Single Nucleotide Polymorphism Markers in Aedes aegypti Populations in Brazil

Single nucleotide polymorphisms (SNPs) were investigated in eighteen genes of sixteen populations of Aedes aegypti in Brazil. Eight SNP markers were selected in nine genes and surveyed in A. aegypti populations of three localities in different geographical locations. SNPs revealed significant genetic differentiation among populations recently analyzed by mitochondria DNA (mtDNA) and represented by a single genetic group (lineage). Results suggest that a haplotype derived from mtDNA analysis could be represented by different Aedes lineages revealed by SNP characterization. Genetic distances (pairwise F(ST)), AMOVA and cluster analyses indicated a high genetic structure for the A. aegypti populations investigated by SNPs. This set of SNP markers represents a useful tool for genetic studies in A. aegypti populations

Characterization of eight single nucleotide polymorphism markers in Aedes aegypti

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Morphometric changes associated with sex and development in the Malpighian tubules of Aedes aegypti

The Malpighian tubules of Aedes aegypti showed significant differences in their diameters between male and female larvae, male and female pupae, male larvae and male adults and male pupae and male adults. in every case, female values were greater than in males. Measurements of mean nuclear areas of the principal and stellate cells from Malpighian tubules, taken in males and females during development, showed that this parameter in both types of cell was significantly greater in females than in male larvae, pupae and adult stages. In males, significant differences between developmental stages were observed only in comparison with the nuclear area of larvae and adults in the principal cells, but in females, every comparison between stages showed significant differences except between pupae and adults in stellate cells. The frequency distribution of nuclear area values, in development, for male stellate and principal cells, were mostly concentrated in the first seven classes among the 30 classes considered in every stage, while for females, the frequency dropped drastically in the same classes from larvae to pupae and adults, moving to classes of higher values. Considering the importance of Malpighian tubules in insect physiology, the meaning of the differences detected are discussed on the basis of different metabolic levels, between sexes and developmental stages.

Effect of phenobarbital on inducing insecticide tolerance and esterase changes in Aedes aegypti (Diptera: Culicidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variation of the oviposition preferences of Aedes aegypti in function of substratum and humidity

Two Aedes aegypti (L.) populations were studied in the laboratory regarding the preference for three types of breeding sites, i.e., flasks containing only water, flasks with a plant and flasks with a stick. Each of these breeding units was placed in one cage and the choice of the oviposition sites was determined for individual females and three females per experimental unit at two humidity levels. Preference for ovipositing on the water surface was observed and varied according to experimental unit and humidity. Mean hatching of eggs in water surface was 46.6%. Experiments with three females showed a more marked difference than when only one female was used. Inter and intrapopulation variability regarding oviposition sites was observed. The discrimination between the different oviposition substrates, hatching in water surface and its implication for mosquito control are discussed.

Effects of caffeine and used coffee grounds on biological features of Aedes aegypti (Diptera, Culicidae) and their possible use in alternative control

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Chitinolytic activities in the gut of Aedes aegypti (Diptera : Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut. (C) 2003 Elsevier B.V. All rights reserved.

X chromosome heterochromatic pattern and nucleolar synthesis in pupal ovaries of Aedes aegypti

C-banding and silver-staining techniques were used to examine pupal ovaries of Aedes aegypti from Sao Jose do Rio Preto (Brazil). Silver staining in ovary cystocytes showed two basic patterns relative to the nucleolar morphology: viz (1) a single, compact small body; and (2) multiple bodies encompassing large nuclear areas. These two types of cystocytes were present in the ratio of 7:1, which is the same as the number of nurse cells and oocytes, respectively, in each follicle. This suggests the possibility of eventually using such a nucleolar morphological difference to recognize both cell types in developmental stages before emergence. Silver nitrate staining in metaphase chromosomes revealed centromeric bands on all six chromosomes. The C-banding pattern in metaphase chromosomes showed an intercalary band in one of the X arms, as described previously in other populations. In ovary cystocytes (pachytene stage) this C-positive band seemed to consist of two chromomeres. Phase contrast microscopy showed that the nucleolus was associated with the distal chromomere of this intercalary C-band, indicating that the nucleolus organizer region was located in that part of the heterochromatic band.

Genetic variation along time in a brazilian population of Aedes aegypti (Diptera : Culicidae), detected by changes in the esterase patterns

Aedes aegypti from the Brazilian cities of Sao Jose do Rio Preto (SJ) and Goiania (GO) were analyzed as to their esterase patterns and the results were compared with data obtained about 5 years before for SJ population. Esterase bands not detected in the previous study were now observed in mosquitoes from both SJ and GO populations, being the last considered a population resistant to insecticides. Other similarities between SJ and GO populations in this study, and some differences in comparison with the previous data on SJ were observed, involving, in addition to changes in band type, changes in frequency of mosquitoes expressing them and differential gene activation during development. As it is generally true for genetic features, changes in the esterase patterns are expected to be the result of factors such as selection by environmental conditions and genetic drift. In the present case, continuous use of insecticides aiming mosquito population size control in SJ by sanitary authorities could be involved in the observed changes. Changed esterases were classified as carboxylesterases and cholinesterases, which are enzymes already shown to take part in the development of resistance in several organisms. In addition, data obtained in the elapsed time by authorities responsible for the mosquito control has shown increasing insecticide resistance of SJ population mosquitoes parallel to increase in the total amount of esterases, reinforcing the mentioned possibility.

Heterochromatic banding pattern in two Brazilian populations of Aedes aegypti

We analysed samples of Aedes aegypti from Sao Jose do Rio Preto and Franca (Brazil) by C-banding and Ag-banding staining techniques. C-banding pattern of Ae. aegypti from Sao Jose do Rio Preto examined in metaphase cells differed from Franca. The chromosomes 2, 3 and X showed centromeric C-bands in both populations, but a slightly stained centromeric band in the Y chromosome was observed only in Sao Jose do Rio Preto. In addition, the X chromosome in both populations and the Y chromosome of all individuals from Sao Jose do Rio Preto showed an intercalary band on one of the arms that was absent in Franca. An intercalary, new band, lying on the secondary constriction of chromosome 3 was also present in mosquitoes of both populations. The comparison of the present data with data in the literature for Ae. aegypti from other regions of the world showed that they differ as to the banding pattern of sex chromosomes and the now described intercalary band in chromosome 3. The observations suggested that the heterochromatic regions of all chromosomes are associated to constitute a single C-banded body in interphase cells. Ag-banding technique stained the centromeric regions of all chromosomes (including the Y) and the intercalary C-band region of the X chromosome in both populations. As Ae. aegypti populations are widespread in a great part of the world, the banding pattern variations indicate environmental interactions and may reveal both the chromosome evolutionary patterns in this species and the variations that may interfere with its vector activity.

Variation of genetic expression during development, revealed by esterase patterns in Aedes aegypti (Diptera, Culicidae)

Polyacrylamide gel electrophoresis was used to analyze esterase patterns during development of Aedes aegypti from the cities of Marília and São José do Rio Preto (SJRP), Brazil. The zymograms showed a total of 23 esterase bands, 22 of which were in the specimens from Marília and 19 in those from SJRP. These esterase bands were considered to be the product of 23 alleles distributed tentatively in eight genetic loci. Most of the alleles were developmentally regulated. The larval stage expressed the greatest number of them (19 alleles, from the eight loci, in Marília; and 17 alleles, from seven loci, in SJRP). The pupal stage expressed 10 alleles from seven loci, in both populations, and the adult stage expressed 8 alleles from five and six loci in SJRP and Marília, respectively. Some alleles that were active in every stage were developmentally controlled at the level of expression (amount of product). A single allele was constitutively and highly expressed, in larvae, pupae, and adults, in both populations. Differences in esterase synthesis among stages are probably due to regulatory mechanisms acting in agreement with the requirements of a variable number of processes in which esterases are involved. The larval stage is the most active in developmental processes and shows very intense intake of food and very high mobility. These features may demand increased esterase production at that stage. Comparison of the two populations examined showed (besides the existence of alleles that they do not share) that they exhibit differences in the control of expression of other alleles. Such findings may reflect genetic differences between founders in each population, but the possibility of involvement of the intensive use of insecticides in SJRP is also discussed.

Standardization of bioassays for monitoring resistance to insecticides in Aedes aegypti

Since the reintroduction of Aedes aegypti in Brazil in the 1980s, insecticide use for its control is routine. The chemical control efficacy is threatened by vectors developing resistance to insecticides. The World Health Organization, recognizing the impact of insecticide resistance in vector control programmes, proposed standardizing bioassays for detecting and monitoring resistance using a diagnostic dose method. As Brazil has a national programme for monitoring the resistance of Ae. aegypti populations to insecticides, this study was designed to compare diagnostic bioassays at WHO suggested concentrations and those estimated for local conditions. Populations were resistant to both temephos doses. But important differences were seen for fenitrothion and malathion, which could lead to under- or over-estimation of resistance respectively. These results and inclusion of a diagnostic dose bioassay standard for larvae are discussed.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): New perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Efficacy of topical permethrin as repellent against aedes aegypti's bites

Mosquitoes are the most important vectors of infectious diseases and their bites are related to several adverse skin reactions. Permethrin impregnated clothes are an efficient strategy against arthropods' bites; however, its topical efficacy as a repellent has not been well established. We studied the response to permethrin lotion 5 percent and N,N-Diethyl-meta-toluamide (DEET) spray 50 percent applied to the unprotected forearms of 10 volunteers. Each arm was exposed to 20 female mosquitoes of Aedes aegypti. We performed 71 bilateral comparative measurements evaluating the timing for the first bites. The average times for the arm without the product, with permethrin 5 percent, and with DEET 50 percent were: 7.9 seconds, 336.2 seconds and 7512.1 seconds. The results showed a significant difference between repellency times between either product and unprotected controls. In addition, there was a significant difference in time to first bite between permethrin and DEET treated arms (p<0.01). Permethrin affords some repellent activity against Aedes aegypti bites in this experimental setting. However, permethrin's profile of repellency was significantly inferior to that of DEET. © 2008 Dermatology Online Journal.