Prolixus Females Research Articles

Octopamine is required for successful reproduction in the classical insect model, Rhodnius prolixus.

In insects, biogenic amines function as neurotransmitters, neuromodulators, and neurohormones, influencing various behaviors, including those related to reproduction such as response to sex pheromones, oogenesis, oviposition, courtship, and mating. Octopamine (OA), an analog of the vertebrate norepinephrine, is synthesized from the biogenic amine tyramine by the enzyme tyramine β-hydroxylase (TβH). Here, we investigate the mechanisms and target genes underlying the role of OA in successful reproduction in females of Rhodnius prolixus, a vector of Chagas disease, by downregulating TβH mRNA expression (thereby reducing OA content) using RNA interference (RNAi), and in vivo and ex vivo application of OA. Injection of females with dsTβH impairs successful reproduction at least in part, by decreasing the transcript expression of enzymes involved in juvenile hormone biosynthesis, the primary hormone for oogenesis in R. prolixus, thereby interfering with oogenesis, ovulation and oviposition. This study offers valuable insights into the involvement of OA for successful reproduction in R. prolixus females. Understanding the reproductive biology of R. prolixus is crucial in a medical context for controlling the spread of the disease.

Effects of mating on female reproductive physiology in the insect model, Rhodnius prolixus, a vector of the causative parasite of Chagas disease.

The blood-sucking hemipteran Rhodnius prolixus is one of the main vectors of Chagas disease, a neglected tropical disease that affects several million people worldwide. Consuming a blood meal and mating are events with a high epidemiological impact since after each meal, mated females can lay fertile eggs that result in hundreds of offspring. Thus, a better knowledge of the control of R. prolixus reproductive capacity may provide targets for developing novel strategies to control vector populations, thereby reducing vector-host contacts and disease transmission. Here, we have used a combination of gene transcript expression analysis, biochemical assays, hormone measurements and studies of locomotory activity to investigate how mating influences egg development and egg laying rates in R. prolixus females. The results demonstrate that a blood meal increases egg production capacity and leads to earlier egg laying in mated females compared to virgins. Virgin females, however, have increased survival rate over mated females. Circulating juvenile hormone (JH) and ecdysteroid titers are increased in mated females, a process mainly driven through an upregulation of the transcripts for their biosynthetic enzymes in the corpus allatum and ovaries, respectively. Mated females display weaker locomotory activity compared to virgin females, mainly during the photophase. In essence, this study shows how reproductive output and behaviour are profoundly influenced by mating, highlighting molecular, biochemical, endocrine and behavioral features differentially expressed in mated and virgin R. prolixus females.

Silencing of Mitochondrial Trifunctional Protein A Subunit (HADHA) Increases Lipid Stores, and Reduces Oviposition and Flight Capacity in the Vector Insect Rhodnius prolixus.

Rhodnius prolixus is an obligatory hematophagous insect, vector of Chagas disease. After blood meal, lipids are absorbed, metabolized, synthesized, and accumulated in the fat body. When necessary, stored lipids are mobilized, transported to other organs, or are oxidized to provide energy. Mitochondrial β-oxidation is a cyclic conserved pathway, where degradation of long-chain fatty acids occurs to contribute to cellular energetic demands. Three of its reactions are catalyzed by the mitochondrial trifunctional protein (MTP), which is composed by hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunits alpha and beta (HADHA and HADHB, respectively). Here, we investigated the role of HADHA in lipid metabolism and reproduction of Rhodnius prolixus females. The expression of HADHA gene (RhoprHadha) was determined in the organs of starving adult insects. The flight muscle and ovary had higher expression levels when compared to the anterior and posterior midguts or the fat body. RhoprHadha gene expression was upregulated by blood meal in the flight muscle and fat body. We generated insects with RNAi-mediated knockdown of RhoprHadha to address the physiological role of this gene. RhoprHadha deficiency resulted in higher triacylglycerol content and larger lipid droplets in the fat body during starvation. After feeding, lifespan of the knockdown females was not affected, but they exhibited a decrease in oviposition, although hatching was the same in both groups. Silenced females showed lower forced flight capacity than the control ones, and their fat bodies had lower gene expression levels of Brummer lipase (RhoprBmm) and long-chain acyl-CoA synthetase 2 (RhoprAcsl2). Taken together, these findings indicate that HADHA is important to guarantee successful reproduction and efficient mobilization of lipid stores during starvation and flight.

Transcriptomic analysis of regulatory pathways involved in female reproductive physiology of Rhodnius prolixus under different nutritional states

The triatomine Rhodnius prolixus, a vector of the etiological agent of Chagas disease, has long been used as model to understand important aspects of insect physiology. Despite this history, the impact of the nutritional state on regulatory pathways associated with reproductive performance in triatomines has never been studied. The insulin-like peptide/target of rapamycin (ILP/ToR) signaling pathway is typically responsible for detecting and interpreting nutrient levels. Here, we analyzed transcriptomes from the central nervous system, fat bodies and ovaries of adult females in unfed and fed conditions, with a focus on the ILP/ToR signaling. The results show an up-regulation of transcripts involved in ILP/ToR signaling in unfed insects. However, we demonstrate that this signaling is only activated in tissues from fed insects. Moreover, we report that FoxO (forkhead box O) factor, which regulates longevity via ILP signaling, is responsible for the up-regulation of transcripts related with ILP/ToR signaling in unfed insects. As a consequence, we reveal that unfed females are in a sensitized state to respond to an increase of ILP levels by rapidly activating ILP/ToR signaling. This is the first analysis that correlates gene expression and protein activation of molecules involved with ILP/ToR signaling in R. prolixus females in different nutritional states.

Females of a blood‐sucking bug may adjust their mating decisions according to the risk of ovipositing infertile eggs

AbstractFemales should adjust their mating preferences when the costs of being selective vary. Here, we focus on the cost of laying unfertilized eggs. Oviparous species lose their fertilizable oocytes if remain virgin for too long. Thus, females who are at a high risk of laying infertile eggs should be selected against rejecting a mating opportunity under such circumstances. We test this prediction using the blood‐sucking bug Rhodnius prolixus females, which exhibit stereotyped discrimination behaviour to undesired copula. Under laboratory conditions, we compared the copulatory rejection behaviour between females at their first ovarian cycle (12 days old adults, at low risk of ovipositing infertile eggs) and females at their second ovarian cycle (26 days old adults, at high risk of ovipositing infertile eggs). Since R. prolixus female rejection behaviour is mainly exhibited in the presence of potential mates, we also manipulated the presence and absence of additional males. We found that rejection behaviour significantly decreases both when females enter their second ovarian cycle (and are at high risk of losing unfertilized eggs) and under the absence of additional males. We discuss the possible effects of the risk of losing fertilizable eggs on female mating preferences.

Female choosiness and mating opportunities in the blood-sucking bug Rhodnius prolixus

We investigated the relationship between female choosiness and mating opportunities in the blood sucking bug Rhodnius prolixus. Rhodnius prolixus females exhibit active discrimination behaviour to male mating attempts which delays or prevents copulation. Female rejection behaviours can be beneficial if mating opportunities are readily available and the pool of males varies in their quality. Thus, the benefits of finding a better quality male may override the cost of rejecting a mating opportunity. Since the availability of mating opportunities is affected by the ratio of sexually active males to females, we randomly assigned focal pairs to arenas with a sex ratio biased toward males, females or without other individuals. More females exhibited rejection behaviour when conspecifics were present, however, no differences were found when the sex ratio was biased toward either males or females. We discuss possible explanations for these results and hypothesize about the adaptive function of female rejection behaviour.

Chitin deposition on the embryonic cuticle of Rhodnius prolixus: The reduction of CHS transcripts by CHS–dsRNA injection in females affects chitin deposition and eclosion of the first instar nymph

In a previous study, we found that the embryonic cuticle of Rhodnius prolixus is a chitin-based structure that helps the first instar nymph to hatch from the chorion. Here, we investigated how the reduction of transcripts induced by CHS dsRNA injection affects R. prolixus embryogenesis and eclosion. Deposition of chitin in the embryonic cuticle begins later at embryogenesis, around day 8, and ends approximately at day 15, when the insects are ready for eclosion. In R. prolixus, chitin deposition follows pari passu with the synthesis of the chitin synthase mRNA, indicating a regulation at the transcriptional level. The reduction of the chitin synthase gene transcripts by the injection of CHS dRNA prevented chitin deposition during embryonic cuticle formation, being lethal to hatching nymphs, which end up dying while stuck in the chorionic border trying to leave the chorion. The successful eclosion rates were reduced by 60% in animals treated with CHS dsRNA when compared to animals injected with a control (dsRNA no related gene or water). We found that the harmful effects on oviposition and eclosion are possibly due to changes in the structure of the embryonic cuticle, as observed by directly comparing the morphology of control and chitin-deficient embryonic cuticles under the transmission electron microscope. The lack of chitin and changes in its morphological characteristics appears to alter the embryonic cuticle physiology and functionality. Additionally, we observed that the effects of CHS dRNA treatment on R. prolixus females lasted up to 3 egg-laying cycles (∼100 days), pointing to R. prolixus as a useful model for developmental studies.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host–pathogen associations and the origin of protease activity that led to yolk resorption are discussed.

Flight Initiation by Male Rhodnius prolixus is Promoted by Female Odors

Several triatomine bug species utilize chemical cues for sexual communication. We tested whether female or male Rhodnius prolixus, a vector of Chagas disease, produce volatile chemicals that elicit flight responses from conspecifics, and then isolated the source of the chemical. Males confronted with an airstream containing female odors showed a significantly greater take-off frequency compared to a blank airstream or an airstream with male odors. In contrast, females exhibited similar take-off frequencies to male or female odor as to a clean airstream. Occlusion of female metasternal glands with paraffin wax resulted in a significant decrease in male take-off frequency compared to that of intact females. Additionally, excised female metasternal glands elicited a similar take-off frequency from males as did intact females, both significantly greater than the take-off frequency to clean air. These results show that R. prolixus females release a pheromone from their metasternal glands that causes upwind flight in conspecific males.

Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

BackgroundHematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.Methodology/Principal FindingsHz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.Conclusions/SignificanceHerein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: The involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57±17μg/fat body, it increased almost five times and at fourth day it was 244±35μg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31±4.9μg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. 3H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.

Flight‐oogenesis syndrome in a blood‐sucking bug: Biochemical aspects of lipid metabolism

Lipophorin (Lp), either labeled in diacylglycerol moiety with [(3)H]-Palmitic acid or in phospholipid moiety with (32)Pi, was injected into Rhodnius prolixus females. Insects were induced to flight for different times. In just a few minutes of flight, the transfer of radioactivity to ovaries decreased, accompanied by its increase to flight muscles. After one hour of flight, Lp density was higher (1.132 g/mL) than before flight (1.116 g/mL). Lp purified from insects after flight was analyzed by gel filtration chromatography and a polyacrylamide gel pore limit electrophoresis. Both analyses demonstrated a decrease in Lp molecular mass after flight but no changes in apoLp-III amounts were observed. Time-course experiments showed that only 30 min of flight are required for the detection of changes in Lp density and molecular mass. About the same time of rest is necessary for Lp density and molecular mass to return to the baseline value. The lipid content from Lp particles, determined by high-performance thin-layer chromatography (HPTLC), showed a decrease in total lipids after flight. At the same time, an increase of many classes of lipids was observed in flight muscles except for triacylglycerol, which was reduced. The increase of flight muscle lipids was accompanied by a decrease of the ovaries lipid content. The insects subjected to daily exhaustive flight showed a significant decrease in total number of eggs produced. But insects subjected to a single exhaustive flight showed only a small reduction in total number of eggs. Lp density variation during the flight activity of Rhodnius prolixus females is discussed in association with physiological events such as oogenesis.

Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus

Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS–PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.

Two different vibratory signals in Rhodniusprolixus (Hemiptera: Reduviidae)

In this study the substrate-borne stridulatory vibrations produced by Rhodnius prolixus females were recorded and analysed in two different behavioural contexts. In the context of sexual communication females spontaneously stridulated to reject copulatory attempts performed by males. These male-deterring stridulations were fully effective: out of 61 attempts, no copulation occurred. These stridulations consisted of short series of repetitive syllables, each one composed by a single chirp. In the context of defensive behaviour, bugs stridulated if they were clasped or restrained. These disturbance stridulations consisted of long series of repetitive syllables, each one composed by a series of short chirps and a long one. Male-deterring and disturbance stridulations differed in their temporal pattern and frequency spectra, having a main carrier frequency of about 1500 and 2200 Hz, respectively. As no differences in the inter-ridge distances along the whole stridulatory organ were found, the differences in the frequency between both signals could be explained on the basis of a different velocity of rubbing of the proboscis against the prosternal stridulatory organ. It was found that R. prolixus and the related species Triatoma infestans rubbed only the central region of the stridulatory groove (around 1/3 of the total length) to produce disturbance stridulations. The results are discussed in relation to previous work on vibrational sensitivity in R. prolixus and are also compared with results reported for T. infestans.

Fatty acid incorporation by Rhodnius prolixus midgut.

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.

Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus.

32P-Labeled fat bodies (32P-fat bodies) of Rhodnius prolixus females were incubated in the presence of non radioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin is a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of pre-labeled 32P-lipophorin by the tissue. Analysis of 32P-phospholipids transferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximal at about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32P-phospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.