Elsevier

Aquaculture

Volume 523, 30 June 2020, 735220
Aquaculture

Central and peripheral clocks in Atlantic bluefin tuna (Thunnus thynnus, L.): Daily rhythmicity of hepatic lipid metabolism and digestive genes

https://doi.org/10.1016/j.aquaculture.2020.735220Get rights and content

Highlights

  • Daily rhythmicity was demonstrated in central and peripheral tissues in tuna.

  • Clock and lipid metabolism genes showed strong daily rhythmicity in brain and liver.

  • Several digestive enzyme genes studied also displayed rhythmicity.

  • The novel results could facilitate the development of efficient feeding protocols.

  • The knowledge will further support the aquaculture of this iconic species.

Abstract

Atlantic bluefin tuna (ABT; Thunnus thynnus) is a highly regarded and consumed species, but farming is still in its infancy. Currently, nothing is known about the presence of circadian rhythmicity at central or peripheral tissues, or if there are daily rhythms in expression of genes involved in lipid metabolism. In order to elucidate clock gene regulation of genes of lipid metabolism in ABT, six clock genes (bmal1, clock, cry1, cry2, per1 and per2) were sequenced and 24 h expression of these genes determined in brain and liver of fish acclimated to a light:dark (L:D) photoperiod. Additionally, the daily expression of lipid metabolism and digestive enzyme genes in ABT was also determined in liver. All six clock genes displayed rhythmicity in the brain and liver, other than cry2, which did not show an acrophase in liver. In liver, all the transcription factors analysed other than srebp1 and srebp2 displayed rhythmicity, with lxr and pparα displaying diurnal expression, whereas pparγ was highly expressed at the end of the scotophase. Some of the target genes of lxr such as elovl5 and lpl also oscillated rhythmically, with acrophases during the photophase. In contrast, only three of the eight digestive enzyme genes studied displayed rhythmicity, at different times of the day, suggesting that either ABT display different feeding periods or the digestion of some nutrients (e.g. lipids) is prioritized over others. The present study showed that clock and lipid metabolism genes displayed strong daily rhythmicity in ABT brain and liver, which could be an area of considerable interest for the establishment of efficient feeding protocols in this new aquaculture species.

Introduction

Organisms have developed biological clocks that regulate physiological processes in response to cyclic environmental conditions that vary in a regular manner (Panda et al., 2002). These clocks need to be adjusted daily by exogenous cues (zeitgebers, ZT) such as light-dark cycles in order to synchronize the molecular clocks that, in turn, generate the circadian rhythms (DeCoursey, 2004). In addition to this central circadian clock mechanism, there are peripheral clock systems that provide targeted rhythmic control to specific processes. The liver peripheral clock appears to play a key role in the entrainment of the circadian timing system in mammals (Yang et al., 2006; Le Martelot et al., 2009), with some recent studies focussing on teleosts (Vera et al., 2013; Betancor et al., 2014; Paredes et al., 2014, Paredes et al., 2015).

The molecular mechanisms of the circadian clock is composed of intracellular transcriptional-translational feedback loops that are, in turn, regulated by clock genes and corresponding proteins (Vatine et al., 2011). The positive arm of the circadian clock consists of two genes: the Circadian Locomotor Output Cycles Kaput (Clock) and aryl hydrocarbon receptor nuclear translocator-like protein-1 (also known as Brain and Muscle ARNT-Like Bmal1) that operate as a dimer, whereas the negative arm is controlled by period (Per) and cryptochrome (Cry) genes (Cahill, 2002). In mammals, an interplay between clock genes and the regulation of lipid and cholesterol metabolism has been described (Le Martelot et al., 2009) involving the activation of nuclear receptors such as sterol regulatory element binding proteins (Srebp; Sato, 2010), liver X receptor (Lxr; Evans et al., 2004) and peroxisome proliferator – activated receptors (Pparα and Pparγ; Cruz-García et al., 2009) and their respective target genes.

Fish in the wild display feeding rhythms that are related to food availability or the presence of predators (López-Olmeda and Sánchez-Vázquez 2010a). Under farming conditions when they are provided with a continuous supply of feed, fish continue to display feeding rhythms (Lopez-Olmeda and Sánchez-Vázquez, 2011). Given that the main operating cost in intensive aquaculture farms is feed, it is pivotal to know the feeding habits of the different fish species in order to minimise this cost, as the feeding regime applied could greatly impact on feed efficiency and waste. In this respect, the domestication and farming of Atlantic bluefin tuna (ABT; Thunnus thynnus) is in its infancy and little is known in this iconic species about their feeding habits or metabolic patterns throughout the day, including lipid metabolism or digestion. Given that this species is a top predator, it is not appropriate to simply extrapolate feeding protocols used for other species such as European sea bass (Dicentrarchus labrax) or gilthead sea bream (Sparus aurata).

A fuller understanding of all the physiological mechanisms is essential for the successful domestication of new fish species. The overall objective of the present study was to evaluate the relationships between the daily hepatic expression patterns of genes of the circadian clock, and genes involved in lipid metabolism and digestion in ABT. In this respect, daily patterns of expression of transcription factors and some of their target genes were studied. In addition, the daily expression pattern of the main components of the molecular clock were studied in the brain of ABT in a 24 h cycle. As this is the first study to report the expression patterns of the clock genes in ABT, firstly the complete or partial open reading frames of the six genes (bmal1, clock, per1, per2, cry1 and cry2) were identified and their expression determined during a daily cycle (14 L:10D) by real time quantitative PCR (qPCR). By evaluating the rhythmic daily regulation of lipid metabolism and digestive genes in ABT liver, deeper insight into the mechanisms involved in the regulation of absorption, transport and utilization of lipids and other nutrients will be achieved.

Section snippets

Isolation of clock genes

Sequences of genes encoding for clock mechanism genes (bmal1, clock, per1, per2, cry1 and cry2) were obtained by identifying the sequences from Sequence Read Archives (SRA) specific to T. thynnus (SRX2255758, ERX555873 and ERX555874). Once the contiguous sequences for each gene were obtained, these were assembled using CAP3 (Huang and Madan, 1999), confirming the identity of the deduced amino acid (aa) sequences by BLASTp sequence analysis service of the National Centre for Biotechnology

Brain gene expression

In the brain, all the clock genes studied displayed statistically significant daily rhythms in ABT subjected to a photoperiod of 14 L:10D (Fig. 1; Table 2). Three of the genes (bmal1, clock and cry1) peaked in the second half of the photophase (ZT12:44 h, ZT11:32 h and ZT12:29 h, respectively; Fig. 2). The acrophase of per1 was observed 20 min after lights were switched on (ZT0:20 h), whereas the acrophase of per2 was delayed more than three hours (ZT3:50 h; Fig. 1). In the case of cry2, the

Discussion

The present study described for the first time the comparison between the central and peripheral (liver) clock mechanisms in ABT. Moreover, daily rhythmic expression patterns of specific genes of lipid metabolism and digestion in liver of ABT were demonstrated, which indicated which molecular control mechanisms were involved. Here, partial cDNA sequences of six core clock genes (bmal1, clock, per1, per2, cry1 and cry2) were obtained in ABT. The results showed that, other than cry2 in liver, all

Declaration of Competing Interest

The authors declare no conflict of interest exist.

Acknowledgements

We thank the technical staff at the Laboratory of Marine Aquaculture (IEO), Puerto de Mazarrón (Murcia), Spain and Nutritional Analytical Services (NAS), Institute of Aquaculture, University of Stirling, UK that contributed to this work. This work was supported by an AquaExcel Transnational Action (TUNATIME; AE120023). Further funding was obtained through the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía, Proyecto de Excelencia de Promoción General del Conocimiento [Ref.

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