Goldfish Tissues Research Articles

Copper-oxide nanoparticles effects on goldfish (Carassius auratus): Lethal toxicity, haematological, and biochemical effects.

The advancement of nanotechnology and the widespread use of nanoparticles (NPs) in various industries have highlighted the importance of studying the potential harmful effects of nanomaterials on organisms. This study aimed to evaluate the lethal toxicity thresholds of Copper Oxide Nanoparticles (CuO-NPs). The investigation focused on examining the sub-lethal toxicity effects of CuO-NPs on blood parameters, as well as their influence on the gill tissue and liver of goldfish (Carassius auratus). Goldfish were exposed to varying concentrations of CuO-NPs (10, 20, 30, 40, 60, 80, and 100mg/L) for 96h. The Probit software was employed to determine the LC50 (lethal concentration causing 50% fish mortality) by monitoring and documenting fish deaths at 24, 48, 72, and 96-hour intervals. Subsequently, sub-lethal concentrations of 5% LC50 (T1), 10% LC50 (T2), and 15% LC50 (T3) of CuO-NPs were administered based on the LC50 level to investigate their effects on haematological parameters, encompassing the number of red blood cells and white blood cells, hematocrit and haemoglobin levels, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Additionally, histopathological examinations were conducted on the gill and liver tissues of the studied fish. Results indicated concentration-response of fish mortalities. In general, changes in the blood biochemical parameters of fish exposed to sub-lethal concentrations of CuO-NPs included a significant decrease in leukocyte count and glucose level and an increase in protein and triglyceride levels. Furthermore, an escalation in tissue damage such as gill apical and basal hyperplasia, lamellae attachment, squamous cell swelling, blood cell infiltration, and cellular oedema in gills tissue. and bleeding, increased sinusoidal space, necrosis, lateralization of the nucleus, cell swelling, and water retention in the liver. The findings showed dose-dependent increasing toxicity in goldfish specimens exposed to CuO-NPs.

The potential mechanism of concentrated mannan-oligosaccharide promoting goldfish’s (Carassius auratus Linnaeus) resistance to Ichthyophthirius multifiliis invasion

Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1β, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.

SAT049 Goldfish LEAP2: Molecular Cloning, Functional Characterization, And Novel Regulation By IGF And Insulin At The Hepatic Level

Abstract Disclosure: F. Jin: None. X. Qin: None. C. Ye: None. M. He: None. A.O. Wong: None. Liver-expressed antimicrobial peptide 2 (LEAP2) is a peptide highly expressed in the liver and its secretion induced by bacterial infection constitutes an integral component of innate immunity in vertebrate species. Recently, LEAP2 has been confirmed to be the endogenous antagonist of the feeding regulator ghrelin, implying that the peptide may play a role in appetite control and energy homeostasis. However, whether LEAP2 can be regulated by metabolic/endocrine signals is still unclear and the signal transduction involved is totally unknown. Using goldfish as a model, three isoforms of LEAP2, namely LEAP2a, 2b & 2c, have been cloned and phylogenetic analysis confirmed that they could be clustered into the clade of fish LEAP2. Comparative synteny also revealed that the three isoforms were derived from separated genes with similar structural organization but located in different chromosomes of goldfish genome. The a.a. sequences and 3D protein structure of LEAP2a-c were found to be highly comparable with LEAP2 of other vertebrates and tissue expression profiling by RT-PCR also showed that they were expressed at high levels in the liver but to a lower extent in other tissues. As members of antimicrobial peptides, goldfish LEAP2a-c not only could exhibit antimicrobial activity against Staphylococcus epidermidis but also play important role in fish innate immunity, which is evidenced by their transcript expression induced by LPS & cytokines at hepatic level as well as in immune tissues in goldfish naturally infected with Aeromonas hydrophila. In goldfish liver cells, transcript expression of LEAP2a-c could be up-regulated in a time- and dose-dependent manner by IGF-I, IGF-II and insulin. Using a pharmacological approach, the stimulatory effects of IGF-I/-II and insulin on the three isoforms of LEAP2 were confirmed to be mediated by IGF-I receptor (IGF1R) but not insulin receptor activation. Furthermore, the PI3K/Akt, MEK/ERK and P38 MAPK pathways were shown to be involved in IGF-I induced LEAP2a-c gene expression. Our findings for the first time demonstrate that the endocrine signals targeting the liver (insulin) or produced locally within the liver (IGF-I & -II) can play a role in regulating LEAP2 expression at the hepatic level, which may shed light on a novel aspect of the functional crosstalk between innate immunity and feeding control/energy balance in fish model. Presentation: Saturday, June 17, 2023

Molecular characterization, gene expression and functional analysis of goldfish (Carassius auratus L.) macrophage colony stimulating factor 2.

Macrophage colony-stimulating factor 2 (MCSF-2) is an important cytokine that controls how cells of the monocyte/macrophage lineage proliferate, differentiate, and survive in vertebrates. Two isoforms of MCSF have been identified in fish, each exhibiting distinct gene organization and expression patterns. In this study, we investigated a goldfish MCSF-2 gene in terms of its immunomodulatory and functional properties. In this study, goldfish were acclimated for 3 weeks and sedated with TMS prior to handling. Two groups of fish were used for infection experiments, and tissues from healthy goldfish were collected for RNA isolation. cDNA synthesis was performed, and primers were designed based on transcriptome database sequences. Analysis of gfMCSF-2 sequences, including nucleotide and amino acid analysis, molecular mass prediction, and signal peptide prediction, was conducted. Real-time quantitative PCR (qPCR) was used to analyze gene expression levels, while goldfish head kidney leukocytes (HKLs) were isolated using standard protocols. The expression of gfMCSF-2 in activated HKLs was investigated, and recombinant goldfish MCSF-2 was expressed and purified. Western blot analysis, cell proliferation assays, and flow cytometric analysis of HKLs were performed. Gene expression analysis of transcription factors and pro-inflammatory cytokines in goldfish head kidney leukocytes exposed to rgMCSF-2 was conducted. Statistical analysis using one-way ANOVA and Dunnett's post hoc test was applied. We performed a comparative analysis of MCSF-1 and MCSF-2 at the protein and nucleotide levels using the Needleman-Wunsch algorithm. The results revealed significant differences between the two sequences, supporting the notion that they represent distinct genes rather than isoforms of the same gene. Sequence alignment demonstrated high sequence identity with MCSF-2 homologs from fish species, particularly C. carpio, which was supported by phylogenetic analysis. Expression analysis in various goldfish tissues demonstrated differential expression levels, with the spleen exhibiting the highest expression. In goldfish head kidney leukocytes, gfMCSF-2 expression was modulated by chemical stimuli and bacterial infection, with upregulation observed in response to lipopolysaccharide (LPS) and live Aeromonas hydrophila. Recombinant gfMCSF-2 (rgMCSF-2) was successfully expressed and purified, showing the ability to stimulate cell proliferation in HKLs. Flow cytometric analysis revealed that rgMCSF-2 induced differentiation of sorted leukocytes at a specific concentration. Moreover, rgMCSF-2 treatment upregulated TNFα and IL-1β mRNA levels and influenced the expression of transcription factors, such as MafB, GATA2, and cMyb, in a time-dependent manner. Collectively, by elucidating the effects of rgMCSF-2 on cell proliferation, differentiation, and the modulation of pro-inflammatory cytokines and transcription factors, our findings provided a comprehensive understanding of the potential mechanisms underlying gfMCSF-2-mediated immune regulation. These results contribute to the fundamental knowledge of MCSF-2 in teleosts and establish a foundation for further investigations on the role of gfMCSF-2 in fish immune responses.

Absorption and Distribution of Imidacloprid and Its Metabolites in Goldfish (Carassius auratus Linnaeus).

Imidacloprid (IMI) is the first-generation neonicotinoid insecticide. But, the long-term use of IMI as a pesticide has caused severe water pollution. Recently, the toxicity of IMI to aquatic organisms has received increasing attention. This study aimed to investigate the absorption and distribution of IMI in various tissues (gills, intestine, liver, muscle, brain and gonads) of goldfish through short-term and continuous exposure tests over 28 days. The results of short-term exposure indicated that the concentration of IMI and its metabolites in tissues at the transfer stage decreased steadily after 1 day of 40 mg/L IMI water treatment and was below the detection limit after 3 days. Continuous exposure for 28 days at various treatment concentrations showed that the concentrations of IMI and its metabolites differed significantly among the different tissues of the goldfish. In the 20 mg/L treatment group (S1), the highest concentration of IMI was found in the liver (12.04 μg/gtissue), followed by the intestine (9.91 μg/gtissue), muscle (6.20 μg/gtissue), gill (6.11 μg/gtissue), gonads (5.22 μg/gtissue) and brain (2.87 μg/gtissue). In the 40 mg/L treatment group (S2), the order of the tissue concentrations was similar to that of the S1 group, with the highest concentration observed in the liver (12.04 μg/gtissue), followed by the intestine (9.91 μg/gtissue), muscle (6.20 μg/gtissue), gill (6.11 μg/gtissue), gonads (5.22 μg/gtissue) and brain (2.87 μg/gtissue). Furthermore, the study detected 5-hydroxy-IMI, IMI urea and 6-chloronicotinic acid in IMI metabolites in all tissues, while IMI was detected only in the intestine and liver. Overall, the results of this study contribute to a better understanding of the metabolic behavior of IMI in organisms and provide new data to support the assessment of IMI toxicity in fish.

Evaluation of a chronic exposure to nanoplastics in goldfish (Carassius auratus): Analytical validation of automated assays for the measurement of biochemical markers

The bulk of plastic pollution is mainly composed of small fragments, micro- and nanoplastics (NPs). Although many studies are currently published on NPs, research on the effects of NPs in fish after a chronic exposure is still scarce. The present study aimed to validate a series of automated assays to be used in the monitoring of fish challenged with a chronic exposure to NPs, using Carassius auratus (goldfish) as model species. For this purpose, adult C. auratus were exposed to 100 µg/L polystyrene (PS)-NPs for a 30-day period. Total oxidative status (TOS), total antioxidant capacity (TAC), esterase activity (EA) and adenosine deaminase activity (ADA) were measured in the gills, brain and muscle of fish. In addition, acetylcholinesterase activity (AChE) and creatinine kinase (CK) were measured in the muscle. All biomarkers were successfully validated in goldfish tissues and consequently used to assess the effects of NPs following a chronic exposure. Results showed that EA and TAC significantly increased in gills, while EA decreased significantly in the brain, and no effects were observed in any of these parameters in muscle. These results indicate that both immune and antioxidant responses were triggered by NPs in gills, but not in the brain nor muscle. This suggests that gills may be a primary target for NPs, potentially leading to a cascading effect on gas exchange, or osmo- and ionic regulation that should be further investigated.

Comprehensive Transcriptome Analysis of Different Skin Colors to Evaluate Genes Related to the Production of Pigment in Celestial Goldfish.

Skin color is an important phenotypic feature of vertebrate fitness under natural conditions. Celestial goldfish, a common goldfish breed in China, mainly shows three kinds of skin colors including white, yellow and brown. However, the molecular genetic basis of this phenotype is still unclear. In this study, high-throughput sequencing was carried out on the back skin tissues of celestial goldfish with different skin colors. About 58.46 Gb of original data were generated, filtered and blasted, and 74,297 mRNAs were obtained according to the reference transcriptome. A total of 4653 differentially expressed genes were screened out among the brown, yellow and white groups, and the expression of melanogenesis related genes in brown goldfish was significantly higher than the other two groups. There are 19 common differentially expressed genes among three groups, of which eight genes are related to pigment production, including tyrp1a, slc2a11b, mlana, gch2, loc113060382, loc113079820, loc113068772 and loc113059134. RT-qPCR verified that the expression patterns of randomly selected differentially expressed transcripts were highly consistent with those obtained by RNA sequencing. GO and KEGG annotation revealed that these differentially expressed genes were mostly enriched in pathways of the production of pigment, including melanogenesis, tyrosine metabolism, Wnt signaling pathway, MAPK signaling pathway etc. These results indicated that the external characteristics of goldfish are consistent with the analysis results at transcriptome level. The results of this study will lay a foundation for further study on the expression characteristics and gene network analysis of pigment related genes.

Experimental Infection of Goldfish (Carassius auratus L.) and Tilapia (Oreochromis niloticus L.) with Koi Ranavirus (KIRV)

Background: Ranaviruses are now emerging as serious pathogens for the amphibians, reptiles and fishes. The aim of this study was the determination of the susceptibility of goldfish (Carassius auratus L.) and tilapia (Oreochromis niloticus L.) to experimental infection with Koi ranavirus (KIRV). Methods: The experimental fishes were challenged with an infective dose of KIRV (106.8 TCID50/ml) by intraperitoneal injection (IP), while the control groups were injected with L-15 medium. Target tissues were collected from the experimental fishes infected with virus at 3, 7, 14, 21 and 28 dpi. Tissue samples were subjected to detection of KIRV by polymerase chain reaction (PCR), cell culture assay and histopathological technique. Result: This study reports no specific clinical signs and mortality due to virus infection during the experimental period. Experimentally infected goldfish and tilapia were tested positive for KIRV by PCR at 3 dpi, while target viral DNA was also detected in tilapia at 7 dpi by PCR. However, specific DNA of KIRV was not detected in tissues of experimental fish sampled at later experimental study periods. Cell culture assay revealed no cytopathic effect in the Epithelioma Papulosum Cyprini (EPC) cell line at all sampling time points. Histopathological study revealed focal necrosis, shrunken glomerulus and detached epithelium of tubules in kidney tissue at 3 and 7 dpi in gold fish and melanomacrophage cells and focal necrosis of glomerulus and detachment of the epithelium of tubules in kidney tissue at 3 and 7 dpi in tilapia. Melanomacrophage centers and necrosis were observed in spleen tissues of goldfish and tilapia at 3 and 7 dpi. The challenge study indicated that goldfish and tilapia are not the targeted species for KIRV.

Nuclear Receptors (PPARs, REV-ERBs, RORs) and Clock Gene Rhythms in Goldfish (Carassius auratus) Are Differently Regulated in Hypothalamus and Liver.

The circadian system is formed by a network of oscillators located in central and peripheral tissues that are tightly linked to generate rhythms in vertebrates to adapt the organism to the cyclic environmental changes. The nuclear receptors PPARs, REV-ERBs and RORs are transcription factors controlled by the circadian system that regulate, among others, a large number of genes that control metabolic processes for which they have been proposed as key genes that link metabolism and temporal homeostasis. To date it is unclear whether these nuclear receptors show circadian expression and which zeitgebers are important for their synchronization in fish. Therefore, the objective of this study was to investigate whether the two main zeitgebers (light-dark cycle and feeding time) could affect the synchronization of central (hypothalamus) and peripheral (liver) core clocks and nuclear receptors in goldfish. To this aim, three experimental groups were established: fish under a 12 h light-12 h darkness and fed at Zeitgeber Time 2; fish with the same photoperiod but randomly fed; and fish under constant darkness and fed at Circadian Time 2. After one month, clock genes and nuclear receptors expression in hypothalamus and liver and circulating glucose were studied. Clock genes displayed daily rhythms in both tissues of goldfish if the light-dark cycle was present, with shifted-acrophases of negative and positive elements, as expected for proper functioning clocks. In darkness-maintained fish hypothalamic clock genes were fully arrhythmic while the hepatic ones were still rhythmic. Among studied nuclear receptors, in the hypothalamus only nr1d1 was rhythmic and only when the light-dark cycle was present. In the liver all nuclear receptors were rhythmic when both zeitgebers were present, but only nr1d1 when one of them was removed. Plasma glucose levels showed significant rhythms in fish maintained under random fed regimen or constant darkness, with the highest levels at 1-h postprandially in all groups. Altogether these results support that hypothalamus is mainly a light-entrained-oscillator, while the liver is a food-entrained-oscillator. Moreover, nuclear receptors are revealed as clear outputs of the circadian system acting as key elements in the timekeeping of temporal homeostasis, particularly in the liver.

TBBPA downregulates thyroid receptor and estrogen receptor mRNA levels in goldfish gonadal tissue

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant that has been shown to be a potential thyroid disrupting chemical. Currently, TBBPA is not included in the UN’s list of endocrine disruptors and adverse effects of TBBPA in mammals has not been fully investigated. However, there is clear evidence that TBBPA exerts adverse health effects on reproduction of aquatic species. Therefore, it is important to provide more information on potential endocrine disruptive effects of TBBPA in vertebrate species. In this study we investigated the effect of TBBPA on transcript levels of estrogen receptors (ERs) and thyroid receptors (TRs) in the gonadal tissue of goldfish in vivo and in vitro. ERβ mRNA levels were significantly lower in testis and ovary following exposure to TBBPA. TRα mRNA levels were also downregulated in testis tissue. Importantly, these phenotypic effects occurred at lower, environmentally relevant, concentrations in vivo. Furthermore, exposure to TBBPA also reduced ERβ and TRα mRNA abundance in goldfish testes and ovaries in vivo, which is similar to previously observed T3 responses in these tissues. These findings suggest that TBBPA may be a thyroid hormone mimic, capable of disrupting reproduction by affecting steroid hormone receptors. Our findings suggest that it is important to study TBBPA as an endocrine disruptor in aquatic organisms as it may have implications for both conservation and aquaculture.

Epigenetic and post-transcriptional repression support metabolic suppression in chronically hypoxic goldfish

Goldfish enter a hypometabolic state to survive chronic hypoxia. We recently described tissue-specific contributions of membrane lipid composition remodeling and mitochondrial function to metabolic suppression across different goldfish tissues. However, the molecular and especially epigenetic foundations of hypoxia tolerance in goldfish under metabolic suppression are not well understood. Here we show that components of the molecular oxygen-sensing machinery are robustly activated across tissues irrespective of hypoxia duration. Induction of gene expression of enzymes involved in DNA methylation turnover and microRNA biogenesis suggest a role for epigenetic transcriptional and post-transcriptional suppression of gene expression in the hypoxia-acclimated brain. Conversely, mechanistic target of rapamycin-dependent translational machinery activity is not reduced in liver and white muscle, suggesting this pathway does not contribute to lowering cellular energy expenditure. Finally, molecular evidence supports previously reported chronic hypoxia-dependent changes in membrane cholesterol, lipid metabolism and mitochondrial function via changes in transcripts involved in cholesterol biosynthesis, β-oxidation, and mitochondrial fusion in multiple tissues. Overall, this study shows that chronic hypoxia robustly induces expression of oxygen-sensing machinery across tissues, induces repressive transcriptional and post-transcriptional epigenetic marks especially in the chronic hypoxia-acclimated brain and supports a role for membrane remodeling and mitochondrial function and dynamics in promoting metabolic suppression.

A peek into mass mortality caused by antimicrobial resistant Edwardsiella tarda in goldfish, Carassius auratus in Kerala

Edwardsiella tarda is one of the serious threats affecting the worldwide aquaculture. In the present study, four isolates were recovered from diseased goldfish, showing hemorrhages, reported with 60% mass mortality in an ornamental fish farm, Ernakulam, Kerala. Based on the phenotypic and genotypic analysis, the bacteria were identified as Edwardsiella tarda, Citrobacter freundii, Acinetobacter junii and Comammonas testosteronii. Experimental challenge studies using healthy goldfish revealed that among the four isolates, E. tarda alone leads to 100% mortality of experimental fish within 175 degree days and the pathogen could be successfully re-isolated from the moribund fish. The LD50 value of E. tarda was calculated as 9.9 × 105 CFU/fish. The histopathology of the infected tissues of goldfish had shown the typical features of E .tarda infection. The pathogen was found positive for the virulence genes viz., hly, etfA, etfD and eseD as detected using PCR. Thus E. tarda was confirmed as the real causative agent of the disease outbreak. Multiple antimicrobial resistance (AMR) exhibited by the pathogen towards 19 tested antibiotics with the MAR index of 0.46 highlighted the exposure of antibiotics to the fish in the farm. The existence of antibiotic resistant genes within the plasmid as revealed through plasmid curing studies pointed out the possibility of rapid dissemination of AMR in aquaculture. Hence proper surveillance and appropriate diagnostic methods need to be implemented at regular intervals to mitigate the menace.

Establishment, characterization, and transfection potential of a new continuous fish cell line (CAM) derived from the muscle tissue of grass goldfish (Carassius auratus).

A new continuous fish cell line (CAM) has been successfully derived from the muscle tissues of grass goldfish, Carassius auratus. The primary cell cultures were initiated by incomplete trypsinization first and then explant culture in a Leibovitz-15 medium supplemented with 15% fetal bovine serum and 10% fish muscle extract. It was found that the CAM cells were very sensitive to trypsinization and needed to be sub-cultured at a low trypsin concentration of 0.0625% to be able to go through the crisis of spontaneous immortalization transformation, and afterward a total of five derivative cell strains were isolated from the original CAM cell line. This spontaneous immortalization transformation event was recorded successively at passages 44-47, beginning with a large-scale apoptosis and senescence and followed by mitosis arrest and re-activation, thus designated as cell strain CAM-44A, 44B, 45A, 44B, and 47A. Now both the CAM cell line and strains had been sub-cultured for more than 89 times and could be well cryopreserved in the growth medium containing 5% dimethylsulfoxide. Chromosome analysis and COI gene analysis had confirmed the grass goldfish origin of these CAM cells. Transfection potential analysis indicated that Lipofectamine LTX and Xfect were two suitable transfection reagents to be used in the gene delivery of CAM cells with a transfection efficiencies up to 11±6% and 8±3% in the CAM cell lines, respectively. Among the five cell strains, CAM-47A showed the highest transfection potential with a transfection efficiency up to 28 ± 5%. This work will provide a useful cell source for works on the cell-based artificial fish meat production and functional studies of fish myogenesis-related genes.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Comparative sensitivity of three new cell lines developed from gill, liver and brain tissues of goldfish, Carassius auratus (L.) to cyprinid herpesvirus-2 (CyHV-2)

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of Goldfish herpesviral haematopoietic necrosis (GHVHN) in goldfish. In this study, three new cell lines including Fantail goldfish gill (FtGG), Fantail goldfish liver (FtGL) and Fantail goldfish brain (FtGB) had been established and characterized from the gill, liver and brain tissue of C. auratus respectively. Cell lines were optimally grown at 28 °C in Leibovitz-15 (L-15) medium supplemented with 10 % fetal bovine serum (FBS). The PDT during exponential growth of FtGG, FtGL and FtGB cells were determined to be 41.47 h, 63.43 h and 79.79 h respectively. Karyotyping analysis of cell lines remained diploid (2n = 100). The revival rate was 82 %, 72 % and 70 % in FtGG, FtGL and FtGB cells respectively after 6 months of cryopreservation. All the three cells showed similar cytopathic effect (CPE) between 3–5 days post-infection (dpi) with CyHV-2 and complete destruction of the monolayer was observed at 8–10 dpi. The viral titers of CyHV-2 in FtGG, FtGL and FtGB reached 107.375±0.35 TCID50 ml−1, 104·55±0.070 TCID50 ml−1 and 106.45±0.070 TCID50 ml−1 respectively. These newly established cell lines will be a useful diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in future.

Accumulation of C-CTX1 in Muscle Tissue of Goldfish (Carassius auratus) by Dietary Experience.

Simple SummarySome marine microalgae usually present in warm waters can produce ciguatoxins (CTXs); these toxins can accumulate in fish through the trophic chain, causing the food poisoning known as ciguatera in humans. It is important to understand how these compounds could be incorporated into fish muscle. For this purpose, this study was conducted using goldfish, an omnivorous freshwater species, daily fed raw fish flesh contaminated with a known toxicity concentration of CTX, seeking the accumulation profile in muscle and any signs of intoxication. Toxicity was detectable from day eight of the toxic diet and reached its maximum after two weeks. Signs of poisoning were observed after two weeks in all treated fish. However, two individuals developed strong symptoms, and one of them was separated and fed non-toxic food for 60 days; it showed recovery signs after the first week, and no toxicity was observed at the end of that non-toxic period. These results demonstrate that this toxin can accumulate in the muscle tissue of goldfish and produce associated symptomatology. Moreover, goldfish can recover and eliminate the CTX from its muscle if the toxin source is not available.Ciguatoxins (CTXs) are produced by dinoflagellates usually present in tropical and subtropical waters. These toxins are bioaccumulated and transformed in fish causing ciguatera fish poisoning (CFP) in humans. Few trials have been performed to understand how CTXs are incorporated into fish. This study developed an experimental model of goldfish (Carassius auratus) fed flesh contaminated with Caribbean ciguatoxin (C-CTX1). Fourteen goldfish were fed 0.014 ng CTX1B (Eq. g−1 of body weight) daily, and control goldfish received non-toxic flesh. CTX presence was determined by a cell-based assay on days 1, 8, 15, 29, 36, 43, and 84. Toxicity was detected in muscle from the second sampling and then seemed to stabilize at ~0.03 ng CTX1B Eq. g−1. After two weeks, all experimental goldfish developed lethargy and loss of brightness, but only two of them displayed erratic swimming and jerking movements near the sixth sampling. One of these fish had its toxic diet replaced by commercial food for 60 more days; the fish showed recovery signs within the first weeks and no CTX activity was detected. These results indicate that C-CTX1 could accumulate in goldfish muscle tissue and produce toxic symptoms, but also remarked on the detoxification and recovery capacity of this species.

Global tissue transcriptomic analysis to improve genome annotation and unravel skin pigmentation in goldfish

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf, ednrb, mc1r, tyr, mlph and gch1, and xanthophore differentiation such as pax7, slc2a11 and slc2a15. These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

Research Article: A rapid approach to assess estrogenic transcriptional activity of bisphenol A in the liver of goldfish

Bisphenol A (BPA) is a synthetic compound widely used in types of consumer goods and medical tools. It has been shown that BPA acts as an endocrine disruptor chemical casuing negative impact on reproductive functions. The present study used an in vitro approach to assess estrogenic transcriptional activity of BPA as a rapid and sensitive method. The cultured pieces of hepatic tissue of goldfish (Carassius auratus) were exposed to 1, 5, 25 μg/L BPA and 1 µg/L E2 for comparison over 24 h. Compared to control, the mRNA transcript of erβ-I showed a significant increase in the hepatic tissues exposed to all doses of BPA and E2. The mRNA transcript of erβ-II was signifincantly increased in hepatic tissues exposed to 1 and 5 μg/L BPA and E2. The mRNA transcripts of vtg in the hepatic tissues treated with 5 μg/L BPA and E2 were also increased, significantly. Finally, cyp1a mRNA transcript showed a significant increase in the hepatic tissues exposed to 5 and 25 μg/L BPA and E2. These results show an estrogenic activity of BPA similar to E2, and suggest that in vitro approach can be used as an alternative to in vivo test to detect estrogenic effects of BPA.

Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2).

BackgroundHerpesviral 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.MethodsCaudal fin tissue samples were collected from goldfish without killing the fish. Cell culture of goldfish caudal fin cells was carried out using Leibovitz’s L-15 (L-15) medium containing 20% FBS and 1X concentration of antibiotic antimycotic solution, incubated at 28 °C. Cells were characterized and origin of the cells was confirmed by sequencing fragments of the 16S rRNA and COI genes. CyHV-2 was grown in the FtGF cells and passaged continuously 20 times. The infectivity of the CyHV-2 isolated using FtGF cells was confirmed by experimental infection of naïve goldfish.ResultsThe cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30th, 40th and 56th passage indicated that modal chromosome number was 2n = 104. Species authentication of FtGF was performed by sequencing of the 16S rRNA and COI genes. The cell line was used for continuous propagation of CyHV-2 over 20 passages with high viral titer of 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th dpi. An experimental infection of naïve goldfish using supernatant from infected FtGF cells caused 100% mortality and CyHV-2 infection in the challenged fish was confirmed by the amplification of DNA polymerase gene, histopathology and transmission electron microscopy. These findings provide confirmation that the FtGF cell line is highly permissive to the propagation of CyHV-2.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.