The Physiological Effects of Kinin and CAPA Analogs in Rhodnius Prolixus

Neuropeptide Mimetics: The Physiological Effects of Kinin and CAPA Analogs in Rhodnius prolixus

Vishal Sangha

A thesis submitted in conformity with the requirements for the degree of Master of Science Cell and Systems Biology University of Toronto

Neuropeptide Mimetics: The Physiological Effects of Kinin and CAPA Analogs in Rhodnius prolixus

Vishal Sangha

Master of Science

Cell and Systems Biology University of Toronto

2019 Abstract

In the Chagas disease vector Rhodnius prolixus, the kinin and CAPA neuropeptides modulate a host of feeding and diuresis-related behaviours that are implicated in disease transmission. CAPA and kinin neuropeptide analogs have been developed to elicit potent changes in physiology, to be later incorporated in novel pest-control strategies. Here, the effects of kinin and CAPA analogs were investigated on feeding and diuresis-related tissues, with the kinin and

CAPA analogs inducing physiological changes in vivo and in vitro. Within the hindgut, novel intracellular interactions were uncovered between RhoprCAPA, Rhopr-kinin, and serotonin [5- hydroxytryptamine (5-HT)]. Following identification and sequencing of the Rhopr-kinin receptor, the receptor transcript was observed throughout the gut, with RNA interference (RNAi)-mediated knockdown of the receptor causing reductions in hindgut contractions and increasing the size of blood meal consumed. Overall, these findings highlight the role of kinin and CAPA within R. prolixus, and the promise of their neuropeptide analogs to be used as lead compounds in pest- control strategies.

Acknowledgments

I am truly grateful to Dr. Lange for providing me with the opportunity to conduct research in the esteemed Lange/Orchard lab. Your guidance and endless support throughout these past two years has allowed me to grow as a researcher, and as a person. To Dr. Orchard, who essentially served as a co-supervisor, thank you for taking the time out of your busy schedule to provide direction and feedback on my work, and answering every possible silly question of mine. Yours and Dr.

Lange’s passion for science is truly inspiring.

I would like to thank Dr. Senatore for being a part of my committee and providing me with feedback throughout my research, and Dr. Westwood for serving as an external examiner

To past and present members of the Lange/Orchard lab who I have had a chance to work with, thank you for welcoming me with open arms and being wonderful colleagues. We have created some delightful memories, and I am glad to know that I am leaving this lab with some great friends.

Lastly, I’d like to thank my family for always supporting me regardless of what I decided to pursue.

To my parents, I am grateful for your encouragement and love, and emphasizing the value of hard work and perseverance from a young age. To my brother Deepak, thank you for being a wonderful role model, and a great brother. To Jaskaran, my best friend and partner in crime, thank you for all of your love and support, and always believing in me even when I doubted myself. I am thankful for all of you.

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Table of Contents

Abstract…………………………………………………………………………….…………...ii

Acknowledgments…………………………………………………………………………...…iii

Organization of Thesis…………………………………………………………....…………....vi

List of Figures and Tables…………….……………………………………….…...…………vii

List of Abbreviations……………………………………….………………………...……...... ix

Chapter 1: General Introduction……………………………………………………...... 1

Rhodnius prolixus…………………………………………………………………………...... 1

Neuropeptides……………………………………………………………………………….…...2

G Protein-Coupled Receptors (GPCRs) ……………………………………………….….….....4

Kinin……………………………………………………………………………………………..7

CAPA……………………………………………………………………………………...…....11

Post-Feeding Physiology………………………………………………………………...……..13

Diuresis…………………………………………………………………………….…...13

Excretion………………………………....………………………………………….….15

Neuropeptide Analogs………………………………………………………………………….16

Objectives of Thesis……………………………………………………………………………18

References………………………………………………………………………………….…..20

Chapter 2: Physiological Effects of Kinin and CAPA Analogs in the Chagas Disease Vector, Rhodnius Prolixus…………………………………………………….29

Abstract………………………………………………………………………………………...30

Introduction ……………………………………………………………………………………31

Materials and Methods……………………………………………………………………...... 34

Results ……………………………………………………………………………………...... 37

Discussion……………………………………………………………………………….……..60

References…………………………………………………………………………….….…….66

Chapter 3: Identification and Cloning of the Kinin Receptor in the Chagas Disease Vector, Rhodnius Prolixus…………………………………………………….71

Abstract………………………………………………………………………………………...72

Introduction ……………………………………………………………………………………73

Materials and Methods……………………………………………………………...………….77

Results.………………………………………………………………………………………....82

Discussion………………………………………………………………….….……………….98

References……………………………………………………………………………………..104

Chapter 4: General Discussion………………………………………….……………..113

Feeding………………………………………………………………………………………...113

Kinin …………………………………………………………………………………..113

CAPA……………………………...…………………………………………………...115

Myotropic Effects……………………………………………………………………………...115

Kinin…………………………………………………………………………………...115

CAPA....…………………………………….………………………………………….117

Diuretic Effects…………………………………………………...……………………………126

Summary of Physiological Effects of Analogs…………...……………………………………129

References…………………………………………………………………………………...…131

Organization of Thesis

This thesis consists of four chapters. Chapter 1 is a general introduction, providing background information for the proceeding chapters. Chapter 2 is organized as a research article examining the physiological effects of kinin and CAPA analogs and has been submitted to Insect

Biochemistry and Molecular Biology. Chapter 3 is also organized as a research article focusing on the cloning of the R. prolixus kinin receptor, examining its expression profile, and RNAi- mediated knockdown of the receptor transcript. Chapter 4 is a general discussion that discusses the research findings of this thesis, and future directions.

List of Figures and Tables

Chapter 1: General Introduction

Table 1: Amino acid sequences of Rhopr-kinins……………………………………………..…9

Chapter 2: Physiological Effects of Kinin and CAPA Analogs in the Chagas Disease Vector, Rhodnius Prolixus

Table 1: Structure of kinin and CAPA analogs………………………………………………...41

Figure 1: Effects of injection of Rhopr-kinin 2 and kinin analog on in vivo feeding and diuresis………………………………………………………………………………...……….42

Figure 2: Effects of injection of Rhopr-kinin 2 and CAPA analog on in vivo feeding and diuresis…………………………………………………………………………………………44

Figure 3: Effects of Rhopr-kinin 2 and kinin analog on hindgut basal tonus……………...….46

Figure 4: Effects of RhoprCAPA-2 and CAPA analog on hindgut basal tonus…………...….48

Figure 5: Potentiation effects of RhoprCAPA-2 and Rhopr-kinin 2 on hindgut basal tonus…50

Figure 6: Effects of co-application of Rhopr-kinin 2 and CAPA analog on the hindgut …….52

Figure 7: Potentiation effects of 5-HT and CAPA analog on the frequency of hindgut contractions…………………………………………………………………………………….54

Figure 8: Effects of co-application of 5-HT and RhoprCAPA-2 on the hindgut………….…..56

Figure 9: Effects of CAPA analog on Malpighian tubule secretion…………………………..58

Chapter 3: Identification and Cloning of the Kinin Receptor in the Chagas Disease Vector, Rhodnius Prolixus

Figure 1: RhoprKR cDNA sequence and exon map…………………………………………..86

Figure 2: Multiple sequence alignment of invertebrate kinin receptors and R. prolixus receptors……………………………………………………………….…………………...... 88

Table 1: Identity within transmembrane domains of invertebrate kinin receptors…………….91

Figure 3: Expression profile of the RhoprKR transcript…………………………………...... 92

Figure 4: Effects of RNAi-mediated RhoprKR knockdown on hindgut contractions………...94

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Figure 5: Effects of RNAi-mediated RhoprKR knockdown on in vivo feeding and diuresis……………………………………………………………………………………...….96

Supplemental Figure 1: Knockdown efficiency of RhoprKR in dsRhoprKR R. prolixus insects…………………………………………………………………………………………109

Table S1: Primers used for amplification of RhoprKR cDNA fragments…………………………………………………………………………….…….….109

Table S2: 5’ and 3’ RACE primers for RhoprKR……………………………………………110

Table S3: 5’ and 3’ primers used for dsRNA synthesis………………………………....…...110

Table S4: Primers used for qPCR analysis of the RhoprKR transcript……………………....111

Chapter 4: General Discussion

Figure 1: Model of kinin, CAPA, and 5-HT receptor activation……………………………..120

Figure 2: Model showing the intracellular interactions between RhoprCAPA-2 and Rhopr-kinin 2/5-HT…………………………………………………………………………………………122

Figure 3: Model showing the intracellular interactions between CAPA analog and Rhopr-kinin 2/5-HT……………………………………………………………………….………………...124

Figure 4: Summary of physiological effects of kinin and CAPA in the alimentary canal of R. prolixus………………………………………………………………………………………...127

Table 1: Summary of future directions………………………………………………...……...130

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List of Abbreviations

5-HT: Serotonin (5-hydroxytryptamine)

AC: Adenyl cyclase

ACE: Angiotensin converting enzyme

ACN: Acetonitrile

Aib: Alpha-aminoisobutyric acid

AMG: Anterior midgut

ANOVA: Analysis of variance

ARG: Ampicillin resistance gene

ATP: Adenosine triphosphate

BLAST: Basic local alignment search tool

Ca2+: Calcium

CaCl2: Calcium chloride cAMP: Cyclic adenosine monophosphate

CAP: Cardioactive peptide

CCAP: Crustacean cardioactive peptide cDNA: Complementary DNA cGMP: Cyclic guanosine monophosphate

CHS: Chitin synthase

CNS: Central nervous system (CNS),

CRF/DH: Corticotropin-releasing factor-like diuretic hormone

CRZ: Corazonin

DH31: Calcitonin-like diuretic hormone dsRNA: Double-stranded RNA

DV: Dorsal vessel

EC: Extracellular loop ix

ER: Endoplasmic reticulum

FB: Fat body

FG: Foregut

GDP: Guanosine diphosphate

GPCR: G protein-coupled receptor

GTP: Guanosine triphosphate

HEK293/CNG: Human embryonic kidney cells stably expressing a cyclic nucleotide gated channel

HG: Hindgut

HPLC: High-performance liquid chromatography

IC: Intracellular Loop

IP3: Inositol triphosphate

KCl: Potassium chloride

MgCl2: Magnesium chloride

MT: Malpighian tubules

MTGM: Mesothoracic ganglionic mass

NaCl Sodium choloride

NaHCO3: Sodium bicarbonate

NEP: Neprilysin

ORF: Open reading frame

PBS: Phosphate-buffered saline

PIP2: Phosphatidylinositol 4,5-bisphosphate

PK: Pyrokinin

PLC: Phospholipase C

PMG: Posterior midgut

PVK: Periviscerokinin

x qPCR: Quantitative polymerase chain reaction

RACE: Rapid amplification of CDNA ends

RNAi: RNA Interference

SEM: Standard error of measure

SG: Salivary glands

SOG: Suboesophageal ganglion

TFA: Trifluoroacetic acid

TM: Transmembrane domains

UTR: Untranslated region

Chapter 1 General Introduction Rhodnius prolixus

Rhodnius prolixus is a blood-gorging hemipteran of the Reduviidae family, domestic to

Central and South America (Dujardin et al., 1998; Buxton, 1930). Within the R. prolixus life cycle, five nymphal stages (or instars) and a final adult stage exist, with the blood meal serving as a requirement for successful moulting to occur between each stage and for reproductive processes

(Wigglesworth, 1934). This requirement of a blood meal to initiate developmental and reproductive processes made R. prolixus an essential model insect in the very origins of insect physiology and endocrinology (Davey, 2007). R. prolixus only requires one blood meal for the transition through each life stage, surviving for months between feeds (Uribe, 1926; Wigglesworth,

1934). The transition period between each stage is predictable, during which essential physiological processes of growth, development, ecdysis and reproduction can be thoroughly examined (Azambuja et al., 2017). Native R. prolixus can be divided into two populations: domiliciary, and sylvan. Sylvan populations tend to exist within palm tree leaves and pteridophytes and are often found hidden with various species such as mammals, marsupials, and reptiles (Davey,

2007). The domiciliary population is more closely associated with humans, existing in damp, dark spaces within houses, and tend to feed at night on humans or domestic animals (Davey, 2007;

Garcia et al., 2007). During feeding, R. prolixus consumes a blood meal that can be up to 8-10 times it’s body weight. This feeding strategy requires a tight regulation of osmotic balance, which is subject to control by the neuroendocrine system (Orchard, 2006; Coast et al., 2002).

From a medical perspective, R. prolixus is an organism of interest, as it is a carrier of the

Trypanosoma cruzi parasite, making it a vector of Chagas disease (Dujardin et al., 1998; Moncayo,

2 2003). Rhodnius prolixus is one of 12 Triatominae species that act as a vector for the T. cruzi parasite (Schofield, 1988). Chagas disease, originally discovered by the Brazilian physician Carlos

Chagas, is principally found in the continental part of Latin America but has been recently detected in the U.S, and Canada (WHO, 2019; Steverding, 2014). Six to seven million people worldwide are infected with the T. cruzi parasite, with most cases in Latin America (WHO, 2019). Chagas disease can be divided into an acute and chronic phase based on its symptoms. In the chronic phase, progressive heart failure is caused by the deterioration of heart muscle and the nervous system

(WHO, 2019; CDC, 2019). Within R. prolixus, T. cruzi exists in the highly infectious life stage within the hindgut and is expelled during excretion after diuresis (Bern et al., 2011). Following this excretion, T. cruzi can enter the host through bite wounds, or through mucous membranes

(CDC, 2019). Since there is no vaccine for Chagas disease, the most effective method of preventing the spread of this disease is vector control (WHO, 2019). It is essential to examine the mechanisms by which T. cruzi is spread by R. prolixus, which in turn will aid in the development of strategies to decrease the spread of R. prolixus, and thereby the spread of Chagas disease.

Neuropeptides

Within nervous systems, there are several types of chemical messengers that function to direct various essential processes that are critical for normal growth and development (Altstein &

Nässel, 2010). Amongst these chemical messengers are neuropeptides, that are synthesized and released from neurons or neuroendocrine cells (Yeoh et al., 2017). They can function as neuromodulators, neurotransmitters, or neurohormones (Orchard, 2009). This diversity in the mode of action by which neuropeptides can function allows them to govern many physiological processes. As neurotransmitters, their release is localized to the synaptic cleft, initiating rapid changes via receptors on post-synaptic membranes. The effects of neurotransmitters are short term

3 as they are subject to degradation, diffusion, and reuptake from the synaptic cleft (Orchard, 2009).

As neurohormones, their signal is much more global as they are released from neurosecretory cells into the circulatory system and act upon peripheral target tissues that express the receptor. These effects are longer lasting since neurohormones typically function through G protein-coupled receptors (GPCRs) resulting in downstream effects. Neuropeptides that do not fit within these specified roles are termed neuromodulators, which includes any neuroactive compound that modulates its target (Orchard, 2009; Schoofs et al., 2017). Bioactive neuropeptides are synthesized from larger precursor molecules, known as prepropeptides. They are then targeted via the regulatory secretory pathway to intracellular electron dense granules, where they are stored until secretion (Elphick et al., 2018). Prepropeptides comprise of a signal peptide, propeptides (which later become mature peptides), spacer peptides, and cleavage sites (Yeoh et al., 2017). The signal peptide directs the prepropeptide to the secretory pathway, with monobasic and dibasic cleavage sites surrounding the mature peptides, which are targets for various neuropeptidases (Yeoh et al.,

2017; Veenstra, 2000). Following processing, the neuropeptides can also be subject to post- translational modifications, such as C-terminal amidation. (Yeoh et al., 2017). Mature neuropeptides function by being released from granules and then typically binding onto specific

GPCRs on the cell membranes of target cells, initiating further downstream signaling pathways

(Elphick et al., 2018).

Within insects, and indeed other animals, neuropeptides represent the largest class of chemical compounds involved in physiological processes such as development, reproduction, metabolism, and behaviour (Altstein & Nässel, 2010). The first insect neuropeptide to be identified was proctolin in Periplaneta americana, and there are currently approximately 50 identified neuropeptide families in insects (Yeoh et al., 2017). Genes can encode for prepropeptides that vary

4 in the number of mature peptides, and in some cases the mature peptides may be structurally and functionally distinct (Yeoh et al., 2017) An example of this is the Drosophila capability gene, which encodes for two CAPA neuropeptides, and a third pyrokinin neuropeptide (Kean et al.,

2015).

G Protein-Coupled Receptors (GPCRs)

Neuropeptides function on their target tissues by binding onto their specified GPCRs, resulting in the activation of a second messenger cascade (Grimmelikhuijzen & Hauser, 2012).

GPCRs, which comprise the largest group of membrane receptors, all share a similar structure which includes an intracellular C-terminus, an extracellular N-terminus, and seven hydrophobic transmembrane domains (TM1-TM7) (Gether, 2000; Pierce et al., 2002). These membrane domains are linked by intracellular (ICL1-ICL3) and extracellular loops (ECL1-ECL3) (Gether,

2000). GPCRs can bind a variety of ligands, including peptides, biogenic amines, lipids, and proteases (Gether, 2000). They can be classified into six classes (class A-F), with the possible ligands varying within these classes. The rhodopsin-like (class A) family, which is the largest and most studied family of GPCRs, includes receptors for various small molecules, neurotransmitters, peptides and hormones (Munk et al., 2016). Some conserved regions of family A GPCRs include the Asp-Arg-Tyr (DRY) motif, which is located on the intracellular side of TM3, and the Asp-Pro- xx-Tyr (NPxxY) domain which is located within TM7 (Munk et al., 2016). The DRY (sometimes

DRH or ERY) motif is critical in inducing conformational changes that are required for receptor activation, while the NPxxY domain is implicated in maintaining structural integrity (Fritze et al.,

2003; Capra et al., 2004; Rovati et al., 2007).

5 GPCRs are first synthesized within the endoplasmic reticulum (ER), and are then transported to the cell membrane, which is its final target. During this transport, GPCRs undergo various post-translational modifications to ensure biological activity (Duvernay et al., 2005).

GPCRs are often subject to phosphorylation within various sites at the C-terminus and intracellular loops by protein kinases, which is essential for various mechanisms within GPCRs, such as desensitization and internalization of the receptor (Ferguson, 2001; Tobin, 2008; Yang et al.,

2017). GPCRs are also targets of N-glycosylation, which is critical for trafficking and expression to the cell surface and overall function (Michineau et al., 2005; Chen et al., 2010). Palmitoylation, which is a lipid modification, involves the modification of Cys residues within the intracellular loops, and C-terminus of GPCRs through the addition of a palmitic acid (Qanbar & Bouvier, 2003;

Goddard & Watts, 2002). This post-translational modification modulates various aspects of GPCR function (Goddard & Watts, 2002).

GPCRs are activated through the binding of ligands, involving the function of heterotrimeric G-proteins, which are a family of proteins made up of an α, β, and γ subunit.

Heterotrimeric G-proteins can be further divided into classes, depending on the type of α subunit.

Some examples are Gq, Gs, and Gi/o (Hamm, 1998; Caers et al., 2012). Gq induces the activation of phospholipase C (PLC), activating the inositol triphosphate (IP3) pathway. This causes the

2+ intracellular release of IP3 which binds onto an IP3 sensitive Ca channel on the endoplasmic reticulum and causing Ca2+ release. Ca2+ functions as a second messenger to further initiate various downstream effects. Gs/Gi/o G-proteins are associated with adenyl cyclase (AC), an enzyme responsible for the synthesis of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) (Sadana & Dessauer, 2009; Caers et al., 2012). Gs is known to activate AC, while Gi/o inhibits it (Hamm, 1998; Caers et al., 2012). Activation of the GPCR induces a

6 conformational change with its associated intracellular G-protein subunit, releasing guanosine diphosphate (GDP), followed by the binding of guanosine triphosphate (GTP) to the α subunit of the G-protein (Munk et al., 2016). The GTP-bound α subunit then dissociates from the receptor, causing the release of the β-γ dimer. Both the GTP-bound α subunit and β-γ dimer can interact with various intracellular mechanisms, eventually leading to the activation or inhibition of signaling pathways. Investigating the structure and function of GPCRs provides value in the development of next-generation pesticides, whereby neuropeptide GPCRs can be targeted in order to disrupt critical processes within relevant species (Audsley & Down, 2015).

GPCRs have been observed to elicit their intracellular changes even in the absence of ligand binding, known as constitutively active GPCRs. GPCR activity can be described by the two-state model of GPCR activation, where GPCRs exist in two separate states, an inactive state

(R), and an active state (R*) (Seifert, 2002). The transition from R to R* state involves a conformational change within the GPCR, which is induced by an agonist (Seifert, 2002). In constitutively active GPCRs, transition to the R* state occurs independent of an agonist, stably inducing basal G protein activity and its associated downstream effects (Steifert, 2002). Ligands can function as full agonists that induce maximal activity of the constitutively active GPCR, or as partial agonists that maintain the R* state of the GPCR to a lesser degree (Steifert, 2002). Inverse agonists function to maintain the GPCR in its R state, in turn reducing basal G protein activity

(Steifert, 2002). Like agonists, inverse agonists can also function as full or partial inverse agonists

(Steifert, 2002). GPCR mutants causing constitutive activity or loss of constitutive activity have been implicated in various diseases and are often used as pharmacological targets in drug development (Chalmers & Behan, 2002).

7 Kinin

The kinin (or leucokinin) family of cephalomyotropic peptides was first identified through high-performance liquid chromatography (HPLC) of head extracts of the cockroach, Leucophaea maderae, and are known for their ability to stimulate hindgut contractions (Holman et al., 1986a, b; Holman et al., 1987a, b). Following this, kinin-like neuropeptides were discovered in various insect species, all sharing the C-terminal pentapeptide sequence FX1X2WG- amide where X1 can be Ser, Phe, His, Asn, or Tyr and X2 can be Ser, Pro or Ala (Torfs et al., 1999). This core pentapeptide sequence is essential for the biological activity of the neuropeptide (Nachman et al.,

1991; Coast et al., 1990). Kinins have been identified in various arthropods, with multiple isoforms often being present within these species (Coast et al., 2002). Kinins also stimulate fluid secretion from isolated Malpighian tubules (MTs) of Aedes aegypti females, suggesting a secondary diuretic role of these neuropeptides (Hayes et al., 1989). In addition to their myotropic and diuretic effects, kinins have also been implicated in a diverse set of functions, such as ecdysis-related behaviours in Manduca sexta (Kim et al., 2006), and more recently, locomotor activity and metabolic rate in

Drosophila melanogaster (Zandawala et al., 2018).

Within R. prolixus, the Rhopr-kinin transcript encodes eighteen predicted kinins and precursor associated peptides, the most found in any species (Table 1) (Te Brugge et al., 2011;

Bhatt et al., 2014). As seen in other species, Rhopr-kinins are primarily known for stimulating hindgut contractions, with Rhopr-kinin 2 producing a potent effect (Bhatt et al., 2014). Kinins have also been shown to have myotropic effects on the anterior midgut and salivary glands in R. prolixus

(Orchard & Te Brugge, 2002; Te Brugge et al., 2009). To observe the distribution of kinins within

R. prolixus, immunohistochemical analyses were performed, with kinin-like immunoreactive staining observed in cell bodies and processes within the central nervous system (CNS), gut,

8 peripheral nerves, and peripheral neurons (Te Brugge et al., 2001). Kinins may also be implicated in feeding-related behaviors within R. prolixus, due to their co-localization with the corticotropin- releasing factor (CRF)-like diuretic hormone (Rhopr-CRF/DH). Rhopr-CRF/DH has been shown to influence feeding as injection of Rhopr-CRF/DH into 5th instar insects resulted in the intake of significantly smaller blood meals (Mollayeva et al., 2018). Kinin-like and CRF-like staining is observed within the CNS, and endocrine cells of the midgut of R. prolixus, with a decrease in staining observed in neurosecretory cells up to 2.5 hours after feeding. Levels of the kinin-like and

CRF-like staining are restored 1 day after feeding (Te Brugge et al., 1999; Te Brugge et al., 2001;

Te Brugge et al., 2002, Mollayeva et al., 2018).

The first kinin GPCR, belonging to the family A of GPCRs, was characterized in the snail,

Lymnaea stagnalis (Cox et al., 1997). Since then, kinin receptors have been functionally characterized in a small number of insect species including D. melanogaster, A. aegypti, and

Anopheles stephensi (Radford et al., 2002; Radford et al., 2004; Pietrantonio et al., 2005). It is hypothesized that all invertebrate kinin receptors may utilize intracellular Ca2+ as a second

2+ messenger, as release of intracellular Ca through activation of the IP3 pathway was observed in

L. Stagnalis, D. melanogaster, and A. aegypti (Cady and Hagedorn, 1999; Radford et al., 2002;

Pietrantonio et al., 2005). Currently, the kinin GPCR has not yet been characterized within R. prolixus, therefore its downstream signaling pathway is unknown.

Table 1: Amino Acid sequences of Rhopr-kinins (K) processed from the kinin-precursor. Table modified from (Te Brugge et al., 2011)

Neuropeptide name Neuropeptide sequence K-1 TNNRGNFAGNPRMRFSSWAa K-2 AKFSSWGa K-3 ANKFSSWAa K-4 AKFSSWAa K-5 DEDRQKFSHWAa K-6 GAKFSSWAa K-7 AKFNSWGa K-8 LSINPWKKIDDNGa K-9 AKFSSWGa K-10 ADDDWLKKARFNSWGa

K-109–15 ARFNSWGa K-11 SAAAYTPLSWKRKPIFSSWGa

K-111–11 SAAAYTPLSW-OH

K-1113–20 KPIFSSWGa

K-1114–20 PIFSSWGa K-12 RGPDFYAWGa

K-122–9 GPDFYAWGa KPP-6 FSNEFMNDNNDIEKNIVEE-OH

11 CAPA

CAPA neuropeptides, a family of neuropeptides belonging to the PRXamide superfamily are characterized by their consensus carboxyl terminal sequence A/PFPRV-NH2 (Paluzzi, 2012;

Alford et al., 2019a). The first CAPA neuropeptide was originally isolated in ventral nerve chord extracts of M. sexta (Huessman et al., 1995). Initially named CAP2b, this neuropeptide was found to increase heart rate in M. sexta, hence it’s designation as a cardioactive peptide (CAP). CAP2b is one of the many cardioactive peptides isolated from M. sexta (CAP1a, CAP1b, CAP2a, CAP2b), but shares no sequence homology, making it a novel neuropeptide (Tublitz & Truman, 1985a–d;

Huessman et al., 1995). CAP2b was also found to have diuretic effects, as it increased fluid secretion within D. melanogaster (Huessman et al., 1995). Separately from CAP2b, a myotropic neuropeptide was isolated from the perisympathetic organs of P. americana, named periviscerokinin (Pea-PVK) (Predel et al., 1995). Due to structural similarities between CAP2b and periviscerokinin, they can be grouped together into the CAPA family of neuropeptides (Predel &

Wegener, 2006). Following sequencing of the CAPA gene in M. sexta, CAP2b was later named

ManseCAPA-1 (Loi & Tublitz, 2004). In D. melanogaster, a gene named capability was identified that encoded two ManseCAPA-1 related peptides (capa-1, capa-2), and a third pyrokinin-related neuropeptide (Kean et al., 2015). Like ManseCAPA-1, capa-1 and capa-2 also stimulated MT fluid secretion within D. melanogaster (Kean et al., 2015).

As seen in other insects, the CAPA neuropeptides within R. prolixus are encoded by the

RhoprCAPA transcript, named RhoprCAPA-1 (SPISSVGLFPFLRA-NH2), RhoprCAPA-2

(EGGFISPRV-NH2), and Rhopr-pk1 (NGGGGNGGGLWFGPRL-NH2) (Paluzzi et al., 2008).

RhoprCAPA-1 lacks the characteristic PRVamide sequence found in other CAPA neuropeptides

(Paluzzi et al., 2008). A second CAPA gene paralog was identified in R. prolixus that shares a very

12 high sequence similarity with the first RhoprCAPA transcript, named RhoprCAPA-β (Paluzzi &

Orchard, 2010). The CAPA neuropeptides encoded by RhoprCAPA-β are named RhoprCAPA-

β1, RhoprCAPA-β2, and RhoprCAPA-βpk1. As a second gene paralog for the RhoprCAPA transcript was identified, the first transcript is referred to as RhoprCAPA-α, with the encoded neuropeptides renamed to the following: RhoprCAPA-α1, RhoprCAPA-α2, and Rhopr-αpk1

(Paluzzi et al., 2009). CAPA neuropeptides are distributed in various neurons within the nervous system of R. prolixus, including the brain, suboesophageal ganglion (SOG), prothoracic ganglion, and in the abdominal neuromeres of the mesothoracic ganglionic mass (MTGM) (Paluzzi et al,

2006; Paluzzi & Orchard, 2010; Paluzzi et al., 2008). RhoprCAPA-α2 was identified as the first anti-diuretic hormone within R. prolixus, as it inhibited serotonin [5-hydroxytryptamine (5-HT)] stimulated effects on the MT, and anterior midgut (Paluzzi et al., 2006; Ianowski et al., 2009). The effects of RhoprCAPA-α2 may be related to post-feeding diuresis, as immunohistochemical staining of three pairs of neurosecretory cells within the abdominal neuromeres is significantly decreased three to four hours post-blood meal (Paluzzi et al., 2006).

CAPA receptors, belonging to family A GPCRS, have been characterized in insects such as D. melanogaster, A. gambiae, and Tribolium castaneum (Iversen et al., 2002; Olsen et al., 2007;

Jiang et al., 2015). The R. prolixus CAPA receptor (capa-r) has been characterized, and it was identified as the first antidiuretic hormone receptor in insects, with cyclic guanosine monophosphate (cGMP) likely acting as a second messenger (Paluzzi et al., 2010). Expression of capa-r is highest in the MTs and anterior midgut of R. prolixus, which are known targets of

RhoprCAPA-α2 (Paluzzi et al., 2010). Expression of capa-r has also been confirmed on the hindgut, suggesting a role of CAPA neuropeptides on this tissue (Paluzi et al., 2010). Two transcript variants of capa-r were identified, named capa-r1 and capa-r2, however capa-r2

13 encodes an atypical GPCR (Paluzzi et al., 2010). As RhoprCAPA- α1 lacks the characteristic

PRVamide sequence, it is unable to activate the capa-r1 receptor (Paluzzi et al., 2010).

Post-Feeding Physiology

Feeding within R. prolixus has been a widely studied phenomenon, due to the dependence of a blood meal to successfully transition to the next instar and to initiate many crucial developmental and reproductive processes. The feeding strategy of R. prolixus can add a great deal of osmotic stress, as feeding on such a large blood meal results in the consumption of excess water and salts. This large blood meal also causes a large increase in the body weight of the insect, leaving it in a state of susceptibility to predation (Orchard, 2006; Orchard, 2009). The blood meal serves as a signal to initiate short-term endocrinological and physiological changes to lower the insect’s mass and return to a homeostatic state (Maddrell, 1976).

Diuresis

Within the digestive tract of R. prolixus, the anterior midgut, upper and lower MTs are the tissues primarily responsible for the rapid absorption and secretion of water and salts, resulting in the production of primary urine (Coast et al., 2002; Maddrell, 1969). Once blood has entered the midgut, absorption of water and NaCl occurs from the anterior midgut into the haemolymph.

Following this, the upper MTs secrete a fluid containing a high NaCl and KCl content. The final modification of this fluid occurs by the lower MTs, which reabsorb KCl, resulting in the production of primary urine (Orchard, 2009). This ionic movement that occurs within the anterior midgut and

MTs is fast-acting, occurring within minutes following feeding (Maddrell, 1976; Orchard, 2009).

The cells in the epithelium of MTs within R. prolixus have been described as “the fastest fluid

14 secreting cells known”, further emphasizing how quickly post-feeding diuresis occurs to rid the insect of excess fluids (Maddrell, 1991).

The absorption and secretion mediated by the anterior midgut and MTs occurs in a highly timed and coordinated manner, and these tissues are under direct control by a host of diuretic and antidiuretic factors (Coast et al., 2002). The diuretic neurohormones 5-HT, CRF/DH, and calcitonin-like diuretic hormone (Rhopr-DH31) are primarily responsible for the diuretic action of the anterior midgut and the MTs (Maddrell et al., 1971; Te Brugge et al., 2011, Te Brugge et al.,

2005), with evidence of their release observed through immunohistochemical staining and analysis of haemolymph composition. There is a reduction in 5-HT-like immunoreactive staining in central and peripheral tissues during feeding in 5th instar insects, with a rapid rise in serotonin within the haemolymph (Lange et al., 1988; Orchard et al., 1989). A decrease of CRF-like and kinin-like immunoreactive staining in neurosecretory cells is seen up to 2.5 hours after feeding, with levels being restored 1 day after feeding (Te Brugge et al., 1999; Te Brugge et al., 2001; Te Brugge et al., 2002; Mollayeva et al., 2018). In addition, there is a reduction in DH31-like staining within nerve processes post-feeding (Te Brugge et al., 2005). These reductions in immunoreactive staining post-feeding is likely due to the release of these neurohormones during post-feeding diuresis.

5-HT increases the rate of fluid transport across the anterior midgut and increases the frequency of contractions in both in vitro and in vivo studies within 5th instar R. prolixus (Te

Brugge et al., 2009; Barrett et al., 1993). Within the MTs, 5-HT has been shown to stimulate upper

MT fluid secretion and stimulates reabsorption within the lower MTs (Te Brugge et al., 2009;

Donini et al., 2008). In the upper and lower MTs, 5-HT also increases cAMP content, which

15 suggests that cAMP is acting as a second messenger to facilitate this process (Te Brugge et al.,

2002). When tested on the anterior midgut, Rhopr-DH31 failed to have any effects on fluid absorption but was found to increase the frequency of contractions and cAMP levels (Te Brugge et al., 2009). Within the MTs, Rhopr-DH31 only induces minor increases in fluid secretion in the upper MTs but has no effect on the lower MTs (Te Brugge et al., 2002; Donini et al., 2008). Rhopr-

CRF/DH stimulates fluid transport and has myotropic effects within the anterior midgut, and stimulates upper MT fluid secretion (Te Brugge et al., 2002; Te Brugge et al., 2009). However, within the lower MTs, Rhopr-CRF/DH does not have any effects on reabsorption (Donini et al.,

2008). An increase in cAMP levels is also exhibited within these tissues following treatment with

Rhopr-CRF/DH (Te Brugge et al., 2002). Within the MTs, it is suggested that there is a synergistic interaction between Rhopr-CRF/DH and 5-HT to increase fluid secretion rates, highlighting the coordination required during post-feeding diuresis (Maddrell et al., 1971; Barrett & Orchard,

1990). Following the production of primary urine, the termination of diuresis occurs to prevent any further fluid loss (Maddrell, 1964). As found within diuresis, this termination is under the control of the anti-diuretic hormone, RhoprCAPA-2 (Paluzzi et al., 2008; Orchard & Paluzzi,

2009). RhoprCAPA-2 inhibits 5-HT stimulated fluid secretion in the upper MTs, and inhibits 5-

HT-stimulated fluid absorption in the anterior midgut (Paluzzi, 2006; Paluzzi et al., 2008; Ianowski et al., 2009)

Excretion

Following the production of primary urine, the final step occurs within the hindgut, whereby contractions of the hindgut result in excretion of this urine (Maddrell, 1964). In addition to its role in post-feeding diuresis, the hindgut is a tissue of epidemiological relevance, as the T. cruzi parasite exists in its highly infective stage in the hindgut, and so hindgut contractions result

16 in the transmission of the parasite along with urine onto the host (Bern et al, 2011). The myotropic action of the hindgut is under control by many neuroendocrine factors such as 5-HT, Rhopr-DH31,

Rhopr-CRF/DH, kinins and tachykinins (Te Brugge et al., 2002; Bhatt et al., 2014; Haddad et al.,

2018). As seen in the MTs, interactions between different neuropeptide families within the hindgut has been observed, with kinins, tachykinins and CRF/DH exhibiting additive or co-operative effects on hindgut contraction stimulation (Bhatt et al., 2014; Haddad et al., 2018).

Neuropeptide Analogs

From an agrochemical and medical perspective, neuropeptide signaling is a field of interest, as many critical physiological processes and behaviors are under direct control of neuropeptides.

They can be used in insecticidal strategies to interfere with the normal functioning of pests and disease vectors, thereby preventing the detrimental effects of these insect species (Gäde &

Goldsworthy, 2003). As many insects have developed resistance to many traditional insecticides, neuropeptides provide a promising alternative to combat particularly damaging species (Nachman

& Smagghe, 2011). Neuropeptides have been studied as lead compounds in the development of more environmentally friendly pest control strategies, due to their specificity and activity at low concentrations (Nachman & Smagghe, 2011). Some of the limitations with the use of native neuropeptides is the susceptibility to a variety neuropeptidases within the insect’s haemolymph and tissues, which results in the inactivation of the neuropeptide, reducing its bioavailability. In addition, neuropeptides in their native form are unable to penetrate an insect’s exoskeleton, thus severely limiting the possible forms of delivery (Isaac et al., 2009; Menn & Bořkovec, 1989). The study of neuropeptides, their receptors and the biochemical features required for successful interaction has allowed the development of compounds that function as agonists or antagonists to specific neuropeptide receptors (Menn & Bořkovec, 1989; Keeley & Hayes, 1987). These

17 compounds, known as neuropeptide analogs, are synthesized with modifications to their amino acid sequence to overcome the limitations associated with native neuropeptides, so they can be successfully used in pest control strategies (Nachman, 2009). Various analogs of many well- studied neuropeptide families have been synthesized (eg. kinins, CAPA) and have been examined for their effects on tissues within several insect species (Bhatt et al., 2014; Lange et al., 2016;

Smagghe et al., 2010; Alford et al., 2019a; Alford et al., 2019b)

Within kinins, the C-terminal pentapeptide core region between the Ser (or Pro) and conserved Trp residues is susceptible to primary hydrolysis. A secondary site of hydrolysis is also found outside of the core region, at the neuropeptide bond N-terminal to Phe (Nachman et al.,

1997a; Nachman et al., 1997b). The fly angiotensin converting enzyme (ACE) can cleave the primary hydrolysis site, with neprilysin (NEP) cleaving the primary and secondary hydrolysis sites.

Replacement of the Ser (or Pro) with an Aib residue blocks ACE or NEP hydrolysis, while mimicking a critical conformation required for activity (Nachman et al., 1997a; Nachman et al.,

1997b; Nachman et al., 2002; Xiong et al., 2018). The Aib-kinin analog induces potent changes in physiology, and in some cases disrupt essential processes. Within the aphids Myzus persicae and

Macrosiphum rosae, decreased survival was exhibited under cold stress exposure after kinin analog treatment. In Acyrthosiphon pisum, the kinin analog induced antifeedant activity and high mortality (Smagghe et al., 2010). Aib-containing kinin analogs also induced physiological changes within R. prolixus, as they were found to have antifeedant effects, and disrupted ecdysis (Lange et al., 2016). Aib-containing analogs had potent myotropic effects in R. prolixus, as they induced stronger hindgut contractions than native Rhopr-kinins (Bhatt et al., 2014).

18 More recently, analogs from the PRXamide family of neuropeptides have been synthesized, with addition of hydrophobic moieties to the N-terminus to increase greater in vivo stability (Jurenka, 2015; Zhang et al., 2011). Second generation analogs for CAPA neuropeptides have also been synthesized with steric hinderances adjacent to the C-terminal position, thus biasing it’s binding to CAPA receptors, since cross-reactivity has been exhibited on CAPA receptors with

CAPA and pyrokinin neuropeptides (Jiang et al., 2015; Paluzzi et al, 2010; Paluzzi & O’Donnell,

2012). Within D. melanogaster and D. suzukii, flies microinjected with a CAPA analog had an increased survival rate under desiccation stress (Alford et al., 2019a). In the aphids M. persicae and M. rosae, the CAPA analogs accelerated aphid mortality under desiccation and starvation stress, with the analog causing enhanced mortality under cold stress in M. persicae (Alford et al.,

2019b). The ability of these neuropeptide analogs to disrupt physiological processes, and in some cases cause mortality within insects highlights their promise in pest control strategies. It is imperative to continue investigating the use of neuropeptide analogs to prevent the further spread of pests and disease vectors.

Objectives of Thesis

Within R. prolixus, feeding and diuresis are epidemiologically-relevant behaviours, as they are implicated in the transmission of the T. cruzi parasite. Previous studies have investigated the role of kinins and CAPA, with Rhopr-kinins primarily exerting myotropic effects on target tissues, and RhoprCAPA-2 functioning as an anti-diuretic hormone. Kinin and CAPA neuropeptides will be further investigated on feeding and diuresis-related tissues, which will provide insight into their roles as neuroendocrine factors in R. prolixus. Neuropeptide analogs have been developed for

Rhopr-kinin and RhoprCAPA-2, that may influence processes related to disease transmission.

These analogs will be investigated for their efficacy in inducing changes in physiology, to

19 determine whether they are lead compounds for the development of pesticides. The physiological approaches to investigate these neuropeptides and analogs are listed below:

• Analyze the in vivo effects of injected kinin, CAPA, and their analogs on feeding and

post-feeding diuresis.

• Determine the effects of Rhopr-kinin 2, the Aib-containing kinin analog 2139[Ф1]wp-

2, CAPA neuropeptides, and CAPA analog 2129-SP3[Ф3]wp-2 on the hindgut of R.

prolixus.

• Determine the effects of 2129-SP3[Ф3]wp-2 on fluid secretion in the MTs of R.

prolixus.

Currently, the R. prolixus kinin receptor has not yet been characterized. Identifying, cloning, and sequencing the Rhopr-kinin receptor will aid in the development of next-generation pest control strategies. The molecular approach to characterizing this receptor is listed below:

• Isolate, clone and sequence the Rhopr-kinin GPCR.

• Analyze and predict the biochemical features and structural characteristics of the

kinin GPCR using online tools.

• Determine the spatial expression of the kinin receptor transcript within the CNS and

tissues involved in feeding, diuresis and excretion.

• Target the kinin receptor through RNA interference (RNAi) and examine effects of

knockdown through hindgut contraction assays and feeding bioassays.

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Chapter 2 Physiological Effects of Biostable kinin and CAPA Analogs in the Chagas Disease Vector, Rhodnius prolixus

30 Abstract

In the Chagas disease vector Rhodnius prolixus, the kinin and CAPA family of neuropeptides are implicated in feeding and diuresis-related behaviours, with Rhopr-kinins stimulating contractions of the midgut, salivary glands, and hindgut, with RhoprCAPA-2 functioning as an anti-diuretic hormone. The current study examined the effects of kinin and

CAPA neuropeptides and their analogs on feeding and diuresis, and on hindgut contractions and

MT fluid secretion in R. prolixus. The biostable Aib-containing kinin analog 2139[F1]wp-2 was found to have antifeedant effects, and to be more potent than Rhopr-kinin 2 in stimulating hindgut contractions. The CAPA analog 2129-SP3[F3]wp-2 induced the intake of a larger blood meal, and increased the rate of post-prandial rapid diuresis. RhoprCAPA-2, but not its analog, potentiated hindgut contractions induced by Rhopr-kinin 2. Potentiation was observed with the CAPA analog on 5-HT-stimulated increases in frequency of hindgut contractions, whereas RhoprCAPA-2 inhibited this 5-HT-mediated stimulation. The CAPA analog induced hindgut contractions and prevented the inhibition induced by RhoprCAPA-2 on 5-HT-stimulated MT secretion. These results demonstrate novel interactions between Rhopr-kinin and RhoprCAPA-2 on the hindgut, possibly influencing post-feeding excretion. The kinin analog is a potent agonist of the kinin receptor, and the CAPA analog an antagonist of the CAPA receptor. The use of neuropeptide mimetics is a promising approach to vector control as they can disrupt behaviours, and the effects of these neuropeptide analogs highlight their value as lead compounds, given their ability to interfere with epidemiologically-relevant behaviours.

31 Introduction

The blood-gorging hemipteran Rhodnius prolixus is a domestic vector of Chagas disease in Central and South America, caused by the transmission of Trypanosoma cruzi, a flagellate parasite (Moncayo, 2003; Dujardin et al., 1998). R. prolixus consumes a blood meal that can be up to 10 times its body weight during each instar, following which the insect rids itself of the excess water and salt from the blood meal. The transmission of the parasite to its host occurs during this post-prandial diuresis, and so an understanding of the physiology of feeding and diuresis-related behaviours is essential in understanding disease transmission (Orchard, 2006; Orchard & Paluzzi,

2009). The systems controlling diuresis include the Malpighian tubules (MTs), midgut and hindgut, which are subject to control by various diuretic and antidiuretic hormones and myotropic factors (Coast et al., 2002; Orchard, 2006; Orchard & Paluzzi, 2009).

Many essential processes within insects are regulated by neuropeptides which act upon target tissues through their release as neurohormones into the haemolymph or as neuromodulators

(Schoofs et al., 2017). Insect kinins, first isolated from Leucophaea maderae head extracts, share the C-terminal pentapeptide sequence FX1X2WG-amide (where X1 can be Ser, Phe, His, Asn, or

Tyr and X2 can be Ser, Pro or Ala) (Holman et al., 1986a, b; Holman et al., 1987a, b). Kinins have been implicated in various behaviours, such as hindgut contraction (Bhatt et al., 2014), MT fluid secretion (O’Donnell et al., 1998; Terhzaz et al., 1999; Rosay et al., 1997), ecdysis (Kim et al.,

2006), and more recently locomotor activity and metabolic rate (Zandawala et al., 2018). The R. prolixus kinin (Rhopr-kinin) transcript encodes for eighteen predicted kinins and precursor associated peptides (Te Brugge et al., 2011a; Bhatt et al., 2014) and these kinins stimulate R. prolixus hindgut, anterior midgut, and salivary gland contractions (Orchard & Te Brugge, 2002;

Te Brugge et al., 2009; Te Brugge et al., 2011; Bhatt et al., 2014). Interestingly, co-localization of

32 Rhopr-kinin has been shown with corticotropin-releasing factor (CRF)-like diuretic hormone

(Rhopr-CRF/DH) within the R. prolixus central nervous system (CNS) and endocrine cells of the midgut. A decrease of CRF-like and kinin-like immunoreactive staining in neurosecretory cells is seen up to 2.5 hours after feeding, with levels being restored 1 day after feeding, suggesting a role of CRF and kinin in feeding-related behaviours (Te Brugge et al., 1999; Te Brugge et al., 2001;

Te Brugge et al., 2002; Mollayeva et al., 2018). In the R. prolixus excretory system, the MTs play a dominant role in the regulation of urine volume and composition (Coast, 2009), primarily under control by the diuretic neurohormones serotonin [5-hydroxytryptamine (5-HT)], calcitonin-like diuretic hormone (DH31), and Rhopr-CRF/DH (Maddrell, 1971; Te Brugge et al., 2005; Te Brugge et al., 2011b), and the antidiuretic hormone R. prolixus CAPA (Rhopr-CAPA) (Orchard & Paluzzi,

2009; Ianowski et al., 2009; Paluzzi et al., 2008). CAPA peptides are encoded by the capability genes, initially identified in Drosophila melanogaster (Kean et al., 2002). The first two CAPA peptides encoded by the gene contain the consensus carboxyl terminal sequence A/PFPRV-NH2, with the third peptide containing the consensus carboxyl terminal sequence G/MWFGPRL-NH2, typically referred to as a pyrokinin (PK)-related peptide (Paluzzi, 2012; Paluzzi et al., 2008).

CAPA peptides were originally discovered in Manduca sexta for their cardioacceletatory effects and display diuretic, anti-diuretic, and myotropic effects in a variety of species (Huesmann et al.,

1995; Predel & Wegener, 2006; Paluzzi et al., 2008). The RhoprCAPA transcript encodes three

CAPA peptides, as found in other species: RhoprCAPA-1, RhoprCAPA-2, and RhoprCAPA-pk1.

RhoprCAPA-2 has been found to inhibit 5-HT-stimulated secretion by the MTs and absorption from anterior midgut (Ianowski et al., 2010; Orchard & Paluzzi, 2009; Paluzzi et al., 2008). The effects of the CAPA peptides are mediated by the RhoprCAPA G protein-coupled receptor

(GPCR) (capa-r) (Paluzzi et al., 2010). Expression of the capa-r transcript has been confirmed in

33 the hindgut of R. prolixus, suggesting that CAPA peptides may influence hindgut contractions

(Paluzzi et al., 2010).

From an agrochemical and medical perspective, neuropeptides and neuropeptide analogs are compounds of interest for the disruption of critical functions as a means of pest control (Jiang et al., 2015). Neuropeptide analogs have been synthesized with a modified chemical structure in order to overcome limitations associated with delivery of the compound (eg. movement through the cuticle, endopeptidases, exopeptidases) (Nachman et al., 1997). In Musca domestica, addition of alpha-aminoisobutyric acid (Aib) to a kinin analog resulted in resistance to hydrolysis by angiotensin converting enzyme (ACE) and neprilysin (NEP) and the analog was found to be potent in inducing myotropic activity in L. maderae hindguts (Nachman et al., 1997). Recently, the kinin analog 2139 was shown to stimulate fluid secretion in D. melanogaster, and significantly reduced survival under desiccation stress (Alford et al., 2019a). Decreased survival was also exhibited in

Myzus persicae and Macrosiphum rosae under cold stress exposure after kinin analog treatment

(Alford et al., 2019b). In R. prolixus, an Aib-containing kinin analog was found to have antifeedant effects, as insects only consumed 60% of a blood meal that contained the analog (Lange et al.,

2016).

The CAPA analog 2129-SP3[F3]wp-2 has been designed with the addition of hydrophobic moieties to the N-terminus to increase greater in vivo stability, and also possesses a steric hinderance adjacent to the alpha carbon in the C-terminal position, directing its binding to a CAPA receptor whilst interfering with any PK receptor binding (Zhang et al., 2011; Jurenka, 2015; Alford et al., 2019a). This CAPA analog also influences desiccation and starvation survival, as it significantly improved D. suzukii desiccation survival, while significantly increasing the

34 desiccation and starvation mortality in M. persicae and M. rosae (Alford et al., 2019a; Alford et al., 2019b).

In this study, we examine the effects of the biostable Aib-containing kinin analog

2139[F1]wp-2 and an antagonist of the CAPA receptor, CAPA analog 2129-SP3[F3]wp-2 (Table

1) on feeding and diuresis-related behaviours, including changes in blood meal size, hindgut contractions, and excretion rate. Due to the presence of CAPA receptors on the hindgut (Paluzzi et al., 2010), we also further investigated the role of members of the RhoprCAPA family of peptides on hindgut contractions. Examining the effects of the kinin and CAPA analogs on the physiology of R. prolixus will assist in determining the potential value of these analogs in wide scale pest control strategies.

Materials and Methods

Animals

5th instar male and female R. prolixus were obtained from an established colony at the

University of Toronto Mississauga. Insects were reared at 25oC and 50% humidity in incubators, and were fed defibrinated rabbit blood (Cedarlane Laboratories, Burlington, ON, Canada) once in each instar. Tissues were dissected from 5th instar R. prolixus 3-5 weeks post-feeding as 4th instars.

All tissue dissections were performed in R. prolixus physiological saline, consisting of 150 mM

NaCl, 8.6 mM KCl, 2.0 mM CaCl2, 4.0 mM NaHCO3, 8.5 MgCl2 , 0.02 mM HEPES and 34 mM glucose in pH 7.0.

35 Chemicals

Rhopr-kinin 2, RhoprCAPA-1, RhoprCAPA-2, and RhoprCAPA-pk1 were custom synthesized by Genscript (Piscataway, NJ, USA). The peptides were then reconstituted in double- distilled water into stock solutions at 10-3 M and stored at -20oC. Stock solutions of the Aib- containing insect kinin analog (2139[F1]wp-2) with the amino acid sequence Phe-Phe-Aib-Trp-

Gly-NH2 (Nachman et. al., 1997), and insect CAPA analog (2129-SP3[F3]wp-2) with the sequence 2Abf-Suc-ATPRIa synthesized as previously described (Nachman et al., 1997; Alford et al., 2019b) were prepared in 80 % aqueous acetonitrile (ACN) containing 0.01% trifluoroacetic acid (TFA), and stored at 4oC at a concentration of 10-3 M. Peptides and analogs were diluted in physiological saline to various concentrations to be used during physiological assays

Hindgut Contraction Assay via Force Transducer

The R. prolixus hindgut was isolated under physiological saline, along with the cuticle at the posterior end and fixed onto a Sylgard-coated dish using minuten pins through the cuticle and bathed in 200 μl of physiological saline. One end of a fine silk thread was tied to the anterior end of the hindgut, with the other end tied to a Grass FT03 force transducer (Astro-Nova Inc., Rhode

Island, USA). The amplitude of basal tonus changes were recorded using the PicoScope 2204

Oscilloscope (Pico Technology, Cambridgeshire, UK). Tissues were equilibrated in saline for 10 minutes. Peptides, including their analogs, were applied by addition of 100 μl of various concentrations of the peptides in saline concurrent with removal of 100 μl of the bath saline to ensure the bath volume remained constant. The preparations were washed with saline between test doses of peptide and the bath volume was maintained at 200 μl of saline. The recorded traces were analyzed for changes in basal tonus.

36 Hindgut Contraction Assay via Impedance

The R. prolixus hindgut was isolated under physiological saline, along with the cuticle at the posterior end and fixed onto a Sylgard-coated dish using minuten pins through the cuticle and the anterior end and bathed in 200 μl of physiological saline. Electrodes were placed on either side of the anterior region of the hindgut. Peristaltic contractions were monitored through a UFI impedance converter (Model 2991, Morro Bay, CA, USA). The frequency of the hindgut contractions was recorded using the PicoScope 2204 Oscilloscope (Pico Technology,

Cambridgeshire, UK). Tissues were equilibrated with saline for 10 minutes. Peptides and analogs were applied onto the tissue by addition of 100 μl of various concentrations in saline concurrent with removal of 100 μl of the saline, ensuring that the volume of saline within the bath remained constant. To validate the recorded contractions, the tissue was observed visually to correlate which deflections represent contractions. The frequency of contractions was measured for 3 minutes, and the recorded traces were analyzed.

Feeding Bioassay

Unfed 5th instar insects (3-5 weeks post feeding as 4th instars) were separated into 3 groups of 20 with similar average weights. Each group was injected through the membrane at the junction of the hind leg with the abdomen with one of the following: 1 μl Rhopr-kinin 2 (10-4 M), 1 μl kinin analog 2139[F1]wp-2 (10-4 M), 1 μl RhoprCAPA-2 (10-4 M), 1 μl CAPA analog 2129-

SP3[F3]wp-2 (10-4 M), or 1 μl physiological saline. After a 2-hour recovery period, each group was placed in a 10 cm diameter glass jar and fed on 20 mL of warm defibrinated rabbit blood for

20 minutes. Insects from each group were individually weighed immediately after feeding (time

0) and maintained in individual cubicles. Weights were later recorded at 1, 2, 3, and 4-hour time points. Since 5th instar R. prolixus tend to take a blood meal 8-10 times their initial body weight,

37 insects that fed less than 1 times their body weight were considered not fed and excluded from the data, as were those punctured during the weighing process.

Malpighian Tubule Secretion Assay

Whole MTs from 5th instar insects were dissected under saline using glass probes and transferred to a Sylgard-coated Petri dish containing 20 µl drops of saline overlaid with water- saturated mineral oil. The proximal end of the tubule was pulled out of the saline and wrapped around a minuten pin. Excess tubule from the proximal end was cut prior to wrapping, and the tubules were nicked gently at the pin. The equilibrating saline was removed and replaced with 10-

8 M 5-HT (Sigma, Oakville, ON, Canada), the CAPA analog 2129-SP3[F3]wp-2, a mixture of 10-

8 M 5-HT and 10-7 M RhoprCAPA-2, or a mixture of 10-8 M 5-HT, 10-7 M RhoprCAPA-2 and different concentrations of the CAPA analog 2129-SP3[F3]wp-2. Tubules were allowed to secrete for 30 minutes. Droplets of secreted fluid from the nicked end of the tubule were then collected using an oil filled micropipette tip, and the diameter of the droplet was measured using an eyepiece micrometer on the bottom of the Sylgard-coated Petri dish. The droplet volume was then calculated using the equation V=(π/6)d3 where d is the diameter of the droplet measured. At the end of the experiments, tubules were stimulated with 10-6 M 5-HT and the maximal rate of secretion was measured to check viability of the tissues.

Results

In Vivo Effects of Kinin and CAPA Analogs on Feeding and Diuresis

Injection of Rhopr-kinin 2 prior to feeding did not alter the size of blood meal consumed over a 20-minute feed as compared to saline injected insects (Fig. 1A). On the other hand, injection of the kinin analog 2139[F1]wp-2 prior to feeding led to a significant decrease in the size of the

38 blood meal consumed over a 20 minute period (Fig. 1A). Rapid post-feeding diuresis occurs over the subsequent 3-4 hours and this can be monitored by measuring the loss of weight due to excretion over time. Rhopr-kinin 2 and the kinin analog 2139[F1]wp-2 did not alter the rate of diuresis over 4 hours (Fig. 1B,1C).

The effects of RhoprCAPA-2 and the CAPA analog, 2129-SP3[F3]wp-2, were also examined on feeding and rate of diuresis in 5th instar insects. Injection of RhoprCAPA-2 had no effect on the size of the blood meal, whereas injection of the CAPA analog 2129-SP3[F3]wp-2, resulted in a larger blood meal being consumed (Fig. 2A). Injection of RhoprCAPA-2 did not influence the rate of diuresis over 4 hours (Fig. 2B); however, the CAPA analog, 2129-

SP3[F3]wp-2, which resulted in a larger blood meal taken appeared to have an increased rate of diuresis over the first hour after feeding (Fig. 2C).

In Vitro Effects of Kinin and CAPA Analogs on Hindgut Contractions

To further investigate the analogs, we turned to in vitro preparations of tissues associated with feeding, namely the hindgut and MTs. Both Rhopr-kinin 2 and the kinin analog, 2139[F1]wp-

2, resulted in dose-dependent increases in basal tonus of the hindgut with threshold at 10-10 M for

Rhopr-kinin 2 and 10-14 M for 2139[F1]wp-2, illustrating the potency of the analog (Fig. 3). In addition, 2139[F1]wp-2 induced stronger contractions than Rhopr-kinin 2. The EC50 value of

-10 2139[F1]wp-2 is approximately 5.5 x 10 M, whereas the EC50 value for Rhopr-kinin 2 is approximately 5.5 x 10-9 M. (Fig. 3C, 3D) The effects of 2139[F1]wp-2 were more difficult to reverse and required more washes in saline than Rhopr-kinin 2. Neither RhoprCAPA-1 (not shown), RhoprCAPA-2 (Fig. 4A) nor RhoprPK-1 (not shown) altered contractions of hindgut.

39 Interestingly, the CAPA analog, 2129-SP3[F3]wp-2, stimulated dose-dependent increases in

-10 -8 hindgut contractions (Fig. 4B, 4C). The threshold is at 10 M, EC50 of approximately 10 M, and maximum tension at 10-6 M (Fig. 4C). In order to examine for any interaction between Rhopr- kinin 2 and RhoprCAPA-2, the two peptides were applied simultaneously on the hindgut (Fig. 5,

6). Application of Rhopr-kinin 2 along with RhoprCAPA-2 resulted in statistically significant increases in hindgut contractions relative to Rhopr-kinin 2 alone (Fig. 5A-5C). Interestingly, this potentiation of hindgut contractions was not observed with the CAPA analog, 2129-SP3[F3]wp-

2 (Fig. 6).

To further examine any co-operative effects of RhoprCAPA-2 and the CAPA analog 2129-

SP3[F3]wp-2, changes in 5-HT-stimulated increases in the frequency of hindgut contractions were measured. As the contractions induced by 5-HT are not easily monitored by the force transducer

(Te Brugge et al., 2002; Bhatt et al., 2014), an impedance monitor was used to assess changes in the frequency of hindgut contractions. Varying concentrations of RhoprCAPA-2 and 2129-

SP3[F3]wp-2 were each applied simultaneously with 10-8 M 5-HT on the hindgut (Fig. 7, 8). 2129-

SP3[F3]wp-2 potentiated the effects of 10-8 M 5-HT, with a statistically significant increase in hindgut frequency observed at a concentration of 10-7 M 2129-SP3[F3]wp-2 (Fig. 7B, 7C).

However, Rhopr-CAPA 2 was found to inhibit this 5-HT-mediated increase in frequency, with a statistically significant reduction in frequency at 10-6 M (Fig. 8B, 8C).

In Vitro Effects of CAPA Analogs on Malpighian Tubule Secretion

To determine the effects of the CAPA analog 2129-SP3[F3]wp-2 on diuresis in vitro, varying concentrations of the analog were tested on unstimulated tubules, and 5-HT-stimulated tubules. The analog had no effect on unstimulated tubules (not shown) and failed to have any

40 potentiation effect on tubules stimulated with 10-8 M 5-HT (Fig. 9A). Varying concentrations of the CAPA analog 2129-SP3[F3]wp-2 were mixed with 10-8 M 5-HT and 10-7 M RhoprCAPA-2.

As previously shown, RhoprCAPA-2 inhibits 5-HT stimulated secretion (Fig. 9B). The analog prevented the anti-diuretic effect of RhoprCAPA-2 with a statistically significant difference observed at 10-6 M but failed to return the tubules to its initial rate of 5-HT-stimulated secretion

(Fig. 9B).

Table 1: Structure of kinin and CAPA analogs

Analog Structure

Kinin: 2139[Ф1]wp-2 FF[Aib]WGa

CAPA: 2129-SP3[Ф3]wp-2 2Abf-Suc-ATPRIa

Fig. 1: A) The effects of injection of 1 µl saline, 1 µl Rhopr-kinin 2 (10-4 M), and 1 µl of the kinin analog, 2139[Ф1]wp-2 (10-4 M), on the size of blood meal taken by 5th instar R. prolixus. Weight of insects was measured after 20 minutes of blood-feeding (time 0). The effects of injection of 1

µl of saline and 1 µl of B) Rhopr-kinin 2 (10-4 M) and C) kinin analog 2139[Ф1]wp-2 (10-4 M) on the rate of diuresis of 5th instar R. prolixus. Weight of insects were measured at time 0 and at 1 hour increments post-feeding for 4 hours. (One-way ANOVA followed by Tukey’s post-hoc test, slopes tested for significance using an F-test, *=p<0.05. Data are means ± SEM of n=16-20).

Fig. 2: A) The effects of injection of 1 µl saline, 1 µl RhoprCAPA-2 (10-4 M) and 1 µl CAPA analog, 2129-SP3[Ф3]wp-2 (10-4 M), on the size of blood meal taken by 5th instar R. prolixus.

Weight of insects was measured after 20 minutes of blood-feeding (time 0). The effects of injection of 1 µl of saline and 1 µl of B) Rhopr-CAPA 2 (10-4 M) and C) the CAPA analog, 2129-SP3[Ф]wp-

2 (10-4 M), on the rate of diuresis of 5th instar R. prolixus. Weight of insects was measured at time

0 and at 1 hour increments post-feeding for 4 hours. (One-way ANOVA followed by Tukey’s post- hoc test, slopes tested for significance using an F-test, *=p<0.05. Data are means ± SEM of n=18-

20).

Fig. 3: Example traces of changes in basal tonus of hindgut contractions in response to A) 10-8 M

Rhopr-kinin 2 and B) 10-8 M kinin analog, 2139[Ф1]wp-2. Downward arrowheads denote application of peptide, upward arrowheads denote the start of saline wash, and circles denote vertical deflections due to wash. Dose-response curves displaying changes in basal tonus of hindgut contractions in response to C) Rhopr-kinin 2 and D) kinin analog, 2139[Ф1]wp-2. (One- way ANOVA followed by Dunnett’s multiple comparison test, *=p<0.05, **=p<0.01,

***=p<0.001. Data are means ± SEM of n=5).

Fig. 4: Example traces of changes in basal tonus of hindgut contractions in response to A) 10-8 M of RhoprCAPA-2 and B) 10-8 M of CAPA analog, 2129-SP3[Ф3]wp-2. Downward arrowheads denote application of peptide, upward arrowheads denote the start of saline wash, and circles represent vertical deflections due to wash. C) Dose-response curve displaying changes in basal tonus of hindgut contractions in response to the CAPA analog, 2129-SP3[Ф3]wp-2. (One-way

ANOVA followed by Dunnett’s multiple comparisons test, *=p<0.05, **=p<0.01. Data are means

± SEM of n=5).

Fig. 5: A) Example traces of changes in basal tonus of hindgut contractions in response to 10-8 M

Rhopr-kinin 2 and a mixture of 10-8 M Rhopr-kinin 2+10-8 M RhoprCAPA-2. Downward arrowheads denote application of peptide, upward arrowheads denote the start of saline wash, and circles denote vertical deflections due to wash. B) 10-8 M RhoprCAPA-2 and C) 10-7 M

RhoprCAPA-2 potentiates the change in basal tonus elicited by varying concentrations of Rhopr- kinin 2. Change in tension is represented as a percent of maximum tension induced by 10-8 M

Rhopr-kinin 2 on each preparation. (Two-way ANOVA followed by Tukey’s post-hoc test,

*=p<0.05, **=p<0.01. Data are means ± SEM of n=5).

Fig. 6: The effects of varying concentrations of the CAPA analog, 2129-SP3[Ф3]wp-2, on the changes in basal tonus elicited by 10-8 M Rhopr-kinin 2. No statistically significant differences were found. (One-way ANOVA followed by Dunnet’s multiple comparison test, p>0.05. Data are means ± SEM of n=5).

Fig. 7: Example traces of changes in the frequency of hindgut contractions in response to A) 10-8

M 5-HT and B) a mixture of 10-8 M 5-HT and 10-7 M of the CAPA analog 2129-SP3[Ф3]wp-2.

Downwards triangles denote vertical deflections of hindgut contractions. C) The effects of varying concentrations of the CAPA analog 2129-SP3[Ф3]wp-2 on the frequency of hindgut contractions elicited by 10-8 M 5-HT over a 2-minute period. (One way-ANOVA followed by Dunnett’s multiple comparison test, *=p<0.05. Data are means ± SEM of n=8).

Fig. 8: Example traces of changes in the frequency of hindgut contractions in response to A) 10-8

M 5-HT and B) a mixture of 10-8 M 5-HT and 10-6 M RhoprCAPA-2. Downwards triangles denote contractions. C) The effects of varying concentrations of RhoprCAPA-2 on the frequency of hindgut contractions elicited by 10-8 M 5-HT over a 2-minute period. (One way-ANOVA followed by Dunnett’s multiple comparison test, *=p<0.05. Data are means ± SEM of n=8).

Fig. 9: A) The effects of varying concentrations of the CAPA analog 2129-SP3[Ф3]wp-2 on the fluid secretion rate of MTs stimulated by 10-8 M 5-HT. No statistically significant differences were found. B) The antagonist effects of various concentrations of the CAPA analog 2129-SP3[Ф3]wp-

2 on RhoprCAPA-2 (10-7 M) resulted in blocking of the inhibitory effect of Rhopr-CAPA-2 on

MTs stimulated with 10-8 M 5-HT. (One-way ANOVA followed by Dunnet’s multiple comparison test, *=p<0.05. Data are means ± SEM of n=6).

60 Discussion

In R. prolixus, the precisely timed events that occur during post-prandial rapid diuresis are governed by the neuroendocrine system, therefore investigating the neuroactive chemicals that may be associated with these specific behaviours and physiology provides insight into how these processes occur. Also, from an agrochemical perspective, the endocrine system can be used as a target to disrupt epidemiologically-relevant behaviours in order to prevent disease transmission.

Neuropeptides show great promise in the development of next-generation insecticides, due to their specificity in terms of function and binding to GPCRs (Audsley & Down, 2015).

As blood feeding is a requirement for the initiation of many developmental and reproductive processes and the transition to the next instar within R. prolixus, interference with these events would prove to be quite detrimental (Lange et al., 2016). Insects that were injected with the kinin analog 2139[F1]wp-2 prior to feeding had a significantly reduced blood meal compared to saline injected insects which was consistent with previous work where Aib-containing analogs were found to have antifeedant effects on 5th instar R. prolixus (Lange et al., 2016). In the hemipteran Acyrthosiphon pisum, while the presence of Aib-containing analogs within the aphid diets resulted in reduced feeding, and aphicidal activity, 2139[F1]wp-2 failed to reduce aphid fitness under desiccation and survival stress in M. persicae and M. rosae (Alford et al., 2019b,

Smagghe et al., 2010). These differences may be due to the specificity of the kinin analogs, as species-specific effects of neuropeptide analogs have been previously observed in D. melanogaster

(Alford et al., 2019a). Injection of the kinin analog did not influence the rate of post-prandial rapid diuresis which is consistent with the fact that kinins do not play a direct role in post-prandial rapid diuresis (Lange et al., 2016, Te Brugge et al., 2002; Te Brugge et al., 2009).

61 In contrast to Rhopr-kinins, RhoprCAPA functions as an anti-diuretic hormone through the inhibition of MT fluid secretion and anterior midgut fluid transport (Paluzzi et al., 2008, Ianowski et al., 2009). Insects injected with the CAPA analog 2129-SP3[F3]wp-2 prior to feeding took a significantly increased blood meal compared to saline injected insects and had a significantly greater rate of post-prandial rapid diuresis within the first hour. As the CAPA analog 2129-

SP3[F3]wp-2 is likely acting upon Rhopr-CAPA receptors as an antagonist (Jiang et al., 2015), this increased rate of rapid diuresis is likely due to the blocking of the CAPA receptor, thus preventing the anti-diuretic effects of RhoprCAPA-2. The changes in blood meal size upon injection of 2129-SP3[F3]wp-2 suggests that RhoprCAPA may also influence feeding. Once an insect has successfully fed, it has obtained enough nutrients required to moult into the next instar, and so does not require another blood meal (Buxton, 1930). Since RhoprCAPA-2 is released towards the end of diuresis, it may also serve as a signal to prevent additional feeding events.

Within R. prolixus, multiple neurohormones such as sulfakinin (Rhopr-SK-1) and Rhopr-CRF/DH have also been identified as influencing feeding (Al-Alkawi et al., 2017; Mollayeva et al., 2018), and therefore may co-operatively function in regulating satiety and the motivation to feed. The injection of RhoprCAPA-2 did not have an impact on the size of blood meal, which suggests that

RhoprCAPA-2 might not influence satiety, but is more a signal to prevent additional feeding events.

The hindgut of R. prolixus plays an essential role during post-feeding diuresis, as it is responsible for the excretion of accumulated urine (Maddrell, 1976). In addition, the T. cruzi parasite is present in the hindgut in its highly infectious stage, and so contraction of the hindgut results in the release of the parasite along with urine onto the host (Bern et al., 2011). Aib- containing analogs have previously been shown to be more effective in eliciting myotropic and

62 diuretic effects than their endogenous counterparts in L. maderae and Acheta domesticus

(Nachman et al., 1997; Taneja-Bageshwar et al., 2009). Aib-containing analogs tested on R. prolixus hindgut were found to be more biologically active than Rhopr-kinins, and in some cases eliciting irreversible changes in basal tonus (Bhatt et al., 2014). Similar results were obtained with the kinin analog 2139[F1]wp-2, which caused dose-dependent increases in basal tonus of the hindgut and was active at concentrations as low as 10-14 M. As described for other Aib-containing analogs in R. prolixus, the effects of 2139[F1]wp-2 were more difficult to wash off. As these analogs were synthesized to prevent degradation by endogenous peptidases, the analog may have a prolonged effect on its target tissue by also influencing the binding to the receptor (Nachman et al., 2003). These potent changes in physiology induced by the kinin analogs highlight their promise in future studies for pesticide development.

Despite the presence of CAPA receptors (and indeed pyrokinin receptors) (Paluzzi et al.,

2012; Paluzzi et al., 2008) on the hindgut, none of the three CAPA neuropeptides (RhoprCAPA-

1, RhoprCAPA-2, or Rhopr-pk1) were found to have any direct effect on hindgut contractions.

However, the CAPA analog 2129-SP3[F3]wp-2 induced hindgut contraction in a dose-dependent manner, but its effects were not as intense as Rhopr-kinin 2 or the kinin analog 2139[F1]wp-2.

Interestingly though, RhoprCAPA-2 potentiated the effect of Rhopr-kinin 2. Since RhoprCAPA-

2 is released as a signal to terminate diuresis, it may also assist the hindgut in excretion of the remaining urine. This potentiation effect was not observed with RhoprCAPA-1 or Rhopr-pk1, nor was it seen with the CAPA analog 2129-SP3[F3]wp-2.

63 The potentiation effect of RhoprCAPA-2 may be due to the interaction of separate second messenger pathways after GPCR activation. As the kinin receptor has not yet been characterized within R. prolixus, the associated second messenger pathway is currently unknown. Within other insects, the kinin receptor has been associated with an increase in intracellular Ca2+ via the inositol phosphate (IP3) pathway. Activation of the IP3 pathway increases IP3 levels within the cytoplasm

2+ which later binds to an IP3-sensitive Ca channel on the endoplasmic reticulum. Following release, Ca2+ induces muscle contraction by enabling cross-bridge cycling (Radford et al., 2002;

Tehrzaz, et al., 1999; Beyenbach, 2003; Pietrantonio et al., 2005; Kuo & Ehrlich, 2015). In R. prolixus, activation of the CAPA receptor in the MTs results in an increase in cGMP, which in turn activates a phosphodiesterase that degrades cAMP, thereby lowering cAMP levels. (Paluzzi

& Orchard, 2006; Paluzzi et al., 2013). Here, we propose that the CAPA receptor may be constitutively active within the hindgut, with cGMP stably keeping cAMP levels low. Upon activation of both the kinin and CAPA receptors, the increase in cGMP may participate in the IP3 pathway to increase intracellular Ca2+ resulting in stronger contractions. In vertebrates, cGMP signaling is implicated in both stimulating or inhibiting contraction (Fischmeister et al., 2005;

Fellner & Arendshorst, 2009). Within vertebrate cardiac muscle, cGMP has shown to have excitatory effects through the stimulation of Ca2+ channels, resulting in an increase of intracellular

Ca2+. These Ca2+ channels are responsible for the excitation-contraction coupling within the muscle

(Wang et al., 1999; Fischmeister et al., 2005). In contrast are the stimulatory effects of the CAPA analog 2129-SP3[F3]wp-2. This analog appears to be an antagonist of the CAPA receptor and so blocks the cGMP-mediated cAMP degradation via activation of a phosphodiesterase, in turn allowing cAMP to increase and stimulate hindgut contractions. In order to further investigate the possible mechanism by which this kinin/CAPA interaction occurs, the effects of RhoprCAPA-2 and 2129-SP3[F3]wp-2 were assessed with 5-HT. 5-HT has myostimulatory effects on the

64 hindgut, via an increase in cAMP levels (Orchard, 2006). A potentiation effect was observed with co-application of 5-HT and 2129-SP3[F3]wp-2 on the frequency of hindgut contractions. This is likely due to the antagonist action on the CAPA receptors, preventing the RhoprCAPA-mediated cAMP decrease. Conversely, the effects of 5-HT were inhibited by RhoprCAPA-2, due to the increase in cGMP, activation of a phosphodiesterase, and degradation of cAMP.

The MTs are critical in allowing R. prolixus to return to a homeostatic state following a blood meal. As diuretic hormones such as 5-HT, Rhopr-DH31, and Rhopr-CRF/DH function to stimulate secretion within the tubules, Rhopr-CAPA 2 is required as a signal to abolish this secretion to prevent excess ion and water loss (Paluzzi et al., 2008; Orchard, 2006). The CAPA analog 2129-SP3[F3]wp-2 was found to interfere with Rhopr-CAPA 2’s ability to inhibit 5-HT- stimulated MT secretion, that is to say, it is an antagonist of the CAPA receptor. Within D. suzukii,

2129-SP3[F3]wp-2 had a protective effect as females injected with the analog had a significantly increased survival rate. Within D. melanogaster, CAPA functions as a diuretic hormone (Kean et al., 2002), with desiccation survival linked to the regulation of fluid secretion (Terhzaz et al.,

2015). This supports the function of 2129-SP3[F3]wp-2 as a CAPA receptor antagonist, as it prevents CAPA-stimulated fluid secretion in D. melanogaster, thereby increasing survivability under desiccation stress (Alford et al., 2019a). Within M. persicae and M. rosae, however, injection of 2129-SP3[F3]wp-2 resulted in accelerated mortality under desiccation and starvation stress (Alford et al., 2019b). These effects of the CAPA analog may be due to the lack of MTs within aphids, therefore CAPA may not play a direct role in desiccation (Jing et al., 2015).

In summary, these results display the efficacy in which the biostable neuropeptide analogs are able to induce potent changes in physiology, thus showing potential use in the development of

65 pest control strategies. The need for highly coordinated release of neuroactive chemicals within R. prolixus is a necessity in order for the insect to successfully gorge on blood. Given the bugs susceptibility to predators in its engorged state, rapid elimination of water and salt is required

(Orchard, 2006). These analogs are able to successfully disrupt this coordination. The novel interaction between Rhopr-kinin 2 and RhoprCAPA-2 further highlights the importance of coordinated release of these neuropeptides throughout the life cycle of R. prolixus. Investigating the intracellular mechanisms by which the actions of the neuroactive chemicals function provides insight into the mode of action of epidemiologically-relevant behaviours, which can also aid in the development of next-generation pest control strategies.

Acknowledgements

The authors would like to thank Stuti Joshi for maintaining the colony and Allison Strey

(ARS-USDA) for technical assistance. This work was financially supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant [RGPIN 2014-06253 to AL, and

RGPIN 8522-12 to IO].

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Chapter 3 Identification and Cloning of the Kinin Receptor in the Chagas Disease Vector, Rhodnius Prolixus

72 Abstract

Within invertebrates, the kinin family of neuropeptides is responsible for the modulation of a host of physiological and behavioural processes. In Rhodnius prolixus, kinins are primarily responsible for eliciting myotropic effects on various feeding and diuresis-related tissues. Kinins function through binding to G-protein coupled receptors (GPCRs), likely utilizing intracellular

Ca2+ as a second messenger. Here, the R. prolixus kinin receptor (RhoprKR) has been successfully isolated, cloned and sequenced from the central nervous system (CNS) and hindgut of R. prolixus.

Sequence analysis show high similarity between RhoprKR with other cloned invertebrate kinin receptors, along with the presence of various highly conserved residues. The expression profile of

RhoprKR shows expression of the RhoprKR transcript throughout the R. prolixus gut, suggesting a role of Rhopr-kinins in various aspects of feeding and digestion. RNA interference (RNAi)- mediated knockdown of the RhoprKR transcript resulted in a significant reduction of hindgut contractions in response to Rhopr-kinin 2 and an Aib-containing kinin analog. dsRhoprKR- injected insects also consumed a significantly larger blood meal, suggesting a role of Rhopr-kinins in satiety. Overall, these findings highlight the role of the kinin signaling system in R. prolixus, and the effectiveness of RhoprKR as a target in RNAi-mediated pest control strategies.

73 Introduction

Neuropeptides comprise the largest family of neuroactive chemicals and are involved in many essential physiological and behavioural processes within insects. The effects of these neuropeptides are directed through activation of G protein-coupled receptors (GPCRs), the largest group of membrane receptors (Altstein & Nässel, 2010; Lismaa & Shine, 1992). In insects, studies have examined the potential use of these neuropeptides in pest control strategies, since they are more environmentally-friendly than traditional pesticides and can elicit effects at low concentrations (Nachman & Smagghe, 2011; Gäde & Goldsworthy, 2003). In the context of pharmaceutical development, GPCRs are often used as targets for novel drugs, with 40-50% of drugs acting upon GPCRs (Garland, 2013). However, GPCRs have only recently been targeted in pest control strategies within insects, despite their potential use in the development of next- generation pesticides (Audlsey & Down, 2015). Increasing our understanding of neuropeptide families and their GPCRs can provide great insight into how crucial processes can be disrupted, preventing the spread of relevant pest species and disease vectors (Audsley & Down, 2015;

Nachman & Smagghe, 2011; Nachman, 2009).

In the Chagas disease vector, Rhodnius prolixus, post-feeding physiology has been widely studied and is under regulation by various neuroendocrine factors (Orchard, 2006; Coast et al.,

2002; Orchard, 2009). Diuresis and excretion are considered to be epidemiologically-relevant behaviours, as they are implicated in the transmission of Chagas disease (Orchard, 2006; Orchard

& Paluzzi, 2009). Excretion occurs through contractions of the hindgut, where the Trypanosoma cruzi parasite exists in the highly infectious stage of its life cycle (Bern et al., 2011). Contractions of the hindgut result in the transmission of the parasite onto the host, and therefore possible

74 infection (Bern et al., 2011). From a medical perspective, neuropeptides that modulate hindgut contractions are compounds of interest, as they can be targeted in an effort to disrupt excretion.

The kinins, first identified for their myotropic effects in the cockroach Leucophaea maderae, are a family of neuropeptides with the following conserved C-terminal pentapeptide sequence: FX1X2WG-amide, where X1 can be Ser, Phe, His, Asn, or Tyr, and X2 can be Ser, Pro or Ala (Holman et al., 1986a, b; Holman et al., 1987a, b). This core pentapeptide sequence is required for the neuropeptide to be biologically active (Coast et al., 1990; Nachman et al., 1991).

Kinins have now been identified in a number of species and are implicated in a diverse set of functions (Rosay et al., 1997; O’Donnell et al., 1998; Tehrzaz et al., 1999; Kim et al., 2006; Bhatt et al., 2014; Zandawala et al., 2018). The R. prolixus kinin (Rhopr-kinin) transcript encodes for the most kinins found in any species, with eighteen predicted kinins and precursor associated species (Te Brugge et al., 2011; Bhatt et al., 2014). Within R. prolixus, Rhopr-kinins are primarily known for their myotropic effects, eliciting contractions of the hindgut, midgut, and salivary glands

(Orchard & Te Brugge, 2002; Te Brugge et al., 2009; Te Brugge et al., 2011; Bhatt et al., 2014).

Given the ability of Rhopr-kinins to stimulate hindgut contractions, they are a neuropeptide of interest, as they are directly implicated in the transmission of the T. cruzi parasite, and Chagas disease (Bern et al., 2011).

Kinin GPCRs belong to the family A GPCRs, which is the largest and most studied family of GPCRs, with the first kinin receptor characterized in the snail, Lymnaea stagnalis (Cox et al.,

1997; Munk et al., 2016). Following this, kinin receptors have been characterized in a small number of insects including Drosophila melanogaster, Aedes aegypti, and Anopheles stephensi

(Radford et al., 2002; Radford et al., 2004; Pietrantonio et al., 2005). Kinin receptors may function

2+ by increasing intracellular Ca through the inositol phosphate (IP3) pathway, as activation of this pathway was observed within the Malpighian tubules (MTs) of D. melanogaster and A. aegypti

(Cady and Hagedorn, 1999; Radford et al., 2002; Pietrantonio et al., 2005). Interestingly, the kinin receptor within A. aegypti functions as a multiligand receptor for the three Aedes kinins, with the three kinins utilizing different intracellular pathways (Pietrantonio et al., 2005). Currently, the kinin receptor within R. prolixus has not yet been characterized, but analysis of potential GPCRs within the R. prolixus transcriptome has identified a possible kinin receptor candidate

(RPRC000494) (Ons et al., 2016).

Advances in molecular biology have allowed the development of various tools that can be used in the creation of novel strategies to combat pests and disease vectors. RNA interference

(RNAi) is a common research tool in insects, and can be used in pest control development, since it can specifically target species (Vogel et al., 2019). RNAi has been successful at inducing phenotypic changes that decrease overall fitness, such as reduction of cardiac output through silencing of the crustacean cardioactive peptide (CCAP) transcript in A. gambiae (Estévez-Lao et al., 2013) and reduced fecundity after silencing of the kinin receptor in Rhipicephalus microplus

(Brock et al., 2019). Within R. prolixus, RNAi has been utilized in a similar manner, with silencing of the CCAP receptor leading to a decrease in basal heartbeat frequency and disrupting ecdysis

(Lee et al., 2013). In addition, R. prolixus injected with chitin synthase (CHS) double-stranded

RNA (dsRNA) had severe cuticle deformations which interfered with mobility and longevity, and adult females injected with CHS dsRNA had overall reduced oviposition (Mansur et al., 2014).

In the case of larger insects such as R. prolixus, dsRNA is often delivered through injection which can induce phenotypic changes that may not be associated with the intended knockdown

76 target, or cause trauma due to the injection wound (Whitten et al., 2016). In Acyrthosiphon pisum, injected of dsRNA resulted in unexpected changes in gene expression and disruptive effects resulting in mortality (Jauber-Possamai et al., 2007). A novel form of RNAi delivery, known as symbiont-mediated RNAi aims to overcome the limitations of large-scale dsRNA delivery. This form of delivery involves the integration of dsRNA into the symbiotic bacteria of a target species, thus evoking RNAi within the host (Whitten et al., 2016). Symbiont-mediated RNAi has been successfully tested on R. prolixus, inducing various knockdown phenotypes. As these recombinant bacteria can be delivered through a blood meal or through coprophagy within R. prolixus populations, large-scale application of this RNAi strategy can prove to be quite feasible (Whitten et al., 2016).

In this study, the R. prolixus kinin receptor (RhoprKR) was isolated and cloned. The expression profile of the RhoprKR transcript within the tissues of 5th instar R. prolixus using quantitative PCR (qPCR) was determined. As kinins are responsible for the contraction of hindgut muscle within R. prolixus (Bhatt et al., 2014; Te Brugge et al., 2011), RNAi was utilized to knockdown the RhoprKR transcript followed by hindgut contraction assays to confirm knockdown. In addition, the effects of RhoprKR knockdown was assessed on in vivo feeding and diuresis. Identification of RhoprKR will assist in the development of next-generation pest control strategies where this receptor can be targeted to disrupt the overall physiology of R. prolixus and therefore release of the parasite onto the host.

77 Materials and Methods

Animals

5th instar male and female R. prolixus were obtained from a colony at the University of

Toronto Mississauga. Insects were reared in incubators at 25oC and 50% humidity and were fed defibrinated rabbit blood (Cedarlane Laboratories, Burlington, ON, Canada) once in each instar.

Chemicals

Rhopr-kinin 2 was custom synthesized by Genscript (Piscataway, NJ, USA). The neuropeptides were then reconstituted in double-distilled water in stock solutions at 10-3 M, and later stored at -20oC. The Aib-containing insect kinin analog (2139[F1]wp-2) with the following amino acid sequence Phe-Phe-Aib-Trp-Gly-NH2 (Nachman et. al., 1996) was prepared in 80% aqueous acetonitrile (ACN) at a concentration of 10-3 M and stored at 4oC. Rhopr-kinin 2 and

2139[F1]wp-2 were later diluted in physiological saline (150 mM NaCl, 8.6 mM KCl, 2.0 mM

CaCl2, 4.0 mM NaHCO3, 8.5 MgCl2, 0.02 mM HEPES and 34 mM glucose in pH 7.0) to various concentrations for use in bioassays.

Identification and Cloning of cDNA Sequences Encoding the R. prolixus Kinin Receptor

The genome, transcriptome, and peptidome of R. prolixus was obtained from vectorbase.org and uploaded into Geneious 8.1 (Auckland, New Zealand). A BLAST search was performed against the R. prolixus transcriptome using the D. melanogaster kinin receptor

(Q9VRM0) and A. aegypti kinin receptor (Q5EY37) as templates, giving hits for an annotated transcript sequence (RPRC00494), which has been previously predicted to be a Rhopr-kinin receptor (Ons et al., 2016). Following this, a nucleotide sequence alignment was performed with the D. melanogaster and A. aegypti kinin receptors, and highly conserved cDNA sequences within

78 the predicted Rhopr-kinin receptor (RhoprKR) were amplified by gene-specific primers (Table

S1). OneTaq ® DNA Polymerase (NEB, Whitby, ON, Canada) was used for all PCRs, and reactions were performed using Bio-Rad’s s100 thermocycler (Bio-Rad Laboratories, Mississauga,

ON, Canada). PCR products were extracted from 1.2% agarose gel using an EZ-10 Spin Column

DNA Gel Extraction Kit (Bio Basic, Markham, ON, Canada) and later cloned using the pGEM-T

Easy Vector (Promega, Madison, WI, USA). Bacteria that had successfully taken up the insert were inoculated and left to grow overnight. Using the EZ-10 Spin Column Plasmid DNA MiniPrep

Kit (Bio Basic, Markham, ON, Canada), inserts were extracted, and sent for Sanger sequencing at

Macrogen USA (Macrogen, Brooklyn, NY, USA).

To successfully amplify the 5’ and 3’ regions of RhoprKR, 5’ and 3’ RACE was performed using the SMARTer® RACE 5’/3’ Kit (Takara Bio USA, Mountain View, CA, USA). The CNS and hindgut was dissected and placed in nuclease-free phosphate-buffered saline (PBS) (Sigma

Aldrich, Oakville, ON, Canada). Following this, total RNA extraction was performed using the

EZ-10 Spin Column Total RNA Miniprep Super kit (Bio Basic, Markham, ON, Canada). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems,

Mississauga, ON, Canada) to be later used to synthesize 5’ and 3’ RACE-ready cDNA. Gene specific forward and reverse primers were developed for the 5’ and 3’ end of the RhoprKR transcript, to be amplified using the 5’ and 3’ RACE-ready cDNA (Table S2). Using the initial 5’ and 3’ RACE products as DNA templates, a nested PCR was performed with the additional downstream 5’ and 3’ primers to ensure specificity of the amplified sequence. 5’ and 3’ RACE reactions were performed using the SMARTer® RACE 5’/3’ Kit protocol, and products of the

RACE reactions were later purified using the High Pure PCR Product Purification Kit (Roche

Applied Science, Penzberg, Germany), cloned, and sequenced as described above.

79 Sequence Analysis

Following sequencing of RhoprKR, the structural and biochemical features of the receptor were analyzed using various online tools. The seven transmembrane domains for RhoprKR were predicted by TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The potential N- linked glycosylation sites were predicted using the NetNGlyc 1.0 Server

(http://www.cbs.dtu.dk/services/NetNGlyc/) with potential phosphorylation sites predicted using the NetPhos 3.1 Server http://www.cbs.dtu.dk/services/NetPhos/). Lipid modification sites for palmitoylation of cysteine residues were predicted by GPS-Lipid

(http://lipid.biocuckoo.org/webserver.php). To predict the exon-intron boundaries within the sequence, BLAST was performed, following confirmation with a fruit fly splice site prediction tool. (http://www.fruitfly.org/seq_tools/splice.html). Alignment of RhoprKR was performed with cloned kinin receptors of invertebrate species and other R. prolixus receptors using the MUSCLE alignment tool (https://www.ebi.ac.uk/Tools/msa/muscle/).

Spatial Expression of Rhopr-kinin Receptor in Feeding and Diuresis-Related Tissues

Expression of RhoprKR was examined in 5th instar R. prolixus, 3-5 weeks post-feeding as

4th instars. The CNS, fat body (FB), dorsal vessel (DV), salivary glands (SG), foregut (FG), posterior midgut (PMG), anterior midgut (AMG), hindgut (HG), and MTs were dissected using nuclease-free PBS. Tissues were dissected and pooled into three different biological replicates

(n=3). Total RNA extraction was performed using an EZ-10 Spin Column Total RNA Miniprep

Super kit with cDNA synthesized using a High-Capacity cDNA Reverse Transcription Kit. cDNA was used for quantitative PCR (qPCR) reactions. cDNA was diluted by 10-fold and amplified using qPCR primers for RhoprKR (Table S4) and reference genes (α-tubulin, β-actin, ribosomal protein 49) (Table S1). qPCR reactions were performed using Bio-Rad’s CFX96 TouchTM Real-

80 Time PCR Detection System (Bio-Rad Laboratories, Mississauga, ON, Canada). Three technical replicates were performed per tissue, with a non-template control for each biological replicate.

Expression of the transcript levels in each tissue were calculated relative to an average of the reference genes using the ∆Ct method.

Double Stranded RNA Synthesis

Gene-specific primers were developed for a partial cDNA sequence from the RhoprKR transcript and the ampicillin resistance gene (ARG) (to be used as a control). These primers were also conjugated with 23 bases of the T7 RNA polymerase promoter on the 5’ end (Table S3). The cDNA sequence for RhoprKR was amplified using R. prolixus CNS cDNA, with ARG amplified from the pGEM-T Easy Vector system, both via PCR. These PCR products were purified using the High Pure PCR Product Purification Kit, to be used as a DNA template for the following T7

PCR reactions. The sequences possessing the T7 promoters were amplified via PCR, and later purified with the High Pure PCR Product Purification Kit. Double stranded RNA (dsRNA) was synthesized using the purified products of the T7 reactions using the T7 RiboMAX Express RNAi

System (Promega, Madison, WI, USA). Following synthesis, the dsRNA was precipitated with isopropanol, eluted in InvitrogenTM UltraPureTM DNase/RNase-Free Distilled Water (Thermo

Fisher Scientific, Waltham, MA, USA) then quantified using a nanodrop at 260nm wavelength to assess its concentration and quality. The dsRNA was later resuspended in InvitrogenTM

UltraPureTM DNase/RNase-Free Distilled Water at a final concentration of 2 μg/ml.

dsRNA Delivery

5th instar R. prolixus were injected through the membrane at the junction of the hind leg with the abdomen with 1 μl of 2 μg/ml RhoprKR dsRNA (dsRhoprKR) or ARG dsRNA (dsARG)

81 using a 10 μl Hamilton syringe (Hamilton Company, Reno, Nevada, USA). Following injection,

R. prolixus were left to recover at room temperature for two hours, then placed in incubators at

25oC and 50% humidity.

Knockdown Verification using Quantitative PCR

Three pools of CNS and hindgut tissue (n=3) were dissected using nuclease-free PBS from

R. prolixus at 2 days and 7 days post-injection with dsRhoprKR or dsARG, and RNA extraction was performed with the EZ-10 Spin Column Total RNA Miniprep Super kit. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit and diluted 10-fold to be used for qPCR. To verify the knockdown efficiency, qPCR was performed as described above, with changes in transcription levels measured using the 2-∆∆Ct method. Insects from day 7 post- injection (highest knockdown efficiency) were selected to be used for hindgut contraction assays and feeding bioassays.

Hindgut Contraction Assay via Force Transducer

The R. prolixus hindgut was isolated under physiological saline along with the cuticle at the posterior end and fixed onto a Sylgard-coated dish using minutien pins through the cuticle. The isolated hindgut was bathed in 200 μl of physiological saline. One end of a fine silk thread was tied to the anterior end of the hindgut with the other end tied to a Grass FT03 force transducer

(Astro-Nova Inc., Rhode Island, USA). Using a Picoscope 2204 Oscilloscope (Pico Technology,

Cambridgeshire, UK), the amplitude of basal tonus changes was recorded. Tissues were first equilibrated in physiological saline for 10 minutes followed by application of Rhopr-kinin 2 or the kinin analog 2139[F1]wp-2. 100 μl of various concentrations of Rhopr-kinin 2 or the kinin analog were applied to the bath concurrent with removal of 100 μl of saline to ensure the bath volume

82 remained constant. In between doses the preparations were washed with saline and the bath volume was maintained at 200 μl.

Feeding Bioassay

On day 7 post-injection, dsARG-injected and dsRhoprKR-injected insects were weighed before feeding, then placed in a 10 cm diameter glass jar and fed on 20 mL of a warm saline consisting of 0.15 M NaCl and 10-3 M ATP (Lange et al., 1988; Friend, 1965) for 20 minutes.

Following feeding, insects were individually weighed immediately after feeding (time 0), and then placed in individual cubicles. To measure the rate of diuresis, the weights of individual insects were then recorded at 1,2,3, and 4-hour time points. Insects that fed less than 1 times their initial body weight were excluded as they were not considered to have fed successfully. Insects that were also punctured during the weighing process were omitted from the data.

Results

Structure and Sequence Analysis of RhoprKR

Following an in silico analysis of potential kinin GPCRs within the R. prolixus transcriptome, partial sequences of the candidate kinin receptor were amplified using PCR. 5’ and

3’ RACE was then utilized to amplify the open reading frame (ORF) and the 5’ and 3’ untranslated regions (UTR). The length of the ORF of RhoprKR is 1285 bp, which translates to 415 amino acids (Fig. 1A). The ORF spans 8 exons, separated by 7 introns (Fig. 1B). The 5’ UTR is 40 bp, and the 3’ UTR is 387 bp, with two stop codons observed in the 5’ UTR (Fig. 1A). Following synthesis, GPCRs undergo post-translational modifications to ensure complete functionality which include N-glycosylation, palmitoylation of Cys residues, and phosphorylation (Kristiansen, 2004;

Duvernay et al., 2005). Within the RhoprKR transcript, N-glycosylation is predicted to occur at

83 Asn2, and Asn25 (Fig. 1A). Phosphorylation sites are predicted on 4 Thr residues, 2 Tyr residues, and 15 Ser residues, with palmitoylation predicted to occur on Cys338 (Fig. 1A).

Alignment of cloned GPCRs of the kinin family with other R. prolixus receptors reveals considerable identity and conserved features across the kinin receptors but not against GPCRs for other peptide families (Fig 2), including other family A and family B receptors (Lee et al., 2016).

Amongst the aligned kinin receptors there is approximately 42% pairwise identity across the ORFs.

Sequence similarity is high within TM1, TM2, TM3, TM6, and TM7, with TM4 and TM5 showing less similarity amongst the receptors (Table 1). Family A GPCRs are characterized by a DRY (and in some cases DRH and ERY) motif, and an NPxxY domain (Holmes et al., 2000; Capra et al.,

2004; Radford et al., 2004; Pietrantonio et al., 2005; Munk et al., 2016). This motif is seen in all of the aligned family A receptors, while it exists as DRY/DRH in the kinin receptors, but as ERY in the sulfakinin receptors, Rhopr-SKR-1 and Rhopr-SKR-2, and in the CAPA receptor, capa-r1

(Fig. 2). The NPxxY domain is also observed within all of the family A receptors but exists as

NPFIY in the kinin receptors (Fig. 2). Conserved Cys residues present in the first and second extracellular loops, predicted to form a disulphide bridge, are conserved in all of the kinin receptors except for L. stagnalis (Holmes et al., 2000; Radford et al., 2004; Pietrantonio et al., 2005) (Fig.

2). Unlike the TMs, a high degree of similarity is not observed within the N-terminus and C- terminus. However, some conserved Ser and Thr residues within the C-terminus may represent conserved phosphorylation sites across the receptors (Radford et al., 2004).

Spatial Expression of RhoprKR

Transcript expression level of RhoprKR was measured in the CNS and various tissues of

5th instar R. prolixus using qPCR. High expression of the RhoprKR transcript is observed in the

84 CNS and HG (Fig. 3). There are similar transcript expression levels within the FG and PMG, with slightly lower expression in the AMG (Fig 3). Expression of the RhoprKR transcript is also observed in the SG, but expression levels are lower than those seen in the gut (Fig. 3). Low expression is observed in the FB, DV, MTs (Fig 3).

Knockdown of RhoprKR Transcript

To verify the likelihood that this RhoprKR transcript is a kinin GPCR, RNAi was utilized to reduce the transcript expression within 5th instar R. prolixus. Changes in RhoprKR transcript expression was verified in the CNS and hindgut using qPCR, with a reduction in transcript expression of 13% observed in the CNS, and 18% in the hindgut on day 2 post-dsRNA injection.

On day 7 post-dsRNA injection, a 76% reduction in transcript expression was observed in the CNS and a 70% reduction in transcript expression in the hindgut (Supp. Fig.1).

Effects of RhoprKR knockdown on Hindgut Contractions

Following RNAi-mediated knockdown of the RhoprKR transcript in R. prolixus, hindgut contraction assays were performed on insects 7 days-post injection. Both Rhopr-kinin 2 and the kinin analog 2139[F1]wp-2 elicited smaller changes in basal tonus of hindgut contraction from dsRhoprKR-injected insects than dsARG-injected insects (Fig. 4). Relative to dsARG, dsRhoprKR-injected insects had a 43 % reduction in response to 10-8 M Rhopr-kinin 2, with a 54

% reduction in response to a dose of 10-8 M 2139[F1]wp-2 (Fig 4).

Effects of RhoprKR Knockdown on in vivo Feeding and Diuresis

To observe the effects of RhoprKR knockdown on feeding and diuresis, the blood meal size and excretion rate for dsARG-injected and dsRhoprKR-injected insects was examined. In

85 comparison to dsARG-injected insects, dsRhoprKR-injected insects had consumed a significantly larger meal, feeding an average of 12 times their initial body weight (Fig 5A). In addition, a higher number of dsRhoprKR-injected insects fed on a meal much greater than their body weight, with

17 insects feeding over 10 times their body weight. For dsARG-injected insects, 9 insects fed over

10 times their body weight, while 7 insects fed 2-4 times their body weight (Fig. 5B). As post- feeding diuresis occurs over a time period of 3-4 hours following feeding, the weight loss due to excretion was monitored for the dsARG-injected and dsRhoprKR-injected insects to examine changes in diuresis rate. Despite a difference in artificial blood meal size, there was no difference in the rate of diuresis over the measured 4 hours (Fig 5B)

Fig. 1: A) The cDNA sequence of RhoprKR with the predicted amino acid sequence. Nucleotide number is on the right of the sequence, with amino acid number bolded below the nucleotide numbers. Predicted transmembrane domains within the sequence are highlighted in grey. Stop codons in the 5’UTR are boxed in green. Predicted N-glycosylation sites are shaded light blue and underlined, potential phosphorylation sites boxed in black, and a potential palmitoylation of Cys residue in green. B) The exon map of RhoprKR, with the open reading frame (ORF) and exons/introns. The ORF of RhoprKR is denoted by the solid black box spanning the exons, with numbers above exons representing exon length and numbers below introns representing intron length.

A) 5’-tactatctgtcatgtgtaatacgtcatctaactaaagatt 40 atg aac tgc agt ttt cta gaa gac gaa cta ggg cct tta cca cca agc gca aat tgt tca 100 M N C S F L E D E L G P L P P S A N C S 20 tgg ctc ctt cac aat caa tca gtg tat ttc tat gaa gaa agt ctg tac gaa gtt cct gcc 160 W L L H N Q S V Y F Y E E S L Y E V P A 40 ggt gta ata gta tta cta tca gta ttc tat ggc acg ata tct gtg gtg gca gtt ggc gga 220 G V I V L L S V F Y G T I S V V A V G G 60 aat ttt ctg gtt atg tgg att gtg gcc act agt aga cgc atg cag aat gtt act aac tgt 280 N F L V M W I V A T S R R M Q N V T N C 80 ttt ata gca aat cta gcc ttg gct gat ata gtt atc ggc cta ttc gct ata cca ttt caa 340 F I A N L A L A D I V I G L F A I P F Q 100 ttc caa gca gca ctg ttg cag agg tgg aac ctg cca aat ttt atg tgc cct ttt tgc cca 400 F Q A A L L Q R W N L P N F M C P F C P 120 ttc gtc cag gtg ctc agc gtc aat gtt agt gta ttc aca ctg acg gcc att gct gta gat 460 F V Q V L S V N V S V F T L T A I A V D 140 aga cat cgg gca gtc ctt aac cca tta agt gct cca ccc tca aaa tta aga gct aaa gct 520 R H R A V L N P L S A P P S K L R A K A 160 tta ttg ggc gct att tgg att ttg gct gct atc ctt gca aca cca atg gct gta gca tta 580 L L G A I W I L A A I L A T P M A V A L 180 aat gta act tat gtt gaa gaa aat gat cat gtt ggt cat gtt tac acc aaa cca ttt tgt 640 N V T Y V E E N D H V G H V Y T K P F C 200 ata aat aca aaa ctg tca aat aac cat atg atg gcc tac agg atg ata ttg gta tca gtc 700 I N T K L S N N H M M A Y R M I L V S V 22 760 caa tat ctg aca ccg ttg tgt gtc ata tca tat gcc tat gca aaa atg gca ctc agg ttg Q Y L T P L C V I S Y A Y A K M A L R L 240 tgg gga tca aga gct cct ggt aat gct caa cat tct agg gat gca aat tta atg aga aat 820 W G S R A P G N A Q H S R D A N L M R N 260 aag aaa aag gta atc aaa atg tta gtt ata gtg gtg gct cta ttt gca ata tgt tgg ctt 880 K K K V I K M L V I V V A L F A I C W L 280 cca tta caa act tac aat gtt tta caa gac ata ttt cca caa att aat ggg tac aga tat 940 P L Q T Y N V L Q D I F P Q I N G Y R Y 300

att aat ata att tgg ttt tgt tgt gat tgg ctg gca atg agt aat tcc tgt tac aat ccg 1000 I N I I W F C C D W L A M S N S C Y N P 320 ttt ata tac ggt att tac aat gaa aag ttt aag caa gaa ttt caa caa cga tgc ccg ttc 1060 F I Y G I Y N E K F K Q E F Q Q R C P F 340 agt aga aga aga aag tgg act cat ggt ttt ggt gcc ggt ggt agt gac agt tta gat cta 1120 S R R R K W T H G F G A G G S D S L D L 360 gat aaa aca ata cat cgt ttt ggt agt gtt aac cgt aat tcg tct cgc tgg ata aga tac 1180 D K T I H R F G S V N R N S S R W I R Y 380

tca tcc cgt gtg caa tat act cca gca caa cat tac att tac cac tgt gcc aat tcc aat 1240 S S R V Q Y T P A Q H Y I Y H C A N S N 400 act gta cac cac agt tcc caa tca gaa ata gaa gag ctt tgt ctg taa 1288 T V H H S S Q S E I E E L C L - 416

atactattattacaattaacttgtgaacatcttgcttcagaaatattatgcagtgtaacaataacttttggacattttttatgaaccaaaat ttttacaaatatctgtagatttagtgacgaaattattgtcggaattcacaattgtattttcacattttcaattggaattctaaaatgaaatg aattaggtattcgtggtacgtttccttatattttcctgcttctgaagttaggtgtgcataccctcgaaaatcattaacaagtgtgatgtatt accctaaagagctagaaaatctctaatggacagcatttaactgaatataatttttggggcacatttaaataaaaccatttatctcaaatgag 16751675 tcgctgtacaaatgtaaaa-3’

35 105 194 150 126 217 102 92 654 bp

bp 107098 19390 14682 1348 177 851 105 187

Fig. 2: Multiple sequence alignment of cloned invertebrate kinin receptors and various cloned R. prolixus receptors. Sequences shaded in black represent identical amino acids across sequences and 60% similar sequences are shaded in grey. Bolded red lines above sequences represent predicted RhoprKR transmembrane domains. The conserved DRY/DRH motif, NPxxY domain, and conserved Cys residues are boxed in red. Abbreviations: Rhopr-CRF/DH-R2: R. prolixus

CRF/DH receptor 2 (A0A191UP91); L. stagnalis: Lymnaea stagnalis kinin receptor (P92045); D. melanogaster: Drosophila melanogaster kinin receptor (Q9VRM0); A. stephensi: Anopheles stephensi kinin receptor (Q69V6); A. aegypti: Aedes aegypti kinin receptor (Q5EY37); R. microplus: Rhipicephalus microplus kinin receptor (Q9NHA4); RhoprCAPA-R1: R. prolixus

CAPA receptor variant A (D6P3E5): Rhopr-SKR-1: R. prolixus sullfakinin receptor 1

(MK513659); SKR-2: R. prolixus sullfakinin receptor 2 (MK513660).

89 Rhopr-CRF/DH-R2 1 -----GLLCWPNTPPGVTAYLPCV------AEIDNVKYDTNQNASRICYENGTWANQTD L. stagnalis 1 MSQIESMSEQA---AVI--FIEQANQDLDNVSGNDVSSFFYNET------TTLF---- D. melanogaster 1 ------MAMDLIEQE--SR------LE-FLP-- A. stephensi 1 MQ------ATDITAYHTAYN------YTLNQS-- A. aegypti 1 MR------AVDGIAFHYANN------NTLNGS-- R. microplus 1 MTSLPGMTLDPSAPPPL--LLDSS------YVSPDYGN------LSLLSS-- RhoprKR 1 ------MNCS------FLEDELGPLPPSAN------CSWLLH-- RhoprCAPA-R1 1 ------MNSFDIIETVT-NS------TPVNVS-- Rhopr-SKR-1 1 ------Rhopr-SKR-2 1 ------MR-NN------TEATVQ--

Rhopr-CRF/DH-R2 49 YGLCSELHTLTSNQILSDE--G-IIVQSTIYAVGYGFSLTALGLAVWIFLYY------L. stagnalis 44 ------PGSNESFVMPYDVPTGLICLLAFLYGSISLLAVIGNGLVILVIVKNRRMHTVTN D. melanogaster 17 -----GAEEEAEFERLYAAPAEIVALLSIFYGGISIVAVIGNTLVIWVVATTRQMRTVTN A. stephensi 21 -----DVRIVLEDENLYKVPIGLLVLLSIFYGTISILAVIGNSLVIWIVITTKQMQTITN A. aegypti 21 -----DVEIVKEQDALYDVPVGLVVLLSIFYGTISIIAVIGNSLVIWIVLTTKQMQTITN R. microplus 37 -----LPAANISSNKLYQVPVGFIVLLSIFYGIISLVAVAGNFMVMWIVATSRRMQTVTN RhoprKR 25 -----NQSVYFYEESLYEVPAGVIVLLSVFYGTISVVAVGGNFLVMWIVATSRRMQNVTN RhoprCAPA-R1 20 -----LEEYLIIVRGPKFLPLKILLPITFTYGILFISGLFGNLAVCIVIAYNKSMHNATN Rhopr-SKR-1 1 ------MLPNESWWEAGKVQIPTYSIIFLLGLVGNILVILVLVKNKGMRTVTN Rhopr-SKR-2 11 -----PKSTSTTNTGGSDNGSGISELMIPLYMLIFILAVVGNSLVLATLTRNRRMRTVTN

Rhopr-CRF/DH-R2 98 ------KDLWCLRNTIHTNLMCTYILAD------LMWILSSIQ L. stagnalis 98 IFIPNLAVSDVIIGLFSIPFQFQAA-LLQRWVLANFMSSLPPFVQVVTVNLTIFTLRVIA D. melanogaster 72 MYIANLAFADVIIGLFCIPFQFQAA-LLQSWNLPWFMCSFCPFVQALSVNVSVFTLTAIA A. stephensi 76 MFIANLALADVTIGVFAIPFQFQAA-LLQRWNLPEFMCPFCPFVQLISVNVSVFTLTAIA A. aegypti 76 MFIANLALADVTIAVFAIPFQFQAA-VLQRWNLPEFMCPFCPFVQLLSVNVSVFTLTAIA R. microplus 92 FFIANLAVADIIIGLFSIPFQFQAA-LLQRWVLPEFMCAFCPFVQVLSVNVSIFTLTAIA RhoprKR 80 CFIANLALADIVIGLFAIPFQFQAA-LLQRWNLPNFMCPFCPFVQVLSVNVSVFTLTAIA RhoprCAPA-R1 75 YYLFSLAMSDLVLLLLGLPNDLSVFWQQYPWILGLLVCKLRALVSEMSSYVSVLTIVAFS Rhopr-SKR-1 48 VFLLNLAVSDILLGVLCMPFTLVGS-LLKDFVFGHFMCRLIPYMQACSVAVSGWTLVCLS Rhopr-SKR-2 66 VYLFNLAVADILLGVFCMPFTLIGQ-LLRNFVFGRIMCKLIPYFQAVSVSVAVWTLVAIS

Rhopr-CRF/DH-R2 129 VYVKTDPAICMVLFI-----LLHYLILTNYFWMFVEGLYL------L. stagnalis 157 --VDRYIAVIHPFKAGCSK--KRAAIIISIIWAVGIGAALPVPLFYWVEDLTE------D. melanogaster 131 --IDRHRAIINPLRARPTK--FVSKFIIGGIWMLALLFAVPFAIAFRVEELTER-F-REN A. stephensi 135 --VDRHRAIINPLRARTSK--NISKFVISSIWMLSFVLAAPILFALRVRPVSYIALGGMN A. aegypti 135 --VDRHRAIINPLRARASK--NISKFVISAIWMMSFALAAPTLFALRVVPVSIVSLGETN R. microplus 151 --LDRYRAVMSPLKARTTK--LRAKFIICGIWTLAVAAALPCALALRVETQV-----E-- RhoprKR 139 --VDRHRAVLNPLSAPPSK--LRAKALLGAIWILAAILATPMAVALNVTYVE-----END RhoprCAPA-R1 135 --VERYTAICYPLKSYTTDKLNRVIKVIGTLWLISLGFAAPFAIYTTIDYVDFPPG---- Rhopr-SKR-1 107 --VERYYAICHPLRSRTWQTLTHAYRLIGAIWVCSLLLMTPISVLSELIPT---S----- Rhopr-SKR-2 125 --LERYFAICRPLKSRRWQTQFHAYKMIAIVWAMSLVWNSPILFVSRLLAM---GG----

Rhopr-CRF/DH-R2 164 ------YMLVVETFTRENINLRAYLAIGWG L. stagnalis 206 ---NNIVIPRCDWHAPDNWLDFHLYYNTLLVCFQYLLPLVIITYCYCRIAWHIWGSRRPG D. melanogaster 185 NETYNVTRPFCMNKNL--SDDQLQSFRYTLVFVQYLVPFCVISFVYIQMAVRLWGTRAPG A. stephensi 191 DTYTNITVPFCKVVNF--EDGEILLYRYVLVLVQYFIPLFVISFVYIQMALRLWGSKTPG A. aegypti 191 ETYINMTKPFCQVVNF--EESEMLLYRYILTLVQYFVPLCVISFVYIQMALRLWGSKTPG R. microplus 200 SHALNLTKPFCHEVGI--SRKAWRIYNHVLVCLQYFFPLLTICFVYARMGLKLKESKSPG RhoprKR 190 HVGHVYTKPFCINTKL--SNNHMMAYRMILVSVQYLTPLCVISYAYAKMALRLWGSRAPG RhoprCAPA-R1 189 SGKAVIESAFCAMLKQ--NVPADVPLYELSCTLFFICPAVILIFLYVRIGL------Rhopr-SKR-1 157 GGHRK-----CRELWP--NEDIEKTYNLLLDFLLLVIPLIVMVTTYTLVAKTLWRVMKTQ Rhopr-SKR-2 176 KGRHK-----CREVWP--GRRSEGAYIIFLDIVLLMIPLLIMSLAYSLIVLKLWKGLQRE

Rhopr-CRF/DH-R2 188 IPVIIVIPSCLARA------FISDDYE-- L. stagnalis 263 ------A------D. melanogaster 243 ------N------A. stephensi 249 ------N------A. aegypti 249 ------N------R. microplus 258 ------N------RhoprKR 248 ------N------RhoprCAPA-R1 238 ------Rhopr-SKR-1 210 KPG---NEMGLK------DNRVT Rhopr-SKR-2 229 LKH---SNSCLQTVDRSASLPTMTEVVISKNLNTNSEAIHRVIPADSQQGQFCNKNPVQM

Rhopr-CRF/DH-R2 209 YVL------ITKLRSSNNA-ETQQYRKATKALLVLIPLL L. stagnalis 264 ------HVTTEDV-RGRNKRKVVKMMIIVVCLF D. melanogaster 244 ------AQDSRDITLLKNKKKVIKMLIIVVIIF A. stephensi 250 ------AQDSRDITMLKNKKKVIKMLIIVVALF A. aegypti 250 ------AQDSRDMTMLKNKKKVIKMLIIVVALF R. microplus 259 ------AQGARDAGILKNKKKVIKMLFVIVALF RhoprKR 249 ------AQHSRDANLMRNKKKVIKMLVIVVALF

RhoprCAPA-R1 238 ------TIKNNTKL------RGNVHGELQSIQSKKSIVSMLMAVVVAF Rhopr-SKR-1 224 WKQN------SRG------SPHLRRSNTEKALKKKKRVVKMLFAVVLEF Rhopr-SKR-2 286 WLLKVKLEEGIAQRSGTPLEPLESPGPKFTRHAIRSNYMDKS IEAKKKVIRMLFVVVAEF

Rhopr-CRF/DH-R2 241 GVTYI------LFIAGPTEGPYAYLFS---YIRAFLLSTQGLMVALLYCFLNTEVQNTV L. stagnalis 290 VLCWLPLQMYNLLHNINPLINHYHYINI-IWFSSNWLAMSNSCYNPFIYGLLNEKFKREF D. melanogaster 271 GLCWLPLQLYNILYVTIPEINDYHFISI-VWFCCDWLAMSNSCYNPFIYGIYNEKFKREF A. stephensi 277 GVCWFPLQLYNILHVTWPEINEYRFINI-IWFVCDWLAMSNSCYNPFIYGIYNEKFKREF A. aegypti 277 GICWFPLQLYNILHVTWSEVNEYRYINI-IWFVCDWLAMSNSCYNPFIYGIYNEKFKREF R. microplus 286 AFCWLPYQLYNILREVFPKIDKYKYINI-IWFCTHWLAMSNSCYNPFIYAIYNERFKREF RhoprKR 276 AICWLPLQTYNVLQDIFPQINGYRYINI-IWFCCDWLAMSNSCYNPFIYGIYNEKFKQEF RhoprCAPA-R1 274 FICWAPFHMQRLIYVYMSDYPWYGIVNVWLYYISGIFYYFSATINPILYNLMSLKYRKAF Rhopr-SKR-1 261 FVCWTPLYVINTITLFAPQAVYERLGYK-GISFLQLLAYSSSCCNPITYCFMNYRFRRAF Rhopr-SKR-2 346 FICWAPLHVLNTWYQFRPDLVHQYVGST-GVSLVQLLAYISSCCNPITYCFMNYRFRQAF

Rhopr-CRF/DH-R2 291 RHHFTRWKESRNLGARRYTCSRDWSPNTRTESVRLCSKHDVMPYRKRESVASENTTMTLV L. stagnalis 349 HQLFVMCPCWKARVDYYT-----EY---FSEDANICRRANTNGHCPANRHGAVGTTSTET D. melanogaster 330 NKRFAACFCKFKTSM------DAHERT------FSMHTRASSIRSTY A. stephensi 336 RKRYPFKR-DQTYNHNHE------SDKT------SSIFTRVSSIRSTY A. aegypti 336 HKRYPFRGRNQSYHQEQL------TDKT------LSMFTRVSSIRSNY R. microplus 345 ATRCTCGGHRYKSPKS------RFASYEQEDNST RhoprKR 335 QQRCPFSRRRKWTHGFGA-----GGSDSLDLDKT------IHRFGSVNRNSSRW RhoprCAPA-R1 334 KQTLWCRKYNRIIKTPGL-----RETNSTSRQVNK------SIKSMNMQHNQS----LL Rhopr-SKR-1 320 LKLFGCLREEKGSS------Rhopr-SKR-2 405 ISLFNFPRLCCWCGIPVE-----SKLAQRTDTAN------EPNSLSANDS---TLY

Rhopr-CRF/DH-R2 351 GGSTNLARLS------L. stagnalis 401 TRKSMLSR------SRCKGTRRR- D. melanogaster 365 ANSSMRIRSNLFGPARGGVNNGKPGLHMPRVHGSGANSGIYNGSSGQ--NNNVNGQHHQH A. stephensi 371 ATSSIRNKLST-NRYSASKQFKFPPPNHHFQHQPG---GHHNATGGAHLHELAFGTSK-- A. aegypti 372 ATSSIRNKLYT-GPIGGGSGN------G---GTHVGSGY---SSNAFYQNQNS R. microplus 373 IIVSM------RHS------FRLSFKN--SAPLKASTQV------RhoprKR 378 IRYSS------RVQYTPAQHYIYHCAN--SNTVHHSSQSEIEELCL------RhoprCAPA-R1 378 ANN------IED------Rhopr-SKR-1 ------Rhopr-SKR-2 447 AGRANRSEVMVLEK------EER------

Rhopr-CRF/DH-R2 ------L. stagnalis 418 --RQTYDERRETSS------D. melanogaster 423 QSVVTFAATPGVSAPGVGVAMPPWR-----RNNFKPLHPNVIECEDDVAL------MELP A. stephensi 425 KGPVNFDGTVT---TTFATNHPREKKMDHRLVE---HDQLIASCIERLD-----HELACS A. aegypti 412 HHQQSYKSPNTNSVAGYQRNSTTDRNSSRKTAAGAPWDPKCCPCRQNSTRTSTAAASACP R. microplus ------RhoprKR ------RhoprCAPA-R1 384 ---IT------Rhopr-SKR-1 ------Rhopr-SKR-2 464 ---V------

Rhopr-CRF/DH-R2 ------L. stagnalis ------D. melanogaster 472 STTPPSEELASG------AGVQLALLSRESSSCICEQEFGSQTECDGTCILSE A. stephensi 474 STVDSSEDHRNGEPRTLNRPDIDGNGTGRAAKLRNGSS-----RE----CGLSIASNYAD A. aegypti 472 YRMPLPAVASDGDSGSEGGP-CNSAGGGQSPMINNDER-----QLLGADDNYGSAAQKLE R. microplus ------RhoprKR ------RhoprCAPA-R1 ------Rhopr-SKR-1 ------Rhopr-SKR-2 ------

Rhopr-CRF/DH-R2 ------L. stagnalis ------D. melanogaster 519 VSRVHLPGSQAK------DKDAGKSLWQPL------A. stephensi 525 RMALKHPHPDSGGESGDGEPKPGQRSSEERDSGGHLYCNDLEELGPYYD------A. aegypti 526 VISLDHPHPDSADDENGVAETPHSRTANGQEQDERLQLTSFISSGNGRHERFHFHINNL R. microplus ------RhoprKR ------RhoprCAPA-R1 ------Rhopr-SKR-1 ------Rhopr-SKR-2 ------

Table 1: Similarity within transmembrane domains (TMs) of cloned invertebrate kinin receptors based on sequence alignments carried out using the MUSCLE alignment tool and later scored on the BLOSUM62 matrix.

TM1 TM2 TM3 TM4 TM5 TM6 TM7 Percent Pairwise 71% 76% 75% 43% 52% 69% 87% Identity

Percent Identical 47% 56% 47% 21% 26% 50% 73% Sites

Fig. 3: Expression profile of the RhoprKR transcript in the CNS and various feeding-related tissues. Transcript expression is shown for the following tissues: CNS, fat body (FB), dorsal vessel

(DV), salivary glands (SG), foregut (FG), anterior midgut (AMG), posterior midgut (PMG), hindgut (HG), and Malpighian tubules (MT). Transcript expression is relative to an average of the three reference genes (α-tubulin, β-actin, ribosomal protein 49). Data represents the mean ± SEM of three biological replicates per tissue, with three technical replicates for each biological replicate.

Fig. 4: The effects of RhoprKR transcript knockdown on R. prolixus hindgut contractions in response to Rhopr-kinin 2 and the kinin analog 2139[F1]wp-2. Hindgut contractions were performed on R. prolixus 7 days post-dsRNA injection. A) Changes in basal tonus in response to varying concentrations of Rhopr-kinin 2 in dsARG-injected and dsRhoprKR-injected insects. B)

Changes in basal tonus in response to varying concentrations of 2139[F1]wp-2 in dsARG-injected and dsRhoprKR-injected insects (One-way ANOVA followed by Tukey’s post-hoc test, *=p<0.05.

Data are means ± SEM of n = 5).

Fig. 5: The effects of RhoprKR transcript knockdown on in vivo feeding and diuresis in R. prolixus.

Weights of insects were measured after 20 minutes of feeding on saline supplemented with ATP

(time 0), and at 1 hour increments post-feeding for 4 hours. A) Weights of dsARG-injected and dsRhoprKR-injected insects before and after artificial blood-feeding (time 0). B) The amount of meal size times body weight consumed by dsARG-injected and dsRhoprKR-injected insects. C)

The rate of post-feeding diuresis of dsARG-injected and dsRhoprKR-injected insects. (Student’s t-test, slopes tested for significance using an F-test. *=p<0.05. Data are means ± SEM of n=19-

23).

98 Discussion

The R. prolixus kinin receptor (RhoprKR) has been successfully cloned, and its sequence, structure, and expression profile analyzed. Examining the kinin signaling pathway within R. prolixus is quite significant, due to its direct role in Chagas disease transmission (Bern et al., 2011).

For example, Rhopr-kinin 2, and its analog 2139[F1]wp-2 have been previously shown to be potent in inducing hindgut contractions, a behaviour required for the excretion of urine (Bhatt et al., 2014; see chapter 2).

Following analysis of its sequence and predicted structure, the RhoprKR transcript codes for a kinin GPCR, belonging to the rhodopsin-like (family A) family of GPCRs. In comparison with other invertebrate kinin receptors, there is a high degree of similarity between the sequences.

The predicted transmembrane domains within the kinin receptors are highly conserved in size and spacing, but this conservation is not observed within TM4 and TM5, which was also observed by

Radford et al. (2004). The DRY/DRH/ERY motif is characteristic of all family A GPCRs which is important in conformational changes for receptor activation (Capra et al., 2004; Rovati et al.,

2007). This motif is present as DRH within all of the insect kinin receptors analyzed, while in L. stagnalis and R. microplus, it is present as DRY (Cox et al., 1997; Radford et al., 2002; Holmes et al., 2002; Radford et al., 2004; Pietrantonio et al., 2005). In Rhopr-SKR-1, Rhopr-SKR-2 and capa-r1 this motif exists as ERY (Paluzzi et al., 2010). The NPxxY domain within TM7, required for inducing key structural changes within the receptor, is found in all family A GPCRs (Fritze et al., 2003) and is observed in all of the kinin receptors as NPFIY but extends into the C-terminus in R. prolixus and D. melanogaster. In addition, the first extracellular loop may be involved in the binding of the kinin ligand, as it is also highly conserved (Radford et al., 2004). Disulphide bridges are essential for the structural integrity of the receptor (Knudsen et al., 1997), with the Cys residues

99 that likely form this disulphide bridge conserved across all of the analyzed kinin receptors but is not seen in the receptor sequence of the snail, L. stagnalis (Radford et al., 2004). Phosphorylation, mediated by various protein kinases, are essential for various functions within GPCRs such as internalization of the receptor and desensitization (Ferguson, 2001; Tobin, 2008). Ser and Thr residues, which are common sites of phosphorylation by these kinases, appear to be fairly conserved suggesting that these are common phosphorylation sites in kinin GPCRs (Radford et al.,

2004). In many invertebrate kinin receptors, receptor activation results in increases in intracellular

Ca2+ (O’ Donnell et al., 1998; Cady & Hagedorn, 1999; Tehrzaz et al., 1999; Cox et al., 1997). It is expected that RhoprKR utilizes intracellular Ca2+ as a second messenger at the hindgut of R. prolixus, but further functional studies need to be performed to confirm this.

The feeding strategy of R. prolixus requires the coordination of various neuroendocrine factors to undergo post-prandial diuresis and excretion, and thereby rid the insect of excess water and salts following a large blood meal (Orchard, 2006; Coast et al., 2002; Orchard, 2009). During the production of primary urine, various diuretic and antidiuretic hormones act upon the anterior midgut and MTs in order to facilitate the fluid and ion absorption and secretion required to maintain osmotic balance (Coast et al., 2002; Maddrell, 1969). Kinins have been identified to have myostimulatory effects on the anterior midgut but have no role in fluid transport within R. prolixus

(Te Brugge et al., 2009). An increase in the frequency of anterior midgut contraction that occurs during feeding may assist in the mixing of the blood meal within the gut and flow of haemolymph, thereby assisting in fluid absorption that occurs from the anterior midgut to the haemolymph

(Maddrell, 1964; Te Brugge et al., 2009). Expression of the RhoprKR transcript was observed in the anterior midgut, which supports this myotropic role of Rhopr-kinins. While not directly involved in the post-feeding diuresis or excretion, the salivary glands are responsible for the

100 secretion of substances during feeding into the host to ensure successful blood uptake, as these substances prevent platelet aggregation, coagulation, and vasoconstriction within the host’s blood vessels (see Orchard & Te Brugge, 2002). Kinins have been shown to induce dose-dependent increases in salivary contractions, mediating the release of these substances during blood feeding

(Orchard & Te Brugge, 2002). As expected therefore, transcript expression of RhoprKR is seen within the salivary glands.

Expression of the RhoprKR transcript was also observed in the foregut and posterior midgut, and is higher in these tissues than that observed in the anterior midgut. During feeding, the foregut functions to assist in the movement of the blood meal to the anterior midgut, where the initial fluid absorption in the diuretic process occurs (Cooper & He, 1994; Te Brugge et al., 2009).

Like many other feeding and diuresis-related visceral muscle tissues, contraction and relaxation of the foregut has been found to be under neuroendocrine control within various insects (Audsley &

Weaver, 2009). In Periplaneta americana, tachykinins were found to induce foregut contractions, with proctolin and serotonin [5-hydroxytryptamine (5-HT)] inducing foregut contractions in

Teleogryllus commodus (Nässel et al., 1998; Cooper & He, 1994). In contrast, FGLamide-related allostatins were shown to inhibit foregut contractions in L. maderae and L. migratoria (Duve et al., 1995). The neuroactive chemicals that may be involved in modulating foregut contractions have not been studied in R. prolixus. As expression of RhoprKR has been identified in the foregut,

Rhopr-kinins may also play a role in modulating movement of the foregut.

In contrast to the anterior midgut, the posterior midgut does not have a direct diuretic function within R. prolixus. It instead aids in the digestion of the blood meal, as various digestive enzymes have been identified within this tissue that aid in nutrient breakdown (Billingsley &

101 Downe, 1983). The transcript expression profile of RhoprKR suggests that Rhopr-kinins also act upon the posterior midgut, possibly inducing contraction or aiding in blood digestion. As seen in other tissues within R. prolixus, Rhopr-kinins likely elicit myotropic effects on this tissue. Kinin- like immunoreactivity has been previously observed within endocrine cells in the posterior midgut

(Te Brugge et al., 2001). Within insects, contractions of the posterior midgut have also been found to be under the influence of various neuroendocrine factors. Within Diploptera punctata, proctolin and leucomyosuppressin alter contractions of the posterior midgut, albeit to a lesser degree than the anterior midgut (Fusé & Orchard, 1998). Similarly, in Schiscostera migratoria, innervation using FMRFamide-related peptides in seen in the posterior midgut, with SchistoFLRFamide, leucomyosuppressin, and ManducaFLRFamide inhibiting spontaneous and proctolin-induced contractions of midgut muscle (Lange & Orchard, 1998). Within D. melanogaster larvae, immunoreactivity for a kinin receptor has also been observed in cells of the posterior midgut

(Veenstra, 2009), suggesting a possible role of kinins in posterior midgut function. The presence of RhoprKR throughout the entire alimentary canal suggests that Rhopr-kinins may have a broader role in feeding regulation. Relative to other analyzed tissues, expression levels of the RhoprKR transcript was negligible in the MTs, fat body and dorsal vessel.

From an agrochemical perspective, identification of the RhoprKR sequence allows the utilization of novel strategies to target the kinin signaling system within R. prolixus. Successful

RNAi-mediated knockdown of the RhoprKR transcript was found to have a significant impact on hindgut contractions, as insects injected with dsRhoprKR had a reduced response to Rhopr-kinin

2 and the analog 2139[F1]wp-2. In addition, dsRhoprKR-injected insects had a significantly larger meal compared to dsARG-injected insects, with a majority of dsRhoprKR-injected insects consumed a meal that was over 10 times their body weight. These results suggest that Rhopr-kinins

102 may also function as satiety factors, as Aib-containing kinin analogs have been previously described to induce antifeedant effects in R. prolixus (Lange et al., 2016). Within R. prolixus, several neuroendocrine factors such as sulfakinin (Rhopr-SK-1) and corticotropin-releasing factor

(CRF)- like diuretic hormone (Rhopr-CRF/DH) have been identified in influencing feeding (Al-

Alkwai et al., 2017; Mollayeva et al., 2018) As co-localization of Rhopr-kinins and Rhopr-

CRF/DH has been observed within the CNS and midgut of R. prolixus, they may co-operatively function to induce satiety following a blood meal (Te Brugge et al., 1999; Te Brugge et al., 2001;

Te Brugge et al., 2002; Mollayeva et al., 2018). However, despite the presence of kinin receptors throughout the gut of R. prolixus, there were no significant differences in excretion rate between dsRhoprKR-injected and dsARG-injected insects. The lack of significant differences on excretion rate may be due to a partial knockdown of the RhoprKR transcript. The RhoprKR transcript is one of many targets within R. prolixus that result in altered phenotypes due to the disruption of critical signaling systems. Successful RNAi has been observed in a diverse set of tissues within R. prolixus, all of which have resulted in physiological and behavioural changes that may interfere with normal functioning. RNAi-mediated knockdown of the adipokinetic hormone receptor resulted in an increased level of lipid content in the fat body of R. prolixus insects, and a decrease in lipid levels in the haemolymph, due to the disruption of lipid mobilization (Zandawala et al.,

2015). The receptor for corazonin (CRZ), a neuropeptide implicated in dorsal vessel regulation in insects, was targeted using RNAi, resulting in a reduced basal heartbeat, with a reduced response to CRZ (Hamoudi et al., 2016). As the dorsal vessel is responsible for the circulation of haemolymph within the insect, this knockdown can compromise the timely circulation of essential endocrine factors. Development of novel pest-control strategies surrounding RNAi can prove to be quite efficacious in preventing the spread of Chagas disease. On a larger scale, utilizing symbiont-mediated RNAi can prove to be a successful next-generation pest control strategy

103 (Whitten et al., 2016). The efficacy of dsRhoprKR makes it an excellent candidate to be integrated into essential gut bacteria, thereby knocking down signaling pathways that could ultimately prevent the spread of the T. cruzi parasite through excretion (Whitten et al., 2016; Bern et al.,

2011).

In summary, based on its sequence similarity and expression profile, the RhoprKR transcript encodes for an insect kinin receptor. Expression of RhoprKR in tissues such as the foregut and posterior midgut indicate that Rhopr-kinins may have novel myotropic effects on these tissues within R. prolixus. The effective knockdown of RhoprKR through RNAi, and the resultant influence on kinin-stimulated hindgut contractions and feeding make dsRhoprKR a viable dsRNA that can be incorporated in pest-control strategies, such as symbiont-mediated RNAi. Identification of RhoprKR allows further investigation of the Rhopr-kinin signaling pathway, a neuropeptide pathway that is closely associated with behaviours related to Chagas disease transmission.

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110 Supplementary Material

Supp. Fig. 1: Analysis of RhoprKR transcript expression following dsRhoprKR injection.

Reduction in transcript expression within the CNS and hindgut is shown for dsRhoprKR-injected insects 2 days and 7 days post-dsRNA injection. Reduction in transcript expression is relative to

RhoprKR transcript expression in dsARG-injected insects. Data represents the mean ± SEM of three biological replicates per tissue, with three technical replicates for each biological replicate.

Table S1: Primers used for the amplification of RhoprKR sequence fragments

Primer Name Primer Sequence (5’ to 3’)

KRFW1 GCGCAAATTGTTCATGGCTC

KRFW2 AAAGTCTGTACGAAGTTCCTGC

KRFW3 AAGCTTTATTGGGCGCTATTTG

KRFW4 GGTGGCTCTATTTGCAATATGT

KRFW5 AGCAAGAATTTCAACAACGATG

111 KRRV1 GCAAAAAGGGCACATAAAATTT

KRRV2 CATTTAATGCTACAGCCATTGG

KRRV3 ATGCGTCTACTAGTGGCCAC

KRRV4 TGGCCGTCAGTGTGAATACA

KRRV5 GCCATTGGTGTTGCAAGGAT

Table S2: RACE Primers for the amplification of 5’ and 3’ regions of RhoprKR

Primer Name Primer Sequence (5’ to 3’)

KR5RACE1 ATTCTGCATGCGTCTACTAGTGGCCAC

KR5RACE2 TTACACCGGCAGGAACTTCGTACAGA

KR3RACE1 GTCCAGGTGCTCAGCGTCAATGTTAGT

KR3RACE2 GACGGCCATTGCTGTAGATAGACATCG

KR3RACE3 GCTATCCTTGCAACACCAATGGCTGTAG

Table S3: Primers used for RhoprKR dsRNA synthesis

Primer Name Primer Sequence (5’ to 3’)

dsRhoprKR

KRRNAIFW CGGCCTATTCGCTATACC

KRRNAIFWT7 TAATACGACTCACTATAGGGAGACGGCCTATTCGCTATACC

KRRNAIRV GGAATTACTCATTGCCAGCC

KRRNAIRVT7 TAATACGACTCACTATAGGGAGAGGAATTACTCATTGCCAGCC

dsARG

ARGFW AATAGTTTGCGCAACGTTG

112 ARGRV ATGAGTATTCAACATTTCCGTGTC

ARGFWT7 AATAGTTTGCGCAACGTTGTAATACGACTCACTATAGGGAGA

ARGRVT7 ATGAGTATTCAACATTTCCGTGTCTAATACGACTCACTATAGGGAGA

Table S4: Primers used for qPCR analysis

Primer Name Primer Sequence (5’ to 3’)

RhoprKR

KRQPCRFW TGCTCCACCCTCAAAATTAAGA

KRQPCRR ACCAACATGATCATTTTCTTCAACA

Rhopr-α-tubulin

FW GTGTTTGTTGATTTGGAACCTACAG

RV CCGTAATCAACAGACAATCTTTCC

Rhopr-β-actin

FW AGAGAAAAGATGACGCAGATAATGT

RV ATATCCCTAACAATTTCACGTTCG

Rhopr-ribosomal protein 49 FW GTGAAACTCAGGAGAAATTGGC

RV AGGACACATGCGTATC

Chapter 4 General Discussion Feeding

The regulation of feeding behaviour is under the control of various neuropeptides within insects (Audsley & Weaver, 2009), and here, the kinin and CAPA neuropeptides and their analogs have been examined on feeding in Rhodnius prolixus

Kinin

Within R. prolixus, kinins are co-localized with corticotropin-releasing factor (CRF)- like diuretic hormone (Rhopr-CRF/DH) within neurosecretory cells, with a decrease in staining at 2.5 hours after feeding (Te Brugge et al., 1999; Te Brugge et al., 2001; Te Brugge et al., 2002;

Mollayeva et al., 2018). As CRF/DH has been associated with feeding regulation in R. prolixus

(Mollayeva et al., 2018), these staining patterns suggested a role of kinins in feeding-related behaviours. In Chapter 2, it was found that insects injected with the Aib-containing kinin analog

2139[Ф1]wp-2 took a significantly reduced blood meal relative to insects injected with a saline control. These results complemented previous studies describing the antifeedant effects of Aib- containing kinin analogs (Lange et al., 2016; Smagghe et al., 2010). This change in meal size may be due to the disruption of feeding behaviour by interrupting the usual timely release of Rhopr- kinins (Lange et al., 2016).

As transcript expression of RhoprKR was also confirmed in the hindgut (Chapter 3), these receptors may be involved in modulating feeding behaviour, with the potent kinin analogs interfering with this feeding signal (Te Brugge et al., 2001). In addition, dsRhoprKR-injected insects consumed a significantly greater meal than dsARG-injected insects, suggesting that Rhopr-

113

114 kinins may possibly function as satiety factors (Chapter 3). Kinin-like immunoreactivity is present in the abdominal nerves branching out to the abdominal regions, with additional peripheral neurons that possess kinin-like staining within this area, suggesting the role of Rhopr-kinins in a gut-brain feedback mechanism (Te Brugge et al., 2001). Neuropeptide release upon activation of a stretch receptor has been previously demonstrated in crustaceans, as activation of a stretch-induced mechanoreceptor in Homarus americanus resulted in a significant release of proctolin (Pasztor et al., 1988). In D. melanogaster, the kinin pathway is implicated in meal regulation, as mutations in the kinin/kinin receptor genes resulted in increased meal sizes and decreased meal frequency (Al-

Anzi et al., 2010). These phenotypic differences were due to impaired gut-brain signaling, as neurons expressing the kinin and kinin receptor gene are expressed in the brain, with kinin- containing neurons innervating the gut (Al-Anzi et al., 2010). A similar mechanism may exist in

R. prolixus, as kinin-like immunoreactivity and expression of the Rhopr-kinin transcript is present within the CNS and digestive system (Te Brugge et al., 2001; Garima et al., 2014), with expression of RhoprKR also seen within the CNS and digestive system (Chapter 3). More specifically, kinin- like immunoreactivity has also been observed in neuropile throughout the CNS including the subesophageal ganglion (SOG), which is primarily responsible for feeding regulation (Te Brugge et al., 2001; Gllferin, 1972). Examining immunoreactivity of RhoprKR and transcript expression through functional in situ hybridization (FISH) will assist in determining the specific localization of RhoprKR within the CNS and digestive tissues. This will determine whether RhoprKR also exists within these peripheral neurons near the abdomen, and whether similar staining patterns are observed that have been seen with kinins.

115 CAPA

To further examine the neuropeptides that may modulate feeding behaviour, RhoprCAPA-

2 and its analog were also examined on in vivo feeding behaviour. As RhoprCAPA-2 is responsible for the cessation of diuresis (Paluzzi et al., 2008; Paluzzi et al., 2010), it’s role in feeding regulation was examined. R. prolixus that were injected with the CAPA analog 2129-SP3[Ф3]wp-2 had a significantly higher blood-meal relative to the saline control (Chapter 2). As the CAPA analog is an antagonist to the CAPA receptor (Jiang et al., 2015; Alford et al., 2019), this increase in blood meal size suggest that RhoprCAPA-2 may have an additional role in serving as a feeding signal.

RhoprCAPA-2 appears to be released three to four hours after feeding, coinciding with the end of diuresis (Orchard & Paluzzi, 2009). CAPA may activate a signaling cascade that possibly induces satiety, or prevents additional feeding events. In addition, this feeding signal can be examined through injection of the CAPA analog a few days following a blood meal, or at different states of satiety and assessing feeding behaviour, as R. prolixus insects only feed once during each instar

(Buxton, 1930).

Following in vivo feeding assays, in vitro physiological assays on relevant feeding and diuresis-related tissues were performed with the kinin and CAPA neuropeptides and their analogs to uncover their role in these behaviours, and the efficacy of the analogs on the tissues.

Myotropic effects

Kinin

Rhopr-kinins, and Aib-containing kinin analogs have been previously shown to stimulate hindgut contractions in R. prolixus (Te Brugge et al., 2002; Bhatt et al., 2014). These kinin analogs are more biologically active in inducing hindgut contractions than the endogenous Rhopr-kinins

116 (Bhatt et al., 2014). In Chapter 2, the Aib-containing analog 2139[Ф1]wp-2 was potent in inducing hindgut contractions. Following RNAi-mediated knockdown of the RhoprKR transcript, the effects of Rhopr-kinin 2 and kinin analog were also examined (Chapter 3). The hindguts from dsRhoprKR-injected insects had a reduced response to Rhopr-kinin 2 and 2139[Ф1]wp-2, but

2139[Ф1]wp-2 was still found to induce stronger hindgut contractions than Rhopr-kinin 2.

Analysis of the expression profile of the RhoprKR transcript showed high expression levels in the hindgut, supporting the likelihood that RhoprKR indeed codes for a kinin receptor (Chapter 3).

In addition to its myotropic effects on the hindgut, kinins also stimulate contractions of the anterior midgut and salivary glands, highlighting the multifunctional role of kinins in feeding and diuresis-related tissues (Orchard & Te Brugge, 2002; Te Brugge et al., 2009). As the Aib- containing kinin was potent in inducing hindgut contractions (Chapter 2, 3) examining its effects on the anterior midgut and salivary glands would assist in determining their efficacy to be used as lead compounds.

Following analysis of the expression profile of RhoprKR, high expression was also confirmed in the foregut and posterior midgut (Chapter 3). However, the physiological role of

Rhopr-kinins on the foregut and posterior midgut have not been thoroughly examined. Within insects, contractions of the foregut and posterior midgut are regulated by a host of neuropeptides

(Audsley & Weaver, 2009). The presence of RhoprKR within these tissues suggests that Rhopr- kinins may regulate the alimentary canal as a whole, in turn regulating various aspects of the digestive process following blood-feeding (Chapter 3). The kinin-like staining patterns support the likelihood that Rhopr-kinins modulate foregut and posterior midgut function, as kinin-like immunoreactivity has been detected in endocrine cells within the posterior midgut, and corpus

117 cardiacum (CC) (Te Brugge et al., 2001). The CC serves as a neurohemal organ that is located above the foregut, therefore Rhopr-kinins may be released into the haemolymph as neurohormones to modulate foregut contractions and salivary gland muscle contractions. (Orchard & Te Brugge,

2002).

CAPA

CAPA neuropeptides have been studied extensively across insects and elicit both myotropic and diuretic effects (Predel & Wegener, 2006). As transcript expression of a

RhoprCAPA receptor was detected in the hindgut (Paluzzi et al., 2010), the effects of the

RhoprCAPA neuropeptides (RhoprCAPA-1, RhoprCAPA-2, Rhopr-pk1) were examined on hindgut contractions (Chapter 2). Interestingly, in the insect species studied, CAPAs have shown to be mutually exclusive in their myotropic or diuretic effects (Predel & Wegener, 2006). In R. prolixus, none of the three CAPA neuropeptides on their own yielded any changes in hindgut contractions (Chapter 2).

Within R. prolixus, various neuroendocrine factors have been found to act in concert to elicit changes in basal tonus of the hindgut (Bhatt et al., 2014; Haddad et al., 2018). This was seen with RhopCAPA-2 and Rhopr-kinin 2, as RhoprCAPA-2 potentiated the excitatory effects of

Rhoprkinin-2 (Chapter 2). It is possible that this interaction may occur at the intracellular level, through separate second messenger pathways. Activation of the CAPA receptor results in increases in cGMP, which in turn activates a phosphodiesterase that degrades cGMP (Paluzzi & Orchard,

2006; Paluzzi et al., 2013). In invertebrates, kinin receptors have been shown to cause intracellular

2+ Ca increases through the inositol triphosphate (IP3) pathway (O’ Donnell et al., 1998; Cady &

Hagedorn, 1999; Tehrzaz et al., 1999; Cox et al., 1997). It is likely that RhoprKR activates this

118 second messenger pathway also, due to the high sequence similarity in comparison to other invertebrate kinin receptors (Chapter 3). It is also hypothesized that the CAPA receptor may be constitutively active, stably increasing levels of cGMP and decreasing levels of cAMP (Chapter

2), with the CAPA analog serving as an inverse agonist. Serotonin [5-hydroxytryptamine (5-HT)], which utilizes cAMP as a second messenger, causes increases in the frequency of hindgut contractions (Orchard, 2006). The CAPA analog was found to potentiate the effects of 5-HT on contraction frequency, while RhoprCAPA-2 inhibited them. The inhibitory effects of

RhoprCAPA-2 may be due to the elevated levels of cGMP, in turn activating the phosphodiesterase that degrades cAMP, thereby reducing muscle stimulation. Figures 1,2, and 3 are models proposing the mechanism of these intracellular interactions. Within invertebrates, constitutively active

GPCRs modulate a host of behaviours. In Caenorhabditis elegans, a constitutively active 5-HT7- like GPCR is involved in pharyngeal pumping, while in Schistosoma mansoni, a constitutively active acetycholine GPCR regulates larval motility (Hobson et al., 2003; MacDonald et al., 2015).

In addition, interaction of separate second messenger pathways has been found to occur in insects, as cAMP potentiates the release of intracellular Ca2+ from the endoplasmic reticulum through the

IP3 pathway in the salivary glands of Calliphora vicina (Schmidt et al., 2008).

To further investigate this interaction, a calcium mobilization assay must be performed to confirm RhoprKR ligand binding and its associated second messenger pathway. Utilizing

HEK293/CNG cells will assist in determining whether luminescence that occurs through activation

2+ of RhoprKR is through the activation of cytosolic cAMP or intracellular Ca through the IP3 pathway. Measuring levels of intracellular Ca2+ through in vivo calcium imaging (Russell, 2011) during sole application of Rhopr-kinin 2 and a mixture of Rhopr-kinin 2 and RhoprCAPA-2 will assist in determining whether higher levels of intracellular Ca2+ are achieved during this

119 interaction. In addition, measuring levels of cGMP via cGMP radioimmunoassay (Paluzzi &

Orchard, 2006) within the hindgut upon application of the CAPA analog will confirm whether this analog is functioning as an inverse agonist on CAPA receptors within the hindgut.

120

Fig. 1: Model describing the possible second messenger pathways associated with the activation of the R. prolixus kinin and 5-HT receptors, and the constitutively active CAPA receptor in the hindgut

A) Activation of the kinin receptor increases hindgut contractions through intracellular Ca2+ increases mediated by the IP3 pathway (PIP2: Phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C)

B) Activation of the 5-HT receptor stimulates increases in frequency of hindgut contraction through activation of an adenyl cyclase (AC) causing increases in cAMP, resulting in activation of protein kinase A (PKA) and eliciting further downstream effects leading to hindgut contraction stimulation.

C) A constitutively active CAPA receptor stably increases cGMP levels, activating a phosphodiesterase (PDE) that degrades cAMP D) Activation of the CAPA receptor through

RhoprCAPA-2 (agonist) contributes to greater increases in cGMP content, resulting in more cAMP degradation.

121

A) Rhopr-kinin 2

(Agonist)

U U

Y U

U U U β PIP2 √ PLC α GTP

IP3 Sensitive IP3 Endoplasmic Ca2+ Channel Reticulum Increase in Hindgut Contraction

Ca2+

B) 5-HT

(Agonist)

U U

Y U

U U U β AC √ α GTP

[cAMP] ATP

Endoplasmic Reticulum PKA

Ca2+ Increase in Frequency of Hindgut Contraction

C) CAPA Receptor (Constitutively

active) U U

Y U

U U U β α √

[cGMP]

Endoplasmic PDE [cAMP] Reticulum Ca2+

D) RhoprCAPA-2

(Agonist)

U U

Y U

U U U β α √

[cGMP]

Endoplasmic PDE [cAMP] Reticulum

Ca2+

122

Fig. 2: Model describing the possible intracellular interactions between RhoprCAPA-2 and Rhopr- kinin 2 and 5-HT in the hindgut of R. prolixus

A) Activation of the CAPA receptor by RhoprCAPA-2 causes increases in cGMP levels, which interact with the IP3 pathway (activated by Rhopr-kinin 2), causing potentiated increases in hindgut contraction.

B) Activation of the CAPA receptor by RhoprCAPA-2 causes increases in cGMP levels, thereby decreasing cAMP levels through action of a phosphodiesterase, in turn reducing the stimulatory effects of 5-HT on the frequency of hindgut contractions.

123

A) RhoprCAPA-2 Rhopr-kinin 2

(Agonist) (Agonist)

U U U U U

Y Y U

U U U β U U U β PIP2 α √ √ PLC α GTP

[cGMP]

IP3 Sensitive IP3 Endoplasmic Ca2+ Channel Reticulum Potentiated Increase in Hindgut Contraction Ca2+

B) RhoprCAPA-2 5-HT

(Agonist) (Agonist)

U U U U U

Y Y U

U U U β U U U β AC α √ √ α GTP

[cGMP] PDE [cAMP] ATP

Endoplasmic Reticulum PKA

Ca2+ Reduction of 5-HT-Stimulated Increase in Frequency of Hindgut Contraction

124

Fig. 3: Model describing the possible effects of the CAPA analog 2129-SP3[Ф3]wp-2 and its intracellular interactions with Rhopr-kinin 2 and 5-HT in the hindgut of R. prolixus

A) The CAPA analog acts as an inverse agonist on the CAPA receptor, resulting in decreased levels of cGMP, thereby preventing the degradation of cAMP and resulting in increases of hindgut contraction.

B) The CAPA analog decreases cGMP levels, preventing an interaction with the IP3 pathway

(activated by Rhopr-kinin 2), resulting in no potentiation

C) The CAPA analog decreases cGMP levels, thereby preventing degradation of cAMP by a phosphodiesterase, increasing cAMP levels, and potentiating the 5-HT-stimulated increases in the frequency of hindgut contractions.

125

A) 2129-SP3[F3]wp-2

(Inverse agonist)

U U

Y U

U U U β α √

[cGMP]

Endoplasmic PDE [cAMP] Reticulum

Ca2+ PKA

Increase in Hindgut Contraction

B) 2129-SP3[F3]wp-2 Rhopr-kinin 2

(Inverse agonist) (Agonist)

U U U U U

Y Y U

U U U β U U U β PIP2 α √ √ PLC α GTP

[cGMP]

IP3 Sensitive IP3 Endoplasmic Ca2+ Channel Reticulum No Potentiated Increase in Hindgut Contraction Ca2+

C) 2129-SP3[F3]wp-2 5-HT

(Inverse agonist) (Agonist)

U U U U U

Y Y U

U U U β U U U β AC α √ √ α GTP

[cGMP] PDE [cAMP] ATP

Endoplasmic Reticulum PKA

Ca2+ Potentiated Increases in Frequency of Hindgut Contraction

126 Diuretic effects

As RhoprCAPA-2 was identified as the first anti-diuretic hormone in R. prolixus, the effects of the CAPA analog were tested on MT fluid secretion (Paluzzi & Orchard, 2006; Paluzzi et al., 2008). Interestingly, Rhopr-kinins have no role in diuresis within R. prolixus, in contrast to their diuretic effects in many other insects (Torfs et al., 1999). The CAPA analog was found to prevent the inhibitory effects of RhoprCAPA-2 on 5-HT-stimulated tubules, supporting its function as a CAPA receptor antagonist within the MTs (Chapter 2). The in vitro effects of the

CAPA analog support the differences in the rate of diuresis that was observed in R. prolixus insects injected with the CAPA analog, as they had a significantly greater rate of post-feeding diuresis in the first hour (Chapter 2). These effects of the CAPA analog highlight its promise as a lead compound for pest-control development, as it can induce excess fluid loss in R. prolixus by preventing the anti-diuretic signal of RhoprCAPA-2. As RhoprCAPA-2 also abolishes the synergism that occurs between 5-HT and Rhopr-CRF/DH on stimulating upper MT secretion

(Paluzzi et al., 2011), examining the CAPA analog’s effects on the abolishment of this synergism would provide more insight on its efficacy. In addition to the MTs, RhoprCAPA-2 has anti-diuretic effects on the anterior midgut, as it inhibits 5-HT-stimulated fluid absorption (Ianoswki et al.,

2010). This would be an additional tissue that the CAPA analog can be tested on for its effects as an antagonist.

Figure 4 provides a summary of the roles of kinin and CAPA in R. prolixus feeding and diuresis-related behaviours.

127

Fig 4: The physiological roles of kinin and CAPA in the alimentary canal of R. prolixus.

Highlighted regions represent novel research findings.

128

CAPA Kinin Foregut RhoprKR expression confirmed Unknown physiological role Anterior Midgut capa-r expression confirmed (Paluzzi et al., Salivary Glands 2010) RhoprKR expression confirmed Downregulates 5-HT- Increases basal tonus (Orchard & stimulated fluid Te Brugge, 2002) absorption (Ianowski et al., 2010)

Upper Malpighian Anterior Midgut Tubules RhoprKR expression capa-r expression confirmed confirmed (Paluzzi et al., Increases frequency of 2010) contractions (Te Brugge et Inhibits 5-HT-stimulated al., 2009) fluid secretion (Paluzzi et al., 2008) Abolishes 5-HT/DH synergism (Paluzzi et al., 2011)

Posterior Midgut Lower Malpighian RhoprKR expression Tubules confirmed capa-r expression Unknown physiological confirmed (Paluzzi et al., role 2010) Unknown physiological role

Hindgut Hindgut capa-r expression confirmed RhoprKR expression (Paluzzi et al., 2010) confirmed RhoprCAPA-2 potentiates Increases basal tonus, Rhopr-kinin 2 hindgut frequency of contractions contraction stimulation (Te Brugge et al., 2002; (no individual effects of Garima et al., 2014) RhoprCAPA neuropeptides)

129 Summary of Physiological Effects of Analogs

Following the investigation of the neuropeptide analogs on feeding and diuresis-related behaviours, they were found to cause potent changes in physiology that can potentially interfere with the normal functioning of R. prolixus. The kinin analog 2139[Ф1]wp-2 stimulated dose- dependent increases in basal tonus of the hindgut and was more effective than the endogenous

Rhopr-kinin 2, and also had anti-feedant effects. The CAPA analog 2129-SP3[Ф3]wp-2 also stimulated dose-dependent increases in hindgut contraction and potentiated the effects of 5-HT on the frequency of hindgut contractions (not seen by endogenous RhoprCAPAs), increases the size of blood meals, and prevented RhoprCAPA-2’s inhibition on 5-HT stimulated MTs.

The objective of this research project was to investigate the roles of Rhopr-kinins and

RhoprCAPAs on the feeding and post-feeding physiology of R. prolixus. Analogs that were developed for kinin and CAPA were assessed for their efficacy to be used as compounds in pest- control development. The neuropeptide analogs had a significant impact in vivo and in vitro and show promise for use in various pest-control strategies. Novel roles of CAPA and kinins within feeding, diuresis and excretion have been uncovered using a physiological and molecular approach. Lastly, RhoprKR has been successfully identified, cloned and sequenced, showing high susceptibility to RNAi-mediated knockdown. Table 1 provides a summary of the future directions in light of these research findings. Overall, this thesis highlights the importance of neuropeptide signaling within R. prolixus, and its role in physiology and behaviours related to disease transmission.

130

Table 1: A summary of future directions

Physiological Approach Molecular Approach Investigate the effects on 2139[Ф1]wp-2 on Functionally characterize RhoprKR and contractions of salivary glands and anterior confirm its second messenger pathway midgut Investigate the effects of 2129-SP3[Ф3]wp-2 Perform FISH to determine RhoprKR on anterior midgut absorption, and 5-HT/DH transcript localization synergism in upper MTs Using functional receptor assay to determine Measure levels of second messengers the binding efficiency of the kinin analogs involved in Rhopr-kinin/RhoprCAPA and any possible interference by the CAPA interaction within the hindgut neuropeptides and the CAPA analog Perform immunohistochemistry for RhoprKR-like staining in the CNS and digestive system Perform additional immunohistochemistry for

RhoprCAPA-2 like innervation

Examine effects of RhoprCAPA-2 on various states of satiation during R. prolixus life cycle

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