The Role of Food Nutrition: Effects on Foraging Behavior, Dealing with Plant Secondary Metabolites and Parasites in Helicoverpa Armigera Larvae

The role of food nutrition: effects on foraging behavior, dealing with plant secondary metabolites and parasites in Helicoverpa armigera larvae

PENG WANG B.Sc., M.Sc. (Ag.)

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 School of Biological Sciences

Abstract Nutrition has a fundamental influence on all living organisms. The amount and balance of different nutrients can impact the behaviour and physiology of animals. When offered nutritionally complementary diets, animals tend to regulate their intake of nutrients to a point that is optimal for their requirements. This mixing behaviour was investigated in early instar larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) using a putative optimal artificial diet (OP) and high protein (HP) and high carbohydrate (HC) artificial diets based on protein (p) and carbohydrate (c) ratios. Larvae were allowed to choose between the same kind of diet cubes (effectively no-choice), or diet cubes with different protein-to-carbohydrate (p: c) ratios (choice test). In no-choice tests, I found that first instar larvae remained longest on OP diet and spent least time on HC diet, whilst third instar larvae remained longest on HC diet and spent least time on OP diet. First instar larvae moved most when provided with HC diet, while third instar larvae moved most when provided with OP diet. However, both stages moved least when allowed to choose between diet cubes with different p: c ratios. The relative growth rate decreased when larvae moved often, but this influence was not evident when larvae fed on HC diet. Larvae that fed only on HC diet had the highest relative growth rate, followed by larvae with access to all diets simultaneously, indicating a behaviour to mix nutrient intake.

Insect herbivores encounter secondary metabolites when feeding on host plants. How insects deal with plant secondary metabolites in a complex nutrient environment is unclear. The influence of a classic plant secondary metabolite – allyl glucosinolate (sinigrin), and its hydrolysed product allyl isothiocyanate (AITC) on the development of H. armigera when fed on diets with different p: c ratios was investigated, and metabolized products in the frass were analysed. As expected, AITC had a greater effect than sinigrin on H. armigera survival, development, and pupal weight. However, AITC at low doses appeared to have a positive effect on the parameters above. Both sinigrin and AITC can induce detoxification activity in the gut, and the reaction was related to diet protein concentration. High protein diet can provide adequate free amino acid, especially cysteine, that is part of the detoxification process. Nutrient content of the diet plays an important role influencing how plant secondary metabolites are handled.

Infection by parasites is another stressor that insects may encounter. A new parasite that is similar to Ophryocystis elektroscirrha (McLaughlin & Myers) (Neogregarinorida:

Ophryocystidae) (OE) was found on H. armigera. OE is a relatively benign neogregarine protozoan parasite that infects monarch (Danaus plexippus (Linnaeus) (Lepidoptera: Nymphalidae)), queen ((Danaus gilippus (Cramer) (Lepidoptera: Nymphalidae)) and lesser wanderer (Danaus petilia (Stoll) (Lepidoptera: Nymphalidae)) butterflies. Spores of this new OE-like parasite were inspected under a microscope and compared with spores recovered from infected D. plexippus and D. petilia. There were clear morphological differences between the spores from the three sources. Larvae of H. armigera were challenged with spores from the three sources. Even at the highest dose tested (1000 spores per larva), spores from Danaus spp. did not infect H. armigera. The virulence of the OE-like spore from H. armigera and interactions with diets with different p: c ratios were investigated. Infection was positively related with spore dose, but did not significantly affect fitness of H. armigera. Developmental traits between infected and uninfected larvae did not differ across diet treatments, but larvae fed on high protein diet showed the lowest rate of infection.

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis.

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Publications included in this thesis

Wang, P., Furlong, M. J., Walsh, T. K., & Zalucki, M. P. (2019). Moving to keep fit: feeding behavior and movement of Helicoverpa armigera (Lepidoptera: Noctuidae) on artificial diet with different protein: carbohydrate ratios. Journal of Insect Science, 19 (5), 20. https://doi.org/10.1093/jisesa/iez098.

This paper has been incorporated as Chapter 2.

Submitted manuscripts included in this thesis No other manuscripts submitted for publication

Other publications during candidature Peer-reviewed papers: Wang, P., Furlong, M. J., Walsh, T. K., & Zalucki, M. P. (2019). Moving to keep fit: feeding behavior and movement of Helicoverpa armigera (Lepidoptera: Noctuidae) on artificial diet with different protein: carbohydrate ratios. Journal of Insect Science, 19(5), 20. https://doi.org/10.1093/jisesa/iez098.

Gao, K., Muijderman, D., Nichols, S., Heckel, D. G., Wang, P., Zalucki, M. P., & Groot, A. T. (2020). Parasite-host specificity: A cross-infection study of the parasite Ophryocystis elektroscirrha. Journal of Invertebrate Pathology. https://doi.org/10.1016/j.jip.2020.107328.

Katsikis, C. I., Wang, P., Zalucki, M. P. (2020). Life history traits of a key agricultural pest, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae): are laboratory settings appropriate? Austral Entomology. https://doi.org/10.1111/aen.12441.

Contributions by others to the thesis Professor Myron Zalucki and Associate Professor Michael Furlong made contributions to the development of the concept and design of the experiments. They have monitored the research, contributed to experimental design and data analysis and provided guidance on writing and editorial comments on the thesis.

The team lead by Dr. Daniel Vassao from Max Planck Institute for Chemical Ecology, Germany, undertook the analyses of larval frass samples mentioned in chapter 3.

Dr. Lynda Perkins, Dr. Patrick Ward and Dr. Simon Blomberg from School of Biological Sciences, the University of Queensland, provided help in data analysis.

Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects No animal or human subjects were involved in this research.

Acknowledgements I would like first sincerely thank my principle supervisor professor Myron Zalucki, whose professional knowledge, optimistic personality and efficient working habit guided and encouraged me throughout my entire PhD life.

I would also like to thank my co-supervisor associate professor Michael Furlong for his insightful perspective in all the meetings and discussions. His support and help is invaluable.

A special thanks to Dr. Lynda Perkins, who guided and supported me the first time I stepped into the lab, and helped a lot in my experiment and data analysis. She is the person I can always count on.

I would like to thank my readers Dr. Bronwen Cribb and associate professor David Merritt, who gave me critical advices and suggestions to the direction of my research. To Dr. Tom Walsh in CSIRO Black Mountain Laboratory, Canberra, Australia, who supported me doing experiment in his lab. Ashley Tessnow, from Texas A &M University, Texas, USA, taught me the technique of making diet with defined nutrient ratios during my stay in Tom’s lab. The technique became one of the foundations of this thesis. To Dr. Daniel Vassão from Max Planck Institute for Chemical Ecology, Germany, who helped analyse my samples and gave professional advices to my project, and professor David Heckel, who supported my brief study in Max Planck Institute for Chemical Ecology in 2016. To professor James De Voss and Dr. Jeanette Stok, who from School of Chemistry and Molecular Biosciences, UQ, allowed and helped me to use the GC-MS in their lab. To Dr. Patrick Ward and Dr. Simon Blomberg, from School of Biological Sciences, UQ, provided help in data analysis.

A heartfelt thanks to all the lab members Dr. Rehan Silva, Dr. Gurion Ang, Dr. Md Mahbubur Rahman, Leyun Wang, Rafeya Akhtar Munia for your help and friendship during this wonderful journey.

Last but not the least, to my partner Zheng Xiaoxiao, who is my biggest happiness and source of love. I could not have done this without you. To my beloved family. Your understanding and encouragement is always my strongest support.

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Financial support

This research was supported by CSC-UQ PhD Scholarship

Keywords Helicoverpa armigera, artificial diet, nutrition, glucosinolate, isothiocyanate, metabolism, parasite, infection

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Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060201, Behavioural Ecology, 35% ANZSRC code: 060806, Animal Physiological Ecology, 40% ANZSRC code: 060808, Invertebrate Biology, 25%

Fields of Research (FoR) Classification

FoR code: 0602, Ecology, 35% FoR code: 0608, Zoology, 65%

Table of content

Abstract ...... i Declaration by author ...... iii Publications included in this thesis ...... iv Submitted manuscripts included in this thesis ...... v Other publications during candidature ...... v Contributions by others to the thesis ...... v Statement of parts of the thesis submitted to qualify for the award of another degree ...... vi Research Involving Human or Animal Subjects ...... vi Acknowledgements ...... vii Financial support ...... viii Keywords ...... viii Australian and New Zealand Standard Research Classifications (ANZSRC) ...... ix Fields of Research (FoR) Classification ...... ix Table of content ...... x List of figures and tables ...... xiii List of abbreviations used in the thesis ...... xviii Chapter 1 General introduction ...... 1 1.1 Literature review ...... 1 1.1.1 Nutrition self-selection ...... 1 1.1.2 Plant secondary metabolite and insect detoxification activity ...... 5 1.1.3 The interaction between nutrition and toxin ...... 6 1.1.4 Pathogen and host insect in diverse nutritional environment ...... 7 1.2 Aims and perspectives ...... 9 Chapter 2 Moving to keep fit: feeding behavior and movement of Helicoverpa armigera (Lepidoptera: Noctuidae) on artificial diet with different protein: carbohydrate ratios ...... 11 2.1 Abstract ...... 11 2.2 Introduction ...... 11 2.3 Materials and Methods ...... 13 2.3.1 Insects ...... 13 2.3.2 Artificial diets ...... 14 2.3.3 Feeding and behavioral studies in a no choice experiment ...... 14 2.3.4 Feeding and behavioral studies in a choice experiment ...... 15 x

2.3.5 Statistical analyses ...... 16 2.4 Results ...... 16 2.4.1 Feeding and behavioral studies in a no choice experiment ...... 16 2.4.2 Feeding and behavioral studies in a choice experiment ...... 21 2.5 Discussion ...... 23 2.6 Acknowledgments ...... 27 Chapter 3 The challenge of eating: balancing nutrients in a toxic environment ...... 28 3.1 Abstract ...... 28 3.2 Introduction ...... 28 3.3 Materials and Methods ...... 30 3.3.1 Insect ...... 30 3.3.2 Diet ...... 30 3.3.3 AITC encapsulation ...... 30 3.3.4 Total AITC determination ...... 31 3.3.5 Release characteristics of AITC from diet...... 31 3.3.6 Diet preparation and feeding larvae with sinigrin and encapsulated AITC .. 32 3.3.7 Frass analysis ...... 32 3.3.8 Statistical analyses ...... 36 3.4 Results ...... 36 3.4.1 Diet analysis ...... 36 3.4.2 AITC encapsulation ...... 37 3.4.3 Release characteristic of AITC from diet ...... 37 3.4.4 Feeding assay with sinigrin ...... 39 3.4.5 Frass analysis of sinigrin feeding larvae ...... 41 3.4.6 Feeding assay with AITC ...... 46 3.4.7 Frass analysis of AITC feeding larvae ...... 50 3.5 Discussion ...... 53 Chapter 4 What a parasite needs: effect of an Ophryocystis elektroscirrha (Neogregarinorida: Ophryocystidae)-like spore in Helicoverpa armigera ...... 59 4.1 Abstract ...... 59 4.2 Introduction ...... 59 4.3 Materials and Methods ...... 61 4.3.1 Insect ...... 61 4.3.2 Diet ...... 61

4.3.3 Spore extraction and measurement ...... 61 4.3.4 Infection ...... 61 4.3.5 Statistical analyses ...... 62 4.4 Result ...... 63 4.4.1 Morphology and size of spores from Helicoverpa armigera, Danaus plexippus and Danaus petilia...... 63 4.4.2 The effect of OE like spores from H. armigera and two other spores on H. armigera ...... 64 4.4.3 Infection vs. diet ...... 68 4.5 Discussion ...... 74 Chapter 5 General discussion ...... 78 5.1 Introduction ...... 78 5.2 Foraging behavior of Helicoverpa armigera on diets with different protein-to- carbohydrate ratios ...... 79 5.3 Interaction between diet nutrients and plant secondary metabolites...... 80 5.4 Interaction between an OE-like parasite and diets ...... 83 5.5 Conclusion ...... 84 List of references ...... 85 Appendix A ...... 105 Appendix B ...... 106

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List of figures and tables Figure 1. 1 A hypothetical two-dimensional nutrient space showing the position of intake target (IT) and the rails of two nutrients (A and B). Arrows show one possible way animal could choose between the two food sources to achieve IT...... 3

Figure 2. 3 Scatter plot of individual larval RGR versus the number of moves in the first two days. Data from the first instar larvae (A) and the third instar larvae (B) were showed with different shapes and colors to represent different diets. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Lines indicate regression lines of the same colored data...... 20

Figure 2. 5 The influence of dish types (A) and instar (B) on RGR over two days. Asterisks represent significance between groups. OP, HC, HP, MIX represent optimal diet, high carbohydrate diet, high protein diet and mixed-diet dish types respectively (*, p < 0.05; **, p < 0.01; ***, p < 0.001).23Figure 3. 1 Protein and digestible carbohydrate analysis of the xiii experimental diets. Position of points showed dry compositions of protein and digestible carbohydrate in the diets (three repeats each). The three rails represent three p:c ratios of diet (indicated on each rail). OP, HC, HP, UQ represent optimal, high carbohydrate high protein and UQ standard rearing diet respectively...... 37

Figure 3.2 The change of AITC concentration over time in three diets, with the added doses at 4.96 μmoL/g, 3.37 μmoL/g, 1.79 μmoL/g. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 38

Figure 3.3 The change in weight (±SE) of 300 mg fresh diet for three diet types over time. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 38

Figure 3. 4 Box and whiskers plot of developmental time of larvae fed on OP, HC and HP diet with different concentrations of sinigrin. Different letters represent significant difference between treatments. Lowercase letters represent difference within the same diet of different doses. Capital letters represent difference between diets of the same dose. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 39

Figure 3. 5 Box and whiskers plot of pupal weight of larvae fed on OP, HC and HP diet at different concentrations of sinigrin. Different letters represent significant difference between treatments. Lowercase letters represent difference within the same diet of different doses. Capital letters represent difference between diets of the same dose. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 40

Figure 3. 6 Box and whiskers plot of pupal developmental time of larvae fed on OP, HC and HP diet with different concentrations of sinigrin. No significant difference was found between treatments. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively ...... 41

Figure 3. 7 The levels of free amino acid in frass of larvae fed on different diets with (20 μmoL/g) or without sinigrin (A), and the total free amino acid value (except cysteine) in different diet and dose treatments (B). OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Asterisks represent significance between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001)...... 44

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Figure 3. 8 The levels of uric acid in frass of larvae fed on different diet with (20 μmoL/g) or without sinigrin. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Asterisks represent significance between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001)...... 45

Figure 3. 9 The levels of AITC conjugates (log transformed): Allyl-Cys (A), Allyl-CG (B) and Allyl-GSH (C) in frass from larvae fed on different diet with (20 μmoL/g) or without sinigrin. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Asterisks represent significance between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001)...... 46

Figure 3. 10 Survival curve of larvae fed on OP (A), HC (B), and HP (C) diets with different AITC concentrations. P value on each plot indicate overall significance of influence of different concentrations of AITC to survival curves. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 47

Figure 3. 11 Box and whiskers plot of developmental time of larvae fed on OP, HC and HP diet with different concentrations of AITC. Different letters represent significant difference between treatments. Lowercase letters represent differences within the same diet of different doses. Capital letters represent differences between diets of the same dose. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively ...... 48

Figure 3. 12 Box and whiskers plot of pupal weight of larvae fed on OP, HC and HP diet at different concentrations of AITC. Different letters represent significant difference between treatments. Lowercase letters represent difference within the same diet of different doses. Capital letters represent difference between diets of the same dose. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively ...... 49

Figure 3. 13 Box and whiskers plot of pupal developmental time of larvae fed on OP, HC and HP diet with different concentrations of AITC. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Asterisks represent significance between groups when tested in GLM (*, p < 0.05; **, p < 0.01; ***, p < 0.001)...... 50

Figure 3. 14 The levels of free amino acid in frass of larvae fed on different diets with (0.14, 0.3, or 0.48 μmoL/g) or without AITC (A and B), and the total free amino acid value (except xv cys) in different diet and dose treatments (C). OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Different letters represent significant difference within the same diet between doses...... 52

Figure 3. 15 The levels of uric acid in frass of larvae fed on different diets with (0.14, 0.3, or 0.48 μmoL/g) or without AITC. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Different letters represent significant difference within the same diet between doses...... 53

Figure 4. 1 The method for inoculating first instar larvae with spores. Spore suspension was first mixed with diet in a 10ul pipette tip. Each larva was confined near the tip by sealing the pipet with tissue paper. By consuming all the diet, larva can escape from the tip into a 2mL Eppendorf tube...... 62

Figure 4. 2 Spores recovered from Helicoverpa armigera (A), Danaus plexippus (B) and Danaus petilia (B) and the comparisons of their length (D) and width (E). Different letter represents significant difference between species...... 64

Figure 4. 3 Infectious status of H. armigera after ingesting different number of spores from H. armigera, D. plexippus and D. petilia...... 65

Figure 4. 4 Developmental time of larvae accomplished larval stage in different spore and dosage treatments. Different letters represent significant different between treatments. ... 66

Figure 4. 5 Pupal weight of larvae exposed to different spore types and dosage treatments...... 67

Figure 4. 6 Pupal duration of larvae exposed to different spore and dosage treatments. .. 68

Figure 4. 7 Larval survival probability when fed on OP (A), HC (B), and HP (C) diet with different doses of OE like spores from H. armigera...... 69

Figure 4. 8 Infection status of adult H. armigera after feeding on different dose of spores from H. armigera on different diets. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 70

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Figure 4. 9 Larval developmental time when assorted by different statuses (A) and different doses (B) when fed on three diets. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Different letters represent significant different between treatments...... 72

Figure 4. 10 Helicoverpa armigera pupal weight after larval stage. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Different letters represent significant differences between treatments...... 73

Figure 4. 11 Helicoverpa armigera pupal duration that resulted in infected and uninfected adults. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively...... 74

Table 2 MS spectrometry parameters for multiple reaction monitoring (MRM) of AITC mercapturic acid conjugates. DP = declustering potential, CE = collision energy ...... 36

Table 3 Standard Diet ...... 105

Table 4 *Anti-fungal solution ...... 105

Table 5 Adult Diet ...... 105

Table 6 Vitamin Solution ...... 106

Table 7 Vitamin Mix ...... 107

Table 8 Diet with Different Protein: Carbohydrate Ratio ...... 107

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List of abbreviations used in the thesis AITC- Allyl isothiocyanate AMPK- adenosine monophosphate-activated kinase ANOVA- Analysis of Variance Cys- cysteine CysGly- cysteinylglycine FMOC- Fluorenylmethyloxycarbonyl GF- geometric framework GLM- generalized linear model GOPOD- glucose oxidase/ peroxidase GSH- Glutathione GSS- glucosinolate sulfatase GST- glutathione-S-transferase HC- high carbohydrate diet HP- high protein diet HPLC- high performance liquid chromatography IT- intake target ITC- isothiocyanate KM- Kaplan-Meier LPS- lipopolysaccharide MRM- multiple reaction monitoring NAC- N-acetylcysteine OE- Ophryocystis elektroscirrha OP- optimal diet PEP- peptidoglycan RGR- Relative growth rate TOR- target of rapamycin β-CD- β-cyclodextrin

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Chapter 1 General introduction 1.1 Literature review 1.1.1 Nutrition self-selection Nutrition is one of the most basic and important requirements for all living organisms. For insects, the requirement for nutrition is highly diverse and complicated (Cohen 2015). A better understanding of insect nutritional needs has a profound influence on agricultural and ecological research. The work on insect nutrient requirements can be sourced from Fraenkel, who discussed the function of specific nutrients from food in a series papers (Fraenkel and Blewett 1942b, a, 1943, 1947, Fraenkel and Printy 1954, Lipke and Fraenkel 1956, Fraenkel 1959b). But nutrient availability in the natural world is highly diverse spatially and temporally, and between species and even within an individual (Berendse 1985, Diawara et al. 1995, Müller et al. 2000, Denno and Fagan 2003, Deans et al. 2016b). The complexity of nutritional requirements and what is available in nature bring up an obvious question: how do insect make decisions of what to eat and balance the different nutrients in food.

The first critical steps in insects involve chemosensory systems to detect food nutrients. Most insects depend on gustatory and olfactory receptors in foraging activity, but the ability to discriminate different nutrients is limited (Chapman 2003). For phytophagous insects, sugars are the most important phagostimulants, because they are commonly present in green plants as the end product of photosynthesis, and all studied phytophagous insects have gustatory cells responding to sugars (Chapman 2003). No evidence has shown phytophagous insects can taste protein, but most of them can respond to amino acids (Blaney 1974, Mitchell and Gregory 1979, van Loon and van Eeuwijk 1989, Simpson et al. 1990, Chyb et al. 1995, Schoonhoven and van Loon 2002). Studies have also demonstrated that insects can respond to NaCl and KCl (Wei-Chun 1972, Mitchell and Gregory 1981, White and Chapman 1990), but the evidence for minor nutrients like vitamins and fatty acids is limited.

Another important factors that insect use to identify food are plant secondary metabolites. Many plant secondary metabolites are classified as toxins or deterrents for phytophagous insects. Both specialist and generalist insects need to identify their host plants by detecting taxon-specific chemicals, and avoid non-host plants at the same time (Dethier 1973, Chapman 2003, Elsayed 2011). Although single plant secondary metabolite can stimulate feeding behavior, it is believed that phytophagous insects are more likely identifying food

1 plants by recognizing a mixture of compounds, which represent the fingerprint of a host plant (Bernays and Chapman 2001, Schoonhoven and van Loon 2002, Chapman 2003).

Even though phytophagous insects can react differently to food plant chemicals, it is still largely “adventitiously” that they acquire a balanced nutrient intake, because leaves have an appropriate chemical composition (Chapman 2003). After eating a nutritionally unbalanced food, postingestive regulation may step in to digest excessive nutrients, accelerating the absorption of deficient nutrients, and the resultant physiological status may motivate insect through the central nervous system to find compensatory food (Behmer 2009).

A Geometric Framework (GF) model (Figure 1. 1) has been proposed to explain the interaction between animals and their environment through nutrition (Raubenheimer and Simpson 1993, Simpson and Raubenheimer 1993a). In the GF model, the optimal nutrient requirement (both the amount and balance) of an animal is called an intake target (IT). The IT can be placed in a two or more dimensional graph known as a nutrient space, where each axis represents the amount of one food component ingested. Depending on the ratios of different nutrient components, one food can be represented as a rail in the graph. The hypothesis is animal tend to ingest different foods or digest each nutrient differently to achieve or at least approach the IT (Simpson and Raubenheimer 2012) (Figure 1. 1).

The GF has been tested in a wide range of organisms including many insect species (Raubenheimer and Simpson 1993, Abisgold et al. 1994, Lee et al. 2008, Cook et al. 2010, Jensen et al. 2012, Tessnow et al. 2018), spider (Wilder 2011), rat (Simpson and Raubenheimer 1997), chicken (Shariatmadari and Forbes 1993), and primates (Simpson et al. 2003, Raubenheimer et al. 2015). The wide application of the GF model suggests balancing nutrient intake may account for foraging behaviors of many animals.

Figure 1. 1 A hypothetical two-dimensional nutrient space showing the position of intake target (IT) and the rails of two nutrients (A and B). Arrows show one possible way animal could choose between the two food sources to achieve IT.

Most insects feed selectively when given the opportunity. On whole plants, early instars insects tend to eat soft tissues and avoid consuming tissues containing high dose of plant secondary metabolites (Reese 1981, Cohen et al. 1988, Parrott 1990, Cízek 2005). In diet choice experiments, insects offered nutritionally complementary diets tend to mix diets to regulate consumption of each nutrient in a rigid manner (Waldbauer et al. 1984, Simpson et al. 2002, Lee et al. 2004, Deans et al. 2015). Such food selection behavior can benefit insect survival, growth, longevity and fecundity (Lewis 1984, Lewis and Bernays 1985, Cohen et al. 1987a, Lee et al. 2008).

The IT is not consistent for insects over their life span. Abiotic factors like temperature (Coggan et al. 2011, Clissold and Simpson 2015) and water (Clissold et al. 2014), biotic factors like insect age (stage) (Chyb and Simpson 1990), plant secondary metabolites (Simpson and Raubenheimer 2001), pathogens (Lee et al. 2006) and parasites (Thompson et al. 2001), can all influence the IT, suggesting a dynamic adjustment in response to internal physiological changes and external environmental stimuli (Simpson and Raubenheimer 2012).

The mechanism of nutrient regulation is complex. In general, an insect detects and assesses the nutrient quality of available food, then assesses its own nutritional state, and “compares” these two to make appropriate responses (Simpson and Raubenheimer 2012). Most insects detect nutrient using mouthpart chemoreceptors (Chapman 2003), which can active the nervous system or modulate key factor (like amino acids and sugars) levels in hemolymph directly (Abisgold and Simpson 1988, Friedman et al. 1991, Simpson and Simpson 1992). The hemolymph plays a central role and provides a constantly updated summary of nutritional state (Simpson and Raubenheimer 1993b); TOR (target of rapamycin) and AMPK (adenosine monophosphate-activated kinase) signaling pathways are emerging as central coordinators (Simpson and Raubenheimer 2009).

Learning is another way insects can regulate nutrient acquisition. Fifth instar Locusta migratoria could associate food nutrient composition with color and odor (Simpson and White 1990, Raubenheimer and Tucker 1997), and fifth instar larvae of Schistocerca gregaria (Forsskål) (Orthoptera: Acrididae) can relate odor to food quantity (Behmer et al. 2005). Learning can benefit growth rate of Schistocerca americana (Drury) (Orthoptera: Acrididae) by enabling more efficient food choice (Dukas and Bernays 2000).

Most studies have manipulated macronutrients like protein, carbohydrate, and fat in GF analysis, because they can explain a good deal of the physiology, behavior and performance responses of animals (Simpson and Raubenheimer 2012). However, other nutrients like vitamins and minerals also play important roles in animal behavior and physiology (Denton et al. 1993, Trumble et al. 1998, Markison 2001, Mogren and Trumble 2010).

The boundary of what constitutes a nutrient is fuzzy. Plant secondary metabolites can be repellent for some insect species, but for host plant specialists, it can also be a feeding stimulant (Bernays and Chapman 2007). The ingestion of plant toxins can even be a self- medication behavior; Grammia incorrupta (H. Edwards) (Lepidoptera: Erebidae) ingesting pyrrolizidine alkaloids improved the survival of caterpillars parasitized by tachinid flies Exorista mella (Walker) (Diptera: Tachinidae) (Singer et al. 2009). Larvae of Danaus plexippus (Linnaeus) (Lepidoptera: Nymphalidae) that fed on milkweeds with higher concentrations of cardenolides experienced lower parasite infection and growth (De Roode et al. 2008).

1.1.2 Plant secondary metabolite and insect detoxification activity In the evolutionary “war” between plants and insect herbivores, both defensive and counter- defense measures have been developed over millions of years (Wikström et al. 2001, Braby et al. 2006). Plants have evolved a vast array of chemicals, including furanocoumarin, monoterpenes, gossypol, alkaloid, cyanogenic glucosides, glucosinolates, cardenolides, etc. that defend them against insect herbivores (Heckel 2018). These chemicals are supposed to be effective against the majority of herbivores, but a few insect species that have developed countermeasures to the chemicals can successfully survive feeding on these plants. This evolutionary army race was recognized in1950s (Fraenkel 1959a), and has been extensively investigated since then (Voelckel and Jander 2014)

One of the most extensively studied plant secondary metabolites are glucosinolates, which are found mostly in the Capparales (Rodman et al. 1996). Depending on precursor amino acids, derived compounds can be classified into aliphatic glucosinolates (from Alanine, Leucine, Isoleucine, Methionine, or Valine), aromatic glucosinolates (from Phenylalanine or Tyrosine), and indole glucosinolates (from Tryptophan). More than 100 described glucosinolates share a chemical structure consisting of a β-D-glucopyranose residue linked via a sulfur atom to a (Z )-N-hydroximinosulfate ester, plus a variable R group (Halkier and Gershenzon 2006). Glucosinolates and their hydrolytic enzymes, myrosinases, are stored in separate compartments in plant tissue. Upon tissue damage, glucosinolate and myrosinase are mixed and react rapidly, to form toxic isothiocyanates, nitriles, thiocyanates, etc., known as the “mustard oil bomb” (Halkier and Gershenzon 2006).

Isothiocyanates are the major by-products that are directly responsible in defending plants with this chemistry from herbivory (Li et al. 2000, Buskov et al. 2002, Agrawal and Kurashige 2003). But for insect specialists like Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae), the production of isothiocyanates can be circumvented by producing a glucosinolate sulfatase (GSS) that convert glucosinolates to harmless desulfo-glucosinolates before they react with myrosinase (Ratzka et al. 2002), or insects that redirect the hydrolysis reaction with the presence of a nitrile-specifier protein in gut, to form nitriles instead of isothiocyanates, as in Pieris rapae (Linnaeus) (Lepidoptera: Pieridae) (Wittstock et al. 2004). The green peach aphids Myzus persicae (Sulzer) (Hemiptera: Aphididae) can feed on host plant phloem without disturbing the glucosinolate–myrosinase system by inserting their flexible stylet intercellularly, but intact glucosinolates can be ingested (Kim et al. 2008).

Glucosinolates can act as feeding stimulant to larvae of specialists like P. xylostella (van Loon et al. 2002). Larvae fed on leaf disks showed significantly higher preference to the disks containing sinigrin. For adults, isothiocyanates and nitriles are involved in attracting adults to their host plants for oviposition, and the responses can be specific to particular isothiocyanates and nitriles, and different combinations of these compounds, indicating a recognition pattern to a particular host plant (Wittstock et al. 2003).

For generalists, broad-spectrum detoxification abilities are needed to deal with toxins in diverse plant species. One exception is in desert locust S. gregaria, which possesses a glucosinolate sulfatase in the gut like P. xylostella, and can hydrolyze glucosinolates to their corresponding desulfonated forms. This detoxification system can be greatly activated when locusts were fed on glucosinolates containing food, but remained at low activity when no glucosinolates are available (Falk and Gershenzon 2007).

A glutathione-S-transferases (GSTs) mediated conjugates with isothiocyanate was proposed as a major detoxification mechanism for generalist herbivories (Schramm et al. 2012). Glutathione (GSH)-, cysteinylglycine (CysGly)- and cysteine (Cys)-ITC-conjugates were detected in frass from Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), Mamestra brassicae (Linnaeus) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae after feeding on glucosinolate-containing plants (Schramm et al. 2012). Only a small proportion of ingested ITCs were excreted as conjugates in S. littoralis, but a much larger proportion of conjugates was thought to be formed initially and then dissociated during digestion (Schramm et al. 2012). This association and dissociation process in the gut can re-release free ITCs, which could go back into epithelium cells causing intracellular and overall GSH depletion. A supplement of cysteine in diet relieved insect from the pressure of forming ITCs that caused growth and protein reduction, increased excretion of GSH conjugates and their derivatives in frass, indicating the importance of diet protein or more precisely, the availability of specific amino acids in generalist detoxification activity (Jeschke et al. 2016).

1.1.3 The interaction between nutrition and toxin Singer et al. (2002) suggested that plant secondary metabolites predominate throughout the process of switching between different food types. In a test using two secondary metabolites,

6 larvae of Grammia geneura (Strecker) (Lepidoptera: Arctiidae) recently fed on one secondary metabolite was more phagostimulatory to diet with a different secondary metabolite, indicating a toxin dilution behavior. In addition, nutrition with co-occurring toxin may influence the total amounts of toxin ingested. Larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) fed with nutrient diluted diet without changing concentration of caffeine compensated by eating more food, resulting in ingesting of more caffeine to a pharmacologically effective dose, interfering with food utilization, slowing growth, reducing subsequent feeding and lowering survival (Slansky Jr and Wheeler 1992). Water content in food may affect deterrence of plant secondary metabolites. As the dietary water increased, the deterrent effect of caffeine and linamarin on larvae of the oligophagous A. gemmatalis and the polyphagous Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) increased as well, suggesting a broad range of this water effect (Glendinning and Slansky Jr 1994). Simpson and Raubenheimer (2001) demonstrated that the balance of macronutrients in food can affect toxicity of tannic acid. Larvae of Locusta migratoria (Fairmaire & L.J. Reiche) (Orthoptera: Acrididae) that ingested a balanced food had the lowest mortality, but the mortality rose markedly with increasing tannic acid level when protein-to-carbohydrate (p: c) ratio become more biased. The high carbohydrate food with tannic acid reduced the rate of intake, while the high protein food with tannic acid reduced nitrogen utilization efficiency.

1.1.4 Pathogen and host insect in diverse nutritional environment Animals have the ability to exploit plant properties to combat parasites (Hutchings et al. 2003). In insects, this has been shown in a limited number of studies. Lee et al. (2006) showed when offered a choice, S. littoralis larvae infected with nucleopolyhedrovirus had higher demand for protein than digestible carbohydrate compared with control and the larvae died of infection. Insects survival probability and developmental rate decreased with decreased diet protein ratio in a no choice test. Similarly, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae) larvae infected with bacterium Bacillus subtilis (Ehrenberg) (Bacillales: Bacillaceae) had increased survival probability with increasing dietary p: c ratio. In a choice test, larvae injected with a sub-lethal dose of bacteria increased protein intake compared with controls, whilst maintaining similar carbohydrate intake levels (Povey et al. 2009).

The performance of insects self-selecting nutrients under pathogen challenge was associated with immune responses. Spodoptera littoralis larvae fed with high protein diet showed higher in lysozyme-like antimicrobial activity, encapsulation response and, marginally, for PO activity (Lee et al. 2006). An increase in antibacterial activity, PO activity and protein levels in the hemolymph of S. exempta was detected when feeding on higher dietary protein levels. However, when challenged with lipopolysaccharide (LPS) and peptidoglycan (PEP), the elevated antibacterial activity was accompanied with a decrease of PO activity, this physiological trade-off was ascribed to a limited protein resource (Povey et al. 2009). Cotter et al. (2011) found that only one concentration of total digestible nutrients differing in p: c ratios could not tell the whole story. By giving larvae of S. littoralis 20 diets with four total macronutrient concentrations and five p: c ratios, a more complex relationship between larval performance, hemolymph protein levels and immune traits was shown in response surfaces. Each trait was affected by a specific combination of nutritional treatment. While cuticular melanism, hemolymph protein and lysozyme levels increased with the increase of dietary protein, PO activity remained relatively stable, though peaking in more carbohydrate-biased intake. Larval performance peaked at intermediate protein levels. The fact that no combination of specific nutrients can simultaneously optimize all immune responses suggested a compromise between the nutritional requirements of growth and immune responses. In contrast with previous studies, diet choice was not affected in larvae challenged with a gram-positive bacteria lyophilised cells, but nutrients were re-allocated to improve their immune response. This difference was attributed to the immune elicitor used in the studies (Cotter et al. 2011).

The change in behavior of self-selection for nutrients under immune challenge may fit the criteria of “self-medication” proposed by Singer et al. (2009): First, self-medication behavior should improve the fitness of animals infected by parasites or pathogens. Second, self- medication behavior in the absence of infection should decrease fitness. Third, infection should induce self-medication behavior. This behavior has been documented in insects when they ingest plant secondary metabolites (Abbott 2014, de Roode and Hunter 2019).

The shifted acquisition for nutrients may represent competition for resources between an insect and pathogen or parasite, but it does not assure the insect will benefit the most from this behavior. Larvae of Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) parasitized by Cotesia congregata (Say) (Hymenoptera: Braconidae) altered food selection from a p: c

8 ratio of approximately 2:1, to a ratio of approximately 1:1. However, instead of reducing parasite load, the diet with a 1:1 p: c ratio supported the largest parasite population (Thompson et al. 2001).

1.2 Aims and perspectives Nutrition is one of the most fundamental components in ecology. Nutrients are produced, transmitted, and consumed by different individuals and species, connecting and affecting each other, ensuring the growth, development and reproduction of life. The geometric framework approach provides a powerful tool to investigate the effect of nutrient interactions in animals. Research has demonstrated that the balance of nutrients in food is more important than energy alone in affecting animal life history, defending from pathogens and parasites, metabolizing toxins, and determining the distribution and abundances of organisms at higher level (Simpson and Raubenheimer 2012). Here I use a GF approach to investigate how diet nutrients shape foraging behavior of H. armigera larvae, and how different diet nutrients affect insects in coping with plant secondary metabolites and parasite infection. Specifically, I undertake experiments that determined behavior of neonates and third instar caterpillars on diets with different P: C ratios (Chapter 2). This work is novel in that almost all work in this space used later instar caterpillars. In Chapter 3 I report studies that caterpillars fed on diets with different P: C ratios responded differently to allyl glucosinolate and allyl isothiocyanate challenge, and the detoxification process was discussed through analysis of frass samples. Finally, a new neogregarine protozoan parasite was identified in H. armigera, which allowed me to test the effect of different diet nutrition to parasite infection (Chapter 4).

The work in this thesis demonstrated that nutrients have profound influence on larval foraging behavior at different instars, and larval detoxification capability and resistance to a parasite were greatly affected by the p: c ratios in diets. This research has extended our understanding of the roles of different nutrients in insect life history and under immune system challenges, which may represent the circumstances that an insect encounters in the field.

Chapter 2 Moving to keep fit: feeding behavior and movement of Helicoverpa armigera (Lepidoptera: Noctuidae) on artificial diet with different protein: carbohydrate ratios

Published as: Wang, P., Furlong, M. J., Walsh, T. K., & Zalucki, M. P. (2019). Moving to keep fit: feeding behavior and movement of Helicoverpa armigera (Lepidoptera: Noctuidae) on artificial diet with different protein: carbohydrate ratios. Journal of Insect Science, 19(5), 20. https://doi.org/10.1093/jisesa/iez098.

1. Conception and design of the project. Peng Wang: (60%) Michael J Furlong: (15%) Thomas K Walsh: (10%) Myron P Zalucki: (15%) 2. Analysis and interpretation of the research data Peng Wang: (70%) Michael J Furlong: (15%) Thomas K Walsh: (0%) Myron P Zalucki: (15%) 3. Drafting the publication and reviewing Peng Wang: (70%) Michael J Furlong: (15%) Thomas K Walsh: (0%) Myron P Zalucki: (15%)

Chapter 2 Moving to keep fit: feeding behavior and movement of Helicoverpa armigera (Lepidoptera: Noctuidae) on artificial diet with different protein: carbohydrate ratios 2.1 Abstract Insect herbivores can modify their foraging behavior to obtain a balanced food intake, and they tend to move between food sources with different nutrient values. We investigated this movement in early instar larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) using a putative optimal artificial diet (OP) and high protein (HP) and high carbohydrate (HC) artificial diets based on protein (p) and carbohydrate (c) ratios. Larvae were allowed to choose between the same kind of diet cubes (effectively no-choice), or diet cubes with different p: c ratios. In no-choice tests, we found that first instar larvae remained longest on OP diet and spent the least time on HC diet, whilst third instar larvae remained longest on HC diet and spent least time on OP diet. First instar larvae moved the most when provided with HC diet, while third instar larvae moved most when provided with OP diet. However, both stages moved the least when allowed to choose between diet cubes with different p: c ratios. The relative growth rate decreased when larvae increased their movement, but this influence was not evident when larvae fed on HC diet. Larvae that fed only on HC diet had the highest relative growth rate, followed by larvae with access to all diets simultaneously, indicating a behavior to mix nutrient intake. We relate these findings to behavior of this major pest species under field conditions. 2.2 Introduction Many insect herbivores have the ability to regulate their intake of specific nutrients to meet their physiological demands, thereby enhancing growth and development, as well as fitness and performance (Simpson and Raubenheimer 1999, Lee et al. 2002, Simpson et al. 2004, Deans et al. 2015, Simpson et al. 2015). The movement of insects between food sources to balance intake of different nutrients has been documented in the laboratory (Simpson et al. 2004, Simpson et al. 2015), and changes in these behaviors allow insects to respond to changes in the relative abundance of different food resources (Behmer et al. 2001), as well as changes in their relative proximity (Behmer et al. 2003). This control of food intake suggests regulation of foraging behavior in imbalanced nutritional environments.

Nutritionally imbalanced environments are very common in nature. Host plants offer a highly heterogeneous nutrient landscape at various scales (Woodwell et al. 1975, Eigenbrode and Espelie 1995, Low et al. 2014) even in agricultural monocultures (Deans et al. 2016b). The

11 quality of food an insect eats can be different from plant to plant (Sánchez et al. 2004, Tao et al. 2014), tissue to tissue (Deans et al. 2016b), and mouthful to mouthful (Shroff et al. 2008), influencing insect movements, development and survival (Zalucki et al. 2002, Perkins et al. 2013, Zalucki et al. 2017).

In addition to variation in what foods are available, the nutrients needed to complete development in different instars of a given insect herbivore can also be different, and the altered foraging strategies exhibited by some caterpillars as they grow may reflect this changed demand. Cohen et al. (1988) showed that first and second instar Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) larvae tended to feed on silk of maize, but that this tendency was lost early in the third instar when the larvae fed almost exclusively on the kernels. When first instars were offered only silk or only kernels, they molted after approximately three days. Subsequently, silk-fed larvae took another 12 days to complete the next three instars while kernel fed larvae took only 5 days. Deans et al. (2018) showed total macronutrient content (soluble protein and digestible carbohydrate content) in corn was always higher in kernels than in silk, which may be the reason why the larvae grow faster when fed on kernels rather than on silk in later instars. Larvae of Eldana saccharina (Walker) (Lepidoptera: Pyralidae) switch from feeding on leaf sheath to stalks of maize after the third instar (Kantiki and Ampofo 1989). This behavior was ascribed to the high sugar content of the maize stalks as well as the ability of larger larvae to penetrate stalks (Scheltes 1978). Gaston et al. (1991) found that 200 of 1137 species of British microlepidoptera make a single marked change in feeding habit as they grow. They suggested a number of reasons that may influence these changes, including food availability, an increase in body size that allows different foods to be exploited, and the risk of being exposed to predators.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a highly polyphagous insect which can feed and develop on a wide variety of native and cultivated plant species (Zalucki et al. 1986, 1994), and the behavior of H. armigera has been studied extensively on individual plants and in the field (Zalucki et al. 2002, Johnson and Zalucki 2007, Amin et al. 2014), these attributes make it an appropriate model insect to investigate the influence of diet on feeding behavior, and to compare with results of the field experiments. Johnson and Zalucki (2007) reported that both first and third instar larvae move extensively on whole Vigna radiata ((L.) R. Wilczek) (Fabales: Fabaceae) (mung bean) plants and that they feed at different locations. However, neonates tended to move to, and stay longer on, the terminal leaves and flower

12 buds of a plant where food was soft and moist, while third instars were less likely to stay on the terminal plant parts and were less selective in their choice of food. Third instars fed at fewer sites and in longer bouts than first instars, but spent less time searching and resting (Johnson and Zalucki 2007).

For insect herbivores, protein (p) and carbohydrate (c) are the most intensively studied nutrient groups (Simpson and Raubenheimer 1999, Simpson et al. 2004), but only a few studies have shown that movement of immature Lepidoptera between food resources is related to the nutrient composition of the food available. Cohen et al. (1987b) showed final instar larvae of H. zea switched between diets more frequently when offered two nutritionally complementary diets in one Petri dish than when only offered nutritionally complete diets. However, the nutritionally incomplete diets used were extreme (p: c ratios were 100: 0 or 0: 100) and unlikely to be found in nature. A less extreme set of diets could lend additional information and insights to the current body of knowledge.

In this study, we examined the influence of defined diets that differ in p: c ratio on the movement of caterpillars in different instars without the confounding influence of other plant characteristics (secondary plant compounds, leaf hairs, waxes etc.). A priori we might expect less movement and more sustained feeding and weight gain on the putative optimised diet and that ‘vulnerable” neonates may move more cautiously and feed rapidly for shorter bouts than larger third instars, as described by Johnson and Zalucki (2007). We compared neonates and newly moulted third instars on supposed optimised diet and diets defined as sub-optimal based on either their p or c content. Different diets were provided in Petri dishes in either no-choice or choice experiments. The no-choice test investigated how often larvae leave a food resource of a particular nutrient content and the choice test investigated movement and feeding between food resources with different nutrient contents and the consequences of these behaviors, as measured by short-term weight gains, final weight and time to complete development to a given stage. 2.3 Materials and Methods 2.3.1 Insects The H. armigera (Mahon et al. 2007) used in the no choice tests were obtained from CSIRO, Black Mountain Laboratory, ACT, Australia. The same H. armigera strain was used in choice tests, but was sourced from the Australian Cotton Research Institute, Narrabri, NSW,

Australia. Insects were reared as described by Teakle and Jensen (1985) and later modified in Perkins et al. (2010). 2.3.2 Artificial diets For general rearing a standard soyflour-based artificial diet which is described in Perkins et al. (2010) was used. For experiments, the artificial diet used was first developed by Ritter and Nes (1981), and modified by Jing et al. (2013). All diets had a total macronutrient (p+c) content of 42% with different protein to carbohydrate ratios (p: c), while all other nutrients remained the same (see appendix 2). Three diets were prepared: the optimal diet (OP), p24: c18 (Tessnow et al. 2018); the high carbohydrate diet (HC), p12: c30; and the high protein diet (HP), p30: c12. The p: c ratios of the HC and HP diets were developed following empirical determination of total macronutrient content (%) and p: c ratios for different cotton tissues grown under both field and greenhouse conditions (Deans et al. 2016b). 2.3.3 Feeding and behavioral studies in a no choice experiment Experiments were conducted using neonates and third instar larvae. Neonates were obtained by allowing eggs to hatch in the absence of food and used in experiments within 8 h of hatching. Third instar larvae used in experiments were reared on standard diet until the end of the second instar, and then isolated until they moulted. Neonates were weighed (Mettler Toledo Excellence XS Balance) and then directly transferred to diet using a paintbrush. Similarly, third instar larvae were weighed but then cooled on ice before placement on the central cube of treatment diet.

For each developmental stage, five 1cm3 cubes (1 cm x 1 cm x 1cm) of a given diet were placed on damp filter paper and positioned in the lid of a Petri dish (9 cm diameter); one cube was placed in the centre of the lid and four cubes were placed 1 cm away, at the cardinal points. A weighed neonate was then placed on the central cube of diet and the bottom section of the Petri dish then placed over the diet and insects. Forty-five replicates of each diet type (OP, HC and HP) were prepared and these were divided randomly into three groups of 15 dishes. In the first group, larvae were weighed again 24 h after placement on a diet cube, in the second group larvae were weighed again after 48 h, and the third group larvae were re-weighed as soon as they moulted to the next instar. These measurements allowed growth rates (weight gain) to be calculated both within and over the entire instar. All larvae in a group were observed semi-continuously for 6 hours (3 h in the morning and 3 h in the afternoon) at approximately 20 min intervals on the day before they were to be re-weighed. The location of each larva (on diet, noting the specific cube; or off

14 the diet) was recorded. A move onto another diet cube or a move off diet cubes was counted as one movement event. After being re-weighed and placed back on the diet, larvae were not subject to further detailed observations.

In total 270 larvae, 135 first instars and 135 third instars, were used. Experiments were conducted under uniform light conditions (L: D, 14: 10) at 25°C± 2°C, relative humidity was 80%. Relative growth rate (RGR) was calculated as the difference in the natural log wet weights between two time periods divided by time elapsed (Kogan and Cope 1974):

RGR = (ln(wt1) - ln(wt0)) / (t1 - t0),

where wt1 and wt0 are the wet weights at times t1 and t0.

2.3.4 Feeding and behavioral studies in a choice experiment A choice experiment was conducted to investigate how larvae perform in a heterogeneous nutritional environment; three complementary no-choice experiments were run concurrently as controls. To set up each choice arena, three cubes (1cm3) of each diet (OP, HC, HP) were randomly selected and placed in the upturned lid of a Petri dish (9 cm dimeter) in a 3 x 3 cube “checkerboard” pattern; within the checkerboard, diet cubes were positioned 1 cm apart and randomized to make sure larvae had equal opportunity to initiate feeding on each diet type. No-choice controls, which were prepared for each diet type, were set up in the same way but contained 9 cubes of a single diet type in a Petri dish lid. For the first instars, 1 larva was placed on each diet cube (9 larvae per dish). There were 10 replicates of mixed diet Petri dishes and a total of 9 control dishes (3 dishes containing just OP diet, 3 dishes containing just HC diet and 3 dishes containing just HP diet). For the third instars, 30 Petri dishes containing mixed diet were prepared along with 30 dishes containing just OP diet, 30 dishes containing just HC diet and 30 dishes containing just HP diet. A single larva was placed on the central diet cube in a Petri dish. All larvae were observed at 15 min intervals for 6 hours a day (3 h in the morning and 3 h in the afternoon) for two days. The location of larvae (on diet, noting the diet type and the specific cube, or off the diet) was recorded. All larvae were re-weighed after two days to calculate RGR.

2.3.5 Statistical analyses Two categories of behavior were evaluated: the presence of larvae on diet and the frequency of larvae transferring between cubes of diet. Only data from the first two days were included in the movement analysis to avoid the influence of moulting on larval behavior.

All data analysis was conducted in R, version number 3.2.5 (R Core Team 2016). In the no choice test, a Generalized Linear Model (GLM) based on a binomial response distribution was fitted to the data of the presence larvae on diet, with predictors of diet and instar. A Generalized Linear Model with the same predictors based on a Poisson response distribution was fitted to the data of larval movement between diets. In the developmental test, one-way ANOVA was used to analyse the effect of diet. When appropriate, multiple comparisons were made using Tukey’s HSD post-hoc test following ANOVA. Comparisons of regression lines (see Figure 2.3) for differences were made using ANCOVA, with number of moves as the predictor variable and diet as the co-variate. In choice tests, the proportion of first instar larvae on different diets was analysed by the Friedman test and then by Wilcoxon test for multiple comparisons. The other movement tests and RGR analysis were the same as in the no choice tests. 2.4 Results 2.4.1 Feeding and behavioral studies in a no choice experiment 2.4.1.1 Movement We found a significant effect of diet on the presence of first instar larvae on diet. Larvae spent a significantly higher proportion of time on OP diet than on HC diet (glm: p < 0.01; Figure 2. 1 A). Diet had a significant effect on the transfer frequency between diet cubes; larvae fed on HC diet alone were more likely to transfer between cubes of diet compared with larvae feeding on OP diet or HP diet alone (glm: OP vs. HC, p = 0.015; HP vs. HC, p = 0.049; Figure 2. 1 C).

We found a significant effect of diet on the presence of third instar larvae on diet. Among the three diets, larvae spent the least amount of time on OP diet, and there were significant differences in the time spent on OP diet compared with HC diet and HP diet (glm: OP vs. HC, p < 0.001; OP vs. HP, p < 0.001; Figure 2. 1 A). Diet also had a significant effect on the transfer frequency between cubes; larvae on OP diet transferred significantly more frequently than larvae fed on HC diet (glm: OP vs. HC, p = 0.003; Figure 2. 1 C).

When data of both first instar and third instar were compared, instar significantly affected both the presence of larvae on diet and the transfer frequency between diets; first instar larvae spent more time on diet (glm: first vs. third, p < 0.001; Figure 2. 1 B) and transferred less between diets (glm: first vs. third, p < 0.001; Figure 2. 1 D) than third instar larvae.

Figure 2. 1 Probability that first instar and third instar Helicoverpa armigera larvae were observed on diet (A, B) and the mean number of moves larvae made between diet cubes (C, D). Asterisks represent significance between groups when tested in GLM (*, p < 0.05; **, p < 0.01; ***, p < 0.001). OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively; First represents first instar larvae; Third represents third instar larvae. 2.4.1.2 Development and growth Neonates fed on HC diet were significantly heavier at the end of the instar than larvae fed on OP or HP diet (Tukey’s HSD test: OP vs. HC, p = 0.045; HP vs. HC, p = 0.013; HP vs. 17

OP, p = 0.877; Figure 2. 2 A), but there was no effect of diet on the weight of larvae completing development through the third instar (ANOVA: F2,38 = 0.754, p = 0.477; Figure 2. 2 B). Larval developmental time in both instars showed no difference between diet treatments (ANOVA: first instar, F2,30 = 2.235, p = 0.125; third instar, F2,38 = 2.059, p = 0.142; Figure 2. 2 A, B).

Diet had a significant impact on RGR of first instar (ANOVA: F2,42 = 4.054, p = 0.025; Figure

2. 2 C) and third instar larvae on the first day (ANOVA: first day, F2,42 = 4.551, p = 0.016), and on third instar larvae over the first two days (ANOVA: F2,42 = 10.94, p < 0.001; Figure 2. 2 D) of the experiment. First instar larvae that fed on OP diet had a significantly higher RGR than larvae fed on HC diet on the first day (Tukey’s HSD test: OP vs. HC, p = 0.019; Figure 2. 2 C). On the first day of the third instar, larvae that fed on HC diet had a significantly higher RGR than larvae that fed on HP diet (Tukey’s HSD test: HP vs. HC, p = 0.013), and over the first two days of the third instar, larvae that fed on HC diet had a significantly higher RGR than larvae that fed on OP or HP diet (Tukey’s HSD test: OP vs. HC, p = 0.001; HP vs. HC, p < 0.001; Figure 2. 2 D).

Developmental stage had a significant impact on RGR of larvae; first instars always had higher RGRs than third instars (ANOVA: over first day, F1,88 = 77.291, p < 0.001; over first two days, F1,87 = 74.53, p < 0.001; and over the instar, F1,63 =96.523, p < 0.001; Figure 2. 2 E).

Figure 2. 2 Larval weight and developmental time when fed on different diets in first (A) and third (B) instars; effect of diet on RGR of (C) the first instar larvae, and (D) the third instar larvae; instar effect on RGR in both first and third instar larvae (E). OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Means with different letters were significantly (P < 0.05) different. 2.4.1.3 Number of moves and RGR The number of moves made by each individual larva was plotted against its RGR. Larvae in both instars had lower RGRs when they made more moves between cubes of diet (linear regression: first instar, p < 0.001; third instar, p < 0.001; Figure 2.3). When fed on single diets, the regression of number of moves against RGR had the shallowest slope on HC diet for both larval instars, but there was no significant difference when compared with 0 (linear regression: first instar, slope = -0.022, SE = 0.015, p = 0.155; third instar, slope = -0.012, SE = 0.009, p = 0.206). Larvae fed on OP diet had the steepest regression line slope (linear regression: first instar, slope = -0.035, SE = 0.013, p = 0.013; third instar, slope = -0.038, SE = 0.008, p < 0.001). The regression slope for larvae fed on HP diet was intermediate between these extremes (linear regression: first instar, slope = -0.029, SE = 0.022, p = 0.198; third instar, slope = -0.025, SE = 0.009, p = 0.008). The only significant difference between

19 regression lines was for larvae fed on HC diet and larvae fed on OP diet in third instar

(ANCOVA: F2,1 = 4.356, p = 0.041).

first instar larvae (A) and the third instar larvae (B) were showed with different shapes and colors to represent represent to colors and shapes different with showed were (B) larvae instar third the and (A) larvae instar first regression lines of the same colored data. same of colored the lines regression OP, diets. different HC, HP optimal, carbohydrate represent and highhigh protein diet 2. Figure

Scatter plot of individual larval RGR versus th versus RGR larval individual of plot Scatter

e number of moves in the first two days. Data from the the from Data days. two first the in movesof number e respectively. Lines indicate

2.4.2 Feeding and behavioral studies in a choice experiment 2.4.2.1 Movement In the first instar, larvae were likely to be on diet most of the time, and in mixed-diet Petri dishes they were more likely be found on OP diet and less likely be found on HC diet (Wilcoxon test: OP vs. HC, P = 0.001; OP vs. HP, P = 0.038; HP vs. HC, P = 0.161; Figure 2. 4 A). When the number of movements across treatments was compared, larvae on HC diet only moved most, and larvae with access to all three diet types (MIX) moved least; this was the only significant difference between treatments (glm: MIX vs. HC, p = 0.026; Figure 2. 4 C).

In the third instar (Figure 2. 4 B), larvae were more likely to be found on HC diet than on the other two diets in mixed-diet Petri dishes and OP diet was the least favoured amongst the three diets (glm: OP vs. HC, p < 0.001; OP vs. HP, p < 0.001; HC vs. HP, p = 0.007). When the number of movements across treatments was compared, larvae on OP-diet only moved most, and larvae on mixed-diet moved least. Significant differences were detected between the OP-diet only treatment and the mixed-diet treatment (glm: MIX vs. OP, p = 0.001; Figure 2. 4 D).

Figure 2. 4 Proportion of times that first instar (A) and probability of third instar (B) H. armigera larvae were observed on diet in choice experiment and the mean number of moves larvae made per dish between diet cubes across different dish types (C, D). OP, HC, HP represent optimal, high carbohydrate, and high protein diet respectively (A, B). OP, HC, HP, MIX represent optimal diet, high carbohydrate diet, high protein diet and mixed-diet dish types respectively (C, D). Asterisks represent significance between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 2.4.2.2 Relative growth rate When RGRs were compared across different treatments in both instars, larvae presented with mixed-diet had a significantly higher RGR than larvae presented with only HP-diet (Tukey’s HSD test: first instar, MIX vs. HP, p = 0.018; third instar, MIX vs. HP, p = 0.012), and larvae presented with only HC-diet had significantly higher RGR than larvae presented with only OP-diet or only HP-diet (Tukey’s HSD test: first instar, HC vs. HP, p = 0.006; HC vs. OP, p = 0.033; third instar, HC vs. HP, p < 0.001; HC vs. OP, p = 0.001; Figure 2. 5 A).

Overall, there was no difference between the RGR of first and third instar larvae over the first two days in the given instar (ANOVA: F1,1 = 1.272, p = 0.261; Figure 2. 5 B).

Figure 2. 5 The influence of dish types (A) and instar (B) on RGR over two days. Asterisks represent significance between groups. OP, HC, HP, MIX represent optimal diet, high carbohydrate diet, high protein diet and mixed-diet dish types respectively (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 2.5 Discussion Generalist herbivores, such as highly mobile locusts and caterpillars of some species of Lepidoptera, are considered to have many plant food choices in heterogeneous landscapes, and experiments on chemically defined diet have confirmed they can “maintain” their intake by ingesting complementary nutrients from different food sources (Abisgold et al. 1994, Lee et al. 2002, Simpson et al. 2004, Deans et al. 2015, Simpson et al. 2015). However, why insects make a “decision” to move between different food sources and how this may change across instars as insects develop, is unclear.

With a simplified single diet environment and a mixed diet environment, we found a clear correlation between diet and movement in first and third instar H. armigera larvae. Whether a larva stayed at a feeding location (cube of diet) and the degree of movement (from one cube to another) were significantly affected by diet treatment (Figure 2. 1 A, C; Figure 2. 4). Normally when larvae stayed longer on a particular type of diet, they were also less likely to

23 change locations. Generally, first instar larvae stayed the longest on OP diet, and the third instar larvae stayed longest on HC diet (Figure 2. 1 A; Figure 2. 4 A, B). Accordingly, the frequency with which larvae transferred between diet cubes was the least when they fed on OP diet in the first instar and when they fed on HC diet in the third instar (Figure 2. 1 C; Figure 2. 4 C, D). However, in the choice tests, across different diet treatments, larvae made the least moves in mixed-diet environments in both instars (Figure 2. 4 C, D). This is contrary to the finding of Cohen et al. (1987b) who showed that final instar larvae of H. zea switched between diets more often when offered nutritionally complimentary food than when offered nutritionally complete food. We assume when offered nutritionally complimentary food, early instar H. armigera larvae in our experiment would tend to move less because if the nutrient content is “complete”, there was higher evolutionary risk associated with movement (e.g. predation) than remaining in the same place. However, comparisons with H. zea are difficult as the diet, instar and species, though closely related (Behere et al. 2007), are different .

In the no choice test, larvae took the least time to complete the first instar when fed on OP diet (Figure 2. 2 A), and the least time to complete the third instar when fed on HC diet (Figure 2. 2 B), which were the diets they were more likely to be found on (Figure 2. 1). Diet had a significant effect on RGR of first instar and third instar larvae over the first day, and on the RGR of third instar larvae over the first two days (Figure 2. 2 A, B). The first instar larvae always had the highest RGR when fed on OP diet, but the differences between diets were less obvious over time (Figure 2. 2 A). However, in the choice test, the first instar larvae had the highest RGR over the first two days when fed on HC diet (Figure 2. 5 A), even though they made the highest number of moves (Figure 2. 4 C). This difference may be because of the different cultures kept in the two laboratories that we used in our experiments. The third instar larvae consistently had the highest RGR when fed on HC diet (Figure 2. 2 B; Figure 2. 5 A).

Movement and growth rate appear to be related. In general, larvae that move more had lower RGRs (Figure 2.3). The effect was least for larvae fed on HC diet; regression of RGR against the number of moves had the lowest slope (Figure 2.3), however the number of moves between cubes of diet were not always the fewest (Figure 2. 1 C). The implication is that larvae that fed on HC diet had more energy to compensate for the respiration costs of high frequency movement between foods when compared with larvae fed on other diets.

Larvae on all three diets, including the assumed optimal (OP) diet, made movements between diet cubes, indicating other reasons for this behavior. The supposed OP diet was developed from work on final instar larvae to assess intake target and had not been tested on other instars of H. armigera previously. Furthermore, an intake target is not necessarily the same for larvae in all situations, so this ‘optimal’ diet might not be the preferred choice for all instars. Various studies have demonstrated that larvae in different instars have different demands for nutrients. When larvae of Lymantria dispar (Linnaeus) (Lepidoptera: Erebidae) were provided with choices of artificial diets differing in protein and lipid concentrations, their preference shifted away from high protein, low lipid cubes toward low protein, high lipid cubes across third instar to final instar (Stockhoff 1993). Cohen et al. (1987b) also found that fifth instar H. zea increased sucrose intake at the end of the larval period and they suggested that this was due to the metabolic costs of pupation and adult eclosion. The digestive enzymes in the larval mid-gut were shown to be correlated with this behavior (Kotkar et al. 2009, Clissold et al. 2010, Lwalaba et al. 2010, Kotkar et al. 2012, Sarate et al. 2012). First instar larvae of H. armigera had much lower amylase levels than third and later instars (Kotkar et al. 2009), which may explain the lack of interest in carbohydrate in first instars. On the other hand, the level of digestive enzymes can be adjusted to meet the nutrient deficiency, by releasing less enzymes for nutrients that are in excess while maintaining or boosting levels of enzymes for nutrients that are in deficit (Clissold et al. 2010, Sarate et al. 2012). This may help explain the decreasing difference in RGR between first instar larvae fed on HC and OP diets over time (Figure 2. 2 C).

The macronutrient composition in standard colony diet was similar to that in HP diet (Wang P. unpublished data). Third instar larvae previously raised on standard diet in our experiment had “experienced” high protein diet. However, third instar larvae in choice tests sill preferred HC diet over HP diet (Figure 2. 4 B). In a separate study, when larvae were provided with both diets (p35:c7 and p14:c28) from first to third instar, H. armigera larvae changed their preference from high protein diet in first instar to high carbohydrate diet in third instar (Katsikis et al. 2020), indicating carbohydrate is a more important nutrient in this stadium.

Sugars are the most important phagostimulants for phytophagous insects, and there is no evidence that herbivores can taste protein directly (Chapman 2003). However, third instars preferred HP diet over OP diet (Figure 2. 1 A; Figure 2. 4 B), even though OP diet has more sugar than HP diet. Katsikis et al. (2020) and Tessnow et al. (2018) showed a preference of

H. armigera for p35:c7 over p7:c35 diet in the third and final instars, for p14:c28 over p35:c7 diet in third instar, but no obvious preference when offered p28:c14 and p14:c28 diets in both third and final instars. The choice for diet seems based on a combination of protein and sugar, not sugar alone, but little is known about the physiological basis of how this apparent feedback operates.

The performance of larvae and their behaviors changed when offered diverse diets rather than only one. It has been suggested that feeding on different foods is beneficial for insects because it allows them to obtain target nutrient intake in nutritionally imbalanced environments (Lee et al. 2002, Simpson et al. 2004, Deans et al. 2015, Simpson et al. 2015). Larvae in our experiments had a higher RGR when given access to the three diet types than when they had access to only HP diet or only OP diet, but not when they had access to only HC diet (Figure 2. 5). HC only diet is normally considered a sub-optimal food because of its low protein content. Roeder and Behmer (2014) also showed high carbohydrate diet slowed down the development of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) larvae, and resulted in lower fecundity. By mixing diets in our study, larvae were given the chance to amend a biased nutrient intake to achieve optimized development.

The amount of food that larvae consume varies within each instar. Usually larvae do not feed for a short period after hatching or ecdysis, and this is followed by a period during which food consumption is either constant or, more usually, it increases progressively. The feeding rate will then decline and then cease at some time before ecdysis (Browne 1995). Browne and Raubenheimer (2003) showed increased food ingestion within the fourth instar and fifth instar in H. armigera. Johnson and Zalucki (2007) found a decreased RGR with time in the first instar and a relatively stable RGR in the third instar H. armigera larvae. Here we found a decreased RGR in larvae exposed to different diet regimes over time within both instars (Figure 2. 2).

First and third instars showed differences in movement between diets. Contrary to Johnson and Zalucki (2007), the number of times larvae moved between diets changed dramatically from the first instar (5.24 moves/ larva) to the third instar (8.01 moves/ larva) over the first two days in the no choice test. First instar larvae were less likely to move between diets, suggesting a more conservative strategy in food selection compared to the third instar larvae. This may reflect a greater vulnerability in neonates to extreme environments (Terry et al.

1989, Kobori and Amano 2003, Leonard et al. 2016) and predators (Zalucki et al. 2002) than in the third instar larvae. Consistent with other studies (Johnson and Zalucki 2007, Quintero and Bowers 2018), first instar larvae had higher RGR than the larvae in the third instar (Figure 2. 2 C; Figure 2. 5 B).

In summary, our study directly associated the movement of caterpillars and the nutrient composition of their diet, in two early instars of H. armigera larvae. We present evidence that diet has a clear influence on caterpillar movement, as does instar. However, our study of caterpillar foraging behavior was under relatively simplified laboratory conditions. Singer et al. (2002) suggested that food mixing behavior in nature may frequently involve both nutrient balancing and toxin dilution, and showed that secondary metabolites were more important factors affecting behavior of Grammia geneura (Strecker) (Lepidoptera: Arctiidae) larvae than nutrient balancing. Behmer et al. (2001) showed learning may also influence insect food mixing behavior in fifth-instar nymphs of Locusta migratoria (Linnaeus) (Orthoptera: Acrididae) (Behmer et al. 2003). Such factors introduce exciting interactions with nutrient which will be explored in future experiments.

2.6 Acknowledgments We thank Sharon Downes for providing us Helicoverpa armigera culture, the help from Ashley Tessnow with artificial diet protocol, and Lynda Perkins, Patrick Ward and Simon Blomberg for help in data analysis. This research was partially supported by the program of China Scholarship Council (No. 201508410154).

Chapter 3 The challenge of eating: balancing nutrients in a toxic environment 3.1 Abstract Insect herbivores can regulate their food intake by mixing food sources with different nutrient content, but they also face the challenge of plant secondary metabolites. How insects deal with toxins in a complex nutrient environment is unclear. I investigated the influence of a classic plant secondary metabolite, allyl glucosinolate (sinigrin), and its hydrolysed product allyl isothiocyanate (AITC), on the development of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) when fed on diets with different protein-to-carbohydrate (p: c) ratios. Further, I examined how these toxins were metabolized by analysing the constituents of frass produced by insects feeding on different diets. As expected, AITC had a greater effect than sinigrin on H. armigera survival, development, and pupal weight. However, AITC at low doses appeared to have a positive effect on the parameters above. Both sinigrin and AITC can induce detoxification activity in gut, and the reaction was related to diet protein concentration. High protein diet can provide adequate free amino acid, especially cysteine, that is part of the detoxification process. Nutrient content of the diet plays an important role influencing how plant secondary metabolites are handled. 3.2 Introduction The presence of plant secondary metabolites poses a dilemma for insect herbivores: To eat more to get more nutrition, and ingest a greater dose of toxin that may kill them; or to eat less to reduce toxin effects, but slow down their development due to the lack of nutrients. Many insects can select from a range of food sources to achieve their intake target of basic nutrients (Simpson et al. 2004, Simpson et al. 2015), but to what extent plant secondary metabolites can influence insect feeding behaviour in different nutritional environments and how they cope with the toxins is not clear.

The glucosinolate–myrosinase system found in the Brassicales is a classic example of plant chemical defence. Glucosinolates and their hydrolytic enzymes, myrosinases, are stored in separate compartments in intact plant tissue. Upon tissue disruption, myrosinases come into contact with glucosinolate substrates, and glucosinolate hydrolysis results in the formation of toxic isothiocyanates and other biologically active products (Winde and Wittstock 2011). Isothiocyanates (ITCs) have a characteristic pungent odour and are toxic to a broad range of organisms (Winde and Wittstock 2011). Experiments have shown that ITCs reduce herbivore survival and growth, and increase developmental time, each in a dose-dependent manner (Li et al. 2000, Agrawal and Kurashige 2003).Glucosinolates alone are normally

28 considered non-toxic, however they can influence insect development at high doses, suggesting hydrolyse may be occurring in the gut (Li et al. 2000).

The generalist herbivore Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) does not have a glucosinolate specialized detoxification system as found in the diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) (Ratzka et al. 2002), but the species is recorded as a pest on Brassica plants (Cunningham and Zalucki 2014). Adult female H. armigera tend to lay few eggs on common cabbage, Brassica oleracea (Linnaeus) (Brassicales: Brassicaceae) (Firempong and Zalucki 1989, Zalucki et al. 2012) and larvae tend to avoid glucosinolate containing food when foraging (Shroff et al. 2008), however, they can survive (albeit poorly) on Brassicales, like common cabbage (Zalucki et al. unpublished) and Arabidopsis thaliana ((L.) Heynh.) (Brassicales: Brassicaceae) (Zalucki et al. 2017). The ability to feed on glucosinolate containing plants has been attributed to formation of glutathione-conjugates of ITCs in the gut of larvae, enabling the toxin to be excreted (Schramm et al. 2012).

In addition to the glucosinolate–myrosinase system, diets with different protein-to- carbohydrate (p: c) ratios have been used to explore the interaction between nutrients and plant secondary metabolites. The detoxification process in insects is costly (Lindroth et al. 1990, Despres et al. 2007, Petschenka and Agrawal 2016), and insects cope better when fed on certain diets. Simpson and Raubenheimer (2001) showed with increasing percentage of tannic acid, mortality of fifth-instar Locusta migratoria (Fairmaire & L.J. Reiche) (Orthoptera: Acrididae) decreased with the increasing protein proportion in the diet; survival was highest when p: c ratio was 21:21 (which was away from the optimal ratio p19: c23 without tannic acid), but dropped when with further increases in protein ratio. Bacillus thuringiensis (Bt) toxin susceptibility may also be influenced by diet in H. armigera and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae); larvae fed on protein biased diet were less susceptible to Bt toxin (Deans et al. 2016a, Tessnow et al. 2018, Luong et al. 2019).

Here I investigated the performance of H. armigera larvae on diets of different p:c ratios containing both sinigrin (allyl glucosinolate) and its hydrolysed product allyl isothiocyanate (AITC). Given the rich knowledge of these compounds from previous research, and the performance of H. armigera on glucosinolate containing plants, it will be interesting to investigate how this generalist herbivore deals with these plant secondary metabolites.

Although AITC is volatile, I encapsulated it into β-cyclodextrin (β-CD), and carefully monitored the release of AITC over time to ensure a “known” dose was delivered to feeding larvae. Life-history traits of larvae reared on different diets with and without toxins were recorded and frass was collected to determine potential detoxification activity during digestion by caterpillars. 3.3 Materials and Methods 3.3.1 Insect The H. armigera was reared at UQ, and sourced from the Australian Cotton Research Institute, Narrabri, NSW, Australia. The rearing method is the same as described in chapter 2. 3.3.2 Diet The standard rearing diet (Appendix A) and nutritionally defined artificial diets (Appendix B): optimal (OP), high carbohydrate (HC) and high protein (HP) were the same as described in chapter 2 (Wang et al. 2019).

All diets were sent to the School of Agriculture and Food Sciences, The University of Queensland, for further analysis of protein and digestible carbohydrate concentrations using standard techniques. Dumas method was used to analyse protein constituents (Simonne et al. 1997). Soluble carbohydrates were hydrolysed to reducing sugars and reacted with ferricyanide, while starches were broken down enzymatically to glucose and reacted with GOPOD (glucose oxidase/ peroxidase) glucose reagent and evaluated spectrophotometrically. The results for soluble carbohydrate and starches were combined as digestible carbohydrate (Weir et al. 1977, Karkalas 1985, McCleary and Codd 1991). 3.3.3 AITC encapsulation The method used to encapsulate AITC (Sigma, catalog no. w203408, ≥ 95%) was as described by Li et al. (2007) with minor modification. β-CD (5 g) was dissolved in 150 ml of distilled water at 60°C on a hot plate. After cooling the β-CD solution to 40°C , AITC in ethanol (1:1, v/v) was slowly added to the solution with continuous agitation. The vessel was sealed and the solution continuously stirred for 3 h with a magnetic stirrer, and then the resulting slurry was refrigerated overnight at 4°C . The cold precipitate was recovered by vacuum- filtration, and dried in an oven at 70°C for 24 h. The final dry encapsulated powder was stored in an airtight tube at room temperature.

3.3.4 Total AITC determination The total content of AITC in the powder was measured as described by Li et al. (2007). Encapsulated powder (0.10 g) was weighed into a 50 ml flask and mixed with 5 ml of distilled water and 7 ml of n-hexane. The flask was connected to an upright glass condenser cooled by tap water. Then the mixture was heated in a water bath at 85°C for 20 min with intermittent shaking. On heating, a glass lid was attached to the top of the condenser, to avoid the loss of AITC. After the first extraction, the flask was cooled to room temperature and the inner wall of the condenser was washed with 3 ml of hexane in order to collect the maximum amount of AITC. After that, the upper hexane, containing AITC, was separated by decantation. Finally, the volume (V, ml) and the absorbance at 248 nm of the combined hexane extracts were measured. The concentration of AITC (C, g/mL) of the extracts was assessed against a calibration curve of AITC standards. The final amount of AITC complexed in the powder was calculated using the product of V and C: AITC content = V ∗ C/0.1 3.3.5 Release characteristics of AITC from diet Diet was microwaved and cooled down to about 40°C, then mixed with encapsulated AITC with a pellet pestle motor (Kontes, Vineland, NJ).

In order to determine how much AITC was left in the diet over time, three doses of encapsulated AITC (1.224 μmoL/g, 1.836 μmoL/g and 2.448 μmoL/g) were mixed with all three diets. Once solid, 3g of diet was placed in a 9 cm diameter Petri dish covered with a lid at room temperature. 300mg of diet from all three concentrations was removed at 0h, 12h, 36h and 60h, and put into a 50 mL flask and processed using the same procedure for determining the AITC in the complex above. The weight of the leftover diets at each sampling time were recorded.

A gas chromatograph mass spectrometer (Shimadzu GCMS-QP2010 Plus) was used to analyse the samples. The applied column was a ZB5-MS 0.25 mm x 30 m x 0.25 μm. The oven temperature was programmed from 50ºC for 5 min, for 110ºC with a rate increase of 5ºC/min and 300ºC with a rate increase of 20ºC/min for 3.5 min. The temperature of the ion source was set to 200ºC, for the interface at 250ºC. The injection was split, and the injector temperature was set to 250ºC. The volume of the injected sample was 1 µL. The flow rate of the helium carrier gas in the column was 3.14 mL/min. Quantification was made by

31 selective ion monitoring of the 99 m/z fragment for AITC, 119 m/z for internal standard tert- Butylbenzene.

A series of concentrations (0.0010 μmoL/mL, 0.0051 μmoL/mL, 0.0102 μmoL/mL, 0.0204 μmoL/mL, 0.0510 μmoL/mL) of pure AITC in n-hexane were prepared to make a standard curve. The concentration of internal standard was 0.00025μL/mL in all samples. 3.3.6 Diet preparation and feeding larvae with sinigrin and encapsulated AITC The presence of glucosinolates and isothiocyanates in plants can be up to 3.52 μmoL/g (Rosen et al. 2005) and 1.7 μmoL/g (Agrawal and Kurashige 2003), respectively. Here sinigrin (Sigma, catalog no. 85440, ≥ 99%) at doses of 0, 0.25 μmoL/g, 5 μmoL/g, 10 μmoL/g, 20 μmoL/g, and AITC at doses of 0, 0.14 μmoL/g, 0.3 μmoL/g, 0.48 μmoL/g (concentration at 12 h) were added into all three diets (concentrations determined from preliminary experiment). The control diet of AITC feeding assay contained the same amount of β-CD as the highest dose.

Sinigrin is a stable product, so diets containing sinigrin were freshly made before feeding experiments. However, AITC is volatile and release is a dynamic process. Based on the experiment above, all diets containing encapsulated AITC were made 12 hours before feeding, and were changed every day until pupation. AITC concentration was determined at 12h.

All treatments started from the first instar, except for the highest AITC dose treatment, which started from the third instar; AITC is so toxic it can kill first instar larvae overnight at this concentration. All sinigrin treatments started with 20 larvae, and all AITC treatments started with 30 larvae. Larval frass was collected at the fourth instar for further analysis. Larval developmental time, pupal weight, and pupal duration were recorded. Insects were checked every day. Pupae were weighed one day after pupation. 3.3.7 Frass analysis Frass analysis was conducted by Daniel Vassão’s team in Max Planck Institute for Chemical Ecology, Germany.

Extracts for uric acid and amino acid measurements were prepared from freeze-dried frass samples. Approximately 10 mg of each frass sample was extracted with 400 µL of aqueous tris buffer (50 mM, pH 7,5) in 2 mL Eppendorf tubes under vigorous shaking (2 x 4 min) at

32 room temperature. After centrifugation (20 min under 4300 rpm at 4 °C), the clear supernatants were transferred to new vials, and aliquots separated for further analyses as described below.

Amino acids were measured as their Fluorenylmethyloxycarbonyl (FMOC) derivatives using a LC-MS/MS system (Jeschke et al. 2016). The FMOC-derivatization was carried out as follows: 10 µL of the aqueous extract was mixed with 90 µL 13C- and 15N- labeled amino acid standard solution (Isotec (Miamisburg, Ohio, USA), 20 µg/mL) and 100 µL borate buffer (0.8 M, pH 10). 200 µL FMOC-reagent (30 mM FMOC-Cl in acetonitrile) was added and the reaction was gently mixed and incubated for 5 min. Excess FMOC-Cl was removed by extraction with hexane (800 µL). After phase separation, 200 µL of the bottom aqueous phase were carefully collected and transferred to a glass vial. FMOC-derivatized amino acids were analyzed by employing an Agilent 1260 HPLC (Agilent Technologies, Böblingen, Germany) coupled to an API5000 tandem mass spectrometer (Applied Biosciences, Darmstadt, Germany). The HPLC was equipped with a C18 reversed phase column (XDB C18, 1.8 mm, 4.6 x 50 mm; Agilent Technologies, Böblingen, Germany) and the separation was achieved with a gradient of water/0.05% formic acid (solvent A) - acetonitrile (solvent B) at a flow rate of 1.1 mL * min-1 at 25 °C with the following gradient: 10 % B (0.5 min), 10 – 90 % B (4 min), 90 – 100 % B (1.5 min), 100 % B (0.5 min), 100 – 10 % (0.1 min), 10 % (2.5 min). The ionspray voltage was maintained at -4.5 keV. The turbo gas temperature was set at 700 °C. Nebulizing gas was set at 70 psi, curtain gas at 35 psi, heating gas at 70 psi and collision gas at 2 psi. MS parameters for detection were as in Table 1 below. Quantification relied on the isotopically labelled amino acids added to individual samples (cysteine not present, so it is not possible to quantify it in absolute concentrations, only to compare peak areas in different groups).

Table 1 Mass spectrometry parameters for amino acid and uric acid detection for multiple reaction monitoring (MRM) of the compounds analyzed. Amino acids are listed as their FMOC-derivatives. The MRM of the respective labelled standard is listed in brackets [XX]. DP = declustering potential, CE = collision energy. Compound Analyte Q1 (Da) Q3 (Da) DP (volts) CE (volts) Class Ala 310 [314] 88 [92] -25 -10 Arg 395 [405] 173 [183] -45 -18 Asn 353 [359] 157 [163] -35 -12 Asp 354 [359] 158 [163] -40 -16 Cystine 683 [691] 152 [156] -60 -34 Gln 367 [374] 145 [152] -35 -12 Glu 368 [374] 172 [178] -40 -14 Gly 296 [299] 74 [77] -30 -10 Amino acids His 598 [607] 154 [163] -40 -18 Leu+Ile 352 [359] 130 [137] -30 -10 Lys 589 [597] 145 [153] -45 -20 Met 370 [376] 174 [180] -30 -18 Phe 386 [396] 164 [174] -30 -10 Pro 336 [342] 114 [120] -30 -10 Ser 326 [330] 130 [134] -25 -14 Thr 340 [345] 144 [149] -35 -14 Val 338 [344] 116 [122] -30 -10 Uric acid 167 124 -30 -18

Uric acid was measured directly using an Agilent HP1200 (Agilent Technologies, Böblingen, Germany) Series instrument coupled to an API3200 tandem mass spectrometer (Applied Biosciences, Darmstadt, Germany) (Jeschke et al. 2016). The HPLC was equipped with a C18 reversed phase column (XDB C18, 1.8 mm, 4.6 x 50 mm; Agilent Technologies, Böblingen, Germany) and the separation was achieved with a gradient of water/0.05% formic acid (solvent A) - acetonitrile (solvent B) at a flow rate of 1.1 mL * min-1 at 25 C with the following gradient: 3 % B (1 min), 3 – 100% B (1.7 min), 100 % B (0.3 min), 100 – 3 %

B (0.1 min), 3 % B (2.9 min). The ionspray voltage was maintained at -4.2 keV. The turbo gas temperature was set at 700 C. Nebulizing gas was set at 70 psi, curtain gas at 35 psi, heating gas at 70 psi and collision gas at 2 psi. Quantification relied on an external calibration curve using an authentic standard.

Extracts for analysis of AITC metabolites were prepared from freeze-dried frass samples. Approximately 10 mg of each frass sample was extracted with 250 µL of 1:1 methanol: water pH 3 in 2 mL Eppendorf tubes under vigorous shaking (2 x 4 min) with metal beads at room temperature. After centrifugation (20 min under 4300 rpm at 4 °C), aliquots of the clear supernatants (200 µL) were transferred to new vials for HPLC-MS analyses.

HPLC analyses were performed in an Agilent 1260 HPLC (Agilent Technologies, Böblingen, Germany) coupled to an API5000 tandem mass spectrometer (Applied Biosciences, Darmstadt, Germany) operating in MRM (multiple reaction monitoring) mode. The HPLC was equipped with a C18 reversed phase column (XDB C18, 1.8 mm, 4.6 x 50 mm; Agilent Technologies, Böblingen, Germany) and the separation was achieved with a gradient of water/0.05% formic acid (solvent A) - acetonitrile (solvent B) at a flow rate of 1.1 mL * min-1 at 20 C with the following gradient: 3 – 15 % B (0.5 min), 15 – 85 % B (2 min), 85 – 100 % B (0.1 min), 100 % B (0.9 min), 100 – 3 % (0.1 min), 3 % (2.4 min). The ionspray voltage was maintained at -5.5 keV. The turbo gas temperature was set at 550 C. Nebulizing gas was set at 70 psi, curtain gas at 35 psi, heating gas at 70 psi and collision gas at 4 psi. Parameters for analysis of the mercapturic acid conjugates of AITC were optimized using crude reaction mixtures containing these products, prepared following the protocol of Kassahun et al. (1997) with modifications as follows. Conjugates of AITC were prepared by mixing a solution of 9.8 µL AITC in 990 µL ethanol with a solution of either glutathione (GSH, 32.62 mg), cysteinylglycine (CG, 16.6 mg), cysteine hydrochloride (Cys, 14.09 mg) or N- acetylcysteine (NAC, 19.35 mg) in 2.5 mL 1:1 ethanol: water pH 7.7. Solutions were stirred under a nitrogen atmosphere at room temperature for 18h, then concentrated to about 1 mL under a nitrogen flow and used as-is for HPLC-MS analysis for product identification. MS parameters for detection were as in Table 2. Absolute quantification was not possible, as pure standards were not available. NAC conjugate was not detected above noise in any frass sample, and values are therefore not included in final table.

Table 2 MS spectrometry parameters for multiple reaction monitoring (MRM) of AITC mercapturic acid conjugates. DP = declustering potential, CE = collision energy Compound Analyte Q1 (Da) Q3 (Da) DP (volts) CE (volts) Class Allyl-GSH 407,2 278,1 131 17 Allyl-CG 278,1 75,7 61 33 ITC conjugates Allyl-Cys 221,2 204,0 46 15 Allyl-NAC 285,1 152 91 19 (Na+)

3.3.8 Statistical analyses All data analysis was conducted in R, version number 3.2.5 (R Core Team 2016). Frass from fourth instar larvae was analyzed to examine free amino acids, uric acid and AITC conjugates levels via LC-MS/MS. Only the extreme treatments, control and 20 μmoL/g sinigrin treated groups, were included to obtain the most significant difference. Data of AITC conjugates was log transformed for analysis. One-way ANOVA was used to analyse the difference between different concentrations of treatments. When appropriate, multiple comparisons were made using Tukey’s HSD post-hoc test following ANOVA. Kaplan-Meier method was used to estimate survival probability. 3.4 Results 3.4.1 Diet analysis The three artificial diets with different p: c ratios were all around expected rails, except HC diets were a little higher than the 12: 30 expected rail, which means they had a higher protein ratio than calculated. UQ standard diets was similar to HP diet in terms of ratios of protein and digestible carbohydrate compositions. Even though the three repeat samples were from the same batch of diet, all test results stand out individually indicating diets were not totally homogeneous (Figure 3. 1).

Figure 3. 1 Protein and digestible carbohydrate analysis of the experimental diets. Position of points showed dry compositions of protein and digestible carbohydrate in the diets (three repeats each). The three rails represent three p:c ratios of diet (indicated on each rail). OP, HC, HP, UQ represent optimal, high carbohydrate high protein and UQ standard rearing diet respectively.

3.4.2 AITC encapsulation Several batches of encapsulated AITC were made during the experiment. All batches were tested before use to determine the total amount of AITC in the product. A small difference was detected between batches but all were about 470-550μmoL/g. 3.4.3 Release characteristic of AITC from diet In all three diets, AITC content had a steep decline in the first 12 hours in all concentrations, and then remained relatively stable in following hours (Figure 3.2). On the other hand, the weight of 300 mg fresh diets went down steadily after calibration, indicating water loss from the diet (Figure 3.3). Weight losses were consistent between diets.

2.5 OP 4.96 2 HC 4.96 HP 4.96 1.5 OP 3.37 HC 3.37 1 HP 3.37

OP 1.79 Concentration (umol/g) Concentration 0.5 HC 1.79 HP 1.79 0 0 12 24 36 48 60 72 Time (h)

350

300

250 OP 200 HC HP

150 Weight (mg) Weight 100

0 0 10 20 30 40 50 60 70 Time (hour)

Figure 3.3 The change in weight (±SE) of 300 mg fresh diet for three diet types over time. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively.

3.4.4 Feeding assay with sinigrin Most larvae survived in this experiment even at the highest sinigrin dose (≤3 died for each group), but some differences in development time were found between doses and diets. When fed on OP diet, larvae took significantly less time to complete the larval stage when fed diet containing 20 μmoL/g sinigrin than all other treatments (Tukey’s HSD test: OP diet, 20 μmoL/g vs. 0 μmoL/g, p = 0.002; 20 μmoL/g vs. 0.25 μmoL/g, p < 0.001; 20 μmoL/g vs. 5 μmoL/g, p = 0.021; 20 μmoL/g vs. 10 μmoL/g, p = 0.001). When fed on HP diet, larvae took significantly less time to complete development when fed on diet containing 5 μmoL/g sinigrin than 0.25 μmoL/g sinigrin (Tukey’s HSD test: 5 μmoL/g vs. 0.25 μmoL/g, p = 0.017). No significant difference was found between treatments when larvae fed on HC diet. Within control and 0.25 μmoL/g sinigrin treated groups, larval developmental time was significantly longer when fed on HC diet than when fed on OP or HP diet (Tukey’s HSD test: control, HC vs. OP, p = 0.004; HP vs. HC, P = 0.002; 0.25 μmoL/g sinigrin, HC vs. OP, p < 0.001; HP

39 vs. HC, P = 0.005). At 10 μmoL/g sinigrin, larvae fed on HC diet took significantly more time to develop than larvae fed on OP diet (Tukey’s HSD test: HC vs. OP, p = 0.025) (Figure 3. 4).

For pupal weight, no significant difference was found between treatments within a diet, except when fed on OP diet, larvae treated with 5 μmoL/g sinigrin resulted in significantly heavier pupae than larvae treated with 0.25 μmoL/g sinigrin (Tukey’s HSD test: 5 μmoL/g vs. 0.25 μmoL/g, p = 0.029). At 0.25 μmoL/g and 10 μmoL/g, larvae fed on HC diet resulted in significantly heavier pupae than larvae fed on OP diet (Tukey’s HSD test: 0.25 μmoL/g, HC vs. OP, p = 0.028; 10 μmoL/g, HC vs. OP, p = 0.009). Similarly, at 5 μmoL/g, larvae fed on HC diet resulted in significantly heavier pupae than larvae fed on HP diet (Tukey’s HSD test: HC vs. HP, p = 0.021) (Figure 3. 5).

No significant difference between pupal developmental times were found between treatments (Figure 3. 6).

3.4.5 Frass analysis of sinigrin feeding larvae Free amino acid analysis showed diverse levels between amino acids and within an amino acid group between diets. Some significances were seen between diet treatments in alanine, glycine, proline, threonine, glutamate, arginine, lysine, histidine and cysteine groups. All the significant effects showed a higher relative value in sinigrin treated than control groups in protein biased diets (OP or HP diet), except for the relative values of cysteine in the sinigrin- treated groups were significantly smaller than control groups in both OP and HP diets treatments. When fed on HC diet, most free amino acids had lower relative value in sinigrin treated group than control group, but no significant difference was detected (Figure 3. 7 A).

The concentration of total free amino acids (excluding cysteine) in frass showed significant differences between insects fed on sinigrin treated diet and control diet when the diet was

OP or HP, but not for HC diet (ANOVA: OP, F1,38 = 8.636, p = 0.006; HC, F1,27 = 0.864, p =

0.361; HC, F1,25 = 4.930, p = 0.036). There was a significant interaction between diet and sinigrin dose (ANOVA: F2,90 = 3.824, p = 0.025). When treated with sinigrin, total free amino acid in frass only increased when insects fed on protein biased diets (OP and HP diet), but decreased when insects fed on low protein diet (HC diet) (Figure 3. 7 B).

Uric acid is one of the main products of protein digestion (Chapman and Chapman 1998). The levels of uric acid in frass were significantly higher in control groups than 20 μmoL/g sinigrin treated groups in all three diet treatments. Across diets, the uric acid was correlated with the protein concentration in control diets (HP > OP >HC), but not in diets containing 20 μmoL/g sinigrin. (Figure 3. 8)

Glutathione conjugate derivatives were demonstrated to be the major metabolites of isothiocyanates in several generalist lepidopterans (Schramm et al. 2012). To assess the detoxification capability of larvae fed on diets with different p: c ratios and sinigrin, the levels of Allyl-Cys, Allyl-CG and Allyl-GSH in frass were measured in all groups.

Even though larvae ingested a glucosinolate (sinigrin), AITC conjugates were detected in the frass. The Allyl-Cys level in frass from OP diet treatment, and Allyl-GSH level in frass from HP diet treatment were significantly higher when larvae were treated with sinigrin compared with larvae in control groups (ANOVA: OP, F1,38 = 6.636, p = 0.014; HP, F1,23 = 7.484, p = 0.012). The Allyl-CG level in frass was significantly lower in the sinigrin treated group than in the control group when larvae were fed HC diet (ANOVA: F1,25 = 15.89, p < 0.001). The most abundant conjugate was Allyl-Cys (Figure 3. 9), and when correlated with free amino acid analysis, cysteine levels were always lower in sinigrin treated groups than control groups (Figure 3. 7).

3.4.6 Feeding assay with AITC As the hydrolysed product of sinigrin, AITC is more toxic and larval performance was much more dramatically affected by addition of AITC into diet than the addition of sinigrin into diet. Sometimes only one larva developed to the pupal stage (Figure 3. 12).

Survival probability was analysed by the Kaplan-Meier (KM) method in each diet treatment (highest dose groups were not included because they started from third instar). Different concentrations of AITC in OP diet and HC diet had a significant influence on larval survival probability, but in HP diet, no significant concentration effect was detected (Log-Rank test: OP, p = 0.007; HC, p = 0.005; HP, p = 0.097). In pairwise comparisons, larvae fed on OP diet with 0.14 μmoL/g of AITC had significantly higher survival probability than larvae fed on control diet (Log-Rank test: control vs. 0.14 μmoL/g, p < 0.001; control vs. 0.3 μmoL/g, p = 0.358; 0.14 μmoL/g vs. 0.3 μmoL/g, p = 0.078). In HC diet groups, significantly higher survival probability was detected when larvae fed on diet with 0.14 μmoL/g of AITC than the

46 other two concentrations (Log-Rank test: control vs. 0.14 μmoL/g, p = 0.002; 0.14 μmoL/g vs. 0.3 μmoL/g, p = 0.002; control vs. 0.3 μmoL/g, p = 0.575). (Figure 3. 10)

For all three diets, larvae took significantly less time to complete the larval stage when diet contained 0.14 μmoL/g AITC than all other treatments (Tukey’s HSD test: OP diet, 0.14 μmoL/g vs. 0 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.3 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.48 μmoL/g, p < 0.001; HC diet, 0.14 μmoL/g vs. 0 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.3 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.48 μmoL/g, p < 0.001; HP diet, 0.14 μmoL/g vs. 0 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.3 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.48 μmoL/g, p < 0.001), but no significant differences were found between other dose combinations (Figure 3. 11).

The only difference, when comparing different diets at the same AITC concentration, was that larvae took significantly less time to complete development when fed on HC control diet than when fed on OP control diet (Tukey’s HSD test: control, HC vs. OP, p = 0.003). (Figure 3. 11)

As for pupal weight, when fed on OP diet, larvae treated with 0.14 μmoL/g AITC had significantly heavier pupae than larvae treated with control or 0.3 μmoL/g AITC (Tukey’s HSD test: OP, 0.14 μmoL/g vs. control, p < 0.001; 0.14 μmoL/g vs. 0.3 μmoL/g, p < 0.001). When fed on HC diet, larvae treated with 0.14 μmoL/g AITC had significantly heavier pupae than larvae treated with control or 0.48 μmoL/g AITC (Tukey’s HSD test: OP, 0.14 μmoL/g vs. control, p = 0.040; 0.14 μmoL/g vs. 0.48 μmoL/g, p = 0.011). When fed on HP diet, larvae treated with 0.14 μmoL/g AITC had significantly heavier pupae than all other three treatments (Tukey’s HSD test: OP, 0.14 μmoL/g vs. control, p < 0.001; 0.14 μmoL/g vs. 0.3 μmoL/g, p < 0.001; 0.14 μmoL/g vs. 0.48 μmoL/g, p < 0.001). Comparing pupal weight between control groups, larvae fed on OP diet had significantly lighter pupae than larvae fed on HC and HP diet (Tukey’s HSD test: control, OP vs. HC, p = 0.001; OP vs. HP, p = 0.048). No significant differences were found between diets at other AITC concentrations (Figure 3. 12).

Pupal durations in all treatment groups were similar except in OP diet treatment, insects treated with 0.3 μmoL/g AITC took significantly longer than insect treated with 0.14 μmoL/g AITC (Tukey’s HSD test: OP, 0.14 μmoL/g vs. 0.3 μmoL/g, p = 0.001). This difference was caused by a few outliers in 0.3 μmoL/g AITC treatment group.

3.4.7 Frass analysis of AITC feeding larvae Similar to the sinigrin test, free amino acids levels in frass varied greatly and were influenced by AITC concentrations. Almost all diet treatments in each amino acid analysis showed significant differences between different doses of AITC (Figure 3. 14 A, B).

When pooled together, both diet and AITC dose had a significant influence on total amino acids level in the frass (excluding cysteine) (ANOVA: diet, F2,160 = 11.071, p < 0.001; dose,

F3,160 = 6.434, p < 0.001), however there was no interaction. When fed on protein biased diets (OP and HP), total values showed similar changes with increased AITC dose: values decreased at first then jumped higher than control at the highest dose. In all three diet

50 treatments, the smallest total free amino acids values occurred when larvae were fed diet treated with 0.3 μmoL/g AITC (Figure 3. 14 C).

Figure 3. 14 The levels of free amino acid in frass of larvae fed on different diets with (0.14, 0.3, or 0.48 μmoL/g) or without AITC (A and B), and the total free amino acid value (except cys) in different diet and dose treatments (C). OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively. Different letters represent significant difference within the same diet between doses. 52

Diet and dose of AITC significantly influenced uric acid detected in larval frass (ANOVA: diet,

F2,159 = 74.844, p < 0.001; dose, F3,159 = 4.425, p = 0.005), and thier was a significant interaction (ANOVA: F6,159 = 3.841, p = 0.001). The abundance of uric acid in frass was correlated with diet protein concentration, which was highest in HP diet, and lowest in HC diet. Protein biased diets (OP and HP) shared a similar pattern of uric acid levels, with the highest uric acid levels when larvae fed on control diet, and the lowest uric acid levels when fed on diet with 0.14 μmoL/g AITC. On the other hand, larvae fed on low protein diet (HC) had the most uric acid in frass when treated with diet containing 0.14 μmoL/g AITC (Figure 3. 15).

The AITC conjugates analysis in this test is not shown because the data obtained was not reliable due to high variance. 3.5 Discussion The glucosinolates-myrosinase system is one of the most important defence mechanisms in cruciferous plants (Winde and Wittstock 2011). During the coevolution of plant-insect

53 interactions, insect herbivores that consume these plants have developed different strategies to cope with the toxins generated by the “mustard oil bomb”. Specialists can accomplish this feat by converting glucosinolates to harmless compounds that are not substrates for myrosinases (Ratzka et al. 2002), yielding nitriles instead of toxic isothiocyanates (Wittstock et al. 2004), by avoiding cell disruption (Kim et al. 2008) and some even sequester intact glucosinolates as a defence measure (Bridges et al. 2002). On the other hand, how generalist insect herbivores cope with this system is not well understood. The detoxification process of ITCs in generalist insects was suggested to be by conjugation with the tripeptide L-glutathione (GSH), as was found in frass of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) after feeding on A. thaliana plants (Schramm et al. 2012). During this process, protein content, especially GSH in insects, was reduced dramatically, and insects actively attempted to supply cysteine for GSH biosynthesis by induction of the expression of the gene encoding glutamatecysteine ligase, the rate-limiting enzyme in GSH biosynthesis. The negative growth and protein effects were relieved by dietary supplementation with cystine (Jeschke et al. 2016). By providing insect diets with different p: c ratios, I investigated how diet nutrient levels can influence the detoxification activity, when fed either sinigrin or its hydrolysed product isothiocyanate, AITC.

The adverse effect of isothiocyanates (ITCs) on insects has been noted in previous research (Li et al. 2000, Ratzka et al. 2002, Burow et al. 2006, Mumm et al. 2008, Rohr et al. 2011), however, due to the characteristics of these chemicals, ITCs are mostly volatile, which makes it difficult to address this topic directly. The use of microencapsulated AITC was first introduced by Agrawal and Kurashige (2003) to investigate the interaction in Pieris rapae (Linnaeus) (Lepidoptera: Pieridae). I found this is not a definitive approach, because AITC can still evaporate into the air but at a slower rate. When added to the diet, AITC reduced by more than 1/2 during the mixing process, and the concentration kept dropping during the first 12 hours. However, in the following hours, AITC concentration remained relatively stable (Figure 3.2), partially because the drying process concentrated the remaining AITC in the diet, but the loss does not stop (Figure 3.3). That is why in my experiments the diet mixed with AITC was only used after 12 hours for 24 hours. Agrawal and Kurashige (2003) did not report on this problem.

As a substrate that reacts with myrosinases, sinigrin alone is considered nontoxic (Donkin et al. 1995, Jeschke et al. 2016). However, Li et al. (2000) recorded a LC50 ± 95% CI at 6.73

± 1.39 μmoL/g for sinigrin in diet in a generalist Spodoptera eridania (Stoll) (Lepidoptera: Noctuidae). In a four day feeding assay, H. armigera neonates had a 50% mortality when fed on diet with 50 μmoL/g sinigrin concentration (data not shown), and indeed AITC conjugates were identified in frass of sinigrin fed larvae (Figure 3. 9). These results indicate a hydrolysis process of sinigrin in the gut without myrosinases, which confirmed a previous report (Agnihotri et al. 2018). In rats and human, gut bacteria can hydrolyse glucosinolate but in a very inefficient way (Krul et al. 2002, Angelino et al. 2015).

The influence of 20 μmoL/g sinigrin on larval development, pupal weight and pupal duration was not very obvious when compared with control, except for developmental time in the OP diet group, which might be due to a few abnormal values (outliers) in this group (Figure 3. 4). The tolerance to toxins is associated with increasing larval size; bigger instars can tolerate bigger doses (Yu 1983, Jeschke et al. 2017). Larvae were given constant dose of sinigrin throughout all instars, so even though a small influence appeared at early instars, it is possible they can overcome and catch up with control group in later instars.

On the other hand, addition of encapsulated AITC into the diets had a significant impact on larval survival and development. In most cases, 0.14 μmoL/g AITC treated groups had the highest survival probability (Figure 3. 10). Larval developmental time and pupal weight both fluctuated with increased dose of AITC, with the shortest developmental time and biggest pupal weight in 0.14 μmoL/g AITC treated groups across diets (Figure 3. 11 and Figure 3. 12). The shorter development time and increased pupal weight at a low dose of AITC was attributed to a hormesis effect, a dose–response relationship characterized by a reversal in response between low and high doses of a stressor (some insecticides for example) (Guedes and Cutler 2014). The hormesis phenomenon is widely recognized in various organisms, and insecticide-induced hormesis is the most studied for arthropods (Knutson 1955, Ouye and Knutson 1957, Luckey 1968, Morse and Zareh 1991, Guedes et al. 2009, Rabhi et al. 2014). The prevalent explanation for hormesis is the principle of resource allocation theory: the stress-exposed individual shifts the balance between potentially energy-conflicting physiological trade-offs, favoring one (e.g. reproduction) at the expense of another (e.g. longevity) (Guedes and Cutler 2014). At the transcript level, hormesis was observed in H. armigera at the low dose of gossypol; genes involved in energy acquisition such as β-fructofuranosidases were up-regulated in the gut, and genes involved in cell adhesion were down-regulated in the body (de la Paz Celorio-Mancera et al. 2011).

The influence of AITC dose on amino acids levels is very interesting. Though 0.14 μmoL/g AITC showed a benefit to H. armigera, total free amino acid in frass did not change significantly compared with controls in all diet treatments at this concentration. But individually, relative values of proline and cysteine were always highest at 0.14 μmoL/g (even though sometimes not significant), which suggests they were less utilised compared with other AITC doses (Figure 3. 14). It is difficult to conclude the function of a single amino acid in a living organism, but some evidence suggested that proline is one of the most soluble amino acids and can act as substrate of tricarboxylic acid cycle to provide energy for flight muscle (Bursell 1981). Cysteine has been recognised as an important amino acid in detoxification process which will be addressed below. So, basically at this concentration, larvae of H. armigera spent less energy and were less involved in detoxification activity compared with larvae treated with other doses of AITC. Total free amino acid levels and individual levels of free amino acid started to increase at the highest dose of AITC, but compared with control, the increases were not significant most of the time. Larvae at the highest dose of AITC suffered very high mortality, the insignificant influence of AITC at amino acid level may imply there was a limitation for this generalist insect to cope with the AITC challenge.

Diets with different p: c ratios had a profound influence on larvae when exposed to potential toxins. Even at a relatively low dose of sinigrin, the larval detoxification system responded actively and showed clear difference between diet treatments. The total free amino acid levels in frass were elevated in OP and HP diets treated groups, and decreased in HC diet treated group, but cysteine decreased in all groups (Figure 3. 7). The levels of AITC conjugates in three diet treatments was irregular when compared between control and sinigrin treated groups. But in protein biased diet treatments, the only two significant effects were higher AITC conjugate levels in sinigrin treated groups than control groups; and in HC diet treatments, there was a significantly lower AITC conjugate level in the sinigrin treated group than in the control group (Figure 3. 9). Much more Allyl-Cys was excreted than the other two conjugates in sinigrin treated groups compared with control groups, which partially explained the decreased relative value of free cysteine in sinigrin treated group compared with control group (Figure 3. 7 and Figure 3. 9).

Larvae fed on HC and OP diets were more likely to be influenced by AITC dose, compared with larvae fed on HP diet, in survival probability (Figure 3. 10). HP diet group normally had

56 higher total free amino and uric acid levels in frass than the other two diets, indicating more protein digestion in this group (Figure 3. 14). However this result cannot be confirmed with elevated AITC conjugates level in frass, given a previous study in S. littoralis, where conjugates excreted in frass were elevated in a dose dependent manner when treated with ITCs (Jeschke et al. 2016).

As was suggested by Jeschke et al. (2016), generalist herbivores detoxify ITCs via conjugation to GSH, and further hydrolysis via the mercapturic acid pathway leads to the corresponding cysteinylglycine (CysGly) and cysteine (Cys) conjugates. This conjugate and disassociation reaction is likely to be a dynamic process, which will eventually release free AITC. Free AITC can once again conjugate with GSH, leading to the depletion of GSH pool (Equation 1). In order to restore homeostatic level of free GSH, insect need to synthesis more GSH. High protein diets can help replenish the shortage of cysteine and other amino acids that can help to overcome AITC, but low protein diet that cannot provide enough amino acids may jeopardize the detoxification process. Lindroth et al. (1990) showed low dietary protein reduced glutathione transferase in Lymantria dispar (Linnaeus) (Lepidoptera: Noctuidae), and cytochrome P-450 and cytochrome P-450 reductase are also likely be reduced under low protein stress (Campbell 2012). HC diet with sinigrin treatment in this experiment showed no significant increase for the levels of AITC conjugates in frass, and even worse, decreased the level of Allyl-CG and amino acids in total (Figure 3. 7 B and Figure 3. 9). +GSH −Glu −Gly AITC → Allyl − GSH → Allyl − CysGly → Allyl − Cys Equation 2 Larval detoxification process in gut after ingestion AITC.

Uric acid is the final product in protein metabolism, and is related to the level of protein catabolism. Excretion of a large quantity of Allyl-Cys increased the demand for cysteine in GSH biosynthesis, therefor increased protein catabolism. Excess amino acids will go through an amino acid deamination process to form uric acid (Weihrauch et al. 2012). It is logical to conclude that elevated free amino acid in frass stimulated by sinigrin will be accompanied by higher levels of uric acid, which have been reported in a few studies (Horie and Inokuchi 1978, Horie and Watanabe 1983, Jeschke et al. 2016). Interestingly in the sinigrin feeding experiment, uric acid levels in all diet treatments were significantly lower in sinigrin fed groups than in controls (Figure 3. 8). In the AITC feeding experiment, uric acid levels in frass were positively correlated with total amino acid levels in control and 0.14 57

μmoL/g AITC dose, but negatively correlated with total amino acid levels at higher doses (Figure 3. 15). These results indicate a complicated relationship between uric acid level, sinigrin/ AITC dose and diet type. Uric acid level in frass does not always relate to total amino acid, and different toxins and doses can end up with completely different results. Further investigation with more comprehensive test involving measurement of total body nitrogen and hemolymph protein concentration, are needed to understand this phenomenon.

Even though the effect of sinigrin in gut was subjected to hydrolysed product–AITC, they still showed different consequences in frass analysis. The control groups between the sinigrin and AITC feeding assays were not consistent. Most groups of HC diet treatment showed the highest level of free amino acid in sinigrin feeding assay, but lowest level of amino acid in AITC feeding assay amongst the three diets (Figure 3. 7 and Figure 3. 14). The only difference between control diets in the two assays was additional β-CD in controls of AITC feeding assay, but this theoretically should not change amino acid concentration in diet. Logically, HC diet should have the lowest amino acid concentration among the three diets. Higher mortality in controls of AITC feeding assay than in sinigrin feeding assay also showed this inconsistency (Figure 3. 10). I suggest it is not appropriate to compare the results of the two assays, but this should not influence comparisons within an assay.

In conclusion, the influence of sinigrin and AITC on development of a generalist herbivory H. armigera, and the detoxification capability in a diverse diet nutrient context were addressed in this chapter. Although AITC showed higher toxicity than sinigrin as expected, they both triggered active detoxification responses, highlighted with a hormesis effect at low dose of AITC treatment across all diets. Diets with different p: c ratios were the first time related with AITC detoxification process, and demonstrated that protein could be a crucial factor in the process as well as to their development and survival probability. Further research may need to involve analysis of gene expression, enzyme activity, and amino acid measurement inside larvae, to shed more light on the interaction of diet, toxins and insect feeding and physiology.

Chapter 4 What a parasite needs: effect of an Ophryocystis elektroscirrha (Neogregarinorida: Ophryocystidae)-like spore in Helicoverpa armigera 4.1 Abstract Ophryocystis elektroscirrha (McLaughlin & Myers) (Neogregarinorida: Ophryocystidae) (OE) is a relatively benign neogregarine protozoan parasite that infects monarch (Danaus plexippus (Linnaeus) (Lepidoptera: Nymphalidae)) and queen (Danaus gilippus (Cramer) (Lepidoptera: Nymphalidae)) butterflies, but may have some negative effects on the fitness of adults. A new OE-like spore was found on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Morphometric measurements and infection tests of this OE-like spore and the spores recovered from infected D. plexippus and Danaus petilia (Stoll) (Lepidoptera: Nymphalidae) showed a clear difference among them. The virulence of this OE-like spore and interactions with diets with different protein-to-carbohydrate (p: c) ratios were investigated. Infection was positively related with dose of the OE-like spore, but did not significantly affect fitness of H. armigera. Development between infected and uninfected larvae did not differ when treated with diets with different nutrient levels, but larvae fed on high protein diet showed the lowest rate of infection. 4.2 Introduction The neogregarine protozoan parasite Ophryocystis elektroscirrha (McLaughlin & Myers) (Neogregarinorida: Ophryocystidae) (OE), was first recovered from monarch (Danaus plexippus (Linnaeus) (Lepidoptera: Nymphalidae)) and queen (Danaus gilippus (Cramer) (Lepidoptera: Nymphalidae)) butterflies in Florida in 1966 (McLaughlin and Myers 1970) and has been reported in monarch populations worldwide since then (Barriga et al. 2016). Parasite life history is closely correlated with host development. OE spores ingested by larvae lyse within their gut, and emerging sporozoites penetrate the intestinal wall, infect hypodermal tissue where they replicate and complete morphogenesis resulting in spores found on the exterior of adult butterflies. Infected females scatter spores onto the egg chorion and host plant surface during oviposition (McLaughlin and Myers 1970). Neonate larvae are the most susceptible instar and receive OE by consuming egg chorion. One spore appears sufficient to produce a detectable spore load in the adult, and older instars are less susceptible and have fewer opportunities to encounter sufficient viable spores for infection to occur (Leong et al. 1997).

Monarchs exposed to a high spore density experienced decreased larval survival, smaller adult size, and shorter adult lifespans (Altizer and Oberhauser 1999). Host dispersal

59 distance was negatively associated with parasite prevalence in a population. Average parasite loads of summer-breeding adults in western North America decreased with increasing distance from overwintering sites (Altizer et al. 2000).

As a parasite on insects, OE is not as virulent as a pathogen. It has been suggested that in a vertical transmission system, any severe negative effect on host fitness will result in a failure of pathogen to propagate (Altizer and Oberhauser 1999). Maybe that’s why only until recently, an OE-like spore in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) cultures was identified in laboratories, but may potentially be wide spread in the field (Gao et al. 2020). This parasite at a low dose showed no difference with a clean culture, but caused decreased host adult activity and fecundity when there was a high dose (personal observation).

When fighting against pathogens, nutrients and energy are consumed differently (Thompson et al. 2001, Lee et al. 2006). Nutritionally defined diets give us the opportunity to evaluate the function of each ingredient. Higher dietary protein content for nucleopolyhedrovirus challenged Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) larvae had a higher survival rate than larvae fed on diet with low protein content. And in a choice test, insects that survived viral challenge increased their relative intake of protein compared with controls and those larvae that died of infection (Lee et al. 2006). When parasitized by Cotesia congregata (Say) (Hymenoptera: Braconidae), Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) larvae exhibited altered food selection from a protein: carbohydrate (w/w) ratio of approximately 2:1, to a ratio of approximately 1:1. However, when offered several isocaloric diets with a varying ratios of protein and carbohydrate, larvae fed on the diet with a ratio of 1:1 of these nutrients supported the largest parasite population, but the population decreased as protein: carbohydrate ratio increased (Thompson et al. 2001). Although nutritional regulation in H. armigera and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) has been reported to affect insect susceptibility to Bt toxin challenge (Deans et al. 2016a, Tessnow et al. 2018), it is unknown if different nutrient compositions can influence this OE- like parasite infection.

I investigated the effect of different doses of OE-like spore to H. armigera larvae, and whether two other kinds of spores isolated from D. plexippus and D. petilia can infect H. armigera. The second aim was to investigate how different diet nutrients may shape the

60 fitness of H. armigera infected by OE-like spores. I used newly hatched first instar larvae as this is the stage likely to be infected. Larval developmental time, pupal weight, pupal duration, mortality and infectious status were recorded. 4.3 Materials and Methods 4.3.1 Insect The H. armigera was reared in UQ, and sourced from the Australian Cotton Research Institute, Narrabri, NSW, Australia. The rearing method is the same as described in chapter 2. D. plexippus and D. petilia adults were collected at the University farms at Pinjarra Hills and at Mt Crosby, Queensland, Australia.

A clean H. armigera culture without this OE-like spore infection was obtained by soaking eggs with 0.2 % sodium hypochlorite solution for 8 minutes (coats of spores are dissolved by the solution), and then eggs were washed three times with distilled water. 4.3.2 Diet The standard rearing diet and nutritionally defined artificial diets: optimal (OP), high carbohydrate (HC) and high protein (HP) were the same as described in chapter 2 (Wang et al. 2019). 4.3.3 Spore extraction and measurement Spores were extracted from adults of infected H. armigera, D. plexippus and D. petilia by vigorously agitating their abdomens (2- 4 abdomens) in water, then the suspension was filtered through four layers’ gauze to remove large scales etc., and centrifuged at 4000×g for 10 minutes before the resulting pellet was resuspended in water (~ 200 μL). The concentrations of spores were determined by gradient dilution of the suspension obtained above, until ~10 spores/ μL. Five drops (3 μL/ drop) of diluted and well agitated suspension were put on a glass slide, and spores counted under a Zeiss Axioskop FS2 microscope (× 150). The original spore concentration can be obtained by calculation. Pictures were taken (× 750) to measure the size of spores. Some 30 well developed spores from each species were selected and measured for lengths and widths. 4.3.4 Infection Different concentrations of spores (0, 10, 100 and 1000 spores/ μL) were mixed with diet in a 10μL pipette tip. This was done by taking up 1μL of each suspension of spores with micropipette, and inserting the tip into diet till 2mm of the tip was filled with diet and mixed with the remaining spore suspension.

A neonate first instar larva (0- 12h old) was placed into the 10ul pipette tip and blocked with a small tissue ball. The tip was then sealed in a 2mL Eppendorf tube. Larvae that finished the spore containing diet in tips were able go through the tunnel but still confined in the tube, were then transferred to cups with spore-free diet. Thirty larvae were included in each dose in all experiments.

Infection status was determined by placing a 1-cm2 sticky tape on the ventral side of adult abdomen to remove a sample of scales, which were then examined under a microscope (Altizer and Oberhauser 1999). Larval developmental time, pupal weight and pupal duration were recorded.

Two infection tests were conducted with different diets. A standard diet was used in the first test with four concentrations of spore (0, 10, 100 and 1000 spores/ μL) from H. armigera, and with OE spores from D. plexippus and D. petilia at the highest concentration (1000 spores/ μL). Three nutrient defined diets were used in the second infection test, with three concentrations of spore (0, 100 and 1000 spores/ μL) isolated from H. armigera. 4.3.5 Statistical analyses All data analysis was conducted in R, version number 3.5.1 (R Core Team 2018). The status of all larvae at the end of the experiment were used as data in stack bar plots (Figure 4. 3, Figure 4. 8) and survival probability analysis (Figure 4. 7). As for developmental time (Figure 4. 4 and Figure 4. 9) and pupal weight analysis (Figure 4. 5 and Figure 4. 10), only individuals 62 that reached pupal stage were included. Pupae that emerged as adults were included in pupal duration analysis (Figure 4. 6 and Figure 4. 11). A Generalized Linear Model (GLM) based on a binomial response distribution was fitted to the data of mortality, with predictors of diet or dose. In the spore length, spore width, developmental time, pupal weight and pupal duration analysis, one-way ANOVA was used to analyse the effect of species, diet or dose. When appropriate, multiple comparisons were made using Tukey’s HSD post-hoc test following ANOVA. Kaplan-Meier method was used to analyse survival probability. 4.4 Result 4.4.1 Morphology and size of spores from Helicoverpa armigera, Danaus plexippus and Danaus petilia. Spores from three different species showed similar oval shape and amber coloration. However, spores from H. armigera showed a black belt in the middle, and spores from D. plexippus and D. petilia showed a groove on flattened side of spore (Figure 4. 2 A, B and C). OE spores from D. plexippus were a little bigger than the other two. I found OE spores were around 14 µm in length and spores from H. armigera and D. petilia were around 12 µm. ANOVA analysis confirmed that the spores from D. plexippus were significantly longer than spores from H. armigera and D. petilia (Tukey’s HSD test: D. plexippus vs. H. armigera, p < 0.001; D. petilia vs. H. armigera, p = 0.159; D. petilia vs. D. plexippus, p < 0.001); and the width of spores from D. petilia were significantly smaller than width of spores from D. plexippus (Tukey’s HSD test: D. plexippus vs. H. armigera, p = 0.157; D. petilia vs. H. armigera, p = 0.061; D. petilia vs. D. plexippus, p < 0.001) (Figure 4. 2 D and E).

4.4.2 The effect of OE like spores from H. armigera and two other spores on H. armigera On standard diet, H. armigera larvae infected per os with different doses of OE like spores showed no significant difference in mortality (glm: control vs. 10, p = 0.351; control vs. 100, p = 1; control vs. 1000, p = 0.227; 10 vs. 100, p = 0.351; 10 vs. 1000, p = 0.774; 100 vs. 1000, p = 0.227;). But dose had a significant effect on infection status; larvae fed with 1000 spores had significantly higher rate of infection than larvae fed with 10 or 100 spores (glm: 10 vs. 1000, p < 0.001; 100 vs. 1000, p < 0.001). (Figure 4. 3)

No infection was found when H. armigera larvae were fed with a 1000 spores from D. plexippus or D. petilia. (Figure 4. 3)

Figure 4. 3 Infectious status of H. armigera after ingesting different number of spores from H. armigera, D. plexippus and D. petilia.

When treated with spores from H. armigera, no difference was detected between infected and uninfected groups within a dose treatment in larval developmental time, pupal weight or pupal duration (Figure 4. 4 A, Figure 4. 5 and Figure 4. 6). However, dose had significant effect on larval developmental time. Larvae fed with spores from H. armigera took significantly longer to pupate than larvae in control group (Tukey’s HSD test: control vs. 10, p < 0.001; control vs. 100, p = 0.006; control vs. 1000, p < 0.001) (Figure 4. 4 B). Larvae that failed to emerge from pupae took significantly longer to develop (ANOVA: F1,104 =

16.528, p < 0.001) and were significantly lighter in pupal weight (ANOVA: F1,104 = 32.497, p < 0.001) than larvae that resulted in emerged adults.

Figure 4. 4 Developmental time of larvae accomplished larval stage in different spore and dosage treatments. Different letters represent significant different between treatments. 66

Figure 4. 5 Pupal weight of larvae exposed to different spore types and dosage treatments.

Figure 4. 6 Pupal duration of larvae exposed to different spore and dosage treatments. 4.4.3 Infection vs. diet Larvae fed with nutritionally defined diet suffered higher mortality with or without spore infection in this experiment than in previous experiments (chapters 3). Kaplan-Meier analysis showed no significant effect of OE-like spores on larval survival probability (Figure 4. 7).

Figure 4. 7 Larval survival probability when fed on OP (A), HC (B), and HP (C) diet with different doses of OE like spores from H. armigera.

There were no significant differences in mortality within a diet treatment between doses (glm: OP, control vs. 100, p = 0.433; control vs. 1000, p = 0.407; 100 vs. 1000, p = 0.111; HC, 69 control vs. 100, p = 0.603; control vs. 1000, p = 1; 100 vs. 1000, p = 0.603; HP, control vs. 100, p = 0.795; control vs. 1000, p = 0.302; 100 vs. 1000, p = 0.199). But at dose 1000, larvae fed on OP diet had significantly higher mortality than larvae fed on both HC diet and HP diet (glm: OP vs. HC, p = 0.011; OP vs. HP, p = 0. 011; HC vs. HP, p = 1).

Only larvae treated with 1000 spores were found to be infected as adults in all three diet groups, and at this dose, larvae fed on HP diet had a significantly lower infection rate than larvae fed on OP and HC diets (glm: OP vs. HC, p = 0.621; OP vs. HP, p = 0.0.034; HC vs. HP, p = 0.002) (Figure 4. 8).

Within a diet type, larval developmental time was not significantly different between infected and uninfected groups for all three diets (ANOVA: OP, F1,31 = 0.082, p = 0.776; HC, F1,50 =

0.923, p = 0.341; HP, F1,43 = 0.039, p = 0.844) (Figure 4. 9 A). However, when fed with OP diet, larvae that did not complete the pupal stage took significantly longer to develop than those that did successfully emerge, but no differences were found when fed on the other two diets (ANOVA: OP, F1,46 = 9.347, p = 0.004; HC, F1,60 = 2.144, p = 0.148; HP, F1,64 = 2.172, p = 0.145).

Dose had a significant effect on larval developmental time within HC diet only, larvae treated with 100 spores had significantly shorter developmental time than larvae treated with control or 1000 spores (Tukey’s HSD test: HC, control vs. 100, p = 0.032; control vs. 1000, p < 0.998; 100 vs. 1000, p < 0.029) (Figure 4. 9 B). Dose had no significant effect on larval developmental time in the other two diets (ANOVA: OP, F2,45 = 0.118, p = 0.889; HC, F2,63 = 1.058, p = 0.353).

There were no significant differences in pupal weight between infected and uninfected groups in all diet types (ANOVA: OP, F1,31 = 3.526, p = 0.070; HC, F1,50 = 0.031, p = 0.861;

HP, F1,43 = 1.317, p = 0.258) and in pupal duration (ANOVA: OP, F1,31 = 0.9711, p = 0.332;

HC, F1,50 = 1.046, p = 0.311; HP, F1,43 = 1.571, p = 0.217) (Figure 4. 10 and Figure 4. 11). However, the pupae that did not emerge were significantly lighter in all diet treatments

(ANOVA: OP, F1,46 = 18.99, p < 0.001; HC, F1,60 = 30.518, p < 0.001; HP, F1,64 = 11.137, p = 0.001).

Figure 4. 11 Helicoverpa armigera pupal duration that resulted in infected and uninfected adults. OP, HC, HP represent optimal, high carbohydrate and high protein diet respectively.

4.5 Discussion The OE-like spore from H. armigera had similar shape and colour with spores from D. plexippus and D. petilia, but both spores from D. plexippus and D. petilia showed a flattened side and a groove (Barriga et al. 2016), while spores from H. armigera were more rounded and sometimes with a black belt in the middle (Figure 4. 2 A, B and C). Length and width measurements also showed differences between spores from different species. The OE spore from D. plexippus was the largest one (Figure 4. 2 D and E).

Larvae treated with spores from D. plexippus and D. petilia had no sign of infection, and their mortality, developmental time, pupal weight, pupal duration showed no significant difference to controls (Figure 4. 3, Figure 4. 4, Figure 4. 5 and Figure 4. 6). Previous studies have suggested OE and OE-like parasites are host species specific (Gao et al. 2020). Barriga et al. (2016) showed OE spores from D. plexippus were more effective at infecting D. plexippus than D. gilippus, and D. plexippus were less likely to be infected by OE-like spores from D. gilippus and D. petilia. All these three butterflies are Danaines and closely related (Braby et al. 2015) and they all feed on the same milkweed hosts, so there is a high

74 chance they can encounter spores from each other in North America (D. plexippus and D. gilippus) and Australia (D. plexippus and D. petilia). Even though these OE or OE-like spores are species specific, they can still cross infect each other. H. armigera on the other hand is a moth in the Noctuidae, and although highly polyphagous (Cunningham and Zalucki 2014), does not feed on milkweed, but it does overlap geographically with D. plexippus and D. petilia in Australia. The lack of cross infection in this case suggests the spores from these two groups of Lepidoptera may have been parted a long time ago, and went along different evolutionary paths.

Infection assays showed this parasite is relatively benign to H. armigera. Normal spore loads on eggs from an infected culture was about 40 each (unpublished data), but up to 1000 spores were used in the experiments to test the effect in extreme situations. Larvae treated with spores did not show significantly higher mortality than control groups (Figure 4. 3, Figure 4. 7 and Figure 4. 8). Infected individuals showed no significant difference in pupal weight and pupal duration when compared with uninfected individuals (Figure 4. 5, Figure 4. 6, Figure 4. 10 and Figure 4. 11). The effect of this OE-like spore to H. armigera was similar to the effect of OE to D. plexippus, and further confirmed the theory that vertically transmitted parasites are expected to have minor fitness influences to their hosts (Altizer and Oberhauser 1999).

The OE-like spore can prolong larval developmental time, but when compared between infected and uninfected groups, no significant difference was detected (Figure 4. 4). This is partially because some infected larvae died in the pupal stage, so they were not included in the ANOVA analysis of infected group vs. uninfected group.

Food quality has been suggested to have a big influence on insect fitness (Le Gall and Behmer 2014, Borzoui et al. 2017, Helm et al. 2017), and fighting against a pathogen is always costly to the host (Sheldon and Verhulst 1996, Rolff and Siva-Jothy 2003, Schmid- Hempel 2005, Lee et al. 2006). Protein has been shown to be an important ingredient in insect immunology. Larvae of black soldier fly Hermetia illucens (Linnaeus) (Diptera: Stratiomyidae) infected with pathogens expressed the highest number of antimicrobial peptides (AMPs) when diets were supplemented with protein or sunflower oil (Vogel et al. 2018). Survival of bacterially infected larvae of Spodoptera exempta (Walker) (Lepidoptera: Noctuidae) increased with increasing dietary p: c ratio (Povey et al. 2009). Our result did not

75 suggest a higher survival rate, but at the highest spore dose (1000), the lowest infection rate was detected when treated with high protein diet, and the highest number of infections happened in HC diet treated group (Figure 4. 8). The physiological mechanism of how H. armigera suppresses OE-like spore infection when fed on high protein diet was unclear.

The different diet treatments showed some unexpected results. The OP diet contains a medium ratio (24: 18) of protein and carbohydrate. But H. armigera fed on OP diet suffered high mortality in general, and had the highest mortality across diet treatments at highest spore dose tested (Figure 4. 8). Individuals that died at the pupal stage took significantly longer to develop to this stage than those that eventually successfully emerged (Figure 4. 9 A). The HC diet treatment groups showed significantly shorter developmental time at dose of 100 spores when compared with other doses (Figure 4. 9 B). Perhaps the diet treatments stressed the larvae more than the spore treatment, there were problems with handling larvae or an unrecognized infection occurred during the experiment.

Pupae that did not successfully emerge were significantly lighter than the emerged groups in both experiments (Figure 4. 10), suggesting poorly developed insects had smaller chance to reach adult stage. But once emerged, diet and dose had no effect on pupal duration across all tests (Figure 4. 6 and Figure 4. 11).

The research reported here was essentially an exploratory project about this newly discovered OE-like spore on H. armigera, and many interesting questions have been raised, especially the interaction between diet nutrients and the infection process. By switching between food sources with different nutrient ratios, insect can mitigate the pressure of pathogen challenge (Lee et al. 2006, Povey et al. 2009), but this action may not always favour the host. Pathogens may also influence the host to choose the diet to support the largest population of pathogen, as described by Thompson et al. (2001). This hypothesis has never been well tested in other insects and H. armigera, because it feeds well on artificial diets offers an opportunity to do so.

Helicoverpa armigera is an important agricultural pest that rapidly develops resistance to insecticides (Downes et al. 2017) and much research has been directed at finding alternative management tactics. The OE-like spore has the potential to act as a “Trojan horse” to introduce novel toxic elements and reduce the fitness and eventually the population of this

76 pest. The replication and dispersion mechanism of this OE-like spore need to be clarified in the field.

Chapter 5 General discussion 5.1 Introduction One advantage of a generalist insect herbivore is the capability to choose from a large number of host plants to fulfil nutritional needs. Such switching is thought to be restricted to very mobile herbivores such as Orthoptera. However even caterpillars of Lepidoptera move between hosts, particularly in older instars (Dethier 1959, Jones 1977, Steffan-Dewenter and Tscharntke 1997, Wahlberg 2000, Cunningham et al. 2011). Take H. armigera as an example of a widespread (Kriticos et al. 2015) and highly polyphagous lepidopteran (Zalucki et al. 1994, Cunningham and Zalucki 2014, Gregg et al. 2018). Nutrient levels and composition between plant species or even within a plant species can be vastly different (Berendse 1985, Müller et al. 2000, Machado et al. 2015, Deans et al. 2016b). Larvae move extensively within plants (Perkins et al. 2008, Yang et al. 2008, Perkins et al. 2010) and between plants (Zalucki et al. 1986, Zalucki et al. 1994, Cunningham and West 2008). One of the biggest challenges for a generalist insect appears to be how to balance nutrient intake while avoiding predators and resisting plant secondary metabolites, pathogens and parasites. In this study, a nutrient centred approach was taken to investigate the movement behaviour of H. armigera larvae between diets with different protein-to-carbohydrate (p: c) ratios, and how insect perform on these diets under the pressure of plant secondary metabolites as well as a newly discovered neogregarine protozoan parasite.

Insects tend to ingest different nutrients in a ratio that can optimise their fitness and performance, the amount and balance of nutrient is termed the intake target or IT (Raubenheimer and Simpson 1993, Simpson and Raubenheimer 1993a, 1995, Raubenheimer and Simpson 1999, Simpson and Raubenheimer 2001). The theory was first tested on Locusta migratoria (Fairmaire & L.J. Reiche) (Orthoptera: Acrididae) (Raubenheimer and Simpson 1993), and then on other insects (Abisgold et al. 1994, Lee et al. 2002, Merkx-Jacques et al. 2008, Tessnow et al. 2018), and on mammals (Simpson and Raubenheimer 1997, Thompson and Redak 2005, Felton et al. 2009, Rothman et al. 2011). However, the intake target is not consistent. As insects grow, or attempt to mitigate the effect of plant secondary metabolites, or fight against pathogens, the demand for nutrients can be different.

5.2 Foraging behavior of Helicoverpa armigera on diets with different protein-to- carbohydrate ratios All the analysis of macronutrients for insects has used their larger instars (Raubenheimer and Simpson 1993, Lee et al. 2002, Tessnow et al. 2018), but in my study (chapter 2), the performance of first and third instar of H. armigera were tested for the first time on three diets with different p:c ratios. Although the OP diet was determined as optimal diet in the final instar of H. armigera (Tessnow et al. 2018), the first and third instars performed very differently on this diet. First instar larvae were more likely to be found on OP diet, but third instar larvae were least likely to be found on this diet, HC diet being the most visited diet. The number of moves was negatively related with probability of larvae being on diet -- if larvae stayed longer on one kind of diet, they were less likely to make a movement (Figure 2.1). The developmental time and RGR were also influenced by diets in each instar. First instar larvae fed on OP diet, and third instar fed on HC diet resulted the shortest developmental time of the instar and the highest RGR over the first day respectively (Figure 2.2). These results were further confirmed in a choice test: when offered the three diets rather than one, the order of diets larvae chose was the same as the no choice test in both instars (Figure 2.4 A, B). These results show that H. armigera larvae have different nutrient requirements in different developmental stages. Yet larvae continue to move even on the diet they were most likely to be found on, and when offered a choice (Figure 2.4 C, D), larvae moved the least compared with no choice controls, indicating other factors that may influence their foraging behavior, for example the propensity to avoid predation (Bernays 1988, 1989).

Measurements of amylase levels in different instars suggest that there may be a physiology bases explanation for diet choice by different instars. Kotkar et al. (2009) found that first instar larvae of H. armigera had very low amylase activity when fed on artificial diet, and that the activity of this enzyme increased dramatically and peaked at the third instar, then slowly decreased. This may reflect that the need for digestible protein in early instars is more important than carbohydrate. It is commonly reported that in forest pests, protein is usually a scarce food resource, and early instar larvae need more protein than later instars (Montgomery 1982, West 1985, Auerbach and Simberloff 1989). It has also been reported that ozone breaks down tissues of Solanum lycopersicum (Linnaeus) (Solanales: Solanaceae) that release large amounts of free amino acids which greatly increased first

79 instar survival and developmental rate of Keiferia lycopersicella (Walsingham) (Lepidoptera: Gelechiidae) (Trumble et al. 1987).

Apart from diet difference, first and third instar larvae showed different RGR and movement strategy in general. First instar larvae had a higher RGR than third instar larvae, as reported by Johnson and Zalucki (2007), and Quintero and Bowers (2018). Third instar larvae were more often observed to move between diet cubes than first instar larvae (Figure 2.1 D), which is consistent to Johnson and Zalucki (2007), that third instar larvae rested more often and fed more often, and spent less time on a visit to a feeding site on a whole plant than first instar larvae. Larger larvae, with bigger mandibles and better digestion, can feed on more diverse diets than first instars; this allows them to adjust feeding locations not just for nutrients, but also to provide more flexibility to avoid their natural enemies and exposure to damaging environmental conditions. First instar larvae on the contrary, are more constrained to the diet already available, and are more vulnerable to extreme environmental conditions and predators (Zalucki et al. 2002), this is reflected in a more conservative feeding strategy.

Diet, RGR and number of moves were related. More movement resulted lower RGR in general, which is easy to understand because activity can increase respiration cost. But feeding on different diets had different consequences; RGR of HC diet fed larvae were less influenced by movement than those fed on other diets, indicating excess carbohydrate can compensate for the energy cost and support growth at the same time. Put another way, a high carbohydrate diet can support caterpillar making long distance moves without greatly undermining fitness in terms of weight gain. 5.3 Interaction between diet nutrients and plant secondary metabolites Plant secondary metabolites are sometimes an even more important factor than nutrients in shaping foraging behaviour (Singer et al. 2002). Some secondary metabolites are acute toxins for insects; like nicotine, thymol, pyrethrin, Bt toxin (Berdegué et al. 1996, Rattan 2010) and AITC (chapter 3); and insects need to avoid these chemicals to prevent death. The interaction between plant secondary metabolite and nutrients is complicated. Slansky Jr and Wheeler (1992) showed that a nutrient dilute diet can lead larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) to ingest toxic levels of caffeine. Simpson and Raubenheimer (2001) demonstrated that tannic acid in high carbohydrate diets can act as antifeedant on juveniles of L. migratoria, but not in a balanced diet or high protein diets. However, in high protein diets, tannic acid had a toxic effect after ingestion. In a more

80 complex situation, three diets were provided with two of them providing nutritionally complementary foods (high protein or high carbohydrate) and containing tannic acid, the third one was tannic acid free. If the third diet was high carbohydrate diet, locusts were able to mix this diet with tannic acid containing high protein diet, to achieve their intake target. But if the third diet was high protein diet, locusts will abandon the intake target because of the deterrent effect of high carbohydrate diet with tannic acid (Behmer et al. 2002).

The interactions between nutrients and two secondary metabolites, the parent glucosinolate (sinigrin) as well as its hydrolysis product AITC were investigated during the whole larval stage. As was expected, sinigrin was much less toxic compared with AITC. Mortalities of sinigrin fed larvae were low and were not influenced by diets with different p: c ratios. Developmental time, pupal time, and pupal duration had no or only a few significant differences between concentrations within a diet. But between diets, HC diets normally resulted in longer developmental time and heavier pupal weight (Chapter 3; section 3.4.4). On the other hand, AITC caused very high mortality across all diet treatments and sometimes only one larva made it to the pupal stage. Survival probabilities was significantly influenced by AITC concentrations in OP and HC diets treatments, but not in HP diet treatment. Larval developmental time and pupal weight showed a clear hormesis effect with the increase of AITC dose in all three diets, highlighted with the shortest developmental time and the heaviest pupal weight at 0.14 μmoL/g AITC (Chapter 3; section 3.4.6).

The detoxification process was investigated by analysing frass from fourth instar larvae. Surprisingly, even though sinigrin did not show toxicity effects on life history traits, at doses much higher than previously reported (Agnihotri et al. 2018), the detoxification reaction was induced actively and was quite different between diet treatments. Total amino acid levels in frass were significantly higher in sinigrin treated groups than controls when larvae fed on protein biased diets (OP and HP), but not when larvae fed on HC diet. The levels of individual amino acids in frass varied greatly between each diet/dose treatment and all significant effects between sinigrin doses were detected in protein biased diets (Figure 3.7). The free amino acid analysis provided a basis on which to speculate what happened in the gut and how diet can influence the result. AITC conjugates were suggested to be the major detoxification products in gut, and the process relied on cysteine to complex the conjugates (Schramm et al. 2012). The result in this study showed that Allyl-Cys was the most abundant AITC conjugate in frass, suggesting cysteine was the most consumed amino acid in this

81 detoxification process. Sinigrin treated groups had higher Allyl-Cys levels than control groups when larvae fed on protein biased diets, although not significant in HP diet treated group (because the variance was high) (Figure 3.9). Almost all other free amino acids levels were higher in sinigrin treated groups than control groups when fed on protein biased diets, cysteine levels were significant lower in sinigrin treated groups than control groups in these diet treatments (Figure 3.7). The logical inference is compared with other amino acids, cysteine was more used when exposed to sinigrin and the production of AITC in gut. Contrary to protein biased diets, HC diet treatment showed that total amino acids level and almost all free amino acids levels decreased in frass with exposure to sinigrin. Allyl-CG level was significantly decreased but no significant differences were found in the other two AITC conjugates detected (Figure 3.9). Low protein in diet may cause an overall amino acids deficiency and low assimilation efficiency, and under constant pressure, cysteine was lost without sufficient supplement, resulting in excretion of low levels of free amino acids. The high variability of AITC conjugates levels may reflect variations in gut microflora influenced by antibiotics in diets in a lab culture (Thakur et al. 2016, Khaing et al. 2018, Bai et al. 2019).

AITC treated larvae showed quite complicated results in frass analysis, and were different to sinigrin. In all three diet treatments, larvae treated with AITC did not result in significantly higher levels of total free amino acids and most of individual free amino acid in frass than larvae in control groups, even though larvae suffered high mortality at high dose of AITC, indicating a limited detoxification capability. Because of the hormesis effect, low concentration (especially at 0.14 μmoL/g) of AITC benefited larval fitness. The levels of free cysteine in frass increased significantly when fed with protein biased diets, but not HC diet, at 0.14 μmoL/g AITC, indicating AITC conjugates were even less formed compared with control groups. Proline was another elevated free amino acid at this dose, suggested higher energy provision capability (Figure 3.14).

Uric acid level is related to protein catabolism activity, and the levels in frass were normally consistent with protein concentration in diet. Sinigrin and AITC ingestion can both negatively influence the level of uric acid in frass. This result was contrary with a previous study (Jeschke et al. 2016), in which uric acid level increased when third instar larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) were challenged with isothiocyanate. However, the experimental design in this study was different. Larvae were continuously under sinigrin and AITC pressure from the first instar, and were tested in fourth

82 instar, whereas the Jeschke et al. (2016) study exposed third instars for ten days to toxin. More comprehensive assays like measurement of related gene expression, total body nitrogen and hemolymph protein concentration are needed to further investigate this problem. 5.4 Interaction between an OE-like parasite and diets The effects of the OE-like neogregarine protozoan parasite found on H. armigera was investigated at different doses and with different diet. Like OE in Danaus plexippus (Linnaeus) (Lepidoptera: Nymphalidae), this OE-like spore was relatively benign for H. armigera. Larvae inoculated with different doses of this OE-like spore showed no increase in mortality, but the infection ratio increased dramatically at the highest dose (Figure 4.2). Larvae inoculated with OE-like spore showed significant prolonged developmental time, but no effect on pupal weight and pupal duration (Figure 4.3, 4.4 and 4.5).

Different doses of OE-like spore did not affect larval survival probability when fed on diets with different p: c ratios (Figure 4.6, 4.7). Excluding animals that died, infected and uninfected H. armigera (determined at adult stage) showed no significant difference in developmental time, pupal weight and pupal duration across all diet treatments (Figure 4.8, 4.9, 4.10). But at the highest dose (1000 spores per larva), HP diet fed larvae showed significantly lower infectious rates when compared with larvae fed with OP and HC diet (Figure 4.7).

Nutrient acquisition can greatly affect the fitness of insect, and further impact the immune system (Lee et al. 2006, Alaux et al. 2010, Vogel et al. 2018, Wilson et al. 2019). Larval food restriction causes decreased phenoloxidase activity and hemocyte concentration, which mediated increasing spore load on D. plexippus adults after OE infection, but did not increase the probability of parasite infection (McKay et al. 2016). The study here showed evidence than high protein diet may reduce the probability of OE-like parasite infection. The reason needs to be explored from a physiological perspective in the future.

In comparison with spores found on D. plexippus and Danaus petilia (Stoll) (Lepidoptera: Nymphalidae), spores from H. armigera showed differences in size and morphology (Figure 4.1). No infection was detected when H. armigera were inoculated with spores from D. plexippus and D. petilia (Figure 4.2), and inoculated larvae showed no differences in

83 developmental time, pupal weight and pupal duration with control group (Figure 4.3, 4.4, and 4.5), implying these spores are species specific (Gao et al. 2020). 5.5 Conclusion Nutrition is not only essential for growth, development and reproduction of individual organism, but also touches, links, and shapes all aspects of the biological world (Simpson and Raubenheimer 2012). For insects, nutrients in food can be variable spatially and temporally (Lenhart et al. 2015, Deans et al. 2016b), and the requirement for nutrient changes with the development of insect and under different challenges (Simpson and Raubenheimer 2001, Thompson et al. 2001, Deans et al. 2016a, Tessnow et al. 2018)chapter 2, chapter 3 and chapter 4). This complex interaction resulted in a dynamic intake target, and some nutrients can have very different effects on performance of insects depending on developmental stage. One should make generalizations with great caution.

In this thesis, the changing intake target was tested in different instars of H. armigera and under the pressure of plant secondary metabolites and a parasite. The results indicated that larvae had different nutritional requirements at different developmental stages, and different diet composition shaped their foraging behaviour even in the absence of secondary metabolites (chapter 2). Protein is an important component in resisting plant secondary metabolites and parasite, as high protein diet was more beneficial for insect than other diets under these pressures (chapter 3 and chapter 4).

Insect growth, development and reproduction is always accompanied by dynamic interaction with food, and external biotic and abiotic factors also affect insect nutritional acquisition. The study here can only observe and interpret this interaction from a specific time range and angle, more work is needed to clarify the whole picture. To further address this topic from here, plant secondary metabolites and parasites need to be introduced in self-selection behavioural tests. More physiological research in insect relating nutrition and toxin or pathogen stresses is needed. The OE-like spore from H. armigera deserves more attention for its potential in pest control. My studies have been laboratory based, but the theory needs to be tested and confirmed in field research to further extend our understanding in natural or agricultural environments..

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Appendix A General diet Soybean flour was microwaved with water for 3 minutes and stirred between each minute, then mixed with all other solid ingredients except agar. Agar was microwaved with water before added into the mixture. Anti-fungal solution was added at last and then mixed thoroughly all together. Adult diet was prepared by mixing sucrose and ascorbic acid with water (table 3). Table 3 Standard Diet Component Weight (g) Soybean flour 86 g Water for soy flour (ml) 500 Wheatgerm 60 Yeast 50 methyl paraben 3 L-ascorbic acid 3 Sorbic acid 1 Anti-fungal solution* (ml) 2.6 Agar 10.5 Water for agar (ml) 300

Table 4 *Anti-fungal solution Component volumn (ml) Propionic acid 42 Phosphoric acid 4 Water 54

Table 5 Adult Diet Component Weight (g) Water (ml) 1000 Sucrose 100 Ascorbic acid 3

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Appendix B Diet with different protein: carbohydrate ratio Prepare vitamin mix and vitamin solution ahead of time (table 4 and table 5). Pre-mix casein, cellulose, and cholesterol in a 500 ml beaker. The cholesterol and chloroform mixed in a small (30 mL) beaker in a fume hood and then pour into the mixture. Cholesterol will dissolve in chloroform and the beaker should be washed 3 times with chloroform. The mixture should be left in a fume hood overnight.

The next day, add sucrose, Wesson’s salt, cysteine HCl, myo-inositol, choline chloride, torula yeast, dry milk, vitamin mix, sorbic acid, ascorbic acid, methyl paraben, chlorotetracycline, and streptomycin to the 500 mL beaker. Cholecalciferol, menadione, linoleic linolenic acid and tocopherol were first dissolved in 5 ml absolute ethanol before pouring into the mixture. Add 125 mL distilled water, 365 uL formaldehyde and 30 ml vitamin solution to the mixture and mix thoroughly with stirring rod. At last, boil the mixture of 10g agar and 250mL distilled water in microwave, add it to the rest of the ingredients in the 500mL beaker and stir thoroughly. Once stirred, pour into the molds. The diet will cool and set-up in about 30 minutes. Table 6 Vitamin Solution

COMPONENT weight (g)

Nicotinic acid amide 0.5

D-Pantothenic acid hemicalcium salt 0.5

thiamine HCl 0.0125

riboflavin 0.025

pyridoxine HCl 0.0125

folic acid 0.0125

biotin 0.001

vitamin B12 0.0001

zinc acetat 0.025

cobalt chloride 0.0125 sodium molybdate 0.0125 distilled water (mL) 300 (Store in a flask with parafilm on top to prevent evaporation in the refrigerator 4°C .)

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Table 7 Vitamin Mix Vitamin weight(mg) thiamine 75 riboflavin 75 nicotinic acid 300 pyridoxine 75 folic acid 75 myo-inositol 750 calcium pantothenate 150 p-aminobenzoic acid 75 choline 3750 biotin 3 total (mg) 5328 (Seal with parafilm in a tube in -20°C )

Table 8 Diet with Different Protein: Carbohydrate Ratio

COMPONENT Protein: Carbohydrate Ratio 30:12 (g) 12:30 (g) 24:18 (g) cellulose 30 30 30 vitamin-free casein 26.63 7.58 20.18 cholesterol 0.1 0.1 0.1 chloroform (mL) 35 35 35 sucrose 9.82 26.57 15.5 Wesson's salt 5 5 5 cysteine HCl 0.557 0.557 0.557 myo-inositol 0.2 0.2 0.2 choline chloride 0.5 0.5 0.5 torula yeast 5 5 5 dry milk (whole) 3.75 3.75 3.75 vitamin mix 0.8125 0.8125 0.8125 sorbic acid 0.5 0.5 0.5 l-ascorbic acid 1 1 1

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Methyl 4-hydroxybenzoate 0.4875 0.4875 0.4875 chlorotetracycline HCl 0.008675 0.008675 0.008675 streptomycin sulphate 0.008675 0.008675 0.008675 37% formaldehyde (uL) 365 365 365 100% ethanol (mL) 5 5 5 cholecalciferol 0.0025 0.0025 0.0025 menadione (vit K) 0.0025 0.0025 0.0025 linoleic acid (uL) 275 275 275 alpha-linolenic acid (uL) 193 193 193 dl-alpha-tocopherol 0.05 0.05 0.05 acetate vitamin solution (mL) 30 30 30 distilled water (mL) 375 375 375 agar 10 10 10

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