Could edible insects be one solution for a circular economy and food production? : designing feeds for insects using food industry by-products

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Dissertations in Forestry and Natural Sciences

DISSERTATIONS | JAANA M. SORJONEN | COULD EDIBLE INSECTS BE ONE SOLUTION FOR A CIRCULAR ECONOMY AND... | No 444

JAANA M. SORJONEN

COULD EDIBLE INSECTS BE ONE SOLUTION FOR A CIRCULAR ECONOMY AND FOOD PRODUCTION?

Designing feeds for insects using food industry by-products PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

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Could edible insects be one solution for a circular economy and food production?

Designing feeds for insects using food industry by-products

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Jaana M. Sorjonen

Could edible insects be one solution for a circular economy and food production?

Designing feeds for insects using food industry by-products

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 444

University of Eastern Finland Joensuu

2021

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium N100 in the Natura Building at

the University of Eastern Finland, Joensuu, on 14^th of January, 2022, at 12 o’clock noon

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PunaMusta Joensuu, 2021 Editor: Raine Kortet

Sales: University of Eastern Finland Library ISBN: 978-952-61-4388-0 (nid.) ISBN: 978-952-61-4389-7 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)

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Author’s address: Jaana M. Sorjonen

University of Eastern Finland

Depart. of Environmental and Biological Sciences P.O. Box 111

80101 JOENSUU, FINLAND email: jaana.sorjonen@uef.fi

Supervisors: University Researcher Anu Valtonen, Ph.D.

80101 JOENSUU, FINLAND email: anu.valtonen@uef.fi

Professor Emeritus Heikki Roininen, Ph.D.

80101 JOENSUU, FINLAND email: heikki.roininen@uef.fi

Reviewers: Professor Jarkko Niemi, Ph.D.

Natural Resources Institute Finland (Luke) Kampusranta 9

60320 SEINÄJOKI, FINLAND email: jarkko.niemi@luke.fi

Senior Scientist Susanne Heiska, Ph.D.

Natural Resources Institute Finland (Luke) Yliopistokatu 6B

80100 JOENSUU, FINLAND email: susanne.heiska@uef.fi

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Opponent: Associate Professor Cecilia Lalander Swedish University of Agricultural Sciences Depart. of Energy and Technology; Environmental Engineering

Box 7032

75007 UPPSALA, SWEDEN email: cecilia.lalander@slu.se

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7 Sorjonen, Jaana M.

Could edible insects be one solution for a circular economy and food production? Designing feeds for insects using foo industry by-products.

Joensuu: Itä-Suomen yliopisto, 2021

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences; 444 ISBN: 978-952-61-4388-0 (print)

ISSNL: 1798-5668 ISSN: 1798-5668

ISBN: 978-952-61-4389-7 (PDF) ISSN: 1798-5676 (PDF)

ABSTRACT

Edible insects could offer a solution to the increasing global food demand and food security because they can provide a valuable source of proteins, fatty acids, vitamins, and minerals to humans, and in many parts of the world they are culturally accepted and valued as traditional parts of the human diet. Insect rearing is ecologically sustainable; it requires less feed, land, and water to produce the same amount of protein as traditional livestock. In the future, the insect economy could be steered in a more sustainable direction by incorporating insect rearing into strategies for a circular economy. Insect rearing creates interesting opportunities for a circular economy because many edible insect species have the capacity to utilise a wide variety of plant-based materials, making it possible to use food and agricultural by-products as insect feeds. Developing insect feeds from food industry by-products could be a way to reuse, recycle, and valorise materials that are not consumed by humans. However, the bare use of by- products, unprocessed or low-quality materials, are not sufficient to maximise rearing yields or to guarantee insect health and proper development. Insect feeds need to be designed specifically for each species to meet their nutritional needs, to ensure successful growth and

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development, and to ensure they are nutritious and safe for human consumption.

My thesis examines the potential of food industry by-products as feed ingredients for three edible insect species: house cricket Acheta domesticus Linnaeus (Orthoptera: Gryllidae), two-spotted cricket Gryllus bimaculatus De Geer (Orthoptera: Gryllidae), and long-horned grasshopper Ruspolia differens Serville (Orhoptera: Tettigoniidae). These three species are economically valuable as human food. Crickets are already widely reared for human consumption, whereas R. differens is harvested from nature in Eastern Africa, even though mass rearing technologies are currently being developed. My goal was to develop artificial diets i.e., diets that insects would not naturally obtain, for rearing A. domesticus, G. bimaculatus, and R.

differens by combining earlier information on insect feeds and nutritional requirements with the current need to reuse materials from the food industry that are not consumed by humans. Furthermore, this thesis contributed to the scientific literature by increasing the general understanding of insect feeds. My specific study objectives were: (I) to investigate how R. differens grows and develops on by-product diets; to determine if the diets can alter the fatty acid content and composition of R.

differens; and to identify the protein-level requirements for the feed; (II) to investigate how A. domesticus and G. bimaculatus grow and develop on diets including food industry by-products; (III) to examine the effect of by-product diets on the fatty acid composition and content of these crickets, and (IV) to evaluate how the artificial diets for crickets with the most potential perform in small-scale farming conditions.

The experiments included experimental diets with food industry by- products and control diets (with no by-products). The experimental by- product diets were designed to meet the nutritional requirements of insects.

In these diets, the main protein source (e.g., soybeans) was partly or completely replaced with food industry by-products. The by-products were selected among those readily available from the Finnish food industry. They included potato protein, barley mash (brewer’s spent grain), barley feed from ethanol industry, compressed leftover of turnip rape, a mix of broad

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9 bean and pea, and a mix of potato, carrot, and apple. The fatty acid composition and content of the produced insects were analysed, as well as the glycoalkaloid content of insects feeding on potato protein. Finally, the feasibility of the most suitable by-product feeds was examined in a small- scale cricket farm, using current rearing technology.

My thesis showed that the studied insect species accept several food industry by-products in their feed and can successfully grow and develop when fed on by-product diets. For R. differens, the suitable by-product diets included potato protein, barley feed, and barley mash, which resulted in high survival, high individual weight, fast development, and low feed conversion ratios, e.g., the highest feed conversion efficiency. Diets containing 17–22% protein significantly improved the growth performance of R. differens. Furthermore, diets containing barley increased the polyunsaturated fatty acid content in R. differens, i.e., the essential fatty acids for humans. For A. domesticus and G. bimaculatus, several suitable by- product feeds were identified, for example, compressed leftovers of turnip rape from rape seed oil production, barley mash from the beer industry, and barley feed from the ethanol industry. The studied crickets showed high growth performance on these diets when the protein content of the diet ranged between 22.5 and 30.5%. For two-spotted crickets and house crickets, by-product diets modified the fatty acid content more than the composition, i.e., whereas the total fatty acid content of crickets can be modified by the diet, the fatty acid composition tends to remain unchanged.

The small amounts of glycoalkaloids in potato protein by-products do not affect the growth performance of the studied insects, and both crickets and R. differens feeding on potato by-products were safe for human consumption.

My thesis also showed that diets that maximise the by-product content might cost less but are not necessarily suitable to maximise farming yields.

Overly simplified by-product diets, where by-products replace traditional feed ingredients, might potentially lower the overall farming yields, although they may not impact the development time or feed conversion ratio of insects.

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The findings of this thesis emphasise that there are new ways to utilise food industry by-products as insect feed ingredients and encourage further research to identify potential locally available side streams that could have a use for insect farmers and companies. The global food production system is constantly progressing, and new practical solutions and methods are necessary to meet future food security and food demand challenges. The results of this study are a step towards a circular economy and sustainable insect production, promoting the use of local resources and avoiding the use of feed ingredients that could be consumed directly by humans.

Universal Decimal Classification: 595.72; 638.4

Keywords: Insects; Edible insects; Orthoptera; Gryllidae; Tettigoniidae;

House cricket; Gryllus bimaculatus; Conocephalus; By-products; Sustainable development; Animal feeding; Artificial feeding; Insect rearing; Growth;

Insects - Life cycles; Survival; Weight gain; Feed utilization efficiency; Fatty acids; Alkaloids; Edible insects - Nutritional aspects

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Acknowledgements

Throughout this project, I have received a great deal of support and assistance. First, I would like to thank my supervisors, emeritus Professor Heikki Roininen and Dr. Anu Valtonen. The determination, admirable enthusiasm, and encouragement of Heikki made everything seem possible and his insightful feedback guided me in the right direction when I felt lost.

Thank you for making this journey possible. Thank you Anu for always finding time for my endless questions and travelling to Joensuu whenever I needed your help. Your constant and helpful support during the final steps of this thesis was essential, which I am very grateful. I want to thank my supervisors for all the great stories, experiences, and memories we created together on our adventures in China and Uganda.

I wish to thank all my co-authors. Maija Karhapää, thank you for your patience, for answering my questions and sharing your expertise. I would also like to thank the fellow researchers from Natural Resources Institute Finland: Maria Tuiskula-Haavisto, Miika Tapio, Hilkka Siljander-Rasi, Maarit Mäki, Pertti Marnila and Elina Hirvisalo for their collaboration at the beginning of this project. I am thankful for Minna Hiltunen for helping me to understand the fascinating world of fatty acids. Vilma, thank you for being such a great support during this project and your company made it much more fun. You truly set big boots to fill. Thank you Sille for being a great coworker and made me smile even when I struggled with my writing.

I sincerely wish to acknowledge the reviewers Jarkko Niemi and Susanne Heiska for their contribution.

I wish to express my gratitude to many funding organizations for making this research possible. The Ministry of Agriculture and Forestry, the Academy of Finland, Olvi foundation, Finnish Food and Drink Industries’ Federation, University of Eastern Finland Doctoral School and the North Carelian Regional fund of the Finnish Cultural Foundation enabled me to focus on research fulltime. Many thanks for Bugbox Ltd. and Erlend Sild for the

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financial help. I also want to thank Hermanni Nieminen and Sanne Seppälä for offering their insect rearing facilities for my experiments. I also wish to thank Entocube, Panimo Honkavuori, Kankaisten öljykasvit Oy and Vilomix Oy, for offering their products for research purposes. My warm thank you go to all the bold insect farmers who have brought the industry forward.

My warm thanks go to everyone who has helped me in the laboratory, especially our master students Patrick Mooney, Joni Immonen, Josefina Lindgren and Maija Erkilä. Thank you for all the helpers in the lab for taking care of our precious little insects. Special thank you for Lauri Jauhiainen for the help with the statistical analyses and Toni Vesala for preparing the experimental diets. In addition, my gratitude goes to our fellow researchers in Uganda: Dr. Philip Nyeko (I can still taste the sweetest mangos like it was yesterday), Geoffrey, Rutaro and Robert. Thank you, Markus, for your help with proofreading my manuscripts. My warmest thank you go to the whole staff of the Department of Environmental and Biological Sciences, especially Pyry Pihlasvaara and Marja Noponen. I am grateful for Riikka and Meeri for showing great examples and other fellow PhD students for valuable peer support. Thank you for our department’s work wellbeing group, Annalaura, Anu and Saskia for making sure we had fun aside work.

To all my dear friends and relatives: You’re the best. Thank you for walking through this project with me. Thank you Miika for your support and encouragement to start this project. Jenni, Heidi, Lotta, thank you for all the memorable trips we have done together, laughs are guaranteed when we are together. Thank you Iina for taking me for long walks, stand up paddling, diving, or other nature adventures when I needed to rest my mind. The warmest hug to Petra, for being a sympathetic ear and an honest friend.

Thank you Jonna and Emma for reminding me what is important in life and my godson Aapo for all the happy moments. Thank you, Emma and Mani for being the best roommates and shoulders. I wish to thank all my family members, including aunties, uncles, and cousins for being there. Thank you, Manna and Peter, for showing great examples and for your valuable guidance on the academic road.

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13 Finally, I want to express my gratitude to my dear family. I want to thank Jukka and Oku for your brotherly support and showing interest in what I was doing. Mom and dad, the words can’t express how grateful I am for your support. Thank you for your wise counsels and always reminding me that everything will be all right. Thank you, Urho, for bringing fluffiness and happiness to my life. Vincent, thank you for keeping me fed while I was busy feeding my crickets. I could not have asked for better support, thank you for being my rock.

A special thank you go to all the little insects who were sacrificed in the name of the science.

Joensuu, 29th July 2021 Jaana Sorjonen

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LIST OF ABBREVIATIONS

ANOVA Analysis of variance

ECI Efficiency of conversion of ingested food FAME Fatty acid methyl ester

GC-FID Gas chromatography with flame ionisation detection GC-MS Gas chromatography mass spectrometer

GLM General linear model FCR Feed conversion ratio GHG Greenhouse gas LD Light dark

LMM Linear mixed model

LSD Least significance difference MUFA Monounsaturated fatty acids

NMDS Non-metric multidimensional scaling

PERMANOVA Permutational multivariate analysis of variance PUFA Polyunsaturated fatty acids

RGR Relative growth rate RH Relative humidity SFA Saturated fatty acids SE Standard error

SIMPER Similarity percentage analysis TFA Total fatty acids

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15 LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referred to by the Roman Numerals I-IV.

I Sorjonen JM, Lehtovaara VJ, Immonen J, Karhapää M, Valtonen A, Roininen H. (2020). Growth performance and feed conversion of Ruspolia differens on plant-based by-product diets. Entomologia Experimentalis et Applicata, 168: 460–471.

https://doi.org/10.1111/eea.12915

II Sorjonen JM, Valtonen A, Hirvisalo E, Karhapää M, Lehtovaara VJ, Lindgren J, Marnila P, Mooney P, Mäki M, Siljander-Rasi H, Tapio M, Tuiskula-Haavisto M, Roininen H. (2019). The plant-based by-product diets for the mass-rearing of Acheta domesticus and Gryllus

bimaculatus. PLoS ONE 14(6): e0218830.

https://doi.org/10.1371/journal.pone.0218830

III Sorjonen JM, Valtonen A, Hiltunen M, Erkilä M, Lehtovaara VJ, Karhapää M, Roininen H. Fatty acids of Acheta domesticus and Gryllus bimaculatus fed on by-product diets. Submitted manuscript.

IV Sorjonen JM, Karhapää M, Holm S, Valtonen A, Roininen H. (2021).

Performance of the house cricket (Acheta domesticus) on by-product diets in small-scale production. Journal of Insects as Food and Feed.

Accepted manuscript.

The above publications have been included at the end of this thesis with their copyright holders’ permission.

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AUTHOR’S CONTRIBUTION

I) The author participated in the development of ideas and planned the study together with the co-authors. The author was responsible for organizing the laboratory work and conducted the statistical analyses with the assistance of Anu Valtonen. The author was the main author and wrote the first draft of article.

II) The author participated in the development of ideas and planned the study together with the co-authors. The author was responsible for organizing the laboratory work and conducted the statistical analyses with the assistance of Anu Valtonen. The author was the main author and wrote the first draft of article.

III) The author participated in the development of ideas and planned the study together with the co-authors. The author was responsible for organizing the laboratory work and carrying out laboratory work to collect data in cooperation with co-authors. The author conducted the statistical analyses with the assistance of Anu Valtonen. The author was the main author and wrote the first draft of article.

IV) The author participated in the development of ideas and planned the study together with the co-authors. The author was responsible for organizing the laboratory work and conducted the statistical analyses with the assistance of Anu Valtonen. The author was the main author and wrote the first draft of article.

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1 Introduction

1.1 The future challenges of food production

The past decades of development in the food production sector have had a major impact on the fight against hunger (Godfray et al., 2010). The number of people suffering malnutrition and hunger has decreased significantly since the 1990’s. Nevertheless, a sufficient level of food security is not globally achieved, and many factors such as food price fluctuations, climate change, changes in food consumption patterns, and economic instability are obstacles to the global aim of reducing hunger. The global food system also faces challenges in responding to the future increase in food demand. The United Nations estimates that the global human population will increase up to 9 billion by 2050 (United Nations, 2017). In addition, food consumption per person is increasing due to increased wealth in households and the decrease in food prices (Guyomard et al., 2012). The changes in food consumption patterns are creating a greater demand for processed food and animal proteins, but such changes can also have negative outcomes for human health and the environment (Guyomard et al., 2012).

At the same time, climate change, land degradation, urban expansion, water scarcity, overfishing of seas, non-food production in agriculture (which is consuming space from food production), and pest infestations are threatening the current food production system (Nelleman, 2009). These factors pose risks to the different aspects of food security: 1) the availability of food through production, food supplies, or food imports, 2) access to adequate resources for acquiring foods and maintaining healthy diets, 3) the utilisation of safe and nutritious foods through sanitation, 4) clean water and health care to meet physiological needs and reach nutritional wellbeing, and 5) stability in the availability of food at all times (Food and Agriculture Organization of the United Nations [FAO], 2008; Wheeler & von Braun, 2013).

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The future challenge is also to produce food for the continuously growing human population with fewer resources (Godfray et al., 2010). Moreover, food needs to be produced in a socially, environmentally, and economically sustainable way to match the food demand with fewer resources.

Sustainable diets require decreasing the environmental impact so that they support food security for current and future generations; they should be affordable, culturally accepted, and accessible, as well as nutritionally adequate, safe, and respectful of the environment, biodiversity, and ecosystems (FAO & World Health Organization [WHO], 2019). While the global food system is a complex network with heterogeneous food producers, it is one of the main contributors to climate change. Therefore, the current food production system needs to be re-evaluated; food waste should be minimised, side streams should be recycled and reused, and new technologies need to be developed.

The increasing demand for animal protein challenges attempts to improve the sustainability of global food production systems. While livestock production is one of the main contributors to food production-derived greenhouse gas (GHG) emissions (FAO, 2009; Poore & Nemecek, 2018) and excessive consumption of meat is related to many health problems (Richi et al., 2015), the demand for animal-based proteins is increasing (FAO, 2009).

One of the most efficient ways to reduce global GHG emissions from agriculture and land use sectors is to reduce meat consumption (Tilman &

Clarke, 2014). The development of new alternative proteins to replace conventional livestock could overcome both health and environmental problems of meat production. Many alternatives, such as cultured meat, mycoproteins, algae, cereal and legume proteins, or edible insects, could provide a more sustainable food production system (Van Huis and Tomberlin 2017; Schweiggert-Weisz et al., 2020; Ullmann & Grimm, 2021).

1.2 Edible insects in a sustainable food production system Eating insects and producing them for food and feed purposes has gained attention in recent years. Insects are now promoted as healthy alternatives

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21 for traditional meat products, such as chicken, pork, beef, and fish (Van Huis et al., 2013). Insects are a good source of protein, and they contain complete amino acid profiles for human diets. Many insect species contain polyunsaturated fatty acids, especially essential omega 3 fatty acids (Finke, 2015; Lehtovaara, 2017). Insects are also rich in iron and other minerals, and they are a good source of vitamin B12 (Finke, 2015). However, there are many factors that influence the nutritional composition of insects, such as their diet or environmental factors (Finke & Oonincx, 2014). Nevertheless, edible insects could offer a nutritious alternative for animal proteins, especially in regions that are challenged by food insecurity and malnutrition and where insects are culturally accepted and valued as traditional parts of the diets.

Traditionally, edible insects are harvested from nature (Van Huis et al., 2013). Their consumption is deeply rooted in human evolution; edible insects formed an important part of the diet in early hominins, and the fats consumed from insects could have served as a major benefit for brain development (Lesnik, 2018). Currently, over 2,111 different insect species are reported to be consumed globally (Jongema, 2017). Most of them belong to the groups of beetle larvae (Coleoptera 31%), caterpillars, wasps, bees, and ants (15%), crickets, grasshoppers, and locusts (13%), and true bugs (11%). Edible insects are consumed especially in tropical regions, where they are abundant and harvesting is easy (van Huis et al., 2013). Harvesting typically takes place seasonally when each species becomes available in nature. Gathering from natural habitats also provides risks, such as overharvesting of the natural populations and not meeting the consumption demand for the growing human population. To alleviate these risks, mass- rearing technologies are being developed for year-round rearing (Rumpold

& Schlüter, 2013; Van Huis & Tomberlin, 2017).

Edible insect production by improved rearing technologies could be one solution for producing high-quality proteins in a more sustainable way but also improving food security. Many edible insect species have the capacity to utilise a variety of plant materials, including side streams from the food industry, making it possible to improve the sustainability of the insect rearing industry. Therefore, insect rearing could be one solution for circular

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economy practices and recycling and reuse of materials. The transition from seasonal harvesting from nature towards year-round production in rearing facilities with by-product feeds could improve food security, especially in areas where malnourishment is common, and insects are culturally accepted and valued as traditional parts of human diets.

1.2.1 Sustainability of edible insects

Edible insect rearing is attracting attention as a novel way to produce food because of its environmental benefits. The main reason for this is explained by insect physiology. As poikilotherms, i.e., cold-blooded animals, insects do not maintain their body temperature, which results in a generally higher feed conversion ratio compared to conventional livestock (Nakagaki and DeFoliart 1991; Collavo et al., 2005; Van Broekhoven et al., 2015). For example, insects require 12 times less feed than cattle to produce the equivalent amount of protein (Van Huis et al., 2013). The feed conversion ratio (FCR) and the efficiency of conversion of ingested food to body matter (ECI) are ways to measure this efficiency. Generally, these parameters show the weight gain per unit of food consumed, e.g., the efficiency of food utilisation (Waldbaeur, 1968; Wilkinson, 2011; Nation, 2016). Efficient food utilisation is directly linked with lower land and water use (Oonincx and De Boer, 2012; Herrero et al., 2015).

Another aspect of sustainability is GHG emissions. The global food system is responsible for 26% of anthropogenic GHG emissions, with livestock production being one of the largest contributors (Poore & Nemecek, 2018).

Insects emit less direct GHGs and ammonia than conventional livestock (Oonincx et al., 2010). Some species do not emit methane, including the house cricket (Acheta domesticus Linnaeus, (Orthoptera: Gryllidae), yellow mealworm (Tenebrio molitor Linnaeus, Coleoptera: Tenebrionidae), and migratory locust (Locusta migratoria Linnaeus, Orhoptera: Acrididae).

However, Argentinean cockroaches (Blaptica dubia Serville, Blattodea:

Blaberidae) and sun beetles (Pachnoda marginate Kolbe, Coleoptera:

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23 Scarabaeidae) emit methane on similar levels as pigs but less than cattle (Oonincx et al., 2010).

The overall environmental impact of producing insects for food or feed purposes has been studied in small-scale farming systems (Halloran et al., 2016; Nikkhah et al., 2021) and for a few insect-derived products (Oonincx, 2017). Life cycle assessment is a tool to estimate the environmental impact of a product throughout the life cycle; it quantifies the materials, resources, and services needed to produce the final product (Guinée et al., 2002). Many of the life cycle assessments carried out for edible insect products have found, for example, that mealworms have a lower global warming potential than traditional livestock, and they also require less land to produce one kilogram of edible meal worm protein (Oonincx & De Boer, 2012). However, insect rearing requires high rearing temperatures, which is also associated with higher energy use (Oonincx & De Boer, 2012; Nikkhah et al., 2021).

Edible insect production is still in its infancy, but the biology and physiology of insects suggests that further development of insect production in a more ecologically sustainable way is possible.

1.2.2 Insects as bio converters in a circular economy

Many edible insect species are polyphagous and can consume side streams, which creates interesting opportunities to recycle and reuse different kinds of plant-based materials. The principal of a circular economy is to recycle and reuse materials and maximise waste prevention (Velenturf & Purnell, 2021). At the same time, the use of raw natural resources can be minimised.

In food production systems, this could mean the utilisation of materials such as food and agroindustry side streams, biowaste, manure, municipal waste, or plastic, reducing waste and recycling nutrient flows (Jurgilevich et al., 2016; Vilariño et al. 2017; Urbanek et al., 2020). Many of these materials can be difficult to recycle, whereas some agroindustry by-products have been used for livestock feed (Ellis & Bird, 1950; Walker, 2000). The use of edible insects as bio converters can steer the insect economy in a more sustainable direction. Leftover materials can be converted back to the food production

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cycle in the form of feed sources for insects, which in turn can be consumed either by humans or as feeds for conventional livestock.

Among edible insects, there are a wide variety of feeding specialisms and potentially acceptable feed sources. For example, the black soldier fly (Stratiomyidae: Hermetia illucens Linnaeus) and house fly (Muscidae: Musca domestica Linnaeus) have high reproductive and bioconversion capacities, and both species can utilise organic waste and manure (Ocio et al. 1979;

Čičková et al., 2015; Oonincx et al., 2015b). In particular, black soldier fly larvae have a high capacity to live on different waste materials. There have been many attempts to incorporate black soldier fly production as part of waste management systems; the larvae could be used as protein source in animal feeds (Lohri et al., 2017; Van Huis & Tomberlin, 2017). Mealworms (Coleoptera: T. molitor) and crickets (Gryllidae) are typically reared for human consumption. Additionally, these species can consume a wide variety of unused plant materials, such as biowaste and side streams from food production, e.g., vegetable remains, dried distillers’ grains, or weeds (Ramos- Elorduy et al., 2002; Oonincx et al. 2015a; Miech et al. 2016). However, even though insects have the potential to recycle and reuse unused materials, it needs to be done in a way that also considers animal health and welfare. A detailed, species-specific feed design is key to such attempts.

1.3 From side streams to insect feeds

The careful selection of appropriate animal feeds ensures the growth and health of animals, but they are also important for sustainability (Makkar &

Ankers, 2014). Animal feeds also determine production costs, and they have an important role in the composition of the end-product (Lehtovaara et al., 2017). Many by-products from the food industry can be utilised as livestock feeds, and sometimes, their use is cost-effective (Pinotti et al., 2019). For example, in cricket rearing systems, feed is the most costly expense; hence, the use of local vegetable remains or other food industry by-products have been proposed to reduce costs in cricket rearing systems (Durst &

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25 Hanboongsong, 2015). The use of side streams has appealing potential in insect rearing, since many insect species can utilise a wide variety of plant- based side streams that are not consumed by humans. However, studies of how edible insects perform while feeding on carefully designed feeds from side streams are still rare (Morales-Ramos et al., 2020).

1.3.1 Nutritional requirements of insects

Insects generally require the same nutritional components from their feeds as other conventional livestock animals (Chapman, 2013; Nation, 2016).

However, differences between insect species can be found, and development stage, sex, or physiological stress factors can modify the nutritional requirements on an individual level (Woodring et al., 1977). The appropriate balance of nutritional components is a critical aspect for feed design and meeting the nutritional requirements of each insect species. The nutritional requirements of an insect species can be established by quantifying insect performance, e.g., survival, growth, development rate, and reproduction, on chemically defined diets (Chapman, 2013). These artificial diets can vary; therefore, they lack or contain specific nutrients to determine whether these nutrients are required. However, suitable chemically defined diets for each currently used edible insect species are still lacking. Meridic diets are semi-defined artificial diets that include many complex ingredients (Nation, 2016). With thorough examination, they can also be used to define the nutritional requirements of insects and nutrient interactions.

Sources of nitrogen are important nutritional requirements for all insect species. Whole proteins (peptides) from the diets are used as the main source of nitrogen, and they are broken down into amino acids (Cohen, 2004). Amino acids are needed to produce proteins that are required for structural purposes, such as enzymes, transportation, storage, and receptor molecules (Chapman, 2013). Insects typically require 10 essential amino acids, namely arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Cohen, 2004; Nation,

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2016). Other amino acids (serine, asparagine, aspartic acid, glutamine, glutamic acid, alanine, cysteine, glycine, tyrosine, and proline) are considered non-essential because insects can synthesise these amino acids in their metabolic pathways. The potential for non-essential amino acid synthesis differs among insect species, and even though non-essential amino acids can be synthetised, their deficiencies can decelerate insect growth (Chapman, 2013). Hence, insects grow better when their diet is supplemented with non-essential amino acids, especially aspartic and glutamic acids (Dadd, 1973). For example, the addition of aspartic and glutamic acids improved the growth of the silkworm Bombyx mori Linnaeus (Lepidoptera: Bombycidae). The adequate protein level is species dependent, and it may also change during insect development when physiological needs are different (Woodring et al., 1977; Nation, 2016; Reifer, et al. 2018). For example, juvenile Jamaican field crickets Gryllus assimilis Fabricius (Orhoptera: Gryllidae) reached the adult stage faster and were larger in size when they were provided with a protein-rich diet (Reifer et al., 2018). Female insects require protein for the ovary maturation and egg production. The lack of dietary amino acids can affect the serine/threonine kinase TOR pathway that regulates the biosynthesis of the juvenile hormone. Juvenile hormones control vitellogenesis in Hemimetabola and are important for ovary and egg development (Smykal & Raikhel, 2015).

Carbohydrates serve as the main energy source for most insects. Even so, insects do not have specific requirements for carbohydrates, and in fact, insects can generally form carbohydrates, glucose sugar, from amino acids and lipids by gluconeogenesis (Nation, 2016). Carbohydrates (polysaccharides, oligosaccharides, and monosaccharides) are used as fuel, transformed to lipids, or used to form the skeleton for amino acid synthesis (Cohen, 2004; Chapman 2013). The insect cuticle forms an exoskeleton that is formed from chitin (Merzendorfer & Zimoch, 2003). Chitin is a polysaccharide formed from glycogen and trehalose (Muthukrishnan et al., 2012). Some insect species need carbohydrates from their feeds to reach maturity, while other species do not have such requirements (Crompton &

Birt, 1967; Nation, 2016). Some insects can digest cellulose, such as termites

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27 (Isoptera) and some beetles (Coleoptera); however, cellulose digestion is rare in insects (Martin, 1983; Cohen, 2004). Nevertheless, cellulose can be used as a filler to help digestion (Cohen, 2004).

Lipids are a group of biological molecules, including sterols, oils, fats, and phospholipids. Most insects can convert carbohydrates into lipids with lipogenesis and store them in the fat body (Arrese & Soulages, 2010). Unlike vertebrates, insects cannot build sterols, and therefore, insects have a sterol requirement from their food (Behmer & Nes, 2003). Sterols work as membrane components in lipid biostructures, and they regulate developmental processes. Sterols are also important for waxes of the cuticula cortex and for the synthesis of hormones that are part of moulting (Klowden, 2007). Insects also have a dietary requirement for polyunsaturated fatty acids, more specifically linoleic or alpha-linolenic acids (Canavoso et al., 2001). Fatty acids are present in the form of triglycerides and diacylglycerides. Fatty acids contain a carbon chain with a carboxyl group at the end (Britannica, 2020). Depending on the type and amount of carbon-to-carbon bonds, fatty acids are categorised as saturated, monounsaturated, or polyunsaturated. Insects can synthesise many fatty acids; however, the dietary requirement of polyunsaturated fatty acids is well known among Lepidotera, Coleoptera, and Orthoptera (DeRenobales et al., 1987; Stanley-Samuelsson et al., 1988; Chapman, 2013).

Vitamins are either water soluble or lipid soluble compounds, and many of them are required for the development of insects (Cohen, 2004;

Chapman, 2013). Water soluble vitamins stay for a shorter time in insect bodies, whereas lipid soluble vitamins tend to accumulate in fat storage. Of the water-soluble vitamins, insects require C and B vitamins. Livestock animals’ vitamin deficiency causes certain diseases, such as beriberi and scurvy; however, similar diseases or specific growth defects have not been reported for insects (McDowell, 2005). Of the lipid-soluble vitamins, insects require the vitamin A complex (β-carotene and related carotenoids) and vitamin E (tocopherols). Vitamin A is important for the formation of eye pigments, and vitamin E is necessary for reproduction, spermatogenesis, and egg maturation (Cohen, 2004).

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Insects also require specific minerals for coenzymes and metalloenzymes (Cohen, 2004). Minerals, different metal ions, are usually available in artificial diets as impurities; however, insects require the addition of sodium, potassium, phosphate, and chloride, which are required for cellular ion balance (Chapman, 2013).

1.3.2 Artificial diets for insect rearing

The development of artificial diets for insects started in the 1960’s and has been a prerequisite for maintaining laboratory insect populations (Panizzi &

Parra, 2012). There are over 1,300 insect species that are reared on artificial diets for research purposes or for pest control (Singh & Moore, 1985). The development of artificial diets has provided important information about insects’ nutritional requirements. Fulfilling the nutritional requirements of insects has been the main goal in the development of these diets; however, their sustainability has not attracted much attention. While the rearing of insects for human consumption has recently gained attention in the Europe and the US and the mass-rearing technologies, which are currently being developed, will benefit from earlier research and development of rearing practices, including acceptable feeds in rearing conditions (Rumpold &

Schlüter, 2013).

Artificial diets for insects have traditionally included many purified protein sources. Examples are casein from milk, gluten from wheat albumin from eggs, and soybeans or peanut protein (Nation, 2016). Artificial diets typically include one or more different protein sources since single purified protein sources rarely satisfy the amino acid requirements of insects.

Soybeans are used widely in animal nutrition because of their nutritional and economic values (Cohen, 2004). They have a high protein content and balanced amino acids and are a valuable source of vitamins, minerals, and fatty acids. In addition, the use of soybeans in animal feeds has been economically affordable. However, the use of soybean comes with many environmental issues, including their negative contribution to climate change and deforestation (da Silva et al., 2010; Richards et al., 2012).

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29 Patton (1967, 1978) developed several artificial diets for rearing the house cricket (A. domesticus). These included 16 oligidic diets differing in the nutrient content of proteins, carbohydrates, and fats, mixing commercial feed ingredients. Patton’s diet no. 16 was referred to as an optimal diet that allows crickets to grow and develop consistently in optimal rearing conditions. This diet contains typical components of artificial diets: soybean, wheat, skimmed milk, brewer’s yeast, and powdered animal liver. The diet contains 30% protein, 37% carbohydrates, and 5% fat. The observations of McFarlane (1964) also suggested a 20% or higher protein content for the optimal growth of A. domesticus.

Many artificial feeds that were originally developed for conventional livestock have been used in insect rearing, even though they include many ingredients that are not required by insects (Patton, 1978). Insect mass- rearing production is still in its infancy, and therefore, the commercial production of insect feeds in large quantities is an underdeveloped field. For example, chicken feeds that contain up to 21% protein are commonly used in cricket rearing (Hanboonsong, 2013). Many studies have used chicken feed as a control diet while investigating the suitability of new feed components for insect rearing. In addition, chicken feed (Lundy & Parrella, 2015; Dobermann et al., 2018; Bawa et al., 2020; Ng’ang’a et al., 2020), dog feed (Malinga et al., 2018b), and Patton’s diet are typically used in research.

Although many animal feeds allow insects to grow and develop well, some might include feed components, such as minerals, in unnecessary amounts (Chapman, 2013). In addition, many animal feeds may include feedstuff, such as additives or harmful substances that are not currently permitted to use in the insect feeds (Commission Regulation (EU) No 68/2013).

1.3.3 By-products as insect feeds

Recently, there have been attempts to increase the sustainability of insect rearing by using fewer resources (Van Huis & Tomberlin, 2017). By incorporating different food and agroindustry by-products in insect feeds, the environmental impact of insect rearing could be decreased (Smetana et

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al., 2016). A variety of plant-based by-products could replace traditional feed ingredients in insect feeds, and such adjustments can also be cost-effective (Makkar & Ankers, 2014; Pinotti et al., 2019). However, the feeds need to be designed specifically for each insect species to enhance their welfare and healthy development (Makkar & Ankers, 2014). Currently, incorporating different reused plant materials into insect feeds, such as food waste or by- products from agriculture and the food industry, is gaining attention (Čičková et al., 2015; Vaga et al., 2021). Farm weeds (Ng’ang’a et al., 2020), soy extract, corn meal, dried cowpea (Straub et al., 2019), rice bran, and spent grains (Orinda et al., 2017; Dobermann et al., 2018) have been under investigation for G. bimaculatus feed. Buckwheat, dry cabbage (Morales- Ramos et al., 2020), kale, sweet potato vines, banana leaves (Oloo et al., 2019), rice bran (Orinda et al., 2017), potato peels, beer yeast (Oonincx et al., 2015a), food waste, and crop residues (Lundy & Parrella, 2015) have been studied for A. domesticus feed. Rice bran, water spinach, cassava plant tops (Miech et al., 2016), taro parts, and cashew leaves (Caparros Megido et al., 2016) have been under investigation for Teleogryllus testacus Walker (Orthoptera: Gryllidae) cricket feeds.

Even though offering by-products from agriculture or the food industry is an appealing way to reuse different kinds of plant materials, they also need to ensure animal welfare and the normal growth and development of insects. The bare use of by-products, unprocessed or low-quality materials, or food waste might not be adequate for insect growth and development, or they can lower the feed conversion ratios (Lundy & Parrella, 2015; Ooninxc et al., 2015a) if the nutritional requirements of insects are not met. Further research is necessary to screen adequate feed mixtures that would maximise the use of by-products.

1.4 Studied edible insects

Globally, there are many edible insect species that are reared for either food or feed purposes. Cricket rearing has long traditions, especially in Thailand (Hanboonsong, et al., 2013; van Huis, 2020). Insects are not only produced

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31 for human food, but they have also been reared for pet feeds. House cricket (A. domesticus) and two-spotted crickets (G. bimaculatus) are the most important and economically profitable species. However, in many regions of the world, edible insects are mainly harvested and gathered from nature.

For example, R. differens is an edible insect species collected from the wild in Sub-Saharan Africa (van Huis et al., 2013). There have been attempts to develop mass-rearing methods for this species for small-scale farm rearing (Lehtovaara et al., 2018; Malinga et al., 2018a, 2018b; Rutaro et al., 2018a, 2018b, 2018c; Lehtovaara et al., 2019). Such methods could strengthen the economic status of local people and support food security in Sub-Saharan Africa. Thus, it is necessary to find new practical solutions for rearing several economically valuable edible insects and to reduce the ecological footprint by finding alternative feed ingredients for these species specifically.

1.4.1 House cricket (Gryllidae: Acheta domesticus)

The house cricket A. domesticus is a commercially reared cricket species for both food and feed consumption (Figure 1A). This species originates from Southwestern Asia, but today, it is distributed all over the world (Ghouri, 1961). House cricket rearing has long traditions in Thailand, where it is popular for human consumption (Hanboongsong et al., 2013; Van Huis, 2020). House crickets are generally easy to rear, and it has been possible to upscale their production from small-scale farming systems to large-scale cricket farms (Durst & Hanboonsong, 2015). The life cycle of the house cricket is, on average, 30 to 49 days at 28°C (Booth & Kiddell, 2007). A short life cycle enables farmers to make quick profits for their investments. House crickets are omnivorous species, and they can utilise both animal and plant- based food sources. Typically, house crickets are reared in large quantities with chicken feed and vegetable remains. Many studies have focused on finding new alternative feed components for this species, especially different food or agroindustry by-products (Collavo et al., 2005; Lundy &

Parrella, 2015; Oonincx et al., 2015a). With a well-balanced diet that contains

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by-products, house cricket rearing could be more environmentally sustainable, and the feed conversion ratios could be enhanced.

1.4.2 Two-spotted cricket (Gryllidae: Gryllus bimaculatus)

The two-spotted cricket G. bimaculatus (Figure 1B), also called the African field cricket, Mediterranean field cricket, or black cricket, is reared both for human consumption and for livestock feeds, for example, for fish and poultry (Van Huis, 2020). Many cricket farmers prefer G. bimaculatus over A.

domesticus because they develop faster and are larger in size but are also easier to sell (Halloran et al., 2017). In standardised conditions, the life cycle of G. bimaculatus lasts 42 days, which is shorter than the 49-day life cycle of A. domesticus (Halloran et al., 2017).

1.4.3 African bush cricket (Tettigoniidae: Ruspolia differens)

Ruspolia differens Serville (Orthoptera: Tettigoniidae) (Figure 1C), known as the African edible bush cricket or ‘nsenene,’ is a popular edible insect in Eastern Africa, especially in Lake Victoria basin (van Huis et al., 2013). It is a nocturnal long-horned grasshopper that swarms during and after rainy seasons (Bailey & McCrae, 1978). Swarms are attracted to lights, and they are commercially harvested using bright light traps (Mmari et al., 2017).

There have been attempts to develop mass-production systems for this species (Lehtovaara, 2019; Malinga et al., 2018a, 2018b; Rutaro et al., 2018a, 2018b) that could prevent overharvesting of natural wild populations in the long term. R. differens is an oligophagous grass specialist (Valtonen et al., 2018), and it prefers certain grasses and inflorescences (Opoke et al., 2019).

In the laboratory, R. differens has been reared with many African grasses but also with artificial feeds or locally available ingredients, such as whey, finger millet, rice, chicken feed, and sorghum (Lehtovaara et al., 2017; Valtonen et al., 2018; Malinga et al. 2018a, 2018b). Feed development for R. differens is one step in developing mass-rearing technology for this valuable edible insect species. Incorporating food industry by-products in their feed is an

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33 appealing possibility to avoid the use of ingredients in their feeds that could be consumed by humans. Additionally, the nutritional requirements of this species, e.g., its protein requirement, are not well understood. Such information is necessary for future feed design.

Figure 1. The studied edible insect species in adult stage: A. house cricket (Acheta domesticus), B. two-spotted cricket (Gryllus bimaculatus) and C.

Ruspolia differens. Photos: Jaana Sorjonen.

1.4.4 Objectives of this study

Since many edible insect species can utilise a variety of plant-based materials as their food, we could incorporate materials that are not consumed by humans into insect feeds to increase the ecological sustainability of insect rearing. The goal of this study was to develop artificial diets for rearing A. domesticus, G. bimaculatus, and R. differens by combining earlier information on insect feeds and nutritional requirements with the current need to reuse materials from food industries that are not consumed by humans. Furthermore, the goal was to improve our general understanding of insect feeds and to determine practical solutions for insect production. Artificial diets that contain side streams from the food industry have been previously lacking or they have not been sufficient for the growth and development for these species.

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The specific study objectives were:

1. To investigate how R. differens grows and develops on by-product diets;

to determine if the diets can alter the fatty acid content and composition of R. differens; and to identify the protein-level requirement in the feed (I).

2. To investigate how crickets A. domesticus and G. bimaculatus grow and develop on diets including food industry by-products (II).

3. To examine the effect of by-product diets on the fatty acid composition and content of crickets (III).

4. To evaluate how artificial diets with the most potential for crickets perform in small-scale farming conditions (IV).

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2 Materials and methods

2.1 Source populations

R. differens laboratory populations (I) were derived from natural populations from Kabanyolo, Kampala, Uganda. Individuals were reared at the Department of Environmental and Biological Sciences, University of Eastern Finland, with methods described in detail in Lehtovaara et al. (2019). Insects were fed with oatmeal and oat seeds and reindeer feed pellets (Poro-Elo 1, Suomen Rehu, Hyvinkää, Finland), and water absorbed in cotton was offered in plastic cylinder jars. A piece of tissue paper was placed over the top of the container to offer a moulting site and hiding place.

Cricket populations (II, III) were derived from laboratory populations maintained at the Department of Environmental and Biological Sciences, University of Eastern Finland. Crickets were feed ad libitum on an equal mix of three commercial animal feeds, two chicken feeds Punaheltta Paras Poikanen and Punaheltta Paras Kana (Suomen Rehu, Finland) and one reindeer feed Poro-Elo 1 (Suomen Rehu, Hyvinkää, Finland). Additionally, we offered water absorbed in tissue paper, and pieces of fresh carrot. Both A.

domesticus and G. bimaculatus populations were kept in large plastic rearing containers. For egg-laying, we placed smaller plastic containers filled with peat and vermiculate, with a metal mesh lid, in the rearing containers.

During rearing, crickets were kept in LD 12:12 photoperiod.

A. domesticus crickets for the small-scale farming experiment (IV) were derived from the cricket population at the Niittykumpu farm, Joensuu, Finland. They were fed cricket feed (Rehux, Finland, distributer Entocube Ltd.) and maintained at 29°C, with a constant photoperiod of 12:12 L:D.

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2.2 Feed design

All experiments included experimental diets with by-products and control diets (with no by-products). Control diets for R. differens (I) included two types of Suomalainen (1999) mealworm diet, chicken feed and reindeer feed. Control diets for crickets (II) included two types of modified Patton’s diet no. 16 (Patton, 1967) and two types of chicken feeds. Control diets in the small-scale farm experiment (IV) included commercial cricket feed (manufacturer Rehux, distributer Entocube).

For crickets, the experimental diets with by-products (I, III, IV) were based on Patton’s diet no 16 (Patton, 1967), and for R. differens (II), based on Patton’s diet or the Suomalainen mealworm diet (Suomalainen, 1999). In these diets, the main protein source (e.g., soybeans in Patton’s diet) was partly or completely replaced with food industry by-products. The by- products were selected among those readily available from the Finnish food industry. They included potato protein, barley mash (brewer’s spent grain), barley feed from ethanol industry, compressed leftover of turnip rape, a mix of broad bean and pea, and a mix of potato, carrot, and apple. Details of each experimental diet and the sources of by-products are given in I—IV.

The experimental diets with by-products were designed with the WinOpti program, which is a feed design and optimisation program for the traditional livestock (AgroSoft WinOpti A/S, Agrosoft Ltd, Tørring, Denmark). The nutritional information of each diet ingredient was inserted as input data into the program, and each diet was designed to reach a certain protein level, while fat and carbohydrate levels were allowed to vary. Since the nutritional composition of diet ingredients, including by-products differed, the by-product proportions also differed in the experimental diets.

2.3 Experimental design

In study I, we evaluated the effect of diet on the survival, development time, weight, FCR, and fatty acid composition of R. differens. The experiment included 16 diet treatments, 12 of which included by-products (potato

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37 protein, barley mash, barley feed, turnip rape, a mix of broad bean and pea, and a mix of potato, carrot, and apple). The experimental diets were designed to reach six different protein levels: 7.23, 9.75, 15.0, 17.4, 22.5, and 30.5%. Four control diets were used: chicken feed, reindeer feed, and two mealworm diets (Suomalainen, 1999). In this study, 4th instar R. differens individuals were reared until adulthood so that the experiment was terminated one week after half of the individuals reached adulthood. Four R. differens nymphs were placed in each plastic rearing container (replicate), and each container was randomly subjected to the experimental diets. Each experimental diet had ten replicates. The experiment was conducted over the course of two experimental rounds (times), and the Suomalainen diet was applied on both experimental times. The performance of R. differens was evaluated as weight, survival, development time, and FCR. Additionally, the effect of diet on the fatty acid composition and the content of TFA, SFA, MUFA, and PUFA were evaluated.

In study II, we evaluated the effect of diet on the yield, performance, and ECI of A. domesticus and G. bimaculatus in a laboratory experiment. The study included 18 diet treatments, 14 of which contained food industry by- products. The by-products were potato protein, barley mash, barley feed, turnip rape, and a mix of broad bean and pea. The diets were designed to reach three different protein levels: 15.0, 22.5, and 30.5%. The control diets included two types of modified Patton’s diet no. 16 and two types of chicken feed. In this experiment, 15-day-old nymphs were reared until adulthood so that the experiment was terminated one week after half of the individuals reached adulthood. Crickets were reared in plastic containers (replicates), each containing ten crickets. Containers were randomly subjected to each experimental diet, with each diet having ten replicates. The experiment took place in three experimental rounds (times), and one control diet (chicken feed) was applied during each experimental time. The effect of diet on performance was evaluated using five performance traits: survival, weight, RGR, developmental rate, and ECI.

In study III, we determined the fatty acid content and composition of A.

domesticus and G. bimaculatus crickets that were fed on by-product diets

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(study I). The effect of diet was determined on the content of TFA, SFA, MUFA or PUFA, and FA composition of crickets. A total of 56 samples of A.

domesticus individuals, representing 14 diet treatments, and 24 samples of G. bimaculatus individuals, representing seven diet treatments, were analysed. We also examined the similarity among the fatty acid composition of cricket individuals and their feed.

In study IV, we tested cricket performance on four by-product diets, including turnip rape and barley mash, in the small-scale farming system compared to the laboratory. A commercial cricket feed was used as a control diet, and two experimental diets with by-products, including mixtures of turnip rape and barley mash by-products, were included to determine whether the diet influenced the yield, development time, or FCR of A.

domesticus. The experiment was conducted at a local small-scale cricket farm in Joensuu and in the laboratory at the Department of Environmental and Biological Sciences (with the same equipment and conditions). In this experiment, crickets were reared in 80 L plastic containers (N = 45). We also calculated a feed cost estimate for the by-product diets.

In all experiments, a randomised block design was used to determine the effect of diet on the performance variables. This design was selected because it controls for environmental variation and more efficiently reveals the effect of diet treatments. In experiments I–III, blocks represented the rearing chambers, where the temperature was controlled. In experiment IV, the rearing containers were spatially grouped as blocks sharing similar conditions. In all experiments, a 12:12 L:D photoperiod was used, and insects were allowed to feed ad libitum. Water was absorbed in paper tissue (I–III) or an upside-down glass-water system (IV). When experiments were terminated, the insects were killed by freezing them at -18°C.

2.4 Chemical analyses

Samples (insect individuals) were prepared for chemical analyses by first freeze drying for 22 h, and then final drying 1 h (Alpha 1-4 LD Plus, Christ, Osterode am Harz, Germany) (I, III). A composite of feed samples from each

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39 diet treatment were freeze dried with the following set-ups: main drying for 24 h and final drying for 6 h (III).

2.4.1 Fatty acid analyses

We analysed the fatty acid composition and content from four to five R.

differens females from each diet treatment (n = 76) (I). In study III, we analysed fatty acid composition and content from four A. domesticus females from each diet treatment (n = 56) and four G. bimaculatus females from seven diet treatments (n = 24) (i.e., two control diets and five by-product diets that did not contain soybean). Additionally, one composite sample of each diet treatment was analysed (III). The R. differens and G. bimaculatus individuals and the feed samples were analysed at the Center of Food and Fermentation Technologies in Tallinn, Estonia with a method described by Sukhija and Palmquist (1988) with minimal modifications. To quantify the fatty acid methyl esters (FAME), gas chromatography with flame ionisation detection (GC-FID) was used. Common fatty acids were identified by comparing the retention times of sample peaks with FAME standards. A.

domesticus crickets were analysed at the Department of Environmental and Biological Sciences, University of Eastern Finland, using an extraction method by Folch et al. (1957). A gas chromatograph-mass spectrometer (GC- MS) was used to quantify the FAMEs. FAME identification was based on their retention times and mass spectra.

2.4.2 Glycoalkaloid analyses

Glycoalkaloid analyses were performed for A. domesticus (I) and R. differens (II) that were subjected to potato protein diets. Four A. domesticus individuals subjected to the two potato protein diets (n = 8) (I) and four female R.

differens (II) from each potato protein diet (n = 16) were randomly selected for glycoalkaloid analyses. Glycoalkaloid analyses were carried out in the Center of Food and Fermentation Technologies in Tallinn, Estonia. The α-

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solanine and α-chaconine concentrations were determined using UPLC-MS internal standard method.

2.5 Statistical analyses

In study I, a general linear model (GLM) was fitted to test whether development time, weight, or FCR of R. differens differed among diet treatments (for details of models, see II). To model survival, a binary logistic generalised linear model for events/trials data was fitted. We used Spearman’s rank correlation was used to test for the association between the protein level of the diet and each performance variable. The similarities in fatty acid composition (%) among individuals were visualised with nonmetric multidimensional scaling (NMDS). The permutational multivariate analysis of variance (PERMANOVA) was used to test for the differences in the fatty acid composition among diet treatments. PERMDISP routine was conducted to determine if the degree of variability differed among diet treatments. Finally, the routine RELATE was used to test if there was an association between the similarity matrices of fatty acid composition in R. differens individuals and their feeds.

In study II, linear mixed models were fitted to analyse the effect of diet on the weight, survival, relative growth rate (RGR = fresh weight gain during feeding period (g) ∕ (duration of feeding (d) × mean fresh weight during the feeding period (g) (Waldbauer 1968), ECI, and yield of crickets (for details of models, see II). To model survival, a binary logistic model for events/trials data was fitted. To determine which diet treatments differed from each other, the least significant difference (LSD) pairwise test was used.

In study III, we fitted one-way ANOVA models to determine if the diet modified the TFA, SFA, MUFA, and PUFA content of crickets. If there were significant differences among diet treatments, Bonferroni pairwise tests were used to determine which treatments differed significantly from each other. PERMANOVA was used to test if the diets differed in the produced fatty acid composition of insects. We also used PERMDISP routine to analyse differences in the degree of within-treatment variability among the diet

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41 treatments, and SIMPER routine to evaluate which fatty acids explained the dissimilarities in fatty acid composition among diet treatments. Finally, we conducted RELATE routine to test matching multivariate patterns in cricket individuals and their feed.

In study IV, three linear mixed models (LMM) to analyse, if diet influenced yield, development time, and FCR (for details of models, see IV).

Multivariate analyses were conducted with Primer-E version 6 and PER- MANOVA+ expansion (Clarke & Gorley, 2006; Anderson et al., 2008). Other statistical analyses were conducted with IBM SPSS statistics version 23–27.

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Could edible insects be one solution for a circular economy and food production? : designing feeds for insects using food industry by-products

Dissertations in Forestry and Natural Sciences

JAANA M. SORJONEN

COULD EDIBLE INSECTS BE ONE SOLUTION FOR A CIRCULAR ECONOMY AND FOOD PRODUCTION?

Could edible insects be one solution for a circular economy and food production?

Could edible insects be one solution for a circular economy and food production?

Acknowledgements

Table of contents

1 Introduction

2 Materials and methods