Influence of Biotope and Biotic Factors on Cyanobacteria Abundance, Genotype and Toxin

31 Environmental genetics-related modern methods are shown as important indicators of various cyanotoxins syntheses, and 32 their knowledge and use are critically analyzed. Microcystins and other cyanotoxins loads and syntheses are related to 33 different drivers, like various chemical elements and compounds (especially nutrients, such as nitrogen and phosphorus, 34 and their ratio), then to the light, conductivity, temperature, and other climatical and hydrological factors, to which spatial 35 and geographical features (such as the surface of the water bodies) have to be added. The biotic relationships include 36 different specific and supraspecific, uniand bilateral links between the cyanobacteria, and subsequently their synthesized 37 toxins, and protozoans (or protoctists), chromists, macrophytes, different systematical and ecological groups of 38 zooplankton, and others. The importance of, but also the gaps in, the knowledge and the scarcity of studies involving 39 ectocrines mediated interactions between different groups of algae and plants are highlighted. The paper ends with an 40 interesting classification of lakes' trophicity, illustrated with conceptual diagrams, based on possible scenarios 41 of cyanobacteria behavior. 42 43


Introduction
Light conditions in shallow lakes may change on a time scale of days to weeks due to changes in cloudiness or 119 wind-induced resuspension of sediments. It was demonstrated that light intensity is a critical factor influencing the 120 production of cyanotoxins (33). Changes in light conditions may profoundly affect the microcystin composition and thereby 121 the toxicity of cyanobacteria (34); the transcription of two genes responsible for microcystin production was already shown 122 to be influenced by light quality (33). For example, the harmful cyanobacterium Planktothrix agardhii, a species that 123 prefers mostly shallow, turbid lakes, produces a more toxic variant during periods of sunny weather, when recreational 124 activities in lakes are most attractive (34). It has been documented that the excessive growth of cyanobacteria can reduce 125 water transparency with light penetration to only few centimeters, and thus having important effects on both pelagic and 126 benthic communities (35). The reduction of the euphotic zone together with the excessive increase in the ratio between the 127 epilimnetic mixing layer and the euphotic depth is an unfavorable factor for other organisms (35).

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Another physical parameter of the water, which has exhibited a high positive correlation with MCs concentration,

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but not with the number of mcyE gene copies, was the conductivity. Conductivity is a parameter related to the ability of 130 electric conduction of water, and can indicate the ion concentration. Microcystis utilize various inorganic ions such as 131 macronutrients and trace metal for growth (36). This confirms previous studies showing that alkaline pH (7. 5 -8.5), 132 electrical conductivity from 241 to 367 µS/cm, and temperature ranging from 24.8 to 32ºC, are promoting microcystin 133 development (37).

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The pH of water may also influence toxin production. For M. aeruginosa, higher MC production occurred at pH 135 values above and below their optimum growth threshold (33).

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Temperature is a crucial parameter of the environment that influences the survival, metabolism, growth and 137 reproduction of all cyanobacteria, as well as the interactions between them and other species (38) as well a fundamental 138 relationship with cyanotoxin production (33). For example, some cyanotoxins are often found at temperatures that would 139 be considered sub-optimum for cell growth, with maximum reported at 20 °C and production ceasing at temperatures 140 exceeding 35 °C, namely cylindrospermopsin (33). Anatoxin-a production has also been shown to be highest at 20 °C, 141 whereas maximum production of MC and nodularin has been reported to occur between 18 and 25 °C (33). Other studied 142 showed that temperature, DO and Microcystis biomass positively correlated with MC accumulation as well (39).

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Temperature alone may only partly determine bloom formation and it is accepted that a combination of factors are 144 responsible for a bloom to develop: increasing temperatures, decreasing nutrients and increased water column stability 145 (40); temperature has the most pronounced effect on toxicity; the highest toxicity was found at 20°C, but reduced at 146 temperatures in excess of 28°C.

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Toxin production in non-nitrogen fixing cyanobacteria, such as Microcystis and Planktothrix, has been shown to 160 peak with high concentrations of nitrogen (33). According to a recent study (51)

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The presence of N can contribute to both increased and decreased MC production, depending on the genera (33).

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Nitrogen and phosphorus are the main elements for the matter and energy metabolism of the algae (52). Toxin production 169 in non-nitrogen fixing cyanobacteria, such as Microcystis and Planktothrix, has been shown to peak with high 170 concentrations of nitrogen (33). When attempting biomanipulation, the omission of nitrogen causes approximately ten fold 171 decrease in toxicity (40). High levels of nitrogen and phosphorus in freshwaters favour the growth of toxic strains 172 over nontoxic ones (53). Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE 173 genes, whereas total phosphorus alone accounted for MC concentrations (54).

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Historically, phosphorus was seen as the primary limiting nutrient in freshwaters (

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The findings related to phosphorous also demonstrate the importance of this nutrient to toxin production (33

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Cyanotoxin production is dependent on a number of environmental conditions. Predominantly, these could include 199 nutrient concentration, light intensity, and temperature (33). Eutrophication increased the co-occurrence of potentially

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MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation (54).In general, studies considering the 201 effects of nutrients on toxin production find a positive correlation between the nutrient of interest and cellular toxin content.

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Research focusing on nitrogen, cyanobacterial growth, and subsequent toxin production, report that increased nitrogen 203 corresponds to increased toxin production (65-67). Recent studied showed that physical and chemical parameters did not  For some organisms that accumulate microcystins, total MC concentrations declined after October and began to 208 increase in May , from the season point of view, that give us also a clue about the toxin production in the ecosystem (39).

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microcystins production did not correlate with the high level of nutrients (t test, p > 0.1). In fact, microcystins were never 210 detected in the more eutrophic reservoirs (68). The cyanobacterial biomass in water, pH, and temperature explain the  (68). It is speculated that N and P nutrients and the associated genes (e.g., mcy) may jointly drive MC 215 concentration and toxigenicity (59). Also species biomass was the best predictor of MC concentrations (51)

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When the density of phytoplankton was too high it was concluded that the multiplication of zooplankton was Daphnia. Therefore, at times when these colonial forms dominate the phytoplankton, Daphnia populations might be 227 expected to show decreased growth and fecundity in response to food limitation or toxicity. Is very probably that any effect 228 of cyanobacteria on zooplankton could be influenced by evolutionary mechanisms in natural systems with a long history 229 of cyanobacterial blooms: zooplankton that co-occur with dense biomass of cyanobacteria have better chance to adapt than 230 others that were not exposed to these conditions (41). It was already demonstrated that, if the proportion of any toxic 231 cyanobacteria and any edible algae studied is in concordance with tested microorganism needs, the survival rate is growing 232 (64,(73)(74)(75)(76). This suggests that in nature, at least some of the total amount of M. aeruginosa is consumed, but only when 233 the strain is not highly toxic. Therefore changes in the cyanobacterial abundances in nature are not only related to physical 234 and chemical variables but are also likely due to grazing from zooplankton (64). There were species-specific differences 235 in the filtration and feeding rates of zooplankton when offered mixed diets of green algae and toxic cyanobacteria.

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These probably explain the coexistence of different zooplankton species in Microcystis-dominant waterbodies. (76) 237

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There are also strong intraspecific differences in the tolerance of different Daphnia clones to toxic/non-toxic cyanobacteria,

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and therefore the dynamics of the daphnid populations vary significantly in the presence of these microalgae in their diet 317 (90).

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Zooplankton groups may act as vector of the toxin uptake in the aquatic food web (91)

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There is one study that shows that a golden algae have a great potential to biodegrading microcystin-LR (MC-345 LR) (98). They reported that the alga Poterioochromonas sp was able to degrade MC-LR in cells of M. aeruginosa while 346 digesting the whole cells; degradation process was determined to be carried out inside the algae cell. As well, another study 347 showed that Ochromonas sp., a mixotrophic chrysophyte, was able to feed on all four cyanobacterial strains tested,

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Notably is that symbiosis between diatoms and cyanobacterial colonies may also occur in natural water 350 ecosystems (99). Cyanobacterial toxin production can be regulated by complex growth phase dependent and environmental   In conclusion, since we cannot discuss about one or few direct factors that trigger the cyanobacteria mass 357 development, target genes involved in cyanotoxin production and toxin production and release into environment, we 358 formulate in this short review chapter five possible scenarios of cyanobacteria behavior in any freshwater ecosystem, 359 especially shallow lakes ( Fig. 1-5).

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Cyanobacteria from here don't need to release toxins to any of other trophic compartment because: (i) there exists enough 363 zooplankton that will eat microalgae, so those are not a stress factors for our cyanobacteria; (ii) there exists enough fish 364 that will eat the zooplankton so that is not a stress factor for our cyano community; (iii) there are enough plants that will 365 keep under control an algal bloom so that our zooplankton and fish could survive, but not that many that could disturb our Scenarios 2 (Fig. 2). Interpretation\explanation: in second case, in our lake ecosystem we have also fish, 372 zooplankton, (especially daphnia), bacterioplankton and aquatic plants but less phytoplankton, because we have a 373 cyanobacteria blooming. Cyanobacteria from here don't need to release toxins to any of other trophic compartment 374 because:(i) exists enough zooplankton that will eat cyano (because those are the main food source), but will not survive 375 enough to become a stress factor for our cyano population; (ii) there is enough fish that will eat plants and the zooplankton 376 so that will be kept under control limit and not eat very much cyano; (iii) there are some plants that will keep under control 377 an massive algal bloom so that our zooplankton and fish could survive, but not that many that could disturb our  Scenarios 3 (Fig. 3). Interpretation\explanation: in this ideal case, in our lake ecosystem there are also fish, 386 phytoplankton, bacterioplankton and aquatic plants, but no cladocerans for example. Cyanobacteria from here don't need 387 to release toxins to any of other trophic compartment because: (i) there is enough fish that will eat other microalgae and 388 zooplankton so that not represent as much an stress factor for our cyano community; (ii) there are enough plants that will 389 keep under control an algal bloom by others microalgae so that our zooplankton and fish could survive, but not that many 390 that could disturb our cyanobacteria community.Conclusion: this represents the ideal picture of a typical lake ecosystem 391 and a scenario were cyanobacteria could live in "medium stress". Lake type: meso-eutrophic.

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Scenarios 4 (Fig. 4). Interpretation\explaination: in this ideal case, in our lake ecosystem are also fish,  Scenarios 5 (Fig. 5). Interpretation\explanation: in this ideal case, in our lake ecosystem there are also fish, 408 zooplankton, phytoplankton, bacterioplankton and aquatic plants, but the diversity is low regarding species. We don't have 409 a cyano bloom. Cyanobacteria from here began to release toxins to any of other trophic compartment because: (i) there 410 exists too much zooplankton that will eat microalgae, but also cyanobacteria so those are a stress factors for our 411 cyanobacteria; (ii) it doesn't exist enough fish that will eat the zooplankton so that is a stress factor for our cyano 412 community; (iii) there are enough plants that will keep under control an algal bloom so that why the release allelopathyc 413 substance and stress our cyanobacteria community. Conclusion: this represents the ideal picture for instability of a typical 414 lake ecosystem and a scenario were cyanobacteria live in "high stress" and became toxic. Lake type: eu-hypetrophic.