Proteomic analysis of stress responses in Daphnia

Organisms respond to changes in their environment affecting their
physiological or ecological optimum by reactions called stress
responses. These stress responses may enable the organism to
survive by counteracting the consequences of the environ- mental
change, the stressor, and usually consist of plastic alterations of
traits related to physiology, behaviour, or morphology. In the
ecological model species Daphnia, the waterflea, stressors like
predators or parasites are known to have an important role in
adaptive evolution and have been therefore studied in great detail.
However, although various aspects of stress responses in Daphnia
have been analysed, molecu- lar mechanisms underlying these traits
are not well understood so far. For studying unknown molecular
mechanisms, untargeted ‘omics’ approaches are especially suit-
able, as they may identify undescribed key players and processes.
Recently, ‘omics’ approaches became available for Daphnia. Daphnia
is a cosmo- politan distributed fresh water crustacean and has been
in research focus for a long time because of its central role in
the limnic food web. Furthermore, the responses of this organism to
a variety of stressors have been intensively studied e.g. to
hypoxic conditions, temperature changes, ecotoxicological relevant
substances, parasites or predation. Of these environmental factors,
especially predation and interactions with parasites have gained
much attention, as both are known to have great influence on the
structure of Daphnia populations. In the work presented in this
thesis, I characterised the stress responses of Daphnia using
proteomic approaches. Proteomics is particularly well suited to
analyse bio- logical systems, as proteins are the main effector of
nearly all biological processes. However, performing Daphnia
proteomics is a challenging task due to high proteolytic activity
in the samples, which most probably originate from proteases
located in the gut of Daphnia, and are not inhibited by proteomics
standard sample pre- paration protocols. Therefore, before
performing successful proteomic approaches, I had to optimise the
sample preparation step to inhibit proteolytic activity in Daph-
nia samples. After succeeding with this task, I was able to analyse
stress responses of Daphnia to well-studied stressors like
predation and parasites. Furthermore, I stud- ied their response to
microgravity exposure, a stressor not well analysed in Daphnia so
far. My work on proteins involved in predator-induced phenotypic
plasticity is de- scribed in chapter 2 and 3. Daphnia is a textbook
example for this phenomenon and is known to show a multitude of
inducible defences. For my analysis, I used the system of Daphnia
magna and its predator Triops cancriformis. D. magna is known to
change its morphology and to increase the stability of its carapace
when exposed to the pred- ator, which has been shown to serve as an
efficient protection against T. cancriformis predation. In chapter
2, I used a proteomic approach to study predator-induced traits in
late-stage D. magna embryos. D. magna neonates are known to be
defended against Triops immediately after the release from the
brood pouch, if mothers were exposed to the predator. Therefore,
the formation of the defensive traits most probably oc- curs during
embryonic development. Furthermore, embryos should have reduced
protease abundances, as they do not feed inside the brood pouch
until release. To study proteins differing in abundance between D.
magna exposed to the predator and a control group, I applied a
proteomic 2D-DIGE approach, which is a gel based method and
therefore enables visual monitoring of protein sample quality. I
found differences in traits directly associated with known defences
like cuticle proteins and chitin-modifying enzymes most probably
involved in carapace stability. In addition, enzymes of the energy
metabolism and the yolk protein vitellogenin indicated alterations
in energy demand. In chapter 3, I present a subsequent study
supporting these results. Here, I analysed responses of adult D.
magna to Triops predation at the proteome level using an optimised
sample preparation procedure, which was able to generate adult
protein samples thereby inhibiting proteolysis. Furthermore, I
established a different proteomic approach using a
mass-spectrometry based label- free quantification, in which I
integrated additional genotypes of D. magna to create a more
comprehensive analysis. With this approach, I was able to confirm
the results of the embryo study, as similar biological processes
indicated by cuticle proteins and vi- tellogenins were involved.
Furthermore, additional calcium-binding cuticle proteins and
chitin-modifying enzymes and proteins involved in other processes,
e.g. protein biosynthesis, could be assigned. Interestingly, I also
found evidence for proteins in- volved in a general or a genotype
dependent response, with one genotype, which is known to share its
habitat with Triops, showing the most distinct responses. Genotype
dependent changes in the proteome were also detectable in the study
which I present in chapter 4. Here, I analysed molecular mechanisms
underlying host-parasite interactions using the well characterised
system of D. magna and the bacterial endoparasite Pasteuria ramosa.
P. ramosa is known to castrate and kill their host and the
infection success is known to depend strongly on the host’s and the
para- site’s genotype. I applied a similar proteomic approach as in
chapter 3 using label- free quantification, but contrastingly, I
did not use whole animal samples but only the freshly shed cuticle.
It has been shown, that the genotypic specificity of P. ramosa
infection is related to the parasite’s successful attachment to the
cuticle of the host and is therefore most probably caused by
differences in cuticle composition. Hence, I analysed exuvia
proteomes of two different genotypes known to be either suscept-
ible to P. ramosa or not. Furthermore, I compared exuvia proteomes
of susceptible Daphnia exposed to P. ramosa to a control group for
finding proteins involved in the infection process and in the
stress response of the host. The proteomes of the different
genotypes showed indeed very interesting abundance alterations,
connected either to cuticle proteins or matrix metalloproteinases
(MMPs). Additionally, the cuticle pro- teins more abundant in the
susceptible genotype showed a remarkable increase in predicted
glycosylation sites, supporting the hypothesis that P. ramosa
attaches to the host’s cuticle by using surface collagen-like
proteins to bind to glycosylated cuticle proteins. Most
interestingly, in all replicates of the susceptible genotype
exposed to P. ramosa, such a collagen-like protein was found in
high abundances. Another group of proteins found in higher
abundance in the non-susceptible genotype, the MMPs, are also
connected to this topic, as they may have collagenolytic
characteristics and therefore could interfere with parasite
infection. Furthermore, the data indicate that parasite infection
may lead to retarded moulting in Daphnia, as moulting is known to
reduce the infection success. Contrastingly to the work presented
so far, the study described in chapter 5 invest- igated the protein
response of Daphnia to a stressor not well studied on other levels,
namely microgravity. As gravity is the only environmental parameter
which has not changed since life on earth began, organisms usually
do not encounter alterations of gravity on earth and cannot adapt
to this kind of change. Daphnia has been part of one mission to
space, however, responses of the animals to microgravity are not
well described so far. In addition, as Daphnia are an interesting
candidate organisms for aquatic modules of biological life support
systems (BLSS), more information on their response to microgravity
is necessary. For this reason, proteomics is an interesting ap-
proach, as biological processes not detectable at the morphological
or physiological level may become apparent. Therefore, a
ground-based method, a 2D-clinostat, was used to simulate
microgravity, as studies under real microgravity conditions in
space need high technical complexity and financial investment.
Subsequently, a proteomic 2D-DIGE approach was applied to compare
adult Daphnia exposed to microgravity to a control group. Daphnia
showed a strong response to microgravity with abundance alterations
in proteins related to the cytoskeleton, protein folding and energy
meta- bolism. Most interestingly, this response is very similar to
the reactions of a broad range of other organisms to microgravity
exposure, indicating that the response to altered gravity
conditions in Daphnia follows a general concept. Altogether, the
work of my thesis showed a variety of examples of how a proteomic
approach may increase the knowledge on stress responses in an
organisms not well- established in proteomics. I described both,
the analysis of molecular mechanisms underlying well-known traits
and the detection of proteins involved in a response not well
characterised. Furthermore, I gave examples for highly genotype
dependent and also more general stress responses. Therefore, this
thesis improves our understanding of the interactions between
genotype, phenotype and environment and, moreover, offers
interesting starting points for studying the molecular mechanisms
underlying stress responses of Daphnia in more detail.