Illumina Based Analysis of Bacterial and Fungal Flora in Foreguts and Hindguts of Crucian Carp (Carassius Aumtus) in Retail Markets

The intestinal microbiota of fi sh are the major cause for spoilage. To elucidate the bacterial and fungal community of guts, the bacterial and fungal taxa in foreguts and hindguts of crucian carp (Carassius aumtus) were analyzed by Illumina-based sequencing. Results suggested that the foreguts contained more diverse bacteria than those in hindguts; nevertheless, the hindguts contained more diverse fungi than foreguts. The anaerobic bacterial genera Cetobacteri Desulfovibrio and Shewanella in foreguts were still detected in hindguts. The fungal taxa in foreguts were different from those in hindguts. The dominant fungal genera Alternaia (78.6%), Massarina (0.8%) and Fusarium (0.2%) were only detected in hindguts. It was considered that the Alternaria, Emericella, and Cochliobolus might derive from the diets of crucian carp. The activities of Desulfovibrio might be responsible for the changes in odour, fl avor and texture of the fi sh meat. The H2S produced by Desulfovibrio is potentially a major toxin to the fi sh gut epithelium and promoted the fi sh spoilage processes. The results are helpful for manipulation of intestinal fl ora to preserve fresh crucian carp in tanks. Research Article Illumina Based Analysis of Bacterial and Fungal Flora in Foreguts and Hindguts of Crucian Carp (Carassius Aumtus) in Retail Markets Mingyue Wang, Miaomiao Yin, Hongming Tan, and Lixiang Cao* School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China Dates: Received: 30 Decmber, 2016; Accepted: 20 January, 2017; Published: 23 January, 2017 *Corresponding author: Lixiang Cao, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China, Tel: 8620-84110238, Fax: 862084036215; E-mail:


Introduction
As with good survival rate, high reproduction rate, and disease resistance, crucian carp (Carassius auratus) is widely bred across Eurasia and America [1]. In China, crucian carp is one of the most economically important freshwater-cultured fi sh species. The production yield reached nearly 2,000,000 tons in 2009 [2]. However, fi sh are more perishable than other muscle foods, and a considerable number of fi sh are spoiled due to lack of good preservation. In world, 30% of landed fi sh are spoiled and lost [3].
Fresh fi sh spoilage can be very rapid after it is caught [3].
Crucian carp is an easily perishable product because of its relatively high quantities of volatile basic nitrogen as well as free amino acids, high water activity, and presence of autolytic enzymes [2]. As one of the most important food fi sh in China, only alive crucian carp fi sh is accepted in market [4]. The spoilage of fi sh is a complicated process in which microbial, physical and chemical variations interact. Activity of the fi sh's own enzymes and chemical reactions are usually responsible for the initial loss of fi sh freshness, whereas the metabolic activities of microorganisms are involved in the whole spoilage [2]. Gutting of the fi sh immediately after capture can avoid the invasion of digestive tract proteases through the abdominal cavity to the tissue and prevent or slow degradation [3]. However, there may be chances of bacterial crosscontamination of fi sh during the gutting procedure. Microbial growth and metabolism are the major cause for food spoilage, the microbial populations may shift during storage and only a small fraction of fi sh microbiota is responsible for spoilage, known as "specifi c spoilage organisms" [5]. These specifi c spoilage organisms are present in low number in fresh fi sh and can eventually become dominant in spoilage microorganisms [6].
Bacterial fl ora isolated from eggs, skins, gills, and intestines have been described for some fi sh species. Bacteria recovered from the skin and gills may be transient rather than resident on the fi sh surfaces [7]. The gastrointestinal microbiota in fi sh is constituted of facultative and obligate anaerobes, which may vary among fi sh species with different digestive apparatus [8].
However, the zebrafi sh intestinal habitat select for specifi c bacterial taxa despite radical differences in host provenance and domestication status [9]. So far, the microbial community in fi sh guts has not been systematically characterized [10].
Most previous studies used traditional culture-dependent or DGGE, clone library methods to investigate the fi sh intestinal Citation: Wang  bacterial diversity [11][12][13][14][15][16][17]. With the development of the next-generation sequencing technologies, high-throughput sequencing platform is available for characterizing the bacterial community in the gut of fi sh [10,[18][19][20]. More comprehensive information on fi sh gut microbiota would be obtained by highthroughput sequencing approaches. Illumina has fewer errors than 454 sequencing and it could provide a higher phylogenetic resolution than 454 based approaches [21]. The advantage of Illumina to provide 30 times more reads would enable us to perform in depth sequencing of samples in one run, making it an excellent tool for gut microbial diversity.
The objective of this study was to elucidate the bacterial and fungal fl ora in foreguts and hindguts of crucian carp. Specially, total DNAs of foreguts and hindguts were purifi ed and analyzed by Illumina-based sequencing. The bacterial and fungal communities in foreguts and hindguts were further compared, the spoilage bacterial and fungal taxa in foreguts and hindguts were elucidated in the study.

Fish sample
Ten live commercial-sized crucian carp with average weight of 250 ± 20 g were purchased from aquatic market in March, 2015. They were kept alive before being processed. The fi sh were killed by slurry ice and gutted under sterile conditions. The portion of intestinal tract posterior from bile duct to the fi rst distal loop (foreguts) and the intestinal tract anterior from anus to last anterior loop (hindguts) were removed for DNA extraction.

DNA Extraction
The foreguts (designated as QianC), hindguts (designated as HouC) were used for total DNA extraction. The total DNA was extracted using PowerSoil ® DNA Isolation Kit (Mol Bio) according to the manufacture's instruction. Total DNA concentration and purity were monitored on 1% agarose gels.

Amplicon Generation and Illumina MiSeq sequencing
The primers S-D-Bact-0341-b-S-17 (5'-CCTACGGGNG-GCWGCAG -3') and S-D-Bact-0785-a-A-21 (5' -GACTACH-VGGGTATCTAATCC -3') targeting the V3-V4 hyper variable regions of bacterial 16S rRNA genes were selected for analyze bacterial taxa [22]. The primers ITS5-1737F: GGAAG-TAAAAGTCGTAACAAGG; ITS2-2043R: GCTGCGTTCTTCATC-GATGC targeting the ITS2 regions of fungal rRNA genes were adopted to analyze fungal taxa [23]. Both forward and reverse primers were tagged with adapter, pad and linker sequencing. Each barcode sequence was added to the reverse primer for pooling multiple samples into one run of sequencing. All PCR reactions were performed in a total volume of 30μL containing 15μL Phusion ® High-Fidelity PCR Master Mix (New England Biolabs) and 0.5 units of AccuPrimer TM Taq DNA Polymerase (Life Technologies, USA), 0.2 μM of forward and reverse primers, and 10 ng template DNA. Thermal cycling conditions were as follows: an initial denaturation at 98 ºC for 1 min, each of 30 cycles at 98 ºC for 10 s, 50 ºC for 30 s, and 72 ºC for 60 s, with a fi nal extension at 72 ºC for 5 min.
Following amplifi cation, 2 μL of PCR product was used to verify successful amplifi cation by 2% agarose gel electrophoresis. The products of triplicate PCR reaction from one sample were combined and the pooled mixtures were purifi ed with GeneJET Gel Extraction Kit (Thermo Scientifi c) and analyzed on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Chips (Agilent Technologies, Germany) for size distribution. The sequencing libraries were generated using NEB Next ® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientifi c) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Germany). Finally, the library was sequenced on an Illumina MiSeq platform at Magigen biotechnology Co. Ltd, Guangzhou, China.

Combination and data preprocessing
Forward and reverse sequences were merged by overlapping paired-end reads using FLASH (V1.2.7, http://ccb.jhu.edu/ software/FLASH/) [24]. All sequence reads with the same tag were assigned to the same sample according to the unique barcodes (raw tags). The raw tags were further strictly fi ltered by previous methods (clean tags) [25] and the quality of clean tags were detected by Qiime (V1.7.0 http://qiime.org/index. html) [22]. The low quality tags were removed. The tags with chimera were detected and removed using UCHIME Algorithm (http://www.drive5.com/usearch/manual/uchime_algo.html) [26,27]. The effective sequences were then clustered into operational taxonomic units (OTU) at 97% sequence similarity using the UPARSE-OTU and UPARSE-OTUref algorithms of UPARSE software package (Uparse v7.0.1001 http://drive5.com/ uparse/), the indices of alpha diversity were calculated [28]. Finally, the RDP classifi er was used to assign representative sequence to the microbial taxa [29]. Sequence data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number SRP062743.

Statistical analysis
Cluster analysis was preceded by principal component analysis (PCA) using the QIIME software package. QIIME calculates both weighted and unweighted unifrac distances, which are phylogenetic measures of beta diversity [30], the phylogenetic relations among different microbial taxa were further displayed by KRONA [31]. Alpha diversity indices Chao1, ACE, Shannon, Simpson and coverage were calculated to refl ect the diversity and richness of the endophytic community in different samples [32].  indicated that the foreguts contained more diverse bacteria than those in hindguts; nevertheless, the hindguts contained more diverse fungi than foreguts (Table 2).

Fungal and bacterial species richness and diversity
Total 229 bacterial OTUs were detected in foreguts, 148 OTUs were still detected in hindguts (Figure 1). Contrary to bacteria, the hindguts contained more fungal OTU than foreguts, and 42 OTUs were detected in foreguts simultaneously (Figure 2).   * Both Chao1 and ACE described an estimate of the total number of phylotypes in a source environment, and Chao1 is particularly appropriate for data sets in which most phylotypes are relatively rare in the community, ACE is appropriate for data sets in which some phylotypes occur more frequently. Both Shannon and Simpson index comprehensively refl ect the richness and evenness of community, Shannon index is more sensitive to the richness of the community, and Simpson index is more sensitive to the evenness of the community. Coverage is a non-parametric estimator of the proportion of phylotypes in a library of infi nite size that would be represented in a smaller library.  The bacterial phyla Proteobacteria (55.6%), Fusobacteria (27.5%) and Bacteroidetes (7.7%) in phylum level with high abundance were detected in two types of samples ( Figure 5). The fungal phyla Ascomycota (50.4%) and Basidiomycota(0.3%) were detected in the two types of samples ( Figure 6).

Discussion
Crucian carp is one of the most important food fi sh in China, and only alive fi sh is accepted in markets. Aquariums and tanks displaying live crucian carp caught in supermarkets and retail outlets are becoming increasingly common in China [4]. The water body in tanks is different from that in aquaculture, the effects of water body on intestinal fl ora of live crucian carp caught is still unknown. Our results illustrated that the foreguts contained more diverse bacteria than those in hindguts; nevertheless, the hindguts contained more diverse fungi than those in foreguts. The bacterial diversity distributed in foreguts and hindguts of crucian carp is consist with that in indigenous planktivorous gizzard shad, contratry to that in invasive Asian siler carp in Mississippi river basin, USA [10]. Although gut microbiota has become an integral component of the host, and received increasing attention [18], the fungal diversity in fi sh gut fl ora is not still reported [10]. The fi sh gut microbiota is only focused on bacteria [7][8][9][10][11][12][13][14][15][16][17][18][19]. Our results showed that the hindguts contained more diverse fungi than those in foreguts. The fungal phyla Ascomycota and Basidiomycota with high abundance were detected in guts of crucian carp. Fungal orders Pleosporales, Eurotiales, Saccharomycetales in foreguts were still detected in hindguts. The fungal taxa in foreguts were different from those in hindguts. The dominant fungal genera Alternaia (78.6%), Massarina (0.8%) and Fusarium (0.2%) were only detected in hindguts. Most Alternaria species are saprophytes that are commonly found in soil or on decaying plant tissues. Some species are opportunistic plant pathogens that cause a range of disease (stem cancer, leaf blight, or leaf spot) with economic impact on the variety of important agronomic crops [33]. Alternaria species have been frequently isolated as endophytes from leaves and stems but not from roots [34][35][36].
Alternaria alternate has been isolated as the most dominant species in leaves [35]. So it was considered that the Alternaria, Emericella, and Cochliobolus might derive from the diets of crucian carp.    More diverse bacteria in foreguts were detected than those in hindguts. The anaerobic bacterial genera Cetobacterium Desulfovibrio and Shewanella in foreguts were still detected in hindguts. The Cetobacterium spp. had been detected in the intestinal contents of goldfi sh (Carassius auratus) [8]. The Shewanella also been found in intestinal contents of goldfi sh, yellow catfi sh, rainbow rout [8,11,17]. The sulphate-reducing bacteria Desulfovibrio had not been detected in fi sh guts [7][8][9][10][11][12][13][14][15][16][17][18][19], although the Desulfovibrio are the most routinely recovered from animal and human faeces [37]. The results illustrated that the intestinal fl ora varied after the live crucian carp was caught and maintained in the tanks. The Desulfovibrio served as terminal oxidizers in the anaerobic degradation of organic matter entering the gastrointestinal tract and reduce sulphur and suphur-containing compounds to hydrogen sulphide (H 2 S) [37]. The production of H 2 S by the Desulfovibrio is potentially a major toxin to the fi sh gut epithelium and promoted the fi sh spoilage processes. The activities of Desulfovibrio might be responsible for the changes in odour, fl avor and texture of the fi sh meat. Our results were consistent with the fact that fresh fi sh spoilage can be very rapid after it is caught [3]. So the manipulation of intestinal fl ora to preserve fresh crucian carp in tank would be further studied.