The cyanobacterial species Raphidiopsis raciborskii (Wołoszyńska) Aguilera et al. has a high invasiveness potential, which in less than a century leads to its cosmopolitan spread. In the temperate climate of Europe, R. raciborskii has been reported in many countries, but there is still a lack of detailed information about the current status of its distribution in lakes of Bulgaria, as a part of the southeastern range of its spread in Europe. We investigated the distribution of the species using data on the phytoplankton of 122 lakes surveyed during 13-years period (2009-2022). The species was found in 33 lakes (up to 324 m asl), and 14 new localities were registered during the studied period. The results reveal that the number of lakes with the presence of R. raciborskii (27 % of all research lakes) and its contribution to the total phytoplankton biomass, has increased significantly over the last decade. The species has successfully adapted and dominated the phytoplankton in 9 lakes, forming a bloom in 8 of them. The dominant position of R. raciborskii causes loss of species and functional diversity of phytoplankton and displaces the native bloom-forming cyanobacteria. Lakes with and without the species were compared based on the available data on bioclimatic and local environmental variables. Statistically significant differences were established with respect to water transparency, conductivity, maximum depth and maximum air temperature in the warmest month. Species distribution models (SDMs) were used to identify lakes in high risk of future invasion by R. raciborskii. The results of the SDMs implementation confirmed the high maximum air temperature and low water transparency to be important predictors of the occurrence of R. raciborskii in freshwater lakes in Bulgaria. In the areas with high summer temperatures the most suitable for R. raciborskii development were found to be shallow polymictic or medium deep lakes with small surface area and low water transparency. In areas with a suitable climate, the large, deep reservoirs with high transparency as well as macrophyte dominated lakes have a low probability of occurrence of R. raciborskii. Future colonization of lakes above 500 m asl (but most likely below 700 m asl) is also possible, especially in the conditions of global warming. SDMs account for climatic and biogeographic differences of lakes and could help in elucidating the underlying factors that control the occurrence and adaptation of R. raciborskii in a given area.

Feeding effects are crucial for evaluating the capacity of zooplankton to regulate phytoplankton populations within freshwater ecosystems. To examine the impact of the bloom-forming cyanobacteria Raphidiopsis raciborskii, which occurs in tropical and subtropical freshwaters, on the growth of zooplankton Daphnia in relation to toxins, filament length and fatty acid content, we fed D. magna with R. raciborskii only (cylindrospermopsin (CYN)-producing and non-CYN-producing, as the negative controls), Chlorella pyrenoidosa only (as the positive control) and a mixed diet containing R. raciborskii (CYN-producing and non-CYN-producing) and C. pyrenoidosa. Consequently, our findings revealed that the toxic effect of CYN-producing R. raciborskii strains on Daphnia was mitigated by the coexistence of C. pyrenoidosa containing stearidonic acid (SDA, C18:4 ω3) in mixed diets. This was evident in the elevated survival rate compared that from diets containing only R. raciborskii and a significantly higher reproduction and population intrinsic increase rate compared to diets consisting of only R. raciborskii or C. pyrenoidos. Additionally, a strong positive correlation was observed between arachidonic acid (ARA, 20:4ω6) and the population intrinsic increase rate of Daphnia; notably, R. raciborskii strains were found to be rich in the ω6 polyunsaturated fatty acid ARA. These outcomes reinforce the crucial role of polyunsaturated fatty acids in predicting the population increase of crustacean zooplankton, which has long been neglected. Furthermore, our results underscore the potential effectiveness of zooplankton, particularly in temperate lakes, in controlling CYN-producing R. raciborskii populations.

As a tropical filamentous cyanobacterium, Raphidiopsis raciborskii has attracted much attention due to its expansion and toxin production. However, the mechanisms of its expansion to temperate regions have not been studied in detail. To address the potential strategies, the physiological and metabolomic profiles of R. raciborskii FACHB 1096 isolated from a temperate lake in China were determined and measured at different temperatures (10 °C, 15 °C, 20 °C, 25 °C, and 32 °C). The results demonstrated that temperature significantly changed cell viability, chlorophyll a content, specific growth rate, Chl a fluorescence, and filamentous shape of R. raciborskii. Low temperature decreased cell viability, specific growth rate, and photosynthetic efficiency, while the proportion of akinete and carbon fixation per unit cell were significantly increased compared with high temperature (32 °C). A constructed unimodal model indicated that filament length, cell volume, and cell length/width of R. raciborskii were significantly reduced in both high and low temperature environments. Under low-temperature conditions, R. raciborskii suffered different degrees of oxidative damage and produced corresponding antioxidant substances to resist oxidative stress, suggesting that low temperature changes the metabolic level of the cells, causing the cells to gradually switch from development to defense. Metabolomic data further confirmed that temperature change induced shifts in metabolic pathways in R. raciborskii, including starch and sucrose metabolic pathways, glutathione metabolic pathways, and the pentose phosphate pathways (PPP), as well as metabolic pathways related to the tricarboxylic acid (TCA) cycle. Our results indicated that the trade-offs of R. raciborskii cells among the growth, cell size, and metabolites can be significantly regulated by temperature, with broad implications for its global expansion in temperate waterbodies.

This study was aimed primarily at describing the planktonic assemblages with special attention to invasive and toxin-producing cyanobacterial species in the context of ecological and health threats. The second aim was to analyze the aspect of recreational pressure, which may enhance the cyanobacterial blooms, and, as a consequence, the negative changes and loss of planktonic biodiversity. This study was carried out in recreationally used Lake Sztynorckie throughout the whole growing season of 2020 and included an assessment of the abundance and biomass of phytoplankton (cyanobacteria and algae) in relation to environmental variables. The total biomass was in the range of 28-70 mg L-1, which is typical for strong blooms. The dominant filamentous cyanobacteria were Pseudanabaena limnetica, Limnothrix redekei, Planktolyngbya limnetica, and Planktothrix agarhii, and three invasive nostocalean species Sphaerospermopsis aphanizomenoides, Cuspidothrix issatschenkoi, and Raphidiopsis raciborskii. They can pose a serious threat not only to the ecosystem but also to humans because of the possibility of cyanobacteria producing cyanotoxins, such as microcystins, saxitoxins, anatoxin-a, and cylindrospermopsins, having hepatotoxic, cytotoxic, neurotoxic, and dermatoxic effects. The water quality was assessed as water bodies had bad ecological status (based on phytoplankton), were highly meso-eutrophic (based on zooplankton), and had very low trophic efficiency and low biodiversity.

The increasing occurrence of cyanobacteria blooms is of global concern, and is often associated with environmental and socio-economic problems, such as degenerated ecosystems and aquaculture impairment. The diazotrophic cyanobacterium Raphidiopsis raciborskii (R. raciborskii) grows rapidly in the tropics, and produces the toxin, cylindrospermopsin (CYN), which has harmful effects on aquatic organisms. Thus, to protect water quality and ecosystem, it is essential to have rapid and reliable methods for cyanobacteria and R. raciborskii detection and prediction so that early warning can be provided for management. Molecular assays, such as PCR, real-time quantitative PCR (qPCR), two-step PCR assays are accurate and widely used, but still require several hours from sample preparation to data analysis. In this study, insulated isothermal PCR (iiPCR) assays in conjunction with fast DNA extraction method, were developed and verified as a rapid detection assay in detecting cyanobacteria and R. raciborskii within 50 min, and also with high detection accuracy (98.8%) and the overall high agreement level (98.8%, k = 97.5%)) comparing to conventional qPCR assay. However, the limitation of the iiPCR assay is that it only generates qualitative results. Therefore, the quantified iiPCR assay, named as A-iiPCR, by coupling iiPCR device with fluorescence signal catching and interpretation instrument (Andor spectrometer with Solis spectroscopy software) was developed and verified with in situ environmental samples. The fluorescence intensity decreased accordingly with the drop of DNA concentration until reaching 1.32 ng/μL. Also, Delft 3D modelling was established to simulate R. raciborskii change in predicting spatial and temporal variabilities for reservoir management, as the simulated R. raciborskii concentration was the highest at sampling site 1, as well as temporally highest in April and October, posing as the most high-risk location and time periods for R. raciborskii bloom-forming requiring corresponding governance measures.

Cyanobacteria are a bloom-forming ancient group of photosynthetic prokaryotes. A rise in temperature is a major contributor to its massive proliferation, namely on freshwater ecosystems, with social and economic impacts. Thus, reliable and cost-effective tools can permit the fast surveillance and assessment of temperature effects on potentially toxic cyanobacteria distribution and impacts. The occurrence of three potentially toxic cyanobacteria species was assessed on seven sampling points across three sampling years. Moreover, the association between the occurrence of those cyanobacteria species with climate change events was addressed. Here, we combined molecular and statistical methods to study the impacts of temperature on the occurrence of three globally occurring cyanotoxin-producing cyanobacteria species-Microcystis aeruginosa (microcystins), Raphidiopsis raciborskii (cylindrospermopsins and saxitoxins) and Planktothrix agardhii (microcystins and saxitoxins). Samples were collected on seven European temperate freshwater systems located on the North and Centre regions of Portugal, across three distinct sampling years with distinct ranges of air temperature. Data support that M. aeruginosa is still a common inhabitant of Portuguese freshwater ecosystems and a new trend was found on R. raciborskii recent invasion and establishment on the colder north ecosystems of Portugal. Additionally, the highest frequency of detection of both cyanobacteria was associated with warmer years. P. agardhii also revealed a new trend, being reported for the first time on North and Centre Regions of Portugal, however with no statistical relation with air temperature, demonstrating a higher ecological fitness. Distinct profiles of the statistical analysis on the three tested cyanobacteria species contribute to deepen the studies on other species as well as of our analyzed species on a global level. This assessment may help to anticipate possible repercussions on water quality and public health due to most probable alterations on cyanotoxins profile given the ecological fitness established among air temperature and PCR detection of potentially toxic cyanobacteria.

To elucidate the interspecies connectivity between cyanobacteria and other bacteria (noncyanobacteria), microbial diversity and composition were investigated through high-throughput sequencing (HTS) in a drinking water reservoir in Chongqing city, Southwest China, during Raphidiopsis raciborskii blooms. Significant temporal changes were observed in microbial community composition during the sampling period, primarily reflected by variations in relative bacterial abundance. The modularity analysis of the network demonstrated that the bacterial community forms co-occurrence/exclusion patterns in response to variations in environmental factors. Moreover, five modules involved in the dynamic phases of the R. raciborskii bloom were categorized into the Pre-Bloom, Bloom, Post-Bloom, and Non-Bloom Groups. The reservoir was eutrophic (i.e., the average concentrations of total nitrogen (TN) and total phosphorus (TP) were 2.32 and 0.07 mg L-1, respectively) during the investigation; however, Pearson\'s correlation coefficient showed that R. raciborskii was not significantly correlated with nitrogen and phosphorus. However, other environmental factors, such as water temperature, pH, and the permanganate index, were positively correlated with R. raciborskii. Importantly, Proteobacteria (α-, γ-Proteobacteria), Acidobacteria, Chloroflexi, and Firmicutes were preferentially associated with increased R. raciborskii blooms. These results suggested that the transition of R. raciborskii bloom-related microbial modules and their keystone species could be crucial in the development and collapse of R. raciborskii blooms and could provide a fundamental basis for understanding the linkage between the structure and function of the microbial community during bloom dynamics.

In freshwater habitats, invasive species and the increase of cyanobacterial blooms have been identified as a major cause of biodiversity loss. The invasive cyanobacteria Raphidiopsis raciborskii a toxin-producing and bloom-forming species affecting local biodiversity and ecosystem services is currently expanding its range across Europe. We used species distribution models (SDMs) and regional bioclimatic environmental variables, such as temperature and precipitation, to identify suitable areas for the colonization and survival of R. raciborskii, with special focus on the geographic extent of potential habitats in Northern Europe. SDMs predictions uncovered areas of high occurrence probability of R. raciborskii in locations where it has not been recorded yet, e.g. some areas in Central and Northern Europe. In the southeastern part of Sweden, areas of suitable climate for R. raciborskii corresponded with lakes of high concentrations of total phosphorus, increasing the risk of the species to thrive. To our knowledge, this is the first attempt to predict areas at high risk of R. raciborskii colonization in Europe. The results from this study suggest several areas across Europe that would need monitoring programs to determine if the species is present or not, to be able to prevent its potential colonization and population growth. Regarding an undesirable microorganism like R. raciborskii, authorities may need to start information campaigns to avoid or minimize the spread.

Phosphorus (P) is a vital macronutrient associated with the growth and proliferation of Raphidiopsis raciborskii, an invasive and notorious bloom-forming cyanobacterium. However, the molecular mechanisms involved in P acclimation remain largely unexplored for Raphidiopsis raciborskii. Here, transcriptome sequencing of Raphidiopsis raciborskii was conducted to reveal multifaceted mechanisms involved in mimicking dipotassium phosphate (DIP), β-glycerol phosphate (Gly), 2-aminoethylphosphonic acid (AEP), and P-free conditions (NP). Chlorophyll a fluorescence parameters showed significant differences in the NP and AEP groups compared with the DIP and Gly-groups. Expression levels of genes related to phosphate transportation and uptake, organic P utilization, nitrogen metabolism, urea cycling, carbon fixation, amino acid metabolism, environmental information, the ATP-synthesis process in glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway were remarkably upregulated, while those related to photosynthesis, phycobiliproteins, respiration, oxidative phosphorylation, sulfur metabolism, and genetic information were markedly downregulated in the NP group relative to the DIP group. However, the expression of genes involved in organic P utilization, the urea cycle, and genetic information in the Gly-group, and carbon-phosphorus lyase, genetic information and environmental information in the AEP group were significantly increased compared to the DIP group. Together, these results indicate that Raphidiopsis raciborskii exhibits the evolution of coordination of multiple metabolic pathways and certain key genes to adapt to ambient P changes, which implies that if P is reduced to control Raphidiopsis raciborskii bloom, there is a risk that external nutrients (such as nitrogen, amino acids, and urea) will stimulate the growth or metabolism of Raphidiopsis.

Raphidiopsis raciborskii (previously Cylindrospermopsis raciborskii) can produce cylindrospermopsin (CYN) which is of great concern due to its considerable toxicity to human and animals. Its CYN-producing (toxic) and non-CYN-producing (non-toxic) strains co-exist commonly in natural water bodies, while how their relative dominance is regulated has not been addressed. In this study, we combined field investigation with laboratory experiments to assessed the relationship between toxic and non-toxic R. raciborskii abundances under different nutrient levels. The rpoC1- and cyrJ-based qPCR was applied for quantifying total and toxic R. raciborskii abundances, respectively. The field survey showed that toxic R. raciborskii was detected in 97 of 115 reservoirs where its proportion ranged from 0.3% to 39.7% within the R. raciborskii population. Both total and toxic R. raciborskii abundances increased significantly with trophic level of these reservoirs, consistent with our monoculture and co-culture experiments showing in an increase in R. raciborskii growth with increasing nitrogen (N) or phosphorus (P) concentrations. In the monoculture experiments, growth rates of non-toxic and toxic strains from Australia or China were not significantly different under the same culture conditions. On the other hand, in the co-culture experiments, the toxic strains displayed a significantly faster growth than non-toxic strains under nutrient-replete conditions, resulting in an obvious shift toward the dominance by toxic strains from day 3 to the end of the experiments, regardless of the strain originating from Australia or China. The reverse was found under N- or P-limited conditions. Our results indicated that the toxic strains of R. raciborskii have a competitive advantage relative to the non-toxic strains in a more eutrophic world. In parallel to an increase in dominance, both toxic strains grown in the mixed population significantly increased CYN production under nutrient-replete conditions as compared to nutrient-limited conditions, suggesting that CYN may be of significance for ecological advantage of toxic R. raciborskii. These results highlight the importance of nutrient availability in regulating abundances and strain dominance of two genotypes of R. raciborskii. Our findings demonstrated that elevated nutrients would favor the growth of CYN-producing R. raciborskii and CYN production, leading to more blooms with higher toxicity at global scale.