Research

Our research aims to define the integrative neuroendocrine control of reproductive structures and associated visceral tissues (eg. digestive, cardiac) in the agricultural pest Locusta  migratoria and in the medically-important vector of Chagas’ disease, Rhodnius prolixus; and to define the integrative control of ecdysis behaviour (shedding of the exoskeleton) through the coordinated action of neuropeptides on CNS and associated peripheral systems (cardiac, digestive, skeletal muscle) in R. prolixus.

Our scientific approach is holistic and incorporates multi-facetted aspects from gene to behaviour.  We define the neural substrates and circuits (including their neuroactive chemicals and receptors) used in the integrative control of these behaviours using a variety of techniques including molecular biology, pharmacology, neurophysiology, physiological assays, peptide isolation, neural mapping, and immunohistochemistry. 

Novelty and expected significance: Our long term goal is to study how cells communicate with one another in order to produce an appropriate physiological and behavioral output. We identify and define the interactions between the diverse neurochemicals which are used in our model systems, and also look at interactions / coordination of organ systems. The importance of research into insects lies increasingly in the testing and demonstration of universal principles of neural organization and functioning, while identifying unique features for target of novel pest control strategies. For example, just like the pharmaceutical industry, the agrochemical industry is targeting neuroactive chemicals and GPCRs for novel pest control strategies due to the ever increasing resistance to the current generation of pesticides.  The future is hopeful, especially for neuropeptides, where the intrinsic problems associated with their use have been overcome. This need for control is particularly relevant for our model insects, L. migratoria and R. prolixus.  Locusts are agricultural pests, eating or destroying crops and affecting over 20% of the earth’s land and more than 65 of the world’s poorest countries.  Their swarms lead to devastating consequences in many developing countries.  The medically-important insect, R. prolixus, is the vector of human Chagas disease, and whilst once considered to be confined to the Americas, Chagas disease now occurs throughout the World due to human migration and blood transfusions.  For example, it is estimated that 300,000 people are infected in the USA, 50,000 in Spain and 2,000 in Canada.  Therefore, this research has the potential to discover lead compounds that can be developed into bio-pesticides in the future.

Research Excellence Award 2006/2007:  http://www.utm.utoronto.ca/vp-research/research-campus/u-t-mississauga-excellence-awards/desmond-morton-research-excellence-award

Teaching Excellence Award 2004/2004: http://www.utm.utoronto.ca/vp-research/teaching-excellence-award-faculty

Examples of Significant Contributions to Research; Students and Post-docs indicated in Bold.  For more complete list see https://www.researchgate.net/profile/Angela_Lange2/contributions and https://loop.frontiersin.org/people/20990/publications

  1. Defferrari MS, Orchard I, Lange AB. (2016). Identification of the first insulin-like peptide in the disease vector Rhodnius prolixus: Involvement in metabolic homeostasis of lipids and carbohydrates. Insect Biochem. Molec. Biol. 70:148; An insulin-like growth factor in R. prolixus is involved in post-feeding nutrient balance and growth. Front. Neurosci. 10: 566.

The insulin signaling pathway is a conserved regulator of metabolism, and in insects, as well as in other animals, can modulate physiological functions associated with the metabolism of lipids and carbohydrates. In the present studies, we have identified the first insulin-like peptide (ILP) and insulin-like growth factor (IGF) from Rhodnius prolixus (termed Rhopr-ILP and Rhopr-IGF).Rhopr-ILP is only present in neurosecretory cells in the brain and using RNA interference (RNAi), we have shown that Rhopr-ILP is a modulator of lipid and carbohydrate metabolism and storage through signaling the presence of available energy and nutrients in the hemolymph.Rhopr-IGF is found in a variety of tissues including the fatbody and CNS and silencing the expression of the transcript using RNAi, also altered nutrient balance but was found to modulate growth in Rhodnius prolixus. Recently, we have characterized the first insulin receptor (IR) to be discovered in R. prolixus. Rhopr-IR is a tyrosine kinase and signals through activation of protein kinase Akt and phosphorylation of its downstream targets glycogen synthase kinase 3 β (GSK3β) and the transcription factor FOZO. Our results suggest that Rhopr-IR signaling is involved in hemolymph nutrient homeostasis and fat body storage. These metabolic effects are likely regulated by the activation of Akt and downstream cascades similar to mammalian insulin signaling pathways.

  1.  Hana S, Lange AB. 2017. Cloning and Functional Characterization of Octβ2-Receptor and Tyr1-Receptor in the Chagas Disease Vector, Rhodnius prolixus. Frontiers in Physiology. 8: 744; and Hana S, Lange AB. 2017; Octopamine and tyramine regulate the activity of reproductive visceral muscles in the adult female blood-feeding bug, Rhodnius prolixus. Journal of Experimental Biology 220: 1830-1836.  

There have been many pioneering studies, including those from my own research laboratory, which show that the biogenic amines octopamine (OA) and tyramine (TA) modulate myogenic activity of a variety of visceral muscles in insects, including tissues of the reproductive system (see ref list 39 - 41). The role of OA and TA in regulating reproductive tissues was examined for the first time in the female R. prolixus in our two papers. Both OA and TA inhibit visceral muscle contraction of reproductive tissues. Octopamine’s mode of action is via a cAMP-mediated pathway and TA was via Ca2+.The cDNA sequences of two G-protein coupled receptor (GPCRs), RhoprOctβ2-R, and RhoprTyr1-R, were cloned and their pharmacological profiles were analyzed using a heterologous receptor assay. RhoprOctβ2-R is specifically activated by OA, whereas RhoprTyr1-R is specifically activated by TA. The spatial distribution of these two receptor transcripts in the female reproductive system suggest their importance in modulating reproductive processes. The RhoprOctβ2-R expressed in the oviducts of R. prolixus is likely involved in the relaxation of the oviducts, which may be a vital step in the process of ovulation. RhoprOctβ2-R and RhoprTyr1-R knockdown studies are needed to further elucidate the role of these receptors in ovulation and other reproductive processes in R. prolixus.

 

  1.  Sedra L, Paluzzi J.-P, and Lange, AB. 2018. Characterization and expression of a long neuropeptide F (NPF) receptor in the Chagas disease vector Rhodnius prolixus.  PLoS ONE 13(8): e0202425; SEDRA L and Lange, AB. 2016. Cloning and expression of long neuropeptide F and the role of FMRFamide-like peptides in regulating egg production in the Chagas vector Rhodnius prolixus. Peptides 82:1-11; SEDRA L and Lange AB. 2014. The female reproductive system of the kissing bug, Rhodnius prolixus: Arrangements of muscles, distribution and myoactivity of two endogenous FMRFamide-like peptides.  Peptides 53:140-147.

This series of 3 papers examines the varied roles of members of the FMRF-amide like peptide (FLP) superfamily on the function of adult female reproductive system of R. prolixus. Members of this peptide family stimulate muscle contraction of the ovarioles, ovary, oviduct and bursa.Interestingly, the myosuppressin, RhoprMS, does not have any myogenic effect on the reproductive tract although it has been shown in most insects to be a myoinhibitor. The RhoprNPF (neuropeptide F) and RhoprNPF receptor (RhoprNPFR) transcript have been cloned. RhoprNPFR has been classified as a rhodopsin-type GPCRs. The RhoprNPFR transcript is expressed in putative pre-follicular cells of the R. prolixus telotrophic ovariole suggesting that the transcript is supplied to the developing oocyte during vitellogenesis and the receptor most likely aids in the differentiation of pre-follicular cells into mature follicle cells surrounding the growing oocyte and helps regulate the supply of nutrients. Screening members of the FLP family in an egg-laying assay showed that N-terminally extended FM/L/IRFamides and short NPF stimulate oogenesis, whereas MS inhibits it. This series of papers show that functions within one family of peptides are varied and that the individual peptides need to be precisely controlled to lead to coordination of a behavior – in this case vitellogenesis, oogenesis and oviposition.

 

  1.  Mesquita RD , …Lange, A.B.…. et al. 2015. Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection. Proceedings of the National Academy of Sciences. 112(48): 14936-41.

This publication came about from funding from NIH in response to a White paper written by a consortium of Rhodnius researchers. We provided insects for determining the best colony to use for the sequencing, provided libraries and transcriptomes, disseminated research in progress, and participated in neuropeptide transcript annotation. This genome publication has contributed to significant advances in biomedical and biological research in R. prolixus and the consortium has provided a wonderful vehicle for collaborative research and research performed in my lab (see CCV).For example, we have received requests from the UK (Sevanar wines), US (Paul Taghert) for access to our doing at UTM and collaborations.

  1.  Lee DH, Vanden Broeck J and Lange AB. 2013. Identification and expression of the CCAP receptor in the Chagas’ disease vector, Rhodnius prolixus, and its involvement in cardiac control. PLoS ONE 8(7): e68897; Lee DH, Orchard I and Lange AB 2013.  Evidence for a conserved CCAP-signaling pathway controlling ecdysis in a hemimetabolous insect, Rhodnius prolixus.  Frontiers in Neuroendocrine Science 7:207.    

Crustacean cardioactive peptide (CCAP) is an important neuropeptide in insects which has multiple physiological roles such as controlling heart rate and modulating ecdysis behaviour. In this series of papers, we cloned the cDNA sequence of the CCAP receptor (RhoprCCAPR) from R. prolixus and found it to be a GPCR. A functional receptor expression assay confirms that the RhoprCCAPR is activated by CCAP but not by other peptides. In vivo, the basal heartbeat frequency is decreased by 31% in bugs treated with dsCCAPR. Knocking down the receptor in dsCCAPR-treated bugs also resulted in loss of function of applied CCAP in vitro. The significance of this work is that this was the first report of a GPCR knock-down in R. prolixus and the first report showing that a reduction in CCAPR transcript levels leads to a reduction in cardiac output in any insect. A vital feature in the success of Ecdysozoa is their ability to shed their exoskeleton (a process called ecdysis) allowing them to grow. The behaviour of ecdysis is very well studied in holometabolous insects, such as Drosophila melanogaster and Manduca sexta, and these behaviours are orchestrated by the actions of neuropeptides. Little is known about the control of ecdysis in hemimetabolous insects. Here, we report that CCAP is an essential neuropeptide for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The critical importance of the CCAP signaling pathway in ecdysis was demonstrated by knockdown (as determined by qPCR and immunohistochemistry) of the CCAP and CCAPR transcripts utilizing dsRNA. Insects with these transcripts knocked down had very high mortalitytypically at the expected time of ecdysis, or had a delay in the time to ecdysis. This is the first report of the susceptibility of R. prolixus to dsRNA knockdown of both neuropeptide and receptor transcripts, and the data clearly demonstrates for the first time the conserved nature of the CCAP signaling pathway in ecdysis between holometabolous and hemimetabolous insects.