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The present study aimed to identify the triatomine species and evaluate Trypanosoma cruzi infection in insects captured in endemic areas of Pará State, Brazil. Triatomines were captured in nine rural communities in the municipality of São Domingos do Capim in August, September and December 2014 using active searches and Malaise and Noireau traps. Additionally, from 2014 to 2018, residents and community health agents submitted captured triatomines to the study team. The analysis of T. cruzi infection in the insects was performed by direct parasitological examination and nested-PCR. A total of 225 triatomines were captured and identified: Rhodnius robustus (n = 111), Rhodnius pictipes (n = 54), Panstrongylus geniculatus (n = 44), Eratyrus mucronatus (n = 11), Panstrongylus lignarius (n = 4), and Panstrongylus rufotuberculatus (n = 1). Direct parasitological examination was performed in 27 living triatomines R. robustus (n = 14), P. geniculatus (n = 7) and R. pictipes (n = 6) and metacyclic trypomastigote forms similar to those of T. cruzi were observed in 66.6% (18/27) samples. Of 174 samples analysed by nested-PCR, 81.6% were positive for T. cruzi DNA: R. robustus (84.7%; 72/85), R. pictipes (84.1%; 37/44), P. geniculatus (69.4%; 25/36), P. lignarius (100%; 4/4), E. mucronatus (75%; 3/4) and P. rufotuberculatus (100%; 1/1). R. robustus, R. pictipes and P. geniculatus were the main vectors of T. cruzi in the studied areas; however, the detection of infections in P. lignarius, E. mucronatus and P. rufotuberculatus indicated that these species can also act as potential vectors of T. cruzi in the study areas.

Fatty acid synthase is a multifunctional enzyme involved in the formation of fatty acids. Despite the role of fatty acids in cell signalling and energy metabolism, and as precursors to pheromones and hydrocarbons that waterproof the cuticle, the insect fatty acid synthases have been scarcely studied. Here we perform the molecular characterization of three fatty acid synthase genes (fatty acid synthase RPRC000123, RPRC000269 and RPRC002909) in the Chagas disease vector, Rhodnius prolixus. Gene expression screening by reverse transcription quantitative PCR showed that RPRC000123 and RPRC002909 are expressed almost exclusively in the integument tissue whilst RPRC000269 is mostly expressed in the fat body and also in several body organs. Phylogenetic analysis, together with gene expression results, showed that RPRC000269, RPRC002909 and RPRC000123 are orthologues of Drosophila melanogaster fatty acid synthase 1 (FASN1), FASN2 and FASN3 genes, respectively. After RNA interference-mediated knockdown of RPRC000123, insects died immediately after moulting to the next developmental stage. However, mortality was prevented by placing the insects under saturated humidity conditions, suggesting that dehydration might play a role in the insects’ death. Lipid analyses in RPRC000123-silenced insects showed reduced amounts of integument fatty acids and methyl-branched hydrocarbons, compared to controls. These data support an important role for FASN3 in the biosynthesis of the precursors to hydrocarbons that waterproof the insect cuticle. Fil: Moriconi, Debora Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Dulbecco, Andrea Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Calderón Fernández, Gustavo Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina

The mammalian gonadotropin-releasing hormone is evolutionarily related to the arthropod adipokinetic hormone and the recently discovered adipokinetic hormone/corazonin-related peptide (ACP). The function of the ACP signaling system in arthropods is currently unknown. In the present study, we identify and characterize the ACP signaling system in the kissing bug Rhodnius prolixus. We isolated the complete cDNA sequence encoding R. prolixus ACP (Rhopr-ACP) and examined its expression pattern. Rhopr-ACP is predominantly expressed in the central nervous system. In particular, it is found in both the brain and corpus cardiacum (CC)/corpora allata (CA) complex. To gain an insight into its role in R. prolixus, we also isolated and functionally characterized cDNA sequences of three splice variants (Rhopr-ACPR-A, B and C) encoding R. prolixus ACP G protein-coupled receptor (Rhopr-ACPR). Rhopr-ACPR-A has only five transmembrane domains, whereas Rhopr-ACPR-B and C have all seven domains. Interestingly, Rhopr-ACPR-A, B and C were all activated by Rhopr-ACP, albeit at different sensitivities, when expressed in Chinese hamster ovary cells stably expressing the human G-protein G16 (CHO/G16). To our knowledge, this is the first study to isolate a truncated receptor cDNA in invertebrates that is functional in a heterologous expression system. Moreover, Rhopr-ACPR-B and C but not Rhopr-ACPR-A can be coupled with Gq α subunits. Expression profiling indicates that Rhopr-ACPR is highly expressed in the central nervous system, as well as the CC/CA complex, suggesting that it may control the release of other hormones found in the CC in a manner analogous to gonadotropin-releasing hormone. Temporal expression profiling shows that both Rhopr-ACP and Rhopr-ACPR are upregulated after ecdysis, suggesting that this neuropeptide may be involved in processes associated with post-ecdysis.

The Triatominae subfamily is comprised of 18 genera and six tribes. The tribe Rhodniini is comprised of two genera (Rhodnius and Psammolestes). Nucleolar persistence is defined by the presence of the nucleolus or nucleolar corpuscles during the meiotic metaphase. To date, this phenomenon has been described for 13 species of triatomine that are included in the genera Triatoma, Rhodnius, and Panstrongylus. Thus, because the phenomenon of nucleolar persistence has been described in only two species of the genus Rhodnius, we have analyzed the nucleolar behavior during spermatogenesis of eight species of the genus Rhodnius (R. colombiensis, R. montenegrensis, R. nasutus, R. neglectus, R. neivai, R. pictipes, R. prolixus, and R. robustus), with a focus on nucleolar persistence. By means of cytogenetic analysis with silver ions, nucleolar behavior during spermatogenesis is described in the eight species of Rhodnius analyzed. In all of them nucleolar behavior was similar and the phenomenon of nucleolar persistence was often observed. Therefore, we confirm nucleolar persistence as a peculiarity of the genus Rhodnius. However, it is emphasized that new cytogenetic analysis should be performed in the Triatominae subfamily, more specifically among the 15 genera that do not exhibit the nucleolar behavior described, in order to assess whether this phenomenon is truly a synapomorphy of these hematophagous insects.

The rhythmic phenomena expressed by organisms change over their lifetimes, but little is known of accompanying reorganization of the central circadian timing system in the brain. Especially dramatic changes in overt rhythms and morphology occur during transformation of larval insects into the adult form (metamorphosis). In Rhodnius prolixus, both the physiology of metamorphosis and its hormonal control are known in detail. Here we report changes in the brain timing system as revealed by pigment dispersing factor immunohistochemistry and confocal microscopy. Most of the features of the larval system are retained, but new clock cells differentiate and the arborizations of their axons increase in complexity, as do pathways connecting the lateral (LNs) and dorsal (DNs) groups of clock neurons. Early in metamorphosis, the LNs increase from 8 to 11 in number, becoming five small and six large LNs. Two large LNs then migrate to new positions in the protocerebrum. Another clock cell differentiates in the posterior protocerebrum. Each change occurs at a characteristic concentration of the ecdysteroid molting hormones that regulate metamorphosis. Clock cell axons invade the mushroom body and corpus allatum and travel down the ventral nerve cord. New overt rhythms develop during metamorphosis, in which these structures participate. The neuroendocrine cells of the brain receive more extensive branches of clock cell axons than in larvae. These increases in size and complexity of the circadian system during metamorphosis imply a greater complexity and diversity of outputs from it to both behavioral and hormonal rhythms in the adult.

OBJECTIVES To determine (i) whether peridomestic Attalea phalerata palms in fragmented human-occupied areas of the Alto Beni, Bolivia, are infested by triatomines; (ii) the specific status of triatomines captured in the area; and (iii) the rate of natural Trypanosoma cruzi infection among those triatomines. METHODS One hundred and twenty-five live-bait traps were used to sample 47 A. phalerata palms in three Alto Beni localities. Active search for vectors was also performed in 10 chicken coops and three rice storage units. Only Rhodnius specimens were found. As nymphs of closely related Rhodnius species are morphologically undistinguishable, and because of controversy in the literature regarding which Rhodnius species occur in Bolivia, collected insects were identified through molecular taxonomy. Phylogenetic analyses of DNA sequences obtained for a fragment of the mitochondrial cytochrome b gene and for the nuclear ITS-2 ribosomal region were used as molecular markers. Natural infection rates were determined using a pair of primers that PCR-amplify a 330-bp fragment of the parasite's kDNA. RESULTS Twelve nymphs were captured in five A. phalerata palms (from two of the three localities studied), and an adult was collected from a chicken coop in Iniqua (and morphologically identified as Rhodnius stali). All nymphs (as well as the adult) were molecularly identified as R. stali based on the two molecular markers used. A single nymph was found to be infected with T. cruzi. CONCLUSIONS Attalea phalerata palms represent an important sylvatic ecotope occupied by R. stali in the Alto Beni region of Bolivia, where there are signs of T. cruzi transmission to humans, despite the preliminary indication of low level of natural infection of the vectors.

Proctolin is a neuroactive pentapeptide first isolated from the cockroach Periplaneta americana in which it has an excitatory effect on contractions on visceral muscles of the hindgut. Subsequently, proctolin is reported in a wide variety of invertebrates, and considerable efforts have been made to determine its mode of action. Its primary role appears to be that of a neuromodulator rather than a classical neurotransmitter, and it may also serve as a neurohormone, depending on the muscles examined. The present study identifies the vagina muscles of the blood-sucking insect Rhodnius prolixus (Stal) as a proctolinergic system. Physiological doses of proctolin generate prolonged contractions that closely mimic the effects of motor nerve stimulation. This preparation is convenient and robust, warranting its use as an experimental system to further understand the role of proctolin in the regulation of muscle contractions in insects. Moreover, these muscles are innervated by an identifiable inhibitory component providing a means to investigate the interaction between proctolin excitation and neural inhibition.

We show a sensitive and straightforward off-line nano-LC-MALDI-MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF-amide-related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF-amide-related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin-like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C-terminal domain among insect neuropeptides identified so far.

Adult Rhodnius take smaller blood meals than do fifth stage larvae and excrete less fluid afterwards. The total amount of diuretic hormone and active precursors that can be extracted from their nervous system is also smaller. However, more diuretic hormone is released in adult than in fifth stage insects when the hormone release sites are depolarized for short periods with potassium-rich solutions. This shows that the adult can release diuretic hormone at a higher rate than can the fifth stage larva, a feature that can be correlated with the larger volume of haemo-lymph which the adult insect has to load with hormone in order to promote diuresis. In vivo, the concentration of diuretic hormone in the haemo-lymph of adult and fifth stage insects rises at the same rate. Detectable amounts of hormone are found in samples of haemolymph from insects which have been feeding for periods as short as 15 s.

To make reliable interpretations about evolutionary relationships between Trypanosoma rangeli lineages and their insect vectors (triatomine bugs of the genus Rhodnius) and, thus, about the determinant factors of lineage segregation within T. rangeli, we compared phylogenies of parasite isolates and vector species. Sixty-one T. rangeli isolates from invertebrate and vertebrate hosts were initially evaluated in terms of polymorphism of the spliced-leader gene (SL). Further analysis based on SL and SSUrRNA sequences from 33 selected isolates, representative of the overall phylogenetic diversity and geographical range of T. rangeli, supported four phylogenetic lineages within this species. By comparing the phylogeny of Rhodnius species with that inferred for T. rangeli isolates and through analysis of the geographical range of the isolates, we showed that there is a very significant overlap in the distribution of Rhodnius species and T. rangeli lineages. Congruence between phylogeographical analysis of both T. rangeli lineages and complexes of Rhodnius species are consistent with the hypothesis of a long coexistence of parasites and their vectors, with lineage divergence associated with sympatric species of Rhodnius apparently without association with particular vertebrate hosts. Separation of T. rangeli isolates from vectors of distinct complexes living in sympatry favours the absence of gene flow between the lineages and suggests evolution of T. rangeli lineages in independent transmission cycles, probably associated to specific Rhodnius spp. ecotopes. A polymerase chain reaction assay based on SL intergenic sequences was developed for simultaneous identification and lineage genotyping of T. rangeli in epidemiological surveys.

Lipophorin (Lp), either labeled in diacylglycerol moiety with [3H]-Palmitic acid or in phospholipid moiety with 32Pi, 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. Arch Insect Biochem Physiol 62:164–175, 2006. © 2006 Wiley-Liss, Inc.

Most Rhodnius species (Triatominae) are primarily associated with palm trees. They maintain enzootic Trypanosoma cruzi transmission and are responsible for human infection (causing Chagas disease) through the Neotropics. Assessing whether individual palm traits (ecological and/or botanical) may increase the risk of palm infestation by triatomines is relevant in areas where bugs invade houses flying from peridomestic palms. We developed a novel fieldwork approach with that objective, and applied it to study infestation by sylvatic Rhodnius ecuadoriensis in 110 tagua palms (Phytelephas aequatorialis). Palm infestation (23% overall) was non-randomly distributed in our sample. Palms located in anthropic landscapes were frequently infested (>27%, n=92), whereas no bugs were collected from palms surveyed within forest remnants (n=18; P=0.01). The presence of abundant decaying vegetable matter (P=0.001) and (to a lesser extent) epiphytic plants (P=0.049) on palm crowns and stems increased the probability of infestation and was positively correlated with the apparent density of bug colonies (R2=0.68). A trend towards higher infestation rates in male palms (34% vs. 18%) could relate to female palm management (removal of infrutescences and vegetable debris) in areas where palm seeds are harvested. An outline of 'risk palm ecotopes' and environmental management-based strategies for the control of peridomestic, palm tree-living vector populations are proposed.

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic beta-hematin (betaH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to betaH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.

Two sampling methods (manual capture and live-baited adhesive traps) were compared for collecting the bug Rhodnius neglectus Lent (Hemiptera: Reduviidae: Triatominae) from palm trees, Mauritia flexuosa L. (Arecaceae), in the savanna of Brasilia DF. R. neglectus was found in 19/50 (38%) of palm trees sampled. The detection rate was much higher by visual inspection and manual capture (18/50 = 36%) than by our trapping method (5/50 = 10%), although one tree was found to be positive by trapping but not by manual capture. Bugs collected manually were mostly (146/154 = 95%) found among the dead organic material in palm crowns. In combination, these sampling techniques are useful for quick detection of triatomine bug infestation in palm trees, especially in areas of high ecological value where the palms should not be cut and dissected, but arboreal Rhodnius are suspected to transmit enzootic Trypanosoma cruzi that might represent a risk of causing human cases of Chagas disease.

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.

Summary We report a systematic collection of Triatominae inside houses and in the peridomestic environment of Alto Beni, department of La Paz, Bolivia. This area is free of Triatoma infestans and although we detected previously seropositivity for Trypanosoma cruzi, the Alto Beni region is not officially considered as endemic for Chagas disease. From 11 houses of five localities, we collected adults, nymphs and eggs of a Rhodnius species, which was confirmed by morphological and morphometric analysis as Rhodnius stali. This little-known species has long been confused with R. pictipes, and was originally described from museum specimens labelled as R. pictipes. Our data show that R. stali is able to establish colonies in domestic and peridomestic habitats in Bolivia, and it is probably the vector responsible for Chagas disease seropositivity observed in the indigenous population of Alto Beni.

We present evidence for the putative role of Rhodnius robustus as extradomestic vector of Chagas disease in Western Venezuela. First, we assessed the validity of this triatomine species by genetic characterization in relation with some other species of the prolixus group. Random amplified polymorphic DNA data showed a clear separation between this species and R. prolixus and indicated a probable genetic heterogeneity within R. robustus. Faeces and gut contents were microscopically examined in 54 of 137 R. robustus collected in palm trees. According to this morphological examination, 18% were positive for Trypanosoma cruzi, 11% harboured T. rangeli and 11% showed mixed infection. Five of the seven samples examined gave a polymerase chain reaction major band of 270 bp specific of T. cruzi. The hybridization probes showed that R. robustus may transmit clones 20 and 39 (or genetically related ones) in Venezuela. Such a transmission might occur when, in absence of domestic R. prolixus and attracted by artificial light, R. robustus enters houses and feeds on humans, or when people are bitten outdoors. The lack of bugs inside houses could mean that the insects leave houses after feeding, or die without reproducing there.

Genetic relationships among 10 species of bugs belonging to the tribe Rhodniini (Hemiptera: Reduviidae), including some important vectors of Chagas disease, were inferred from allozyme analysis of 12 enzyme loci (out of 21 enzyme systems examined), using agarose gel electrophoresis. These species formed two clusters: one comprising Rhodnius brethesi, R. ecuadoriensis, R. pallescens and R. pictipes; the other with Psammolestes tertius, Rhodnius domesticus and the Rhodnius prolixus group comprising R. nasutus, R. neglectus, R. prolixus and R. robustus. The resulting tree was [((R. ecuadoriensis, R. pallescens) R. brethesi) R. pictipes], [R. domesticus (P. tertius [(R. nasutus, R. neglectus) (R. prolixus, R. robustus)])]. Rhodnius nasutus and R. neglectus differed by only one locus, whereas no diagnostic loci were detected between R. prolixus and R. robustus (22 loci were analysed for these four species), despite considerable DNA sequence divergence between species in each of these pairs. Allozymes of the R. prolixus group showed greater similarity with Psammolestes tertius than with other Rhodnius spp., indicating that Rhodnius is paraphyletic and might include Psammolestes.

The hemiptera Rhodnius prolixus is a blood-feeding insect and a primary vector of Trypanosoma cruzi, the etiological agent of the Chagas disease. Over the past century, Rhodnius has been the subject of intense investigations, which have contributed to unveil important aspects of metabolism and physiology in insects. Recent technological innovations are helping dissect the genetic and molecular underpinnings of Rhodnius embryogenesis and organogenesis, thus fostering the use of this important species in the fields of developmental and evolutionary biology. Rhodnius represents also an excellent system to study development under stressful conditions, since the embryo must develop in the presence of a large amount of blood-derived reactive oxygen species. With a recently sequenced genome, small among other Hemiptera, and the identification of basic elements for all classical development pathways, functional studies in this species are revealing novel aspects of insect development and evolution. Here we review early studies on this model insect and how this paved the way for recent functional studies using the kissing bug.

Knowledge of chemical ecology of haematophagous triatomine bugs (Hemiptera: Heteroptera: Reduviidae) could be of practical value because this polyphyletic subfamily includes species of medical importance, such as vectors of Trypanosoma cruzi Chagas (Kinetoplastida: Trypanosomatidae), the aetiological agent of Chagas disease. For particular species of Dipetalogaster, Panstrongylus, Rhodnius and Triatoma, therefore, we review information on exocrine glands and products, interpreting their apparent roles as semiochemicals in the aggregation, alarm, defence, host-finding and sexual behaviour of these Triatominae.

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517–518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.

An active corpus allatum (CA) in the blood feeding insect, Rhodnius prolixus, releases a substance that inhibits metamorphosis in larvae, and activates egg production in adults. In adults, transecting the nervus corporis cardiacum II's (NCCII), which are attached posteriorly to each protocerebral lobe, greatly increases egg production indicating that the adult CA is activated and receives neural inhibition from cells associated with the NCCII [Chiang, Arch. Insect. Biochem. Physiol. 39:126-131 (1998)]. In the present study, the NCCII's in fifth instar larvae were transected immediately before or after feeding to determine if these nerves normally inhibit CA activity in the last larval instar. Approximately 20 to 25 days following ingestion of a blood meal, L5's with transected NCCII's emerged as fully-formed adults with no larval characteristics. Examination of the brain in these recently emerged adults revealed that the NCCII's were absent. Since fifth instar larvae with transected NCCII's emerged with no juvenile characteristics, cutting the NCCII's did not activate the CA, indicating that the mechanism for inhibition of the CA differs in the last larval instar and adult animals.

Insect salt and water homeostasis is controlled by diuretic and antidiuretic hormones that can act upon the Malpighian tubules, midgut, and hindgut. Much is known about the physiological events stimulating diuresis and their signaling cascades; however, the factors inhibiting diuresis are less well known. An endogenous peptide in Rhodnius prolixus inhibits absorption by the anterior midgut and secretion from the Malpighian tubules. We have cloned the gene, sequenced this antidiuretic peptide, and examined gene expression via in situ hybridization. The antidiuretic peptide targets multiple tissues, and its discovery could facilitate the development of effective pest control agents to impede the transmission of Trypanosoma cruzi, the causative agent of Chagas' disease.

Expression within insects of foreign antiparasitic gene products via microbial symbionts could be used to prevent transmission of vector-borne pathogens to vertebrate hosts. Genetically transformed symbiotic bacteria Rhodococcus rhodnii expressed functional antibody fragments (rDB3 encoding murine V(H)/K which binds progesterone) that were exported into the gut lumen of the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae), a vector of Chagas disease. Transgenic symbionts were maintained in successive nymphal instars and adults of Rhodnius prolixus despite competition with native untransformed Rhodococcus rhodnii. This is the first description of a functional mammalian antibody fragment expressed in an insect. Our system is a model for constructing paratransgenic insects (insects carrying transformed symbionts) with compromised ability to transmit pathogens.

The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from oocyte was labeled with 125 I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [ 125 I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [ 125 I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [ 125 I]RHBP was linear at least up to 60 min. The uptake was dependent on [ 125 I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [ 125 I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed. Arch. Insect Biochem. Physiol. 39:133‐143, 1998. © 1998 Wiley-Liss, Inc.

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations od lipoproteins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophorin of different densities.

Rhodnius prolixus, a blood-sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132,331 Da. The primary structures of mammalian NOS, including the putative cofactor-recognition sites for heme, tetrahydrobiopterin (BH4), calmodulin. FMN, FAD and NADPH are all conserved in salivary-gland NOS. Recombinant salivary-gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N-terminal domain and an N-terminal myristylation sequence, respectively. Salivary-gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary-gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary-gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.

Applied topically to larvae of Rhodnius prolixus, Triatoma infestans, Triatoma Vitticeps, Panstrongylus herrera and Panstrongylus megistus a novel, synthetic, furan-containing anti-juvenile hormonal compound, 2-(2-ethoxyethoxy)ethyl furfuryl ether (EEFE) induced a variety of biological responses attributable to an enforced cessation of juvenile hormone secretion, including precocious metamorphosis into diminutive adultoids and retardation of molting. The highest percentage of precocious adultoids was obtained when EEFE was applied prior to feeding. In decreasing order of sensitivity to this anti-juvenile hormone (AJH) were T. infestans, R. prolixus, P. herrera, P. megistus and T. vitticeps. The ED 50 of EEFE in R. prolixus compared well with that of the botanically derived AJH, precocene II, but T. infestans was significantly more sensitive to the furanyl antihormone than to precocene II. The significance of these findings is discussed in terms of the complexity of expression of the anti-juvenile hormonal activity during the post-embryonic development of triatomines.

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite.

32P-Labelled midguts (32P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-midguts were incubated in the absence of lipophorin, no 32P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of 32P-phospholipids derived from metabolically labelled 32P-midgut or lipophorin particles after incubation with 32P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of 32P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of 32P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A2 was incubated with 32P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.

Myogenic contractions of the heart of the female blood-feeding insect, Rhodnius prolixus (Stal), are inhibited by crude extracts of testes applied directly to isolated dorsal vessels. Dorsal vessels were observed with a stereo microscope and heart beats timed with a stopwatch. In normal Rhodnius saline, hearts contract at 14.8 ± 7.1 beats per minute (n= 45). Crude extracts of the testes and the two male reproductive accessory organs (the opaque and transparent accessory glands) were prepared from previously frozen tissue by homogenizing 5–20 glands in a small glass homogenizer containing Rhodnius saline, centrifuging for 5 min at 2 000 g, and collecting the supernatant. Testes extract as low as 1.0 glands per mL inhibit contractions whereas crude extracts of the opaque or transparent accessory glands have no consistent effect. We refer to this cardiac inhibitor as rhodtestolin (Rhodnius testis inhibitory factor), and discuss its possible effects on the female during copulation.

An organism's saliva has a direct “intimacy” with the environment. Saliva is an oral secretion from salivary glands that can be a watery or an oily matrix of substances that interfaces with the outside world. Insect saliva has numerous functions and properties such as digestion, maintenance of mouth parts, water balance, pathogen transmission, acts as a vehicle for pheromones, provides anti-predator defenses, has anti-microbial activity, and can aid in circumventing animal-host defenses and plant-host defenses. This Special Issue of Archives of Insect Biochemistry and Physiology is based on papers presented during a symposium held at the 51st Annual Meeting of the Entomological Society of America, October 2003. The symposium was entitled “Insect Saliva: An Integrative Approach”. The goal of the symposium was to foster discussion among scientists who study insect saliva in relation to animal and plant hosts. The presentations and thus this Special Issue were heavily focused on the function of insect saliva in relation to the interactions of the insect and their host. All articles in this Issue have undergone a rigorous review process. This special Insect Saliva edition nicely complements the symposium presentations. James Tumlinson's and Cameron Lait's article discusses the biosynthesis of fatty acid amide elicitors of plant defenses. These elictors are synthesized by enzymes embedded in the membranes of the insect's crop and anterior midgut. John Labavitch, Kenneth Shackel, Elaine Backus, and colleagues provide a nice contribution showing that much of plant damage by Lygus is due to polygalacturonase activity found in the saliva. Cesar Rodriguez-Saona, Livy Williams, and colleagues contribute an article that investigates volatile emissions of plants responding to piercing-sucking herbivores. A report by John Andersen and colleagues provides a look at the role of the salivary protein family, lipocalin, found in the blood-sucking bug Rhodnius prolixus. Gary Felton and colleague demonstrate in their study that glucose oxidase levels in Helicoverpa zea are altered due to the host plant that the caterpillar feeds on. Ken Korth, Susana Gomez, and colleagues show that terpene synthases, which form the important terpene plant defenses, are elicited in Medicago truncatula by oral secretions of the caterpillar Spodoptera exigua. Richard O. Musser, Gary Felton, and colleagues provide two contributions demonstrating that glucose oxidase has two functions that increase the survival of the caterpillar Helicoverpa zea. The first report further confirms that glucose oxidase is the primary salivary factor to suppress the induction of the anti-herbivore defense, nicotine, in tobacco plants. The second report shows that the salivary enzyme has anti-microbial properties that suppress the infectivity of potential bacterial pathogens. Insect saliva holds exciting potential to better understand the co-evolution of insect-host interactions. Studying the complex properties and functions of insect saliva will require integrative and collaborative effort of scientists specializing in various areas.

Stimulation of left atrial receptors causes a diuresis partly through a blood borne agent. Malpighian tubules of Rhodnius prolixus can detect a blood borne agent during the diuresis. It has been suggested that the agent is ADH but this is in dispute. In the present investigation, vasopressin (1-300 mu i.u. cm-3) was found to have no effect on Malpighian tubule secretion when added to plasma, Rhodnius Ringer and Rhodnius Ringer + 5-HT. In addition, it was shown that incubation with sodium thioglycollate of plasma samples obtained from anaesthetized dogs, before and during stimulation of atrial receptors by distension of a balloon in the lumen of the left atrium, did not abolish the differences between test and control plasma samples detected by the Malpighian tubules. It is concluded that differences in plasma detected by the Malpighian tubules and related to the diuresis which results from left atrial stimulation are not attributable to changes in concentration of ADH.

We describe, for the first time in insects, the presence of serotonin-like immunoreactive dorsal unpaired median (DUM) neurons. In unfed, untreated Rhodnius prolixus, the cell bodies of these DUM neurons displayed only faint serotonin-like immunofluorescence without any detectable fluorescence in the axons. There was, however, an extensive serotonin-like immunoreactive peripheral complex. We have enhanced the immunostaining of the cell bodies and axons of these DUM neurons by using 5,7-dihydroxytryptamine (5,7-DHT) and nerve transection, and have further defined their morphology with cobalt backfilling and Lucifer yellow injection. Injection of 5,7-DHT resulted in enhanced serotonin-like immunostaining, after 24 hours, of five DUM neurons in the mesothoracic ganglionic mass. Each DUM neuron possessed bifurcating serotonin-like immunoreactive axons projecting to the periphery via one of the five bilaterally paired abdominal nerves. Concomitant with the enhanced immunostaining in the cell body and axons of these DUM neurons was the depletion of a serotonin-like immunoreactive neurohaemal area lying on each of the abdominal nerves and the depletion of serotonin-like immunoreactive processes on the body wall. Enhancement of cell body and axon immunostaining was also observed in preparations in which the abdominal nerves had been transected 24 hours previously. The location and morphology of these DUM neurons were studied in further detail by using cobalt backfilling of the abdominal nerves and intracellular injection of Lucifer yellow followed by immunohistochemistry. The five serotonin-like immunoreactive DUM neurons were found to represent a sub-population in a group of ten DUM neurons located in the mesothoracic ganglionic mass and associated with the abdominal nerves. Each abdominal nerve received projections from two DUM neurons, only one of which was serotonin-like immunoreactive. Electrophysiological recordings revealed that the serotonin-like immunoreactive DUM neurons of Rhodnius had similar properties to previously described octopaminergic DUM neurons of insects. The five DUM neurons in Rhodnius appear to represent a subpopulation of DUM neurons containing serotonin.

The paired spermathecae of Rhodnius are simple tubular out-pocketings of the common oviduct. Each consists of a short muscular proximal duct and the distal glandular region with a blind tapering end. The spermathecal wall has a cuticular intima, slender columnar epithelial cells and ensheathing longitudinal striated muscle, connective tissue, tracheoles, and nerves. Glandular epithelial cells possess an elaborate apical secretion-filled tubular inpocketing with an extensively folded plasma membrane. Laterally, cells interact by desmosomes, septate desmosomes, and extensive interdigitations. The cytoplasm is rich in longitudinally oriented microtubules associating with membrane densities along the invagination, lateral, and basal plasmalemmae. Apical concentration of mitochondria suggests their role in secretion or ion transport. The possible role of the spermathecae in maintaining the stored luminal sperm and its role in transmitting the mating stimulus is considered in light of the epithelial ultrastructure. The ultrastructure of the spermathecae of Rhodnius differs significantly from that of other insects.

The diuretic response to stimulation of left atrial receptors, by distending balloons located either at the pulmonary vein-left atrial junctions or in the body of the left atrium, was studied during blockade of the generation of angiotensin II using a continuous infusion of angiotensin converting enzyme inhibitor, SQ 14,225, in anaesthetized dogs. Despite blockade of angiotensin conversion, known also to potentiate the activity of bradykinin, a diuretic response was obtained in each dog; the response was similar to that observed in dogs without blockade of angiotensin conversion. Angiotensin II and bradykinin did not significantly influence the rate of secretion of Malpighian tubules of Rhodnius prolixus, which have been shown capable of detecting the blood-borne agent known to mediate the diuretic response. It is concluded that neither angiotensin II nor bradykinin is likely to be the blood-borne agent released by stimulation of atrial receptors.

Rhodnius prolixus nymphs fed 7-ethoxy-6-methoxy-2,2-dimethylchromene (ethoxyprecocene II, EPII) show a variety of responses, including precocious molting to diminutive adults, severe retardation of molting, or a condition of permanent ecdysial stasis. The latter two conditions are reversible by subsequent treatment with 20-hydroxyecdysone. Ecdysteroid titers in the hemolymph of individual insects, determined by radioimmunoassay (RIA), show that the ecdysteroid cycle in nymphs undergoing precocious metamorphosis is similar to that of untreated fifth stage nymphs during normal imaginal molting. Nymphs in ecdysial stasis, following EPII treatment, were found to have very low ecdysteroid titers. Analysis of ecdysteroid synthesis by the prothoracic glands (PG), cultured in vitro, showed that: 1) only traces of ecdysteroid were detectable in PG from nymphs treated in vivo with EPII; 2) the PG from untreated nymphs incubated in culture medium with EPII possessed significantly lower ecdysteroid synthesis compared with controls. These studies sought to determine if the inhibition of ecdysteroid biosynthesis observed in Rhodnius, following exposure to EPII in vivo and in vitro, is due to a direct action on the PG or result as an indirect effect perhaps mediated by the neuroendocrine system.

Trypanosoma (Herpetosoma) tamiasi sp. n. is described from blood and organs of the eastern chipmunk, Tamias striatus, and the least chipmunk, Eutamias minimus. In experimentally infected Tamias striatus and E. minimus, Trypanosoma tamiasi reproduced by equal binary fission in the trypo-, sphaero-, epi-, or amastigote form, mainly in lymphoid organs. Trypomastigotes developed from amastigotes through epimastigotes, or from sphaeromastigotes, mainly in the same organs. The mean lengths of body and flagellum of trypomastigotes increased linearly until the organisms reached a maximum length of similar to 37-39 mum. The distance between the nucleus and kinetoplast increased to a mean total length of similar to 33 mum, and thereafter remained constant. Parasitemias of up to 2.5 times 10-7 trypanosomes/ml were produced in Tamias striatus by inoculation with bloodstream, culture, or flea gut forms of Trypanosoma tamiasi; natural parasitemias in Tamias striatus peaked at up to 2.1 times 10-7 trypanosomes/ml. One E. minimus and 2 Spermophilus tridecemlineatus developed parasitemias of more than 1 times 10-6 trypanosomes/ml after inoculation of bloodstream forms of Trypanosoma tamiasi; other experimentally inoculated rodents, including mice, rats, and Spermophilus richardsonii, proved refractory. In chipmunk fleas, Megabothris acerbus, Megabothris quirini, and Tamiophila grandis, bloodstream forms of Trypanosoma tamiasi developed to metacyclic forms in 4-9 days; in rat fleas, Xenopsylla cheopis and Nosopsyllus fasciatus, they survived with little development for up to 2 days, and in kissing bugs, Rhodnius prolixus, up to 10 days. Apparently, the speciation of this trypanosome involved adaptation to certain species of both fleas and rodents.

Metacyclic forms of Trypanosoma cruzi isolated from the hindgut of infected insect vectors (Rhodnius prolixus) were found to be immunologically cross-reactive with cultured epimastigote, amastigote, and metacyclic stages of the parasite as well as with bloodstream trypomastigote forms by direct agglutination and indirect immunofluorescence techniques. Sera specific for each of these forms of the parasite systematically yielded maximal antibody titers when measured against the homologous antigen, indicating that antigenic determinants are shared by all of the developmental forms used in this work. Supporting this conclusion were the significant reductions in anti-insect-derived metacyclic antibody titer caused by absorption with any of the other life stages of T. cruzi. These results are relevant to the potential use of laboratory-grown forms of T. cruzi in vaccination against a natural infection with this parasite.