Transgenerational disrupting impacts of atrazine in zebrafish: Beneficial effects of dietary spirulina.

In a range of fish species, offspring sustainability is much dependent to their mother's investment into the egg yolk. A healthy environment helps broodfish to produce normal quality offspring. However, deviation from optimal conditions can disturb body functions that effect the next generation. Here, zebrafish (Danio rerio) was employed to investigate the transgenerational impacts of an immunotoxic and endocrine disruptor, atrazine (AZ). In addition, the possible ameliorated effects of a nutraceutical, Arthrospira platensis (spirulina- SP), was considered. Adult females were either exposed to 0 (Cn), 5 (AZ5), and 50 (AZ50) µg/L AZ or fed SP-supplemented diet (10 g/kg; SP). In combination treatments, fish were also exposed to AZ and fed SP (SP-AZ5 and SP-AZ50). Embryos were obtained after 28 d of exposure. Exposure to AZ50 caused females to produce eggs with significantly lower fertilization and hatching. No changes were observed in the concentrations of thyroid hormones. AZ significantly increased cortisol response and reduced levels of immunoglobulin, lysozyme and complement activities in females and their offspring. SP-AZ5 and SP-AZ50 females, however, resisted to the toxic effects of AZ, produced embryos with lower cortisol content and higher immunity competence. Bactericidal activity of the embryos also showed the transgenerational antimicrobial effects of SP along with the AZ immunotoxicity. Overall, these results indicate that AZ could have long lasting toxic effects on fish, and that dietary SP-supplementation could ameliorate AZ induced transgenerational toxic effects.

Behavioral and immunotoxic effects of Prograf® (tacrolimus) in the male Siamese fighting fish.

Siamese fighting fish (Betta splendens) has been extensively exploited in the behavioral and physiological toxicology studies of drugs. Tacrolimus is an immunosuppressant drug largely used in liver and renal transplantations. Here we found that a 7-day exposure of male B. splendens to concentrations of 0.05 and 0.1 µg/mL Prograf® (tacrolimus) caused alterations in aggression and immunity indexes. Tacrolimus exposed fish presented lower opercular display in a mirror test which is indicative of reduced aggression. In addition, serum levels of lysozyme, IgM, alternative complement, and bactericidal activity of subjects exposed to 0.1 µg/mL tacrolimus were lower than those from the control treatment. These results showed the behavioral impairment and immunotoxic impacts of tacrolimus in a model of aquatic toxicology. The results suggest fishes provide a possible model for better understanding of the drug action in vertebrates, and possible consequences for the environment via its effects on non-target organisms in an ecotoxicology context.

"Not tonight zebrafish": the effects of Ruta graveolens on reproduction.

CONTEXT: There is growing interest in the pharmacological evaluation of Rue due to its potential to treat a variety of clinical diseases. The plant seems to present potent endocrine disrupting effects, and its excretion and disposal are not a concern. OBJECTIVE: The effects of Ruta graveolens L. (Rutaceae) ethanol extract (RE) on reproductive behaviour, fertility, and steroid and thyroid hormone levels in zebrafish were investigated. MATERIAL AND METHODS: We exposed subjects to varying concentrations of RE, and one-tenth the LC50 concentration (2.37 ppm) was established as the sublethal dose. After 2 weeks exposure, reproductive behaviour, cumulative number of eggs laid, percentage of fertilized eggs, and whole body steroid and thyroid hormones were measured. RESULTS: Reproductive association behaviour did not differ between control and RE-exposed animals, but spawning attempts were reduced in RE exposed animals. Cumulative egg production between days 9 to 14, RE exposed fish laid 672 eggs while control fish laid 1242 eggs. Also, percentage of fertilized eggs was higher for the control than for the RE exposed fish. Estradiol-17ß (E2) levels were reduced in females exposed to RE and testosterone (T) was statistically lower in both males and females treated with RE. Furthermore, thyroid hormones (T3 and T4) declined in fish treated with RE. CONCLUSION: RE has endocrine disrupting potential in fish, which has important implications for studying the effects of unintentional pharmaceutical exposure. Moreover, the results demonstrate that drug exposure may affect more than just the overall level of behaviour, emphasizing the relevance of examining the effects of individual exposure. We reinforce the use of zebrafish as a model organism in physiology and behaviour, and raise concerns about the toxic effects of RE in non-target organisms such as aquatic vertebrates, which may ultimately affect human health.

Effects of the prebiotic mannan-oligosaccharide on the stress response of feed deprived zebrafish (Danio rerio).

Feed deprivation has deleterious effects on fish behavior and stress physiology which may susceptible them to disease outbreak. Functional ingredients in diets may substantially impact the physiology and stress responses of host organisms. Here, we hypothesized that the administration of a dietary prebiotic might attenuate the negative influences of feed deprivation on the behavioral profile of anxiety and physiological responses to stress in zebrafish (Danio rerio). Fish were fed with either basal or mannan-oligosaccharide supplemented (0.4% MOS/kg diet) diets, once per day (normal-control: CN, and normal-prebiotic: PN) or once every other day (starved-control: CS, and starved-prebiotic: PS) for 8weeks. Afterwards, fish were subjected to a novel tank test to measure anxiety. Fish from the CS treatment exhibited more pronounced bottom-dwelling behavior than the other treatments. The number of transitions from the bottom to the top third of the novel tank was significantly higher in PN fish than the CS specimens. No significant differences were found between the CN and PS treatments in all of the anxiety behaviors. CS fish showed higher baseline cortisol levels than the other treatments, which was in line with higher expression of CRH gene in fish subjected to this treatment. Cortisol levels and CRH gene expression of the subjects were also measured after induction of two routine aquaculture stressors. CN and PS fish exhibited similar patterns of cortisol responses at most of the sampling times after stress, and PN specimens showed a significantly lower concentration of cortisol than the other treatments in most cases. Expression of the CRH gene was higher in feed deprived fish immediately after stress induction. Overall, the results show that feed deprivation in some cases influenced anxiety-like behaviors and elevated stress response in zebrafish juveniles; however, the addition of MOS to the diet helped deprived fish exhibit behaviors more typical of normally fed animals.

Prozac impacts lateralization of aggression in male Siamese fighting fish.

Previous studies have shown that Siamese fighting fish, Betta splendens, preferentially use right-eye during the aggressive displays. However, administration of antidepressant drugs may disrupt eye-use preference in association with a reduction in aggression; a phenomena that has not been explored in fish. The objective of the current study was to examine the effects of exposure to the antidepressant drug, fluoxetine, on lateralization in eye-use during aggressive displays in male Siamese fighting fish. Baseline aggression and lateralization in eye use of thirty fish were assessed toward live conspecifics, following which experimental subjects (n=15) were then exposed to fluoxetine (540ng/L) in a static renewal water system. Behavior was quantified again after 9 days of exposure. All of the subjects preferentially used the right-eye during aggressive responses before the exposure experiments. Fluoxetine exposed subjects showed a reduction in the time spent gill flaring as has previously been reported, indicative of a reduction in the level of aggression. Fluoxetine also had a significant effect on the lateralization in preferred eye-use while looking at their opponent. Fish exposed to fluoxetine switched from a preferential use of the right-eye during aggressive encounters prior to exposure to using their left-eye after exposure to fluoxetine. The results are discussed with regard to asymmetrical distribution of serotonin between the two brain hemispheres.

Individual boldness traits influenced by temperature in male Siamese fighting fish.

Temperature has profound effects on physiology of ectothermic animals. However, the effects on temperature variation on behavioral traits are poorly studied in contrast to physiological endpoints. This may be important as even small differences in temperatures have large effects on physiological rates including overall metabolism, and behavior is known to be linked to metabolism at least in part. The primary aim of this study was to determine the effects of ambient temperature on boldness responses of a species of fish commonly used in behavioral experiments, the Siamese fighting fish (Betta splendens). At 26°C, subjects were first examined for baseline behaviors over three days, using three different (but complementary) 'open field' type assays tested in a fixed order. Those same fish were next exposed to either the same temperature (26°C) or a higher temperature (30°C) for 10days, and then the same behavioral assays were repeated. Those individuals exposed to increased temperatures reduced their latency to leave the release area (area I), spent more time in area III (farthest from release area), and were more active overall; together we infer these behaviors to reflect an increase in general 'boldness' with increased temperature. Our results add to a limited number of studies of temperature effects on behavioral tendencies in ectotherms that are evident even after some considerable acclimation. From a methodological perspective, our results indicate careful temperature control is needed when studying behavior in this and other species of fish.

Quantity discrimination in parental fish: female convict cichlid discriminate fry shoals of different sizes.

Numerical abilities have been found to be adaptive in different contexts, including mating, foraging, fighting assessment and antipredator strategies. In species with parental care, another potential advantage is the possibility to adjust parental behavior in relation to the numerosity of the progeny. The finding that many fish vary their parental investment in relation to brood size advocates the existence of a mechanism for appraising offspring number, an aspect that has never been directly investigated. Here we tested the ability of parental female convict cichlid (Amatitlania nigrofasciata) to discriminate between two fry groups differing in number by measuring time spent attempting to recover groups of fry experimentally displaced from the next. Females spent more time trying to recover the fry from larger groups when tested with contrasts 6 versus 12 (1:2) and 6 versus 9 fry (2:3); however, they showed no preference in the 6 versus 8 (3:4) contrast, suggesting that this task exceeds their discrimination capacity.

The toxicological effect of Ruta graveolens extract in Siamese fighting fish: a behavioral and histopathological approach.

The effects of pharmacological waste on aquatic ecosystems are increasingly highlighted in ecotoxicology research. Many of these products are designed for human physiology but owing to the conservative nature of vertebrate evolution they also tend to have effects on aquatic organisms and fishes in particular when they find their way into aquatic systems via wastewater effluent. One area of research has focused on reproductive control and the associated hormone treatments. Many of these hormones affect the reproductive physiology of fishes and may cause feminization of male reproductive traits. Alternative medicines have also been widely used particularly in traditional cultures but few of these alternative treatments have been assessed with respect to their potential impact on aquatic ecosystems. Rue (Ruta graveolens) has been used as a male contraceptive in traditional medicines but its effects on fish behavior and reproductive anatomy have yet to be established. Here we show that treating Siamese fighting fish, Betta splendens, with extract of rue has a significant effect on key aggressive/reproductive behaviors and the propensity to explore novel objects (boldness). In all cases the respective behaviors were reduced relative to controls and sham injected fish. Histological analysis of the testes revealed that rue exposure reduced the number of spermatozoa but increased the number of spermatocytes relative to controls.

Fluoxetine inhibits aggressive behaviour during parental care in male fighting fish (Betta splendens, Regan).

The increasing presence of aquatic contaminants, such as the pharmaceutical fluoxetine, has raised concerns over potentially disrupting effects on several aspects of fish reproduction. However, the effects of fluoxetine on reproductive and paternal behavior in fish remain understudied, particularly at environmentally relevant concentrations. In the current study, we therefore tested the hypothesis that waterborne fluoxetine at an environmentally relevant concentration (540 ng/l), disrupts specific reproductive and paternal behaviors in male Siamese fighting fish at distinct reproductive phases. A pre-post test design was adopted to investigate specific behavioral responses at the individual fish level in response to male conspecific intruders at two different distances from the nest across four distinct reproductive phases (before bubblenest construction, following bubblenest construction, after spawning and after hatching of the larvae). In the control specimens, the measured behaviours were not different between the spawning times and among the interactions in either distance to nest at the different reproduction phases. Our results indicate that fluoxetine specifically disrupts characteristic paternal territorial aggression behaviour only after spawning and hatching of the larvae, while male behaviour in previous reproductive phases is unaffected by fluoxetine exposure. Results of comparison between males at 1st spawning and specimens exposed to fluoxetine at 2nd spawning showed that the first reaction of the nest-holding males to the intruders, duration of fin spreading, number of bites, and 90° turn, and the frequency of sweeps were different between the spawning times after spawning or hatching of embryos. However, interaction of spawning time and reproduction phase was significant on biting behaviour. These results demonstrate that fluoxetine exposure at environmental concentrations negatively affects territorial defense behaviour in fighting fish during parental care after larval hatching, which may have possible implications on reproductive success and population dynamics.