Silencing the odorant receptor co-receptor RproOrco affects the physiology and behavior of the Chagas disease vector Rhodnius prolixus

Olfaction is one of the main sensory modalities that allow insects to interpret their environment. Several proteins, including odorant-binding proteins (OBPs) and odorant receptors (ORs), are involved in this process. Odorant receptors are ion channels formed by a binding unit OR and an odorant receptor co-receptor (Orco). The main goal of this study was to characterize the Orco gene of Rhodnius prolixus (RproOrco) and to infer its biological functions using gene silencing. The full-length RproOrco gene sequence was downloaded from VectorBase. This gene has 7 introns and is located in the genome SuperContig GL563069: 1,017,713–1,023,165. RproOrco encodes a protein of 473 amino acids, with predicted 7 transmembrane domains, and is highly expressed in the antennae during all R. prolixus developmental stages. The RNAi technique effectively silenced RproOrco, reducing the gene's expression by approximately 73%. Interestingly, the effect of gene silencing persisted for more than 100 days, indicating a prolonged effect of dsRNA that was maintained even after molting. The phenotypic effects of silencing involved the following: (1) loss of the ability to find a vertebrate host in a timely manner, (2) decreased ingested blood volume, (3) delayed and decreased molt rate, (4) increased mortality rate, and (5) decreased egg laying. Our data strongly suggest that dsOrco disrupts R. prolixus host-finding behavior, which is further reflected in the blood ingestion, molting, mortality, and egg laying data. This study clearly demonstrates that Orco is an excellent target for controlling triatomine populations. Thus, the data presented here open new possibilities for the control of vector-borne diseases.     

Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi

American trypanosomiasis, or Chagas disease, is transmitted by both domestic and sylvatic species of Triatominae which use sensory cues to locate their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of different species of Triatomines, but to date a comparative functional study of the olfactory system is lacking. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of Rhodnius prolixus and the sylvatic Rhodnius brethesi using an electrophysiological approach. In electroantennogram (EAG) recordings, we first showed that the antenna of R. prolixus is highly responsive to carboxylic acids, compounds found in their habitat and the headspace of their vertebrate hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species, as these are tuned to these compounds using single-sensillum recordings (SSRs). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic sibling, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and the likely shift towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future.     

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX₁X₂WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX₁X₂WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.     

Molecular phylogeography of the Amazonian Chagas disease vectors Rhodnius prolixus and R. robustus

The phylogeographical structure of the closely related species Rhodnius prolixus and R. robustus is presented based on a 663-base pair (bp) fragment of the mitochondrial cytochrome b gene. Twenty haplotypes were recovered from 84 samples examined, representing 26 populations from seven Latin American countries. The resulting phylogenetic tree is composed of five major reciprocally monophyletic clades, one representing R. prolixus and four representing R. robustus. While R. prolixus is a very homogeneous assemblage, R. robustus has deeper clades and is paraphyletic, with the clade comprising R. robustus from Venezuela (Orinoco region) more closely related to the R. prolixus clade than to the other R. robustus populations from the Amazon region. The R. robustus paraphyly was supported further by the analysis of a nuclear gene (D2 region of the 28S RNA) for a subset of specimens. The data support the view that R. robustus represents a species complex. Levels of sequence divergence between clades within each region are compatible with a Pleistocene origin. Nucleotide diversity (pi) for all R. prolixus populations was extremely low (0.0008), suggesting that this species went through a recent bottleneck, and was subsequently dispersed by man.     

Trypanosoma rangeli interactions within the vector Rhodnius prolixus: a mini review

This article is an integrative mini review of the research on the interactions between Trypanosoma rangeli and the insect vector, Rhodnius prolixus. Special attention is given to the interactions of these parasites with the gut environment, gut walls, with hemolymph invasion, hemocytes, hemocyte microaggregations, prophenoloxidase-activating system, superoxide, and nitric acid generation and eicosanoid pathways. We described factors affecting vectorial capacity and suggested that T. rangeli may modulate the hemocoelic invasion and the survival of the parasites by overcoming the cellular and humoral defense reactions of the insect vector at different physiological events. The mechanisms of these interactions and their significance for parasite transmission are discussed.     

Lethal and sublethal effects of eucalyptol on Triatoma infestans and Rhodnius prolixus,vectors of Chagas disease

Eucalyptol is the common name for a cyclic ether monoterpene found in essential oils from Eucalyptus species and other plants. Several reports showed its insecticidal activity. In this work, visible symptoms of intoxication, effect on locomotor activity, knock‐down, and repellence produced by eucalyptol were evaluated on nymphs of Triatoma infestans Klug and Rhodnius prolixus Ståhl (both Hemiptera: Reduviidae). Both insects are among the main vectors of Chagas disease in Latin America. Visible symptoms of intoxication were similar to those observed for neurotoxic insecticides. A video tracking technique was used to evaluate locomotor activity and repellence by exposing the nymphs to impregnated papers. Hyperactivity (a non‐directional increase in locomotor activity) is a symptom of intoxication that is used to detect triatomines in rural houses, because it causes the insects to leave their refuges. Eucalyptol produced hyperactivity only in T. infestans at a concentration 1 000× higher than the positive control, deltamethrin [(S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐(2,2‐dibromoethenyl)‐2,2‐dimethylcyclopropanecarboxylate]. It also produced repellence on both species at a concentration 10× higher than the positive control, DEET (N,N‐diethyl‐3‐methylbenzamide). Knock‐down effect was evaluated by exposing the nymphs to impregnated papers in closed containers (contact and fumigation simultaneously). Values of knock‐down time for 50% of exposed nymphs (KT₅₀) were calculated for various concentrations of eucalyptol. The onset of knock‐down occurred more rapidly as the concentration increased. In the best cases, eucalyptol was 12–15× less toxic than the positive control dichlorvos (2,2‐dichlorovinyl dimethyl phosphate). After these results, eucalyptol seems discouraged as a hyperactivant agent for monitoring insects in rural houses. Nevertheless, its knock‐down and repellence effect on vectors of Chagas disease deserve further investigation.     

Comparative kinetics of bloodmeal intake by Triatoma infestans and Rhodnius prolixus, the two principal vectors of Chagas disease

Chagas disease vector insects Triatoma infestans and Rhodnius prolixus (fifteen stage III nymphs per 4 litre cage) were allowed to feed on anaesthetized mice for 1 h (control group), or on active non-anaesthetized mice (NAM) for 2, 4 or 8 h exposure. The bloodmeal size (weight increase) for both species was proportional to the duration of contact with NAM, due to ingestion of multiple small bloodmeals, up to 142% of control weight for T. infestans with 8 h exposure to NAM. The mean weight increase of T. infestans nymphs after 4 h contact with NAM was similar to that of the control group, whereas for R. prolixus , 8 h contact with NAM gave only 64% of the control value. For both species of insect, within 4 h of feeding, >20% of the bloodmeal weight was lost by defaecation and diuresis. The proportions of unfed nymphs and mortality during 2 h contact with NAM were significantly higher for R. prolixus , demonstrating better exploitation of the host blood source by T. infestans , apparently because during blood-feeding the latter insect species caused less irritation to the host.     

Chromosome variability in the Chagas disease vector Rhodnius pallescens (Hemiptera, Reduviidae, Rhodniini)

Rhodnius pallescens is the main vector of Trypanosoma cruzi in Panama and one of the most relevant secondary vectors in Colombia. Despite the importance of this species, there is limited knowledge about the genetic variability along its geographical distribution. In order to evaluate the degree of karyotype variability we analyzed the meiotic behavior and banding pattern of the chromosomes of 112 males of R. pallescens coming from different regions of Colombia and Panama. Using the C-banding technique we identified two chromosomal patterns or cytotypes characterized by differences in the amount, size and distribution of constitutive heterochromatic regions in the chromosome complement (2n = 20 autosomes plus XY in males). The individuals can be easily classified in each cytotype by the analysis of the chromosomes during first meiotic prophase. The frequencies of the cytotypes are variable according to the geographic origin of the populations. This chromosomal divergence together with morphological data supports the existence of three genetically different populations of R. pallescens and provides new information to understand the distribution dynamics of this species.     

Rhodnius prolixus smells repellents: Behavioural evidence and test of present and potential compounds inducing repellency in Chagas disease vectors

Insect repellents are known since many decades ago and constitute a major tool for personal protection against the biting of mosquitoes. Despite their wide use, the understanding of why and how repellents repel is relatively recent. In particular, the question about to what extent insects other than mosquitoes are repulsed by repellents remains open. We developed a series of bioassays aimed to test the performance of well established as well as potential repellent molecules on the Chagas disease vector Rhodnius prolixus. Besides testing their ability to prevent biting, we tested the way in which they act, i.e., by obstructing the detection of attractive odours or by themselves. By using three different experimental protocols (host-biting, open-loop orientation to odours and heat-triggered proboscis extension response) we show that DEET repels bugs both in the presence and in the absence of host-associated odours but only at the highest quantities tested. Piperidine was effective with or without a host and icaridine only repelled in the absence of a living host. Three other molecules recently proposed as potential repellents due to their affinity to the Ir40a⁺ receptor (which is also activated by DEET) did not evoke significant repellency. Our work provides novel experimental tools and sheds light on the mechanism behind repellency in haematophagous bugs.     

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.     

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae)

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.