PhD in Agricultural and Environmental Sciences CYCLE XXXII COORDINATOR Prof. Giacomo Pietramellara Innovative protein sources in feed for salmonids: Effects on lipid metabolism, gut microbiota and fillet quality Academic Discipline (SSD) AGR/20 Doctoral Candidate Supervisor Dr. Leonardo Bruni Prof. Giuliana Parisi ___________________ ___________________ Coordinator Prof. Giacomo Pietramellara ____________________ Years 2016/2019 In memory of my father La filosofia è scritta in questo grandissimo libro che continuamente ci sta aperto innanzi a gli occhi (io dico l'universo), ma non si può intendere se prima non s'impara a intender la lingua, e conoscer i caratteri, ne' quali è scritto Galileo Galilei, Il Saggiatore Table of contents Table of contents................................................................................. 5 Acknowledgements .............................................................................. 7 Summary ........................................................................................... 9 Riassunto .......................................................................................... 11 List of abbreviations ........................................................................... 13 List of figures..................................................................................... 15 List of tables ...................................................................................... 17 List of papers .................................................................................... 19 Introduction ...................................................................................... 21 Aquaculture production .................................................................... 21 Salmonids: anatomy, digestive physiology and lipid metabolism ............23 Animal protein derived from farmed insects ....................................... 25 Effect of diets on fish: gut microbiota................................................. 27 Methodology .............................................................................. 30 Effect of diets on fish: nutritional quality ............................................32 Focus: EPA and DHA shortage ...................................................... 33 Aims of the study .............................................................................. 35 Materials and methods ........................................................................ 37 Ethical statement ............................................................................ 37 Overview of the studies ................................................................... 37 Diets ............................................................................................ 39 Methods ....................................................................................... 43 qPCR and microbiota data analysis and bioinformatics ......................... 44 Statistics ...................................................................................... 45 References ....................................................................................... 47 Papers 1 to 8 .................................................................................... 55 Abstracts in Conference proceedings ................................................... 283 Conclusion ...................................................................................... 287 Outlook ........................................................................................... 289 5 Acknowledgements My PhD project was carried out from 2016 to 2019 mainly in the Aquaculture Group of the Department of Agriculture, Food, Environment and Forestry at the University of Florence (Italy). During these years, I also performed analyses in the Laboratories of the Nutrition Group at the Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo (Norway), of the Development and Reproduction Biology Group, Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona (Italy), and of the Food Microbiology Group, Department of Agriculture, Food and Environmental Science, Polytechnic University of Marche, Ancona (Italy). Samplings were carried out at the Gildeskål Research Station in Langholmen, Inndyr (Norway), at the Mach Foundation, San Michele all’Adige, TN (Italy), at the Department of Agricultural, Food, Environmental and Animal Science, University of Udine (Italy), and at the Department of Agricultural, Forest, and Food Sciences, University of Turin (Italy). I am grateful to all those who gave me the opportunity, the support and the knowledge to carry out my work. I would like to thank my supervisor Professor Giuliana Parisi for her meticulous attention and accuracy that she used to guide me. I thank my “second boss” Dr Giulia Secci, for being strict and maternal in both work and personal matters, and my colleagues Yara Husein and Adja Medeiros for being such good lab colleagues and for bringing some exotic relax in the three hectic years. They all helped me whenever necessary in the lab and during drafting. The people I met during these three years at work are many and I apologise for leaving someone behind. First of all, I owe gratitude to Professor Åshild Krogdahl and her big Nutrition Group in Oslo (Trond M. Kortner, Alex Jaramillo, Karina Gajardo, Ellen K. Hage, Kirsti Præsteng, Amr Gamil, Yanxian Li, Jie Wang, Weiwen Zhou, Jintao Liu, Anne Sundby); they helped me through the dark Norwegian days with bright research and they became more than just a working Group. Then, thanks to Prof Ike Olivotto’s Group in Ancona with Arturo Vargas, Matteo Zarantoniello, Basilio Randazzo and all the other numerous people that made me love their molecular biology laboratory. I would also like to thank Dr Filippo Faccenda, Dr Francesca Fava and all their collaborators at the Mach Foundation, Prof Lucia Aquilanti and her microbiology Group in Ancona. I acknowledge the internal reviewers Prof Riccardo Bozzi and Prof Luciana Giovannetti for giving some good advice in the first half of my PhD. I would like to thank the seven colleagues that undertook my same path in the very same moment. We seemed to share frustration, mostly, but in fact it was the step to get satisfaction. Thanks to all my Friends around Europe for being there when I raised my hand, especially during these last three months of permanent lack of concentration. Each of you should be the first one to be mentioned. The Italian group in Oslo 7 definitely helped during my stay with some “mediterraneanity”, food, chat and also help at work: Elia, Giulia, Francesca, Alessandro and Chiara. My long- standing friends: in Firenze Irene, James, Luca, Sgri and Guglielmo made me grow the bonds with my Country; in Berlin Ianne, the creative link between present, past, home and abroad. Then, Beatrice&Daniel and, obviously, Piotr, far away yet so close and present in my life, made me feel part of Europe. Many thanks to my Personal Proofreader and to the people of the “lunch group” for relieving the long days in Firenze! Last but not least, thank you Federico for everything you instilled in me and for having reinforced my scientific method with the right rigour. Finally, I would like to express my gratitude to my relatives, who have always been attentive asking how I was, and to my family, for the patience they had with me. Counting the times in which each of them supported me along all these years would result in an endless list. Quindi grazie mamma, Emi, Lella, Sina, il Tipo e le pupette Ida e Anna. 8 Summary The purpose of aquaculture is to contribute to feed an increasing world population and to become more sustainable. Consequently, the aquafeed sector must adapt to supply more feed with less expensive and more eco-friendly ingredients. Insects have been studied as one of the promising and innovative protein sources. Evidence showed that their production leads to a circular bioeconomy, and generates positive societal externalities. Moreover, insects are considered fairly nutritious as aquafeed ingredients. Administering a new diet to fish rest on the assumption that animal welfare and final eating quality are preserved. The present thesis analyses the effects of feeding the Salmonidae Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) with diets containing the larvae of Hermetia illucens, belonging to the Diptera order. The ultimate goal of the present research is to provide a meaningful insight into the decision making process for feeding and farming in order to optimise the overall production process. To fulfil this ambition, three topics were taken into consideration: • gut microbiota, for its countless functions and widespread impacts that it can have on the hosts; • lipid metabolism, for better understanding the laws behind the lipid constitution in the final product; • final product quality, specifically addressing its fatty acid composition, for the importance of fish fatty acids in human nutrition. The results on gut microbiota of either A. salmon or rainbow trout indicated that microbiota composition is modulated by dietary insect and that it differs depending on sample origin (mucosa or digesta). In addition to changes in composition, microbiota of fish fed diets containing insects was also marked by higher alpha- and beta-diversity. Additionally, mucosa samples tended to show lower alpha-biodiversity in comparison to digesta samples, fact that could be explained by positing that mucosa