235mended as a routine procedure (Bettarel et al. 2000 ). Besides,
the fl uorochrome - based methods described previously used
low pressure membrane-concentration. They are well known
to provide accurate counts of microbes and viruses, but
resulted in a highly poor description of diversity.
An accurate description of the diversity of pelagic viruses
usually needs highly concentrated aliquots. The current
method available to date is ultrafi ltration that allows concen-
trating viruses contained in several tens of liters of water into
small volumes of a few hundreds of milliliters (Suttle et al.
1991 ; Wommack et al. 1995 ). However, the application of
this procedure requires further concentration by ultracentri-
fugation to obtain viral pellets necessary for TEM observa-
tions and for estimating viral diversity. To avoid
ultracentrifugation and the underlined disadvantages, we
conducted, for the fi rst time, a methodological comparative
study with samples collected in Lake Pavin and other fresh-
water sites located in the Massif Central region of France,
and have proposed a simple, cheaper and effi cient alternative
protocol using polyethylene glycol (PEG) to obtain virio-
plankton precipitates that can be used for different purposes,
primarily for TEM observations, electrophoretic plugs, and
the downstream molecular genetic analyses (Colombet et al.
2007 ; Colombet and Sime-Ngando 2012b ). The process,
named “pegylation”, yields the greatest recovery effi ciently
of free-occurring viruses, allowing the recovery of > two-fold
more viruses compared to ultracentrifugation and ultrafi l-
tration. In addition, the diversity of virioplankton, based on
genomic size profi ling using pulsed fi eld gel electrophore-
sis , was higher and better discriminated when we used the
PEG method which both concentrates and purifi es viral
material in water samples. The effects of potential virucidal
compounds (e.g. enzymes, antibodies, and other inhibitory
substances) are probably minimized or avoided during
pegylation. This property of PEG molecules is currently
used in medicine to protect drug-carrier viruses, interferons
of immunitary system, or antibodies (Harris and Chess
2003 ). PEG can thus also be used as a conservative for plank-
tonic viruses, thereby avoiding the use of toxic fi xatives and
the related disadvantages such as losses during storage of
samples.
14.4 Diversity and Endemicity of Viruses
14.4.1 Phenotypic Diversity
The fi rst descriptions of the global diversity of viruses were
based on their morphological discrimination with transmis-
sion electron microscopy (Børsheim et al. 1990 ; Proctor and
Fuhrman 1990 ; Weinbauer and Höfl e 1998 ). To our knowl-
edge, the phenotypic diversity of viruses in Lake Pavin is
apparently highly endemic, with viral morphotypes that typi-
cally are different and highly constraint by depth (Fig. 14.2 ).
In the surface mixed waters (i.e. mixolimnion ) , viral pheno-
types are limited, mainly including tailed or untailed parti-
cles with capsid heads, characteristics of bacteriophages.
This is typical in the world aquatic ecosystems : tailed phages
belong to the order Caudovirales , all of which are double-
stranded DNA viruses that generally represent 10 to 40 % of
the total abundance of viruses (Sime-Ngando and Colombet
2009 ). Within Caudovirales , three families emerge as quan-
titatively dominant: Siphoviridae with long non-contractile
tails (e.g. Phage lambda), Podoviridae with a short non-
contractile tail (e.g. Phage T7), and Myoviridae with con-
tractile tails of variable length (e.g. Phage T4). In Lake
Pavin, non-tailed capsids dominate viral abundance in the
mixolimnion (Colombet and Sime-Ngando 2012a ) in accor-
dance with results obtained from metagenomic analysis
(Roux et al. 2012 ). A recent global morphological analysis
of marine viruses also suggested that non-tailed viruses,
which comprised 50–90 % of the viral particles observed,
might represent the most ecologically important component
in natural viral communities (Brum et al. 2013 ). However,
we cannot completely exclude a signifi cant effect of
mechanic shocks from handling resulting in losses of tails,
because 96 % of the 5500 specimens of described bacterio-
phages are tailed particles (Ackermann 2007 ).
Our seasonal depth-related studies in Lake Pavin
(Colombet et al. 2006 , 2009 ; Colombet and Sime-Ngando
2012a ) clearly demonstrated substantial changes in the mor-
phological diversity of pelagic viruses in relation to the
depth-related gradients. Indeed, viral communities in the
permanently anoxic monimolimnion of Lake Pavin com-
prised a substantial number of atypical bacteriophage mor-
photypes, larger in capsid size (>60 nm), and with more
complex spatial conformation (Fig. 14.2 ), compared to the
mixolimnic viruses, which were more typical of the world
aquatic free-occurring bacteriophages dominated by small
(capsid size < 60 nm) Caudovirales (see above). In addition,
an analysis of viral diversity based on the distribution fre-
quency of capsid sizes through the whole water column of
Lake Pavin has indicated that viruses were apparently typical
and more diversifi ed in the monimolimnion than in the sur-
face waters.
This spatial pattern was further enhanced by the recent
discovery of the highest diversity and complexity of viral
communities in the deep-dark permanently anoxic sediments
of Lake Pavin, with the occurrence of unexpected and novel
viruses (Borrel et al. 2012 ). Indeed, the examination of sedi-
ment cores encompassing 130 years of sedimentation of the
lake has unveiled exceptional morphotypes of viruses, previ-
ously never reported in freshwater systems (Fig. 14.2 ). Some
of these resembled dsDNA viruses of hyperthermophilic
and hyperhalophilic archaea. Moreover, unusual types of
spherical and cubic virus-like particles (VLPs) were14 Ecology of Viruses in Lake Pavin