Maternal exposure to bisphenol S reduces anxiety and impairs collective antipredator behavior of male zebrafish (Danio rerio) offspring through dysregulation of their serotonergic system.

Bisphenol S (BPS) is a common endocrine-disrupting chemical globally used in several consumer and industrial products. Although previous studies suggested that BPS induces multiple effects in exposed organisms, very little is known about its intergenerational effect on offspring behavior and/or the potential underlying mechanisms. To this end, adult female zebrafish Danio rerio were exposed to BPS (0, 10, 30 µg/L) and 1 µg/L of 17-ß-estradiol (E2) as a positive control for 60 days. Afterwards, female fish were bred with untreated males, and their offspring were raised to 6 months old in control water. Maternal exposure to BPS decreased male offspring anxiety and antipredator behaviors while boldness remained unaffected. Specifically, maternal exposure to 10 and 30 µg/L BPS and 1 µg/L E2 were found to impact male offspring anxiety levels as they decreased the total time that individuals spent in the dark zone in the light/dark box test and increased the total track length in the center of the open field test. In addition, maternal exposure to all concentrations of BPS and E2 disrupted antipredator responses of male offspring by decreasing shoal cohesion in the presence of chemical alarm cues derived from conspecifics, which communicated high risk. To elucidate the possible molecular mechanism underlying these neuro-behavioral effects of BPS, we assessed the serotonergic system via changes in mRNA expression of serotonin receptors, including the 5-HT1A, 5-HT1B, and 5-HT1D subtypes, the serotonin transporter and monoamine oxidase (MAO). The impaired anxiety and antipredator responses were associated with reduced levels of 5-HT1A subtype and MAO mRNA expression within the brain of adult male offspring. Collectively, the results of this study demonstrate that maternal exposure to environmental concentrations of BPS can interfere with the serotonergic signaling pathway in the developing brain, subsequently leading to the onset of a suite of behavioral deficits in adult offspring.

Transgenerational plasticity of exploratory behavior and a hidden cost of mismatched risk environments between parental sexes.

We require a better understanding of the relative contribution of different modes of non-genetic inheritance in behavioral trait development. Thus, we investigate variation in exploratory behavior, which is ecologically relevant and a target of selection. The metabolic hypothesis predicts exploratory behavior to be size-dependent across taxa. This size-dependency is cancelled out under high perceived risk, allowing us to determine the transgenerationally integrated estimated level of risk. Using fathead minnows Pimephales promelas, we manipulated perceived risk in mothers, fathers, caring males and offspring through continuous exposure to either conspecific alarm cues or to a control water treatment. In 1000 four-month old offspring, we determined body sizes and exploratory behavior. Perceived high risk in mothers, followed by personal risk, was most effective in eliminating size-dependent behavior whereas effects of paternal risk on offspring behavioral development were substantially weaker. When maternal risk is high, environmental mismatches between parents prevented offspring from responding appropriately to personal high risk. The environment of the caring male also impacted offspring behavior to a greater extent than that of its genetic parents. Our study highlights the high relative importance of maternal, personal and caring male risk environments and showcases potential costs of an environmental mismatch between parental sexes.

On the use of antibiotics in plasticity research: Gastropod shells unveil a tale of caution.

Through phenotypic plasticity, individual genotypes can produce multiple phenotypes dependent on the environment. In the modern world, anthropogenic influences such as man-made pharmaceuticals are increasingly prevalent. They might alter observable patterns of plasticity and distort our conclusions regarding the adaptive potential of natural populations. Antibiotics are nowadays nearly ubiquitous in aquatic environments and prophylactic antibiotic use is also becoming more common to optimize animal survival and reproductive output in artificial settings. In the well-studied plasticity model system Physella acuta, prophylactic erythromycin treatment acts against gram-positive bacteria and thereby reduces mortality. Here, we study its consequences for inducible defence formation in the same species. In a 2 × 2 split-clutch design, we reared 635 P. acuta in either the presence or absence of this antibiotic, followed by 28-day exposure to either high or low predation risk as perceived through conspecific alarm cues. Under antibiotic treatment, risk-induced increases in shell thickness, a well-known plastic response in this model system, were larger and consistently detectable. Antibiotic treatment reduced shell thickness in low-risk individuals, suggesting that in controls, undiscovered pathogen infection increased shell thickness under low risk. Family variation in risk-induced plasticity was low, but the large variation in responses to antibiotics among families suggests different pathogen susceptibility between genotypes. Lastly, individuals that developed thicker shells had reduced total mass, which highlights resource trade-offs. Antibiotics thus have the potential to uncover a larger extent of plasticity, but might counterintuitively distort plasticity estimates for natural populations where pathogens are a part of natural ecology.

Effects of common antiepileptic drugs on teleost fishes.

Antiepileptic drugs (AEDs) are globally prescribed to treat epilepsy and many other psychiatric disorders in humans. Their high consumption, low metabolic rate in the human body and low efficiency of wastewater treatment plants (WWTPs) in eliminating these chemicals results in the frequent occurrence of these pharmaceutical drugs in aquatic systems. Therefore, aquatic organisms, including ecologically and economically important teleost fishes, may be inadvertently exposed to these chemicals. Due to their physiological similarity with humans, fishes may be particularly vulnerable to AEDs. Almost all AED drugs are detectable in natural aquatic ecosystems, but diazepam (DZP) and carbamazepine (CBZ) are among the most widely detected AEDs to date. Recent studies suggest that these drugs have a substantial capacity to induce neurotoxicity and behavioral abnormality in fishes. Here we review the current state of knowledge regarding the potential mode of action of DZP and CBZ as well as that of some other AEDs on teleosts and put observable behavioral effects into a mechanistic context. We find that following their intended mode of action in humans, AEDs also disrupt the GABAergic, glutamatergic and serotonergic systems as well as parasympathetic neurotransmitters in fishes. Moreover, AEDs have non-specific modes of action in teleosts ranging from estrogenic activity to oxidative stress. These physiological changes are often accompanied by dose-dependent disruptions of anxiety, locomotor activity, social behaviors, food uptake, and learning and memory, but DZP and CBZ consistently induced anxiolytic effects. Thereby, AED exposure severely compromises individual fitness across teleost fish species, which may lead to population and ecosystem impairment. We also showcase promising avenues for future research by highlighting where we lack data when it comes to effects of certain AEDs, AED concentrations and behavioral endpoints.

Evidence for selfing in a vertebrate from whole-genome sequencing.

A growing number of recent genomic studies report asexual parthenogenetic reproduction in a wide range of taxa, including vertebrate species from the reptile, bird, and fish lineages. Yet, self-fertilization (selfing) has been recorded only in a single vertebrate, the mangrove killifish Kryptolebias marmoratus In cichlid fishes, sex determination is notably diverse and can be influenced by the environment, and sequential hermaphroditism has been reported for some species. Here, we present evidence for a case of facultative selfing in the cichlid fish Benitochromis nigrodorsalis, which is otherwise known as biparentally reproducing ovophilic mouthbrooder from Western Africa. Our laboratory observations revealed that a wild-caught individual produced repeatedly viable offspring in absence of a mating partner. By analyzing genome-wide single-nucleotide polymorphism (SNP) data, we compare that individual and two of its offspring to shed light on its reproductive mode. First, our results confirm uniparental reproduction. Second, overall heterozygosity is reduced in the offspring compared with outbred individuals. Retained maternal heterozygosity in the offspring is ∼51%, which is close to the theoretically expected value of a heterozygosity reduction of 50% by selfing. Heterozygosity patterns along individual chromosomes do not point to alternative parthenogenetic reproductive mechanisms like automixis by terminal or central fusion. Facultative selfing may represent an adaptive strategy ensuring reproduction when mating partners are absent and, hence, contribute to the cichlids' enormous evolutionary success.

Maternal exposure to bisphenol S induces neuropeptide signaling dysfunction and oxidative stress in the brain, and abnormal social behaviors in zebrafish (Danio rerio) offspring.

Recent studies show that bisphenol S (BPS) induces multiple adverse effects in exposed organisms; however, the maternal effects of BPS exposure remain poorly understood. Here, we expose adult female zebrafish to environmentally relevant concentrations of BPS (0, 1, 10, 30 µg/L) and 1 µg/L of 17-ß-estradiol (E2) as a positive control for 60 days. Females were then paired with BPS-unexposed males and their offspring were raised in control water for 6 months. Maternal exposure to BPS was found to alter social behavior and anxiety response in a dose-specific manner in male offspring. Group preferences and social cohesion were significantly reduced by maternal exposure to 1 and 10 µg/L BPS, respectively. Additionally, maternal exposure to 1 and 30 µg/L BPS and E2 decreased offspring stress responses during the novel tank test. The impaired social behavior was associated with elevated arginine-vasotocin (AVT) level as well as with the altered expression of genes involved in AVT signaling pathway (AVT, avpr1aa) and enzymatic antioxidant genes (cat and Mn-sod) in the brain. Collectively, these results suggest that maternal exposure to environmentally relevant concentrations of BPS alters social behavior in zebrafish offspring, which is likely mediated by oxidative stress and disruption of neuropeptide signaling pathways in the brain.

Proximate causes and ultimate effects of common antidepressants, fluoxetine and venlafaxine, on fish behavior.

Antidepressant (AD) drugs are widely prescribed for the treatment of psychiatric disorders, including depression and anxiety disorders. The continuous use of ADs causes significant quantities of these bioactive chemicals to enter the aquatic ecosystems mainly through wastewater effluent discharge. This may result in many aquatic organisms being inadvertently affected by these drugs. Fluoxetine (FLX) and venlafaxine (VEN) are currently among the most widely detected ADs in aquatic systems. A growing body of experimental evidence demonstrates that FLX and VEN have a substantial capacity to induce neurotoxicity and cause behavioral dysfunctions in a wide range of teleost species. At the same time, these studies often report seemingly contradictory results that are confounding in nature. Hence, we clearly require comprehensive reviews that attempt to find overarching patterns and establish possible causes for these variable results. This review aims to explore the current state of knowledge regarding the neurobehavioral effects of FLX and VEN on fishes. This study also discusses the potential mechanistic linkage between the neurotoxicity of ADs and behavioral dysfunction and identifies key knowledge gaps and areas for future research.

Paternal care effects outweigh gamete-mediated and personal environment effects during the transgenerational estimation of risk in fathead minnows.

BACKGROUND: Individuals can estimate risk by integrating prenatal with postnatal and personal information, but the relative importance of different information sources during the transgenerational response is unclear. The estimated level of risk can be tested using the cognitive rule of risk allocation, which postulates that under consistent high-risk, antipredator efforts should decrease so that individual metabolic requirements can be satisfied. Here we conduct a comprehensive study on transgenerational risk transmission by testing whether risk allocation occurs across 12 treatments that consist of different maternal, paternal, parental care (including cross-fostering) and offspring risk environment combinations in the fathead minnow Pimephales promelas, a small cyprinid fish with alloparental care. In each risk environment, we manipulated perceived risk by continuously exposing individuals from birth onwards to conspecific alarm cues or a control water treatment. Using 2810 1-month old individuals, we then estimated shoaling behaviour prior to and subsequent to a novel mechanical predator disturbance. RESULTS: Overall, shoals estimating risk to be high were denser during the prestimulus period, and, following the risk allocation hypothesis, resumed normal shoaling densities faster following the disturbance. Treatments involving parental care consistently induced densest shoals and greatest levels of risk allocation. Although prenatal risk environments did not relate to paternal care intensity, greater care intensity induced more risk allocation when parents provided care for their own offspring as opposed to those that cross-fostered fry. In the absence of care, parental effects on shoaling density were relatively weak and personal environments modulated risk allocation only when parental risk was low. CONCLUSIONS: Our study highlights the high relative importance of parental care as opposed to other information sources, and its function as a mechanism underlying transgenerational risk transmission.

Early-life and parental predation risk shape fear acquisition in adult minnows.

Exposure to predation risk can induce a fearful baseline state, as well as fear reactions toward novel situations (i.e., neophobia). Some research indicates that risk exposure during sensitive periods makes adults more prone to acquiring long-term fearful phenotypes. However, chronic risk can also lead to ignoring threats in order to maintain other activities. We sought to assess how a relatively long period of low risk, experienced either early in life or by the previous generation, influences fear behaviour acquired from a short period of high risk as adults. We used fathead minnows as study subjects and simulated predation risk with repeated exposures to conspecific chemical alarm cues. The period of high risk experienced by adults induced typical fear behaviour (baseline freezing and neophobia), whereas the early-life low-risk period 1 year prior caused only a reduction in baseline foraging. We found no evidence that the early-life risk significantly altered the fear acquired from the adult-risk period. However, in a second experiment, a low-risk period during the parental generation interacted with a high-risk period experienced by the adult offspring. The combination of both risk periods heightened baseline freezing despite parental risk having little effect independently. Hence, our study provides evidence that parental risk exposure can lead to an additive intergenerational effect on fear acquisition in minnows.

Chronic exposure to bisphenol S induces oxidative stress, abnormal anxiety, and fear responses in adult zebrafish (Danio rerio).

Bisphenol S (BPS) is increasingly used in a wide range of industrial and consumer products, resulting in its ubiquitous distribution across the environment, including aquatic ecosystems. Although it is commonly known as a weak/moderate estrogenic compound, there has been a growing acknowledgment of the potential of BPS to cause toxicity by inducing oxidative stress. Oxidative stress is a major participant in the development of anxiety-like behaviors in humans and animals. Therefore, the present study was designed to examine the impact of BPS on anxiety-like behavior and fear responses in adult zebrafish and also to elucidate the possible linkage between the BPS neurotoxicity and oxidative status of the brain. To this end, adult male and female zebrafish were exposed to 0 (control), 1, 10, and 30 µg/L of BPS and 1 µg/L of 17-ß-estradiol (E2) for 75 days. Following exposure, changes in anxiety and fear-related responses were evaluated by applying a novel tank test and by exposing focal fish to chemical alarm cues. Additionally, we evaluated the expression of multiple antioxidant genes in the zebrafish brain. Our results indicate that BPS, irrespective of exposure concentration, and E2 significantly decreased bottom-dwelling behavior and the latency to enter the upper water column. Furthermore, exposure to the highest concentration of BPS and E2 induced a significant decrease in fear-related responses. The impaired anxiety and reduced fear-related responses were associated with a down-regulation in the transcription of genes involved in enzymatic antioxidant defense. Taken together, our results suggest that chronic exposure to BPS impairs anxiety and fear responses in adult zebrafish, possibly by inducing oxidative stress in the brain.

Anticipatory plastic response of the cellular immune system in the face of future injury: chronic high perceived predation risk induces lymphocytosis in a cichlid fish.

Vertebrate cellular immunity displays substantial variation among taxa and environments. Hematological parameters such as white blood-cell counts have emerged as a valuable tool to understand this variation by assessing the immunological status of individuals. These tools have long revealed that vertebrate cellular immune systems are highly plastic and respond to injury and infection. However, cellular immune systems may also be able to anticipate a high risk of injury from environmental cues (e.g., predation-related cues) and respond plastically ahead of time. We studied white blood-cell (leukocyte) profiles in African cichlids Pelvicachromis taeniatus that were raised for 4 years under different levels of perceived predation risk. In a split-clutch design, we raised fish from hatching onwards under chronic exposure to either conspecific alarm cues (communicating high predation risk) or a distilled water control treatment. Differential blood analysis revealed that alarm cue-exposed fish had twice as many lymphocytes in peripheral blood as did controls, a condition called lymphocytosis. The presence of a higher number of lymphocytes makes the cellular immune response more potent, which accelerates the removal of invading foreign antigens from the bloodstream, and, therefore, may be putatively beneficial in the face of injury. This observed lymphocytosis after long-term exposure to conspecific alarm cues constitutes first evidence for an anticipatory and adaptive plastic response of the cellular immune system to future immunological challenges.

Increased Levels of Perceived Competition Decrease Juvenile Kin-Shoaling Preferences in a Cichlid Fish.

Inclusive fitness theory predicts that individuals can increase their indirect fitness by grouping with kin. However, kin grouping also increases competition between kin, which potentially outweighs its benefits. The level of kin competition is contingent on environmental conditions and thus highly variable. Hence, individuals should benefit from plastically adjusting kin discrimination according to the expected level of kin competition. Here, we investigate whether perceived high competition affects juvenile kin-shoaling preferences in the cichlid Pelvicachromis taeniatus. Juveniles were given the choice between two shoals consisting of either kin or nonkin. Levels of perceived competition were manipulated through food limitation in the face of the differential energy expenditure of differently sized fish. The preference to shoal with kin decreased with increasing levels of perceived competition; small food-deprived individuals avoided kin. Shoaling with kin under strong competition may reduce individual indirect fitness. Hence, individuals can likely improve their inclusive fitness by plastically adjusting their kin-grouping preferences.

Effects of chronic exposure to bisphenol-S on social behaviors in adult zebrafish: Disruption of the neuropeptide signaling pathways in the brain.

Bisphenol S (BPS), considered to be a safe alternative to Bisphenol A, is increasingly used in a wide variety of consumer and industrial products. However, mounting evidence suggests that BPS can act as a xenoestrogen targeting a wide range of neuro-endocrine functions in animals. At present, very little is known about the impacts of BPS on social behaviors and/or the potential underlying mechanisms. To this end, we exposed adult male and female zebrafish to environmentally relevant concentrations of BPS (0 (control), 1, 10, and 30 µg/L), as well as to 17ß-estradiol (E2; 1 µg/L; as positive control) for 75 days. Subsequently, alterations in social behaviors were evaluated by measuring shoal cohesion, group preferences, and locomotor activity. Furthermore, to elucidate the possible molecular mechanism underlying the neuro-behavioral effects of BPS, we also quantified the changes in the mRNA abundance of arginine vasotocin (AVT), isotocin (IT), and their corresponding receptors in the zebrafish brain. The results showed that E2 and BPS (30 µg/L) decreased shoal cohesion in both males and females. Moreover, a marked decline in group preferences was observed in all treatment groups, while locomotor activity remained unaffected. Alterations in the social behaviors were associated with sex-specific changes in the mRNA expression of genes involved in IT and AVT signaling. Taken together, the results of this study suggest that chronic exposure to BPS can impair zebrafish social behaviors via disruption of isotocinergic and vasotocinergic neuro-endocrine systems.

Predation risk induces age- and sex-specific morphological plastic responses in the fathead minnow Pimephales promelas.

Although comprehending the significance of phenotypic plasticity for evolution is of major interest in biology, the pre-requirement for that, the understanding of variance in plasticity, is still in its infancy. Most researchers assess plastic traits at single developmental stages and pool results between sexes. Here, we study variation among sexes and developmental stages in inducible morphological defences, a well-known instance of plasticity. We raised fathead minnows, Pimephales promelas, under different levels of background predation risk (conspecific alarm cues or distilled water) in a split-clutch design and studied morphology in both juveniles and adults. In accordance with the theory that plasticity varies across ontogeny and sexes, geometric morphometry analyses revealed significant shape differences between treatments that varied across developmental stages and sexes. Alarm cue-exposed juveniles and adult males developed deeper heads, deeper bodies, longer dorsal fin bases, shorter caudal peduncles and shorter caudal fins. Adult alarm cue-exposed males additionally developed a larger relative eye size. These responses represent putative adaptive plasticity as they are linked to reduced predation risk. Perhaps most surprisingly, we found no evidence for inducible morphological defences in females. Understanding whether similar variation occurs in other taxa and their environments is crucial for modelling evolution.

Neglected Patterns of Variation in Phenotypic Plasticity: Age- and Sex-Specific Antipredator Plasticity in a Cichlid Fish.

The ability of organisms to plastically respond to changing environments is well studied. However, variation in phenotypic plasticity during ontogeny is less well understood despite its relevance of being an important source of phenotypic variation in nature. Here, we comprehensively study ontogenetic variation in morphological antipredator plasticity across multiple traits in Pelvicachromis taeniatus, a western African cichlid fish with sexually dimorphic ornamentation. In a split-clutch design, fish were raised under different levels of perceived predation risk (conspecific alarm cues or distilled water). Morphological plasticity varied substantially across ontogeny: it was first observable at an early juvenile stage where alarm cue-exposed fish grew faster. Subsequently, significant plasticity was absent until the onset of sexual maturity. Here, alarm cue-exposed males were larger than control males, which led to deeper bodies, longer dorsal spines, larger caudal peduncles, and increased eye diameters. Sexual ornamentation emerged delayed in alarm cue-exposed males. In later adulthood, the plastic responses receded. Despite small effect sizes, these responses represent putative adaptive plasticity, as they are likely to reduce predation risk. In females, we did not observe any plasticity. In accordance with theory, these results suggest fine-tuned expression of plasticity that potentially increases defenses during vulnerable developmental stages and reproductive output.

Predator-induced neophobia in juvenile cichlids.

Predation is an important but often fluctuating selection factor for prey animals. Accordingly, individuals plastically adopt antipredator strategies in response to current predation risk. Recently, it was proposed that predation risk also plastically induces neophobia (an antipredator response towards novel cues). Previous studies, however, do not allow a differentiation between general neophobia and sensory channel-specific neophobic responses. Therefore, we tested the neophobia hypothesis focusing on adjustment in shoaling behavior in response to a novel cue addressing a different sensory channel than the one from which predation risk was initially perceived. From hatching onwards, juveniles of the cichlid Pelvicachromis taeniatus were exposed to different chemical cues in a split-clutch design: conspecific alarm cues which signal predation risk and heterospecific alarm cues or distilled water as controls. At 2 months of age, their shoaling behavior was examined prior and subsequent to a tactical disturbance cue. We found that fish previously exposed to predation risk formed more compact shoals relative to the control groups in response to the novel disturbance cue. Moreover, the relationship between shoal density and shoal homogeneity was also affected by experienced predation risk. Our findings indicate predator-induced, increased cross-sensory sensitivity towards novel cues making neophobia an effective antipredator mechanism.

Visual prey detection by near-infrared cues in a fish.

Many animal species are able to perceive light wavelengths beyond those visible to humans. While numerous species are additionally sensitive to short wavelengths (UV), long wavelengths such as the near-infrared spectrum (NIR) are supposed to be unsuitable for visual perception. Here, we experimentally show that under exclusive NIR illumination, the cichlid fish Pelvicachromis taeniatus displays a clear foraging response towards NIR reflecting prey. Additional control experiments without prey indicate that the observed behavior is not a mere response to the NIR environment. These results give first evidence for NIR visual sensitivity in a functional context and thus challenge the current view about NIR perception.