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Please use this identifier to cite or link to this item: http://hdl.handle.net/10277/290

Authors: Corà, Samuela
Internal Tutor: TEROVA, GENCIANA
Title: Molecular cloning and expression analysis of genes involved in the compensatory growth of sea bass (Dicentrarchus labrax).
Abstract: In natural aquatic systems, many fish species experience periods of fasting during their life cycle because of the high variation in food availability. Although fish are highly tolerant to relatively long periods of food deprivation, energy storage and growth rate is reduced in those that experience different degrees of starvation, redirecting this energy towards maintenance metabolism. Fasting-associated growth retardation is completely overcome, or at least reduced, if an abundant food supply becomes available after a prolonged period of food shortage. Then, fish display an exceptionally rapid growth rate known as “compensatory growth”. There are several descriptions of compensatory growth in fishes, but the mechanisms involved in such rapid recovery from fasting are still not fully understood. Such mechanisms have principally been searched for at the level of total fish growth where a number of circulating hormones are thought to be involved. Some attention has also been paid to muscle growth and to locally produced paracrine/autocrine factors, which can profoundly affect tissue growth and development. Only little information is available at the molecular level. Accordingly, this research focused on identifying candidate genes whose expression contributes to the compensatory growth induced by refeeding in sea bass (Dicentrarchus labrax), a marine fish of great interest for Mediterranean aquaculture. In the course of the study, we firstly isolated the complete cDNAs encoding diverse physiologically relevant proteins involved in lipid and protein metabolism, and then assessed the impact of chronic feed deprivation and subsequent refeeding on their mRNA abundance levels in different tissues, with the aim to relate these expression levels to fish feeding status. The molecular cloning and sequencing strategy resulted in the isolation of cDNAs that encode the following proteins: - Δ6 desaturase, which has a recognized capacity to desaturate the fatty acids with 18 carbon atoms, into highly unsaturated fatty acids (HUFA) with 20 and 22 carbon atoms. - Lipin, an enzyme required for triacylglycerol and phospholipid biosynthesis, and a transcriptional coactivator in regulating lipid metabolism genes. - Peroxisome proliferator-activated receptor (PPARγ), a central factor in the control of the expression of genes involved in lipid homeostasis. - Oligopeptide transporter (PepT1), an integral plasma membrane protein responsible for the uptake of dietary di- and tri-peptides from the intestinal lumen into the enterocytes. The isolated sequences were then deposited in GenBank databases with the following accession numbers: EU647692 for Δ6 desaturase; EU644089 for lipin; FJ237043 for PepT1. The evolutionary relationship of all target genes with respect to other publicly available related genes in other teleosts, amphibian, avian, and mammalian species, was also studied. After the sequence isolation, for the quantification by real-time RT-PCR of each gene transcript levels, in response to the nutritional status of the animals, a fasting and refeeding trial was conducted at the marine water recirculating system at our department in Varese. Briefly, 140 sea bass were stocked into four tanks of 2 m3 each, with 35 fish per tank. At the start of the experiment, all the fish were weighed, and two of the tanks were randomly assigned to each of two treatments. Fish in these two tanks were fed to apparent satiety (control), whereas fish in the other two tanks were deprived of food for 35 days and then refed to apparent satiety for 21 days with the same type of feed utilized before fasting. Five fish from each of the experimental groups were sampled at the following time points: before fasting (day 0), 4 days after fasting, at the end of fasting, and then sequentially at 4, 14, and 21 days following refeeding. For the molecular biology analysis, the whole digestive tract, liver, gill, heart, kidney, ovary, brain, muscle and spleen were dissected out. After 35 days of fasting body weight of fasted fish were lower than the fed controls, whereas during the subsequent refeeding period, previously unfed fish were able to increase body weight sufficiently to overcome weight loss imposed by the 35 days feed restriction. This is the evidence that the compensatory growth was in play. The results of the real-time RT-PCR absolute quantification using the mRNA standard curve method, revealed that the nutritional status significantly influenced E6 desaturase, lipin and PPARγ mRNA copy number in the liver of sea bass, inducing an up-regulation during prolonged fasting (35 days) and a downregulation during the recovery from fasting (21 days of refeeding). In the proximal intestine, 35 days of fasting contributed to a significant decrease in E6 desaturase transcript levels, whereas the subsequent recovery from fasting was associated with an increase in E6 desaturase mRNA copy number, which return to normal (control) levels after 21 days of refeeding. The abundance of lipin and PPARγ mRNA levels in the proximal intestine were not affected by the availability of food, as they remained similar between fed, fasted and refed fish. The nutritional status significantly influenced PepT1 mRNA copy number in the proximal intestine of sea bass, inducing a down-regulation during 35 days of fasting and an up-regulation during the subsequent 21 days of refeeding. This pattern of expression seems to be in complete support of the compensatory growth. The increase in the transcripts of the intestinal oligopeptide transporter Pept1 might be correlated to the great quantity of protein taken in with the food in the initial days of refeeding. In fact, refeeding of sea bass after a long starvation period was marked by hyperphagia, as early as the first day. In conclusion, we have isolated the cDNA sequences encoding Δ6 desaturase, lipin, PPARγ and Pept1in sea bass, and also demonstrated that the nutritional state of the animal influences their levels of expression. The present study is the first one to investigate the behavior of these transcripts over a long period of fasting and subsequent refeeding of fish. We recognize that mRNA levels in our study do not measure physiological effects produced by the proteins. Due to this, our hypothesis that the aforementioned genes are important triggers of the fasting response in the sea bass, will have to be confirmed by future studies.
Keywords: compensatory growth, gene expression, Δ6 desaturase, lipin, peroxisome proliferator-actoivated receptors (PPAR), oligopeptide transporter (PEPT1), real time PCR
Issue Date: 2010
Doctoral course: Analisi, Protezione e Gestione delle Biodiversità
Academic cycle: 23
Publisher: Università degli Studi dell'Insubria
Citation: Corà, S.Molecular cloning and expression analysis of genes involved in the compensatory growth of sea bass (Dicentrarchus labrax). (Doctoral Thesis, Università degli Studi dell'Insubria, 2010).

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