Dinâmica temporal das cianobactérias em um reservatório urbano hipereutrófico: uma abordagem morfológica e molecular

Abstract

Cyanobacteria are an important phytoplankton group that is favored in conditions of water eutrophication. Given their potential toxicity, blooms of cyanobacteria not only contaminate the water, but they also turn it unsuitable for human consumption. Additionally, their presence alters many ecological interactions in the aquatic ecosystem, thus making its prevention and control extremely relevant. Pampulha reservoir, known as Pampulha lake, is a small urban reservoir located in Belo Horizonte city (MG, Brazil) that has undergone a long process of eutrophication due to the continuous input of industrial and domestic sewage. The high nutrient supply in the reservoir has favored the occurrence of cyanobacterial blooms along the year and for this reason studies on the dynamics of these communities become essential. The objectives of this study were: (1) to evaluate the dynamics of the cyanobacterial community in the Pampulha reservoir during three years, aiming to detect factors influencing the proliferation of dominant species and their alternate occurrence and (2) to study the intraspecific diversity of the bloom forming species Cylindrospermopsis raciborskii in two different periods, since its genetic variability could favor its dominance. In order to achieve objective 1, monthly samplings were performed from 2010 to 2012 in Pampulha reservoir, water physical and chemical characteristics were measured and the cyanobacterial community was accessed through morphological and molecular approaches. Microscopic identification and counting of all samples by the Utermöhl (1958) technique were performed and, additionally, the Automated Ribosomal Intergenic Spacer Analysis (ARISA) was performed on thirteen most representative samples. Both approaches were able to detect three different blooms: one promoted by C. raciborskii (Nostocales) predominantly in the rainy season, another by the Chroococcales Microcystis aeruginosa and Sphaerocavum brasiliense at the end of the dry season and a bloom of Planktothix isothrix (Oscillatoriales) at the end of 2012, changing the alternating pattern between Nostocales and Chroococcales in the reservoir. The Non-Metric Multidimensional Scaling (NMDS) and the Analysis of Similarity (ANOSIM) revealed a clear division between rainy and dry periods in the 36 phytoplankton samples, suggesting that changes in the environmental parameters between seasons may modify the cyanobacterial community composition. The Principal Component Analysis (PCA) showed strong correlation between C. raciborskii biomass and increased temperature, water resistance, nitrite concentration and total precipitation. The Chroococcales species, on the other side, were unsuccessful in periods of water stratification while the dominance of Oscillatoriales was positively correlated to total phosphorus and ammonia concentration. The molecular approach through ARISA allowed a more robust study of the cyanobacterial community, since it was more efficient than microscopy to differentiate close related species. The rapidity and precision of ARISA suggest its use as a potential method for environmental monitoring studies. Aiming to achieve objective 2, two samples of bloom periods in the reservoir were chosen: in the first C. raciborskii represented almost 100% of the total phytoplanktonic biomass and in the second it was codominant with Chroococcales. The genetic variability of the intergenic transcribed spacer (ITS) between the 16S rRNA and the 23S rRNA in C. raciborskii populations was studied through restriction analyses of the amplified DNA and Next Generation Sequencing (NGS) analysis. Both approaches revealed a larger variability during the monospecific bloom of C. raciborskii, suggesting that a selective pressure resulting from the interspecific competition leads to a reduced diversity within the population. The detection of intraspecific variability in nature is an evidence that different ecotypes of the species coexist during the bloom and may contribute to its stability in the environment. More polymorphisms in the ITS 16S-23S fragment were detected through NGS than through restriction analyses of the amplified DNA, revealing better accuracy of the NGS and the subsequent bioinformatics analyses. Further studies will be done aiming to detect the percentage of genetic variants present in each NGS sample and to evaluate the phylogenetic relationships between them. The present work has contributed to a better understanding on the dynamics of the dominant cyanobacteria in Pampulha reservoir and on the ecological and genetic factors that can influence or favor their blooms under different conditions. This work also exemplifies the importance of the molecular approach while studying environmental microbial communities.