Host-parasite genomics and ecology: linking genes and transcriptomes to disease and contemporary selection

Infectious diseases in natural populations are important areas of research in ecology and evolution as they describe how parasites influence the host fitness. A host may undergo adaptive evolution against the parasite by acquiring either resistance or tolerance through developing intricate biochemical and molecular defence strategies. However, the knowledge about the genes associated with these traits remains limited. Furthermore, the strength of ongoing natural selection on transcript abundance has not been studied directly, despite the fact that gene regulation has a major role in adaptive evolution. In this thesis, I applied genomic and transcriptomic approaches to study the host parasite system of anadromous brown trout (Salmo trutta) infected with a myxozoan parasite, Tetracapsuloides bryosalmonae. In salmonids, T. bryosalmonae causes an emerging temperature-dependent disease, proliferative kidney disease (PKD). The parasite infects the kidney and spleen of juvenile fish, and at elevated temperatures, causes strong inflammatory response, anaemia and kidney hypertrophy. In this thesis, I performed one of the first association mapping attempts on parasite resistance and tolerance in brown trout and demonstrated the possibilities as well as limitations of association analysis in natural populations. As there was very limited genomic information available for T. bryosalmonae, I also generated an annotated assembly of the parasite transcriptome. Furthermore, by combining -omic approaches with genetic mark-recapture and classical regression-based selection analysis, I demonstrated the effect of temperature-driven parasite-induced contemporary natural selection on transcript abundance and co-regulated gene networks in this wild vertebrate species. I identified several promising candidate genes involved in PKD resistance and severity in brown trout. I also characterized more than three thousand transcripts of T. bryosalmonae. Among these, I also identified four novel protein drug targets, which can help in curing the infected fish. I also showed that the myxozoan parasite induces massive cell proliferation in the fish host whose variation is associated with the survival selection on the co-regulated gene networks. The directional selection on the individual transcript abundances was weak, similar to the published selection estimates on phenotypic traits. Finally, I also discovered many transcripts exhibiting widespread signal of disruptive selection, related to host immune defence, host–pathogen interactions, cellular repair and maintenance. Overall, my thesis showcases the power of integrating ecological and genomic perspectives to gain novel insights into the functional genomic basis of resistance against a parasite, health damage (i.e., anaemia) caused by the parasite and the ongoing associated natural selection in the wild. Altogether, my thesis combines multiple levels of biological complexities and represents a significant step forward towards understanding the molecular basis of T. bryosalmonae and PKD.Isäntä-loissuhteen genomiikka ja ekologia: geenien ja transkriptomien yhdistäminen sairauksiin ja valintaan arttuvat taudit luonnonpopulaatioissa ovat tärkeitä ekologian ja evoluution tutkimuskohteita koska ne kuvaavat, kuinka loinen vaikuttaa isäntänsä kelpoisuuteen. Isännässä voi kehittyä evolutiivisia adaptaatioita loista vastaan joko resistenssin tai toleranssin kautta. Tällaisten piirteiden voidaan katsoa olevan isännässä kehittyneitä biokemiallisia ja molekyylisiä puolustusstrategioita loisia vastaan. Näihin piirteisiin liittyviä geenejä tunnetaan heikosti. Lisäksi luonnonvalinnan voimakkuutta transkription määrään ei ole tutkittu suoraan, huolimatta siitä, että geenisäätelyllä on tärkeä rooli adaptiivisessa evoluutiossa. Tässä väitöskirjassa käytin genomisia ja transkriptomisia lähestymistapoja Tetracapsuloides bryosalmonae -loistartunnan saaneen anadromisen taimenen (Salmo trutta) isäntäloisjärjestelmän tutkimiseen. Lohikaloissa T. bryosalmonae aiheuttaa lämpötilasta riippuvan sairauden, proliferatiivisen munuaissairauden (PKD). Tämä Myxozoa-pääjaksoon kuuluva loinen infektoi nuorien kalojen munuaiset ja pernan, ja aiheuttaa korkeissa lämpötiloissa voimakkaan tulehdusvasteen, anemiaa ja munuaisten liikakasvua. Tein ns. ”association mapping”-analyysin liittyen taimenen resistenssiin ja toleranssiin T. bryosalmonae-loista kohtaan, ja osoitin sekä assosiaatioanalyysin käytön mahdollisuudet että rajoitukset tutkittaessa luonnonpopulaatioita. Koska T. bryosalmonaesta oli saatavilla hyvin vähän genomista tietoa, loin myös annotoidun koosteen loisen transkriptomista. Lisäksi, yhdistämällä kolme metodia: ns. ”-omics”-lähestymistavan, geneettisen merkintäjälleenpyynnin ja klassiseen regressioon perustuvan valinta-analyysin, pystyin osoittamaan lämpötilasta riippuvaisen loisinfektion indusoiman luonnonvalinnan vaikutuksen transkription määrään ja yhteissäädeltyihin geeniverkostoihin tässä luonnossa elävässä selkärankais-lajissa. Tunnistin useita lupaavia kandidaattigeenejä, jotka liittyvät PKD-resistenssiin ja taudin vaikeusasteeseen taimenessa. Kuvasin myös yli kolmetuhatta T. bryosalmonaen transkriptia. Näiden joukosta tunnistin myös neljä uutta proteiinilääke-kohdetta, joiden avulla voidaan parantaa tartunnan saaneita kaloja. Lisäksi osoitin, että tämä Myxozoan-pääjaksoon kuuluva loinen indusoi massiivista solujen lisääntymistä kalaisännässä, ja jonka vaihtelu liittyy selviytymisvalintaan yhteissäädellyissä geeniverkostoissa. Yksittäisten transkriptien runsauteen kohdistuva suuntaava valinta oli heikkoa, ollen samantasoista kuin kirjallisuudessa esitetyt arviot fenotyyppisiin ominaisuuksiin kohdistuvasta valinnasta. Löysin myös monia transkripteja, joihin kohdistui hajoittavaa valintaa. Nämä liittyvät isännän immuunipuolustukseen, isännän ja patogeenin väliseen vuorovaikutukseen, solujen korjaamiseen ja ylläpitoon. Väitöskirjani tuo hyvin esille ekologisten ja genomisten lähestymistapojen yhdistämisestä avautuvat uudet tutkimusmahdollisuudet ja näkökulmat loisen vastustuskyvyn genomiseen ja toiminnalliseen perustaan, loisen aiheuttamaan terveyshaittaan (eli anemiaan) ja siihen liittyvään luonnonvalintaan. Väitöskirjani yhdistää monia biologian tasoja ja on merkittävä askel kohti T. bryosalmonaen ja PKD:n molekyyliperustan ymmärtämistä

The strength and form of natural selection on transcript abundance in the wild

Gene transcription variation is known to contribute to disease susceptibility and adaptation, but we currently know very little about how contemporary natural selection shapes transcript abundance. Here, we propose a novel analytical framework to quantify the strength and form of ongoing natural selection at the transcriptome level in a wild vertebrate. We estimated selection on transcript abundance in a cohort of a wild salmonid fish (Salmo trutta) affected by an extracellular myxozoan parasite (Tetracapsuloides bryosalmonae) through mark-recapture field sampling and the integration of RNA-sequencing with classical regression-based selection analysis. We show, based on fin transcriptomes of the host, that infection by the parasite and subsequent host survival is linked to upregulation of mitotic cell cycle process. We also detect a widespread signal of disruptive selection on transcripts linked to host immune defence, host-pathogen interactions, cellular repair and maintenance. Our results provide insights into how selection can be measured at the transcriptome level to dissect the molecular mechanisms of contemporary evolution driven by climate change and emerging anthropogenic threats. We anticipate that the approach described here will enable critical information on the molecular processes and targets of natural selection to be obtained in real time

Tonsillar transcriptional profiles in atopic and non‐atopic subjects

Background: Emerging research suggests that local lymphatic tissue such as tonsils have important role in regulating the immune responses. However, allergen sensitization-induced alterations in transcriptome of tonsils are not known. Objectives: To examine the key differences in tonsillar gene expression between atopic and non-atopic subjects and further by type of sensitization. Methods: RNA-sequencing was performed on 52 tonsillar samples from atopic and non-atopic tonsillectomy patients. Sensitization to common food- and aero-allergen was defined by allergen specific IgE. Following groups were studied: (1) aero- and food-allergen sensitized (AS+FS) versus non-sensitized (NS), (2) aeroallergen-sensitized (AS) versus food-allergen sensitized (FS), (3) AS versus NS, (4) FS versus NS. Bioinformatics analysis was done using DESeq2(v3.10.2), WGCNA and GATK pipeline in R software (v3.3.1). Protein-protein interaction network was made from String database. Results: We studied 13 aeroallergen-sensitized, 6 food-allergen sensitized, 4 both food-and aero-allergen-sensitized and 29 non-sensitized tonsillectomy patients. Overall, 697 unique differentially expressed genes (DEGs) were detected in all sensitized subgroups including chemokines (CXCL2, CXCL8, CXCL10, CXCL11), IL-20RA, MUC1 and MUC20. When comparing different groups, the gene expression profiles overlapped except the AS versus FS group comparison, suggesting significantly different gene expression between the two sensitization subgroups. Furthermore, aeroallergen-sensitized subjects had more prominent immune responses compared with non-sensitized and food-allergen sensitized subjects including gene expression for IL-17 pathway and Toll-like receptor signalling pathway. Conclusion: Allergic sensitization is associated with extensive tonsillar transcriptomic alterations and changes in immune related genes and pathways. Distinct differences were found between aero-allergen and food-allergen sensitization

Tonsillar transcriptional profiles in atopic and non-atopic subjects

Abstract Background: Emerging research suggests that local lymphatic tissue such as tonsils have important role in regulating the immune responses. However, allergen sensitization-induced alterations in transcriptome of tonsils are not known. Objectives: To examine the key differences in tonsillar gene expression between atopic and non-atopic subjects and further by type of sensitization. Methods: RNA-sequencing was performed on 52 tonsillar samples from atopic and non-atopic tonsillectomy patients. Sensitization to common food- and aero-allergen was defined by allergen specific IgE. Following groups were studied: (1) aero- and foodallergen sensitized (AS+FS) versus non-sensitized (NS), (2) aeroallergen-sensitized (AS) versus food-allergen sensitized (FS), (3) AS versus NS, (4) FS versus NS. Bioinformatics analysis was done using DESeq2(v3.10.2), WGCNA and GATK pipeline in R software (v3.3.1). Protein–protein interaction network was made from String database. Results: We studied 13 aeroallergen-sensitized, 6 food-allergen sensitized, 4 both food-and aero-allergen-sensitized and 29 non-sensitized tonsillectomy patients. Overall, 697 unique differentially expressed genes (DEGs) were detected in all sensitized subgroups including chemokines (CXCL2, CXCL8, CXCL10, CXCL11), IL-20RA,MUC1 and MUC20. When comparing different groups, the gene expression profiles overlapped except the AS versus FS group comparison, suggesting significantly different gene expression between the two sensitization subgroups. Furthermore, aeroallergen-sensitized subjects had more prominent immune responses compared with non-sensitized and food-allergen sensitized subjects including gene expression for IL-17 pathway and Toll-like receptor signalling pathway. Conclusion: Allergic sensitization is associated with extensive tonsillar transcriptomic alterations and changes in immune related genes and pathways. Distinct differences were found between aero-allergen and food-allergen sensitization. KEYWORDS aeroallergen, atopy, IL-17, tonsil, transcriptomePeer reviewe

Comparative High-Density Linkage Mapping Reveals Conserved Genome Structure but Variation in Levels of Heterochiasmy and Location of Recombination Cold Spots in the Common Frog

By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog, Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis. We confirmed marked heterochiasmy between sexes and detected non-recombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.</p

Know your enemy - Transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.</p

Highly Continuous Genome Assembly of Eurasian Perch (Perca fluviatilis) Using Linked-Read Sequencing

The Eurasian perch (Perca fluviatilis) is the most common fish of the Percidae family and is widely distributed across Eurasia. Perch is a popular target for professional and recreational fisheries, and a promising freshwater aquaculture species in Europe. However, despite its high ecological, economical and societal importance, the available genomic resources for P. fluviatilis are rather limited. In this work, we report de novo assembly and annotation of the whole genome sequence of perch. The linked-read based technology with 10X Genomics Chromium chemistry and Supernova assembler produced a draft perch genome approximate to 1.0 Gbp assembly (scaffold N-50 = 6.3 Mb; the longest individual scaffold of 29.3 Mb; BUSCO completeness of 88.0%), which included 281.6 Mb of putative repeated sequences. The perch genome assembly presented here, generated from small amount of starting material (0.75 ng) and a single linked-read library, is highly continuous and considerably more complete than the currently available draft of P. fluviatilis genome. A total of 23,397 protein-coding genes were predicted, 23,171 (99%) of which were annotated functionally from either sequence homology or protein signature searches. Linked-read technology enables fast, accurate and cost-effective de novo assembly of large non-model eukaryote genomes. The highly continuous assembly of the Eurasian perch genome presented in this study will be an invaluable resource for a range of genetic, ecological, physiological, ecotoxicological, functional and comparative genomic studies in perch and other fish species of the Percidae family

The strength and form of natural selection on transcript abundance in the wild

Gene transcription variation is known to contribute to disease susceptibility and adaptation, but we currently know very little about how contemporary natural selection shapes transcript abundance. Here, we propose a novel analytical framework to quantify the strength and form of ongoing natural selection at the transcriptome level in a wild vertebrate. We estimated selection on transcript abundance in a cohort of a wild salmonid fish (Salmo trutta) affected by an extracellular myxozoan parasite (Tetracapsuloides bryosalmonae) through mark-recapture field sampling and the integration of RNA-sequencing with classical regression-based selection analysis. We show, based on fin transcriptomes of the host, that infection by the parasite and subsequent host survival is linked to upregulation of mitotic cell cycle process. We also detect a widespread signal of disruptive selection on transcripts linked to host immune defence, host-pathogen interactions, cellular repair and maintenance. Our results provide insights into how selection can be measured at the transcriptome level to dissect the molecular mechanisms of contemporary evolution driven by climate change and emerging anthropogenic threats. We anticipate that the approach described here will enable critical information on the molecular processes and targets of natural selection to be obtained in real time

Humic-acid-driven escape from eye parasites revealed by RNA-seq and target-specific metabarcoding

Background: Next generation sequencing (NGS) technologies are extensively used to dissect the molecular mechanisms of host-parasite interactions in human pathogens. However, ecological studies have yet to fully exploit the power of NGS as a rich source for formulating and testing new hypotheses.Methods: We studied Eurasian perch (Perca fluviatilis) and its eye parasite (Trematoda, Diplostomidae) communities in 14 lakes that differed in humic content in order to explore host-parasite-environment interactions. We hypothesised that high humic content along with low pH would decrease the abundance of the intermediate hosts (gastropods), thus limiting the occurrence of diplostomid parasites in humic lakes. This hypothesis was initially invoked by whole eye RNA-seq data analysis and subsequently tested using PCR-based detection and a novel targeted metabarcoding approach.Results: Whole eye transcriptome results revealed overexpression of immune-related genes and the presence of eye parasite sequences in RNA-seq data obtained from perch living in clear-water lakes. Both PCR-based and targeted-metabarcoding approach showed that perch from humic lakes were completely free from diplostomid parasites, while the prevalence of eye flukes in clear-water lakes that contain low amounts of humic substances was close to 100%, with the majority of NGS reads assigned toTylodelphys clavata.Conclusions: High intraspecific diversity ofT. clavataindicates that massively parallel sequencing of naturally pooled samples represents an efficient and powerful strategy for shedding light on cryptic diversity of eye parasites. Our results demonstrate that perch populations in clear-water lakes experience contrasting eye parasite pressure compared to those from humic lakes, which is reflected by prevalent differences in the expression of immune-related genes in the eye. This study highlights the utility of NGS to discover novel host-parasite-environment interactions and provide unprecedented power to characterize the molecular diversity of cryptic parasites.</div