As an ancient allotetraploid species, goldfish (Carassius auratus) have two sets of subgenomes. In this study, immunoglobulin heavy-chain (IGH) genes were cloned from the red crucian carp (Carassius auratus red var.), and the corresponding loci were identified in the gynogenetic diploid red crucian carp (GRCC) genome as well as the genomes of three other goldfish strains (Wakin, G-12, and CaTCV-1). Examination showed that each goldfish strain possessed two sets of parallel IGH loci: a complete IGHA locus and a degenerated IGHB locus that was nearly 40 × smaller. In the IGHA locus, multiple τ chain loci were arranged in tandem between the μ&δ chain locus and the variable genes, but no τ-like genes were found in the IGHB locus.

Distant hybridization leads to obvious changes in genotypes and phenotypes, giving rise to species with novel capabilities. However, the fusion of distinct genomes also polymerizes the DNA or gene variations that occur during the course of evolution. Knowledge of the early stages of post-hybridization evolution is particularly important. Here, we investigated the full-length (FL) transcriptomes and the sequences resulting from the genome resequencing of the red crucian carp-like homodiploid fish (RCC-L) and goldfish-like homodiploid fish (GF-L) derived from the interspecific hybridization of koi carp (KOC) and blunt snout bream (BSB) to provide molecular evidence for the hybrid origin of the goldfish (GF). We compared the orthologous genes in the transcriptomes of RCC-L and GF-L with those of KOC and BSB. We also mapped the orthologous genes to the common carp (CC) and BSB genomes and classified them into eight gene patterns in three categories (chimaera, mutant, and biparental origin genes). The results showed that 48.20% and 46.50% of the genes were chimaera and that 3.70% and 8.30% of the genes were mutations of orthologous genes in RCC-L and GF-L, respectively. In RCC-L and GF-L, 63.70% and 68.20% of the genetic materials were from KOC, and 12.30% and 11.90% of the genetic materials were from BSB. The sequences from the genome resequencing of RCC-L and GF-L were mapped to the genome sequences of CC and BSB, revealing that the similarities of both RCC-L and GF-L to the CC genome (92.52%, 90.18%) were obviously higher than to the BSB genome (50.33%, 49.18%), supporting the suggestion that the genomes of both RCC-L and GF-L were mainly inherited from KOC but had some DNA fragments from BSB. Overall, our results provide molecular biological evidence for the hybrid origin of red crucian carp (RCC) and GF.

Previous work has shown that the neural retina in adult goldfish can regenerate. Following retinal damage elicited by surgical or cytotoxic lesions, missing neurons are replaced by foci of proliferating neuroepithelial cells, which previous studies have suggested are derived from rod precursors. In the intact retina, rod precursors proliferate but produce only new rods. The regenerative responses observed previously have involved replacement of neurons in all retinal layers; selective regeneration of specific neuronal types (except for rod photoreceptors) has not been reported. In the experiments described here, we specifically destroyed either cones alone or cones and rods with an argon laser, and we found that both types of photoreceptors regenerated within a few weeks. The amount of cone regeneration varied in proportion to the degree of rod loss. This is the first demonstration of selective regeneration of a specific class of neuron (i.e., cones) in a region of central nervous tissue where developmental production of that class of neuron has ceased. Selective regeneration may be limited to photoreceptors, however, because when dopaminergic neurons in the inner retina were ablated with intraocular injections of 6-hydroxydopamine, in combination with laser lesions that destroyed photoreceptors, the dopaminergic neurons did not regenerate, but the photoreceptors did. These data support previous studies which showed that substantial cell loss is required to trigger regeneration of inner retinal neurons, including dopaminergic neurons. New observations here bring into question the presumption that rod precursors are the only source of neuronal progenitors during the regenerative response. Finally, a model is presented which suggests a possible mechanism for regulating the phenotypic fate of retinal progenitor cells during retinal regeneration.

A set of cytological studies performed in the utricle and saccule of Astronotus ocellatus (Teleostei, Percomorphi, Cichlidae) identified two basic types of hair cells and others with some intermediate characteristics. This paper reports on applying the same techniques to the saccule of Carassius auratus (Teleostei, Otophysi, Cyprinidae) and demonstrates similar types of hair cells to those found in Astronotus. Since Carassius and Astronotus are species of extreme taxonomic distance within the Euteleostei, two classes of mechanoreceptive hair cells are likely to represent the primitive condition for sensory receptors in the euteleost inner ear and perhaps in all bony fish ears.

We recently established a spontaneously proliferating macrophage cell line from the goldfish (GMCL), and in this report demonstrate the production of a macrophage-specific growth factor(s) (MGFs) by these cells. The supernatants from GMCL cultures induced proliferation and differentiation of macrophage-like cells from kidney hematopoietic tissues of goldfish. Kidney leukocytes cultured at 6.25 x 10(4)cells/ml in the presence of GMCL-derived MGFs proliferated during two weeks of cultivation, whereas those cultured without the MGFs did not. Leukocytes cultured at higher densities (2.5 x 10(5) cells/ml) proliferated in the absence of exogenous growth factor, but not to the same extent as those stimulated with GMCL-derived MGFs, suggesting that kidney leukocytes may produce endogenous MGFs. At higher cell density (1 x 10(6) cells/ml), kidney leukocytes multiplied extensively over a two-week cultivation period in the absence of exogenous GMCL-derived MGFs. The supernatants from these cultures restored the proliferative ability of leukocytes cultured at low densities, providing direct evidence of MGFs production by kidney leukocytes. The predominant cell-type in cultures grown in the presence of GMCL or kidney leukocyte-MGFs was the macrophage based on the following criteria: (1) non-specific esterase staining; (2) morphologic similarity to GMCL; (3) phagocytosis of the bacterium, A. salmonicida; (4) production of reactive oxygen and nitrogen intermediates in response to stimulation with macrophage activating factors and/or bacterial lipopolysaccharide; and (5) flow cytometric analyses. Both in vitro-derived kidney macrophage (IVDKM) and GMCL cultures contained three distinct populations of cells, (determined by flow cytometry), suggesting that these macrophage cultures are comprised of cells arrested at distinct differentiation junctures in macrophage development. Production of MGFs by macrophages and kidney leukocytes may play an important role in regulating macrophage hematopoiesis in fish.

Herpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, Carassius auratus. Permissive cell lines for the isolation and propagation of CyHV-2 have been established from various goldfish tissues by sacrificing the fish. Here, we report the development of a cell line, FtGF (Fantail Goldfish Fin), from caudal fin of goldfish using non-lethal sampling. We also describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continuous propagation of CyHV-2 with high viral titer using this cell line.

Although domesticated goldfish strains exhibit highly diversified phenotypes in morphology, the genetic basis underlying these phenotypes is poorly understood. Here, based on analysis of transposable elements in the allotetraploid goldfish genome, we found that its two subgenomes have evolved asymmetrically since a whole-genome duplication event in the ancestor of goldfish and common carp. We conducted whole-genome sequencing of 27 domesticated goldfish strains and wild goldfish. We identified more than 60 million genetic variations and established a population genetic structure of major goldfish strains. Genome-wide association studies and analysis of strain-specific variants revealed genetic loci associated with several goldfish phenotypes, including dorsal fin loss, long-tail, telescope-eye, albinism, and heart-shaped tail. Our results suggest that accumulated mutations in the asymmetrically evolved subgenomes led to generation of diverse phenotypes in the goldfish domestication history. This study is a key resource for understanding the genetic basis of phenotypic diversity among goldfish strains.

Xenin, a highly conserved 25 amino acid peptide cleaved from the N-terminus of the coatomer protein alpha (COPA), is emerging as a food intake regulator in mammals and birds. To date, no research has been conducted on xenin biology in fish. This study aims to identify the copa mRNA encoding xenin in goldfish (Carassius auratus) as a model, to elucidate its regulation by feeding, and to describe the role of xenin on appetite. First, a partial sequence of copa cDNA, a region encoding xenin, was identified from goldfish brain. This sequence is highly conserved among both vertebrates and invertebrates. RT-qPCR revealed that copa mRNAs are widely distributed in goldfish tissues, with the highest levels detected in the brain, gill, pituitary and J-loop. Immunohistochemistry confirmed also the presence of COPA peptide in the hypothalamus and enteroendocrine cells on the J-loop mucosa. In line with its anorexigenic effects, we found important periprandial fluctuations in copa mRNA expression in the hypothalamus, which were mainly characterized by a gradually decrease in copa mRNA levels as the feeding time was approached, and a gradual increase after feeding. Additionally, fasting differently modulated the expression of copa mRNA in a tissue-dependent manner. Peripheral and central injections of xenin reduce food intake in goldfish. This research provides the first report of xenin in fish, and shows that this peptide is a novel anorexigen in goldfish.

Owning to the extreme difficulty in identifying the primary generation (G0), the common ancestor of various twin-tail goldfish strains remains unclear. However, several authors have hypothesized that this ancestor may have been the crucian carp (Carassius auratus). Previously, we generated an experimental hybrid goldfish (EG) from the interspecific hybridization of red crucian carp (Carassius auratus ♀, RCC) × common carp (Cyprinus carpio ♂, CC). Unlike either parent, EG possessed twin caudal fins similar to those of natural goldfish (Carassius auratus, NG). The genetic characteristics of EG, as well as the mechanisms underlying its formation, are largely unknown. Here, we identified the genetic variation in the chordin gene that was associated with the formation of the twin-tail phenotype in EG: a stop codon mutation at the 127th amino acid. Furthermore, simple sequence repeat (SSR) genotyping indicated that, among the six alleles, all of the EG alleles were also present in female parent (RCC), but alleles specific to the male parent (CC) were completely lost. At some loci, EG and NG alleles differed, showing that these morphologically similar goldfish were genetically dissimilar. Collectively, our results demonstrated that genetic variations and differentiation contributed to the changes of morphological characteristics in hybrid offspring. This analysis of genetic variation in EG sheds new light on the common ancestor of NG, as well as on the role of hybridization and artificial breeding in NG speciation.

While serotonin (5-HT) can stimulate the hypothalamic-pituitary-interrenal stress axis in fish, the specific site(s) of 5-HT action are poorly understood. In this study, goldfish (Carassius auratus) were injected intraperitoneally with either saline or the 5-HT1A/7 receptor agonist 8-OH-DPAT at a dose of 100 or 400 μg/kg body weight and sampled 1.5 and 8 h post-injection. Relative to unhandled controls, the saline and 100 μg/kg 8-OH-DPAT treatments elicited similar transient 5- to 7-fold increases in plasma cortisol and the 400 μg/kg 8-OH-DPAT dosage resulted in a sustained 16-fold increase in cortisol levels. Although the 5-HT1A receptor is expressed in the brain preoptic area (POA), the pituitary and the head kidney, neither the saline nor the 8-OH-DPAT treatments affected the mRNA abundance of POA corticotropin-releasing factor and pituitary pro-opiomelanocortin or plasma adrenocorticotropic hormone (ACTH) levels. To assess the direct actions of 5-HT on cortisol secretion relative to those of ACTH, head kidney tissue were superfused with 10(-7)M 5-HT, ACTH or a combined 5-HT/ACTH treatment. Overall, the ACTH and 5-HT/ACTH treatments resulted in higher peak cortisol and total cortisol release than in the 5-HT treatment but the response time to peak cortisol release was shorter in the combined treatment than in either the 5-HT or ACTH alone treatments. Both 8-OH-DPAT and cisapride, a 5-HT4 receptor agonist, also stimulated cortisol release in vitro and their actions were reversed by selective 5-HT1A and 5-HT4 receptor antagonists, respectively. Finally, double-labeling with anti-tyrosine hydroxylase and anti-5-HT revealed that the chromaffin cells of the head kidney contain 5-HT. Thus, in goldfish, 5-HT can directly stimulate cortisol secretion from the interrenals via multiple 5-HT receptor subtypes and the chromaffin cells may be involved in the paracrine regulation of cortisol secretion via 5-HT.

When gametes and embryos are not available, cryobanking of somatic tissues is one possibility to keep a genetic record of fish valuables in a context of biodiversity conservation and animal breeding management. Cryopreservation of whole fin pieces would be more advantageous than the commonly used cryopreservation of cells after fin culture, as it would allow extensive sampling without immediate need for laboratory facilities. The objective of this work was to assess the cryopreservation ability of fin pieces from goldfish (Carassius auratus) and to test whether a laboratory procedure could be adapted to field conditions. Caudal fin explants were cryopreserved in culture medium with 125mM sucrose and 10% Me(2)SO. After 14days of culture, the frozen-thawed explants showed the same cell growth rate and grew the same somatic cell number as the fresh ones. Cells proliferated inside and around the explants as shown by BrdU labeling. Neither the size of the fin pieces nor the freezer type, -70 degrees C upright or -20 degrees C chest, influenced the outcome of cryopreservation. Fin pieces were stored 4days at 4 degrees C in dry conditions prior to cryopreservation without alteration of the fin explant culture success. This study demonstrated that field collecting of goldfish fin pieces is possible as whole fin pieces can be stored in standard fridge or be shipped at subzero temperature before they are frozen into a plain -20 degrees C chest freezer. After incorporation in cryobanks in liquid nitrogen, thawed fin pieces reliably produce somatic cells in cell culture conditions.

Otophysine fishes are characterized by Weberian ossicles connecting the swimbladder to the ear acoustically. In order to determine the degree to which these ossicles contribute to auditory sensitivity, the tripus was unilaterally or bilaterally extirpated in goldfish and hearing thresholds determined. The auditory evoked potential (AEP) recording technique was used to measure auditory sensitivity between 100 and 4000 Hz. Bilateral extirpation resulted in a hearing loss at all frequencies ranging from 7 dB at 100 Hz to 33 dB at 2 kHz; no AEPs were detectable at 4 kHz. In contrast to bilateral extirpation, unilateral tripus removal caused no sensitivity change. Pre-exposure to intense white noise caused different threshold shifts in unilaterally versus bilaterally extirpated goldfish. Thresholds increased at all frequencies in unilaterally extirpated goldfish but only at 100 and 200 Hz after bilateral extirpation. The comparison between the hearing generalist Neolamprologus brichardi (family Cichlidae) and the tripus-extirpated otophysine revealed that the latter is still more sensitive than the cichlid. Higher sensitivity in the goldfish after bilateral extirpation as compared to swimbladder elimination indicates that swimbladder oscillations might partly be transmitted to the inner ear independently of the ossicular chain. This suggests that the auditory system in otophysines improves with increasing frequency due to a more efficient connection between the swimbladder and inner ear ensured by the Weberian ossicles.

The structure of goldfish (Carassius auratus) Tgf2 transposase is still poorly understood, although it can mediate efficient gene transfer in teleost fish. We hypothesized the existence of a nuclear localization signal (NLS) within Tgf2 transposase to assist transport into the nucleus. To explore this, 15 consecutive amino acid residues (656-670 aa) within the C-terminus of Tgf2 transposase were predicted in silico to be a NLS domain. The pEGFP-C1-Tgf2TP(△31C) plasmid encoding the NLS-domain-deleted Tgf2 transposase fused to EGFP was constructed, and transfected into 293T cells. After transfection with pEGFP-C1-Tgf2TP(△31C), EGFP was not detected in the nucleus alone, while 67.0% of cells expressed EGFP only in the cytoplasm. In contrast, after transfection with control plasmids containing C- or N-terminal truncated Tgf2 transposases with an intact NLS domain, EGFP was not detected in the cytoplasm alone, while approximately 40% of cells expressed EGFP only in the nucleus, and the remaining 60% expressed EGFP in both the nucleus and cytoplasm. Our results demonstrated that loss of the NLS domain results in expression in the cytoplasm but not in the nucleus. These findings suggest that 15 aa residues located from 656 to 670 aa within the C-terminus of Tgf2 transposase can function as a NLS to assist the transfer of the transposase into the nucleus where it mediates DNA transposition.

The spectral sensitivities of retinal cones isolated from goldfish (Carassius auratus) retinas were measured in the range 277-737 nm by recording membrane photocurrents with suction pipette electrodes (SPE). Cones were identified with lambda max (+/- S.D.) at 623 +/- 6.9 nm, 537 +/- 4.7 nm, 447 +/- 7.7 nm, and about 356 nm (three cells). Two cells (lambda max 572 and 576 nm) possibly represent genetic polymorphism. A single A2 template fits the alpha-band of P447(2), P537(2), and P623(2). HPLC analysis showed 4% retinal:96% 3-dehydroretinal. Sensitivity at 280 nm is nearly half that at the lambda max in the visible. The lambda max of the beta-band (in nm) is a linear function of the lambda max of the alpha-band and follows the same relation as found for A1-based cone pigments of a cyprinid fish.

A fluorometric procedure to image release of the neurotransmitter glutamate from living retinal slices is described. Patterns of endogenous glutamate efflux were imaged with a cooled CCD camera in goldfish retinal slices as NADH fluorescence produced by a cycling glutamate dehydrogenase (GDH). Basal and potassium evoked glutamate effluxes were strongly localized to the outer and inner plexiform layers, supporting the model that photoreceptors and bipolar cells release glutamate as their prime fast neurotransmitter.

Cardiac physiology of fish models is an emerging field given the ease of genome editing and the development of transgenic models. Several studies have described the cardiac properties of zebrafish (Denio rerio). The goldfish (Carassius auratus) belongs to the same family as the zebrafish and has emerged as an alternative model with which to study cardiac function. Here, we propose to acutely study electrophysiological and systolic Ca2+ signaling in intact goldfish hearts. We assessed the Ca2+ dynamics and the electrophysiological cardiac function of goldfish, zebrafish, and mice models, using pulsed local field fluorescence microscopy, intracellular microelectrodes, and flash photolysis in perfused hearts. We observed goldfish ventricular action potentials (APs) and Ca2+ transients to be significantly longer when compared to the zebrafish. The action potential half duration at 50% (APD50) of goldfish was 370.38 ± 8.8 ms long, and in the zebrafish they were observed to be only 83.9 ± 9.4 ms. Additionally, the half duration of the Ca2+ transients was also longer for goldfish (402.1 ± 4.4 ms) compared to the zebrafish (99.1 ± 2.7 ms). Also, blocking of the L-type Ca2+ channels with nifedipine revealed this current has a major role in defining the amplitude and the duration of goldfish Ca2+ transients. Interestingly, nifedipine flash photolysis experiments in the intact heart identified whether or not the decrease in the amplitude of Ca2+ transients was due to shorter APs. Moreover, an increase in temperature and heart rate had a strong shortening effect on the AP and Ca2+ transients of goldfish hearts. Furthermore, ryanodine (Ry) and thapsigargin (Tg) significantly reduced the amplitude of the Ca2+ transients, induced a prolongation in the APs, and altogether exhibited the degree to which the Ca2+ release from the sarcoplasmic reticulum contributed to the Ca2+ transients. We conclude that the electrophysiological properties and Ca2+ signaling in intact goldfish hearts strongly resembles the endocardial layer of larger mammals.

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin's physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish.

There is general agreement that the hippocampus of vertebrates, from fish to mammals, is involved in map-like spatial memory. However, in mammals the role of the hippocampus goes beyond the spatial domain as it is also involved in binding the temporally separate events that compose episodic memories. In this regard, the hippocampus of mammals is essential for trace classical conditioning, in which a stimulus-free time gap separates the conditioned stimulus (CS) and the unconditioned stimulus (US), but not for delay conditioning, in which both stimuli coincide in time. Although the involvement of the hippocampus in encoding relational memories based on a temporal frame-work has been extensively studied in mammals, there is scarce evidence about the possible contribution of the hippocampus of non-mammalian vertebrates to the temporal, non-spatial dimension of relational memories. The present work was aimed to determine if the ventral part of the lateral division of the area dorsalis telencephali (Dlv) of goldfish, proposed as homologous to the hippocampus of mammals, is also involved in trace classical conditioning. With this purpose, goldfish with lesions in Dlv, complete telencephalon ablation and sham operation, were trained in delay and trace heart rate classical conditioning. Dlv lesions severely impaired the acquisition of the conditioned response when a stimulus-free time gap was elapsed between the CS and the US (trace conditioning), but not when both stimuli overlapped in time (delay conditioning), revealing that this region, like the hippocampus of mammals, is essential to form the temporal associative memories required by trace conditioning. Present data suggest that the presence of a hippocampal pallium involved in relational, episodic-like memory that preserves both the spatial and the temporal dimensions of past events, could be a primitive feature of the vertebrate brain that has been conserved through evolution.

By immunohistochemistry VIP-like immunoreactive cells were observed in the second segment of some renal proximal tubules of Carassius auratus. Immunoreactivity displayed a granular appearance all over the cells. VIP-like immunoreactive cells showed a shape of an over-turned flask whose globe-like part looks like the tubular lumen. No immunoreactive material was observed in other tracts of the nephron and in the interstitial cells. The presence of VIP-like immunoreactive cells is discussed in relation to their possible function.

Complementary DNAs encoding two major osteoclastic markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Cath K) were cloned from the scales of a teleost, the goldfish. This is the first report of the full coding sequence of TRAP and Cath K molecules in fish. In the goldfish scale both TRAP and Cath K mRNAs were expressed in the multinucleate osteoclasts, which showed large numbers of mitochondria and lysosomes, and a well developed ruffled border. These characteristic features of osteoclasts in the scales are similar to those in mammals. Most teleosts use the scale as an internal calcium reservoir during the reproductive season. The expression of TRAP and Cath K mRNAs in the scale significantly increased in April, which is a reproductive season, compared with that in October, a non-reproductive season. Thus, both of these molecular markers should be useful for the study of osteoclasts in the teleost scale.