When does a parasite become a disease? eDNA unravels complex host-pathogen dynamics across environmental stress gradients in wild salmonid populations.

Infectious diseases stem from disrupted interactions among hosts, parasites, and the environment. Both abiotic and biotic factors can influence infection outcomes by shaping the abundance of a parasite's infective stages, as well as the host's ability to fight infection. However, disentangling these mechanisms within natural ecosystems remains challenging. Here, combining environmental DNA analysis and niche modelling at a regional scale, we uncovered the biotic and abiotic drivers of an infectious disease of salmonid fish, triggered by the parasite Tetracapsuloides bryosalmonae. We found that the occurrence and abundance of the parasite in the water-i.e., the propagule pressure- were mainly correlated to the abundances of its two primary hosts, the bryozoan Fredericella sultana and the fish Salmo trutta, but poorly to local abiotic environmental stressors. In contrast, the occurrence and abundance of parasites within fish hosts-i.e., proxies for disease emergence-were closely linked to environmental stressors (water temperature, agricultural activities, dams), and to a lesser extent to parasite propagule pressure. These results suggest that pathogen distribution alone cannot predict the risk of disease in wildlife, and that local anthropogenic stressors may play a pivotal role in disease emergence among wild host populations, likely by modulating the hosts' immune response. Our study sheds light on the intricate interplay between biotic and abiotic factors in shaping pathogen distribution and raises concerns about the effects of global change on pathogen emergence.

A longitudinal survey in the wild reveals major shifts in fish host microbiota after parasite infection.

Recent studies have highlighted associations between diseases and host microbiota. It remains extremely challenging - especially under natural conditions - to clarify whether host microbiota promote future infections, or whether changes in host microbiota result from infections. Nonetheless, deciphering between these two processes is essential for highlighting the role of microbes in disease progression. We longitudinally surveyed, in the wild, the microbiota of individual fish hosts (Leuciscus burdigalensis) both before and after infection by a crustacean ectoparasite (Tracheliastes polycolpus). We found a striking association between parasite infection and the host microbiota composition restricted to the fins the parasite anchored. We clearly demonstrated that infections by the parasite induced a shift in (and did not result from) the host fin microbiota. Furthermore during infection, the microbiota of infected fins got similar to the microbiota of the adult stage, and the free-living infective stage of the parasite with a predominance of the Burkholderiaceae bacteria family. This suggests that some Burkholderiaceae bacteria are involved in a coinfection process and possibly facilitate T. polycolpus infection. In this study, we reveal novel mechanistic insights for understanding the role of the microbiota in host-parasite interactions, which has implications for predicting the progression of diseases in natural host populations.

Changes in fish skin microbiota along gradients of eutrophication in human-altered rivers.

The skin microbiota plays a major role in health of organisms but it is still unclear how such bacterial assemblages respond to changes in environmental conditions and anthropogenic perturbations. In this study, we investigated the effects of the eutrophication of freshwater ecosystems on the skin microbiota of fish. We sampled wild gudgeon Gobio occitaniae from 17 river sites along an eutrophication gradient and compared their skin microbiota diversity and composition, using a 16s rRNA gene metabarcoding approach. Results showed a tendency for higher taxonomic and phylogenetic diversity in highly eutrophic sites linked to the presence of suspended organic matters. We also highlighted significant links between eutrophication and skin microbiota taxonomic composition and beta-diversity. In contrast, skin microbiota characteristics did not correlate with host factors such as age or sex, although microbiota beta-diversity did vary significantly according to host parasite load. To conclude, our study highlights the importance of environmental factors, especially eutrophication, on the diversity and composition of skin mucus bacterial communities. Because changes in the skin microbiota may induce potential deleterious consequences on host health and population persistence, our results confirm the importance of accounting for host-microbiota interactions when examining the consequences of anthropogenic activities on aquatic fauna.

Functional variability of rhesus macaque (Macaca mulatta) NAT2 gene for drug-metabolising arylamine N-acetyltransferase 2.

Human NAT2 is a polymorphic pharmacogene encoding for N-acetyltransferase 2, a hepatic enzyme active towards arylamine and arylhydrazine drugs, including the anti-tubercular antibiotic isoniazid. The isoenzyme also modulates susceptibility to chemical carcinogenesis, particularly of the bladder. Human NAT2 represents an ideal model for anthropological investigations into the demographic adaptation of worldwide populations to their xenobiotic environment. Its sequence appears to be subject to positive selection pressures that are population-specific and may be attributed to gene-environment interactions directly associated with exogenous chemical challenges. However, recent evidence suggests that the same evolutionary pattern may not be observed in other primates. Here, we report NAT2 polymorphism in 25 rhesus macaques (Macaca mulatta) and compare the frequencies and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified, including one nonsense mutation. The missense SNVs were demonstrated to affect enzymatic function in a substrate-dependent manner, albeit more moderately than certain NAT1 SNVs recently characterised in the same cohort. Haplotypic and functional variability of NAT2 was comparable to that previously observed for NAT1 in the same population sample, suggesting that the two paralogues may have evolved under similar selective pressures in the rhesus macaque. This is different to the population variability distribution pattern reported for humans and chimpanzees. Recorded SNVs were also different from those found in other primates. The study contributes to further understanding of NAT2 functional polymorphism in the rhesus macaque, a non-human primate model used in biomedicine and pharmacology, indicating variability in xenobiotic acetylation that could affect drug metabolism.

Patterns of Epigenetic Diversity in Two Sympatric Fish Species: Genetic vs. Environmental Determinants.

Epigenetic components are hypothesized to be sensitive to the environment, which should permit species to adapt to environmental changes. In wild populations, epigenetic variation should therefore be mainly driven by environmental variation. Here, we tested whether epigenetic variation (DNA methylation) observed in wild populations is related to their genetic background, and/or to the local environment. Focusing on two sympatric freshwater fish species (Gobio occitaniae and Phoxinus phoxinus), we tested the relationships between epigenetic differentiation, genetic differentiation (using microsatellite and single nucleotide polymorphism (SNP) markers), and environmental distances between sites. We identify positive relationships between pairwise genetic and epigenetic distances in both species. Moreover, epigenetic marks better discriminated populations than genetic markers, especially in G. occitaniae. In G. occitaniae, both pairwise epigenetic and genetic distances were significantly associated to environmental distances between sites. Nonetheless, when controlling for genetic differentiation, the link between epigenetic differentiation and environmental distances was not significant anymore, indicating a noncausal relationship. Our results suggest that fish epigenetic variation is mainly genetically determined and that the environment weakly contributed to epigenetic variation. We advocate the need to control for the genetic background of populations when inferring causal links between epigenetic variation and environmental heterogeneity in wild populations.

Quantifying the individual impact of artificial barriers in freshwaters: A standardized and absolute genetic index of fragmentation.

Fragmentation by artificial barriers is an important threat to freshwater biodiversity. Mitigating the negative aftermaths of fragmentation is of crucial importance, and it is now essential for environmental managers to benefit from a precise estimate of the individual impact of weirs and dams on river connectivity. Although the indirect monitoring of fragmentation using molecular data constitutes a promising approach, it is plagued with several constraints preventing a standardized quantification of barrier effects. Indeed, observed levels of genetic differentiation GD depend on both the age of the obstacle and the effective size of the populations it separates, making comparisons of the actual barrier effect of different obstacles difficult. Here, we developed a standardized genetic index of fragmentation (F INDEX), allowing an absolute and independent assessment of the individual effects of obstacles on connectivity. The F INDEX is the standardized ratio between the observed GD between pairs of populations located on either side of an obstacle and the GD expected if this obstacle completely prevented gene flow. The expected GD is calculated from simulations taking into account two parameters: the number of generations since barrier creation and the expected heterozygosity of the populations, a proxy for effective population size. Using both simulated and empirical datasets, we explored the validity and the limits of the F INDEX. We demonstrated that it allows quantifying effects of fragmentation only from a few generations after barrier creation and provides valid comparisons among obstacles of different ages and populations (or species) of different effective sizes. The F INDEX requires a minimum amount of fieldwork and genotypic data and solves some of the difficulties inherent to the study of artificial fragmentation in rivers and potentially in other ecosystems. This makes the F INDEX promising to support the management of freshwater species affected by barriers, notably for planning and evaluating restoration programs.

Population variability of rhesus macaque (Macaca mulatta) NAT1 gene for arylamine N-acetyltransferase 1: Functional effects and comparison with human.

Human NAT1 gene for N-acetyltransferase 1 modulates xenobiotic metabolism of arylamine drugs and mutagens. Beyond pharmacogenetics, NAT1 is also relevant to breast cancer. The population history of human NAT1 suggests evolution through purifying selection, but it is unclear whether this pattern is evident in other primate lineages where population studies are scarce. We report NAT1 polymorphism in 25 rhesus macaques (Macaca mulatta) and describe the haplotypic and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified and experimentally demonstrated to compromise enzyme function, mainly through destabilization of NAT1 protein and consequent activity loss. One non-synonymous SNV (c.560G > A, p.Arg187Gln) has also been characterized for human NAT1 with similar effects. Population haplotypic and functional variability of rhesus NAT1 was considerably higher than previously reported for its human orthologue, suggesting different environmental pressures in the two lineages. Known functional elements downstream of human NAT1 were also differentiated in rhesus macaque and other primates. Xenobiotic metabolizing enzymes play roles beyond mere protection from exogenous chemicals. Therefore, any link to disease, particularly carcinogenesis, may be via modulation of xenobiotic mutagenicity or more subtle interference with cell physiology. Comparative analyses add the evolutionary dimension to such investigations, assessing functional conservation/diversification among primates.

Environmental conditions and neutral processes shape the skin microbiome of European catfish (Silurus glanis) populations of Southwestern France.

Teleost fishes interact with diverse microbial communities, playing crucial functions for host fitness. While gut microbiome has been extensively studied, skin microbiome has been overlooked. Specifically, there is no assessment of the relative impact of host and environmental factors on microbiome variability as well as neutral processes shaping fish skin microbiome. Here, we assessed the skin microbiome of a Siluriforme, the European catfish (Silurus glanis) sampled in four sites located in Southwestern France. We assessed the relative roles of individual features (body size and genetic background), local environment and neutral processes in shaping skin microbiome. Catfish skin microbiome composition was distinct to that of other freshwater fish species previously studied with high abundances of Gammaproteobacteria and Bacteroidetes. We found no effect of catfish individual genotype and body size on the structure of its associated skin microbiome. Geographical location was the best catfish skin microbiome structure predictor, together with neutral models of microbiome assembly.

Systematic conservation planning for intraspecific genetic diversity.

Intraspecific diversity informs the demographic and evolutionary histories of populations, and should be a main conservation target. Although approaches exist for identifying relevant biological conservation units, attempts to identify priority conservation areas for intraspecific diversity are scarce, especially within a multi-specific framework. We used neutral molecular data on six European freshwater fish species (Squalius cephalus, Phoxinus phoxinus, Barbatula barbatula, Gobio occitaniae, Leuciscus burdigalensis and Parachondrostoma toxostoma) sampled at the riverscape scale (i.e. the Garonne-Dordogne river basin, France) to determine hot- and coldspots of genetic diversity, and to identify priority conservation areas using a systematic conservation planning approach. We demonstrate that systematic conservation planning is efficient for identifying priority areas representing a predefined part of the total genetic diversity of a whole landscape. With the exception of private allelic richness (PA), classical genetic diversity indices (allelic richness, genetic uniqueness) were poor predictors for identifying priority areas. Moreover, we identified weak surrogacies among conservation solutions found for each species, implying that conservation solutions are highly species-specific. Nonetheless, we showed that priority areas identified using intraspecific genetic data from multiple species provide more effective conservation solutions than areas identified for single species or on the basis of traditional taxonomic criteria.

Inferring Causalities in Landscape Genetics: An Extension of Wright's Causal Modeling to Distance Matrices.

Identifying landscape features that affect functional connectivity among populations is a major challenge in fundamental and applied sciences. Landscape genetics combines landscape and genetic data to address this issue, with the main objective of disentangling direct and indirect relationships among an intricate set of variables. Causal modeling has strong potential to address the complex nature of landscape genetic data sets. However, this statistical approach was not initially developed to address the pairwise distance matrices commonly used in landscape genetics. Here, we aimed to extend the applicability of two causal modeling methods-that is, maximum-likelihood path analysis and the directional separation test-by developing statistical approaches aimed at handling distance matrices and improving functional connectivity inference. Using simulations, we showed that these approaches greatly improved the robustness of the absolute (using a frequentist approach) and relative (using an information-theoretic approach) fits of the tested models. We used an empirical data set combining genetic information on a freshwater fish species (Gobio occitaniae) and detailed landscape descriptors to demonstrate the usefulness of causal modeling to identify functional connectivity in wild populations. Specifically, we demonstrated how direct and indirect relationships involving altitude, temperature, and oxygen concentration influenced within- and between-population genetic diversity of G. occitaniae.

Elucidating the spatio-temporal dynamics of an emerging wildlife pathogen using approximate Bayesian computation.

Emerging pathogens constitute a severe threat for human health and biodiversity. Determining the status (native or non-native) of emerging pathogens, and tracing back their spatio-temporal dynamics, is crucial to understand the eco-evolutionary factors promoting their emergence, to control their spread and mitigate their impacts. However, tracing back the spatio-temporal dynamics of emerging wildlife pathogens is challenging because (i) they are often neglected until they become sufficiently abundant and pose socio-economical concerns and (ii) their geographical range is often little known. Here, we combined classical population genetics tools and approximate Bayesian computation (i.e. ABC) to retrace the dynamics of Tracheliastes polycolpus, a poorly documented pathogenic ectoparasite emerging in Western Europe that threatens several freshwater fish species. Our results strongly suggest that populations of T. polycolpus in France emerged from individuals originating from a unique genetic pool that were most likely introduced in the 1920s in central France. From this initial population, three waves of colonization occurred into peripheral watersheds within the next two decades. We further demonstrated that populations remained at low densities, and hence undetectable, during 10 years before a major demographic expansion occurred, and before its official detection in France. These findings corroborate and expand the few historical records available for this emerging pathogen. More generally, our study demonstrates how ABC can be used to determine the status, reconstruct the colonization history and infer key evolutionary parameters of emerging wildlife pathogens with low data availability, and for which samples from the putative native area are inaccessible.

Rapid birth-and-death evolution of the xenobiotic metabolizing NAT gene family in vertebrates with evidence of adaptive selection.

BACKGROUND: The arylamine N-acetyltransferases (NATs) are a unique family of enzymes widely distributed in nature that play a crucial role in the detoxification of aromatic amine xenobiotics. Considering the temporal changes in the levels and toxicity of environmentally available chemicals, the metabolic function of NATs is likely to be under adaptive evolution to broaden or change substrate specificity over time, making NATs a promising subject for evolutionary analyses. In this study, we trace the molecular evolutionary history of the NAT gene family during the last ~450 million years of vertebrate evolution and define the likely role of gene duplication, gene conversion and positive selection in the evolutionary dynamics of this family. RESULTS: A phylogenetic analysis of 77 NAT sequences from 38 vertebrate species retrieved from public genomic databases shows that NATs are phylogenetically unstable genes, characterized by frequent gene duplications and losses even among closely related species, and that concerted evolution only played a minor role in the patterns of sequence divergence. Local signals of positive selection are detected in several lineages, probably reflecting response to changes in xenobiotic exposure. We then put a special emphasis on the study of the last ~85 million years of primate NAT evolution by determining the NAT homologous sequences in 13 additional primate species. Our phylogenetic analysis supports the view that the three human NAT genes emerged from a first duplication event in the common ancestor of Simiiformes, yielding NAT1 and an ancestral NAT gene which in turn, duplicated in the common ancestor of Catarrhini, giving rise to NAT2 and the NATP pseudogene. Our analysis suggests a main role of purifying selection in NAT1 protein evolution, whereas NAT2 was predicted to mostly evolve under positive selection to change its amino acid sequence over time. These findings are consistent with a differential role of the two human isoenzymes and support the involvement of NAT1 in endogenous metabolic pathways. CONCLUSIONS: This study provides unequivocal evidence that the NAT gene family has evolved under a dynamic process of birth-and-death evolution in vertebrates, consistent with previous observations made in fungi.

Combining genetic and demographic data for prioritizing conservation actions: insights from a threatened fish species.

Prioritizing and making efficient conservation plans for threatened populations requires information at both evolutionary and ecological timescales. Nevertheless, few studies integrate multidisciplinary approaches, mainly because of the difficulty for conservationists to assess simultaneously the evolutionary and ecological status of populations. Here, we sought to demonstrate how combining genetic and demographic analyses allows prioritizing and initiating conservation plans. To do so, we combined snapshot microsatellite data and a 30-year-long demographic survey on a threatened freshwater fish species (Parachondrostoma toxostoma) at the river basin scale. Our results revealed low levels of genetic diversity and weak effective population sizes (<63 individuals) in all populations. We further detected severe bottlenecks dating back to the last centuries (200-800 years ago), which may explain the differentiation of certain populations. The demographic survey revealed a general decrease in the spatial distribution and abundance of P. toxostoma over the last three decades. We conclude that demo-genetic approaches are essential for (1) identifying populations for which both evolutionary and ecological extinction risks are high; and (2) proposing conservation plans targeted toward these at risk populations, and accounting for the evolutionary history of populations. We suggest that demo-genetic approaches should be the norm in conservation practices. We combined genetic and demographic data from a threatened freshwater fish species (Parachondrostoma toxostoma) at the river basin scale for conservation purposes. Genetic diversity and effective population sizes are very low, probably due to the strong genetic bottlenecks detected in this study. The species spatial distribution and abundance also decreased during the last decades.