Major histocompatibility (MH) polymorphism of common carp : link with disease resistance

The impact of diseases caused by a wide range of pathogens (viruses, bacteria and parasites) is the most important problem in aquaculture of common carp (Cyprinus carpio L.). Genetic selection aimed at obtaining population of more resistant common carp is potential and sustainable approach to disease control in semi-intensive carp pond farming. Genes of the major histocompatibility complex (MHC) are candidate marker genes for studies on association with disease resistance. The MHC contains some of the most polymorphic genes known to date and are considered crucial to adaptive immunity. MHC molecules bind both self and foreign peptides and present them to T lymphocytes (T cells). MHC class I molecules present endogenously derived peptides to CD8+ T cells, while MHC class II molecules present exogenously derived peptides to CD4+ T cells. Each MHC molecule has the ability to bind and present different groups of peptides in more or less successful ways. This can influence the immune response of an organism since the peptides derived from a certain pathogen may either not be presented by specific MHC molecules, which can result in higher susceptibility or, may be bound with a high affinity by specific MHC molecules which could lead to increased resistance to the pathogenic organism. In teleosts, unlike to humans, tetrapods and cartilaginous fish, class I and class II genes are not linked and segregate independently, which allows for association studies of only class I or only class II MH genes with disease resistance. MHC class II molecules generally have a broader spectrum of action in the immune system than the MHC class I genes. There are also observations that suggest a more intense selection pressure and a more rapid evolution of MH class II than class I alleles in fish. Although the expression of both MH class II chains is equivalent, the beta chain generally has a higher degree of polymorphism than the alpha chain. This thesis addressed possible implementations of MH class II B genes for selection aimed at improving of a common carp resistance in semi-intensive pond farming. In common carp there are two paralogous groups of MH class II B genes, Cyca- DAB1-like and Cyca-DAB3-like genes. In a preliminary study, we examined the polymorphism for the Cyca-DAB1-like and Cyca-DAB3-like genes in different European common carp lines (chapter 2). These carp lines were of various geographical origins and part of carp live gene bank, which is maintained at the Institute of Ichthyobiology and Aquaculture in Golysz. Previous observations over a period of at least 15 years revealed significant differences between lines in survival rate and parasite load under natural conditions. Also, differences in resistance to atypical Aeromonas salmonicida in laboratory based challenge tests was observed, suggesting genetic differences in resistance between the carp lines. Analysis of polymorphism of MH class II B genes in selected carp lines revealed a ubiquitous presence and high polymorphism of Cyca-DAB1-like but not Cyca-DAB3-like genes. The observed allelic polymorphism for Cyca-DAB1-like genes rather than Cyca-DAB3-like genes stimulated further studies into the association of Cyca-DAB1-like allelic polymorphism and disease resistance of common carp. In order to study association between Cyca-DAB1-like gene polymorphism and resistance of common carp we optimized a technique designated polymerase chain reaction -restriction fragments- single strand conformation polymorphism (PCR-RFSSCP) to be able to screen and type large numbers of individual carp (chapter 3). The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RFSSCP analysis of n = 79 carp individuals from 8 lines challenged with Aeromonas hydrophila revealed the presence of different genotypes consisting of unique combinations of Cyca-DAB1 and Cyca-DAB2 sequences. We found four alleles for the Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02). We noted that the Cyca-DAB2 gene was either homozygous or absent. The degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes clearly correlated with the number of SSCP bands. Thus, we proved that PCR-RF-SSCP is a reliable technique that can be used for screening large number of individuals for investigating the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. Previously, we performed a long-term divergent selection of common carp for antibody production, which successfully resulted in carp lines with a different immune response. We studied the segregation of Cyca-DAB genes with the DNP-specific antibody response and we showed that the presence of Cyca-DAB1-like, but not Cyca- DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp (chapter 4). Background genes other than Cyca-DAB genes also influenced the level of antibody response. We also studied the transcription of both Cyca-DAB1-like and Cyca- DAB3-like genes in different organs of carp. The constitutive transcription for both Cyca-DAB1-like and Cyca-DAB3-like genes was high, although Cyca-DAB1-like genes consistently showed slightly higher mRNA transcription than Cyca-DAB3-like genes in some immunological relevant organs. Sequence information, constitutive transcription levels and co-segregation data indicated that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. We then proceeded to study association of Cyca-DAB1-like genotypes with resistance to four different pathogens; the bacterium Aeromonas hydrophila, the ectoparasite Argulus japonicus, and the blood parasite Trypanoplasma borreli (chapter 5) and the viral pathogen Cyprinid herpesvirus-3 (CyHV-3) (chapter 6). We used a large number of individuals of different carp lines and revealed, using PCR-RF-SSCP, the presence of n = 9 unique Cyca-DAB1-like genotypes, of which three genotypes (B, D, and E) were most common (chapter 5). In general, Cyca-DAB2 was often homozygous or absent while allelic polymorphism was detected in Cyca-DAB1 gene. We could detect significant associations between genotype E and abundance of A. japonicus and between genotype D and higher level of parasitaemia after T. borreli infection. We also observed a significant association between Cyca-DAB1 heterozygosity and lower level of parasitaemia after T. borreli infection. In chapter 6, we showed a strong association between Cyca-DAB1-like genotypes and resistance or susceptibility to CyHV-3. One genotype (E) performed significantly better, resulting in carp more resistant to CyHV-3, while three other genotypes (B, H and J) could be linked to higher susceptibility to the virus. Subsequent analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. The resistant genotype E did not comprise the Cyca-DAB2 gene and consisted of a homozygous Cyca-DAB1*05 allele. Phylogenetic analysis of all Cyca-DAB1 alleles showed that the Cyca-DAB1*05 allele represents the oldest allele in our study (chapter 7). We discussed the possibility for using Cyca-DAB1 allelic polymorphism as a potential genetic marker in future breeding programs of common carp (chapter 7). We expect that selection of carp for particular MH class II B genotypes or alleles could allow for an increased survival upon challenge with selected pathogens and possibly, increased survival rate under pond conditions.