Genetic variability and differentiation between populations of Rhodnius prolixus and R. pallescens, vectors of Chagas' Disease in Colombia

Enzyme polymorphism in Rhodnius prolixus and R. pallescens (Hemiptera, Reduviidae), principal vectors of Chagas' disease in Colombia, was analyzed using starch gel electrophoresis. Three geographic locations were sampled in order to determine gene flow between populations and to characterize intra- and interspecific differences. Of 25 enzymes assayed 10 were successfully resolved and then used to score the genetic variation. The enzymes PEPD, GPI, PGM and ICD were useful to differentiate these species and PGD, PGM and MDH distinguished between sylvatic and domiciliary populations of R. prolixus. Both polymorphism and heterozygosity indicated greater genetic variability in sylvatic habitats (H = 0.021) compared to domiciliary habitats (H = 0.006) in both species. Gene flow between sylvatic and domiciliary populations in R. prolixus was found to be minimal. This fact and the genetic distance between them suggest a process of genetic isolation in the domiciliary population.

Analysis of genetic relatedness between populations of Aedes aegypti from different geographic regions of São Paulo state, Brazil

RAPD markers have been used for the analysis of genetic differentiation of Aedes aegypti, because they allow the study of genetic relationships among populations. The aim of this study was to identify populations in different geographic regions of the São Paulo State in order to understand the infestation pattern of A. aegypti. The dendrogram constructed with the combined data set of the RAPD patterns showed that the mosquitoes were segregated into two major clusters. Mosquitoes from the Western region of the São Paulo State constituted one cluster and the other was composed of mosquitoes from a laboratory strain and from a coastal city, where the largest Latin American port is located. These data are in agreement with the report on the infestation in the São Paulo State. The genetic proximity was greater between mosquitoes whose geographic origin was closer. However, mosquitoes from the coastal city were genetically closer to laboratory-reared mosquitoes than to field-collected mosquitoes from the São Paulo State. The origin of the infestation in this place remains unclear, but certainly it is related to mosquitoes of origins different from those that infested the West and North region of the State in the 80's.

Genetic variability of Triatoma flavida and Triatoma bruneri (Hemiptera: Reduviidae) by RAPD-PCR technique

The Triatominae (Hemiptera:Reduviidae) contains the principal and potential Chagas disease vectors present in Mexico, Central America and South America. Triatoma flavida and T. bruneri are Cuban species. These species are closely related according to morphology and were considered synonyms until 1981, when they were separated on the grounds of external characters of the body and the morphology of male genitalia. The present study seeks to analyze genetic polymorphism of T. flavida and T. bruneri populations using RAPD techniques, and to assess the genetic relationship between these species. Ten random primers were used to evaluate the genetic variability among species using RAPD-PCR. The genetic flow among them was calculated. The dendrogram based on calculated Jaccard distances showed two clearly distinguishable clusters which coincided with the studied species. Within each species, moderate genetic differentiation (Fst 0.05-0.15) and migration rates (N > 1) were found among populations, that reveal gene flow and genetic homogeneity. Between species, the Fst value showed a high genetic differentiation and the migration rate was insufficient to maintain genetic homogeneity, and confirmed the absence of gene flow between them. Our results confirm the genetic variability among T. flavida and T. bruneri species.

Genetic Control of Mosquitoes: population suppression strategies

Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquito's great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.

Induction of male sterility through manipulation of genetic mechanisms present in vector species of Triatominae. II. Partial restoration of male fertility

The development of integrated measures which involve sterile mate release to supplement the conventional insecticidal techniques used in controlagainst insects of medical importance, raised the question, whether the vectors of Chagas'disease possess the natural mechanisms by manipulation of which they may be controlled. Results of earlier expenments, that had been published previously, were restricted to fragmentary information that raised various questions, the answer to which became available in the study herein described. Interspecific hybrids were produced from reciprocal crosses between T. pseudomaculata and T. sórdida and from unilateral crosses between female T. pseudomaculata and male. T. infestans. These females mated with males, laid less than the normal complement of eggs, but offspring was relatively abundant. When T. pseudomaculata females were paired with T. brasiliensis males, hybridization was more difficult because few of the females mated and those that did had a strongly reduced fertility. Adults emerged from ali crosses but exhibited sex disproportion, females predominating in all populations but one. The two Rhodnius species tested were also found to cross, but only when female R. prolixus were paired with male R. neglectus. These females laid a relatively high complement o f eggs, had a strongly reduced fertility, but 50% of the fertile eggs developed into vigorous adults, males predominating females. Neither type of hybrid male elicited fertilized eggs from either parental type of female, through their vesicula seminal is were found to be packed with spermatozoa, some normal looking and moving, others underdeveloped and motionless. Although, no artificial insemination was performed, the sperm in itself did not appear to be the prime inducer of sterility. Females paired with these hybrids did mate, sperm was transfered, as evidenced by the discharged spermatophores smeared with sperm, but did notcontain spermatozoa in their spermatecae. The failure of the sperm to migrate to the spermatecae indicate prezygotic pos-copulation incompatibility, thus the hybrid male can't be used to suppress populations. The female hybrids mated with parent males of either species had reduced fertility and ther sons were sterile as were those of their fertile daughters. However, continous backcrossing of the hybrid females and their female progeny to parental males partially restored fertility of the males and increased fertility of females, as scored by egg hatchability. Fertility of hybrid females, measured by the yield of adults capable to reproduce, indicated that the reproductive perfomance decreased when hybrid females and their daughters were backcrossed additional generations to parental males. It is tentatively suggested that hybrid females could be used for suppression if they compete efficiently with wild females.

High similarity of Trypanosoma cruzikDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

IntroductionDetermining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease.MethodsAn epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture).ResultsT. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected.ConclusionsLSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

Molecular biological approaches to the study of vectors in relation to malaria control

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology wich is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress wich has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

Integrated Genetic Epidemiology of Infectious Diseases: The Chagas Model

Genetic typing of pathogenic agents and of vectors has known impressive developments in the last 10 years, thanks to the progresses of molecular biology, and to the contribution of the concepts of evolutionary genetics. Moreover, we know more and more on the genetic susceptibility of man to infectious diseases. I propose here to settle a new, synthetic field of research, which I call `integrated genetic epidemiology of infectious diseases' (IGEID). I aim at evaluating, by an evolutionary genetic approach, the respective impact, on the transmission and pathogenicity of infectious diseases, of the host's, the pathogen's and the vector's genetic diversity, and their possible interactions (co-evolution phenomena). Chagas' disease constitutes a fine model to develop the IGEID methodology, by both field and experimental studies.

Fipronil Insecticide: Novel Application against Triatomine Insect Vectors of Chagas Disease

We investigated the efficacy and the residual effect of fipronil® against two species of triatomine bugs, Triatoma infestans and Rhodnius neglectus, in laboratory conditions measuring concentration-response and residual activity on different surfaces (dried mud and lime coated mud). Lethal concentrations (LC50,90) were determined on filter paper. The higher insecticide efficacy against R. neglectus when compared to T. infestans may be partially attributed to the differences in their biological cycles and genetic structures. Comparison with lambdacyhalothrin wettable powder showed that fipronil mortality rates (above 50%) were observed on mud blocks and lime-coated mud blocks up to 3 months when fipronil was sprayed at 100 and 200 mg a.i./m². Residual effect deeply decayed after 3 months; and at 6 months post treatment mortality was not observed. In contrast, lambdacyhalothrin showed a long lasting residual effect on both surfaces up to 6 months. Also, it should be mentioned that fipronil had a slow, but lethal activity on the triatomine bugs when wettable formulations were used on porous surfaces.

HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection.

Genetic engineering of baker's and wine yeasts using formaldehyde hyperresistance-mediating plasmids

Yeast multi-copy vectors carrying the formaldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker's and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use

Main features of DNA-based immunization vectors

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Regulation of transgene expression in genetic immunization

The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-g on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.