcoding Analysis of the CMC Gut MicrobiomeThe 16S rDNA of the gut microbiome of the CMCs from three different habitats was sequenced. A total of 1,977,372 reads were generated from HD, whereas 1,778,436 and 1,645,806 reads were produced from TD and GD, respectively. The total read bases range between 495.4 Mbp and 595.2 Mbp. Table 1 shows the raw data statistics for each sampling site. The read length from all the three habitats was 301 bp, and a total of about 76% of the reads passed the input filter. These data indicate that the sequencing results were reliable and that all three datasets could be compared and analyzed. Subsequently, a total of 305, 570, and 565 ASVs were identified from the gut microbiota of GD, HD, and TD habitats, respectively. A total of 46 ASVs were shared by all samples, whereas the number of unique ASVs for GD, HD, and TD were 236, 497, and 503, respectively (Figure 2). Rarefaction analysis suggested that an adequate sampling depth was achieved for each sampling location (Figure S1). Additionally, in order to assess and compare the bacterial diversity in each location, bacterial richness and diversity indices were calculated from the proportion of ASVs. The microbial complexities in the guts of CMCs were estimated based on alpha-diversity indices (Chao1 and Shannon indices). The results indicate that the bacterial community in crabs from HD and TD showed higher alpha-diversity indices than those from GD (Table S4). 3.3. Microbial Community Composition and StructureThe gut microbiota of CMCs from three different sites mainly comprised bacteria belonging to four phyla, Pseudomonadota (31.3%), Mycoplasmatota (20.2%), Bacillota (19.6%), and Bacteroidota (19.4%). About 7% remained unclassified, and taxa with abundance