Association of Fungi in the Intestine of Black Carp and Grass Carp Compared with their Cultured Water

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Introduction
Intestinal microbiota plays an important role in host health and nutrition and has attracted increasing attention recently [1].As an important source of human protein, fsh is undoubtedly the focus of research [2].A large number of microorganisms are distributed in the intestinal tract of fsh, which have formed a complex symbiotic relationship with the host in the long-term natural evolution process [3,4].Fish gut provides a reproductive environment for intestinal microorganisms, which in turn play an irreplaceable role in the development, nutrition, immune metabolism, and physiological health of the host [5,6].
Sustainable development of aquaculture requires full consideration of the interaction between the environment and aquatic organisms [7][8][9].As one of the most important aquaculture models, pond aquaculture plays an irreplaceable role in aquaculture.Tere are a large number of microorganisms such as bacteria and fungi in the pond ecosystem.As an important microorganism, fungi have a large number, diverse forms, and complex community structure [10,11].On the one hand, fungi participate in the decomposition of organic matter and provide nutrients for aquatic plants and fsh [12]; on the other hand, many pathogenic fungi afect the growth and reproduction of plants, thus afecting the health of fsh [13,14].Terefore, comparative studies of fungal microbial community characteristics in aquaculture waters and intestinal tract of aquatic animals are benefcial to the regulation of aquaculture water quality.
Grass carp (Ctenopharyngodon idella) and black carp (Mylopharyngodon piceus), both of which belong to the "four major Chinese carps," are widely distributed in China and have been extended to more than 100 countries [15].Grass carp is an herbivorous freshwater fsh belonging to Cyprinidae (the carp family) and the genus Ctenopharyngodon.It often lives in the middle water body and mainly feeds on the stems and leaves of aquatic plants [16].Black carp is a carnivorous freshwater fsh belonging to Cyprinidae and the genus Mylopharyngodon.It often lives in the lower water body.Juvenile fsh live on zooplankton, while adult fsh feed on mollusks such as snails and clams [17].With the excellent characteristics of large yield, delicious meat quality and high nutritional value, it is deeply loved by the majority of consumers [18,19].According to the statistics, the annual production of black carp was about 6.8 × 10 5 tons and the annual production of grass carp was about 5.53 × 10 6 tons [20].
At present, researchers have conducted a large number of studies on the structural characteristics of bacteria and archaea in the aquatic environment [21][22][23][24].Te studies on intestinal microorganisms of fsh mainly focus on growth and metabolism, nutrient absorption, immune regulation, and the efect of bait on intestinal tract [25][26][27].Te correlation between intestinal fungi of two diferent predatory fshes and fungi in reproductive water environment has not been reported.In this study, we selected grass carp, black carp, and cultured water samples from the same aquatic culture environment.Based on Illumina Hiseq 2500 high-throughput sequencing platform, we analyzed the diversity and community structure of fungi in grass carp, black carp and their cultured water, and explored their correlation between fungi.Tese results will provide basic data for the tolerance of fsh with diferent feeding habits to aquaculture water, so as to provide theoretical basis for the regulation and improvement of aquaculture water quality and the realization of healthy and green aquaculture.the same size without disease symptoms were randomly selected from the fsh caught in the net and brought back to the laboratory together with water samples, and the others were put back into the pond.Te fsh used in the experiment came from natural ponds and were not fed any food.At the same time, 8 sampling points were randomly selected in the pond, and 10 mL equal volume water samples were collected at a depth of about 1.0 m under the water surface.After mixing, the samples were loaded into sterilization centrifuge tubes.

Materials and Methods
In order to minimize sample contamination, the fsh surface was washed with sterile water and 70% ethanol successively before dissection.Grass carp intestinal contents samples (CY1-CY5), black carp intestinal contents samples (QY1-QY5), and water samples (SY1-SY5) were collected in a sterile operating tray with sterilized centrifuge tubes and refrigerated at −80 °C for later use.

PCR Amplifcation and Illumina HiSeq Sequencing.
Te MN NucleoSpin 96 Soi kit was used to extract DNA from collected chyme samples.Te PCR amplifcation was performed with ITS1_F and ITS2_R primers of fungal ITS, and high-throughput sequencing was performed using Illumina HiSeq 2500 platform.Te primer, reaction system, and amplifcation conditions are as follows: Primer synthesis and sequencing were completed by Beijing Biomarker Technologies Co., Ltd (Beijing, China).

Microbial Composition Analysis.
Quality fltering was performed on the original data (Trimmomatic, version 0.33) [28], primer sequences were identifed and removed (Cutadapt, version 1.9.1) [29], and double-ended reads were spliced (FLASH, version 1.2.11) [30].Finally, we removed chimeras using UCHIME version 8.1 to obtain high-quality sequences for subsequent analysis [31].Te sequences were clustered at the 97% similarity level (USEARCH, Version 10.0) [32], and operational taxonomic units (OTUs) were fltered with 0.005% of all sequenced sequences as the threshold [33].QIIME2 software (https://qiime2.org/)was used to calculate alpha and beta diversity in the samples to comprehensively assess the overall diversity and reveal diferences between samples.Alpha diversity includes Chao1 richness estimator and Ace richness estimator to measure the richness of the microbiota and Shannon−Wiener diversity index and Simpson diversity index to measure the diversity of the microbiota.Beta diversity analysis includes principal component analysis (PCA), principal coordinates analysis (PcoA), and nonmetric multidimensional scaling (NMDS), all belonging to ordination analysis, which is about rearrange these samples in a visualized low-dimensional space or plane, so that the distance between samples can refect the relationship information between samples in the plane scatter plot to the maximum extent.Based on the four distance matrices obtained from beta diversity analysis, unweighted paired average (UPGMA) was used to perform hierarchical clustering of samples by R language tool to assess the similarity of species composition among samples.

2
Aquaculture Research LefSe analysis [34] (https://huttenhower.sph-harvard.edu/lefse/), namely, the analysis of species with signifcant differences between groups, used linear discriminant analysis (LDA) to estimate the impact of each species abundance on the diference efect size and searched for species with signifcant diferences between all groups.
2.4.Statistical Analysis.SPSS 25.0 statistical software (IBM Corp., Armonk, NY, USA) was used to analyze the data, and the measurement data was represented by means ± standard deviations, and independent sample T test was used for pair comparison.Diferences between groups were considered statistically signifcant at P < 0.05 [35].

Data Storage.
Te original sequences obtained in this study have been submitted to the NCBI sequence read archive (accession number is PRJNA802701 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA802701).

Sequencing Characteristics and Microbial Diversity.
After quality control, a total of 1193261 high quality sequences were obtained from 15 samples in CY, QY, and SY, and the proportion of efective sequences in each sample was between 0.9748 and 0.9911.Te average sequence length was between 235-251 bp (Table 1).Te average coverage index of each sample was 0.9986, between 0.9978 and 0.9997, which could refect the real situation of species in the community (Table 2).As shown in the species Venn diagram, a total of 1099 OTUs were obtained in the three groups, and 651, 825, and 670 OTUs were found from the CY, QY, and SY groups.Among them, 282 were identical (Figure 1).It indicated that the richness and diversity of fungi in QY and SY were higher than that in CY, but there was no statistical diference.Chao1 index, Ace index, Shannon index, and Simpson index were calculated to illustrate the diversity and richness of CY, QY, and SY.From the calculation results of richness index (Table 2), the Chao1 index in QY was the highest, the Ace index in SY was the highest, and the Chao1 index and Ace index in CY were the lowest.From the calculation results of diversity index, the Simpson index and Shannon index in QY were the highest, the Simpson index in SY was the lowest, and the Shannon index in CY was the lowest.Compared with SY, there was no signifcant diference in the other indexes except Shannon diversity index in CY (P < 0.05).Tese results indicated that the richness of fungi in cultured water was the highest, and the diversity of fungi in the intestinal tract of black carp was the highest, with no signifcant diference between them.Tese results indicated that the richness of fungi in SY was the highest, while the diversity of fungi in QY was the highest, with no signifcant diference between them.

Overall Microbial Community
Structure.A total of 9 phyla and 288 genera were identifed from 15 samples collected from CY, QY, and SY groups.Nine phyla and 210 genera were identifed in CY, 9 phyla, and 253 genera in QY and 8 phyla and 188 genera in SY.Among the identifed phyla, Ascomycota, Basidiomycota, Chytridiomycota, and Mortierellomycota had higher relative abundance.Te total abundance of these four phyla accounted for 94.34%, 91.54%, and 66.77% in CY, QY, and SY, respectively (Figure 2).

Characteristic of Fungal Community Composition at
Genus Level.A total of 288 genera were detected in 15 samples of the three groups.210, 253, and 188 genera were detected in CY, QY, and SY, respectively.Te unclassifed genera in the SY were the most, accounting for 66.87%.Of the 288 identifed genera, the top 10 genera in relative abundance among these three groups of samples were Mortierella, Termoascus, Termomyces, Aspergilleus, Penicillium, Fusarium, Saitozyma, Archaeorhizomyces, Alternaria, and Cladosporium (Figure 4, Table 4).Compared with SY sample, there were 5 genera in the CY and QY samples that were signifcantly diferent from the SY sample (P < 0.01 or P < 0.05).In addition, Aspergillus and Cladosporium had signifcant diferences in CY and QY samples (P < 0.05).
Further, as shown in Figure 5(a), UPGMA analysis showed that the microbial community similarity of each group was relatively high.According to LefSe analysis results, there were more fungal species with statistical differences in the CY but fewer in the QY, and only phylum Chytridiomycota and class Chytridiomycetes had statistical diferences in the SY (Figure 5(b)).

Discussion
Pond aquaculture has become the most important and broadest breeding mode in China and the most important source of aquatic products [36].Te quality of aquatic products is closely related to the cultured environment, and the microbial composition in the cultured environment has become one of the most important factors.Terefore, the Aquaculture Research aquaculture environment of aquatic products should be highly valued.In the intestinal tract of freshwater fsh, the dominant bacteria belong to Pseudomonas, Bacteroides, and Aeromonas, whose main functions can be summarized as improving the absorption and utilization of nutrients, promoting the maturation of the immune system, and protection against pathogenic microorganisms [37][38][39][40].Its community structure varies with fsh species, feeding habits, bait, and various environmental factors [41][42][43].Under the same conditions, the composition of microbial community in reproductive water environment will directly afect the community structure of intestinal microorganisms of fsh [43][44][45][46].
Fungi, as an important component of intestinal microorganisms, play an important role in maintaining the   balance of intestinal microecology [47].Under normal circumstances, there is a stable synergistic, antagonistic, or symbiotic relationship between intestinal fungi, intestinal bacteria, and other intestinal microorganisms to jointly stabilize the intestinal microbiota environment and maintain the intestinal mucosal barrier function [16].However, when intestinal microbiota is disturbed, fungi will play a negative role and become the source of pathogenic bacteria for fungal infection [48].Studies have confrmed that pathogenic fungi Fusarium, Aphanomyces, and Lagenidium were identifed in Oreochromis niloticus aquaculture pond [49].
In this study, we applied high-throughput sequencing technology for the frst time to fungal microbial communities in cultured water bodies and the intestinal tracts of aquatic animals.Te results showed that 9 phyla were identifed from the DNA metabarcodes, among which Ascomycota, Basidiomycota, and Mortierellomycota were the dominant in CY and QY samples.Ascomycota, Basidiomycota, Mortierellomycota, and Chytridiomycota were the dominant phyla in SY samples.At the phylum level, the fungal microbial composition in CY and QY samples was similar, and there was no signifcant diference in abundance.However, Ascomycota, Chytridiomycota, Glomeromycota, and    and foreign scholars [50][51][52].Surprisingly, Ascomycota was enriched in CY and QY samples.Ascomycota is the largest category in the fungal community in the study, mainly by the Dothideomycetes, Sordariomycetes, and Eurotiomycetes.Tese three fungi were widely distributed in all habitat types, among which Dothideomycetes had diverse ecological functions and environmental adaptability [50].Basidiomycota is the main component of the terrestrial fungal community, and the abundance of Basidiomycota in sediments is closely related to the input of exogenous organic matter [51,53].Chytridiomycota feeds mainly on aquatic plant residues [54], which may be the reason for its relatively high abundance in SY.Furthermore, Ascomycota and Basidiomycota are not only the main decomposers of organic matter in the soil fungal community but also participate in the nitrogen cycle and play an extremely important role in the material cycle and energy fow of the biosphere [55,56].
At the genus level, a total of 288 genera were detected, of which 210 were detected in CY samples, 253 in QY samples, and 188 in SY samples.Mortierella, Termoascus, and Termomyces were the three genera with the highest relative abundance in each group.Among the unclassifed fungi detected, CY and QY samples accounted for 25.68% and 26.65%, respectively.Tere was little diference between the two groups of samples, but they accounted for 66.87% in SY samples.Tese results indicated that the fungal species of QY sample were richer than CY sample, and the fungal diversity of SY sample was richer than CY and QY samples, mainly belonging to unclassifed fungi.Compared with SY samples, 5 of the 10 dominant fungal genera in CY and QY samples showed signifcant diferences, but these genera did not show signifcant diferences in CY and QY.Tese results indicated that the species and abundance of intestinal fungi in QY and CY samples were basically the same, which were related to cultured water.Mortierella is an important component of soil microbial community.Some species form symbiotic relationships with plants [57,58], while some are hosts themselves and beneft from the symbiotic bacteria in the process of growth and reproduction [59][60][61].Both Termoascus and Termomyces are thermophilic fungi with unique survival ability and can secrete a variety of enzymes such as amylase and cellulase, which can provide nutrients for the growth of microorganisms [62,63].
In conclusion, based on the community characteristics of fungi in CY, QY, and SY samples, our work identifed the similarity of fungal microbes in CY and QY, and the correlation between fungal microbes in CY, QY, and SY.Te present study showed that species composition of abundance of intestinal fungi in two cyprinid species were remarkably similar despite their contrasting feeding habits (herbivorous vs. carnivorous).Tis suggests that intestinal fungi have similar roles to the digestive ability of the two carp species.Tese results will provide a theoretical basis for the regulation and improvement of aquaculture water quality, the realization of healthy and green fsh aquaculture, and provide reference for the research and development of probiotic products.

Figure 3 :
Figure 3: Relative abundance of fungi identifed at phylum level in each group.Note: CY stands for grass carp, QY stands for black carp; SY stands for water sample.

Figure 4 :
Figure 4: Relative abundance of fungi identifed in each group for the most abundant 10 genera.Note: CY stands for grass carp, QY stands for black carp; SY stands for water sample.

Figure 5 :
Figure 5: Similarity and diference analysis of fungi in each group.(a) UPGMA analysis, (b) LefSe analysis.Note: CY stands for grass carp, QY stands for black carp; SY stands for water sample.

Table 2 :
Coverage and diversity indices of fungal species in each group.
Note.CY stands for grass carp, QY stands for black carp; SY stands for water sample; compared with water sample.* stands for P < 0.05, * * stands for P < 0.01.

Table 1 :
Characteristics of ITS sequences in each sample.Note.CY stands for grass carp, QY stands for black carp; SY stands for water sample.

Table 3 :
Relative abundance of fungi identifed at phylum level in each group.Figure Te fungal community in all samples at phylum level.Note: CY stands for grass carp, QY stands for black carp; SY stands for water sample.

Table 4 :
Relative abundance of fungi in each group for the 10 most abundant genera.Note.CY stands for grass carp, QY stands for black carp; SY stands for water sample; compared with water sample.* stands for P < 0.05, * * stands for P < 0.01.