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.