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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

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