Composição de cianobactérias e análise de genes responsáveis pela biossíntese de cianotoxinas provenientes do Corpo Central I da Represa Billings (São Paulo/SP – Brasil)
Data
2023-06-27
Autores
Ribeiro, Matheus Santos Freitas 
Orientadores
Niero, Cristina Viana
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Atualmente, as cianobactérias são consideradas os organismos fotossintetizantes mais
difundidos em corpos de água doce mundiais. Algumas linhagens produzem metabólitos
secundários tóxicos que afetam eucariotos, as cianotoxinas. A Represa Billings, local do estudo,
apresenta frequentes florações de cianobactérias e relatos da presença de microcistina e saxitoxina,
o que leva à preocupação sanitária, uma vez que se trata de um reservatório utilizado para
abastecimento de água potável para as cidades do entorno. Desta forma, este trabalho teve como
objetivos: i) identificar as espécies de cianobactérias presentes nas amostras, ii) verificar a
densidade dos táxons encontrados, iii) realizar a detecção de genes do cluster gênico responsáveis
pela produção de microcistinas e saxitoxinas. Para a coleta, foram escolhidos quatro pontos na
Represa Billings, todos localizados no Corpo Central I, compartimento hipereutrófico. As coletas
foram realizadas em 2019. Após identificação das espécies e quantificação destes organismos pelo
método de Utermöhl, foram calculados os biovolumes (mm³/L) dos táxons. Amostras foram
utilizadas para extração de ácidos nucleicos para posterior amplificação do cluster gênico das
microcistinas e saxitoxinas por PCR, PCR em tempo real e hibridação, além de quantificação de
microcistina por HPLC/MS e de saxitoxina por ELISA. Os resultados mostraram que o biovolume
de cianobactérias foi maior do que o observado para os organismos fotossintetizantes eucariotos em
todas as amostras, com dominância das cianobactérias variando de 1,19 a 46,2 mm³/L. As espécies
Microcystis aeruginosa e Woronichinia naegeliana contribuíram com grande parte do biovolume
observado em todas as amostras. Houve amplificação dos genes mcyA, mcyB, mcyE, mcyF, mcyG
e mcyJ pela técnica de PCR convencional, enquanto os genes mcyC e mcyD foram detectados
somente por PCR em tempo real e hibridação, respectivamente. As microcistinas foram observadas
em todas as amostras estudadas, variando de 0,33 a 50,7 ug/L. As variantes de microcistinas mais
abundantes nas amostras foram MC-RR, -LR e -YR respectivamente sendo que -LY foi relatada
pela primeira vez em águas brasileiras. Com relação as saxitoxinas, os genes sxtA, sxtB, sxtC, sxtD,
sxtG, sxtH, sxtT e sxtW foram amplificados por PCR convencional nas doze amostras. Os genes
sxtIJKL, sxtN, sxtS, sxtU e sxtX foram avaliados por hibridação e apenas duas amostras (P1 e P2,
Coleta 3) não ocorreram a deteção dos genes sxtU e sxtS pelas técnicas utilizadas. As concentrações
de saxitoxina variaram de 0,05 a 0,087 ug/L nas amostras analisadas. Espécies conhecidamente
produtoras de microcistina, como Microcystis aeruginosa, e de saxitoxina, como Raphidiopsis
raciborskii, foram observadas durante o monitoramento dos táxons presentes de cianobactérias.
Este trabalho reforça a possibilidade da utilização de técnicas de biologia molecular para
monitoramento de cianotoxinas associadas ou não à métodos químicos ou bioquímicos.
Currently, cyanobacteria are considered the most widespread photosynthetic organisms in freshwater bodies worldwide. Some strains produce toxic secondary metabolites that affect eukaryotes, cyanotoxins. The Billings Reservoir, the study site, has frequent cyanobacterial blooms and reports of the presence of microcystin and saxitoxin, which leads to health concerns, since it is a reservoir used to supply drinking water to the surrounding cities. Thus, this work aimed to: i) identify the species of cyanobacteria present in the samples, ii) verify the density of the taxa found, iii) perform the detection of genes of the gene cluster responsible for the production of microcystins and saxitoxins. For the collection, four points were chosen in the Billings Reservoir, all located in the Central Body I, hypereutrophic compartment. The collections were carried out in 2019. After identifying the species and quantifying these organisms by the Utermöhl method, the biovolumes (mm³/L) of the taxa were calculated. Samples were used for nucleic acid extraction for subsequent amplification of the microcystin and saxitoxin gene cluster by PCR, real-time PCR and hybridization, in addition to quantification of microcystin by HPLC/MS and saxitoxin by ELISA. The results showed that the biovolume of cyanobacteria was greater than that observed for eukaryotic photosynthetic organisms in all samples, with dominance of cyanobacteria ranging from 1.19 to 46.2 mm³/L. The species Microcystis aeruginosa and Woronichinia naegeliana contributed with a large part of the biovolume observed in all samples. There was amplification of the mcyA, mcyB, mcyE, mcyF, mcyG and mcyJ genes by the conventional PCR technique, while the mcyC and mcyD genes were only detected by real-time PCR and hybridization, respectively. Microcystins were observed in all samples studied, ranging from 0.33 to 50.7 ug/L. The most abundant microcystin variants in the samples were MC-RR, -LR and -YR were, respectively, the most abundant and -LY was reported for the first time in Brazilian waters. Regarding saxitoxins, the sxtA, sxtB, sxtC, sxtD, sxtG, sxtH, sxtT and sxtW genes were amplified by conventional PCR in the twelve samples. The sxtIJKL, sxtN, sxtS, sxtU and sxtX genes were evaluated by hybridization and only two samples (P1 and P2, Collection 3) did not detect the sxtU and sxtS genes by the techniques used. Saxitoxin concentrations ranged from 0.05 to 0.087 ug/L in the analyzed samples. Species known to produce microcystin, such as Microcystis aeruginosa, and saxitoxin, such as Raphidiopsis raciborskii, were observed during the monitoring of present taxa of cyanobacteria. This work reinforces the possibility of using molecular biology techniques to monitor cyanotoxins associated or not with chemical or biochemical methods.
Currently, cyanobacteria are considered the most widespread photosynthetic organisms in freshwater bodies worldwide. Some strains produce toxic secondary metabolites that affect eukaryotes, cyanotoxins. The Billings Reservoir, the study site, has frequent cyanobacterial blooms and reports of the presence of microcystin and saxitoxin, which leads to health concerns, since it is a reservoir used to supply drinking water to the surrounding cities. Thus, this work aimed to: i) identify the species of cyanobacteria present in the samples, ii) verify the density of the taxa found, iii) perform the detection of genes of the gene cluster responsible for the production of microcystins and saxitoxins. For the collection, four points were chosen in the Billings Reservoir, all located in the Central Body I, hypereutrophic compartment. The collections were carried out in 2019. After identifying the species and quantifying these organisms by the Utermöhl method, the biovolumes (mm³/L) of the taxa were calculated. Samples were used for nucleic acid extraction for subsequent amplification of the microcystin and saxitoxin gene cluster by PCR, real-time PCR and hybridization, in addition to quantification of microcystin by HPLC/MS and saxitoxin by ELISA. The results showed that the biovolume of cyanobacteria was greater than that observed for eukaryotic photosynthetic organisms in all samples, with dominance of cyanobacteria ranging from 1.19 to 46.2 mm³/L. The species Microcystis aeruginosa and Woronichinia naegeliana contributed with a large part of the biovolume observed in all samples. There was amplification of the mcyA, mcyB, mcyE, mcyF, mcyG and mcyJ genes by the conventional PCR technique, while the mcyC and mcyD genes were only detected by real-time PCR and hybridization, respectively. Microcystins were observed in all samples studied, ranging from 0.33 to 50.7 ug/L. The most abundant microcystin variants in the samples were MC-RR, -LR and -YR were, respectively, the most abundant and -LY was reported for the first time in Brazilian waters. Regarding saxitoxins, the sxtA, sxtB, sxtC, sxtD, sxtG, sxtH, sxtT and sxtW genes were amplified by conventional PCR in the twelve samples. The sxtIJKL, sxtN, sxtS, sxtU and sxtX genes were evaluated by hybridization and only two samples (P1 and P2, Collection 3) did not detect the sxtU and sxtS genes by the techniques used. Saxitoxin concentrations ranged from 0.05 to 0.087 ug/L in the analyzed samples. Species known to produce microcystin, such as Microcystis aeruginosa, and saxitoxin, such as Raphidiopsis raciborskii, were observed during the monitoring of present taxa of cyanobacteria. This work reinforces the possibility of using molecular biology techniques to monitor cyanotoxins associated or not with chemical or biochemical methods.
Descrição
Citação
RIBEIRO, Matheus S. Freitas. Composição de cianobactérias e análise de genes responsáveis pela biossíntese de cianotoxinas provenientes do Corpo Central I da Represa Billings (São Paulo/SP – Brasil). 2023. 76 f. Tese (Doutorado em Biologia Química) - Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, 2023.