Molecular variations in Vibrio alginolyticus and V. harveyi in shrimp-farming systems upon stress

Abstract

A study was performed to investigate the genomic variations in the shrimp farm isolates of Vibrio alginolyticus and V. harveyi when the isolates were subjected to environmental stress. Samples of shrimps, water and sediment were collected from Southern Indian coastal shrimp farms. Vibrio isolates were biochemically identified and confirmed using 16S rDNA and gyrB gene specific PCR. The bacterial strains were genotyped by PCR fingerprinting using GTG(5) and IS (Insertion Sequence) primers. Seven strains each of V. alginolyticus and V. harveyi were subjected to 10 passages through trypticase soya broth (TSB), which contained different NaCl concentrations (3, 6 and 8%) and trypticase soya agar (TSA). V. alginolyticus was also passaged through TSB with a 12% NaCl concentration. PCR fingerprinting, which was performed on the strains that were passaged through different salt concentrations, confirmed that V. alginolyticus and V. harveyi could affect the genomic variations, depending on the environmental conditions of the culture. The study highlights the complex genotypic variations that occur in Vibrio strains of tropical aquatic environment because of varied environmental conditions, which result in genetic divergence and/or probable convergence. Such genetic divergence and/or convergence can lead to the organismal adaptive variation, which results in their ability to cause a productive infection in aquatic organisms or generation of new strains.

Figure 1. Fingerprint patterns generated using GTG PCR amplification of the genomic DNA in 1.2% agarose gel (a) V. harveyi strains at 0 passage; lane 1- DS134, lane 2- DS149, lane 3- DS158, lane 4- DS165, lane 5- DS184, lane 6- DS218, lane 7- DS260. (b) V. alginolyticus strains at 0 passage. Lane1- DS29, lane 2- DS199, lane 3- DS200, lane 4-DS246, lane 5- DS263, lane 6- DS334, lane 7- DS350.

Figure 2. Fingerprint patterns generated using IS PCR amplification of the genomic DNA of V. harveyi with primers, which were targeted at insertion sequences in 1.2% agarose gel. The number of strains is indicated in each gel. Lane 1- 0 passage, lane 2 to 4- after 10 passages in 3, 6 and 8% NaCl containing medium, respectively, lane 5- after 10 passages in TSA for strains DS134, DS158, DS184 and DS260. Lane 2- 0 passage, lane 3 to 5- after 10 passages in 3, 6 and 8% NaCl containing medium, respectively, lane 6- after 10 passages in TSA for strains DS149, DS165 and DS218.

Figure 3. Fingerprint patterns generated using GTG (5) PCR amplification of the genomic DNA of V. harveyi in 1.2% agarose gel. The number of strains is indicated in each gel. Lane 1- 0 passage, lane 2 to 4- after 10 passages in 3, 6 and 8% NaCl containing medium, respectively, lane 5- after 10 passages in TSA for strains DS134, DS158, DS184 and DS260. Lane 2- 0 passage, lane 3 to 5- after 10 passages in 3, 6 and 8% NaCl containing medium, respectively, lane 6- after 10 passages in TSA for strains DS149, DS165 and DS218.

Figure 4. Fingerprint patterns generated using IS PCR amplification of the genomic DNA of V. alginolyticus with primers, which were targeted at the insertion sequences in 1.2% agarose gel. The number of strains is indicated in each gel. Lane 1- 0 passage, lane 2 to 5- after 10 passages in 3, 6, 8 and 12% NaCl containing medium, respectively, lane 6- after 10 passages in TSA for strains DS29, DS199, DS246, DS263, DS334 and DS350. For strain 200, the order starts from 3- 8, respectively.

Figure 5. Fingerprint patterns generated using GTG (5) PCR amplification of the genomic DNA of V. alginolyticus in 1.2% agarose gel. The number of strains is indicated in each gel. Lane 1- 0 passage, lane 2 to 5- after 10 passages in 3, 6, 8 and 12% NaCl containing medium, respectively, lane 6- after 10 passages in TSA for strains DS29, DS200, DS263 and DS350. Lane 2- 0 passage, lane 3 to 6- after 10 passages in 3, 6, 8 and 12% NaCl containing medium, respectively, lane 7- after 10 passages in TSA for strains DS199, DS246 and DS334.