Università Politecnica Delle Marche Research Doctorate in Life and Environmental Sciences Curriculum Marine Biology and Ecology Cycle XXXI The use of unconventional products in aquaculture, use of insects and macroalgae as feed additives for teleosts Jorge Arturo Vargas Abúndez Prof. Ike Olivotto PhD candidate Tutor Ancona, Italy Content Acknowledgments ............................................................................................................ 3 Abstract ............................................................................................................................. 5 Sommario ......................................................................................................................... 6 Introduction ...................................................................................................................... 7 Scope of the thesis .......................................................................................................... 27 Chapter 1 ........................................................................................................................ 47 Chapter 2 ........................................................................................................................ 92 Chapter 3 ...................................................................................................................... 130 Chapter 4 ...................................................................................................................... 176 General conclusions ...................................................................................................... 209 Appendix ...................................................................................................................... 212 2 Acknowledgments First of all I want to express my sincere gratitude to UNIVPM for financially supporting my studies in Italy. I wish to thank my supervisor, Prof. Ike Olivotto, for the support and patience during these three years of PhD. Thanks for the opportunity to work in your team and for closely following my work in Ancona. More particularly, thanks for offering me the project on insects as feed in aquaculture; for enriching my experience by inviting me to seminars, meetings, congresses and workshops; for sending me to Oslo to be part of a great research group and amazing country and for the great help in writing papers. I really appreciate all this help that enriched my academic career. I learned a lot directly from you and I hope to have the opportunity to collaborate in the near future on projects related to the ornamental aquaculture. I want also to express my gratitude to Prof. Oliana Carnevali for the warm welcome to her lab and for the academical and administrative support. Thanks to Francesca Maradonna and Giorgia Gioacchini for the support in several lab analyses. Thanks to my close colleagues Basilio Randazzo, Matteo Zarantoniello and Giulia Chemello for teaching me techniques applied for this project and the help during all phases of experimentation. Without their help, this work would not have happened. This was a multidisciplinary work and several people were in charge of specific analyses that I could not do alone. In particular, million thanks to Prof. Cristina Truzzi and the PhD student Matteo Antonucci for the gas chromatography–mass spectrometry analysis, which were of central importance in my work. Many thanks to Prof. Elisabetta Giorgini and Carla Conti for their valuable collaboration in the Fourier-transformed infrared spectroscopy analysis. The insect production in chapter one was enthusiastically done by the PhD student Nino Loreto under the supervision of Prof. Paola Riolo and I am immensely grateful for their help. Thanks to researchers Gloriana Cardinaletti and Francesca Tulli from the University of Udine and Prof. Laura Gasco from the University of Turin for their collaboration in feed formulation and production. Thanks to the master students directly involved in this project- Sara Polverini, Andrea D'Addazio, Anna Grill, Marco Foddai and Lorenzo Sanchini. Thanks also to the international PhD student from Vietnam, Huyen Kieu, who visited us for six months and 3 contributed to this project by his valuable help in the experiments and also in histological and molecular analysis. I had the opportunity to spent six months in the Norwegian University of Life Sciences. I want to express my sincere gratitude to Prof. Åshild Krogdahl and the whole research group for their warm welcome and for making me feel part of their group in Oslo. I learned significantly from the Monday meetings and during lunch time, as well as from their exemplary work. Special thanks goes to Guro Løkka who patiently and committedly supervised my work there; to Trond Kortner for sharing his expertise in molecular and fish biology; to Alex Jaramillo for his valuable help in understanding molecular biology; and, to Elvis Mashingaidze for helping me finish histological analysis. Thanks to the whole cheerful research group- Nicole, Hellen, Kirsti, Yanxian, Jie, Weiwen, Abdelazis and Yintaw - for making my work possible in more than one way. Thanks to the technical staff and didactic unit for their kind help. In particular, I appreciate the support of Monica Ferraioli, Simone Bellagamba, Lambertucci Luca, and Giardi Paola and Giardini Mara from the administrative and accountant office. I warmly thank my close friends in the office for the great time and company, as well as mutual support: Valentina Notarstefano, Isabel Forner, Michela Kandelma, Martina Api, Danilo Basili, Andrea Pessina, Jerry Maria Sojan, Andrea Zimbelli, Giulia Chemelo, Silvia Falcinelli, Chiara Piccinetti and Leonardo bruni. I want to mention and thank Mara Gaspari who supported me with wise advice during difficult periods in this journey. Thanks Yas for your company and love despite the long distance, and finally thanks to my family for their unconditional love. 4 Abstract One of the major challenges of the aquaculture sector is to replace most of fish meal and fish oil with more sustainable, cost-effective and environmental friendly ingredients. Insects and seaweeds are interesting candidates. Insects are extremely diverse nutritious organisms capable of massively growing on a variety of compounds, including organic wastes. Seaweeds are nutritious crops rich in bioactive compounds, which culture is associated with positive impacts to the environment. Early research on these unconventional ingredients is promising but in its infancy. The major goal of the present doctoral thesis was to evaluate the effects of insect based diets and seaweeds on key fish biological responses and to improve the understanding of the mechanisms involved in fish nutrition and growth. A comprehensive knowledge at the histological, biochemical, spectroscopic, physiological and molecular level was achieved through a multidisciplinary approach. Taking advantage of fish models, that included zebrafish (Danio rerio), clownfish (Amphiprion ocellaris) and medaka (Oryzias latipes), information obtained in the present thesis may be used to generalize how several biological processes occur in related organisms. Results from the first three experiments, on zebrafish and clownfish, revealed a good potential of insect based diets at even high inclusion levels, up to 100% in zebrafish larval rearing. Fish growth and survival were not affected by the new diets and no signs of intestinal inflammation were detected. In fact, clownfish fatty acid profile was profoundly affected by the novel ingredient, unlike zebrafish which showed molecular and biochemical capacity to compensate essential fatty acid deficiencies. Finally, results from the fourth chapter revealed that the seaweeds Palmaria palmata and Saccharina latissima did not induce any functional effect on the fish growth and gut health parameters of medaka. 5 Sommario Una delle maggiori sfide del settore dell’acquacoltura è la sostituzione di farina ed olio di pesce con ingredienti più sostenibili, vantaggiosi dal punto di vista economico e sostenibili dal punto di vista ambientale. Gli insetti e le alghe si presentano come candidati interessanti da questo punto di vista. Gli insetti sono degli organismi estremamente eterogenei e capaci di crescere in maniera massiva su una varietà di composti, inclusi i rifiuti organici. Le alghe sono vegetali ricchi in composti bioattivi, la cui coltura è associata ad impatti positivi sull’ambiente. I primi studi su questi ingredienti non convenzionali hanno mostrato risultati promettenti, ma sono tuttavia ancora agli albori. Lo scopo principale della presente tesi di dottorato è stato quello di valutare gli effetti di diete basate su insetti ed alghe sulle risposte chiave dei pesci per migliorare la comprensione dei meccanismi coinvolti nella nutrizione e nella crescita. Attraverso un approccio multidisciplinare è stato possibile ottenere una conoscenza di insieme a livello istologico, biochimico, spettroscopico, fisiologico e molecolare. Avvalendosi di pesci come modello sperimentale, come lo zebrafish (Danio rerio), il pesce pagliaccio (Amphiprion ocellaris) ed il medaka (Oryzias latipes), le informazioni ottenute nella presente tesi possono essere utilizzate per comprendere come diversi processi biologici avvengono in organismi simili. I risultati ottenuti dai primi tre esperimenti, su zebrafish e pesce pagliaccio, hanno mostrato un buon potenziale di diete a base di insetti nell’allevamento larvale di zebrafish anche ad alte inclusioni e