RESUMO
We compared rates of feeding on human hosts for blood-engorged female Anopheles quadrimaculatus species A, B and C1 collected from daytime resting sites in Manatee Springs State Park, Levy County, Florida during 1992-1993. Quick-blot DNA probes were used to identify mosquito taxa and also the presence of human blood in the mosquito gut. In collections from a campground area, human blood-feeding rates differed significantly among mosquito species (10.7% [19 of 177], 0%, [0 of 62], and 1.2%, [4 of 327]), respectively for species A, B and C1). In collections from a woodland site (1 km from the campground), 1.5% (2 of 129) of the species B females had fed on humans, whereas none of 19 species A or 159 species C1 females had done so. Of the three species in this study area, species A appears the most likely to be a biting pest of humans and a vector of human malaria.
Assuntos
Anopheles/fisiologia , Animais , Anopheles/classificação , Comportamento Apetitivo , Sondas de DNA , Comportamento Alimentar/fisiologia , Feminino , Florida , Humanos , Especificidade da EspécieRESUMO
A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis.
Assuntos
Anopheles/genética , RNA Ribossômico/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Anopheles/classificação , Anopheles/parasitologia , Sequência de Bases , Sondas de DNA/genética , DNA Ribossômico/genética , Feminino , Genes/genética , Biblioteca Genômica , Insetos Vetores/classificação , Insetos Vetores/genética , Insetos Vetores/parasitologia , Malária/transmissão , Dados de Sequência Molecular , RNA Ribossômico 28S/genética , Homologia de Sequência do Ácido Nucleico , América do Sul , Especificidade da EspécieRESUMO
The nucleotide sequence was determined for a portion of a 12-kb genomic DNA clone specific for Anopheles quadrimaculatus species A. Four short, internally repeated sequences were identified. Synthetic oligonucleotide probes were prepared based on these four repeats. The oligonucleotides are highly specific and can be reliably used to separate individuals of An. quadrimaculatus species A from members of other species of the complex.
Assuntos
Anopheles/genética , Sondas de DNA/química , Animais , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/química , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
Methods are described for the isolation of mitochondrial and total cellular DNA from mosquitoes. The mitochondrial and ribosomal DNA restriction patterns could be detected in the total DNA of an individual mosquito by the use of cloned probes. DNA restriction analysis may prove to be a useful alternative to isozyme electrophoresis for the study of insect population genetics.
Assuntos
Anopheles/análise , DNA Mitocondrial/isolamento & purificação , DNA Ribossômico/isolamento & purificação , Animais , Anopheles/genética , Sondas de DNA , DNA Mitocondrial/genética , DNA Ribossômico/genética , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
Temperature effects on Anopheles albimanus larval survival were investigated. Larvae were exposed to 30 min heat shocks at various temperatures. Almost no mortality was observed at 40 degrees C, but was complete at 43 degrees C. Increased larval thermotolerance could be induced by higher rearing temperature or by a 30 min exposure to 37 degrees C.
Assuntos
Anopheles/fisiologia , Temperatura Alta , Adaptação Fisiológica/fisiologia , Animais , Ecologia , Proteínas de Choque Térmico/biossíntese , Larva/fisiologia , Estações do AnoRESUMO
The quick blot protocol is an improved technique for preparing crude insect homogenates for hybridization to nucleic acid probes. Individual insects are ground in wells of a microtiter plate and transferred to a dot blot manifold. This allows preparation of multiple filters and provides uniformity and an orderly arrangement of samples. The high background detection resulting from use of crude insect homogenates with nonradioactive detection systems was eliminated by incubating quick blot filters in a laundry stain remover containing proteases. We used mosquito species-specific DNA probes to demonstrate the effectiveness of nonradioactive DNA labeling systems with quick blots.
Assuntos
Culicidae/genética , Sondas de DNA/análise , Mapeamento de Nucleotídeos/métodos , Animais , Hibridização de Ácido Nucleico , Traçadores Radioativos , Especificidade da EspécieAssuntos
DNA Recombinante , Drosophila melanogaster/genética , Controle de Insetos/métodos , Animais , Mapeamento Cromossômico/métodos , Clonagem Molecular , Contenção de Riscos Biológicos , DNA/genética , Elementos de DNA Transponíveis , Genes , Vetores Genéticos , Meiose , Hibridização de Ácido Nucleico , Seleção Genética , Análise para Determinação do Sexo , Transformação GenéticaRESUMO
A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.
Assuntos
Anopheles/isolamento & purificação , DNA/análise , Animais , Anopheles/genética , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Especificidade da EspécieRESUMO
Most of the Dictyostelium ribosomal nontranscribed spacer DNA has been mapped using Eco R1, Hind III, and Sal I restriction digests and recombinant plasmids containing parts of the rDNA (ribosomal DNA). The repeat units are at least 42 kb (1 kilobase = 1000 base pairs = 1 kb) long; of this, 8 kb codes for the 36S rRNA precursor. The repeat units give a homogeneous pattern of Sal I, Eco R1, and Hind III restriction fragments. The repeats could be linked by a heterogeneous region in the nontranscribed spacer, not tandemly repeated, or linked in a more complicated structural arrangement. The total rDNA comprises at least 18% of the nuclear DNA.
Assuntos
DNA Recombinante , Dictyostelium/metabolismo , Genes , Mixomicetos/metabolismo , RNA Ribossômico/biossíntese , Animais , Mapeamento Cromossômico , DNA , Enzimas de Restrição do DNA , Hibridização de Ácido Nucleico , PlasmídeosRESUMO
The entire 15 kilobase (kb) Anopheles quadrimaculatus mitochondrial DNA (mtDNA) was cloned as three EcoRI fragments in a bacteriophage vector and then subcloned into plasmid vectors. The cloned DNA was physically mapped with restriction endonucleases, and the maps were compared to the restriction patterns of native A. quadrimaculatus mtDNA. Several genes were mapped by sequencing the ends of A. quadrimaculatus mtDNA subclones and by hybridization with the previously characterized Aedes albopictus mtDNA clones. These portions of the genetic map were identical in gene order to those of Drosophila yakuba. The predicted amino acid sequence of the protein coding regions that were sequenced were between 72% and 98% homologous to D. yakuba. The cloned mtDNA will be useful as a probe for population genetic analysis of mosquitoes.
Assuntos
Anopheles/genética , Clonagem Molecular , DNA Mitocondrial/genética , Sequência de Aminoácidos , Animais , Bacteriófagos , Sequência de Bases , Sondas de DNA , DNA Mitocondrial/química , Desoxirribonuclease EcoRI , Biblioteca Gênica , Vetores Genéticos , Hibridização de Ácido Nucleico , Plasmídeos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The complete sequence (15,455 bp) of the mitochondrial DNA of the mosquito Anopheles quadrimaculatus species A is reported. This genome is compact and very A+T rich (77.4% A+T). It contains genes for 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement is the same as in Drosophila yakuba, except that the positions of two contiguous tRNAs are reversed and a third tRNA is transcribed from the complementary strand. Protein-coding genes, rRNAs, and most tRNAs were similar to D. yakuba. Two tRNAs had nonstandard secondary structures comparable with those of nematode mitochondrial tRNAs. The very small putative control region (625 bp) contains no sequence motifs similar to those used in vertebrates and other insects for initiation of transcription and replication.
Assuntos
Anopheles/genética , DNA Mitocondrial/genética , Genes de Insetos , Sequência de Aminoácidos , Animais , Composição de Bases , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Códon/genética , Drosophila/genética , Genoma , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Proteínas/genética , RNA Ribossômico/genética , RNA de Transferência Aminoácido-Específico/química , RNA de Transferência Aminoácido-Específico/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Populations of the malaria vector Anopheles aquasalis from Venezuela, Trinidad, and Brazil were analyzed using restriction enzyme digestion of mitochondrial DNA (mtDNA). The five enzymes surveyed yielded 12 mtDNA haplotypes. Three restriction endonuclease profiles (Bcl I, Eco RI, and Hind III) differentiated the Venezuelan and Trinidadian A. aquasalis from two Brazilian populations. Estimates of mtDNA sequence divergence between the Venezuelan/Trinidadian populations and each Brazilian population (0.023-0.032), as well as divergence between the two Brazilian populations (0.030), were within the range of interspecific distances calculated for members of anopheline species complexes. Ribosomal DNA (rDNA) restriction profiles of the A. aquasalis populations examined were identical.
Assuntos
Anopheles/genética , Insetos Vetores/genética , Malária , Animais , Brasil , DNA Mitocondrial/genética , Feminino , Variação Genética , Haplótipos , Humanos , Malária/transmissão , Polimorfismo de Fragmento de Restrição , Especificidade da Espécie , Trinidad e Tobago , VenezuelaRESUMO
The bacterial parathion hydrolase gene (opd) was expressed in transformed D. melanogaster under the control of an hsp70 promoter. Transformed lines carrying chimaeric genes designed for either cytoplasmic or secretory expression exhibited high- or low-level heat-shock-inducible transient resistance to paraoxon respectively. Greatest levels of resistance occurred approximately 12-16 h after heat shock and well after periods of maximal transcription. Insecticide resistance conferred by the cytoplasmic form of opd is expressed as a semidominant trait.
Assuntos
Drosophila melanogaster/genética , Esterases/genética , Resistência a Inseticidas/genética , Paraoxon/toxicidade , Animais , Arildialquilfosfatase , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Esterases/biossíntese , Esterases/metabolismo , Expressão Gênica , Genótipo , Temperatura Alta , Dados de Sequência Molecular , Mortalidade , Paration/toxicidade , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
We analyzed variation in mitochondrial DNA (mtDNA) of two neotropical mosquitoes, Anopheles rangeli (n = 181) and A. trinkae (n = 45), with very different distribution patterns in Latin America, to assess species boundaries for these putative sister taxa and to examine population genetic structure. Phylogenetic analyses revealed (1) support for the monophyletic origin of each species; (2) diagnostic restriction site differences between the species; (3) geographic partitioning of haplotypes by country in A. rangeli from Bolivia, Ecuador, and Venezuela compared with considerable overlap in haplotypes of A. trinkae from Bolivia and Ecuador; and (4) similar levels of mean haplotype and nucleotide diversity in both species, but lower levels of mean nucleotide divergence in A. trinkae compared with A. rangeli. We hypothesize that higher maternal gene flow and lower divergence in A. trinkae are most likely due either to a distinctive matrilineal history or to a smaller effective population size, which may have been influenced by a smaller, essentially linear geographic range along the eastern flank of the Andes. In the cladistic analysis of A. rangell, the Bolivian haplotypes appear to be more derived than those from Ecuador or Venezuela, yet there is no evidence to support the hypothesis of a recent range expansion from Ecuador into Bolivia.
Assuntos
Anopheles/genética , DNA Mitocondrial , Genes de Insetos , Variação Genética , Animais , Anopheles/classificação , Haplótipos , FilogeniaRESUMO
Restriction enzyme mapping of snap-back DNA has been used to show that the ribosomal DNA (rRNA genes and spacers) from Dictyostelium discoideum exists as 88 kb (kb=1,000 base pairs) linear palindromic dimers. Analysis of the mobility of total cell DNA in 0.15% agarose gels indicates that the majority of the rDNA is not covalently attached to chromosomal DNA.
Assuntos
Dictyostelium/genética , Herança Extracromossômica , RNA Ribossômico/genética , Eletroforese em Gel de ÁgarRESUMO
Four Hsp70 genes of the malaria vector Anopheles albimanus were isolated from a genomic DNA library as two non-overlapping clones each containing a pair of divergently transcribed genes having 75% DNA sequence similarity to the protein-coding regions of the Drosophila melanogaster Hsp70 genes. The clones were assigned to two loci on chromosome 2R by in situ hybridization. These clones hybridize strongly to heat-shock but only weakly to non-shocked mosquito RNA. The Hsp70 gene family of A. albimanus is undergoing concerted evolution probably by gene conversion. The general arrangement of the genes suggests that divergently transcribed pairs of genes at two loci is an ancient Dipteran arrangement predating the Nematocera/Cyclorrapha divergence.
Assuntos
Anopheles/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Insetos/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Genes de Insetos , Proteínas de Choque Térmico HSP70/metabolismo , Dados de Sequência Molecular , Família Multigênica , RNA/análise , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Silicon carbide fiber-mediated delivery of DNA into intact plant cells was investigated. Black Mexican Sweet (BMS) maize (Zea mays) and tobacco (Nicotiana tabacum) suspension culture cells were vortexed in the presence of liquid medium, plasmid DNA encoding ß-glucuronidase (GUS), and silicon carbide fibers. Penetration of BMS cells by the silicon carbide fibers was observed by scanning electron microscopy of vortexed cells. Following fiber and DNA treatment, BMS cells transiently expressed GUS activity at a mean frequency of 139.5 units (one unit = one blue cell or one colony of blue cells) per sample. Treated tobacco cells expressed an average of 373 GUS units per sample. Untreated controls did not exhibit GUS activity. These results indicate that the silicon carbide fibers-vortex procedure can be used to rapidly and inexpensively deliver foreign DNA into intact plant cells for investigations of transient gene expression.