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      Ontogenetic Characterization of the Intestinal Microbiota of Channel Catfish through 16S rRNA Gene Sequencing Reveals Insights on Temporal Shifts and the Influence of Environmental Microbes

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          Abstract

          Aquaculture recently overtook capture fisheries as the largest producer of food fish, but to continue increasing fish production the industry is in search of better methods of improving fish health and growth. Pre- and probiotic supplementation has gained attention as a means of solving these issues, however, for such approaches to be successful, we must first gain a more holistic understanding of the factors influencing the microbial communities present in the intestines of fish. In this study, we characterize the bacterial communities associated with the digestive tract of a highly valuable U.S. aquaculture species, channel catfish Ictalurus punctatus, over the first 193 days of life to evaluate temporal changes that may occur throughout ontogenetic development of the host. Intestinal microbiota were surveyed with high-throughput DNA sequencing of 16S rRNA V4 gene amplicons derived from fish at 3, 65, 125, and 193 days post hatch (dph), while also characterizing the environmental microbes derived from the water supply and the administered diets. Microbial communities inhabiting the intestines of catfish early in life were dynamic, with significant shifts occurring up to 125 dph when the microbiota somewhat stabilized, as shifts were less apparent between 125 to 193 dph. Bacterial phyla present in the gut of catfish throughout ontogeny include Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria; with the species Cetobacterium somerae and Plesiomonas shigelloides showing the highest abundance in the catfish microbiota after 3 dph. Comparisons of the gut microbiota to the environmental microbes reveals that the fish gut is maintained as a niche habitat, separate from the overall microbial communities present in diets and water-supply. Although, there is also evidence that the environmental microbiota serves as an inoculum to the fish gut. Our results have implications for future research related to channel catfish biology and culture, and increase our understanding of ontogenetic effects on the microbiota of teleost fish.

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          Most cited references33

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          Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis.

          Symbiotic bacteria often help their hosts acquire nutrients from their diet, showing trends of co-evolution and independent acquisition by hosts from the same trophic levels. While these trends hint at important roles for biotic factors, the effects of the abiotic environment on symbiotic community composition remain comparably understudied. In this investigation, we examined the influence of abiotic and biotic factors on the gut bacterial communities of fish from different taxa, trophic levels and habitats. Phylogenetic and statistical analyses of 25 16S rRNA libraries revealed that salinity, trophic level and possibly host phylogeny shape the composition of fish gut bacteria. When analysed alongside bacterial communities from other environments, fish gut communities typically clustered with gut communities from mammals and insects. Similar consideration of individual phylotypes (vs. communities) revealed evolutionary ties between fish gut microbes and symbionts of animals, as many of the bacteria from the guts of herbivorous fish were closely related to those from mammals. Our results indicate that fish harbour more specialized gut communities than previously recognized. They also highlight a trend of convergent acquisition of similar bacterial communities by fish and mammals, raising the possibility that fish were the first to evolve symbioses resembling those found among extant gut fermenting mammals. © 2012 Blackwell Publishing Ltd.
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            Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection.

            The gut microbiotas of zebrafish and mice share six bacterial divisions, although the specific bacteria within these divisions differ. To test how factors specific to host gut habitat shape microbial community structure, we performed reciprocal transplantations of these microbiotas into germ-free zebrafish and mouse recipients. The results reveal that communities are assembled in predictable ways. The transplanted community resembles its community of origin in terms of the lineages present, but the relative abundance of the lineages changes to resemble the normal gut microbial community composition of the recipient host. Thus, differences in community structure between zebrafish and mice arise in part from distinct selective pressures imposed within the gut habitat of each host. Nonetheless, vertebrate responses to microbial colonization of the gut are ancient: Functional genomic studies disclosed shared host responses to their compositionally distinct microbial communities and distinct microbial species that elicit conserved responses.
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              Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota.

              Animals have developed the means for supporting complex and dynamic consortia of microorganisms during their life cycle. A transcendent view of vertebrate biology therefore requires an understanding of the contributions of these indigenous microbial communities to host development and adult physiology. These contributions are most obvious in the gut, where studies of gnotobiotic mice have disclosed that the microbiota affects a wide range of biological processes, including nutrient processing and absorption, development of the mucosal immune system, angiogenesis, and epithelial renewal. The zebrafish (Danio rerio) provides an opportunity to investigate the molecular mechanisms underlying these interactions through genetic and chemical screens that take advantage of its transparency during larval and juvenile stages. Therefore, we developed methods for producing and rearing germ-free zebrafish through late juvenile stages. DNA microarray comparisons of gene expression in the digestive tracts of 6 days post fertilization germ-free, conventionalized, and conventionally raised zebrafish revealed 212 genes regulated by the microbiota, and 59 responses that are conserved in the mouse intestine, including those involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune responses. The microbial ecology of the digestive tracts of conventionally raised and conventionalized zebrafish was characterized by sequencing libraries of bacterial 16S rDNA amplicons. Colonization of germ-free zebrafish with individual members of its microbiota revealed the bacterial species specificity of selected host responses. Together, these studies establish gnotobiotic zebrafish as a useful model for dissecting the molecular foundations of host-microbial interactions in the vertebrate digestive tract.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                15 November 2016
                2016
                : 11
                : 11
                : e0166379
                Affiliations
                [1 ]Center for Fisheries, Aquaculture, and Aquatic Sciences, Department of Animal Science, Southern Illinois University, Carbondale, IL, United States of America
                [2 ]Warmwater Aquaculture Research Unit, USDA-ARS, Stoneville, MS, 38776, United States of America
                [3 ]Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois-Urbana Champaign, Urbana, IL, United States of America
                [4 ]Aquaculture Research Institute, Department of Fish and Wildlife Sciences, University of Idaho, Hagerman, ID, United States of America
                University of North Carolina at Chapel Hill, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: JWB BCP BCS.

                • Data curation: JWB BCS.

                • Formal analysis: JWB.

                • Funding acquisition: BCP BCS.

                • Investigation: JWB.

                • Resources: BCP BCS KSS.

                • Supervision: BCS.

                • Writing – original draft: JWB.

                • Writing – review & editing: KSS BCP BCS.

                [¤]

                Current address: Aquaculture Research Institute, Department of Fish and Wildlife Sciences, University of Idaho, Hagerman, ID, United States of America

                Article
                PONE-D-16-29717
                10.1371/journal.pone.0166379
                5113000
                27846300
                2bb35018-9411-4c24-8e5c-93d1f86d6907

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 29 July 2016
                : 27 October 2016
                Page count
                Figures: 5, Tables: 3, Pages: 22
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100007917, Agricultural Research Service;
                Award ID: 6066-31000-013-00
                Award Recipient :
                The project was funded by the United States Department of Agriculture, Agricultural Research Service, Warmwater Aquaculture Research Unit (6066-31000-013-00; http://www.ars.usda.gov/research/projects/projects.htm?accn_no=428191).
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbiome
                Biology and Life Sciences
                Genetics
                Genomics
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Microbiology
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Osteichthyes
                Catfish
                Biology and Life Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Biology and Life Sciences
                Nutrition
                Diet
                Medicine and Health Sciences
                Nutrition
                Diet
                Biology and Life Sciences
                Zoology
                Fish Biology
                Fish Physiology
                Biology and Life Sciences
                Zoology
                Animal Physiology
                Vertebrate Physiology
                Fish Physiology
                Biology and Life Sciences
                Organisms
                Bacteria
                Gut Bacteria
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Freshwater Fish
                Custom metadata
                All sequencing files are available from the NCBI database (accession number PRJNA329560).

                Uncategorized
                Uncategorized

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