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Doctoral thesis aquaculture with focus on the use of insects for feeding Atlantic salmon
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Thesis for the Degree of Philosophiae Doctor (PhD) at the University of Bergen 2020
Acknowledgements First, I would like to express my gratitude to my supervisors, Heidi Amlund, Erik-Jan Lock, and Rune Waagbø for guiding me through the project since day one. You have always supported me, while giving me the freedom to express my ideas when conducting the work. This allowed me to grow as a researcher in so many ways. I also thank Bente Torstensen for designing the PhD project, and giving me the opportunity to be part of it. I want to express my gratitude to my colleague Kristin Hamre for giving me the opportunity to be her assistant at the University: you gave me a great chance to experience teaching and to fell in love with it! I am also thankful to the technicians at the laboratories, in particular Bashir Abdulkader , Joar Breivik , Nina Magrethe Steinsvik and Snorri Gunnarson who taught me the methods (very patiently!). Special thanks to all my colleagues at IMR, for bringing a very positive atmosphere at work and outside work. In particular, thanks to my friends Anne-Catrin (my forever “Germana”) , Ikram (my “sister”, according to many people), Nina (the most talkative Norwegian I know), Marta (the loudest laugh in Bergen) and Stig (one of my dearest “bergenser”). I also want to thank Karina Bjørnes and Eva Mykkeltvedt for holding my hand through the hardest times in these past 5 years. I could not manage all these years in the rainy Bergen without the support of my Italian- Spanish crew, the so called “Cintas y secretos”: Sissi, Vale, Nico, Antonio, Jose’, Willy, Noel, Ugo, Raquel and Mauricio. Thank you for all the laughs which saved me from winter depression! I want to thank my parents who supported me financially and mentally in the whole career. Thanks also to my partner, Eleonora. You have always believed in me and you truly support me in every choice I make. My biggest gratitude goes to my beloved daughter, Gaia. You gave me a hard time very often (and you still do) but you also bring all the joy I need to keep going every day. Last but not least, I thank myself for always chasing dreams, despite all the difficulties I meet on the way. “ Ad astra per aspera” , a rough road leads to the stars.
Abstract Traditionally, major sources of protein and lipid in aquaculture fish feeds have been fish meal and fish oil. However, fish stocks used for fish meal and fish oil production are fully exploited, therefore prices of these ingredients continue to increase. In recent years, substantial progress has been made by the research community and feed producers to test novel sources of protein and lipid to replace marine feed ingredients in aquaculture. Insects have been identified as feed ingredients of great potential for farmed fish. In particular, being high in energy and protein content, they seem a good source of ingredients in compound feeds for Atlantic salmon ( Salmo salar ). However, insects reared on terrestrial feedstuff are not a source of the essential marine omega- 3 fatty acids, which Atlantic salmon has a dietary requirement for. The AquaFly project aimed to develop novel insect feed ingredients for Atlantic salmon, contributing essential nutrients to produce robust and healthy fish. To achieve this, tailoring of the nutrient composition of the insect feed ingredients towards fish nutrient requirements was investigated through the use of seaweed as feeding substrate for the insects. Seaweeds are known to contain marine omega-3 fatty acids and essential minerals (like iodine) which are generally absent in terrestrial feedstuff for insects. At the same time, seaweeds can contain undesirable substances, especially heavy metals and arsenic, which could be transferred to the insects, therefore enter the food production chain. The focus of this PhD project, as part of AquaFly , was to evaluate the suitability and safety of the seaweed-insect-fish food production chain, by studying the transfer of both nutrients and undesirable substances along the food chain. Several species of seaweeds from Norwegian waters were screened for their chemical profile. The seaweed species studied contained both nutrients and undesirable substances (heavy metals and arsenic); the concentrations thereof were highly dependent on species and taxonomic group. Based on the data obtained, the brown alga Ascophyllum nodosum was chosen for insect rearing. This seaweed species showed the highest lipid content and the lowest concentrations of undesirable substances among the species investigated. In the insect feeding trial, larvae of the black soldier fly ( Hermetia
Abbreviations AB ARA Arenobetaine Arachidonic acid BSF DGI Black soldier fly Daily growth index DHA Docosahexaenoic acid (22:6n-3) DMA Dimethylarsinate DW Dry weight EAA Essential amino acid EPA Eicosapentaenoic acid (20:5n-3) FA Fatty acid FCR Feed conversion ratio FM Fish meal FO HSI Fish oil Hepatic somatic index LC-PUFA Long-chained polyunsaturated fatty acid LOQ Limit of quantification IM Insect meal IL Insect lipid ML Maximum level N-Prot Nitrogen-to-protein NPN Non-protein nitrogen PAP Processed animal protein PUFA SGR Polyunsaturated fatty acid Specific growth rate SPC Soy protein concentrate VO Vegetable oil WW Wet weight
List of Publications Paper I Biancarosa I ., Espe M., Bruckner C.G., Heesch S., Liland N.S, Waagbø R., Torstensen B.E. and Lock E-J. (2017). “Amino acid composition, protein content, and nitrogen-to-protein conversion factors of 21 seaweed species from Norwegian waters”. Journal of Applied Phycology 29(2): 1001-1009. Paper II Biancarosa I ., Belghit I., Bruckner C.G., Liland N.S., Waagbø R., Amlund H., Heesch S. and Lock E-J. (2018). “Chemical characterization of 21 species of marine macroalgae common in Norwegian waters: benefits of and limitations to their potential use in food and feed”. Journal of the Science of Food and Agriculture 98(5): 2035-2042. Paper III Liland N. S., Biancarosa I ., Araujo P., Biemans D., Bruckner C.G., Waagbø R., Torstensen B.E. and Lock E-J. (2017). “Modulation of nutrient composition of black soldier fly ( Hermetia illucens ) larvae by feeding seaweed-enriched media”. PLoS One 12(8): e0183188. Paper IV Biancarosa I ., Liland N.S., Biemans D., Araujo P., Bruckner C.G., Waagbø R., Torstensen B.E., Lock E-J. and Amlund H. (2018). “Uptake of heavy metals and arsenic in black soldier fly ( Hermetia illucens ) larvae grown on seaweed- enriched media”. Journal of the Science of Food and Agriculture 98(6): 2176-
Paper V Belghit I., Liland N.S., Waagbø R., Biancarosa I ., Pelusio N., Li Y., Krogdahl Å. and Lock E.-J. (2018). “Potential of insect-based diets for Atlantic salmon (S almo salar )”. Aquaculture 491: 72-81.
Figure 1. Development of world capture fisheries and aquaculture production (expressed as million tonnes), 1990- 2030 © FAO (FAO 2018). Orange line: total capture fisheries; dark blue line: aquaculture for human consumption; light blue line: capture fisheries for human consumption. So far, plant-based feed ingredients like soy-based proteins have been the main replacement of FM and FO in aquafeeds for salmonids, but also other farmed species. This is mainly because of lower prices for the raw materials compared to marine feed ingredients, and an all year-round availability ( Figure 2 ). However, using plant-based feed ingredients for fish nutrition has led to adverse effects on fish health, due to nutrient deficiencies or imbalances, and occurrence of anti-nutrients and toxic substances in plant-based ingredients (Francis et al. 2001; Waagbø et al. 2013). Additionally, the replacement of marine ingredients with plant-based ingredients shifted resource demand from oceans to lands, adding pressure to the terrestrial environment and food production systems for human consumption. Today, research is therefore focused on finding novel sources of feed ingredients to replace both marine and plant ingredients, among which single cell proteins, microalgae and insects, have received the highest attention.
Figure 2. Fish meal and soybean meal prices (expressed as USD/tonne) trend in Germany and the Netherlands (above), fish oil and soybean oil prices trend in the Netherlands (below) 1983- 2017 © FAO (FAO 2018). Orange line: soybean oil; blue line: fish oil. The interest in using insects in animal nutrition has increased tremendously in the last decade. Insects are natural components of the diet of many animal species, therefore the inclusion of insect ingredients in feed formulas seems self-evident. They are typically protein-rich and have a well-balanced profile of amino acids, which makes insect meal (IM) highly digestible for animals (Gasco et al. 201 9 ; Sogari et al. 2019). The nutritional composition of insects strongly reflects the composition of the insect feeding media (Meneguz et al. 2018; Pinotti et al. 2019). This is a major advantage that allows insect producers to tailor the nutrient profile of the IM towards a desired profile, through the insect feeding media.
68/2013) (EU 2002a; EU 2009; EU 2013). However, insect producers have also shown interest in using other media for insect rearing. Figure 3. Estimated volumes of production of insect protein until 2025 in Europe (in thousands of tonnes) (IPIFF 2018). In the last decade, seaweeds have been studied as potential fish feed ingredients in aquaculture, due to their richness in both macro- and micronutrients (Satoh et al. 1987; Wassef et al. 2013; Norambuena et al. 2015). Low inclusion of seaweed meal in fish feed (<10 % of the diet) seemed to have positive effects, while high seaweed inclusion levels could be detrimental for fish (Soler-Vila et al. 2009; Wassef et al. 2013; Norambuena et al. 2015). As such, the use of seaweed in aquaculture is most likely limited to the inclusion of seaweed-based additives, rather than as a realistic replacement of marine resources (i.e. FM and FO) in aquafeeds. In Norway, large volumes of seaweed naturally grow along the Norwegian coastline, of which only 1 % is harvested every year, mainly for the industrial production of thickeners (e.g. agar and alginates) (Olafsen et al. 2012; Skjermo et al. 2014). As such, seaweed represents an under-exploited marine resource for the Norwegian economy and the interest in exploiting this resource for other applications has increased. In this PhD work, seaweed was used as feeding substrate for rearing BSF larvae. BSF is considered one of the most suitable species for feeding salmonids as the larvae of this fly are protein- and lipid-rich and have an EAA profile similar to FM (Barroso et al. 2014 ; Henry et al. 2015; Lock et al. 2018). However, BSF larvae are naturally absent in
n-3 LC PUFAs, like most terrestrial insect species (Henry et al. 2015). By feeding seaweed to BSF larvae, we aimed to tailor the nutritional composition of the insect larvae towards a “marine profile” which better suits the nutritional requirements of Atlantic salmon. The suitability of seaweed as rearing substrate for insect larvae was not known in the literature. Growth and performance of BSF larvae reared on seaweed- enriched media were investigated, while assessing the potential transfer of nutrients from seaweed to larvae. The BSF larvae fed seaweed were therefore utilized to develop IM and insect lipid (IL), which were later included in the diet of farmed Atlantic salmon. We investigated nutrient digestibility of the insect-based diets as well as growth and health parameters of the fish. A major aim in food production chains is to produce safe food for consumers which means to ensure food safety. When food comes from farmed animals, it is also necessary to provide safe feed for the animals, that is to guarantee feed safety. Novel feed ingredients may bring undesirable substances to aquafeeds. Feed and food safety regulations are therefore in place to ensure that feed and food stuff do not represent a danger to human health, animal health or the environment. The EU legislation has set maximum levels (MLs) for undesirable substances in feed and food stuff (EC Directive 2002/32 and amendments; Commission Regulation (EC) No 1881/2006 and amendments) (EU 2002; EU 2006). This covers a wide range of compounds such as heavy metals, arsenic, polychlorinated biphenyls, dioxins, pesticides, plant and fungal toxins. This PhD project evaluated availability, access and utilization of nutrients as well as challenges with undesirable substances along the seaweed-insect-fish food production chain ( Figure 4 ). My focus was put on heavy metals (cadmium, lead and mercury) and the metalloid arsenic, as they are major potential chemical hazards associated with seaweed (Almela et al. 2006; Chakraborty et al. 2014). Therefore they were expected to enter the food chain when seaweed was fed to insect larvae. I evaluated the potential transfer of these undesirable substances from seaweed to insects to feed to fish. In this PhD thesis, I discuss the overall suitability and safety of the seaweed-insect-fish food production chain in Atlantic salmon farming.
3. Background 3.1 Insects for Atlantic salmon nutrition. For wild Atlantic salmon, insects comprise more than 50 % of the diet in freshwater (Gabler and Amundsen 1999); as such, the inclusion of insect feed ingredients in the diet of salmon appears appropriate. When choosing which insect species to incorporate into a fish diet, it is pivotal to determine the nutritional composition of the insect, as it should meet the nutrient requirements of the fish. Carnivorous fish like salmonids require between 40 and 55 % protein, and EAA for optimal growth (NRC 2011). The protein content of most insect species range from 50 to 82 % on dry weight (DW) basis (Barroso et al. 2014), which is comparable to the average protein content of FM (70 %) and higher than that of soybean meal (45 %) (NRC 2011). Moreover, while plant-based feed ingredients have some EAA imbalances and deficiencies, the EAA profile of most insects meets the EAA requirements of most fish species and, in some cases, even exceed these requirements (Henry et al. 2015). Besides protein and amino acids, fish require lipids as a source of energy. Commercial feeds for Atlantic salmon typically contain 20 % and 30 % of lipids in freshwater and seawater diets, respectively. Dietary lipids above these levels can lead to reduction of fish growth and fat deposition (New and Wijkstroem 2002). The lipid content of insects ranges from 6 to 38 % (DW basis) and it is higher than lipid levels typically found in FM (8.2 %) and soybean meal (3 %) (St-Hilaire et al. 2007; Barroso et al. 2014). Besides energy, lipids provide essential fatty acids (FAs) such as the n- 3 LC-PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Most insects species lack n-3 LC-PUFAs (Barroso et al. 2014), which could hamper the use of insect feed ingredients in the diet of Atlantic salmon. However, the FA profile of insects is highly affected by the FA profile of their feeding substrate, and there are evidences that feeding insects on substrates rich in n-3 LC-PUFAs (e.g. fish offal) enriches the larvae with such nutrients (Sealey et al. 2011; St-Hilaire et al. 2007a). Sealey et al. (2011) highlighted the importance of the insect rearing substrate on the quality of the IM in a trial with rainbow trout ( Onchorhynchus mykiss ). Fish fed IM produced from BSF larvae reared on subsrates enriched with fish offal, performed better (in terms of fish
growth) than fish fed IM produced from BSF larvae fed substrates without the fish offal enrichment (Sealey et al. 2011). Besides macronutrients, insects are a good source of other essential nutrients such as minerals (i.e. potassium, calcium, iron, magnesium, zinc, selenium) (Finke 2002; Banjo et al. 2006; Schabel 2010; Rumpold and Schluter 2013 ). However, calcium and phopshorus levels in insects are usually lower than the ones found in FM (Makkar et al. 2014 ). 3.2 The black soldier fly. The BSF belongs to the order of Diptera (the true flies) and originates from the American continent, although its diffusion is nowadays ubiquitous in tropical and temperate regions. The larvae of BSF typically contain high amounts of both protein (~40 %) and lipids (~35 %) (of DW) (Barroso et al. 2014). BSF has a relatively short lifecycle including four stages: eggs (4 days), larvae (18 days), pre- pupae (14 days) and finally adults (9 days) (Sheppard et al. 2002). BSF larvae are scavengers and can grow on a wide range of organic substrates, while adults do not feed and rely on the fat storage from the larval stage (Diclaro and Kufman 2009). BSF larvae are able to withstand extreme environmental conditions such as drought, dehydration, shortage of food and oxygen deficiency, and this robust nature makes rearing activities quite simple (Diener et al. 2011). Rearing of BSF has been done since the 1990s, mainly for biodegradation of organic waste by the larvae (Diener et al. 2009; Čičková et al. 2015 ; Fowles and Nansen 2020). To date, BSF larvae are also reared for feed purposes and biodiesel production (Diener et al. 2009; Čičková et al. 2015 ; Fowles and Nansen 2020 ). Studies investigating the effects of IM for salmonids are only a few, as reviewed by Lock et al. (2018). St-Hilaire et al. (2007) investigated the use of a full fat meal from BSF pre-pupae as substitution of FM and FO in the diet of rainbow trout. In this study, the inclusion of 15 % of BSF meal in the diet of rainbow trout (corresponding to 25 % and 36 % substitution of FM and FO, respectively) was possible without significant impacts on growth performances. Renna et al. (2017) showed that a partially defatted IM from BSF larvae can be included in the diet of rainbow trout up to 40 % (50 % of
