The involvement of Rhopr-CRF/DH in feeding and reproduction in the blood-gorging insect Rhodnius prolixus

Abstract

Rhodnius prolixus is a blood-gorging insect and a vector for human Chagas disease. The insect transmits the disease following feeding, when it excretes urine and feces contaminated with the Trypanosoma cruzi parasite. A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH); however, its distribution throughout the insect's central nervous system (CNS) and the expression of its receptor in feeding-related tissue as well as the female reproductive system suggests a multifaceted role for the hormone beyond that of diuresis. Here we investigate the involvement of Rhopr-CRF/DH in feeding and reproduction in R. prolixus. Immunohistochemistry of the CNS showed diminished CRF-like staining in neurosecretory cells (NSCs) of the mesothoracic ganglionic mass (MTGM) immediately following feeding, and partial restocking of those same cells two hours later, indicating Rhopr-CRF/DH stores in this regions are involved in feeding. The results of the temporal qPCR analysis were consistent with the immunohistochemical findings, showing an increase in Rhopr-CRF/DH transcript expression in the MTGM immediately after feeding, presumably capturing the restocking of Rhopr-CRF/DH in the lateral NSCs following release of the peptide during feeding. Elevating haemolymph Rhopr-CRF/DH titres by injection of Rhopr-CRF/DH prior to feeding resulted in the intake of a significantly smaller blood meal in 5th instars and adults without an apparent effect on the rate of short-term diuresis. When adult females were injected with Rhopr-CRF/DH, they also produced and laid significantly fewer eggs. Finally, in vitro oviduct contraction assays illustrate that Rhopr-CRF/DH inhibits the amplitude of contractions of the lateral oviducts, highlighting a potential mechanism via which the hormone diminishes reproductive capacity. To conclude, the study of the Rhopr-CRF/DH pathway, its components and mechanisms of action, has implications for vector control by highlighting targets to alter feeding, diuresis, and reproduction of this disease vector.

What happens after a blood meal? A transcriptome analysis of the main tissues involved in egg production in Rhodnius prolixus, an insect vector of Chagas disease

“…Adult female R . prolixus , injected with Rhopr-CRF/DH produce and lay significantly fewer eggs [ 152 ]. In addition, in locusts, CRF/DH inhibits oocyte growth and reduces ecdysteroid levels [ 153 ].…”

“…A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH) [151]; however, its distribution throughout the CNS and the expression of its receptor in feeding-related tissues as well as the female reproductive system suggests a multifaceted role for the neuropeptide. Adult female R. prolixus, injected with Rhopr-CRF/DH produce and lay significantly fewer eggs [152]. In addition, in locusts, CRF/DH inhibits oocyte growth and reduces ecdysteroid levels [153].…”

What happens after a blood meal? A transcriptome response from the main tissues involved in egg production inRhodnius prolixus, an insect vector of Chagas disease

“…A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH) [103]; however, its distribution throughout the CNS and the expression of its receptor in feeding-related tissues as well as the female reproductive system suggests a multifaceted role for the neuropeptide. Adult female R. prolixus, injected with Rhopr-CRF/DH produce and lay significantly fewer eggs [104]. In addition, in locusts, CRF/DH inhibits oocyte receptor mRNA in OV and FB from unfed insects ( Fig 8B and S2 Table), where vitellogenesis is inhibited, supporting its effects as a negative regulator of reproduction.…”

SIFamide Influences Feeding in the Chagas Disease Vector, Rhodnius prolixus

“…Insects (fifth instars and adults) were dissected in physiological saline (150 mM NaCl, 8.6 mM KCl, 2 mM CaCl 2 , 4 mM NaHCO 3 , 34 mM glucose, 8.5 mM MgCl 2 , 5 mM HEPES, pH 7). Fixation and staining were performed as described by Mollayeva et al (2018), but using a primary anti-SIFamide antiserum raised in rabbit (antiserum kindly provided by Dr. J. A. Veenstra, Bordeaux University, France) at a concentration of 1:10,000 in 0.4% Triton-X and normal goat serum (NGS) for 48 h at 4 • C. The secondary antibody, a 1:600 Cy3-labeled sheep anti-rabbit immunoglobulin (Jackson ImmunoResearch Laboratories, West Grove, PA, United States) was made up in phosphate-buffered saline (PBS) containing 10% NGS for 24 h. Following washes in PBS, an ethanol series (starting with 25% ethanol diluted in distilled water, and increasing in 25% increments) was performed and tissues were mounted in methyl salicylate.…”

“…After injection, insects were kept in a chamber with 12:12 h light/dark cycle, and were fed 3 h after injection on defibrinated rabbit blood for 20 min. This is the typical time for blood-gorging (Mollayeva et al, 2018) and whilst diuresis is underway during this time, the insects do not eliminate any urine until feeding has ceased (Maddrell, 1964). Immediately after feeding, insects were weighed to measure the weight of the meal.…”