The liver-X-receptors (LXRs) act as cholesterol sensors and participate in the regulation of lipid and cholesterol metabolism. The objective of this study was to determine the role of LXR during development using the zebrafish model. By in situ hybridization we showed distinct expression of lxr in the brain and the retina in the developing and adult zebrafish. Lxr ligand activation affected the expression of genes involved in lipid metabolism in zebrafish adult brain and eye as well as in zebrafish embryos. Morpholino knock down of lxr resulted in an overall impaired lipid deposition as determined by oil red O staining particularly in the head and around the eyes, and to significantly elevated levels of both total and free cholesterol in the yolk of lxr morphant embryos. The expression of genes involved in lipid and cholesterol metabolism was also changed in the lxr morphants. Furthermore, alcian blue staining revealed malformation of the pharyngeal skeleton in the lxr morphant. Our data show that Lxr is an important component of the regulatory network governing the lipid homeostasis during zebrafish development, which in turn may support a role of Lxr for normal development of the central nervous sytem, including the retina.

Oestrogens are biosynthesised by cytochrome p450-aromatase (Cyp19). Brain oestrogens serve several important functions of which nerve protection, cell proliferation, nerve development and behaviour control are a few. Teleost brain aromatase activity is exceptionally high (between 100-1000 times) compared to mammals and birds. We have successfully cloned and sequenced a 950 bp long partial fragment of the guppy CYP19B gene (PrCyp19b) derived from adult brain mRNA. Sequence alignment of translated amino acid sequence shows PrCYP19b having high sequence similarity to teleost brain aromatase. Anatomical distribution of PrCYP19b expression in adult guppy brains was studied using in situ-hybridisation with an antisense riboprobe synthesised from the cloned PrCYP19b gene. Expression of brain aromatase appeared at ventricular surfaces of the ventral telencephalic zones and the pre-optic area, in the hypothalamus, the pituitary gland, optic tectum and the cerebellum. This pattern is similar to previously reported distribution of brain aromatase in other teleosts. Measurement of brain aromatase activity in telencephalon, mesencephalon/diencephalon and rhombencephalon revealed female guppies producing the most oestrogens in mesencephalon/diencephalon, whereas males produces the most oestrogens in both telencephalon and mesencephalon/diencephalon. This indicates that brain oestrogen production is sexually dimorphic in the guppy and may serve different functions in the two genders.

In mammalian and avian vertebrate groups, androgens act as controlling agents on male aggression and courtship behaviour by their conversion to oestrogens by cytochrome P450 aromatase in well-defined brain regions. Despite the fact that bony fishes have exceptionally high brain aromatase activity, little is known about it's possible regulatory effects on the reproductive behaviours of teleosts. In this study, Endler guppy males (Poecilia reticulata) were subjected to 26-29 days of 24-h exposure to two different concentrations (15 and 100 pg/L) of the aromatase inhibitor fadrozole in the water. Compared with the control males, two of three courtship activities in males exposed to the higher concentration were reduced when they were paired with receptive stimulus females. Reduction in brain aromatase activity was confirmed in both exposed groups with the use of the tritiated water assay.

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen androstenedione (A), the synthetic estrogen 17 alpha-ethinylestradiol (EE2), and the estrogenic surfactant 4-nonylphenol (NP), at high (50 mu g/L) and at environmentally relevant concentrations (10 ng/L EE2, 5 mu g/L NP, and 0.7 mu g/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE2 at 50 mu g/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE2 treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short-term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors.

Understanding the neuroendocrinological mechanisms behind reproductive behaviour is fundamental when studying endocrine disruption. Neuroestrogen production is a key step in the activation of reproductive behaviours among vertebrates. The actions of estrogens are transmitted through estrogen receptors (ERs) in distinct brain nuclei. Here we report alterations in reproductive behaviours in guppy males following 55-day food treatments with the antiestrogen fulvestrant (ICI) and the synthetic oestrogen 17α-ethinylestradiol (EE2). Male courtship and mating attempts were severely reduced in EE2 treated males after both 27-30 and 41-55 days of treatment when paired with females. ICI treatment gave a significant reduction in the frequency of sigmoid display behaviour after 27-30 days of treatment, and an almost 2.5-fold increase in gonopodium thrusting after 41-55 days of treatment. ICI treated males also decreased their frequency in successful mating attempts in comparison to the control males. The neurological effects of ICI were confirmed by Real Time-PCR analysis for brain aromatase and ERα gene expression. ICI treatment suppressed aromatase expression to 64% and stimulated ERα gene expression by over 300%. These results indicate that oestrogen action via ERs may play an important role for the complete display of male courtship and mating behaviour in the guppy. The results also suggest that local steroids are involved in regulating brain aromatase expression and that the negative effects of EE2 on sexual behaviour are linked via endocrine disruption of gonadal function.

Behaviour studies are used in toxicology research as they are excellent tools to measure physiological end-points caused by exogenous chemicals. In mammals both reproductive and non-reproductive behaviours have been used for a long period of time, whereas in teleost fishes non-reproductive behaviours have received little attention compared to reproductive behaviours. Recent advances in measuring stress related behaviours in zebrafish have provided additional tools to understand behaviour toxicology in fish. One species with well documented reproductive behaviour disturbed by different toxicants is the guppy, which is better suited than zebrafish for reproductive behaviour studies and therefore might be a better model organism for comparative behaviour studies in fish toxicology. Here we report new applications for non-reproductive behaviours in guppy and test these behaviours on males treated with the endocrine disruptor 17α-ethynylestradiol at environmentally relevant concentrations. 17α-ethynylestradiol increased freezing and bottom-dwelling when fish were placed in a non-familiar aquarium, but did not significantly affect shoaling behaviour. These results are similar to the anxiogenic behaviours seen in rats treated perinatally with 17α-ethynylestradiol and add more concern to the impacts of endocrine disruptors on aquatic wildlife.

The processes underlying divergent selection and genetic adaptation have been on the evolutionary biologists agenda for a long time. In this study we used the three-spined stickleback (Gasterosteus aculeatus) study system, a perfect system to study the evolution of similar traits in different lineages exposed to similar environmental conditions. Lind and Grahn (2011) have found directional selection caused by pulp mill effluent on populations of three-spined stickleback along the Swedish coast. In their study, they identified 21 AFLP- outlier loci indicated to be under selection. Here we converted some of these anonymous AFLP loci into sequenced markers and aligned them to the stickleback genome. Four out of five loci, aligned within or close to coding regions, on chromosome I, chromosome VIII, chromosome XIX and chromosome XX. One of the locus, located on chromosome VIII, have been identified to be under selection for fresh water adaption in other studies, including Baltic Sea stickleback populations (Mäkinen et al. 2008a,b). We believe that this is feasibly method that can be used as a starting point for identification of genes and genomic regions possible involved in adaptation, both for model and non-model organisms. 

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1μgL^-1. In two experiments exposing both sexes to 0.2, 2.3 or 15μgL^-1 for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15μgL^-1. They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15μgL^-1 significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25ng/L EE2for 14days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25ng/L, but not 5ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41-48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.

Exposure to estrogenic endocrine disruptors (EDCs) during of development affects fertility, reproductive and non-reproductive behavior in mammals and fish. These effects can also be transferred to coming generations. In fish, the effects of developmental EDC exposure on non-reproductive behavior is less well studied. Here, we analyze the effects of 17α-Ethinylestradiol (EE2) on anxiety, shoaling behavior and fertility in zebrafish after developmental treatment and remediation in clean water until adulthood. Zebrafish embryos were exposed from day 1 to day 80 post fertilization to actual concentrations of 1.2 and 1.6ng/L EE2. After remediation for 82days non-reproductive behavior and fertilization success were analyzed in both sexes. Males and females from the 1.2ng/L group, as well as control males and females, were bred, and behavior of the untreated F1 offspring was tested as adults. Developmental treatment with 1.2 and 1.6ng/L EE2 significantly increased anxiety in the Novel Tank test and increased shoaling intensity in both sexes. Fertilization success was significantly reduced by EE2 in both sexes when mated with untreated fish of opposite sex. Progeny of fish treated with 1.2ng/L EE2 showed increased anxiety in the Novel tank test and increased light avoidance in the Scototaxis test compared to control offspring. In conclusion, developmental exposure of zebrafish to low doses of EE2 resulted in persistent changes in behavior and fertility. The behavior of unexposed progeny were affected by their parents' exposure, which might suggest transgenerational effects.

The effects of endocrine disruptors may vary with the timing of exposure. The physiological implications of adult exposure are present during and shortly after exposure while embryonic exposure can imprint changes manifested in adulthood. In this study, guppy (Poecilia reticulata) embryos were exposed to 2 ng/L and 20 ng/L of 17α-ethinylestradiol during development via the mother and reared in clean water from gestation until 6 months of age. As adults, fish exposed to 20ng/L during development showed significantly altered behaviour in the Novel Tank test, where anxiety is determined as the tendency to remain at the bottom upon introduction into an unfamiliar tank. 17α-ethinylestradiol treatment increased the latency time before swimming to the upper half of the tank and decreased the number of transitions to the upper half. In control females the basal stress behaviour responses were significantly higher than in males, as indicated by longer latency period and fewer and shorter visits to the upper half, supporting the importance of gonadal hormones for the behaviour. The anxiety increased, however, with treatment in both sexes, suggesting that the observed response is not entirely due to feminization of the males. Shoaling behaviour, analyzed as tendency to leave a shoal of littermates, was neither sex-differentiated nor changed by treatment. Also male reproductive behaviour, brain aromatase activity and testes histology, previously shown to respond to oestrogen exposure in adult guppy, were unaffected by the developmental treatment. This suggests that the stress system in the guppy is very sensitive to 17α-ethinylestradiol, which possibly causes an early organisational imprint on the brain circuit that regulates stress reactions.