Impact of lighting conditions and feeding cycles on rhythms of early development, behaviour and sexual differentiation in zebrafish "Danio rerio"

Abstract

The aim of the present thesis was to investigate the effect of light of different wavelength and photoperiod on early development, hatching rhythms and locomotor activity of zebrafish larvae. Morover, the study of an additional zeitgeber (food) permitted to evaluate the feeding rhythms synchronized to different feeding period in adult zebrafish and cavefish blind specie. Finally we aims to describe in adult zebrafish, the daily rhythms of genes involved on sexual differentiation. In the present Doctoral thesis the specific objectives were established: 1 To determine the ontogeny of zebrafish larvae clock genes expression (clock1, bmal1, per1b, per2 dbp) under light-dark cycle of 12:12 hours and different spectrum (LDW, LDB, LDR, DD). 2 To investigate the influence of several lighting conditions of different wavelength on the ontogeny of locomotor activity of zebrafish larvae. 3 To determine the effect of illumination changes of different light spectrum (LDW, LDB, LDR) on locomotor activity of zebrafish larvae and the presence of photo receptors capable to mediate the response to light. 4 To describe the existence of hatching rhythms and embryo synchronization to different light cycle (LD, DL 1, DL2, LL, DD). 5 To evaluate the existence of feeding anticipatory activity when food is periodically provided in zebrafish and cavefish under continuous dark conditions. Evaluate the ability of the fish to re-synchronize when feeding time is delayed. 6 To describe the daily variation in the expression of genes involved in sexual differentiation into male and female zebrafish adults. Methodology: 1. Embryos were maintained under LD 12:12 LDW (white), LDB (blue) and LDR (red). Larvae were sampled at 0,3,7 dpf and different time point ZT3,9,15,21. RNA was extracted and the gene expression analyzed with qPCR. 2. Embryos were kept in LDW, LDB, LDR. The larvae were recorded with a video camera and analyzed with specialized tracking software. 3.The larvae kept under LDW, LDB, LDR were recorded until 11 dpf with a camera. At 3 dpf were sampled and the presence of gene expression analyzed by PCR. 4.Embryos under LD, DL, DL, LL and DD at 28 ° C. They were visually analyzed every two hours to calculate hatching rate. 5.Zebrafish and cavefish were fed periodically with automatic feeder. Photocell with infrared beam were used to record the locomotor activity and a software "El Temps" used for data representation. 6 Zebrafish males and females maintained under cycle LD 12:12, and 28 degrees, were sampled during a day at different time point ZT2,6,10,14,18,22. The expression genes were measured in gonads and brain of both sexes with qPCR. Conclusions. 1.The ontogeny of clock genes expression develops differently depending on the light cycles and the wavelength of the light. The genes of the negative loop (per1b and per2) develop rhythmicity before, during development, respect those positive loop (clock and bmal). 2.The locomotor activity is strongly influenced by light cycles, showing no rhythmicity under continuous darkness. The different spectrum of light induces the asynchronous start of locomotor rhythmicity in larvae. 3.The locomotor activity increment as response of illumination change of different spectrum of the light cycles is related to the presence of several non visual photoreceptors already at 3 dpf. 4. Zebrafish hatch rhythmically during the light phase, appearing strongly synchronized to light-dark cycles. The phase of this rhythm at constant temperature depends on the lighting cycle. 5. The feeding anticipatory activity has endogenous origin in both species. The oscillator appears to posses: high plasticity in the synchronization of its rhythm to feeding cycle different of 24-hour and ability to re-synchronize when feeding time is delayed. 6. The expression of genes involved in sexual differentiation in adults varies during the day and the phase depends on the light cycle and sex.