Genotypic, size and morphological diversity of virioplankton in a deep oligomesotrophic freshwater lake (Lac Pavin, France)

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Abstract

We examined changes in morphological and genomic diversities of viruses by means of transmission electronic microscopy and pulsed field gel electrophoresis (PFGE) over a nine-month period (April–December 2005) at four different depths in the oligomesotrophic Lac Pavin. We found that the majority of viruses in this lake belonged to the family of Siphoviridae or were untailed, with capsid sizes ranging from 30 to 60 nm, and exhibited genome sizes ranging from 15 to 45 kb. On average, 12 different genotypes dominated each of the PFGE fingerprints. The highest genomic viral richness was recorded in summer (mean = 14 bands per PFGE fingerprint) and in the epilimnion (mean = 13 bands per PFGE fingerprint). Among the physico-chemical and biological variables considered, the availability of the hosts appeared to be the main factor regulating the variations in the viral diversity.

Introduction

It is now widely accepted that viruses are the most abundant entities in aquatic ecosystems, where their abundance is about an order of magnitude greater than the number of bacteria (Weinbauer, 2004, Suttle, 2007). Viruses are ubiquitous entities in natural environments, infecting virtually every living form of life, from cellular prokaryotes to animals and plants. A given host probably has a range of different co-infectious viruses, resulting in enhanced gene exchanges between phages and hosts (Moineau et al., 1994). For example, it was suggested that bacteria and their co-infecting phages function as ‘phage factories’ and produce a variety of chimeric or mosaic phages that increase viral diversity (Ohnishi et al., 2001). Undoubtedly, environmental viruses are the largest reservoir of genetic diversity on the planet (Suttle, 2005, Suttle, 2007). For viruses there are no universal molecular markers that are shared among all viruses, similar to molecular RNA genes for cellular organisms, that could be used to assess their diversity (Hendrix et al., 1999). In aquatic environments, only genes belonging to specific groups or families can be targeted (Culley et al., 2003). This renders the study of global viral diversity difficult by PCR-based methods. In addition to the use of transmission electronic microscopy (TEM) for estimating viral diversity based on phenotypic traits (Auguet et al., 2006, Brum and Steward, 2010, Liu et al., 2006, Weinbauer, 2004, Wommack and Colwell, 2000), metagenomic and other molecular techniques have been developed and extensively used (Angly et al., 2006, Breitbart et al., 2002, Hambly and Suttle, 2005, Williamson et al., 2008, Winget and Wommack, 2008, Wommack and Colwell, 2000). Among these, pulsed field gel electrophoresis (PFGE) is based on size fractionation of intact DNA and has the advantage of allowing easy and rapid estimation of the whole-genome fingerprint of natural communities of viruses (Steward and Azam, 2000, Wommack et al., 1999a, Wommack et al., 1999b).

Results obtained with this method have showed that between 7 and 35 bands can be distinguished by applying PFGE to individual samples, as a minimum viral richness, with a dominance of < 100 kb genomes in both marine (Jamindar et al., 2012, Weinbauer, 2004) and freshwater plankton (Auguet et al., 2006, Filippini and Middelboe, 2007, Tijdens et al., 2008). Some studies have shown that viral genomic diversity, determined from PFGE and the relative intensity of individual bands, can vary seasonally (Castberg et al., 2001, Jamindar et al., 2012, Larsen et al., 2001, Larsen et al., 2004, Sandaa and Larsen, 2006; Wommack et al., 1999b, Zhong et al., 2014) and spatially (Auguet et al., 2006, Filippini and Middelboe, 2007, Jiang et al., 2004, Riemann and Middelboe, 2002, Steward et al., 2000, Tijdens et al., 2008; Wommack et al., 1999a). However, little is known about the factors controlling these variations, because only rare works have simultaneously studied the viral genomic composition (VGC) and the microbial community structure and environmental factors. A few authors have hypothesized that VGC changes with the structure of host communities (Auguet et al., 2006, Steward et al., 2000, Tijdens et al., 2008). Sandaa and Larsen (2006) have shown that seasonal changes in viral community composition can be related to changes in the abundances of cyanobacteria, autotrophic nano- and picoeukaryotes and heterotrophic bacteria, whereas Riemann and Middelboe (2002) and Tijdens et al. (2008) reported no clear link between temporal changes in VGC and bacterial community composition and/or phytoplankton abundance. With the exception of the work of Tijdens et al. (2008), the few data available were conducted in marine waters, but without taking into account phytoplanktonic species and protistan flagellates. Potential links between VGC and microbial community structure thus remain to be established, because there are many routes of interaction between viruses and microbial communities (Pradeep Ram and Sime-Ngando, 2008, Sime-Ngando and Pradeep-Ram, 2005). For example, in addition to representing a potential host reservoir for viruses (Massana et al., 2007), protistan flagellates could also graze bacteria and/or viruses (Bettarel et al., 2005), thereby influencing directly or indirectly the viral diversity (Weinbauer, 2004). Viral lytic or lysogenic activity could also affect VGC (Weinbauer, 2004). Parada et al. (2008) suggested that viral richness changes at time scales of hours to days linked with the lysing of specific bacterioplankton phylotypes. Since the genetic exchanges are probably higher among lysogenic than among lytic phages, the diversity of lysogenic phages is important (Chen and Lu, 2002), and higher viral diversity could be recorded during lysogen induction events. However, at the community level, no study to our knowledge has considered simultaneous changes in VGC together with lytic and lysogenic activities and with potential grazers as well. Finally, Jiang et al. (2004) have shown that VGC changed with oxygen conditions in the hypersaline Lake Mono (California, USA). Weinbauer (2004) suggested that certain physico-chemical parameters may define the niches for phages and thus influence viral diversity.

Many biological or physico-chemical factors could thus potentially affect VGC, but very few attempts have been made to study these effects, leaving open many questions of ecological interest concerning the dynamics of the biodiversity within virioplankton communities and the related environmental forcing factors. In this study, we have investigated the spatio-temporal dynamics of viral community diversity (based on genomic and morphological characteristics) and activity (lytic and lysogenic), concurrently with physico-chemical parameters (temperature, oxygen, chlorophyll a) and microbial community variables (bacteria, picocyanobacteria, autotrophic picoeukaryotes, autotrophic and heterotrophic nanoflagellates). We sought empirical evidence of the potential connections between the diversity of viruses and both abiotic and biotic environmental factors, at consistent seasonal and depth scales in aquatic systems. This study complements companion papers in which the standing stock of virus and microbial communities (Colombet et al., 2009) as well as grazing and viral activities (Colombet and Sime-Ngando, 2012) were examined.

Section snippets

Study site and sample collection

Samples were collected in Lake Pavin (altitude 1197 m), a meromictic and dimictic oligomesotrophic lake located in the French Massif Central, that experiences partial overturn. It is a typical crater mountain lake characterized by a maximum depth of 92 m and low surface (44 ha) and catchment (50 ha) areas. A characteristic feature of the physical structure of Lake Pavin is the existence of an oxic/anoxic interface (i.e., oxycline) between 50 and 60 m depth. Samples were collected monthly

Frequency of distribution of capsid size

The mean (± standard deviation) capsid size for all samples was 49 ± 4 nm, and fluctuated weakly from 47 ± 4 nm in the metalimnion to 50 ± 5 nm in the oxycline. No clear vertical trend was recorded (Table 1). Seasonal mean values were relatively lower in spring and summer than in autumn (Table 1). Particles with capsid size ranging from 30 to 60 nm largely dominated the viral communities, representing from 55% to 78% of the total viral abundance (Fig. 1). Viruses in the capsid size classes of < 30 nm and

Methodological considerations

Critical assumptions for the use of pulsed field gel electrophoresis (PFGE) to assess viral diversity have previously been made in various papers (Filippini and Middelboe, 2007, Riemann and Middelboe, 2002, Steward et al., 2000, Weinbauer, 2004, Wommack et al., 1999b). Among these, it is important to keep in mind that only quantitatively dominant viral genome sizes may be analyzed, because of the sensitivity of the method (≥ 106 viruses per band (Wommack et al., 1999b)), which means that some

Conclusions

In the present study, we have shown that the main viruses in Lake Pavin had a capsid size between 30 and 60 nm, and belonged to the family of Siphoviridae or were untailed, with a genome size in one of two major size classes: 25–40 and 55–65 kb. Although the composition of the viral community based on these criteria showed a tendency to be relatively stable, interesting spatio-temporal variations and empirical correlations were recorded. The composition of the viral community was more strongly

Acknowledgments

JC was supported by a PhD Fellowship from the Grand Duché du Luxembourg (BFR04/047, Ministry of Culture, High School, and Research). The study was partly supported by the French National Program ACI/FNS “ECCO” (VIRULAC research grant awarded to TSN, coordinator), and by the French ANR Program “Biodiversité” (AQUAPHAGE research grant to TSN, PI).

References (66)

  • F.E. Angly et al.

    The marine viromes of four oceanic regions

    PLoS Biol.

    (2006)
  • J.C. Auguet et al.

    Structure of virioplankton in the Charente Estuary (France): transmission electron microscopy versus pulsed field gel electrophoresis

    Microb. Ecol.

    (2006)
  • Y. Bettarel et al.

    Low consumption of virus-sized particles by heterotrophic nanoflagellates in two lakes of the French Massif Central

    Aquat. Microb. Ecol.

    (2005)
  • M. Breitbart et al.

    Genomic analysis of uncultured marine viral communities

    PNAS

    (2002)
  • J.R. Brum et al.

    Morphological characterization of viruses in the stratified water column of alkaline, hypersaline Mono Lake

    Microb. Ecol.

    (2010)
  • C.P.D. Brussaard

    Viral control of phytoplankton populations—a review

    J. Eukaryot. Microbiol.

    (2004)
  • T. Castberg et al.

    Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta)

    Mar. Ecol. Prog. Ser.

    (2001)
  • F. Chen et al.

    Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phage

    Appl. Environ. Microbiol.

    (2002)
  • J. Colombet et al.

    Seasonal depth-related gradients in virioplankton: lytic activity and comparison with protistan grazing potential in Lake Pavin (France)

    Microb. Ecol.

    (2012)
  • J. Colombet et al.

    Depth-related gradients of viral activity in lake Pavin

    Appl. Environ. Microbiol.

    (2006)
  • J. Colombet et al.

    Seasonal depth-related gradients in virioplankton: standing stocks and relationships with microbial communities Lake Pavin (France)

    Environ. Microbiol.

    (2009)
  • A.I. Culley et al.

    High diversity of unknown picorna-like viruses in the sea

    Nature

    (2003)
  • R. Danovaro et al.

    Determination of virus abundance in marine sediments

    Appl. Environ. Microbiol.

    (2001)
  • J. Demuth et al.

    Direct electron evidence study on the morphological diversity of bacteriophage populations in Lake Plußsee

    Appl. Environ. Microbiol.

    (1993)
  • M. Filippini et al.

    Viral abundance and genome size distribution in the sediment and water column of marine and freshwater ecosystems

    FEMS Microbiol. Ecol.

    (2007)
  • R. Hendrix et al.

    Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage

    PNAS

    (1999)
  • K.P. Hennes et al.

    Significance of bacteriophages for controlling bacterioplankton growth in a mesotrophic lake

    Appl. Environ. Microbiol.

    (1995)
  • K.P. Hennes et al.

    Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy

    Limnol. Oceanogr.

    (1995)
  • S. Jamindar et al.

    Evaluation of two approaches for assessing the genetic similarity of virioplankton populations as defined by genome size

    Appl. Environ. Microbiol.

    (2012)
  • S. Jiang et al.

    Abundance, distribution, and diversity of viruses in alkaline, hypersaline Mono Lake

    Microb. Ecol.

    (2004)
  • A. Larsen et al.

    Population dynamics and diversity of phytoplankton, bacteria and viruses in a seawater enclosure

    Mar. Ecol. Prog. Ser.

    (2001)
  • A. Larsen et al.

    Spring phytoplankton bloom dynamics in Norwegian coastal waters: microbial community succession and diversity

    Limnol. Oceanogr.

    (2004)
  • Y.M. Liu et al.

    Spatial distribution and morphologic diversity of virioplankton in Lake Donghu, China

    Acta Oecol.

    (2006)
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