More than one million barriers fragment Europe's rivers.

Rivers support some of Earth's richest biodiversity1 and provide essential ecosystem services to society2, but they are often fragmented by barriers to free flow3. In Europe, attempts to quantify river connectivity have been hampered by the absence of a harmonized barrier database. Here we show that there are at least 1.2 million instream barriers in 36 European countries (with a mean density of 0.74 barriers per kilometre), 68 per cent of which are structures less than two metres in height that are often overlooked. Standardized walkover surveys along 2,715 kilometres of stream length for 147 rivers indicate that existing records underestimate barrier numbers by about 61 per cent. The highest barrier densities occur in the heavily modified rivers of central Europe and the lowest barrier densities occur in the most remote, sparsely populated alpine areas. Across Europe, the main predictors of barrier density are agricultural pressure, density of river-road crossings, extent of surface water and elevation. Relatively unfragmented rivers are still found in the Balkans, the Baltic states and parts of Scandinavia and southern Europe, but these require urgent protection from proposed dam developments. Our findings could inform the implementation of the EU Biodiversity Strategy, which aims to reconnect 25,000 kilometres of Europe's rivers by 2030, but achieving this will require a paradigm shift in river restoration that recognizes the widespread impacts caused by small barriers.

Phylogenomics reveals extensive introgression and a case of mito-nuclear discordance in the killifish genus Kryptolebias.

Introgression is a widespread evolutionary process leading to phylogenetic inconsistencies among distinct parts of the genomes, particularly between mitochondrial and nuclear-based phylogenetic reconstructions (e.g., mito-nuclear discordances). Here, we used mtDNA and genome-wide nuclear sites to provide the first phylogenomic-based hypothesis on the evolutionary relationships within the killifish genus Kryptolebias. In addition, we tested for evidence of past introgression in the genus given the multiple reports of undergoing hybridization between its members. Our mtDNA phylogeny generally agreed with the relationships previously proposed for the genus. However, our reconstruction based on nuclear DNA revealed an unknown lineage - Kryptolebias sp. 'ESP' - as the sister group of the self-fertilizing mangrove killifishes, K. marmoratus and K. hermaphroditus. All individuals sequenced of Kryptolebias sp. 'ESP' had the same mtDNA haplotype commonly observed in K. hermaphroditus, demonstrating a clear case of mito-nuclear discordance. Our analysis further confirmed extensive history of introgression between Kryptolebias sp. 'ESP' and K. hermaphroditus. Population genomics analyses indicate no current gene flow between the two lineages, despite their current sympatry and history of introgression. We also confirmed introgression between other species pairs in the genus that have been recently reported to form hybrid zones. Overall, our study provides a phylogenomic reconstruction covering most of the Kryptolebias species, reveals a new lineage hidden in a case of mito-nuclear discordance, and provides evidence of multiple events of ancestral introgression in the genus. These findings underscore the importance of investigating different genomic information in a phylogenetic framework, particularly in taxa where introgression is common as in the sexually diverse mangrove killifishes.

MHC class I-α population differentiation in a commercial fish, the European sea bass (Dicentrarchus labrax).

Identifying population structuring in highly fecund marine species with high dispersal rates is challenging, but critical for conservation and stock delimitation for fisheries management. European sea bass (Dicentrarchus labrax) is a commercial species of fisheries and aquaculture relevance whose stocks are declining in the North Atlantic, despite management measures to protect them and identifying their fine population structure is needed for managing their exploitation. As for other marine fishes, neutral genetic markers indicate that eastern Atlantic sea bass form a panmictic population and is currently managed as arbitrarily divided stocks. The genes of the major histocompatibility complex (MHC) are key components of the adaptive immune system and ideal candidates to assess fine structuring arising from local selective pressures. We used Illumina sequencing to characterise allelic composition and signatures of selection at the MHC class I-α region of six D. labrax populations across the Atlantic range. We found high allelic diversity driven by positive selection, corresponding to moderate supertype diversity, with 131 alleles clustering into four to eight supertypes, depending on the Bayesian information criterion threshold applied, and a mean number of 13 alleles per individual. Alleles could not be assigned to particular loci, but private alleles allowed us to detect regional genetic structuring not found previously using neutral markers. Our results suggest that MHC markers can be used to detect cryptic population structuring in marine species where neutral markers fail to identify differentiation. This is particularly critical for fisheries management, and of potential use for selective breeding or identifying escapes from sea farms.

Quantitative assessment of fish larvae community composition in spawning areas using metabarcoding of bulk samples.

Accurate assessment of larval community composition in spawning areas is essential for fisheries management and conservation but is often hampered by the cryptic nature of many larvae, which renders them difficult to identify morphologically. Metabarcoding is a rapid and cost-effective method to monitor early life stages for management and environmental impact assessment purposes but its quantitative capability is under discussion. We compared metabarcoding with traditional morphological identification to evaluate taxonomic precision and reliability of abundance estimates, using 332 fish larvae from multinet hauls (0-50 m depth) collected at 14 offshore sampling sites in the Irish and Celtic seas. To improve quantification accuracy (relative abundance estimates), the amount of tissue for each specimen was standardized and mitochondrial primers (12S gene) with conserved binding sites were used. Relative family abundance estimated from metabarcoding reads and morphological assessment were positively correlated, as well as taxon richness (RS  = 0.81, P = 0.007) and diversity (RS  = 0.90, P = 0.002). Spatial patterns of community composition did not differ significantly between metabarcoding and morphological assessments. Our results show that DNA metabarcoding of bulk tissue samples can be used to monitor changes in fish larvae abundance and community composition. This represents a feasible, efficient, and faster alternative to morphological methods that can be applied to terrestrial and aquatic habitats.

Filling the gaps: phylogeography of the self-fertilizing Kryptolebias species (Cyprinodontiformes: Rivulidae) along South American mangroves.

Mangrove killifishes of the genus Kryptolebias have been historically classified as rare because of their small size and cryptic nature. Major gaps in distribution knowledge across mangrove areas, particularly in South America, challenge the understanding of the taxonomic status, biogeographical patterns and genetic structuring of the lineages composing the self-fertilizing "Kryptolebias marmoratus species complex." In this study, the authors combined a literature survey, fieldwork and molecular data to fill major gaps of information about the distribution of mangrove killifishes across western Atlantic mangroves. They found that selfing mangrove killifishes are ubiquitously distributed across the Caribbean, Central and South American mangroves and report 14 new locations in South America, extending the range of both the "Central clade" and "Southern clade" lineages which overlap in the Amazon. Although substantial genetic differences were found between clades, the authors also found further genetic structuring within clades, with populations in Central America, north and northeast Brazil generally showing higher levels of genetic diversity compared to the clonal ones in southeast Brazil. The authors discuss the taxonomic status and update the geographical distribution of the Central and Southern clades, as well as potential dispersal routes and biogeographical barriers influencing the distribution of the selfing mangrove killifishes in the western Atlantic mangroves.

DNA Methylation Changes in the Sperm of Captive-Reared Fish: A Route to Epigenetic Introgression in Wild Populations.

Interbreeding between hatchery-reared and wild fish, through deliberate stocking or escapes from fish farms, can result in rapid phenotypic and gene expression changes in hybrids, but the underlying mechanisms are unknown. We assessed if one generation of captive breeding was sufficient to generate inter- and/or transgenerational epigenetic modifications in Atlantic salmon. We found that the sperm of wild and captive-reared males differed in methylated regions consistent with early epigenetic signatures of domestication. Some of the epigenetic marks that differed between hatchery and wild males affected genes related to transcription, neural development, olfaction, and aggression, and were maintained in the offspring beyond developmental reprogramming. Our findings suggest that rearing in captivity may trigger epigenetic modifications in the sperm of hatchery fish that could explain the rapid phenotypic and genetic changes observed among hybrid fish. Epigenetic introgression via fish sperm represents a previously unappreciated mechanism that could compromise locally adapted fish populations.

Environmental enrichment induces intergenerational behavioural and epigenetic effects on fish.

Parental effects influence offspring phenotypes through pre- and post-natal routes but little is known about their molecular basis, and therefore their adaptive significance. Epigenetic modifications, which control gene expression without changes in the DNA sequence and are influenced by the environment, may contribute to parental effects. We investigated the effects of environmental enrichment on the behaviour, metabolic rate and brain DNA methylation patterns of parents and offspring of the highly inbreed mangrove killifish (Kryptolebias marmoratus). Parental fish reared in enriched environments had lower cortisol levels, lower metabolic rates and were more active and neophobic than those reared in barren environments. They also differed in 1,854 methylated cytosines (DMCs). Offspring activity and neophobia were determined by the parental environment. Among the DMCs of the parents, 98 followed the same methylation patterns in the offspring, three of which were significantly influenced by parental environments irrespective of their own rearing environment. Our results suggest that parental environment influences the behaviour and, to some extent, the brain DNA methylation patterns of the offspring.

Environmental plasticity and colonisation history in the Atlantic salmon microbiome: A translocation experiment.

Microbial communities associated with the gut and the skin are strongly influenced by environmental factors, and can rapidly adapt to change. Historical processes may also affect the microbiome. In particular, variation in microbial colonisation in early life has the potential to induce lasting effects on microbial assemblages. However, little is known about the relative extent of microbiome plasticity or the importance of historical colonisation effects following environmental change, especially for nonmammalian species. To investigate this we performed a reciprocal translocation of Atlantic salmon between artificial and semi-natural conditions. Wild and hatchery-reared fry were transferred to three common garden experimental environments for 6 weeks: standard hatchery conditions, hatchery conditions with an enriched diet, and simulated wild conditions. We characterized the faecal and skin microbiome of individual fish before and after the environmental translocation, using a BACI (before-after-control-impact) design. We found evidence of extensive microbiome plasticity for both the gut and skin, with the greatest changes in alpha and beta diversity associated with the largest changes in environment and diet. Microbiome richness and diversity were entirely determined by environment, with no detectable effects of fish origin, and there was also a near-complete turnover in microbiome structure. However, we also identified, for the first time in fish, evidence of historical colonisation effects reflecting early-life experience, including ASVs characteristic of captive rearing. These results have important implications for host adaptation to local selective pressures, and highlight how conditions experienced during early life can have a long-term influence on the microbiome and, potentially, host health.

More than meets the eye: syntopic and morphologically similar mangrove killifish species show different mating systems and patterns of genetic structure along the Brazilian coast.

Different mating systems can strongly affect the extent of genetic diversity and population structure among species. Given the increased effects of genetic drift on reduced population size, theory predicts that species undergoing self-fertilisation should have greater population structure than outcrossed species; however, demographic dynamics may affect this scenario. The mangrove killifish clade is composed of the two only known examples of self-fertilising species among vertebrates (Kryptolebias marmoratus and Kryptolebias hermaphroditus). A third species in this clade, Kryptolebias ocellatus, inhabits mangrove forests in southeast Brazil; however, its mating system and patterns of genetic structure have been rarely explored. Here, we examined the genetic structure and phylogeographic patterns of K. ocellatus along its distribution, using mitochondrial DNA and microsatellites to compare its patterns of genetic structure with the predominantly selfing and often-syntopic, K. hermaphroditus. Our results indicate that K. ocellatus reproduces mainly by outcrossing, with no current evidence of selfing, despite being an androdioecious species. Our results also reveal a stronger population subdivision in K. ocellatus compared to K. hermaphroditus, contrary to the theoretical predictions based on reproductive biology of the two species. Our findings indicate that, although morphologically similar, K. ocellatus and K. hermaphroditus had remarkably different evolutionary histories when colonising the same mangrove areas in southeastern Brazil, with other factors (e.g., time of colonisation, dispersal/establishment capacity) having more profound effects on the current population structuring of those species than differences in mating systems.

Comparative transcriptomics reveal conserved impacts of rearing density on immune response of two important aquaculture species.

Infectious diseases represent an important barrier to sustainable aquaculture development. Rearing density can substantially impact fish productivity, health and welfare in aquaculture, including growth rates, behaviour and, crucially, immune activity. Given the current emphasis on aquaculture diversification, stress-related indicators broadly applicable across species are needed. Utilising an interspecific comparative transcriptomic (RNAseq) approach, we compared gill gene expression responses of Atlantic salmon (Salmo salar) and Nile tilapia (Oreochromis niloticus) to rearing density and Saprolegnia parasitica infection. Salmon reared at high-density showed increased expression of stress-related markers (e.g. c-fos and hsp70), and downregulation of innate immune genes. Upon pathogen challenge, only salmon reared at low density exhibited increased expression of inflammatory interleukins and lymphocyte-related genes. Tilapia immunity, in contrast, was impaired at low-density. Using overlapping gene ontology enrichment and gene ortholog analyses, we found that density-related stress similarly impacted salmon and tilapia in key immune pathways, altering the expression of genes vital to inflammatory and Th17 responses to pathogen challenge. Given the challenges posed by ectoparasites and gill diseases in fish farms, this study underscores the importance of optimal rearing densities for immunocompetence, particularly for mucosal immunity. Our comparative transcriptomics analyses identified density stress impacted immune markers common across different fish taxa, providing key molecular targets with potential for monitoring and enhancing aquaculture resilience in a wide range of farmed species.

Crayfish plague affects juvenile survival and adult behaviour of invasive signal crayfish.

The spread of invasive, non-native species is a key threat to biodiversity. Parasites can play a significant role by influencing their invasive host's survival or behaviour, which can subsequently alter invasion dynamics. The North American signal crayfish (Pacifastacus leniusculus) is a known carrier of Aphanomyces astaci, an oomycete pathogen that is the causative agent of crayfish plague and fatal to European crayfish species, whereas North American species are considered to be largely resistant. There is some evidence, however, that North American species, can also succumb to crayfish plague, though how A. astaci affects such 'reservoir hosts' is rarely considered. Here, we tested the impact of A. astaci infection on signal crayfish, by assessing juvenile survival and adult behaviour following exposure to A. astaci zoospores. Juvenile signal crayfish suffered high mortality 4-weeks post-hatching, but not as older juveniles. Furthermore, adult signal crayfish with high-infection levels displayed altered behaviours, being less likely to leave the water, explore terrestrial areas and exhibit escape responses. Overall, we reveal that A. astaci infection affects signal crayfish to a much greater extent than previously considered, which may not only have direct consequences for invasions, but could substantially affect commercially harvested signal crayfish stocks worldwide.

Smell of Infection: A Novel, Noninvasive Method for Detection of Fish Excretory-Secretory Proteins.

Chemical signals are produced by aquatic organisms following predatory attacks or perturbations such as parasitic infection. Ectoparasites feeding on fish hosts are likely to cause release of similar alarm cues into the environment due to the stress, wounding, and immune response stimulated upon infection. Alarm cues are often released in the form of proteins, antimicrobial peptides, and immunoglobulins that provide important insights into bodily function and infection status. Here we outline a noninvasive method to identify potential chemical cues associated with infection in fish by extracting, purifying, and characterizing proteins from water samples from cultured fish. Gel free proteomic methods were deemed the most suitable for protein detection in saline water samples. It was confirmed that teleost proteins can be characterized from water and that variation in protein profiles could be detected between infected and uninfected individuals and fish and parasite only water samples. Our novel assay provides a noninvasive method for assessing the health condition of both wild and farmed aquatic organisms. Similar to environmental DNA monitoring methods, these proteomic techniques could provide an important tool in applied ecology and aquatic biology.

Transcriptomic response to parasite infection in Nile tilapia (Oreochromis niloticus) depends on rearing density.

BACKGROUND: Captive animal populations, be it for food production or conservation programmes, are often maintained at densities far beyond those in natural environments, which can have profound effects on behaviour, immune and stress levels, and ultimately welfare. How such alterations impact transcriptional responses to pathogen infection is a 'different kettle of fish' and remains poorly understood. Here, we assessed survival and gene expression profiles of infected fish reared at two different densities to elucidate potential functional genomic mechanisms for density-related differences in disease susceptibility. RESULTS: Utilising a whole-transcriptome sequencing (RNAseq) approach, we demonstrate that rearing density in tilapia (Oreochromis niloticus) significantly impacts susceptibility to the oomycete Saprolegnia parasitica, via altered transcriptional infection responses. Tilapia held at low densities have increased expression of genes related to stress, likely due to increased aggressive interactions. When challenged with Saprolegnia, low-density fish exhibit altered expression of inflammatory gene responses and enhanced levels of adaptive immune gene suppression compared to fish reared at higher density, resulting in significantly increased mortality rates. In addition, Saprolegnia infection substantially perturbs expression of circadian clock genes, with fish reared at low-density having higher levels of molecular clock dysregulation. CONCLUSIONS: Our results reveal the wide-scale impact of stocking density on transcriptional responses to infection and highlight the need to incorporate circadian biology into our understanding of disease dynamics in managed animals.

Interpopulation Variation in the Atlantic Salmon Microbiome Reflects Environmental and Genetic Diversity.

The microbiome has a crucial influence on host phenotype and is of broad interest to ecological and evolutionary research. Yet, the extent of variation that occurs in the microbiome within and between populations is unclear. We characterized the skin and gut microbiomes of seven populations of juvenile Atlantic salmon (Salmo salar) inhabiting a diverse range of environments, including hatchery-reared and wild populations. We found shared skin operational taxonomic units (OTUs) across all populations and core gut microbiota for all wild fish, but the diversity and structure of both skin and gut microbial communities were distinct between populations. There was a marked difference between the gut microbiomes of wild and captive fish. Hatchery-reared fish had lower intestinal microbial diversity, lacked core microbiota found in wild fish, and showed altered community structure and function. Skin and gut microbiomes were also less varied within captive populations, reflecting more uniform artificial rearing conditions. The surrounding water influenced the microbiome of the gut and, especially, the skin, but could not explain the degree of variation observed between populations. For both gut and skin, we found that there was greater difference in microbiome structures between more genetically distinct fish populations, and that population genetic diversity was positively correlated with microbiome diversity. However, diet is likely to be the major factor contributing to the large differences in gut microbiota between wild and captive fish. Our results highlight the scope of interpopulation variation in the Atlantic salmon microbiome and offer insights into the deterministic factors contributing to microbiome diversity and structure.IMPORTANCE Variation in the microbiome has a fundamental influence on host health, ecology, and evolution, but the scope and basis of this variation are not fully understood. We identified considerable variation in skin and gut microbial communities between seven wild and captive populations of Atlantic salmon, reflecting divergent environmental conditions and fish genetic diversity. In particular, we found very pronounced differences in the intestinal microbiomes of wild and hatchery-reared fish, likely reflecting differences in diet. Our results offer an insight into how the microbiome potentially contributes to the generation of local adaptations in this species and how domestication alters intestinal microbial communities, highlighting future research directions in these areas.

Ultrasensitive environmental assessment of xeno-estrogens in water samples using label-free graphene immunosensors.

There is a growing interest in the possible environmental health impact posed by endocrine-disrupting chemicals (EDCs). A challenge to the field of endocrine disruption is that these substances are diverse and may not appear to share any structural similarity other than usually being low molecular mass (<1000 Da) compounds. Here we demonstrate the effectiveness of sensor device for the detection of low molecular weight, poorly water soluble, estrogenic compounds E1, E2 and EE2, fabricated by electropolymerization over graphene screen printed electrode (SPE). The PANI/Gr-SPE-devices displayed linear responses to estrogenic substances, in EIS assays, from 0.0975 ng/L to 200 ng/L in water samples, with a detection limit of 0.043 pg/L for E1, 0.19 ng/L for E2 and 0.070 pg/L for EE2 which is lower than other current biosensing techniques. This portable, disposable immunosensor offers a solution for immediate measurement at sample collection sites, due to its excellent sensitivity and selectivity when testing water samples obtained directly from rivers and waste water treatment facilities. The simple screen printing production method will enable the low cost, high volume production required for this type of environmental analysis.

Epigenetic-induced alterations in sex-ratios in response to climate change: An epigenetic trap?

We hypothesize that under the predicted scenario of climate change epigenetically mediated environmental sex determination could become an epigenetic trap. Epigenetically regulated environmental sex determination is a mechanism by which species can modulate their breeding strategies to accommodate environmental change. Growing evidence suggests that epigenetic mechanisms may play a key role in phenotypic plasticity and in the rapid adaptation of species to environmental change, through the capacity of organisms to maintain a non-genetic plastic memory of the environmental and ecological conditions experienced by their parents. However, inherited epigenetic variation could also be maladaptive, becoming an epigenetic trap. This is because environmental sex determination can alter sex ratios by increasing the survival of one of the sexes at the expense of negative fitness consequences for the other, which could lead not only to the collapse of natural populations, but also have an impact in farmed animal and plant species.

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates.

Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus. We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus, with naturally inbred populations, represents a good vertebrate model for epigenetic studies.

Patterns of natural selection acting on the mitochondrial genome of a locally adapted fish species.

BACKGROUND: Mitochondrial DNA (mtDNA) is frequently used in population genetic studies and is usually considered as a neutral marker. However, given the functional importance of the proteins encoded by the mitochondrial genome, and the prominent role of mitochondria in cellular energy production, the assumption of neutrality is increasingly being questioned. RESULTS: We tested for evidence of selection on the mitochondrial genome of the Atlantic salmon, which is a locally adapted and widely farmed species and is distributed across a large latitudinal cline. We analysed 20 independent regions of the salmon mtDNA that represented nine genes (ND1, ND2, ND3, COX1, COX2, ATP6, ND4, ND5, and CYTB). These 20 mtDNA regions were sequenced using a 454 approach from samples collected across the entire European range of this species. We found evidence of positive selection at the ND1, ND3 and ND4 genes, which is supported by at least two different codon-based methods and also by differences in the chemical properties of the amino acids involved. The geographical distribution of some of the mutations indicated to be under selection was not random, and some mutations were private to artic populations. We discuss the possibility that selection acting on the Atlantic salmon mtDNA genome might be related to the need for increased metabolic efficiency at low temperatures. CONCLUSIONS: The analysis of sequences representing nine mitochondrial genes that are involved in the OXPHOS pathway revealed signatures of positive selection in the mitochondrial genome of the Atlantic salmon. The properties of the amino acids involved suggest that some of the mutations that were identified to be under positive selection might have functional implications, possibly in relation to metabolic efficiency. Experimental evidence, and better understanding of regional phylogeographic structuring, are needed to clarify the potential role of selection acting on the mitochondrial genome of Atlantic salmon and other locally adapted fishes.

Microbiome and epigenetic variation in wild fish with low genetic diversity.

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity.