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Analysis of Size and Shape in Early Larval Stages of European Seabass (Dicentrarchus Labrax): Effects of Egg Disinfection, Axenity and Bacterial Load

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Analysis of Size and Shape in Early Larval Stages of European Seabass (Dicentrarchus Labrax): Effects of Egg Disinfection, Axenity and Bacterial Load Analysis of size and shape in early larval stages of European seabass (Dicentrarchus labrax): effects of egg disinfection, axenity and bacterial load Spyros Nikolakakis Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Promoters: Prof. Dr. Peter Bossier ([email protected]) Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience Engineering, Department of Animal Production, Ghent University, Belgium Prof. Dr. Dominique Adriaens ([email protected]) Research Group of Evolutionary Morphology of Vertebrates Faculty of Sciences, Department of Biology, Ghent University, Belgium Dean: Prof. Dr. Marc Van Meirvenne Rector: Prof. Dr. Rik Van De Walle Members of the Examination and Reading Committee: Prof. Dr. Nico Boon (Chairman) Department of Microbial and Biochemical Technology, Faculty of Bioscience Engineering, Ghent University, Belgium [email protected] Prof. Dr. Wim Van Den Broeck Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Belgium [email protected] Prof. Dr. Peter Goethals Department of Applied Ecology and Environmental Biology, Faculty of Bioscience Engineering, Ghent University, Belgium [email protected] Prof. Emeritus Dr. Patrick Sorgeloos Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Belgium [email protected] Prof. Dr. Olivier Thas Department of Mathematical Modelling, Statistics and Bio-informatics, Faculty of Bioscience Engineering, Ghent University, Belgium [email protected] Prof. Dr. Giorgos Koumoundouros Department of Biology, University of Crete, Greece [email protected] Dutch translation of the title: Analyse van grootte en vorm in vroege larvale stadia van de Europese zeebaars (Dicentrarchus labrax): effecten van desinfectie van eieren, axeniciteit en bacteriologische belasting To cite this work: Nikolakakis, S. (2017) Analysis of size and shape in early larval stages of European seabass (Dicentrarchus labrax): effects of egg disinfection, axenity and bacterial load. PhD thesis, Ghent University, Belgium. ISBN number: 9789463570565 The author and the promoters give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Illustration on the cover page: photo of a European seabass larva of day after hatching 5 by Spyros Nikolakakis This study was funded by a doctoral grant from the Greek State Scholarships Foundation. “It is a good life. Though you will not know how good until you come to the end. The destination cannot be described; you will know very little until you get there; you will journey blind. But the way leads towards possession of what you had sought for in the wrong place”. T. S. Eliot, The Cocktail Party Obtaining my PhD was a long journey. There were many times I knew little of where I was going, or where it was leading. But there were many people who believed in me, supported me immensely, and helped me reach its end. Their faith fuelled mine, and kept me going. I would like to dedicate this thesis to them; especially to my mother, my sisters Garyfallia and Greta, my brother-in-law Bernd, and my beloved father who passed away, but I know that he is feeling now more proud and happy than any words could ever describe. LIST OF ABBREVIATIONS AND UNITS °C Degree Celsius DAH Day after hatching l Litre m Meter g Gram k kilo (103) c centi (10−2) m milli (10−3) μ micro (10−6) n nano (10−9) h Hour min Minute s Second cd sr m-2 Candela steradian per square meter CFU Colony forming unit p Probability value of statistical hypothesis pH Measure of the acidity of solution F Formalin E Ethanol G Glutaraldehyde DX Disinfected and Xenic DA Disinfected and Axenic NX Non-disinfected and Xenic A Antibiotics NA No Antibiotics F F-test statistic SD Standard deviation PCA Principal Component Analysis DA/CVA Discriminant/Canonical Variate Analysis ANOVA Analysis Of Variance ANCOVA Analysis of Covariance MANOVA Multivariate Analysis of Variance PERMANOVA Permutation Multivariate Analysis of Variance 1 1 CONTENTS 1. Chapter 1: Introduction and thesis outline ............................................................... 10 1.1 The value of aquaculture today............................................................. 11 1.2 Abnormalities and deformities: importance and financial loss .............. 13 1.3 Scientific goals of the thesis ................................................................. 15 1.4 The choice of European seabass as a case study ................................... 19 2. Chapter 2: Literature review ..................................................................................... 22 2.1 European seabass - biology and aquaculture life stages ........................ 23 2.2 The importance of the early larval stages.............................................. 27 2.3 Abnormalities, deformities and malformations ..................................... 28 2.3.1 Definition and types..................................................................... 28 2.3.2 Causative factors.......................................................................... 33 2.4 Methods for identification of deformities, and their limitations............. 36 2.4.1 Morphometric size analysis, radiography and staining ................. 36 2.4.2 Geometric morphometrics: the landmark and outline methods ..... 39 2.4.3 Limitations originating from fixation, mounting, handling & anesthetisation ............................................................................. 42 2.5 Bacterial load, egg disinfection and axenic studies ............................... 44 2.5.1 Effects of bacterial load and disinfection in aquaculture............... 44 2.5.2 Bacterial control with microbial maturation……….…………...….45 2.5.3 Axenic and gnotobiotic studies .................................................... 47 2.5.4 Associations with deformities ...................................................... 48 3. Chapter 3: Protocol for quantitative shape analysis of deformities in early larval European seabass Dicentrarchus labrax L........................................................................ 52 3.1 Introduction ........................................................................................ 54 3.2 Materials and Methods........................................................................ 57 3.2.1 Fixation treatments, procedure and sample selection criteria ........ 57 3.2.2 Optical distortion testing.............................................................. 60 3.2.3 Staining and mounting................................................................. 61 3.2.4 Size analyses ............................................................................... 63 3.2.5 Shape analyses............................................................................. 63 2 3.3 Results ................................................................................................ 65 3.3.1 Size effects .................................................................................. 65 3.3.2 Shape effects ............................................................................... 68 3.3.3 Mounting..................................................................................... 71 3.4 Discussion........................................................................................... 72 Appendix...................................................................................................... 78 4. Chapter 4: Phenotypic effects of antibiotic-induced axenity and egg disinfection in early larval European seabass (Dicentrarchus labrax L.) ................................................ 80 4.1 Introduction ........................................................................................ 82 4.2 Materials and methods ........................................................................ 84 4.2.1 Rearing and sampling .................................................................. 84 4.2.2 Hatching percentages................................................................... 85 4.2.3 Mortalities ................................................................................... 86 4.2.4 Analysis of distance & angle morphometry.................................. 86 4.2.5 Outline-based shape analysis...…...…..................…...............….....91 4.3 Results ................................................................................................. 92 4.3.1 Hatching percentages................................................................... 92 4.3.2 Mortalities ................................................................................... 92 4.3.3 Distance and angle analysis ......................................................... 94 4.3.4 Outline-based shape analysis ..................................................... 100 4.4 Discussion......................................................................................... 105 4.4.1 Hatching and mortality percentages ........................................... 105 4.4.2 Effects on distances, angles and yolk sac areas .......................... 107 4.4.3 Outline-based shape effects........................................................ 110 5. Chapter 5: Effects of antibiotic-induced differences in bacterial load on growth and shape of early larval European seabass (Dicentrarchus labrax L.)...............................
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