Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) in blunt snout bream (Megalobrama amblycephala): cDNA cloning, tissue distribution and mRNA expression changes responding to fasting and refeeding

Accepted Manuscript Ghrelin, Neuropeptide Y (NPY) and cholecystokinin (CCK) in blunt snout bream (Megalobrama amblycephala): cDNA cloning, tissue distribution and mRNA expression changes responding to fasting and refeeding Wei Ji, Hai-Chao Ping, Kai-Jian Wei, Gui-Rong Zhang, Ze-Chao Shi, Rui-Bin Yang, Gui-Wei Zou, Wei-Min Wang PII: DOI: Reference:

S0016-6480(15)00228-2 http://dx.doi.org/10.1016/j.ygcen.2015.08.009 YGCEN 12164

To appear in:

General and Comparative Endocrinology

Received Date: Revised Date: Accepted Date:

27 January 2015 18 August 2015 22 August 2015

Please cite this article as: Ji, W., Ping, H-C., Wei, K-J., Zhang, G-R., Shi, Z-C., Yang, R-B., Zou, G-W., Wang, WM., Ghrelin, Neuropeptide Y (NPY) and cholecystokinin (CCK) in blunt snout bream (Megalobrama amblycephala): cDNA cloning, tissue distribution and mRNA expression changes responding to fasting and refeeding, General and Comparative Endocrinology (2015), doi: http://dx.doi.org/10.1016/j.ygcen.2015.08.009

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1

Ghrelin, Neuropeptide Y (NPY) and cholecystokinin (CCK) in blunt

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snout bream (Megalobrama amblycephala): cDNA cloning, tissue

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distribution and mRNA expression changes responding to fasting and

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refeeding

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Wei Ji1,2†, Hai-Chao Ping 1†, Kai-Jian Wei1,2*, Gui-Rong Zhang1,2*, Ze-Chao Shi3,

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Rui-Bin Yang 1,2, Gui-Wei Zou3 and Wei-Min Wang1

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1– Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture of China,

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College of Fisheries, Huazhong Agricultural University, Wuhan, P. R. China

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2– Freshwater Aquaculture Collaborative Innovation Centre of Hubei Province, Wuhan, P.R.

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China

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3– Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of

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China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences,

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Wuhan, P. R. China

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*–Corresponding author.

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Mailing address for Kai-Jian Wei: College of Fisheries, Huazhong Agricultural University,

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Wuhan 430070, P. R. China. Tel: +86 27 87282113; Fax: +86 27 87282114. E-mail:

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[email protected]

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Mailing address for Gui-Rong Zhang: College of Fisheries, Huazhong Agricultural

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University, Wuhan 430070, P. R. China. Tel: +86 27 87282113; Fax: +86 27 87282114.

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E-mail: [email protected]

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† – These authors contributed equally to this work.

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Abstract

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Blunt snout bream (Megalobrama amblycephala Yih, 1955) is an endemic freshwater fish in

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China for which the endocrine mechanism of regulation of feeding has never been examined.

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Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) play important roles in the

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regulation of fish feeding. In this study, full-length cDNAs of ghrelin, NPY and CCK were

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cloned and analyzed from blunt snout bream. Both the ghrelin and NPY genes of blunt snout

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bream had the same amino acid sequences as grass carp, and CCK also shared considerable

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similarity with that of grass carp. The three genes were expressed in a wide range of adult

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tissues, with the highest expression levels of ghrelin in the hindgut, NPY in the

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hypothalamus and CCK in the pituitary, respectively. Starvation challenge experiments

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showed that the expression levels of ghrelin and NPY mRNA were up-regulated in brain and

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intestine after starvation, and the expression levels of CCK were down-regulated after

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starvation. Refeeding could bring the expression levels of the three genes back to the control

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levels. These results indicated that the feeding behavior of blunt snout bream was regulated

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by the potential correlative actions of ghrelin, NPY and CCK, which contributed to the

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defense against starvation. This study will further our understanding of the function of

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ghrelin, NPY and CCK and the molecular mechanism of feeding regulation in teleosts.

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Key words: ghrelin; neuropeptide Y; cholecystokinin; Megalobrama amblycephala; fasting;

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refeeding

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1. Introduction Food intake plays an important role in fish growth and production performance, which is

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regulated by both central and peripheral signals, involving the central nervous system (CNS),

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gastrointestinal (GI) tract, adrenals, pancreas and adipose tissue (Naslund and Hellstrom,

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2007). Like other vertebrates, fish regulate their feeding by key appetite-stimulating

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(orexigenic) and appetite-inhibiting (anorexigenic) endocrine factors. Orexigenic factors

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include ghrelin, neuropeptide Y (NPY), galanin, orexins and agouti-related protein (AgRP).

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Anorexigenic

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amphetamine-regulated transcript (CART), and corticotropin-releasing factor (CRF)

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(Volkoff et al., 2010). Ghrelin, NPY and CCK are important endocrine factors in the

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regulation of feeding, as well as growth and reproduction of fish.

factors

include

cholecystokinin

(CCK),

leptin,

cocaine-

and

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Ghrelin is a brain-gut peptide first purified from rat stomach as an endogenous ligand for

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the growth hormone secretagogue receptor (GHS-R). It has two main physiological actions:

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regulation of appetite and hormone (including GH (growth hormone)) release (Kaiya et al.,

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2008; Kojima et al., 1999). So far, ghrelin has been isolated from several teleost and

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elasmobranch fish species (Kaiya et al., 2011). Previous studies showed that Ghrelin mRNA

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was mainly expressed in the fish stomach and in the intestine of stomachless fish, but it was

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also detected in the hypothalamus, spleen, gill, skin, kidney, heart and liver of fish (Feng et

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al., 2012; Xu and Volkoff, 2009). The tissue distribution patterns vary from species to

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species in fish and other vertebrates (Kaiya et al., 2011). Ghrelin can stimulate GH release.

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For example, Unniappan and Peter (2004) reported that intracerebroventricular (ICV) or

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intraperitoneal (IP) injections of ghrelin stimulated the release of GH in 15 minutes in the

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goldfish (Carassius auratus). In addition, both IP and ICV injections of ghrelin stimulated

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food intake in goldfish (Unniappan et al., 2004a) and this effect was mediated by NPY and

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orexin pathways (Miura et al., 2006, 2007). Ghrelin mRNA expression increased during 3

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starvation in goldfish (Unniappan et al., 2004b), sea bass (Dicentrarchus labrax) (Terova et

77

al., 2008) and zebrafish (Danio rerio) (Amole and Unniappan, 2009). In addition, many

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studies have shown that ghrelin is a multifunctional hormone involved in the regulation of

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various physiological processes in fish (Kaiya et al., 2008).

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NPY is a peptide with 36 amino acid residuesbelonging to the NPY family, which is

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present in the central and peripheral nervous systems and controls the appetite and blood

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pressure (Kalra et al., 1999). NPY is highly conserved among vertebrates, including fish,

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and it is one of the most potent orexigenic agents in mammals (Halford et al., 2004; Hoyle,

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1999). Numerous studies show that NPY is involved in the regulation of feeding in fish.

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Both central and peripheral injections of NPY increase food intake in goldfish

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(López-Patiño et al., 1999), channel catfish (Ictalurus punctatus) (Silverstein and

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Plisetskaya, 2000) and tilapia (Oreochromis mossambicus) (Kiris et al., 2007). Fasting

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induces an increase of NPY expression in the hypothalamus of goldfish (Narnaware and

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Peter, 2001) and salmon (Oncorbyncbus spp.) (Silverstein et al., 1999), and refeeding can

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reverse these effects (Narnaware and Peter, 2001). As reported for some mammals, NPY in

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fish species also interacts with a number of appetite regulators, including CART, leptin,

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orexins, and ghrelin (Mercer et al., 2011; Volkoff, 2006).

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CCK, a peptide hormone which can stimulate pancreatic secretion (Murashita et al.,

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2006), is dominantly distributed in the brain and digestive tract of vertebrates, including fish

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(Johnsen, 1998). In mammals, CCK is released from intestinal endocrine cells during a meal

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and decreases gastric emptying, stimulates pancreatic and gastric secretions and reduces

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food intake via vagal afferent pathways (Chandra and Liddle, 2007; Rehfeld et al., 2007).

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CCK also influences appetite in fish which has been identified in several fish, including red

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drum (Sciaenops ocellatus) (Webb et al., 2010), winter flounder (Pseudopleuronectes

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americanus) (MacDonald and Volkoff, 2009a), yellowtail (Seriola quinqueradiata) 4

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(Murashita et al., 2006), winter skate (Raja ocellata) (MacDonald and Volkoff, 2009b) and

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grass carp (Ctenopharyngodon idellus) (Feng et al., 2012). Central or peripheral injection of

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sulfated CCK-8 (CCK-8 has a well conservedC-terminal octapeptide among vertebrates,and

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it is sulphated at the tyrosine residue and is the mostabundant form of CCK) suppressed

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food intake in goldfish (Himick and Peter, 1994), while oral administration of CCK receptor

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antagonists increased food intake in rainbow trout (Gelineau and Boujard, 2001), which

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revealed the suppression role of CCK as an appetite-regulating hormone. In addition, CCK

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mRNA levels increased in the brain of goldfish (Peyon et al., 1999) and in the pyloric caeca

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of yellowtail (Murashita et al., 2007) following a meal.

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The blunt snout bream (Megalobrama amblycephala Yih, 1955) is an endemic freshwater

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fish in China. It was originally distributed in the middle reaches of the Yangtze River and a

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few accessory lakes, of which the Liangzi Lake, Poyang Lake and Yuni Lake are three main

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sources (Li et al., 1991). In addition, it was also introduced to North America, Africa and

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Eurasia (Li et al., 2012). In recent years, it has been intensively cultured in China because of

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its wide range of food sources, fast growth, tender flesh and high larval survival rate. In

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2012, the total production of the bream reached 705,821 tonnes (FBAMC, 2013).

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Our previous study showed that ghrelin, NPY and CCK were all expressed throughout the

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embryonic and larval development stages, and higher expression levels were found in larval

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stages than in embryonic stages. The mRNA expression levels of these three genes in larvae

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varied significantly until 30 days after hatching (Ping et al., 2014). To further investigate the

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regulation of the appetite endocrine factors ghrelin, NPY and CCK in blunt snout bream, we

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cloned the full-length cDNAs of these appetite-regulating hormones, examined the mRNA

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expression in various adult tissues. Because juveniles are harder to culture than adults (and

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therefore the juvenile stage represents an important step in the developmental process for

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producing an aquaculture product), we assessed the effects of fasting and refeeding on gene 5

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expression of these hormones in juvenile brain and intestine. The aim of our research was to

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contribute to the further development of the blunt snout bream aquaculture industry by

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improving our understanding of the effect of fasting and refeeding on the gene expression of

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ghrelin, NPY and CCK in this important fish.

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2. Materials and methods

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2.1 Fish and samples

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For cDNA cloning and mRNA expression analyses of ghrelin, NPY and CCK in various

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tissues, individuals of adult blunt snout bream (~400 g each), were obtained from the Nanhu

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fish breeding base of Huazhong Agricultural University. The blunt snout breams were

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acclimated in indoor tanks with freshwater at 20ºC and fed daily at 09:00 for at least 1 week

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before the experiment. Five fish individuals were sampled at 4 h post-feeding and were

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anesthetized with tricaine methanesulfonate (MS-222, 100 mg/L) before dissection. Tissues

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were isolated and immediately immersed in liquid nitrogen and then stored at -80 ºC until

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RNA isolation.

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To evaluate the effects of starvation and refeeding on the gene expression of ghrelin, NPY

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and CCK, approximately 100 individuals of juvenile blunt snout bream (~10 g each) were

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randomly divided into two groups (experimental and control groups, respectively). The two

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groups of breams were respectively maintained in two indoor tanks with sufficient dissolved

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oxygen at 25 ± 1 °C and were fed with a commercial diet (Hubei Haid Feeds Company,

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Wuhan, China) twice a day (08:00 and 16:00) for 2 weeks to acclimate them to the artificial

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culture environment. After acclimation, the individuals of the experimental group were

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starved for 15 days and then were refed to apparent satiety twice a day for 15 days, while

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the individuals of the control group were kept under the same feeding condition as the

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acclimation period. Five individuals were randomly sampled from each group at the

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following time points: 0, 1, 4, 7, 15 days of starvation, and 1, 4, 7, 15 days of refeeding. To 6

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avoid the short-term effects of feeding the fish were sampled 4 h post-feeding (12:00 each

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day). The sampled individuals were anesthetized with MS-222 (50 mg/L), and then the

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whole brain and the intestine (foregut) were rapidly isolated for RNA extraction.

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This study was approved by the Institutional Animal Care and Use Committees (IACUC)

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of Huazhong Agricultural University.

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2.2 Molecular cloning of ghrelin, NPY and CCK cDNAs

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Total RNA was extracted from the brain tissue of adult blunt snout bream using TRIzol

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Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The integrity and

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purity of RNA were assayed by agarose gel electrophoresis and a Nanodrop ND-2000

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spectrophotometer (Thermo Electrom Corporation, USA), respectively. cDNA was reverse

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transcribed from total RNA using a molony murine leukemia virus (M-MLV) Reverse

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Transcriptase kit (Toyobo, Osaka, Japan) following the manufacturer's protocol. Based on

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the partial cDNA sequences of ghrelin, NPY and CCK genes that were previously obtained

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(Ping et al., 2014), the full-length cDNAs of the three genes were obtained by the rapid

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amplification of cDNA ends (RACE) method (Feng et al., 2013). The gene-specific primers

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and adaptor primers are shown in Table 1. All the primers were designed using the software

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Primer premier 5.0. The PCR reactions were carried out in a volume of 10 µL including 1.0

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µL of 10×PCR buffer, 0.2 mM of dNTP, 1.5 mM MgCl2, 0.4 µM for each primer, 0.4 U Taq

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DNA polymerase (TaKaRa, Japan), and 1.0 µL of cDNA. Touchdown PCR was used to

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improve the specificity of SMARTer RACE amplification.The SMARTer RACE primers for

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the 5’ and 3’ RACE PCR were designed according to the core sequences of the three genes

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(Ping et al., 2014) and were synthesized by Sangon Biotech (Shang hai, China). The target

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products were obtained by nested PCR. The obtained products were isolated using an

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agarose purification kit (Axygen, USA) and ligated into the pMD18-T vector (TaKaRa,

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Japan). Following transfection into Escherichia coli DH5α competent cells, recombinants 7

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were identified by blue-white spot screening. Finally, three positive clones were confirmed

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by sequencing (Sangon Biotech Company, Shanghai, China).

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2.3 Structural analysis

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Open reading frame (ORF) and protein prediction were performed using the ORF finder

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software (www.ncbi.nlm.nih.gov/gorf/gorf.html). The deduced amino acid sequences of

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ghrelin, NPY and CCK were analyzed with the BLAST program on the National Center for

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Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/). The protein

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molecular weight, theoretical isoelectric point and other basic properties were predicted

184

using the ProtParam tool (http://web.expasy.org/protparam/). The cleavage site of the signal

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peptide was estimated using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). The Tyr

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sulfation site in deduced CCK amino acid sequences was predicted by the SulfoSite

187

program (http://sulfosite.mbc.nctu.edu.tw/). Multiple alignments of amino acid sequences

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were performed using Clustal X 1.83 software, and a phylogenetic tree was constructed

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using the neighbor-joining (NJ) method by MEGA 5.1.

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2.4 Real-time quantitative PCR and expression analysis

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To estimate the tissue distributions of ghrelin, NPY and CCK in blunt snout bream,

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real-time quantitative PCR (qPCR) was carried outusing a Rotor-Gene 6500 Thermocycler

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(Corbett Research, Sydney, Australia). Following Ping et al. (2014), total RNA was

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extracted from muscle, spleen, foregut, midgut, hindgut, pituitary, hypothalamus and other

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parts of the brain of adult blunt snout breams. The different parts of the brain were dissected

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under anatomical lens. According to the anatomical structure of the intestine in adult grass

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carp (Ni and Wang 1999), we divided the intestine of blunt snout bream into three different

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parts—foregut, midgut, and hindgut. The foregut is from gut sphincter to the first turn of

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thecoiled intestine, and the hindgut is from the last turn tothe anus. The cDNAs were then 8

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diluted 1:2 in double-distilled water (ddH2O) for subsequent experiments.

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The qPCR primers for ghrelin, NPY and CCK genes were the same as those designed in

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our previous study (Ping et al., 2014) (Table 2). Blunt snout bream β-actin was used as an

203

internal control (Ming et al., 2010). The standard cDNA plasmids and the standard curves

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were made according to the previous studies conducted in our lab (Feng et al., 2013).

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SYBR®Premix Ex TaqTM (Takara, Japan) was used for qPCR following the manufacturer's

206

protocols. Each PCR was performed in a total volume of 20 µL, including 10 µL of

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SYBR®Premix Ex TaqTM, 2 µL of cDNA, 0.2 µM of each sense and antisense primer, and

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7.2 µL ddH2O. Reactions were performed by a three-step method, 95 ºC for 1 min initially,

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followed by 40 cycles, 95 ºC for 5 s, 60 ºC for 20 s and 72 ºC for 20 s. Melt curve analysis

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was carried out over a range from 55°C to 99 °C at the end of each PCR run. For each sample,

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quantitative PCR was performed in triplicate. A negative control (template was replaced by

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ddH2O) was set for each primer pair. The Ct (cycle threshold) values of amplification

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products were calculated by the standard curve. The relative mRNA expression of ghrelin,

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NPY and CCK was determined with a two standard curve method (Ruan et al., 2010).

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2.5 Effects of starvation and refeeding on the expression of ghrelin, NPY and CCK

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Total RNA was isolated from the whole brain and intestine (foregut) of the experimental

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and control groups of juvenile blunt snout bream. Then the first-strand cDNA synthesis and

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the qPCR were carried out as described above (see section 2.4).

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2.6 Statistical analysis

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For the purposes of comparing gene expression amongst as many different tissue types as

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possible, we included previously published data for liver, kidney, gill and heart (Ping et al.

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2014). To this data set we added the new data for foregut, midgut, hindgut, pituitary, 9

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hypothalamus, other parts of brain, muscle and spleen to give a total of 12 different tissue

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types. All data were expressed as the mean ± SE. A one-way analysis of variance (ANOVA)

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was conducted to compare the differences of relative mRNA expression of ghrelin, NPY and

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CCK in various adult tissues using STATISTICA 6.0. The one-way ANOVA was also used

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to test the effects of starvation and refeeding on the mRNA expression changes of these

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three genes in brain and intestine. Newman-Keuls test was used to evaluate the differences

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after checking for data normality and homogeneity of variances. Student’s t-test was used to

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compare the difference of expression levels between experimental and control groups on a

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given day in the fasting/refeeding experiment. The level of statistical significance was set at

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P< 0.05 for all analyses.

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3. Results

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3.1 Cloning and sequence analysis of the ghrelin, NPY and CCK cDNAs

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3.1.1 Ghrelin

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The full-length cDNA sequence of blunt snout bream ghrelin (GenBank Accession No.

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JQ301476) was 494 bp, and contained 59 bp of 5'-untranslated region (5'-UTR), 123 bp of

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3'-untranslated region (3'-UTR) and 312 bp of open reading frame (ORF) encoding a

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preproghrelin with 103 amino acids (aa) (Figure 1). The preproghrelin included a putative

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signal peptide of 26 aa and a mature peptide of 19 aa. There were two putative cleavage and

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amidation signals (GRR) after 12 and 19 aa of the mature peptide region. ProtParam

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analysis predicted the molecular formula of preproghrelin to be C510H809N137O150S7; its relative

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molecular mass, isoelectric point and the parameter of instability were 11484.2, 5.62 and

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64.98, respectively. 10

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To examine the structural similarities at the protein level, the amino acid sequence of

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blunt snout bream ghrelin was aligned with ghrelins of other vertebrates (Figure 2A). The

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ghrelin amino acid sequence of blunt snout bream was exactly the same as that of grass carp,

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and the similarities to goldfish and zebrafish were 88% and 71%, respectively. A lower

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sequence similarity was observed in the obestatin region of ghrelin between mammals and

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teleosts.

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To investigate the evolutionary relationships of blunt snout bream ghrelin with those of

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other vertebrates, a phylogenetic tree based on the deduced amino acid sequences of ghrelin

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was constructed by the neighbor-joining method using MEGA5.1 software (Supplementary

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Figure 1). According to the tree, blunt snout bream ghrelin clustered with grass carp,

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common carp, goldfish and other cyprinid fishes. The teleostean ghrelin was separated from

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amphibian, reptilian, avian and mammalian, whereas elasmobranch ghrelin was clustered to

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an independent branch that was separated from other vertebrates. The phylogenetic tree

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analysis corresponded with conventional systematics.

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3.1.2 NPY

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The full-length cDNA of Blunt snout bream NPY (GenBank Accession No. JQ301475)

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was 760 bp, and contained 65 bp of 5'-UTR, 404 bp of 3'-UTR and 291 bp of ORF encoding

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a prepro-NPY with 96 aa (Figure 3). The predicted prepro-NPY peptide contained a putative

263

signal peptide of 28 aa and a mature peptide of 36 aa. The molecular formula of prepro-NPY

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was predicted to be C494H749N131O144S5; the relative molecular mass, isoelectric point and

265

the parameter of instability were 10987.4, 5.72 and 60.34, respectively.

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Multiple alignments of the amino acid sequence indicated that the NPY of blunt snout 11

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bream was exactly the same as that of grass carp and common carp and also had high

268

similarity to other vertebrates (Figure 2B).

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To reveal the molecular phylogenetic position of NPY, a phylogenetic tree was

270

constructed by the neighbor-joining method, as shown in Supplementary Figure 2. The NPY

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gene of blunt snout bream and the NPYs of other cyprinid fishes (grass carp, common carp,

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goldfish and zebrafish) were grouped in the same cluster, whereas teleost NPY was

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differentiated from the NPYs of other vertebrates. In addition, peptide YY (PYY) was

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clustered into an independent branch from other vertebrates.

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3.1.3 CCK

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Blunt snout bream CCK (GenBank Accession No. JQ290110) cDNA sequence was 770

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bp in length, containing 49 bp of 5'-UTR, 309 bp of 3'-UTR and 372 bp of ORF encoding a

278

prepro-CCK with 123 aa (Figure 4). The predicted prepro-CCK peptide contained a putative

279

signal peptide of 19 aa and a mature peptide of 104 aa, including a C-terminal octapeptide

280

CCK-8 (DYLGWMDF). A potential sulfation site was predicted at the seventh amino acid

281

(tyrosine) of CCK-8 from the C-terminus using the SulfoSite program. The predicted

282

molecular formula of preprocholecystokinin was C575H933N175O197S6; the relative molecular

283

mass, isoelectric point and the parameter of instability were 13642.1, 6.41, and 71.38,

284

respectively.

285

To analyze structural similarities at the protein level, the amino acid sequence of blunt

286

snout bream CCK was aligned with CCKs from other vertebrates (Figure 2C). The

287

similarity of CCK between blunt snout bream and grass carp was 98%, while lower

288

similarity was found between blunt snout bream and other vertebrates. The octapeptide 12

289

CCK-8 sequence was highly conserved in vertebrates.

290

A phylogenetic tree based on the deduced amino acid sequences of CCK was constructed

291

by the neighbor-joining method (Supplementary Figure 3). The results indicated that teleost

292

CCK was differentiated from the CCK of other vertebrates. Teleost CCK can be divided into

293

two distinct branches: fish CCK-1 and CCK-2. The blunt snout bream CCK shared the

294

highest homology with grass carp and goldfish, and the blunt snout bream CCK belonged to

295

the fish CCK-2 type.

296

3.2 Expression of ghrelin, NPY and CCK mRNA in adult tissues

297

The general tissue distributions of ghrelin, NPY and CCK mRNA in blunt snout bream

298

were analyzed by qPCR (Figure 5). Ghrelin mRNA was mainly expressed in the intestine,

299

and weakly expressed in other tissues. The relative expression levels of ghrelin increased

300

significantly (P< 0.05) from foregut to hindgut in the intestine. NPY mRNA was mostly

301

expressed in the brain, of which the hypothalamus had the highest expression level. The

302

relative expression level of CCK was the highest in the pituitary, followed by the

303

hypothalamus and then the other parts of the brain.

304

3.3 Effects of starvation and refeeding on the expression of ghrelin, NPY and CCK

305

For ghrelin, the mRNA expression level was gradually increased during starvation in the

306

brain, and was significantly higher than that of the control group after fasting for 1 day.

307

Refeeding could decrease the expression level of ghrelin gradually, and after 7 days of

308

refeeding (day 22 in Figure 6), the expression level almost declined to the same level as that

309

of control group (Figure 6A). The ghrelin mRNA expression levels in the intestine also 13

310

increased significantly after fasting and rose to the highest level on day 7 after starvation

311

(Figure 6B). After refeeding for 7 days, the ghrelin mRNA expression decreased to the same

312

levels as the control group.

313

For NPY, the mRNA expression level was increased during starvation in the brain, and

314

was significantly higher than the control group after fasting for 1 day. The expression level

315

decreased to the same level asthe control group after refeeding for 4 days (Figure 7A). In the

316

intestine, the NPY mRNA expression was not significantly changed (P> 0.05) (Figure 7B).

317

The CCK mRNA expression levels in the brain and intestine fell to their lowest levels

318

after starvation for 4 days, and a modest rebound was observed on day 7 and 15, but these

319

levels were still lower than those of the control group (Figure 8). Refeeding reversed the

320

decrease of the CCK expression, and the expression levels recovered to the same levels as

321

the control group after refeeding for 7 days.

322

4. Discussion

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Ghrelin and NPY are important appetite-stimulating factors. Previous studies have

324

shown that over-expression of these two factors can increase the up-take of food in animals

325

(Kaiya et al., 2008; Volkoff, 2006). Ghrelin consists of 28 amino acids, of which Ser3 is

326

found n-octanoylated in mammals, and studies on animals have shown that this modification

327

is essential for the activity of ghrelin. The non-acylated form of ghrelin is also present in

328

mammals in both stomach and blood at higher levels(Matsumoto et al., 2001). However,

329

non-acylated ghrelin can neither bind GHS-R nor exhibit GH-releasing activity in rats

330

(Hosoda et al., 2000) and similar results were observed in humans (Broglio et al., 2003).

331

Among teleosts, two isoforms of ghrelin (Ghrelin-1 and 2) have been found in rainbow trout

332

and Atlantic salmon (Kaiya et al., 2011). For blunt snout bream and other fishes, only one 14

333

form of ghrelin has been found. Ghrelin is primarily produced in the GI tract in all

334

vertebrates that have been studied to date (Kaiya et al., 2008). In this study, ghrelin mRNA

335

was mainly expressed in the intestine, and had low expression levels in other tissues. The

336

relative expression levels increased significantly (P< 0.05) from foregut to hindgut in the

337

intestine. This result was similar to the studies of many other fish species without a stomach,

338

such as goldfish and common carp, whose ghrelin mRNA was predominantly expressed in

339

the gut (Kono et al., 2008; Unniappan et al., 2002). Feng et al. (2013) studied the tissue

340

distribution of ghrelin in grass carp and found the highest expression of ghrelin in the

341

foregut, suggesting that ghrelin expression has species-specific differences in tissue

342

distribution. High expression levels of ghrelin mRNA in the intestine of blunt snout bream

343

may be related to its biological function of appetite regulation. However, for fish species

344

with stomachs, such as rainbow trout (Kaiya et al., 2003), channel catfish (Kaiya et al.,

345

2005), sea bass (Terova et al., 2008) and Atlantic cod (Xu and Volkoff, 2009), ghrelin was

346

primarily detected in the stomach. In addition, expression of ghrelin mRNA was found in

347

various organs of other non-mammalian vertebrates (Kaiya et al., 2008).

348

Many researchers have demonstrated that NPY has a role in the regulation of food intake,

349

and in mammals NPY is recognized as one of the most effective appetite regulators (Kalra et

350

al., 1999). High concentrations of NPY were found in the central and peripheral nervous

351

systems of mammals (Gray et al., 1986; Kashihara et al., 2008). Many studies of fish have

352

also shown that NPY is mainly expressed in the central nervous system, pituitary and some

353

peripheral tissues (Chen et al., 2005; Leonard et al., 2001; Liang et al., 2007; Murashita et

354

al., 2009a). In blunt snout bream, NPY is mainly expressed in the pituitary, hypothalamus

355

and other parts of the brain, in agreement with the results mentioned above. Additionally,

356

NPY mRNA expression has also been detected in the spleen, liver and intestine, in addition

357

to the brain inChinese perch (Siniperca chuatsi) (Liang et al., 2007). In the Brazilian 15

358

flounder (Paralichthys orbignyanus), NPY mRNA was expressed in the brain, liver, spleen,

359

gill, intestine, heart and kidney (Campos et al., 2010). The tissue-specific distributional

360

differences of NPY mRNA expression may be attributed to variation among fish species or

361

to sensitivity of the detection methods. Regardless, NPY is mostly expressed in the central

362

nervous system and some peripheral tissues (e.g., intestine and heart) of most fishes.

363

CCK is distributed in both the brain and GI tract in vertebrates (Johnsen, 1998),

364

including fish (Kurokawa et al., 2003; Rojas-García and Rønnestad, 2002). A high

365

abundance of CCK in the brain was also observed in rainbow trout (Jensen et al., 2001),

366

yellowtail (Murashita et al., 2006) and Atlantic salmon (Murashita et al., 2009b). For grass

367

carp, CCK mRNA was mainly expressed in the hypothalamus and pituitary (Feng et al.,

368

2012). In the present study, blunt snout bream CCK was mainly expressed in the brain,

369

followed by the intestine. This result suggests that CCK might act as a brain-gut peptide

370

which distribute in the GI tract and nerve system in blunt snout bream. Additionally, we

371

found that CCK mRNA had moderate expression levels in heart, gill and muscle but the

372

function of CCK in the three tissues is unknown.

373

Ghrelin, NPY and CCK are neuropeptides involved in the regulation of appetite and

374

feeding in vertebrates. There was evidence that ghrelin functioned in controlling energy

375

homeostasis and increasing food intake (Volkoff, 2006). Intracerebroventricular (ICV)

376

injection of ghrelin significantly raised food intake in rats (Nakazato et al., 2001). Both

377

central and peripheral injections of ghrelin stimulated food intake in goldfish (Unniappan et

378

al., 2004a; Unniappan et al., 2004b; Unniappan et al., 2002). Food deprivation increased

379

ghrelin mRNA expression in the hypothalamus and gut of goldfish and in the stomach of sea

380

bass (Terova et al., 2008; Unniappan et al., 2004a). In the present study, the ghrelin gene

381

expression in the brain significantly increased after starvation for 1-15 days; it then

382

decreased after refeeding and returned to the control level after 7 days of refeeding. In the 16

383

intestine, ghrelin gene expression reached the highest level after 7 days of starvation, and

384

decreased after refeeding. These results were similar to those observed in grass carp and

385

black sea bream (Feng et al., 2013; Ma et al., 2009). For Atlantic salmon, ghrelin-1 gene

386

expression levels increased after 6 days of starvation, while the ghrelin-2 gene expression

387

did not change significantly (Murashita et al., 2009a). In conclusion, gene expression levels

388

of ghrelin usually increased when the fish were not fed at a meal time and decreased after

389

food intake, which revealed the role of ghrelin as a hunger signal.

390

NPY is a vital orexigenic hormone to promote appetite in fish (Cerdá-Reverter and

391

Larhammar, 2000). Central and peripheral injections of NPY increased food intake in

392

goldfish, channel catfish and tilapia (Kiris et al., 2007; López-Patiño et al., 1999; Silverstein

393

and Plisetskaya, 2000). In the present study, NPY mRNA expression was gradually

394

up-regulated during starvation in the brain of blunt snout bream. And there were noticeable

395

differences between the fed-group and unfed-group after starvation in the brain tissue

396

(Figure 6A), while there was no significant difference in NPY expression between the fed-

397

and unfed-group over the whole study period in the intestine tissue. In the goldfish, NPY

398

mRNA expression increased during starvation in the brain tissue (Narnaware and Peter,

399

2001). However, there was no significant difference in NPY expression after fasting in

400

Atlantic cod (Kehoe and Volkoff, 2007). NPY mRNA level was not affected by 6 days

401

fastingin brain of Atlantic salmon (Murashita et al., 2009a). In the winter skate, NPY mRNA

402

expression increased in the telencephalon but not the hypothalamus after 2 weeks of fasting

403

(MacDonald and Volkoff, 2009b). Our data suggest that food intake is controlled by central

404

NPY rather than peripheral NPY in blunt snout bream. In the brain tissue of blunt snout

405

bream, the expression levels of NPY increased after starvation and decreased after refeeding,

406

exhibiting a similar change to that of ghrelin. These results indicate that NPY and ghrelin

407

function as orexigenic factors and may have mutul association in the regulation of feeding 17

408

in blunt snout bream.

409

Based on studies in mammals and teleosts, CCK acts as an anorexigenic peptide that

410

suppresses food intake, which has the opposite role to ghrelin and NPY. CCK is released

411

after feeding in the intestine (Aldman and Holmgren, 1995). Previous studies showed that

412

both central and peripheral injections of CCK inhibited food intake in goldfish (Himick and

413

Peter, 1994; Volkoff et al., 2003). In addition, CCK expression increased in the brain of

414

goldfish at 2 hours after feeding (Peyon et al., 1999) and decreased in the intestine of

415

yellowtail during starvation (Murashita et al., 2006). In grass carp, CCK mRNA expression

416

decreased during starvation, and was up-regulated after refeeding (Feng et al., 2012). In

417

Atlantic salmon, CCK gene expression levels in the brain decreased after starvation, but

418

those of pyloric caeca levels did not change significantly (Murashita et al., 2009a). In our

419

study, both brain and intestine CCK mRNA expression decreased during starvation, and

420

then increased after refeeding. These results indicate that CCK plays an anorectic role in

421

fish comparable to that in mammals.

422

In the present study, ghrelin was maily distibuted in the intestine, while NPY and CCK

423

were maily distributed in the brain of blunt snout bream. For the fasting-refeeding

424

experiments, expression levels of ghrelin and NPY in the brain increased after fasting, and

425

decreased after refeeding, showing that ghrelin and NPY functioned as orexigenic factors to

426

increase food intake. In the intestine, expression levels of ghrelin also increased after fasting,

427

and decreased after refeeding, whereas expression levels of NPY showed no significant

428

differences after fasting and refeeding. These results show that ghrelin has responses to

429

fasting and refeeding both in the brain and in the intestine tissuess and acts as a brain-gut

430

peptide to stimulate appetite (Kaiya et al., 2008), whereas NPY can response to fasting and

431

refeeding in the brain tissues but not in the intestine. CCK expression levels decreased after

432

fasting, and increased after refeeding both in the brain and in the intestine, indicating that 18

433

CCK can suppress food intake and has an antergic role to ghrelin and NPY (Volkoff, 2006).

434

In conclusion, we report the full-length cDNAs of ghrelin, NPY and CCK in blunt snout

435

bream and these three genes were characterized with respect to their expression in various

436

tissues. Ghrelin was predominantly expressed in the intestine, whereas NPY and CCK were

437

mainly expressed in the brain. Fasting and refeeding experiments showed that ghrelin and

438

NPY had the opposite function to CCK, and all of the three genes were involved in the

439

regulation of feeding in blunt snout bream. These findings will help us to understand the

440

role of ghrelin, NPY and CCK in regulation of appetite in blunt snout bream and will

441

provide basic information for the reasonable aquaculture of blunt snout bream.

442

Acknowledgments

443

We thank the editor and the anonymous reviewers for their helpful comments and

444

suggestions for the manuscript. We also thank Prof. Jonathan Gardner (Victoria University

445

of Wellington) for his helpful edits on the manuscript. This work was supported by the

446

Major Science and Technology Program for Water Pollution Control and Treatment (Grant

447

No. 2014ZX07203010-4), the

448

Construction Projects of China entitled “Staple Freshwater Fishes Industry Technology

449

System” (Grant No. CARS-46-05), and the National R&D Infrastructure and Facility

450

Development Program of China (Grant No. 2006DKA30470-002-03).

Modern Agriculture Industry Technology System

451

452

453

454

19

455

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456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497

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617 618 619 620 621 622 623 624 23

625

Tables

626

Table 1. Primers used for cloning of ghrelin, CCK and NPY from blunt snout bream Name

Sequence (5'-3')

Application

Oligo dT adaptor GACTC GAGTC GACAT CGA(T)17 Adaptor primers for RACE Adaptor

GACTC GAGTC GACAT CGA

G-3' outer

GCGGCACCAGCTTTCTCAG

G-3' inter

CTCAGAAACCACAGGGTC

C-3' outer

ATGAACGCTGGAATCTGTG

C-3' inter

TGCTGTCTAAACAAGAGGATG

N-3' outer

CTCTTGTTYGYCTGCTTGG

N-3' inter

GYCTGCTTGGGAACTCTAAC

G-5' outer

GATGAGTGCTCCGTTCGA

G-5' inter

CAGAAACCACAGGGTCGAAGG

C-5' outer

CAACCTGATTAGCCAATCAT

C-5' inter

AACTGAGCCTGCTGTCTA

N-5' outer

GTGAAGTACCACAGCCAT

N-5' inter

CATCAACCTAATAACAAGGCAG

627

628

629

630

631

632

24

Ghrelin 3' RACE cloning

CCK 3' RACE cloning

NPY 3' RACE cloning

Ghrelin 5' RACE cloning

CCK 5' RACE cloning

NPY 5' RACE cloning

633

Table 2. Primers designed for expression analysis of ghrelin, NPY and CCK Name

Sequence (5'-3')

Application

ghr-F

CTCAGAAACCACAGGGTC

Real-time PCR for ghrelin

ghr-R

TCGAACGGAGCACTCATC

NPY-F

CTGCTTGGGAACTCTTAC

NPY-R

ATGGTCCTCATATCTGGT

CCK-F

CACTCACAGAATAAAGGACAGA Real-time PCR for CCK

Real-time PCR for NPY

CCK-R ATGATTGGCTAATCAGGTTG actin-F

TCGTCCACCGCAAATGCTTCTA

actin-R

CCGTCACCTTCACCGTTCCAGT

634 635 636 637 638 639 640 641 642 643 644 645 646 647 25

Real-time PCR for β-actin

648

Figure legends

649

Figure 1. The complete cDNA and deduced amino acid sequence of ghrelin in blunt

650

snout bream.

651

5' and 3' untranslated regions are shown in lowercase; coding region is shown in uppercase,

652

where the upper sequence indicates the nucleotide and the lower shows the corresponding

653

amino acids; asterisk indicates stop codon; putative polyadenylation signal (aataaa) is boxed;

654

the region of putative signal is underlined by the dotted line; the mature peptide is

655

underlined；the putative cleavage sites and amidation signals are indicated by the shadow

656

region.

657 658

Figure 2. Alignment of the deduced animo acid sequences of ghrelin, NPY and CCK.

659

(A) Amino acid sequence alignment for ghrelin, the region corresponding to the obestain

660

peptide in mammals is underlined; (B) Amino acid sequence alignment for NPY; (C) Amino

661

acid sequence alignment for CCK, the underlined region indicates the octapeptides of CCK.

662

Shadow region indicates identical amino acids.

663 664

Figure 3. The complete cDNA and deduced amino acid sequence of NPY in blunt snout

665

bream.

666

5' and 3' untranslated regions are shown in lowercase; coding region is shown in uppercase,

667

where the upper sequence indicates the nucleotide and the lower shows the amino acids;

668

asterisk indicates stop codon; putative polyadenylation signal (aataaa) is boxed; the region

669

of putative signal is underlined by the dotted line; the mature peptide is indicated by the 26

670

shadow region.

671 672

Figure 4. The complete cDNA and deduced amino acid sequence of CCK gene of blunt

673

snout bream.

674

5' and 3' untranslated regions are shown in lowercase; coding region is shown in uppercase,

675

where the upper sequence indicates the nucleotide and the lower shows the amino acids;

676

asterisk indicates stop codon; putative polyadenylation signal (aataaa) is boxed; the region

677

of putative signal is underlined by dotted line; the octapeptides of CCK are underlined; the

678

predicted Tyr sulfation site is indicated by the shadow region.

679 680

Figure 5. Tissue distribution of ghrelin (A), NPY (B) and CCK (C) mRNAs in blunt

681

snout bream.

682

Different letters above the bars indicate significant difference（P< 0.05）. Mu, muscle; L,

683

liver; H, heart; Sp, spleen; G, gill; K, kidney; Fg, foregut; Mg, midgut; Hg, hindgut; Pi,

684

pituitary; Hy, hypothalamus; B, other parts ofbrain (excluding pituitary and hypothalamus).

685 686

Figure 6. The expression changes of ghrelin in the brain (A) and intestine (B) of blunt

687

snout bream responding to fasting and refeeding experimental conditions.

688

Different italic lowercase letters above the bars indicate significant differences in the control

689

group and bold lowercase letters for experimental group (P< 0.05). “*” indicates a

690

significant difference between experimental and control groups on the given day (P< 0.05).

691 27

692

Figure 7. The expression changes of NPY in the brain (A) and intestine (B) of blunt

693

snout bream responding to fasting and refeeding.

694

Different italic lowercase letters above the bars indicate significant differences in the control

695

group and bold lowercase letters for experimental group (P< 0.05). “*” indicates a

696

significance difference between experimental and control groups on the given day (P<

697

0.05).

698 699

Figure. 8 The expression changes of CCK in the brain (A) and intestine (B) of blunt

700

snout bream responding to fasting and refeeding.

701

Different italic letters above the bars indicate significant differences in the control group and

702

bold lowercase letters for experimental group (P< 0.05). No significant difference was

703

observed in the control group of (A). “*” indicates a significance difference between

704

experimental and control groups on the given day (P< 0.05).

705 706

Supplementary Figure 1. NJ phylogenetic tree depicting the evolutionary relationships

707

of blunt snout bream ghrelin with other vertebrates. The phylogenetic tree was

708

constructed based on the amino acid sequences of different species. The numbers at tree

709

nodes refer to percentage bootstrap after 1000 replicates. “▲” represents blunt snout bream.

710

GenBank accession numbers of the sequences used are available in Supplementary Table 1.

711 712

Supplementary Figure 2. NJ phylogenetic tree depicting the evolutionary relationships

713

of blunt snout bream NPY with other vertebrates. The phylogenetic tree was constructed 28

714

based on the amino acid sequences of different species. The numbers at tree nodes refer to

715

percentage bootstrap after 1000 replicates. “▲” represents blunt snout bream. GenBank

716

accession numbers of the sequences used are available in Supplementary Table 1.

717 718

Supplementary Figure 3. NJ phylogenetic tree depicting the evolutionary relationships

719

of blunt snout bream CCK with other vertebrates. The phylogenetic tree was constructed

720

based on the amino acid sequences of different species. The numbers at tree nodes refer to

721

percentage bootstrap after 1000 replicates. “▲” represents blunt snout bream. GenBank

722

accession numbers of the sequences used are available in Supplementary Table 1.

723 724 725 726

29

727 728 729

Fig. 1

730 731

30

732 733 734

Fig. 2

735 736

31

737 738 739

Fig. 3

740 741

32

742 743 744

Fig. 4

745 746

33

747 748 749

Fig. 5

750 751

34

752 753 754

Fig. 6

755 756

35

757 758 759

Fig. 7

760 761

36

762 763 764

Fig. 8

765 766

37

767

Highlights

768


Full-length cDNAs of ghrelin, NPY and CCK were cloned in blunt snout bream.

769


Deduced amino acid sequences of ghrelin, NPY and CCK were analyzed.

770


Ghrelin was mainly expressed in the intestine, and NPY and CCK in the brain.

771


Ghrelin and NPY had opposite role of CCK responding to fasting and refeeding.

772 773 774 775

38