Spatial match between Planktothrix rubescens and whitefish in a mesotrophic peri-alpine lake: Evidence of toxins accumulation

Lake Bourget (France) provides drinking water and is a place for professional and recreational fishing. Since the mid 1990s, the lake has been exhibiting blooms of the filamentous cyanobacterium Planktothrix rubescens. This species is able to produce microcystin-LR and RR, toxins that contaminate different fish tissues and, if concentrated in the liver, can induce fish mortality. However, data on fish exposure to these toxins in a natural environment are scare and comparisons of spatial distribution between P. rubescens and exploited fish are needed to determine whether these fish avoid or converge in zones affected by the cyanobacteria. From June to November 2009, diurnal data on P. rubescens and whitefish (Coregonus lavaretus) spatial distributions have been monitored by hydroacoustic and BBE probe sampling. For all water samples, intracellular microcystin concentration of P. rubescens was quantified by HPLC/PDA. Furthermore, an arbitrary sample of eight whitefish captured at the moment of highest P. rubescens concentration in the lake were analyzed to investigate both the presence of this cyanobacterium in the gastrointestinal tract by optical microscopy and the possible bioaccumulation of the microcystin in their tissues by liquid chromatography–tandem mass spectrometry. Results show that P. rubescens abundance was at a maximum between the end of July and the beginning of September. During this period, P. rubescens abundance was vertically stratified with a maximum around 14–22 m, depending on the sampling station. The horizontal distributions of P. rubescens were heterogeneous at the scale of the lake. Results indicate that the presence of P. rubescens, for the observed cyanobacterial abundance, does not exert a significant pressure on the distribution pattern of the whitefish. Whitefish were present in the same areas as P. rubescens maxima during daytime, and they do not avoid or seek out the zone of high P. rubescens abundance. Filaments of P. rubescens have been observed in intestinal tracts of whitefish and the presence of microcystin-LR has been detected in their intestine and liver. Consequently, because of a direct contact between these organisms, toxins can be incorporated into whitefish by ingestion of P. rubescens filaments, leading to potential adverse effects on the health of this species.     

The trophic state of shallow lakes in The Netherlands

A study has been carried out on the development of an ecological assessment method for shallow lakes in The Netherlands. Analyses of eutrophication characteristics of 93 lakes with, in total, 127 sampling localities gave insight into some of the steering variables, such as total-P, total-N, chlorophyll-a and transparency. On the basis of phytoplankton species composition, three main groups of lakes could be distinguished. The first group, characterized by persistent filamentous Cyanobacteria, had the highest summer means of chlorophyll-a and total-P. The second group was characterized by non-persistent filamentous Cyanobacteria, occurring in low abundancies, and at lower chlorophyll-a and total-P concentrations. The third group of lakes was characterized by the absence or very low abundance of the filamentous Cyanobacteria. As a result of these analyses, criteria for the abatement of eutrophication in shallow lakes could be refined.     

Detection of <Emphasis Type="Italic">Planktothrix rubescens</Emphasis> (Cyanobacteria) associated with microcystin production in a freshwater reservoir

So far, the presence of microcystins in Portuguese freshwater resources has always been attributed to the bloom-forming cyanobacteria Microcystis aeruginosa. In 2005, however, microcystins were detected at the Beliche reservoir (Algarve, South Portugal), following the development of a bloom dominated by Planktothrix rubescens. The identity of the causative organism was confirmed by combining both morphological and 16S rRNA gene sequence analysis. Its ability to produce microcystins was confirmed by HPLC and MALDI-TOF MS. Unlike M. aeruginosa that usually accumulates near the water surface, P. rubescens found at the Beliche reservoir accumulated only at deep water levels. Being invisible from the surface, the occurrence of toxic P. rubescens in freshwater resources requires special attention when designing site inspection and sampling procedures for the correct risk assessment and management of cyanobacterial blooms in the field. Handling editor: D. Hamilton     

Automated Quantification and Sizing of Unbranched Filamentous Cyanobacteria by Model-Based Object-Oriented Image Analysis

Quantification and sizing of filamentous cyanobacteria in environmental samples or cultures are time-consuming and are often performed by using manual or semiautomated microscopic analysis. Automation of conventional image analysis is difficult because filaments may exhibit great variations in length and patchy autofluorescence. Moreover, individual filaments frequently cross each other in microscopic preparations, as deduced by modeling. This paper describes a novel approach based on object-oriented image analysis to simultaneously determine (i) filament number, (ii) individual filament lengths, and (iii) the cumulative filament length of unbranched cyanobacterial morphotypes in fluorescent microscope images in a fully automated high-throughput manner. Special emphasis was placed on correct detection of overlapping objects by image analysis and on appropriate coverage of filament length distribution by using large composite images. The method was validated with a data set for Planktothrix rubescens from field samples and was compared with manual filament tracing, the line intercept method, and the Utermöhl counting approach. The computer program described allows batch processing of large images from any appropriate source and annotation of detected filaments. It requires no user interaction, is available free, and thus might be a useful tool for basic research and drinking water quality control.Automated quantification and sizing of single cells by microscopy and image analysis are routinely used to determine microbial biomass (2, 6, 18). In contrast, fully automated quantification and measurement of the length of filamentous organisms are considerably more difficult, and not all problems have been solved satisfactorily yet, even though there is a strong demand for such approaches both in microbial ecology and in drinking water quality control (5). The filamentous cyanobacterium Planktothrix rubescens is a prominent organism in this context. It produces a variety of potent toxins (3, 4, 11, 12, 14) that may threaten animal and human health. Its ability to form blooms in some freshwater habitats makes this organism highly relevant in terms of public health and drinking water control (8, 9, 13, 23). Moreover, P. rubescens has recently been reported to unexpectedly invade drinking water reservoirs (15). Not surprisingly, monitoring of filamentous cyanobacteria is a standard task in many laboratories. Cyanobacterial biomass is often used as a proxy for estimating toxin concentration, as determination of this biomass is less costly than chemical analysis. Determination of abundance and biomass is routinely performed by microscopy using either the Utermöhl sedimentation method (20) or collection of cells on filters (22). These methods involve manual microscopy and are thus labor-intensive and time-consuming.A major improvement in filament quantification occurred when charge-coupled device (CCD) cameras and image analysis software became available. Several automated or semiautomated methods have been proposed for measuring total filament length, either indirectly (e.g., the line-intercept method) (16, 17) or directly by filament detection via image analysis of either fluorescence (10, 22) or bright-field images (1). However, the filamentous nature of P. rubescens (i.e., its remarkable length-to-width ratio) poses problems for determination of the number and length of individual filaments by conventional image analysis. P. rubescens filaments are around 5 to 8 μm wide (7, 23), whereas their length ranges from less than 50 μm to more than 2,500 μm. If the length of individual filaments is measured, filaments have to be located entirely within a field of view (FOV), which means that large areas have to be imaged. Larger FOV areas may be viewed by using lower magnifications, but this comes at the cost of lower levels of fluorescence intensity and resolution, which are important for both efficient imaging and precise measurement. A second problem results from the fact that filamentous organisms often overlap in a microscopic preparation. Overlapping objects in a two-dimensional image, like the images generated by wide-field microscopy, cannot be correctly separated by conventional image analysis that is based on thresholding and binarization; two overlapping objects typically are merged into a single object. While the effect of this artifact on the automated determination of abundance is obvious, the corresponding error in the length measurements depends greatly on the method used. These problems have been discussed in great detail by Walsby and Avery (22), but no automated solution has been proposed yet.The aim of this work was to develop a computer program that (i) counts filaments, (ii) measures the length of individual filaments, and (iii) determines the cumulative lengths of filaments in large composite images generated by epifluorescence microscopy. The probability that a filament is located only partially in an FOV and thus cannot be measured and the probability of filament overlaps were assessed theoretically by using a Monte Carlo simulation approach. Here we specifically address the problem of overlapping filaments and describe a strategy to recognize and correctly count them. The performance of the algorithm was tested by using measurements for filaments in images obtained by the manual filament tracing, line-intercept (16), and Utermöhl methods.     

Abundance and toxicity of Planktothrix rubescens in the pre-alpine Lake Ammersee,Germany

Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm³ m^?2 were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml^?1 (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion, possibly additionally benefiting from high nitrogen loads.Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp³]-MC-RR and [Asp³,Dhb⁷]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.     

Coupling high-resolution measurements to a three-dimensional lake model to assess the spatial and temporal dynamics of the cyanobacterium Planktothrix rubescens in a medium-sized lake

In a medium-sized pre-alpine lake (North Italy) the cyanobacterium Planktothrix rubescens has strongly dominated the phytoplankton assemblage since 2000, similar to many pre-alpine lakes, despite improvements in water quality. The objective of this study was to determine the factors governing the spatial distribution of P. rubescens, including the major hydrodynamic processes and the influence of long-term reduction in nutrient concentrations during a period of climate warming. We used an intensive field campaign conducted from February 2010 to January 2011, to evaluate distributions of phytoplankton phyla, as well as P. rubescens, using spectrally resolved fluorescence measurements. These data provided highly spatially and temporally resolved phytoplankton population data suitable to calibrate and validate a coupled three-dimensional hydrodynamic (ELCOM) and ecological model (CAEDYM) of the lake ecosystem. The simulations revealed the fundamental role of physiological features of P. rubescens that led to observed vertical patterns of distribution, notably a deep chlorophyll maximum, and a strong influence of lake hydrodynamic processes, particularly during high-discharge inflows in summer stratification. The simulations are used to examine growth-limiting factors that help to explain the increased prevalence of P. rubescens during re-oligotrophication.     

In-Situ Optical and Acoustical Measurements of the Buoyant Cyanobacterium P. Rubescens: Spatial and Temporal Distribution Patterns

Optical (fluorescence) and acoustic in-situ techniques were tested in their ability to measure the spatial and temporal distribution of plankton in freshwater ecosystems with special emphasis on the harmful and buoyant cyanobacterium P. rubescens. Fluorescence was measured with the multi-spectral FluoroProbe (Moldaenke FluoroProbe, MFP) and a Seapoint Chlorophyll Fluorometer (SCF). In-situ measurements of the acoustic backscatter strength (ABS) were conducted with three different acoustic devices covering multiple acoustic frequencies (614 kHz ADCP, 2 MHz ADP, and 6 MHz ADV). The MFP provides a fast and reliable technique to measure fluorescence at different wavelengths in situ, which allows discriminating between P. rubescens and other phytoplankton species. All three acoustic devices are sensitive to P. rubescens even if other scatterers, e.g., zooplankton or suspended sediment, are present in the water column, because P. rubescens containing gas vesicles has a strong density difference and hence acoustic contrast to the ambient water and other scatterers. After calibration, the combination of optical and acoustical measurements not only allows qualitative and quantitative observation of P. rubescens, but also distinction between P. rubescens, other phytoplankton, and zooplankton. As the measuring devices can sample in situ at high rates they enable assessment of plankton distributions at high temporal (minutes) and spatial (decimeters) resolution or covering large temporal (seasonal) and spatial (basin scale) scales.     

Distribution and synonymy of Plagiochila punctata (Taylor) Taylor, with hypotheses on the evolutionary history of Plagiochila sect. Arrectae (Plagiochilaceae, Hepaticae)

For a long time, Plagiochila (sect. Arrectae) punctata (Taylor) Taylor has been treated as an endemic of Atlantic Europe. Studies of larger specimen sets demonstrated that the species is widespread in mountainous regions of the Neotropics where it is known under several names including P. stolonifera Lindenb. & Gottsche, P. choachina Gottsche and P. patzschkei Steph. In tropical Africa P. punctata is established as Plagiochila subalpina Steph., nom. illeg. The sporophyte of P. punctata is described for the first time, based on material from Costa Rica. A maximum likelihood analysis of a nrITS sequence alignment with sequences of P. sects. Fuscoluteae (outgroup), Arrectae, and Rutilantes as well as of P. rubescens (Lehm. & Lindenb.) Lindenb. results in a tree with well bootstrap supported Arrectae and Rutilantes clades. ITS sequences of Plagiochila punctata from the Comoros and Zaire are placed in an unsupported monophyletic lineage together with P. punctata sequences from the British Isles and Ecuador. The species is nested in a robust clade with P. spinulosa (Dicks.) Dumort. and P. stricta Lindenb. The weak genetic separation of these three species indicates a recent diversification; the disjunct ranges may be the result of long range dispersal events. Plagiochila rubescens from southern South America is placed sister to P. sect. Arrectae. The latter section possibly originated from southern South American ancestors, diversified in tropical America after the uplift of the Andes and reached the Holarctics and tropical Africa by long range dispersal of diaspores.We are grateful to the curators and owners of the herbaria cited in the text for the loan of specimens, especially to Tamás Pócs, Volker Buchbender and Eberhard Fischer for their continuous support with recent collections from tropical Africa. David Rycroft kindly sent a duplicate of P. rubescens. Financial support of the Deutsche Forschungsgemeinschaft (grant HE 3584/1) is gratefully acknowledged.     

Assessment of the metabolic activity of Acremonium chrysogenum using Acridine Orange

A method is described for the assessment of the metabolic activity of the filamentous fungus Acremonium chrysogenum using the fluorescent dye Acridine Orange. Changes in metabolic activity are indicated by a reversible red-green shift in the colour of the dye, quantifiable by image analysis. A. chrysogenum mycelia exhibited an overwhelmingly green colour in circumstances leading to high substrate uptake, while under carbon starvation they appeared orange-red. It is believed these colour changes reflected changes in internal pH. The method provides a visual tool for the investigation of the metabolic behaviour of filamentous fungi.     

Diets of reef‐dwelling labrids (Choerodon species) vary with body size,season and habitat: influence of foraging ability,specialization and opportunism

Contemporary multivariate statistics were used to test the hypotheses that the dietary compositions of three populations of labrids on the west Australian coast are related to body size and undergo seasonal changes and to elucidate the relative extents and basis for any dietary differences within and between those populations. Gut content analyses determined the dietary compositions of Choerodon rubescens in marine waters of the outer reefs in the World Heritage Area of Shark Bay (26° S; 114° E) and of Choerodon schoenleinii in inner protected reefs of that large embayment. The dietary compositions of C. rubescens and C. schoenleinii differed significantly among length classes, progressed serially with increasing body size, both overall and almost invariably in each season and were more closely related to body size than season, whose effect was at best minimal. The size‐related dietary change in C. rubescens involved, in particular, a shift from crustaceans and non‐mytilid bivalves to mytilid bivalves and echinoid echinoderms. Although the diet of C. schoenleinii followed similar size‐related changes, it contained a greater volume of gastropods when the fish were small and mytilids when large and only a small volume of echinoids. The dietary composition of C. rubescens in the Abrolhos Islands, 300 km to the south of Shark Bay, was related both to length class and season and differed from that of this labrid in Shark Bay with the ingestion of lesser volumes of mytilids and greater volumes of echinoids. The size‐related changes in diet imply that these species shift from foraging over soft substrata to over reefs as their very well‐developed jaws become sufficiently strong to remove attached and larger prey. The dietary compositions of C. rubescens and C. schoenleinii in Shark Bay and of C. rubescens at the Abrolhos Islands were related far more to habitat–locational differences than to length class and season. The above intraspecific and interspecific differences in diet are consistent with qualitative accounts of the relative abundances of the main prey in their respective environments, supporting the view that, despite specializations in their feeding apparatus, these labrids can feed opportunistically to a certain extent and could thus potentially respond to moderate changes in the composition of their prey caused by climate change and other anthropogenic effects.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

Fungal infection for cyanobacterium <Emphasis Type="Italic">Anabaena smithii</Emphasis> by two chytrids in eutrophic region of large reservoir Lake Shumarinai,Hokkaido, Japan

Fungal infection of the filamentous cyanobacterium Anabaena smithii was observed in Lake Shumarinai in 2004–2006. Two fungal species were found to parasitize the specialized cells of A. smithii. These fungi might not correspond to the chytrid species that the previous studies reported as the parasites for Anabaena species. One fungus showed selective attachment to the akinete (akinete type). The filaments parasitized by this fungus increased in August 2004 and October 2006, when akinete and filament densities also increased. The maximum percentage of parasitized filaments was 3.2% of all filaments in October 2006. The other fungus was usually attached to the heterocyst (heterocyst type). The filaments parasitized by this fungus increased in October from 2004 to 2006. The maximum percentage of parasitized filaments was 20.6% in October 2004. The biomass of A. smithii was not suppressed by akinete-type fungus because of the low percentage of parasitized filaments. The heterocyst-type fungus might disturb the nitrogen fixation, but its effect was negligible due to a high concentration of available nitrogen for planktonic algae in Lake Shumarinai.     

Photoheterotrophy and light-dependent uptake of organic and organic nitrogenous compounds by Planktothrix rubescens under low irradiance

1. Planktothrix rubescens is the dominant photoautotrophic organism in Lake Zürich, a prealpine, deep, mesotrophic freshwater lake with an oxic hypolimnion. Over long periods of the year, P. rubescens accumulates at the metalimnion and growth occurs in situ at irradiance near the photosynthesis compensation point. Experiments were conducted to evaluate the contribution of photoheterotrophy, heterotrophy and light‐dependent uptake of nitrogenous organic compounds to the carbon and nitrogen budget of this cyanobacterium under conditions of restricted availability of light quanta. 2. We used both purified natural populations of P. rubescens from the depth of 9 m and an axenic culture grown under low irradiance at 11 μmol m^?2 s^?1 on a light : dark cycle (10 : 14 h) to determine the uptake rates of various amino acids, urea, glucose, fructose, acetate and inorganic carbon. The components were added to artificial lake water in low amounts that simulated the naturally occurring potential concentrations. 3. The uptake rates of acetate and amino acids (glycine, serine, glutamate and aspartate) were strongly enhanced at low irradiance as compared with the dark. However, no difference was observed in the uptake of arginine, which was taken up at high rates under both treatments. The uptake rates of glucose, fructose and urea were very low under all conditions. Similar results were obtained for both axenic P. rubescens and for purified natural populations of P. rubescens that were separated from bacterioplankton and other phytoplankton. 4. Metalimnetic P. rubescens that was stratified at low irradiance for weeks exhibited much higher uptake rates than filaments that were entrained in the deepening surface mixed layer and experienced higher irradiance. The added organic compounds contributed up to 62% to the total carbon uptake of metalimnetic P. rubescens. On the basis of a molar C : N ratio of 4.9, the nitrogen uptake as organic compounds satisfied up to 84% of the nitrogen demand. 5. The experiments indicate that photoheterotrophy and light‐dependent uptake of nitrogenous organic compounds may contribute significantly to the carbon and nitrogen budget of filaments at low irradiance typical for growth of P. rubescens in the metalimnion and at the bottom of the surface mixed layer.     

Isolation,characterization, and abundance of filamentous members of <Emphasis Type="Italic">Caldilineae</Emphasis> in activated sludge

Chloroflexi are currently believed to serve as backbone forming agents in the activated sludge of wastewater treatment plants (WWTPs). In this study, we isolated and characterized filamentous bacteria in the class Caldilineae of the phylum Chloroflexi in municipal WWTPs. Diversity analysis using Chloroflexi-specific 16S rRNA gene clone libraries showed that 97% of the clones belonged to the subdivision Anaerolineae comprising the two classes Anaerolineae (95%) and Caldilineae (2%). Clones of Caldilineae were related to a thermophilic filament Caldilinea aerophila with 93% 16S rRNA gene sequence similarity. We obtained filamentous isolates classified into the class Caldilineae showing the best match to C. aerophila with 89% 16S rRNA gene sequence similarity. Isolates showed no ability to assimilate glucose or N-acetylglucosamine or to degrade biopolymers which were observed in filamentous Chloroflexi of WWTPs. The assessment of relative abundance based on quantitative PCR of the 16S rRNA gene indicated that members of the class Caldilineae comprised 12–19% of the Chloroflexi in the activated sludge. Additionally, fluorescence in situ hybridization experiments showed that diverse filamentous Caldilineae inhabit the activated sludge of municipal WWTPs. These findings yield insight into the role of filamentous mesophilic Caldilinea in stabilizing flocs of activated sludge in a wide range of WWTPs.     

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.     

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.     

Variations in the Microcystin Production of Planktothrix rubescens (Cyanobacteria) Assessed from a Four-Year Survey of Lac du Bourget (France) and from Laboratory Experiments

Between 1999 and 2002, a routine survey of water quality in the Lac du Bourget was performed to study the dynamics and microcystin (MC) production of Planktothrix rubescens. Using liquid chromatography coupled to diode array detection and mass spectrometry, we found that two main variants ([d-Asp³] and [d-Asp³, Dhb⁷] microcystin-RR) were produced. The proportion of these two variants was not influenced by the depth or season of sampling. Expressed in microcystin-LR equivalents, high microcystin concentrations were recorded from August to December each year, reaching values of up to 6.7 μg L⁻¹. A significant correlation was found between the microcystin cell content and the cell densities of P. rubescens. Cellular quotas of microcystins ranged from 0.1 to 0.3 pg cell⁻¹. Simultaneously, laboratory experiments were performed on a strain of P. rubescens isolated from the lake to assess the potential impact of various P–PO₄³⁻ concentrations on intra- and extracellular microcystin production. Unlike natural populations, this strain only produced [d-Asp³] MC-RR. The intracellular microcystin content was similarly correlated to the cell density, but the cellular quota was slightly higher (0.3–0.7 pg cell⁻¹) than in the natural population. Again, as in the natural population, a linear relationship was found between growth rate and microcystin production rate. These findings support the hypothesis that environmental factors, such as phosphate concentrations, have no direct impact on microcystin production by P. rubescens, but act indirectly by affecting growth rate.     

The Consequences of Internal Waves for Phytoplankton Focusing on the Distribution and Production of Planktothrix rubescens

Consequences of internal wave motion for phytoplankton and in particular for the distribution and production of the harmful and buoyant cyanobacterium Planktothrix rubescens were investigated based on data from two field campaigns conducted in Lake Ammer during summer 2009 and 2011. In both years, P. rubescens dominated the phytoplankton community and formed a deep chlorophyll maximum (DCM) in the metalimnion. Internal wave motions caused vertical displacement of P. rubescens of up to 6 m and 10 m, respectively. Vertical displacements of isotherms and of iso-concentration lines of P. rubescens from the same depth range coincided, suggesting that P. rubescens did not or could not regulate its buoyancy to prevent wave-induced vertical displacements. Diatoms dominated the phytoplankton community in the epilimnion and were vertically separated from P. rubescens. The thickness of the diatom layer, but not the diatom concentrations within the layer, changed in phase with the changes in the thickness of the epilimnion caused by internal wave motions. Seiche induced vertical displacements of P. rubescens caused fluctuations in the light intensity available at the depth of the P. rubescens layer. The interplay between seiche induced vertical displacements of the P. rubescens layer and the daily cycle of incident light lead to differences in the daily mean available light intensity between lake ends by up to a factor of ∼3. As a consequence, the daily mean specific oxygen production rate of P. rubescens differed by up to a factor of ∼7 between lake ends. The horizontal differences in the specific oxygen production rate of P. rubescens were persistent over several days suggesting that the associated production of P. rubescens biomass may lead to phytoplankton patchiness. The effect of internal seiches on the spatial heterogeneity and the persistence of horizontal differences in production, however, depend on the timing and the synchronization between internal wave motion and the daily course of incident light intensity. Vertical displacements caused by internal waves could be distinguished from other factors influencing the distribution of P. rubescens (e.g. active buoyancy control, production, vertical mixing) by a temperature-based data transformation. This technique may be of general use for separating wave-induced transport from other processes (e.g. sedimentation, vertical mixing) that affect the distributions of dissolved substances and suspended particles.     

Effects of Daphnia exudates and sodium octyl sulphates on filament morphology and cell wall thickness of Aphanizomenon gracile (Nostocales), Cylindrospermopsis raciborskii (Nostocales) and Planktothrix agardhii (Oscillatoriales)

Grazing is recognized as one of the selective factors shaping the morphology and physiology of cyanobacteria. A recent study has shown that the filamentous cyanobacterium Aphanizomenon gracile strain SAG 31.79 thickened in the presence of Daphnia (Cladocera) and its exudates. The aims of our study were: (1) to determine whether this type of response to Daphnia cues is common for other strains of A. gracile, and other species of filamentous cyanobacteria, (2) to test whether the response is due to nutrients recycled by Daphnia, or kairomone induced, and (3) whether it is related to toxin production. Prior to the experiment, cyanobacterial strains were inspected using chromatographic methods for the presence of two toxins, cylindrospermopsin (CYN) and three homologues of microcystin (MC-RR, MC-YR, MC-LR). HPLC analyses showed that all strains were free of cylindrospermopsin, whereas microcystins were detected only in one strain (Planktothrix agardhii). We then tested whether Daphnia exudates can cause thickening of cyanobacterial filaments, which would suggest the morphological changes in cyanobacterial filaments are caused by recycled nutrients. Cyanobacteria were also exposed to sodium octyl sulphate (a commercially available Daphnia kairomone). Transmission electron microscopy (TEM) was used to check whether Daphnia exudates and sodium octyl sulphate trigger thickening of cyanobacterial cell walls, which would be a defence mechanism against grazing. The TEM analysis revealed no significant effect of either Daphnia exudates or kairomone (sodium octyl sulphate) on the cell wall thickness of cyanobacteria. However, our study showed that Daphnia exudates triggered filament thickening in nostocalean cyanobacteria, while filaments of the oscillatorialean strain P. agardhii did not show this response. It was also demonstrated that sodium octyl sulphate alone can also cause filament thickening, which suggests that this might be a specific defence response to the presence of grazers.     

Response of Tetraselmis suecica to nutrient and grazer manipulation

Three methods of algal quantification (direct cell counts, chlorophyll a extraction, in vivo fluorescence) were used to evaluate the response of the unicellular green flagellate Tetraselmis suecica to nutrients and grazers. Nutrient enrichment enhanced total cell counts, chlorophyll a concentration and in vivo and DCMU-fluorescence. Photosynthetic efficiency was reduced in the complete F2 medium as indicated by the high level of in vivo fluorescence, whereas photosynthetic efficiency was increased by the introduction of mussels to the F2 medium. The addition of mussels significantly increased the proportion of non-motile cells, but did not reduce the total cell count. The effect of mussel grazing on algae could be underestimated if only total cells were counted or only the chlorophyll a concentration was measured. The results indicate that these three methods measure different properties of an algal culture and are complementary to each other in assessing the quality and quantity of an algal population. Direct algal counting offers a reliable numerical assessment for cell population abundance. Chlorophyll a concentration was closely correlated to the total cell count. In the presence of mussels, in vivo fluorescence did not correlate with either algal cell counts or chlorophyll a concentration, indicating that the measurement of in vivo fluorescence may be misleading for estimating algal abundance under different culture conditions. This revised version was published online in August 2006 with corrections to the Cover Date.