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.

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco.

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 +/- 3.1 mg a.i./m2 in walls and 11.9 +/- 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 +/- 6.9 mg a.i./m2 and 34.7 +/- 10.4 mg a.i./m2 in walls and 28.8 +/- 19.2 mg a.i./m2 and 24.9 +/- 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 ± 3.1 mg a.i./m2 in walls and 11.9 ± 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 ± 6.9 mg a.i./m2 and 34.7 ± 10.4 mg a.i./m2 in walls and 28.8 ± 19.2 mg a.i./m2 and 24.9 ± 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.

Comparative evaluation of pyrethroid insecticide formulations against Triatoma infestans (Klug): residual efficacy on four substrates

We investigated the residual efficacy of four insecticide formulations used in Chagas disease vector control campaigns: cyfluthrin 12.5 percent suspension concentrace (SC), lambda-cyhalothrin 10 percent wettable powder (WP), deltamethrin 2.5 percent SC, and 2.5 percent WP on four types of circular blocks of wood, straw with mud, straw with mud painted with lime, and mud containing 5 percent of cement. Three concentrations of these insecticides were tested: the LC90 (previously determined on filter paper), the double of the LC90, and the recommended operational dose. For each bioassay test, 15 third-stage nymphs of Triatoma infestans (Klug) (Hemiptera: Reduviidae) were exposed for 120 h to each treatment at 24 h, 30, 60, 90, and 180 days post-spraying. Mortality rates, moulting history and behaviour were recorded at 24, 48, 72, and 120 h of exposure. Mortality rates were highest during the first 30 days post-spraying. Highest mortality rates (above 50 percent) were observed for deltamethrin 2.5 percent SC and lambda-cyhalothrin 10 percent WP on wood blocks up to three months post-spraying. Mud was the substrate on which treatments showed lowest persistence, with the other two substrates showing intermediate residual efficacy of all treatments. During the first 30 days WP formulations were not as effective as SC flowable formulations but, overall in the longer term, WP gave grater mortality rates of T. infestans nymphs exposed at up to six months post-spraying. Porous surfaces, especially mud, showed most variability presumably due to absorption of the insecticide. In contrast the less porous surfaces (i.e. wood and lime-coated mud) kept mortality rates high for longer post-treatment, irrespective of the insecticide concentration used.

Comparative evaluation of pyrethroid insecticide formulations against Triatoma infestans (Klug): residual efficacy on four substrates.

We investigated the residual efficacy of four insecticide formulations used in Chagas disease vector control campaigns: cyfluthrin 12.5% suspension concentrace (SC), lambda-cyhalothrin 10% wettable powder (WP), deltamethrin 2.5% SC, and 2.5% WP on four types of circular blocks of wood, straw with mud, straw with mud painted with lime, and mud containing 5% of cement. Three concentrations of these insecticides were tested: the LC90 (previously determined on filter paper), the double of the LC90, and the recommended operational dose. For each bioassay test, 15 third-stage nymphs of Triatoma infestans (Klug) (Hemiptera: Reduviidae) were exposed for 120 h to each treatment at 24 h, 30, 60, 90, and 180 days post-spraying. Mortality rates, moulting history and behaviour were recorded at 24, 48, 72, and 120 h of exposure. Mortality rates were highest during the first 30 days post-spraying. Highest mortality rates (above 50%) were observed for deltamethrin 2.5% SC and lambda-cyhalothrin 10% WP on wood blocks up to three months post-spraying. Mud was the substrate on which treatments showed lowest persistence, with the other two substrates showing intermediate residual efficacy of all treatments. During the first 30 days WP formulations were not as effective as SC flowable formulations but, overall in the longer term, WP gave grater mortality rates of T. infestans nymphs exposed at up to six months post-spraying. Porous surfaces, especially mud, showed most variability presumably due to absorption of the insecticide. In contrast the less porous surfaces (i.e. wood and lime-coated mud) kept mortality rates high for longer post-treatment, irrespective of the insecticide concentration used.

Molecular cloning and sequencing of salivary gland-specific cDNAs of the blood-sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae).

Haematophagous insects produce pharmacological substances in their saliva to counteract vertebrate host haemostasis events such as coagulation, vasoconstriction and platelet aggregation. To investigate the bioactive salivary molecules of the triatomine bug Triatoma brasiliensis, we produced subtraction-enriched cDNAs of salivary-gland specific genes using suppression subtractive hybridization. Six full-length differentially expressed cDNAs (Tb113, Tb125, Tb152, Tb169, Tb180 and Tb198) were selected, cloned and sequenced. Sequence similarity searches of the databases using the putative amino acid sequence of our clones gave the following results: Tb152 - Triabin, an antithrombin induced platelet aggregation factor found in salivary gland extracts of T. pallidipennis. Tb169 - Pallidipin, an anticollagen induced platelet aggregation factor also found in T. pallidipennis salivary homogenates. Tb180 - Procalin, the major allergen of T. protracta saliva. The other three salivary-gland specific cDNAs produced no obvious homologies. Comparison of these salivary gland-specific cDNAs of with those of other triatomines combined with functional studies using recombinant proteins will allow a better understanding of the co-evolutionary process occurring between these insects and their vertebrate hosts, and may also lead to the discovery of novel antihaemostatic agents.

An immunocytochemical investigation of trypsin secretion in the midgut of the stablefly, Stomoxys calcitrans.

Musca domestica trypsin antibody cross-reacts with polypeptide bands of M(r) 25,000 and 30,000 showing proteolytic activity from Stomoxys calcitrans midgut extracts. Secretory granules from the main enzyme-secreting region, the opaque zone, stained heavily with the trypsin antibody in both unfed and blood-fed flies. Heterogeneous staining of granules suggests the unequal distribution of trypsin in secretory granules. This is also consistent with the occurrence of non-parallel secretion, which is also suggested by the possible preferential release of smaller, heavily stained secretory granules in fed flies. The predigestive, anterior midgut region responsible for rapid dehydration of the blood meal, the reservoir zone, contains a different population of secretory granules which stain heavily with trypsin antibody. This zone contains 20% of the midgut trypsin activity in unfed flies; trypsins are held here as proenzymes which are probably only activated postsecretion. In the midgut lumen of both unfed and blood-fed flies, trypsin is mainly immunolocalized in the ectoperitrophic space. Enzyme assays suggest that 5-15% of the lumenal trypsin is associated with the peritrophic matrix. The finding of intact secretory granules plus cell debris in the ectoperitrophic space of opaque and lipoid zones of blood-fed flies supports the contention that some trypsin is released by apocrine secretion in this insect.

Dispersive flight by Triatoma infestans under natural climatic conditions in Argentina.

Male and female T. infestans were released on two consecutive nights in the salinas of Cordoba Province, Argentina, when air temperatures during the flying period averaged 28.5 degrees C. 136 males (43% of the total released) and 170 females (57%) flew on the first night, and 6 males (18%) and 7 females (27%) on the second. Of these, we recovered 23 males and 14 females within 100 m, and a further female within 200 m, all from the first night's release. The remainder appear to have flown more than 100 m and possibly more than 200 m. In support of this conclusion 4 male and 5 female bugs were recovered in an abandoned brick house 550 m from the release point. Since the proportion of bugs apparently flying more than 200 m is considerably greater than that deduced from previous experiments at lower temperatures (Lehane & Schofield, 1981) it is possible that temperature influences not only the proportion of bugs flying but also the distance flown. Flight appears to be affected by wind speed. On the first night, when there was negligible wind, 43% of male and 57% of female bugs flew. On the second night, with winds gusting at 4-5 m/s, only 18% of the male and 27% of the female bugs flew.

Field experiments of dispersive flight by Triatoma infestans.

Using marked male Triatoma infestans allowed to escape and fly on a little-used airport in Brazil and salt flats in Argentina, 33 of 169 flew in the former and 28 of 528 in the latter experiment. Many of the bugs which flew had been classed as poorly fed. The proportion of bugs which flew was in reasonable agreement with predictions based on laboratory observations. It was shown that in the field male bugs are capable of flights in excess of 100 m. The method described served the purpose of this experiment well but would not be suitable for distances greater than 100 to 200m.