Effect of salivary CYP4EM1 and CYP4EM2 gene silencing on the life span of Chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae) exposed to sublethal dose of deltamethrin.

Control of Chagas disease in endemic countries is primarily accomplished through insecticide spraying for triatomine vectors. In this context, pyrethroids are the first-choice insecticide, and the evolution of insect resistance to these insecticides may represent an important barrier to triatomine control. In insects, cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals that are encoded by genes divided into different families. In this work, we evaluated the role of three Rhodnius prolixus CYP4EM subfamily genes during blood meal and after deltamethrin exposure. CYP4 gene members were expressed in different insect organs (integument, salivary glands (SGs), midgut, fat body and malpighian tubules) at distinct transcriptional levels. CYP4EM1 gene was highly expressed in the SG and was clearly modulated after insect blood meal. Injection of CYP4EM1dsRNA promoted significant reduction in mRNA levels of both CYP4EM1 and CYP4EM2 genes and induced deleterious effects in R. prolixus nymphs subsequently exposed to sublethal doses of deltamethrin (3.4 or 3.8 ng/nymph treated). The higher dose reduced the survival over time and increased susceptibility of R. prolixus nymphs to deltamethrin. A better understanding of this mechanism can help in developing of more efficient strategies to reduce Trypanosoma cruzi vector transmission in Americas.

Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms.

The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source.

Susceptibility to chemical insecticides of two Brazilian populations of the visceral leishmaniasis vector Lutzomyia longipalpis (Diptera: Psychodidae).

OBJECTIVES: To investigate the insecticide susceptibility of two geographically separated Lutzomyia longipalpis populations (Lapinha and Montes Claros) with different histories of insecticide exposure (i.e. no exposure and repeated exposure, respectively). METHODS: (i) Bioassay monitoring of sand fly survival over time when exposed to a range of insecticides; and (ii) analysis of the level of insecticide detoxification enzymes in individual sand flies caught at both study sites. Insecticides tested were the organophosphates malathion and fenitrothion and the pyrethroids lambda-cyhalothrin, permethrin and deltamethrin. RESULTS: Survival analyses showed that whilst there was no overall significant difference in susceptibility of both populations to organophosphates, Lapinha sand flies were significantly more susceptible to pyrethroids than those from Montes Claros. Multiple regression analyses also showed that insecticide susceptibility in both locations varied with sand fly sex. The relative susceptibilities of the two sand fly populations to tested insecticides were also compared. Thus, Montes Claros sand flies were most susceptible to malathion, followed by fenitrothion, deltamethrin and permethrin. Those from Lapinha were most susceptible to lambda-cyhalothrin, followed by malathion, permethrin, deltamethrin and fenitrothion. Biochemical analyses demonstrated that Montes Claros sand flies had significantly lower insecticide detoxification enzyme activity than Lapinha sand flies. CONCLUSIONS: Our results are the first record of significantly reduced susceptibility to the insecticides used in control of wild populations of Lu. longipalpis. They demonstrate the importance of evaluating chemicals against this species by conventional bioassay and microplate assays before and during spraying programmes.

Brasiliensin: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake.

Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15mug of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of approximately 18- and approximately 3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight.

RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection.

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

Leishmania amazonensis: chemotaxic and osmotaxic responses in promastigotes and their probable role in development in the phlebotomine gut.

Taxic responses may play a role in development of Leishmania in their phlebotomine sand fly vectors. They are possibly responsible for movement of the parasites towards the anterior regions of the gut, from where they would be transmitted to the vertebrate host. A methodology capable to distinguish chemotaxic from osmotaxic responses was described and used to characterise taxic responses in Leishmania promastigotes. These were able to respond to chemotaxic as well as to osmotaxic stimuli. Like bacteria, promastigotes were capable to undergo "adaptation," a phenomenon by which they stop responding to a continuos stimulus. A model capable to explain how a relatively small number of different receptors works to perceive gradients in chemotaxic responses was proposed. According to this model, these receptors possess low specificity and a wide range of affinities varying from high to low. A low specificity makes the same receptor able to bind to a large number of different but structurally related molecules and; a wide range of affinities (considering a population of receptors), implies that the number of receptors "occupied" by attractant molecules along a gradient would go growing step by step.

Anti-complement activity in the saliva of phlebotomine sand flies and other haematophagous insects.

The saliva of haematophagous insects has a series of pharmacological activities which may favour blood feeding. In the present study, an inhibitory effect on the complement system was observed in salivary extracts obtained from the phlebotomine sand flies Lutzomyia longipalpis and Lu. migonei. Saliva from Lu. longipalpis was capable of inhibiting both the classical and alternative pathways, while that from Lu. migonei acted only on the former. Other haematophagous insect species were screened for inhibition of the classical pathway. The triatomine bugs Panstrongylus megistus, Triatoma brasiliensis and Rhodnius prolixus were also able to inhibit the classical pathway whereas the mosquito Aedes aegyti and flea Ctenocephalides felis were not. The activity of Lu. longipalpis saliva on the classical pathway was partially characterized. The inhibitor is a protein of Mr 10000-30000 Da, which is very resistant to denaturation by heat. The inhibition of the complement system by phlebotomine sand flies may have a role in the transmission of Leishmania to the vertebrate hosts. The inhibitor molecule is thus a promising component of a vaccine to target salivary immunomodulators.

Identification of morphologically similar Rhodnius species (Hemiptera: Reduviidae: Triatominae) by electrophoresis of salivary heme proteins.

We examined intraspecific variability in the genus Rhodnius using starch gel electrophoresis of salivary heme proteins. Salivary protein profiles of 8 Rhodnius species (R. prolixus, R. robustus, R. neglectus, R. nasutus, R. ecuadoriensis, R. pallescens, R. pictipes, and R. domesticus) were compared. All species could be distinguished by this technique. The greatest protein polymorphism was found in R. ecuadoriensis, R. nasutus, R. robustus, and R. pictipes, followed by R. prolixus, R. neglectus, R. pallescens, and R. domesticus. This approach was able to distinguish R. prolixus from R. robustus and R. neglectus from R. nasutus, species with extreme phenotypical similarity.

A sensitive method for assaying chemotaxic responses of Leishmania promastigotes.

This article describes a sensitive, cheap, and easy method for assaying chemotaxic responses of Leishmania promastigotes. A gradient of the substance to be assayed was produced inside a series of commercially available capillary tubes submerged in a promastigote suspension. After an incubation period, the attractiveness of the substance under test was measured by counting the number of parasites in the capillaries in a Neubauer chamber. Different responses were detected in two strains of Leishmania amazonensis and one strain of L. chagasi after standardization of the method to assay attraction to carbohydrates. Very different responses were obtained when the test was performed using promastigotes of the same strain in two different physiological states (log and stationary phase). The stationary phase cells showed an enhanced chemotaxic capability, which can be explained by the fact that the metacyclic forms commonest in stationary phase cultures have greater mobility than other promastigotes. This method will permit studies to be made of the attractive response to different substances in Leishmania species and other trypanosomatids and facilitate characterization of the potential receptors involved in the chemotaxic response. An adaptation of the method to assay the response to repellent substances is also provided.

Lutzomyia longipalpis: pH in the gut, digestive glycosidases, and some speculations upon Leishmania development.

Screening for digestive glycosidases in different parts of the gut and associated organs of Lutzomyia longipalpis is reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an alpha-glucosidase, an N-acetyl-beta-d-glucosaminidase, an N-acetyl-beta-d-galactosaminidase, and an alpha-l-fucosidase. Their possible role and importance for Leishmania development are discussed and the alpha-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data and Leishmania development is proposed.

Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae).

Rhodnius prolixus interpopulation variability was studied based on a new approach using salivary heme proteins (nitrophorins) electrophoresis in starch gel. We compared salivary proteins profiles of R. prolixus from three different laboratory colonies from Honduras, Venezuela, Brazil and Rhodnius robustus from Venezuela, constructing a UPGMA. The Honduran and Venezuelan populations could not be distinguished from each other, but the Brazilian population was well separated from the others. The high similarity between Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus may have been introduced from a Venezuelan origin. The low polymorphism shown by the Honduran specimens is in agreement with a possible founder effect. This new approach also distinguished R. prolixus populations from R. robustus, species with extreme phenotypical similarity.

Glycosidases in Leishmania and their importance for Leishmania in phlebotomine sandflies with special reference to purification and characterization of a sucrase.

Culture forms of Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) produce an extracellular enzyme that hydrolyzes sucrose molecules into their component monosaccharides. This is important because phlebotomine sand flies, the invertebrate hosts of Leishmania, ingest plant sap or aphid and coccid honeydew rich in sucrose between blood meals and Leishmania promastigotes cannot uptake sucrose. The sucrase was purified and characterized; its molecular weight, estimated by gel filtration chromatography and SDS-PAGE electrophoresis, was about 73 kDa. K(m) and V(max) measured with sucrose as substrate were respectively 4.4 mM and 6.9 mumole glucose.min-1 (mg sucrase)-1, with maximum pH activity at pH 5.5. A series of natural and p-nitrophenyl-derived substrates were assayed, characterizing the enzyme as a highly specific beta-D-fructofuranoside fructohydrolase. When 11 species of Leishmania and 7 genera of trypanosomatids were screened, only the species of the genus Trypanosoma did not produce an enzyme with saccharolytic activity. These data are in agreement with the fact that the latter vectors do not acquire sucrose or raffinose in their meals. Searching for glycolytic enzymes other than sucrase, we found an N-acetyl-beta-D-galactosaminolytic activity. This N-acetyl-galactosaminidase, here described for the first time, might have a role in peritrophic membrane disruption. The importance of sucrase and N-acetyl-beta-D-galactosaminidase in the Leishmania life cycle is discussed.

DNA bioprints: simple nonisotopic DNA fingerprints with biotinylated probes.

The discovery of multilocal DNA fingerprinting represented a revolution in criminal identification and paternity testing. However, for routine use in clinical laboratories, the standard DNA fingerprint methodology is too complex. We have been successful in the development of a simplified DNA nonisotopic fingerprinting system using biotin-labeled probes which we have called DNA bioprints. To achieve this we explored three main technical features: utilization of biotinylated nonradioactive probes as a simpler substitute for 32P-labeled probes, utilization of oligonucleotide probes as a simpler substitute for recombinant probes, and direct hybridization in the dried agarose gel as a simpler substitute for Southern blots. In this article we review our results in the development of DNA bioprints.