Variability in the sxt Gene Clusters of PSP Toxin Producing Aphanizomenon gracile Strains from Norway, Spain, Germany and North America

Paralytic shellfish poisoning (PSP) toxin production has been detected worldwide in the cyanobacterial genera Anabaena, Lyngbya, Scytonema, Cuspidothrix and Aphanizomenon. In Europe Aphanizomenon gracile and Cuspidothrix issatschenkoi are the only known producers of PSP toxins and are found in Southwest and Central European freshwater bodies. In this study the PSP toxin producing Aphanizomenon sp. strain NIVA-CYA 851 was isolated from the Norwegian Lake Hillestadvannet. In a polyphasic approach NIVA-CYA 851 was morphologically and phylogenetically classified, and investigated for toxin production. The strain NIVA-CYA 851 was identified as A. gracile using 16S rRNA gene phylogeny and was confirmed to produce neosaxitoxin, saxitoxin and gonyautoxin 5 by LC-MS. The whole sxt gene clusters (circa 27.3 kb) of four A. gracile strains: NIVA-CYA 851 (Norway); NIVA-CYA 655 & NIVA-CYA 676 (Germany); and UAM 529 (Spain), all from latitudes between 40Ê and 59Ê North were sequenced and compared with the sxt gene cluster of reference strain A. gracile NH-5 from the USA. All five sxt gene clusters are highly conserved with similarities exceeding 99.4%, but they differ slightly in the number and presence of single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels). Altogether 178 variable sites (44 SNPs and 4 In/Dels, comprising 134 nucleotides) were found in the sxt gene clusters of the Norwegian, German and Spanish strains compared to the reference strain. Thirty-nine SNPs were located in 16 of the 27 coding regions. The sxt gene clusters of NIVA-CYA 851, NIVACYA 655, NIVA-CYA 676 and UAM 529, were characterized by 15, 16, 19 and 23 SNPs respectively. Only the Norwegian strain NIVA-CYA 851 possessed an insertion of 126 base pairs (bp) in the noncoding area between the sxtA and sxtE genes and a deletion of 6 nucleotides in the sxtN gene. The sxtI gene showed the highest variability and is recommended as the best genetic marker for further phylogenetic studies of the sxt gene cluster of A. gracile. This study confirms for the first time the role of A. gracile as a PSP toxin producer in Norwegian waters, representing the northernmost occurrence of PSP toxin producing A. gracile in Europe known so far.publishedVersio

Cyanotoxins in Lake Garda? The critical evaluation of chemodiversity

Anthropic pressure and climate changes are the major stressors in lakes. Both factors play an important role in the development of cyanobacteria with the capability of producing toxic metabolites (cyanotoxins). Cyanotoxins have negative effects on aquatic flora and fauna, and can be cause of serious intoxications episodes in animals and humans. Some cyanotoxins moreover can undergo bioaccumulation. Lake Garda hosts some potentially toxic cyanobacteria (Planktothrix rubescens and Dolichospermum lemmermannii) which are known to produce microcystins (hepatotoxins) and anatoxins (neurotoxins). The European Community funded the EULAKES project, which supported a monitoring campaign in 2010-2012 aimed at assessing the presence of cyanotoxins in Lake Garda. In that period the chemical diversity and the abundance of cyanotoxins were investigated by advanced analytical techniques (LC-MS) with a monthly frequency. The survey showed that some cyanotoxins (in particular microcystin RRdm) were present in the lake water with concentrations, however, which did not represent a sanitary risk. The most abundant toxins were microcystins, which five variants have been identified, and anatoxin-a. The highest content of toxins has been registered in spring-summer periods. The data collected, in combination with biological and environmental parameters, provide useful indications for a rational management of possible toxic bloom

Microcystis, Raphidiopsis raciborskii and Dolichospermum smithii, toxin producing and non-toxigenic cyanobacteria in Yezin Dam, Myanmar

6openInternationalInternational coauthor/editorYezin Dam is a man-made reservoir located close to Yezin village in Myanmar. Its water is used for irrigation, domestic purposes and as drinking water for many urban communities in the watershed area. In recent years, increased pollution due to the concurrent development around the dam has led to water quality deterioration. No detailed study on the distribution of cyanobacteria and toxin production has been conducted so far. In order to provide insight into the extent of cyanobacteria and cyanotoxins in the dam, water samples were collected once in January 2014 for the isolation of cyanobacterial strains and eight times between March 2017 and June 2018 for the investigation of physical, chemical and biological parameters. A total of 99 phytoplankton taxa belonging to 50 genera were recorded from Yezin Dam. Microscopic examination showed that a Dolichospermum sp. was the dominant cyanobacterium followed by small numbers of Microcystis, and Raphidiopsis raciborskii in all samples throughout the sampling period. 15 isolated cyanobacterial strains were classified morphologically and phylogenetically as Dolichospermum smithii, R. raciborskii and Microcystis and tested for microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins (STXs) and anatoxins (ATXs) by liquid chromatography-tandem mass spectrometry (LC–MS/MS) and enzyme-linked immunosorbent assay (ELISA). The toxin analysis of all isolated Dolichospermum strains by ELISA and LC–MS did not indicate the presence of ATXs, STXs, CYNs nor MCs. Four of the five isolated Raphidiopsis strains produced CYN and deoxyCYN. One of the isolated Microcystis strains (AB2017/08) from Yezin Dam produced 22 MC congeners. Concentrations of 0.12 μg L−1 CYNs and 0.34 μg L−1 MCs were also found in an environmental sample from Yezin Dam by ELISA. The potential therefore exists for the use of untreated water from Yezin Dam to cause harmful effects on humans, domestic and wild animals.openSwe, T.; Miles, C.O.; Cerasino, L.; Mjelde, M.; Kleiven, S.; Ballot, A.Swe, T.; Miles, C.O.; Cerasino, L.; Mjelde, M.; Kleiven, S.; Ballot, A

Potential toxicity and distribution patterns of Dolichospermum lemmermannii (Cyanobacteria) in European lakes

Dolichospermum lemmermannii (Nostocales) is distributed in temperate and boreal regions. Despite this species is typical of cold environments, many populations were shown to be able to form huge water blooms in summer stratified conditions. Indeed, this species is characterized by high variability to temperature adaptation and some strains show high temperature optima (i.e. between 19°C and 26°C). In the large and deep lakes south of the Alps D.lemmermannii developed only recently. Extended surface bloom occurred firstly in Lake Garda (1990/91) and progressively in lakes Iseo (second half of the 1990s), Maggiore (2005), and Como (2006). The recent spread of this species in Southern Europe emphasizes its ecological heterogeneity. Since all these evidences suggest the existence of different ecotypes adapted to different European climatic regions, a wide research on populations isolated from several European waterbodies was carried out. Several strains of D.lemmermannii were studied applying a multidisciplinary approach, which includes taxonomical, genetic and metabolomic analyses. A phylogenetic study on the 16S rRNA and housekeeping genes (e.g. rpoB,) was integrated by the assessment of the toxic potential, evaluating the presence of cyanotoxins (i.e. microcystins, nodularins, anatoxins, cylindrospermopsins) and cyanotoxins encoding genes. Further studies will allow gaining insight about the phylogeography of this fast spreading species at a continental level, along climatic and trophic gradients

Potentially toxic planktic and benthic Cyanobacteria in Slovenian freshwater bodies: detection by quantitative PCR

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures

Biodiversity patterns of cyanobacterial oligotypes in lakes and rivers: results of a large-scale metabarcoding survey in the Alpine region

In this work, we characterised the cyanobacterial communities in the plankton and littoral biofilm of 38 lakes and in the biofilm of 21 rivers in the Alps and surrounding subalpine regions by 16S rRNA gene metabarcoding. We found little overlap in the distribution of amplicon sequence variants (ASVs) between the three habitats and between water bodies. The differences were caused by environmental filtering acting on the selection of the most abundant ASVs and a high contribution of rare oligotypes. The differentiation of community and genotype composition from specific water bodies was explained to a significant extent by environmental variables and morphometry. The taxonomic consistency of ASVs classified under the same genus name was assessed by phylogenetic analyses performed on three representative dominant genera, namely Cyanobium, Tychonema and Planktothrix. The analyses revealed eco-evolutionary adaptations in lakes and rivers, including some evidence for a polyphyletic nature. Monitoring individual genotypes in relation to environmental conditions will be useful to define the ecological amplitude of these taxa. However, the persistence or ephemeral nature of some of the rarest and most unusual ASVs has remained unknow

Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Toxins

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.Peer reviewe

Metodi chimici per l\u2019analisi delle cianotossine nelle acque di balneazione

Cyanobacteria thrive in many aquatic environments, where they can produce cyanotoxins with different toxicological profiles. This report provides the guidelines for the management of cyanobacterial blooms in bathing water, put together by a group of experts. The first part summarizes the current scientific knowledge on various aspects, including their presence in the Italian lakes, chemical and toxicological characteristics of different cyanotoxins, the observed effects on human health and the risk assessment. The second part defines the guidelines to prevent harmful effects on the health of bathers and manage the risk associated with blooms. It provides recommendations for planning environmental monitoring activities and a health surveillance system in most critical areas. It also introduces an environmental and health reporting system, with the purpose to standardize the information at national level too. The report is supplemented by technical information aimed at territorial authorities in charge