Effects of a dietary administration of purple coneflower (Echinacea purpurea) on growth,antioxidant activities and 8 miRNAs expressions in crucian carp (Carassius auratus)

Echinacea purpurea (EP), a globally popular herbal medicine, has been used to treat various diseases in human and animals. However, little has been reported about its effects in fish. In this study, crucian carp (Carassius auratus) were selected to evaluate the effects of EP on growth performance and antioxidant response and the expressions of microRNAs. The results showed EP could stimulate the growth of crucian carp with the best effect was observed at dose of 4 g kg^?1. In serum, the content of hydroxyl radical (·OH) and malondialdehyde (MDA) decreased by EP supplementation, whereas the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) increased. Similarly, the content of ·OH decreased, and the activities of CAT, GPX and GR increased in liver of crucian carp. Furthermore, in livers of crucian carp, EP supplements upregulated the expressions of the microRNAs (miR‐16, miR‐21, miR‐125b, miR‐146a, miR‐155, miR‐181a and miR‐223), which had been confirmed to participate in regulating antioxidant and immune function in mammals. Our results suggest EP supplements in diets stimulated growth performance and antioxidant response of crucian carp. In liver, the upregulation of specific miRNAs may participate in the antioxidant effects of EP diets.     

Deficient melanin production contributes to the absence of melanophores in early development of red carp

Red carp and red crucian carp are ornamental fish with a red body color. Unlike in red crucian carp, no melanophores are observed in red carp embryos or larvae. To explore the roles of the mitfa gene in body color formation in red carp, we investigated the structural characteristics and physicochemical properties of the mitfa gene in 16 kinds of fish. The mitfa amino acid sequence similarity between red carp and red crucian carp was 95.6%, and this was 91.5% similar between carp and zebrafish. Compared with red crucian carp, red carp showed lower tyrp1 messenger ribonucleic acid (mRNA) expression but similar mitfa mRNA expression in the body pigment stage of the embryo. Moreover, mitfa⁺ cells as well as melanocytes could be observed in cultured embryo cells derived from red carp and red crucian carp. Our data show that the absence of melanophores in red carp is not the result of mitfa gene deletion or mutation, increasing our understanding of the molecular and genetic mechanisms of coloration in cyprinid fish.     

锦鲤墨蝶呤还原酶基因的克隆、表达和定位分析

为探究SPR在锦鲤体色形成中的作用,实验利用RACE技术获得spr cDNA全长序列,并分析其时空表达模式,同时利用Western blot和免疫组织化学方法检测SPR蛋白在皮肤、鳍条和鳞片中的分布和表达情况。结果显示,spr cDNA全长879 bp,包含132 bp和134 bp的5′和3′非编码区,开放阅读框510 bp,编码170个氨基酸残基。氨基酸序列比对和系统进化树分析显示,锦鲤SPR具有保守的adh_short_C2结构域,与金鱼相似性高达97.7%。spr在各组织中均有表达,其中皮肤的表达量最高。spr在锦鲤个体发育的4个阶段表现为先降后升。纯红、纯白及红白3种体色锦鲤皮肤、鳞片和鳍条中spr mRNA和蛋白表达水平基本一致,在纯红锦鲤皮肤中表达量最高,红白锦鲤白色皮肤、鳞片和鳍条的表达量最低。SPR组织定位分析显示,红色锦鲤和白色锦鲤皮肤中均检测到阳性信号,其中红色皮肤阳性信号强度高于白色皮肤。研究表明,spr可能与锦鲤红/黄色素细胞的分化和形成具有一定相关性,参与了锦鲤体色的形成。     

Differential expression of lipid metabolism‐related genes and miRNAs in Ctenopharyngodon idella liver in relation to fatty liver induced by high non‐protein energy diets

To explore the mechanism of fatty liver formation induced by high non‐protein energy diets in grass carp (Ctenopharyngodon idella), basal diet and high‐energy diets were fed to juvenile grass carp for 9 weeks. The experimental groups fed on high‐energy diets which included a high‐lipid diet (H‐LIP), a high‐carbohydrate diet (H‐CHO) and a high‐lipid and carbohydrate diet (H‐CL). The control group fed on basal diet. Growth performance, liver fat accumulation, serum biochemical indexes and the expression levels of lipid metabolism‐related genes (SREBP‐1, PPARγ, FAS, ACC1, and LPL) and miRNAs (miR‐33, miR‐122, and miR‐370) were examined at the end of the feeding trial. There were no significant differences in growth rate and feed efficiency among the four groups. However, significant increase in mesenteric and liver fat contents, and lipid droplets in the liver was induced by high‐lipid and high‐carbohydrate diets. There were significant differences in serum biochemical indicators such as AST/ALT, GLB, TG and TP, and liver fatty acid composition between the control and experimental groups. The expression levels of SREBP‐1, PPARγ, FAS, ACC1 and LPL were upregulated, while CPT‐1 was downregulated with the high‐energy treatments. Additionally, the expression levels of miR‐33, miR‐122 and miR‐370 in the liver were higher in the three high‐energy treatments than those in the control (P < 0.05). The results suggest that modifications of lipid metabolism‐related genes and miRNAs may be involved in fatty liver formation induced by high non‐protein energy diets in grass carp.     

Colouring ornamental fish (Cyprinus carpio and Carassius auratus) with microalgal biomass

Koi carp and goldfish value increases with intensity of skin colour, which is an important quality criterion. Fish cannot fully synthesize their own carotenoid colourings and these must therefore be included in their diet. Two trials were undertaken to investigate skin colour enhancement in ornamental species (i.e. three chromatic varieties of koi carp (Cyprinus carpio), namely Kawari (red), Showa (black and red) and Bekko (black and white) and goldfish (Carassius auratus)) by feeding a dietary carotenoid supplement of freshwater microalgal biomass [Chlorella vulgaris, Haematococcus pluvialis, and also the cyanobacterium Arthrospira maxima (Spirulina)], using a diet containing synthetic astaxanthin and a control diet with no colouring added for comparison. In the first trial, five homogeneous duplicate groups of 25 juvenile koi carp (C. carpio) (initial mean body weight 24.6 ± 0.7 g) were fed, for 10 weeks, one of the four diets containing 80 mg colouring/kg diet. In the second trial, this procedure was repeated for five homogeneous duplicate groups of 25 goldfish (C. auratus) (initial mean body weight of 0.9 ± 0.1 g). Initial and final samples of skin along the dorsal fin were withdrawn, from five fish per group, for subsequent analysis of total carotenoid content (spectrophotometric analysis), and red hue (colorimetric analysis, CIE (1976) L* a* b* colour system). Growth and feed efficiency were not significantly different between groups administered by the various dietary treatments. In both trials, dietary carotenoid supplementation increased total skin carotenoid content. The more efficient colouring for koi carps was found to be C. vulgaris biomass, providing both maximum total carotenoid deposition and red hue for the three chromatic koi carp varieties studied, and particularly for the kawari variety. For goldfish the best colouring obtained, as ascertained by total carotenoid content, was also achieved using C. vulgaris biomass, and red hue was maximum when using H. pluvialis biomass.     

Screening and identification of microRNAs during larval metamorphic development of Chinese mitten crab Eriocheir sinensis

MicroRNAs (miRNAs) represent a class of endogenous small non‐coding RNAs with 18–25 nucleotides that are involved in diverse biological processes. In the present study, differentially expressed miRNAs in four different metamorphic stages of Eriocheir sinensis were comprehensively detected and validated from high‐throughput sequencing miRNA data sets. We identified and characterized 597 known miRNAs and 901 novel miRNAs from the four small RNA libraries. We also demonstrated the evolutionary conservation of miRNAs from Chinese mitten crab with those from other species. Analyses of miRNA expression profiles during brachyurization development of the Chinese mitten crab revealed that 86 mature miRNAs were dysregulated, 47 of which were significantly differentially expressed. The results of real‐time quantitative PCR analyses for a few selected miRNAs were similar to those of deep sequencing analyses. Intriguingly, the results of Gene Ontology annotation and KEGG pathway analyses indicated that miRNA gene clusters and families were consistently dysregulated at four different metamorphic stages, although they were enriched at various levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways) of these miRNAs. The results suggest that these miRNAs might play potential key roles in the regulation of gene expression in the brachyurization development of the Chinese mitten crab. Particularly, miR‐2a‐3p, miR‐305‐5p, miR‐263a‐5p and miR‐7‐5p may play major regulatory roles in the metamorphosis of E. sinensis.     

Effects of dietary Bacillus natto supplementation on growth performance and the growth‐related gene/microRNA expression in the skeletal muscle of grass carp (Ctenopharyngodon idella)

In this study, Bacillus natto NT was evaluated for use as a probiotic supplement in the feeds on the growth performance and the growth‐related genes' and microRNAs' (miRNAs) expression in the skeletal muscle of grass carp (Ctenopharyngodon idella). Grass carps (ave. wt. 43.96 ± 0.27 g) were fed diets supplemented with 1.87 × 10⁹ (Bn1), 3.73 × 10⁹ (Bn2), 5.60 × 10⁹ (Bn3), 7.47 × 10⁹ (Bn4) and 9.33 × 10⁹ (Bn5) B. natto NT cells per 100 g feed for 56 days. The control group (BnC) was not supplemented with the B. natto NT. The fish of Bn3, Bn4 and Bn5 groups displayed better growth performance and lower feed conversion ratio (FCR) than the other groups (P < 0.05). Compared with the fish of BnC group, miR‐1a, miR‐181a, miR‐23a and miR‐206 expressions increased (P < 0.05), and the myostatin and myocyte enhancer factor C (MEF2C) mRNAs were down‐regulated (P < 0.05) in the fish of Bn4 and Bn5 groups. The specific growth rate analysis and apparent expressional regulation of the growth‐related miRNAs and genes stimulated by B. natto suggest the potential application of B. natto in improving the growth performance on the grass carps.     

Antiviral activity of exopolysaccharides from Arthrospira platensis against koi herpesvirus

Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra‐ and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT‐PCR that EPS, applied at a concentration of >18 μg mL^?1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post‐infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.     

Alpinia galanga oil—A new natural source of fish anaesthetic

This study explores the anaesthetic activity of Alpinia galanga oil (AGO) in fish. Cyprinus carpio (koi carp) was used as a fish model. It was found that the induction time to stage 3 anaesthesia and the recovery time of the fish after exposure to AGO were exponentially and polynomially correlated to AGO concentrations. The viability of normal blood cells of koi carp anaesthetized with 500 mg/L AGO was found to be higher than 90% for normal red blood cells and white blood cells and 89% for peripheral blood nuclear cells indicating nontoxicity of AGO to the fish. A concentration of 300 mg/L of AGO was the most suitable for anaesthetizing koi carp due to the safety and effectiveness aspects as being ideally fitted to anaesthetic criteria. This concentration gave the induction time of 205.55 ± 5.07 s and the recovery time of 202.50 ± 9.30 s. Determination of stress biomarker such as blood cortisol and glucose as well as gene expression showed that the blood cortisol level of the fish anaesthetized with AGO was similar to normal levels. Moreover, blood glucose level was significantly less increased than those anaesthetized with tricaine methanesulfonate. Gene expressions of the fish cortisol receptor, cytochrome oxidase subunit1, heat shock protein 70 and Na⁺/K⁺‐ATPaseα₃ were significantly reduced after exposure to AGO indicating the advantages of AGO on fish stress reduction. Thus, AGO is a promising natural source for an alternative fish anaesthetics.     

Antibiotic resistance and repetitive‐element PCR fingerprinting in Aeromonas veronii isolates

This study evaluated antibiotic resistance and the related genes in total 47 Aeromonas veronii isolates from pet fish, eel (Anguilla japonica) and koi (Cyprinus carpio) in Korea. In comparison with the antibiotic susceptibilities of isolates from eel and koi, those of pet fish were more resistant to ceftiofur, aminoglycosides, tetracycline and nitrofurantoin. And isolates from pet fish showed high prevalences of class 1 integron, quinolones and tetracycline resistance determinants than those from eel and koi. Repetitive‐element palindromic PCR (rep‐PCR) showed larger diversities among A. veronii isolates. Collectively, pet fish may be a reservoir for multiple clones of A. veronii involved in antibiotic resistance. In this aspect, imported fish in the aquaculture trade should be steadily and continually screened for bacterial antibiotic resistance and related genes.     

Effects of sodium butyrate stimulation on immune‐related mRNA‐miRNA network in intestine of grass carp

Sodium butyrate is one of the most popular feed additives in animal husbandry. In recent years, sodium butyrate has been increasingly used as supplement in aquaculture. The present study is to investigate the intestinal mRNA and microRNA response to diet with sodium butyrate in grass carp (Ctenopharyngodon idella), an important aquaculture species in China. mRNA and microRNA profiles of intestine of grass carp fed with diet contained 0, 1.0, 2.5, 5.0, 7.5 and 10.0 g/kg sodium butyrate were obtained by RNA‐seq using Illumina Hiseq 2,500 platform. The feeding trial was performed using 18 individuals of 1‐year‐old grass carp (n = 3 for each group) and lasted for 40 days in tanks in laboratory. A total of 349,860,852 sequence reads were generated from six intestinal libraries. Functional analysis of differentially expressed genes showed that genes participated in immune pathways tend to be activated by sodium butyrate supplementation. A total of 700 microRNAs were obtained, including 275 conserved microRNAs and 425 novel microRNAs which are potentially involved in regulating 14,300 genes. Spearman's correlation analysis identified 18 pairs of microRNA‐mRNA associated with immune pathways (p < .01 and R<?0.5). The potential genes targeted by microRNAs include CXCL12, AKT1S1, Cab39 and MHCII which are important genes associated with intestinal immune pathways. To our knowledge, this is the first integrated profiling of both mRNA and microRNA in intestine with supplementation of sodium butyrate in grass carp. The present results suggest that sodium butyrate affects intestinal immune system by regulating microRNA‐mRNA interaction in fish.     

Susceptibility of koi × crucian carp and koi × goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD)

Hybrids of koi, Cyprinus carpio × crucian carp, Carassius carassius and koi × goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV‐3) and developed KHV disease (KHVD). While hybrids of koi × goldfish were partly resistant to mortality following infection by immersion, most koi × crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi × goldfish and koi × crucian carp hybrids as well as in SPF carp.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Potential vector species of carp edema virus (CEV)

During a PCR‐based CEV survey in Poland in 2015–2017, the virus was detected in many farms both in clinical and asymptomatic cases and in common as well as in koi carp (Cyprinus carpio). In order to evaluate the potential carrier role of fish species that share the same habitats with carp, an experimental trial was performed. Investigations carried out on specimens of bleak (Alburnus alburnus), crucian carp (Carassius carassius), European perch (Perca fluviatilis), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) cohabited with CEV‐infected carp yielded positive results. These species of fish were experimentally cohabited with CEV‐infected common carp at a temperature of 16°C ± 1. Material from the brain, gills, spleen, kidneys, intestine and skin was investigated for the presence of CEV DNA. Similar investigations were performed with uninfected fish designated controls. Samples were tested for CEV by qPCR.     

Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

Digestibility,growth and pigmentation of astaxanthin,canthaxanthin or lutein diets in Lake Kurumoi rainbowfish,Melanotaenia parva (Allen) cultured species

New cultured ornamental fish namely Lake Kurumoi rainbowfish Melanotaenia parva (Allen) run into reduced of colour performances when reared in the aquaria, consequently, fish feed must be added with carotenoids as a pigment source. The aim of this study was to evaluate the digestibility, growth and pigmentation of astaxanthin, canthaxanthin and lutein in diet. Apparent digestibility coefficients (ADC) of dry matter, lipid, protein, carotenoids, growth and pigmentation were studied in twenty fish after 14 and 56 days of observation. The single‐dose supplementation of 100 mg/kg of astaxanthin, canthaxanthin, or lutein diets on fish was fed by apparent satiation. The basal diet without carotenoids was used as control. The result showed that the ADC of carotenoids of test diets was higher compared to control. Fish fed astaxanthin diet had higher survival rate (96.67 ± 2.89%), colour measurements of lightness (57.60 ± 7.46%), a*‐values (4.66 ± 1.20), total carotenoids content in skin (33.75 ± 5.02 mg/kg) and muscle (2.16 ± 0.74 mg/kg). Astaxanthin also increased the growth after 14 days (2.00% ± 0.19%/days) but there was no significantly different at the end of experiment. The yellowish‐orange colour performance was more rapidly achieved by fish fed astaxanthin diet after 28 days experimentation. These values suggested that dietary carotenoids were required and astaxanthin diet was superior to other diets for skin pigmentation of Lake Kurumoi rainbowfish.     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

Comparison of food selection and growth performance of koi carp, Cyprinus carpio L., and goldfish, Carassius auratus (L.) in mono- and polyculture rearing in tropical ponds

To compare the effect of polyculture against conventional monoculture on ornamental carp production, investigations on food selection and growth performance of koi carp (K), Cyprinus carpio L. and goldfish (G), Carassius auratus (L.) were conducted in a 11‐week rearing experiment in two monoculture (100% K and 100% G) and five polyculture (90% K–10% G, 70% K–30% G, 50% K–50% G, 30% K–70% G and 10% K–90% G) conditions in tropical ponds. There were three replicates for each treatment. Environmental conditions and food availability were similar in all the treatments. Ivlev's electivity index showed that both fish species avoided phytoplankton and preferred cladocerans to other zooplankton groups (copepods and rotifers) in monotypic conditions. However, in the polyculture treatments, the positive electivity of goldfish towards cladocerans reduced significantly (P<0.05), while the percentage of copepods, rotifers and phytoplankton in the gut content increased. No significant differences in weight gain, specific growth rate and deformities were recorded at harvest for koi carp between the different treatments (P>0.05). Even the survival rate of koi carp recorded above 90% in all the treatments. However, the goldfish recorded significantly better weight gain, specific growth rate and survival in monoculture (100% G), compared with the polyculture treatments (P<0.05). Goldfish deformities were lowest (P<0.05) in the monoculture treatment (2.42%). The number of marketable fish above a set size limit of 4 g total weight was significantly higher in the two monoculture treatments, compared with the five polyculture treatments (P<0.05). Keeping in view of the dietary similarities of koi carp and goldfish, and the aggressive nature of koi carp in polyculture, it is suggested to refrain from polyculture of goldfish and koi carp until further documentations relating to optimum stocking density and management of polyculture of ornamental carps are available.