Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus).

Abstract

The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D:12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10:00 h); (2) fish fed with a 12-h shifted schedule (at 22:00 h), (3) fish fed at 10:00 h throughout the experiment, except the last day when fed at 22:00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, gPer1a, gPer2a, gPer3, and gCry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock.

Interplay between the endocrine and circadian systems in fishes

“…Similarly, gut melatonin in goldfish also exhibits this dependence on scheduled feeding (Vera et al 2007). The close correlation between the acrophases of biosynthetic enzymes (aanat2 and hiomt2; Velarde et al 2010) and clock genes (per1a, per2a and cry3; Nisembaum et al 2012) in the goldfish hindgut supports the idea that gut melatonin rhythmicity is coupled to a gut molecular clock that may work independently of the pineal clock.…”

Performing a hepatic timing signal: glucocorticoids induce gper1a and gper1b expression and repress gclock1a and gbmal1a in the liver of goldfish

“…Clock genes (gper1a, gper1b, gper2a, gper3a, gcry1a, gcry2a, gcry3, gbmal1a, gclock1a and gclock2) expression was measured by RT-qPCR, using gβ-actin as a reference gene as previously described (Nisembaum et al 2012;Nisembaum et al 2014a, b …”

“…In goldfish (Carassius auratus), a rhythmic expression of several clock genes has been reported in the central nervous system and also in peripheral tissues (Velarde et al 2009;Feliciano et al 2011;Nisembaum et al 2012;Sánchez-Bretaño et al 2015), including the liver. Moreover, the hepatic oscillator in this teleost seems to be modulated by feeding time (Feliciano et al 2011), and also by the orexigenic hormone ghrelin (Nisembaum et al 2014b).…”

“…Specific primers and Gene Data Bank Reference Numbers are shown inTable 1. RNA extraction (TRI ® Reagent method, Sigma Chemical, Madrid, Spain), DNase treatment (Promega, Madison, USA), cDNA synthesis (Invitrogen, Carlsbad, USA) and real-time PCR reactions (iTaqTM SYBR ® Green Supermix in a CFX96TM Real-Time System, Biorad Laboratories, Hercules, USA) were carried out following manufacturer instructions with minor modifications(Nisembaum et al 2012). Total RNA (1 μg) was retrotranscribed and PCR reactions were developed in a final volume of 10 μl (1 μl of cDNA per sample).…”

Anatomical distribution and daily profile ofgper1bgene expression in brain and peripheral structures of goldfish (Carassius auratus)

“…Despite the fact that the SCN is neuroanatomically well defined in fish (Rink & Wullimann, 2004), there is no evidence that it acts as a major clock.Using RT-PCR, expression of several clock genes were detected in some areas in the brain of trout (Oncorhynchus mykiss, Mazurais et al, 2000) zebrafish (Danio rerio, Moore & Whitmore, 2014;Whitmore et al, 1998), Atlantic salmon (Salmo salar, Davie et al, 2009), European seabass (Dicentrarchus labrax, del Pozo et al, 2012), Senegal sole (Solea senegalensis, Martín-Robles et al, 2012), threespot wrasse (Halichoeres trimaculatus, Hur et al, 2012) and goldfish (Velarde et al, 2009). In addition, clock genes transcripts were identified in different peripheral tissues including pituitary, heart, kidney, liver, testis, gills, intestine, muscle, ovary and spleen in zebrafish (Cermakian et al, 2000;Whitmore et al, 1998), European seabass (del Pozo et al, 2012), Senegal sole (Martín-Robles et al, 2012) and goldfish (Nisembaum et al, 2012;Velarde et al, 2009). However, there are very few studies dedicated to the accurate localization of clock gene expression in fish brain, and, to the best of our knowledge, none in peripheral tissues.…”