Morphological and ultrastructural analysis of an important place of sexual communication of Rhodnius prolixus (Heteroptera: Reduviidae): the Metasternal Glands.

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the etiological agent of Chagas disease. Insect adults have a pair of Metasternal Glands (MGs) and the secretion emitted by these glands acts as sex pheromone. Recent studies have focused on the chemical composition of this pheromone, electrophysiological responses to MGs compounds and mating behavior assays. Morphological studies of these glands are still scarce. Thus, considering the relevance of MGs in the sex pheromone biosynthesis, we investigated the morphology and ultrastructure of R. prolixus MGs. The glandular apparatus presents a tubular structure containing secretory cells with canalicules that fuse with the central duct which conducts the secretion to a pear-shaped reservoir connected to the exterior by a droplet-shape orifice. The secretory cells are classified as class III, they present a well-developed rough and smooth endoplasmic reticulum. Smooth endoplasmic reticulum is a site of lipid biosynthesis that may be involved in the mevalonate pathway, a probable route of the sex pheromone biosynthesis in this insect. The presence of rough endoplasmic reticulum indicates a possible peptides/proteins secretions site which were still not characterized in MGs. Several mitochondria are scattered in the cytoplasm that may suggest a high metabolic activity. Further studies should be carried out to correlate these data with the sex pheromone biosynthesis in this vector.

Neuropeptide- and serotonin- cells in the brain of Rhodnius prolixus (Hemiptera) associated with the circadian clock.

The neuronal pathways of the circadian clock in the brain of R. prolixus have been described in detail previously, but there is no information concerning the cells or their pathways which relay either inputs to the clock (e.g. for light entrainment), or outputs from it to driven rhythms. Here, we employ antisera to three neuropeptides (type A allatostatin-7, crustacean cardioactive peptide and FMRFamide), and serotonin in confocal laser scanning immunohistochemistry to analyze the distribution of cell bodies and their projections in relation to the principle circadian clock cells (lateral cells, LNs) for all four neuron types. LNs are revealed following labelling with anti- pigment dispersing factor in double labelled preparations. Regions of potential communication between ramifications of the LNs and each of the four other neuron types is described (identified by close superposition of their neurites in various brain regions), as is their detailed projections within the brain. Neuromodulation is sometimes suggested by close, but not intimate, proximity of varicosities of neurites. We infer that some neuron types comprise input pathways to the LNs, some are outputs to neuroendocrine or behavioral rhythms, and others participate in both input and output pathways, sometimes by the same neuron type but in different locations. For example, one retinula cell in each ommatidium is immunoreactive for allatostatin A; its axon projects to the medulla making superpositions with LNs, as do serotonin cells in the optic lobe, indicating roles of both neuron types in light input (entrainment) to the clock. But in other brain areas, these same types appear to mediate outputs from the clock. The accessory medulla has been widely reported as the principle center of integration in other insects; but we found sparse evidence of this in R. prolixus as it contains few neurites other than those from the clock cells. Rather, the importance of neural pathways involving the medulla and the superior protocerebrum is emphasized. We conclude that there is a vast and complex web of interactions in the brain with the LNs, which potentially receive multiple pathways of inputs and outputs that could drive rhythmicity in a multitude of downstream cells, rendering a host of output pathways rhythmic, notably hormone release from neurosecretory cells and behaviors.

The modern morphometric approach to identify eggs of Triatominae.

BACKGROUND: Egg morphometrics in the Triatominae has proved to be informative for distinguishing tribes or genera, and has been based generally on traditional morphometrics. However, more resolution is required, allowing species or even population recognition, because the presence of eggs in the domicile could be related to the species ability to colonize human dwellings, suggesting its importance as a vector. RESULTS: We explored the resolution of modern morphometric methods to distinguish not only tribes and genera, but also species or geographic populations in some important Triatominae. Four species were considered, representing two tribes and three genera: Panstrongylus chinai and P. howardi, Triatoma carrioni and Rhodnius ecuadoriensis. Within R. ecuadoriensis, two geographical populations of Ecuador were compared. For these comparisons, we selected the most suitable day of egg development, as well as the possible best position of the egg for data capture. The shape of the eggs in the Triatominae does not offer true anatomical landmarks as the ones used in landmark-based morphometrics, except for the egg cap, especially in eggs with an evident "neck", such as those of the Rhodniini. To capture the operculum shape variation, we used the landmark- and semilandmark-based method. The results obtained from the metric properties of the operculum were compared with the ones provided by the simple contour of the whole egg, as analyzed by the Elliptic Fourier Analysis. Clear differences could be disclosed between the genera, between the species - among which two very close species (P. chinai and P. howardi), as well as between two allopatric, conspecific populations. The whole egg contour (including the operculum) produced reclassification scores much more satisfactory than the ones obtained using the operculum only. CONCLUSIONS: We propose the outline-based approach as the most convenient characterization tool to identify unknown eggs at the species or population levels.

Structure and postembryonic development of the intersegmental nodules in the non-muscular joints of the antennae in Rhodnius prolixus.

The antennae of Insecta consist of two basal segments and the distal annulated flagellum lacking intrinsic muscles. Non-muscular joints are important to preserve the flexibility and structure of the long heteropteran antennae which bear an intersegmental nodule on each non-muscular joint. Little is known about their properties or function. Here we characterize the structure and postembryonic development of the non-muscular joints of Rhodnius prolixus antennae. Using Scanning Electron Microscopy, we tracked the changes in shape and size of both intersegmental nodules during the course of the hemimetabolous insect life cycle. Using Atomic Force Microscopy, we established a qualitative correlation between the topography of the surface and the rigidity of the joint between pedicel and flagellum. Our results confirmed the presence of two sub-articulations on each non-muscular joint. Also, the two intersegmental nodules have different origins: the one between the two flagellar segments (intraflagelloid) is a sclerite already present from the early nymph, while the nodule between pedicel and flagellum (prebasiflagellite) originates by gradual separation of the proximal end of the basiflagellum during postembryonic development. Various changes occur in the non-muscular joints and segments of the antenna during the life cycle of R. prolixus.

Presence of chromatoid bodies in the Rhodnius genus detected by cytochemical analysis.

In addition to the epidemiological importance regarding the transmission of Chagas disease, triatomines are also important biological models for cellular studies, because they have holocentric chromosomes, post-reductional meiosis for sex chromosomes, and nucleolar persistence. Although the nucleolus is present during spermiogenesis, it was suggested that it is inactivated and all transcriptional activity necessary for cell differentiation is supported by the chromatoid body (CB). Thus, considering the importance of CB to triatomine spermatogenesis, this paper aims to analyze the presence of this organelle in the Rhodnius genus. The testicles of five adult R. neglectus and R. prolixus males were analyzed after semi-fine sections were performed, and the material was subjected to silver ion impregnation. The CB organelle was found in the spermatid of both species. Most CB studies focused on the Triatoma genus. This study described the existence of CB in Rhodnius, confirming thus the importance of this cytoplasmic organelle to spermiogenesis of these insect vectors of Chagas disease.

Nucleolar persistence during spermatogenesis of the genus Rhodnius (Hemiptera, Triatominae).

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.

Epimastigogénesis de Trypanosoma cruzi: método para estudiar in vivo la transformación de tripomastigota en epimastigota / Trypanosoma cruzi epimastigogenesis: a method for studying Trypomastigote to epimastigote transformation in vivo

La epimastigogénesis de Trypanosoma cruzi ocurre naturalmente en el intestino del hospedador invertebrado. Se desconoce si los cambios morfológicos que ocurren durante la transformación de los tripomastigotas sanguíneos en epimastigotas son idénticos para diferentes aislados de T. cruzi. Aquí mostramos un método útil para estudiar los eventos que ocurren durante la epimastigogénesis, comparando parásitos de diferentes procedencias epidemiológicas. Se alimentaron artificialmente ninfas de V estadio de Rhodnius prolixus con una solución ad hoc conteniendo tripomastigotas tipo-sanguíneo y siguiendo los cambios morfológicos por 8 días. Los contenidos del intestino anterior de las ninfas se obtuvieron decapitando y comprimiendo el abdomen lo que permitió obtener rápidamente casi 100% del inóculo con cargas entre 1,9 y 8,9 × 106 tripomastigotas/ninfa. El número de parásitos por ninfa y los cambios morfológicos se determinaron por Microscopia de Contraste de Fases y coloración con Giemsa. Las ninfas ingirieron 7,4 veces su peso de solución infectante, con volúmenes entre 101 y 357 µL (229 ± 66 mg), 50% de esa ingesta se eliminó como orina durante las primeras 24 h. Los tripomastigotas se transformaron en formas redondeadas antes de evolucionar a epimastigotas, siguiendo cinéticas diferentes según el aislado. Proponemos esta metodología para estudiar rápida y cuantitativamente los eventos tempranos de la epimastigogénesis de T. cruzi in vivo.

Trypanosoma cruzi epimastigogenesis naturally occurs in the intestine of the invertebrate host. It is not known whether the morphological changes that occur during transformation of bloodstream trypomastigotes to epimastigotes are identical for different T. cruzi isolates. This research shows a useful method for studying the events that occur during epimastigogenesis, comparing parasites from epidemiological sources. Rhodnius prolixus V stage nymphs were fed artificially with an ad hoc solution containing blood-like trypomastigotes and the morphological changes were examined during eight days. Anterior intestinal contents were removed by decapitation and squeezing the abdomen of the nymphs, which permitted obtaining quickly almost 100% of the inoculate with loads between 1.9 and 8.9 × 106 trypomastigotes/nymph. The number of parasites per nymph and morphological changes were determined using phase microscopy with Giemsa staining. The nymphs ingested 7.4 times their weight of the infecting solution with volumes between 101 e 357 µL (229 ± 66 mg); 50% of the ingest was eliminated as urine during the first 24 hours. The trypomastigotes transformed to rounded forms before evolving into epimastigotes following different kinetics according to the isolate. This method is proposed for rapid, quantitative study of the early events of epimastigogenesis for T. cruzi in vivo.

Metamorphosis of a clock: remodeling of the circadian timing system in the brain of Rhodnius prolixus (Hemiptera) during larval-adult development.

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.

Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl.

BACKGROUND: The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus. METHODOLOGY/PRINCIPAL FINDINGS: Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H(+)-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs. CONCLUSIONS: We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.

Involvement of sulfated glycosaminoglycans on the development and attachment of Trypanosoma cruzi to the luminal midgut surface in the vector, Rhodnius prolixus.

In the present study, we investigated the involvement of sulfated glycosaminoglycans in both the in vivo development and adhesion of T. cruzi epimastigotes to the luminal surface of the digestive tract of the insect vector, Rhodnius prolixus. Pre-incubation of T. cruzi, Dm 28c epimastigotes with heparin, chondroitin 4-sulfate, chondroitin 6-sulfate or protamine chloridrate inhibited in vitro attachment of parasites to the insect midgut. Enzymatic removal of heparan sulfate moieties by heparinase I or of chondroitin sulfate moieties by chondroitinase AC from the insect posterior midgut abolished epimastigote attachment in vitro. These treatments also reduced the labelling of anionic sites exposed at the luminal surface of the perimicrovillar membranes in the triatomine midgut epithelial cells. Inclusion of chondroitin 4-sulfate or chondroitin 6-sulfate and to a lesser extent, heparin, in the T. cruzi-infected bloodmeal inhibited the establishment of parasites in R. prolixus. These observations indicate that sulfated glycosaminoglycans are one of the determinants for both adhesion of the T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine and the parasite infection in the insect vector, R. prolixus.

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of Rhodnius prolixus Stahl.

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18-24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.

Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae) salivary gland cells.

In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae) salivary gland cells

In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

Cytochemical characterization of microvillar and perimicrovillar membranes in the posterior midgut epithelium of Rhodnius prolixus.

Perimicrovillar membranes (PMM) are structures present on the surface of midgut epithelial cells of the hematophagous insect, Rhodnius prolixus. They cover the microvilli and are especially evident 10 days after blood meal, providing the compartmentalization of the enzymatic processes in the intestinal microenvironment. Using an enzyme cytochemical approach, Mg2+-ATPase and ouabain-sensitive Na+K+-ATPase activities were observed in the plasma (or microvillar) membrane (MM) of midgut cells and in the PMM. In contrast, alkaline phosphatase was only detected in MM. Using cationized ferritin and colloidal iron hydroxide particles, anionic sites were found only on the luminal surface of the PMM. Using fluorescein isothiocyanate (FITC)-labeled lectins, residues of alpha-d-galactose, mannose, N-acetyl-neuraminic acid, N-acetyl-d-galactosamine and N-acetyl-galactosamine-alpha-1,3-galactose were detected on the apical surface of posterior midgut epithelial cells. On the other hand, using FITC-labeled neoglycoproteins (NGP) it was possible to detect the presence of carbohydrate binding molecules (CBM) recognizing N-acetyl-d-galactosamine, alpha-d-mannose, alpha-l-fucose and alpha-d-glucose in the posterior midgut epithelium. The use of digitonin showed the presence of sterols in the MM and PMM. These results have led the authors to suggest that for some components the PMM resembles the MM lining the midgut cells of R. prolixus, composing a system which covers the microvilli and stretches to the luminal space.

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities.

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixus Stahl.

This study examined the process of membrane fusion of yolk granules (YGs) during early embryogenesis of Rhodnius prolixus. We show that eggs collected at days 0 and 3 after oviposition contain different populations of YGs, for example day-3 eggs are enriched in large YGs (LYGs). Day-3 eggs also contain the highest free [Ca(2+)] during early embryogenesis of this insect. In vitro incubations of day-0 YGs with [Ca(2+)] similar to those found in day-3 eggs resulted in the formation of LYGs, as observed in vivo. Fractionation of LYGs and small YGs (SYGs) and their subsequent incubation with the fluorescent membrane marker PKH67 showed a calcium-dependent transference of fluorescence from SYGs to LYGs, possibly as the result of membrane fusion. Acid phosphatase and H(+)-PPase activities were remarkably increased in day-3 LYGs and in calcium-treated day-0 LYGs. Both fractions were found to contain vitellins as major components, and incubation of YGs with calcium induced yolk proteolysis in vitro. Altogether, our results suggest that calcium-induced membrane fusion events take part in yolk degradation, leading to the assembly of the yolk mobilization machinery.

Inhibition of hemocyte microaggregation reactions in Rhodnius prolixus larvae orally infected with Trypanosoma rangeli.

Hemocoelic inoculation of epimastigotes of Trypanosoma rangeli strain H14 into 5th-instar larvae of Rhodnius prolixus previously fed on blood containing the same parasites, showed reduced number of hemocyte microaggregates in the hemolymph, enhanced number of flagellates in the hemolymph as well as increased mortality of these insects. All these effects were counteracted by combined inoculation of R. prolixus with T. rangeli and arachidonic acid. In vitro assays using hemolymph taken from insects previously fed on blood containing parasites showed that hemocyte microaggregation reactions were also attenuated when T. rangeli is used as inducer of the reaction, and that simultaneous applying T. rangeli with arachidonic counteracted the hemocyte microaggregation inhibition. We suggest that arachidonic acid pathway can be a mediator of hemocyte microaggregation reactions in the hemolymph of insects inoculated with T. rangeli, and that oral infection with this protozoan inhibits the release of arachidonic acid.

The distribution of a CRF-like diuretic peptide in the blood-feeding bug Rhodnius prolixus.

The blood-feeding bug Rhodnius prolixus ingests a large blood meal, and this is followed by a rapid diuresis to eliminate excess water and salt. Previous studies have demonstrated that serotonin and an unidentified peptide act as diuretic factors. In other insects, members of the corticotropin-releasing factor (CRF)-related peptide family have been shown to play a role in post-feeding diuresis. Using fluorescence immunohistochemistry and immunogold labelling with antibodies to the Locusta CRF-like diuretic hormone (Locusta-DH) and serotonin, we have mapped the distribution of neurones displaying these phenotypes in R. prolixus. Strong Locusta-DH-like immunoreactivity was found in numerous neurones of the central nervous system (CNS) and, in particular, in medial neurosecretory cells of the brain and in posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM). Positively stained neurohaemal areas were found associated with the corpus cardiacum (CC) and on abdominal nerves 1 and 2. In addition, Locusta-DH-like immunoreactive nerve processes were found over the posterior midgut and hindgut. Double-labelling studies for Locusta-DH-like and serotonin-like immunoreactivity demonstrated some co-localisation in the CNS; however, no co-localisation was found in the medial neurosecretory cells of the brain, the posterior lateral neurosecretory cells of the MTGM or neurohaemal areas. To confirm the presence of a diuretic factor in the CC and abdominal nerves, extracts were tested in Malpighian tubule secretion assays and cyclic AMP assays. Extracts of the CC and abdominal nerves caused an increase in the rate of secretion and an increase in the level of cyclic AMP in the Malpighian tubules of fifth-instar R. prolixus. The presence of the peptide in neurohaemal terminals of the CC and abdominal nerves that are distinct from serotonin-containing terminals indicates that the peptide is capable of being released into the haemolymph and that this release can be independent of the release of serotonin.

A subpopulation of dorsal unpaired median neurons in the blood-feeding insect Rhodnius prolixus displays serotonin-like immunoreactivity.

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 subpopulation 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.

Endpolyploidy and digestion in the midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae).

The Feulgen-DNA content of the midgut cell nuclei of Rhodnius prolixus is estimated by microspectrophotometry. Midgut cells contain 23.35-32.16 pg DNA/nucleus, which represents an increase of up to 26c when compared to diploid R. prolixus brain tissue. A high copy number cell population disappears from the anterior intestinal cells after feeding, and the presence of a new population of low copy number cells indicates that there may be some regeneration of the midgut epithelium in adult insects, or that non-mitotic division of the high ploidy cells occurs. Polyploidy of the midgut cells is thought to be important in the rapid post-feeding synthesis of digestive proteinases, whereby enzymes are produced without the immediate need for DNA synthesis.