Abstract
Genetic diversity is a prerequisite for evolution. The genes of the Major Histocompatibility
Complex (MHC) show genetic variation. They are polygenic and contain highly polymorphic
loci. MHC molecules are an important part of the adaptive immune system due to their ability
to bind and present different antigens to the T-lymphocytes. But this high specificity also
implies a risk: the higher the number of recognized antigens, the more likely the similarity of
foreign and auto antigens. This can cause a loss of self tolerance mechanisms due to cross
reactions during molecular mimicry. The MHC might be a key molecule for selection,
adaptive processes and speciation.
Gasterosteus aculeatus shows an intermediate number of MHC IIB alleles. On the one hand,
too few different MHC IIB alleles demonstrate an insufficient diversity to detect a large
number of parasite antigens. But on the other hand, there must be a limiting selection
pressure, which causes this intermediate optimum of MHC IIB alleles. A possible
disadvantage assumes that too many alleles might lead to self-immunoreactivity. Therefore,
this thesis focuses on a possible correlation between certain immunological changes, which
might be associated with autoimmune reactions, and the individual MHC IIB allele diversity of
the three-spined stickleback. It is a pilot study about auto aggressive reactions as a possible
limiting selection pressure on the evolution of the MHC IIB allele diversity. Thus, I used
different methods to examine different aspects of this topic.
First, the MHC class IIB allele configuration of parental fish of 14 different families was
determined. The results indicate five families, whose offspring showed a MHC class IIB allele
diversity of either two or four different alleles. The MHC genotyping of parents and offspring
was done via the Reference Strand-mediated Conformation Analysis (RSCA). The next step
was the isolation of several inflammatory genes. Different primer combinations were tested
via standard PCR and Agarose gelelectrophoresis. After that, primer specificity was checked
via direct sequencing and quantitative Real Time PCR.
I was able to isolate IL 1 beta, MIF, IgM and MHC II in the genome of Gasterosteus
aculeatus. After that, head kidney, spleen, liver, kidney, gut, gonads, heart and brain were
dissected and blood was taken from the caudal vein. The total immunoglobulin level of the
plasma was measured with the help of an Enzyme-linked immunosorbent assay (ELISA).
RNA of head kidney, gut, kidney and spleen was isolated and reverse transcribed into cDNA.
The expression levels of the established inflammatory genes MIF, IL 1 beta, IgM and MHC
class II were measured, relative to those of the housekeeping genes L13 and Ubiquitin. This
was done via the quantitative Real Time PCR, based on the non-specific reporter molecule
SYBR Green I. The raw gene expression data was normalized with the software qBase Plus
and a permutational multivariate analysis of variance was performed with the help of the
software R 2.11.1.
The results indicate the family of origin and sex as influencing factors. The influence of the
dissection day is due to an artefact. Maybe different families contain different repertoires of
immunological factors. This might be due to differences in their genetic background. In
humans, it has been assumed that autoimmunity is correlated with female sex hormones.
Therefore, it might be possible that female sex hormones of teleost fish are correlated with
the occurrence of autoimmune diseases. It would be interesting to examine, whether and
how female sex hormones can influence the adaptive immune system.
Due to a low p-value (p = 0.074), it might be possible that the number of MHC IIB alleles also
affects certain immunological parameters. Therefore, it might be interesting to breed fish with
very high and low numbers of MHC IIB alleles and to expose them to many parasites and
many different types of parasites. Maybe the costs, namely the occurrence of auto
aggressive reactions, are lower than the benefits, namely a high resistance towards
parasites. It might be possible that there is a trade-off between the resistance towards
parasites and auto aggressive reactions
The results of the expression assay indicate that IL 1 beta seems to be an important
component of the adaptive immune response in Gasterosteus aculeatus. It shows a higher
expression in the head kidney of individuals with four MHC IIB alleles. Apart from that, a
positive correlation of IL 1 beta- and MHC II expression is observed in head kidney, spleen
and kidney. It might also be helpful to detect more possible inflammatory genes for a better
understanding of the complex network of associations between several cytokines of the
adaptive immune system. Some studies about cytokines in teleost fish include stimulations of
the adaptive immune system with LPS, FCA and ATH. They resulted in an increase of
expression levels in several organs. Therefore, another interesting project might deal with the
artificial induction of autoimmune reactions due to stimulation of the immune system of
Gasterosteus aculeatus.
