First report of genus Chroococcidiopsis ( cyanobacteria ) from Sri Lanka : a potential threat to human health

A number of studies have been conducted in Sri Lanka for identifying fresh water cyanobacteria based on morphological features, and about 170 cyanobacterial species belonging to 45 genera have been described (Abeywickrama et al., 1986). Microcystis aeruginosa, M. ,. incerta, M. wesenbergi, Cylindrospermopsis, Aphanizomenon, Spirulina, Anabaena aphanizomenoids, , Aphanocapsa, Chroococcus, Gloeocapsa, Nostoc and Coelosphaerium have been frequently recorded from different parts of the country. However, there are no recorded reports on Chroococcidiopsis species from Sri Lanka. Members of the genus Chroococcidiopsis are very primitive, photosynthetic, coccoid cyanobacteria and they have the capability to survive under high radiation, extreme temperatures, osmotic stress and extreme pH values. Genus Chroococcidiopsis have been found in freshwater, marine, hypersaline environments (Dor et al., 1991), hot springs, nitrate caves (Geitler, 1933; Friedmann, 1962), hot and cold deserts, airspaces of porous rocks from Antarctic valleys and in several lichens as cyanobionts (Büdel et al., 2000).

Cyanobacteria (Cyanophytes, Cyanoprokaryotes) are a large group of photosynthetic bacteria and one of the most fascinating groups of organisms on the earth.They show a considerable morphological diversity.Microscopy is the classical method of cyanobacterial to classify cyanobacteria into accurate taxonomic groups Consequently, molecular biological techniques have become a popular tool for phylogenetic analysis of the cyanobacteria.Among the various gene sequences used to assess cyanobacterial biodiversity, 16S rRNA gene has been applied more frequently because it is ubiquitous, function is conserved, the gene is easy to sequence and a large database is available for sequence alignments and et al, 2009).
A number of studies have been conducted in Sri Lanka for identifying fresh water cyanobacteria based on morphological features, and about 170 cyanobacterial species belonging to 45 genera have been described (Abeywickrama et al., 1986) (Geitler, 1933;Friedmann, 1962), hot and cold deserts, airspaces of porous rocks from Antarctic valleys and in several lichens as cyanobionts (Büdel et al., 2000).

While investigating the biodiversity of cyanobacteria in different parts of Sri Lanka, Chroococcidiopsis
Hambantota, Sammanthurai, the Kondawatuwana Tank and the Mahaweli River using molecular techniques.It is interesting to note that until now there have been no reports indicating the occurrence of Chroococcidiopsis species in Sri Lanka.
Water and soil samples were collected into sterilized brown glass containers from Yala, Hambantota, Sammanthurai, the Kondawatuwana Tank and the Mahaweli River (Table 1).Collected water samples were concentrated by centrifugation (3500 rpm, 10 min).An aliquot of 500 µL from the centrifuged pellet and 500 µL from the supernatant and soil samples were inoculated were made from each sample using compound light microscope (Olympus BH2) (400 -1,000×) and standard (5'-GGGGAATYTTCCGCAATGGG-3') and the reverse primer CYA781Rb (5'-GACTA CAGGGGTATCTAATCCCTTT-3').All PCR reactions were performed in a 50 µL reaction mixture containing 0.6 µM of each primer, 100 µM of each deoxynucleoside triphosphate, 10 µL of 5x PCR buffer, 2 mM MgCl 2 , 2.5 U of DNA polymerase (Promega, Madison, Wisconsin, USA) and template DNA.Thermal cycling was performed in a Techine TC 3000 DNA Thermal Cycler.The initial denaturation step at 94 °C for 5 min was followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 1 min and a -1 ethidium bromide and documented through a Gel Documentation System (Syngene, UK).PCR products were carefully excised TM Gel Extraction Kit (SIGMA) according to the manufacturer's instructions and DNA sequencing was carried out by Macrogen Inc., South Korea using ABI 3730XL sequencers with the reverse primer.DNA sequences obtained in this study were deposited in GenBank under accession numbers EU276382, EU276383, EU310420, EU310430, EU310432, GU300772 and GU594024.
Microscopic observation of wet mounts of cultures mature undivided cells were spherical, dark blue green in colour.They formed round or quadratic shaped colonies.
were no considerable morphological variations among the colonies (Figure 1).According to the gel primers yielded the unique fragment of ~ 450 bp.16S rRNA sequences of the tested isolates showed a DNA sequence similarity of 96 to 99 % with previously reported Chroococcidiopsis isolates (Table 1).

Figure 1 :
Figure 1: Light microscopic view of Chroococcidiopsis Details of the sample collection of local isolates of Chroococcidiopsis sp. and comparison of 16S rRNA sequences Table1: