The Role of Octopamine and Tyramine in the Adult Female ......Chapter 2: Octopamine and tyramine regulate the activity of reproductive visceral muscles in the adult female blood-feeding

The Role of Octopamine and Tyramine in the Adult

Female Reproductive System of Rhodnius prolixus

by

Sam Hana

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Cell and Systems Biology

University of Toronto

© Copyright by Sam Hana 2017

ii

The Role of Octopamine and Tyramine in the Adult

Female Reproductive System of Rhodnius prolixus

Sam Hana

Master of Science

Cell and Systems Biology

University of Toronto

2017

Abstract

Octopamine and tyramine are neuroactive chemicals involved in many physiological

processes acting as neurotransmitters, neuromodulators and neurohormones. Octopamine and

tyramine modulate reproduction in insects. In Rhodnius prolixus, octopamine decreased the

amplitude and reduced the RhoprFIRFa-induced oviduct contraction in a dose-dependent manner,

whereas tyramine only reduced the RhoprFIRFa-induced contractions. Also, octopamine and

tyramine reduced the frequency and abolished bursal contractions at higher concentrations.

Octopamine also increased the levels of cAMP in the oviducts, an effect blocked by phentolamine.

Dibutyryl cAMP mimicked the effects of octopamine at the bursa, suggesting that octopamine may

act by an Octβ-receptor, a known GPCR. The cDNA sequences of RhoprOctβ2-R and RhoprTyr1-

R have been cloned and characterized; the receptor transcripts are expressed in all female

reproductive tissues. Injection of octopamine and tyramine into mated and fed adult females

increased oogenesis. Overall, octopamine and tyramine modulate the female reproductive tissues

leading to successful laying of eggs.

iii

Acknowledgments

I would like to first offer my deepest thanks to my mentor, Dr. Angela Lange. Her excellent

guidance and supervision were instrumental in my success. She motivated and inspired me to do

my best throughout this journey. I am fortunate to graduate under your supervision knowing that

I was taught from the best.

I want to acknowledge and thank my committee, Dr. Ian Orchard and Dr. Adriano Senatore for

their input in my research. Dr. Senatore, thank you very much for your helpful tips and feedback

early on in my degree. Dr. Ian Orchard, I would like to thank you for reading, editing, and

providing feedback on the research articles and this thesis.

To all members of the Lange lab, I want to thank you for being part of this journey. I especially

want to acknowledge those that taught, guided and supported me in my research. You have been

great colleagues and I am glad to have known you all. We have created wonderful memories.

And lastly, there was no limit to the amount of love and support my family has given me. Thank

you father and mother for your sacrifices every day, I am blessed to have you. Thank you to my

brothers for the love and support throughout this journey. Thank you God for the countless

blessings.

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Table of Contents

Abstract .......................................................................................................................................... ii

Acknowledgments ......................................................................................................................... ii

Organization of the Thesis ......................................................................................................... vii

List of Figures and Supplementary Tables .............................................................................. viii

List of Abbreviation .......................................................................................................................x

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

Neuroactive chemicals .....................................................................................................................1

Biogenic amines ...............................................................................................................................1

Octopamine ......................................................................................................................................2

Tyramine ..........................................................................................................................................3

Rhodnius prolixus: a vector of Chagas disease ................................................................................7

The female reproductive system ......................................................................................................8

Anatomy ...........................................................................................................................................8

Central nervous system innervation to the reproductive system ...................................................12

Reproductive processes ..................................................................................................................12

Oogenesis ...........................................................................................................................12

Ovulation ............................................................................................................................13

Fertilization and oviposition ..............................................................................................13

Neuroactive chemicals control reproduction in females ................................................................14

Significance....................................................................................................................................15

Thesis objective .............................................................................................................................16

v

References ......................................................................................................................................18

Chapter 2: Octopamine and tyramine regulate the activity of reproductive visceral

muscles in the adult female blood-feeding bug, Rhodnius prolixus. ...................................23

Abstract ..........................................................................................................................................24

Introduction ....................................................................................................................................25

Materials and Methods ...................................................................................................................26

Results ............................................................................................................................................29

Discussion ......................................................................................................................................44

References ......................................................................................................................................47

Chapter 3: Cloning and functional characterization of Octβ2-Receptor and Tyr1-

Receptor in the Chagas disease vector, Rhodnius prolixus. .................................................50

Abstract ..........................................................................................................................................51

Introduction ....................................................................................................................................52


Results ............................................................................................................................................59

Discussion ......................................................................................................................................79

References ......................................................................................................................................84

Supplementary Material .................................................................................................................90

Chapter 4: Octopamine and tyramine induce egg-laying in Rhodnius prolixus. ...................93

Abstract ..........................................................................................................................................94

Introduction ....................................................................................................................................95


Results ..........................................................................................................................................101

vi

Discussion ....................................................................................................................................106

References ....................................................................................................................................108

Chapter 5: General Discussion .................................................................................................111

The role of octopamine and tyramine in the reproductive system ...............................................111

Reproductive visceral muscle ......................................................................................................112

▪ Oviducts ...........................................................................................................................112

▪ Bursa ................................................................................................................................114

Reproductive processes ................................................................................................................117

▪ Direct ................................................................................................................................117

▪ Indirect .............................................................................................................................117

Summary ......................................................................................................................................121

Future directions ..........................................................................................................................122

vii

Organization of the Thesis

The thesis is broken into 5 chapters. Chapter 1 provides a general introduction for the thesis.

Chapter 2 is organized as a research journal article and it focuses on octopamine and tyramine

regulation of reproductive visceral muscles. Chapter 2 has been published in the Journal of

Experimental Biology (Hana and Lange, 2017). Chapter 3 focuses on RhoprOctβ2-R and

RhoprTyr1-R cDNA receptor cloning, functional characterization and expression in the

reproductive system. Chapter 3 is also organized as a research article that has been submitted

for publication in Frontiers in Physiology. Chapter 4 is a short report on the effects of injected

octopamine and tyramine on egg-laying. Chapter 5 is for general discussion and connects all the

chapters.

viii

List of Figures and Supplementary Tables

Chapter 1: General Introduction

Figure 1. The biosynthetic pathway of octopamine and tyramine ..................................................4

Figure 2. The anatomical structures of the adult female reproductive system .............................10

Chapter 2: Octopamine and tyramine regulate the activity of reproductive visceral

muscles in the adult female blood-feeding bug, Rhodnius prolixus

Figure 1. Octopamine and tyramine on rhythmic contractions of the oviducts............................32

Figure 2. Octopamine and tyramine on RhoprFIRFa-induced contractions……………….........34

Figure 3. Octopamine and tyramine on rhythmic contractions of the bursa……………….........36

Figure 4. Phentolamine inhibits octopamine in the lateral oviducts…………………..…...........38

Figure 5. Phentolamine blocks octopamine in the bursa…………………………..………........40

Figure 6. Yohimbine fails to block tyramine in the bursa…………………………..……..........42

Chapter 3: Cloning and functional characterization of Octβ2-Receptor and Tyr1-Receptor

in the Chagas disease vector, Rhodnius prolixus.

Figure 1. Classification of octopamine and tyramine receptors....................................................56

Figure 2. RhoprOctβ2-R cDNA sequence ………………………………..………..……...........64

Figure 3. RhoprTyr1-R cDNA sequence ……………………………………………..…...........66

Figure 4. Phylogenetic tree of insect octopamine and tyramine receptors …………...…….......68

ix

Figure 5. Multiple sequence alignment of insect Octβ2-Rs …………………………..…….......70

Figure 6. Multiple sequence alignment of insect Tyr1-Rs …………………………...……........72

Figure 7. Functional characterization of R. prolixus Octβ2 receptor……………………..……..74

Figure 8. Functional characterization of R. prolixus Tyr1 receptor………………………..……76

Figure 9. Spatial expression of RhoprOctβ2-R and RhoprTyr1-R. ……………………...……..78

Table S1. Primers for the amplification of the receptor fragments ……………….....…..…..….91

Table S2. Primers for the amplification of 3’ region of the receptors…………………….....….91

Table S3. Primers for the amplification of 5’ region of the receptors ……………...……...…...92

Table S4. Primers used for the mammalian expression vector preparation ……………....……92

Table S5. Primers used for RT-qPCR analysis of receptor transcripts………………....….……93

Chapter 4: Octopamine and tyramine induce egg-laying in Rhodnius prolixus

Figure 1. The protocol for the egg-laying assay……………………………………………….101

Figure 2. The result of octopamine injection on the number of eggslaid…………….………..103

Figure 3. The effect of tyramine injection on the number of eggs laid………………...………105

Chapter 5: General Discussion

Figure 1. Model showing the effects of octopamine on the oviducts and the bursa…………...116

Figure 2. Model for the possible effects of octopamine and tyramine on egg production…….120

x

List of Abbreviation

ANOVA analysis of variance

AST Allatostatin

BLAST basic local alignment search tool

bp base pairs

CA corpora allatum

Ca2+ calcium

cAMP 3’-5’-cyclic adenosine monophosphate

CC corpus cardiacum

cDNA complementary deoxyribonucleic acid

CNS central nervous system

EC50 half maximal activation concentration

FLPs FMRFamide-like peptides

GDP guanosine diphosphate

GPCR G-protein coupled receptor

GTP guanosine triphosphate

HEK293/CNG human embryonic kidney cells expressing cyclic nucleotide-gated ion

channel

IP3 inositol triphosphate

JH juvenile hormone

mNSCs median neurosecretory cells

mRNA messenger ribonucleic acid

MS Myosuppressin

xi

MTGM mesothoracic ganglionic mass

NPF Long neuropeptide F

ORF open reading frame

PBS phosphate-buffered saline

PRO prothoracic ganglion

RT-qPCR quantitative reverse transcription Polymerase Chain Reaction

RACE rapid amplification of cDNA ends

SEM standard error of the mean

SOG suboesophageal ganglion

TM transmembrane domain

UTR

untranslated region

1

Chapter 1: General Introduction

Neuroactive chemicals

Insects display a wide range of behaviours important for maintaining survival and

ultimately reproductive success. Insects utilize the nervous system, composed of the brain and the

ventral nerve cord, to monitor external environmental cues and coordinate internal processes. This

complex coordination is enabled by a large network of functionally diverse neurons and supporting

cells. Different neuroactive chemicals are utilized by the nervous system to transmit signals from

neuron to neuron and neuron to target tissues, thereby allowing the flow of information within the

organism. These neuroactive chemicals are classified as neurotransmitters, neurohormones and

neuromodulators (Klowden, 2013). Neurotransmitters are specifically released into the synaptic

cleft causing changes in the post-synaptic membrane potential. Glutamate is known as the classic

excitatory neurotransmitter at neuromuscular junctions in insects (Jan and Jan, 1976). The effect(s)

of neurotransmitters are transient due to re-uptake, enzymatic degradation and diffusion in the

synaptic gap. Neurohormones, synthesized by neurosecretory cells, are released into the

hemolymph and function as circulating hormones. Neurohormones modulate many peripheral

tissues and have longer-lasting effects. For example, adipokinetic hormone released from the

corpus cardiacum regulates energy levels in insects (Orchard and Lange, 1983). Lastly,

neuromodulators, released by neurons, modify the transmission in other synapses and/or modify

activity at target tissues. Neuromodulators can affect the excitability of post-synaptic membranes

and modulate the release of neurotransmitters from the pre-synaptic neurons. For example, at high

concentrations, serotonin can enhance the excitatory responses evoked by electrical stimulation of

the antennal nerves, whereas at low concentrations, serotonin reduces theses responses in Manduca

sexta (Kloppenburg and Hildebrand, 1995).

Biogenic amines

Biogenic amines are a class of organic neuroactive chemicals, mostly obtained from amino

acids and characterized by having low molecular weights and amine functional group(s). In insects,

2

octopamine, tyramine, serotonin (5-HT), dopamine and histamine are examples of the most

common and widely studied biogenic amines. Biogenic amines can be utilized by the nervous

system for the transmission of quick, private and short-lasting messages (Orchard et al., 2001).

Biogenic amines are mainly distributed in the central nervous system (CNS), but are also found in

projections to peripheral tissues suggesting their diverse physiological roles in insects (Blenau and

Thamm, 2011; Monastirioti, 1999; Nässel, 1999; Nässel and Elekes, 1992; Roeder, 2005). They

can also act as neurohormones or neuromodulators. Serotonin is released into the hemolymph

during feeding in Rhodnius prolixus to initiate diuresis by stimulating rapid tubule secretion

(Maddrell et al., 1991). In Periplaneta americana, dopamine stimulates the release of the fluid

component of saliva and serotonin stimulates the release of the proteinaceous components of saliva

(Just and Walz, 1996). Histamine, in Musca domestica and Calliphora erythrocephala, is a

neurotransmitter involved in olfaction as its been shown to be released from photoreceptor cells

(Hardie, 1987). These are just some examples of the physiological effects of these biogenic amines.

Octopamine

Octopamine was discovered in the salivary gland of the octopus, Octopus vulgaris

(Erspamer, 1948). Octopamine is a versatile neuroactive chemical derived from the hydroxylation

of tyramine by tyramine β-hydroxylase (Fig. 1). There is an immense amount of data that confirms

that octopamine acts as a neurotransmitter, neuromodulator and a neurohormone in insects, hence

the its versatility (Orchard, 1982). Octopamine is structurally and functionally similar to

noradrenaline of vertebrates; octopamine is the “adrenergic” of insects (Roeder, 1999; Roeder,

2005). In the CNS, octopamine’s content is 3-7 times higher than tyramine (Lange, 2009). In the

adult D. melanogaster, octopamine is found throughout the CNS with processes innervating

regions such as the ventral nerve cord, subesophageal ganglia, protocerebrum, central complex,

mushroom bodies and the optic lobe (Busch et al., 2009). Multiple groups of neurons containing

octopamine have been characterized, like the ventral median paired (VPM), ventral unpaired

median (VUM) and dorsal unpaired median neurons (DUM) (Busch et al., 2009). In Schistocerca

gregaria and P. americana, octopamine is found in the ganglia of the ventral nerve cord and is

also found in the optic lopes (Evans, 1978). In both species, octopamine was also found to be

associated with the corpora cardiaca (Evans, 1978). Octopamine is involved in a plethora of

3

physiological processes thereby influencing communication, signaling and behaviour in insects.

To briefly state a few roles, octopamine is important in aggression, reproduction, learning and

memory (Farooqui, 2012; Ohta and Ozoe, 2014; Roeder, 1999; Roeder, 2005).

Tyramine

Tyramine was not originally thought be being a neurotransmitter but merely thought to be

the metabolic precursor of octopamine; however, tyramine has been established as an independent

bioactive chemical in insects synthesized via the decarboxylation of the amino acid tyrosine

(Lange, 2009) (Fig. 1). In Drosophila melanogaster, tyrosine decarboxylase 2 is responsible for

synthesizing tyramine in neuronal cells, whereas tyrosine decarboxylase 1 synthesizes non-

neuronal tyramine in peripheral tissues (Cole et al., 2005). The distribution of tyramine specific

neurons, those that do not also contain octopamine, has been described in insects. In S. gregaria,

immunoreactive tyramine neurons are found in the medulla, the protocerebral bridge, the antennal

lobes, subesophageal ganglion and the neuropile (Homberg et al., 2013; Kononenko et al., 2009).

In D. melanogaster larva, tyramine-specific neurons are found in the brain, thoracic ganglia and

abdominal ganglia (Monastirioti et al., 1995). Tyramine-containing neurons that also contain

octopamine, have been found in the suboesophageal ganglia and thoracico-abdominal ganglia

(Nagaya et al., 2002). These neurons are characterized as ventral unpaired medial neurons (VUM)

and the tyramine specific pairs of dorsal lateral neurons in the abdominal ganglia (Nagaya et al.,

2002). VUM and DUM neurons, which also contain octopamine, innervate skeletal muscles and

other peripheral target tissues (Lange, 2009). Physiologically, tyramine along with its metabolic

successor, octopamine, have been shown to have similar effects in invertebrates (Roeder, 1999;

Roeder, 2005); however, tyramine has been reported to exhibit its own physiological actions

independent of octopamine. This was confirmed by the discovery of tyramine specific receptors.

For example, elevation of tyramine attenuates locomotion in D. melanogaster larva lacking

octopamine and octopamine feeding rescues locomotion in mutant flies (Saraswati et al., 2004).

Tyramine has been shown to be involved in the regulation of many physiological processes, such

as reproduction, aggression, feeding and locomotion (see Ohta and Ozoe, 2014).

4

Figure 1. The biosynthetic pathway for the synthesis of octopamine and tyramine. Tyramine is

produced by the decarboxylation of tyrosine, while octopamine is produced by the hydroxylation

of octopamine. Figure obtained from (Lange, 2009)

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G-protein-coupled receptors (GPCRs)

G-protein-coupled receptors (GPCRs), also known as seven-transmembrane domain

receptors (7TMs), are a large family of receptor proteins that bind ligands extracellularly leading

to receptor activation and thereby eliciting intracellular signal transduction (Kristiansen, 2004).

GPCRs are coupled to specific G-proteins which are an integral component in initiating signal

transduction pathways. GPCR ligands include biogenic amines, neuropeptides, glycoproteins,

photons, taste molecules, odorants, hormones + pheromones and odorants (Kristiansen, 2004). All

GPCRs are characterized by having an extracellular N-terminus, 7TMs creating three extracellular

loops along with three intracellular loops and an intracellular C-terminus. The N-terminus is

glycosylated, and this is critical for cell surface expression (Kristiansen, 2004). The 7TM domains,

7 α-helices, span the plasma membrane usually creating a pocket in which ligands interact with

the side chain amino acids (Kristiansen, 2004). The extracellular loops can also participate in

ligand interaction and/or binding (Kristiansen, 2004). The cytosolic loops contain serine and

threonine residues which are potential phosphorylation sites by protein kinases. Protein kinases

serve as a method of GPCR desensitization (Kristiansen, 2004). The C-terminus contains cysteine

residues which serve as a site for palmitoylation. Palmitoylation anchors the C-terminus to the

cytosolic plasma membrane. Palmitoylation allows for normal processing of the receptor and

accessibility of the C-terminus to kinases and regulatory proteins (Kristiansen, 2004). G-proteins

are divided into two main classes, heterotrimeric G-proteins and small cytoplasmic G-proteins.

Heterotrimeric G-protein complexes are composed of α, β and γ subunits. G-proteins are closely

associated with GPCRs at the amphiphatic α-helices of TM5 and TM6 (Kristiansen, 2004). They

are also believed to interact with the intracellular loops (ICL2 and ICL3) and the C-terminus

(Kristiansen, 2004).

The binding of a ligand to the GPCR causes receptor activation and conformational change

leading to an increased affinity for the G-protein (Kristiansen, 2004). This causes the release of

guanosine diphosphate (GDP) from the α subunit and the binding of guanosine triphosphate (GTP)

(Kristiansen, 2004). The α subunit, bound to GTP, dissociates from the βγ complex and leads to

the activation of target proteins, initiating signaling cascades (Kristiansen, 2004). The activated

GPCR lasts until GTP in α subunit is hydrolyzed to GDP and the heterotrimeric complex reforms

(Kristiansen, 2004). There are many classes of Gα subunits. Different subunit lead to different

7

activation or inhibition of downstream effectors (Ellis, 2004). Gαs subunit is known to activate

adenylate cyclase and lead to cAMP elevation, whereas, Gαi/Gαo inhibits adenylate cyclase (Ellis,

2004).

There are six different subfamilies of GPCRs. Biogenic amines belong to the rhodopsin-

like (Class A) subfamily of GPCRs. It is the largest family of GPCRs and members of this family

are characterized by having DRY motif at the end of TM3, and NPxxY domain in TM7 (Rovati

et al., 2007; White et al., 2012). These residues are important for protein stabilization and/or G-

protein activation. Biogenic amines noncovalently bind to the upper part of the 7TM domains. The

binding of these small neuroactive chemicals lies deep between TM3, TM4, TM5, TM6 and TM7

(Kristiansen, 2004). Many insect biogenic amines receptors have been characterized and

functionally analyzed. A focus on octopamine and tyramine receptors is presented in Chapter 3.

The function of these receptors is integral in all animals. Therefore, these GPCRs are potential

targets of many insecticides.

Rhodnius prolixus: a vector of Chagas disease

Rhodnius prolixus, generally known as one of many kissing bugs, is a blood-feeding

hemipteran mainly found in South and Central America (Bern et al., 2011). R. prolixus undergoes

incomplete metamorphosis developing through five nymphal instars and finally molting into a

sexually capable adult stage (Nunes-da-fonseca et al., 2017). A blood meal is required for every

incomplete metamorphosis; those that do not obtain a blood meal attenuate development and

remain in the nymphal stage. R. prolixus typically feed on mammalian, bird, marsupial and

reptilian blood (Davey, 2007). There are two known populations of R. prolixus, sylvan and

domestic (Davey, 2007). Sylvan populations occur among the leaves of palm trees, pteridophytes,

and their hosts’ burrows and nests (Davey, 2007). Domestic R. prolixus are in close association

with human habitats and living spaces. They are found in crevices, thatched roofs and small damp

dark spaces (Davey, 2007). R. prolixus are resilient insects, and nymphal instar stages have been

observed to survive for several months without a blood meal in colonies grown and maintained in

the laboratory (WHO, 2002).

8

R. prolixus are nocturnal, they sense CO2 levels and are attracted to the mouth and eye

regions of the human host (CDC, 2013). Feeding behaviour is initiated with the insertion of the

proboscis into the host. A cocktail of chemicals are injected into the host via the proboscis to

prevent detection, blood coagulation and vasoconstriction (WHO, 2002). Fifth-instar R. prolixus

have been observed to feed for 20 minutes thereby consuming 10 times their unfed body mass

(Orchard, 2006). Taking such a massive blood meal causes a major physiological disturbance in

R. prolixus. While feeding, diuresis is initiated for the expulsion of excess water and salts to

compensate for the large blood meal (Orchard, 2006). Trypanosoma cruzi, a protozoan carried in

the gut of Rhodnius prolixus, is also expelled in the process of diuresis (Garcia et al., 2007). T.

cruzi is introduced into the host by mucosal membrane or through the blood stream by scratching

of the punctured area (WHO, 2002).

T. cruzi is known to cause Chagas disease which infects 6 to 7 million people worldwide

(WHO, 2017). Most cases of the disease have been reported in Latin America (WHO, 2017). In

the United States, the Center for Disease Control and Prevention estimates that 300,000 people are

carriers of the parasite; these individuals include immigrants from Chagas disease native regions

(CDC, 2013). In the acute phase of the disease, the parasite is found in the circulating blood and

fever and swelling at the site of infection are reported. In the chronic phase, 20 to 30% of infected

individuals can develop life-threatening conditions due to cardiac and digestive problems

(Kirchhoff and Pearson, 2007). No vaccines have been developed yet, however there are

treatments for the acute phase of the disease that can eliminate T. cruzi from the blood of the host

(CDC, 2013).

The female reproductive system

Anatomy

The adult female reproductive system is composed of two ovaries, two lateral oviducts, a

common oviduct, spermatheca, bursa and the cement gland (Wigglesworth, 1972) (Fig. 2). The

terminal filament attaches to the body wall. Each ovary contains seven meroistic telotrophic type

ovarioles connected to a terminal filament. The ovarioles within the ovary are held together by a

muscular layer known as the peritoneal sheath (Sedra and Lange, 2014). The ovary is the site of

9

egg growth and development, oogenesis. Mature eggs in the ovary are deposited into the oviducts

by a process known as ovulation. Eggs are then carried to the common oviduct by oviduct

peristaltic contractions. Sperm stored in the spermatheca, released by spermathecal contractions,

fertilizes the eggs at the common oviduct (Davey, 1958). The fertilized eggs move to the bursa for

oviposition. The eggs are first coated with secretions from the cement gland for attachment to

substrates and then laid by strong bursal phasic contractions (Lococo and Huebner, 1980).

All muscle in insects are striated. Insect muscles can be divided into skeletal muscle,

visceral muscle, and cardiac muscles (Wigglesworth, 1972). Skeletal muscles are attached to the

cuticle and serve for locomotion and moulting. Visceral muscles have only one or commonly no

attachments to the cuticle. Visceral muscles serve to move the visceral organs, one of which is the

reproductive system. Visceral muscles in the insect are myogenic, they commonly have slow

rhythmic contractions (Orchard and Lange, 1986). Contractions can also be neurogenic, these

contraction are initiated by the nervous system enable fine control of visceral processes (Orchard

and Lange, 1986).

The reproductive system of the adult female R. prolixus is made of myogenic visceral

muscle and has been well described by Sedra and Lange 2014. The terminal filament is made up

of a thick muscular structure. The ovary itself is encircled with a network of muscle fibers. Each

ovariole is surrounded with a criss-cross network of muscle fibers. The lateral oviducts are

surrounded with first a layer of longitudinal and then circular muscle fibers. Thicker circular

muscle fibers also found at the common oviduct and the spermatheca. Thick longitudinal muscles

arranged in a chevron make up the bursa. The cement gland is largely non-muscular except at the

proximal end of the gland.

10

Figure 2. The anatomical structures of the adult female reproductive system of R. prolixus.

Illustration created by Paul Hong.

12

Central nervous system innervation to the reproductive system

The CNS of R. prolixus is made of a dorsal brain connected to a ventral suboesophageal

ganglion (SOG) located in the narrow head region (Tsang and Orchard, 1991). The SOG is

connected to the prothoracic ganglion (PRO) which is then connected to the mesothoracic

ganglionic mass (MTGM) (Tsang and Orchard, 1991). The MTGM is a large ganglion because it

houses the fused mesothoracic ganglion, metathoracic ganglion and abdominal ganglion. The

MTGM sends nerve fibers that innervate regions in the insect’s abdomen (Insausti, 1994). The

reproductive system is innervated by the trunk nerve (Chiang and O’Donnell, 2009; Insausti, 1994;

Sedra and Lange, 2014). Various neuroactive chemicals are known to innervate the reproductive

system of R. prolixus. Immunoreactivity to FMRFamide-like peptides was detected in process the

ovaries, oviducts, spermatheca and the bursa. In D. melanogaster, octopamine and tyramine

innervate the peritoneal sheaths in the ovary, oviducts, spermatheca and the uterus (Middleton et

al., 2006; Rodriguez-Valentin et al., 2006).

Reproductive processes

Oogenesis

As previously stated, hemipterans have meriostic teleotrophic type ovarioles (Huebner and

Anderson, 1972). Meriostic type ovarioles are characterized by having nurse cells, nutritive cells,

and germ cells that contribute to the nourishment of the developing oocyte (Huebner and

Anderson, 1972). Telotrophic type, a subgroup of meriostic ovarioles, are characterized by having

nurse cells restricted to the apex of the ovariole and provide nutrients to the developing oocyte by

a nutritive cord (Wigglesworth, 1972). These ovarioles are subdivided into four main regions: the

terminal filament, the germarium, the vitellarium and the ovariole stalk (Nunes-da-fonseca et al.,

2017). The germarium region is located below the terminal fiber that attaches the ovariole. The

germarium is composed of undifferentiated oogonia (germline cells) and nurse cells

(Wigglesworth, 1972). The oogonia differentiate into an oocytes and nurse cells. The developing

oocyte, nourished by nurse cells via the nutritive cord and the surrounding follicular cells, moves

down the ovariole into the vitellarium (Bonhag, 1955). The vitellarium is where vitellogenesis

occurs. Vitellogenin, egg yolk, is made in the fat body and is deposited into the hemolymph

(Davey, 1981). Vitellogenin in the hemolymph is transferred to the oocyte via gaps between the

13

follicular cells. Vitellogenin is also synthesized in the follicular cells and directly deposited into

the oocyte (Davey, 1981). Nurse cells also provide vitellogenin via the nutritive cord. After the

developing egg enlarges, the follicular gaps close and vitellogenin uptake ends (Patchin and

Davey, 1968). The follicular cells secrete an elastic vitelline membrane and then form the chorion

(Patchin and Davey, 1968).

Ovulation

After the formation of the chorion, the follicular cells that surround the mature egg

degenerate. This leaves the egg free to move and in direct contact with the peritoneal sheath.

Various hormones circulating in the hemolymph and neurochemicals such as myotropins secreted

by the CNS stimulate contractions in the ovary and oviducts (Davey, 1967). Contractions in the

ovary are synchronized with relaxation of the lateral oviducts (Hana and Lange, 2017; Middleton

et al., 2006). This coordination between the ovary and the lateral oviducts allows the movement of

eggs from the ovary to the lateral oviducts. Peristaltic contractions aided with lumen secretions in

the lateral oviducts allows the movement of ovulated eggs into the common oviduct (Masetti et

al., 1994; Sun and Spradling, 2013). Once the eggs are ovulated, they are immediately fertilized

and laid (Kriger and Davey, 1982).

Fertilization and oviposition

Before the process of fertilization of eggs, spermatozoa must be first taken up and stored

in the spermatheca (Davey, 1958). The spermatophore, containing spermatozoa, is deposited into

the bursa of the female during copulation (Davey, 1958). Male accessory gland secretions leads to

contractions in the common oviduct leading to the movement of the spermatozoa to the

spermatheca (Davey, 1958). The spermatozoa does not play an active role in the migration (Davey,

1958). When the egg arrives at the common oviduct, contractions in the spermatheca cause the

release of spermatozoa onto the chorion coated egg. The spermatozoa fertilizes the egg via narrow

passages within the chorion known as micropyles (Okasha et al., 1970). The fertilized egg moves

to the bursa via contractions in the common oviduct. Secretions from the cement gland coats the

egg and the egg is then deposited onto a substrate by strong phasic bursal contractions (Lococo

and Huebner, 1980).

14

Neuroactive chemicals control reproduction in females

Rhodnius prolixus, like all blood-feeding insects, require blood meals for reproductive

maturity. Despite having a fully functioning reproductive system, female adults must generally be

fed in order to mate (Buxton, 1930). Once they are fed, they can synthesize and lay viable eggs.

Mating introduces male factors that accelerate reproductive processes in females (Davey, 1965).

At the germarium, an oocyte’s growth is directly regulated by its genome and the content

of its species-specific information is known as euplasm; the euplasm is synthesized in the nurse

cells (Chapman, 2013). At the vitellarium, yolk uptake supresses DNA transcription and nuclear

processes. Juvenile hormone (JH) secreted from the corpus allatum acts on the fat body to

synthesize vitellogenin and leads to the formation of gaps between follicular cells (patency)

(Davey, 2007; Davey et al., 1974). These follicular gaps are needed for vitellogenin uptake by the

oocyte (Davey et al., 1974). The synthesis and release of JH is under the control of allatotropins

and allatostatins in the brain (Davey, 1987; Teal, 2002). The brain, acting via neurosecretory cells,

can signal for JH release when mating and feeding have occurred (Davey, 2007). Antigonadotropin

released from neurosecretory cells in the abdomen counteract the effects of JH on follicle cells and

prevent the formation of gaps between follicular cells (decrease patency) (Davey and Kuster,

1981). Recently, it was shown that injected RhoprShortNPF (NNRSPQLRLRFamide),

RhoprFMRFa (GNDNFMRFamide) and RhoprFIRFa (AKDNFIRFamide) increases the number

of eggs produced, i.e. increases oogenesis (Sedra and Lange, 2016). In contrast, injections of

RhoprMS (pQDIDHVFMRFamide) and RhoprAST-2 (LPVYNFGLamide) reduced egg

production (Sedra and Lange, 2016). Therefore, FMRFamide-like peptides (FLPs) are important

for controlling the rate of oogenesis. The mechanisms where this occurs and not known.

Ecdysteroids, produced from the ovary, do not affect vitellogenin content in the hemolymph and

do not increase egg production (Davey, 2007).

At the end of vitellogenesis, ecdysteroids synthesized from the follicular cells are thought

to be essential for ovulation (Chapman, 2013). Circulating ecdysteroids elicit the release of a

myotropic ovulation hormone from the corpus cardiacum of the brain (Ruegg et al., 1981). A

myotropin was found to be synthesized from ten neurosecretory cells in the pars intercerebralis

(Ruegg et al., 1981). This myotropin leads to an increase in muscle contractions in the ovary and

thereby increases the rate of ovulation (Davey, 1967; Ruegg et al., 1981). A FLP has been

suggested to the ten neurosecretory cells (Sevala et al., 1992). The peak of FLP release, five days

15

post-feeding, parallels the peak of myotropin release in the gonadotrophic cycle (Sevala et al.,

1992). Therefore, the myotropin is thought to be related to FLPs (Sevala et al., 1992). Furthermore,

other neuropeptides and biogenic amines can modulate ovulation by controlling lateral oviduct

contractions. For example RhoprFIRFa and RhoprFMRFa have been shown to increase ovariole

and lateral oviduct contractions (Sedra and Lange, 2014). The myoinhibitors, RhoprAST-2 and

RhoprMIP-4, inhibited oviduct contractions (Sedra et al., 2015). Octopamine’s role in oviduct

relaxation has been shown in other insects. In D. melanogaster, octopamine stimulated peritoneal

sheath contractions and relaxed the oviducts (Middleton et al., 2006; Rodriguez-Valentin et al.,

2006). In Locusta migratoria, serotonin, acting as a neuromodulator, elicits an increase in

amplitude of oviduct contractions (Lange, 2004). After ovulation, the eggs are moved through the

oviducts by peristaltic and phasic contractions. For fertilization to occur, the spermatheca must

contract to release the sperm. Tyramine and octopamine cause an increase in the amplitude and

frequency of spermathecal contractions in the locust, L. migratoria (da Silva and Lange, 2008). In

R. prolixus, RhoprFIRFa and RhoprFMRFa have also been shown to cause strong contractions in

the bursa, thereby playing a role in oviposition. Proctolin has also been shown to increase the tone

of bursal contractions in R. prolixus (Chiang et al., 2010). Therefore, a cocktail of neuroactive

chemicals acting as neurotransmitters, neurohormones and neuromodulators are important for the

movement of eggs in the reproductive tract.

Significance

As stated earlier, R. prolixus is one of the primary vectors of Chagas disease. Millions of

people are diagnosed with Chagas disease in Latin America. Chagas disease does not only cripple

the health and wellbeing of those infected, but also causes economic losses due to vector control

initiatives. It is known that a mated, regularly fed kissing bug can lay up to 600 eggs in it’s life

span of 1.5 years (WHO, 2002). This impressive reproductive capability translates to a massive

number of nymphs arising from a few females. Therefore, it is very critical to control the

population of the vector to eradicate Chagas disease in epidemic regions. Octopamine has been

shown to be an important neurochemical associated with the reproduction and the laying of viable

eggs. Disruption of octopamine signaling leads to reproductive sterility. The role of octopamine

and tyramine has not been elucidated yet in R. prolixus. Investigating basic physiology of the

reproductive system will provide information on the role of octopamine and tyramine signaling

pathways, while studying molecular components will provide possible targets for octopamine and

16

tyramine signaling disruption. Perhaps the octopamine and tyramine GPCRs could be used for

developing novel, species-specific and biostable insecticides targeting kissing bugs

Thesis objectives

Octopamine and less so tyramine have been implicated in the regulation of reproductive

processes in many insects such as D. melanogaster, L. migratoria, Stomoxy calcitrans,

Leucophaea maderae, Gryllus biamaculatus, Nilaparvata lugens, Periplaneta americana. Most of

the studies available have focused on octopamine’s ability to modulate contractions of visceral

reproductive muscles. Recently, molecular tools have enabled the discovery of octopamine

receptors in the female reproductive system of D. melanogaster and N. lugens. These receptors

were critical for egg-laying. Interestingly, it has been shown that flies lacking octopamine are

reproductively sterile and retain eggs in the ovary. The purpose of this thesis is to determine the

role of octopamine and tyramine in modulating the reproductive system of the adult female R.

prolixus. This will be investigated by first examining the effects of octopamine and tyramine on

the reproductive musculature and second utilizing the recently sequenced genome to identify and

functionally characterize octopamine and tyramine GPCRs in the reproductive system. The aims

of the physiological and molecular approaches are listed below:

Physiological approach

□ Decipher the effects of octopamine and tyramine on the rhythmic contractions of the

oviducts and the bursa.

□ Determine the mode of action of octopamine and tyramine at the oviducts and the bursa.

□ Analyze the effects of injected octopamine and tyramine on egg-laying.

Molecular approach

□ Isolate and clone octopamine and tyramine GPCRs involved in signaling reproductive

system.

□ Using online tools, analyze and predict the structural characteristics of the octopamine and

tyramine receptors.

□ Deorphan the octopamine and the tyramine receptors through a functional receptor assay.

17

□ Analyze the spatial expression of octopamine and tyramine receptor transcripts in the CNS

and in specific reproductive tissues.

18

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23

Chapter 2: Octopamine and tyramine regulate the activity of reproductive

visceral muscles in the adult female blood-feeding bug, Rhodnius prolixus

Sam Hana, Angela B. Lange

University of Toronto Mississauga, Department of Biology, Mississauga, ON, Canada L5L1C6.

* Correspondence:

Sam Hana

[email protected]

Keywords: Oviducts, bursa, inhibition, contractions, cyclic AMP

*** The proceeding chapter is reproduced/adapted with permission from the Journal of

Experimental Biology.

Octopamine and tyramine regulate the activity of reproductive visceral muscles in the adult

female blood-feeding bug, Rhodnius prolixus.

Hana, S., and Lange, A. B. (2017).

The Journal of Experimental Biology 220, 1830–1836.

doi:10.1242/jeb.156307.

24

Abstract

The role of octopamine and tyramine in regulating spontaneous contractions of

reproductive tissues was examined in the female Rhodnius prolixus. Octopamine decreased the

amplitude of spontaneous contractions of the oviducts and reduced RhoprFIRFa-induced

contractions in a dose-dependent manner, whereas tyramine only reduced the RhoprFIRFa-

induced contractions. Both octopamine and tyramine decreased the frequency of spontaneous

bursal contractions and completely abolished the contractions at 5×10−7 mol l−1 and above.

Phentolamine, an octopamine receptor antagonist, attenuated the inhibition induced by octopamine

on the oviducts and the bursa. Octopamine also increased the levels of cAMP in the oviducts, and

this effect was blocked by phentolamine. Dibutyryl cyclic AMP mimicked the effects of

octopamine by reducing the frequency of bursal contractions, suggesting that the octopamine

receptor may act by an Octβ receptor. The tyramine receptor antagonist yohimbine failed to block

the inhibition of contractions induced by tyramine on the bursa, suggesting that tyramine may be

acting on the Octβ receptor in the bursa.

25

Introduction

The biogenic amine octopamine acts as a neurotransmitter, neuromodulator and

neurohormone in invertebrates (Orchard, 1982). Octopamine and its precursor tyramine are both

derivatives of the amino acid tyrosine, and octopamine and tyramine are believed to function

analogously to adrenaline (epinephrine) and noradrenaline (norepinephrine) in vertebrates

(Roeder, 2005). Thus, tyramine is now considered to be a neuroactive chemical in its own right,

independent of octopamine (Kononenko et al., 2009; Lange, 2009). Octopamine and tyramine

regulate diverse physiological and behavioural processes such as courtship, locomotion, learning

and memory, and reproduction (Avila et al., 2012; Huang et al., 2016; Roeder, 1999; Selcho et al.,

2012). Female Drosophila melanogaster with mutated tyrosine decarboxylase show reproductive

sterility due to the lack of octopamine (Cole et al., 2005). In tyrosine decarboxylase mutant flies,

supplementation with octopamine restored reproductive viability (Cole et al., 2005). Similarly,

tyramine β-hydroxylase mutant flies that are found to only lack octopamine are also reproductively

sterile (Monastirioti, 2003; Monastirioti et al., 1996). Octopamine and tyramine signal via G-

protein coupled receptors (GPCRs), leading to changes in second messenger levels. The recently

updated receptor classification (Farooqui, 2012) divides the receptors into Octα-R, Octβ-Rs

(Octβ1-R, Octβ2-R, Octβ3-R), TYR1-R and TYR2-R. In general, Octβ-Rs lead to elevation of

cAMP while Octα-R and TYR-Rs lead to an increase in Ca2+ (Farooqui, 2012).

The movement of eggs in the reproductive system of Rhodnius prolixus starts at the ovaries,

the site of egg maturation. Upon ovulation, mature eggs are released into the oviducts

(Wigglesworth, 1942). Eggs are then guided, via oviductal peristaltic and phasic contractions, to

the common oviduct, where spermatozoa are released through spermathecal contractions, leading

to fertilization (Davey, 1958). Fertilized eggs are coated with secretions from the cement gland

(Lococo and Huebner, 1980). The bursa deposits the fertilized eggs via strong phasic contractions.

These activities are under the direct control of the central nervous system (CNS) and branches of

the trunk nerves innervate the reproductive tissues of R. prolixus (Insausti, 1994). The lateral

oviducts are made up of two layers of visceral muscle, an inner circular and an outer longitudinal

layer, whilst the bursa is made up of thicker muscle fibres arranged longitudinally (Sedra and

Lange, 2014). The oviducts and the bursa spontaneously contract (Sedra and Lange, 2014) but the

site of the intrinsic pacemaker(s) in the reproductive system has not been identified.

26

Octopamine and tyramine modulate the myogenic activity of a variety of visceral muscles

in insects, including tissues of the reproductive system. Octopamine decreases the basal tonus, and

reduces the amplitude and frequency of neurally evoked contractions of the lateral oviducts of the

locust Locusta migratoria (Lange and Orchard, 1986). Also, octopamine has been shown to

decrease the amplitude of proctolin-induced contractions in a dose-dependent manner (Lange and

Orchard, 1986; Nykamp and Lange, 2000). These effects appear to be mediated by an Oct/Tyr

receptor shown to be expressed in the oviducts of locusts (Molaei et al., 2005). In Drosophila and

the stable fly Stomoxys calcitrans, octopamine reduces the amplitude and frequency of

contractions, and reduces basal tonus of the oviducts in a dose-dependent manner (Cook and

Wagner, 1992; Middleton et al., 2006; Rodríguez-Valentín et al., 2006). These physiological

effects could be linked to two receptors: the octopamine receptor in the mushroom bodies (OAMB)

and Octβ2-R, which have been shown in Drosophila to be expressed in the epithelial and muscle

cells of the oviducts (Lee et al., 2003; Li et al., 2015; Lim et al., 2014). These receptors are involved

in ovulation and fertilization of eggs, whereby mutant constructs of these receptors show

reproductive sterility in females, accumulation of eggs in the ovary and reduction in the number

of eggs laid (Lee et al., 2003; Li et al., 2015; Lim et al., 2014). In contrast, octopamine has also

been shown to increase the frequency and the amplitude of myogenic contractions in the lateral

oviducts of the cricket Gryllus bimaculatus (Tamashiro and Yoshino, 2014). In the cockroach

Leucophaea maderae, the action of octopamine and tyramine is unclear; both stimulated oviduct

contractions in some preparations but inhibited oviduct contractions in other preparations (Cook

et al., 1984). Tyramine decreases the basal tonus and attenuates proctolin-induced contractions in

locusts (Donini and Lange, 2004); however, tyramine has no effect on the amplitude of

contractions or basal tonus of the oviducts in D. melanogaster (Middleton et al., 2006).

The purpose of this study was to determine the role of octopamine and tyramine in

modulating myogenic contractions of the oviducts and the bursa of the adult female R. prolixus

and to investigate the mechanism by which octopamine and tyramine mediate these effects.

Materials and Methods

Animals

Adult R. prolixus Stål 1859 were maintained on a 12 h:12 h light:dark cycle at

approximately 50% humidity and 28°C. Rhodnius prolixus were fed defibrinated rabbit's blood

27

(Hemostat Laboratories, Dixon, CA, USA; supplied by Cedarlane Laboratories Inc., Burlington,

ON, Canada) once in every instar. Four- to five-week-old unfed adult females were used for all

experiments.

Chemicals

D,L-Octopamine hydrochloride and tyramine hydrochloride were made as 10−2 mol l−1

stocks and stored at −20°C. Phentolamine hydrochloride and dibutyryl cAMP were freshly made

in physiological saline prior to use. Aliquots of AKDNFIRFamide (RhoprFIRFa, 10−3 mol l−1;

GenScript USA, Inc., Piscataqay, NJ, USA) were stored at −20°C. Stock solution of yohimbine

was prepared in 95% ethanol; the final percentage of ethanol in the experimental treatments was

≤0.1%. Physiological saline (NaCl 150 mmol l−1, KCl 8.6 mmol l−1, CaCl2 2 mmol l−1, NaHCO3 4

mmol l−1, glucose 34 mmol l−1, MgCl2 8.5 mmol l−1, Hepes 5 mmol l−1, pH 7.2) was prepared in

double distilled water and used to dilute all chemicals. All chemicals were obtained from Sigma

Aldrich (Oakville, ON, Canada) unless otherwise stated.

Contraction assays

Oviduct bioassay

The wings were cut off and the dorsal cuticle along with the gut of a female adult R.

prolixus were removed to expose the reproductive system. Using a fine silk thread, a double knot

was tied at the posterior end of the common oviduct and the other end of the silk was double

knotted onto the hook of the force displacement signal transducer (Aksjeselskapet Mikro-

elektronikk, Horten, Norway). The oviducts (lateral and common) were dissected out and placed

in a Sylgard-coated dish filled with 200 µl of physiological saline at room temperature. The

anterior end of each lateral oviduct was pinned to the dish with minutien pins. The signal generated

was amplified, converted into a digital signal by Picoscope 2200 (Pico Technology, St Neots, UK)

and analysed by the PicoLog program (Pico Technology).

To examine the effects of octopamine and tyramine on contraction of the oviducts, 100 µl

of the bath saline was removed and replaced with 100 µl of 2×10−8 mol l−1 to 2×10−3 mol l−1

octopamine/tyramine. A final volume of 200 µl was maintained at all times. The tissue was washed

between amine applications. For the inhibitor assays, phentolamine or yohimbine was mixed with

octopamine or tyramine before application. The amplitude of three to four contractions (over ∼2

28

min) was averaged and presented as a percentage relative to contractions of the tissue in saline

(control). To examine the effects of octopamine or tyramine on a peptide-induced contraction, 10−6

mol l−1 RhoprFIRFa was used. RhoprFIRFa produced a standard change in basal tonus which was

then compared with the change in basal tonus produced when the peptide was applied with the

amine.

Bursa bioassay

The dorsal cuticle was removed followed by the gut, exposing the reproductive system.

Using a fine silk thread, a double knot was made at the junction of the bursa and the oviducts. A

cut was made above the double knot and the bursa was left attached to the ventral cuticle and the

fine silk thread was attached to the force transducer. The bursa was secured in place by pinning

the ventral cuticle to the Sylgard-coated dish. The amplitude and the frequency of three to four

contractions were averaged (over ∼2 min) and presented as a percentage relative to contractions

of the bursa in saline (control). Amines were added to the preparations as described above for the

oviduct bioassay.

cAMP determination assay

cAMP content in the oviducts of 4- to 6-week-old adult female R. prolixus was measured.

A total of 50 oviducts were dissected and placed in a dish containing saline. Two oviducts were

pooled and placed in Eppendorf tubes containing saline. Using a dispensing pipette, 10 µl of

5×10−3 mol l−1 3-isobutyl-1-methylaxanthine (IBMX) was added to all tubes followed by the

addition of either octopamine or phentolamine, or both. The tubes were gently mixed and left to

incubate for 10 min. The reaction was stopped by adding 400 µl boiling ELISA buffer (Cyclic

AMP ELISA Kit, Cayman Chemical, Ann Arbor, MI, USA). The tubes were boiled for 10 min

and sonicated for 15 s at output 3 and constant duty cycle with a Branson Sonifier 250 (VWR,

Mississauga, ON, Canada). The homogenates were centrifuged for 15 min at 13,000 rpm. Two 50

µl samples of supernatant from each tube were assayed for cAMP with the Cyclic AMP ELISA

Kit (Cayman Chemical) according to the manufacturer's instructions. The pellets were dissolved

in 100 µl of 1 mol l−1 sodium hydroxide and boiled for 10 min. The resulting solution was used

for protein determination using Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific,

Waltham, MA, USA).

29

Statistical analysis

GraphPad Prism version 5.03 (www.graphpad.com) was used to create and statistically analyse all

graphs in this paper.

Results

Effect of octopamine and tyramine on lateral oviduct contractions

The lateral oviducts contracted spontaneously in vitro as a result of the myogenic activity

of the reproductive musculature (Sedra and Lange, 2014). A strong phasic contraction was initiated

by one lateral oviduct and was shortly followed by contraction of the other lateral oviduct. Both

oviducts then relaxed concurrently, causing a single burst (Fig. 1A,B). Twin or triple peaks in the

trace were observed when the two lateral oviducts were not in sync with each other. Stable

rhythmic activity was maintained for a few hours in physiological saline. Octopamine reduced the

amplitude of the oviductal contractions in a dose-dependent manner (Fig. 1C). The amplitude

started to decrease between 10−7 and 10−6 mol l−1 octopamine, with a significant decrease in

amplitude at 10−5 mol l−1 octopamine (one-way ANOVA followed by Dunnett's multiple

comparison test compared with the saline group at 100%, P

30

induced contraction (one-way ANOVA followed by Tukey multiple comparisons test, P

31

10−5 mol l−1 also antagonized the effect of 10−5 mol l−1 tyramine in the bursa and restored the

amplitude of contractions to 102.3±6.3% relative to the saline control.

Yohimbine does not inhibit the effects of tyramine on bursal contractions

Yohimbine is an α2-adrenergic receptor antagonist known to block tyramine receptors

(Broeck et al., 1995; Saudou et al., 1990). Yohimbine did not alter the amplitude of contractions

when applied on its own and did not inhibit the effects of tyramine on bursal contractions (Fig. 6).

Yohimbine at 10−5 mol l−1 failed to restore bursal contractions abolished by 10−5 mol l−1

octopamine.

32

Figure. 1. The effects of octopamine and tyramine on rhythmic contractions of the oviducts of an

adult female Rhodnius prolixus. (A) Application of octopamine (OA, 10−4 mol l−1) inhibits of the

amplitude of contractions. (B) Tyramine (TA, 10−4 mol l−1) does not affect contractions. The black

bar indicates the period of application of the neurochemical and the white bar indicates the wash

period. (C) Dose–response curve for the effects of octopamine and tyramine relative to the

amplitude of contractions in saline prior to the addition of neurochemicals. Octopamine inhibits

the amplitude of contractions, while tyramine does not affect contraction amplitude (one-way

ANOVA followed by Dunnett's multiple comparison test; *P

34

Figure. 2. Octopamine and tyramine effectively antagonize AKDNFIRFamide (RhoprFIRFa)-

induced contraction of the oviducts of R. prolixus. (A) Octopamine (10−4 mol l−1) significantly

reduces the amplitude of the RhoprFIRFa (10−6 mol l−1)-induced contraction. (B) Tyramine (10−4

mol l−1) significantly reduces the RhoprFIRFa (10−6 mol l−1)-induced contraction. The black bar

indicates the period of application of the neurochemical and the white bar indicates the wash

period. (C) Inhibition of RhoprFIRFa-induced contraction by octopamine and tyramine is dose

dependent (one-way ANOVA followed by Tukey multiple comparisons test; *P

36

Figure. 3. Octopamine and tyramine abolish the rhythmic contractions of the bursa in R. prolixus.

(A–D) Application of 10−6 mol l−1 octopamine (A) or tyramine (B) abolishes contractions of the

bursa. Note that contractions are unchanged at 10−7 mol l−1 octopamine (C) and tyramine (D). The

black bar indicates the period of application of the neurochemical and the white bar indicates the

wash period. (E) Dose–response curve showing the sudden abolishment of rhythmic contractions

at concentrations greater than 5×10−7 mol l−1 neurochemical (n=5–9). (F) Octopamine and

tyramine both significantly decrease the burst frequency relative to the saline control. (G)

Dibutyryl cAMP reduces the frequency of contractions significantly at 10−2 mol l−1 relative to the

saline control. (F and G: one-way ANOVA followed by Dunnett's multiple comparison test;

*P

38

Figure. 4. Phentolamine blocks the inhibitory effect of octopamine on rhythmic contractions of

the oviducts. (A) Octopamine (10−4 mol l−1) reduces the amplitude of spontaneous contraction. (B)

The effect of octopamine (10−4 mol l−1) is inhibited by application of phentolamine (10−7 mol l−1,

Phen). The black bar indicates the period of application of the neurochemical and the white bar

indicates the wash period. (C) Phentolamine alone does not affect the amplitude of contraction.

Phentolamine is capable of reversing the inhibition of oviduct contraction by octopamine. (D)

Phentolamine attenuates the octopamine-induced rise in cAMP levels in the oviducts.

Concentrations in C and D are mol l−1. (C and D: one-way ANOVA followed by Tukey multiple

comparisons test; *P

40

Figure. 5. Phentolamine blocks abolishment of rhythmic contractions in the bursa by octopamine.

(A) Octopamine abolishes bursal contractions at 10−5 mol l−1. (B) Phentolamine (10−5 mol l−1)

blocks the inhibition induced by octopamine (10−5 mol l−1) on bursal contraction. The black bar

indicates the period of application of the neurochemical and the white bar indicates the wash

period. (C) Phentolamine at 10−5 mol l−1 does not significantly increase the amplitude of bursal

contractions when compared with saline. Phentolamine significantly blocks the inhibitory effect

of octopamine on bursal contractions (concentrations are mol l−1; one-way ANOVA followed by

Tukey multiple comparisons test; ***P

42

Figure. 6. Yohimbine fails to block tyramine inhibition of rhythmic contractions of the bursa. (A)

Tyramine (10−5 mol l−1) inhibits bursa contractions. (B) Yohimbine (Yhm, 10−5 mol l−1) does not

block the inhibitory effect of tyramine on bursal contractions. The black bar indicates the period

of application of the neurochemical and the white bar indicates the wash period. (C) Yohimbine at

10−5 mol l−1 does not block the inhibitory effect of tyramine on the amplitude of bursal contractions

(one-way ANOVA followed by Tukey multiple comparisons test; not significant, P>0.05).

Concentrations are mol l−1; means±s.e.m. of n samples noted at the bottom of each bar; for 10−5

mol l−1 TA, n=4.

44

Discussion

Octopamine reduced the amplitude of spontaneous rhythmic contractions in R. prolixus

oviducts. This phenomenon is consistent with results previously obtained in L. migratoria, D.

melanogaster and S. calcitrans (Cook and Wagner, 1992; Lange and Orchard, 1986; Middleton et

al., 2006; Rodríguez-Valentín et al., 2006): octopamine reduced the amplitude of contractions in

S. calcitrans and L. migratoria and reduced neurally evoked contractions of L. migratoria oviducts

(Cook and Wagner, 1992; Lange and Orchard, 1986). In R. prolixus, there was no change in

frequency of the oviductal contractions whereas in L. migratoria and S. calcitrans, octopamine led

to a decrease in basal tonus and frequency in oviduct contractions. In contrast, in G. bimaculatus,

octopamine increased the amplitude and frequency of rhythmic contractions in a dose-dependent

manner despite L. migratoria and G. bimaculatus belonging to the order Orthoptera (Tamashiro

and Yoshino, 2014). In addition, octopamine decreased the amplitude of the RhoprFIRFa-induced

contraction of R. prolixus oviducts, confirming it as an inhibitor of oviduct contractions. In locusts,

it was also shown that octopamine reduces proctolin-induced contractions in the oviducts (Nykamp

and Lange, 2000). Proctolin was also shown to reduce octopamine-induced cAMP levels in

oviducts, suggesting that the components of octopamine and proctolin signalling pathways interact

to modulate oviduct contraction (Nykamp and Lange, 2000). The interaction of RhoprFIRFa and

octopamine is not known, although it is not likely that octopamine interacts with the RhoprFIRFa

signalling pathway according to a recent study in Drosophila (Milakovic et al., 2014). Milakovic

et al. (2014) found that FMRFamide-induced muscle contraction is independent of the well-known

intracellular players such as calmodulin kinase II, IP3, cAMP, etc.; however, we do not know the

pathway used by RhoprFIRFa in this preparation.

Phentolamine, an α-adrenergic receptor antagonist, is an effective OctβR antagonist in the

L. migratoria oviduct (Lange and Orchard, 1986; Orchard and Lange, 1986). Thus, the ability of

phentolamine to block the effects of octopamine on R. prolixus oviducts suggests that octopamine

is working via an OctβR. This is supported by the fact that octopamine increases cAMP levels (a

characteristic of OctβRs) in the oviducts, an effect also blocked by phentolamine (Lange and

Orchard, 1986; Orchard and Lange, 1986). The physiological implications of these findings in R.

prolixus are that octopamine plays an essential role in the process of ovulation. Relaxation of the

oviducts would allow the ovary to release more eggs into the oviducts. In D. melanogaster,

octopamine was found to increase the contractions of the peritoneal sheath, a contractile meshwork

45

that surrounds the ovary, and to relax the oviducts, thereby enabling the release of eggs into the

oviducts (Middleton et al., 2006). Moreover, Octβ2R and OAMB have been found to be the

receptors associated with the process of ovulation and fertilization in Drosophila (Lee et al., 2003;

Li et al., 2015; Lim et al., 2014). Deletions in the oamb locus and mutant constructs of Octβ2R

resulted in accumulation of eggs in the ovary and a significant decrease in the number of eggs laid

(Lee et al., 2003; Li et al., 2015; Lim et al., 2014). Similarly, in R. prolixus, octopamine reduced

the amplitude of oviduct contractions, probably by binding to OctβR, leading to an elevation in

cAMP levels and muscle relaxation.

The process by which tyramine regulates the oviducts seems more modulatory. Tyramine,

when applied to the oviducts at a wide range of doses did not elicit any changes in spontaneous

contractions; however, tyramine inhibited RhoprFIRFa-induced contractions in a dose-dependent

manner. This is not the case in L. migratoria, where tyramine mimicked octopamine and decreased

the basal tonus and attenuated proctolin-induced contractions (Donini and Lange, 2004). A

possible explanation for this phenomena in R. prolixus is that tyramine is co-released with

octopamine; octopamine works on the oviducts to reduce the amplitude of spontaneous

contractions induced by a pacemaker, whereas octopamine and tyramine modify the effects of

myogenic stimulators such as RhoprFIRFa.

The effects of octopamine and tyramine on the bursa are similar. Both biogenic amines

completely abolish contractions of the bursa at high concentrations. In addition, octopamine and

tyramine at low concentrations decrease the frequency of contractions. These effects seem to be

mediated by cAMP, as application of the membrane-permeable cAMP analogue dibutyryl cAMP

decreased the frequency of contractions. Phentolamine antagonized the effects of octopamine and

tyramine, suggesting that both are likely to work via an OctβR. Yohimbine did not antagonize the

effects of tyramine and octopamine on the bursa. This suggests that tyramine acts via the OctβR at

high concentrations, as shown in the locust oviducts and foregut (Britain, 1990; Donini and Lange,

2004). Further studies are needed to understand why the contractions are abolished with no

a

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The Role of Octopamine and Tyramine in the Adult Female ......Chapter 2: Octopamine and tyramine regulate the activity of reproductive visceral muscles in the adult female blood-feeding