Too stressed to eat: Investigating factors associated with appetite loss in subordinate rainbow trout.

Juvenile rainbow trout (Oncorhynchus mykiss) form dominance hierarchies in which subordinates experience chronic social stress and suppression of food intake. Here we tested the hypothesis that inhibition of food intake reflects increased expression of anorexigenic (appetite inhibiting) signals and decreased expression of orexigenic (appetite stimulating) signals. Trout were confined in pairs for 1 or 4 days, or were confined in pairs for 4 days and then allowed to recover from social interactions for 2 or 4 days; sham fish were handled identically but held alone. Subordinates did not feed during social interaction and had lower food intake than dominants or shams during recovery. In parallel, plasma cortisol (∼18-26x) and liver leptin (lep-a1) transcript abundance (∼10-14x) were elevated in subordinates during social interaction but not recovery, suggesting that these factors contributed to the suppression of food intake. Fish deemed likely to become subordinate based on inhibition of food intake in response to a mild stressor also showed elevated liver lep-a1 transcript abundance (∼5x). The moderate response in these fish coupled with a correlation between liver lep-a1 and cortisol suggest that stress-induced elevation of cortisol increased liver lep-a1 transcript abundance in subordinate trout, contributing to stress-induced suppression of food intake.

Stimulatory effect of the secretogranin-ll derived peptide secretoneurin on food intake and locomotion in female goldfish (Carassius auratus).

Secretoneurin (SN) is a conserved peptide derived by proteolytic processing from the middle domain of the ∼600 amino acid precursor secretogranin-II (SgII). Secretoneurin is widely distributed in secretory granules of endocrine cells and neurons and has important roles in reproduction as it stimulates luteinizing hormone release from the pituitary. A potential new role of SN in goldfish feeding is the subject of this study. Firstly, we established that acute (26 h; p<0.0001) and short-term (72 h; p=0.016) fasting increased SgIIa precursor mRNA levels 1.25-fold in the telencephalon, implicating SN in the control of feeding. Secondly, we determined that intracerebroventricular injections of the type A SN (SNa; 0.2 and 1 ng/g BW) increased food intake and locomotor behavior by 60 min. Fish injected with the lower and higher doses of SNa (0.2 and 1 ng/g) respectively exhibited significant 1.77- and 2.58-fold higher food intake (p<0.0001) than the saline-injected control fish. Locomotor behavior was increased by 1.35- and 2.26-fold for 0.2 ng/g SNa (p=0.0001) and 1 ng/g SNa (p<0.0001), respectively. Injection of 1 ng/g SNa increased mRNA levels of hypothalamic neuropeptide Y 1.36-fold (p=0.038) and decreased hypothalamic cocaine-and amphetamine-regulated transcript by 33% (p=0.01) at 2h and 5h post-injection, respectively. These data suggest interactions of SNa with stimulatory and inhibitory pathways of food intake control in fish.

Sperm traits in farmed and wild Atlantic salmon Salmo salar.

Differences in sperm metabolism and morphology between wild and non-local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates.

Cloning and tissue distribution of appetite-regulating peptides in pirapitinga (Piaractus brachypomus).

Pirapitinga (or red-bellied pacu, Piaractus brachypomus, Characiforme, Serrasalmidae) is an economically important South American fish for which the endocrine mechanism of the regulation of feeding has never been examined. To better understand these mechanisms, cDNAs encoding the appetite-regulating peptides orexin, cocaine- and amphetamine-regulated transcript (CART), apelin, cholecystokinin (CCK), peptide YY (PYY), leptin and ghrelin were isolated in pirapitinga and their mRNA distributions examined in peripheral tissues and brain. When compared to other fish, the sequences obtained for all peptides were most similar to those of other Characiforme fish (i.e. Mexican cavefish) and Siluriformes (catfish) as well as Cypriniformes (i.e. goldfish, zebrafish). All peptides were widely expressed within the brain. With the exception of CART, which was only expressed in brain, the mRNAs of all peptides were present in several peripheral tissues, including gastrointestinal tract, kidneys and gills. The widespread and peptide-specific distributions suggest that each peptide might have distinct physiological actions in the brain and on peripheral tissues, in particular on the gastrointestinal tract, which include feeding regulation. This preliminary study opens new avenues for further functional studies on the endocrine regulation of feeding in Serrasalmidae fish, including pirapitinga.

Effect of calcitonin gene-related peptide (CGRP), adrenomedullin and adrenomedullin-2/intermedin on food intake in goldfish (Carassius auratus).

The purpose of the present study was to elucidate the possible role of calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) on food intake regulation in goldfish (Carassius auratus). We examined the effects of intracerebroventricular (ICV) administration of these related hormones on food intake. Food-deprived goldfish were subjected to ICV injections of CGRP, AM and IMD and their food intake were quantified. CGRP at 10ng/g body weight (bw) significantly decreased food intake as compared to saline-treated fish. IMD at 10 and 50ng/g bw both significantly decreased food intake as compared to saline group. No significant differences were observed after AM administration. Our results suggest, for the first time in fish, a role for both CGRP and IMD in the central regulation of feeding in fish.

Molecular characterization of calcitonin gene-related peptide (CGRP) related peptides (CGRP, amylin, adrenomedullin and adrenomedullin-2/intermedin) in goldfish (Carassius auratus): cloning and distribution.

To further characterize the structure and function of calcitonin gene-related peptide (CGRP) related peptides in fish, we have cloned cDNA sequences for CGRP, amylin, adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) in goldfish (Carassius auratus) and examined their tissue distribution. CGRP, amylin, AM and IMD cDNAs were isolated by reverse transcription (RT) and rapid amplification of cDNA ends (RACE). The cloned sequences contain the complete four mature peptides, which present a high degree of identity with mature peptide sequences from other fish. Phylogenetic analyses show that goldfish AM and IMD form a sub-family within the CGRP-related peptides that is distinct from the CGRP/amylin sub-family. The distribution of goldfish CGRP-like peptides mRNA expression in different tissues and within the brain was studied by RT-PCR. CGRP, IMD and AM are detected throughout the brain, in pituitary and in most peripheral tissues examined. Amylin mRNA is mostly expressed in the brain, in particular posterior brain, optic tectum and hypothalamus, but is also present in pituitary, gonad, kidney and muscle. Our results suggest that goldfish CGRP, amylin, AM and IMD are conserved peptides that show the typical structure characteristics present in their mammalian counterparts. The widespread distributions of CGRP, AM and IMD suggest that these peptides could be involved in the regulation of many diverse physiological functions in fish. Amylin mRNA distribution suggests possible new roles for this peptide in teleosts, including the control of reproduction.

Effects of tebufenozide on some aspects of lake trout (Salvelinus namaycush) immune response.

Tebufenozide is a nonsteroid ecdysone agonist that causes premature and incomplete molting in Lepidoptera and is used on crops and in forest spray programs. Lake trout (Salvelinus namaycush) were exposed to one pulse of 0.25 ppm tebufenozide every 3 days (12 pulses in total). Cell ratios and respiratory burst responses of circulating white blood cells (WBCs) as well as head-kidney (HK) WBCs were investigated by using cell observation, Nitro-Blue Tetrazolium tests, and flow cytometry. Endpoints studied suggest a difference in sensitivity between HK and circulating blood cells to tebufenozide and show a stimulation of fish cell function and changes in percentages of cell types. Responses are not associated with a strong stress response as highlighted by the absence of effect on cortisol and blood protein levels. These results and tebufenozide persistence in water warrant further studies on pesticide impact on fish immunity when used on crops or in forest spray programs near lakes and streams.

Neuropeptides and the control of food intake in fish.

The brain, particularly the hypothalamus, integrates input from factors that stimulate (orexigenic) and inhibit (anorexigenic) food intake. In fish, the identification of appetite regulators has been achieved by the use of both peptide injections followed by measurements of food intake, and by molecular cloning combined with gene expression studies. Neuropeptide Y (NPY) is the most potent orexigenic factor in fish. Other orexigenic peptides, orexin A and B and galanin, have been found to interact with NPY in the control of food intake in an interdependent and coordinated manner. On the other hand cholecystokinin (CCK), cocaine and amphetamine-regulated transcript (CART), and corticotropin-releasing factor (CRF) are potent anorexigenic factors in fish, the latter being involved in stress-related anorexia. CCK and CART have synergistic effects on food intake and modulate the actions of NPY and orexins. Although leptin has not yet been identified in fish, administration of mammalian leptin inhibits food intake in goldfish. Moreover, leptin induces CCK gene expression in the hypothalamus and its actions are mediated at least in part by CCK. Other orexigenic factors have been identified in teleost fish, including the agouti-related protein (AgRP) and ghrelin. Additional anorexigenic factors include bombesin (or gastrin-releasing peptide), alpha-melanocyte-stimulating hormone (alpha-MSH), tachykinins, and urotensin I. In goldfish, nutritional status can modify the expression of mRNAs encoding a number of these peptides, which provides further evidence for their roles as appetite regulators: (1) brain mRNA expression of CCK, CART, tachykinins, galanin, ghrelin, and NPY undergo peri-prandial variations; and (2) fasting increases the brain mRNA expression of NPY, AgRP, and ghrelin as well as serum ghrelin levels, and decreases the brain mRNA expression of tachykinins, CART, and CCK. This review will provide an overview of recent findings in this field.

Characterization of two forms of cocaine- and amphetamine-regulated transcript (CART) peptide precursors in goldfish: molecular cloning and distribution, modulation of expression by nutritional status, and interactions with leptin.

Complementary DNAs encoding two forms of cocaine- and amphetamine-regulated transcript (CART) peptide precursors were identified from goldfish brain and named CART I and CART II. Each cDNA contains a signal peptide sequence, the putative CART-like peptide, and a carboxy-terminal extension peptide. Form I encodes a 117-amino acid pro-CART, whereas form II encodes a 120-amino acid pro-CART. Both forms resemble mammalian CART peptides. Each goldfish CART precursor is encoded by three exons interrupted by two introns within genomic DNA. RT-PCR, slot blot, and Northern blot analysis showed that the mRNAs for form I and II precursors have a widespread distribution. Form I and II are present in the brain, pituitary, eye, gonads, and kidney. Form I is also present in the gill. In the brain, form I is predominant in the olfactory bulb and hypothalamus, and form II is predominant in the optic tectum. Food deprivation for 96 h induced a decrease in form I mRNA levels in the telencephalon-preoptic region, hypothalamus, and olfactory bulb and in form II mRNA expression in the olfactory bulb. An increase in mRNA levels was observed 2 h following a meal in the olfactory bulbs and hypothalamus for form I whereas no postprandial changes in form II mRNA levels were observed. Intracerebroventricular injections of human CART alone induced a significant decrease in food intake. Injections of leptin reinforced the inhibition of feeding behavior and food intake seen in CART-treated fish. Central injection of leptin induced an increase in CART I mRNA in the optic tectum, hypothalamus, and olfactory bulbs but had no effect on CART II mRNA expression in the brain. These results suggest that CART peptides act as leptin-regulated satiety factors in goldfish and that they might have other physiological roles besides feeding, possibly in sensory information processing.

Interactions between orexin A, NPY and galanin in the control of food intake of the goldfish, Carassius auratus.

The neuropeptides orexin A (OXA), neuropeptide Y (NPY) and galanin (GAL) have been shown to play a role in the regulation of food intake in mammals. They also significantly stimulate feeding in goldfish. In order to assess the interactions between these peptides in the control of feeding in goldfish, we investigated the effects of central injection of specific receptor antagonists for NPY (BIBP 3226) and GAL (M40) on OXA-induced feeding and the effects of desensitization of orexin receptors on NPY- and GAL-induced feeding. We investigated the effects of BIBP 3226 on GAL-induced feeding and the effects of M40 on NPY-induced feeding. We also examined the effects of coinjection of each pair of neuropeptides on feeding behavior. Injections of 10 ng/g OXA, 5 ng/g NPY and 10 ng/g GAL each induced an increase in feeding. Fish treated with 5 ng/g BIBP or 20 ng/g M40 had food consumption similar to saline controls. BIBP at 5 ng/g significantly reduced NPY- and OXA-induced feeding. Injections of 20 ng/g M40 significantly decreased GAL-induced feeding, but had no effect on OXA-induced feeding. Blocking of orexin receptors by treatment with high doses of OXA (100 ng/g) resulted in a decrease in both NPY- and GAL-induced feeding. Coinjection with 0.5 ng/g OXA and either 0.5 ng/g NPY or 0.5 ng/g GAL resulted in a food intake higher than that observed in saline control fish and in fish treated with NPY or GAL alone at 0.5 ng/g. NPY mRNA expression was increased in the telencephalon and in the hypothalamus compared to saline-treated fish, following injection of OXA. These results indicate that both NPY and GAL are at least, in part, dependent on coaction with OXA for the stimulation of food intake and feeding behavior in goldfish. In addition, the effects of OXA are mediated, in part, by the NPY pathway. This suggests a functional interdependence between these three peptidergic systems in the control of energy balance in goldfish.

Brain regulation of feeding behavior and food intake in fish.

In mammals, the orexigenic and anorexigenic neuronal systems are morphologically and functionally connected, forming an interconnected network in the hypothalamus to govern food intake and body weight. However, there are relatively few studies on the brain control of feeding behavior in fish. Recent studies using mammalian neuropeptides or fish homologs of mammalian neuropeptides indicate that brain orexigenic signal molecules include neuropeptide Y, orexins, galanin and beta-endorphin, whereas brain anorexigenic signal molecules include cholecystokinin, bombesin, corticotropin-releasing factor, cocaine- and amphetamine-regulated transcript, and serotonin. Tachykinins may also have an anorectic action in fish. The brain hypothalamic area is associated with regulation of food intake, while sites outside the hypothalamus are also involved in this function. There is correlation between short-term changes in serum growth hormone levels and feeding behavior, although possible mechanisms integrating these functions remain to be defined.

Molecular cloning and expression of cDNA encoding a brain bombesin/gastrin-releasing peptide-like peptide in goldfish.

A complementary DNA (cDNA) of 928 bp encoding a bombesin (BBS)/gastrin-releasing peptide (GRP) precursor was identified from goldfish brain. Goldfish BBS/GRP messenger RNA (mRNA) encodes a 157 amino acid precursor, which contains a signal peptide sequence, the 22 amino acid putative BBS/GRP-like peptide, and a carboxy-terminal extension peptide. Reverse transcription-polymerase chain reaction (PCR) (RT-PCR) demonstrated that the mRNA for this precursor has a widespread distribution in goldfish brain, and is also present in skin, gastrointestinal tract, gonad, and gill. Phylogenetic analysis of BBS/GRP-like peptide precursors in vertebrates shows that goldfish BBS/GRP is more closely related to the known GRP precursors than to BBS precursors.

Effects of CART peptides on food consumption, feeding and associated behaviors in the goldfish, Carassius auratus: actions on neuropeptide Y- and orexin A-induced feeding.

CART (cocaine- and amphetamine-regulated transcript) peptides are novel brain neuropeptides that have been shown to have a role in the control of feeding behavior in mammals. The effects of intracereboventricular (i.c.v.) administration of two CART fragments, CART (62-76) and CART (55-102) on feeding and behavioral activity of goldfish (Carassius auratus) were examined. Both CART peptide fragments inhibited food intake in goldfish. Co-injection of CART peptide and neuropeptide Y (NPY) reduced the increase in feeding caused by injection of NPY alone. CART (55-102) was more potent than CART (62-76). Co-treatment with CART (55-102) and orexin A inhibited the feeding response induced by orexin A alone. Feeding behavior was stimulated relative to saline controls following injection of CART (62-76) alone and co-injection of NPY and CART (62-76), but was not affected by treatment with CART (55-102) alone, NPY and CART (55-102) or orexin A and CART (55-102). Total behavioral activity was increased with NPY, orexin A, and both CART fragments alone as compared to saline controls, as well as in fish co-treated with NPY and CART (62-76) or NPY and CART (55-102) as compared to saline controls and NPY-treated fish. Tremors were seen in fish treated with CART (55-102) alone, and in fish co-treated with NPY and CART (55-102) and orexin A and CART (55-102). Co-treatment of the fish with NPY but not with orexin A significantly lowered the frequency of fish showing tremors as compared to fish treated with CART alone. These results indicate that CART peptides are involved in the regulatory pathways of feeding and behavioral activity in goldfish.

Actions of two forms of gonadotropin releasing hormone and a GnRH antagonist on spawning behavior of the goldfish Carassius auratus.

The central effects of two native forms of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), and a GnRH antagonist, ¿Ac-delta 3-Pro(1), 4FD-Phe(2), D-Trp(3, 6)mGnRH (analog E), on the spawning behavior of sexually recrudescent female goldfish were investigated. The effects of analog E were also observed in mature males. Female spawning behavior was induced by intramuscular injection of females with prostaglandin F(2alpha) and placing them in the presence of mature males. Behavioral responses were quantified by recording the numbers of spawning acts performed by each pair of fish for 2 h following brain intracerebroventricular (icv) injection of different dosages of peptide or saline as control. For males, the time spent courting the female was recorded. Each pair of fish was pretested to determine their level of spawning behavior, for comparison to spawning behavior following icv treatment. Icv injection of analog E caused a significant decrease in the number of spawning acts performed by females, suggesting a role of endogenous GnRH in modulating female spawning behavior. icv injection of 0.5 ng/g of sGnRH or cGnRH-II significantly stimulated female spawning behavior, whereas doses of 1 ng/g and higher resulted in an almost complete inhibition of spawning, reflecting a down-regulation as a result of the excessive dosages. Analog E suppressed the actions of exogenous sGnRH and cGnRH-II on spawning behavior, as both the sGnRH- and cGnRH-II-induced increases in the number of spawning acts were inhibited by concomitant treatment with analog E. Analog E-injected males showed no alteration in courtship behavior. These results indicate that GnRH peptides play a major role in the control of female reproductive behavior in goldfish, but have little or no role in the control of male behavior.

Stimulation of feeding behavior and food consumption in the goldfish, Carassius auratus, by orexin-A and orexin-B.

The neuropeptides, orexin-A and orexin-B, have been demonstrated to have a physiological role in the regulation of food intake in mammals. The effects of human orexin-A and orexin-B intracerebroventricular (i.c.v.) injection on the feeding behavior of goldfish (Carassius auratus) were investigated. I.c.v. injection of orexin-A and orexin-B both caused a significant increase in appetite, as indicated by an increased number of feeding acts. Orexin-A and orexin-B both significantly stimulated food consumption, as indicated by increased total food intake during a 60-min observation period; the actions of orexin-A were dose dependent. Orexin-A was more potent than orexin-B in stimulation of both feeding behavior and food intake. These results indicate that orexin peptides are involved in the hypothalamic regulatory pathways of feeding behavior in goldfish.

Structure of the thyroid gland, serum thyroid hormones, and the reproductive cycle of the Atlantic stingray, Dasyatis sabina.

This study examines the role of the thyroid gland in the control of reproduction in the viviparous Atlantic stingray, Dasyatis sabina. Thyroid activity in individuals in different reproductive stages was assessed both by microscopic examination of the gland, and by analysis of circulating levels of thyroid hormones from the same individuals. The thyroid gland is a cylindric organ, embedded in a connective tissue capsule, and composed of follicles, i.e., monolayer spheres of thyroid epithelial cells. Stingray follicular cells possess several characteristic features, namely apical cilia and a well-developed endoplasmic reticulum. Cells vary in size and shape, according to the activity of the gland. No structural differences were observed between the thyroid glands of the two sexes. Both thyroid hormones, triiodothyronine, [T(3)], and thyroxine, [T(4)], were detected in the serum of all animals examined. Levels ranged from 1.3-2.6 microg/100 ml for total T(4), and from 1.2-2.6 ng/ml for total T(3). The T(4) levels did not vary significantly in any group. Immature individuals and females undergoing oogenesis had the lowest levels of circulating T(3) and mature females from ovulation throughout gestation had high thyroid gland activity and high levels of circulating T(3). J. Exp. Zool. 284:505-516, 1999.