Cytoskeleton elements mediate the inhibition of the (Na++K+)atpase activity by PKC in Rhodnius prolixus malpighian tubules during hyperosmotic shock

In a previous paper, we observed that the specific activity of (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus is inhibited by protein kinase C (PKC) during hyperosmotic shock [Arenstein et al., J Membr Biol 146:47-57 [1995]; Caruso-Neves et al., Z Naturforsch 53c:911-917 [1998]). In the present paper, we study the involvement of the cytoskeleton in this process using isolated Malpighian tubules of Rhodnius prolixus. We observed that pre-incubation of the Malpighian tubule cells in hyperosmotic media decreases the specific activity of (Na++K+)ATPase by 90%. This effect was completely reversed when colchicine, which disrupts microtubules, or cytochalasin B, an inhibitor of actin microfilament polymerization, were added to the media in a dose-dependent manner. The maximal reversion was obtained with colchicine 7.0 microM or cytochalasin B 5.0 microM. The simultaneous addition of sphingosine 50 ng/mL, an inhibitor of PKC, to 10 microM colchicine or 5 microM cytochalasin B, in hyperosmotic media, did not change the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase. On the other hand, the co-incubation of TPA 20 ng/mL, an activator of PKC, to colchicine or cytochalasin B within hyperosmotic media, abolished the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase to a similar extent as hyperosmotic shock. These results suggest that inhibition of the (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus by PKC during hyperosmotic shock is mediated by cytoskeletal elements.     

Inhibition of the SAPK/JNK pathway blocks the stimulatory effects of glutamine on fluid secretion by the Malpighian tubules of Rhodnius prolixus

Physiological levels of amino acids such as glutamine, glutamate, aspartate and proline increase the rates of fluid secretion and ion transport by serotonin-stimulated Malpighian tubules (MTs) of Rhodnius prolixus. Here, we examine the proposal that the effects of glutamine are mediated through activation of specific kinases to produce the observed increases in fluid secretion. The glutamine-dependent increase in MT fluid secretion rate was blocked by two chemically unrelated inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumarol. Inhibitors of phosphatidyl inositol-3 kinase, p38 mitogen activated protein kinase (MAPK), extracellular-signal regulated kinases and MAPK kinase did not block glutamine's effects on fluid secretion rate when applied at commonly used concentrations. Inhibitors of protein kinase A or C reduced fluid secretion rates of serotonin-stimulated MTs, but did not block the response to glutamine. The glutamine-dependent increase in fluid secretion was also insensitive to cytoskeletal disrupting agents and protein synthesis inhibitors. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect MTs.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K⁺ or Cl^? or HCO ₃ ^? by Na⁺, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na⁺ by tetramethylammonium (TMA⁺) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10^?4 m, anthracene-9-COOH 5×10^?4 m, furosemide 5×10^?4 m or ethacrynic acid 5×10^?5 m, also abolished RVI. On the other hand, addition of either Ba²⁺ 10^?3 m, SITS 5× 10^?4 m, ouabain 10^?3 m or vanadate 10^?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10^?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na⁺, K⁺, Cl^?, Ca²⁺ and HCO ₃ ^? transport pathways and a ouabain-insensitive ATPase stimulated by Na⁺. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.     

The paracellular channel for water secretion in the upper segment of the Malpighian Tubule of Rhodnius prolixus

Lumen to bath J ₁₂/C ₁ and bath to lumen J ₂₁/ C ₂ fluxes per unit concentration of 19 probes with diameters (d ^m) ranging from 3.0–30.0 Å (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with d ^m 9.1–30.0 Å) were measured during volume secretion (J _v ) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with ¹⁴C or ³H-labeled probes. J _net=(J ₁₂/C ₁ – J ₂₁/C ₂) was studied as a function of J _v · J _v was varied by using different concentrations of 5-hydroxy tryptamine. J _net for ³H-water was not different from J _v We found: (i) A strong correlation between J _net and J _v for 8 probes d ^m =3.0–11.8 Å (group a probes), indicating that the convective component of J _net is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium, (ii) There is no correlation between J _net and J _v for 11 probes with d ^m=11.8–30 Å (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag, (iii) In a plot of J _net/J _v vs. d ^m group a probes show a steep linear relation with a slope = –0.111, while for group b probes the slope is –0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 Å-wide slit, (ii) Most of J _v must follow an extracellular noncytosolic pathway, (iii) Group b probes must diffuse through a 42 Å-wide slit, (iv) A cylindrical pathway does not fit the data.E.G. is a Visiting Scientist at IVIC. It is a pleasure to thank Drs. A.E. Hill and Bruria Shachar-Hill for their suggestion of the use of dextrans, their instruction and help with the dextran separation technique, and their extensive discussions. Dr. R. Apitz, Mr H. Rojas and Mrs. Fulvia Bartoli were most helpful with suggestions during the course of the experimental work. Mr. Jose Mora was fundamental help with the equipment. Mrs. Lelis Ochoa and Mr. Luis F. Alvarez helped with some of the drawings. This work was partially supported by CONICIT, Fundación Polar and CDCH of UCV. It is a pleasure to thank Dr. H. Passow and Dr. K.J. Ullrich at the Max Planck Institut für Biophysik (Frankfurt/Main) where this work was initiated.     

Serotonin has kinin-like activity in stimulating secretion by Malpighian tubules of the house cricket Acheta domesticus

Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC₅₀ of 9.4 nmol L⁻¹, although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca²⁺ from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT₂-like receptor.     

Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus

Trypanosoma cruzi epimastigotes adhere in vivo to the luminal surface of their triatomid vector digestive tract by molecular mechanisms, as yet, unknown. Here, we show that the administration of 0.5 microM epimastigote major surface glycoinositolphospholipids (GIPLs) to the infected bloodmeal inhibits up to 90% parasite infection in Rhodnius prolixus. The parasite behavior was investigated in vitro using fragments of the insect midgut. The addition of GIPLs in concentration as low as 50-100 nM impaired 95% the attachment of epimastigotes. Previous treatment of GIPLs with trifluoroacetic acid to remove the terminal beta-galactofuranosyl residues reversed 50% the epimastigote in vitro attachment. The binding sites of purified GIPLs on the luminal surface of the posterior midgut were exposed by immunofluorescence microscopy. These observations indicate that GIPLs are one of the components involved in the adhesion of T. cruzi to the luminal insect midgut surface and possibly one of the determinants of parasite infection in the insect vector.     

A FMRFamide-like peptide is associated with the myotropic ovulation hormone in Rhodnius prolixus.

The protocerebral neurosecretory cells previously shown to be the source of the myotropin controlling ovulation in Rhodnius prolixus react in an immunocytochemical assay using an antiserum against FMRFamide. When the same antiserum was injected into fed mated females at the appropriate time the timing of oviposition was delayed, but the total number of eggs developed was unaffected compared to controls injected with pre-immune rabbit serum. The titer of FMRFamide-like peptide (assayed by RIA) in the hemolymph of mated and virgin females was found to fluctuate with the egg laying cycle, and to reflect earlier determinations of the titer of myotropic activity. Western blots of SDS-PAGE revealed a FMRFamide-immunoreactive peptide of approximately 8.5 kDa in both hemolymph and extracts of the ovulation hormone cells.     

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

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

The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study

The Malpighian tubules of Tenebrio molitor provide a model system for interpreting the actions of endogenous diuretic and antidiuretic peptides. The effects of diuretic (Tenmo-DH(37)) and antidiuretic (Tenmo-ADFa) peptides and their respective second messengers (cyclic AMP and cyclic GMP) on basolateral (V(bl)) and transepithelial (V(te)) potentials of Tenebrio Malpighian tubules were determined using conventional microelectrodes. In the presence of 6 mmol l(-1) Ba(2+), Tenmo-DH(37) (100 nmol l(-1)) reversibly hyperpolarized V(bl) and depolarized V(te). A similar response was seen with the addition of 1 mmol l(-1) cyclic AMP; however, the apical membrane potential (V(ap)) then showed a hyperpolarization, whereas a depolarization of V(ap) was observed with Tenmo-DH(37). Bafilomycin A(1) (5 micromol l(-1)) inhibited fluid secretion of stimulated tubules and reversed the hyperpolarization of V(bl) in response to Tenmo-DH(37). In response to 100 nmol l(-1) Tenmo-ADFa or 1 mmol l(-1) cyclic GMP, V(bl) and V(te) depolarized, although cyclic GMP affected membrane potentials somewhat differently by causing an initial hyperpolarization of V(bl) and V(te). In high [K(+)]-low [Na(+)] Ringer, 1 mmol l(-1) amiloride decreased fluid secretion rates, and depolarized both V(bl) and V(te). Amiloride significantly decreased luminal pH in paired experiments, indicating the presence of a K(+)/nH(+) exchanger in tubule cells of Tenebrio. The results suggest that the endogenous factors and their second messengers stimulate/inhibit fluid secretion by acting on the apical V-ATPase, basolateral K(+) transport, and possibly Cl(-) transport.     

The haemoxisome: a haem-iron containing structure in the Rhodnius prolixus midgut cells

Rhodnius prolixus midgut was analysed using transmission electron microscopy and electron spectroscopic imaging in order to localize the cellular structures involved in haem metabolism. In the posterior midgut, special cellular electron-dense structures were observed. These structures are here designated haemoxisomes. Haemoxisomes are present in the epithelial cells at various time points after a blood meal. Several days after the blood meal, some of them become less electron-dense. By electron spectroscopic imaging, large amounts of iron and oxygen were detected in these cellular structures. The iron is probably bound to the porphyrin ring as an iron-protoporphyrin IX complex, as detected using the diaminobenzidine technique. An interesting observation was the presence of endoplasmic reticulum surrounding the haemoxisomes during some special periods. Iron content was monitored in the posterior midgut epithelium and was found to be constant at the initial days after a blood meal, but slightly higher at the end of the digestive process (from 13th up to 20th day). These results are in agreement with the observation that the appearance of the haemoxisomes changes at the end of the digestive process. The ability to degrade haem seems to depend on the presence of endoplasmic reticulum as observed using a haem degradation assay in the presence of an endoplasmic reticulum-enriched fraction. Taken together these results suggest that haemoxisomes may play a role in intracellular haem detoxification.     

Temporal modulation and adaptive control of the behavioural response to odours in Rhodnius prolixus

It has been demonstrated in several insect species that a circadian clock makes the whole of antennal chemoreceptors more sensitive during a particular temporal window every day. This assessment raises the question about how insects exhibiting bimodal activity handle their sensitivity to odours which are relevant at different moments of the day. To shed some light on this problem, we studied in Rhodnius prolixus the daily dynamics of their responsiveness to CO₂ (host-associated cue) and aggregation cues (refuge-associated), which are relevant at dusk and dawn, respectively. We analysed: (1) whether a temporal modulation of the responsiveness to odours does exist in R. prolixus, (2) if this modulation is a general one or it is specific for each type of volatile, and (3) if it is controlled by exogenous or endogenous mechanisms. We found that the responsiveness to CO₂ only occurs at dusk and that to assembling odours is restricted to dawn. Experiments under free-running conditions revealed that only the responsiveness to CO₂ is controlled by a circadian clock, but not that to assembling signals. Thus, by combining endogenous and exogenous mechanisms, sensitivities to different odours are adjusted according to their associated behavioural context and moment of the day.     

Effects of eicosanoid biosynthesis inhibitors on the prophenoloxidase-activating system and microaggregation reactions in the hemolymph of Rhodnius prolixus infected with Trypanosoma rangeli

Investigations on the effects of eicosanoid biosynthesis inhibitors on the hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, parasitemia and mortality of Rhodnius prolixus infected with Trypanosoma rangeli were performed. Hemocoelic injection of live T. rangeli epimastigotes into fifth-instar larvae of R. prolixus that previously fed on blood containing an inhibitor of phospholipase A(2) (dexamethasone), a specific inhibitor of the cyclooxygenase pathway (indomethacin), and a non-selective lipoxygenase inhibitor (NDGA) (i) reduced the hemocyte microaggregation, (ii) attenuated the proPO system in the hemolymph and (iii) enhanced parasitemia and mortality induced by the parasite challenge in these insects. The effects obtained by dexamethasone administered orally were counteracted by inoculation of the insects with arachidonic acid. We suggest that the infectivity of T. rangeli can be increased by interference with the R. prolixus immune system. This is the first demonstration that the triatomine's immune responses to a parasite infection are modulated by a physiological system that includes eicosanoid biosynthesis.     

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.     

Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.     

Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin

The objective of this work was to study the pattern of salivation of triatomines during feeding in mouse skin. Rhodnius prolixus was fed with a solution of the dye acridine orange or fluorescein. The saliva was efficiently labelled with acridine orange, probably due to the difference in pH between the salivary gland (6.0) and the hemolymph (6.5-7.0). This procedure was not effective at labelling the saliva of Triatoma infestans, however, fluorescent labelling of R. prolixus saliva allowed us to demonstrate that salivation occurs during entire feeding process. The saliva is released soon after the bite. In the probing phase, saliva is pumped continuously in the host skin, including around the blood vessels. During the engorgement phase, saliva is observed in a bolus within the blood vessel and some of it is sucked up by the insect, together with blood. The frequency of saliva emission inside the vessels was low (0.51+/-0.18 Hz). The saliva deposition in the microcirculation is continuous and modulated by the frequency of the cibarial pump because, when functioning at high frequency, cibarial pump sucks almost all saliva to the insect gut. This mechanism would determine the quantity of saliva deposited in the microcirculation as necessary, and consequently minimizing the host's immune response to salivary antigens.     

Adaptation of the repellency response to DEET in Rhodnius prolixus

For many years it has been accepted that DEET interferes with the detection of odours from the host instead of having a repellent effect. However, recent work showed that DEET acts as an odorant molecule and elicits a behavioural response in the absence of other stimuli. Therefore, DEET must promote some phenomenon connected with the stimuli-sensory system interaction, such as a sensory adaptation, where the sensory system regulates its sensitivity to different stimuli intensities during continuous or repetitive exposure. In this work, we studied different aspects of the insect-DEET interaction through behavioural observations. Previous exposure of fifth instar Rhodnius prolixus nymphs to DEET decreased the behavioural response to this repellent. We observed a decrease in repellence after different times of continuous stimulation with DEET in a time-dependent manner. The response to DEET was recovered 10 min after exposure, when insects were continuously stimulated during 5 or 10 min; maximum repellence was recovered 20 min after exposure when insects were stimulated for 20 min. DEET produced a repellent effect when nymphs were exposed only to its vapours. These results suggest that exposure to DEET produces adaptation in R. prolixus nymphs, and that the behavioural response elicited by DEET occurs via olfaction when no other stimuli are present.     

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+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/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. (3)H-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.     

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.