Whole-genome analysis reveals phylogenetic and demographic history of Eurasian perch.

The contemporary diversity and distribution of species are shaped by their evolutionary and ecological history. This can be deciphered with the help of phylogenetic and demographic analysis methods, ideally combining and supplementing information from mitochondrial and nuclear genomes. In this study, we investigated the demographic history of Eurasian perch (Perca fluviatilis), a highly adaptable teleost with a distribution range across Eurasia. We combined whole-genome resequencing data with available genomic resources to analyse the phylogeny, phylogeography, and demographic history of P. fluviatilis populations from Europe and Siberia. We identified five highly diverged evolutionary mtDNA lineages, three of which show a strong signal of admixture in the Baltic Sea region. The estimated mean divergence time between these lineages ranged from 0.24 to 1.42 million years. Based on nuclear genomes, two distinct demographic trajectories were observed in European and Siberian samples reflecting contrasting demographic histories ca. 30,000-100,000 years before the present. A comparison of mtDNA and nuclear DNA evolutionary trees and AMOVA revealed concordances, as well as incongruences, between the two types of data, most likely reflecting recent postglacial colonization and hybridization events. Overall, our findings demonstrate the power and usefulness of genome-wide information for delineating historical processes that have shaped the genome of P. fluviatilis. We also highlight the added value of data-mining existing transcriptomic resources to complement novel sequence data, helping to shed light on putative glacial refugia and postglacial recolonization routes.

DNA metabarcoding reveals spatial and temporal variation of fish eye fluke communities in lake ecosystems.

Eye flukes (Diplostomidae) are diverse and abundant trematode parasites that form multi-species communities in fish with negative effects on host fitness and survival. However, the environmental factors and host-related characteristics that determine species diversity, composition, and coexistence in such communities remain poorly understood. Here, we developed a cost-effective cox1 region-specific DNA metabarcoding approach to characterize parasitic diplostomid communities in two common fish species (Eurasian perch and common roach) collected from seven temperate lakes in Estonia. We found considerable inter- and intra-lake, as well as inter-host species, variation in diplostomid communities. Sympatric host species characterization revealed that parasite communities were typically more diverse in roach than perch. Additionally, we detected five positive and two negative diplostomid species associations in roach, whereas only a single negative association was observed in perch. These results indicate that diplostomid communities in temperate lakes are complex and dynamic systems exhibiting both spatial and temporal heterogeneity. They are influenced by various environmental factors and by host-parasite and inter-parasite interactions. We expect that the described methodology facilitates ecological and biodiversity research of diplostomid parasites. It is also adaptable to other parasite groups where it could serve to improve current understanding of diversity, distribution, and interspecies interactions of other understudied taxa.

Subtle transcriptomic response of Eurasian perch (Perca fluviatilis) associated with Triaenophorus nodulosus plerocercoid infection.

Determining the physiological effects of parasites and characterizing genes involved in host responses to infections are essential to improving our understanding of host-parasite interactions and their ecological and evolutionary consequences. This task, however, is complicated by high diversity and complex life histories of many parasite species. The use of transcriptomics in the context of wild-caught specimens can help ameliorate this by providing both qualitative and quantitative information on gene expression patterns in response to parasites in specific host organs and tissues. Here, we evaluated the physiological impact of the widespread parasite, the pike tapeworm (Triaenophorus nodulosus), on its second intermediate host, the Eurasian perch (Perca fluviatilis). We used an RNAseq approach to analyse gene expression in the liver, the target organ of T. nodulosus plerocercoids, and spleen which is one of the main immune organs in teleost fishes. We compared perch collected from multiple lakes consisting of individuals with (n = 8) and without (n = 6) T. nodulosus plerocercoids in the liver. Results revealed a small number of differentially expressed genes (DEGs, adjusted p-value ≤0.05) in both spleen (n = 22) and liver (n = 10). DEGs in spleen consisted of mostly upregulated immune related genes (e.g., JUN, SIK1, THSB1), while those in the liver were often linked to metabolic functions (e.g., FABP1, CADM4, CDAB). However, Gene Ontology (GO) analysis showed lack of functional enrichment among DEGs. This study demonstrates that Eurasian perch displays a subtle response at a gene expression level to T. nodulosus plerocercoid infection. Given that plerocercoids are low-metabolic activity transmission stages, our results suggest that moderate T. nodulosus plerocercoid infection most likely does not provoke an extensive host immune response and have relatively low physiological costs for the host. Our findings illustrate that not all conspicuous infections have severe effects on host gene regulation.

Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

Spatial and intra-host distribution of myxozoan parasite Tetracapsuloides bryosalmonae among Baltic sea trout (Salmo trutta).

Proliferative kidney disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae has been actively studied in juvenile salmonids for decades. However, very little is known about parasite prevalence and its geographical and intra-host distribution at older life stages. We screened T. bryosalmonae among adult sea trout (Salmo trutta) (n = 295) collected along the Estonian Baltic Sea coastline together with juvenile trout from 33 coastal rivers (n = 1752) to assess spatial infection patterns of the adult and juvenile fish. The parasite was detected among 38.6% of adult sea trout with the prevalence increasing from west to east, and south to north, along the coastline. A similar pattern was observed in juvenile trout. Infected sea trout were also older than uninfected fish and the parasite was detected in sea trout up to the age of 6 years. Analysis of intra-host distribution of the parasite and strontium to calcium ratios from the otoliths revealed that (re)infection through freshwater migration may occur among adult sea trout. The results of this study indicate that T. bryosalmonae can persist in a brackish water environment for several years and that returning sea trout spawners most likely contribute to the parasite life cycle by transmitting infective spores.

Whole-genome sequencing illuminates multifaceted targets of selection to humic substances in Eurasian perch.

Extreme environments are inhospitable to the majority of species, but some organisms are able to survive in such hostile conditions due to evolutionary adaptations. For example, modern bony fishes have colonized various aquatic environments, including perpetually dark, hypoxic, hypersaline and toxic habitats. Eurasian perch (Perca fluviatilis) is among the few fish species of northern latitudes that is able to live in very acidic humic lakes. Such lakes represent almost "nocturnal" environments; they contain high levels of dissolved organic matter, which in addition to creating a challenging visual environment, also affects a large number of other habitat parameters and biotic interactions. To reveal the genomic targets of humic-associated selection, we performed whole-genome sequencing of perch originating from 16 humic and 16 clear-water lakes in northern Europe. We identified over 800,000 single nucleotide polymorphisms, of which >10,000 were identified as potential candidates under selection (associated with >3000 genes) using multiple outlier approaches. Our findings suggest that adaptation to the humic environment may involve hundreds of regions scattered across the genome. Putative signals of adaptation were detected in genes and gene families with diverse functions, including organism development and ion transportation. The observed excess of variants under selection in regulatory regions highlights the importance of adaptive evolution via regulatory elements, rather than via protein sequence modification. Our study demonstrates the power of whole-genome analysis to illuminate the multifaceted nature of humic adaptation and provides the foundation for further investigation of causal mutations underlying phenotypic traits of ecological and evolutionary importance.

A century of genetic homogenization in Baltic salmon-evidence from archival DNA.

Intra-species genetic homogenization arising from anthropogenic impacts is a major threat to biodiversity. However, few taxa have sufficient historical material to systematically quantify long-term genetic changes. Using archival DNA collected over approximately 100 years, we assessed spatio-temporal genetic change in Atlantic salmon populations across the Baltic Sea, an area heavily impacted by hydropower exploitation and associated with large-scale mitigation stocking. Analysis was carried out by screening 82 SNPs in 1680 individuals from 13 Swedish rivers. We found an overall decrease in genetic divergence and diminished isolation by distance among populations, strongly indicating genetic homogenization over the past century. We further observed an increase in genetic diversity within populations consistent with increased gene flow. The temporal genetic change was lower in larger wild populations than in smaller wild and hatchery-reared ones, indicating that larger populations have been able to support a high number of native spawners in relation to immigrants. Our results demonstrate that stocking practices of salmon in the Baltic Sea have led to the homogenization of populations over the last century, potentially compromising their ability to adapt to environmental change. Stocking of reared fish is common worldwide, and our study is a cautionary example of the potentially long-term negative effects of such activities.

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.

Climate change-driven disease in sympatric hosts: Temporal dynamics of parasite burden and proliferative kidney disease in wild brown trout and Atlantic salmon.

Global climate change is altering the abundance and spread of various parasites, which has important consequences not only for host-parasite interactions but also for the relationships between different host species. Here, we focus on the myxozoan endoparasite Tetracapsuloides bryosalmonae that causes temperature-dependent proliferative kidney disease (PKD) in salmonids. We characterized the temporal changes in the parasite load and the severity of PKD signs (renal hyperplasia, haematocrit) in two sympatric populations of wild brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). We found that both the parasite load and disease signs vary considerably between individuals, species, rivers and sampling periods. We showed that Atlantic salmon was able to slow down the initial parasite proliferation rate and subsequently tolerate high parasite burden without obvious disease signs. In contrast, the initial parasite proliferation rate was much higher in brown trout, which was followed by the development of severe PKD signs. Thus, the speed of parasite proliferation, rather than the absolute number of the parasites in the host kidney, may play an important role in interspecific variation in PKD susceptibility. To conclude, this study illustrates the usefulness of temporal perspective for understanding host defence mechanisms and climate change-mediated impacts in the wild.

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.

Draft Genome Assembly of the Freshwater Apex Predator Wels Catfish (Silurus glanis) Using Linked-Read Sequencing.

The wels catfish (Silurus glanis) is one of the largest freshwater fish species in the world. This top predator plays a key role in ecosystem stability, and represents an iconic trophy-fish for recreational fishermen. S. glanis is also a highly valued species for its high-quality boneless flesh, and has been cultivated for over 100 years in Eastern and Central Europe. The interest in rearing S. glanis continues to grow; the aquaculture production of this species has almost doubled during the last decade. However, despite its high ecological, cultural and economic importance, the available genomic resources for S. glanis are very limited. To fulfill this gap we report a de novo assembly and annotation of the whole genome sequence of a female S. glanis The linked-read based technology with 10X Genomics Chromium chemistry and Supernova assembler produced a highly continuous draft genome of S. glanis: ∼0.8Gb assembly (scaffold N50 = 3.2 Mb; longest individual scaffold = 13.9 Mb; BUSCO completeness = 84.2%), which included 313.3 Mb of putative repeated sequences. In total, 21,316 protein-coding genes were predicted, of which 96% were annotated functionally from either sequence homology or protein signature searches. The highly continuous genome assembly will be an invaluable resource for aquaculture genomics, genetics, conservation, and breeding research of S. glanis.

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 to Tylodelphys clavata. CONCLUSIONS: High intraspecific diversity of T. clavata indicates 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.

Population Connectivity Predicts Vulnerability to White-Nose Syndrome in the Chilean Myotis (Myotis chiloensis) - A Genomics Approach.

Despite its peculiar distribution, the biology of the southernmost bat species in the world, the Chilean myotis (Myotis chiloensis), has garnered little attention so far. The species has a north-south distribution of c. 2800 km, mostly on the eastern side of the Andes mountain range. Use of extended torpor occurs in the southernmost portion of the range, putting the species at risk of bat white-nose syndrome, a fungal disease responsible for massive population declines in North American bats. Here, we examined how geographic distance and topology would be reflected in the population structure of M. chiloensis along the majority of its range using a double digestion RAD-seq method. We sampled 66 individuals across the species range and discovered pronounced isolation-by-distance. Furthermore, and surprisingly, we found higher degrees of heterozygosity in the southernmost populations compared to the north. A coalescence analysis revealed that our populations may still not have reached secondary contact after the Last Glacial Maximum. As for the potential spread of pathogens, such as the fungus causing WNS, connectivity among populations was noticeably low, especially between the southern hibernatory populations in the Magallanes and Tierra del Fuego, and more northerly populations. This suggests the probability of geographic spread of the disease from the north through bat-to-bat contact to susceptible populations is low. The study presents a rare case of defined population structure in a bat species and warrants further research on the underlying factors contributing to this. See the graphical abstract here. https://doi.org/10.25387/g3.12173385.

Association Mapping Based on a Common-Garden Migration Experiment Reveals Candidate Genes for Migration Tendency in Brown Trout.

A better understanding of the environmental and genetic contribution to migratory behavior and the evolution of traits linked to migration is crucial for fish conservation and fisheries management. Up to date, a few genes with unequivocal influence on the adoption of alternative migration strategies have been identified in salmonids. Here, we used a common garden set-up to measure individual migration distances of generally highly polymorphic brown trout Salmo trutta from two populations. Fish from the assumedly resident population showed clearly shorter migration distances than the fish from the assumed migratory population at the ages of 2 and 3 years. By using two alternative analytical pipelines with 22186 and 18264 SNPs obtained through RAD-sequencing, we searched for associations between individual migration distance, and both called genotypes and genotype probabilities. None of the SNPs showed statistically significant individual effects on migration after correction for multiple testing. By choosing a less stringent threshold, defined as an overlap of the top 0.1% SNPs identified by the analytical pipelines, GAPIT and Angsd, we identified eight candidate genes that are potentially linked to individual migration distance. While our results demonstrate large individual and population level differences in migration distances, the detected genetic associations were weak suggesting that migration traits likely have multigenic control.

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness - Implications for brown trout conservation.

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction-site-associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half- and full-siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual-level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual-level genotype information, such as quantifying relatedness and individual-level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.

Mapping of quantitative trait loci for life history traits segregating within common frog populations.

The evolution of complex traits is often shaped by adaptive divergence. However, very little is known about the number, effect size, and location of the genomic regions influencing the variation of these traits in natural populations. Based on a dense linkage map of the common frog, Rana temporaria, we have localized, for the first time in amphibians, three significant and nine suggestive quantitative trait loci (QTLs) for metabolic rate, growth rate, development time, and weight at metamorphosis, explaining 5.6-18.9% of the overall phenotypic variation in each trait. We also found a potential pleiotropic QTL between development time and size at metamorphosis that, if confirmed, might underlie the previously reported genetic correlation between these traits. Furthermore, we demonstrate that the genetic variation linked to fitness-related larval traits segregates within Rana temporaria populations. This study provides the first insight into the genomic regions that affect larval life history traits in anurans, providing a valuable resource to delve further into the genomic basis of evolutionary change in amphibians.

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 ∼1.0 Gbp assembly (scaffold N50 = 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.

Comparison of Migratory and Resident Populations of Brown Trout Reveals Candidate Genes for Migration Tendency.

Candidate genes associated with migration have been identified in multiple taxa: including salmonids, many of whom perform migrations requiring a series of physiological changes associated with the freshwater-saltwater transition. We screened over 5,500 SNPs for signatures of selection related to migratory behavior of brown trout Salmo trutta by focusing on ten differentially migrating freshwater populations from two watersheds (the Koutajoki and the Oulujoki). We found eight outlier SNPs potentially associated with migratory versus resident life history using multiple (≥3) outlier detection approaches. Comparison of three migratory versus resident population pairs in the Koutajoki watershed revealed seven outlier SNPs, of which three mapped close to genes ZNF665-like, GRM4-like, and PCDH8-like that have been previously associated with migration and smoltification in salmonids. Two outlier SNPs mapped to genes involved in mucus secretion (ST3GAL1-like) and osmoregulation (C14orf37-like). The last two strongly supported outlier SNPs mapped to thermally induced genes (FNTA1-like, FAM134C-like). Within the Oulujoki, the only consistent outlier SNP mapped close to a gene (EZH2) that is associated with compensatory growth in fasted trout. Our results suggest that a relatively small yet common set of genes responsible for physiological functions associated with resident and migratory life histories is evolutionarily conserved.

The complete mitochondrial DNA sequence of Pectenocypris sp. (Actinopterygii: Cyprinidae) from Serkap River, Sumatra, Indonesia.

The whole mitochondrial genome of a small cyprinid freshwater fish Pectenocypris sp. collected from Serkap River, Central Sumatra, Indonesia was sequenced. This mitochondrial genome consisted of 16,589 bp and included 37 genes in the same order as in many other vertebrates including the human. Phylogenetic analysis suggested that this taxon clusters with Boraras maculatus among several Rasbora species.