Prolixus Midgut Research Articles

Azadirachtin interferes with basal immunity and microbial homeostasis in the Rhodnius prolixus midgut

Rhodnius prolixus is an insect vector of two flagellate parasites, Trypanosoma rangeli and Trypanosoma cruzi, the latter being the causative agent of Chagas disease in Latin America. The R. prolixus neuroendocrine system regulates the synthesis of the steroid hormone ecdysone, which is essential for not only development and molting but also insect immunity. Knowledge for how this modulates R. prolixus midgut immune responses is essential for understanding interactions between the vector, its parasites and symbiotic microbes. In the present work, we evaluated the effects of ecdysone inhibition on R. prolixus humoral immunity and homeostasis with its microbiota, using the triterpenoid natural product, azadirachtin. Our results demonstrated that azadirachtin promoted a fast and lasting inhibitory effect on expression of both RpRelish, a nuclear factor kappa B transcription factor (NF-kB) component of the IMD pathway, and several antimicrobial peptide (AMP) genes. On the other hand, RpDorsal, encoding the equivalent NF-kB transcription factor in the Toll pathway, and the defC AMP gene were upregulated later in azadirachtin treated insects. The treatment also impacted on proliferation of Serratia marcescens, an abundant commensal bacterium. The simultaneous administration of ecdysone and azadirachtin in R. prolixus blood meals counteracted the azadirachtin effects on insect molting and also on expression of RpRelish and AMPs genes. These results support the direct involvement of ecdysone in regulation of the IMD pathway in the Rhodnius prolixus gut.

A rhamnose-binding lectin from Rhodnius prolixus and the impact of its silencing on gut bacterial microbiota and Trypanosoma cruzi

Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.

The NF-κB Inhibitor, IMD-0354, Affects Immune Gene Expression, Bacterial Microbiota and Trypanosoma cruzi Infection in Rhodnius prolixus Midgut

Rhodnius prolixus is an insect vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Nuclear factor-κB (NF-κB) transcription factors (TF) are conserved components of the innate immune system in several multicellular organisms including insects. The drug IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is a selective inhibitor of IκB kinases. It blocks IκBα phosphorylation thus preventing nuclear translocation of the NF-κb TF. In humans, NF-κB is involved in several biological processes such as inflammation, cell proliferation and immunity. In insects, the activation of the immune system upon microbial challenge can be controlled by signaling pathways such as the immune deficiency (IMD) and Toll, to combat infection. These activated pathways signal to downstream NF-κB TF to stimulate specific immune genes, triggering the synthesis of several molecules such as the antimicrobial peptides. In Drosophila melanogaster, the activation and regulation of NF-κB TF have been elucidated, while in triatomines these mechanisms are not fully understood Therefore, the present study investigated the effects of oral administration of the drug IMD-0354 on the R. prolixus immune response to challenge with bacteria and T. cruzi, as well as the impact on the gut bacterial microbiota. R. prolixus were fed with rabbit blood containing IMD-0354 and Escherichia coli, Staphylococcus aureus, or T. cruzi. The effects of IMD-0354 on insect mortality and antimicrobial activity in insect midgut samples, as well as the relative expression of R. prolixus immune genes were recorded. The bacterial microbiota was analyzed, and viable parasites were counted in insect midgut samples. The IMD-0354 treatment modulated antibacterial activity and the gene expression patterns of defensin A, defensin B, defensin C, and prolixicin, and the genes involved in the IMD and Toll pathways. Additionally, there was an increase of bacterial microbiota in treated insects. Insects treated with IMD-0354 and concomitantly infected with bacteria or T. cruzi through the blood meal had increased mortality, while the T. cruzi population in R. prolixus midgut was reduced. The inhibitory effect of IMD-0354 indicates the importance of NF-κB TF in the innate immune responses involved in the control of bacteria and parasite infections in the R. prolixus midgut.

Amino acids trigger down-regulation of superoxide via TORC pathway in the midgut of Rhodnius prolixus.

Sensing incoming nutrients is an important and critical event for intestinal cells to sustain life of the whole organism. The TORC is a major protein complex involved in monitoring the nutritional status and is activated by elevated amino acid concentrations. An important feature of haematophagy is that huge amounts of blood are ingested in a single meal, which results in the release of large quantities of amino acids, together with the haemoglobin prosthetic group, haem, which decomposes hydroperoxides and propagates oxygen-derived free radicals. Our previous studies demonstrated that reactive oxygen species (ROS) levels were diminished in the mitochondria and midgut of the Dengue fever mosquito, Aedes aegypti, immediately after a blood meal. We proposed that this mechanism serves to avoid oxidative damage that would otherwise be induced by haem following a blood meal. Studies also performed in mosquitoes have shown that blood or amino acids controls protein synthesis through TORC activation. It was already proposed, in different models, a link between ROS and TOR, however, little is known about TOR signalling in insect midgut nor about the involvement of ROS in this pathway. Here, we studied the effect of a blood meal on ROS production in the midgut of Rhodnius prolixus We observed that blood meal amino acids decreased ROS levels in the R. prolixus midgut immediately after feeding, via lowering mitochondrial superoxide production and involving the amino acid-sensing TORC pathway.

Chitin is a component of the Rhodnius prolixus midgut

Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insect's gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm1. Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium, the region facing the midgut and projections towards the intestinal lumen when evaluated microscopically. The relative reduction in CHS transcripts by approximately 80% using an RNAi assay supports the phenotypical alterations in the midgut observed using fluorescence microscopy assays. These data show that chitin is present in the R. prolixus midgut epithelium and in its surface projections facing the lumen. The CHS gene expression and the presence of chitin in the R. prolixus midgut may suggest a target for controlling Chagas disease vectors and addressing this public health problem.

Rhodnius prolixus interaction with Trypanosoma rangeli: modulation of the immune system and microbiota population.

BackgroundTrypanosoma rangeli is a protozoan that infects a variety of mammalian hosts, including humans. Its main insect vector is Rhodnius prolixus and is found in several Latin American countries. The R. prolixus vector competence depends on the T. rangeli strain and the molecular interactions, as well as the insect’s immune responses in the gut and haemocoel. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus infected with T. rangeli Macias strain, considering the influence of the parasite on the intestinal microbiota.MethodsThe population density of T. rangeli Macias strain was analysed in different R. prolixus midgut compartments in long and short-term experiments. Cultivable and non-cultivable midgut bacteria were investigated by colony forming unit (CFU) assays and by 454 pyrosequencing of the 16S rRNA gene, respectively. The modulation of R. prolixus immune responses was studied by analysis of the antimicrobial activity in vitro against different bacteria using turbidimetric tests, the abundance of mRNAs encoding antimicrobial peptides (AMPs) defensin (DefA, DefB, DefC), prolixicin (Prol) and lysozymes (LysA, LysB) by RT-PCR and analysis of the phenoloxidase (PO) activity.ResultsOur results showed that T. rangeli successfully colonized R. prolixus midgut altering the microbiota population and the immune responses as follows: 1 - reduced cultivable midgut bacteria; 2 - decreased the number of sequences of the Enterococcaceae but increased those of the Burkholderiaceae family; the families Nocardiaceae, Enterobacteriaceae and Mycobacteriaceae encountered in control and infected insects remained the same; 3 - enhanced midgut antibacterial activities against Serratia marcescens and Staphylococcus aureus; 4 - down-regulated LysB and Prol mRNA levels; altered DefB, DefC and LysA depending on the infection (short and long-term); 5 - decreased PO activity.ConclusionOur findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition.

Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut.

BackgroundThe triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract.MethodsIn the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding.ResultsOur results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated.ConclusionOur findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection.

Unsaturated Glycerophospholipids Mediate Heme Crystallization: Biological Implications for Hemozoin Formation in the Kissing Bug Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9–17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

Cruzipain Promotes Trypanosoma cruzi Adhesion to Rhodnius prolixus Midgut

Background Trypanosoma cruzi is the etiological agent of Chagas' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction.Methodology/Principal FindingsHere, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain.Conclusions/SignificanceThese data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.

On the physico-chemical and physiological requirements of hemozoin formation promoted by perimicrovillar membranes in Rhodnius prolixus midgut

Triatomine insects are obligatory blood-feeders that detoxify most of the hemoglobin-derived heme through its crystallization into hemozoin (Hz). Previous evidence demonstrates the key role of midgut perimicrovillar membranes (PMVM) on heme crystallization in triatomines. Here, we investigated some of the physico-chemical and physiological aspects of heme crystallization induced by Rhodnius prolixus PMVM. Hz formation in vitro proceeded optimally at pH 4.8 and 28 °C, apparently involving three kinetically distinct mechanisms along this process. Furthermore, the insect feeding status and age affected PMVM-induced heme crystallization whereas pharmacological blockage of PMVM formation by azadirachtin, reduced hemoglobin digestion and Hz formation in vivo. Mössbauer spectrometry analyses of R. prolixus midgut showed that Hz represents the only measurable iron species found four days after a blood meal. Autocatalytic heme crystallization to Hz is revealed to be an inefficient process and this conversion is further reduced as the Hz concentration increases. Also, PMVM-derived lipids were able to induce rapid Hz formation, regardless of the diet composition. These results indicate that PMVM-driven Hz formation in R. prolixus midgut occurs at physiologically relevant physico-chemical conditions and that lipids derived from this structure play an important role in heme crystallization.

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

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

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

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

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.

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus

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.

Biological effects of canatoxin in different insect models: evidence for a proteolytic activation of the toxin by insect cathepsinlike enzymes.

Canatoxin is a toxic protein isolated from the jackbean, Canavalia ensiformis. The toxin injected intraperitoneally is lethal for mice and rats; however, it is inactive if given orally. In this study, Manduca sexta (L.) (Lepidoptera), Schistocerca americana (Drury) (Orthoptera), Drosophila melanogaster (L.) (Diptera), Aedes aegypti (L.) (Diptera), Rhodnius prolixus (Stål) (Hemiptera), and Callosobruchus maculatus (F.) (Coleoptera) were fed on canatoxin-containing diets. No effects were seen in M. sexta, S. americana, D. melanogaster, or A. aegypti. No traces of canatoxin were found in their feces, suggesting that the protein was digested completely by these insects, which characteristically have a trypsin-based digestion. In contrast, canatoxin was lethal for insects displaying cathepsin-based digestion. Thus, for C. maculatus, a diet containing 0.25% wt:wt canatoxin caused complete inhibition of larval growth. When R. prolixus were fed on canatoxin, 2 effects were seen: impairment of water excretion and increased lethality 48-96 h after feeding. The lethal effect of canatoxin in R. prolixus was blocked partially or completely when the digestion of the toxin by R. prolixus midgut enzymes was impaired. The data showed that canatoxin is highly toxic when ingested by some species of insects but not affecting others, probably in correlation with the characteristics of the digestive process of the insect.

Origin, distribution, properties and functions of the major Rhodnius prolixus midgut hydrolases

Soluble β-acetylglucosaminidase, α-galactosidase, α-glucosidase and α-mannosidase predominate in anterior midgut lumen; soluble aminopeptidase, membrane-bound α-glucosidase and membrane-bound α-mannosidase, in posterior midgut tissue; and proteinases, sb-glucosidase and β-mannosidase, in posterior midgut lumen. Ultracentrifugation data suggest that there is only one major molecular species of the following soluble enzymes: aminopeptidase ( M r 61,000), with a broad specificity toward aminoacyl-β-naphthylamides; β-glucosidase ( M r 64,000), active on several β-glycosides; and acid phosphatase ( M r 82,000), which hydrolyzes many substrates and is competitively inhibited by tartrate ( K i 2 μM), molybdate ( K i 9 nM) and fluoride (three ligands per active site). There are major (85% of total) and minor (15%) thiol-activated hemoglobin hydrolase resolved by ultracentrifugation. The major activity ( M r 40,000) is cathepsin B-like hydrolyzing α-N- benzoyl- dl-arginine -p- nitroanilide , whereas the minor activity ( M r 88,000) is cathepsin d-like, being more active at pH 3.5 and being inhibited by pepstatin. The symbiont, Nocardia rhodnii, possesses an amylase which is found in Rhodnius prolixus midgut and an aminopeptidase and a BAPA hydrolase different from those of R. prolixus. The results suggest that digestion starts in the lumen of posterior midgut and ends with the action of enzymes trapped between extracellular membranes or on the surface of midgut cells. The use of cathepsin-like proteases is thought to be derived character evolved from a putative seed-sucker ancestor.

Proteolytic enzymes in theRhodnius prolixus midgut

Sequential chromatographic fractionation of Rhodnius prolixus midgut homogenate yielded only one endopeptidase, but revealed the presence of carboxypeptidase-A and B-like enzyme, di- and tripeptidases, as well as aminopeptidase activities.

Purification and characterization of a sulfhydryl-dependent protease from Rhodnius prolixus midgut

The characterization and partial purification of one protease from Rhodnius prolixus midgut are described. Enzyme preparations show a high degree of instability under several conditions, such as handling, freezing, and thawing, and at 4°C. This spontaneous inactivation of either crude or purified enzyme is completely protected by 10 mM tetrathionate. Sulfhydryl compounds such as β-mercaptoethanol, dithiothreitol, cysteine, thioglycolic acid, and reduced glutathione are able to activate the enzyme, whereas iodoacetate, mercuric chloride, p -aminophenylmercuric chloride, tetrathionate, and 5,5-dithiobis(2-nitrobenzoic acid) inhibit the enzyme. Partial inhibition was obtained by hydrogen perioxide, oxygen bubbling, and performic acid. Besides casein, the enzyme hydrolyzed benzoyl- l -tyrosine ethyl ester (BTEE), but showed no esterolytic activity upon p -tosyl- l -arginine methyl ester and benzoyl- l -arginine ethyl ester. Purifications of 210- and 187-fold with yields of 80 and 71% respectively, were obtained for the caseinolytic and esterolytic activities by using a Sepharose-mercurial column as a thiol-group affinity adsorbent. The molecular weight was estimated as 21,500 by gel filtration. α-Chymotrypsin and the midgut enzyme show similar abilities to hydrolyze casein, however, the midgut protease hydrolyzes casein 2.7-fold faster than trypsin and about 3-fold slower than pepsin. Soybean trypsin inhibitor, black-eyed pea chymotrypsin inhibitor, tosyl- l -lysine chloromethyl ketone, and tosyl-amide-phenylethyl chloromethyl ketone do not inhibit the protease activity, whereas, pepstatin showed a 100% inhibitory effect at an inhibitor/enzyme molar ratio of 0.36. Midgut protease and α-chymotrypsin showed similar K m values for BTEE (1.06 and 1.15 m ×10 −4 , respectively), however, the former showed a 9-fold lower catalytic efficiency ( K cat / K m ). Dialysis did not affect the enzyme, whereas Cu 2+ , Pb 2+ , and Hg 2+ showed an inhibitory effect; a slight activation was obtained in the presence of Ca 2+ .

Control of protease secretion in the intestine of fifth instar larvae of Rhodnius prolixus

Groups of four Rhodnius prolixus larvae in the 5th-instar received no food or were given once only through a special feeder, similar weights of food solutions. These were either protein solutions (whole human blood, its plasma or erythrocytes, egg albumin, bovine serum albumin, haemoglobin) or non-protein solutions (saline, casein hydrolysate, dextran, maltose). Following feeding, the protein content and caseinolytic activity of the larval midgut homogenates, determined between the 3rd and 14th days, were practically the same in starved larvae and in larvae receiving non-protein solutions. In the protein-fed larvae the protein content and the specific protease activity increased. These findings exclude neurosecretory control and indicate that ingested protein stimulate the proteolytic activity of R. prolixus midgut.