Dissertations in Forestry and Natural Sciences › pub › urn_isbn_978-952-61... · 2020-06-30 · ISBN: 978-952-61-3321-8 (PDF) ISSN: 1798-5676 (PDF) ABSTRACT Consumption of edible

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371

KARLMAX RUTARO

FATTY ACID PROFILES OF THE EDIBLE KATYDID, RUSPOLIA DIFFERENS (SERVILLE) (ORTHOPTERA:

TETTIGONIIDAE) AFTER FEEDING ON DIVERSIFIED DIETS


Rearing of edible insects is seen as one

solution to ensure a sufficient food production for the increasing human population.

A successful mass-rearing programme for edible insects such as the edible katydid, Ruspolia differens, in rural Africa would require affordable but reliable feeds. This

thesis provides novel insights on how feeds influence the fatty acid profiles of R. differens, with emphasis on the essential fatty acids that

are needed for a healthy human diet.

KARLMAX RUTARO

FATTY ACID PROFILES OF THE EDIBLE KATYDID, RUSPOLIA DIFFERENS (SERVILLE)

(ORTHOPTERA: TETTIGONIIDAE) AFTER FEEDING ON DIVERSIFIED DIETS

Karlmax Rutaro

FATTY ACID PROFILES OF THE EDIBLE KATYDID, RUSPOLIA DIFFERENS (SERVILLE)

(ORTHOPTERA: TETTIGONIIDAE) AFTER FEEDING ON DIVERSIFIED DIETS

Publications of the University of Eastern Finland


No 371

University of Eastern Finland

Joensuu

2020

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry, University of

Eastern Finland

for public examination in the Conference room in the School of Forestry at

Makerere University, Kampala, on March, 26, 2020, at 12 noon

Grano Oy

Jyväskylä, 2020

Editor: Professor Raine Kortet

Distribution: University of Eastern Finland / Sales of publications

www.uef.fi/kirjasto

ISBN: 978-952-61-3320-1 (Print)

ISBN: 978-952-61-3321-8 (PDF)

ISSNL: 1798-5668

ISSN: 1798-5676

ISSN: 1798-5676 (PDF)

Author’s address: Karlmax Rutaro


Depart. of Environmental and Biological Sciences

P.O. Box 111

80101 JOENSUU, FINLAND

email: [email protected]

Supervisors: Professor Heikki Roininen, Ph.D.



P.O. Box 111



Anu Valtonen, Ph.D.



P.O. Box 111



Professor Philip Nyeko, Ph.D.

Makerere University

Depart. of Forestry, Biodiversity and Tourism

P.O. Box 7062 KAMPALA, UGANDA


Geoffrey Maxwell Malinga, Ph.D.

Gulu University

Depart. of Biology

P.O. Box 166 GULU, UGANDA


Reviewers: Dennis Oonincx, Ph.D

Wageningen University and Research Centre

Animal Nutrition Group

PO 338, 6700AH, Wageningen, the Netherlands


Robert Fungo, Ph.D

Makerere University

Depart. of Food Technology and Human Nutrition

P.O. Box 7062

KAMPALA, UGANDA


Opponent: John N. Kinyuru, PhD

Jomo Kenyatta University of Agriculture and Technology

Depart. of Food Science and Technology

P.O. Box 62000-00200, Nairobi, Kenya


7

Rutaro, Karlmax

Fatty acid profiles of the edible katydid, Ruspolia differens (Serville) (Orthoptera:

Tettigoniidae) after feeding on diversified diets.

Joensuu: University of Eastern Finland, 2020

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences 2020; 371

ISBN: 978-952-61-3320-1 (print)

ISSNL: 1798-5668

ISSN: 1798-5676

ISBN: 978-952-61-3321-8 (PDF)

ISSN: 1798-5676 (PDF)

ABSTRACT

Consumption of edible insects is seen as a major solution to address the looming

food shortage (due to the increasing human population) and for improving human

nutrition. For example, in Africa and Asia, edible insects have been a traditional

part of the human diet, although with limited documentation. However, the majori-

ty of edible insects in Africa are seasonally harvested from the wild, which unfor-

tunately is unpredictable. Currently, there is growing interest in mass rearing edi-

ble insects to meet the increased food demand and supplement mostly carbohy-

drate-rich diets. A successful mass-rearing programme for edible insects such as the

edible katydid, Ruspolia differens, in rural Africa would require affordable but relia-

ble feed. However, before mass-rearing programmes can commence, there is a great

need to understand accepted feeds and their influence on R. differens nutritional

profiles.

The aim of this dissertation was to investigate how natural (i.e., grass inflo-

rescence) and artificial diets influence the fatty acid content and composition of R.

differens, with emphasis on the essential fatty acids that are needed for a healthy

human diet. Based on earlier studies, R. differens performance is enhanced with

mixed diets. The first objective (study I) was to examine the effect of a diversified

gradient of grass inflorescence diets on the lipid content and fatty acid composition

in R. differens. Here, R. differens sixth instar nymphs were reared on inflorescences

from one grass species or mixtures of inflorescences from two, three or six grass

species for two weeks. The results from this study indicated that the fatty acid

composition and the proportion of essential fatty acids significantly differed among

the diets. The proportion of essential fatty acids was highest in the highly diversi-

fied, six-feed diet but low in less diversified (one-to-three-feed) diets. Total R. dif-

ferens lipid content did not significantly differ among the diets. The nine common

8

fatty acids found across all treatments in this study were (in decreasing order):

palmitic, oleic, palmitoleic, linoleic, stearic, myristic, myristoleic, α-linolenic and

arachidic acid. These findings demonstrated that the R. differens fatty acid composi-

tion can be modified through diet.

The second objective (study II) was to examine the influence of diversified

mixtures of the most accepted natural feeds (grass inflorescences) on the fatty acid

content and composition of R. differens when they are reared from neonatal nymphs

to adults. The results indicated that the contents of saturated, monounsaturated and

polyunsaturated fatty acids, the omega-6/omega-3 ratio and adult body weight did

not differ among insects based on the dietary treatments. However, the composi-

tion of rare (> C20) fatty acids differed significantly among the insects fed on the

six-diet treatment. Furthermore, the omega-6/omega-3 fatty acids ratio was general-

ly low compared to when artificial diet (study III) was used, data that suggest R.

differens reared entirely on grass inflorescences may be suitable for a healthy human

diet.

The third objective (study III) was to assess the influence of diversified lo-

cally sourced, processed and non-processed (artificial) diets on R. differens fatty acid

content and composition. Here, neonatal nymphs were reared on mixtures of six

gradually diversified diets of two, three, four, six, eight or nine feeds. The findings

indicated that the contents of saturated, monounsaturated and polyunsaturated

fatty acids differed significantly among the diets; more diverse diets increased the

polyunsaturated fatty acid content. Furthermore, the omega-6/omega-3 ratio dif-

fered significantly among the diets and between the sexes. R. differens fed on the

four-feed diet had a higher omega-6/omega-3 ratio than those fed on other diets.

Again, the fatty acid composition differed significantly among the diets, and diet

diversification corresponded with the polyunsaturated fatty acid proportions, espe-

cially linoleic acid. The findings of study (III) demonstrated that higher essential

fatty acid levels can be achieved when R. differens is reared on highly diversified

artificial diets.

Overall, the study concluded that the R. differens fatty acid content and

composition was significantly influenced by both grass inflorescences and artificial

diets. The study further revealed that high-quality essential fatty acids needed for

human health can be achieved through dietary manipulations of both grass inflo-

rescence and artificial diets; high essential fatty acid levels are achieved through

highly diversified diets. Highly diversified diets may help balance the ingested

nutrients. Further studies are needed to determine the extent of diet diversification

vis-à-vis the quality of fatty acids, especially essential fatty acids.

9

Universal Decimal Classification: 591.53.063, 591.613, 595.720, 638.4

CAB Thesaurus: insects as food; Orthoptera; Tettigoniidae; Ruspolia differens; nutritive

value; lipids; fatty acids; essential fatty acids; saturated fatty acids; unsaturated fatty acids;

monoenoic fatty acids; polyenoic fatty acids; animal feeding; feeds; diets; diversification;

grasses; inflorescences; manufactured feeds; body weight

Yleinen suomalainen ontologia: hyönteiset; hyönteisruoka; suorasiipiset; hepokatit;

ravintoarvo; lipidit; rasvahapot; omegarasvahapot; ruokinta; rehut; monipuolisuus

10

“Your attitude, not your aptitude, will determine your altitude.” Zig Ziglar

11

ACKNOWLEDGEMENTS

My heartfelt thanks go to my supervisors Professors Heikki Roininen and Philip

Nyeko, Dr. Anu Valtonen and Dr. Geoffrey M. Malinga, first for selecting me

among many to undertake this study and for guiding me this far. Your invaluable

comments have greatly helped to shape this work. Thesis reviewers, Dr. Dennis

Oonincx and Dr. Robert Fungo are greatly thanked for their insights on the thesis.

To the various ‘anonymous’ reviewers and journal editors, thank you for your

comments some a bit harsh, but very insightful. I feel proud that my publications

already have been cited severally.

I thank the administration of the Department of Environmental and Biolog-

ical Sciences, University of Eastern Finland, Joensuu for providing a good working

environment during my stay at Joensuu and for coordinating the study activities

while in Uganda. In addition, I wish to thank the Uganda National Council of Sci-

ence and Technology (UNCST) for permission to conduct this study and the Mak-

erere University Agricultural Research Institute, Kabanyolo (MUARIK) for provid-

ing laboratory space.

Also, I extend my gratitude to Makerere University Council through the of-

fice of the Vice Chancellor for offering me a study leave and providing me other

related support whenever needed throughout the study period. To my colleagues

at the department of Biochemistry and Sports science, especially the HOD Dr. Peter

Vuzi, thank you for the support. Dr. Joseph Kyambadde, Dean SBS, together with

Professor JYT. Mugisha, the Principal, CONAS, your constant follow ups on my

progress gave this work focus, resulting in its record time completion. I really ap-

preciate your support.

I ‘am thankful to my colleagues, VJ. Lehtovaara and R. Opoke for helpful

comments during the writing of the manuscripts, on which this thesis is based.

Robert, our overnight field and laboratory ‘visits’ have not gone to the waste, and

coming this far amidst challenges well known to you confirms that ‘’faith is the only

antidote for failure’’. VJ. Lehtovaara, thank you for ice skiing lessons during the visit

to your summer cottage. The whole experience of skiing and walking on a frozen

lake is still alive and fresh in my mind, kiitos. Furthermore, my gratitude goes to

Mirja Roininen for her hospitality during my visit to her home, and Dr. Jaakko Ha-

verinen for introducing me to many places within Joensuu and for being a wonder-

ful housemate. For this, I say kiitos. Ms. Tuula Toivanen, thank you very much for

the care and for quickly fixing financial issues, whenever approached.

Lastly, I ’am grateful to my family especially my dear wife for her kind

love, patience, and for taking good care of the children during several long and

short absences both within and out of the country in pursuit of this study. To my

12

children, Jo, Gabi, Esther and Jysen you have been the source of strength, some-

times through your jokes and the unending ‘homework issues at times disrupting

my own’, but your ‘necessary disruption’ always relaxed my mind amidst the

tough journey this was. I could not give up, because in me, I knew that completing

this thesis ’’wasn’t about me, but us’’, and I want you to draw your current and

future strength from this triumph.

Funding for this study was provided by the Academy of Finland (grant no

14956) and Bugbox Limited supported fatty-acid analysis in Estonia. Emeritus Pro-

fessor Heikki Roininen, and Mr. Erlend Sild, thank you so much for the logistical

support.

Kampala, January 2020

Karlmax Rutaro

13

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

BAME Bacterial acid methyl ester

CVD Cardiovascular diseases

DHA Docosahexaenoic acid

FA Fatty Acids

FAME Fatty acid methyl ester

FAO Food Agriculture Organization

FID Flame ionization detector

GC-MS Gas chromatography-mass spectrometry

LC-PUFA Long chain polyunsaturated fatty acid

MUFA Monounsaturated fatty acid

n-3 Omega-3 fatty acids

n-6 Omega-6 fatty acids

NADPH Nicotinamide Adenine Dinucleotide Phosphate (reduced)

NMDS Non-metric multidimensional scaling

PEG Poly ethylene glycol

PERMDISP Permutational analysis of multivariate dispersions

PUFA Polyunsaturated fatty acid

SFA Saturated fatty acid

SIMPER Similarity of percentages analysis

14

15

LIST OF ORIGINAL PUBLICATIONS This thesis is based on data presented in the following articles, referred to by the

Roman Numerals I-III.

I Rutaro K, Malinga GM, Lehtovaara VJ, Opoke R, Valtonen A, Kwetegyeka J,

Nyeko P, Roininen H. (2018). The fatty acid composition of edible

grasshopper Ruspolia differens (Serville) (Orthoptera: Tettigoniidae) feeding

on diversifying diets of host plants. Entomological Research, 48: 490-498.

II Rutaro K, Malinga GM, Opoke R, Lehtovaara VJ, Nyeko P, Roininen H,

Valtonen A. (2018). Fatty acid content and composition in edible grasshopper,

Ruspolia differens feeding on mixtures of natural food plants. BMC Research

Notes, 11: 687.

III Rutaro K, Malinga GM, Opoke R, Lehtovaara VJ, Omujal F, Nyeko P,

Roininen H, Valtonen A. (2018). Artificial diets determine fatty acid

composition in edible grasshopper Ruspolia differens (Orthoptera:

Tettigoniidae). Journal of Asia-Pacific Entomology, 21: 1342-1349.

The above publications have been included at the end of this thesis with their copyright

holders’ permission.

16

17

AUTHOR’S CONTRIBUTION The author participated in the planning and design of all the three studies (I-III)

alongside the supervisors and took a leading role in data collection. He was also

responsible for analyzing the data in all papers (I-III) with the assistance of co-

authors and wrote the first drafts of all the manuscripts with subsequent inputs

from co–authors.

18

19

CONTENTS

ABSTRACT ........................................................................................................... 7 ACKNOWLEDGEMENTS ...................................................................................11 1 INTRODUCTION ..........................................................................................21

1.1 THE EDIBLE KATYDID RUSPOLIA DIFFERENS ...................................21 1.2 WHAT DETERMINES THE FATTY ACID COMPOSITION OF EDIBLE

INSECTS? ................................................................................................22 1.3 FATTY ACID BIOSYNTHESIS IN INSECTS ...........................................23

1.3.1 Fatty acids ....................................................................................23 1.3.2 Fatty acid biosynthesis .................................................................25 1.3.3 Lipids ............................................................................................27 1.3.4 Fat body ........................................................................................28

1.4 AIMS OF THE PRESENT STUDY ...........................................................28 1.5 STUDY HYPOTHESES ...........................................................................29

2 MATERIALS AND METHODS .....................................................................31 2.1 STUDY AREA ..........................................................................................31 2.2 EXPERIMENTAL SET-UP .......................................................................31 2.3 LIPID AND FATTY ACID ANALYSIS .......................................................32 2.4 STATISTICAL ANALYSES ......................................................................32

3 RESULTS AND DISCUSSION .....................................................................35 3.1 EFFECT OF DIVERSIFIED DIETS ON R. DIFFERENS LIPID CONTENT

AND FATTY ACID COMPOSITION .........................................................35 3.2 DIVERSIFIED DIETS ALTER THE N-6/N-3 FATTY ACID RATIO IN R.

DIFFERENS .............................................................................................37 3.3 INFLUENCE OF SEX ON THE R. DIFFERENS FATTY ACID

COMPOSITION ........................................................................................38 3.4 BODY WEIGHT OF R. DIFFERENS WAS NOT INFLUENCED BY GRASS

INFLORESCENCE DIET .........................................................................38 4 CONCLUSIONS AND FUTURE PROSPECTS ...........................................41 5 BIBLIOGRAPHY ..........................................................................................43

20

21

1 INTRODUCTION

1.1 THE EDIBLE KATYDID RUSPOLIA DIFFERENS

Consumption of edible insects is seen as a major solution to address the looming

food shortage due to the increasing human population (van Huis et al., 2013)—

projected to be 9.7 billion by 2050 (United Nations, 2019)—and for improving hu-

man nutrition (Shantibala et al., 2014). For example, in Africa and Asia, edible in-

sects are a traditional part of the human diet. Globally, approximately 2000 edible

insect species are documented (Ramos‐Elorduy, 1997; Jongema, 2017). Most belong

to six orders: Lepidoptera (butterflies and moths), Coleoptera (beetles), Isoptera

(termites), Hymenoptera (ants, bees and wasps), Hemiptera (the true bugs) and

Orthoptera (katydids, crickets and grasshoppers; Bukkens, 1997; Dobermann et al.,

2017).

The katydid Ruspolia differens (Serville) (Orthoptera: Tettigoniidae), with

common names such as ‘the edible grasshopper’, ‘the African edible bush cricket’,

‘nsenene’, ‘senesene’ and ‘Nshonkonono’ (Siulapwa et al., 2014), is a popular edible

insect in sub-Saharan Africa. It has the potential to mitigate the nutritional and eco-

nomic challenges of vulnerable communities (van Huis et al., 2013; Siulapwa et al.,

2014; Mmari et al., 2017). Its geographical distribution includes tropical Africa and

some Indian Ocean islands (Bailey & McCrae, 1978; Massa, 2015). They occur in

eight colour morphs (Bailey & McCrae, 1978; Nyeko et al., 2014), mainly in tropical

grasslands and open bushvelds, and are mostly nocturnal (Nonaka, 2009; Matojo &

Hosea, 2013). In the wild, R. differens feeds on a range of grass and sedge species

(predominantly Panicum maximum, Brachiaria ruziziensis, Chloris gayana, Hyparrhenia

rufa, Cynodon dactylon, Sporobolus pyramidalis and Pennisetum purpureum; Opoke et

al., 2019). Furthermore, they prefer anthers or the setting seed at the 'milk stage' of

crops such as rice, sorghum, millet and maize (Bailey & McCrae, 1978; McCrae,

1982). In captivity, R. differens can accept a wide range of diets, including grass

leaves and inflorescences (Valtonen et al., 2018) and seeds or flours of rice seed,

finger millet and sorghum. They can also consume wheat bran, chicken super feed

egg booster, germinated finger millet, simsim cake, crushed dog biscuit pellets,

dried blood, Lucerne meal and a high protein cereal meal (Brits & Thornton, 1981;

Nyeko et al., 2014; Malinga et al., 2018a).

R. differens is particularly valuable due to its fats. Its fat content is 47-49%

based on dry weight (Kinyuru et al., 2010), and it is rich in essential polyunsaturat-

ed fatty acids, i.e., linoleic acid (29.5-31.2%) and α-linolenic acid (3.2-4.2% of the

total fatty acid content). Previous studies on both wild harvested (Kinyuru et al.,

2010; Opio, 2015; Fombong et al., 2017) and laboratory-reared R. differens

(Lehtovaara et al., 2017) demonstrated that the species also contains palmitic acid

(11-35%), stearic acid (5-12%) and oleic acid (19-45%). The R. differens fatty acid con-

22

tent and composition can be modified through diet (Lehtovaara et al., 2017); this

manipulation can produce high‐quality fatty acids, including essential fatty acids

for humans (van Huis et al., 2013). Additionally, R. differens is rich in both macro-

and micronutrients. On a dry weight basis, it contains 35-37% protein, 2.6-2.8% ash,

3.9-4.9% fibre as well as 259.7-370.6 mg/100 g potassium, 121.0-140.9 mg/100 g

phosphorous and 13.0-16.6 mg/100 g iron, among others (Kinyuru et al., 2010).

Despite its enormous potential in sub-Saharan Africa, utilisation of this ed-

ible katydid is currently based on wild harvesting (Mmari et al., 2017). R. differens

are harvested from the wild when they swarm, usually during peaks in rainy sea-

sons (i.e., April-May and November-December; Bailey & McCrae, 1978; Okia et al.,

2017). This practice of seasonal R. differens harvesting is generally unpredictable

(Okia et al., 2017). Consequently, there is a great need to develop mass-rearing

methods for this species to improve food and nutrition security in rural African

communities and prevent overexploitation due to wild harvesting (Agea et al.,

2008; Kinyuru et al., 2010; Kelemu et al., 2015). However, suitable diet mixtures for

mass rearing developed from commonly available local African feeds are not well

understood (but see Malinga et al., 2018a, 2018b).

This study evaluated the influence of a diversified gradient of locally sourced

artificial and natural (tropical grass inflorescence) diets in Uganda, on R. differens

fatty acid content and composition. The diversified feed gradients were studied

because R. differens is known to benefit from diet mixing (Malinga et al., 2018b).

Generally, diet mixing is considered beneficial to insect growth and development

because it allows for a better balance of nutrients (Miura & Ohsaki, 2004a; Unsicker

et al., 2008). Diet mixing could also be beneficial in mass-rearing conditions where

insects are offered feeds that they would not encounter in nature. In R. differens, diet

mixing improves performance, including shortened developmental time, increased

fresh adult weight and improved female fecundity (Malinga et al., 2018b).

However, the effect of diversifying locally sourced diets on R. differens fatty acid

content and composition is not well understood.

1.2 WHAT DETERMINES THE FATTY ACID COMPOSITION OF EDIBLE INSECTS?

The nutritional composition of edible insects is largely determined by the insects’

diet (Barker et al., 1998; Oonincx & van der Poel, 2011; Komprda et al., 2013; Alves

et al., 2016; Adámková et al., 2017; Lehtovaara et al., 2017), although other factors

such as the insect species, developmental stage, rearing conditions, sex and meta-

bolic activity are also important determinants (Yang, Siriamornpun, & Li, 2006;

Oonincx et al., 2015; van Broekhoven et al., 2015; Pino Moreno & Ganuly, 2016;

Sönmez et al., 2016). For example, in a study by Lehtovaara et al. (2017), R. differens

supplied with different diet ingredients that varied in their fat, protein and carbo-

hydrate contents greatly differed in their fatty acid composition. The diets used in

23

Lehtovaara et al. (2017) were designed from linseed, sunflower, sesame seed, oat,

sugar beet fibre, fructose, coconut flour, rice flour, wheat flour, maize starch,

TetraMin (aquarium) fish food, casein, wheat germ, pea protein and milk whey.

Additionally, earlier studies by Oonincx et al. (2015) and van Broekhoven et al.

(2015) that used Argentinian cockroaches and edible mealworms, namely Tenebrio

molitor L., Zophobas atratus and Alphitobius diaperinus, also found that the nutrient

composition, including fatty acids, greatly differs when the insects are reared on

formulated artificial diets. The diets were formulated from by-products of food

manufacturing and bioethanol production, including organic by-products from

beer brewing, bread/cookie baking and potato stem peelings.

Sex is another major factor that influences fatty acid composition. Lease

and Wolf (2011) showed that female insects have higher lipid and/or fatty acid

conents compared to males and varying fatty acid composition (Subramanyam &

Cutkomp, 1987). These differences are attributed to sexual dimorphism that results

in physiological and reproductive differences (Subramanyam & Cutkomp, 1987;

Zhou et al., 1995). The females lay eggs, and as such may require certain fatty acids

in greater proportions than their male counterparts. For example, oleic acid is

thought to play an important role in egg laying (Sönmez et al., 2016).

1.3 FATTY ACID BIOSYNTHESIS IN INSECTS 1.3.1 Fatty acids

Fatty acids are of basic significance in living organisms due to their roles in meta-

bolic energy storage, cell and bio-membrane structure and regulatory physiology,

among others (Stanley-Samuelson et al., 1988, and references therein). Fatty acids

are an important lipid component, and in nature, they exist in combination with

glycerol to form triglycerides (Figure 1). Fatty acids consist of a straight chain (rep-

resented by R in Figure 1), usually with an even number of carbon atoms, including

hydrogen atoms with a carboxyl group (―COOH) at one end. They are categorised

based on the carbon-to-carbon bonds, the nature of which results in saturated and

unsaturated fatty acids. If all the carbon-carbon bonds are single, the fatty acid is

saturated, but if any of the bonds is double or triple, the fatty acid is unsaturated.

Unsaturated fatty acids are further subdivided into monounsaturated and polyun-

saturated fatty acids. Monounsaturated fatty acids contain only one double bond,

while polyunsaturated fatty acid structure contain two or more double bonds.

24

Figure 1. Generalised structure of a fatty acid molecule; R refers to an aliphatic chain.

Polyunsaturated fatty acids (PUFAs) are further categorised as omega-3 and

omega-6 fatty acids. Omega-3 fatty acids are characterised by a double bond three

atoms away from the terminal methyl group. Comparatively, omega-6 fatty acids

have the last double bond six carbons from the omega (methyl) end of the fatty acid

molecule. Both omega-3 and omega-6 fatty acids are classified as essential fatty

acids to animals (including insects). They can only be obtained from dietary

sources, although a few insect species can biosynthesise their own (Cripps,

Blomquist, & de Renobales, 1986; Stanley-Samuelson et al., 1988). α-linolenic and

linoleic acids are the most common omega-3 and omega-6 fatty acids, respectively,

and can be metabolised into other fatty acids, such as eicosapentaenoic (EPA) and

docosahexaenoic (DHA) acids (from omega-3) and arachidonic acid (ARA) from

omega-6 (Figure 2; Stanley-Samuelson et al., 1988). In humans, the balance between

omega-3 and omega-6 is an important determinant for brain development and in

decreasing the risk for coronary heart disease (CHD), hypertension, cancer, diabe-

tes, arthritis and other autoimmune and neurodegenerative diseases (Simopoulos,

2002, 2010). In insects, the balance between omega-3 and omega-6 fatty acids (i.e.,

omega-6/omega-3 ratio of approximately 1-2) enhances olfaction and cognitive

functions, as observed in Apis mellifera (Arien, Dag, & Shafir, 2018). A study by

Hixson et al. (2016) found that the appropriate balance of omega-3 and omega-6

fatty acids is crucial for adult metamorphosis and wing development in the cabbage

white butterfly (Pieris rapae). Furthermore, the lack of PUFA in the developing in-

sect is associated with impaired pupal eclosion (Turunen, 1974; Stanley-Samuelson

et al., 1988). An insufficient PUFA quantity in the diet retards growth; however,

supplementation of the diet with linseed oil, which is high in α-linolenic acid, can

reverse that condition (Turunen, 1974; Komprda et al., 2013).

25

Figure 2. Structures of omega-3 and omega-6 fatty acids, as developed through ChemDraw

Ultra 8.0 software (A = α-linolenic acid, B = eicosapentaenoic acid, C = docosahexaenoic

acid, D = linoleic acid, E = arachidonic acid).

1.3.2 Fatty acid biosynthesis

The majority of insects can biosynthesise de novo certain fatty acids (de Renobales,

Cripps, Stanley-Samuelson, Jurenka, & Blomquist, 1987a; Stanley-Samuelson et al.,

1988 and references therein; Blomquist, Borgeson, & Vundla, 1991). This process is

achieved through a series of reactions (Figure 3) that involve the conversion of sug-

ars and other carbohydrates into fatty acids. When excess carbohydrates are availa-

ble, the pyruvate generated through glycolysis is used to produce acetyl coenzyme

A (acetyl-CoA). Subsequently, the carboxylation of acetyl-CoA through acetyl-CoA

carboxylase forms malonyl-CoA, which is used to form fatty acids, notably a 16-

carbon palmitic acid, using the fatty acid synthase enzyme complex in the presence

of the reduced nicotinamide adenine dinucleotide phosphate (NADPH). Palmitic

acid (C16:0) can then be elongated (through elongase enzymes), into stearic acid

26

(C18:0). The action of desaturases on C18:0 generates oleic acid (C18:1; Downer &

Matthews, 1976; Stanley-Samuelson et al., 1988; Visser et al., 2017). There are some

exceptions to these processes. In some insects, such as the order Diptera, lauric acid

(C12:0) and myristic acid (C14:0) are generated from acetyl CoA (Stanley-

Samuelson et al., 1988). However, some insect species that lack the Δ12 desaturase

enzyme in the biosynthetic pathway are unable to biosynthesise certain fatty acids,

particularly linoleic acid (C18:2; Cripps et al., 1986; de Renobales et al., 1987a;

Stanley-Samuelson et al., 1988). The lack of and inability to biosynthesise certain

fatty acids (such as linoleic acid) leads to the differences in fatty acid composition

observed among insect species such as Musca domestica, Acyrthosiphon pisum and

Blatella germanica (Blomquist et al., 1982; Beenakkers et al., 1985; Stanley-Samuelson

et al., 1988). Notably, in most insect species, fatty acids are fairly similar in qualita-

tive terms, with palmitic, stearic, oleic, linoleic and α-linolenic acids among the

common fatty acids (Stanley-Samuelson et al., 1988; Ogg et al., 1993). However,

there are exceptions; for example, most dipterans are associated with high propor-

tions of palmitoleic acid (C16:1), and some aphids have high myristic acid (C14:0)

proportions, whereas coccids are characterised by high capric acid (C10:0) and lau-

ric acid (C12:0) contents (Stanley-Samuelson et al., 1988, and references therein).

27

Figure 3. Schematic representation of fatty acid biosynthesis; adapted from Takahashi

(2018).

1.3.3 Lipids

Lipids are biological molecules that are soluble in non-polar solvents. In addition to

fatty acids, they include phospholipids, sterols, sphingolipids and terpenes, among

others (Fahy, Cotter, Sud, & Subramaniam, 2011). They provide the energy source

for numerous body processes and act as a source of essential fatty acids (Stanley-

Samuelson et al., 1988; Raksakantong et al., 2010). Lipids are also used for the

transportation and absorption of fat-soluble vitamins and nutrients, as well as the

synthesis of hormones, cellular membrane, structural elements in cells and vital

organ protection (Haunerland, 1997). Lipids are further classified as neutral (tri-

glycerides), which constitute approximately 90% of the total lipid component,

phospholipids (7%) and glycolipids (3%; Turunen, 1974; Kinyuru et al., 2010). In

insects, lipids are generally stored in the fat body, an organ equivalent to the liver

and adipose tissues in vertebrates (Arrese et al., 2001; Azeez, Meintjes, &

Chamunorwa, 2014).

28

1.3.4 Fat body

The fat body is a large, multifunctional organ that is distributed throughout the

insect body, from the terminal abdominal segment to the head capsule (Downer &

Matthews, 1976; Arrese & Soulages, 2010). The fat body is in a close contact with the

hemolymph for easy exchange of metabolites (Downer & Matthews, 1976). Lipids

form the major fat body component; more than 90% are triglycerides (Downer &

Matthews, 1976; Kinyuru et al., 2010). Functionally, the fat body exhibits marked

biosynthetic and metabolic activity, and it plays an essential role in energy storage

and utilisation, as well as excess nutrient storage (Arrese et al., 2001; Azeez et al.,

2014).

The fat body achieves its function through lipogenesis, which results in the

synthesis of triglyceride from diglyceride units as precursors (Arrese et al., 2001).

The precursor diglyceride units can be formed from a number of pathways, i.e.,

phosphatidic acid produced by the glycerophosphate pathway, the monoacylglyc-

erol pathway, degradation of phospholipids or triglyceride diacylation catalysed by

lipases (Arrese et al., 2001; Canavoso et al., 2001; Arrese & Soulages, 2010). The

diglyceride is further esterified through a diacylglycerol acyltransferase in a reac-

tion catalysed by the fatty-acyl-CoA to yield a triglyceride molecule.

When required, the fatty acids stored in the fat body are either mobilised to

provide energy to flight muscles or used for maintenance of metabolic activities for

other insect tissues, including the fat body itself (Downer & Matthews, 1976; Arrese

et al., 2001). This use can be achieved through mobilisation of diglycerides,

trehalose or proline (Arrese & Soulages, 2010). Mobilisation requires fat-body-based

triglyceride lipases (TG-lipase), which catalyse triglyceride hydrolysis. TG-lipases

include insect adipose triglyceride lipase and triglyceride lipase (Arrese & Soulages,

2010, and references therein). The lipids are mobilised and utilised to directly

support flight. This phenomenon has been demonstrated in a number of insect

species, including R. differens (Karuhize, 1972), and for the synthesis of trehalose

and proline (used for ready energy provision) as well as during starvation,

embryogenesis and the immune response (Arrese et al., 2001; Arrese & Soulages,

2010).

1. 4 AIMS OF THE PRESENT STUDY

The goal of this study was to assess how diversified natural diets that included the

inflorescences of selected grass species (i.e., B. ruziziensis, Setaria megaphylla, Setaria

sphacelata, Echinochloa pyramidalis, P. purpureum, C. gayana, E. indica and P. maxi-

mum), as well as artificial diets (formulated from rice seed head, finger millet seed

head, wheat bran, superfeed chicken egg booster, sorghum seed head, germinated

finger millet, simsim cake, crushed dog biscuit pellet and shea butter) influenced

29

the fatty acid content and composition of the edible katydid R. differens, one of the

most nutritionally and economically important edible insects in Africa.

The specific objectives were:

1. To evaluate the effect of diet, represented by a diversified gradient of natu-

ral host plants, on the lipid content and fatty acid composition in R. differens

sixth instar nymphs harvested from the wild and reared for two weeks

(study I).

2. To evaluate the effect of diet, represented by a diverse gradient of natural

host plants, on the content and fatty acid composition in R. differens indi-

viduals, when reared from neonatal nymphs to adults (study II).

3. To assess the influence of diet, represented by a diverse gradient of locally

sourced, processed and non-processed artificial diets, on the fatty acid con-

tent and composition of R. differens (study III).

1.5 STUDY HYPOTHESES

The study hypotheses were:

1. If R. differens are fed on a multi-species diet, the fatty acid content of the in-

sects will be higher than when fed on a less-diversified diet. Similar to oth-

er generalist herbivores, a multi-species diet might allow for ‘compulsive’

switching behaviour (Bernays, Bright, Howard, Raubenheimer, &

Champagne, 1992; Bernays & Bright, 1993) in R. differens. The phenomenon

would result in eating diverse food with more variable nutrient sources, in-

cluding fatty acids and other constituent nutrients (e.g., proteins and car-

bohydrates), than when fed on a mono-species diet.

2. Diet diversification will lead to differences in the fatty acid composition

among R. differens. More diversified diets will offer a more variable fatty ac-

id formulation compared to less diversified diets and thus promote differ-

ences in the R. differens fatty acid composition. Diversified diets will also

lead to alterations in fat content, because this measure reflects the diet fat

content (Turunen, 1974; Stanley-Samuelson et al., 1988; Lease & Wolf, 2011;

Malinga et al., unpublished manuscript).

3. The fatty acid content and composition will be influenced by R. differens

sex, regardless of the dietary levels of the diversified diet, because insects

exhibit sexual dimorphism in their lipid and fatty acid metabolism

(Turunen, 1974; Arrese & Soulages, 2010).

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31

2 MATERIALS AND METHODS

This section provides a general outline of the materials and methods. A detailed

methodology is provided in the original papers I-III, which are attached.

2.1 STUDY AREA

Ruspolia differens used in this work originated from a wild population of the farm-

lands in and around the Makerere University Agricultural Research Institute

(MUARIK), Uganda. MUARIK is located in central Uganda, approximately 20 km

north of Kampala; it lies at 0°27'03.0"N and 32°36'42.0"E, with an average altitude of

1,200 m above sea level (asl). MUARIK has a wet-and-dry climate typical of tropical

regions. The mean annual rainfall (1,160 mm) is distributed bi-modally from March

to June and September to November each year (Tenywa et al., 2000). While the rest

of the insects were hatched and reared to maturity over the course of the experi-

ments at the Animal Science Laboratory, MUARIK (II-III), for study I, sixth instar

nymphs were harvested from the fields prior to using them in experiments. In

study I, fresh samples were used, and for II and III, the samples were freeze-dried

prior to fatty acid analysis. For study I, the analysis was performed at Kyambogo

University and Makerere University, Uganda. For studies II and III, the analysis

was performed at the Bio-Competence Centre of Healthy Dairy Products, Tartu,

Estonia (Accreditation EN ISO/IEC 17025:2005).

2.2 EXPERIMENTAL SET-UP

To test whether a diverse gradient of natural plants as a food source (I & II) affected

fatty acid content and composition, in study I, we reared R. differens individuals on

four dietary treatments. These treatments included one, or mixtures of two, three,

or six host plant inflorescences for two weeks after wild sixth instar harvesting. In

study II, we reared individuals on six dietary treatments that consisted of one, or

mixtures of two, three, five, six or eight host plant inflorescences from neonate

nymphs to adults (II). In both cases, freshly opened host plant inflorescences were

used. In study III, the effect of mixtures of locally sourced artificial diets on R. dif-

ferens fatty acid content and composition was investigated by rearing neonatal indi-

viduals to maturity on six levels of gradually diversified diets of two, three, four,

six, eight or nine feeds. The diets were formulated from rice seed head, finger millet

seed head, wheat bran, superfeed chicken egg booster, sorghum seed head, germi-

nated finger millet, simsim cake, crushed dog biscuit pellet and shea butter. In all

the studies, the insects were reared in the laboratory (temperature 22–28°C, 12 h

light:12 h dark photoperiod and relative humidity 50–60%) using transparent flat-

bottom plastic jars (1,000 mL, Thermopak Limited, Nairobi) that measure 12.5 cm

32

(diameter) × 8 cm (height). For study I, one male and one female were reared to-

gether in each jar for two weeks, whereas in studies II and III, neonatal nymphs

were reared singly per jar for 2-4 months. Moistened tissue paper was provided as

a source of water, and food was provided ad libitum, with regular replenishment

every 3-4 days. Upon maturity, the insects were harvested and frozen at -80°C

(studies II and III) until use in fatty acid analysis. For fatty acid and statistical anal-

ysis in study I, 30 individuals—four male and four female R. differens from each diet

treatment, except in treatment one (three males) and treatment two (three fe-

males)—were analysed, in addition to eight fresh samples (i.e., four males and four

females) collected from the wild. For studies II and III, a total of 30 individuals—

five from each diet treatment—were used.

2.3 LIPID AND FATTY ACID ANALYSIS

Lipid extraction in study I followed the method of Folch et al. (1957). The fatty acid

composition was determined using gas chromatography-mass spectrometry (GC-

MS; Agilent 6890-version N.05.05, GC-System, Santa Clara, CA, USA) fitted with an

electronic pressure control and mass selective detection (ionising energy, 70 eV;

temperature, 250°C) analysis. The fatty acids were identified in the samples using a

standard mixture and mass spectrometry and quantified using an internal standard

(C19:0). In experiments II and III, fatty acid analysis followed a direct transesterifi-

cation method (Sukhija & Palmquist 1988), with minor modifications (Lehtovaara et

al. 2017). Fatty acid methyl esters (FAMEs) were analysed on an Agilent 6890A GC

(Agilent Technologies Inc.), equipped with a flame ionisation detector (FID) detec-

tor and an autosampler. In all cases, the fatty acid peak areas were quantified using

ChemStation chromatography software, and the relative amounts of each fatty acid

were calculated based on their relative retention times and peak areas. Values are

expressed as a percentage (%) of the total analysed fatty acids and as content (mg

fatty acid per g R. differens dry weight).

2.4 STATISTICAL ANALYSES

For all studies, analysis of variance (ANOVA) and permutational multivariate

analysis of variance (PERMANOVA) were applied. ANOVA models (type III sums

of squares) were fitted in SPSS (IBM SPSS Statistics, version 23) to test whether the

content of SFAs, MUFAs, PUFAs or omega-6/omega-3 (n-6/n-3) fatty acid ratio of R.

differens were explained by diet and sex (fixed factors) or their interaction (studies II

and III). The PUFA content, n-6/n-3 ratio and MUFA content (study III) were natu-

ral log or square root transformed to improve normality prior to analysis. In study

III, Duncan’s post hoc test was used for pairwise comparisons, because for some

variables, the more conventional pairwise Tukey test (used in studies I and II) was

too conservative to detect any significant differences (Williams & Abdi, 2010), even

33

when ANOVA indicated significant differences among the diets. PERMANOVA

was performed to test for differences in fatty acid compositions (fatty acid propor-

tions) among diets, between the sexes and the interaction between these two factors

using type III sums of squares and 999 permutations (Anderson, 2001). A permuta-

tional analysis of multivariate dispersions (PERMDISP) was also conducted to as-

sess the degree of variability in the relative fatty acid proportions among samples in

each treatment and to test the dispersions within factor groups based on deviations

from the group centroids (Anderson, Gorley, & Clarke, 2008). Furthermore, a simi-

larity percentage analysis (SIMPER; Clarke & Gorley, 2006) was performed to iden-

tify which fatty acids contributed most to differences in fatty acid composition

among the diets. Non-metric multidimensional scaling (NMDS), with 50 restarts,

was applied to visualise fatty acid patterns among individuals fed with different

diet treatments. In all cases, Bray-Curtis was used as a measure of similarity. As a

response dataset, the proportions of each fatty acid out of the total fatty acid con-

tent in study I, and those with levels of 0.05% and above in a sample in studies II

and III, were used in the multivariate analysis. In study III, PERMANOVA was run

using both untransformed and forth-root transformed fatty acid proportional data

sets (the latter lessens the influence of the most common fatty acids and emphasises

the rare fatty acids). Branched chain (iso/anteiso) fatty acids (studies II and III) were

combined before inclusion in the analysis. PRIMER-E version 6.0 and PERMANO-

VA+ add-on were used for multivariate statistical analyses.

34

35

3 RESULTS AND DISCUSSION

3.1 EFFECT OF DIVERSIFIED DIETS ON R. DIFFERENS LIPID CONTENT AND FATTY ACID COMPOSITION

The study I results demonstrated that when wild sixth instar R. differens nymphs

were reared for two weeks, the fatty acid composition significantly differed among

the individuals fed with distinct natural host plant diets (for diet formulation, see

Table 1; study I). However, when R. differens were reared from neonatal nymphs to

adults, the fatty acid content and composition was not significantly different among

individuals fed with different diets (see Table 1; study II). The reason for this dis-

crepancy is unclear. However, it could be due to seasonal differences in the food

quality in diet mixtures offered to R. differens (i.e., study I was performed during

the wet season in November, whereas study II was executed in March during the

dry season). Food quality and the nutrient levels in grass species vary or fluctuate

with seasons due environmental factor variations, e.g., nutrient availability, solar

radiation, temperature and water deficiency, even when forages are harvested at

the same maturity stage (Buxton, 1996; Warly, Fariani, Ichinohe, & Fujihara 2004).

However, a study by Guil-Guerrero and Rodriguez-Garcia (1999) argued that most

plants are very similar in their fatty acid compositions, especially in leaf tissues, but

may differ in other constituent nutrients (e.g., proteins and carbohydrates) and

other physical characteristics such as plant tissue toughness. These combined fac-

tors could influence the palatability of the diet offered to R. differens, and this phe-

nomenon may have affected nutrient accumulation, including the fatty acids. In

study I, the insects were harvested as wild sixth instar nymphs with relatively well-

developed mandibles that they could possibly use to chew any offered grass type.

Comparatively, in study II, the growing nymphs were less developed and had

more fragile mandibles (Miura & Ohsaki, 2004b). Additionally, restriction in ac-

ceptance of plant species diets due to lack of experience, as suggested by Jermy

(1987), could have led to non-utilisation of some of the offered feeds. A recent study

by Opoke et al. (2019) showed that P. maximum is the preferred host for the young-

est R. differens nymphs. This finding suggests that in study II, the growing nymphs

may have only utilised a limited range of feeds from the mixture, a factor that

would underlie the similarity in their fatty acid profiles. Several studies (Turunen,

1974; Oonincx & van der Poel, 2011; Komprda et al., 2013; Lehtovaara et al., 2017)

argue that insect fatty acid profiles often reflect dietary fatty acids.

Study III results demonstrated that when R. differens individuals were fed

over the full life cycle (neonatal nymph to adult), a diversified gradient of local

(processed and unprocessed) artificial feeds in Uganda (see Table 1 for diet formu-

lations) strongly modified fatty acid content and composition. In particular, the

PUFA content was approximately 3.5-fold higher in R. differens that received the

36

most compared to the least diversified diet. Several reasons may explain the high

PUFA levels in the most diversified (eight- and nine-feed) diets. For example, the

highly diversified diets contained shea butter and simsim seed cake, both of which

are generally rich in PUFAs (Shea butter, 6–8%; simsim cake, 22–46% of the total

fatty acid content; Okullo et al., 2010; Honfo et al., 2014; Gharby et al., 2017). Thus,

R. differens possibly incorporated the dietary PUFAs, a phenomenon that may have

led to the observed high PUFA levels compared to the low PUFA levels in R. dif-

ferens offered the least diversified diets (Table 1; study III). This assertion of incor-

poration of unaltered dietary fatty acids into body tissues is also shared with a pre-

vious R. differens study (Lehtovaara et al., 2017) and in other edible insects, includ-

ing the mealworm Tenebrio molitor (Dreassi et al., 2017) and migratory locusts Lo-

custa migratoria L. (Oonincx & van der Poel, 2011). In the present study, the PUFA

content in R. differens fed with mixtures of artificial feed diet was 10% of the total fat

content, whereas in individuals offered the most diversified natural diet and in the

wild harvested individuals, it was 17% and 21%, respectively (see study I). In the

wild, herbivorous insects can exhibit complementarity effects as well as ‘compul-

sive’ switching on a wide range of plant species (Bernays et al., 1992; Unsicker et al.,

2008). This behaviour may allow accumulation of higher fatty acid levels, in partic-

ular PUFAs, compared to when the insects are offered a limited range of feeds in

artificial diets. Therefore, for fast growth and PUFA accumulation during mass

rearing, diet switching (i.e., the act of alternate feeding on a variety of food types in

polyphagous individuals; see Bernays et al., 1992) between the natural and artificial

diets is suggested. Diet switching can be advantageous to polyphagous insects,

because mixing foods increases the quality of the overall diet through improved

nutrient balance (Bernays et al., 1992; Bernays & Bright, 1993; Bernays, Bright,

Gonzalez, & Angel, 1994).

The results of these studies (I-III) showed that palmitic, stearic, oleic, linole-

ic and α-linolenic acids contributed > 90% of the total fatty acids across all dietary

treatments. These fatty acids were also found to be the most common in previous

studies that analysed composite samples of R. differens harvested from the wild

(Kinyuru et al., 2010; Opio, 2015; Fombong et al., 2017). The reason for this similari-

ty is unclear, but it might be related to de novo biosynthesis (Stanley-Samuelson et

al., 1988). In study III, oleic acid was exceptionally the most predominant fatty acid

(43-53% of the total fatty acid content across the dietary treatments), and this find-

ing could be attributed to oleic-acid-rich cereal feeds, such as rice and wheat

(Weihrauch & Matthews, 1977), that formed a major diet component for R. differens

in this study.

Finally, the results revealed that the lipid content was not altered when R.

differens was offered the different grass inflorescence diets (study I). The results in

study I suggest that R. differens lipid requirements may be fulfilled even with sim-

ple diets. Animals, and particularly insects, utilise lipids for various morphogenetic

and physiological functions, including flight (Karuhize, 1972; Downer & Matthews,

37

1976). In Bailey and McCrae (1978), the fat content of individuals harvested from

swarming R. differens varied from 26-29 g/100 g (based on wet weight) compared to

10 g/100 g in the study I (i.e., the R. differens harvested after two weeks on diet

treatments). However, a study by Lehtovaara, Roininen, and Valtonen (2018) noted

that R. differens weight continues to increase and could be up to 50% higher approx-

imately 10 days after adult moulting. This observation suggests that R. differens

continue to accumulate lipids in its fat body, a phenomenon that increases weight

after maturity is attained. Thus, leveraging the nutritional benefits of R. differens

during mass-rearing requires maintaining the insects after they reach maturity.

However, the lack of differences in the lipid content despite the diets in study I

could be attributed to the insects being confined in the rearing jars with reduced

movements and other energy-consuming processes. It is also possible that lipid

content differences could appear with further maturation, especially as they reach

their reproductive roles.

3.2 DIVERSIFIED DIETS ALTER THE N-6/N-3 FATTY ACID RATIO IN R. DIFFERENS

The results indicated that the n-6/n-3 fatty acid ratio was influenced by the diet

(studies I-III). When R. differens was fed with grass inflorescences, the n-6/n-3 fatty

acid ratio was reduced, i.e., 4.1-6.6 (study I; Table 3) and 1.45-2.03 (study II; Table

2). Several studies demonstrated that reduced n-6/n-3 fatty acid ratio is favourable

for both human health and insect growth and development. In humans, a reduced

n-6/n-3 fatty acid ratio (< 5) is important for brain development and decreasing the

risk for CHD, hypertension, cancer, diabetes, arthritis and other autoimmune and

neurodegenerative diseases (Simopoulos, 2002, 2010). For insects, a reduced n-6/n-3

fatty acid ratio is associated with better olfaction and cognitive functions, adult

metamorphosis, wing development and improved pupal eclosion (Turunen, 1974;

Stanley-Samuelson et al., 1988; Hixson et al., 2016; Arien, Dag, & Shafir, 2018).

On the contrary, when R. differens was reared on an artificial diet, the n-6/n-

3 fatty acid ratio was generally high and variable (range: 14-36; see study III, Table

2). In study III, the n-6/n-3 fatty acid ratio was unusually high compared to the rati-

os reported in studies I and II. This finding, however, suggests that the offered arti-

ficial feeds may contain higher levels of n-6 relative to n-3 fatty acids. The feed

combinations in study III (see Table I) comprised mostly cereals or cereal-based

feeds, all of which contain higher n-6 fatty acid levels (Weihrauch & Matthews,

1977). The n-6 fatty acids from the cereals were possibly incorporated into R. dif-

ferens tissues, a process that would result in the high n-6/n-3 fatty acid ratio. A high

n-6/n-3 fatty acid ratio has also been reported in a study by Lehtovaara et al. (2017),

where R. differens was reared on artificial diets with manipulated fatty acid, carbo-

hydrate and protein contents. Overall, on the basis of the n-6/n-3 fatty acid ratio, to

produce healthy food for humans, it would be important to rear R. differens with

38

grass-inflorescence-based diets or develop artificial diets supplemented with n-3-

rich feeds to balance the high n-6 fatty acids associated with the cereal-based diets.

3.3 INFLUENCE OF SEX ON THE R. DIFFERENS FATTY ACID COMPOSITION

The study showed that sex affected the R. differens fatty acid composition. The com-

positional differences observed between male and female R. differens could be the

result of sexual dimorphism, as reported for insects species from other families

(Subramanyam & Cutkomp, 1987). Females lay eggs, and thus they likely require

greater proportions of certain fatty acids (such as oleic acids) compared to their

male counterparts, as observed in Acanthoscelides obtectus (Sönmez et al., 2016). To

satisfy such physiological requirements, the different sexes may have consumed

different amounts of feeds, a phenomenon that would ultimately modify the overall

fatty acid proportions in their tissues.

3.4 BODY WEIGHT OF R. DIFFERENS WAS NOT INFLUENCED BY GRASS INFLORESCENCE DIET

In contrast to the study hypothesis, adult R. differens weight was not affected by

grass inflorescence diets (study I & II), although the reasons behind these data are

unclear. However, these findings corroborate the results from a related study that

also offered R. differens a grass inflorescence diet (Malinga et al., unpublished data

set). In a related study that used artificial diets (Malinga et al., 2018b), R. differens

weight was on average 0.40-0.65 g, compared to the weight in R. differens offered

the natural diets of 0.33-0.45 g and 0.41-0.45 g in study I and II, respectively. A pre-

vious study by Lehtovaara et al (2017) found that the final R. differens weight dif-

fered significantly among diet treatments when individuals were fed with artificial

diets with varied fat, protein and carbohydrate contents. These study findings sug-

gest that artificial feeds possibly offer R. differens better and more varied nutrients

compared to natural diets. However, based on the previous study by Lehtovaara et

al. (2018), R. differens individuals can achieve up to 50% higher weight approximate-

ly 10 days after final moulting compared to if they are harvested immediately after

adult moulting. Thus, the low weights observed in studies I and II may have result-

ed from early R. differens harvesting, which according to Lehtovaara et al. (2018) is a

limitation for this part of this study.

Furthermore, the study showed that more diversified diets resulted in R.

differens with relatively higher weights than those fed on single or less diversified

diets (studies I and II). In more diversified diets, the insects are considered to be

nutritionally advantaged due to diet complementation (Hagele & Rowell-Rahier,

1999; Miura & Ohsaki, 2004a; Unsicker et al., 2008). Indeed, many generalist and

specialist herbivores are known to perform best when offered a mixed rather than

39

less-diversified or single-food diet (Miura & Ohsaki, 2004a; Unsicker et al., 2008;

Malinga et al., 2018b). Nevertheless, in some cases, as highlighted by Hägele &

Rowell-Rahier (1999), some insects can perform well on less-diverse diets. Accord-

ing to Loveridge (1973), insect weight is largely determined by the offered diet.

Overall, the findings in this study suggest that the quality of the grass inflorescence

diets offered to the insects (in studies I and II) was inferior and could not build a

heavy fat body compared to the artificial diets (Malinga et al., 2018b).

40

41

4 CONCLUSIONS AND FUTURE PROSPECTS

This thesis investigated the influence of diversified diets that contained selected

artificial components and grass inflorescences on the fatty acid content and compo-

sition of R. differens. The results provide new knowledge on the effect of a diversi-

fied diet on fatty acid content and composition in R. differens. This information will

be useful when designing rearing methods and technology for this edible insect.

The main conclusions and the future prospects from this study are summarised

below.

1. R. differens fatty acid content and composition can be influenced by diet.

Thus, using plants (grass inflorescences) and artificial diets, it is possible to

produce R. differens with elevated fatty acid levels, including the preferred

high-quality essential fatty acids that are important in human health.

2. The study I results further showed that it is possible to rear R. differens har-

vested in the wild as sixth instar nymphs and modify their fatty acid com-

position based on mixtures of its natural diet (grass inflorescences).

3. Furthermore, the study III results revealed that when fed from neonatal

nymph to adult, a diversified gradient of local artificial feeds strongly mod-

ified the fatty acid content and composition in R. differens.

4. The most common fatty acids in R. differens included palmitic, stearic, oleic,

linoleic and α-linolenic acids, which collectively contributed > 90% of the

total fatty acids irrespective of diet treatment.

5. The lipid content was not altered when R. differens is offered the grass inflo-

rescence diet (study I).

6. The n-6/n-3 fatty acid ratio in R. differens was influenced by the diet.

7. Despite being offered similar diets, there were notable proportional differ-

ences in fatty acids among female and male R. differens.

8. Finally, adult R. differens weight was not affected by host plant inflo-

rescence diets.

Overall, the study provides important information regarding the influence of diver-

sified diets on the fatty acid content and composition in R. differens. This infor-

mation will be useful for designing nutritious feeds for future R. differens mass-

rearing programmes. However, additional studies are recommended to establish

the extent of diet diversification vis-à-vis the quality of fatty acids, especially the

essential fatty acids.

42

43

5 BIBLIOGRAPHY

Adámková A., Mlček J., Kouřimská L., Borkovcová M., Bušina T., Adámek M., Bednářová M.

& Krajsa J. 2017. Nutritional potential of selected insect species reared on the island of

Sumatra. International Journal of Environmental Research and Public Health 14: 521.

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KARLMAX RUTARO

FATTY ACID PROFILES OF THE EDIBLE KATYDID, RUSPOLIA DIFFERENS (SERVILLE) (ORTHOPTERA:

TETTIGONIIDAE) AFTER FEEDING ON DIVERSIFIED DIETS


Rearing of edible insects is seen as one

solution to ensure a sufficient food production for the increasing human population.

A successful mass-rearing programme for edible insects such as the edible katydid, Ruspolia differens, in rural Africa would require affordable but reliable feeds. This

thesis provides novel insights on how feeds influence the fatty acid profiles of R. differens, with emphasis on the essential fatty acids that

are needed for a healthy human diet.

KARLMAX RUTARO

Documents

Dissertations in Forestry and Natural Sciences › pub › urn_isbn_978-952-61... · 2020-06-30 · ISBN: 978-952-61-3321-8 (PDF) ISSN: 1798-5676 (PDF) ABSTRACT Consumption of edible