Corina P. D. Brussaard

Optimization of Procedures for Counting Viruses by Flow Cytometry

ABSTRACT The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at −80°C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 × 10−5 dilution of commercial stock), incubated for 10 min in the dark at 80°C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least −80°C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Global-scale processes with a nanoscale drive: the role of marine viruses

Viruses, the smallest and most numerous of all biotic agents, represent the planets largest pool of genetic diversity. The sheer abundance of oceanic viruses results in ~1029 viral infections per day, causing the release of 108–109 tonnes of carbon per day from the biological pool (Suttle, 2007). Still, how and to what extent virus-mediated nanoscale processes are linked to global-scale biodiversity and biogeochemistry is poorly defined.

Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes

Large dsDNA viruses are involved in the population control of many globally distributed species of eukaryotic phytoplankton and have a prominent role in bloom termination. The genus Phaeocystis (Haptophyta, Prymnesiophyceae) includes several high-biomass-forming phytoplankton species, such as Phaeocystis globosa, the blooms of which occur mostly in the coastal zone of the North Atlantic and the North Sea. Here, we report the 459,984-bp-long genome sequence of P. globosa virus strain PgV-16T, encoding 434 proteins and eight tRNAs and, thus, the largest fully sequenced genome to date among viruses infecting algae. Surprisingly, PgV-16T exhibits no phylogenetic affinity with other viruses infecting microalgae (e.g., phycodnaviruses), including those infecting Emiliania huxleyi, another ubiquitous bloom-forming haptophyte. Rather, PgV-16T belongs to an emerging clade (the Megaviridae) clustering the viruses endowed with the largest known genomes, including Megavirus, Mimivirus (both infecting acanthamoeba), and a virus infecting the marine microflagellate grazer Cafeteria roenbergensis. Seventy-five percent of the best matches of PgV-16T–predicted proteins correspond to two viruses [Organic Lake phycodnavirus (OLPV)1 and OLPV2] from a hypersaline lake in Antarctica (Organic Lake), the hosts of which are unknown. As for OLPVs and other Megaviridae, the PgV-16T sequence data revealed the presence of a virophage-like genome. However, no virophage particle was detected in infected P. globosa cultures. The presence of many genes found only in Megaviridae in its genome and the presence of an associated virophage strongly suggest that PgV-16T shares a common ancestry with the largest known dsDNA viruses, the host range of which already encompasses the earliest diverging branches of domain Eukarya.

AUTOLYSIS KINETICS OF THE MARINE DIATOM DITYLUM BRIGHTWELLII (BACILLARIOPHYCEAE) UNDER NITROGEN AND PHOSPHORUS LIMITATION AND STARVATION1

Autolysis kinetics in axenic cultures of the diatom Ditylum brightwellii (West) Grunow were studied under nutrient limitation in continuous cultures and under nutrient starvation in batch‐mode cultures obtained by switching off nutrient supply in the continuous cultures. Under N limitation, the specific algal autolysis rates (δ, day−1) were found constant at 0.014 ± 0.002 day−1over a broad range of specific dilution rates (D, day−1) (0.09–0.56 day−1), implying an intrinsic death factor independent of the physiologzc state of the algal cells. Under P limitation, 8 was inversely related to D and ranged between 0.067 and 0.005 day−1 at D = 0.17–0.44 day−1. Under conditions of nutrient stamation, the degree of algal nutrient deficiency prior to stamation affected autolysis rates (δb, day−1) and subsequently survival of the algal cultures. Nitrogen‐starved D. brightwellii showed highest δb (maximum, 0.10 day−1) when precultured at the higher growth rates. Phosphorus stamation led to highest δb (maximum, 0.21 day−1) in the cultures preconditioned at the lower steady state growth rates. The lower death rates for D. brightwellii under limitation and starvation of N compared to P suggest that D. brightwellii was better equipped to handle N than P deficiency. The present results showed that cell lysis induced by nutrient stress was a significant cause of mortality in D. brightwellii and provided more insight into the field distribution of this neritic diatom.

Factors affecting virus dynamics and microbial host-virus interactions in marine environments

Marine microorganisms constitute the largest percentage of living biomass and serve as the major driving force behind nutrient and energy cycles. While viruses only comprise a small percentage of this biomass (i.e., 5%), they dominate in numerical abundance and genetic diversity. Through host infection and mortality, viruses affect microbial population dynamics, community composition, genetic evolution, and biogeochemical cycling. However, the field of marine viral ecology is currently limited by a lack of data regarding how different environmental factors regulate virus dynamics and host-virus interactions. The goal of the present minireview was to contribute to the evolution of marine viral ecology, through the assimilation of available data regarding the manner and degree to which environmental factors affect viral decay and infectivity as well as influence latent period and production. Considering the ecological importance of viruses in the marine ecosystem and the increasing pressure from anthropogenic activity and global climate change on marine systems, a synthesis of existing information provides a timely framework for future research initiatives in viral ecology.

Isolation and phylogenetic analysis of novel viruses infecting the phytoplankton Phaeocystis globosa (Prymnesiophyceae)

ABSTRACT Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.

Re-examination of the relationship between marine virus and microbial cell abundances.

Marine viruses are critical drivers of ocean biogeochemistry, and their abundances vary spatiotemporally in the global oceans, with upper estimates exceeding 108 per ml. Over many years, a consensus has emerged that virus abundances are typically tenfold higher than microbial cell abundances. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5,671 microbial cell and virus abundance estimates from 25 distinct marine surveys and find substantial variation in the virus-to-microbial cell ratio, in which a 10:1 model has either limited or no explanatory power. Instead, virus abundances are better described as nonlinear, power-law functions of microbial cell abundances. The fitted scaling exponents are typically less than 1, implying that the virus-to-microbial cell ratio decreases with microbial cell density, rather than remaining fixed. The observed scaling also implies that viral effect sizes derived from ‘representative’ abundances require substantial refinement to be extrapolated to regional or global scales.

Central Role of Dynamic Tidal Biofilms Dominated by Aerobic Hydrocarbonoclastic Bacteria and Diatoms in the Biodegradation of Hydrocarbons in Coastal Mudflats

ABSTRACT Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.

FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON 1

The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein‐AM], 5‐chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2′,7′‐dichlorofluorescein diacetate [H2DCFDA]; and two membrane probes: bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and SYTOX‐Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold‐water, 26 temperate, and four warm‐water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein‐AM and H2DCFDA (P < 0.001). Of the two membrane probes, DIBAC4(3) stained rhodophytes and euglenophytes much better than SYTOX‐Green. The 13 algal strains best stainable (high live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC4(3), and SYTOX‐Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.

First day of an oil spill on the open sea: Early mass transfers of hydrocarbons to air and water

During the first hours after release of petroleum at sea, crude oil hydrocarbons partition rapidly into air and water. However, limited information is available about very early evaporation and dissolution processes. We report on the composition of the oil slick during the first day after a permitted, unrestrained 4.3 m(3) oil release conducted on the North Sea. Rapid mass transfers of volatile and soluble hydrocarbons were observed, with >50% of ≤C17 hydrocarbons disappearing within 25 h from this oil slick of <10 km(2) area and <10 μm thickness. For oil sheen, >50% losses of ≤C16 hydrocarbons were observed after 1 h. We developed a mass transfer model to describe the evolution of oil slick chemical composition and water column hydrocarbon concentrations. The model was parametrized based on environmental conditions and hydrocarbon partitioning properties estimated from comprehensive two-dimensional gas chromatography (GC×GC) retention data. The model correctly predicted the observed fractionation of petroleum hydrocarbons in the oil slick resulting from evaporation and dissolution. This is the first report on the broad-spectrum compositional changes in oil during the first day of a spill at the sea surface. Expected outcomes under other environmental conditions are discussed, as well as comparisons to other models.