Himali Patel. Copyright by Himali Patel 2013

Transcription

1 Distribution and Physiological Effects of Adipokinetic Hormone (AKH), Corazonin and AKH/corazonin-Related Peptide (ACP) in the Kissing Bug, Rhodnius prolixus by Himali Patel A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Cell and Systems Biology University of Toronto Copyright by Himali Patel 2013

2 Distribution and Physiological Effects of Adipokinetic Hormone (AKH), Corazonin (CRZ) and AKH/corazonin-Related Peptide (ACP) in the Kissing Bug, Rhodnius prolixus Abstract Himali Patel Master of Science Cell and Systems Biology University of Toronto 2013 Rhodnius prolixus is a medically-important hemipteran that serves as a vector of Chagas disease. The distribution and physiological effects of three sequence-related neuropeptides, adipokinetic hormone (AKH), corazonin (CRZ) and AKH/corazonin-related peptide (ACP) have been investigated in R. prolixus. Immunohistochemistry revealed that AKH, CRZ and ACP are not co-localized but are found within a different subset of cells and processes within the central nervous system. Physiologically, CRZ significantly increased the heartbeat rate whereas AKH and ACP did not. AKH significantly increased haemolymph lipid levels whereas CRZ and ACP did not. There is no known function for ACP. Preliminary studies indicated that CRZ may be involved in gating of ecdysis. ii

3 Acknowledgments First and foremost I would like to give a huge thanks and gratitude to my supervisor, Dr. Angela Lange for the opportunity to work in her lab for the past two years as well as for all the support and guidance over that time. You have vouched for me during committee meetings, conference talks, poster presentations, graduate courses, providing me with constant positive reinforcement and in having a huge amount of patience with me -- your dedication to teaching and your passion for science was a constant source of inspiration for me. I want to extend my appreciation to Dr. Ian Orchard as he has been an integral part of the research, for all the wisdom and advice regarding injections for lipid assays, camera lucida drawings, and for constant feedback throughout my time here. I would also like to say thanks to Dr. Joel Levine for being part of supervisory committee and giving positive and constructive advice. I consider myself extremely blessed and fortunate to have this experience and believe that a great deal of my current and future success will stem from the impact Dr. Angela Lange has had on me. I also owe an immeasurable amount of gratitude to members of the Lange and Orchard labs, which is my academic family. In particular I d like to thank Rosa, Jean-Paul, Marina, Meet (especially for your witty humor), Amir, Garima, Hei Ree, Maryam (may the force be with you), Dasha and Hang. A special thanks to Lisa Robertson who initially took many of my calls over weekends to answer my questions, and for showing me the fascinating world of locusts. I am also grateful to Jenny and Felicia, who have helped me often times with the locusts, and picking up insect jars when I was away at conferences. Two active contributors iii

4 to my work were Nikki who has been feeding bugs as per my special instructions for the past year and a half, and DoHee who has been kind enough to collaborate and teach me how to design the ecdysis part of my thesis. Special acknowledgment must be given to Laura, who will understand the following random words: sea horses, epinephrine, fettuccine, Fourier transformation, imgoinhome and mango cheeks. I went through majority of my grad-school life with you, and you know very well that it is true when I say that I couldn't have done it without you. A special mention goes to what I would consider the "honorary" members of our lab-- the stick insects; the kissing bugs, whose biology is the focus of my thesis; and finally the locusts- which I've spent countless hours videotaping and watching in fascination. This work is dedicated to my loving family. My mom, although not understanding what I did at school, still had warm meals ready for me when I got home. My father, who has often driven me to school even after long night shifts at his work. My sister and brother-in-law who has provided positive reinforcement and support. My baby brother, who always makes me smile when I was down and has often listened to me practice and ramble on about my research. Finally, Nigam, for always being there for me, and for being my knight in shining armour to save the day way more times than I can count. I love each and every one of you, and this thesis is an indirect product of your love and support you have given me and thus, I would like to dedicate it to you. iv

5 Table of Contents Abstract... ii Acknowledgments... iii Table of Contents...v List of Tables... vii List of Figures... viii Organization of Thesis...x CHAPTER I : General Introduction...1 Rhodnius prolixus...1 Neuropeptides...1 The central nervous system (CNS) of R. prolixus...2 The circulatory system of R. prolixus...4 Adipokinetic hormone (AKH)...4 Corazonin (CRZ)...5 AKH/corazonin-related peptide (ACP)...7 Receptor/ligand co-evolution...8 Insect Metamorphosis: Ecdysis...8 Thesis Focus...10 References...12 v

6 CHAPTER II : The distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus: Adipokinetic hormone, corazonin and adipokinetic hormone/corazonin-related peptide Abstract...17 Introduction...18 Materials and methods...21 Results...26 Discussion...44 Acknowledgements...50 References...51 CHAPTER III : Possible involvement of corazonin in ecdysis of 4 th instar Rhodnius prolixus: A preliminary analysis Abstract...55 Introduction...56 Methods and Materials...59 Results...61 Discussion...78 References...81 CHAPTER IV: General Discussion...85 Future Directions...90 References...93 vi

7 List of Tables Table 1. Amino acid sequences and similarities between CRZ, RhoprACP and RhoprAKH in R. prolixus.. 10 vii

8 List of Figures CHAPTER II Figure 1. Confocal microscope images and camera lucida composite of CRZ-like immunoreactivity in the CNS of Rhodnius prolixus...31 Figure 2. Confocal microscope images and camera lucida composite of ACP-like immunoreactive cell bodies and processes in the CNS of Rhodnius prolixus...33 Figure 3. Confocal microscope image and camera lucida composite of AKH -like immunoreactivity in the corpus cardiacum and dorsal vessel of Rhodnius prolixus...35 Figure 4. Effects of CRZ, RhoprACP and RhoprAKH on heartbeat frequency of 5 th instar Rhodnius prolixus...37 Figure 5. Time course and dose-response curve showing changes in haemolymph lipid levels following injection of saline or RhoprAKH...39 Figure 6. Neither CRZ nor RhoprACP alter haemolymph lipid levels of adult male Rhodnius prolixus tested 90 minutes after injection...41 Figure 7. RhoprAKH does not alter haemolymph carbohydrate (CHO) levels of adult male Rhodnius prolixus...43 viii

9 Chapter III Figure 1. Number of insects ecdysing from 4th instar to 5th instar on days after feeding...65 Figure 2. Gating of ecdysis to 5 th instar...67 Figure 3. Confocal microscope images of CRZ-like immunoreactivity in the CNS during ecdysis from 4 th to 5th instar R. prolixus Figure 4. Confocal microscope images of CRZ-like immunoreactivity in CNS from 4 th instars injected with double stranded (ds) RNA...71 Figure 5. Percent mortality of insects injected with double stranded RNA or no treatment..73 Figure 6. Gating of insects ecdysing from 4th instars to 5th instars after injection of double stranded RNA or no treatment Figure 7. Gating of ecdysis of insects injected with double stranded RNA or no treatment..77 ix

10 Organization of Thesis CHAPTER I serves as a general introduction to Rhodnius prolixus and the role of neuropeptides on physiological processes concentrating on circulation, lipid metabolism, and ecdysis. Chapter 1 also introduces the three neuropeptide families adipokinetic hormone (AKH), corazonin (CRZ) and AKH/corazonin-related peptide (ACP). CHAPTER II is currently submitted to General and Comparative Endocrinology and is coauthored by Drs. Angela Lange and Ian Orchard. I examined the distribution and physiological effects of the three peptide families introduced in chapter 1. I completed all the experimentation, compiled the results, and wrote the paper. CHAPTER III is preliminary results of experiments investigating the role of CRZ in ecdysis. The determining of the cdna sequence of CRZ and the construction of dscrz was performed by Dr. DoHee Lee. This project was done in collaboration and thus injections, dissections and processing of the immunohistochemistry and confocal imaging were done by me, and the molecular aspect was performed by DoHee Lee. CHAPTER IV serves as a general discussion for the thesis and examines the roles of all of the three peptides in the physiology of R. prolixus. x

11 1 CHAPTER I : General Introduction Rhodnius prolixus Rhodnius prolixus, commonly known as the kissing bug, is a blood-feeding hemipteran that is medically-important and predominantly resides in Central and South America (Koberle, 1968). R. prolixus is the principal vector of Trypanosoma cruzi, the protozoan which causes Chagas disease in humans - an incurable illness damaging the heart, nervous system and often producing chronic symptoms. The most recent estimate indicates that this disease exists in 9.8 million people with an estimated 50,000 people dying annually (Remme et al., 2006; Prata, 2001) Infection occurs after R. prolixus releases protozoans in its urine/feces immediately following a blood meal. It is interesting to note that, despite its flat appearance, R. prolixus can ingest almost 10 times its bodyweight per blood-meal (Remme et al., 2006).The blood intake stimulates short-term and long-term physiological changes associated with osmotic homeostasis, growth and reproduction. The changes associated with ingesting a large blood meal include rapid diuresis, growth and development and depend on the appropriate communication of cells and systems through chemical messengers, such as neuropeptides (Gäde and Goldsworthy, 2003). Neuropeptides Neuropeptides are short chains of amino acids used by neurons to communicate with each other and with their target tissues. They are expressed and released by neurons, and modulate

12 2 neuronal communication by acting on cell surface receptors. They are a class of chemical messengers that mediate neural events associated with behaviour, development, reproduction and homeostasis (see Gäde and Goldsworthy, 2003; Nässel and Winther, 2010; Kim et al., 2004). Neuropeptides can exert their effect in a number of ways acting as neurotransmitters, neuromodulators or neurohormones (see Orchard et al., 2001). Neurotransmitters are chemical messengers that transmit their message from a neuron to a target cell across a synapse. In contrast, neurohormones release their chemical messenger into the haemolymph to work on distant target sites. Neuromodulators are a very broad category of chemical messengers and can be released more locally onto a target tissue to modify or modulate the activity of the target tissue. Neuropeptides are stored in electron-dense granules at their release sites in axon terminals or in en passant varicosities until a depolarization induces release by exocytosis. Release can occur either at typical synapses or at non-synaptic sites. Thus, neuropeptides may act at a short distance in a localized fashion as a neurotransmitter/neuromodulator or at some distance to peripheral tissues as a neurohormone. The central nervous system (CNS) of R. prolixus The R. prolixus CNS consists of four main parts: the brain, the subesophageal ganglion (SOG), the prothoracic ganglion (PRO) and the mesothoracic ganglionic mass (MTGM). The brain is composed of the optic lobes at the most anterior end of the brain and three fused ganglia producing regions known as the protocerebrum, deutocerebrum and tritocerebrum. The term neurosecretory cell denotes a neuron that displays synthesis of secretory products such as neuropeptides in the cell body and axonal transport of the product to a release site

13 3 (Orchard and Loughton, 1985; Steel and Harmsen, 1971). In R. prolixus, there are 5 distinct neurosecretory cell types in the brain that have been well documented and described: type 1, type 2(large), type 2 (small), type 3 and type 4 (Wigglesworth, 1959b; Steel and Harmsen, 1971). It was also established that these cells can be found in different anatomical parts of the brain constituting five different groups: 1) anterior 2) lateral 3) median 4) posterior and 5) ventral neurosecretory regions. The location where lateral and medial neurosecretory cell groups regionalize in the protocerebrum is also called the pars lateralis and are of special interest in this study (Steel and Harmsen, 1971). It is known that neurosecretory cells are present throughout the CNS and express a variety of neuropeptide families (see Nässel and Winther, 2010). From the dorsal side of each of the protocerebral lobes two thin nerves emerge (nervi corporis cardiaci I and II) that project to the corpus cardiacum (CC), a neurohaemal organ in insects (Wigglesworth, 1959a, b). The anterior part of the dorsal vessel, known as the aorta (AO), is closely associated with the CC. The CC consists of intrinsic neurosecretory cells and storage and release sites for products from neurosecretory cells in the brain. The corpora allata (CA) are oval glands that attach to the CC. The brain-cc-ca complex of insects is the physiological equivalent of the brain-hypophysis axis of vertebrates. Joined via connectives to the tritocerebrum the ventral nerve cord then consists of the SOG, the PRO and the MTGM (Wigglesworth, 1959b). Neurosecretory cells are found throughout these ganglia and produce their neurohaemal sites on a variety of peripheral nerves, especially the abdominal nerves.

14 4 The circulatory system of R. prolixus The open insect circulatory system functions in nutrient transport, waste removal, hormone delivery, immune surveillance, thermoregulation and respiration (Esteves-Lao et al., 2013). The dorsal vessel is responsible for pumping haemolymph from the posterior of the insect to the anterior region and is composed of the aorta and the heart (Chiang et al., 1990; McCann, 1970). The haemolymph enters the heart through four pairs of ostia (valves). There are seven pairs of alary muscles connecting the heart to the posterior abdominal segments, which create the flow of haemolymph by contractions that expand and constrict the heart (Chiang et al., 1990) Since there are many neurohormones / endocrine factors that affect heart rate in insects, and in many insects the dorsal vessel also receives direct innervation from the CNS to control heart rate (Chiang et al., 1990), the effect of a peptide on the dorsal vessel may give researchers insight into the function of peptides. Adipokinetic hormone (AKH) The adipokinetic hormones are one of the most studied insect neurohormones with family members isolated from over 70 different species (Gäde et al., 1997). The name adipokinetic hormone was first coined by Mayer and Candy in 1969 who reported the presence of a factor in the CC of Locusta migratoria and Schistocerca gregaria, responsible for the stimulation of lipid release from the fat body (Mayer and Candy, 1969). Since its discovery, numerous studies using immunohistochemical, biochemical and molecular analyses have confirmed that AKH is expressed in the intrinsic cells of the CC in insects (see Gäde et al., 1997; Nässel and Winther, 2010). The action of AKH is comparable to that of glucagon in mammals. It

15 5 contributes to haemolymph carbohydrate homeostasis, but it is also involved in the mobilization of lipids from the fat body during energy-requiring activities, such as flight or other forms of locomotion (see Bednářová et al., 2013). Vertebrate glucagon activity is directed to the liver, whereas AKH function is targeted mainly to the fat body, an insect analogue of the liver tissue (Bednářová et al., 2013). In insects, various biological activities have been attributed to AKH, including mobilization of carbohydrate levels, stimulation of heartbeat frequency and inhibition of protein synthesis (see Gäde and Marco, 2013); however, the primary function of AKH is attributed to lipid mobilization. Lipid mobilization takes place when AKH is released into the circulation from the CC and is transported to the fat body, where it binds to its G protein-coupled receptor (GPCR) and triggers the release of diacylglycerols, that are transported via lipophorins to muscles to fuel high physical activity of the insect (Arrese and Soulages, 2010). Therefore, the concentration of lipid levels in the haemolymph can be controlled by AKH (Siegert and Ziegler, 1983). Based on mass spectrometry and in silico prediction from the R. prolixus genome database, a short eight amino acid sequence (pqltfstdw-nh 2 ) for AKH in R. prolixus has been determined (Ons et al., 2011; Marco et al., 2013). Recently, this RhoprAKH was shown to stimulate small increases in haemolymph lipid levels in 5 th instar of R. prolixus (Marco et al., 2013). Corazonin (CRZ) Corazonin (CRZ) is a highly conserved peptide that is eleven amino acids in length (pqtfqysrgwtnamide) (Veenstra, 1989). It was found to have cardioacceleratory

16 6 effects on the heart of the American cockroach, Periplaneta americana (Veenstra, 1989). Since its discovery, six different CRZ isoforms are known to occur in insect species with variation of only one or two amino acids (Predel et al., 2007; Veenstra, 1993). Currently, CRZ has been shown in the CNS of thirteen insect orders (Roller et al., 2003). The distribution of CRZ-like immunoreactivity (IR) is species specific; however, general patterns of CRZ-like IR can be inferred (see Nässel and Winther, 2010). Basically, three small cell bodies are located in the protocerebrum near the optic lobes while another set of three larger neurosecretory cell bodies are located in the middle of each of the lobes of the protocerebrum. These larger cells have axons projecting to the CC and another pair of bilaterally descending axons project down the ventral nerve cord. The presence of CRZ-like IR in processes on the CC suggests that it might be released into the haemolymph and may act as a neurohormone. It has also been shown in various studies that CRZ-like immunoreactive cells share localization with other neuropeptide pathways. For instance, in Drosophila, CRZ-producing cells in the brain and ventral nerve cord are co-expressed with two different diuretic hormone receptors, DH31R1 and DH44R1 whose respective peptide ligands have been shown to be involved in increasing fluid secretion rates by raising camp levels in the principal cells of the Malpighian tubule (Johnson et al., 2005). Corazonin in the brain is also expressed in clock neurons, neurons that express the period gene (PER) in the hawkmoth, Manduca sexta, which may indicate the involvement of CRZ in circadian rhythm regulation (Wise et al., 2002). Recent research has shown that CRZ has diverse physiological effects in different species of insects (Tawfik et al., 1999; Roller et al., 2003; Hua et al., 2000; Tanaka, 2000, 2001; Tanaka et al., 2002; Boerjan et al., 2010). For example, CRZ increases heart beat frequency in P.

17 7 americana (Veenstra, 1989; Tawfik et al., 1999), and [His] 7 - CRZ acts as a hormonal factor that can induce dark pigmentation in albino Locusta migratoria and green nymphs of Schistocerca gregaria (Hua et al., 2000; Tanaka, ). In addition, extracts and transplantations of brain-cc complexes from 47 species belonging to 10 insect orders induced cuticle pigmentation in the albino locusts (Tanaka, 2000). In Bombyx mori, the domesticated silkworm, the spinning period was significantly prolonged even at a low dose of 1 pmol CRZ (Tanaka et al., 2002). The presence of CRZ in R. prolixus has been confirmed by a neuropeptidome survey (Ons et al., 2009). AKH/corazonin-related peptide (ACP) A structural intermediate of AKH and CRZ was found and given the name AKH/corazoninrelated peptide (Hansen et al., 2010). The authors claim that this finding is a prominent example of receptor/ligand co-evolution. In the flour beetle larvae, Tribolium castaneum, ACP has been confirmed to be localized in a small group of cell bodies in the brain; specifically, in one large and two smaller cell bodies located bilaterally and in axons running down the ventral nerve cord. Furthermore, the authors show that ACP and its receptor are present in eight different orders of insects, suggesting that it is an important insect signaling system. Although it is widespread, nothing is known about the function of ACP or its localization in insects other than mosquitoes and T. castaneum (Hansen et al., 2010). Prior to this thesis ACP had yet to be biochemically identified in R. prolixus but had been predicted by genome search by Hansen et al. (2010).

18 8 Receptor/ligand co-evolution It is interesting that not only are the structures of the peptides, AKH, ACP and CRZ, related, but also their respective receptors. Although all three share sequence similarity, none of the peptide ligands cross-reacts with the receptors for the other ligands (Hansen et al., 2010; Grimmelikhuijzen and Hauser, 2012; Park et al., 2002). Thus, it may be an example of receptor/ligand co-evolution, where an ancestral receptor gene and its ligand gene have been duplicated twice during evolution, yielding three independent neuropeptide/gpcr signaling systems. This relationship was analyzed further in context with another structurally-similar vertebrate hormone, gonadotropin-releasing hormone (GnRH) which is released from the pituitary gland in vertebrates. Through phylogenetic sequence analysis, it was found that all four peptides might all share a common ancestor (Roch et al., 2011). It was further suggested that AKH, CRZ and ACP belong to the GnRH super family. This sheds light on the fact that GnRH already originated before the split of Proto- and Deuterostomia, it also shows that the ancestral ligand, which probably had a mixture of the AKH and GnRH amino acid sequences, might have controlled carbohydrate and lipid release, reproduction, or both (Osada and Treen, 2013). Thus, investigating AKH, CRZ and ACP can help to unravel further functions of the neurosecretory system of the CNS and the CC -- the brain and the pituitary gland analogues of invertebrates and vertebrates, respectively. Insect Metamorphosis: Ecdysis When it comes to evolution of animals, the insects are set on a pedestal as the most successful. There are many attributes of insects in general that gives rise to their success in the face of environmental obstacles. In brief, these attributes include an exoskeleton, small

19 9 body size, the ability to fly, a high reproductive potential, and adaptability in an everchanging environment. Of all the different advantageous features, the most dramatic has to be the process of metamorphosis. The physical growth of insects is restricted by tough sclerotized cuticle; insects solve this problem by intermittent molts in which the old cuticle is shed and a new cuticle is laid down, allowing for growth (Smith and Rybczynski, 2012). There are two different types of metamorphosis that can be categorized in insects. Incomplete (hemimetabolous) metamorphosis, in which immature stages are called nymphs and development proceeds in repeated stages of growth an ecdysis with only minor changes in morphology (other than the appears of wings and mature sexual organs in the adult), and complete (holometabolous) metamorphosis in which insects pass through a larval stage, then enter an inactive pupal state, or chrysalis and finally emerge as adults (Truman and Riddiford, 1999). Each of these stages are morphologically distinct. It is now known that a suite of hormones coordinates these episodes of rapid developmental change, which is referred to in a generic fashion as the molting cycle. In this cycle, the final sequences of events is referred to as "ecdysis" and consists of a suite of characteristic behaviours which include antennal wiping, head bobbing, air swallowing, abdominal contractions, shedding of the old cuticle, and the emergence into the next stage (Žitňan, 2012). Recent literature on M. sexta ecdysis, a holometabolous insect, revealed that Inka cells produce pre-ecdysis and ecdysis-triggering hormones (PETH and ETH) which activate the ecdysis sequence through receptor-mediated actions on neurons in the CNS (Žitňan et al.,

20 ). It was shown that CRZ elicits a graded low level release of ETH (Kim et al., 2004). The discovery of CRZ receptors on Inka cells, and release of CRZ just before pre-ecdysis onset, provides a new perspective about the physiological functions of CRZ (Kim et al., 2004; Žitňan et al., 1996; Žitňan et al., 2007). Thus, it would be appropriate to examine the involvement of CRZ in ecdysis in R. prolixus. Thesis Focus In this study, the distribution and physiological effects of these three evolutionarily and sequence-related peptides, AKH, CRZ and ACP, in the kissing bug R. prolixus was examined. The most intriguing feature about the three previously mentioned peptides is that their peptide sequences are striking similar to one another (Table 1). Table 1. Amino acid sequences and similarities between CRZ, RhoprACP and RhoprAKH in R. prolixus Neuropeptide Amino acid sequence CRZ (Ons et al.2009) pq - T F Q Y S R G W T N NH 2 RhoprACP (Hansen et al., 2010) pq V T F - - S R D W N A NH 2 RhoprAKH (Hansen et al., 2010) pq L T F - - S T D W - NH 2 Since these peptides seem to have evolved with similar sequences, one might assume that these peptides may have some overlap in their physiological roles. Previous studies in insects have implicated CRZ in modulating heart rate, AKH in mobilization of lipid and ACP has no described function. Therefore, the main focus of my research is to understand the relationship

21 11 between all three peptides with regard to their distribution and physiological function in R. prolixus. In addition, the possible involvement of CRZ in ecdysis was also examined, with some preliminary experimentation.

22 12 Specific Objectives 1) Distribution of ACP, AKH and CRZ producing cells in the central nervous system and peripheral tissues of R. prolixus. (Chapter 2) Aim: to determine the distribution of each peptide in the CNS, and peripheral tissues using immunohistochemistry. 2) Physiological role of each peptide on heart rate of R. prolixus. (Chapter 2) Aim: To determine the effects of CRZ, RhoprAKH, RhoprACP on the heart rate using an impedance converter. 3) Physiological role of each peptide on mobilization of haemolymph lipid and carbohydrate levels of R. prolixus. (Chapter 2) Aim: To determine the effects of RhoprAKH, CRZ and RhoprACP on the mobilization of haemolymph 4) Possible involvement of CRZ in the ecdysis cycle of R. prolixus. (Chapter 3) Aim: To observe the behaviours involved in ecdysis of 4th instar R. prolixus. Aim: To examine changes in the amount of CRZ in cells and processes during developmental time points between 4th and 5th instar using immunohistochemistry. Aim: To determine if CRZ is important for ecdysis using RNA interference to inhibit expression of CRZ transcript. References Arrese, E.L., Soulages, J.L., Insect fat body: Energy, metabolism, and regulation. Annu. Rev. Entomol. 55, Bednářová, A., Kodrik, D., Krishnan, N., Unique roles of glucagon and glucagon-like peptides: Parallels in understanding the functions of adipokinetic hormones in stress responses in insects. Comp. Biochem. Physiol. 164,

23 13 Boerjan, B., Verleyen, P., Huybrechts, J., Schoofs, L., De Loof, A., In search for a common denominator for the diverse functions of arthropod corazonin: A role in the physiology of stress? Gen. Comp. Endocrinol. 166, Chiang, R.G., Chiang, J.A., Davey, K.G., Morphology of the dorsal vessel in the abdomen of the blood-feeding insect Rhodnius prolixus. J. Morphol. 204, Estevez-Lao, T.Y., Boyce, D.S., Honegger, H., Hillyer, J.F., Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae. J. Exp. Biol. 216, Gäde, G., Goldsworthy, G., Insect peptide hormones: a selective review of their physiology and potential application for pest control. Pest Manag. Sci. 59, Gäde, G., Marco, H., AKH/RPCH Peptides. In: Handbook of Biologically Active Peptides, Ed. Abba Kastin, Academic Press, Boston, pp Gäde, G., Hoffmann, K., Spring, J.H., Hormonal regulation in insects: Facts, gaps, and future directions. Physiol. Rev. 77, Grimmelikhuijzen, C.J.P., Hauser, F., Mini-review: The evolution of neuropeptide signaling. Regul. Pept. 177, S6-S9. Hansen, K.K., Stafflinger, E., Schneider, M., Hauser, F., Cazzamali, G., Williamson, M., Kollmann, M., Schachtner, J., Grimmelikhuijzen, C.J.P., Discovery of a novel insect neuropeptide signaling system closely related to the insect adipokinetic hormone and corazonin hormonal systems. J. Biol. Chem. 285, Hua, Y., Ishibashi, J., Saito, H., Tawfik, A., Sakakibara, M., Tanaka, Y., Derua, R., Waelkens, E., Baggerman, G., De Loof, A., Schoofs, L., Tanaka, S., Identification of [Arg(7)] corazonin in the silkworm, Bombyx mori and the cricket, Gryllus bimaculatus, as a factor inducing dark color in an albino strain of the locust, Locusta migratoria. J. Insect Physiol. 46, Johnson, E.C., Shafer, O.T., Trigg, J.S., Park, J., Schooley, D.A., Dow, J.A., Taghert, P.H., A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J. Exp. Biol. 208, Kim, Y.J., Spalovska-Valachova, I., Cho, K.H., Žitňanova, I., Park, Y., Adams, M.E., Žitňan, D., Corazonin receptor signaling in ecdysis initiation. Proc. Natl. Acad. Sci. U. S. A. 101,

24 14 Koberle, F., Chagas' disease and Chagas' syndromes: the pathology of American trypanosomiasis. Adv. Parasitol. 6, Marco, H.G., Simek, P., Clark, K.D., Gäde, G., Novel adipokinetic hormones in the kissing bugs Rhodnius prolixus, Triatoma infestans, Dipetalogaster maxima and Panstrongylus megistus. Peptides 41, Mayer, R.J., Candy, D.J., Control of haemolymph lipid concentration during locust flight - an adipokinetic hormone from corpora cardiaca. J. Insect Physiol. 15, 611-&. McCann, F., Physiology of insect hearts. Annu. Rev. Entomol. 15, Nässel, D.R., Winther, A.M.E., Drosophila neuropeptides in regulation of physiology and behavior. Prog. Neurobiol. 92, Ons, S., Sterkel, M., Diambra, L., Urlaub, H., Rivera-Pomar, R., Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus. Insect Mol. Biol. 20, Ons, S., Richter, F., Urlaub, H., Rivera Pomar, R., The neuropeptidome of Rhodnius prolixus brain. Proteomics 9, Orchard, I., Loughton, B. G. (1985) Neurosecretion. In: Comprehensive Insect Physiology, Biochemistry, and Pharmacology, eds. Kerkut, G. A., Gilbert, L. I., Pergamon Press, Oxford, Vol. 7, pp Orchard, I., Lange, A.B., Bendena, W.G., FMRFamide-related peptides: a multifunctional family of structurally related neuropeptides in insects. Adv. Insect Physiol., 28, Osada, M., Treen, N., Molluscan GnRH associated with reproduction. Gen. Comp. Endocrinol. 181, Park, Y., Kim, Y.J., Adams, M.E., Identification of G protein coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc. Natl. Acad. Sci. U. S. A. 99, Prata, A., Clinical and epidemiological aspects of Chagas disease. Lancet Infectious Diseases 1, Predel, R., Neupert, S., Russell, W.K., Scheibner, O., Nachman, R.J., Corazonin in insects. Peptides 28, 3-10.

25 15 Remme J.H.F., Feenstra, P., Lever, P.R., et al., Tropical diseases targeted for elimination: Chagas disease, lymphatic filariasis, onchocerciasis, and leprosy. In: Infectious Diseases: Disease control priorities in developing countries. Eds. Jamison DT, Breman JG, Measham AR, et al., Washington (DC), World Bank. Pp Roch, G.J., Busby, E.R., Sherwood, N.M., Evolution of GnRH: Diving deeper. Gen. Comp. Endocrinol. 171, Roller, L., Tanaka, Y., Tanaka, S., Corazonin and corazonin-like substances in the central nervous system of the Pterygote and Apterygote insects. Cell Tissue Res. 312, Siegert, K., Ziegler, R., A hormone from the corpora cardiaca controls fat-body glycogen-phosphorylase during starvation in tobacco hornworm larvae. Nature 301, Smith, W., Rybczynski, R., Prothoracicotropic Hormone. In: Insect Endocrinology, Ed. Gilbert, L., Academic Press, London, pp Steel, C., Harmsen, R., Dynamics of neurosecretory system in brain of an insect, Rhodnius-prolixus, during growth and molting. Gen. Comp. Endocrinol. 17, Tanaka, S., Induction of darkening by corazonins in several species of Orthoptera and their possible presence in ten insect orders. Appl. Entomol. Zool. 35, Tanaka, S., Endocrine mechanisms controlling body color polymorphism in locusts. Arch. Insect Biochem. Physiol. 47, Tanaka, Y., Hua, Y., Roller, L., Tanaka, S., Corazonin reduces the spinning rate in the silkworm, Bombyx mori. J. Insect Physiol. 48, Tawfik, A., Tanaka, S., De Loof, A., Schoofs, L., Baggerman, G., Waelkens, E., Derua, R., Milner, Y., Yerushalmi, Y., Pener, M., Identification of the gregarizationassociated dark-pigmentotropin in locusts through an albino mutant. Proc. Natl. Acad. Sci. U. S. A. 96, Truman, J., Riddiford, L., The origins of insect metamorphosis. Nature 401, Veenstra, J.A., Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. FEBS Lett. 250,

26 16 Veenstra, J.A., Davis, N.T., Localization of corazonin in the nervous system of the cockroach Periplaneta americana. Cell Tissue Res. 274, Wigglesworth, V., 1959a. The histology of the nervous system of an insect, Rhodnius prolixus (Hemiptera). I. The peripheral nervous system. Q. J. Microsc. Sci. 100, Wigglesworth, V., 1959b. The histology of the nervous system of an insect, Rhodnius prolixus (Hemiptera). II. The central ganglia. Q. J. Microsc. Sci. 100, Wise, S., Davis, N.T., Tyndale, E., Noveral, J., Folwell, M.G., Bedian, V., Emery, I.F., Siwicki, K.K., Neuroanatomical studies of period gene expression in the hawkmoth, Manduca sexta. J. Comp. Neurol. 447, Žitňan, D., Kim, Y., Žitňanova, I., Roller, L., Adams, M.E., Complex steroid-peptidereceptor cascade controls insect ecdysis. Gen. Comp. Endocrinol. 153, Žitňan, D., Kingan, T.G., Hermesman, J.L., Adams, M.E., Identification of ecdysistriggering hormone from an epitracheal endocrine system. Science 271, Žitňan, D., Adams, M. E., Neuroendocrine regulation of ecdysis. In: Insect Endocrinology, Ed. Gilbert, L., Academic Press, London, pp

27 17 CHAPTER II : The distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus: Adipokinetic hormone, corazonin and adipokinetic hormone/corazoninrelated peptide. Abstract We have examined the distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in R. prolixus. These neuropeptides, adipokinetic hormone (RhoprAKH), corazonin (CRZ) and adipokinetic hormone/corazonin-related peptide (RhoprACP) are present in distinct, non-overlapping neuronal subsets in the central nervous system (CNS), as determined by immunohistochemistry. Corazonin-like immunoreactive cell bodies are present in the brain and ventral nerve cord, whereas ACP-like immunoreactive cell bodies are only present in the brain, and AKH-like immunoreactive cell bodies only present in the corpus cardiacum. The immunoreactivity to ACP, CRZ and AKH in R. prolixus suggests that ACP and CRZ are released within the CNS, and that CRZ and AKH are released as neurohormones from the CC. Injection of RhoprAKH into adult males elevated haemolymph lipid levels, but injection of corazonin or RhoprACP failed to have any effect on haemolymph lipid levels. Corazonin stimulated an increase in heart-beat frequency in vitro, but RhoprAKH and RhoprACP failed to do so. Currently there is no known biological function known for RhoprACP. Thus, although all three neuropeptides share sequence similarity, the AKH and corazonin receptors only respond to their own ligand.

28 18 Introduction Adipokinetic/red pigment concentrating hormones (AKH/RPCHs) are amongst the most studied arthropod neurohormones with family members isolated from over 70 different species (see Gäde and Marco, 2013). More than 50 members of this AKH/RPCH family are known by their primary structures throughout Crustacea and Insecta (see Gäde and Marco, 2013). In insects, biological activities attributed to AKHs include mobilization of lipid and carbohydrate haemolymph levels, stimulation of heartbeat rate and inhibition of protein synthesis (see Gäde and Marco, 2013; Gäde et al., 1997; Kodrik, 2008; Lorenz and Gäde, 2009; Siegert, 1999). More recently, in Drosophila, ablation of AKH-producing cells implicates AKH in stress responses; altering life span under starvation conditions (Isabel et al., 2005). Immunohistochemistry and peptide isolation indicates that AKHs are predominantly associated with intrinsic neurosecretory cells of the corpus cardiacum (CC) of insects (see Schooneveld et al., 1987; Noyes et al., 1995; Nässel and Winther, 2010); however, a small number of cells immunoreactive to AKH antisera have also been reported within the brain (Schooneveld et al., 1985; Kaufmann and Brown, 2006; Siegert, 1999). Subsequent work using in situ hybridization and other molecular techniques, however, have confirmed that the AKH transcript is only expressed in CC neurosecretory cells, and so the immunohistochemical staining reported earlier is now considered to be cross-reactivity with another peptide family (Noyes et al., 1995; Isabel et al., 2005; Hansen et al., 2010). Two peptides have subsequently been sequenced from insect brains that were referred to as AKHs (Siegert, 1999; Kaufmann and Brown, 2006), although the one isolated from Locusta migratoria was inactive in the lipid mobilization assay. In addition, to further complicate the story, a cardioacceleratory peptide, corazonin (CRZ), was isolated from Periplaneta

29 19 americana, and identified as having interesting similarities with some members of the AKH/RPCH family (Veenstra, 1989). CRZ is now known to be a highly conserved undecapeptide with diverse physiological effects in different insect species (see Roller et al., 2003), including induction of dark pigmentation in albino L. migratoria (Hua et al.,2000; Tanaka,2001;Tawfik et al., 1999) and prolonged spinning period in the domesticated silkworm, Bombyx mori (Tanaka et al.,2002). A comprehensive review of CRZ suggested that the evolutionary ancient function of CRZ may have been "to prepare animals for coping with the environmental stressors of the day" (Veenstra, 2009; Boerjan et al., 2010). The interesting similarity between CRZ, AKH/RPCH and the brain AKHs has recently been resolved. Thus, Hansen and colleagues de-orphanized a G protein-coupled receptor (GPCR) that had high sequence similarity to the AKH receptor in Anopheles gambiae (Hansen et al., 2010). The peptide ligand was observed to share similarities with both AKH/RPCH and with CRZ. Since this de-orphanized receptor and its ligand held a high sequence similarity to both the AKH/RPCH and CRZ the authors named this peptide AKH/corazonin-related peptide (ACP; Hansen et al., 2010). The authors also suggested that the previously reported brain AKHs (Siegert, 1999; Kaufman and Brown, 2006) were in fact ACPs. This finding presents a prime example of receptor/ligand co-evolution (see Park et al., 2002; Roch et al., 2011; Sun and Tsai, 2011; Grimmelikhuijzen and Hauser, 2012). In Tribolium castaneum, ACP was shown to be localized in a small number of neurons in the brain, with projections throughout the entire central nervous system (CNS), but none leaving the CNS. ACP and its receptor were predicted from genome sequences in eight different orders of insects, suggesting that it is an important insect signaling system. Although ACP appears to have a widespread presence in insects, nothing is known about its physiological

30 20 function or localization in insects other than A. gambiae and T. castaneum (Hansen et al., 2010). Although all three peptides share sequence similarity none of the A. gambiae peptides crossreact with the receptors for the other ligands (Hansen et al, 2010). Thus, receptor/ligand coevolution, where the ancestral receptor gene and its ligand gene have been duplicated twice during evolution yielding three independent neuropeptide/gpcr signaling systems (Park et al., 2002; Grimmelikhuijzen and Hauser, 2012; Hansen et al., 2010), has been suggested for these peptide families. Recently, Ons et al. (2011) and Marco et al. (2013) sequenced the Rhodnius prolixus AKH and CRZ, and the R. prolixus ACP was predicted from genome mining (Hansen et al., 2010). Rhodnius prolixus is a medically-important blood-gorging bug, acting as the vector of Chagas disease. It is an important model insect for studies of physiology and endocrinology. In this study, we have examined the distribution and physiological effects of these three evolutionarily and sequence-related peptides in R. prolixus and have also confirmed the sequence of RhoprACP using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS). The distribution of CRZ, AKH and ACP in distinct cell bodies and processes throughout the CNS has been investigated through immunohistochemistry, and the effects of each neuropeptide on heartbeat rate and lipid mobilization assessed.

31 21 Materials and methods Insects All insects were bred at the University of Toronto Mississauga. Colonies of R. prolixus were housed in an incubator at 26 C with 60% humidity. Insects were fed on defibrinated rabbit blood (Cedarlane Laboratories, Burlington, ON, Canada) once each instar to initiate development to the next instar. Synthetic peptides and antibodies Corazonin, RhoprACP and RhoprAKH (see Table 1 for sequences) were purchased from Genscript Laboratories (Piscataway, USA) and stock solutions of 10 µl aliquots of 10-3 M CRZ and 10-3 M RhoprACP were made using double-distilled water. RhoprAKH is not water soluble and so stock solutions of 5 µl aliquots of 2 X 10-3 M RhoprAKH were made using 10% ammonium hydroxide as per recommendation of Genscript Laboratories. All peptides were stored at -20 C. Further dilutions of all peptides were made using physiological saline (150mM NaCl, 8.6mM KCl, 2mM CaCl 2, 4mM NaHCO 3, and 8.5mM MgCl 2, 5 mm HEPES, ph 7.0). Mouse anti-acp was generated against the N-terminus of A. gambiae ACP by conjugating pglu-val-thr-phe-ser-asp-trp-asn-lys (purchased from GL Biochem, Shanghai, China), through its Lys residue to bovine serum albumin (BSA) using difluorodinitrobenzene (Tager, 1976). Four, six week old female C57B1/J6 mice were immunized with 50 µg of the ACP-conjugate in complete Freuds adjuvant, followed by three booster injection of 25 µg in incomplete Freuds adjuvant at six week intervals. Mice were bled two weeks after each booster injection and sera tested on the CNS of Zophobas morio.

32 22 The serum obtained after the last booster injection of mouse #143 was used here. Rabbit anti-crz (Veenstra and Davis, 1993) and rabbit anti-akh (generous gift from Mark Brown, University of Georgia, USA, see Kaufmann and Brown, 2006) were used throughout. Trichloroacetic acid (TCA) was purchased from Sigma (Oakville, ON, Canada) and triglyceride standard was purchased from Stanbio Laboratory (Texas, USA). Peptide sequencing Two hundred CNS of 5 th instar R. prolixus were dissected under saline and placed into 500 µl of methanol/acetic acid/water (90:9:1, by volume) and stored at -20 C. The samples were then processed through Sep-Pak as described in Lee and Lange (2011) and the fraction eluting with 50% acetonitrile in 0.1% Trifluoroacetic acid (TFA) was sequenced through MALDI-TOF MS/MS at the Advanced Protein Technology Centre (Hospital for Sick Children, Toronto, ON). Immunohistochemistry Unfed 5 th instar or adult female and male R. prolixus CNS, dorsal vessel, and other peripheral tissues were subjected to immunohistochemistry. Immunohistochemistry was performed as previously described in Lee and Lange (2011). Tissues were incubated with primary antiserum (1:1000 rabbit anti-crz or 1:500 mouse anti-acp or 1:500 rabbit anti-akh), made up in 0.4% Triton-X 100 and 2% normal sheep serum in phosphate buffered saline (2.1mM NaH 2 PO 4, 8.3mM NaH 2 PO 4 H 2 O, 150mM NaCl, ph 7.2). Controls were run whereby the primary antiserum was preabsorbed overnight at 4 C with either 10-5 M synthetic CRZ, 10-4 M RhoprAKH or 2 X 10-5 M RhoprACP prior to use. Immunoreactivity

33 23 was observed through a Zeiss confocal laser microscope (Carl Zeiss, Jena,Germany). Images were analyzed using ImageJ viewing software. Camera lucida was used to map out the processes and cells. Heart contraction assays Unfed 5 th instar R. prolixus (previously fed as 4 th instars four weeks prior to experimentation) were used. The ventral cuticle, digestive and reproductive systems were dissected under physiological saline and removed, thereby exposing the dorsal vessel. The dorsal cuticle was then attached dorsal side down to a Sylgard-coated dissecting dish using minutien pins. Electrodes attached to an impedance converter (UFI model 2991, Morro Bay, CA, USA) were placed between the 5 th and 6 th abdominal segments on either side of the dorsal vessel anterior to the alary muscles. The preparation was washed with saline and then allowed to stabilize in 50 µl saline for 20 min at room temperature. Various concentrations of peptide in 50 µl aliquots were exchanged for 50 µl of saline and the preparation monitored for 3 min. Heartbeat frequency was recorded on a Linear Flat-bed single channel chart recorder. The preparation was washed with saline frequently between applications of peptide. Heartbeat frequency was determined for 1 min before and after the application of different concentrations of the peptide. The response was quantified by measuring the frequency in peptide relative to the frequency in saline. One-way ANOVA followed by Bonferroni s post test was conducted for significant differences. Results are shown as mean ± standard error of the mean (SEM) of 4-5 replicates. Lipid and carbohydrate mobilization assay

34 24 Individual male adults fed 15 days prior to experimentation were used. Insects were immobilized by a short exposure to CO 2 and then injected with 2 µl of varying concentrations of CRZ, RhoprAKH or RhoprACP diluted in saline or just saline (control) between the meso- and metathoracic segments mid-ventrally using a 10 µl Hamilton syringe. The injected bugs were then placed into a glass container for 90 minutes, following which a haemolymph sample of 5 µl was withdrawn using a marked glass capillary (Drummond Scientific Company, Broomall, PA) from the clipped wing base. The 5 µl haemolymph samples were each placed in 50 µl of 10% trichloroacetic acid (TCA) to precipitate the lipoprotein. All samples were then centrifuged for 10 min at 25 C (Eppendorf Centrifuge 4513). To measure carbohydrate 20 µl of the supernatant was added to 1 ml of anthrone solution (72% H 2 SO 4 containing 50 mg anthrone and 1g thiourea, heated to C and then cooled), boiled for 15min, cooled and then read in a spectrophotometer at 620 nm as described by Roe (1955). A standard curve from 0 to 15 µg trehalose was run concurrently with the experimental samples. To measure lipid the pellets were dissolved in 400 µl of isopropanol and 100 µl of potassium hydroxide was added to the samples, which were then incubated at 60 C for 10 minutes. After cooling, 100 µl of sodium periodate (11.6mM sodium periodate in 2N glacial acetic acid) was added and the samples left at room temperature for 10 minutes. 600 µl of chromogenic solution (40mL 2M ammonium acetate, 40mL isopropanol, 150µL acetyl acetone) was added, the samples vortexed and then incubated at 60 C for 30 minutes. A standard curve of triglycerides ranging from 0 to 60 µg was run concurrently with the

35 25 experimental samples (Fletcher, 1968). The resultant colour was measured using a spectrophotometer (Spectronic 88, Bausch and Lomb) at 410 nm. Calculations of averages, graphical representations and statistical analysis were performed using GraphPad Prism (Version 5.0). One way ANOVA was utilized to determine the level of significance. Results are shown as mean lipid level ± standard error of the mean (SEM) of 4-15 replicates.

36 26 Results Peptide sequencing Extracts of R. prolixus CNS were subjected to MALDI-TOF MS/MS and found to contain a peak with a mass of and the sequence, pqvtfsrdwamide. This sequence confirmed the predicted sequence of ACP (Hansen et al., 2010). Thus, R. prolixus CNS does phenotypically express RhoprACP, as well as CRZ (Ons et al., 2011) and RhoprAKH (Marco et al., 2013). Immunohistochemistry CRZ-like immunoreactivity (IR) was present in cell bodies and processes in the CNS of R. prolixus (Figure 1). The brain possessed approximately 12 neurons containing CRZ-like IR (Figure 1A and 1E). There are two clusters of 3 bilaterally-paired neurons located dorsally in the protocerebrum of the brain. One of these clusters of 3 bilaterally-paired neurons, the lateral anterior cells (LACs), is located in the lateral anterior region of the protocerebrum near the optic lobes (Figure 1A, E). The second cluster of 3 bilaterally-paired neurons appear to be lateral neurosecretory cells (LNSCs) (Figure 1A, E) with at least one of the cells in each cluster sending an axon out of the brain via the nervi corporis cardiac II (NCCII) to produce multiple-branched processes containing CRZ-like IR over the CC (Figure 1A, B and E). These processes also extend along the anterior of the dorsal vessel as well as projecting to the anterior portion of the esophagus (not shown). A pair of axons containing CRZ-like IR were also observed to project from the brain through the subesophageal ganglion (SOG) and the prothoracic ganglion (PRO), and ending in the mesothoracic ganglionic mass (MTGM).

37 27 These produce an extensive neuropile of CRZ-like IR in the SOG, PRO and MTGM. No cell bodies were immunoreactive in the SOG or in the PRO (Figure 1C). The MTGM contained three bilaterally-paired cell bodies with CRZ-like IR, with one pair located in the mesothoracic neuromere and two pairs located in the abdominal neuromeres of the MTGM. These project axons to the brain. No CRZ-like IR was observed in processes leaving the CNS through any of the peripheral nerves, except for the NCCII. In preparations where the CRZ antiserum was preabsorbed for 24 h with 2 X10-5 M CRZ no staining was seen indicating that the staining was specific for CRZ. The anti-acp and the anti-akh antisera each stains cell bodies in the brain and also in the CC due to the similarity in the amino acid sequence of these two peptides (Table 1). Since AKH is only found in the CC of insects, we can assume the staining in the brain by both the ACP and the AKH antisera is ACP. ACP-like IR is present in two bilaterally-paired cell bodies located in the anterior protocerebrum at the junction with the optic lobes, one of which always stains very faintly, and one large bilaterally-paired cell body located in the posterior region of the protocerebrum (Figure 2A). Interestingly, the axons from the anterior pair of cells cross the medial commissure and end in neuropilar varicosities in the contralateral lobe (Figure 2B and 2E) of the brain. The more posteriorly located cell bodies have axons that project to the median line of the brain, branch, and then project ventrally and posteriorly out of the brain and throughout the CNS ending in the MTGM (Figure 2C-D; Figure 2E). No projections from these cells leave the CNS. All staining was greatly reduced by preabsorption with RhoprACP (2 X 10-5 M) or RhoprAKH (2 X 10-5 M).

38 28 AKH is only found in the CC of insects and staining with the AKH and ACP antisera reveals cell bodies with multiple branched processes and varicosities in the CC (Figure 3). The AKH- like immunoreactive processes project out of the CC and onto the anterior portion of the dorsal vessel. Staining was either eliminated or greatly reduced by pre-absorbing the AKH antisera with either RhoprAKH (2 X 10-5 M) or RhoprACP (2 X 10-5 M). These patterns of immunoreactivity for CRZ, ACP and AKH appear to be consistent between 5 th instar and adult R. prolixus. No staining was present on any peripheral tissues examined, including the heart, foregut, midgut, hindgut, salivary glands, Malphigian tubules, or the reproductive system of adult males and females (data not shown). Heart contraction assays Heartbeat frequency increased in a dose-dependent manner in the presence of CRZ with maximum increase observed at about 10-8 M (14.8 ± 2.1 beats per minute greater than saline control which had a basal rate of 10.9 ± 1.7 beats/minute) (one way ANOVA, P< ; Bonferroni's post-test, p-value< 0.05) (Figure 4A and 4D). In contrast, heartbeat frequency was not affected by RhoprACP (one way ANOVA, p= 0.69) (Figure 4B and 4E) or RhoprAKH (one way ANOVA, p= 0.41) (Figure 4C and 4F). Lipid mobilization assay A time course of action of RhoprAKH on mobilization of haemolymph lipid in vivo was performed. The lipid levels rose from 5.2 ± 0.4 µg/µl in saline injected bugs to 9.7±1.0 µg/µl in bugs injected with 15 pmol of RhoprAKH at 90 minutes (Figure 5A). This time period (90 min) was used in subsequent experiments.

39 29 RhoprAKH significantly increases haemolymph lipid levels in adult male R. prolixus in a dose-dependent manner with maximum increase at approximately 15 pmol RhoprAKH (Oneway ANOVA, Bonferroni's post-test, p< ) (Figure 5B). The threshold concentration of RhoprAKH was seen between 1 pmol to 2.5 pmol. Neither CRZ (Figure 6A) nor RhoprACP injection (Figure 6B) resulted in any significant increase in the haemolymph lipid levels (One-way ANOVA, p< 0.4 and p<0.3, respectively) at any of the concentrations tested. Carbohydrate mobilization assay Haemolymph carbohydrate levels were found to be quite low in R. prolixus (resting levels approximately µg/µl). These levels were not elevated by injection of 15 pmol RhoprAKH (Figure 7).

40 30 Figure 1. Confocal microscope images and camera lucida composite of CRZ-like immunoreactivity in the CNS of Rhodnius prolixus. Whole mount preparations show clusters of bilaterally-paired CRZ-like immunoreactive cells in A) the brain, in which 3 pairs of lateral neurosecretory cells (LNSCs) are located in the protocerebrum and 3 pairs of lateral anterior cells (LACs) are located at the junction of the protocerebrum and optic lobes. Axons project from the LNSCs to the corpus cardiacum (CC) via the nervi corpora cardiaca II (NCCII) (open arrows). No immunoreactive cells are seen in the sub-esophageal ganglion (SOG). B) Higher magnification showing axons in the NCCII (open arrows), and branched processes within the CC. C) Immunoreactive axons project through the prothoracic ganglion (PRO) and continue out through connectives. Branches project into the neuropile. D) Axons from the prothoracic ganglion project into the mesothoracic ganglionic mass (MTGM) where they branch and result in the neuropile. There are also three pairs of bilaterally-paired cell bodies. E) Camera lucida composite showing the CRZ-like immunoreactive cell bodies and processes. Neuropile regions are shown as stippled and axons are shown as solid lines. Scale bars: A-D, 100 µm; E, 200 µm, n= 20.

41 31

42 32 Figure 2. Confocal microscope images and camera lucida composite of ACP-like immunoreactive cell bodies and processes in the CNS of Rhodnius prolixus. Whole mount preparations show 3 bilaterally-paired immunoreactive cell bodies. A) Brain, where two bilaterally-paired cell bodies (one always stains faintly) are present in the anterior portion of the protocerebrum near the optic lobes with axons projecting towards the central commissure and across the midline of the brain into the contralateral hemisphere, where they produce varicosities. B) Bilaterally-paired cell bodies in the medial portion of the protocerebrum with axons projecting towards the central commissure and then descending ventrally and posteriorly to project throughout the CNS. C) The axons from the brain project through the sub-esophageal ganglion (SOG) and then into the prothoracic ganglion (PRO) where they branch and produce varicosities. D) The descending axons continue into the mesothoracic ganglionic mass (MTGM) and end in varicosities and branched processes. E) Camera lucida drawing showing the ACP-like immunoreactivity in the CNS. Neuropile regions are shown as stippled and axons are shown as solid lines. Scale bars: A-D, 100 µm; E, 200 µm, n= 20.

43 33

44 34 Figure 3. Confocal microscope image and camera lucida composite of AKH -like immunoreactivity in the corpus cardiacum and dorsal vessel of Rhodnius prolixus. Whole mount preparations show clusters of bilaterally-paired AKH-like immunoreactive cell bodies in the corpus cardiacum (CC). These cell bodies result in multiple branched processes with varicosities. The processes extend onto the dorsal vessel (DV). No processes are seen in the nervi coporis cardiaci I (NCCI). Scale bar: 100 µm.

45 35

46 36 Figure 4. Effects of CRZ, RhoprACP and RhoprAKH on heartbeat frequency of 5 th instar Rhodnius prolixus. Traces illustrate the effect of varying concentrations of A) Corazonin (CRZ), B) RhoprACP and C) RhoprAKH on heartbeat frequency. Each trace shows heartbeat frequency over 60s in saline and following the application of the peptide. D) The increase in heartbeat frequency induced by is dose-dependent. No effect was observed for E) RhoprACP or F) RhoprAKH. Results are expressed as difference in average heartbeat rate compared to the average heartbeat rate in saline. Symbols represent mean ± standard error of mean (n= 4-5). Treatments with different letters denote significant difference. (Bonferroni's post-test, P < 0.005).

47 37

48 38 Figure 5. Time course and dose-response curve showing changes in haemolymph lipid levels following injection of saline or RhoprAKH. A) RhoprAKH (squares) increase haemolymph lipid levels over a 90 min period. Saline (circles) has no effect. Symbols represent mean ± standard error of mean (SEM) (n= 4-6). B) The effect of RhoprAKH on lipid levels is dose-dependent. The maximum increase can be seen at 15 pmol. The threshold concentration is between 1 to 2.5 pmol. Bonferroni's post-test showed a p-value < Data represents mean ± standard error of mean (n=5-11).

49 39

50 40 Figure 6. Neither CRZ nor RhoprACP alter haemolymph lipid levels of adult male Rhodnius prolixus tested 90 minutes after injection. Symbols represent mean ± standard error of mean (SEM) (n= 4-6).

51 41

52 42 Figure 7. RhoprAKH does not alter haemolymph carbohydrate (CHO) levels of adult male Rhodnius prolixus. Symbols represent mean ± standard error of mean. (n= 4-6).

53 43

54 44 Discussion The evolutionary-related peptides CRZ, AKH and ACP are phenotypically-expressed in R. prolixus and occur in distinct, non-overlapping neuronal subsets within the CNS of both 5 th instar and adults. CRZ-like IR is present in bilaterally-paired cell bodies throughout the CNS, including brain neurosecretory cells that project to the CC. These results are consistent in a general sense with previous studies on insects from six different orders (Roller et al., 2003), although there is some variability between different taxa. Thus, a subgroup of lateral neurosecretory cells in the brain with ipsilateral projections to the CC is a common feature, along with collaterals that project into the protocerebral neurpil region. Corazonin would therefore appear to be a neurohormone in insects. Bilaterally-paired cell bodies are typically observed in the thoracic and abdominal ganglia in insects, although in R. prolixus these appear to be confined to the MTGM and not present in the SOG and PRO. These ventral interneurons project prominent axons throughout the ventral nerve cord to the brain and their axonal tracts produce extensive arborization in neuropiles of all ganglia. This pattern of CRZ-like IR in the CNS of R. prolixus is consistent with the pattern in another Hemipteran, which also is a vector of Chagas disease, Triatoma infestans (Settembrini et al., 2012). We also examined R. prolixus CNS using an antiserum generated against A. gambiae ACP and an antiserum generated against Drosophila AKH. The R. prolixus amino acid sequences for RhoprACP and RhoprAKH are very similar, and so it is not surprising that each antiserum might identify neurons containing ACP or AKH. We believe the staining in the R. prolixus brain to represent ACP because of the similarities with the staining profile of T. castaneum. There is only one published report on the distribution of ACP within an insect,

55 45 that of 5 th instar larvae of T. castaneum (Hansen et al., 2010), although that paper did not mention if there was staining in the CC. Only bilaterally-paired neurons (one of which was weakly stained) are present in the brain of T. castaneum (none in the ventral nerve cord). These neurons have axons that project throughout the CNS, remaining in the mid-line of each ganglion. Hansen et al (2010) observed the highly varicose appearance of these projections and suggested a neurosecretion role for ACP. In a similar fashion within R. prolixus only 3 bilaterally-paired neurons are observed within the brain, with the anti-acp antiserum and the anti-akh antiserum, and these also produce axons projecting posteriorly throughout the ventral nerve cord. The branching pattern seems very similar to that described for T. castaneum, with axons medially-located within the ventral nerve cord and quite blunt, varicose terminals within the more medial neuropiles. Again, no axons exit the CNS. This cross-reactivity in staining was anticipated from earlier work since the primary antiserum used for detecting AKH-like IR recognizes the A. gambiae AKH I found in the CC, and also the AKH II (now identified as ACP) in the brain (Kaufmann and Brown, 2006). Interestingly, in A. gambiae, the brain neurons appear to be neurosecretory with axons projecting to the CC. Similarly, the inactive AKH in L. migratoria (Siegert, 1999) which is also an ACP, was biochemically identified in the storage lobe of the CC, suggestive of synthesis in brain neurosecretory cells. The true insect AKH is now considered to be only synthesized in intrinsic neurosecretory cells of the CC (Bednářová et al., 2010; Staubli et al., 2002), and thus the CC in R. prolixus contains numerous AKH-like immunoreactive cell bodies and extensive varicosities, consistent with that shown in other insects, and consistent with AKH being released as a neurohormone from the CC.

56 46 The immunoreactivity to ACP, CRZ and AKH in R. prolixus suggests that ACP and CRZ are released within the CNS, and that CRZ and AKH are released as neurohormones from the CC. Hansen et al (2010) consider that ACP is a structurally intermediate peptide between AKH and CRZ, but ACP does not activate the A. gambiae AKH or CRZ receptor. Indeed, each peptide only activates its own receptor in A. gambiae. This was examined using bioassay in R. prolixus. Corazonin increases the frequency of heartbeat in R. prolixus in a dose-dependent manner. Among the cardioacceleratory peptides explored in R. prolixus, the most effective peptides known are CCAP (crustacean cardioacceleratory peptide) and proctolin (Lee and Lange, 2011; Orchard et al., 2011). CCAP elicited maximum increase in heart rate at low concentrations (10-10 M) whereas higher concentrations of 10-7 M were needed to reach maximum with proctolin and CRZ (Lee and Lange, 2011; Orchard et al., 2011). Interestingly, the cardioacceleratory effect of CRZ has previously only been shown in P. americana, and not in other insects (see Nässel and Winther, 2010; Hillyer et al., 2012); however, it is always possible that in other insects there may be an influence on stroke volume, thereby stimulating haemolymph flow. Such has been shown for CCAP where CCAP does not increase heartbeat frequency in L. migratoria but does increase stroke volume (da Silva et al., 2011). Interestingly, fly atlas shows expression of the CRZ receptor in Drosophila aorta ( In contrast to CRZ s positive effect on heartbeat frequency in R. prolixus, heartbeat frequency was not affected by AKH or ACP. This is interesting since AKHs have been observed to be cardioacceleratory in several insects, including Baculum extradentatum and Drosophila pupae (Malik et al., 2012; Gäde and Marco, 2013; Noyes et al., 1995). This result in R. prolixus shows that despite the sequence similarity between RhoprAKH, CRZ and RhoprACP, CRZ is unique in its

57 47 cardioacceleratory abilities in R. prolixus, and therefore the CRZ receptor is not activated by RhoprAKH or RhoprACP. In a similar fashion, we examined whether RhoprAKH had lipid mobilizing ability and also whether RhoprACP and CRZ could activate the AKH receptor in R. prolixus. RhoprAKH induces a dose-dependent increase in haemolymph lipid content in vivo. RhoprAKH has previously been shown to elevate haemolymph lipid levels in R. prolixus following injection (Marco et al., 2013); however, quite small increases were observed. Here, we found RhoprAKH to increase haemolymph lipid levels in adult male R. prolixus with maximum increase at 15 pmol RhoprAKH and threshold between 1 pmol to 2.5 pmol. These results are consistent with those of Marco et al. (2013) although the increases observed here are much larger. One possible explanation could be that RhoprAKH is quite insoluble in H 2 O, and in the earlier study RhoprAKH was made up in H 2 O (Marco et al., 2013). Thus the effective concentration of RhoprAKH may have been less than predicted. Nonetheless, the increase in lipid mobilization in the haemolymph of R. prolixus over 90 min is relatively small (7 µg/µl to 11.4 µg/µl), when compared to well-known model insects, such as the migratory locust, L. migratoria where, for example, injection of 2 pmol of AKH (Lom-AKH I) raises the concentration of haemolymph lipid level from 16 µg/µl to 63 µg/µl in 90 min. Flight for 60 min by R. prolixus results in an increase of approximately 57% in diacylglycerol concentration in the haemolymph but no increase in trehalose (Ward et al., 1982). This suggests that AKHs are likely released during flight to elevate haemolymph lipid levels, and so our increase in haemolymph lipid levels of 62% following injection of RhoprAKH is consistent with this notion.

58 48 In R. prolixus, neither CRZ nor RhoprACP were capable of elevating haemolymph lipid levels suggesting that they do not activate the AKH receptor. At present there is no function assigned to ACP and therefore no bioassay available. Although CRZ does not activate AKH receptors in the lipid mobilizing bioassay in R. prolixus, the effect of CRZ on lipid metabolism has been explored via RNA interference of the peptide and its receptor in Drosophila where CRZ was found to extend survival and alter the carbohydrate and lipid metabolism (Kapan et al., 2012). Ablation of CRZ-containing lateral neurosecretory cells resulted in a slight but statistically significant decrease in trehalose levels suggesting that CRZ might modulate the activity of AKH cells (Lee et al., 2008). It is interesting that the structures of these three peptides are related, as are their respective receptors (see Hansen et al., 2010). Although all three share sequence similarity, none of the peptide ligands cross reacts with the receptors for the other ligands in A. gambiae (Hansen et al., 2010). Similarly, in R. prolixus, neither RhoprAKH nor RhoprACP activate the CRZ receptor that elevates heartbeat frequency; and neither CRZ nor RhoprACP activate the AKH receptor that results in haemolymph lipid elevation. Thus, this appears to confirm the notion of receptor/ligand co-evolution, where an ancestral receptor gene and its ligand gene have been duplicated twice during evolution, yielding three independent neuropeptide/gpcr signaling systems (Hansen et al., 2010). Through phylogenetic sequence analysis, it was found that AKH, CRZ, and ACP share a common ancestor and belong to the GnRH superfamily (Park et al., 2002; Roch et al., 2011; Grimmelikhuijzen and Hauser, 2012). This shows that GnRH already originated before the split of Proto- and Deuterostomia. Although the components of this signaling system are well conserved, their functions in the different groups has evolved considerably. Whereas in vertebrates, GnRH plays an essential role in

59 49 reproduction, in mollusks and insects, this does not appear to be the case (Veenstra, 2009; Roch et al., 2011; Sun and Tsai, 2011; Sun et al., 2012). Thus, elucidating the functions of AKH, CRZ and ACP may provide clues as to how functions change during evolution of neuropeptides.

60 50 Acknowledgements We would like to thank Prof. Mark Brown (University of Georgia, U.S.A.) for the gift of the AKH antiserum. This work was supported by the Natural Sciences and Engineering Research Council of Canada.

61 51 References Bednářová, A., Kodrik, D., Krishnan, N., Unique roles of glucagon and glucagon-like peptides: Parallels in understanding the functions of adipokinetic hormones in stress responses in insects. Comp. Biochem. Physiol. 164, Boerjan, B., Verleyen, P., Huybrechts, J., Schoofs, L., De Loof, A., In search for a common denominator for the diverse functions of arthropod corazonin: A role in the physiology of stress? Gen. Comp. Endocrinol. 166, da Silva, S.R., da Silva, R., Lange, A.B Effects of crustacean cardioactive peptide on the hearts of two Orthopteran insects, and the demonstration of a Frank Starling-like effect. Gen. Comp. Endocrinol. 171, Fletcher, M., A colorimetric method for estimating serum triglycerides. Clin. Chim. Acta 22, Gäde, G., Marco. H.G., AKH/RPCH Peptides, in: Kastin, A. (Ed.), Handbook of Biologically Active Peptides. Academic Press, San Diego, pp Gäde, G., Hoffmann, K.H., Spring, J.H., Hormonal regulation in insects: Facts, gaps, and future directions. Physiol. Rev. 77, Grimmelikhuijzen, C.J.P., Hauser, F., Mini-review: The evolution of neuropeptide signaling. Regul. Pept. 177, S6-S9. Hansen, K.K., Stafflinger, E., Schneider, M., Hauser, F., Cazzamali, G., Williamson, M., Kollmann, M., Schachtner, J., Grimmelikhuijzen, C.J.P., Discovery of a novel insect neuropeptide signaling system closely related to the insect adipokinetic hormone and corazonin hormonal systems. J. Biol. Chem. 285, Hillyer, J.F., Estévez-Lao, T.Y., Funkhouser, L.J., Aluoch, V.A., Anopheles gambiae corazonin: gene structure, expression and effect on mosquito heart physiology. Insect Mol. Biol. 21, Hua, Y., Ishibashi, J., Saito, H., Tawfik, A., Sakakibara, M., Tanaka, Y., Derua, R., Waelkens, E., Baggerman, G., De Loof, A., Schoofs, L., Tanaka, S., Identification of [Arg(7)] corazonin in the silkworm, Bombyx mori and the cricket, Gryllus bimaculatus, as a factor inducing dark color in an albino strain of the locust, Locusta migratoria. J. Insect Physiol. 46,

62 52 Isabel, G., Martin, J., Chidami, S., Veenstra, J.A., Rosay, P., AKH-producing neuroendocrine cell ablation decreases trehalose and induces behavioral changes in Drosophila. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, R531-R538. Kapan, N., Lushchak, O.V., Luo, J., Nässel, D.R., Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by co-expressed short neuropeptide F and corazonin. Cell. Mol. Life Sci. 69, Kaufmann, C., Brown, M.R., Adipokinetic hormones in the African malaria mosquito, Anopheles gambiae: Identification and expression of genes for two peptides and a putative receptor. Insect Biochem. Mol. Biol. 36, Kodrik, D Adipokinetic hormone functions that are not associated with insect flight. Physiol. Entomol. 33, Lee, D.H., Lange, A.B., Crustacean cardioactive peptide in the Chagas' disease vector, Rhodnius prolixus: Presence, distribution and physiological effects. Gen. Comp. Endocrinol. 174, Lee, G., Kim, K., Kikuno, K., Wang, Z., Choi, Y., Park, J.H., Developmental regulation and functions of the expression of the neuropeptide corazonin in Drosophila melanogaster. Cell Tissue Res. 331, Lorenz, M.W., Gäde, G., Hormonal regulation of energy metabolism in insects as a driving force for performance. Integr. Comp. Biol. 49, Malik, A., Gäde, G., Lange, A.B., Sequencing and biological effects of an adipokinetic / hypertrehalosemic peptide in the stick insect, Baculum extradentatum. Peptides 34, Marco, H.G., Simek, P., Clark, K.D., Gäde, G., Novel adipokinetic hormones in the kissing bugs Rhodnius prolixus, Triatoma infestans, Dipetalogaster maxima and Panstrongylus megistus. Peptides 41, Nässel, D.R., Winther, A.M.E., Drosophila neuropeptides in regulation of physiology and behavior. Prog. Neurobiol. 92, Noyes, B.E., Katz, F.N., Schaffer, M.H., Identification and expression of the Drosophila adipokinetic hormone gene. Molec. Cell. Endocrinol. 109,

63 53 Ons, S., Sterkel, M., Diambra, L., Urlaub, H., Rivera-Pomar, R., Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus. Insect Molec. Biol. 20, Orchard, I., Lee, D.H., da Silva, R., Lange, A.B., The proctolin gene and biological effects of proctolin in the blood-feeding bug, Rhodnius prolixus. Front. Endocrinol. 2, Park, Y., Kim, Y.J., Adams, M.E., Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc. Natl. Acad. Sci. U. S. A. 99, Roch, G.J., Busby, E.R., Sherwood, N.M., Evolution of GnRH: Diving deeper. Gen. Comp. Endocrinol. 171, Roe, J.H., The determination of sugar in blood and spinal fluid with anthrone reagent. J. Biol. Chem. 212, Roller, L., Tanaka, Y., Tanaka, S., Corazonin and corazonin-like substances in the central nervous system of the Pterygote and Apterygote insects. Cell Tissue Res. 312, Schooneveld, H., Romberg-Privee, H.M., Veenstra, J.A., Adipokinetic hormoneimmunoreactive peptide in the endocrine and central nervous system of several insect species: A comparative immunocytochemical approach. Gen. Comp. Endocrinol. 57, Schooneveld, H., Romberg-Privee, H.M., Veenstra, J.A., Phylogenetic differentiation of glandular cells in corpora cardiaca as studied immunocytochemically with regionspecific antisera to adipokinetic hormone. J. Insect Physiol. 33, Settembrini, B.P., de Pasquale, D., Postal, M., Pinto, P.M., Carlini, C.R., Villar, M.J., Distribution and characterization of corazonin in the central nervous system of Triatoma infestans (Insecta: Heteroptera). Peptides 32, Siegert, K., Locust corpora cardiaca contain an inactive adipokinetic hormone. FEBS Lett. 447, Staubli, F., Jorgensen, T.J.D., Cazzamali, G., Williamson, M., Lenz, C., Sondergaard, L., Roepstorff, P., Grimmelikhuijzen, C.J.P., Molecular identification of the insect adipokinetic hormone receptors. Proc. Natl. Acad. Sci. U. S. A. 99,

64 54 Sun, B., Tsai. P.S., A gonadotropin-releasing hormone-like molecule modulates the activity of diverse central neurons in a gastropod mollusk, Aplysia californica. Front. Endocrinol. 2:36. Sun, B., Kavanaugh, S.I., Tsai, P.S., Gonadotropin-releasing hormone in protostomes: insights from functional studies on Aplysia californica. Gen. Comp. Endocrinol. 176, Tager, H.S., Coupling of peptides to albumin with difluorodinitrobenzene. Anal. Biochem. 71, Tanaka, S., Endocrine mechanisms controlling body-color polymorphism in locusts. Arch. Insect Biochem. Physiol. 47, Tanaka, Y., Hua, Y., Roller, L., Tanaka, S., Corazonin reduces the spinning rate in the silkworm, Bombyx mori. J. Insect Physiol. 48, Tawfik, A., Tanaka, S., De Loof, A., Schoofs, L., Baggerman, G., Waelkens, E., Derua, R., Milner, Y., Yerushalmi, Y., Pener, M., Identification of the gregarizationassociated dark-pigmentotropin in locusts through an albino mutant. Proc. Natl. Acad. Sci. U. S. A. 96, Veenstra, J.A., Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. FEBS Lett. 250, Veenstra, J.A., Davis, N.T., Localization of corazonin in the nervous system of the cockroach Periplaneta americana. Cell Tissue Res. 274, Veenstra, J.A., Does corazonin signal nutritional stress in insects? Insect Biochem. Molec. Biol. 39, Ward, J.P., Candy, D.J., Smith, S.N Lipid storage and changes during flight by Triatomine bugs (Rhodnius prolixus and Triatoma infestans). J. Insect Physiol. 28,

65 55 CHAPTER III : Possible involvement of corazonin in ecdysis of 4 th instar Rhodnius prolixus: A preliminary analysis. Abstract Insects must periodically shed their cuticle so that they can increase in size. This process is called ecdysis and is highly controlled, involving many neuropeptides and hormones. Here, we investigated the possible involvement of corazonin (CRZ) in ecdysis of 4th instar into 5th instar Rhodnius prolixus. The timing of ecdysis for 4 th instar R. prolixus is gated and occurs between days after a blood meal and between 8-12 hours after lights off. Immunohistochemistry performed on the central nervous system (CNS) at different developmental times indicated a reduction in CRZ-like immunoreactivity (IR) at 2 hours post-ecdysis. This indicates that CRZ may be involved in ecdysis. Preliminary RNA interference experiments show that there was a reduction in CRZ-like IR in cell bodies and processes within the CNS after injection of double stranded CRZ (dscrz) suggesting that CRZ was successfully knocked down. Preliminary experiments reveal that the dscrzinjected insects successfully ecdysed between days post-feeding; however their timing was delayed until hours after lights off. This data suggests that CRZ might play a role in gating of ecdysis.

66 56 Introduction Although the detailed mechanisms of metamorphosis vary between different species of insects, the general pattern of hormone action is usually similar. In the 1930s, Sir Vincent Wigglesworth made extensive advances in the physiology of metamorphosis in the hemimetabolous insect, Rhodnius prolixus. Due to his observations and experimentation, ecdysis, the periodic shedding of the old cuticle, in R. prolixus can be accurately predicted from the time of feeding. His study of changes in the cuticle during ecdysis and release of hormonal factors from the corpora allata were breakthroughs in the understanding of this phenomenon (Wigglesworth, 1933, 1934, 1940). Ampleford and Steel (1982a,b) described the stereotypical behaviours of ecdysis which included preparatory, pre-ecdysial, and ecdysial phases in R. prolixus. In insects, the behaviour characterized in the preparatory phase is variable in duration, but eventually culminates in the ecdysis attempt (Carlson, 1977; Hughes, 1980; Ampleford and Steel, 1982a). The ecdysis phase starts with abdominal peristaltic waves that begin to move the old cuticle posteriorly. These are coordinated with other behavioural routines to dis-attach the appendages from the old cuticle as it is shed. Changes in blood distribution with air-swallowing expands the anterior end of the animal and aids in rupturing the old cuticle (Reynolds, 1980). Since these early descriptions on R. prolixus, a hemimetabolous (incomplete metamorphosis) insect, most studies concerning ecdysis have been done on species with complete metamorphosis (holometabolous insects) such as Manduca sexta (Žitňan et al., 2007; Mesce and Fahrbach, 2002; Kim et al., 2004; Gammie and Truman, 1997), Drosophila melanogaster (McNabb et al., 1997, Zhao et al., 2010; Choi et al., 2006) and Bombyx mori

67 57 (Sakurai, 1983; Tanaka et al., 2002; Žitňan et al., 2002). The discovery of the ecdysistriggering hormone (ETH) sparked an era of important advances in our understanding of the regulation of molting (Žitňan et al., 1996; Klein et al., 1999). Since then, the relationship in the release of cascades of regulatory peptides from the neural circuitry has been confirmed to be the ultimate driver of the behaviors associated with ecdysis (Mesce and Fahrbach, 2002). Žitňan et al. (1996) surveyed peptide expression during molting in M. sexta and revealed that Inka cells (peripheral cells located on trachea) produce three crucial neuropeptides to "initiate" the entire ecdysis-related sequence. Two of these neuropeptides are recognized to be pre-ecdysis and ecdysis-triggering hormones (PETH and ETH) which activate the ecdysis sequence through receptor-mediated actions on neurons in the central nervous system (CNS) (Žitňan et al., 2007). It is interesting that in M. sexta, corazonin (CRZ) elicits a graded low level release of ETH (Kim et al., 2004). The discovery of CRZ receptors on Inka cells in this species, and the release of CRZ just before pre-ecdysis onset provides a new perspective on additional functions of CRZ other than the initial role of a cardioacceleratory (Veenstra and Davis, 1993). In B. mori, 1 pmol of CRZ induced a significant prolongation of the spinning period and more than 50% of the larvae could not shed their cuticle and died when 100 pmol CRZ was injected (Tanaka et al., 2002). Additionally, in the larvae injected with CRZ, the increase in haemolymph ecdysteroid level was delayed during the spinning stage. Thus, a delay in ecdysteroid level increases might cause a delay in pupal ecdysis and CRZ might have inhibitory effects on the release of prothoracicotropic hormone (PTTH) that stimulates the prothoracic glands to secrete ecdysteroids (Ishizaki and Suzuki, 1994; Smith and Rybscynski, 2012). It was also recently reported that B. mori CRZ receptor expression was detectable in most tissues, with the silk gland a major site of expression (Yang et al., 2013).

68 58 Since little is known about the involvement of CRZ in hemimetabolous insects, exploring the involvement of CRZ in R. prolixus should reveal information about the peptide's physiological function and involvement in ecdysis. Rhodnius prolixus is not only a medically-important vector of Chagas disease but also an excellent model for the study of physiological processes due to its tightly regulated growth and development. It also provides a great opportunity to examine ecdysis in a hemimetabolous insect since little is known about the hormonal orchestration involved in ecdysis compared to holometabolous counterparts. This investigation provides preliminary information investigating the physiological function of this highly conserved neuropeptide, CRZ, in the process of ecdysis. The initial observations examined the pre-ecdysis, ecdysis and post-ecdysis behaviours of 4th instars molting to 5th instars. Fourth instars will go through ecdysis days after their last blood meal. It was also confirmed through observations that ecdysis is tightly regulated by a circadian rhythm and cues from the environment, such as lights off. Corazonin-like immunoreactivity (IR) is decreased in the blebs, varicosities and neurohemal sites in the CNS from R. prolixus 24hr prior to the expected ecdysis and up to 2hrs after ecdysis. This indicates that CRZ may be released between these time points to facilitate or initiate ecdysis. Thus, preliminary evidence that CRZ may be released 24 hr before ecdysis enabled the design of knock-down RNA interference experiments, in which double stranded CRZ (dscrz) was injected into 4th instars and the effect on ecdysis studied. Immunohistochemistry revealed that the knock-down of CRZ was successful since there was a great reduction in CRZ-like IR in cell bodies and processes in the brain and mesothoracic ganglionic mass (MTGM) collected 5 days after dscrz injections. In addition, preliminary results suggest that insects treated with dscrz had their timing of ecdysis disrupted.

69 59 Methods and Materials Animals Forty mixed male and female 4th instars were kept in a 16hr:8hr light/dark cycle at 28ºC and 30-40% humidity. They were observed daily following a blood meal of defibrinated rabbit blood until ecdysis. The timing and behaviour of pre-ecdysis, ecdysis and post-ecdysis was recorded. Immunohistochemistry The changes in staining intensity of CRZ-like IR in cell bodies and processes during developmental time points between 4th and 5 th instar were done using immunohistochemistry. The CNS was dissected at 6 specific developmental time points: 1) unfed 4th instars 2) six days post fed 4th instars 3) twelve days post fed 4th instars 5) 2 hours after ecdysis 6) six days post ecdysis. Immunohistochemistry was performed as previously described in Lee and Lange (2011). Tissues were incubated in 1:1000 rabbit anti- CRZ (Veenstra and Davis, 1993) in 0.4% Triton-X 100 and 2% normal sheep serum in phosphate buffered saline (PBS; 2.1mM NaH 2 PO 4, 8.3mM NaH 2 PO 4 H 2 O, 150mM NaCl, ph 7.2). Tissues collected at different developmental times were stored in PBS and were processed for immunohistochemistry all at the same time. RNA interference RNA interference experiments was performed as described in Lee et al. (2013). A double stranded construct for CRZ was made to down regulate CRZ mrna. Briefly, 4th instar R. prolixus were anesthetized with CO 2 for 10 sec and 1 µl of 2 µg of dscrz was injected into

70 60 the thorax using a 5 µl Hamilton syringe. As a control, groups of R. prolixus were injected with either 1 µl of double stranded ampicillin resistance gene (dsarg) or had no injection. Injected insects were left for 1 hour at room temperature to recover and then placed into an incubator at 28 C on a 16hr:8hr light/dark cycle. To check for the presence of the CRZ transcript immunohistochemistry was used. Five CNSs were collected on the 1) first day after injection, 2) third day after injection and 3) fifth day after injection.

71 61 Results Circadian control of Ecdysis Preliminary data suggests that 4th instars take days after feeding to ecdyse (Figure 1) with day 13 and 4 post-feeding being the prominent days when the majority of the insects ecdyse. Figure 2 shows that the process of ecdysis is gated and takes place 8hr to 16 hr after lights off. This gated ecdysis allowed for observations of pre-ecdysis, ecdysis and postecdysis behaviours. The majority of insects (78%) complete ecdysis during this gate, i.e hr after lights off. There were a small percentage of insects (about 5%) that ecdysed overnight when they were not being observed (i.e. after 18:00 hr and before 8 hr). Behaviour of Ecdysis The total duration of pre-ecdysis behaviours ranged from min and included antennae rubbing, lodging the body upside down, air swallowing which was followed by head and body bobbing. The ecdysis phase had a duration of min and commenced immediately after the pre-ecdysis phase. If the insect going through pre-ecdysis was disturbed or interrupted, the pre-ecdysis stage was recommenced, starting with the insect looking for a location to lodge. In the ecdysal phase, the characteristic behaviours that were observed included splitting of the exocuticle, emergence of the wings, emergence of the prothoracic legs, the head, the meso- and metathoracic legs, the proboscis, and the abdomen. The end of ecdysis marks the beginning of the post-ecdysis phase in which the cuticle of the insect is still pale and fragile. At this time, the insect is mostly immobile, and seeks shelter away from light. In this phase, the newly emerged insect often avoids interaction with other insects, and

72 62 waits until the soft cuticle has hardened and darkened. The process of post-ecdysis can take from min. Corazonin-like immunoreactivity during ecdysis Corazonin-like IR is present in cell bodies, processes and neuropile throughout the CNS (see Chapter 2 of thesis). The CRZ-like IR is bright in cell bodies, processes and neuropile of unfed 4 th instar CNS (Figure 3A) and this intensity is greatly diminished or absent in CNS taken 2 hr post-ecdysis (Figure 3B). This absence of staining is most dramatic in the processes and neuropile within the CNS with some CRZ-like IR remaining in the cell bodies. Staining intensity increases by 6 days post-ecdysis with the processes and neuropile staining for CRZ-like IR (Figure 3C). RNA interference The presence of CRZ-like IR for insects injected with dscrz served as a control to test if the knock down of the CRZ peptide was successful. Figure 4a shows CRZ-like IR in cell bodies, processes and neuropile of the CNS from the control group (No treatment). The No Treatment insects were not injected and the CRZ-like IR in the CNS is characteristic of CRZ-like IR seen earlier (Chapter 2 of thesis). To control for the act of injection, another control group injected with dsarg were used. Figure 4b shows CRZ-like IR in blebs, axons and cell bodies in CNS collected 1 day after injection of dsarg and this pattern is similar to that seen in the No Treatment group (Figure 4a). The experimental group was injected with dscrz and CNS from days 1, 3 and 5 post-injection were processed for CRZlike IR (Figure 4C, D and E). As can be seen the staining intensity decreases after dscrz

73 63 injection and is nearly absent 5 days post-injection. Some staining is still evident in the cell bodies of the brain and MTGM but the staining of processes and neuropile are mostly absent. The mortality for each of the treatment groups was monitored daily until all insects either completed ecdysis or died (Figure 5). As can be seen, injection increased mortality with the dsarg-injected insects having a higher mortality (10%) than those not injected (5%). Insects injected with dscrz had the highest mortality with 15% dying during the observation period compared to the 10% mortality of the dsarg-injected insects. All of the insects died prior to the time of ecdysis. Injections were performed 4 days after feeding. The dsarg-injected insects underwent ecdysis over a 6 day period (day post-feeding) but were delayed by a day compared to the No Treatment group (day post-feeding) (Figure 6A and B). Insects injected with dscrz were not delayed and underwent ecdysis between day postfeeding (Figure 6C). The circadian gating at which ecdysis occurs in the No treatment insects occurred between 8-14hr after lights off (Figure 7A). This was similar to what was seen in insects injected with dsarg although the gate was shorter with only an 8-12hr time interval after lights off (Figure 7B). Insects injected with dscrz appeared to have a delay in the time of day that they ecdyse with ecdysis occurring between 14-16hr after lights off (Figure 7C). The observations for ecdysis were done between 8:00 and 18:00 hr after lights off and therefore the insects that ecdysed overnight were not timed and added to the graph. For this experiment the insects that ecdysed overnight were 23% from the No treatment group, 54% of insects from the dsarg-injected group and 44% of insects from dscrz-injected group.

74 64 Figure 1. Number of insects ecdysing from 4th instar to 5th instar versus days after feeding. 4th instars (n=40) were fed on day 0 and kept in a 16h:8h light/dark cycle at 28ºC with 30-40% humidity. The majority of insects ecdysed on day 13 and day 14.

75 65

76 66 Figure 2. Gating of ecdysis to 5 th instar. 4th instars (n=38) were kept in an 8 hr dark (gray bar) and 16 hr light (white bar) cycle at 28ºC with 30-40% humidity. Behaviour was observed between 11 to 17 days after feeding as 4th instars, between 8hr to 18hr. The majority of the insects (78%) underwent ecdysis between 8hr to 12hr after lights off. 5% (2) of the insects underwent ecdysis overnight (after 18:00 hr and before 8:00 hr).

77 67

78 68 Figure 3. Confocal microscope images of CRZ-like immunoreactivity in the CNS during ecdysis from 4 th to 5th instar R. prolixus. Whole mount preparations show presence of brightly stained immunoreactive cell bodies, axons and blebs in the CNS and the corpora cardiaca (CC) in unfed 4 th instars and 6d (days) post-ecdysis 5 th instars. Lack of brightly staining immunoreactive cell bodies, axons and blebs are evident 2hr post-ecdysis. Scale bar = 100µm. (PRO, prothoracic ganglion; MTGM, mesothoracic ganglionic mass.

79 69

80 70 Figure 4. Confocal microscope images of CRZ-like immunoreactivity in CNS from 4 th instars injected with double stranded (ds) RNA. Whole mount preparation of CNS showing immunoreactivity in cell bodies, axons and blebs in A) no injection control group, B) 1d (day) post-injection group with double stranded ampicillin gene (dsarg) and C) 1d postinjection with dscrz. A reduction in CRZ-like immunoreactivity in cell bodies, axons and blebs can be seen D) 3d post-injection with dscrz and E) 5d post-injection with dscrz. Scale bar= 100µm

81 71

82 72 Figure 5. Percent mortality of insects injected with double stranded RNA or no treatment. 4th instars were fed on day 0 and injected on day 4 with 2μg of dscrz (n=20), 2μg of dsarg ( n=20) or with no treatment (control, n=20). Insects were kept on a controlled 16h:8h light/dark cycle at 28 C with 30-40% humidity.

83 73

84 74 Figure 6. Gating of insects ecdysing from 4th instars to 5th instars after injection of double stranded RNA or no treament. A) non-injected control group; B) 2 μg of dsarg, and; C) 2 μg of dscrz injected on 4 th day after a bloodmeal. dsarg injected insects had a 1 day delay in ecdysis. Day 13 and day 14 after feeding had the highest frequency of insects ecdysing in the no treatment and dscrz-injected group.

85 75

86 76 Figure 7. Gating of ecdysis of insects injected with double stranded RNA or no treatment. 4th instars were kept on a 8hr dark (gray bar) and16hr light (white bar) cycle at 28ºC with 30-40% humidity. Behaviour was observed between 11 to 17 days after feeding as 4th instars and between 8hr to 18hr after lights off. A) no treatment group (n=22); B) 2 μg of dsarg (n=11); and C) 2 μg of dscrz (n=9) injected on the 4 th day after a bloodmeal. dscrz-injected insects displayed a 2 hr delay in ecdysis. 23% of insects from no treatment group, 54% of insects from dsarg-injected group and 44% of insects from dscrz-injected group completed ecdysis overnight (after 18:00 hr and before 8:00 hr).

87 77

88 78 Discussion Physiologically, ecdysis is constructed as an elaborately orchestrated series of events that are tightly regulated by environmental cues. Based on these preliminary results, it can be confirmed that R. prolixus development, in particular the process of ecdysis between 4th to 5th instars, is under a circadian rhythm. The precise timings and behaviours associated with R. prolixus ecdysis have been an item of fascination since the early works of Sir Vincent Wigglesworth in the early 1930s. His reports are the earliest of many that show that R. prolixus is a haematophagus hemimetabolous insect that will only go through metamorphosis if there is a sufficient amount of blood meal (Wigglesworth, 1934). In his investigations, he also stated that 4 th instars will go through ecdysis at about 15 days post-feeding at 24ºC. More controlled observations were conducted by Ampleford and Steel (1982b) at 28ºC with various L:D regimes, and it was discovered that ecdysis occurred between the days post-feeding and within a 4-6 hr period each day. These gates recur at intervals of 24 hr, producing a rhythm of ecdysis in populations. It was also shown that, in all regimes tested, the median ecdysis time occurs at 10 h after lights off. These results are comparable to the results achieved in our laboratory with 16hr:8hr light/dark cycle at 28ºC, in which 4th instars took days post-feeding to emerge as 5th instars and went through ecdysis between 8-10 hour after lights off. Ampleford and Steel (1982a) also described some characteristic behaviours that marked pre-ecdysis, ecdysis and post-ecdysis behaviours. Although the described behaviours were for 5th instars to adult stage, the behaviours observed for 4 th to 5 th instars are consistent. Any differences in timing between the two colonies may be due to differences in temperature, humidity and the hematocrit percentage of the blood fed. The

89 79 differences in time of ecdysis shows that it is a very delicate procedure and that changes in the temperature and humidity can change the results (Luz et al., 1999). Using immunohistochemistry to draw inferences about the amount of a peptide present has been used before to detect changes during development by Sevala et al. (1992). The CRZlike IR seen in cell bodies and processes of unfed 4 th instars and 6 day old 5th instars (6 days post-ecdysis) is similar in distribution and intensity. The reduction in CRZ-like IR 1hr after ecdysis implies that CRZ release occurred in the process of ecdysis. Since in holometabolous insects such as M. sexta, CRZ has been reported to be released just before the onset of ecdysis, our assumption is that CRZ may be used in similar fashion in R. prolixus. The test of CRZ-like IR was also important because CRZ-containing neurosecretory cells have been identified as Ia1 neurons in M. sexta, which also express the circadian clock protein PER (Copenhaver and Truman, 1986; Homberg et al., 1991; Wise et al., 2002). These cells are also involved in regulation of circadian rhythms in the silkmoth Antheraea pernyi (Sauman and Reppert, 1996) and could be involved in the circadian rhythm of adult eclosion (Truman and Riddiford, 1970; Žitňan and Adams, 2012). Thus CRZ may also be associated with circadian rhythms associated with ecdysis. Decreasing gene expression of CRZ using RNAi at critical time points was used to elucidate if CRZ plays a role in ecdysis. The reduction in CRZ-like IR in tissues collected 5 days after dscrz injection implies the successful transfer of RNAi and resultant reduction in posttranscriptional CRZ coding mrna. This experiment was also done using dsccap injected into 4th instars, and the results revealed a dramatic disruption in the process of ecdysis and reduction in CCAP-like IR in Day 5 post injection insects (Lee, 2013). Overall, the results of

90 80 reduction in CRZ-like IR are preliminary and need further trials to confirm the effects of eliminating CRZ-like mrna on the behaviour of ecdysis between 4th to 5th instars. However, preliminary observation found that the timing of ecdysis was disrupted in R. prolixus with the gating of ecdysis altered by 2hrs. This is unlike the typical behaviour of R. prolixus going through ecdysis since if the insect is unable to ecdyse within the normal range (8hr to 14hr) of gating times, the insect waits for 24 hours, to attempt ecdysis again (Ampleford and Steel, 1982b). This indicates that knock-down of CRZ peptide may have altered the circadian machinery in the insect. The effects observed for CRZ-knockdown in a hemipteran insect is still a novel concept. Future studies will need to be done to more accurately examine the behaviour specific differences in knock-down insects, such as gating of ecdysis, quantitative PCR (polymerase chain reaction) to determine the expression levels of the CRZ transcript during ecdysis, and RNAi knock-down of the CRZ receptor to examine specific changes in the insects physiology. These investigations will help elucidate why the CRZ system has been conserved in so many different orders of insects, including both holometabolous and hemimetabolous insects (Truman and Riddiford, 2002). It would also further our understanding of the hormonal network that is established in 4th instar R. prolixus before going through ecdysis, the overall process of metamorphosis in this important vector of Chagas disease, and the possible development of pest control agents (Maule et al., 2002).

91 81 References Ampleford, E.J., Steel, C.G.H., 1982a. The behavior of Rhodnius prolixus during the imaginal ecdysis. Can. J. Zoo. 60, Ampleford, E.J., Steel, C.G.H., 1982b. Circadian control of ecdysis in Rhodnius prolixus (Hemiptera). J. Comp. Physiol. 147, Carlson, J.R., Imaginal ecdysis of cricket (Teleogryllus oceanicus).1. Organization of motor programs and roles of central and sensory control. J. Comp. Physiol. 115, Choi, Y., Lee, G., Park, J., Programmed cell death mechanisms of identifiable peptidergic neurons in Drosophila melanogaster. Devel.133, Copenhaver, P.F., Truman, J.W., Identification of the cerebral neurosecretory cells that contain eclosion hormone in the moth Manduca sexta. J. Neuro. 6, Gammie, S.C., Truman, J.W., Neuropeptide hierarchies and the activation of sequential motor behaviors in the hawkmoth, Manduca sexta. J. Neuro. 17, Homberg, U., Davis, N.T., Hildebrand, J.G., Peptide immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Manduca sexta. J. Comp. Neurol. 303, Hughes, T.D., Imaginal ecdysis of the desert locust, Schistocerca gregaria.1. Description of the behavior. Physiol. Entomol. 5, Ishizaki, H., Suzuki, A., The brain secretory peptides that control molting and metamorphosis of the silkmoth, Bombyx mori. Int. J. Dev. Biol. 38, Kim, Y.J., Spalovska-Valachova, I., Cho, K.H., Žitňanova, I., Park, Y., Adams, M.E., Žitňan, D., Corazonin receptor signaling in ecdysis initiation. Proc. Natl. Acad. Sci. U. S. A. 101, Klein, C., Kallenborn, H.G., Radlicki, C., The 'Inka cell' and its associated cells: ultrastructure of the epitracheal glands in the gypsy moth, Lymantria dispar. J. Insect Physiol. 45, Lee, D.H., Lange, A.B., Crustacean cardioactive peptide in the Chagas' disease vector, Rhodnius prolixus: presence, distribution and physiological effects. Gen. Comp. Endocrinol. 174,

92 82 Lee, D., Vanden Broeck, J., Lange, A.B., Identification and expression of the CCAP receptor in the Chagas' disease vector, Rhodnius prolixus, and its involvement in cardiac control. Plos One 8, e Lee, D A conserved CCAP-signaling pathway controlling ecdysis in a hemimetabolous insect, Rhodnius prolixus. Ph.D. thesis, University Luz, C., Fargues, J., Grunewald, J., Development of Rhodnius prolixus (Hemiptera: Reduviidae) under constant and cyclic conditions of temperature and humidity. Mem. Inst. Oswaldo Cruz 94, Maule, A.G., Mousley, A., Marks, N.J., Day, T.A., Thompson, D.P., Geary, T.G., Halton, D.W., Neuropeptide signaling systems: Potential drug targets for parasite and pest control. Cur, Top. Med.Chem. 2, McNabb, S.L., Baker, J.D., Agapite, J., Steller, H., Riddiford, L.M., Truman, J.W., Disruption of a behavioral sequence by targeted death of peptidergic neurons in Drosophila. Neuron 19, Mesce, K.A., Fahrbach, S.E., Integration of endocrine signals that regulate insect ecdysis. Front. Neuroendocrinol. 23, Reynolds, S.E., Integration of behavior and physiology in ecdysis. Berridge, M.J., J.E.Treherne and V.B.Wigglesworth (Ed.). Advances in Insect Physiology, Vol.15.Vii+624p.Academic Press, Inc.: London, England; New York, N.Y., Usa.Illus, P Sakurai, S., Temporal organization of endocrine events underlying larval ecdysis in the silkworm, Bombyx mori. J. Insect Physiol. 29, Sauman, I., Reppert, S.M., Circadian clock neurons in the silkmoth Antheraea pernyi: Novel mechanisms of period protein regulation. Neuron 17, Sevala, V.M., Sevala, V.L., Loughton, B.G., Davey, K.G., Insulin-like immunoreactivity and molting in Rhodnius prolixus. Gen. Comp. Endocrinol. 86, Smith, W., Rybczynski, R., Prothoracicotropic hormone. In: Insect Endocrinology, Ed. Gilbert, L., Academic Press, London, pp Tanaka, Y., Hua, Y., Roller, L., Tanaka, S., Corazonin reduces the spinning rate in the silkworm, Bombyx mori. J. Insect Physiol. 48,

93 83 Truman, J.W., Riddiford.Lm, Neuroendocrine control of ecdysis in silkmoths. Science 167, Truman, J.W., Riddiford, L.M., Endocrine insights into the evolution of metamorphosis in insects. Annu. Rev. Entomol. 47, Veenstra, J.A., Davis, N.T., Localization of corazonin in the nervous system of the cockroach Periplaneta americana. Cell Tissue Res. 274, Wigglesworth, V.B., The physiology of the cuticle and of ecdysis in Rhodnius prolixus (Triatomidae, Hemiptera); with special reference to the function of the oenocytes and of the dermal glands. Q. J. Microsc. Sci. 76, Wigglesworth, V.B., The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling molting and 'metamorphosis'. Q. J. Microsc. Sci. 77, Wigglesworth, V.B., Local and general factors in the development of "pattern" in Rhodnius prolixus (hemiptera). J. Exp. Biol. 17, Wise, S., Davis, N.T., Tyndale, E., Noveral, J., Folwell, M.G., Bedian, V., Emery, I.F., Siwicki, K.K., Neuroanatomical studies of period gene expression in the hawkmoth, Manduca sexta. J. Comp. Neurol. 447, Yang, J., Huang, H., Yang, H., He, X., Jiang, X., Shi, Y., Alatangaole, D., Shi, L., Zhou, N., Specific activation of the G protein-coupled receptor BNGR-A21 by the neuropeptide corazonin from the Silkworm, Bombyx mori, dually couples to the G(q) and G(s) signaling cascades. J. Biol. Chem. 288, Zhao, Y., Bretz, C.A., Hawksworth, S.A., Hirsh, J., Johnson, E.C., Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila. Plos One 5, e9141. Žitňan, D., Hollar, L., Spalovska, I., Takac, P., Žitňanova, I., Gill, S., Adams, M., Molecular cloning and function of ecdysis-triggering hormones in the silkworm Bombyx mori. J. Exp. Biol. 205, Žitňan, D., Kim, Y., Žitňanova, I., Roller, L., Adams, M.E., Complex steroid peptidereceptor cascade controls insect ecdysis. Gen. Comp. Endocrinol. 153, Žitňan, D., Kingan, T.G., Hermesman, J.L., Adams, M.E., Identification of ecdysis triggering hormone from an epitracheal endocrine system. Science 271,

94 Žitňan, D., Adams, M. E., Neuroendocrine regulation of ecdysis. In: Insect Endocrinology, Ed. Gilbert, L., Academic Press, London, pp

95 85 CHAPTER IV: General Discussion It is no accident that insects are the most plentiful and most diverse group of organisms on earth. They have sustained a position of evolutionary supremacy for over 400 million years: they have witnessed the rise and fall of dinosaurs; they have survived major catastrophes that resulted in planet-wide extinctions; and they continue to thrive despite our best efforts at eradication. While no single ecological or physiological attribute can account for this unparalleled success, the insects do have a unique combination of characteristics which, as a whole, have given them an unusual survival advantage. Insects have gained great amount of attention from scientists due to their fast generations, low maintenance, and high fecundity rates making them ideal for studies of genomics, molecular biology, neuroendocrinology, physiology, evolution and development. These physiological processes including growth, development, metabolic regulation and cardiovascular control are modulated and controlled by numerous neuropeptides that are released in a need dependent manner via constant communication between the central nervous system and the body. Neuropeptides are short amino acid chains that act as chemical messengers in the form of a neurotransmitter, neurohormone, or a neuromodulater (Gäde and Goldsworthy, 2003; Nässel et al., 2010; Mykles et al., 2010). Although there's a plethora of information about actions of neuropeptides on Drosophila, an often under looked model organism is the blood gorging vector of Chagas disease, Rhodnius prolixus. In this thesis, I have investigated the roles of neuropeptides, and their possible

96 86 implications on the physiology of R. prolixus in regards to the distribution in central nervous system, circulatory system, energy metabolism, and ecdysis. In this project, immunohistochemical data served as a stepping stone to determine in which tissues there may be presence of the peptide and where in the nervous system to these peptides localize. This gave us a starting lead as to where we could investigate possible physiological roles of peptides in the insect body. The presence of corazonin (CRZ)-like IR was able to show that CRZ may be released from the neurosecretory cells into the CC, and act as a neurohormone in R. prolixus. The potential roles of CRZ as a neuromodulatory and neurohormone has been suggested by many other studies and the lack of CRZ-like IR in the dorsal vessel and other peripheral tissues put forward that CRZ may not be directly involved in heart beat regulation; and although the myo-modulatory properties of CRZ and preliminary effects of CRZ knock-down on ecdysis in R. prolixus are evident from this study, the true physiological relevance of the presence and distribution of CRZ-like material in R. prolixus may still be undetermined. The presence of this peptide adds R. prolixus into the long list of species of insect with a conserved CRZ system, indicating that CRZ may have played an essential innate role that is common in insects ( Roller et al., 2003,). The confirmation of the presence of AKH(adipokinetic hormone)-like IR cells and the stimulatory effect on the lipid levels in R. prolixus suggests that AKH has a conserved physiological role among the insect species with a major role played in energy metabolism. Although ACP (AKH/corazonin-related peptide)-like IR has been confirmed to be present in the CNS, it is still a question as to what kind of role ACP plays in physiological processes. Since there is a great overlap in the peptide sequence of both CRZ, ACP and AKH; it was

97 87 surprising that ACP did not show any neuromodulating roles on AKH or CRZ release, as applications of ACP had no effect on lipid metabolism or heart rate. Finding a generalized function for ACP in insects is also a mystery as only one known study has investigated ACP function. Hansen et al. in 2010 indicated that ACP may have a function in development as RT-PCR results revealed in Tribolium castaneum, a high expression level of ACP and its receptor shortly before and after hatching. However, injecting dsacp into larvae did not alter any physiological processes observed including egg count, time of hatching, physical appearance or mortality (Hansen et al., 2010). Due to the lack of literature on ACP, it is difficult to make inferences and one can only entertain the idea of this peptide and its receptor being associated in function to CRZ and AKH peptide and their receptors. Energy homeostasis is a elemental characteristic of animal life, providing a determined balance between fat storage and mobilization. In humans, the importance of body fat regulation is emphasized by dysfunctions resulting in, diabetes, obesity and lipodystrophy. In mammals hunger, satiety and metabolism is under complex regulation of various peptide hormones. These hormones signals participate in a complex network of cellular communication between the brain, endocrine organs, the intestine and adipose tissues. Fascinatingly, in Drosophila many of the peptide hormones and their receptors involved in packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, feeding and metabolism appear to be related to those in mammals (Park et al., 2002). Among these are peptides which are very similar in function to insulin and glucagon that play different roles in feeding, growth and metabolism in vertebrates.

98 88 Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR (AKH receptor) is as important for chronic accumulation and acute mobilization of storage fat. Loss of AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. These data demonstrate that AKHs are essential to adjusting normal body fat content and ensure lifelong fat-storage homeostasis (Grönke et al., 2007). Even more interestingly, cells that express CRZ also express receptors for two unrelated diuretic hormones, DH44 and DH31 (Johnson et al., 2005) in Drosophila. The significance of this finding is that, DH44 and DH31 are related to CRF (corticotropin releasing hormone) and CGRP (Calcitonin-Gene Related Peptide) respectively. In the mammalian hypothalamus, GnRH (gonadotropin releasing hormone) cells express the receptors for both CRF and CGRP and are involved in the stress-induced suppression of GnRH release in rats (Li et al., 2004). Moreover, the DH31 family of peptides in insects is highly conserved and is involved in ion and water balance as well as muscle contractions in insects (Johnson et al., 2005; Park et al.,2002). In 2010, it was reported that in Drosophila CRZ transcript expression levels are altered under starvation and osmotic stress, and that lipid levels are impacted when CRZ neurons are altered and that these conditions are dependent on the sex of the organism.(zhao et al., 2010). Ablation of CRZ neurons confers conflict to metabolic, osmotic as measured by survival. Knock-down and activation of CRZ neurons lead to differential lifespan under stress, and these effects showed a dependence on sex. Additionally, altered CRZ neuron physiology leads to fundamental differences in activities involving locomotion, and these effects were also dependent on the sex of the organism(zhao et al., 2010).

99 89 Choi and colleagues showed that ablation of CRZ in LNCs in Drosophila resulted in a slight but statistically significant decrease in trehalose levels, suggesting that CRZ modulates AKH-cell functions (Choi et al., 2008). It could be possible that, ablation of CRZ containing cells had an effect on the functions of DH31 and DH44 peptides. The native R. prolixus DH31, Rhopr-DH31, plays an integral role in diuresis, stimulating low levels of secretion by the Malpighian tubules, and stimulating contractions of both the heart and hindgut ( TeBrugge, 2008). Therefore, the interaction of Rhopr-DH31 with other factors, in particular CRZ, and its role on the digestive system can be of importance in future studies regarding diuresis. It can also be proposed that DH 31R and DH44R are activated during digestion of a meal, which then can signal CRZ cells to release CRZ into the haemolymph to increase the heart rate and facilitate movement of waste material and nutrients. Additionally, metabolic regulation is very important in insects, since metamorphosis is an energetically costly event. This is especially the case for the obligate blood feeder - R. prolixus, since a blood meal is a necessity for the insect to molt (Wigglesworth, 1934). Ingestion of a sufficient blood meal triggers surge of peptides and amines to co-ordinate and enable the size of the R. prolixus to increase and allow for initiation of molting. The metabolic regulation can also be affected by the circulatory system since in insects, the circulatory system functions to spread the nutrients and metabolites to different parts of the body, facilitating signaling and communication between tissues for expansion upon a blood meal (Chiang and Davey, 1990; McCann, 1970). A comprehensive review of CRZ by Boerjan et al. (2010) suggested that the evolutionary ancient function of CRZ may have been "to prepare animals for coping with the

100 90 environmental stressors of the day." The various associations of CRZ and AKH in ecdysis, energy metabolism, circadian clock, diuresis, cuticle melanisation, and its high degree of structural and immunolocal conservation in insects suggest that these peptides play a dynamic role in insect physiology. Along with similarities in function of CRZ and AKH and their receptors with mammalian hormones and localization within the neuroendocrine system; evolutionarily, it has been established that receptors for CRZ and AKH are ancestrally related to GnRH receptors and are presumably arthropod homologs of GnRHs (Park et al., 2002; Cazzamali et al., 2002). It was further suggested that AKH, CRZ and ACP belong to the GnRH superfamily. Currently, through phylogenetic sequence analysis, it was found that all four peptides might all share a common ancestor (Roch et al., 2011). it also shows that the ancestral ligand, which probably had a mixture of the AKH and GnRH amino acid sequences, might have controlled carbohydrate and lipid release, reproduction, or both (Osada et al., 2013). Thus, investigating AKH, CRZ and ACP can help to unravel further functions of the neurosecretory system of the CNS and the CC - the brain and the pituitary gland analogues of invertebrates and vertebrates. Future Directions The results obtained within the duration of this thesis has opened many exciting doors. A proximate future direction that begs investigation is determining the complete function of CRZ in the development of fourth instars to fifth instars. The preliminary data suggested in this thesis shows that although CRZ does not alter the day at which ecdysis occurs, it does alter the time of day at which ecdysis occurs, indicating that perhaps CRZ is acting on a

101 91 specific behaviour that takes place between the prepatory or pre-ecdysis, ecdysis and postecdysial phases (Chapter 3). The first of the steps would be to repeat experiments with better suited controls to compare immunohistochemistry results with dsrna injected insects; for example, collecting non injected insects at the same as the experimental dsrna injected insects, so that comparison of immunostaining intensity is more reliable. It was also not anticipated that there would be many deaths in the dscrz injected group before the critical day range when ecdysis occurs; due to many deaths occurring before days after feeding, there were only a small group of animals left to observe the behaviour of ecdysis. Another reason why repeating this experiment is important is to determine the exact timing differences of ecdysis gating in knock-down insects. The specific steps to elucidate this information, qpcr (quantitative polymerase chain reaction) must be performed to determine any changes in expression levels of CRZ during the development of fourth instars to fifth instars. Since it is known that CRZ acts as a cardioaccelerator in R. prolixus fifth instars ( Chapter 2), one could also look at the effects of injecting dscrz and down regulation of this peptide to see if the heart rate of the insect has been modified. There is evidence that RNAi technique used to down regulate CRZ in mosquitoes, Anopheles gambiae did not have any significant changes in the heart rate (Hillyer et al., 2012) compared to RNAi performed for CCAP peptide in the same species, which led to a significant reduction in the heart rate frequency (Estévez-Lao et al., 2013). An ultimate future route could include characterization and determination of localization of CRZ, AKH and ACP receptors in R. prolixus. It would also be worth considering if receptors of these three peptides are similar to those in T. castanuem, in that they do not cross

102 92 react with other peptides and are specific to their own ligand despite the similarity in sequence in both peptides and receptors. Although we must take caution in generalizing the roles of neuropeptides as just one function as evolutionary divergence has allowed insects to modify the functions of these peptides according to their adaptations. One must be careful in drawing conclusions and be open to the idea that different neuropeptides may have a completely new role in another species. Perhaps that is why the pinnacle of evolution must be given to the lowly insects. The insect clan is ancient and has survived the worst cataclysmic events in planetary history - meteors, ice ages, droughts, and floods. They are capable of living months without food, remaining alive headless for weeks at a time and even showing great resistance to radiation. Learning and exploring insect biology and physiology, especially of the ever so romantic and elegant R. prolixus, can unravel questions about the origin of the central nervous system and evolutionary relationships between vertebrates and invertebrates.

103 93 References Boerjan, B., Verleyen, P., Huybrechts, J., Schoofs, L., De Loof, A., In search for a common denominator for the diverse functions of arthropod corazonin: A role in the physiology of stress? Gen. Comp. Endocrinol. 166, Brugge, V.A.T., Schooley, D.A., Orchard, I., Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus. J. Exp. Biol. 211, Cazzamali, G., Saxild, N.P.E., Grimmelikhuijzen, C.J.P., Molecular cloning and functional expression of a Drosophila corazonin receptor. Biochem. Biophys. Res. Commun. 298, Chiang, R.G., Chiang, J.A., Davey, K.G., Morphology of the dorsal vessel in the abdomen of the blood-feeding insect Rhodnius prolixus. J. Morphol. 204, Choi, S., Lee, G., Monahan, P., Park, J.H., Spatial regulation of Corazonin neuropeptide expression requires multiple cis-acting elements in Drosophila melanogaster. J. Comp. Neurol. 507, Estévez-Lao, T.Y., Boyce, D.S., Honegger, H., Hillyer, J.F., Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae. J. Exp. Biol. 216, Gäde, G., Goldsworthy, G.J., Insect peptide hormones: a selective review of their physiology and potential application for pest control. Pest Manag. Sci. 59, Grönke, S., Müller, G., Hirsch, J., Fellert, S., Andreou, A., Haase, T., Jäckle, H., Kühnlein, R.P., Dual lipolytic control of body fat storage and mobilization in Drosophila. Plos Biology 5, Hansen, I.A., Sehnal, F., Meyer, S.R., Scheller, K., Corazonin gene expression in the waxmoth Galleria mellonella. Insect Mol. Biol. 10, Hillyer, J.F., Estévez-Lao, T.Y., Funkhouser, L.J., Aluoch, V.A., Anopheles gambiae corazonin: gene structure, expression and effect on mosquito heart physiology. Insect Mol. Biol. 21, Johnson, E.C., Shafer, O.T., Trigg, J.S., Park, J., Schooley, D.A., Dow, J.A., Taghert, P.H., A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J. Exp. Biol. 208,

104 94 Li, X.F., Bowe, J.E., Mitchell, J.C., Brain, S.D., Lightman, S.L., O'Byrne, K.T., Stressinduced suppression of the gonadotropin releasing hormone pulse generator in the female rat: A novel neural action for calcitonin gene related peptide. Endocrinol. 145, McCann, F.V., Physiology of insect hearts. Annu. Rev. Entomol. 15, Mykles, D.L., Adams, M.E., Gäde, G., Lange, A.B., Marco, H.G., Orchard, I., Neuropeptide Action in Insects and Crustaceans. Physiol. Bioch. Zool. 83, Nässel, D.R., Winther, A.M.E., Drosophila neuropeptides in regulation of physiology and behavior. Prog. Neurobiol. 92, Osada, M., Treen, N., Molluscan GnRH associated with reproduction. Gen. Comp. Endocrinol. 181, Park, Y., Kim, Y.J., Adams, M.E., Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution (vol 99, pg 11423, 2002). Proc. Natl. Acad. Sci. U. S. A. 99, Roch, G.J., Busby, E.R., Sherwood, N.M., Evolution of GnRH: Diving deeper. Gen. Comp. Endocrinol. 171, Roller, L., Tanaka, Y., Tanaka, S., Corazonin and corazonin-like substances in the central nervous system of the Pterygote and Apterygote insects. Cell Tissue Res. 312, Veenstra, J.A., Does corazonin signal nutritional stress in insects? Insect Biochem. Mol. Biol. 39, Wigglesworth, V.B., The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling molting and 'metamorphosis'. Q. J. Microsc. Sci. 77, 191-U12. Zhao, Y., Bretz, C.A., Hawksworth, S.A., Hirsh, J., Johnson, E.C., Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila. Plos One 5, e9141.