Using the hidden isotopic heterogeneity in phyto- and zooplankton to unmask disparity in trophic carbon transfer

In this study, we show that natural phototrophic populations can be probed individually for their in situ d13C signature by linking fluorescence-activated cell sorting and isotope-ratio mass spectrometry (IRMS) using in-line pyrolytic methylation. This novel methodology greatly improved the resolution in discriminating and tracing the differential carbon (C) pathways at the base of the pelagic food web in the cyanobacteria-dominated Lake Loosdrecht (The Netherlands). Our analysis revealed the co-occurrence of phytoplankton taxa differing by 6–10‰ in d13C. Predominant micro- and mesozooplankton species reflected this difference as the result of preferential grazing and/or selective digestion. Flow cytometric (FCM) retrieval of phytoplankton d13C signatures, applied in conjunction with 13C-carbonate labeling, also enabled an assessment of in situ population-specific growth rates. Diatoms and green algae exhibited up to ninefold higher growth rates than those for cyanobacterial species. The coexistence of phytoplankton populations widely differing in d13C, standing stock, and turnover time has important implications for the interpretation of C transfer in pelagic food webs. Our approach disclosed a disproportional impact on trophic cascades by numerically minor phototrophs that otherwise would have gone unnoticed. Despite the abundance of cyanobacterial-derived C, the zooplankton largely rely on eukaryotic algae for growth. Rotifers take a central position in passing on this algal C to the cyclopoid copepod populations in the lake. The bosminid-dominated cladoceran population uses both the cyanobacterial- and algal-derived C in approximately equal shares

Optical changes associated with cyanobacterial bloom termination by viral lysis

Optical changes that accompanied a collapse of the population of filamentous cyanobacteria from a shallow, eutrophic lake were studied in laboratory-scale enclosures (LSEs). The experimental conditions are known, from previous work on these systems, to cause a dramatic collapse of the dominant algal or cyanobacterial species, which in turn can be associated with viral activity. Within 2 weeks of continuous addition of nutrient-rich growth medium, near-complete collapse of the dominant population occurred over the span of a few days. The collapse was repeatedly and reproducibly observed and was primarily characterized by a marked increase in water transparency. Scattering of light decreased by 80%, absorption decreased by 20-80%. There was high similarity in optical changes between several experiments, carried out in different seasons. An increase of dissolved material and submicron-sized particles (SMP) that showed chlorophyll a (Chl a) absorption was observed during the collapse. The phycocyanin (PC): Chl a ratio and phaeopigment : Chl a ratio proved to be good indicators of the observed collapse. Reflectance spectra that were modelled using a constant volume-scattering function indicated that mass mortality of this magnitude can be detected in natural systems using current remote sensors.

Optical changes associated with cyanobacterial bloom termination by viral lysis

Optical changes that accompanied a collapse of the population of filamentous cyanobacteria from a shallow, eutrophic lake were studied in laboratory-scale enclosures (LSEs). The experimental conditions are known, from previous work on these systems, to cause a dramatic collapse of the dominant algal or cyanobacterial species, which in turn can be associated with viral activity. Within 2 weeks of continuous addition of nutrient-rich growth medium, near-complete collapse of the dominant population occurred over the span of a few days. The collapse was repeatedly and reproducibly observed and was primarily characterized by a marked increase in water transparency. Scattering of light decreased by 80%, absorption decreased by 20-80%. There was high similarity in optical changes between several experiments, carried out in different seasons. An increase of dissolved material and submicron-sized particles (SMP) that showed chlorophyll a (Chl a) absorption was observed during the collapse. The phycocyanin (PC): Chl a ratio and phaeopigment : Chl a ratio proved to be good indicators of the observed collapse. Reflectance spectra that were modelled using a constant volume-scattering function indicated that mass mortality of this magnitude can be detected in natural systems using current remote sensors

Optical signatures of the filamentous cyanobacterium Leptolyngbya boryana during mass viral lysis

The inherent optical properties of absorption and scattering, the population density of virus-like particles, and the particle size distribution (PSD) for particles <0.7 µm of the filamentous cyanobacterium Leptolyngbya boryana were monitored for 72 h at 9-h intervals following infection with cyanophage LPP-1. Lorenz–Mie scattering theory and the anomalous diffraction approximation were used to derive the refractive index representative of the bulk of particles and to model the particulate backscattering coefficient [bbp(l)]. Upon lysis, particulate absorption [ap(l)] and scattering [bbp(l)] decreased, the number of free virus-like particles increased drastically, the PSD shifted to relative abundance of small particles, and average trichome length decreased sharply. The complex refractive index of the bulk of particles was comparable with literature values for cyanobacteria. Modeled bbp(l) spectra were lowered upon lysis, while backscattering probability increased. The effect of underrepresentation of particles below the measurement limit of the particle sizer was studied; more small particles in the PSD resulted in higher, but still relatively low, backscattering probability. The consequences of the studied optical behavior on spectral reflectance was explored. Significant spectral changes at longer wavelengths were mostly masked by water absorption at nominal population densities. However, strongly reduced ap(l) in the red pigment absorption bands resulted in a pronounced green peak in reflectance spectra, and it was concluded that reflectance band ratio algorithms targeted at the absorption of the pigments phycocyanin and chlorophyll a can be used to detect the mass mortality caused by viral infection. The integrated intensity of reflected light was too variable to serve as an optical indicator for lytic events.

Dynamic modelling of viral impact on cyanobacterial populations in shallow lakes: Implications of burst size

Laboratory experiments with whole water-columns from shallow, eutrophic lakes repeatedly showed collapse of the predominant filamentous cyanobacteria. The collapse could be due to viral activity, from the evidence of electron microscopy of infected cyanobacterial cells and observed dynamics of virus-like particles. Burst-size effects on single-host single-virus dynamics was modelled for nutrient-replete growth of the cyanobacteria and fixed viral decay rate in the water column. The model combined previously published equations for nutrient-replete cyanobacterial growth and virus–host relationship. According to the model results, burst sizes greater than 200 to 400 virions per cell would result in host extinction, whereas lower numbers would allow coexistence, and even stable population densities of host and virus. High-nutrient status of the host cells might accommodate a large burst size. The ecological implication could be that burst-size increase accompanying a transition from phosphorus to light-limited cyanobacterial growth might destabilize the virus–host interaction and result in the population collapse observed in the experiments.

Linking flow cytometric cell sorting and compound-specific 13C-analysis to determine population-specific isotopic signatures and growth rates in cyanobacteria-dominated lake plankton

A novel methodology was applied to determine the 13C signatures of natural cyanobacterial and algal populations by combined compound-specific isotope ratio mass spectrometry and pyrolytic methylation-gas chromatography (Py-GC-IRMS) of the fatty acids released from phytoplankton fractions collected using fluorescence-activated cell sorting. Py-GC-IRMS provided direct analysis of the very small samples (<200 ng total C) derived from the cell sorting of individual phototrophic populations, while minimizing the chances on contamination and loss in sample handling. Despite trichome lengths exceeding the diameter of the sort droplets, filamentous cyanobacteria were amenable to population-specific cell sorting. In concert with 13C-CO2 labeling, the combined use of flow cytometric cell sorting and Py-GC-IRMS enabled both the assessment of standing stocks and of population-specific growth rates of the predominant cyanobacterial and algal taxa in Lake Loosdrecht (The Netherlands). Filamentous prochlorophytes, formerly the dominant cyanobacterial taxon in the lake, appeared less abundant in recent years and exhibited growth rates 30%-40% lower than the rates recorded for oscillatorioid populations. Diatom and green algal populations grew at rates 4- to 10-fold higher than filamentous cyanobacteria and are thus important for the lake's carbon budget. This approach offers new possibilities in studying plankton dynamics at a resolution not feasible in the past. [KEYWORDS: cell sorting ; fatty acid profile ; oscillatorioids ; pyrolytic methylation ; specific growth rates ; stable isotope labeling]

Phylogenetic characterization of phosphatase-expressing bacterial communities in Baltic Sea sediments

Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is largely unknown. We herein presented a culture-independent method to phylogenetically characterize phosphatase-expressing bacteria in sediments. We labeled whole-cell extracts of Baltic Sea sediments with an artificial phosphatase substrate and sorted phosphatase-expressing cells with a flow cytometer. Their phylogenetic affiliation was determined by Denaturing Gradient Gel Electrophoresis. The phosphatase-expressing bacterial community coarsely reflected the whole-cell bacterial community, with a similar dominance of Alphaproteobacteria

Phylogenetic characterization of phosphatase-expressing bacterial communities in Baltic Sea sediments

Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is largely unknown. We herein presented a culture-independent method to phylogenetically characterize phosphatase-expressing bacteria in sediments. We labeled whole-cell extracts of Baltic Sea sediments with an artificial phosphatase substrate and sorted phosphatase-expressing cells with a flow cytometer. Their phylogenetic affiliation was determined by Denaturing Gradient Gel Electrophoresis. The phosphatase-expressing bacterial community coarsely reflected the whole-cell bacterial community, with a similar dominance of Alphaproteobacteria