Auratus Gibelio Research Articles

Efficient showering vaccination with a live attenuated vaccine against herpesviral hematopoietic necrosis in goldfish

Herpesviral hematopoietic necrosis caused by cyprinid herpesvirus 2 (CyHV-2) frequently affects farmed goldfish Carassius auratus and gibel carp C. auratus gibelio, leading to significant economic damage. In our previous study, a live attenuated vaccine, called the P7-P8 vaccine strain, was developed by propagating virulent CyHV-2 in two non-natural host cell lines. The vaccine demonstrated high protective efficacy in goldfish with no signs of virulence reversion. Methods that reduce the costs of the procedure, including labor costs, are warranted for the practical application of this vaccine in goldfish aquaculture. The aim of this study was to explore alternative vaccination protocols for the P7-P8 strain in goldfish. First, we tested the previous standard administration protocol which is the bath vaccination with 1: 1000 diluted vaccine cultures for 2 h. In the simple first step to reduce vaccine virus culture, we examined the bathing protocol with more-diluted virus culture (ranging from 1: 1000 to 1: 81,000). The relative percentage survival (RPS) after the virulent virus immersion challenge for 2 h at 21 days post-vaccination was remarkably decreased in a dose-dependent manner; a dilution of 1: 3000 resulted in a 19% reduction from that used in the standard bathing protocol. Therefore, alternative methods (dipping and showering vaccination protocols) were tested using a 1: 100 dilution vaccine culture (10-fold more concentrated than the standard protocol) with short exposure times. Dipping vaccination for 10 s, 1 min, 2 min, and 5 min showed sufficient RPS of 82.4%–100%. The 10 s process was repeated six times using the same vaccine solution, resulting in an RPS >89%. In the showering vaccination protocol, the vaccine solution (1: 100 dilution; 1 mL, 3 mL, or 5 mL) was showered onto 20 goldfish on a net and held for 10 s, resulting in an RPS of >89%. We selected the showering vaccination as the suitable vaccination protocol. The effectiveness of this vaccination protocol was tested using the virus challenge by employing the cohabitation method with infected fish. The results showed high protective efficacy with an RPS of 65%–100%. This study demonstrated that the showering vaccination protocol, which uses small amounts of vaccine culture and produces less waste vaccine solution with short labor time, may be considered as a suitable alternative to the standard bath method in goldfish aquaculture.

Assessment of aquatic genotoxicity of the Lake Sevan basin, Armenia using natural bioindicators

Water pollution can cause genomic instability, thereby threatening aquatic wildlife and human health․ For a correct assessment of the impact of the mixture of pollutants, chemical analysis of environmental contaminants should be simultaneously applied with genotoxicity assays. The сomet assay is a sensitive, versatile and extensively used method for genotoxicity assessment in a water environment and was successfully applied in aquatic wild species, including fish and crayfish. Earlier we investigated the genotoxicity of waters of the Lake Sevan basin (Simonyan et al. 2016, Simonyan et al. 2019)․ Lake Sevan is the largest lake of the Caucasus Region, situated in the Republic of Armenia, in the Gegharkunik Province. Water pollution in the lake is a result of both anthropogenic and natural pressures. A combination of comet assay (Tice et al. 2000) and chemical analysis was applied for evaluation of water genotoxicity using gibel carp Carassius auratus gibelio and crayfish Astacus leptodactylus as sentinel organisms. Fish and water samples were collected from the south–southwest of the village Shorzha (40028′33″N and 45014′22″E), Peninsula of Lake Sevan (40033′46″N and 45001′37″E), the estuaries of the Rivers Gavaraget (40025′12″N and 45009′53″E) and Dzknaget (40036′55″N and 44058′13″E). Сrayfish and water samples were collected from the estuary of the River Masrik (40013′25″N and 45038′21″E) and near the villages of Artanish (40027′19″N and 45025′12″E) and Tsapatakh (40024′34″N and 45028′22″E). According to the chemical analysis of waters, the south–southwest of the village Shorzha and the estuary of the River Masrik were chosen as reference sites․ The content of contaminants in water was analyzed according to the standard methods in the Environmental Impact Monitoring Center, Ministry of Environment of the Republic of Armenia. The obtained results showed that the levels of DNA damage were significantly higher in both species in polluted areas than in reference sites. A significant positive correlation between DNA damage in C. auratus gibelio and contents of Al, Fe, Cu and Mn in water was shown. DNA damage in A. leptodactylus correlated with Al, Fe, Cu and Mo. C. auratus gibelio and A. leptodactylus were found to be sensitive mostly to the same pollutants, which indicate that these metals may be of primary concern as contaminants of the studied aquatic environment. Thus, the current study indicates that C. auratus gibelio and A. leptodactylus are sensitive bioindicators for monitoring of water pollution in the Lake Sevan basin.

Cyprinid herpesvirus‐2 (CyHV‐2): a comprehensive review

AbstractCyprinid herpesvirus‐2 (CyHV‐2) is a linear double‐stranded DNA virus in the genus Cyprinivirus of family Alloherpesviridae. The virus is known to be highly pathogenic to ornamental goldfish (Carassius auratus), crucian carp (C. carassius) and Gibel carp (C. auratus gibelio), and also to the hybrids of goldfish and other carps. Cyprinid herpesvirus‐2, having the smallest genome (290.3 kb) among Cyprinivirus, causes herpesviral hematopoietic necrosis disease (HVHND) that results in huge economic losses in aquaculture industry as the disease can cause high mortality (50–100%) among the affected fish. The disease was initially reported as the cause of epizootics in juvenile goldfish of Japan during 1992 and 1993. To date, this disease has been reported around the world including Europe, North America, Oceania and Asia. Huge economic losses due to the CyHV‐2 infection among cultured gibel carp in China, during 2011–2012, mass mortality in crucian carp during 2012 in Italy, 95% mortality in goldfish during 2014 in France, 85% mortality in goldfish during 2016 in Poland had been reported. Strategies for controlling the spread of CyHV‐2 are thus urgently required to limit economic damage. Furthermore, the review will shed light on lacunae in current knowledge as well as on the perspectives that merits further investigations on CyHV‐2 research. The paper forms the first comprehensive overview of CyHV‐2 causing a serious economically significant fish disease and, will be helpful for the researchers to get all related information from a single manuscript.

Acute Exposure to key Aquaculture Environmental Stressors Impaired the Aerobic Metabolism of Carassius auratus gibelio.

Carassius auratus gibelio is an omnivore favored for its flavor and is commonly used as a benthic species in traditional pond polyculture. This study investigated the effects of common aquaculture stressors, such as high ammonia, high nitrite, high pH, and hypoxia on the aerobic metabolism of C. auratus gibelio. The results showed that the standard metabolic rate (SMR) was positively correlated with ammonia, nitrite, and pH, while the maximum metabolic rate (MMR) was negatively correlated with all four stressors. Thus, aerobic scope (AS) was reduced when C. auratus gibelio was exposed to high ammonia, high nitrite, high pH, and hypoxia. The peak of post-prandial O2 consumption was positively correlated with nitrite, pH, and the occurrence of the peak metabolic rate post-prandial was delayed in high ammonia, high nitrite, hypoxia, and high pH conditions. These findings indicated that, in experimental conditions, exposure to these environmental stressors can influence aerobic metabolism in C. auratus gibelio. With more energy required to maintain standard metabolic rates, less will be available for growth. While the C. auratus gibelio is one of the most hypoxia tolerance species, the reduction we observed in AS caused by stressors that commonly occur in ponds and in nature will likely affect growth in ponds and fitness in nature. These data have provided insight into the optimal, fitness-maximizing thresholds for these common stressors in this species of interest.

MicroRNA expression profiling of caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection

As a member of the genus Cyprinivirus in the family Alloherpesviridae, Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss in the aquaculture industry, mainly in C. auratus gibelio and goldfish. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 remain elusive. In this study, high-throughput sequencing technology was employed to explore the miRNA expression profiles of C. auratus gibelio (GiCF) caudal fin cells in response to Cyprinid Herpesvirus-2 (CyHV-2) infection. A total of 631 novel miRNAs and 409 known miRNAs were identified. The expression levels of 7 miRNAs were found as significantly modulated (5 down-regulation and 2 up-regulation; P < 0.01, |logFC|>1, TPM>10) in CyHV-2 infected cells. 7 miRNA and their potential mRNA targets were validated by Real-time PCR (qRT-PCR), respectively. Targets prediction and functional analysis of these 7 miRNAs revealed significant enrichment for several signaling pathways, including PPAR, p53 and FoxO pathways. These studies provided more valuable basis for further study on the roles of miRNAs in CyHV-2 replication and pathogenesis.

Recombinant baculovirus BacCarassius-D4ORFs has potential as a live vector vaccine against CyHV-2

Cyprinid herpesvirus II (CyHV-2) is highly contagious and pathogenic to Carassius auratus gibelio (gibel carp), causing enormous economic losses in aquaculture in Yancheng city, Jiangsu province, China; however, to date, there is no effective way to protect C. auratus gibelio from CyHV-2 infection. In this study, a recombinant baculovirus vector vaccine, BacCarassius-D4ORFs, containing a fused codon-optimized sequence D4ORFs comprising the ORF72 (region 1–186 nt), ORF66 (region 993–1197 nt), ORF81 (region 603–783 nt) and ORF82 (region 85–186 nt) genes of CyHV-2, driven by a Megalobrama amblycephala β-actin promoter, was constructed. Then, qPCR, Western blotting and immunofluorescence assays showed that the fused gene D4ORFs was successfully delivered and expressed in fish cells or tissues by transduction with BacCarassius-D4ORFs. The fused gene D4ORFs could not be detected by PCR in the C. auratus gibelio injected with BacCarassius-D4ORFs after 7 weeks. Specific antibody against ORF72 could be detected in the serum of vaccinated C. auratus gibelio by injection with BacCarassius-D4ORFs. Furthermore, when C. auratus gibelio were vaccinated with BacCarassius-D4ORFs via the oral or injection route, followed by challenge with CyHV-2, the relative survival rate of immunized C. auratus gibelio reached 59.3% and 80.01%, respectively. These results suggested that BacCarassius-D4ORFs has the potential to be used as a vector-based vaccine for the prevention and treatment of disease caused by CyHV-2 infection.

DNA barcoding and species and subspecies classification within genus &lt;I&gt;Carassius&lt;/I&gt;

The classification of Carassius has not been well established due to its great variability and wide distribution. Usually, Carassius is identified as three species: C. carassius, C. cuvieri and C. auratus, the latter including several subspecies, such as goldfish. Out of these subspecies, C. auratus gibelio have recently been thought of as a valid species of Carassius. In this study we collected the 5'end 651 bp segments of the mitochondrial cytochrome c oxidase I (COI) gene from 128 specimens, including C. carassius, C. cuvieri, C. auratus auratus, C. auratus gibelio and C. auratus langsdorfii. All three species of Carassius (C. carassius, C. cuvieri, C. auratus) were found to be valid, meanwhile genetic differentiation between the Eurasian C. auratus and Japanese C. auratus has reached a high level. However, several haplotypes were shared between C. auratus auratus and C. auratus gibelio. Consequently, C. auratus gibelio should be regarded as a subspecies of C. auratus rather than a valid species. Moreover, because both diploids and triploids exist in C. auratus auratus and C. auratus gibelio, ploidy level should not be used as criteria for the classification of species or subspecies in Carassius.

Speciation of polyploid Cyprinidae fish of common carp, crucian carp, and silver crucian carp derived from duplicated Hox genes

Recent studies on comparative genomics have suggested that a round of fish-specific whole genome duplication (3R) in ray-finned fishes might have occurred around 226-316 Mya. Additional genome duplication, specifically in cyprinids, may have occurred more recently after the divergence of the teleosts. The timing of this event, however, is unknown. To address this question, we sequenced four Hox genes from taxa representing the polyploid Cyprinidae fish, common carp (Cyprinus carpio, 2n=100), crucian carp (Carassius auratus auratus, 2n=100), and silver crucian carp (C. auratus gibelio, 2n=156), and then compared them with known sequences from the diploid Cyprinidae fish, blunt snout bream (Megalobrama amblycephala, 2n=48). Our results showed the presence of two distinct Hox duplicates in the genomes of common and crucian carp. Three distinct Hox sequences, one of them orthologous to a Hox gene in common carp and the other two orthologous to a Hox gene in crucian carp, were isolated in silver crucian carp, indicating a possible hybrid origin of silver crucian carp from crucian and common carp. The gene duplication resulting in the origin of the common ancestor of common and crucian carp likely occurred around 10.9-13.2 Mya. The speciations of common vs. crucian carp and silver crucian vs. crucian carp likely occurred around 8.1-11.4 and 2.3-3.0 Mya, respectively. Finally, nonfunctionalization resulting from point mutations in the coding region is a probable fate for some Hox duplicates. Taken together, these results suggested an evolutionary model for polyploidization in speciation and diversification of polyploid fish.

Utilization of tissue habitats by Myxobolus wulii Landsberg &amp; Lom, 1991 in different carp hosts and disease resistance in allogynogenetic gibel carp: redescription of M. wulii from China and Japan

Myxobolus wulii (=Myxosoma magna) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio, was designated as a different species, Myxobolus guanqiaoensis, although the morphological features were almost identical to those of M. wulii. In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii, which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus. Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii. A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.

Inheritance of microsatellite DNA in bisexual gynogenesis complex of Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch)

Five gynogenetic progeny groups of silver crucian carp Carassius auratus gibelio were produced and sex ratios (males:total progeny) of each of the progeny groups were analysed. About 110 males and 366 females were genotyped at 15 microsatellite loci for comparison with their parents to (1) verify the gynogenesis status of Fangzheng C. auratus gibelio, (2) detect the incorporation of paternal genetic material into the offspring and (3) study the possible association of genetic exchange at microsatellite loci with the existence of sex. The sex ratios in progenies of five groups were highly variable, but all had significant female bias. The sex ratio ranged from 0 to 0·37. Significant differences in the sex ratio within and between groups were also found. Microsatellite genotyping at 15 loci showed that 100 and 97% of the progeny shared the same genotype with the mother in four groups and in one group, respectively, confirming that gynogenesis is the general mechanism of reproduction in C. auratus gibelio. However, 0·63% of all offspring did show incorporation of paternal genetic material. No single loci tested were associated with the occurrence of male progeny, indicating unknown genetic mechanisms for sex determination in C. auratus gibelio.

Genetic diversity in crucian carp (Carassius auratus).

A survey of restriction fragment polymorphism in mitochondrial DNA of three subspecies of Carassius auratus throughout four provinces in China was undertaken using 17 restriction enzymes. Two carp, Cyprinus carpio rubbrofuscus and Cyprinus carpio carpio, were included as the outgroup. A total of 16 haplotypes was observed: 5 in tetraploids of C. auratus auratus; 8 in hexaploids of C. auratus auratus; and 2 in C. auratus gibelio and C. auratus cuvieri, respectively. The tetraploids and hexaploids share three common haplotypes as I, V, and VI. C. a. Cuvieri may have diverged first among the three subspecies. Interestingly, C. a. auratus and C. a. cuvieri did not form monophyletic clades, which indicated that the classification of Carassius auratus required further studies. The current hypothesis, that hexaploids originated from tetraploids by a polyploidy event, is less favorable, based on the distribution of haplotypes and the lower diversity in tetraploids than in hexaploids. Our data also indicate that divergence of hexaploids and tetraploids might be recent and mtDNA polymorphism existed before the divergence. Meanwhile, genetic isolation exists between the hexaploids and the tetraploids.

Hoferellosis in goldfish Carassius auratus and gibel carp Carassius auratus gidelio

Hoferellus carassii is a common parasite of goldfish Carassius auratus and gibel carp C. auratus gibelio. Early trophozoite stages of H. carassii were found intracellularly, in epithelial cells of renal tubules. Plasmodial stages, however, had a coelozoic way of life, inhabiting the lumen of renal tubules, ureters and preferentially the urinary bladder. Spores were produced mostly in the latter site. No strict seasonality in development was established. In general, early stages were found during summer and autumn months, while spores occurred primarily from autumn to spring. In natural waters H. carassii infection of gibel carp passed without symptoms and only minor degenerations were recorded in kidney and urinary ducts. In goldfish from a pet fishpond, however, hoferellosis induced kidney enlargement disease, severe cystic transformation of tubules, and degeneration of the renal interstitium. Renal damage was caused by trophozoites invading the epithelial cells of renal tubules.