Behavior of triatomines (Hemiptera, Reduviidae) vectors of Chagas' disease. II. Influence of feeding, lighting and time of day on the number of mating, mating speed and duration of copulation of Panstrongylus megistus (Burm, 1835) under laboratory conditions

To determine in influence of feeding, lighting and time of day on the copulating behavior of Panstrongylus megistus, 480 insect pairs were divided into four groups of 120 each and tested in the following respective situations: without food deprivation (F.D.), with five days of F.D., with ten days of F.D., and with 20 days of F. D. The tests were performed between 9:00 a.m. to 12:00a.m. and 7:00 p.m. to 10:00 p.m., with light (700-1400 lux) and in the dark (1.4-2.8 lux) and behavior was recorded by the time sampling technique. Mating spped (MS) and duration of copulation (DC) were also calculated for each situation. The maximum frequency of copulation was observed after five days of F.D., at night, in the dark (n = 16), and the minimum was observed for recently-fed pairs, at night, with light (n = 4). Males approached females more often than females approached males. MS was lowest in pairs with twenty days of F.D., at night, with light (X = 23.0 ± 16.0 minutes), and highest in recently-fed pairs, during the day, with light (X = 2.9 ± 2.5 minutes). DC was shortest in recently-fed insects, during the day, in the dark (X = 23.5 ± 6.7 minutes), and longest in recently-fed animals, at night, in the dark (X = 38.3 ± 6.9 minutes).

Vectors control importance on leishmaniasis transmission

We reviewed the control of transmission of leishmaniasis regarding chemotherapy, reservoirs elimination, vaccination and insect control through the use of chemical insecticides. We also discussed complementary measures like monitoring traps, impregnated bednets and curtains, repelents, pheromones, biological control, etc. A cost comparison of insecticide interventions through the use of products belonging to the four main chemical groups was also alone, comparing together conventional formulations versus a slow-release insecticide developed by the Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro. We finally did recommendations on the situation that would justify an insecticide intervention to control sandflies.

Interactions between parasites and insects vectors

This review stresses the importance of studies that will provide a basic understanding of the pathology of parasite-infected vector insects. This knowledge should be a vital component of the very focussed initiatives currently being funded in the areas of vector control. Vector fecundity reduction is discussed as an example of such pathology. Underlying mechanisms are being investigated in a model system, Hymenolepis diminuta-infected Tenebrio molitor and in Onchocerca-infected blackflies and Plasmodium-infected Anopheles stephensi. In all cases, host vitellogenesis is disrupted by the parasite and, in the tapeworm/beetle model, interaction between the parasite and the endocrine control of the insect's reproductive physiology has been demonstrated.

An ocellar "pupil" that does not change with light intensity, but with the insect age in Triatoma infestans

The simple eyes (ocelli) of recently emerged adult Triatoma infestans exhibit a narrow elongated "pupil", surrounded by a ring of brown-reddish pigment, the "iris". This pupil does not respond to changes in the illumination, but varies in size after the imaginal ecdysis. This change corresponds, internally, with the growth of the corneal lens and the associated retina up to an age of about 20 days. This has not been previously observed in an insect. The use of this characteristic for recognising young adults of this species is suggested.

Transmission blocking vaccines to control insect-borne diseases: a review

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.

Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction.

Ionotropic glutamate receptors (iGluRs) are a highly conserved family of ligand-gated ion channels present in animals, plants, and bacteria, which are best characterized for their roles in synaptic communication in vertebrate nervous systems. A variant subfamily of iGluRs, the Ionotropic Receptors (IRs), was recently identified as a new class of olfactory receptors in the fruit fly, Drosophila melanogaster, hinting at a broader function of this ion channel family in detection of environmental, as well as intercellular, chemical signals. Here, we investigate the origin and evolution of IRs by comprehensive evolutionary genomics and in situ expression analysis. In marked contrast to the insect-specific Odorant Receptor family, we show that IRs are expressed in olfactory organs across Protostomia--a major branch of the animal kingdom that encompasses arthropods, nematodes, and molluscs--indicating that they represent an ancestral protostome chemosensory receptor family. Two subfamilies of IRs are distinguished: conserved "antennal IRs," which likely define the first olfactory receptor family of insects, and species-specific "divergent IRs," which are expressed in peripheral and internal gustatory neurons, implicating this family in taste and food assessment. Comparative analysis of drosophilid IRs reveals the selective forces that have shaped the repertoires in flies with distinct chemosensory preferences. Examination of IR gene structure and genomic distribution suggests both non-allelic homologous recombination and retroposition contributed to the expansion of this multigene family. Together, these findings lay a foundation for functional analysis of these receptors in both neurobiological and evolutionary studies. Furthermore, this work identifies novel targets for manipulating chemosensory-driven behaviours of agricultural pests and disease vectors.

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained. (c) 2006 Elsevier Inc. All rights reserved.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

ANTS AS POTENTIAL VECTORS OF PATHOGENS IN HOSPITALS IN THE STATE OF SAO-PAULO, BRAZIL

Baiting studies performed in large, medium and small hospitals in Brazil revealed the presence of 14 ant species, with up to nine recorded in one hospital. Dominant species were exotic ants, and in the large hospital, Tapinoma melanocephalum was the most prevalent. Ants were not uniformly spread through the hospitals, but tended to be found in the more critical areas, particularly in nursery, intensive care, obstetrics, neurology and dermatology units. Bacteriological studies using specific media for bacteria associated with intra-hospital infections indicated the potential for the mechanical vectoring of species of Staphylococcus, Serratia, Klebsiella, Acinetobacter, Enterobacter, Candida and Enterococcus by ants. Although T. melanocephalum did not have the highest rate of association with these bacteria, its ubiquitous occurrences resulted in the highest overall potential as a vector of these bacteria. Because of a large number of ant species occurring in Brazilian hospitals, ants pose a potential problem to the spread of diseases in hospitals. Because of the number of associated ant species in hospitals, the control of this potential problem is much more difficult than in registered temperate areas.

Subacute toxicity assessment of diflubenzuron, an insect growth regulator, in adult male rats

Difubenzuron (DFB), an insecticide and acaricide insect growth regulator, can be used in agriculture against insect predators and in public health programs, to control insects and vectors, mainly Aedes aegypti larvae. Due to the lack of toxicological assessments of this compound, the objective of the present study was to evaluate the toxicological effects of subacute exposure to the DFB insecticide in adult male rats. Adult male rats were exposed (gavage) to 0, 2, 4, or 8 mg/kg of DFB for 28 days. No clinical signs of toxicity were observed in the DFB-treated animals of the experimental groups. However, there was an increase in serum levels of alanine aminotransferase in the group that received 8 mg/Kg/DFB/day and urea at doses of 4 and 8 mg/Kg/DFB/day, without altering other biochemical or hematological parameters. The subacute exposure to the lowest dose of DFB caused significant decrease in testis weight, daily sperm production, and in number of sperm in the epididymis in relation to the control group. However, no alterations were observed in the sperm morphology, testicular, epididymis, liver and kindney histology, or testosterone levels. These findings unveiled the hazardous effects of DFB on male reproduction after the subacute exposure and special attention should be addressed to the effects of low doses of this pesticide.

Morphometrical diagnosis of the malaria vectors Anopheles cruzii, An. homunculus and An. bellator

Background: Anopheles (Kerteszia) cruzii is a primary vector of Plasmodium parasites in Brazil's Atlantic Forest. Adult females of An. cruzii and An. homunculus, which is a secondary malaria vector, are morphologically similar and difficult to distinguish when using external morphological characteristics only. These two species may occur syntopically with An. bellator, which is also a potential vector of Plasmodium species and is morphologically similar to An. cruzii and An. homunculus. Identification of these species based on female specimens is often jeopardised by polymorphisms, overlapping morphological characteristics and damage caused to specimens during collection. Wing geometric morphometrics has been used to distinguish several insect species; however, this economical and powerful tool has not been applied to Kerteszia species. Our objective was to assess wing geometry to distinguish An. cruzii, An. homunculus and An. bellator. Methods: Specimens were collected in an area in the Serra do Mar hotspot biodiversity corridor of the Atlantic Forest biome (Cananeia municipality, State of Sao Paulo, Brazil). The right wings of females of An. cruzii (n= 40), An. homunculus (n= 50) and An. bellator (n= 27) were photographed. For each individual, 18 wing landmarks were subjected to standard geometric morphometrics. Discriminant analysis of Procrustean coordinates was performed to quantify wing shape variation. Results: Individuals clustered into three distinct groups according to species with a slight overlap between representatives of An. cruzii and An. homunculus. The Mahalanobis distance between An. cruzii and An. homunculus was consistently lower (3.50) than that between An. cruzii and An. bellator (4.58) or An. homunculus and An. bellator (4.32). Pairwise cross-validated reclassification showed that geometric morphometrics is an effective analytical method to distinguish between An. bellator, An. cruzii and An. homunculus with a reliability rate varying between 78-88%. Shape analysis revealed that the wings of An. homunculus are narrower than those of An. cruzii and that An. bellator is different from both of the congeneric species. Conclusion: It is possible to distinguish among the vectors An. cruzii, An. homunculus and An. bellator based on female wing characteristics.

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.