Genotoxic and mutagenic effects of methyl ether dillapiole on the development of Aedes aegypti (Diptera: Culicidae).

Dillapiole, extracted from Piper aduncum essential oil and its derivatives, has been shown to be a potential alternative to the control of Aedes aegypti, which has become resistant to synthetic insecticides. Methyl ether dillapiole (MED) and temephos (TM) were compared to complement the data on the genotoxicity and developmental changes of Ae. aegypti. Over four generations (G1 -G4 ), third stage larvae were treated with MED at 60, 80 and 100 µg/mL and TM at 0.002, 0.005 and 0.007 µg/mL for 4 h. Adult females were separated to estimate oviposition and hatching rates, and total egg length. Over the four generations, a significant reduction was recorded in oviposition and hatching rates, and in mean egg length (Tukey, P < 0.05), compared with the negative control (NC). Cytological slide preparations were done from adult oocytes and larval neuroblasts. The cumulative effects of genotoxic (bridges, budding and nuclear fragmentation) and mutagenic (micronucleus and chromosomal breakage) damage was observed in the neuroblasts and oocytes of exposed mosquitoes. Developmental changes and damage to the genome of MED-treated Ae. aegypti were greater than those caused by TM. Further studies should focus on understanding the effects of the MED molecule on Ae. aegypti.

GNBP domain of Anopheles darlingi: are polymorphic inversions and gene variation related to adaptive evolution?

Anopheles darlingi is the main malaria vector in humans in South America. In the Amazon basin, it lives along the banks of rivers and lakes, which responds to the annual hydrological cycle (dry season and rainy season). In these breeding sites, the larvae of this mosquito feed on decomposing organic and microorganisms, which can be pathogenic and trigger the activation of innate immune system pathways, such as proteins Gram-negative binding protein (GNBP). Such environmental changes affect the occurrence of polymorphic inversions especially at the heterozygote frequency, which confer adaptative advantage compared to homozygous inversions. We mapped the GNBP probe to the An. darlingi 2Rd inversion by fluorescent in situ hybridization (FISH), which was a good indicator of the GNBP immune response related to the chromosomal polymorphic inversions and adaptative evolution. To better understand the evolutionary relations and time of divergence of the GNBP of An. darlingi, we compared it with nine other mosquito GNBPs. The results of the phylogenetic analysis of the GNBP sequence between the species of mosquitoes demonstrated three clades. Clade I and II included the GNBPB5 sequence, and clade III the sequence of GNBPB1. Most of these sequences of GNBP analyzed were homologous with that of subfamily B, including that of An. gambiae (87 %), therefore suggesting that GNBP of An. darling belongs to subfamily B. This work helps us understand the role of inversion polymorphism in evolution of An. darlingi.

Effect of isodillapiole on the expression of the insecticide resistance genes GSTE7 and CYP6N12 in Aedes aegypti from central Amazonia.

The yellow fever mosquito Aedes (Stegomyia) aegypti is the main vector of dengue arbovirus and other arboviruses. Dengue prevention measures for the control of A. aegypti involve mainly the use of synthetic insecticides. The constant use of insecticides has caused resistance in this mosquito. Alternative studies on plant extracts and their products have been conducted with the aim of controlling the spread of the mosquito. Dillapiole is a compound found in essential oils of the plant Piper aduncum (Piperaceae) which has been effective as a biopesticide against A. aegypti. Isodillapiole is a semisynthetic substance obtained by the isomerization of dillapiole. In the present study, isodillapiole was evaluated for its potential to induce differential expression of insecticide resistance genes (GSTE7 and CYP6N12) in 3rd instar larvae of A. aegypti. These larvae were exposed to this compound at two concentrations (20 and 40 µg/mL) for 4 h during four generations (G1, G2, G3, and G4). Quantitative RT-PCR was used to assess the expression of GSTE7 and CYP6N12 genes. GSTE7 and CYP6N12 relative expression levels were higher at 20 than at 40 µg/mL and varied among generations. The decrease in GSTE7 and CYP6N12 expression levels at the highest isodillapiole concentration suggests that larvae may have suffered from metabolic stress, revealing a potential alternative product in the control of A. aegypti.

Factors associated with changes in retinal microcirculation after antihypertensive treatment.

We report the results of hypertensive treatment over retinal arteriole narrowing in a group of 189 hypertensive patients during a 6-month treatment programme for hypertension. These patients were included in an observational study under clinical practice conditions and analysed using digital photographs of the eye fundus with a previously described semiautomatic snakes method. We analysed the relation between blood pressure control and retinal microcirculation changes during the treatment. We included 189 hypertensive patients; 74.0% of them had BP under control after 6 months of follow-up. The arteriovenous ratio (AVR), measured as the relation between the average retinal arteriole and venous diameter, significantly increased after 6 months of follow-up in both eyes (right eye AVR: 0.769±0.065 vs. 0.799±0.066 (P<0.0001); left eye AVR: 0.770±0.065 vs. 0.796±0.071 (P<0.0001)). AVR changes were caused by increases in arteriole diameter. No linear correlation was found between blood pressure-lowering levels and AVR increase, suggesting that other factors could be involved in the regression of retinal changes. We conclude that in our group of hypertensive patients it was possible to increase the retinal arteriole diameter, expressing an improvement in retinal microcirculation after 6 months of adequate BP control. Although there is an inverse tendency between blood pressure and arteriovenous ratio changes, we could not find a linear correlation between these changes.

Isolation and characterization of polymorphic DNA microsatellite loci for Anopheles triannulatus sensu lato (Diptera: Culicidae) and cross-amplification in congeneric species.

Anopheles (Nyssorhynchus) triannulatus is a complex of 3 species. Thirteen polymorphic microsatellite loci were isolated and characterized in 20 to 25 individuals from Manaus (AM, Brazil). The number of alleles per locus varied from 3 to 10 (mean = 6.0). The observed heterozygosity ranged from 0.250 to 0.875 (mean = 0.680) and expected heterozygosity ranged from 0.376 to 0.844 (mean = 0.698). Two loci exhibited null alleles and all loci were in Hardy-Weinberg equilibrium. No linkage disequilibrium between loci was observed. These loci were used in 4 congeneric species and provide a useful tool for studying population genetics and other aspects of the biology of this and other Anopheles species.

Chromosomal localization of actin genes in the malaria mosquito Anopheles darlingi.

Physical and genetic maps have been used for chromosomal localization of genes in vectors of infectious diseases. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to precisely map genes of interest. We report the physical mapping of two actin genes on polytene chromosomes of the major malaria vector in the Amazon, Anopheles darlingi (Diptera: Culicidae). Clones with actin gene sequences were obtained from a cDNA library constructed from RNA isolated from adult females and males of An. darlingi. Each of the two clones was mapped to a unique site on chromosomal arm 2L in subdivisions 21A (clone pl05-A04) and 23B (clone pl17-G06). The obtained results, together with previous mapping data, provide a suitable basis for comparative genomics and for establishing chromosomal homologies among major malaria vectors.

Isolation and characterization of 25 microsatellite DNA loci for Anopheles albitarsis sensu lato and inter-specific amplification in 5 congeneric species.

The Anopheles albitasis complex includes 6 species, and 3 are considered as malaria vectors in Brazil. Twenty-five polymorphic microsatellite DNA loci were isolated and characterized in 24-36 individuals from the neighborhood of Puraquequara, Manaus, Amazonas State, Brazil. The number of estimated alleles ranged from 2 to 10, the observed heterozygosity ranged from 0.182 to 0.897, and the expected heterozygosity ranged from 0.260 to 0.854. Eleven loci showed significant deviation from Hardy-Weinberg equilibrium. Eleven loci were cross-amplified successfully in 5 Anopheles species. These microsatellite loci will be useful in studies investigating population structure and evolutionary genetics in A. albitarsis sensu lato and other A. albitarsis complex species.

Potential control of Aedes aegypti (Diptera: Culicidae) with Piper aduncum L. (Piperaceae) extracts demonstrated by chromosomal biomarkers and toxic effects on interphase nuclei.

Dillapiol, a phenylpropanoid isolate from essential oils of leaves of Piper aduncum (Piperaceae), has insecticidal, fungicidal and antimicrobial activities. The insecticidal activity of dillapiol was tested in vivo on the larvae and pupae of Aedes aegypti, the mosquito vector of dengue. Specifically, the effect of dillapiol on the formation of micronuclei and chromosome aberrations was analyzed. Dillapiol treatments comprised two concentrations of 200 and 400 micro dissolved in well water, and a pure well water control used to rear four generations of mosquitoes. Micronuclei occurred in mitotic diploid and tetraploid chromosomes of larvae; nuclear abnormalities also occurred in interphase, metaphase, telophase, and single nucleus cells of pupae. Mortality, oviposition, chromosome breakage, and anaphase bridges were significantly greater in the extract treatments than in controls. The genotoxic effects of dillapiol described here suggest that this natural product may be a useful alternative for the control of A. aegypti.

Cytogenetic study of Anopheles albitarsis (Diptera: Culicidae) by C-banding and in situ hybridization.

The C-banding pattern and the size and location of the nucleolar organizer regions (NORs) are described for the first time in Brazilian populations of Anopheles (Nyssorhynchus) albitarsis sensu lato. C-banding revealed variation in the size of the centromeric heterochromatic blocks in autosomal chromosomes and in the acrocentric (X) and puntiform (Y) sex chromosomes. Fluorescence in situ hybridization showed that the NORs were located in the pericentromeric region of the sex (XX/XY) chromosomes and that this coincided with the number and location of centromeric constitutive heterochromatin blocks previously revealed by C-banding. The NORs varied in size among the homologues of the three populations. These findings of the populations studied support the hypothesis that the stability of NORs in the A. albitarsis complex is characterized by the presence of clustered and conserved sites in a unique pair of chromosomes.

Karyotype of Brazilian Anopheles albitarsis sensu lato (Diptera: Culicidae)

Anopheles (Nyssorhynchus) albitarsis sensu lato is an important malaria vector in Brazil, especially in the Brazilian Amazon region. Chromosome preparations of fourth-instar larvae of A. albitarsis from Iranduba and Coari (AM) and Ilha Comprida (SP) were analyzed for karyotype determination and to improve cytogenetic identification of this species. Anopheles albitarsis possesses 2n = 6 chromosomes, with two pairs (submetacentric and metacentric) of autosomes and one pair of sex chromosomes, with X-Y dimorphism. The sex pair is homomorphic and acrocentric in females and heteromorphic in males, with a punctiform Y chromosome. Somatic pairing was detected in the prometaphase and metaphase chromosomes of the three A. albitarsis populations. Apparently, sex chromosome evolution in the Culicidae does not function as does evolution in the Culicidae, since it occurs in the subfamily Anophelinae, which possesses heteromorphic sex chromosomes and is regarded as primitive, based on several criteria. These karyotype data on the albitarsis complex reinforce the hypothesis that sex chromosome evolution in the subfamily Anophelinae is conserved, and the variation revealed in the mean size of chromosomes in three populations indicates that selective pressure in these populations is occurring only at a genetic level.

Ecologic observations on anopheline vectors of malaria in the Brazilian Amazon.

Human intervention in the Brazilian Amazon region promotes contacts between humans and vectors that may favor the propagation of anopheline mosquitoes and the spread of malaria in the absence of planning and infrastructure to control this disease. Vector ecology studies were carried out to determine the risk areas. These data should help in designing appropriate malaria control measures. Data from 14 different regions are reported. Vectors are able to adapt to different environments, which made it necessary to study each area. The parameters studied were Anopheles breeding sites, species distribution, incidence, feeding preferences, hours of maximum activity of adult mosquitoes, seasonality, resting places, and the presence of Plasmodium. Species complexes were also studied. Anopheles darlingi may be responsible for maintaining malaria in human populations in this region. A reduction in the population density of A. darlingi in a particular geographic area can sometimes cause the disappearance of malaria. This species feeds at night but has a peak of activity at the beginning of the evening and another at dawn. Other species are mainly crepuscular and all anophelines demonstrated pronounced exophilia. The timing of feeding activities was found to vary in areas altered by human intervention and also depended on the time of the year and climatic conditions. The larvae were more abundant in the rivers with a less acidic pH and rural areas showed the highest larval index.