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Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

ABSTRACT: Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

SUBMITTER: Martinez Martinez J

PROVIDER: S-EPMC3144233 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

Martínez Martínez Joaquín J Poulton Nicole J NJ Stepanauskas Ramunas R Sieracki Michael E ME Wilson William H WH

PloS one 20110726 7

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Fur ...[more]

PMID: 21818332

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Project description:The species concept in marine phytoplankton is defined based on genomic, morphological, and functional properties. Reports of intraspecific diversity are widespread across major phytoplankton groups but the impacts of this variation on ecological and biogeochemical processes are often overlooked. Intraspecific diversity is well known within coccolithophores, which play an important role in the marine carbon cycle via production of particulate inorganic carbon. In this study, we investigated strain-specific responses to temperature in terms of morphology, carbon production, and carbonate mineralogy using a combination of microscopy, elemental analysis, flow cytometry, and nuclear magnetic resonance. Two strains of the cosmopolitan coccolithophore E. huxleyi isolated from different regions (subtropical, CCMP371; temperate, CCMP3266) were cultured under a range of temperature conditions (10°C, 15°C, and 20°C) using batch cultures and sampled during both exponential and stationary growth. Results for both strains showed that growth rates decreased at lower temperatures while coccosphere size increased. Between 15°C and 20°C, both strains produced similar amounts of total carbon, but differed in allocation of that carbon between particulate inorganic carbon (PIC) and particulate organic carbon (POC), though temperature effects were not detected. Between 10°C and 20°C, temperature effects on daily production of PIC and POC, as well as the cellular quota of POC were detected in CCMP3266. Strain-specific differences in coccolith shedding rates were found during exponential growth. In addition, daily shedding rates were negatively related to temperature in CCMP371 but not in CCMP3266. Despite differences in rates of particulate inorganic carbon production, both strains were found to produce coccoliths composed entirely of pure calcite, as established by solid-state 13C and 43Ca NMR and X-ray diffraction measurements. These results highlight the limitations of the species concept and the need for a trait-based system to better quantify diversity within marine phytoplankton communities.

Dataset Information

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

Publications

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

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