The psychoactive drug Escitalopram affects swimming behaviour and increases boldness in zebrafish (Danio rerio).

Selective serotonin re-uptake inhibitors are pharmaceuticals used to treat a range of psychological disorders. They are frequently found in surface waters in populated areas. In recent years, they have been shown to affect the behaviour of various aquatic organisms in a way that can have ecological effects. In this study, we exposed zebrafish of both sexes to nominally 0.00, 0.15 and 1.50 µg L-1 Escitalopram in flow-through tanks for three weeks. Subsequently, ten swimming behaviour parameters were quantified using high-resolution video tracking. There were noticeable gender differences in the behaviour responses to Escitalopram. Female fish exposed to 1.50 µg L-1 Escitalopram had a lower maximum swimming velocity, stopped less often and exhibited increased boldness (reduced thigmotaxis) compared to controls. Male fish exposed to 1.50 µg L-1 had a lower maximum swimming velocity compared to control fish. At the end of exposures, both length and weight of the females exposed to 1.50 µg L-1 Escitalopram were significantly less than the group of control fish. In addition, males exposed to 1.50 µg L-1 Escitalopram were significantly shorter than control fish. The behaviour, weight and body length of the fish exposed to nominally 0.15 µg L-1 was not significantly different from control fish in either sex. The results of this study demonstrate that Escitalopram can affect subtle but ecologically important aspects of fish behaviour and lends further credibility to the assumption that Escitalopram is an environmentally active pharmaceutical.

Earthworms accumulate alanine in response to drought.

Earthworms have ecologically significant functions in tropical and temperate ecosystems and it is therefore important to understand how these animals survive during drought. In order to explore the physiological responses to dry conditions, we simulated a natural drought incident in a laboratory trial exposing worms in slowly drying soil for about one month, and then analyzed the whole-body contents of free amino acids (FAAs). We investigated three species forming estivation chambers when soils dry out (Aporrectodea tuberculata, Aporrectodea icterica and Aporrectodea longa) and one species that does not estivate during drought (Lumbricus rubellus). Worms subjected to drought conditions (< -2MPa) substantially increased the concentration of FAAs and in particular alanine that was significantly upregulated in all tested species. Alanine was the most important FAA reaching 250-650µmolg(-1) dry weight in dehydrated Aporrectodea species and 300µmolg(-1) dry weight in L. rubellus. Proline was only weakly upregulated in some species as were a few other FAAs. Species forming estivation chambers (Aporrectodea spp.) did not show a better ability to conserve body water than the non-estivating species (L. rubellus) at the same drought level. These results suggest that the accumulation of alanine is an important adaptive trait in drought tolerance of earthworms in general.

Male zebrafish (Danio rerio) courtship behaviour resists the feminising effects of 17alpha-ethinyloestradiol--morphological sexual characteristics do not.

The reproductive success of most fish depends partly on their ability to perform correct courtship behaviour. Alteration of this behaviour due to xenooestrogens can potentially affect the reproductive output and consequently population dynamics. In this lifetime study, zebrafish (Danio rerio) were exposed to environmentally realistic (0.05, 0.50 and 5.00 ng L(-1)) nominal concentrations of 17alpha-ethinyloestradiol (EE2) in a flow-through system for 4 months, from egg until sexual maturity. At 0.05 ng EE2L(-1), the secondary sexual characteristics of the males were significantly feminised with development of urogenital papillae and change in body colour. At 0.50 ng EE2L(-1), the sex ratio was altered from 69% males in the control groups to 59% males. The courtship behaviour of the male zebrafish was not affected by these two lower EE2 concentrations. Only at the highest concentration of 5.00 ng EE2L(-1), where only 5% of the group developed into males, was a change in behaviour recorded, and these few males were unable to induce spawning. Accordingly, the courtship behaviour of zebrafish is more resistant to oestrogenic exposure than secondary sexual characteristics and gonad development. This study provides the first quantitative measure of zebrafish courtship behaviour.

Differences in Reproductive Behavior between Spawning and Non-Spawning Zebrafish Pairs and the Effects of 17α-Ethinylestradiol (EE2).

Reproductive success manifested by spawning and fertilization, in most fish, depends partly on an appropriate courtship behavior by both sexes. The zebrafish reproductive behavior can be resolved in some of its constituent elements by a computerized vision system and described in unbiased quantitative terms. Pairs of adult male and female zebrafish were monitored with automatic video tracking at 16 Hz for 45 min in a tank with a spawning area in one corner. Subsequently, spawning, if any, was registered and the swimming behavior and mutual interactions of the two fish were quantified. Further, temporal and frequency distributions of average velocity and turning rate were produced. It is demonstrated that the courtship behavior in spawning pairs differs markedly from non-spawning pairs with differences in both male and female behavior. EE2 (17α-ethinylestradiol), a contraceptive hormone found in aquatic environments, has only a slight effect on these behavior differences between spawning and non-spawning pairs.

Disrupted reproductive behavior in unexposed female zebrafish (Danio rerio) paired with males exposed to low concentrations of 17α-ethinylestradiol (EE2).

Estrogenic substances, including the contraceptive pharmaceutical 17α-ethinylestradiol (EE2), pose a threat to aquatic wildlife causing endocrine disturbances of physiological processes and reproductive behavior. We have previously demonstrated that the reproductive behavior of male zebrafish, Danio rerio, remain intact after lifelong exposure to low concentrations of EE2 (0.05 and 0.5ngL(-1)), concentrations high enough to cause morphological alterations of the male phenotype. Despite normal courtship behavior, the reproductive output is suppressed when these males were paired with unexposed females. In this study, we include the female courtship behavior in the analyzes of EE2's effects on behavior. Groups of male zebrafish were exposed to nominally 0, 1, 3 and 10ngL(-1) of EE2 from hatching to adulthood and subsequently individually paired with an unexposed female. Eleven distinct elements of the reproductive behavior were then extracted and analyzed using an automated video tracking system. Subsequently, male brains were isolated and the expression of genes encoding estrogenic receptors ERα, ERß1 and ERß2, the androgen receptor AR, and the aromatase cyp19a1b were compared with the expression in brains of unexposed males. (In this article gene expression is taken synonymous to transcription, although it is acknowledged that it can also be regulated by, e.g., mRNA and protein stability, and translation). The results confirmed that the male reproductive behavior was unaffected by the EE2 treatments. Also, the expressions of genes encoding estrogen and androgen receptors were unaffected. Only the gene encoding aromatase was 0.6 fold down-regulated. In contrast, and most surprisingly, nearly all the elements in the female courtship behavior were significantly disturbed, despite the fact that these females had never been exposed to EE2; most likely elicited by differences in male morphology, pheromones or some other unrevealed mechanism.

Accumulation of free amino acids during exposure to drought in three springtail species.

Springtails are closely related to insects, but they differ from these with respect to water balance, in particular because springtails are small and have high integumental permeability to water. Here we report a series of experiments addressing the dynamics of osmoregulation, water content and accumulation of free amino acids (FAAs) in three springtail species during exposure to a gradually increasing environmental desiccation simulating conditions in drought exposed soil. Folsomia candida and Protaphorura fimata (both living in the deeper soil layers; euedaphic species) were active throughout the 3week exposure, with the developing drought regime ending at -3.56MPa (the soil water activity at the permanent wilting point of plants is -1.5MPa) and remained hyperosmotic (having an body fluid osmolality higher than the corresponding environment) to their surrounding air. Sinella curviseta (living in upper soil/litter layers; hemiedaphic species) also survived this exposure, but remained hypoosmotic throughout (i.e. with lower osmolality than the environment). The body content of most FAAs increased in response to drought in all three species. Alanine, proline and arginine were the most significantly upregulated FAAs. By combining our results with data in the literature, we could account for 82% of the observed osmolality at -3.56MPa in F. candida and 92% in P. fimata. The osmolality of S. curviseta was only slightly increased under drought, but here FAAs were considerably more important as osmolytes than in the two other species. We propose that FAAs probably have general importance in drought tolerance of springtails.