333fungus rRNA genes needed to be addressed before future
studies could be conducted (Monchy et al. 2011 ). Therefore,
we used longer and more phylogenetically informative
sequences, such as ITS1, and ITS2 regions, generated using
our cloning-sequencing approach, to design more specifi c
primer sets that will be used to follow the dynamics of par-
ticular fungal species in natural waters.
In a follow-up study we conducted in 2007 in Lake Pavin
and other lakes in the vicinity (Jobard et al. 2012 ), we used
the fungal-specifi c NS1 and ITS4 PCR primers for the ampli-
fi cation of the ribosomal ITS 1 and 2 regions (White et al.
1990 ). Our study yielded 17 OTUs that belonged to three
fungal phyla: Chytridiomycota (50 %), Ascomycota (40 %),
and Basidiomycota. Similar to our previous pyrosequencing
study (Monchy et al. 2011 ), no sequence belonging to the
division Zygomycota was retrieved. This observation is con-
sistent with the current knowledge that in aquatic ecosys-
tems, these fungi are restricted to the group of Trichomycetes
known as obligate symbionts in the gut of arthropods
(Lichtwardt et al. 2003 ). In Lake Pavin, 60 % of the fungal
sequences retrieved were absent from the DNA sequence
databases, and mainly affi liated to zoosporic true fungi (i.e.
Chytridiomycota ), primarily to the genera Rhizophydium and
Zygorhizidium known to contain host-specifi c parasites of
phytoplankton in freshwater lakes (Jobard et al. 2010a ;
Rasconi et al. 2011 ). This clearly confi rms the omnipresence
of these parasites in aquatic systems, and the need to develop
analytical tools for their quantitative and ecological study.
20.3 Methodological Challenges
in the Quantitative Assessment
of the Ecology of Parasitic Chytrids
on Phytoplankton
Until recently, the identifi cation of zoosporic fungi was
mainly based on direct observations and culturing techniques
(i.e., baiting techniques; recently reviewed in Marano et al.
2012 ). The main characteristics used for the identifi cation of
parasitic chytrids were based on the observation of their spo-
rangia using light or phase-contrast microscopy (Ingold
1940 ; Canter 1949 , 1950 , 1951 ) of living samples or samples
preserved with Lugol’s iodine (Rasconi et al. 2011 ; Sen
1988a , b ). Subsequently, transmission electron microscopy
was used to describe zoospore ultrastructure and spore dif-
ferentiation (Rasconi et al. 2011 ; Beakes et al. 1992a , b ). The
chytrid Blastocladiella sp. was the fi rst fungal model for
detailed structural studies on sporogenesis (Lovett 1963 ).
The conformation of the fl agellar rootlets and the spatial dis-
tribution of organelles in zoospores provided the basis for
chytrid taxonomy (review in Gleason and Lilje 2009 ).
Ecological investigations of chytrid population dynamics
in natural environments greatly improved with the use of epi-
fl uorescence microscopy. Based on samples regularly col-
lected in Lake Pavin in 2006 and 2007, we developed a
routine protocol based on size fractionation of pelagic sam-
ples and the use of the fl uorochrome calcofl uor white (CFW;
C 40 H 44 N 12 O 10 S 2 ), a chemofl uorescent agent which binds to
β-1,3 and β-1,4 polysaccharides (Rasconi et al. 2009 ). CFW
is commonly used as a whitening agent in the paper industry
and selectively binds to cellulose and chitin. The dye fl uo-
resces when exposed to UV light and offers a very sensitive
method for direct microscopic examination of fungal ele-
ments on skin scrapings, hairs, nails, and other clinical speci-
men for fungal elements. Our CFW-based protocol for
diagnosing, identifying, and counting chitinaceous fungal
parasites (i.e., the sporangia of chytrids) of phytoplankton is
provided in Sime-Ngando et al. ( 2013b ). Results from the
proposed protocol indicated that CFW penetrates infected
host cells remarkably well and is more effi cient than other
stains such as the protein stain fl uorescein isothiocyanate
(FITC) for the observation and photomicrography of the
complete rhizoidal system of chytrid parasites (Rasconi
et al. 2009 ), which is another important criterion for their
identifi cation. These preliminary results also highlighted a
higher diversity of infected host and parasite communities in
Lake Pavin compared to that described in previous studies.
Moreover, we showed that chytrid parasites were omnipres-
ent, infecting diverse phytoplankton host communities, pri-
marily diatoms, chlorophytes, and colonial and fi lamentous
cyanobacteria. The diversity and numerical abundance of
sporangia and infected hosts, as well as the prevalence of
infection (range, <1–24 % of total host cells), increased from
the oligotrophic Lake Pavin to the eutrophic Lake Aydat. In
addition, we showed that the temporal changes in the abun-
dance and activity of the parasites were mainly infl uenced by
the host community composition (Rasconi et al. 2009 ).
More recently, we developed an improved CFW protocol
using the combination of two fl uorochromes, CFW (chitin
stain) and SYTOX green (nucleic acid stain) coupled with
epifl uorescence microscopy for counting, identifying, and
investigating the fecundity (i.e. the number of zoopores per
sporangium) of parasitic fungi of phytoplankton (Gerphagnon
et al. 2013a ). This double-staining protocol targeting chitin
and nucleic acids allows the visualization of both sporan-
gium and zoospore content and enables the determination of
fungal fecundity (Fig. 20.3 ). The method was applied to
freshwater samples collected over two successive years dur-
ing the end of autumnal cyanobacterial blooms in a eutrophic
lake. The study focused on the uncultured host-parasite pair
Anabaena macrospora (cyanobacterium) and Rhizosiphon
akinetum (Chytridiomycota). Our results showed that up to
37 % of cyanobacterial akinetes were infected by fungi.
Simultaneously, we determined the number of zoospores per
sporangium and found out that both host size and intensity of
infection conditioned the fi nal size and hence the fecundity20 Chytrid Parasites of Phytoplankton in Lake Pavin