Uluabat Gölü toksik siyanobakteri türleri ve toksinlerinin belirlenmesi
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Date
2020-03-03
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Bursa Uludağ Üniversitesi
Abstract
Küresel ısınma ve antropojenik ötrofikasyona bağlı olarak dünyanın pek çok bölgesinde özellikle de tatlısularda yaygın siyanobakteri çoğalmaları meydana gelmektedir. Siyanobakteriler, sularda hem tat ve koku problemlerine yol açarak hem de ürettikleri siyanotoksinler ile sucul ekosistemleri olumsuz etkilemektedir. Bu çalışma ile ülkemiz açısından ekolojik öneme sahip Uluabat Gölü’nün, siyanotoksinler ve potansiyel toksik siyanobakteriler açısından araştırılması amaçlanmıştır. Su kolonu ve plankton çekimi örnekleri, 2015 ve 2016 yıllarının Haziran ve Eylül ayları arasında dört farklı noktadan toplanmıştır. Uluabat Gölü’nden izole edilen siyanobakteri suşlarının kültürü yapılmıştır. Hem alan örneklerinde hem de kültür örneklerinde mikrosistin, saksitoksin, anatoksin-a ve silindrospermopsin toksinlerinin varlığı enzim bağlı immünosorbent deneyi (ELISA) ve reseptör bağlanma analizi (RBA) ile araştırılmıştır. Diğer yandan örneklerde polimeraz zincir reaksiyonu (PCR) ile 16S rRNA ve toksin genlerinin taraması yapılmıştır. Morfolojik karakterizasyon ile izole edilen suşların Microcystis aeruginosa, Dolichospermum cf. sigmoideum, Limnothrix planctonica, Cuspidothrix issatschenkoi, Calothrix elenkinii, Planktothrix isothrix, Dolichospermum ellipsoides, Sphaerospermopsis aphanizomenoides, Synechocystis aquatilis, Aphanocapsa grevillei ve Microcystis smithii taksonlarına ait olduğu belirlenmiştir. Ayrıca 16S rRNA gen dizilerine dayalı neighbor-joining (NJ) ve maximum likelihood (ML) filogenetik ağaçları, dokuz suşun M. aeruginosa, Limnothrix sp., Calothrix sp., S. aphanizomenoides ve C. issatschenkoi taksonlarına ait olduğunu doğrulamıştır. Uluabat Gölü’nde ve gölden izole edilen M. aeruginosa (Aquameb 24 ve 25) suşlarında mcy genleri ve mikrosistin üretimi tespit edilmiştir. Sıvı kromatografi-kütle spektrometresi/kütle spektrometresi (LC-MS/MS) analizi ile Aquameb 24 suşunda 430 μg g-1 kuru ağırlık, 22.07.2015 Nokta 1 plankton çekimi örneğinde 75 μg g-1 kuru ağırlık toplam MC tespit edilmiştir. Ayrıca M. smithii ve A. grevillei suşlarında mcyA ve mcyB genleri saptanmıştır. Plankton çekimi örneklerinden klonlanan mcyA dizileri, filogenetik ağaçlarda dünyadaki M. aeruginosa suşlarıyla birlikte gruplanmıştır. Moleküler ve toksikolojik veriler, M. aeruginosa türünün Uluabat Gölü’nde mikrosistin üretimine katkıda bulunan temel tür olduğunu fakat gölde mikrosistin üreticisi olabilecek Microcystis cinsine ait başka türlerin veya başka cinslerin bulunabileceğini düşündürmüştür. Bu çalışma Uluabat Gölü’nde mikrosistin ve üreticileri ile ilgili bilgi vermesi açısından önem teşkil etmektedir.
In many parts of the world, especially in freshwaters, widespread cyanobacteria proliferation occurs due to global warming and antropogenic eutrophication. Cyanobacteria adversely affects aquatic ecosystems both by causing taste and odor problems in waters and with cyanotoxins they produce. The aim of this study was to investigate cyanotoxins and potentially toxic cyanobacteria in Lake Uluabat, an important ecological site for our country. Water column and plankton tow samples were collected from four different sites between June and September of 2015 and 2016. Cyanobacterial strains isolated from Uluabat Lake were cultured. The presence of cyanobacterial toxins microcystin, saxitoxin, anatoxin-a and cylindrospermopsin in both field samples and culture samples were investigated by enzyme-linked immunosorbent assay (ELISA) and receptorbinding assay (RBA). On the other hand, 16S rRNA and toxin genes were screened by polymerase chain reaction (PCR) in these samples. Morphological characterizations suggested that the isolated strains belonged to Microcystis aeruginosa, Dolichospermum cf. sigmoideum, Limnothrix planctonica, Cuspidothrix issatschenkoi, Calothrix elenkinii, Planktothrix isothrix, Dolichospermum ellipsoides, Sphaerospermopsis aphanizomenoides, Synechocystis aquatilis, Aphanocapsa grevillei and Microcystis smithii. In addition, neighbor-joining (NJ) and maximum likelihood (ML) phylogenetic trees based on 16S rRNA gene sequences confirmed that nine strains belonged to M. aeruginosa, Limnothrix sp., Calothrix sp., S. aphanizomenoides and C. issatschenkoi. Mcy genes and microcystin production was determined in Lake Uluabat and M. aeruginosa (Aquameb 24 and 25) strains isolated from the lake. Total MC determined in Aquameb 24 strain was 430 μg g-1 dry weight, in plankton tow samples of 22.07.2015 Site 1 was 75 μg g-1 dry weight as determined with liquid chromatography-tandem mass spectrometry (LCMS/ MS) analysis. In addition, mcyA and mcyB genes were detected in M. smithii and A. grevillei strains. In the phylogenetic trees, the mcyA sequences cloned from plankton tow samples were grouped together with those from M. aeruginosa strains in the world. Molecular and toxin data suggest that M. aeruginosa is the main species that contributes to the production of microcystins in Lake Uluabat. However, other Microcystis species or other genera may be producing microcystins as suggested by MC variants in the lake samples. This study is important in terms of providing information about microcystin and its producers in Lake Uluabat.
In many parts of the world, especially in freshwaters, widespread cyanobacteria proliferation occurs due to global warming and antropogenic eutrophication. Cyanobacteria adversely affects aquatic ecosystems both by causing taste and odor problems in waters and with cyanotoxins they produce. The aim of this study was to investigate cyanotoxins and potentially toxic cyanobacteria in Lake Uluabat, an important ecological site for our country. Water column and plankton tow samples were collected from four different sites between June and September of 2015 and 2016. Cyanobacterial strains isolated from Uluabat Lake were cultured. The presence of cyanobacterial toxins microcystin, saxitoxin, anatoxin-a and cylindrospermopsin in both field samples and culture samples were investigated by enzyme-linked immunosorbent assay (ELISA) and receptorbinding assay (RBA). On the other hand, 16S rRNA and toxin genes were screened by polymerase chain reaction (PCR) in these samples. Morphological characterizations suggested that the isolated strains belonged to Microcystis aeruginosa, Dolichospermum cf. sigmoideum, Limnothrix planctonica, Cuspidothrix issatschenkoi, Calothrix elenkinii, Planktothrix isothrix, Dolichospermum ellipsoides, Sphaerospermopsis aphanizomenoides, Synechocystis aquatilis, Aphanocapsa grevillei and Microcystis smithii. In addition, neighbor-joining (NJ) and maximum likelihood (ML) phylogenetic trees based on 16S rRNA gene sequences confirmed that nine strains belonged to M. aeruginosa, Limnothrix sp., Calothrix sp., S. aphanizomenoides and C. issatschenkoi. Mcy genes and microcystin production was determined in Lake Uluabat and M. aeruginosa (Aquameb 24 and 25) strains isolated from the lake. Total MC determined in Aquameb 24 strain was 430 μg g-1 dry weight, in plankton tow samples of 22.07.2015 Site 1 was 75 μg g-1 dry weight as determined with liquid chromatography-tandem mass spectrometry (LCMS/ MS) analysis. In addition, mcyA and mcyB genes were detected in M. smithii and A. grevillei strains. In the phylogenetic trees, the mcyA sequences cloned from plankton tow samples were grouped together with those from M. aeruginosa strains in the world. Molecular and toxin data suggest that M. aeruginosa is the main species that contributes to the production of microcystins in Lake Uluabat. However, other Microcystis species or other genera may be producing microcystins as suggested by MC variants in the lake samples. This study is important in terms of providing information about microcystin and its producers in Lake Uluabat.
Description
Keywords
16S rRNA, Lake Uluabat, Uluabat Gölü, Mikrosistin, Saksitoksin, Anatoksin-a, Silindrospermopsin, Siyanobakteri kültürü, ELISA, Filogenetik, PCR, Microcystin, Saxitoxin, Anatoxin-a, Cylindrospermopsin, Culture of cyanobacteria phylogenetic
Citation
Özen, M. (2020). Uluabat Gölü toksik siyanobakteri türleri ve toksinlerinin belirlenmesi. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Fen Bilimleri Enstitüsü.