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BackgroundCircadian rhythms of host immune activity and their microbiomes are likely pivotal to health and disease resistance. The integration of chronotherapeutic approaches to disease mitigation in managed animals, however, is yet to be realised. In aquaculture, light manipulation is commonly used to enhance growth and control reproduction but may have unknown negative consequences for animal health. Infectious diseases are a major barrier to sustainable aquaculture and understanding the circadian dynamics of fish immunity and crosstalk with the microbiome is urgently needed.ResultsHere, using rainbow trout (Oncorhynchus mykiss) as a model, we combine 16S rRNA metabarcoding, metagenomic sequencing and direct mRNA quantification methods to simultaneously characterise the circadian dynamics of skin clock and immune gene expression, and daily changes of skin microbiota. We demonstrate daily rhythms in fish skin immune expression and microbiomes, which are modulated by photoperiod and parasitic lice infection. We identify putative associations of host clock and immune gene profiles with microbial composition. Our results suggest circadian perturbation, that shifts the magnitude and timing of immune and microbiota activity, is detrimental to fish health.ConclusionsThe substantial circadian dynamics and fish host expression-microbiome relationships we find represent a valuable foundation for investigating the utility of chronotherapies in aquaculture, and more broadly contributes to our understanding of the role of microbiomes in circadian health of vertebrates.

The ubiquitous freshwater pathogen Saprolegnia parasitica has long been considered a true generalist, capable of infecting a wide range of fish species. It remains unclear, however, whether different isolates of this pathogen, obtained from distinct geographic locations and host species, display differences in host preference. To assess this, the current study examined the induced zoospore encystment responses of four S. parasitica isolates towards the skin of four fish species. While three of the isolates displayed ‘specialist’ responses, one appeared to be more of a ‘generalist’. In vivo challenge infections involving salmon and sea trout with the ‘generalist’ (salmon isolate EA001) and a ‘specialist’ (sea trout isolate EA016) pathogen, however, did not support the in vitro findings, with no apparent host preference reflected in infection outcomes. Survival of sea trout and salmon though was significantly different following a challenge infection with the sea trout (EA016) isolate. These results indicate that while S. parasitica isolates can be considered true generalists, they may target hosts to which they have been more frequently exposed (potential local adaptation). Understanding host preference of this pathogen could aid our understanding of infection epidemics and help with the development of fish management procedures.

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.

Phenotypic plasticity in ancestral populations is hypothesised to facilitate adaptation, but evidence supporting its contribution is piecemeal and often contradictory. Further, whether ancestral plasticity increases the probability of parallel genetic and phenotypic adaptive changes has not been explored. The most general finding is that nearly all ancestral gene expression plasticity is reversed following adaptation, but this is usually examined transcriptome-wide rather than focused on the genes directly involved in adaptation. We investigated the contribution of ancestral plasticity to adaptive evolution of gene expression in two independently evolved lineages of zinc-tolerant Silene uniflora. We found that the general pattern of reversion is driven by the absence of a widespread stress response in zinc-adapted plants compared to ancestral, zinc-sensitive plants. Our experiments show that reinforcement of ancestral plasticity plays an influential role in the evolution of plasticity in derived populations and, surprisingly, one third of constitutive differences between ecotypes are the result of genetic assimilation of ancestral plasticity. Ancestral plasticity also increases the chance that genes are recruited repeatedly during adaptation. However, despite a high degree of convergence in gene expression levels between independently adapted lineages, genes with ancestral plasticity are as likely to have similar expression levels in adapted populations as genes without. Overall, these results demonstrate that ancestral plasticity does play an important role in adaptive parallel evolution, particularly via genetic assimilation across evolutionary replicates.

Parasites display a wide range of behaviours that are frequently overlooked in favour of host responses. Understanding these behaviours can improve parasite control through a more precise application or development of new behaviour-based strategies. In aquaculture fish lice are an ongoing problem, infections reduce fishery production and control options are limited. Fish lice are distinct in their ability to survive and swim off hosts, allowing the transmission to multiple fish hosts across their lifespan. Here we assessed the off-host behaviour of Argulus foliaceus (a freshwater fish louse) and observed a diurnal rhythmical pattern in their behaviour. This pattern was lost when lice were exposed to constant darkness, indicating that the behaviour is not endogenously driven. Males were consistently active in light with reduced activity in darkness. In contrast, females were active during light and dark phases with peak activity at the start of dark periods. A. foliaceus was also strongly attracted to a light stimulus, preferring white- and blue-coloured lights over green- or red-coloured lights. Light is a strong driver of fish louse activity and could be used to trap parasites. Aquaculture light regimes could also be altered to reduce parasite attraction and activity.

The transport of fish in aquaculture and the ornamental trade exposes fish to multiple stressors that can cause mass mortalities and economic loss. Previous research on fish transport has largely focussed on chemical stress related to deterioration in water quality. However, mechanical disturbance during routine fish transport is unpredictable and is a neglected potential stressor when studying fish welfare. Stress-induced immunosuppression caused by mechanical disturbance can increase the chances of contracting infections and can significantly increase infection burden. Here, using a model host-parasite system (guppy Poecilia reticulata and the monogenean ectoparasite Gyrodactylus turnbulli) and a new method of bagging fish (Breathing Bags™), which reduces mechanical disturbance during fish transport, we investigated how parasite infections contracted after simulated transport impact infection trajectories on a globally important ornamental freshwater species. Guppies exposed to mechanical transport disturbance suffered significantly higher parasite burden compared to fish that did not experience transport disturbance. Unfortunately, there was no significant reduction in parasite burden of fish transported in the Breathing Bags™ compared to standard polythene carrier bags. Thus, transport-induced mechanical disturbance, hitherto neglected as a stressor, can be detrimental to disease resistance and highlights the need for specific management procedures to reduce the impact of infectious diseases following routine fish transport.

Parasites display a wide range of behaviours that are frequently overlooked in favour of host responses. Understanding these behaviours can improve parasite control through a more precise application or development of new behaviour-based strategies. In aquaculture fish lice are an ongoing problem, infections reduce fishery production and control options are limited. Fish lice are distinct in their ability to survive and swim off hosts, allowing the transmission to multiple fish hosts across their lifespan. Here we assessed the off-host behaviour of Argulus foliaceus (a freshwater fish louse) and observed a diurnal rhythmical pattern in their behaviour. This pattern was lost when lice were exposed to constant darkness, indicating that the behaviour is not endogenously driven. Males were consistently active in light with reduced activity in darkness. In contrast, females were active during light and dark phases with peak activity at the start of dark periods. A. foliaceus was also strongly attracted to a light stimulus, preferring white- and blue-coloured lights over green- or red-coloured lights. Light is a strong driver of fish louse activity and could be used to trap parasites. Aquaculture light regimes could also be altered to reduce parasite attraction and activity.

1. The increasing illumination of our world by artificial light at night (ALAN) has created a new field of global change research with impacts now being demonstrated across taxa, biological ranks and spatial scales. Following advances in terrestrial ecology, marine ALAN has become a rapidly growing research area attracting scientists from across all biomes. Technological limitations, complexities of researching many coastal and marine ecosystems and the interdisciplinary nature of ALAN research present numerous challenges. 2. Drawing on expertise from optical oceanographers, modellers, community ecologists, experimental and molecular biologists, we share practical advice and solutions that have proven useful for marine ALAN research. Discussing lessons learnt early on can help in the effective and efficient development of a field. 3. The guide follows a sensory ecology approach to marine light pollution and consolidates physics, ecology and biology. First, we introduce marine lightscapes highlighting how these differ from terrestrial ones and provide an overview of biological adaptations to them. Second, we discuss study design and technology to best quantify ALAN exposure of and impacts on marine and coastal organisms including molecular tools and approaches to scale-up marine ALAN research. 4. We conclude that the growing field of marine ALAN research presents opportunities not only for improving our understanding of this globally widespread stressor, but also for advancing fundamental marine photobiology, chronobiology and night-time ecology. Interdisciplinary research will be essential to gain insights into natural marine lightscapes shaping the ecology and evolution coastal and marine ecosystems.

Gyrodactylids are ubiquitous ectoparasites of teleost fish, but our understanding of the host immune response against them is fragmentary. Here, we used RNA‐Seq to investigate genes involved in the primary response to infection with Gyrodactylus bullatarudis on the skin of guppies, Poecilia reticulata, an important evolutionary model, but also one of the most common fish in the global ornamental trade. Analysis of differentially expressed genes identified several immune‐related categories, including IL‐17 signalling pathway and Th17 cell differentiation, cytokine‐cytokine receptor interaction, chemokine signalling pathway, NOD‐like receptor signalling pathway, natural killer cell‐mediated cytotoxicity and pathways involved in antigen recognition, processing and presentation. Components of both the innate and the adaptive immune responses play a role in response to gyrodactylid infection. Genes involved in IL‐17/Th17 response were particularly enriched among differentially expressed genes, suggesting a significant role for this pathway in fish responses to ectoparasites. Our results revealed a sizable list of genes potentially involved in the teleost‐gyrodactylid immune response.

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 ofAphanomyces 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 howA. astaciaffects such ‘reservoir hosts’ is rarely considered. Here, we tested the impact ofA. astaciinfection on signal crayfish, by assessing juvenile survival and adult behaviour following exposure toA. astacizoospores. 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 thatA. astaciinfection 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.

A lack of environmental enrichment can be severely detrimental to animal welfare. For terrestrial species, including humans, barren environments are associated with reduced cognitive function and increased stress responses and pathology. Despite a clear link between increased stress and reduced immune function, uncertainty remains on how enrichment might influence susceptibility to disease. For aquatic vertebrates, we are only now beginning to assess enrichment needs. Enrichment deprivation in fish has been linked to increased stress responses, agonistic behaviour, physiological changes and reduced survival. Limited data exist, however, on the impact of enrichment on disease resistance in fish, despite infectious diseases being a major challenge for global aquaculture. Here, using a model vertebrate host-parasite system we investigated the impact of enrichment deprivation on susceptibility to disease, behaviour and physiology. Fish in barren tanks showed significantly higher infection burdens compared to those in enriched enclosures and they also displayed increased intraspecific aggression behaviour. Infections caused hosts to have significantly increased Standard Metabolic Rates compared to uninfected conspecifics, but this did not differ between enriched and barren tanks. This study highlights the universal physiological cost of parasite infection and the biological cost (increased susceptibility to infection and increased aggression) of depriving captive animals of environmental enrichment.

Background For healthcare systems, an ageing population poses challenges in the delivery of equitable and effective care. Frailty assessment has the potential to improve care in the intensive care setting, but applying assessment tools in critical illness may be problematic. The aim of this systematic review was to evaluate evidence for the feasibility and reliability of frailty assessment in critical care. Methods Our primary search was conducted in Medline, Medline In-process, EMBASE, CINAHL, PsycINFO, AMED, Cochrane Database of Systematic Reviews, and Web of Science (January 2001 to October 2017). We included observational studies reporting data on feasibility and reliability of frailty assessment in the critical care setting in patients 16 years and older. Feasibility was assessed in terms of timing of evaluation, the background, training and expertise required for assessors, and reliance upon proxy input. Reliability was assessed in terms of inter-rater reliability. Results Data from 11 study publications are included, representing 8 study cohorts and 7761 patients. Proxy involvement in frailty assessment ranged from 58 to 100%. Feasibility data were not well-reported overall, but the exclusion rate due to lack of proxy availability ranged from 0 to 45%, the highest rate observed where family involvement was mandatory and the assessment tool relatively complex (frailty index, FI). Conventional elements of frailty phenotype (FP) assessment required modification prior to use in two studies. Clinical staff tended to use a simple judgement-based tool, the clinical frailty scale (CFS). Inter-rater reliability was reported in one study using the CFS and although a good level of agreement was observed between clinician assessments, this was a small and single-centre study. Conclusion Though of unproven reliability in the critically ill, CFS was the tool used most widely by critical care clinical staff. Conventional FP assessment required modification for general application in critical care, and an FI-based assessment may be difficult to deliver by the critical care team on a routine basis. There is a high reliance on proxies for frailty assessment, and the reliability of frailty assessment tools in critical care needs further evaluation. Prospero registration number CRD42016052073.

For generalist pathogens, host species represent distinct selective environments, providing unique challenges for resource acquisition and defense from host immunity, potentially resulting in host-dependent differences in pathogen fitness. Gene expression modulation should be advantageous, responding optimally to a given host and mitigating the costs of generalism. Batrachochytrium dendrobatidis (Bd), a fungal pathogen of amphibians, shows variability in pathogenicity among isolates, and within-strain virulence changes rapidly during serial passages through artificial culture. For the first time, we characterize the transcriptomic profile of Bd in vivo, using laser-capture microdissection. Comparison of Bd transcriptomes (strain JEL423) in culture and in two hosts (Atelopus zeteki and Hylomantis lemur), reveals >2000 differentially expressed genes that likely include key Bd defense and host exploitation mechanisms. Variation in Bd transcriptomes from different amphibian hosts demonstrates shifts in pathogen resource allocation. Furthermore, expressed genotype variant frequencies of Bd populations differ between culture and amphibian skin, and among host species, revealing potential mechanisms underlying rapid changes in virulence and the possibility that amphibian community composition shapes Bd evolutionary trajectories. Our results provide new insights into how changes in gene expression and infecting population genotypes can be key to the success of a generalist fungal pathogen.

Background: 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. Results: 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. Conclusions: 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.

Life processes of ectothermic vertebrates are intimately linked to the temperature of their environment, influencing their metabolism, reproduction, behaviour and immune responses. In amphibians infected by the generalist chytrid pathogen Batrachochytrium dendrobatidis (Bd), host survival, infection prevalence and infection intensity are often temperature- and/or seasonally dependent. However, the transcriptional underpinnings of thermal differences in infection responses remain unknown. Measuring the impact of temperature on host responses to infection is a key component for understanding climatic influences on chytrid disease dynamics. The Bd-responsive gene pathways in frogs are well documented, but our understanding of salamander immune expression profiles during infection with chytrids remains limited. Here we characterize the transcriptomic responses of Plethodon cinereus using RNA sequencing by comparing skin and splenic gene expression of individuals uninfected, succumbing to Bd infection and naturally cleared of Bd infection at three temperatures. We suggest that amphibian temperature-dependent susceptibility to Bd is probably driven by shifts in expression of the innate and adaptive immune axes. Our study shows increased expression of transcripts associated with inflammation at lower temperatures and a shift towards increased expression of adaptive immune genes, including MHC (major histocompatibility complex), at higher temperatures. In the face of climate change, and as concerns for the spread of emergent chytrid pathogens increase, our results provide important functional genomic resources to help understand how these pathogenic fungi may continue to affect amphibian communities globally in the future.

Cleaning interactions, which involve a cleaner removing ectoparasites and other material from the body of a heterospecific (client), are iconic symbiotic interactions observed on coral reefs worldwide. These small cleaners play a disproportionately large role in the structuring and function of coral reefs, influencing species interaction networks, client health and biodiversity. Cleaning patterns, however, are likely to be context-dependent and highly heterogeneous, and although we have some understanding about their spatial variation, longer-term temporal changes in cleaning interactions have remained understudied. Given that coral reefs are globally threatened and are currently experiencing large shifts in their biodiversity, it is vital that we determine which clients are consistently most important for maintaining cleaning. Using a long-term, 8-yr data set (2010–17) on the cleaning behaviour of the predominant Caribbean cleaner, the sharknose goby (Elacatinus evelynae), we investigated whether cleaner fish from the same reef show consistent patterns in their interactions with client species over time. Here we conclusively show that cleaning behaviour is highly plastic, as no single species or family was cleaned consistently more than others, in terms of cleaning frequency and duration, across all years. Only 40% of the species were cleaned more than others, and we also observed ca. one-third of species experience inconsistent cleaning patterns across years. Our study thus quantifies how dynamic cleaner–client relationships are on the same reef across years and highlights the importance of long-term data.

The major histocompatibility complex (MHC) plays an important role in infectious disease resistance. The presence of certain MHC alleles and functionally similar groups of MHC alleles (i.e., supertypes) has been associated with resistance to particular parasite species. Farmed and domesticated fish stocks are often depleted in their MHC alleles and supertype diversity, possibly as a consequence of artificial selection for desirable traits, inbreeding (loss of heterozygosity), genetic drift (loss of allelic diversity) and/or reduced parasite biodiversity. Here we quantify the effects of depletion of MHC class II genotype and supertype variation on resistance to the parasite Gyrodactylus turnbulli in guppies (Poecilia reticulata). Compared to the descendants of wild‐caught guppies, ornamental fish had a significantly reduced MHC variation (i.e., the numbers of MHC alleles and supertypes per individual, and per population). In addition, ornamental fish were significantly more susceptible to G. turnbulli infections, accumulating peak intensity 10 times higher than that of their wildtype counterparts. Four out of 13 supertypes were associated with a significantly reduced parasite load, and the presence of some supertypes had a dramatic effect on the intensity of infection. Remarkably, the ornamental and wildtype fish differed in the supertypes that were associated with parasite resistance. Analysis with a genetic algorithm showed that resistance‐conferring supertypes of the wildtype and ornamental fish shared two unique amino acids in the peptide‐binding region of the MHC that were not found in any other alleles. These data show that the supertype demarcation captures some, but not all, of the variation in the immune function of the alleles. This study highlights the importance of managing functional MHC diversity in livestock, and suggests there might be some immunological redundancy among MHC supertypes.

The amphibian-killing chytrid fungus Batrachochytriumdendrobatidis (Bd) is one of themost generalist pathogens known, capable of\ud infecting hundreds of species globally and causing widespread population declines and extinctions. However, some host species are\ud seemingly unaffected by Bd, tolerating or clearing infections without clinical signs of disease. Variation in host immune responses is\ud commonly evoked for these resistant or tolerant species, yet to date,we have nodirect comparisonof amphibian species responses to\ud infection at the level of gene expression. In this study,we challenged four CentralAmerican frog species that vary in Bd susceptibility,\ud with a sympatric virulent strain of the pathogen. We compared skin and spleen orthologous gene expression using differential\ud expression tests and coexpression gene network analyses.Wefound that resistant species have reduced skin inflammatory responses\ud andincreased expressionofgenes involved inskin integrity. Incontrast, onlyhighly susceptible species exhibited suppressionof splenic\ud T-cell genes. We conclude that resistance to chytridiomycosis may be related to a species’ ability to escape the immunosuppressive\ud activity of the fungus. Moreover, our results indicate that within-species differences in splenic proteolytic enzyme gene expression\ud may contribute to intraspecific variation in survival. This first comparison of amphibian functional immunogenomic architecture in\ud response to Bd provides insights into key genetic mechanisms underlying variation in disease outcomes among amphibian species.

Parasites vary widely in the diversity of hosts they infect: some parasite species are specialists—infecting just a single host species, while others are generalists, capable of infecting many. Understanding the factors that drive parasite host-generalism is of basic biological interest, but also directly relevant to predicting disease emergence in new host species, identifying parasites that are likely to have unidentified additional hosts, and assessing transmission risk. Here, we use mathematical models to investigate how variation in host body size and environmental temperature affect the evolution of parasite host-generalism. We predict that parasites are more likely to evolve a generalist strategy when hosts are large-bodied, when variation in host body size is large, and in cooler environments. We then explore these predictions using a newly updated database of over 20 000 fish–macroparasite associations. Within the database we see some evidence supporting these predictions, but also highlight mismatches between theory and data. By combining these two approaches, we establish a theoretical basis for interpreting empirical data on parasites' host specificity and identify key areas for future work that will help untangle the drivers of parasite host-generalism. This article is part of the themed issue ‘Opening the black box: re-examining the ecology and evolution of parasite transmission’.

article i nfo Background:Frogs are a diverse group of vertebrates for which limited genomic resources are available. Natural frog populationsface amultitude of threats, including habitat degradation, infectious disease, and environmental change. Characterizing the functional genomics of anuran tissues in general - and the immune system in particular - will enhance our knowledge of genetic and epigenetic responses to environmental threats and inform conservation and recovery efforts. Results: To increase the number of species with genomic datasets and characterize gene expression in immune- related tissues, we sequenced the transcriptomes of three tissues from two frogs (Espadarana prosoblepon and Lithobates yavapaiensis) on the Roche 454 GS FLX platform. Our sequencing produced 8881 E. prosoblepon and 5428 L. yavapaiensis annotated gene products after de novo assembly and Gene Ontology classification. Transcripts of the innate and acquired immune system were expressed in all three tissues. Inflammatory response and acquired immunity transcripts were significantly more diverged between E. prosoblepon and L. yavapaiensis compared to innate immunity and immune system development transcripts. Immune-related transcripts did not show an overall elevated rate of functional evolution, with the exception of glycosyl proteases, which include lysozymes, central bacterial and fungal-killing enzymes of the innate immune system. Conclusions: The three frog transcriptomes provide more than 600 Mbp of new genomic data, and will serve as a valuable framework for future comparative studies of non-model anurans. Additionally, we show that immune gene divergence varies by functional group and that transcriptome studies can be useful in comparing rates of evolutionary change across gene families.

Major histocompatibility complex (MHC) genes encode proteins that present pathogen-derived antigens to T-cells, initiating the adaptive immune response in vertebrates. Although populations with low MHC diversity tend to be more susceptible to pathogens, some bottlenecked populations persist and even increase in numbers despite low MHC diversity. Thus, the relative importance of MHC diversity versus genome-wide variability for the long-term viability of populations after bottlenecks and/or under high inbreeding is controversial. We tested the hypothesis that genome-wide inbreeding (estimated using microsatellites) should be more critical than MHC diversity alone in determining pathogen resistance in the self-fertilizing fishKryptolebias marmoratusby analysing MHC diversity and parasite loads in natural and laboratory populations with different degrees of inbreeding. Both MHC and neutral diversities were lost after several generations of selfing, but we also found evidence of parasite selection acting on MHC diversity and of non-random loss of alleles, suggesting a possible selective advantage of those individuals with functionally divergent MHC, in accordance with the hypothesis of divergent allele advantage. Moreover, we found that parasite loads were better explained by including MHC diversity in the model than by genome-wide (microsatellites) heterozygosity alone. Our results suggest that immune-related overdominance could be the key in maintaining variables rates of selfing and outcrossing inK. marmoratusand other mixed-mating species.

Genetic variation within populations depends on population size, spatial structuring, and environmental variation, but is also influenced by mating system. Mangroves are some of the most productive and threatened ecosystems on earth and harbor a large proportion of species with mixed-mating (self-fertilization and outcrossing). Understanding population structuring in mixed-mating species is critical for conserving and managing these complex ecosystems. Kryptolebias marmoratus is a unique mixed-mating vertebrate inhabiting mangrove swamps under highly variable tidal regimes and environmental conditions. We hypothesized that geographical isolation and ecological pressures influence outcrossing rates and genetic diversity, and ultimately determine the local population structuring of K. marmoratus. By comparing genetic variation at 32 microsatellites, diel fluctuations of environmental parameters, and parasite loads among four locations with different degrees of isolation, we found significant differences in genetic diversity and genotypic composition but little evidence of isolation by distance. Locations also differed in environmental diel fluctuation and parasite composition. Our results suggest that mating system, influenced by environmental instability and parasites, underpins local population structuring of K. marmoratus. More generally, we discuss how the conservation of selfing species inhabiting mangroves and other biodiversity hotspots may benefit from knowledge of mating strategies and population structuring at small spatial scales.

Mixed-mating strategies (i.e., intermediate levels of self-fertilization and outcrossing in hermaphrodites) are relatively common in plants and animals, but why self-fertilization (selfing) rates vary so much in nature has proved difficult to explain. We tested the hypothesis that parasites help maintain mixed-mating using a partially selfing fish (Kryptolebias marmoratus) as a model. We show that outcrossed progeny in the wild are genetically more diverse and less susceptible to multiple parasite infections than their selfed counterparts. Given that outcrossing in K. marmoratus can only be attained by male-hermaphrodite matings, our data provide an explanation for the coexistence of males and hermaphrodites in androdioecious species where hermaphrodites are unable to outcross among themselves. Moreover, our study provides evidence that parasites contribute to maintaining mixed-mating in a natural animal population.

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.

The emergence of the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in dramatic global amphibian declines. Although many species have undergone catastrophic declines and/or extinctions, others appear to be unaffected or persist at reduced frequencies after Bd outbreaks. The reasons behind this variance in disease outcomes are poorly understood: differences in host immune responses have been proposed, yet previous studies suggest a lack of robust immune responses to Bd in susceptible species. Here, we sequenced transcriptomes from clutch-mates of a highly susceptible amphibian, Atelopus zeteki, with different infection histories. We found significant changes in expression of numerous genes involved in innate and inflammatory responses in infected frogs despite high susceptibility to chytridiomycosis. We show evidence of acquired immune responses generated against Bd, including increased expression of immunoglobulins and major histocompatibility complex genes. In addition, fungal-killing genes had significantly greater expression in frogs previously exposed to Bd compared with Bd-naïve frogs, including chitinase and serine-type proteases. However, our results appear to confirm recent in vitro evidence of immune suppression by Bd, demonstrated by decreased expression of lymphocyte genes in the spleen of infected compared with control frogs. We propose susceptibility to chytridiomycosis is not due to lack of Bd-specific immune responses but instead is caused by failure of those responses to be effective. Ineffective immune pathway activation and timing of antibody production are discussed as potential mechanisms. However, in light of our findings, suppression of key immune responses by Bd is likely an important factor in the lethality of this fungus.

Androdioecy (the coexistence of males and hermaphrodites) is considered a transitional state derived from pure hermaphroditism or dioecy, but the processes selecting for this rare breeding system are unclear, particularly in animals. In androdioecious species, the proportion of males in relation to hermaphrodites is usually so reduced that it is not known whether there is scope for mate choice, particularly when simultaneous hermaphrodites can self-fertilize. We investigated the potential role of male mate choice in the persistence of androdioecy in animals using a self-fertilizing androdioecious fish (Kryptolebias marmoratus) as a model. Hermaphrodites preferred to associate with males but showed no preference based on genetic similarity. In contrast, males displayed a strong preference for genetically dissimilar hermaphrodites, based, apparently, on olfactory cues. We suggest that disassortative male mate choice could be a critical factor in stabilizing androdioecy in cases where high selfing rates are associated with inbreeding depression.

Aggressive behaviour plays an important role in securing resources, defending against predators and shaping social interactions. Although aggression can have positive effects on growth and reproductive success, it is also energetically costly and may increase injury and compromise survival. Individual genetic diversity has been positively associated with aggression, but the cause for such an association is not clear, and it might be related to the ability to recognize kin. To disentangle the relationships between genetic diversity, kinship and aggression, we quantified aggressive behaviour in a wild, self-fertilizing fish (Kryptolebias marmoratus) with naturally variable degrees of genetic diversity, relatedness and familiarity. We found that in contrast to captive fish, levels of aggression among wild K. marmoratus are positively associated with individual homozygosity, but not with relatedness or familiarity. We suggest that the higher aggression shown by homozygous fish could be related to better kin discrimination and may be facilitated by hermaphrodite competition for scarce males, given the fitness advantages provided by outcrossing in terms of parasite resistance. It seems likely that the relationship between aggression and genetic diversity is largely influenced by both the environment and population history.

Despite being probably one of the most studied topics in Evolutionary Biology, the mechanisms of maintenance of major histocompatibility complex (MHC) diversity in vertebrates remain also one of the most debated topics [[1][1]]. Ellison et al. [[2][2]] studied MHC class I diversity of a selfing