The Effect of Diet on Midgut and Resulting Changes in Infectiousness of Acmnpv Baculovirus in Trichoplusia Ni

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The effect of diet on midgut and resulting changes in infectiousness of AcMNPV baculovirus in Trichoplusia ni

Elizabeth Chen The University of Western Ontario

Supervisor Dr. Cameron Donly The University of Western Ontario Co-Supervisor Dr. Jeremy McNeil The University of Western Ontario

Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Elizabeth Chen 2017

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Recommended Citation Chen, Elizabeth, "The effect of diet on midgut and resulting changes in infectiousness of AcMNPV baculovirus in Trichoplusia ni" (2017). Electronic Thesis and Dissertation Repository. 4924. https://ir.lib.uwo.ca/etd/4924

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Abstract

The cabbage looper, Trichoplusia ni, a global generalist lepidopteran pest, has developed resistance to many synthetic and biological insecticides, requiring effective and environmentally acceptable alternatives. One possibility is the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). This baculovirus is highly infectious for T. ni, with potential as a biocontrol agent, however, its effectiveness is strongly influenced by dietary context. In this study, microscopy and transcriptomics were used to examine how the efficacy of this virus was affected when T. ni larvae were raised on different diets. Larvae raised on potato host plants had lower chitinase and chitin deacetylase transcript levels and thickened, multilayered peritrophic membranes than those reared on either cabbage or artificial diet. These changes help explain the significantly lower susceptibility of potato reared individuals to baculovirus, underlining the importance of considering the dietary influences on insect susceptibility to pathogens when applying biological control agents in integrated pest management strategies.

Keywords:

Cabbage looper, AcMNPV baculovirus, biocontrol, chitinase, chitin deacetylase, dietary effects, electron microscopy, midgut transcriptome, mRNA sequencing, pathogen resistance, peritrophic membrane, susceptibility

Acknowledgements

First and foremost, I would like to thank my supervisor, Dr. Cam Donly, for granting me the opportunity and honor of working with his research team at Agriculture and Agri-food Canada (AAFC) these past two years. During stretches that were trying or exasperating, you were always patient, encouraging, and insightful. I can’t express how much I appreciate the kindness and guidance shown to a measly graduate student finding her footing. No less deserving in gratitude are Dr. Richard Gardiner and Karen Nygard. I am not yet the microscopist they are, but they have trained me sufficiently well to at least avoid heavy-metal-poisoning myself during my time at the Biotron (and more). They were a joy to work with and their genuine investment in the efforts of their clients was commendable. As well, Dr. Martin Erlandson, Dr. Mike O’Donnell, and Dr. Dennis Kolosov were wonderful collaborators whose help was essential. I would also like to thank my co-supervisor Dr. Jeremy McNeil, and my advisors Dr. Aiming Wang and Dr. Brent Sinclair. The invaluable input and feedback provided was instrumental to my growth as a scientist; I couldn’t ask for a better committee. For those named, you are truly a credit to your profession.

To all current and unofficial members of the Donly lab: Lou Ann Verellen, Angela Kipp, Emine Kaplanoglu, Chelsea Ishmael, Grant Favell, Will Burke, Kaitlyn Ludba, Ren Na, Allan Humphrey, and Arun Kumaran, thank you for the camaraderie, friendship, and laughs. I should consider myself lucky if able to work in another lab of a similar caliber.

Finally, I would like to thank the two pillars of my life: Harry Rusnock and Ana Li. To Harry, you are my dearest partner in greatness regarding all matters, and to my mother Ana, your unwavering and unconditional support all my life has been the most singular factor contributing to my achievements today. I love and thank you both – again and always.

P.S. When all is over and done, I look forward to being able to eat cabbage with peace of mind once more. Thanks, loopers.

Table of Contents

Abstract…………………………………………………………………………………… i

Acknowledgments……………………………………………………………………….. ii

Table of Contents……………………………………………………………………...… iii

List of Tables…………………………………………………………………………...... v

List of Figures………………………………………………………………………….... vi

List of Abbreviations……………………………………………..…………………..... viii

Chapter 1. Introduction……………………………………………………………...…… 1

1.1 Trichoplusia ni: A Cosmopolitan Agricultural Pest……………………...….. 1 1.1.1 Current Control Methods used in Canada...……………………… 2 1.2 Baculoviruses: Sophisticated Insect Pathogens……………………………….3 1.2.1 Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) Baculovirus…………..…………………………..…. 4 1.2.2 Infection Cycle………………………………………………...…. 4 1.3 Plant, Pest, and Pathogen Tritrophic Interactions…………………………..... 7 1.3.1 Plant and Pest…………………………………………………...... 7 1.3.1.1 Brassicaceae: The Mustards………………………...….... 8 1.3.1.2 Solanaceae: The Nightshades………………………...….. 8 1.3.2 Pest and Pathogen……………………………………………...... 9 1.3.2.1 Infection Establishment……………………………...…... 9 1.3.2.2 The Peritrophic Membrane…………………………...….. 9 1.3.2.3 Modification of Host Physiology and Behaviour….…...... 13 1.3.3 Plant and Pathogen…………………………………………...…. 14 1.4 Study Rationale, Thesis Hypothesis, Predictions and Objectives.………….. 15

Chapter 2. Materials and Methods……………………………………………………….16

2.1 Insect Rearing, Plants & Diet, and Virus…………………………………….16

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2.2 Bioassays……………………………..……………………………………....16

2.3 Micro-needle Electrode pH Measurements……………………………….….17

2.4 Scanning Electron Microscopy (SEM)………………………….………..…..17

2.5 Transmission Electron Microscopy (TEM)...………………………….……..18

2.6 RNA Extraction, RNA-seq, and Transcriptomic Analysis…………………...19

Chapter 3. Results………………………………………………………………….……..21

3.1 Diet Based T. ni Bioassay Lethality Curves………………………….……....21

3.2 Measurements of Anterior Midgut pH……………………………………….22

3.3 SEM Characterization of the PM…………………………………………….22

3.4 TEM Characterization of the PM…………………………………………….26

3.5 mRNA Sequencing……………………………………………….…………..31

3.5.1 Read Processing and Quality Control……………………………31

3.5.2 Diet-Induced Transcriptomic Responses in T. ni Midgut………..31

Chapter 4. Discussion…………………………………………………………………....54

4.1 Physiological Differences in Gut pH with Different Diets…….…………….54

4.2 Morphological and Transcriptional Differences in T. ni Midgut PM…..…....54

4.3 Stress Associated with Plant Diet: Triggering of Immune Response………..57

4.4 Detoxification Gene Responses to Plant Allelochemicals……….…………..57

4.5 Future Challenges and Directions…………………………………...…….....58

4.6 Conclusion……………………………………………………………...……59

References………………………………………………………………………………..60

Appendix…………………………………………………………………………………74

Curriculum Vitae………………………………………………………………………....82

List of Tables

Table 3.1. Summary of RNA-Seq data before and after trimming and mapping from midguts of 4th instar T. ni raised on different diets, including information for: total raw reads, reads remaining after trimming, % remaining reads, average read length, mapped reads, % mapped reads, uniquely mapped reads, and % of uniquely mapped reads….…32

Table 3.2. Differentially expressed contigs in midguts of cabbage-raised compared to artificial diet-raised 4th instar T. ni.…………………………………..…….……..….…..36

Table 3.3. Differentially expressed contigs in midguts of potato-raised compared to cabbage-raised 4th instar T. ni.……………………………..………..…………..……….38

Table 3.4. Differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T. ni.………………………………..……………..……...39

Table 3.5. Summary of differentially expressed contigs in midguts between all diet group pairs for 4th instar T. ni. ………………………………………………………….………42

Table 3.6. Top 50 differentially expressed contigs in midguts of cabbage-raised compared to artificial diet-raised 4th instar T.ni……………………………….…………45

Table 3.7. Top 50 differentially expressed contigs in midgut of potato-raised compared to cabbage-raised 4th instar T.ni.……………………………………….…….…………..…48

Table 3.8. Top 50 differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T.ni.……………………………………………………51

List of Figures

Figure 1.1. Life cycle of the cabbage looper, T. ni……………………………………… 2

Figure 1.2. The infection cycle of an AcMNPV baculovirus.…………………………... 6

Figure 1.3. The PM of larval T. ni in its most simplistic form: singular, uniform, compact…………………………………………………………………………………. 10

Figure 1.4. The PM of larval Heliothis virescens in a more disorganized state with laminae and fibrils visible, of various density…………...…………………………….... 11

Figure 1.5. The PM of molting and feeding (non-molting) of Mamestra configurata larvae.…………………………………………………………………………………… 13

Figure 2.1. ImageJ pipeline for ridge detection quantification of SEM PM photos of T. ni…………………………………………………………………………………...…. 18

Figure 3.1. Dose dependent mortality of AcMNPV-treated 4th instar T. ni larvae that were raised on artificial diet, cabbage, or potato………………………….………………….. 21

Figure 3.2. pH values in anterior midguts of 4th instar T. ni raised on artificial diet, cabbage, and potato………………………………..……………………………………. 22

Figure 3.4. Perforations in the anterior midgut region of cabbage-raised 4th instar T. ni resulting from ingestion of 0.5% Calcofluor White M2R.…………………..…………. 24

Figure 3.5. Pores inside the PM of a 4th instar T. ni at 17,000 magnification…………. 24

Figure 3.6. The anterior PM from T. ni larvae raised on different diets: left column = artificial diet; middle column = cabbage; right column = potato.…………….………... 25

Figure 3.7. The 4th instar T. ni anterior PM shown in a bundle, its individual fibrils still visible. ………………………………………..…………..………..……………………27

Figure 3.8. The 4th instar T. ni anterior PM shown as separate and disintegrating laminae……………………………………………………………………………...…... 27

Figure 3.9. The 4th instar T. ni anterior PM shown in a neat, even, singular layer..…… 28

Figure 3.10. The 4th instar T. ni anterior PM shown in separated, multiple laminae..…. 28

Figure 3.11. T. ni microvilli (MV) brush border accompanying the PM in cabbage-raised 4th instar larvae..………………………………………………………………………… 29

Figure 3.12. The 4th instar T. ni anterior PM shown in a singular, thick layer...……….. 29

Figure 3.13. The 4th instar T. ni anterior PM shown in multiple layers, with varying distances between layers, and significantly disorganized.…………...…………………. 30

Figure 3.14. T. ni microvilli (MV) brush border accompanying the PM in potato-raised 4th instar larvae.…...... ……. 30

Figure 3.15. Volcano plots illustrating differentially expressed contigs between midguts of 4th instar T. ni larvae raised on different diets.……………………………………….. 33

Figure 3.16. Venn diagram describing the number of contigs significantly differentially expressed in midguts of 4th instar T. ni raised on three diets…………………..………...34

Figure 3.17. Peritrophic membrane physiology, chitinase expression levels, susceptibility to virus, and anterior midgut pH responses as a function of diet in 4th instar T. ni…….. 56

vii

List of Abbreviations

AcMNPV Autographa californica Multicapsid Nucleopolyhedrovirus Bt Bacillus thuringiensis BV Budded Virions CBD Chitin Binding Domain CDA Chitin Deacetylase EGT Ecdysteroid UDP-Glucosyltransferase FDR False Discovery Rate GST Glutathione transferases IIM Insect Intestinal Mucin IPM Integrated Pest Management JH Juvenile Hormone

LD50 Lethal Dose at 50% of the Population MD Mucin Domain OB Occlusion Body ODV Occlusion Derived Virions PM Peritrophic Membrane REPAT Response to Pathogen RNAi RNA Interference RPKM Reads Per Kilobase of Transcript per Million Mapped Reads SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy UGT UDP-Glucosyl Transferases WGA Wheat Germ Agglutinin

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Chapter 1. Introduction

Pest control efforts have been present since the advent of agriculture, but never more so than with the scale of global agriculture today. Simultaneously, the adverse effects of traditional synthetic pesticides, still the mainstay of pest control, continue to come to light. The continual use of these pesticides is neither environmentally sound nor sustainable. However, without an equally effective, viable alternative, the transition to more responsible strategies of biocontrol will not ensue. Therefore, the urgent study and characterization of possible alternatives is needed for weaning of conventionally harmful pest control preferences. This study examines the interplay of a model host-pathogen system consisting of a baculovirus and its target priority pest.

1.1 Trichoplusia ni: A Cosmopolitan Agricultural Pest

In fields or greenhouses, the most damaging pests are those capable of feeding on a wide range of host plants. The cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae), is a cosmopolitan, lepidopteran pest of more than 160 plant species in 36 different families1. Greenhouse environments are particularly susceptible to infestations of T. ni as warm temperatures allow continual generation cycling and thus persistent crop damage. However, current management strategies rely heavily on chemical controls, which have undesirable ecological effects and are now less effective as T. ni has developed resistance to 13 different synthetic insecticides2. Furthermore, this pest has developed resistance to the bio-insecticide Bacillus thuringiensis (Bt)2 making the possibility of using Bt sprays or transgenic Bt crops for effective future management of T. ni increasingly doubtful.

Trichoplusia ni is a homometabolous species, with four distinct life stages: egg, larva, pupa, adult (Figure 1.1). The female lays 200-350 eggs on the underside of host plant leaves and these hatch 2-3 days later. There are five instars and complete larval development may last 2-4 weeks. Younger instars make small holes in a leaf while later instars may consume the entire leaf. At the end of larval development the larva spins a silken cocoon and then metamorphoses into a pupa from which an adult will emerge 12- 14 days later. The entire cycle averages 35 days under greenhouse conditions, but the duration of the different stages varies with temperature. In the fall, in response to

1 environmental cues (day length and temperature), T. ni enters diapause as a pupa within its silken cocoon and the adult emerges the following spring.

Figure 1.1. Life cycle of the cabbage looper, T. ni. Redesigned after Bohmfalk et al. (2011) 3. Photo permission credits in Appendix.

1.1.1 Current Control Methods used in Canada4

T. ni is a major pest in British Columbia and Quebec every year, while it is more sporadic and often localized in Ontario. A plethora of insecticides are used to control these populations:

i. Benzoylurea: novaluron ii. Carbamates: methomyl, carbaryl iii. Diacylhydrazine: methoxyfenozide iv. Diamides: chlorantraniliprole, cyantraniliprole v. Neonicotinoids: acetamiprid, clothianidin, imidacloprid, thiamethoxam vi. Organochlorines: endosulfan vii. Organophosphates: naled, methamidophos, azinphos-methyl, acephate, chlorpyrifos, diazinon, malathion viii. Pyrethroids: permethrin, deltamethrin, cypermethrin, cyhalothrin-lambda ix. Spinosyns: spinetoram, spinosad

Most of these affect the nervous system or functions as hormonal disruptors. However, organophosphates and carbamates are gradually being phased out while pyrethroids are of limited effectiveness during intense summer heat. The biological insecticide Bacillus thuringiensis is often used in conjunction with chemical pesticides as rotating treatments of different compounds, and can slow down the development of resistance. However, even though there is considerable potential, current growers do not use many biological control agents to control T. ni and such control measures merit further attention as components of acceptable management programmes.

1.2 Baculoviruses: Sophisticated Insect Pathogens

Baculoviruses are entomopathogens, only infecting insects, and Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) more specifically only infects lepidopterans. As polyhedra are released from infected hosts, crop contamination can be high: one study found up to 108 polyhedra on 100 cm2 of leaf material from cabbage purchased at five different supermarkets5. However, this does not pose a problem as there have been no reports of polyhedrosis viruses having negative impacts on organisms other than the pest target. Furthermore, as they play a major natural role in controlling insect populations, together with their high target specificity, baculoviruses are the most ecologically responsible biocontrol option commercially available6. Baculoviruses have been most successfully used as pest control regulatory agents by the forestry industry

3 against pests such as the gypsy moth, Douglas-fir tussock moth, and various species of sawflies7,8,9.

1.2.1 Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) Baculovirus

Baculoviruses have double-stranded DNA genomes, ranging from 80-180kbp, containing between 90-180 genes. Phylogenetic analysis suggests the co-evolution of these viruses alongside the first insects several million years ago, and as insects have evolved, so have these viruses in tight association10,11. Most baculoviruses have a narrow host range of one or a few related lepidopteran species, but AcMNPV (one of the most intensely studied) infects > 30 species in several genera12.

1.2.2 Infection Cycle

Among viruses, baculoviruses are unique as they spend considerable time outside their lepidopteran hosts. To enable this, the virions are encased in a protein matrix polyhedron case, called the occlusion body (OB) that is highly stable under most environmental conditions (with the exception of extreme heat and UV light) and allows the virions to remain viable until and upon ingestion by a caterpillar. The highly alkaline pH environment of the larval midgut dissolves the OB, releasing the occlusion-derived virions (ODV) that within hours pass through the midgut peritrophic membrane (PM) and infect the microvilli of columnar midgut epithelium cells13. However, just as the ODVs attempt to establish infection, the host preventatively defends against it as both the epithelium cells and the PM are constantly being sloughed. Basal regenerative cells replace damaged epithelium columnar cells and the PM is degraded by the abrasive food material, possibly extricating virions before they can reach host tissue.

Once within a microvilli columnar cell, the ODV is transported into the nucleus through the nuclear membrane pores and initiates a transcriptional cascade, creating more viral copies14. The nucleus then drastically re-organizes and expands, taking up most of the cell’s volume15. In a study with Helicoverpa zea as the host, it was estimated that there were 131,000 copies of the viral genome of a baculovirus produced per cell16. It is at this time that the baculovirus transitions to its second phenotype: budded virions (BV),

4 forgoing OB packaging. While the ODV phenotype was necessary for persistence outside the insect host until ingestion and initial infection, the BV phenotype is responsible for internal cell-to-cell infection. This bi-phasic property imparts a combination of durability outside the host, and high infection efficiency inside the host. Since the corrosive environment in a lepidopteran is limited to the midgut, the more benign near-neutral pH within the rest of the insect no longer requires the protective OB. BVs exiting the nucleus to infect other cells likely acquire their envelope from the nuclear membrane17.

For an infection to become systemic BVs need to infect the trachea, the insect respiratory network that connects to all other susceptible host tissues. The fat body, a large and energy rich organ, is also an important site for viral replication and may become so engorged that the texture underneath the cuticle becomes visibly white and puffy prior to larval death. The virus represses the insect innate immune system by infecting the haemocytes within the open circulatory system, encoding inhibitors of apoptosis18, including P35/IAP, and modifying or supressing the RNAi response13,18. During late infection, DNA expression shifts to hyper-expression of late viral genes, especially polyhedrin and P10. Polyhedrin accumulation is involved in the lattice crystal packaging of virions into OBs, and P10 fibrils are involved with the assembly of the polyhedrin envelope on the OB surface, giving it its faceted structure19,20. In preparation for exiting their host, virions are packaged in OBs, producing the initial phenotype that is infectious upon ingestion.

Finally, the occluded viral particles are released into the environment. Disintegration of the host results from tissue liquefaction and cuticle rupture facilitated by viral protease and viral chitinase21. The process is illustrated and summarized in Figure 1.2.

Figure 1.2. The infection cycle of an AcMNPV baculovirus. AcMNPV infection is bi-phasic, with the production of two distinct forms of virus: occlusion derived virus (ODV) and budded virus (BV). ODV is required for primary infection, while BV is then produced for systemic cell-to-cell infection. Copied from Graves (2014)22. Diagram permission credits in Appendix.

1.3 Plant, Pest, and Pathogen Tritrophic Interactions

1.3.1 Plant and Pest

Host plant and pest interactions undoubtedly influence insect growth and survival as plants respond to herbivory and attempt to counter it through morphological, biochemical, or molecular means, directly or indirectly, induced or constitutive.

Secondary metabolites, including their phenolics, flavonoids, and tannins (to name only a few large categories) reduce palatability of plant tissues. Examples include quinones and o-dihydroxy phenolics that can oxidize and form toxic o-quinones and other reactive species23,24 that damage lipid membranes, proteins, and DNA25,26,27. Lepidopterans, in response, have highly alkaline midgut pHs that retain the nutritional quality of ingested plant proteins as well as offset the toxicity of their host plant’s defensive compounds. Foliage proteins are more soluble and easily extracted at alkaline pHs28,29. As well, a pH greater than 8 prevents precipitation of proteins by inactivating hydrogen bonding between tannin-protein-aggregates30,31. Lepidopteran midguts maintain this highly alkaline pH by active secretion of carbonate from epithelial goblet cells and proton pumping at a significant metabolic expense32,33.

The PM is another lepidopteran midgut feature that helps to protect insect gut tissues against toxic plant allelochemicals in two different ways. First, the compounds are bound to the PM and excreted (up to 30% of potentially toxic dietary tannins are attached to and excreted with the PM)34. The second way is known as charge exclusion, whereby passage through the porous PM is inhibited even though molecules should be able to pass based on size35. However, because the PM is both responsive to ingested material, and protects against it, more toxic diets can induce thicker PM formation. The trade-off to this additional protective benefit is digestive efficiency; in a study where the PM was removed, digestive rates were increased along with larval growth rate36,37.

Since I only use cabbage (Brassicaceae) and potato (Solanaceae) as host plants in this project, the following considers biochemical background of these two families. These two economically important plant families also exemplify the roles of secondary metabolites ingested by generalist pests.

1.3.1.1 Brassicaceae: The Mustards

Brassica plants synthesize glucosinolates, a significant class of natural defence compounds, which upon herbivory are hydrolysed by myrosinase in the insect gut. Sometimes known as the “mustard oil bomb38,” they generate isothiocyanates, oxazolidine thiones, epithionitroles, and nitrils, all toxic repellents to insect herbivores39,40,41,42. As glucosinolates and myrosinase are spatially segregated until the mix following plant tissue damage, it is considered both a constitutive and induced defense system43, although glucosinolate concentration increases in response to herbivory44,45.

Mechanisms of counter-adaptation by insect herbivores include: avoidance of cell disruption, rapid absorption of intact glucosinolates, and rapid metabolic conversion of glucosinolates to harmless compounds46. Although the functional basis of glucosinolate detoxification remains to be determined, there is transcriptomic evidence that glutathione S-transferases and cytochrome P450s are involved47,48,49.

Herbivory also induces structural changes in mustards, including increased trichome density, usually from 25-100%, but as much as 1000% on new developing leaves within days or weeks of damage50,51,52.

An indirect method of defence triggered by herbivory is a change in the volatile profile that not only plays a role in the upregulation of induced defenses in other parts of the plant, but may also attract natural enemies of the pest53,54.

1.3.1.2 Solanaceae: The Nightshades

The genus Solanum produce glycoalkaloids, which include pyrrolidines as natural defence compounds. They act by inhibiting acetylcholinesterase and butyrylcholinesterase, both of which catalyze the hydrolysis of the neurotransmitter acetylcholine55. In addition, they interfere with calcium and sodium ion transport across cell membranes55.

Mechanisms of counter-adaptations in insect herbivores seem predominantly carried out in the fat body, which contains detoxification enzymes and ABC transporters56. Evidence that fat body cells are involved in detoxification of potato diet in a lepidopteran consisted

8 of swelling of the nuclear envelope and endoplasmic reticulum, mitochondria vacuolization, and fat droplet fusion within fat body cells57.

Induced defences in Solanum plants also feature volatile chemical cues. In a recent study, tomato plants were treated with methyl jasmonate58, a volatile that is known to promote the upregulation of plant defences, including the synthesis of anti-nutritive compounds such as proteinase inhibitors that interfere with insect digestion59. This significantly increased the incidence of cannibalism of a lepidopteran pest, decreasing pest density leading to decreased herbivory.

1.3.2 Pest and Pathogen

1.3.2.1 Infection Establishment

Even including the porous nature of the PM, the passage through it by baculovirus ODVs is not passively reliant on chance. ODVs are equipped with metalloproteinases called enhancins, which specifically cleave mucin (a major PM component)60,61,62, as well as chitin binding domains – found in viral proteins Ac83, Ac145, and Ac150 – that allow the ODV to rend obstructing mucin or chitin making up the PM63,64.

1.3.2.2 The Peritrophic Membrane

The PM is an ultrafine sieve that acts as a physical screen between the internal insect environment and the external gastrointestinal tract. It consists of a proteinaceous matrix embedded in a chitin substructure produced by the midgut, and serves in the combined roles of digestion and absorption of nutrients, mechanical protection, toxin nullification, and pathogen restriction65. Trichoplusia ni possesses a Type II PM, which is secreted by a special ring of cells called the cardia at the anterior end of the midgut as opposed to being secreted along the length of the midgut by the microvilli (Type I)66,67.

Because a type II PM is not uniformly secreted along the length of the midgut, the anterior and posterior regions are defensively vulnerable. The anterior region is where the PM is thinnest and most porous as it is being formed and its additional components are being added while the posterior region is most worn from the passage of food particles. A

9 recent study established the vast majority of infections are initiated at the anterior end of the midgut68.

Harper and Granados, and Ryerse et al. 69,70, using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), were the first to extensively characterize the lepidopteran PM. Anterior PM layers are 1µm thick or less, increasing to 3-5 µm as accessory proteins are added. Identifying the PM requires recognizing two primary laminae facing the endo and ecto-peritrophic spaces, with thin strands between. Strands and laminae of the PM can fuse or separate (Figures 1.2 and 1.3). The primary laminae are more compact and electron dense than the internal strands.

Figure 1.3. The PM of larval T. ni in its most simplistic form: singular, uniform, compact. (A) Compressed laminae yield a well-defined PM in the middle portion of midgut (magnification 25,400×). (B) PM partitions contents of food bolus from midgut epithelium in posterior mesenteron (5,100×). m, microbe; mv, microvilli; pc, plant cell fragment; pm, peritrophic membrane. Copied from Harper and Granados (1999)69. Figure permission credits in Appendix.

Figure 1.4. The PM of larval Heliothis virescens in a more disorganized state with laminae and fibrils visible, of various density. (a) TEM showing the homogeneous and randomly-oriented fibrils and granules which comprise the laminae of the PM. Note greater compaction of the matrix in the endoperitrophic (top) and ectoperitrophic (bottom) faces and the thinner laminae between the main two limiting laminae. (b) Higher magnification showing randomly-oriented network of 5-10nm wide fibres and 15-20nm diameter granules, as indicated by the arrowheads. Fig. 1.4a 25,000× Bar = 1µm. Fig 1.4b 100,000× Bar = 100nm. Copied from Ryerse et al. (1992)70. Figure permission credits in the Appendix.

An additional assortment of proteins and their encoding genes make up, or contribute to, the PM. The assemblage of components involved in PM architecture, synthesis, and function include structural (peritrophins, mucins, glycoproteins, lipases, response to pathogen [REPAT] proteins), delivery (gelsolin, annexin, microvesicles), framework (chitin synthase, chitinase, chitin deacetylase), and hormonal effectors (ecdysterone, juvenile hormone).

i. Peritrophins are integral structural PM proteins that contain a chitin binding domain (CBD)71. Classification of peritrophins includes four types: single (one or two CBDs), binary (two CBDs with a mucin domain (MD) between), complex (multiple CBDs and MDs), and repetitive (long concatemers of CBDs)72,73,74,75,76. PM peritrophins contribute to its integrity, elasticity, and permeability72,77. ii. Insect intestinal mucins (IIM) are high molecular weight PM proteins with similar biochemical characteristics and protective functions as vertebrate intestinal

mucins; they are also a type of complex peritrophin78,79. IIMs can vary widely in size, ranging from a few hundred to several thousand amino acid residues. They are among the largest known proteins80 and their protective function comes from the ability to form viscous solutions or gels81. iii. Glycoproteins are PM-associated proteins and are embedded in the chitin fibril mesh82. iv. Intestinal lipases are major PM associated enzymes whose activities relate to hydrophobic-hydrophilic interfaces74,83,84. v. Response to pathogen (REPAT) proteins are less well characterized, but induction of expression from REPAT encoding genes occurs in response to pathogen challenge. This and the resulting reduction in virulence of baculovirus, strongly suggests a role in immune response for these proteins85. vi. Structural proteins are delivered to the PM by apocrine secretion from the microvilli; the products of the gelsolin and annexin genes involved in this process are expressed in these cells86,87. The delivery of secretory vesicles is helped by the actin-filament severing activity of gelsolin, while annexin promotes vesicle fusion with the PM. vii. Membranous microvesicles, upon solubilisation, release their contents while the remaining membrane is incorporated into the PM. It’s been suggested that PM peritrophin components are secreted by these microvesicles from the columnar cells88,89. viii. Chitin synthase synthesizes chitin whereas chitinase degrades chitin90. Chitin deacetylase (CDA) is involved in the organization of chitin fibrils by altering their structure and orientation, as well as affecting binding proteins, which in turn affects PM integrity and permeability82,91,92,93. ix. Ecdysterone and juvenile hormone are predominantly involved in the coordination of insect molting: 20-hydroxyecdysone (ecdysterone) induces it, juvenile hormone suppresses it94. Because the PM and insect exoskeleton are both composed of chitin, the PM also undergoes chitin modification. The PM of molting insects is significantly thicker than that of non-molting (feeding) insects (Figure 1.5)95. The reason for this is not entirely clear. Why a newly-synthesized and much thicker

PM would be formed during molt, but eliminated as feeding resumes is not obvious since it would not be for food processing purposes. It has been suggested that maybe this maintains the internal midgut structure.

Figure 1.5. The PM of molting and feeding (non-molting) Mamestra configurata larvae. PM dissected from mid-molt and feeding 4th instar larvae cut into anterior, middle, and posterior sections, incubated with Alexafluor 488-labeled-wheat germ agglutinin (WGA) and examined by confocal microscopy. Scale bar = 30µm. Copied from Toprak et al. (2014)95. Figure permission credits in Appendix.

While the PM affects the interaction between the insect and ingested material, the PM itself can also be influenced by the gut’s contents37,96. Although many aspects of tritrophic interactions have been studied, the last part of this introduction, including the PM’s associated proteins and their encoding genes, is an aspect that has not yet been examined in a tritrophic context incorporating multiple diets.

1.3.2.3 Modification of Host Physiology and Behaviour

The viral ecdysteroid UDP-glucosyltransferase gene (egt) also affects the course of infection by preventing pupation97, as this process places high physiological stress and consequently kills infected insects. Therefore, preventing pupation allows the infected host to grow larger and produce a higher viral load. A wild type baculovirus strain had

30% greater OB yield, than a mutant strain lacking the egt gene98,99. Mutant strains without the egt gene reduce the time it takes for the virus to kill its host, thus reducing damage to crops. However, this is also a tradeoff between infection cycle speed and progeny production.

Viral proteins EGT (Ac15), Ac1, and tyrosine phosphatase are also responsible for the “zombie effect”100,101. Climbing upwards on the plant by heavily infected hosts before dying results in a greater dissemination of dispersed virus OBs in the environment upon dissolution of the host body. Phylogenetic analysis revealed that the genes responsible for this behaviour are of lepidopteran origin, meaning that over evolutionary time they have been incorporated into the viral genome as advantageous accessory genes102.

1.3.3 Plant and Pathogen

The mortality rates resulting from a viral infection can vary considerably depending on a number of host plant factors. For example, host plant chemistry is important, e.g. the phenolic content can result in the aggregation of OBs. In addition, insect salivary enzymes, PM permeability/physiology, and impact on host immune function play important roles103,104,105.

Ingestion of acidic foliage lowers the alkalinity of the midgut environment. Consequently, it has been hypothesized that if the pH changes to a point where the OBs are no longer dissolved, then the virions would not be released, and the host will avoid infection. However, the literature on this possibility is contentious: the results of some studies support this hypothesis106, while others do not107,108.

Phenolic tannins are capable of intercepting the pathogen before infection is initiated by binding to OBs. Hydrogen bonding between tannins and proteins create large, indigestible tannin-protein complexes, which can obstruct virions. Thus, while tannins can negatively affect insect health, they may under certain circumstances offer the herbivore some degree of protection from viral infection. Tannin hydrogen bonding is also affected by pH and these bonds do not form at pH greater than 8.029,30. Larval lepidopteran midguts generate pH levels ranging between 8-12, among the highest of any natural biological

14 system109. Therefore the probability that tannins affect viral infectivity depends on midgut pH, and it is not yet resolved if and to what extent gut pH is affected by plant chemistry.

1.4 Study Rationale, Thesis Objectives, Hypothesis, and Predictions

The increase in the number of pest species that have developed insecticide resistance and the public’s desire to reduce the use of synthetic insecticides requires we develop acceptable alternatives. Pathogens have potential as pest control tools but during the “pesticide era” their broad use was not fully exploited. Furthermore, in the case of polyphagous pests, the molecular basis for differences in susceptibility has not been well studied. Thus, the goal of my thesis is to determine if different diets affect the level of mortality observed, and if yes, to determine if this is associated with changes in the PM and altered transcriptomics. Although I specifically work with a model baculovirus AcMNPV, the findings are relevant to other ingested pathogens that may be used as future biocontrol agents. I hypothesize that:

Hypothesis: Insect diet composition affects pathogen infectivity through alterations of midgut structure, chemistry, and gene expression.

Prediction 1: Raising T. ni larvae on different diets will affect the LD50 for AcMNPV baculovirus infection of the insects.

Objective: Correlate baculovirus susceptibility of insects raised on different diets.

Prediction 2: Raising T. ni larvae on different diets will alter the midgut pH.

Objective: Measure pH levels in midguts of larvae raised on different diets.

Prediction 3: Raising T. ni larvae on different diets will alter physical PM structure.

Objective: Determine PM properties when insects are raised on different diets.

Prediction 4: The larval midgut transcript profiles of T. ni raised on different diets will also be different.

Objective: Generate transcriptomic profiles by sequencing RNA from treatment groups of larvae raised on different diets.

Chapter 2. Materials and Methods

2.1. Insect Rearing, Plants & Diet, and Virus

Insects used were obtained from a T. ni culture reared on cabbage under a 16L:8D photoperiod, 25˚C, and 30% humidity at Agriculture and Agri-Food Canada’s (AAFC) London Centre. All experiments were conducted on 4th instar larvae for consistency, as well as the fact that this stage is amenable to both imaging techniques (which favour larger sized structures of older instars) and bioassays (which favour smaller, younger instars due to increased susceptibility to pathogens)104. Golden Acre Cabbage (Brassica oleraceae) and Kennebec Potato (Solanum tuberosum) were grown under greenhouse settings of 16L:8D photoperiod at 25˚C. McMorran wheat germ-based artificial diet110 was purchased from Insect Production Services (Great Lakes Forestry Centre, Sault Ste. Marie, ON, Canada). AcMNPV baculovirus aliquots (strain: BAC887-ie1-gfp) were obtained from Agriculture Canada’s Saskatoon Research & Development Centre courtesy of Dr. Martin Erlandson.

2.2. Bioassays

Individual 4th instar larvae from each diet treatment were isolated in 5.5 cm diameter petri dishes lined with filter paper and provided a leaf disc of 6mm or an artificial diet cube of 5 mm3 that was treated with either 15, 25, 50, 100, or 200 OBs on cabbage, 50, 100, 150, 250, or 400 OBs on potato, or 25, 50, 100, 200, or 350 OBs on artificial diet. Dose concentration ranges were based on preliminary bioassays to determine the projected

LD50. Concentrations achieved in a serial dilution were confirmed through hemocytometer counts. Controls in all treatments was 0.05% Triton X-100 in water, the solution used to suspend the OBs. In all cases, the virus was applied in a 2 µl droplet on a leaf disc or artificial diet cube. On leaf discs, the droplet was allowed to dry before feeding to insects, while in the case of artificial diet, a square of parafilm was placed underneath to ensure the solution was not absorbed by the filter paper. Leaf discs were cut from leaves of cabbage or potato plants that were approximately a month old. Once the treated food was completely consumed, untreated food was provided until the end of the assay. There were three replicates of ten larvae per concentration per diet treatment and

16 mortality was recorded until pupation or seven days post treatment (as baculovirus infection is evident within four to six days). LD50 curves were calculated and generated using Graphpad Prism; 95% confidence intervals were calculated in R’s drc package using Fieller’s method111,112.

2.3 Micro-needle Electrode pH Measurements

These measurements were made in collaboration with Dr. Mike O’Donnell and Dr. Dennis Kolosov of Hamilton, Ontario, McMaster University. Fourth instar larvae were pinned straight while submerged in saline solution and a longitudinal incision was made along the length of the caterpillar to expose the gut. A micro-needle pH electrode was inserted into the anterior lumen of the midgut and the pH of the gut recorded. There were ten larvae per diet treatment. The data was analyzed using a one-way ANOVA.

2.4. Scanning Electron Microscopy (SEM)

Larvae were reared on one of three diets until the 4th instar (head capsule size 1.0 - 1.3 mm), at which time midguts were dissected, the food bolus removed, and the PM incised lengthwise before being immersed in Sorenson’s phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde as a primary fixative. After triple buffer rinsing, samples were then fixed in 1% osmium tetroxide for 1 h, triple rinsed again, and dehydrated in a graded ethanol series before being dried in a graded hexamethyldisilazane series113. Samples were mounted, gold sputtered, and observed with a Hitachi 3400-N VP-SEM. Three midgut samples per diet category were processed.

To confirm the structure being considered as the PM, starved 4th instar T. ni were fed on a 6 mm leaf disc treated with 5 µL droplets containing 0.05% Calcofluor White M2R (Fluorescent Brightener 28) in 10% sucrose solution and dissected one hour later. This chemical has chitin binding properties that result in the inhibition of PM formation at 0.1% and perforation of PM at 0.05%114,115.

For comparative purposes, each PM was divided into anterior, middle, and posterior regions and each region from the larvae reared on differing diets were compared for texture (qualitative) and degree of folding (quantitative).

Degree of folding of the PM was computed using ImageJ vs. 1.1.7 software from 10 images (2,500×) per region per bioreplicate. The images were analyzed and input commands done in the following sequence: smooth > sharpen > find edges > make binary > de-speckle (×3) > distance map > 8-bit conversion > ridge detection (Figure 2.1). Ridge detection parameters were: line width: 10, high contrast: 230, low contrast: 87, sigma: 3.39, lower threshold: 0, upper threshold 0.85, with boxes ticked for darkline, extend line, show IDs, display results, and add to manager selected. The slope method for overlap resolution was applied. Contour IDs were collected for total edge counts and a one-way ANOVA was used to test the means.

Figure 2.1. ImageJ pipeline for ridge detection quantification of SEM PM photos of T. ni. In order of processing: (A) a sample photo original  (B) smooth, sharpen, find edges  (C) convert to binary file  (D) clean/despeckle  (E) highlight ridges and identify  (F) compile and total based on unique IDs.

2.5. Transmission Electron Microscopy (TEM)

Larvae were reared on one of three diets until 4th instar (head capsule size 1.0 - 1.3 mm) and their midguts dissected, carefully removing the food bolus. The initial steps of TEM are identical to those in SEM, up until and including the fixation in 1% osmium tetroxide and secondary wash. Following that, TEM preparation included dehydration in a graded

18 acetone series before infiltration and embedding in epon-eraldite resin and then baking at 60˚C for 48 h. Coarse trimming was done using a razor blade and finer sectioning done on a Sorval Ultracut with a diamond knife. All sections were from the anterior region of the PM and netted on copper grids. Samples were post stained in the dark for 20 min with uranyl acetate, rinsed in five water droplets, stained for 2 min with lead citrate, and rinsed in three water droplets. Grid samples were examined and images taken using a Philips CM10 TEM 60KV. Three larvae for each diet category were studied.

2.6. RNA Extraction, RNA-seq, and Transcriptomic Analysis

Midguts of feeding larvae at 4th instar from each diet category were dissected in Calpode’s insect saline (pH = 7.2, 10.7 mM NaCl, 25.8 mM KCl, 90 mM glucose, 29 mM

CaCl2, 20 mM MgCl2, and 5 mM HEPES) and immediately suspended in RNAlater buffer (Ambion, Fisher Scientific), and then stored at -20ᵒC until RNA extraction. Total RNA was extracted from five midguts using an RNeasy mini kit (Qiagen), which was replicated three times for each diet treatment. The quality and quantity of RNA were assessed using the 2100 Bioanalyzer (Agilent). RNA samples were diluted to 300 ng/µL in DEPC-treated water and 15 µL per sample was shipped on dry ice to McGill University and Génome Québec Innovation Centre (Montreal, QC, Canada) where libraries were constructed and sequencing performed on the Illumina HiSeq 2000 platform using 100 bp single-end reads protocol.

Messenger RNA-seq reads were mapped to a reference T. ni transcriptome containing 58,200 contigs that was previously assembled de novo from transcriptomic sequence reads (Dr. Martin Erlandson, Agriculture Canada Saskatoon Research & Development Centre). Subsequent analysis and data handling were done using CLC Genomics Workbench vs 10.0.1 (Qiagen Bioinformatics). Before mapping, library reads with less than 50 nucleotides were discarded. Gene expression tracks were then generated from RNA-seq analysis, with reads per kilobase of transcript per million mapped reads (RPKM) as the expression value. Finally, all diet group pairs were tested for differential expression. The output from DESeq analysis included normalized mean number of reads assigned to a contig, log2 fold change, and its statistical significance. A contig was considered differentially expressed if the absolute value of the log2 fold change was ≥ 3

19 and the adjusted p-value was ≤ 0.001 following Benjamini-Hochberg false discovery rate (FDR) correction116. Contigs corresponding to relevant genes were screened from those differentially expressed in all comparisons. Contig IDs without annotation were analyzed by BLAST comparison with the NCBI database.

Chapter 3. Results

3.1 Diet Based T. ni Bioassay Lethality Curves

Larval diet has a significant effect on the efficacy of AcMNPV baculovirus against T. ni with the concentration for LD50 being lowest on artificial diet and highest on potato

(Figure 3.1). The LD50 values are reported along with the 95%. LD50 differences between artificial diet and cabbage-raised larvae: p > 0.05, F-ratio = 2.76. LD50 differences between cabbage and potato-raised larvae: p = 0.0019, F-ratio = 7.81. LD50 differences between potato and artificial diet-raised larvae: p < 0.001, F-ratio = 14.16. Df = 2 for all dosage values.

Figure 3.1. Dose dependent mortality of AcMNPV-treated 4th instar T. ni larvae that were raised on artificial diet, cabbage, or potato.

3.2 Measurements of Anterior Midgut pH

Larval diet had a significant effect on the anterior midgut pH of 4th instar T. ni. Larvae raised on cabbage (n = 4) had significantly less alkaline pH than those raised on artificial diet (n = 10) or potato (n = 10) (Figure 3.2).

Figure 3.2. pH values in anterior midguts of 4th instar T. ni raised on artificial diet, cabbage, and potato. Different letters represent significant differences p < 0.05.

3.3 SEM Characterization of the PM

Because T. ni, possess a type II PM, produced at the junction between fore and midgut, there are considerable differences between the anterior, middle, and posterior sections of the midgut (Figure 3.3). The anterior region has relatively shallow but extensive folds that transition into the tight coils of the middle region before becoming degraded and sloughed at the posterior end. Confirmation that the observed structure is the PM was determined with the optical brightener Calcofluor White M2R (Figure 3.4) as well as the identification of pores characteristic of the PM (Figure 3.5). However, as seen in Figure 3.6, larval diet results in marked differences in the structure of PM although the degree of folding did not differ between diet treatments (p > 0.05, F-ratio = 2.68, Df = 9, n = 10).

Figure 3.3. Morphological variation in the anterior, middle, and posterior sections of the PM of a cabbage-raised T. ni midgut per regional characteristics: left column = anterior; middle column = middle; right column = posterior. Magnification of images are: A = 1,500×; B = 1,400×; C = 1,400×; D, E, F = 2,500×; G, H, I = 1,400×. Images A-I are in spatial, sequential order along the length of the midgut.

Figure 3.4. Perforations in the anterior midgut region of cabbage-raised 4th instar T. ni resulting from ingestion of 0.5% Calcofluor White M2R. Magnification levels of images are: A = 3,500×; B = 3,500×; C = 5,000×; D = 3,500×; E = 15,000×, F = 10,000×.

Figure 3.5. Pores in the PM of a 4th instar T. ni at 17,000× magnification (several bacteria on the right of field view).

Figure 3.6. The anterior PM from T. ni larvae raised on various diets: left column = artificial diet; middle column = cabbage; right column = potato. Magnification of images are: A- F, H-J = 2,500×; G = 1,400×; K =3,000×; L = 3,500×.

3.4 TEM Characterization of the PM

TEM imaging of T. ni larval PM revealed significant variability of PM both within each diet category and between diets. Since the PM was not uniform even within a cross- section of the midgut, the morphological range observed is shown for each diet category.

The most organized PM states observed for artificial diet, cabbage, and potato are shown in Figures 3.7, 3.9, and 3.12 respectively. All three display even laminae, but still vary visually. The most organized PM of artificial diet-raised larvae is a near gossamer bundle, delicate and thin (less than 500nm). The most organized PM of cabbage-raised larvae is thin as well (less than 500nm), but dense and uniform. The most organized PM of potato- raised larvae is sometimes thin as well (less than 500nm) but with its layers separated, spanning several microns.

The most disorganized PM states of artificial diet, cabbage, and potato are shown in Figures 3.8, 3.10, and 3.13 respectively. All three differ in their level of organization. The most disorganized PM of artificial diet-raised larvae feature separated laminae, spanning several microns, ranging to a PM that is almost completely unravelled and with little structural integrity. The most disorganized PM of cabbage-raised larvae show divided laminae and further divided internal fibrils spanning several microns, but with each layer still well defined. The most disorganized PM of potato-raised larvae show bends and kinks of multiple fibrils and divided laminae. Although the PM is flexible, the angle of these bends are sharpest in potato-raised larvae. It is possible that since potato is the most toxic diet, that the PM is affected by the numerous alkaloids and secondary metabolites present in the tissue. In potato-raised larvae, the level of disorganization of the PM surpasses that of artificial diet or cabbage-raised larvae and is seen much more frequently than its organized form.

Among all PM forms from larvae raised on the different diet categories, those raised on potato also distinctly feature numerous microvesicles. Figures 3.11 and 3.14 show microvesicles formed at the microvilli brush border in midguts of cabbage and potato- raised T. ni that look comparable to microvesicles seen in the PM photos of Figures 3.7 (R), 3.9 (L), 3.10 (L), 3.12 and 3.13.

Figure 3.7. The 4th instar T. ni anterior PM shown in a bundle, its individual fibrils still visible. (L) magnification = 19,000× (R) magnification = 13,500×. Bars = 500 nm. A black arrow points to a microvesicle (R).

Figure 3.8. The 4th instar T. ni anterior PM shown as separate and disintegrating laminae. (L) magnification = 10,500× (R) magnification = 10,500×. Bars = 500 nm.

Figure 3.9. The 4th instar T. ni anterior PM shown in a neat, even, singular layer. (L) magnification = 34,000× (R) magnification = 25,000×. Bars = 500 nm. Black arrows point to microvesicles (L).

Figure 3.10. The 4th instar T. ni anterior PM shown in separated, multiple laminae. (L) magnification = 19,000× (R) magnification = 10,500×. Bars = 500 nm. A black arrow points to a microvesicle.

Figure 3.11. T. ni microvilli (MV) brush border accompanying the PM in cabbage-raised 4th instar larvae. (L) magnification = 7,900×, bar = 2 µm. (R) magnification = 10,500×, bar = 500 nm. Black arrows point to microvesicles.

Figure 3.12. The 4th instar T. ni anterior PM shown in a singular, thick layer. (L) magnification = 10,000×. The PM shown in multiple layers, with fairly even laminae. (R) magnification = 10,500×. Bars = 500 nm. Black arrows point to microvesicles.

Figure 3.13. The 4th instar T. ni anterior PM shown in multiple layers, with varying distances between layers, and significantly disorganized. (L) magnification = 10,500× (R) magnification = 13,500×. Bars = 500 nm. Black arrows point to microvesicles.

Figure 3.14. T. ni microvilli (MV) brush border accompanying the PM in potato-raised 4th instar larvae. (L) magnification = 7,900×, bar = 2 µm. (R) magnification = 10,500×, bar = 500 nm. Black arrows point to microvesicles. Some artefactual holes in plastic and knife marks are present.

3.5 mRNA Sequencing

3.5.1 Read Processing and Quality Control

The mRNA sequencing of 9 libraries yielded 652,598,015 reads, with raw reads per library ranging from 57.8 – 80.3 million reads, with a mean of 72.5 million reads. The percentage of reads remaining after trimming ranged between 95.8 – 97.7%, with the average read length being 99.8 bp. The proportion of reads per sample mapping to the reference transcriptome ranged from 94.6 – 96.5%. The number of uniquely mapped reads per sample ranged from 48.4 – 64.2 million, with a mean of 58.8 million. The percentage of unique mapped reads ranged from 81.6 – 87.3%. All statistics stated are reported in Table 3.1.

3.5.2 Diet-Induced Transcriptomic Responses in T. ni Midgut

Transcriptomic changes between midguts of 4th instar T. ni larvae reared on different diets were determined through differential expression analysis and visualised using volcano plots in: potato vs cabbage (Figure 3.15A), cabbage vs artificial diet (Figure 3.15B), and potato vs artificial diet (Figure 3.15C).

Reads Average % Uniquely % of Diet Total Raw Remaining % Read Mapped Mapped Mapped Uniquely Sample Reads After Remaining Length Reads Reads Reads Mapped Trimming Reads Artificial 80,386,252 77,443,284 96.34 % 99.7 73,318,717 94.67 % 63,495,585 81.99 % Biorep 1 Artificial 79,976,456 77,718,130 97.18 % 99.8 73,623,144 94.73 % 64,266,561 82.69 % Biorep 2 Artificial 69,251,276 67,674,296 97.72 % 99.8 64,348,860 95.09 % 56,347,289 83.26 % Biorep 3 Cabbage 67,347,576 65,334,533 97.01 % 99.8 62,591,997 95.80 % 56,762,565 86.88 % Biorep 1 Cabbage 57,847,311 55,430,080 95.82 % 99.8 53,229,585 96.03 % 48,403,516 87.32 % Biorep 2 Cabbage 73,837,403 71,006,689 96.17 % 99.8 67,998,188 95.76 % 61,481,209 86.59 % Biorep 3 Potato 76,250,303 73,155,518 95.94 % 99.8 70,317,800 96.12 % 60,869,160 83.21 % Biorep 1 Potato 69,676,403 66,937,736 96.07 % 99.8 64,623,642 96.54 % 54,645,189 81.64 % Biorep 2 Potato 78,025,035 74,960,998 96.07 % 99.8 72,173,224 96.28 % 62,851,574 83.85 % Biorep 3

A B

Figure 3.15. Volcano plots illustrating differentially expressed contigs between midguts of 4th instar T. ni larvae raised on different diets. A) Comparison between potato and cabbage-raised: the left vertical dotted line represents -3 fold change; the right vertical dotted line represents +3 fold change; the horizontal dotted line represents p-value of 0.001; red dots represent significantly downregulated genes; blue dots represent significantly upregulated genes. B) Comparison between cabbage and artificial-raised. C) Comparison between potato and artificial-raised. All three plots follow similar trends and had the same filtering criteria applied, though only A is colored and delineated.

Using a cut-off of ≥ 3 absolute fold change and a FDR-corrected p-value of ≤ 0.001, the total number of contigs significantly differentially expressed for cabbage vs artificial diet: was 1,985, potato vs cabbage: 1,118, and potato vs artificial diet: 1,753. There were 132 shared between all diets. Figure 3.16 shows the number of uniquely and commonly expressed significantly differential contigs.

Figure 3.16. Venn diagram describing the number of contigs significantly differentially expressed in midguts of 4th instar T. ni raised on three diets.

Within the contigs that were significantly differentially expressed, gene categories corresponding to products involved in the PM’s architecture, synthesis, and function were selected for further analysis: structural (peritrophins, mucins, glycoproteins, lipases, response to pathogen [REPAT] proteins), delivery (gelsolin, annexin, microvesicles), framework (chitin synthase, chitinase, chitin deacetylase), and hormonal effectors (ecdysterone, juvenile hormone). The comparisons between cabbage vs artificial diet, potato vs cabbage, and potato vs artificial diet, together with the overall summary, are presented in Tables 3.2, 3.3, 3.4, and 3.5 respectively.

From the compilation of data in Table 3.5, of all the gene categories of interest, contigs corresponding to chitinase and chitin deacetylase were most uniformly downregulated as a function of increasing diet toxicity. Contigs of peritrophins and genes induced by juvenile hormone were also predominantly downregulated. Contigs of mucins, lipases, and genes induced by ecdysone were a mix of up and down-regulated. The sole contigs for a glycoprotein and REPAT gene were upregulated.

When gene contigs had a combination of up or down-regulation, their cumulative effects on the host organism are unclear, as the extent to which these effects are additive, multiplicative, or antagonistic has yet to be fully studied.

The overall top 50 significantly differentially expressed genes between cabbage vs artificial diet, potato vs cabbage, and potato vs artificial diet are listed in Tables 3.6, 3.7, and 3.8 respectively.

Table 3.2. Differentially expressed contigs in midguts of cabbage raised compared to artificial diet-raised 4th instar T. ni.

Gene Max* Fold FDR p- Category Contig ID Group Change value Sequence Description Regulation Mean Chitinase 8386 0.79 -18.24 0 PREDICTED: chitinase 2 Down

16243 11.88 -11.27 2.34E-14 PsChi-h for chitinase Down

17052 14.47 -10.46 2.34E-14 viral-like chitinase Down

gi|687027960 2.27 -11.07 0 chitinase 7 Down

gi|687056067 17.18 -10.39 4.35E-11 viral-like chitinase Down

gi|687094659 180.42 -17.07 0 chitinase (Cht) Down

Peritrophin 5400 22.26 -3.18 2.83E-06 PREDICTED: peritrophin-1-like Down

gi|687043932 21.34 -4.44 3.30E-09 chitin binding peritrophin-A Down

gi|687108074 22.94 -3.18 4.44E-06 peritrophin-1-like Down endocuticle structural glycoprotein ABD- Glycoprotein 18285 3.52 7.69 3.60E-06 Up 5-like Ecdysone PREDICTED: ecdysone-induced protein 3024 2.58 4.23 3.33E-15 Up Inducibles 74EF isoform A 5946 2.13 -5.97 4.72E-05 PREDICTED: ecdysone receptor Down PREDICTED: ecdysteroid-regulated 16 11211 46.66 -7.82 0 Down kDa protein-like 16286 3.31 -3.95 1.07E-05 3-dehydrecdysone 3b-reductase Down

17757 187.15 54.3 0 ecdysone oxidase Up 20-hydroxy-ecdysone receptor - ecdysone 18136 10.38 -3.1 2.10E-11 Down receptor A isoform gi|687085860 26.81 -3.64 1.31E-13 ecdysone oxidase Down

gi|687100743 20.36 -4.26 2.11E-10 ecdysone oxidase Down

Table 3.2. Differentially expressed contigs in midguts of cabbage-raised compared to artificial diet-raised 4th instar T. ni. (continued)

Max Gene Fold FDR p- Contig ID Group Sequence Description Regulation Category Change value Mean Juvenile Hormone 2789 2.55 -5.64 5.34E-13 juvenile hormone-inducible protein Down Inducibles PREDICTED: juvenile hormone esterase- 9577 75.96 -4.35 0 Down like isoform 11982 37.19 -19.07 0 juvenile hormone esterase precursor (JHE) Down

18269 1.95 -24.2 8.41E-09 juvenile hormone binding-like protein Down

gi|687052097 20.2 -24.87 1.31E-13 juvenile hormone esterase precursor (JHE) Down

Gelsolin 14408 13.91 3.73 7.69E-12 PREDICTED: gelsolin-like Up

Mucins 5553 11.34 -3.62 8.03E-09 PREDICTED: mucin-2-like Down

8946 1.78 -3.91 4.33E-08 PREDICTED: mucin-2-like Down

gi|687085871 0.88 5 3.41E-05 PREDICTED: mucin-2-like Up

Lipases 16375 173.6 -3.22 8.22E-05 insect intestinal lipase 6 Down

16377 190.3 3.17 3.37E-11 PREDICTED: lipase 1-like Up

*Max Group Mean = The maximum of the average group RPKM values between two group types.

Table 3.3. Differentially expressed contigs in midguts of potato-raised compared to cabbage-raised 4th instar T. ni.

Max Gene Fold FDR p- Contig ID Group Sequence Description Regulation Category Change value Mean Chitinase 8386 3.65 -3.74 1.11E-08 probable chitinase 2 Down

15347 2.67 -3.26 1.26E-06 chitinase-related protein 1 Down

gi|687027891 0.49 -4.52 2.55E-09 chitinase-related protein 1 Down

gi|687094659 6.07 -4 6.04E-11 chitinase Down Chitin 16131 7.14 -3.71 9.70E-09 chitin deacetylase 1 Down Deacetylase Ecdysone PREDICTED: ecdysteroid-regulated 16 11211 51.31 13.67 0 Up Inducibles kDa protein-like 17757 187.15 -74.94 0 ecdysone oxidase Down

20-hydroxy-ecdysone receptor - ecdysone 18136 32.17 15.03 0 Up receptor A isoform Juvenile Hormone 4247 255.11 3.55 0 juvenile hormone epoxide hydrolase-like Up Inducibles 13294 41 -5.05 0 juvenile hormone epoxide hydrolase-like Down

15986 154.92 3.15 4.63E-11 juvenile hormone esterase-like Up 16491 110.22 -3.43 1.35E-14 juvenile hormone-inducible protein Down

Mucins 16236 149.06 10.13 0 intestinal mucin Up

16269 150.99 10.31 0 insect intestinal mucin 2 Up

REPAT gi|687115186 1.65 3.78 7.24E-05 REPAT34 Up Lipases 16200 153.46 4.29 9.51E-05 insect intestinal lipase 7 Up

16201 223.05 4.67 6.31E-05 insect intestinal lipase 6 Up

16325 211 4.11 3.14E-06 insect intestinal lipase 6 Up

16375 30.5 -4.66 1.02E-07 insect intestinal lipase 6 Down

Table 3.4. Differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T. ni.

Max Gene Fold FDR p- Contig ID Group Sequence Description Regulation Category Change value Mean Chitin gi|687040850 2.85 -13.43 1.48E-06 chitin synthase 1 Down Synthase gi|687122461 2.03 -13.34 5.96E-05 chitin synthase A Down

Chitinase 8386 115.73 -68.23 0 PREDICTED: chitinase 2 Down

11859 2.38 -5.86 7.11E-06 PREDICTED: chitinase 3 Down

15347 5.12 -3.71 1.77E-08 chitinase-related protein 1 Down

16243 11.88 -9.26 2.11E-13 PsChi-h mRNA for chitinase Down

17052 14.47 -13.27 0 viral-like chitinase Down

17700 4.3 -8.41 4.12E-07 chitinase mRNA Down

gi|687027891 2.18 -11.57 0 chitinase-related protein 1 Down

gi|687027960 2.27 -14.81 0 chitinase 7 Down

gi|687056067 17.18 -20.17 0 viral-like chitinase Down

gi|687094659 180.42 -68.27 0 chitinase Down

gi|687131816 6.23 -3.82 9.37E-13 PREDICTED: endochitinase A1-like Down Chitin 11096 27.73 -5.71 0 chitin deacetylase Down Deacetylase 16131 25.48 -7.66 0 chitin deacetylase 1 Down

gi|687033646 22.13 -4 4.76E-12 chitin deacetylase 1 Down

Table 3.4. Differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T. ni. (continued) Max Gene Fold FDR p- Contig ID Group Sequence Description Regulation Category Change value Mean Peritrophins 5400 22.26 -9.06 0 PREDICTED: peritrophin-1-like Down

16350 38.59 8.59 8.91E-04 peritrophin type-A domain protein 2 Up

gi|687043932 21.34 -10.55 0 chitin binding peritrophin-A Down

gi|687055341 1.28 -8.92 3.50E-05 PREDICTED: peritrophin-1-like Down

gi|687067717 1.4 -24.33 1.42E-05 PREDICTED: peritrophin-1-like Down

gi|687108074 22.94 -7.22 0 peritrophin-1-like Down PREDICTED: endocuticle structural Glycoprotein 18285 3.52 6.98 2.25E-04 Up glycoprotein ABD-5-like Ecdysone 3024 PREDICTED: ecdysone-induced protein 2.61 3.93 5.81E-12 Up Inducibles 74EF isoform A 6995 40.12 -4.55 1.49E-04 ecdysteroid-induced (E75) Down

5946 2.13 -4.15 6.38E-04 PREDICTED: ecdysone receptor Down

16286 3.31 -6.41 1.27E-09 3-dehydrecdysone Down

18136 32.17 4.85 0 20-hydroxy-ecdysone receptor Up

gi|687085860 26.81 -4.1 0 ecdysone oxidase Down

gi|687100743 20.36 -4.19 2.37E-10 ecdysone oxidase Down

gi|687128575 4.7 -3.46 7.01E-05 ecdysone receptor Down

Table 3.4. Differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T. ni. (continued)

Max Gene Fold FDR p- Contig ID Group Sequence Description Regulation Category Change value Mean Juvenile juvenile hormone esterase precursor Hormone 11982 37.19 -26.82 0 Down (JHE) Inducibles juvenile hormone esterase-like; 14365 7.07 -5,811.56 1.19E-05 Down PREDICTED: esterase B1-like juvenile hormone sensitive hemolymph 16406 0.19 -8.01 8.38E-05 Down protein 16491 215.29 -3.96 0 juvenile hormone-inducible protein Down

17075 500.51 -5.25 0 juvenile hormone esterase-like Down PREDICTED: juvenile hormone epoxide 17228 0.59 -25.24 9.61E-08 Down hydrolase-like 18269 1.95 -78.03 5.64E-08 juvenile hormone binding-like protein Down juvenile hormone esterase precursor gi|687052097 20.2 -35.21 0 Down (JHE) Mucins 5197 1.1 -3.89 3.43E-07 PREDICTED: mucin-5AC-like Down

8946 1.78 -6.1 1.32E-13 PREDICTED: mucin-2-like Down

10541 0.79 -13.51 2.86E-06 PREDICTED: mucin-5AC Down

16236 149.06 11 0 intestinal mucin Up

16269 150.99 10.24 0 insect intestinal mucin 2 Up

17180 1.16 -7.66 6.59E-05 PREDICTED: mucin-5AC Down

gi|687093538 0.69 -12.24 7.70E-07 PREDICTED: mucin-5AC Down

Lipases 16375 173.6 -15.02 0 insect intestinal lipase 6 Down

16377 446.23 6.75 0 PREDICTED: lipase 1-like Up

Table 3.5. Summary of differentially expressed contigs in midguts between all diet group pairs for 4th instar T. ni.

Cabbage vs Potato vs Potato vs Gene Category Contig ID Sequence Description Artificial Cabbage Artificial Regulation Regulation Regulation Chitin gi|687040850 chitin synthase A NA NA Down 13x Synthase Chitinase 8386 probable chitinase 2 Down 18x Down 3x Down 68x

11859 probable chitinase 3 NA NA Down 5x

15347 chitinase-related protein 1 (ChiR1) NA Down 3x Down 3x

16243 PsChi-h for chitinase Down 11x NA Down 9x

17052 viral-like chitinase gene Down 10x NA Down 13x

17700 chitinase NA NA Down 8x

gi|687027891 chitinase-related protein 1 (ChiR1) NA Down 4x Down 11x

gi|687027960 chitinase 7 Down 11x NA Down 14x

gi|687056067 viral-like chitinase gene Down 10x NA Down 20x

gi|687094659 chitinase (Cht) Down 17x Down 4x Down 68x

gi|687131816 endochitinase A1-like NA NA Down 3x

11096 chitin deacetylase NA NA Down 5x Chitin 16131 chitin deacetylase 1 (cda1) NA Down 3x Down 7x Deacetylase gi|687033646 chitin deacetylase 1 (cda2) NA NA Down 4x peritrophin-1-like, transcript variant 5400 Down 3x NA Down 9x X1 Peritrophins 16350 peritrophin type-A domain protein 2 NA NA Up 8x

gi|687043932 chitin binding peritrophin-A Down 4x NA Down 10x

gi|687055341 peritrophin-1-like NA NA Down 9x

gi|687067717 peritrophin-1-like NA NA Down 24x

Table 3.5. Summary of differentially expressed contigs in midguts between all diet group pairs for 4th instar T. ni. (continued) Cabbage vs Potato vs Potato vs Gene Contig ID Sequence Description Artificial Cabbage Artificial Category Regulation Regulation Regulation endocuticle structural glycoprotein ABD-5- Glycoprotein 18285 Up 7x NA Up 7x like Ecdysone 3024 ecdysone-induced protein 74EF isoform A Up 4x NA Up 4x Inducibles 6995 ecdysteroid-induced (E75) NA NA Down 4x

5946 ecdysone receptor transcript variant X2 Down 5x NA Down 4x

11211 ecdysteroid-regulated 16 kDa protein-like Down 7x Up 13x NA

16286 3-dehydrecdysone 3b-reductase Down 4x NA Down 6x

17425 ecdysteroid-regulated 16 kda protein Down 8x NA NA

17757 putative ecdysone oxidase Up 54x Down 75x NA

18136 20-hydroxy-ecdysone receptor Down 3x Up 15x Up 4x

gi|687085860 ecdysone oxidase gene Down 3x NA Down 4x

gi|687100743 ecdysone oxidase gene Down 4x NA Down 4x

gi|687128575 ecdysone receptor NA NA Down 3x Juvenile Hormone 2789 juvenile hormone-inducible protein Down 5x NA NA Inducibles juvenile hormone epoxide hydrolase 4247 NA Up 3x NA precursor 9577 juvenile hormone esterase-like isoform Down 4x NA NA

11982 juvenile hormone esterase precursor (JHE) Down 19x NA Down 27x juvenile hormone epoxide hydrolase-like 13294 NA Down 5x NA protein Down 14365 juvenile hormone esterase-like NA NA 5,811x 15986 juvenile hormone esterase-like NA Up 3x NA juvenile hormone sensitive hemolymph 16406 NA NA Down 8x protein

Table 3.5. Summary of differentially expressed contigs in midguts between all diet group pairs for 4th instar T. ni. (continued)

Cabbage vs Potato vs Potato vs Gene Contig ID Sequence Description Artificial Cabbage Artificial Category Regulation Regulation Regulation Juvenile Hormone 16491 juvenile hormone-inducible protein NA Down 3x Down 4x Inducibles 17075 juvenile hormone esterase-like NA NA Down 5x

17228 juvenile hormone epoxide hydrolase-like NA NA Down 25x

18269 juvenile hormone binding-like protein Down 24x NA Down 78x

gi|687052097 juvenile hormone esterase precursor (JHE) Down 25x NA Down 35x

Gelsolin 14408 gelsolin-like Up 3x NA NA

Mucins 5197 mucin-5AC-like transcript variant X2 NA NA Down 4x

5553 mucin-2-like Down 3x NA NA

8946 mucin-2-like Down 3x NA Down 6x

10541 mucin-5AC NA NA Down 13x

16236 intestinal mucin NA Up 10x Up 11x

16269 insect intestinal mucin 2 NA Up 10x Up 10x

17180 mucin-5AC NA NA Down 7x

gi|687085871 mucin-2-like Up 5x NA NA

gi|687093538 mucin-5AC NA NA Down 12x

REPAT gi|687115186 REPAT34 NA Up 3x NA

Lipases 16200 insect intestinal lipase 7 NA Up 4x NA

16325 insect intestinal lipase 6 NA Up 4x NA

16375 insect intestinal lipase 6 Down 3x Down 4x Down 15x

16377 lipase 3-like Up 3x NA Up 6x

Table 3.6. Top 50 differentially expressed contigs in midguts of cabbage-raised compared to artificial diet-raised 4th instar T.ni.

Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean 8807 5.43 4,710.44 3.69E-06 PREDICTED: protein split ends transcript variant X4 Up

11607 10.42 -2,043.91 1.88E-04 rna-binding protein 1 isoform x3 Down

gi|687032876 0.57 -1,344.53 4.72E-04 PREDICTED: mechanosensitive ion channel Down

16439 5,485.97 994.23 0 18S ribosomal RNA gene Up PREDICTED: tyrosine-protein phosphatase non- 4131 0.53 856.88 4.50E-04 Up receptor type 9-like gi|687083130 25.47 -660.96 2.10E-11 PREDICTED: uncharacterized LOC105391589 partial Down

gi|687095027 1.34 647.25 9.54E-04 chromosome: chr8 Up

16585 14.1 -640.74 0 arylphorin mRNA Down

16441 1,404.47 -507.53 0 epididymal secretory protein e1-like Down PREDICTED: uncharacterized LOC106720632 18088 32.34 -375.87 0 Down transcript variant X3 gi|687111713 6.76 -344.95 8.17E-09 PREDICTED: hormone receptor 3 Down

gi|687066127 34.14 -327.14 0 osmosensing histidine protein kinase SLN1 Down

16750 122.57 -311.51 0 facilitated trehalose transporter tret1-like Down

16983 658.95 311.49 0 18S ribosomal RNA Up

9056 5.77 -294.36 8.64E-09 CBS 6284 chromosome 3 Down

7484 23.73 -286.72 0 PREDICTED: nuclear hormone receptor HR3 Down

15414 9.46 -266 0 PREDICTED: organic cation transporter protein-like Down

Table 3.6. Top 50 differentially expressed contigs in midguts of cabbage-raised compared to artificial diet-raised 4th instar T.ni. (continued)

Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean gi|687085342 53.06 -248.77 0 PREDICTED: nuclear receptor subfamily 6 group A Down

gi|687126840 23.06 -227.88 0 PREDICTED: uncharacterized LOC105397725 Down

10749 12.66 226.78 0 glucose oxidase-like enzyme mRNA Up

5184 46.64 226.24 0 UDP-glycosyltransferase 39B4 Up

gi|687042275 16.25 -224.63 3.84E-13 PREDICTED: eisosome protein SEG2 Down

gi|687124701 3.92 -220.6 8.65E-08 PREDICTED: serine proteinase stubble-like Down

16372 2,485.31 217.17 0 lebocin precursor Up

gi|687066166 21.64 -216.92 4.20E-13 PREDICTED: uncharacterized LOC106137673 Down

gi|687070620 36.77 -200.93 0 hormone receptor 4 (HR4) Down

gi|687085654 13.06 190.97 0 genome assembly GPUH_scaffold0035729 Up

2819 1.6 -189.59 2.11E-07 unknown secreted protein sequence id: Px-1534 Down uncharacterized LOC106111980 for unknown secreted gi|687122464 21.16 -178.08 0 Down protein, sequence id: Pp-0370 16101 6,335.94 174.25 0 serine protease, clone SR19, SR110 Up

12888 19.49 -173.9 0 PREDICTED: allantoinase-like Down

gi|687128779 2.61 171.29 7.78E-14 BAC, egg DNA Up

gi|687118721 46.93 -163.53 0 PREDICTED: proline-rich extensin-like protein Down

gi|687044229 25.63 -161.76 0 hormone receptor 4 Down

Table 3.6. Top 50 differentially expressed contigs in midguts of cabbage-raised compared to artificial diet-raised 4th instar T.ni. (continued)

Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean gi|687040727 50.88 157.98 0 scaffold BTMF_scaffold0000231 Up

gi|687075461 21.27 -155.57 0 PREDICTED: uncharacterized LOC105387535 Down

gi|687041211 22.32 -152.29 0 hormone receptor 4 Down

gi|687052737 6.84 -147.78 7.77E-07 PREDICTED: aminoacylase-1-like Down

gi|687088903 24.93 141.11 0 BAC, egg DNA Up

gi|687074443 45.3 -135 0 clone: fepM12H13 Down

gi|687128305 1,375.86 114.6 0 beta-glucosidase precursor Up

16608 501.16 -112.33 0 not available Down

gi|687091878 9.8 -109.2 4.43E-11 UDP-glycosyltransferase Down PREDICTED: nuclear hormone receptor family member gi|687064871 5.8 -109.12 1.65E-07 Down nhr-91-like gi|687099862 0.09 108.92 3.88E-07 BAC clone:520F12 Up

16587 242.16 -107.61 0 18S ribosomal RNA Down

6477 57.78 106.87 0 clone BAC 33J17 cytochrome P450 Up

6491 138.08 -101.14 0 molt-regulating transcription factor HaHR3 Down

gi|687066378 9.46 -100.66 1.84E-13 MHR3 mRNA Down

Table 3.7. Top 50 differentially expressed contigs in midguts of potato-raised compared to cabbage-raised 4th instar T.ni.

Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean 10039 12.57 -3,922.67 3.66E-05 not available Down

18323 2,006.68 -3,567.27 0 cytochrome CYP324A6 Down PREDICTED: tRNA dimethylallyltransferase, 10485 8.4 -3,376.69 2.46E-05 Down mitochondrial mRNA PREDICTED: ABC transporter A family member 2- gi|687104771 6.99 3,114.00 1.08E-04 Up like 5184 46.64 -3,015.11 0 UDP-glycosyltransferase 39B4 Down

17848 1,464.79 -1,896.21 0 cytochrome P450 CYP321A5 Down

gi|687040727 50.88 -1,467.35 0 scaffold BTMF_scaffold0000231 Down PREDICTED: ATP-binding cassette sub-family A gi|687096244 6.7 1,289.23 8.93E-04 Up member 2-like 15414 25.49 1,138.81 0 flavin-dependent monooxygenase FMO3B Up

18929 12.9 -1,085.29 6.57E-11 PREDICTED: reticulon-1-A Down

17635 180.12 -1,079.85 0 scaffold SMTD_scaffold0023335 Down

16610 1,086.64 -1,052.15 0 PREDICTED: solute carrier family 19 member 1 Down

17514 2,199.97 -880.63 0 cytochrome P450 CYP321A9 Down

16439 5,485.97 -669.14 0 18S ribosomal RNA gene, internal transcribed spacer 1 Down

gi|687078584 556.87 607.9 0 PREDICTED: pancreatic triacylglycerol lipase-like Up

gi|687075186 718.46 483.68 0 PREDICTED: interleukin 5 receptor subunit Up

gi|687085460 774.08 468.07 0 PREDICTED: pancreatic triacylglycerol lipase-like Up

Table 3.7. Top 50 differentially expressed contigs in midguts of potato-raised compared to cabbage-raised 4th instar T.ni. (continued) Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean gi|687069664 18.05 449.21 0 chromosome 4 sequence Up PREDICTED: ABC transporter A family member 1- gi|687119103 10.67 432.37 0 Up like 2891 39.39 420.36 0 PREDICTED: DNA-binding protein D-ETS-6-like Up

gi|687125436 1,112.45 -393.89 0 BAC, egg DNA Down

gi|687038127 18.39 387.95 0 chromosome 8 sequence Up

8320 6.32 373.17 0 PREDICTED: serine/threonine-protein kinase 10-like Up

10706 68.93 370.49 0 PREDICTED: cytochrome b5 Up

263 38.36 366.43 0 alcohol dehydrogenase Up

gi|687127134 14.07 352.04 0 PREDICTED: cyanate hydratase Up

10695 14.8 349.06 0 PREDICTED: protein FEV-like Up

18364 48.92 -313.33 0 UDP-glycosyltransferase Down

gi|687121575 13.43 311.51 0 clone L581 gallerimycin Up

17013 256.26 -296.64 0 UDP-glycosyltransferase Down

16983 658.95 -288.86 0 18S ribosomal RNA Down

17974 208.31 -283 0 PREDICTED: UDP-glucuronosyltransferase 2B19-like Down

1727 2.38 278.24 3.95E-14 PREDICTED: DNA-binding protein D-ETS-6-like Up

18023 219.8 -265.8 0 UDP-glycosyltransferase 33F4 Down

gi|687114672 121.34 233.82 0 hypothetical protein Up

18111 18.8 -188.84 0 prophenoloxidase-activating proteinase-3 (PAP-3) Down

gi|687048759 7.64 -182.14 1.27E-11 clone POP002-K09 Down

gi|687119680 0.34 -170 5.63E-07 scaffold SMTD_scaffold0000668 Down

16389 2,321.25 -159.05 0 PREDICTED: pancreatic triacylglycerol lipase-like Down

7640 1.01 157.24 0 clone BA_Ba68O14 Up

18608 19.69 -150.94 1.49E-10 UDP-glycosyltransferase 33F4 (UGT33F4) Down

gi|687067156 10.16 -145.25 6.27E-13 scaffold TCLT_scaffold0000596 Down

gi|687133666 4.48 -143.94 0 PREDICTED: uncharacterized oxidoreductase Down

gi|687081553 3.3 -143.39 1.42E-06 RhoGEF domain containing protein Down

16587 201.07 141.75 0 18S ribosomal RNA Up

gi|687097466 1.54 136.54 2.20E-06 clone AC1_B5 microsatellite sequence Up PREDICTED: ABC transporter A family member 2- gi|687103806 4.08 132.05 0 Up like PREDICTED: ATP-binding cassette sub-family A gi|687110487 6.75 127.68 2.20E-10 Up member 2-like

Table 3.8. Top 50 differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T.ni.

Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean 14365 7.07 -5,811.56 1.19E-05 juvenile hormone esterase-like Down PREDICTED: tyrosine-protein phosphatase non- 4131 2.18 3,310.68 1.44E-05 Up receptor type 9-like gi|687065638 25.11 -2,957.40 2.43E-05 PREDICTED: uncharacterized LOC101744039 Down

8807 2.76 2,203.80 4.28E-05 PREDICTED: protein split ends transcript variant X4 Up

16585 14.1 -1,751.48 0 arylphorin Down

gi|687075461 21.27 -1,629.63 1.29E-04 PREDICTED: uncharacterized LOC105387535 Down

gi|687066166 21.64 -1,621.14 1.27E-04 PREDICTED: uncharacterized LOC106137673 Down PREDICTED: piezo-type mechanosensitive ion gi|687032876 0.57 -1,452.86 1.70E-04 Down channel component gi|687084284 0.41 1,296.74 2.25E-04 cryptochrome 2 mRNA Up

gi|687080545 4.58 -1,180.27 2.97E-04 PREDICTED: uncharacterized LOC106714378 Down

gi|687122464 21.16 -1,135.73 1.22E-13 uncharacterized LOC106111980 Down

7484 23.73 -1,094.86 0 PREDICTED: probable nuclear hormone receptor HR3 Down

gi|687066127 34.14 -954.67 4.08E-13 osmosensing histidine protein kinase SLN1 Down

gi|687070620 36.77 -938.01 3.97E-13 hormone receptor 4 (HR4) Down

gi|687084286 0.25 876.19 6.13E-04 cryptochrome 2 Up

2819 1.6 -811.84 7.59E-04 unknown secreted proteinsequence id: Px-1534 Down

gi|687083130 25.47 -784.14 3.09E-12 PREDICTED: uncharacterized LOC105391589 Down

Table 3.8. Top 50 differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T.ni. (continued)

Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean gi|687040383 31.39 -709.78 2.43E-09 PREDICTED: uncharacterized LOC105389919 Down

gi|687116298 1.85 -483.69 1.86E-10 clone: fepM03P02 Down

813 31.2 -473.75 0 UDP-glycosyltransferase UGT46A4 Down

gi|687118721 46.93 -464.48 0 PREDICTED: proline-rich extensin-like protein EPR1 Down

6343 6.17 -443.63 3.90E-10 PREDICTED: uncharacterized LOC101737971 Down

gi|687111713 6.76 -402.04 1.52E-09 PREDICTED: hormone receptor 3 (Hr3) Down

16258 3.07 -364.36 1.33E-14 arylphorin Down

gi|687044229 25.63 -358.33 0 hormone receptor 4 Down

9056 5.77 -346.02 1.69E-09 CBS 6284 chromosome 3 Down PREDICTED: nuclear receptor subfamily 6 group A gi|687085342 53.06 -294.55 0 Down member 1 6491 138.08 -289.31 0 molt-regulating transcription factor HaHR3 Down

16750 122.57 -281.01 0 facilitated trehalose transporter tret1-like Down

gi|687042275 16.25 -269.78 3.87E-14 PREDICTED: eisosome protein SEG2 Down

gi|687041211 22.32 -258.58 5.11E-14 hormone receptor 4 (HR4) Down

gi|687124701 3.92 -257.43 1.85E-08 PREDICTED: serine proteinase stubble-like Down

18088 32.34 -241.89 0 PREDICTED: uncharacterized LOC106720632 Down

18929 4.21 -206.7 1.73E-06 PREDICTED: reticulon-1-A Down

gi|687074443 45.3 -201.9 0 clone: fepM12H13 Down

1352 2.82 191.13 0 PREDICTED: uncharacterized LOC106093080 Up

beta-hexosaminidase subunit beta-like; beta-N- 2424 48.98 -181.28 0 Down acetylglucosaminidase 3 precursor

Table 3.8. Top 50 differentially expressed contigs in midguts of potato-raised compared to artificial diet-raised 4th instar T.ni. (continued) Max Fold FDR p- Contig ID Group Sequence Description Regulation Change value Mean gi|687038127 18.39 161.34 0 chromosome 8 sequence Up

gi|687125436 754.22 -152.21 0 BAC, egg DNA Down

18364 39.53 -148.32 0 UDP-glycosyltransferase UGT33F1 Down

gi|687060035 42.58 -138.95 0 PREDICTED: UDP-glucuronosyltransferase 2B18-like Down

gi|687088378 12.8 -138.14 7.48E-14 thymosin beta-like protein Down

gi|687133666 6.99 -133.61 0 PREDICTED: uncharacterized oxidoreductase Down

gi|687072573 5.97 -133.44 1.78E-06 molting carboxypeptidase A Down

gi|687091878 9.8 -130.24 3.70E-10 UDP-glycosyltransferase UGT33B12 Down

gi|687101440 1.5 130.19 2.97E-06 43U chromosome 14 sequence Up

16732 220.75 128.69 1.20E-05 clone: fwd-02H12 Up

2891 39.39 123.4 0 PREDICTED: DNA-binding protein D-ETS-6-like Up

gi|687059532 4.91 -123.16 2.84E-06 PREDICTED: glucose dehydrogenase Down

Chapter 4. Discussion

In this thesis I have investigated the effects of diet on infection of T. ni larvae by an AcMNPV baculovirus. I first confirmed that raising T. ni larvae on different diets does alter the efficiency with which the virus infects insects by measuring the LD50 values using three different diets. The results showed that the LD50 for this virus varied from 3.1 to 61.6 OBs when larvae were reared on different food sources (Figure 3.1).

4.1 Physiological Differences in Gut pH with Different Diets

To determine what conditions under the different diet regimes might be responsible for this effect on infection, I tested the pH of anterior midgut contents when the insects were fed different foods since it has been previously suggested that dietary changes in pH may play a role in the resistance to pathogens106.

I found that pH can be significantly altered depending on diet (Figure 3.2). Cabbage- raised larvae had the least alkaline pH in the midgut (mean 8.5) whereas potato and artificial diet-raised larvae had similar, more alkaline pHs (mean ~9.5) characteristic of what is reported in the literature.109 Cabbage is rich in vitamin C: ascorbic acid. However, despite having a significantly lower pH than the other diet categories, cabbage-raised larvae were not the least susceptible to baculovirus having an LD50 between artificial diet and potato-raised larvae (Figure 3.1). Therefore, the prediction that diet would result in alterations to the midgut pH was supported by my data, but the results were not consistent with this pH change contributing to the differences in virus infection observed. Lowering of the pH in the anterior midguts of cabbage-raised insects did not reduce their susceptibility to the virus. It is possible that since the altered pH remained above 8, this is still alkaline enough to dissolve OBs effectively while also preventing tannin-protein aggregation.

4.2 Morphological and Transcriptional Differences in T. ni Midgut PM

To determine what morphological differences might occur with different diets, I examined the PM of larvae raised on different food sources. Previous studies looking at AcMNPV baculovirus infections in T. ni reported that most infections occurred at the

54 anterior end of the PM, with a small percentage at the distal end68. This can be explained by the fact that this species secretes a type II PM66,67 which has weaker structural integrity at the point of origin, becomes thicker and hyper-coiled as additional components are added69,70, and then depleted in the posterior section of the midgut (Figure 3.3). Therefore I focussed my studies on the anterior region of the midgut.

Larvae fed on artificial diet had thin (less than 1µm) and fragile PMs, with midgut RNA profiles showing high levels of chitinase transcribed, the protein products of which would actively degrade chitin material. A thin PM (Figures 3.7 and 3.8) would not provide a very effective barrier against viral infection, potentially explaining the low LD50 for larvae reared on artificial diet: 3.1 OBs (95% CI: 0, 39.1). The PM of larvae reared on cabbage, a preferred host plant, was also thin (less than 1µm) but more uniform and dense than those from larvae fed artificial diet (Figures 3.9 and 3.10). The lower transcript levels of chitinase observed in the midgut of these insects would result in less degradation than seen in the artificial diet treatment, potentially contributing to the significantly lower susceptibility to baculoviral infection of cabbage-raised larvae: LD50 = 16.6 OBs (95%

CI: 8.4, 32.5). The LD50 for larvae raised on potato, a non-preferred and chemically 55,57 defended host plant , was higher than that for the other two treatments: LD50 = 61.6 OBs (95% CI: 37.2, 102.1) (Figure 3.1). This could be explained, at least in part by the fact that the PM in potato-raised larvae had thicker and less organized layers (Figures 3.12 and 3.13). The morphological differences are reflected in the RNA transcript profiles as larvae reared on potato had the lowest chitinase and chitin deacetylase transcript levels, resulting in the thicker less organised layers. Higher mucin and lipase levels in potato- raised larvae compared to cabbage-raised larvae (but inconsistent changes compared to artificial-diet-raised larvae) suggests a PM with greater gel protective layering along the PM. The PM of larvae reared on potato also had more microvesicles. This is consistent with repair or reinforcement of the PM structure as the membranes of the microvesicles become partially soluble in alkaline pH, and when close to the intestinal lumen they release their contents and become incorporated into the PM88,89. Overall, the differentially expressed levels of chitinase, chitin deacetylase, REPAT, mucins, and lipases, considered alongside the TEM images, all indicate a PM that is more robust in potato-raised larvae. The level of diet toxicity and the corresponding effects on 4th instar T. ni PM physiology,

55 chitinase regulation levels, susceptibility to baculovirus, and anterior midgut pH are summarized in Figure 3.17.

Figure 3.17. The peritrophic membrane physiology, chitinase expression levels, susceptibility to virus, and anterior midgut pH responses as a function of diet in 4th instar T. ni. In order of increasing toxicity are artificial diet, cabbage, and potato.

Another feature of interest was the level of ecdysone and JH-inducibles in the midgut environment, as ecdysone and JH work antagonistically to coordinate molting, during which the insect’s chitin content is drastically altered. Ecdysone, among other roles, induces molting and molting is correlated with thicker PM growth95. Inversely, feeding reduces PM thickness. (Larvae molting, or soon to be entering a molting phase, cease feeding, so the two actions are exclusive.) Interestingly, larvae reared on potato also have increased transcript levels of ecdysone receptors and reduced levels of those for ecdysone oxidase. Ecdysone oxidase catalyzes ecdysone into 3-dehydroecdysone, diverting its conversion into the pre-hormone 20-hydroxyecdysone which acts on ecdysone receptors to stimulate molting117. Thus the former increases an inducer while the latter reduces a repressor, so both differential expressions levels could contribute to a PM state closer to that of molting insects in non-molting larvae. However, this is a hypothesis only as RNA transcript levels were measured in the midgut tissue and ecdysone was not directly measured.

4.3 Stress Associated with Plant Diet: Triggering of Immune Response

It is intriguing that REPAT protein transcript levels were significantly upregulated in potato-raised larvae compared to cabbage-raised larvae, even though no pathogen challenge was introduced, suggesting a heightened basal immune system induced by a relatively toxic diet. It is possible that the uptake of sterols, exceptionally abundant in Solanaceous foliage118, can lead to membrane and cell damage. This in turn would trigger general stress responses that induce an immune response when T. ni feed on potato and may contribute to the low susceptibility of potato-raised larvae to AcMNPV. A lepidopteran midgut transcriptomic study comparing the responses of three Spodoptera species also detected considerably higher expression of genes associated with the insect immune response after feeding on maize leaves compared to pinto bean artificial diet119.

4.4 Detoxification Gene Responses to Plant Allelochemicals

The midgut’s ability to detoxify plant toxins is an essential characteristic of insects, especially generalists, for managing diet toxin diversity. Many cytochrome P450s are detoxifying enzymes involved in the functionalization step of detoxification120,121,122. Glutathione transferases (GSTs) convert lipophilic xenobiotics into hydrophilic compounds for excretion or sequestration123,124. UDP-glucosyl transferases (UGTs) detoxify benzoxazinoids by conjugation with a sugar125. All three major detoxifying enzyme families were represented in the top 50 most differentially expressed contig transcripts of midguts from potato-raised larvae versus cabbage-raised larvae (Table 3.7), and more so than in comparison of midguts from cabbage-raised larvae vs artificial diet- raised larvae or potato-raised larvae vs artificial diet-raised larvae (Tables 3.6 and 3.8). These tended to be downregulated in midguts of potato-raised larvae. A similar response consisting of strong gene repression of detoxification enzymes was found using midgut transcriptomics of T. ni fed on tomato (Solanaceae) compared to Arabidopsis (Brassicaceae) – two different plants that share the same family as the plant diets used in this study126. The lower expression levels of detoxifying enzymes may be indicative of how larvae raised on potato are more negatively impacted when it comes to growth rate, as they take longer to reach pupation compared to the other two diet groups. The hypothesis put forth by Herde and Howe126 proposes that anti-nutritive proteins elicit

57 large-scale remodelling of digestive enzymes and that the metabolic costs associated with digestive flexibility constrains an insect’s ability to detoxify secondary metabolites. This hypothesis is supported by the varied defense-related plant compounds present in Solanaceae and by my results. However, the effects of lower detoxification enzyme levels and their impact on growth are difficult to isolate from the thickened PM structure observed in potato-raised larvae. A thicker PM reduces digestive rates and this would also result in decreased growth rates37.

4.5 Future Challenges and Directions

The results of this study underline the importance of taking into account the crop type when determining the dose of viruses used for effective control in an integrated pest management program, with consequences for application, time, and cost. The application of an insufficient dose of virus could result in unacceptable crop losses, especially if the pest is univoltine as there would be no opportunity for the virus to increase over successive generations. Furthermore, as the in vivo production of baculoviruses is expensive, if the dose required on a specific crop is high farmers may reject the option on financial grounds. My research has shown that the effect of food source on gut pH does not appear to be responsible for variation in viral infectiousness of AcMNPV baculovirus in T. ni. However, a very interesting finding was that gut pH did vary with the different diets tested. A useful line of future inquiry could involve testing for changes in other properties of the midgut resulting from different diets, such as the proteases and protease inhibitors present. Proteases can affect the structure of the PM and thereby how efficiently it can provide a barrier to pathogens78,127. Finding additional factors that modulate infectiousness of baculovirus could provide novel means for optimizing virus performance.

Currently, one active field of research is molecular based biocontrol, specifically RNA interference (RNAi)128. However, the successful RNAi knockdown of genes in insects has been quite variable and it is becoming evident that while the overall machinery of RNAi response is generally conserved, specific components differ considerably depending on the class, family and even species level129,130,131. To date, there have as of yet been no successful systemic RNAi knockdowns in T. ni through feeding assays, but other methods

58 for introducing dsRNA such as injection are possible. This method of experimentally altering the levels of targets of interest such as chitin synthase or chitin deacetylase could be used to reduce the expression of these genes in cabbage-fed larvae. Additionally, any of the other proteins of interest from this studythat are associated with the PM could be target options – particularly those that were upregulated on a challenging diet, such as: glycoproteins, mucins, lipases, and REPATs. The knockdown of two essential PM proteins in the red flour beetle resulted in significant mortality and it was concluded that these proteins were essential for regulation of PM permeability, which is essential for survival and fat body maintenance132. By measuring PM thickness and virus susceptibility with such genes silenced, it would be possible to determine if particular genes and their corresponding enzymes contribute to the reduced viral susceptibility I observed in potato- fed larvae. Ultimately, this approach might prove effective in improving the efficacy of a promising oral pathogen for use as a biopesticide and reduce the dosage of pathogen required for effective control, resulting in the desired control at a lower cost.

4.6 Conclusion

Baculoviruses potentially offer an effective alternative to insecticides as co-evolution with their insect hosts has resulted in biological properties that can be advantageous for agricultural purposes. They are also only one specific tool in a developing arsenal of existing and upcoming pest biocontrol possibilities. If used solely, the context in which they are applied needs to be considered for efficacy. If used in concert with other methods, their inclusion should be to counterbalance the drawbacks of those systems. My research offers a detailed study on the factors that underlay infection and susceptibility in a model baculovirus-host system. My findings suggest that in this model system, the effects of diet on virus infectivity are primarily mediated through alterations to the structure of the PM and not through changes to midgut pH. The changes in gene expression associated with these alterations provide leads for further experiments to identify the specific mechanisms involved. It is my hope that a better understanding of this pathogen-host relationship will lead to better informed applications of effective and convenient biocontrol techniques.

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Appendix

Permission to use copyright materials in Figure 1.1.

Photos used in the T. ni life cycle were with their photographers’ permission. Special thanks to Ms. Roberta Gibson, Mr. Ian Maton from domain pages albertanaturephotography.com, getawaymoments.com, blog.growingwithscience.com.

Permission to use copyright material in Figure 1.2.

The diagram used to illustrate the AcMNPV baculovirus infection cycle was used with the permission of its creator Dr. Leo Graves of Oxford Brookes University and the CEO of Oxford Expression Technologies, Dr. Robert Possee.

Permission to use copyright material in Figures 1.3, 1.4, and 1.5.

Licensee: London Research and Development Centre Order Date: Aug 14, 2017 Order Number: 4167461180214 Publication: Tissue and Cell Peritrophic membrane structure and formation in the larva of a moth, Title: Heliothis Type of Use: reuse in a thesis/dissertation Order Total: 0.00 CAD

Licensee: London Research and Development Centre Order Date: Aug 14, 2017 Order Number: 4167461304027 Publication: Tissue and Cell Peritrophic membrane structure and formation of larval Trichoplusia ni Title: with an investigation on the secretion patterns of a PM mucin Type of Use: reuse in a thesis/dissertation Order Total: 0.00 CAD

Licensee: London Research and Development Centre Order Date: Aug 14, 2017 Order Number: 4167460633121 Publication: Insect Biochemistry and Molecular Biology Spatial and temporal synthesis of Mamestra configurata peritrophic Title: matrix through a larval stadium Type of Use: reuse in a thesis/dissertation Order Total: 0.00 CAD

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Curriculum Vitae

Name: Elizabeth Chen

Post-secondary Queen’s University Education and Kingston, Ontario, Canada Degrees: 2011-2015 BSc Honours Specialization in Biology University of Western Ontario London, Ontario, Canada 2015-2017 MSc Cell and Molecular Biology Honours and Western Graduate Research Scholarship, 2015-2017 Awards: Dean’s Honour List 2014-2015

Related Work Graduate Research Assistant, Agriculture and Agri-Food, Experience: London, Ontario, Canada, 2016-2017 Teaching Assistant, University of Western Ontario, 2015-2016 Summer Research Assistant, Agriculture and Agri-Food, London, Ontario, Canada, 2015 Summer Research Assistant, Agriculture and Agri-Food, Ottawa, Ontario, Canada, 2013-2014

Conference Chen, E, and Donly, C. 2017. The effect of diet on midgut and Presentation: resulting changes in infectiousness of AcMNPV baculovirus in Trichoplusia.ni. Oral Presentation, Insect Biotechnology Conference. Niagara, Ontario, Canada.