Cellular and Molecular Approaches in Fish Biology

Cellular and Molecular Approaches in Fish Biology is a highly interdisciplinary resource to bring industry professionals, students and researchers up-to-date with the latest developments and information on fish biology research combining a historical overview of the different research areas in fish biology and detailed descriptions of cellular and molecular approaches with explanations and recommendations for research. The book presents a global perspective of each research area with detailed analytical methodologies on the cellular and molecular mechanisms within fish biology for expermentation. The book provides different points of view on how researchers have addressed timely issues, while describing and dissecting some of the new experimental/analytical approaches used to answer the key questions at cellular and molecular levels, making this a valuable resource to those in industry and academia as well as those entering the field. Provides detailed descriptions of each research approach, highlighting the tricks of the trade for its effective and successful application Includes the latest developments in fish reproduction, fish development and nutrition, fish welfare, fish immunology, ecology and biomedics Presents hot topics of research such as genetics, transcriptomics and epigenetics Front Cover Cellular and Molecular Approaches in Fish Biology Copyright Contents Contributors Editor’s biographies Preface Chapter 1 Technologies and strategies for ex situ conservation of aquatic organisms: The role of cryopreservation in long- ... 1 Factors and steps to consider in setting up a cryopreservation protocol 1.1 Cryopreservation definition 1.2 Vitrification 1.3 Setting up a cryopreservation protocol 1.4 Assay of freezing–thawing process 1.4.1 Cell integrity and functionality 1.4.2 Mitochondria and sperm motility 1.4.3 DNA damage analysis 1.5 Cell types used for genome cryobanking 2 Applications of cryopreservation techniques in aquatic animals 2.1 Historic aspects and current situation 2.1.1 Fish 2.1.2 Invertebrates 2.2 Cryopreservation applications: The use of cryobanks 3 Cryopreservation of different biological materials to create Genome Resource Banks 3.1 Sperm 3.2 Germline stem cells (GSCs) 3.3 Embryos and larvae cryopreservation 4 Examples of sperm cryopreservation in specific species 4.1 Endangered species 4.1.1 The European eel case study 4.2 Aquacultured species 4.2.1 The salmonid species case study 4.3 Model species 5 Conclusions and future perspectives Acknowledgments References Further reading Chapter 2 Molecular approaches on DNA damage evaluation after primordial germ cell cryopreservation in zebrafish 1 Introduction 2 Conventional DNA evaluation methods 3 DNA lesion detection by qPCR: A novel approach 4 Our contribution to DNA damage evaluation in cryopreserved germ cells 5 Specific protocol for DNA damage evaluation in zebrafish PGCs after cryopreservation 5.1 PGC isolation 5.2 Experimental groups 5.2.1 PGC cryopreservation 5.2.2 Without cryoprotectant treatment 5.2.3 Hydrogen peroxide treatment 5.2.4 Fresh samples 5.3 DNA isolation 5.4 Primer design 5.5 qPCR assay 5.6 Lesion rate calculation 6 Summary and future perspectives Acknowledgments References Chapter 3 The role of epigenetics in fish biology and reproduction: An insight into the methods applied to aquaculture 1 Introduction: Concept and importance of epigenetics in terms of evolution 2 Epigenetic mechanisms 2.1 DNA covalent modifications 2.2 Histone modifications 2.3 Noncoding RNAs 3 The role of epigenetics in farmed fish species 3.1 Domestication 3.2 Nutrition 3.3 Reproduction 3.4 Rearing temperature 4 Epigenetic mechanisms in model species: A case study in zebrafish 4.1 The epigenetic events involved in gametogenesis 4.2 Epigenetic changes during embryo development 5 Techniques applied for the evaluation of DNA methylation 5.1 Evaluation of global DNA methylation 5.1.1 Luminometric methylation assay (LUMA): A brief overview 5.2 Analysis of gene promoter methylation 5.2.1 Bisulfite conversion and nested PCR 5.2.2 Sequencing 5.2.3 CpG island identification and primer design 6 Techniques applied for the evaluation of histone modifications 6.1 Global evaluation of epigenetic modifications in histones 6.1.1 Analysis of global histone H3 acetylation by ELISA 6.1.2 Analysis of the enzymes in charge of histone modifications 6.2 Specific measurements of histone epigenetic marks 6.2.1 Whole-mount immunostaining 6.2.2 Analysis of histone acetylation in gene promoters by ChIP-qPCR 7 Application of the epigenetic methods to our research References Further reading Chapter 4 Noncoding RNAs in fish physiology and development: miRNAs as a cornerstone in gene networks 1 Noncoding RNAs 2 Biogenesis and mechanisms of action of miRNAs 3 Methodological approaches applied in miRNA research 3.1 Methodological approaches to quantify miRNA expression 3.2 Methodological approaches to localize the expression of miRNA 3.3 Methodological approaches to predict mRNA targets 3.4 Methodological approaches for functional characterization of miRNA-mRNA interactions 4 miRNAs playing key roles in fish development and physiology 4.1 miRNAs in fish development 4.2 miRNAs in reproduction and sex differentiation 4.3 miRNAs involved in adaptation/response to different stressors and environmental signals 4.4 miRNAs involved in fish immunocompetence and disease resistance 4.5 miRs involved in fish metabolism and nutrition 5 Recent progress and future perspectives Acknowledgments References Chapter 5 Nutritional epigenetics 1 Introduction 2 Epigenetic regulators 2.1 DNA methylation 2.2 Histone tail modifications 2.3 Bioinformatics workflow of high-throughput sequencing data 3 Nutrients and metabolites influencing the epigenetic regulators 3.1 Folate and methionine cycles constitute the 1C metabolism 3.2 Acetyl-CoA and α -ketoglutarate regulate epigenetic writers and erasers 4 Transfer of cell memory utilizing the epigenetic regulators 4.1 Intragenerational inheritance from cell to cell during growth 4.2 Nutritional intergenerational inheritance from broodstock to offspring 4.3 Transgenerational inheritance of meiotically stable marks through generations in fish 5 Where do we go with nutritional epigenetics? Acknowledgements References Chapter 6 Methods for mapping genomic variants associated with production traits in aquaculture species 1 Introduction 2 Finding causative variants through QTL mapping approaches 2.1 Linkage analysis and interval mapping approaches 2.2 Genome-wide association studies 2.3 GWAS and genome complexity 3 Detailed description of state-of-the-art methodology: Step by step on how to perform GWAS 3.1 Genotype files and format conversion 3.1.1 PLINK inputs files: PED and MAP format 3.1.2 Binary format 3.2 Quality controls using the PLINK software 3.2.1 Duplicated samples 3.2.2 Individual and genotyping call rate 3.2.3 Minor allele frequency 3.2.4 Hardy-Weinberg equilibrium 3.3 GWAS with GCTA 3.3.1 GRM construction 3.3.2 Estimation of genetic parameters 3.3.3 GWAS 3.4 Visualization of results: Manhattan plots 3.5 Significance thresholds 4 Pipeline 5 The next generation of association mapping methods: Multimarker models 5.1 GBLUP and single-step GBLUP 5.2 Bayesian stochastic search variable selection approaches 6 Implementation of QTLs and causative variants into breeding programs 7 Funneling candidate variants toward causality 8 Conclusions References Chapter 7 Double-labeling immunofluorescence: A simple and effective tool to study pituitary regulation in teleost fish 1 The importance of gaining knowledge about the neuroendocrine system 2 Neuroendocrine system 3 Double-labeling immunofluorescence 3.1 General immunofluorescence protocols 3.2 Antigen–antibody recognition 3.2.1 Monoclonal versus polyclonal antibodies 3.2.2 Homologous versus heterologous antibodies 3.2.3 The masking effect of fixative 3.2.4 Negative controls 3.3 Immunofluorescence visualization 3.3.1 Autofluorescence background 3.3.2 Fluorescence microscopes 3.3.3 Immunofluorescence interpretation 4 Going deeper on the pituitary hormone regulation 4.1 In vitro assays 4.1.1 Whole pituitary vs pituitary dispersed cells 4.1.2 Static vs perifuse culture 4.1.3 Neuropeptide action over synthesis and release 4.2 Receptor studies 5 How to approach this kind of neuroendocrine study in fish? 5.1 Regulation of somatolactin (Sl) release by melanin concentrating hormone (Mch) and gonadotropin-releasing hormone (Gn ... 5.2 Regulation of growth hormone (Gh) by melanin concentrating hormone (Mch), neuropeptide Y (Npy), and gonadotropin inh ... 6 Summary Acknowledgments References Chapter 8 Mucosal immunology in fish 1 A brief introduction to the immune system in teleost fish 2 Historical perspective and relevance of immunological studies in gilthead seabream 3 Fish mucosal immunology 3.1 Intestine mucosal immunology 3.2 Skin mucosal immunology 3.3 Skin mucosal immunology during wound repair and healing 3.4 Mucosal immunity during infection or vaccination 3.5 Mucosal immunity during exposure to contaminants 3.6 Mucosal immunity during stress 4 How to alter or strengthen the fish mucosal immune system 4.1 Dietary improvement of mucosal immunity 4.1.1 Probiotics 4.1.2 Prebiotics 4.1.3 Plants and herbs 4.1.4 Amino acids 5 Methodologies to collect and preserve fish mucus 5.1 Intestine mucus 5.1.1 Method 1 5.1.2 Method 2 5.1.3 Method 3 5.2 Skin mucus 5.2.1 Sample collection Method 1 Method 2 5.2.2 Sample processing and storage 5.3 Gill mucus 5.3.1 Method 1 5.3.2 Method 2 5.4 General considerations 5.5 Some examples of downstream analysis with mucus samples 6 Main questions still to be addressed Acknowledgments References Chapter 9 Characterization of the neuroendocrine stress status as part of the multiparametric assessment of welfare in fish 1 Animal welfare 1.1 Current knowledge gaps related to fish welfare 1.2 Evaluation of fish welfare 2 The neuroendocrine stress response and its relation to welfare 2.1 General considerations on the use of stress indicators for welfare evaluation 3 A proposed method to determine the stress status of fishes as part of the assessment of their welfare 3.1 Proposed variables 3.2 Technical considerations for experimental design and sampling 3.2.1 Experimental design 3.2.2 Sampling design 3.2.3 Procedures for tissue collection 3.3 Analytical procedures 3.4 Interpretation of the data 3.5 Examples of application of the proposed method 4 Conclusions and future perspectives References Chapter 10 Evolution of ray-finned fish genomes: Status and directions with a primer on microRNA characterization 1 Introduction 2 Resources to study fish genome evolution 3 Overview of fish genome evolution 3.1 WGD events—Introduction 3.2 Types of WGD event—Auto vs. Allo 3.3 WGDs with deep vertebrate origins 3.4 WGD in the teleost ancestor 3.5 Further WGDs in fish evolution 4 Evolutionary consequences of rediploidization—No “one size fits all” 4.1 Rediploidization begins at the chromosomal level 4.2 Lineage-specific outcomes of rediploidization 5 Transposable elements as agents of fish genome evolution 5.1 TEs and their roles in fish evolution 6 Fish phylogenomics 6.1 Phylogenomics to infer species relationships 6.2 Phylogenomics for investigations of WGD events 7 Evolution of “functional” regions in fish genomes 7.1 Protein coding regions 7.2 Noncoding regions 7.3 Evolution of the epigenome 7.4 Ohnologue regulatory evolution 8 A primer on miRNA evolutionary characterization 8.1 miRNAs and their role in fish biology and evolution 8.2 Main steps in miRNA phylogenetic analysis 8.2.1 Step 1: Data collection 8.2.2 Step 2: Sequence alignment 8.2.3 Step 3: Phylogenetic tree reconstruction 8.2.4 Step 4: Inferences on miRNA family evolution 9 Concluding remarks References Chapter 11 Zebrafish xenotransplantation: A versatile platform for cancer and stem cell translational research 1 Introduction 2 Larval zebrafish XT models of cancer 2.1 Advantages and limitations of larval zebrafish XT models 2.2 Transplantation of human cancer cell lines in larval zebrafish 2.2.1 Angiogenesis and the microenvironment 2.2.2 Liquid tumor XTs 2.2.3 Solid tumor XTs 2.3 Patient-derived xenografts (PDX) in larval zebrafish 2.4 Drug screening, novel drug discovery, and radiotherapy enhancement using larval zebrafish 3 Using the zebrafish for translational stem cell research 3.1 Nonmalignant stem cells 3.2 Hematopoietic stem cell transplants 3.3 Regenerative therapies 4 Adult zebrafish XT models of cancer 4.1 Genetic approaches for adult immunosuppression and XT 4.1.1 PDXs using prkdc − / −, il2rga − / − mutant zebrafish 4.1.2 Single cell resolution imaging for XT studies 4.1.3 Pharmacodynamic studies in zebrafish XT models 4.2 Chemical approaches for adult immunosuppression and XT 4.3 Irradiation approaches for adult immunosuppression and XT 5 Conclusion References Chapter 12 Status, challenges, and perspectives of fish cell culture—Focus on cell lines capable of in vitro mineralization 1 A growing interest for cell lines of fish origin 2 Current status of fish cell culture 3 Applications for fish cell lines 3.1 Applications in virology and immunology 3.2 Applications in ecotoxicology and toxicology 3.3 Biotechnological applications 3.4 Applications in functional genomics 3.5 Application in cell biology 3.6 Applications in species conservation 4 Fish cell lines capable of in vitro mineralization 5 Development of bone-derived cells 6 Perspectives for fish cell culture Acknowledgments References Index Back Cover "Cellular and Molecular Approaches in Fish Biology is a highly interdisciplinary resource to bring industry professionals, students and researchers up-to-date with the latest developments and information on fish biology research combining a historical overview of the different research areas in fish biology and detailed descriptions of cellular and molecular approaches with explanations and recommendations for research. The book presents a global perspective of each research area with detailed analytical methodologies on the cellular and molecular mechanisms within fish biology for expermentation. The book provides different points of view on how researchers have addressed timely issues, while describing and dissecting some of the new experimental/analytical approaches used to answer the key questions at cellular and molecular levels, making this a valuable resource to those in industry and academia as well as those entering the field."--Publisher's description Cellular And Molecular Approaches In Fish Biology Is A Highly Interdisciplinary Resource That Will Bring Industry Professionals Up-to-date On The Latest Developments And Information On Fish Biology Research. The Book Combines An Historical Overview Of The Different Research Areas In Fish Biology With Detailed Descriptions Of Cellular And Molecular Approaches And Recommendations For Research. It Provides Different Points-of-view On How Researchers Have Addressed Timely Issues, While Also Describing And Dissecting Some Of The New Experimental/analytical Approaches Used To Answer Key Questions At Cellular And Molecular Levels. Provides Detailed Descriptions Of Each Research Approach, Highlighting The Tricks Of The Trade For Its Effective And Successful Application Includes The Latest Developments In Fish Reproduction, Fish Nutrition, Fish Wellbeing, Ecology And Toxicology Presents Hot Topic Areas Of Research, Including Genetic Editing, Epigenetics And Edna __Cellular and Molecular Approaches in Fish Biology__