Marcelo S. Gonzalez

Effects of the essential oil obtained from Pilocarpus spicatus Saint-Hilaire (Rutaceae) on the development of Rhodnius prolixus nymphae

Bioassays against fifth-instar nymphae of Rhodnius prolixus were conducted with essential oil of Pilocarpus spicatus extracted by hydrodistillation. The main results may be summarized as follows: (i) high levels of toxicity and paralysis together with discrete moulting inhibition were caused by topical application of either 0.5 µL or 1.0 µL per insect of the crude essential oil; (ii) partial fagoinhibition, high moulting inhibition, prolonged intermoulting period and high number of paralyzed insects, but no toxicity were observed after oral treatment using either 5 µL or 10 µL of Pilocarpus spilcatus essential oil per mL of ingested blood meal. The importance of these results in relation to the relevant biological events in R. prolixus is herein discussed.

Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM), and produced large vacuoles and small electrondense granules; insects fed on Ringers saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringers saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringers saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringers saline meal with ecdysone; Ringers saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.

Cytochemical characterization of microvillar and perimicrovillar membranes in the posterior midgut epithelium of Rhodnius prolixus

Perimicrovillar membranes (PMM) are structures present on the surface of midgut epithelial cells of the hematophagous insect, Rhodnius prolixus. They cover the microvilli and are especially evident 10 days after blood meal, providing the compartmentalization of the enzymatic processes in the intestinal microenvironment. Using an enzyme cytochemical approach, Mg2+-ATPase and ouabain-sensitive Na+K+-ATPase activities were observed in the plasma (or microvillar) membrane (MM) of midgut cells and in the PMM. In contrast, alkaline phosphatase was only detected in MM. Using cationized ferritin and colloidal iron hydroxide particles, anionic sites were found only on the luminal surface of the PMM. Using fluorescein isothiocyanate (FITC)-labeled lectins, residues of alpha-d-galactose, mannose, N-acetyl-neuraminic acid, N-acetyl-d-galactosamine and N-acetyl-galactosamine-alpha-1,3-galactose were detected on the apical surface of posterior midgut epithelial cells. On the other hand, using FITC-labeled neoglycoproteins (NGP) it was possible to detect the presence of carbohydrate binding molecules (CBM) recognizing N-acetyl-d-galactosamine, alpha-d-mannose, alpha-l-fucose and alpha-d-glucose in the posterior midgut epithelium. The use of digitonin showed the presence of sterols in the MM and PMM. These results have led the authors to suggest that for some components the PMM resembles the MM lining the midgut cells of R. prolixus, composing a system which covers the microvilli and stretches to the luminal space.

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

Biological factors involving Trypanosoma cruzi life cycle in the invertebrate vector, Rhodnius prolixus

Trypanosoma cruzi, the ethiologic agent ofChagas disease (Chagas 1909), is transmittedthrough triatomine vectors during the meal on theinvertebrate host. The parasite displays quite dis-tinct morphological and functional forms, alternat-ing between replicative stages (epimastigotes in thevector midgut and amastigotes in vertebrate cells)and infective, non-dividing forms (metacyclictrypomastigotes in the vector, which are depositedtogether with feces and urine, and bloodstreamtrypomastigotes in mammals) (for revision seeBrener 1973, Zeledon 1974, Garcia & Azambuja1991, 1996, Gonzalez et al. 1998a).Because of the importance of Chagas diseasein Latin America a great effort has been devotedto develop an experimental model that could imi-tate basic features on T. cruzi life cycle in the in-vertebrate vector. Thus, we developed in our labo-ratory the Rhodnius prolixus model to study sev-eral parameters related to the high degree of inter-action between T. cruzi and invertebrate vector.Basically, this model implies in feeding of larvaeand adults of R. prolixus, through a special mem-brane feeding apparatus, on blood containing dif-ferent strains/clones of T. cruzi and, at differentintervals, determination of the number of parasitein the gut, urine and feces of the vector (see Garcia& Azambuja 1991, 1996, Gonzalez et al. 1998a,1999). Herein, we will describe experiments, us-ing this useful parasite-vector model, to elucidatesome mechanisms involved in the interaction T.cruzi-triatomine insect and also link insect factorswith the success or the failure of strains/clones ofparasites to establish the infection in the digestivetract of the vector.

Effects of azadirachtin on the development and mortality of Lutzomyia longipalpis larvae (Diptera: Psychodidae: Phlebotominae).

Abstract The effects of azadirachtin A added to the standard diet on the development, mortality, and metamorphosis of Lutzomyia longipalpis Lutz & Neiva, 1912 were studied. Concentrations of 0.1, 1.0, and 10.0 μg of azadirachtin/mg of diet significantly increased larval mortality in comparison with nontreated insects. Concentrations 0.1 and 1.0 μg blocked the molt of larvae, which remained as third instars until the end of the experiment. The 10 μg/mg concentration resulted in greater molt inhibition. In this group, all insects stopped their development as second instars. Simultaneous addition of ecdysone (1 μg/mg) to the standard diet containing azadirachtin counteracted the effects of azadirachtin on mortality and inhibition of ecdysis. These results indicate that azadirachtin is a potent growth inhibitor of L. longipalpis.

Development of an insecticidal nanoemulsion with Manilkara subsericea (Sapotaceae) extract

BackgroundPlants have been recognized as a good source of insecticidal agents, since they are able to produce their own defensives to insect attack. Moreover, there is a growing concern worldwide to develop pesticides with low impact to environment and non-target organisms. Hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea and its triterpenes were considered active against a cotton pest (Dysdercus peruvianus). Several natural products with insecticidal activity have poor water solubility, including triterpenes, and nanotechnology has emerged as a good alternative to solve this main problem. On this context, the aim of the present study was to develop an insecticidal nanoemulsion containing apolar fraction from fruits of Manilkara subsericea.ResultsIt was obtained a formulation constituted by 5% of oil (octyldodecyl myristate), 5% of surfactants (sorbitan monooleate/polysorbate 80), 5% of apolar fraction from M. subsericea and 85% of water. Analysis of mean droplet diameter (155.2 ± 3.8 nm) confirmed this formulation as a nanoemulsion. It was able to induce mortality in D. peruvianus. It was observed no effect against acetylcholinesterase or mortality in mice induced by the formulation, suggesting the safety of this nanoemulsion for non-target organisms.ConclusionsThe present study suggests that the obtained O/A nanoemulsion may be useful to enhance water solubility of poor water soluble natural products with insecticidal activity, including the hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea.

Triflumuron Effects on the Physiology and Reproduction of Rhodnius prolixus Adult Females

We evaluated the efficacy of the growth regulator triflumuron (TFM) in inducing mortality and disrupting both oviposition and egg hatching in Rhodnius prolixus adult females. TFM was administered via feeding, topically or by continuous contact with impregnated surfaces. Feeding resulted in mild biological effects compared with topical and impregnated surfaces. One day after treatment, the highest mortality levels were observed with topical surface and 30 days later both topical and impregnated surfaces induced higher mortalities than feeding. Oral treatment inhibited oviposition even at lower doses, and hatching of eggs deposited by treated females was similarly affected by the three delivery modes. Topical treatment of eggs deposited by nontreated females significantly reduced hatching. However, treatment per contact of eggs oviposited by untreated females did not disrupt eclosion. Additionally, oral treatment increased the number of immature oocytes per female, and topical treatment reduced the mean size of oocytes. TFM also affected carcass chitin content, diuresis, and innate immunity of treated insects. These results suggest that TFM acts as a potent growth inhibitor of R. prolixus adult females and has the potential to be used in integrated vector control programs against hematophagous triatomine species.

Effect of Fruit and Leaves of Meliaceae Plants (Azadirachta indica and Melia azedarach) on the Development of Lutzomyia longipalpis Larvae (Diptera: Psychodidae: Phlebotominae) Under Experimental Conditions

ABSTRACT This no-choice, laboratory study focuses on the feeding of dried, ground, homogeneous powdered, unprocessed fruit and leaves of Azadirachta indica and Melia azedarach to Lutzomyia longipalpis larvae to determine the effects on their mortality and metamorphosis. A. indica and M. azedarach fruit and leaves significantly increased larval mortality in comparison to larvae fed the untreated, standard diet. A. indica fruit and leaves blocked the molting of the larvae to the fourth instar, resulting in them remaining as third instars until the end of the experiment. M. azedarach fruit also blocked the molting of larvae, which remained permanently in the fourth instar. Feeding M. azedarach leaves resulted in greater molt inhibition. All insects in this group stopped their development as second-instar larvae. No antifeedant effect was detected for any experimental treatment. The results indicate that nontoxic, unprocessed materials obtained from A. indica and M. azedarach are potent development inhibitors of L. longipalpis larvae.

Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus nymph

Studies were carried out to evaluate the efficacy of the growth regulator, triflumuron (TFM) (Starycide sc 480 Bayer), for disrupting the development of Rhodnius prolixus fifth-instar nymph by oral, topical or continuous treatment. All treatments were able to induce high levels of mortality, delay development and molt inhibition. Oral treatment induced molt inhibition in all insects that survived at doses of 0.25, 0.50 and 5.0 mg/mL of a blood meal. The highest levels of both mortality in 24 h and molt inhibition were always observed after topical treatment. The lowest doses needed to obtain considerable biological effects were always observed after continuous treatment. In this way, the highest levels of mortality within 30 days were detected after continuous treatment, which also induced an extended inter-molting period, a lower number of over-aged nymphs and the highest level of molting in nymphs that survived. Moreover, the effects of TFM on insects were often displayed in a dose response manner. These results indicate that TFM acts as a potent growth inhibitor of R. prolixus nymphs and has the potential to be used in integrated vector control programs against hematophagous triatomine species.