Coinfection by Trypanosoma cruzi and a fungal pathogen increases survival of Chagasic bugs: advice against a fungal control strategy.

Triatomine bugs carry the parasitic protozoa Trypanosoma cruzi, the causal agent of Chagas disease. It is known that both the parasite and entomopathogenic fungi can decrease bug survival, but the combined effect of both pathogens is not known, which is relevant for biological control purposes. Herein, the survival of the triatomine Meccus pallidipennis (Stal, 1872) was compared when it was coinfected with the fungus Metarhizium anisopliae (Metschnikoff) and T. cruzi, and when both pathogens acted separately. The immune response of the insect was also studied, using phenoloxidase activity in the bug gut and hemolymph, to understand our survival results. Contrary to expectations, triatomine survival was higher in multiple than in single challenges, even though the immune response was lower in cases of multiple infection. We postulate that T. cruzi exerts a protective effect and/or that the insect reduced the resources allocated to defend itself against both pathogens. Based on the present results, the use of M. anisopliae as a control agent should be re-considered.

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): Purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes.

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from "classical" cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes.

Acid phosphatase activity distribution in salivary glands of triatomines (Heteroptera, Reduviidae, Triatominae).

Acid phosphatase activity (Gömori technique) in salivary gland cells was investigated in adult insects (males and females) of four species of triatomines: Triatoma infestans, Panstrongylus megistus, Rhodnius neglectus, and Rhodnius prolixus. Binucleated cells with bulky and polyploidy nuclei were detected, with acid phosphatase activity in the heterochromatin and nucleolus, which showed the most intense response. Thus, the activity of these phosphatases during rRNA molecule transcription, possibly in the nucleolar fibrillar center, is suggested. The difference in reactivity found among salivary glands is associated with the cellular metabolism of these regions and, probably, with the biosynthesis of their different secretions. This must be essential in maintaining the hematophagy of triatomines.

Phylogenetic relationships within the oliveirai complex (Hemiptera:Reduviidae:Triatominae).

The oliveirai complex is actually composed of nine related species of Triatominae that occur in the Pantanal ecosystem of Mato Grosso, Brazil. In order to confirm their specific status and infer their phylogenetic relationships, we performed a genetic and morphometric comparison concerning seven of the nine species of the complex. Isoenzyme analysis provided broadly similar phylogenetic information to that derived from discriminant analysis of size-free variables. The derived trees reveal two main species groups, one composed of three closed species (T. jurbergi, T. matogrossensis and T. vandae), and another one subdivided into two different pairs, the T. guazu-T. williami pair and the T. klugi-T. oliveirai pair. The results call the specific status of T. guazu and T. williami into question, support the possibility that the other analyzed species represent distinct taxa and query the existence of the oliveirai complex.

Effects of temperature and pH on hexokinase from the flight muscles of Dipetalogaster maximus (Hemiptera: Reduviidae).

Effects of temperature and pH on the catalytic properties of hexokinase (HK, EC 2.7.1.1) from the flight muscles of Dipetalogaster maximus (Uhler) were studied. The enzyme showed a hyperbolic behavior with its two substrates (glucose and ATP). There was no inhibition by glucose. Apparent Km and Vmax increased as pH increased from 7.0 to 8.5. Catalytic efficiency was lowest at pH 7.0. Km, Vmax, and catalytic efficiency were higher at 37 degrees C than at 30 and 20 degrees C. There was marked inhibition by ATP, which was dependent on pH and temperature. Km values for ATP were reduced and catalytic efficiency increased as pH increased. Lowest Vmax was observed at pH 7.0. At this pH there was 87.3% inhibition by ATP, whereas it was only 5.7% at pH 8.5 (at 30 degrees C). Km, Vmax, and catalytic efficiency were higher at 37 degrees C than at 30 and 20 degrees C. The strong inhibition by ATP detected at 20 degrees C (pH 7.6) almost disappeared at 37 degrees C. Therefore, temperature could regulate hexokinase activity by modulating the inhibition produced by ATP. Glucose utilization and ATP production would be promoted when temperature rises from 30 to 37 degrees C. Because insect thoracic muscles increase their temperature over 30 degrees C during flight, this phenomenon elucidates a mechanism enhancing energy supply for muscle activity.

Enzyme polymorphism among Triatoma infestans (Hemiptera: Reduviidae) colonies.

Allozyme variability in populations of the Chagas's disease vector Triatoma infestans (Klug) was investigated by means of starch gel electrophoresis. Samples were taken from nine laboratory colonies established with individuals collected at different localities across the range of this insect in South America. Zymograms for proteins coded by a total of 17 loci were obtained. Allele frequencies, proportion of polymorphic loci (P), mean heterozygosity per locus (H), similarity (S), and identity (I) indices, genetic distance (D), and gene flow among populations were estimated. Mean values for P = 58.53% and for H = 0.095, indicating an important level of genetic variability. There was remarkable similarity among the colonies (mean I = 0.9946). Estimated gene flow among populations was high. However, on the basis of the known natural history of T. infestans, the uniformity of allele frequencies among populations may be interpreted as the result of the recent and rapid dispersal of the species from the site of origin in the Cochabamba Valley, Bolivia.

Genetic structure of four species of Triatoma (Hemiptera: Reduviidae) from Argentina.

The genetic structure of Triatoma guasayana Wygodzinsky & Abalos, T. sordida (Stål), T. platensis Neiva, and T. infestans (Klug) was compared by starch gel electrophoresis. In total, 17 enzyme loci were analyzed in T. infestans and 14 loci in the other three species. Zymograms were useful in species identification. Icdh-2 and Hk-1 are diagnostic loci, and Icdh-1, Hk-2, Es-1, and Es-2 are helpful in distinguishing these species. The proportion of polymorphic loci for the four species (range, 52.9 to 58.3%) was somewhat higher than that expected from reports for other insects. Mean heterozygosity per locus (range, 0.062 to 0.156) was within the range reported for insects in the literature. The level of heterozygosity was highest for T. guasayana and lowest for T. sordida. Values of genetic distance (Nei's D), identity (Nei's I), and similarity (Rogers's S) indices indicate that the four species are well-defined taxa. T. infestans and T. platensis are the closest (D = 0.45, S = 0.62). T. guasayana and T. sordida (D = 0.77, S = 0.46) form another pair of related species.

Characterization of inducible lysozyme activity in the hemolymph of Rhodnius prolixus.

1. The characterization and partial purification of an induced lysozyme activity in the hemolymph of adult Rhodnius prolixus inoculated with Micrococcus lysodeikticus is described. 2. Little or no activity against M. lysodeikticus appeared in the first hours after inoculation, but the activity increased reaching a maximum 4 days later, which was maintained to day 12. 3. The activity was characterized as lysozyme on the basis of the following considerations: 1) pH optimum and thermostability at acidic pH; 2) rate of lysis negatively dependent on ionic strength; 3) binding to SP-Sephadex at pH 5.5; 4) apparent molecular weight of 15 kDal. 4. Crude or semi-purified enzyme preparations showed a high degree of stability during handling, freezing and thawing, and standing at 5 degrees C. 5. Incubation of abdominal fat bodies from treated insects resulted in the release of activity into the medium. 6. The relationship between induced lysozyme activity and its role as an insect defense mechanism is discussed.

Comparative study of enzymes in testes and ovaries from adult Dipetalogaster maximus (Uhler) and triatoma infestans (Klug) (Hemiptera: Reduviidae). correlation with fine structural organization.

Activities of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), fructose-6-phosphate kinase (F6PK), glutamate dehydrogenase (GlutDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH) and glycerol-3-phosphate dehydrogenase (GPDH) were determined in tissue extracts of testes and ovaries of adult Dipetalogaster maximus (Uhler) and Triatoma infestans (Klug) (Hemiptera: Reduviidae), insect vectors of Chagas disease. The fine structure organization of the same organs were studied by electron microscopy. Results allow the following inferences: in testes from both species, most of the glucose would be utilized through the glycolytic pathway. Amino acid catabolism for energy purposes appears to be unimportant. The number of mitochondria and the development of the rough endoplasmic reticulum in cells of the spermatogenic line indicate the occurrence of active oxidative metabolism and protein synthesis; in ovaries, levels of G6PDH indicate the existence of an active pentose pathway which would supply the NADPH required for fat and ecdysteroid synthesis. Amino acid catabolism appears to be relatively more important in ovary than in testis. Fat and glycogen are stored in follicular cells of D. maximus; oocytes of both species contain numerous fat droplets. Abundant mitocondria are present in follicular cells and oocytes. A well developed rough endoplasmic reticulum and free ribosomes are also conspicuous in these cells. The malate/aspartate H-transfer system seemed to be relatively more important than the glycerophosphate shuttle in ovaries as well in testes.

[Prolixase, a fibrinolytic enzyme isolated from Rhodnius prolixus (Heteroptera, Reduviidae): antigenic properties]. / La prolixase, enzyme fibrinolytique isolée à partir de Rhodnius prolixus (Heteroptera, Reduviidae): propriétés antigéniques.

Antigenic properties of prolixase are studied showing that this fibrinolytic enzyme, induces in the rabbit, the formation of precipitating but slightly, hemagglutinating homologous antibodies.

Description of new fibrinolytic enzyme, isolated from the haemolymph of Rhodnius prolixus Stal (Heteroptera Reduviidae).

A new fibrinolytic enzyme, isolated from the haemolymph of Rhodnius prolixus Stal (Heteroptera, Reduviidae) has been discovered. This enzyme does not act by activating plasminogen to plasmin. However, the split products obtained with this new enzyme inhibit fibrin formation in normal plasma. This enzyme seems to act specifically on the proteins involved in blood coagulation. Its origin has induced us to name it prolixase.

Interactions between intestinal compounds of triatomines and Trypanosoma cruzi.

Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease, a devastating disease that disables and leads to the death of many people in Latin America. In this review, factors from the insect vector are described, including digestive enzymes, hemolysins, agglutinins, microbiota and especially antimicrobial factors, which are potentially involved in regulating the development of T. cruzi in the gut. Differential regulation of parasite populations shows that some triatomine defense reactions discriminate not only between molecular signals specific for trypanosome infections but also between different strains of T. cruzi.

alpha-glycerophosphate dehydrogenase activity in flight muscles of triatomine bugs Panstrongylus megistus and Triatoma sordida.

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.

Distribution and properties of glutathione S-transferase from T. infestans.

The glutathione transferase from T. infestans is able to render aqueous metabolites when incubated in vitro with malathion, parathion and fenitrothion. It is a soluble enzyme present in every developmental stage and widely distributed in all insect organs. The purification procedure applied, consisting of fractionation with ammonium sulfate and Bio-Gel P-60 chromatography, gives an unique molecular form catalytically active using methyl iodide as substrate in polyacrylamide gel electrophoresis (PAGE). One of the most active substrates is the 1-chloro-2,4-dinitrobenzene (CDNB), with an activity maximum at pH 7.5 and at 45 degrees C temperature. Its activation energy calculated from an Arrhenius plot is 14,846 cal mol-1. The enzyme susceptibility to inhibition by thiol reagents shows three degrees of responses; slight, moderate or high, depending on the compounds used. The kinetics of the enzyme catalysed reaction with the purified fraction is complex, and resembles that reported for glutathione S-transferase A from rat liver, showing a biphasic kinetic mechanism in which the reaction pathway depends on the concentration of GSH. In general, the properties of this insect enzyme are similar to those enzymes isolated from vertebrate organisms.

Proteolytic enzymes in the Rhodnius 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.

[Comparative characteristics of the digestive enzymes of blood-sucking insects. II. Invertase and lipase]. / K sravnitel'noi kharakteristike pishchevaritel'nykh fermentov krovososushchikh nasekomykh. II. Invertaza i lipaza.

The activity of invertase and lipase in the midguts of 11 hematophagous insect species have been determined. The highest invertase activity at pH 7-10. Insects feeding on carbohydrate meals as well as with blood have the greatest invertase activity, the obligatory blood feeders have the least one. The lipase activity does not depend on trophic peculiarities of the blood-suckling insects.

Allozyme relationships among ten species of Rhodniini, showing paraphyly of Rhodnius including Psammolestes.

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

Salivary apyrase of Rhodnius prolixus. Kinetics and purification.

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.