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of chytrids. We also established that the relationships linking
host size, fi nal parasite size, and chytrid fecundity were con-
served from year to year and seemed to be host-chytrid pair-
ing specifi c (Gerphagnon et al. 2013a ).
In contrast to sporangia, zoospores lack a chitinaceous cell
wall, are small, and possess very few phenotypic characteris-
tics that can be used for their identifi cation. Therefore, molecu-
lar biology approaches offer an alternative for the quantitative
assessment of both chytrid sporangia and zoospores in nature.
Molecular approaches are commonly used for the reconstruc-
tion of chytrid phylogenies (James et al. 2006 ), but can also be
applied (i.e., oligonucleotide probes and primers ) to the quan-
titative ecological study of aquatic parasites. Fluorescent in
situ hybridization (FISH) with horseradish peroxidase activa-
tion by fl uorescent tyramide (also known as catalyzed reporter
deposition, CARD FISH or TSA FISH) is a reliable approach
to describe the dynamics of chytrid parasites, especially the
zoosporic stage. The optimization of the FISH method requires
the use of positive controls, ideally pure cultures that are chal-
lenging to obtain especially for chytrid parasites. To circum-
vent this issue, we used the clone-FISH approach, which uses
transformed clones of Escherichia coli containing a gene or
DNA region of interest (e.g., 18S rDNA sequence of a parasitic
chytrid obtained from some of our previous environmental
cloning surveys; Jobard et al. 2010b ). Using molecular
sequences generated from our previous 18 rDNA environmen-
tal surveys in Lake Pavin and other lakes in the vicinity, we
designed oligonucleotidic probes targeting members of the
Chytridiales (i.e., of the groups of Chytridiomycota containing
the highest number of described species). We then used the
clone-FISH approach to optimize the hybridization conditions
of our designed probes before application to natural samples
using the CARD-FISH approach (Jobard et al. 2010b ). Our
protocols are presented in a methodological paper by Sime-
Ngando et al. ( 2013a ). Using molecular sequences derived
from 18S rDNA cloning/sequencing surveys in Lake Pavin and
other lakes in the vicinity, we designed oligonucleotidic probes
specifi c for Chytridiales (i.e. the largest group of the true-fun-
gal division of Chytridiomycota ). We adapted a clone-FISH
approach known from prokaryotes to optimize the hybridiza-Fig. 20.3 Mature sporangium of Rhizosiphon akinetum containing zoospores ( A , B , C , D ), stained by the double staining method (CFW and
SYTOX green ) and excited by UV light ( A ), blue light ( B , C ), or both UV and blue light ( D ) as observed by optical microscopy. Z, zoospores
T. Sime-Ngando et al.