The role of the Glucocorticoid Receptor in the Regulation of Diel Rhythmicity.

Virtually all organisms have adapted to the earth's day-night cycles by the evolution of endogenous rhythms that regulate most biological processes. Recent research has highlighted the role of glucocorticoids and the Glucocorticoid receptor (GR) in coordinating clock function across various levels of biological organisation. In the present study, we have explored the role of the GR in the rhythmicity of the biological clock, by comparing 5 day old wildtype zebrafish larvae (gr+) with mutant larvae with a non-functional GR (grs357). The mutants display a weaker rhythmicity in locomotor activity in wildtypes than in mutants, while the rhythmicity of the angular velocity was higher for wildtypes. The melatonin production of the mutants showed a weaker rhythmicity, but surprisingly, there were no differences in the rhythmicity of clock-related gene expression between genotypes that could explain a mechanism for GR functionality at the transcriptional level. Furthermore, our results show that grs357 larvae have a more erratic swimming path, and cover more distance during locomotor activity than wild type larvae, in line with previously described behaviour of this mutant. Therefore, these results suggest that GR affects the diel rhythmicity of zebrafish larvae at the behavioural and endocrine level, but that these effects are not mediated by changes in the expression of clock-related genes.

Profound effects of glucocorticoid resistance on anxiety-related behavior in zebrafish adults but not in larvae.

Previously, adult zebrafish with a mutation in the gene encoding the glucocorticoid receptor (Gr) were demonstrated to display anxiety- and depression-like behavior that could be reversed by treatment with antidepressant drugs, suggesting that this model system could be applied to study novel therapeutic strategies against depression. Subsequent studies with zebrafish larvae from this grs357 line and a different gr mutant have not confirmed these effects. To investigate this discrepancy, we have analyzed the anxiety-like behavior in 5 dpf grs357 larvae using a dark/tapping stimulus test and a light/dark preference test. In addition, grs357 adult fish were subjected to an open field test. The results showed that in larvae the mutation mainly affected general locomotor activity (decreased velocity in the dark/tapping stimulus test, increased velocity in the light/dark preference test). However, parameters considered specific readouts for anxiety-like behavior (response to dark/tapping stimulus, time spent in dark zone) were not altered by the mutation. In adults, the mutants displayed a profound increase in anxiety-like behavior (time spent in outer zone in open field test), besides changes in locomotor activity (decreased velocity, increased angular velocity and freezing time). We conclude that the neuronal circuitry involved in anxiety- and depression-like behavior is largely affected by deficient Gr signaling in adult fish but not in larvae, indicating that this circuitry only fully develops after the larval stages in zebrafish. This makes the zebrafish an interesting model to study the ontology of anxiety- and depression-related pathology which results from deficient glucocorticoid signaling.

Reproductive toxicity of primary and secondary microplastics to three cladocerans during chronic exposure.

Microplastics (<5 mm) are distributed ubiquitously in natural environments. The majority of microplastics in aquatic environments are shown to have rough surfaces due to various weathering processes (secondary microplastics; SMP), while laboratory studies predominantly utilise pristine microplastics (primary microplastics; PMP). Here we present the results from a study comparing the chronic effects of pristine PMP and artificially weathered SMP to three different Cladoceran species (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia). We assessed the impact of PMP and SMP on reproductive output using various measured parameters, including time of first brood, size of first brood, size of first three broods, cumulative number of neonates, total number of broods and terminal length of test animals. Our results show that reproductive output of all species declined in a dose-dependent manner. The No Observed Effect Concentration (NOEC) was less than the lowest tested concentration (102 p/mL) for at least one measured endpoint for all species and both PMP and SMP. Further, it was inferred that species sensitivity varied inversely with body size for most endpoints, resulting in C. dubia being the most sensitive species; and D. magna being the least sensitive species under study. In addition, PMP appeared to have greater toxic potential as compared to SMP. This study is the first to directly compare the chronic toxicity of both pristine and weathered microplastic particles on three freshwater toxicological model organisms. Our results indicate that sensitivity in reproduction and growth to microplastics may differ between species and type of microplastic exposed; highlighting the importance of using multiple species and structural types of particles.

Acute sensitivity of three Cladoceran species to different types of microplastics in combination with thermal stress.

Microplastics (<5 mm, MP) are ubiquitously distributed in the environment, causing increasing concern regarding their potential toxicity to organisms. To date, most research has focussed on the impacts of MPs on marine and estuarine organisms, with fewer studies focussing on the effects of microplastics on freshwater ecosystems, especially under different environmental conditions. In the present study, the sensitivity of two temperate Cladoceran species, Daphnia magna and Daphnia pulex, and a smaller tropical species Ceriodaphnia dubia, to primary microplastics (PMP) and secondary (weathered) microplastics (SMP) was assessed. A prolonged acute toxicity assay (up to 72 or 96 h) was performed at 18°, 22°, and 26 °C, to determine the influence of temperature as an additional stressor and survival data were analysed using toxicokinetic-toxicodynamic (TK-TD) model. Acute sensitivity of D. magna and D. pulex to both PMP and SMP increased sharply with temperature, whereas that of C. dubia remained relatively stable across temperatures. C. dubia was the most sensitive species at 18 °C, followed by D. pulex and D. magna, which were of comparable sensitivity. However, this ranking was reversed at 26 °C as could be seen from the No Effect Concentration (NEC) estimates of the TK-TD model. In addition, SMP and PMP had a similar effect on D. magna and D. pulex, but PMP was more toxic to C. dubia. Effects on survival were strongly time-dependent and became substantially more severe after the standard 48 h test period. Our results indicate that sensitivity to microplastics may differ between species for different types of microplastics, and could be drastically influenced by temperature albeit at high exposure concentrations.