Ecoepidemiology and biology of Eratyrus mucronatus Stål, 1859 (Hemiptera: Reduviidae: Triatominae), a sylvatic vector of Chagas disease in the Brazilian Amazon

Introduction Eratyrus mucronatus Stål, 1859 is a wild triatomine vector of Trypanosoma cruzi Chagas, 1909. However, little is known regarding the biology and ecoepidemiology of this triatomine in the Brazilian Amazon. The present study describes the biology of E. mucronatus grown under laboratory conditions and the epidemiological aspects of its natural breeding sites. Methods Five colonies were monitored in the field for 3 years. Temperature and humidity measurements were taken in the mornings and afternoons at the natural breeding sites, and the behavior and distribution of the nymphs and adults were observed in the wild colony. We also monitored the life cycle under controlled laboratory conditions. Results Some factors that were considered decisive for the establishment of these colonies were present at all of the colonies studied in the field. These factors included an active termite nest, a vertebrate for repast, and dry and shaded substrates with temperatures of 24-28°C and with humidity of 80-90%. A generation was developed in 274 days under these microclimatic conditions in the laboratory. Conclusions The climatic variables described in the field indicate that these environmental parameters have a limiting effect on the dispersal and colonization of E. mucronatus to new environments. In addition, the long period of development to adulthood demonstrates that only one generation can develop per year even under the more favorable laboratory conditions.

Reproductive isolation between different forms of Lutzomyia longipalpis (Lutz & Neiva), (Diptera: Psychodidae), the vector of Leishmania donovani chagasi Cunha & Chagas and its significance to kala-azar distribution in South America

The males of the sandfly Lutzomyia longipalpis occur in two forms, one which bears a single pair of pale spots on tergite 4 and another in which an additional pair of spots characterizes tergite 3. In crosses between laboratory reared stocks of the two forms originating from allopatric and sympatric sites in Brazil nearly all males of one form fail to inseminate females of the other. In addition, insemination failure between some allopatric populaytions of Lu. longipalpis with similar tergal spot patterns is recorded, indicating the existence of additional forms in an apparent species complex. The possibility that Lu. longipalpis sensu latu represents more than a single taxon is discussed and the relevance of these findings to future epidemiological studies on kala-azar is considered.

Vector incrimination and effects of antimalarial drugs on malaria transmission and control in the Amazon Basin of Brazil

World ecosystems differ significantly and a multidisciplinary malaria control approach must be adjusted to meet these requirements. These include a comprehensive understanding of the malaria vectors, their behavior, seasonal distribution and abundance, susceptibility to insecticides (physiological and behavioral), methods to reduce the numbers of human gametocyte carriers through effective health care systems and antimalarial drug treatment, urban malaria transmission versus rural or forest malaria transmission, and the impact of vaccine development. Many malaria vectors are members of species complexes and individual relationship to malaria transmission, seasonal distribution, bitting behavior, etc. is poorly understood. Additionaly, malaria patients are not examined for circulating gametocytes and both falciparum and vivax malaria patients may be highly infective to mosquitoes after treatment with currently used antimalarial drugs. Studies on the physiological and behavioral effects of DDT and other insecticides are inconclusive and need to be evalusted.

An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

Aggregation mediated by faeces and footprints in Triatoma pseudomaculata (Heteroptera: Reduviidae), a Chagas disease vector

The behavioural response of Triatoma pseudomaculata to chemical substances present in their faeces or cuticle (footprints) was analyzed. Groups of larvae were simultaneously exposed to a clean filter paper and to another paper impregnated with a chemical stimulus in a circular arena. In these choice experiments, the insects aggregated significantly around papers impregnated with dry faeces. In addition, the bugs also showed a significant aggregation response to papers impregnated with compounds derived from their cuticle that were deposited by contact on the substrate. These results indicate that chemical compounds that affect the behaviour of T. pseudomaculata are present in the faeces and in the cuticle of this species. Results are discussed in relation to chemical communication in the Triatominae, as well as to the potential use of these substances in traps or sensors for the detection of this species.

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Vector density and the control of kala-azar in Bihar, India

Bihar, India has been in the grip of kala-azar for many years. Its rampant and severe spread has made life miserable in most parts of the state. Such conditions require a comprehensive understanding of this affliction. The numbers coming out of the districts prone to the disease in the north and south Ganges have provided us with several startling revelations, as there are striking uniformities on both sides, including similar vegetation, water storage facilities, house construction and little change in risk factors. The northern areas have been regularly sprayed with DDT since 1977, but eradication of the disease appears to be a distant dream. In 2007 alone, there were as many as 37,738 cases in that region. In contrast, the southern districts of Patna and Nalanda have never had the disease in its epidemic form and endemic disease has been present in only some pockets of the two districts. In those cases, two rounds of spraying with DDT had very positive results, with successful control and no new established foci. In addition, an eleven-year longitudinal study of the man hour density and house index for the vector Phlebotomus argentipes demonstrated that they were quite high in Patna and Nalanda and quite low in north Bihar. Given these facts, an attempt has been made to unravel the role of P. argentipes saliva (salivary gland) in the epidemiology of kala-azar. It was determined that patchy DDT spraying should be avoided for effective control of kala-azar.

Anticoagulant activity in salivary gland homogenates of Thyrsopelma guianense (Diptera: Simuliidae), the primary vector of onchocerciasis in the Brazilian Amazon

In this study, anticoagulant activity was detected in salivary gland homogenates (SGHs) of Thyrsopelma guianense (Diptera: Simuliidae). The SGH yielded 1.07 μg ± 0.03 (n = 15) of total soluble protein per pair of glands. In addition, following SDS-PAGE (12.5% gel) and silver nitrate staining, 12 polypeptides with molecular weights ranging from 14-69 kDa were detected in all physiological ages analyzed (12 h, 24 h, 48 h and 72 h following emergence). Coagulation bioassays showed that the SGHs had activities that interacted at all levels of coagulation (the intrinsic, extrinsic and common pathways), by extending the plasma recalcification time, prothrombin time, thrombin time. This is the first report on the activity of salivary gland proteins from the main vector of onchocerciasis in Brazil. We also suggest detailed studies on the morphology and function of the salivary glands in order to understand the role of these proteins in host/vector interactions.

Multiple insecticide resistance in Aedes aegypti (Diptera: Culicidae) populations compromises the effectiveness of dengue vector control in French Guiana

In French Guiana, pyrethroids and organophosphates have been used for many years against Aedes aegypti. We aimed to establish both the resistance level of Ae. aegypti and the ultra low volume spray efficacy to provide mosquito control services with practical information to implement vector control and resistance management. Resistance to deltamethrin and fenitrothion was observed. In addition, the profound loss of efficacy of AquaK'othrine® and the moderate loss of efficacy of Paluthion® 500 were recorded. Fenitrothion remained the most effective candidate for spatial application in French Guiana until its removal in December 2010. Further investigation of the mechanism of resistance to deltamethrin demonstrated the involvement of mixed-function oxidases and, to a lesser extent, of carboxylesterases. However, these observations alone cannot explain the level of insecticide resistance we observed during tube and cage tests.

Progress with schistosome transgenesis

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.

Effect of cropping systems in no-till farming on the quality of a Brazilian Oxisol

The no-till system with complex cropping sequences may improve the structural quality and carbon (C) sequestration in soils of the tropics. Thus, the objective of this study was to evaluate the effects of cropping sequences after eight years under the no-till system on the physical properties and C sequestration in an Oxisol in the municipality of Jaboticabal, Sao Paulo, Brazil. A randomized split-block design with three replications was used. The treatments were combinations of three summer cropping sequences - corn/corn (Zea mays L.) (CC), soybean/soybean (Glycine max L. Merryll) (SS), and soybean-corn (SC); and seven winter crops - corn, sunflower (Helianthus annuus L.), oilseed radish (Raphanus sativus L.), pearl millet (Pennisetum americanum (L.) Leeke), pigeon pea (Cajanus cajan (L.) Millsp), grain sorghum (Sorghum bicolor (L.) Moench), and sunn hemp (Crotalaria juncea L.). Soil samples were taken at the 0-10 cm depth after eight years of experimentation. Soil under SC and CC had higher mean weight diameter (3.63 and 3.55 mm, respectively) and geometric mean diameter (3.55 and 2.92 mm) of the aggregates compared to soil under SS (3.18 and 2.46 mm). The CC resulted in the highest soil organic C content (17.07 g kg-1), soil C stock (15.70 Mg ha-1), and rate of C sequestration (0.70 Mg ha-1 yr-1) among the summer crops. Among the winter crops, soil under pigeon pea had the highest total porosity (0.50 m³ m-3), and that under sunn hemp had the highest water stable aggregates (93.74 %). In addition, sunn hemp did not differ from grain sorghum and contained the highest soil organic C content (16.82 g kg-1) and also had the highest rate of C sequestration (0.67 Mg ha-1 yr-1). The soil resistance to penetration was the lower limit of the least limiting water range, while the upper limit was air-filled porosity for soil bulk densities higher than 1.39 kg dm-3 for all cropping sequences. Within the SC sequence, soil under corn and pigeon pea increased least limiting water range by formation of biopores because soil resistance to penetration decreased with the increase in soil bulk density.

Physical Properties of a Hapludox after Three Decades under Different Soil Management Systems

ABSTRACT Changes in soil physical properties due to different management systems occur slowly, and long-term studies are needed to assess soil quality. The objectives of this study were to evaluate the effects of soil management systems and liming methods on the physical properties of a Latossolo Bruno Alumínico típico (Hapludox). A long-term experiment that began in 1978 with conventional and no-tillage systems was assessed. In addition, different liming methods (no lime, incorporated lime, and lime on the soil surface) have been applied since 1987 and were also evaluated in this study. Moreover, an area of native forest was evaluated and considered a reference for the natural condition of the soil. Soil physical properties were evaluated in layers to a depth of 1.00 m. Compared to native forest, the conventional tillage and no-tillage systems had higher soil bulk density, penetration resistance, and microporosity, and lower aggregate stability and macroporosity. Compared to the conventional tillage system, long-term no-tillage improved the structure of the Hapludox, as evidenced by increased microporosity and aggregate stability, especially in the soil surface layer. In no-tillage with lime applications sporadically incorporated, soil physical properties did not differ from no-tillage without lime and with lime applied on the soil surface, indicating that this practice maintains the physical quality of soil under no-tillage. Liming in a conventional tillage system improved soil aggregation and reduces penetration resistance in the soil layers near the soil surface. No-tillage was the main practice related to improvement of soil physical quality, and liming methods did not influence soil physical properties in this soil management system.

The challenge of vector development in gene therapy

Gene therapy is the treatment of diseases based on the transfer of genetic information. Agents that carry or deliver DNA to target cells are called vectors (Latin vector: carrier, deliverer). Ideally, a vector should accommodate an unlimited amount of inserted DNA, lack the ability of autonomous replication of its own DNA, be easily manufactured, and be available in concentrated form. Secondly, it should have the ability to target specific cell types or to limit its gene expression to specific cell types, and to achieve sustained gene expression in the long term or in a controlled fashion. Finally, it should not be toxic or immunogenic. Such a vector does not exist and none of the DNA delivery systems so far available for in vivo gene transfer is perfect with respect to any of these points. Gene therapy and the means to promote it depend heavily on the development and improvement of new gene vector systems.

DNA-lipid systems: A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.