335tion conditions of our designed probes before application to
natural samples using the CARD-FISH approach (Jobard et al.
2010b ). When applied to natural samples, our CARD-FISH
assay demonstrated high specifi city and sensitivity, and we
showed that 60 % of the total abundance of heterotrophic fl ag-
ellates were actually fungal zoospores. Although the results
from the CARD-FISH approach were preliminary, our fi ndings
were consistent with ecological considerations known from
pelagic habitats, and recurrent ecological patterns in two con-
trasting lake ecosystems were observed.
We further developed a quantitative real-time PCR
(qPCR) assay for the quantifi cation of fungal zoopsores
(Lefèvre et al. 2010 ; also provided in Sime-Ngando and Jobard
2013 ). QPCR is a very sensitive tool for the detection of less
abundant or rare species. When optimal DNA extraction proto-
col and qPCR conditions were applied, our qPCR assay dem-
onstrated high specifi city and sensitivity, with a detection limit
as low as 100 18S rDNA copies per reaction (Lefèvre et al.
2010 ). Application of the method to samples collected in Lake
Pavin and other natural samples gave us important clues as to
what roles these organisms could have in the pelagic system.
More recently, the application of next generation sequenc-
ing technologies has not only advanced our understanding of
zoosporic fungal diversity, but also provided insights into
their ecological functions through the analysis of their
genomes and gene expression (Monchy et al. 2011 ).
Nevertheless, it is still necessary to complement these
approaches with culturing methods in order to gain a deeper
understanding of the ecological and physiological roles of
zoosporic fungi (Marano et al. 2012 ).
Overall, the methodological diffi culties are increasingly
being overcome, and it is becoming evident that molecular
biology approaches are crucial for a better understanding of
zoosporic fungi ecology. The challenge of matching molecu-
lar sequence to microscopic phenotypes is increasigly being
met (Jobard et al. 2012 ; Monchy et al. 2012 ). Furthermore,
to assess the functional impact of chytrid parasites on host
populations, we applied population dynamic models com-
monly used by parasitologists such as the ones predicting
prevalence and the intensity of infection for the ecological
studies of chytridimycosis (Rasconi et al. 2012 ). Input
parameters for these models are derived from direct micros-
copy and are essential for assessing life cycle, community
structure of parasites, their interactions with hosts, as well as
the potential impact of fungal parasites on the food web
dynamics. Understanding the environmental factors includ-
ing epidemics can also be inferred through empirical correla-
tions or by using epidemiological approaches such as the
changes in incidence rates (i.e. the number of new cases of
infections occurring during a given time) or the occurrence
of epidemics (i.e. a widespread outbreak of an infection)
within host populations (Fox 2003 ).
20.4 Chytrid Life Cycle and Adaptation
to Planktonic LifestyleChytrid species have an atypical life cycle in the context of
the pelagic realm where the two main stages (i.e. sporangium
and zoospore ) have different effects on the food web dynam-
ics. Most members reproduce asexually by releasing swim-
ming zoospores with a single posteriorly-directed whiplash
fl agellum (Sparrow 1960 ; Barr 2001 ). A typical chytrid para-
site life cycle begins with the encystment of a free-swimming
zoospore on a host followed by the development of a rhizoi-
dal system inside the host cell. The encysted zoospore devel-
ops into a mature sporangium drawing its energy from the
host, and new zoospores are eventually released into the
environment. Because water is necessary for chytrids to
complete their life cycle, these water-borne fungi are ubiqui-
tous in bodies of waters, primarily in freshwater environ-
ments, and in wet soils.
In a recent study conducted in the eutrophic Lake Aydat
located in the vicinity of Lake Pavin (Gerphagnon et al.
2013b ), we were able to reconstruct phenotypic stages of
host-chytrid systems using the double staining method men-
tioned above. We sampled at a relatively high frequency
(every 3 days) the fi lamentous cyanobacterium Anabaena
macrospora during a bloom. Based on the morphology of the
sporangium and on the type of host cells, we were able to
identify two species of chytrids parasites of this cyanobacte-
rium, both belonging to the same genus, Rhizosiphon. While
R. crassum infected both vegetative cells and resistant forms
(i.e. akinetes), and was responsible for the death of cells
within host fi laments, R. akinetum , which only infected aki-
netes, may have affected the survival of cyanobacterial hosts
and their proliferations from year to year. We divided the
asexual life cycles of the two parasite species into six stages
grouped in 3 phases: young, maturation, and empty phases
(Fig. 20.4 ). These phases correspond to the growth phases
(i.e. encystement, germination, growth and maturation)
known from the general life cycle of the Chytridiomycota.
Chytridiomycosis epidemics are known to produce mas-
sive numbers of zoospores , now known as valuable food
source for zooplankton (Kagami et al. 2007a , b ). The consid-
eration of the two main development stages (i.e., attached
sporangia and free-swimming zoospores) of chytrids in the
context of the pelagic system thus highlights two overlooked
potential trophic paths in food web dynamics: (i) parasitic
predation of host populations, most of which are inedible
(i.e. unexploited by grazers), and (ii) the subsequent trophic
link via the release of energy-rich zoospores that can serve as
food for zooplankton. In addition, we recently shown that
chytrid parasitism within the fi laments of cyanobacteria dur-
ing bloom events can result in a mechanical fragmentation of
the inedible fi laments into shorter-size edible fi laments20 Chytrid Parasites of Phytoplankton in Lake Pavin