Social experience influences thermal sensitivity: lessons from an amphibious mangrove fish.

Understanding the factors affecting the capacity of ectothermic fishes to cope with warming temperature is critical given predicted climate change scenarios. We know that a fish's social environment introduces plasticity in how it responds to high temperature. However, the magnitude of this plasticity and the mechanisms underlying socially modulated thermal responses are unknown. Using the amphibious hermaphroditic mangrove rivulus fish Kryptolebias marmoratus as a model, we tested three hypotheses: (1) social stimulation affects physiological and behavioural thermal responses of isogenic lineages of fish; (2) social experience and acute social stimulation result in distinct physiological and behavioural responses; and (3) a desensitization of thermal receptors is responsible for socially modulated thermal responses. To test the first two hypotheses, we measured the temperature at which fish emerged from the water (i.e. pejus temperature) upon acute warming with socially naive isolated fish and with fish that were raised alone and then given a short social experience prior to exposure to increasing temperature (i.e. socially experienced fish). Our results did not support our first hypothesis as fish socially stimulated by mirrors during warming (i.e. acute social stimulation) emerged at similar temperatures to isolated fish. However, in support of our second hypothesis, a short period of prior social experience resulted in fish emerging at a higher temperature than socially naive fish suggesting an increase in pejus temperature with social experience. To test our third hypothesis, we exposed fish that had been allowed a brief social interaction and naive fish to capsaicin, an agonist of TRPV1 thermal receptors. Socially experienced fish emerged at significantly higher capsaicin concentrations than socially naive fish suggesting a desensitization of their TRPV1 thermal receptors. Collectively, our data indicate that past and present social experiences impact the behavioural response of fish to high temperature. We also provide novel data suggesting that brief periods of social experience affect the capacity of fish to perceive warm temperature.

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.

Context-dependent relationships between swimming, terrestrial jumping and body composition in the amphibious fish Kryptolebias marmoratus.

Understanding the mechanisms that create phenotypic variation within and among populations is a major goal of physiological ecology. Variation may be a consequence of functional trade-offs (i.e. improvement in one trait comes at the expense of another trait) or alternatively may reflect the intrinsic quality of an organism (i.e. some individuals are simply better overall performers than others). There is evidence for both ideas in the literature, suggesting that environmental context may mediate whether variation results from trade-offs or differences in individual quality. We tested this overarching 'context dependence' hypothesis by comparing the aquatic and terrestrial athletic performance of the amphibious fish Kryptolebias marmoratus captured from two contrasting habitats, a large pond and small burrows. Overall, pond fish were superior terrestrial athletes but burrow fish were better burst swimmers, suggestive of a performance trade-off at the population level. Within each population, however, there was no evidence of a performance trade-off. In burrow fish, athletic performance was positively correlated with muscle content and body condition, consistent with the individual quality hypothesis. In pond fish, there was only a relationship between glycolytic white muscle and aquatic burst performance. Notably, pond fish were in better body condition, which may mask relationships between condition and athletic performance. Overall, our data highlight that population-level trends are insufficient evidence for the existence of phenotypic trade-offs in the absence of similar within-population patterns. Furthermore, we only found evidence for the individual quality hypothesis in one population, suggesting that patterns of phenotypic covariance are context dependent.

Positive feedback promotes terrestrial emergence behaviour in an amphibious fish.

Major ecological transitions such as the invasion of land by aquatic vertebrates may be facilitated by positive feedback between habitat choice and phenotypic plasticity. We used the amphibious fish Kryptolebias marmoratus to test the hypothesis that aquatic hypoxia, emergence behaviour and respiratory plasticity create this type of positive feedback loop that causes fish to spend increasing amounts of time on land. Terrestrially acclimated fish were more sensitive to aquatic hypoxia (emergence at higher PO2) and were less hypoxia tolerant (shorter time to loss of equilibrium) relative to water-acclimated fish, which are necessary conditions for positive feedback. Next, we tested the prediction that exposure to aquatic hypoxia causes fish to emerge frequently, reduce gill surface area, and become less hypoxia tolerant. Indeed, fish exposed to severe aquatic hypoxia spent almost 50% of the time out of water and coverage of the gill lamellae by an inter-lamellar cell mass almost doubled. Fish exposed to aquatic hypoxia that could emerge from water were also more sensitive to subsequent acute aquatic hypoxia and were less hypoxia tolerant than normoxia-exposed controls. These results are opposite those of fish that cannot escape from aquatic hypoxia and presumably arise owing to plastic changes that occur during air exposure. Together, these results indicate that emergence behaviour begets further emergence behaviour, driven by gill remodelling which reduces aquatic respiratory function. This type of positive feedback may explain how amphibious behaviour has repeatedly evolved in fishes that occupy hypoxic aquatic habitats despite the associated challenges of life on land.

Extensive hybridization and past introgression between divergent lineages in a quasi-clonal hermaphroditic fish: Ramifications for species concepts and taxonomy.

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self-fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self-fertilization for 1-3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.

Genomic and physiological mechanisms underlying skin plasticity during water to air transition in an amphibious fish.

The terrestrial radiation of vertebrates required changes in skin that resolved the dual demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Using the amphibious killifish Kryptolebias marmoratus, we found that transcriptional regulation of skin morphogenesis was quickly activated upon air exposure (1 h). Rapid regulation of cell-cell adhesion complexes and pathways that regulate stratum corneum formation was consistent with barrier function and mechanical reinforcement. Unique blood vessel architecture and regulation of angiogenesis likely supported cutaneous respiration. Differences in ionoregulatory transcripts and ionocyte morphology were correlated with differences in salinity acclimation and resilience to air exposure. Evolutionary analyses reinforced the adaptive importance of these mechanisms. We conclude that rapid plasticity of barrier, respiratory and ionoregulatory functions in skin evolved to support the amphibious lifestyle of K. marmoratus; similar processes may have facilitated the terrestrial radiation of other contemporary and ancient fishes.

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.

Prolonged survival out of water is linked to a slow pace of life in a self-fertilizing amphibious fish.

Metabolic rate and life-history traits vary widely both among and within species, reflecting trade-offs in energy allocation, but the proximate and ultimate causes of variation are not well understood. We tested the hypothesis that these trade-offs are mediated by environmental heterogeneity, using isogenic strains of the amphibious fish Kryptolebias marmoratus that vary in the amount of time each can survive out of water. Consistent with pace of life theory, the strain that survived air exposure the longest generally exhibited a 'slow' phenotype, including the lowest metabolic rate, largest scope for metabolic depression, slowest consumption of energy stores and least investment in reproduction under standard conditions. Growth rates were fastest in the otherwise slow strain, however. We then tested for fitness trade-offs between 'fast' and 'slow' strains using microcosms where fish were held either with constant water availability or under fluctuating conditions where water was absent for half of the experiment. Under both conditions the slow strain grew larger and was in better condition, and under fluctuating conditions the slow strain produced more embryos. However, the fast strain had larger adult population sizes under both conditions, indicating that fecundity is not the sole determinant of population size in this species. We conclude that genetically based differences in the pace of life of amphibious fish determine survival duration out of water. Relatively slow fish tended to perform better under conditions of limited water availability, but there was no detectable cost under control conditions. Thus, pace of life differences may reflect a conditionally neutral instead of antagonistic trade-off.

Natural hybridization between divergent lineages in a selfing hermaphroditic fish.

By definition, mating between individuals is infrequent in highly selfing organisms, and so too, therefore, hybridization should be rare between genetically divergent lineages in predominantly self-fertilizing species. Notwithstanding these expectations, here we report a remarkable case of natural hybridization between highly diverged phylogeographic lineages of the mangrove rivulus, a small killifish that reproduces predominantly by self-fertilization and typically is found as highly homozygous lines in most parts of its extensive geographical range. Two distinctive genetic lineages (Kryptolebias marmoratus and a 'Central clade' closely related to K. hermaphroditus) previously were not known in sympatry, but were found by us to co-occur on San Salvador, Bahamas. Genetic analyses of a mitochondrial and multiple nuclear markers determined the direction of a cross producing a hybrid fish. Furthermore, we show that this hybrid individual was viable, as it successfully reproduced by self-fertilization for two generations. Additional sampling of this population will be necessary to determine if backcrossing of hybrids to the parental lineages occurs in nature and to analyse whether such backcross progeny are viable. Application of the biological species concept (BSC) is traditionally difficult in clonally reproducing organisms. Our results show that although mangrove rivulus fish are mostly highly selfing in nature (resulting in isogenic, effectively clonal and homozygous progeny), classification within this taxonomic complex need not be incompatible with the BSC.

By land or by sea: a modified C-start motor pattern drives the terrestrial tail-flip.

Aquatic C-start escape responses in teleost fishes are driven by a well-studied network of reticulospinal neurons that produce a motor pattern of simultaneous contraction of axial muscle on the side of the body opposite the threatening stimulus, bending the fish into the characteristic C shape, followed by a traveling wave of muscle contraction on the contralateral side that moves the fish away from the threat. Superficially, the kinematics of the terrestrial tail-flip resemble the C-start, with the anterior body rolling up and over the tail into a tight C shape, followed by straightening as the fish launches off of the caudal peduncle into ballistic flight. We asked whether similar motor control is used for both behaviors in the amphibious mangrove rivulus, Kryptolebias marmoratus Fine-wire bipolar electrodes were percutaneously inserted into repeatable paired axial locations in five individual fish. Electromyograms synchronized with high-speed video were made of aquatic C-starts, immediately followed by terrestrial tail-flips. Tail-flips took longer to complete than aquatic escapes; correspondingly, muscles were activated for longer durations on land. In the tail-flip, activity was seen in contralateral posterior axial muscle for an extended period of time during the formation of the C shape, likely to press the caudal peduncle against the ground in preparation for launch. Tail-flips thus appear to be produced by modification of the motor pattern driving the aquatic C-start, with differences consistent with the additional requirement of overcoming gravity.

Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus, which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks.

Evolution, ecology and physiology of amphibious killifishes (Cyprinodontiformes).

The order Cyprinodontiformes contains an exceptional diversity of amphibious taxa, including at least 34 species from six families. These cyprinodontiforms often inhabit intertidal or ephemeral habitats characterized by low dissolved oxygen or otherwise poor water quality, conditions that have been hypothesized to drive the evolution of terrestriality. Most of the amphibious species are found in the Rivulidae, Nothobranchiidae and Fundulidae. It is currently unclear whether the pattern of amphibiousness observed in the Cyprinodontiformes is the result of repeated, independent evolutions, or stems from an amphibious common ancestor. Amphibious cyprinodontiforms leave water for a variety of reasons: some species emerse only briefly, to escape predation or capture prey, while others occupy ephemeral habitats by living for months at a time out of water. Fishes able to tolerate months of emersion must maintain respiratory gas exchange, nitrogen excretion and water and salt balance, but to date knowledge of the mechanisms that facilitate homeostasis on land is largely restricted to model species. This review synthesizes the available literature describing amphibious lifestyles in cyprinodontiforms, compares the behavioural and physiological strategies used to exploit the terrestrial environment and suggests directions and ideas for future research.

The amphibious fish Kryptolebias marmoratus uses different strategies to maintain oxygen delivery during aquatic hypoxia and air exposure.

Despite the abundance of oxygen in atmospheric air relative to water, the initial loss of respiratory surface area and accumulation of carbon dioxide in the blood of amphibious fishes during emersion may result in hypoxemia. Given that the ability to respond to low oxygen conditions predates the vertebrate invasion of land, we hypothesized that amphibious fishes maintain O2 uptake and transport while emersed by mounting a co-opted hypoxia response. We acclimated the amphibious fish Kryptolebias marmoratus, which are able to remain active for weeks in both air and water, for 7 days to normoxic brackish water (15‰, ~21kPa O2; control), aquatic hypoxia (~3.6kPa), normoxic air (~21 kPa) or aerial hypoxia (~13.6kPa). Angiogenesis in the skin and bucco-opercular chamber was pronounced in air- versus water-acclimated fish, but not in response to hypoxia. Aquatic hypoxia increased the O2-carrying capacity of blood via a large (40%) increase in red blood cell density and a small increase in the affinity of hemoglobin for O2 (P50 decreased 11%). In contrast, air exposure increased the hemoglobin O2 affinity (decreased P50) by 25% without affecting the number of red blood cells. Acclimation to aerial hypoxia both increased the O2-carrying capacity and decreased the hemoglobin O2 affinity. These results suggest that O2 transport is regulated both by O2 availability and also, independently, by air exposure. The ability of the hematological system to respond to air exposure independent of O2 availability may allow extant amphibious fishes, and may also have allowed primitive tetrapods to cope with the complex challenges of aerial respiration during the invasion of land.

Detection of relatedness in chemical alarm cues by a selfing vertebrate depends on population and the life stage producing the alarm cue.

Aquatic prey have impressive abilities to extract information from a variety of chemical cues. For example, they can use the alarm cues released by wounded individuals during a predator attack to learn about predation risk, and they can also distinguish kin from non-kin individuals during interactions. However, it remains unclear whether animals can combine this information on predation risk with kin recognition of the particular individuals under threat. To examine how the relatedness of the individuals in alarm cue affects behaviour we used the self-fertilizing hermaphroditic mangrove rivulus (Kryptolebias marmoratus), in which lineages produce genetically identical offspring through selfing. We explored this in two populations that differ in their level of outcrossing. We measured activity before and after exposure to alarm cue made from individuals (either adults or embryos) from their own lineage or an unrelated lineage from the same population. Fish responded weakly to embryo alarm cues, but tended to reduce their activity more when the alarm cues were from an unrelated lineage compared to alarm cues from their own lineage, particularly in fish from the outcrossing population. In contrast, there was no effect of cue relatedness on the response to adult alarm cues but there was a strong population effect. Specifically, individuals from the outcrossing population tended to react more strongly to alarm cues compared to individuals from the predominantly selfing population. We discuss the potential roles of the major histocompatibility complex in cue detection, differences between adult vs embryo alarm cues in terms of concentration and information, and underlying differences among populations and genetic lineages in their production and detection of chemical cues. Whether this kin recognition offers adaptive benefits or is simply a consequence of being able to detect relatedness in living individuals would be an exciting area for future research.

Launches, squiggles and pounces, oh my! The water-land transition in mangrove rivulus (Kryptolebias marmoratus).

Mangrove rivulus (Kryptolebias marmoratus) are small fusiform teleosts (Cyprinodontiformes) with the ability to locomote on land, despite lacking apparent morphological adaptations for terrestrial movement. Rivulus will leave their aquatic habitat for moist, terrestrial environments when water conditions are poor, or, as we show here, to capture terrestrial insects. Specimens were conditioned to eat pinhead crickets on one side of their aquaria. After 2 weeks of conditioning, a barrier with a slope of 15 deg was partially submerged in the middle of the tank, forcing the fish to transition from water to land and back into water in order to feed. Kinematics during the transition were recorded using Fastec high-speed video cameras (125-250 frames s(-1)). Videos were analyzed using Didge and ImageJ software programs. Transition behaviors were characterized and analyzed according to their specific type. Body oscillation amplitude and wave duration were quantified for movements along the substrate, along with initial velocity for launching behaviors. Kryptolebias marmoratus used a diverse suite of behaviors to transition from water to land. These behaviors can be categorized as launches, squiggles and pounces. Prey were captured terrestrially and brought underwater for consumption. Kryptolebias marmoratus's suite of behaviors represents a novel solution to non-tetrapodal terrestrial transition, which suggests that fishes may have been able to exploit land habitats transiently, without leaving any apparent evidence in the fossil record.

Genome-wide DNA methylation of the liver reveals delayed effects of early-life exposure to 17-α-ethinylestradiol in the self-fertilizing mangrove rivulus.

Organisms exposed to endocrine disruptors in early life can show altered phenotype later in adulthood. Although the mechanisms underlying these long-term effects remain poorly understood, an increasing body of evidence points towards the potential role of epigenetic processes. In the present study, we exposed hatchlings of an isogenic lineage of the self-fertilizing fish mangrove rivulus for 28 days to 4 and 120 ng/L of 17-α-ethinylestradiol. After a recovery period of 140 days, reduced representation bisulphite sequencing (RRBS) was performed on the liver in order to assess the hepatic genome-wide methylation landscape. Across all treatment comparisons, a total of 146 differentially methylated fragments (DMFs) were reported, mostly for the group exposed to 4 ng/L, suggesting a non-monotonic effect of EE2 exposure. Gene ontology analysis revealed networks involved in lipid metabolism, cellular processes, connective tissue function, molecular transport and inflammation. The highest effect was reported for nipped-B-like protein B (NIPBL) promoter region after exposure to 4 ng/L EE2 (+ 21.9%), suggesting that NIPBL could be an important regulator for long-term effects of EE2. Our results also suggest a significant role of DNA methylation in intergenic regions and potentially in transposable elements. These results support the ability of early exposure to endocrine disruptors of inducing epigenetic alterations during adulthood, providing plausible mechanistic explanations for long-term phenotypic alteration. Additionally, this work demonstrates the usefulness of isogenic lineages of the self-fertilizing mangrove rivulus to better understand the biological significance of long-term alterations of DNA methylation by diminishing the confounding factor of genetic variability.

Hydrogen sulphide sensitivity and tolerance in genetically distinct lineages of a selfing mangrove fish (Kryptolebias marmoratus).

Mangroves are critical marine habitats. High hydrogen sulphide (H2S) is a feature of these important ecosystems and its toxicity creates a challenge for mangrove inhabitants. The mangrove rivulus (Kryptolebias marmoratus) is a selfing, hermaphroditic, amphibious fish that can survive exposure to 1116 µM H2S in the wild. These fish rely on cutaneous respiration for gas and ion exchange when emerged. We hypothesized that the skin surface is fundamentally important in H2S tolerance in these mangrove fish by limiting H2S permeability. To test our hypothesis, we first disrupted the skin surface in one isogenic lineage and measured H2S tolerance and sensitivity. We increased water H2S concentration until emersion as a measure of the ability to sense and react to H2S, which we refer to as sensitivity. We then determined H2S tolerance by preventing emersion and increasing H2S until loss of equilibrium (LOE). The H2S concentration at emersion and LOE were significantly affected by disrupting the skin surface, providing support that the skin is involved in limiting H2S permeability. Capitalizing on their unique reproductive strategy, we used three distinct isogenic lineages to test the hypothesis that there would be genetic differences in H2S sensitivity and tolerance. We found significant differences in emersion concentration only among lineages, suggesting a genetic component to H2S sensitivity but not tolerance. Our study also demonstrated that external skin modifications and avoidance behaviours are two distinct strategies used to tolerate ecologically relevant H2S concentrations and likely facilitate survival in challenging mangrove habitats.

Behavior and gene expression in the brain of adult self-fertilizing mangrove rivulus fish (Kryptolebias marmoratus) after early life exposure to the neurotoxin ß-N-methylamino-l-alanine (BMAA).

ß-N-Methylamino-l-alanine (BMAA), a neurotoxin naturally produced by cyanobacteria, diatoms and dinoflagellates, constitutes a serious environmental and health threat especially during acute blooms, which are becoming more frequent. This neurotoxin is implicated in several neurodegenerative diseases (ND) in humans through contaminated water or food consumption. Even low doses of neurotoxic compounds (NCs) can have lasting effects later in life. In this sense, early stages of development constitute a period of high sensitivity to environmental influence, particularly for the central nervous system. To understand the mechanisms underlying the delayed effects of NCs, newly hatched larvae of the mangrove rivulus fish, Kryptolebias marmoratus, were exposed to two sub-lethal doses of BMAA (20 µg/L and 15 mg/L) for 14 days. This fish naturally produces isogenic lineages due to its self-fertilizing reproduction, which is unique case among vertebrates. It thus provides genetic characteristics that allow scientists to study organisms' true reaction norm, minimizing genetic variability and focusing exclusively on the effects of the environment. Effect assessment was performed at different levels of biological organization to detect inconspicuous effects of BMAA, since this molecule displays long retention in organisms. BMAA effects on life history traits as well as behavioral traits such as boldness and aggressiveness were assessed more than 100 days after exposure. In addition, the relative expression of 7 potential BMAA target genes was studied, given their involvement in neurotransmission or their association with individual variation in boldness and aggressiveness. Selected genes code for reticulon 4 (RTN4), glutamate vesicular transporter 1 (Slc17a7), glutamine synthetase a (Glula), dopamine receptor D4 (DRD4), monoamine oxidase A (MAOA), calmodulin (CaM) and epedymine (Epd). Despite observing no effects of BMAA on growth, reproduction and behavioral traits, BMAA induced a significant increase of the expression of CaM and MAOA genes at 20 µg/L BMAA compared to the control group. A significant decrease of expression was observed between this lowest BMAA dose and 15 mg/L for DRD4, MAOA and CaM genes. Our results suggest disruption of glutamate turnover, intracellular dopamine depletion and activation of astrocyte protective mechanisms, indicating that BMAA might be excitotoxic. Our study revealed that BMAA can have long-lasting effects on the brain that are suspected to affect phenotypic traits with aging. Furthermore, it highlights the importance of studying delayed effects in ecotoxicological studies.

Early-life exposure to the endocrine disruptor 17-α-ethinylestradiol induces delayed effects in adult brain, liver and ovotestis proteomes of a self-fertilizing fish.

Early-life represents a critically sensitive window to endocrine disrupting chemicals, potentially leading to long-term repercussions on the phenotype later in life. The mechanisms underlying this phenomenon, referred to as the Developmental Origins of Health and Disease (DOHaD), are still poorly understood. To gain molecular understanding of these effects, we exposed mangrove rivulus (Kryptolebias marmoratus) for 28 days post hatching (dph) to 4 and 120 ng/L 17-α-ethinylestradiol, a model xenoestrogen. After 28 days, fish were raised for 140 days in clean water and we performed quantitative label-free proteomics on brain, liver and ovotestis of 168 dph adults. A total of 820, 888 and 420 proteins were robustly identified in the brain, liver and ovotestis, respectively. Effects of 17-α-ethinylestradiol were tissue and dose-dependent: a total of 31, 51 and 18 proteins were differentially abundant at 4 ng/L in the brain, liver and ovotestis, respectively, compared to 20, 25 and 39 proteins at 120 ng/L. Our results suggest that estrogen-responsive pathways, such as lipid metabolism, inflammation, and the innate immune system were affected months after the exposure. In addition, the potential perturbation of S-adenosylmethionine metabolism encourages future studies to investigate the role of DNA methylation in mediating the long-term effects of early-life exposures. SIGNIFICANCE: The Developmental Origins of Health and Disease (DOHaD) states that early life stages of humans and animals are sensitive to environmental stressors and can develop health issues later in life, even if the stress has ceased. Molecular mechanisms supporting DOHaD are still unclear. The mangrove rivulus is a new fish model species naturally reproducing by self-fertilization, making it possible to use isogenic lineages in which all individuals are highly homozygous. This species therefore permits to strongly reduce the confounding factor of genetic variability in order to investigate the effects of environmental stress on the phenotype. After characterizing the molecular phenotype of brain, liver and ovotestis, we obtained true proteomic reaction norms of these three organs in adults after early life stages have been exposed to the common endocrine disruptor 17-α-ethinylestradiol (EE2). Our study demonstrates long-term effects of early-life endocrine disruption at the proteomic level in diverse estrogen-responsive pathways 5 months after the exposure. The lowest tested and environmentally relevant concentration of 4 ng/L had the highest impact on the proteome in brain and liver, highlighting the potency of endocrine disruptors at low concentrations.

Behavioral effects of the neurotoxin -N-methylamino- L-alanine on the mangrove rivulus (Kryptolebias marmoratus) larvae.

Mangrove rivulus, Kryptolebias marmoratus, is a hermaphrodite fish capable of self-fertilization. This particularity allows to naturally produce highly homozygous and isogenic individuals. Despite the low genetic diversity, rivulus can live in extremely variable environments and adjust its phenotype accordingly. This species represents a unique opportunity to clearly distinguish the genetic and non-genetic factors implicated in adaptation and evolution, such as epigenetic mechanisms. It is thus a great model in aquatic ecotoxicology to investigate the effects of xenobiotics on the epigenome, and their potential long-term impacts. In the present study, we used the mangrove rivulus to investigate the effects of the neurotoxin b-N-methylamino-L-alanine (BMAA) on larvae behaviors after 7 days exposure to two sub-lethal concentrations. Results show that BMAA can affect the maximal speed and prey capture (trials and failures), suggesting potential impacts on the organism's fitness.