MOLECULAR IDENTIFICATION OF PLANKTONIC MEALS FOR TILAPIA SPECIES BASED ON rRNA GENE ANALYSES

Abstract

Tilapia is a well-known tropical freshwater fish native to Africa. These fishes have been considered as the most economically important warm water fishes used for world aquaculture production. In order to understand the contribution of gut microbiota to the health of tilapia fish, O. niloticus, at Lake Nasser, Egypt, a first molecular genetic study was applied to explore the diversity of fish gut microbiome, based on culture-independent analyses of rRNA genes. Ten individuals of O. niloticus, average weight 1000g,were collected from each of the four different khors namely; KhorKalabsha, KhorWadiAbyad, KhorKorosko and KhorTushka in the Lake Nasser. 1. Metagenomic DNAs extraction from guts microbiota Bulk DNAs were extracted from gut contents of O. niloticus, using DNA isolation kit. All DNAs purities were ranged between 1.6 and 1.95 based on A260/A280 reading. The metagenomic DNA concentrations were ranged from 330.80 ng/µLto 410.60 ng/µL. 2. PCR amplifications The eukaryotic 18S rRNA gene, bacterial and archaeal 16S rRNA genes were positively amplified by PCR, from bulk genomic DNAs of gut microbiota of O. niloticus from each khor. The eukaryotic 18S rRNA gene had amplicon size of 210 bp. The bacterial 16S rRNA gene amplicon had an approximate size of 433 bp, while the archaeal 16S rRNA gene amplicon had size of 590 bp. 3. Denatured Gradient Gel Electrophoresis, DGGE. The 18S rRNA gene amplicons were run on 9% polyacrylamide gel, while the 16S rRNA gene amplicons were run on 6% polyacrylamide gel. Electrophoresis was run for 17 h at 120 V and 35 Amp under a constant temperature of 60°C in a DCodeTM Universal Mutation DetectionSystem. After electrophoresis, the gels were stained with ethidium bromide, washed with distilled water, and visualized under UV light by Gel Doc™ XR+ imager. 4. Statistical analysis based on AlphaView and ProSeq program The DGGE patterns displayed 5, 12 and 5 band groups, phylotypes, for eukaryotic 18S rRNA gene, bacterial and archaeal 16S rRNA genes, respectively, in gut contents of O. niloticus from all studied khors. DGGE showed bands, which were common and specific for each site and could be used as a bar code to certify the origin of the fish. Statistical analyses, using binary matrix, showed that numbers of DGGE bands, 2.Euk.Common, 6.Bac.Common, 8.Bac.Common and 2.Arc.Common, for eukaryotes, bacteria and archaea, respectively, were commonly occurred in all studied khors. Nucleotide diversity between rRNA genes for each phylotype among tilapia guts from the studied khors was estimated using the freeware program ProSeq. 5. Sequencing and construction of phylogenetic tree Sequencing approach was applied on DGGE rRNA gene phylotype products to show the nucleotide sequences of recorded phylotypes. Bioinformatics analyses of rRNA gene phylotypes were done through bioinformatics processes, binary matrix, similarity index, nucleotide diversity and phylogenetic analyses. In total, 22 different phylotypes, eukaryotic, bacterial and archaeal communities were detected. Five eukaryal species were belonged to crustacean, Ostracoda, platyhelminths and acanthocephala, were identified. There were 12 bacterial phylotypes belongedto two phyla, cyanobacteria, and alphaproteobacteria. Most of the recorded bacteria-like phylotypes in the studied tilapia guts had unique phylogenetic lineages within the branch of uncultured environmental bacteria. All archaealphylotypes were located in the branch of uncultured methanogen Euryarchaeota. Some gut eukaryotic phylotypes from Khors, Kalabsha, Tushka and Korosko were recorded belonging to parasitic worms of the genera Neoechinorhynchusand Catenula, respectively. The study presented sharingof rRNA gene phylotypes among the studied khors, suggesting core gut microbiota.