Effects of environmental cycles (light and temperature) on daily rhythms and its application to fish aquaculture- Efectos de los ciclos ambientales (luz y temperatura) en los ritmos diarios y su aplicación a la acuicultura de peces

Objectives, Methodology and Results: 1. To design and prove an automated-recording, low-cost and user- friendly-interface solution for studying thermal preferences and locomotor activity in fish of commercial interest both diurnal and nocturnal and to investigate if this daily thermal preference is driven by endogenous circadian clocks or not (Exp Chapters 1.1 and 1.2). To do so, fish were kept for a period of 28 days in a custom multi-chamber tank in which a thermal gradient was created. During these 28 days, the fish were subjected to three different photic conditions (LD, DL and DD) and their behaviour video recorded. Video analysis showed that all species tested (diurnal and nocturnal) exhibited a daily rhythm of thermal preference that persists even under constant DD condition and is therefore endogenously regulated by the circadian clock. 2. To evaluate the possible daily thermal preference in blind cavefish species pointing out temperature variations as a feasible Zeitgeber for these animals that have evolved in constant conditions environments and if these rhythms are endogenous or not (Exp Chapter 1.2). The methodology applied was already described in section 1. Both cave species tested showed a daily rhythm of thermal preference, but this did not persist in DD condition. 3. To delineate whether fish can exhibit daily rhythms of light selection and determine whether these rhythms are endogenous, testing different wavelengths (white and red) on the experimental zebrafish model (Exp Chapter 2.1). To do this, fish were free to choose a compartment (lit or dark) for a period of 7 days, after acclimation, testing both white and red light. Under white light condition, the fish exhibited a significant circadian endogenous rhythm, choosing the illuminated compartment during subjective day hours and the dark compartment during subjective night hours. 4. To ascertain and characterise the long-term photic entrainment of locomotor activity and circadian endogenous rhythmicity in the blind Mexican tetra and to investigate a possible daily relative expression of selected non- visual opsin genes in the brain of this species (Exp Chapter 2.2). The locomotor activity of the fish was long-term recorded and fish were kept in different photic conditions (LD, 6-hour shift of LD, DD and LLdim). Fish brains were sampled for 24 hours (every 4 hours) and subsequently processed (RNA extraction and cDNA retrotranscription) to study gene expression of non-visual opsins by Real-Time qPCR. Astyanax mexicanus showed a significant daily photic entrainment of locomotor activity. The persistence of this rhythm in DD and LLdim confirms its endogenous nature and that it is controlled by the circadian clock. 3 out of 6 non-visual opsin genes showed a daily rhythm of expression. 5. To describe the possible effects on fish physiology of emergent ingredient diets focusing on the central mechanisms that regulate food intake in Nile tilapia and Gilthead seabream (Exp Chapter 3). Fish were fed three different diets (one control and two experimental containing alternative ingredients to fish oil and meal) for 60 days. At the end of the trial, the brains were sampled and subsequently processed to study brain food intake control genes expression. Nile tilapia negatively accepted an experimental diet (with high inclusion levels of spirulina and quinoa) while sea bream positively accepted all three diets tested. 6. To determine a potential application of microalgal extract treatments in captive fish breeding, by investigating in vitro zebrafish cell proliferation and migration activity, involved in the healing process and to essay if these treatments can be time-dependent (day or night) in the healing process (Exp Chapter 4). To do so, the wound healing technique was performed on a zebrafish caudal fin fibroblast cell line and a microalgae extract was tested (different dilutions and for different times). The wound treated for 30 minutes with a 1:32 dose closed significantly before.