Combining Imaging Flow Cytometry and Molecular Biological Methods to Reveal Presence of Potentially Toxic Algae at the Ural River in Kazakhstan

Abstract

Algal blooms occur in freshwater bodies throughout the world, often leading to fish kills. Cases of these kills along the Ural River were reported in 2018–2019, involving significant amount of sturgeon in fish farming areas. In this study, the analysis of algal samples from the delta of the Ural River up to 100 km inland was carried out from August to December 2019 using imaging flow cytometry (IFC), molecular biological, and microscopic techniques. We identified the filamentous cyanobacteria Cuspidothrix issatschenkoi, Dolichospermum cf. flos-aquae, Dolichospermum cf. macrosporum, Pseudanabaena limnetica, and Planktothrix spp. as the dominant potentially toxic phytoplankton species, and we also found minor quantities of Cylindrospermopsis raciborskii. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Ural River were carried out to establish the taxa of the dominant species and to identify the presence of genes encoding toxins. The complementary analysis with nanopore-based next-generation sequencing overlapped with the results of IFC and was instrumental in revealing minor cyanobacteria taxa. Real-time PCR analysis and sequencing indicated the presence of Microcystis and ADA-clade spp. as well as genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originating from cyanobacteria. These findings suggest that toxin-producing cyanobacteria could become a threat in the Ural River near Atyrau, which can significantly affect aquaculture in the region.

MinION Nanopore Sequencing Accelerates Progress towards Ubiquitous Genetics in Water Research

“…The use of complementary biology methods to validate and/or augment sequencing data was common practice in the reviewed MinION applications. Complementary methods included quantitative PCR (qPCR) [27,38,50,54,58,63,65,66,68,69,71,74], culturing [27,43,47,50,52,54,63,66,68,71,72,76], flow cytometry [54,65], and fluorescence in situ hybridization (FISH) microscopy [51]. Across a range of case studies, we reported significant correlations between MinION sequencing, qPCR, and culturing derived indications of faecal pollution in water [50,63,66,71].…”

“…Amplification of specific genes or genome regions of interest, targeted with appropriate primers, additionally assures that the full capacity of the MinION is devoted to sequencing these genes or regions. Several studies used full-length 16S rRNA gene amplicon sequencing to characterize communities of bacteria and archaea [27,29,38,48,50,54,56,63,65,66,68,71], mostly with ONT's 16S barcoding kit. Long-range PCR amplicons spanning 3 kb of the rDNA cassette were used for the detection of algal bloom organisms [74].…”

“…The reviewed studies used basecalling tools from ONT such as MinKNOW, Metrichor, Albacore, and its replacement, Guppy, to translate the raw signals generated by the MinION into the corresponding nucleotide sequences. MinKNOW allows live basecalling during sequencing runs [48,51,65] for near real-time data generation as envisioned by ubiquitous genetics (Table 1). Guppy is the currently recommended basecaller of choice by ONT and includes high-accuracy and fast basecalling options, where the high-accuracy model improves single read accuracy at the cost of increased computational complexity [32].…”

“…Several 16S rRNA gene amplicon studies used ONT's FASTQ 16S workflow to classify bacteria in drinking water [54], groundwater [50], river water [27,63,65,66,68,71], wastewater [38,66,71], aquaculture ponds [68], and sea lice [29]. Van der Loos et al [59] used EPI2ME and Kraken2 with three different reference databases (SILVA [98], Greengenes [99] and NCBI) and recommended assigning taxonomy of nanopore-derived long-reads with Kraken2 and the SILVA database in seaweed-microbiome studies to avoid misidentifications of cyanobacteria due to chloroplast contamination.…”

Long-term temperature trend in Kamchatka supports expansion of harmful algae

“…On the other hand, the recent development of next-generation sequencing (NGS) technologies led to their availability for field analysis and offered an enormous perspective for HAB monitoring (Jung et al, 2018; Hatfield et al, 2020). Ocean color satellite images is another key to studying the temperature trends and response of marine phytoplankton to climate variability and change (Groom et al, 2019), plankton distribution in ocean, and detecting and predicting HABs (Stumpf et al, 2008; Walsh et al, 2011; Zhong et al, 2019; Tang and Liu, 2020; Mirasbekov et al, 2021; Iwataki et al, 2022). Given the substantial climate-driven shifts in coastal ecological systems, accelerated interdisciplinary efforts are required to understand how climate change will influence the HABs.…”