Proteomic analysis of stress responses in Daphnia Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06

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 indicat