All jawed vertebrates have an adaptive immune system and antigen presentation by MHC proteins is a key step in every adaptive immune response. MHC genes are among the most variable genes known in animals and it is believed that this variation is maintained by selection from...
All jawed vertebrates have an adaptive immune system and antigen presentation by MHC proteins is a key step in every adaptive immune response. MHC genes are among the most variable genes known in animals and it is believed that this variation is maintained by selection from pathogens. However, MHC is mostly known from humans and domestic mammals and when MHC was studied in wild songbirds that are subject to natural selection from pathogens it was clear that the MHC diversity was much higher in songbirds. What is the purpose of this high MHC diversity in wild songbirds, and has it evolved to enable particularly sophisticated adaptive immune responses? Appreciating the diversity of the different kinds of immune systems that exists in wild animals will aid not only in ecological and evolutionary research, but also in medicine and conservation. Importantly, in a much wider perspective, it is my sincere opinion that we must appreciate the diversity on all levels, genes-proteins-species-communities, in the wild. This diversity cannot be studied in humans or in captive animal-model systems in the laboratory, we need to include wild populations. The main objective is to measure and characterize how selection from pathogens affects diversity of MHC genes in wild songbird populations. More specifically I seek to understand why songbirds have such large number of MHC gene copies. Does this high MHC gene copy number really translate into a broader MHC recognition repertoire against pathogens? I will (1) characterize the MHC genomic region for the first time in songbirds, (2) measure expression of different MHC gene copies, (3) investigate characteristics of MHC proteins, and (4) study costs of infection related to MHC diversity in wild birds.
Objective 1 - We have assembled major parts of the core MHC genomic region(s) in a songbird, the great reed warbler Acrocephalus arundinaceus, based on high quality DNA from a single individual. To achieve this, we produced several different types of state-of-the-art genomic data from this individual: long genomic reads by ‘single molecule real-time sequencing (SMRT)’ (PacBio), an optical genome map using Irys long-range next-generation technique (Bionano genomics), linked genomic reads by Chromium genome sequencing (10X Genomics) and short genomic reads by HiSeq (Illumina).
Objective 2 - We have collected blood samples for DNA and RNA analyses from long-distance migratory great reed warblers that bred in our study population at lake Kvismaren the breeding seasons 2016, 2017 and 2018 and we have also collected blood samples for DNA and RNA analyses from sparrows in three different sparrow projects: Sparrow I (Three different populations from three different regions in Europe (Spain in the south, Bulgaria in the center and Sweden in the north) have been sampled every second month during an entire year, September 2016 – September 2017). Sparrow II (21 different populations from 15 different countries within and close to Europe have been sampled in September 2018) and Sparrow III (Samples have been collected every seventh day throughout three different natural avian malaria infections (two set-ups in Spain and one in Bulgaria) in young house sparrows, naïve to avian malaria, that were kept in aviaries with and without mosquito nets).
Objective 3 - We have successfully produced MHC-I proteins based on the amino acid sequences from three different MHC-I alleles from great reed warblers (Acar_3, Acar_5 and Acar 19) and we have also managed to produce the small stabilizing protein needed for expression of the MHC-I molecule, beta-2-microglobulin (B2M). At present we are folding and trying to crystalize the MHC-I protein Acar_3, together with a suitable peptide and B2M.
Objective 4 - We have sampled three different house sparrow populations every second month during an entire year and these samples have been analyzed for prevalence of malaria infections. The overall diversity and frequency of malaria infections were highest in Spain and lowest in Sweden, although the pattern varied considerably over the year. With this information in hand we decided to sample throughout Europe in detail, 23 different locations, in September 2018 and May 2019. The infection period of natural avian malaria infections was monitored in young house sparrows kept in outdoor aviaries where the vectors were free to infect the birds with avian malaria. These birds were primarily infected with two avian malaria infections Plasmodium relictum strain SGS1 and Haemoproteus passeris strain Padom05. Twelve out of the 15 experimental birds were infected with both infections simultaneously. We determined the malaria strain by Sanger sequencing and the infection intensities throughout the infection period using qPCR.
Professor Jochen Wolf at the Ludwig-Maximilian University in Germany offered me to use his unpublished PacBio data and assemble and study the MHC regions in the Hooded crow and the Jackdaw in parallel with the great reed warbler. We have therefore a more solid MHC comparison than outlined in my application including MHC data from four and not two different passerines.
Maria Strandh, employed in my project, has unraveled that repetitive elements are most likely the drivers of the highly duplicated MHC region in great reed warblers. She has developed bioinformatic pipelines together with Verena Kutschera at SciLifeLab to study the distribution of repetitive elements in the entire genome, an up-coming and most likely important parameter in studies of structural genetic variation.
SciLifeLab offered me to run an optical genome map using Irys long-range next-generation technique (Bionano genomics) at an extremely low cost since they thought that we were producing a really high quality genome for the great reed warbler and Maria Strandh was invited to talk at their international workshop December 2017.
Sophisticated combinations of different DNA sequencing techniques make it possible to study the highly repetitive MHC-I loci in the great reed warbler. Now we have a backbone in the PacBio genome, a genome that is purely constructed from a single great reed warbler individual, and then we can map RNA transcripts from Illumina and shorter amplicon reads to study expression and selection within this single individual in great detail.
I expect to publish the comparative genomics paper on passerine MHC in a high profile journal and that this paper will gain a lot of interest from the research community. Moreover, I want to use the great reed warbler genome in the future as ‘a sort of library’ where I can collect information on specific genes, their location etc.
We had recent progress in the protein work on MHC-I in great reed warblers and I hope that we now have a pipeline that will result in that the antigen binding of MHC-I can be studied, not only in great reed warblers but also in house sparrows.
In my project, we are monitoring natural avian malaria infections, meaning that the vectors transmit the infection to the malaria naïve bird. Malaria parasites infect red blood cells and in birds these are nucleated. Hence, it is possible that we will be able to observe adaptive immune responses triggered by red blood cells, this would be fascinating, and cannot be seen in mammals since they do not have nucleated red blood cells.