•There is presence of corazonin in

clusters of cells in the CNS.

•ACP is present in clusters of cells and

processes in the CNS.

•AKH is present in cells inside the CC of

the CNS.

•AKH increased lipid levels. No effect was

observed for ACP and CRZ.

•Corazonin increases heartbeat

frequency. No effect was observed for

ACP and AKH.

Literature Cited:

1. F Koberle, Chagas' disease and Chagas' syndromes: the pathology of American trypanosomiasis. Adv.Parasitol.

1968; 6:63-116.

2. WG Friend, JJB Smith, Feeding in Rhodnius prolixus - Increasing Sensitivity to ATP during Prolonged Food-

Deprivation, J.Insect Physiol. 1975; 21:1081-1084.

3. G Gäde, GJ Goldsworthy, Insect peptide hormones: a selective review of their physiology and potential

application for pest control, Pest. Manage. Sci. 2003; 59: 1063–1075.

4. DR Nässel, Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as

neuromodulators and neurohormones, Prog. Neurobiol. 2002; 68:1–84.

5. DH Lee, AB Lange, Crustacean cardioactive peptide in the Chagas' disease vector, Rhodnius prolixus: presence,

distribution and physiological effects. Gen.Comp.Endocrinol. 2011; 174:36-43.

6. Malik, A, AB Lange, G Gäde, Sequencing and biological effects of an adipkinetic/ hypertrahlosemic peptide in the

stick insect, Baculum extradentatum. Peptides. 2012; 34(1): 51- 56

Acknowledgments

I would like to thank Dr. Ian Orchard for advice and direction during the making of my camera lucida drawings.

Distribution and physiological effects of adipokinetic hormone (AKH), corazonin and

AKH/corazonin related peptide (ACP) in the kissing bug, Rhodnius prolixus

HIMALI PATEL and ANGELA B. LANGE

Department of Biology, University of Toronto Mississauga

Introduction and background

Rhodnius prolixus, is a blood-feeding Hemipteran that is medically important and predominantly resides in South and Central America [1].

Physiological changes to processes are mediated by neuropeptides and hormones present in the central nervous system (CNS) [2].These

chemicals are distributed and released by neurons, and modulate neuronal communication by acting on cell surface receptors [4-5].

The focus of this project is three evolutionarily related neuropeptides with similar amino acid sequences: corazonin (CRZ), adipokinetic

hormone (AKH) and AKH/corazonin-related peptide (ACP). First main goal of this project was to map the distribution and presence of

AKH, corazonin and ACP in the CNS of R. prolixus . The second part was to determine their physiological effects on heart beat frequency

and lipid levels in the haemolymph.

Methods and materials:

Immunohistochemistry: R. prolixus CNS, dorsal vessel, and

peripheral tissues were subjected to immunohistochemitry as

previously described in Lee and Lange (2011) [5]. Tissues were

incubated with a primary antiserum (rabbit anti-CRZ and rabbit

anti-AKH). The primary antibody used for detecting

immunoreactivity for AKH also stained cells containing ACP.

Lipid Mobilization assay: Male adults were injected with 2 µL of

varying concentrations of AKH,CRZ and ACP diluted in saline

with a syringe. Saline was used as a control. After a 90 minute

period, a haemolymph sample of 5µL was withdrawn and

processed as previously described in [6]. The samples were read

against a standard curve of triglyceride with a spectrophotometer.

Corazonin immunoreactivity in the CNS

Figure 1. Confocal microscope images and camera lucida composite of corazonin-like immunoreactivity in the CNS of Rhodnius prolixus.

Whole mount preparations show clusters of bilaterally paired corazonin-like immunoreactive cells in the A) brain in which, six cells are medially located (red arrows) in the protocerebrum and six cells are (blue arrow) laterally located at the junction of the protocerebrum and optic lobes. Axons project from the medially located cells towards the corpus cardiacum (CC) (white arrow) and the subesophageal ganglion (SOG) B). Immunoreactive axons project through the prothoracic ganglion (PRO) C) and terminates in the mesothoracic ganglionic mass (MTGM) D) Six cells are located in the MTGM (green arrows) (scale bar = 100 µm).

Heart Contraction assay:

One electrode is placed on either side of the

heart and the electrical signal is passed

through an impedance converter and

displayed on a chart recorder. Physiological

saline served as a control for normal heart

rate and the effects of a variety of

concentrations of CRZ, AKH and ACP were

measured.

Figure 1. Schematic

diagram of a heart

ACP immunoreactivity in the CNS

Table 1. Comparison between AKH, ACP and CRZ

Figure 2. Confocal image and camera

lucida composite of AKH

immunoreactivity in the CNS of R.

prolixus. Whole mount preparations

show clusters of cells in the corpus

cardiacum, a neurohaemal organ. ( Scale

bar = 100 µm)

AKH Immunoreactivity in the Corpus Cardiacum

Figure 5. Dose response curve showing

the effect of AKH, CRZ and ACP on the

haemolymph lipid level of R. prolixus .

The largest increase can be seen at 15

pmol AKH. Coloured arrow denotes lipid

levels at saline control. Errors bars

represent standard error of mean (n=5-20).

Figure 3. Confocal images and camera lucida composite of corazonin immunoreactivity in

the CNS .Whole mount preparations show clusters of bilaterally paired cells in the A) brain.

Corazonin-like immunoreactive processes are present in the B) corpus cardiacum (CC),C)

prothoracic ganglion (PRO) and D) mesothoracic ganglionic mass (MTGM). (scale bar =100 µm).

The open arrows show nervous corporis cardiaci (NCC). The camera lucida composite is a

representation of corazonin immunoreactivity in the CNS.(scale bar=200 µm)

Figure 4. Confocal images and camera lucida composite of ACP in the CNS of Rhodnius

prolixus. Whole mount preparations show clusters of bilaterally paired cells in the A-B) brain.

ACP-like immunoreactive processes are present in the C) prothoracic ganglion (PRO) and; D)

mesothoracic ganglionic mass (MTGM). (scale bar =100 µm). The camera lucida composite is a

representation of ACP immunoreactivity in the CNS.(scale bar=200 µm)

Figure 6. The effect of CRZ, AKH and ACP on the heart rate of R. prolixus Traces illustrate

the effect of A) corazonin B) AKH C) ACP on heart contractions at concentrations of 10-9 M and

10-7 M. Open arrows represent saline response and closed arrow show the addition of the

respective peptide and dose. D)The change in heart rate frequency by CRZ is dose-dependent.

No effect was observed for ACP or AKH. Results are expressed as difference in average heart rate

during the dose to average heart rate in saline . Error bars represent the standard error of mean.

(n= 4-5)

A) Corazonin AKH ACP

10-9 M

10-7 M

B) C)

AKH increases lipid levels

in the haemolymph

Heart Contraction Assay

D)

Main Findings

30 sec