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Abstract Background Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS). Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454") and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. Results Thirteen types of oligopeptides were uncovered by mass spectrometry (MS) analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded) precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. Conclusion Altogether seven nonribosomal peptide synthetase (NRPS) gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully can identify NRPS gene clusters and the corresponding oligopeptides. The analyses suggest independent evolution of all NRPS gene clusters as functional units. Our data indicate that the Planktothrix genome displays evolution of dual pathways (NRPS and ribosomal) for production of oligopeptides in order to maximize the diversity of oligopeptides with similar but functional discrete bioactivities.

Abstract Background Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer. Results A comparative analysis of the adenylation domains from the first module of McyB (McyB1) and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants. Conclusion We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may influence the ability of a particular strain to interact with its environment.

Abstract Background Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera Microcystis, Planktothrix and Anabaena. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected. Results We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS) cyanopeptolin gene cluster from the Japanese strain Planktothrix NIES 205 (205-oci), showed the 30 kb gene cluster to be highly similar to the oci gene cluster previously described in Planktothrix NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S) and a glyceric acid loading (GA)-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A)-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, ntcA and the phycocyanin cpcBA spacer) were identical, supporting that these geographically separated Planktothrix strains are closely related. Conclusion A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are acting within and around the epimerase insertion while purifying selection conserves the remaining (major) part of the gene cluster. The presence of an epimerase in the gene cluster is in line with the D-configuration of Htyr, determined experimentally in oscillapeptin E in a previous study.

Chytrid parasites are increasingly recognized as ubiquitous and potent control agents of phytoplankton, including bloom-forming toxigenic cyanobacteria. In order to explore the fate of the cyanobacterial toxin microcystins (MCs) and assess potential upregulation of their production under parasite attack, a laboratory experiment was conducted to evaluate short- and long-term variation in extracellular and intracellular MC in the cyanobacteria Planktothrix agardhii and P. rubescens, both under chytrid infection and in the presence of lysates of previously infected cyanobacteria. MCs release under parasite infection was limited and not different to uninfected cyanobacteria, with extracellular toxin shares never exceeding 10%, substantially below those caused by mechanical lysis induced by a cold-shock. Intracellular MC contents in P. rubescens under infection were not significantly different from uninfected controls, whereas infected P. agardhii showed a 1.5-fold increase in intracellular MC concentrations, but this was detected within the first 48 hours after parasite inoculation and not later, indicating no substantial MC upregulation in cells being infected. The presence of lysates of previously infected cyanobacteria did not elicit higher intracellular MC contents in exposed cyanobacteria, speaking against a putative upregulation of toxin production induced via quorum sensing in response to parasite attack. These results indicate that chytrid epidemics can constitute a bloom decay mechanism that is not accompanied by massive release of toxins into the medium., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microbial parasites have only recently been included in planktonic food web studies, but their functional role in conveying dietary energy still remains to be elucidated. Parasitic fungi (chytrids) infecting phytoplankton may constitute an alternative trophic link and promote organic matter transfer through the production of dissemination zoospores. Particularly, during proliferation of inedible or toxic algal species, such as large Cyanobacteria fostered by global warming, parasites can constitute an alternative trophic link providing essential dietary nutrients that support somatic growth and reproduction of consumers. Using phytoplankton-parasites associations grown under laboratory controlled conditions we assessed the fatty acids and biochemical composition of species with different nutritional quality and followed the metabolic pathway from the algal host and their parasites zoospores using compound-specific stable isotope analysis. This study demonstrated that chytrids are trophic upgraders able to retain essential nutrients that can be transferred to upper trophic levels both in terms of organic matter quantity and nutritional quality. Through the production of zoospores, nutritionally important long-chain polyunsaturated fatty acids that can be consequently assimilated by consumers. We conclude that parasitism at the base of aquatic food webs may represent a crucial trophic link for dietary nutrients and essential biomolecules alternative to herbivory or bacterivory, which can be particularly crucial during the proliferation of inedible or nutritionally inadequate algal species fostered by climate change., (Copyright © 2020. Published by Elsevier GmbH.)

Gonyostomum semen is a bloom-forming freshwater raphidophyte that is currently on the increase, which concerns water managers and ecologists alike. Much indicates that the recent success of G. semen is linked to its diel vertical migration (DVM), which helps to overcome the spatial separation of optimal light conditions for photosynthesis at the surface of a lake and the high concentration of phosphate in the hypolimnion. I here present data from a field study conducted in Lake Lundebyvannet (Norway) in 2017-2019 that are consistent with the idea that the DVM of G. semen also allows for a hypolimnetic uptake of ammonium. As expected, microbial mineralization of organic matter in a low-oxygen environment led to an accumulation of ammonium in the hypolimnion as long as G. semen was absent. In contrast, a decreasing or constantly lower concentration of hypolimnetic ammonium was found in presence of a migrating G. semen population. In summer of 2019, a short break in the DVM of G. semen coincided with a rapid accumulation of hypolimnetic ammonium, which was equally rapidly decimated when G. semen resumed its DVM. Taken together, these data support the idea that G. semen can exploit the hypolimnetic pool of ammonium, which may be one reason for the recent success of the species and its significant impact on the structure of the aquatic food web., Competing Interests: Conflict of interest: The author declares no conflict of interests., (© 2020 the Author(s), licensee AIMS Press.)

A number of boreal surface waters have become browner over the last two decades. Recovery from acid rain is regarded as an important driver for this lake color increase, indicating a general browner lake color in preindustrial times. However, the lack of long-term monitoring data makes it challenging to unravel historical changes in lake color. In this study, we estimated long-term development in lake color (1800 to 2015) based on the optical properties of alkaline extractable dissolved organic matter (DOM) from sediment using UV-Vis and fluorescence spectroscopy. We found that the present lake color (2015) was significantly browner (four times higher in absorption coefficient) than for the period from 1800 to 1915 when lake color was at a lower and more stable level. Fluorescence excitation-emission matrices combined with parallel factor analysis (EEM-PARAFAC) indicate that terrestrially derived DOM was the main source of sediment DOM. However, the importance of in-lake source of DOM has significantly increased with time. The long-term trend in DOM burial was not consistent with the anthropogenic sulfur (S) deposition pattern. However, along with the increased sediment DOM, there has been increased precipitation, temperature and forest growth with time, which affect the production and degradation of DOM. Even though S deposition might have delayed the runoff of terrestrial DOM for a certain period, it comes in addition to other color-regulating factors. Thus, there is no single driver for the observed lake browning, but rather an interplay between different drivers varying in strength over time, such as afforestation, changes in areal use, declined S deposition, and increased temperature and precipitation., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Fungal diseases threaten natural and man-made ecosystems. Chytridiomycota (chytrids) infect a wide host range, including phytoplankton species that form the basis of aquatic food webs and produce roughly half of Earth's oxygen. However, blooms of large or toxic phytoplankton form trophic bottlenecks, as they are inedible to zooplankton. Chytrids infecting inedible phytoplankton provide a trophic link to zooplankton by producing edible zoospores of high nutritional quality. By grazing chytrid zoospores, zooplankton may induce a trophic cascade, as a decreased zoospore density will reduce new infections. Conversely, fewer infections will not produce enough zoospores to sustain long-term zooplankton growth and reproduction. This intricate balance between zoospore density necessary for zooplankton energetic demands (growth/survival), and the loss in new infections (and thus new zoospores) because of grazing was tested empirically. To this end, we exposed a cyanobacterial host (Planktothrix rubescens) infected by a chytrid (Rizophydium megarrhizum) to a grazer density gradient (the rotifer Keratella cf. cochlearis). Rotifers survived and reproduced on a zoospore diet, but the Keratella population growth was limited by the amount of zoospores provided by chytrid infections, resulting in a situation where zooplankton survived but were restricted in their ability to control disease in the cyanobacterial host. We subsequently developed and parameterized a dynamical food-chain model using an allometric relationship for clearance rate to assess theoretically the potential of different-sized zooplankton groups to restrict disease in phytoplankton hosts. Our model suggests that smaller-sized zooplankton may have a high potential to reduce chytrid infections on inedible phytoplankton. Together, our results point out the complexity of three-way interactions between hosts, parasites, and grazers and highlight that trophic cascades are not always sustainable and may depend on the grazer's energetic demand., (© 2019 The Authors. Ecology published by Wiley Periodicals, Inc. on behalf of Ecological Society of America.)

The ability to identify drivers responsible for algal community shifts is an important aspect of environmental issues. The lack of long-term datasets, covering periods prior to these shifts, is often limiting our understanding of drivers responsible. The freshwater alga, Gonyostomum semen (Raphidophyceae), has significantly increased distribution and mass occurrences in Scandinavian lakes during the past few decades, often releasing a skin irritating slime that causes discomfort for swimmers. While the alga has been extensively studied, long-term data from individual lakes are often absent or greatly limited and drivers behind this species' success are still not clear. However, if specific and persistent taxa biomarkers for G. semen could be detected in dated sediment cores, long-term data would be improved and more useful. To test for biomarkers, we examined the pigment composition of several G. semen strains in culture. Further, dated sediment core samples from Lake Lundebyvann, Norway, were used to test the pigments' suitability as biomarkers in paleolimnological studies. Modifications to a common analysis allowed for the successful detection of the polar xanthophyll heteroxanthin and the non-polar chlorophyll a, as well as several other algal pigments by using high performance liquid chromatography-photometric diode arrays (HPLC-PDA). Heteroxanthin was confirmed by liquid chromatography-mass spectrometry (LC-MS) and detected by HPLC-PDA in all examined G. semen strains, along with chlorophyll a. Using HPLC-PDA, we also identified and confirmed the presence of the biomarker, xanthophyll heteroxanthin, in sediment core samples up to 60 years of age. The specificity of this xanthophyll was also tested by examining a wide range of algal strains from common Norwegian phytoplankton species. Heteroxanthin was not detected in any species commonly occurring in significant amounts in Norwegian lakes. We therefore conclude that heteroxanthin is a suitable pigment biomarker for G. semen and that this pigment can be successfully used for paleolimnological studies., Competing Interests: The authors have declared that no competing interests exist.

Understanding how individual parasite traits contribute to overall fitness, and how they are modulated by both external and host environment, is crucial for predicting disease outcome. Fungal (chytrid) parasites of phytoplankton are important yet poorly studied pathogens with the potential to modulate the abundance and composition of phytoplankton communities and to drive their evolution. Here, we studied life-history traits of a chytrid parasite infecting the planktonic, bloom-forming cyanobacterium Planktothrix spp. under host genotype and thermal variation. When expressing parasite fitness in terms of transmission success, disease outcome was largely modulated by temperature alone. Yet, a closer examination of individual parasite traits linked to different infection phases, such as (i) the establishment of the infection (i.e. intensity of infection) and (ii) the exploitation of host resources (i.e. size of reproductive structures and propagules), revealed differential host genotype and temperature × host genotype modulation, respectively. This illustrates how parasite fitness results from the interplay of individual parasite traits that are differentially controlled by host and external environment, and stresses the importance of combining multiple traits to gain insights into underlying infection mechanisms.

The human health risks posed by exposure to cyanobacterial toxins such as microcystin (MC) through water and fish consumption remain poorly described. During the last two decades, coastal regions of Lake Victoria such as Nyanza Gulf (Kisumu Bay) have shown severe signs of eutrophication with blooms formed by Microcystis producing MC. In this study, the spatial variability in MC concentration in Kisumu Bay was investigated which was mostly caused by Microcystis buoyancy and wind drifting. Small fish (<6 cm) mainly composed of Rastrineobola argentea were examined for MC content by means of biological methods such as ELISA and protein phosphatase inhibition assay (PPIA) and partly by chemical-analytical methods such as LC-MS/MS. Overall, the MC content in small fish was related to the MC content observed in the seston. When comparing the MC content in the seston in relation to dry weight with the MC content in small fish the latter was found three orders of magnitude decreased. On average, the ELISA-determined MC contents exceeded the PPIA-determined MC contents by a factor of 8.2 ± 0.5 (SE) while the MC contents as determined by LC-MS/MS were close to the detection limit. Using PPIA, the MC content varied from 25⁻109 (mean 62 ± 7) ng/g fish dry weight in Kisumu Bay vs. 14 ± 0.8 ng MC/g in the more open water of L. Victoria at Rusinga channel. Drying the fish under the sun showed little effect on MC content, although increased humidity might indirectly favor photocatalyzed MC degradation.

Experimental evolution can be used to test for and characterize parasite and pathogen adaptation. We undertook a serial-passage experiment in which a single parasite population of the obligate fungal (chytrid) parasite Rhizophydium megarrhizum was maintained over a period of 200 days under different mono- and multiclonal compositions of its phytoplankton host, the bloom-forming cyanobacterium Planktothrix . Despite initially inferior performance, parasite populations under sustained exposure to novel monoclonal hosts experienced rapid fitness increases evidenced by increased transmission rates. This demonstrates rapid adaptation of chytrids to novel hosts and highlights their high evolutionary potential. In contrast, increased fitness was not detected in parasites exposed to multiclonal host mixtures, indicating that cyanobacterial intraspecific diversity hampers parasites adaptation. Significant increases in intensity of infection were observed in monoclonal and multiclonal treatments, suggesting high evolvability of traits involved in parasite attachment onto hosts (i.e., encystment). A comparison of the performance of evolved and unevolved (control) parasite populations against their common ancestral host did not reveal parasite attenuation. Our results exemplify the ability of chytrid parasites to adapt rapidly to new hosts, while providing experimental evidence that genetic diversity in host populations grants increased resistance to disease by hindering parasite adaptation.

Freshwater cyanobacteria are prone to a wide range of highly potent microbial antagonists. Most of these exploit their prey in a frequency-dependent manner and are therefore particularly well suited to prevent any accumulation of cyanobacteria. Mass developments of cyanobacteria, the so-called blooms, should therefore be rare events, which is in striking contrast to what we actually see in nature. Laboratory experiments of the present study showed that the temperature range 5.8-10 °C forms a thermal refuge, inside which the cyanobacterium Planktothrix can grow without being exploited by two otherwise highly potent microbial antagonists. In nature, access of Planktothrix to this refuge was associated with positive net growth and a high probability of bloom formation, confirming that refuge temperatures indeed allow Planktothrix to grow with a minimum of biomass loss caused by microbial antagonists. Contact to higher temperatures, in contrast, was associated with decreases in net growth rate and in probability of bloom formation, with population collapses and with the occurrence of parasite infection. This is in agreement with the finding of laboratory experiments that above 10 °C exploitation of Planktothrix by multiple microbes increases in a temperature-dependent manner. Taken together, above findings suggest that temperature modulates the microbial control of natural Planktothrix populations. Low temperatures form a thermal refuge that may promote Planktothrix bloom formation by shielding the cyanobacterium from otherwise highly potent microbial antagonists., Competing Interests: Conflict of interest: The author declares no conflict of interests in this article.

Chytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as co-evolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)