Strain of Bacillus thuringiensis from Restinga, toxic to Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae) / Estirpe de Bacillus thuringiensis da restinga, tóxico ao Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae)

Abstract Bacillus thuringiensis is the most commonly used entomopathogen in the control of Aedes aegypti, which is a vector for different etiological agents that cause serious infections in humans. Several studies aim to isolate strains of this bacterium from different environments, with the perspective of selecting isolates with larvicidal activity for mosquitoes. Aiming at the insecticidal action of B. thuringiensis, the present study aimed to prospect B. thuringiensis of restinga and mangrove soils from the state of Maranhão, Brazil, with toxic potential for use in the biological control of Ae. aegypti. Bioassays were performed to determine the entomopathogenic activity of the bacilli against Ae. aegypti and lethal concentrations (LC50 and CL90) were estimated after the tests. Polymerase Chain Reaction and SDS-PAGE techniques were performed to verify the gene and protein content of the isolates, respectively. The soil of the mangrove and restinga ecosystems showed potential for obtaining B. thuringiensis. This isolate, in addition to having proteins with molecular mass similar to the toxins Cry and Cyt, also presented several diptera-specific genes cry and cyt, demonstrating that it has high potential to be used in the biological control of Ae. aegypti.

Resumo Bacillus thuringiensis é o entomopatógeno mais utilizado no controle do Aedes aegypti, vetor de diferentes agentes etiológicos que causam infecções graves em humanos. Diversos estudos têm como objetivo isolar cepas dessa bactéria de diferentes ambientes, com a perspectiva de selecionar isolados com atividade larvicida para mosquitos. Visando a ação inseticida de B. thuringiensis, o presente estudo teve como objetivo prospectar B. thuringiensis de solos de restinga e mangue do estado do Maranhão, Brasil, com potencial tóxico para uso no controle biológico de Ae. aegypti. Bioensaios foram realizados para determinar a atividade entomopatogênica do bacilo contra Ae. aegypti e as concentrações letais (CL50 e CL90) foram estimadas após os testes. As técnicas de Reação em Cadeia da Polimerase e SDS-PAGE foram realizadas para verificar o conteúdo de genes e proteínas dos isolados, respectivamente. Os solos dos ecossistemas de mangue e restinga apresentaram potencial para obtenção de B. thuringiensis. O isolado BtMA-750, obtido a partir da amostra de solo da restinga, foi interessantemente distinguido por sua alta toxicidade para Ae. aegypti. Este isolado, além de apresentar proteínas com massa molecular semelhante às toxinas Cry e Cyt, apresentou também diversos genes díptero-específicos cry e cyt, demonstrando que tem alto potencial para ser usado no controle biológico de Ae. aegypti.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae) / Isolados de Bacillus thuringiensis de biomas maranhenses com potencial ação inseticida contra larvas de Aedes aegypti (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae).

Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Strain of Bacillus thuringiensis from Restinga, toxic to Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae).

Bacillus thuringiensis is the most commonly used entomopathogen in the control of Aedes aegypti, which is a vector for different etiological agents that cause serious infections in humans. Several studies aim to isolate strains of this bacterium from different environments, with the perspective of selecting isolates with larvicidal activity for mosquitoes. Aiming at the insecticidal action of B. thuringiensis, the present study aimed to prospect B. thuringiensis of restinga and mangrove soils from the state of Maranhão, Brazil, with toxic potential for use in the biological control of Ae. aegypti. Bioassays were performed to determine the entomopathogenic activity of the bacilli against Ae. aegypti and lethal concentrations (LC50 and CL90) were estimated after the tests. Polymerase Chain Reaction and SDS-PAGE techniques were performed to verify the gene and protein content of the isolates, respectively. The soil of the mangrove and restinga ecosystems showed potential for obtaining B. thuringiensis. This isolate, in addition to having proteins with molecular mass similar to the toxins Cry and Cyt, also presented several diptera-specific genes cry and cyt, demonstrating that it has high potential to be used in the biological control of Ae. aegypti.

Eggs viability of Aedes aegypti Linnaeus (Diptera, Culicidae) under different environmental and storage conditions in Manaus, Amazonas, Brazil / Viabilidade de ovos de Aedes aegypti (Diptera, Culicidae) em diferentes condições de armazenamento em Manaus, Amazônia, Brasil

Abstract The viability of Aedes aegypti eggs was assessed in the Amazon region. The eggs were maintained under different conditions: indoors (insectarium) and outdoors (natural environment), as well as in different storage types (plastic cup, paper envelope, plastic bag) for different days. Egg viability was measured as the mean of hatchings observed from egg-bearing sheets of filter paper immersed in water, using three sheets randomly selected from each storage type and at both sites. There were significant differences in the viability of Ae. aegypti eggs with respect to the location (F=30.40; DF=1; P<0.0001), storage type (F=17.66; DF=2; P<0.0001), and time of storage (F=49.56; DF=9; P<0.0001). The interaction between storage site versus storage type was also significant (F=15.96; DF=2; P<0.0001). A higher hatching mean was observed for the eggs kept in the insectarium than for those outdoors (32.38 versus 7.46). Hatching rates of egg batches stored for 12 to 61 days ranged between 84 and 90%. A reduction was observed between 89 and 118 days, with values of 63 and 48%, respectively. With respect to type of storage, mean egg hatching was higher for the eggs in plastic cups (44.46). It was concluded that the viability of the eggs of Ae. aegypti in the Amazon region remains high up to 4 months, after which it declines drastically, although in this study hatching occurred for up to 8 months in very low percentages.

Resumo Testou-se a viabilidade dos ovos de Ae. aegypti na região amazônica armazenados em copos plásticos, envelopes de papel e sacos plásticos que foram mantidos em área interna e em área externa por dias diferentes. Verificaram-se diferenças significativas na viabilidade dos ovos considerando-se a localização (F=30,40; GL=1; P<0,0001), os tipos de armazenamento (F=17,66; GL=2; P<0,0001) e os tempos (F=49,56; GL=9; P<0,0001) e na interação localização versus tipo de armazenamento (F=15,96; GL=2; P<0,0001). A média de eclosão dos ovos armazenados no insetário foi maior: 32,38, do que a dos lotes armazenados na área externa: 7,46. As taxas de eclosão dos ovos armazenados de 12 a 61 dias ficaram entre 90% e 84% com reduções a partir de 89 e 118 dias, quando atingiu de 63% e 48%, respectivamente. Em relação aos tipos de armazenamento a média de ovos dos copos foi significativa (44,46). Conclui-se que a viabilidade dos ovos de Ae. aegypti na região amazônica é mantida em níveis elevados até 4 meses, e a partir daí ocorrem reduções drásticas, com eclosões até 8 meses em percentuais muito baixos.

Eggs viability of Aedes aegypti Linnaeus (Diptera, Culicidae) under different environmental and storage conditions in Manaus, Amazonas, Brazil.

The viability of Aedes aegypti eggs was assessed in the Amazon region. The eggs were maintained under different conditions: indoors (insectarium) and outdoors (natural environment), as well as in different storage types (plastic cup, paper envelope, plastic bag) for different days. Egg viability was measured as the mean of hatchings observed from egg-bearing sheets of filter paper immersed in water, using three sheets randomly selected from each storage type and at both sites. There were significant differences in the viability of Ae. aegypti eggs with respect to the location (F=30.40; DF=1; P<0.0001), storage type (F=17.66; DF=2; P<0.0001), and time of storage (F=49.56; DF=9; P<0.0001). The interaction between storage site versus storage type was also significant (F=15.96; DF=2; P<0.0001). A higher hatching mean was observed for the eggs kept in the insectarium than for those outdoors (32.38 versus 7.46). Hatching rates of egg batches stored for 12 to 61 days ranged between 84 and 90%. A reduction was observed between 89 and 118 days, with values of 63 and 48%, respectively. With respect to type of storage, mean egg hatching was higher for the eggs in plastic cups (44.46). It was concluded that the viability of the eggs of Ae. aegypti in the Amazon region remains high up to 4 months, after which it declines drastically, although in this study hatching occurred for up to 8 months in very low percentages.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Strain of Bacillus thuringiensis from Restinga, toxic to Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae)

Abstract Bacillus thuringiensis is the most commonly used entomopathogen in the control of Aedes aegypti, which is a vector for different etiological agents that cause serious infections in humans. Several studies aim to isolate strains of this bacterium from different environments, with the perspective of selecting isolates with larvicidal activity for mosquitoes. Aiming at the insecticidal action of B. thuringiensis, the present study aimed to prospect B. thuringiensis of restinga and mangrove soils from the state of Maranhão, Brazil, with toxic potential for use in the biological control of Ae. aegypti. Bioassays were performed to determine the entomopathogenic activity of the bacilli against Ae. aegypti and lethal concentrations (LC50 and CL90) were estimated after the tests. Polymerase Chain Reaction and SDS-PAGE techniques were performed to verify the gene and protein content of the isolates, respectively. The soil of the mangrove and restinga ecosystems showed potential for obtaining B. thuringiensis. This isolate, in addition to having proteins with molecular mass similar to the toxins Cry and Cyt, also presented several diptera-specific genes cry and cyt, demonstrating that it has high potential to be used in the biological control of Ae. aegypti.

Resumo Bacillus thuringiensis é o entomopatógeno mais utilizado no controle do Aedes aegypti, vetor de diferentes agentes etiológicos que causam infecções graves em humanos. Diversos estudos têm como objetivo isolar cepas dessa bactéria de diferentes ambientes, com a perspectiva de selecionar isolados com atividade larvicida para mosquitos. Visando a ação inseticida de B. thuringiensis, o presente estudo teve como objetivo prospectar B. thuringiensis de solos de restinga e mangue do estado do Maranhão, Brasil, com potencial tóxico para uso no controle biológico de Ae. aegypti. Bioensaios foram realizados para determinar a atividade entomopatogênica do bacilo contra Ae. aegypti e as concentrações letais (CL50 e CL90) foram estimadas após os testes. As técnicas de Reação em Cadeia da Polimerase e SDS-PAGE foram realizadas para verificar o conteúdo de genes e proteínas dos isolados, respectivamente. Os solos dos ecossistemas de mangue e restinga apresentaram potencial para obtenção de B. thuringiensis. O isolado BtMA-750, obtido a partir da amostra de solo da restinga, foi interessantemente distinguido por sua alta toxicidade para Ae. aegypti. Este isolado, além de apresentar proteínas com massa molecular semelhante às toxinas Cry e Cyt, apresentou também diversos genes díptero-específicos cry e cyt, demonstrando que tem alto potencial para ser usado no controle biológico de Ae. aegypti.

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.

Comparative in vitro and in vivo antimalarial activity of the indole alkaloids ellipticine, olivacine, cryptolepine and a synthetic cryptolepine analog.

Indole alkaloids ellipticine (1), cryptolepine triflate (2a), rationally designed 11-(4-piperidinamino)cryptolepine hydrogen dichloride (2b) and olivacine (3) (an isomer of 1) were evaluated in vitro against Plasmodium falciparum and in vivo in Plasmodium berghei-infected mice. 1-3 inhibited P. falciparum (IC50≤1.4 µM, order of activity: 2b>1>2a>3). In vitro toxicity to murine macrophages was evaluated and revealed selectivity indices (SI) of 10-12 for 2a and SI>2.8×10² for 1, 2b and 3. 1 administered orally at 50mg/kg/day was highly active against P. berghei (in vivo inhibition compared to untreated control (IVI)=100%, mean survival time (MST)>40 days, comparable activity to chloroquine control). 1 administered orally and subcutaneously was active at 10 mg/kg/day (IVI=70-77%; MST=27-29 days). 3 exhibited high oral activity at ≥50 mg/kg/day (IVI=90-97%, MST=23-27 days). Cryptolepine (2a) administered orally and subcutaneously exhibited moderate activity at 50mg/kg/day (IVI=43-63%, MST=24-25 days). At 50 mg/kg/day, 2b administered subcutaneously was lethal to infected mice (MST=3 days) and moderately active when administered orally (IVI=45-55%, MST=25 days). 1 and 3 are promising compounds for development of antimalarials.

On the use of classic epidemiological formulae for estimating the intensity of endemic malaria transmission by vectors in the Amazon.

Although various reports have described entomological inoculation rates of malaria vector species, most were limited to providing descriptive field data. Here, we report biting rates and survival data for two important malaria vectors in the Amazon, Anopheles darlingi (Root) and Anopheles albitarsis E (Lynch-Arribalzaga) (Diptera: Culicidae), in the state of Roraima, Brazil. We calculated theoretical sporozoite infection rates and critical vector biting rates for these species during 1 year, comprising six bimestrial collections. Anopheles darlingi had higher sporozoite rates and lower critical biting rates, indicating that it would be the more efficient vector at the beginning of epidemic malaria transmission. Our data, together with compiled information from the literature in the Amazon, suggest that epidemic malaria transmission may be initiated by the primary vector, such as A. darlingi, while secondary vectors, such as A. albitarsis E, may only become epidemiologically important when there is an increase in the prevalence of human malaria. We propose that mathematical modeling may be able to quantify the relative importance of secondary vector species in malaria epidemiology.

Reduced levels of genetic variation in Aedes albopictus (Diptera: Culicidae) from Manaus, Amazonas State, Brazil, based on analysis of the mitochondrial DNA ND5 gene.

Aedes albopictus, a mosquito originally from Southeast Asia, is considered to be one of the main vectors of dengue fever, yellow fever and other arboviruses. We examined the genetic variability and population structure of 68 individuals of Ae. albopictus collected from five neighborhoods of the city of Manaus, based on the mitochondrial gene coding for NADH dehydrogenase subunit 5 (ND5). Two haplotypes were found, separated by a single mutational event (T <--> C), with extremely low levels of genetic variability (h = 0.187 +/- 0.059; pi = 0.00044 +/- 0.00014). Based on AMOVA, we concluded that most of the variation (99.08%) occurred within populations, though the levels of variation were not significant. Neutrality tests (Tajima's D and Fu's Fs) were non-significant, indicating that these populations are in genetic equilibrium. The most frequent haplotype (H1) is restricted to Brazilian populations of Ae. albopictus, while the rarer haplotype (H2) is shared with populations from the United States and Asia. We suggest that the reduced variability and low genetic structure identified in our study is a consequence of the recent introduction of this species in Manaus, possibly through a founder effect, followed by expansion throughout the city neighborhoods. Genetic similarity would therefore be due to insufficient time to have accumulated genetic differences between the populations of Ae. albopictus and not to extensive gene flow among them.

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.

Amazonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences.

Species identification of anopheline mosquitoes (Diptera: Culicidae) can be problematic because many of them belong to complexes of morphologically similar species, often with contrasted ecology, behaviour and vectorial importance. The application of DNA-based diagnostics has proved to be useful for distinguishing between such species. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) from samples of 16 species of Anopheles captured in the Amazon Basin, Brazil. Length of the ITS2 varied from 323 to 410 base pairs, with GC content ranging from 50.7% to 66.5% and sequence identity from 25% to 99% between species. Maximum-likelihood paup analysis separated two distinct groups of species conforming with the recognized subgenera Anopheles (represented by eiseni, mattogrossensis, mediopunctatus and peryassui) and Nyssorhynchus (represented by 12 spp.). For the latter group, the neighbour-joining tree generated from rDNA sequence ITS2 relationships is compatible with the morphological taxonomic key established for these Amazonian species: albitarsis, aquasalis, benarrochi, braziliensis, darlingi, deaneorum, dunhami, evansae, nuneztovari, oswaldoi, rangeli and triannulatus. These ITS2 sequence data proved to be a useful tool for species identification and, potentially, to solve taxonomic problems.

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.

Differentiation and genetic variability in natural populations of Anopheles (N.) triannulatus (Neiva & Pinto, 1922) of Brazilian Amazonia.

Populations of Anopheles triannulatus from Macapá (AP), Aripuanã (MT), Ji-Paraná (RO), and Manaus-Janauari Lake (AM) were studied using 16 enzymatic loci. The results of the isozyme analysis showed that the population of Macapá presented higher polymorphism (56.3%). The lowest variability was observed in the population of Manaus (p = 25.0; Ho = 0.077 +/- 0.046). The results of Wright's F statistics showed unbalance due to excess of homozygotes (F(is) > F(st)), denoting a certain intrapopulational differentiation. Although the populations are genetically very close (D = 0.003-0.052), the dendrogram separates the populations in two groups: Macapá separated from that of Manaus, Ji-Paraná, and Aripuanã. This result may suggest a reduction in the genic flow, which possibly had some influence in the substructuration of the populations.

Differentiation and genetic variability in natural populations of Anopheles (N. ) triannulatus (Neiva& Pinto, 1922) of Brazilian Amazonia

Populaçäes de Anopheles triannulatus procedentes de Macapá (AP), Aripuanã (MT), Ji-Paraná (RO) e Manaus-Lago Janauari (AM) foram estudadas utilizando-se 16 locos enzimáticos. Os resultados mostraram maior polimorfismo (56,3%) na população de Macapá. A menor variabilidade foi verificada na população de Manaus (p = 25,0; Ho = 0,077 ñ 0,046). Os resultados das estatísticas F de Wright mostraram desequilíbrio decorrente de excesso de homozigotos (Fis > Fst), denotando certa diferenciação intrapopulacional. Embora as populaçäes sejam geneticamente muito próximas (D = 0,003 - 0,052), o dendrograma separa as populaçäes em dois grupos: Macapá separado de Manaus, Ji-Paraná e Aripuanã. Isto pode ser indicativo de redução no fluxo gênico, que possivelmente influenciou a subestruturação das populaçäes.

Enzymatic analysis in Anopheles nuneztovari Gabaldón (Diptera, Culicidae)

Enzymatic analysis in Anopheles nuneztovari was made using four populations from the Brazilian Amazon and two from Colombia. The enzymes ME and XDH presented a monomorphic locus in all of the studied populations. EST and LAP presented a higher number of loci. In EST, genetic variation was observed in the five loci; LAP presented four loci, with allec variation in two loci. In IDH, three activity regions were stained, with genetic variation for locus Idh-1 in the Brazilian Amazon populations. A locus for MDH was observed, with genetic variation in the six populations. A region was verified for ACON, with four alleles in Sitronela and three in the other populations. PGM constituted one locus, with a high variability in the Brazilian Amazon populations. A locus was observed for 6-PGD with allelic variation in all of the populations with the exception of Tibú. Enzyme PGI presented two loci, both with genetic variability in the Tucuruí population. The enzyme alpha-GPD showed an activity region with polymorphism in the Tucuruí, Tibú and Sitronela populations. The phenotypic variations detected for these enzymes suggest that four (EST, LAP, ACON and PGM) possess monomeric structures and five (IDH, MDH, 6-PGD, PGI and alpha-GPD) dimeric structures in their proteins. These enzymes constitute in important markers to estimate variability and genetic divergence in natural populations of A. nuneztovari