Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance

Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection

Disease resistance and infectivity of virus susceptible and resistant common carp strains

Abstract Infectious diseases challenge health and welfare of humans and animals. Unlike for humans, breeding of genetically resistant animals is a sustainable solution, also providing unique research opportunities. Chances to survive a disease are improved by disease resistance, but depend also on chances to get infected and infect others. Considerable knowledge exists on chances of susceptible and resistant animals to survive a disease, yet, almost none on their infectivity and if and how resistance and infectivity correlate. Common carp (Cyprinus carpio) is widely produced in aquaculture, suffering significantly from a disease caused by cyprinid herpes virus type 3 (CyHV-3). Here, the infectivity of disease-resistant and susceptible fish types was tested by playing roles of shedders (infecting) and cohabitants (infected) in all four type-role combinations. Resistant shedders restricted spleen viral load and survived more than susceptible ones. However, mortality of susceptible cohabitants infected by resistant shedders was lower than that of resistant cohabitants infected by susceptible shedders. Virus levels in water were lower in tanks with resistant shedders leading to lower spleen viral loads in cohabitants. Thus, we empirically demonstrated that disease resistant fish survive better and infect less, with implications to epidemiology in general and to the benefit of aquaculture production

Revisiting the species list of freshwater fish in Israel based on DNA barcoding

Abstract Israel's region forms a continental bridge; hence, the freshwater fish fauna in Israel consists of unique populations of species that originated from Africa, Asia, or Europe and are often endemic or at the edge of their distribution range. Worldwide, fish biodiversity suffers significantly from pressures and disturbances of freshwater habitats, especially in arid regions, such as in parts of Israel. Biodiversity conservation requires efficient tools for monitoring changes in populations. DNA barcoding, by complementing and enhancing species identification, provides such monitoring tools. In this study, over 200 specimens representing over 28 species were DNA barcoded and together with previously available records, a DNA barcoding database for freshwater fish of Israel was established. Of the 71 distinct barcodes generated, 37% were new, attesting to the uniqueness of fish populations in Israel. For most species, morphological and molecular species identifications agreed. However, discrepancies were found for five genera. Based on DNA barcoding, we propose Acanthobrama telavivensis as a junior synonym for Acanthobrama lissneri. In Garra spp., we propose splitting Garra nana into two species and assigning Garra rufa in the region to Garra jordanica, or possibly to two species. Israeli Pseudophoxinus kervillei is not the same species as in Syria and Lebanon. However, Pseudophoxinus syriacus might not be endangered since it is genetically very similar to Pseudophoxinus drusensis. In Israel, instead of five reported Oxynoemacheilus species, combining DNA barcoding with morphology suggests only three. Genetic and geographic separation suggested that Aphanius mento is likely a species complex. The study provides a thorough barcoding database, suggests significant species reconsiderations in the region, and highlights the Sea of Galilee and the Beit She'an valley streams as biodiversity “hotspots.” This study will therefore promote further studying of the fish species in the region and their ecology, as well as the monitoring and conservation of freshwater fish biodiversity in Israel and the region

Establishing and using a genetic database for resolving identification of fish species in the Sea of Galilee, Israel.

Freshwaters are a very valuable resource in arid areas, such as Mediterranean countries. Freshwater systems are vulnerable ecological habitats, significantly disturbed globally and especially in arid areas. The Sea of Galilee is the largest surface freshwater body in the Middle East. It is an isolated habitat supporting unique fish populations, including endemic species and populations on the edge of their distribution range. Using the Sea of Galilee for water supply, fishing and recreation has been placing pressure on these fish populations. Therefore, efficient monitoring and effective actions can make a difference in the conservation of these unique fish populations. To set a baseline and develop molecular tools to do so, in this study, DNA barcoding was used to establish a database of molecular species identification based on sequences of Cytochrome C Oxidase subunit I gene. DNA barcodes for 22 species were obtained and deposited in Barcode of Life Database. Among these, 12 barcodes for 10 species were new to the database and different from those already there. Barcode sequences were queried against the database and similar barcodes from the same and closely related species were obtained. Disagreements between morphological and molecular species identification were identified for five species, which were further studied by phylogenetic and genetic distances analyses. These analyses suggested the Sea of Galilee contained hybrid fish of some species and other species for which the species definition should be reconsidered. Notably, the cyprinid fish defined as Garra rufa, should be considered as Garra jordanica. Taken together, along with data supporting reconsideration of species definition, this study sets the basis for further using molecular tools for monitoring fish populations, understanding their ecology, and effectively managing their conservation in this unique and important habitat and in the region

Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.)

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed

RNA sequencing of susceptible and resistant common carp before and after infection with CyHV-3

Infectious disease outbreaks in aquaculture form major setbacks to fish production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely affecting production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought for a sustainable solution to this problem. To study the genetic basis and cellular pathways underlining CyHV-3 resistance in common carp, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. Understanding resistance mechanisms is important both for development of both vaccines and resistant strains and lays the foundation for future studies in this field

Data_Sheet_1_A method for quick and efficient identification of cichlid species by high resolution DNA melting analysis of minibarcodes.FASTA

Freshwater bodies are key in supporting aquatic and terrestrial life. Ecological balance of freshwater habitats is very vulnerable, hence, often significantly disrupted by climatic changes and anthropogenic acts. In Israel, due to its relatively arid climate, many freshwater resources have been disrupted and still are under great pressure. The Sea of Galilee is the largest surface freshwater body in the Middle East and a habitat to unique populations of several fishes, including six cichlid species. Studies on the ecology of these fish and their conservation require effective monitoring tools. In this study, a simple and efficient molecular method was developed to identify the species of these lake cichlids using high resolution melting analysis of mini DNA barcodes. The species of an individual sample can be identified by a single tube PCR reaction. This assay successfully identified sequence differences both among and within species. Here, this method identified the species for 279 small cichlid fry that could not be morphologically identified, allowing to estimate relative species abundance and map their distribution in time and location. The results are key to understand not only the ecology of young stages but also their recruitment potential to adult fish populations and their sustainability. This method can be readily implemented in further ecological studies and surveys related to these species, in the lake and its surroundings, as a tool to enhance understanding and protection of these species.</p

Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance

Background Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. Results In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar. Conclusions Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture

Symmetric expression of ohnologs encoding conserved antiviral responses in tetraploid common carp suggest absence of subgenome dominance after whole genome duplication

International audienceAllopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp