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      Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health

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          Abstract

          Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.

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          Most cited references87

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          The zebrafish reference genome sequence and its relationship to the human genome.

          Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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            Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

            Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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              Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

              A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                05 February 2020
                2020
                : 11
                : 114
                Affiliations
                [1] 1Cell Biology and Immunology Group, Wageningen University and Research , Wageningen, Netherlands
                [2] 2Aquaculture and Fisheries Group, Wageningen University and Research , Wageningen, Netherlands
                [3] 3Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University , Sendai, Japan
                [4] 4Microbiology, Wageningen University and Research , Wageningen, Netherlands
                [5] 5Skretting Aquaculture Research Centre , Stavanger, Norway
                Author notes

                Edited by: Julio Villena, CONICET Centro de Referencia para Lactobacilos (CERELA), Argentina

                Reviewed by: Zhigang Zhou, Feed Research Institute (CAAS), China; Xiaofei Sun, University of California, San Francisco, United States

                *Correspondence: Sylvia Brugman Sylvia.brugman@ 123456wur.nl

                This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology

                Article
                10.3389/fimmu.2020.00114
                7014991
                32117265
                0e23f4c0-b625-418f-a23b-3761ff6f9aaa
                Copyright © 2020 López Nadal, Ikeda-Ohtsubo, Sipkema, Peggs, McGurk, Forlenza, Wiegertjes and Brugman.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 15 November 2019
                : 16 January 2020
                Page count
                Figures: 2, Tables: 1, Equations: 0, References: 98, Pages: 15, Words: 11850
                Funding
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek 10.13039/501100003246
                Categories
                Immunology
                Review

                Immunology
                zebrafish,immunity,prebiotics,probiotics,microbiota,intestine,gut
                Immunology
                zebrafish, immunity, prebiotics, probiotics, microbiota, intestine, gut

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