Environmental DNA metabarcoding reveals diverse phytoplankton assemblages and potentially harmful algal distribution along the urban coasts of Türkiye.

Marine phytoplankton are widely used to monitor the state of the water column due to their rapid changes in response to environmental conditions. In this study, we aimed to investigate the coastal phytoplankton assemblages, including bloom-forming species using high-throughput sequencing of 18S rRNA genes targeting the V4 region and their relationship with environmental variables along the Istanbul coasts of the Sea of Marmara. A total of 118 genera belonging to six phyla were detected. Among them, Dinoflagellata (36) and Bacillariophyta (26) were represented with the highest number of genera. According to the relative abundance of DNA reads, the most abundant taxa were Dinoflagellata_phylum (18.1%), Emiliania (8.4%), Biecheleria (8.4), and Noctiluca (8.1%). The ANOSIM test showed that there was a significant temporal difference in the assemblages, while the driving environmental factors were pH, water temperature, and salinity. According to the TRIX index, the trophic state of the coasts was highly mesotrophic and eutrophic. In addition, 45 bloom-forming and HAB taxa were detected and two species of Noctiluca and Emiliania, which frequently cause blooms in the area, were recorded in high abundance. Our results provide insight into the phytoplankton assemblages along the urbanized coastlines by analysing the V4 region of 18S rRNA. This data can support future studies that use both traditional methods and metabarcoding, employing various primers and targeting different genes and regions.

Does environmental DNA reflect the actual phytoplankton diversity in the aquatic environment? Case study of marine mucilage in the Sea of Marmara.

The present study was designed to assess the effectiveness of the eDNA metabarcoding approach to determine the phytoplankton composition in the marine environment with a special focus on mucilage episodes in the Sea of Marmara. For this purpose, the samples were collected from 5 different sites located in the Sea of Marmara and the northern Aegean Sea during the mucilage episode in June 2021. The phytoplankton diversity was analyzed morphologically and by 18S rRNA gene amplicon sequencing, and the dataset of both methods was compared, accordingly. The results showed significant differences between methods in terms of composition and the abundance of the phytoplankton groups. While Miozoa was the most abundant group by metabarcoding, light microscopy (LM) indicated a dominance of Bacillariophyta. Katablepharidophyta was found at lower abundances by the metabarcoding (representing < 1% of the community); the members of this phylum were not observed by a microscope. At the lower taxonomic levels, Chaetoceros was the only genus detected in all samples by both methods. Additionally, while mucilage-forming Gonyaulax fragilis, Cylindrotheca closterium, and Thalassiosira rotula were detected to species-level by LM, metabarcoding was able to determine these organisms at the genus level. On the other hand, the genus Arcocellulus was found in all metabarcoding datasets and not detected by microscopy. The results indicated that metabarcoding can detect a greater number of genera and reveal taxa that were overlooked by light microscopy but to develop a complete picture of phytoplankton diversity in the sample, microscopical observations still are in need.

Insights into the bacterial community structure of marine mucilage by metabarcoding.

This prospective study was aimed to explore the bacterial diversity of marine mucilage developed in the Marmara Sea and the North Aegean Sea by metabarcoding. For this purpose, mucilage samples were collected from five different sampling locations, and the bacterial community structure was analyzed by 16S rRNA gene amplicon sequencing. The results highlighted a diverse bacterial community dominated by Proteobacteria and Bacteroidetes species. A negative and significant correlation between pH level and Campylobacterales, Clostridiales, and Vibronales abundances was detected, while a strong positive correlation was determined between total phosphorus (TP) and Campylobacterales. Results revealed that the bacterial community in the mucilage samples was predominated by particle-attached species preferring high-nutrient concentrations. This is the first study evaluating the bacterial diversity in a mucilage outbreak using a metabarcoding approach. Its results may contribute to this growing area of research and provide a database for further studies.

Depth profiles of protein-bound microcystin in Küçükçekmece Lagoon.

Microcystis is the most commonly found toxic cyanobacterial genus around the world and has a negative impact on the ecosystem. As a predominant producer of the potent hepatotoxin microcystin (MC), the genus causes outbreaks in freshwaters worldwide. Standard analytical methods that are used for the detection of microcystin variants can only measure the free form of microcystin in cells. Since microcystin was found as free and protein-bound forms in the cells, a significant proportion of microcystin is underestimated with analytical methods. The aim of the study was to measure protein-bound microcystins and determine the environmental factors that affect the binding of microcystin to proteins. Samples were taken at depths of surface, 1 m, 5 m, 10 m, 15 m, and 18 m in Küçükçekmece Lagoon to analyze depth profiles of two different microcystin forms from June to September 2012 at regular monthly intervals. Our findings suggest that the most important parameter affecting protein-bound microcystin at surface water is high light. Due to favorable environmental conditions such as temperature, light, and physicochemical parameters, the higher microcystin contents, both free and protein-bound MCs, were found in summer periods.

Contrasting the Water Quality and Bacterial Community Patterns in Shallow and Deep Lakes: Manyas vs. Iznik.

The objectives of this study are to monitor the physicochemical properties of two freshwater lakes with different chemical characteristics and trophic status over a year (2019) and assess the bacterial diversity by a high-throughput sequencing method for a certain time. Carlson Trophic Index analysis revealed that, whereas the deep lake, Iznik Lake, (TSImean = 48.9) has mesotrophic characteristics, the shallow lake Manyas Lake (TSImean = 74.2) was found at a hypertrophic status. The most important parameters controlling water qualities in the lakes were temperature, alkalinity, and phosphate levels. Although the bacterial communities were dominated by the same phyla (Cyanobacteria, Bacteroidetes, Actinomicrobia, Proteobacteria, and Verrucomicrobia) in both lakes, the communities differed distinctly at the lower levels. Whereas Sporichthyaceae in Manyas Lake accounted for 10% of the total reads, the major share of the sequences was assigned to Cyanobacteria Family I (8%) in Iznik Lake. The hypertrophic Manyas Lake had a more diverse bacterial community rather than Iznik Lake and contained higher numbers of unique Operational Taxonomic Units.

Heavy Metal Concentrations in Trachurus Mediterraneus and Merlangius Merlangus Captured from Marmara Sea, Turkey and Associated Health Risks.

Rapid industrialization and excessive human population growth may cause deterioration in marine water quality and biodiversity. Heavy metals are one of the most common pollutants in the seas and marine organisms, including demersal and pelagic fish, can accumulate them from the environment. Assessment of the ecological risk of heavy metals from fish has an important role in public health. In this study, some heavy metal (Pb, Cd, As, Cr, Hg, Cu, Zn, and Fe) concentrations were determined in the muscle tissues of two commonly consumed fish species, Trachurus mediterraneus (Mediterranean horse mackerel) and Merlangius merlangus (Whiting), which are the fifth (14,222 tons/year) and sixth (6814 tons/year) highest commercial catches of marine fish species in Turkey, respectively. Heavy metal concentrations of samples collected from four sites (Adalar, Izmit Bay, Yalova, and Tekirdag) in the Marmara Sea were determined using ICP-MS. Fish samples caught at Yalova station were found to have the highest heavy metal concentrations. According to the World Health Organization (WHO), in terms of the mean values, only As, and Cr were higher than permissible limits in T. mediterraneus, while Pb, Cd, As, and Cr were higher in M. merlangus. Arsenic concentrations were higher than maximum limits in both T. mediterraneus and M. merlangus. The estimated weekly intake (EWI) was calculated to assess the potential health impact. The EWI for arsenic, cadmium, lead, and mercury for some sites was above the provisional tolerable weekly intake.

Seasonal dynamics of freshwater pathogens as measured by microarray at Lake Sapanca, a drinking water source in the north-eastern part of Turkey.

Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project µAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.

Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods.

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project µAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654. © 2016 SETAC.

A validated UPLC-MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems.

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2-9.6% and 1.3-12.0% respectively. The method was applied to the analysis of aquatic samples (n=206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n=22), cylindrospermopsin (n=25), microcystin-RR (n=17), microcystin-LR (n=12), microcystin-LY (n=1), microcystin-LF (n=1) and nodularin (n=5). For microcystins, the levels detected ranged from 0.001 to 1.51µg/L, with two samples showing combined levels above the guideline set by the WHO of 1µg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.

First report of cylindrospermopsin production by two cyanobacteria (Dolichospermum mendotae and Chrysosporum ovalisporum) in Lake Iznik, Turkey.

Cylindrospermopsin (CYN) is a cytotoxic alkaloid produced by cyanobacteria. The distribution of this toxin is expanding around the world and the number of cyanobacteria species producing this toxin is also increasing. CYN was detected for the first time in Turkey during the summer months of 2013. The responsible species were identified as Dolichospermum (Anabaena) mendotae and Chrysosporum (Aphanizomenon) ovalisporum. The D. mendotae increased in May, however, C. ovalisporum formed a prolonged bloom in August. CYN concentrations were measured by LC-MS/MS and ranged from 0.12 µg·mg⁻¹ to 4.92 µg·mg⁻¹ as dry weight, respectively. Both species were the only cyanobacteria actively growing and CYN production was attributed solely to these species. Despite CYN production by C. ovalisporum being a well-known phenomenon, to our knowledge, this is the first report of CYN found in D. mendotae bloom.

Diversity of peptides produced by Nodularia spumigena from various geographical regions.

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.

Effects of water quality and hydrologic drivers on periphyton colonization on Sparganium erectum in two Turkish lakes with different mixing regimes.

The aim of this paper is to determine and compare the environmental factors controlling vertical colonisation of periphyton on Sparganium erectum in a shallow eutrophic turbid lake, Manyas Lake, and an oligo-mesotrophic deep lake, Sapanca Lake, Turkey during the July 1997--November 1998. To investigate the effect of the environmental factors on periphyton colonization on S. erectum, the stem was cut above the rhizomes and subdivided into three equal sections. Multivariate statistical analyses have been applied to clarify relationships between environmental variables and periphyton colonization on S. erectum. Results indicated that physical disturbance and trophic level of the lakes influenced the colonization of the periphyton. Among the measured parameters, low light intensity, total suspended solids, temperature and water level fluctuation were observed as driving factors in Manyas Lake whereas nutrient deficiency was found as key factor in Sapanca Lake. The zonation of the periphyton, density, composition and dominant/subdominant taxa were significantly different in these lakes. However, Oedogonium sp., Mougeotia sp., Cylindrocapsa sp., Cladophora glomerata (Linn.), Aulacoseira italica (Ehr.) Simonsen, Melosira varians C. Agardh, Navicula tripunctata (O.F. Müller) Bory and Fragilaria ulna (Nitzsch) Lange-Bertalot were found as dominant species at all sections of S. erectum both in Manyas Lake and Sapanca Lake. It can be thought that these species have a broad range of tolerance to several physical, chemical and hydrologic disturbances. This is the first study to introduce how much the water quality and hydrologic drivers have affected vertical colonization of periphyton on S. erectum in two lakes with different mixing regimes.

Microcystin analysis in single filaments of Planktothrix spp. in laboratory cultures and environmental blooms.

Single filaments of Planktothrix spp. were isolated from laboratory cultures of P. agardhii (NIES 595) and P. rubescens (SL 03) and from four freshwater lakes in England and Turkey. Filament lengths were measured and microcystins were extracted by freeze-thawing and boiling. Microcystin analysis of the isolated single filaments was performed by ELISA using antibodies raised against microcystin-LR with a minimum detection limit (MDL) of 11 pg filament(-1). In some cases a high percentage of the filaments from the environmental samples and laboratory cultures were below the MDL of the assay. Based on the filaments with detectable microcystin contents, P. agardhii from Bassenthwaite Lake (England) had the lowest mean microcystin concentration (0.7 fg microm(-3)), and the highest microcystin concentration (2.9 fg microm(-3)) was measured in P. rubescens from Iznik Lake (Turkey). We investigated the relationship for filaments with microcystin contents above MDL and their biovolume. Relationships varied widely although P. agardhii from Bassenthwaite showed a better (positive) relationship between filament biovolume and microcystin content than P. rubescens from environmental samples. Under culture conditions, P. rubescens showed a good relationship between filament biovolume and toxin content.