Identification and Expression of the CCAP Receptor in the Chagas’ Disease Vector, Rhodnius prolixus, and Its Involvement in Cardiac Control

Abstract

Rhodnius prolixus is the vector of Chagas’ disease, by virtue of transmitting the parasite Trypanosoma cruzi. There is no cure for Chagas’ disease and therefore controlling R. prolixus is currently the only method of prevention. Understanding the physiology of the disease vector is an important step in developing control measures. Crustacean cardioactive peptide (CCAP) is an important neuropeptide in insects because it has multiple physiological roles such as controlling heart rate and modulating ecdysis behaviour. In this study, we have cloned the cDNA sequence of the CCAP receptor (RhoprCCAPR) from 5th instar R. prolixus and found it to be a G-protein coupled receptor (GPCR). The spatial expression pattern in 5th instars reveals that the RhoprCCAPR transcript levels are high in the central nervous system, hindgut and female reproductive systems, and lower in the salivary glands, male reproductive tissues and a pool of tissues including the dorsal vessel, trachea, and fat body. Interestingly, the RhoprCCAPR expression is increased prior to ecdysis and decreased post-ecdysis. A functional receptor expression assay confirms that the RhoprCCAPR is activated by CCAP (EC50 = 12 nM) but not by adipokinetic hormone, corazonin or an extended FMRFamide. The involvement of CCAP in controlling heartbeat frequency was studied in vivo and in vitro by utilizing RNA interference. 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. This is 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.

Evidence for a conserved CCAP-signaling pathway controlling ecdysis in a hemimetabolous insect, Rhodnius prolixus

“…D. melanogaster heartbeat frequency is increased during the last 10 h of adult development and peaks at 1 h before the ecdysis behavioral sequence in the white stage (Baker et al, 1999). Previously, we reported that RhoprCCAPR transcript level was up-regulated 10-fold in a pool of tissues containing the heart (dorsal vessel), trachea and fat body prior to ecdysis and dropped to basal levels post-ecdysis (Lee et al, 2013). This indicates that the heart/dorsal vessel may have increased sensitivity to CCAP during the ecdysis behavioral sequence.…”

“…Tissues were dissected from R. prolixus 4th instar (Day 4–9 post-fed) and 5th instar (Day 1–6 post-ecdysis). Total RNA was isolated from tissues using the Trizol® reagent (Ambion, Austin, TX) and first-strand cDNA was synthesized using 100 ng of total RNA as previously described (Lee et al, 2013). The reaction was diluted 20-fold with nuclease free water and qPCR analyses were carried out on a CFX96Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA) using the Ssofast™ EvaGreen supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA).…”

“…Gene specific primers for RhoprCCAP , RhoprCCAPR , and the reference genes (ribosomal protein 49, rp49, and beta-actin) (Table 1) were designed to amplify target fragments of similar size across all samples. The reference genes have been validated as stable targets and used previously (Lee et al, 2013). Each primer set was designed with one primer over an exon/exon boundary and the primer efficiency was determined for each target.…”

“…Two overlapping fragments of the DNA template for dsRNA synthesis (see Table 2) were prepared with PCR by conjugating the T7 RNA polymerase promoter to the 5′ end (5′-taatacgactcactatagggaga-3′) on RhoprCCAP , RhoprCCAPR , or ampicillin resistant gene ( ARG ) as previously described (Lee et al, 2013). The PCR products were used as template for double stranded RNA (dsRNA) synthesis using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA).…”

“…We have also cloned and characterized the CCAP receptor gene ( RhoprCCAPR ) and have shown that RhoprCCAPR is upregulated prior to ecdysis in the heart and plays a role in regulating heartbeat rate (Lee et al, 2013). In the current study, we have investigated the developmental expression pattern of both RhoprCCAP and RhoprCCAPR during ecdysis in the central nervous system (CNS) and peripheral tissues of R. prolixus .…”