RESUMO
The host decoy trap (HDT) is a surveillance trap that presents a combination of heat, visual and odour stimuli to attract bloodmeal-seeking mosquitoes. Here we employed a semi-field study to demonstrate the role of the visual attributes present on the HDT on the effectiveness of Aedes aegypti capture. Our results show that the HDT is an effective means of capturing Ae. aegypti mosquitoes in semi-field conditions, with a per trial capture rate of up to 69% across four visually distinct HDTs. The solid black coloured HDT captured more mosquitoes than HDTs with black-white stripes, black-white checkerboard patches or solid white colour by a factor of 1.9, 1.7 and 1.5, respectively. In all cases, mosquito capture was not evenly distributed on the HDT surface, with captures on the HDT's outer downwind half, away from the odour delivery, exceeding captures on the inner upwind half. We conclude that the solid black surface of the original HDT design is more effective than the other surfaces (white or black/white patterns) for the capture of Ae. aegypti. Our results demonstrate that mosquito attraction to the thermal and odorant cues of the HDT is modulated by visual information.
Assuntos
Aedes/fisiologia , Comportamento Alimentar , Controle de Mosquitos/métodos , Animais , Cor , Mosquitos Vetores/fisiologia , Odorantes , TemperaturaRESUMO
The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.
Assuntos
Proteínas de Transporte/análise , Proteínas de Drosophila , Proteínas do Olho , Proteínas de Membrana/análise , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico/fisiologia , Western Blotting , Química Encefálica , Cálcio/metabolismo , Radioisótopos de Cálcio , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Membrana Celular/química , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , DNA/metabolismo , Drosophila , Expressão Gênica , Genes/genética , Imuno-Histoquímica , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Neurônios Aferentes/química , Neurônios Aferentes/citologia , Neurônios Aferentes/fisiologia , Proteínas de Transferência de Fosfolipídeos , Fosfolipídeos/metabolismo , Células Fotorreceptoras/química , Células Fotorreceptoras/citologia , Células Fotorreceptoras/fisiologia , Degeneração Retiniana/fisiopatologiaRESUMO
The effect of light on the polypeptide map profile of the Drosophila eye preparation was examined by two-dimensional polyacrylamide gel electrophoresis. The results show (i) that illuminating the living fly reversibly changes the isoelectric points of three classes of polypeptides specific for the photoreceptor layer and (ii) that the norpA mutation, which prevents the generation of the receptor potential, blocks the modifications.
Assuntos
Drosophila/metabolismo , Proteínas do Olho/metabolismo , Luz , Peptídeos/metabolismo , Retina/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Ponto Isoelétrico , Cinética , Mutação , Células Fotorreceptoras/metabolismoRESUMO
Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.
Assuntos
Drosophila/genética , Mutação , Células Fotorreceptoras/fisiologia , Degeneração Retiniana , Pigmentos da Retina/fisiologia , Rodopsina/fisiologia , Envelhecimento , Animais , Mapeamento Cromossômico , Drosophila/fisiologia , Eletrofisiologia/métodos , Luz , Microscopia Eletrônica , Fenótipo , Células Fotorreceptoras/crescimento & desenvolvimento , Células Fotorreceptoras/ultraestrutura , Fosfolipases Tipo C/metabolismoRESUMO
Dominant mutations of the Drosophila ninaE-encoded rhodopsin are described that reduce the expression of wild-type rhodopsin and cause a slow, age-dependent form of retinal degeneration. A posttranslational event subsequent to the requirement for the ninaA-encoded cyclophilin is disrupted by the dominant mutations. Most of these dominant mutations are missense mutations that affect the physical properties of one of the seven transmembrane domains; another affects the cysteine involved in a disulfide linkage. The results indicate that misfolded or unstable mutant rhodopsin can interfere with maturation of wild-type rhodopsin, and that these cellular conditions may trigger retinal degeneration. In addition, these dominant rhodopsin mutations suppress the rapid degeneration seen in rdgC and norpA flies, indicating that high levels of rhodopsin are required.
Assuntos
Drosophila/genética , Mutação , Degeneração Retiniana/genética , Rodopsina/genética , Envelhecimento/fisiologia , Alelos , Sequência de Aminoácidos , Animais , Eletrorretinografia , Deleção de Genes , Expressão Gênica , Genes Dominantes , Genes de Insetos , Genótipo , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Estrutura Secundária de Proteína , Retina/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Rodopsina/biossíntese , Rodopsina/químicaRESUMO
We placed UAA, UAG and UGA nonsense mutations at two leucine codons, Leu205 and Leu309, in Drosophila's major rhodopsin gene, ninaE, by site-directed mutagenesis, and then created the corresponding mutants by P element-mediated transformation of a ninaE deficiency strain. In the absence of a genetic suppressor, flies harboring any of the nonsense mutations at the 309 site, but not the 205 site, show increased rhodopsin activity. Additionally, all flies with nonsense mutations at either site have better rhabdomere structure than does the ninaE deficiency strain. Construction and analysis of a 3'-deletion mutant of ninaE indicates that translational readthrough accounts for the extra photoreceptor activity of the ninaE309 alleles and that truncated opsins are responsible for the improved rhabdomere structure. The presence of leucine-inserting tRNA nonsense suppressors DtLa Su+ and DtLb Su+ in the mutant strains produced a small increase (less than 0.04%) in functional rhodopsin. The opal (UGA) suppressor derived from the DtLa tRNA gene is more efficient than the amber (UAG) or opal suppressor derived from the DtLb gene, and both DtLa and DtLb derived suppressors are more efficient at site 205 than 309.
Assuntos
Drosophila/genética , Rodopsina/genética , Supressão Genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Drosophila/metabolismo , Drosophila/ultraestrutura , Eletrofisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestrutura , Conformação Proteica , Rodopsina/ultraestruturaRESUMO
The somda (sine oculis-medusa) mutant is the result of a P element insertion at position 43C on the second chromosome. somda causes aberrant development of the larval photoreceptor (Bolwig's) organ and the optic lobe primordium in the embryo. Later in development, adult photoreceptors fail to project axons into the optic ganglion. Consequently optic lobe development is aborted and photoreceptor cells show age-dependent retinal degeneration. The so gene was isolated and characterized. The gene encodes a homeodomain protein expressed in the optic lobe primordium and Bolwig's organ of embryos, in the developing adult visual system of larvae, and in photoreceptor cells and optic lobes of adults. In addition, the SO product is found at invagination sites during embryonic development: at the stomadeal invagination, the cephalic furrow, and at segmental boundaries. The mutant somda allele causes severe reduction of SO embryonic expression but maintains adult visual system expression. Ubiquitous expression of the SO gene product in 4-8-hr embryos rescues all somda mutant abnormalities, including the adult phenotypes. Thus, all deficits in adult visual system development and function results from failure to properly express the so gene during embryonic development. This analysis shows that the homeodomain containing SO gene product is involved in the specification of the larval and adult visual system development during embryogenesis.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Olho/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Genes de Insetos , Proteínas de Homeodomínio , Lobo Óptico de Animais não Mamíferos/crescimento & desenvolvimento , Vias Visuais/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Axônios/ultraestrutura , Sequência de Bases , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Desenvolvimento Embrionário , Olho/embriologia , Olho/crescimento & desenvolvimento , Proteínas do Olho/genética , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutagênese Insercional , Lobo Óptico de Animais não Mamíferos/embriologia , Fenótipo , Células Fotorreceptoras de Invertebrados/embriologia , Células Fotorreceptoras de Invertebrados/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/biossíntese , Degeneração Retiniana/embriologia , Degeneração Retiniana/genética , Alinhamento de Sequência , Homologia de Sequência , Vias Visuais/embriologiaRESUMO
Retinal degeneration-B (rdgB) mutants of Drosophila melanogaster undergo rapid light-induced retinal degeneration. We conducted a molecular characterization of the rdgB gene to examine the nature of the gene product. Through the isolation and analysis of X-ray-induced rdgB alleles, the cytogenetic position of the gene was determined to be the 12C1 salivary region. Genomic DNA corresponding to this region was isolated by a chromosomal walk. The chromosomal aberrations associated with the three X-ray-induced rdgB alleles were shown to be within a 5-kb genomic region. A single transcription unit was affected by the alleles, identifying it as the rdgB gene. RNA-RNA Northern hybridization indicated the rdgB gene transcribed five mRNAs ranging in size from 3.9 to 9.5 kb. These mRNAs were expressed in adult heads, but not detected in bodies. Analysis of RNA isolated from wild-type and eyes absent heads indicated that rdgB mRNA expression was not restricted to the retina. DNA sequence analysis of the transcription unit revealed an open reading frame capable of encoding a 116-kD transmembrane protein. The deduced protein shows no overall homology to previously described proteins, but has sequences in common with proposed functional domains of Ca(2+)-ATPase.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Olho , Proteínas de Membrana/genética , Células Fotorreceptoras/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Aberrações Cromossômicas , Mapeamento Cromossômico , Clonagem Molecular , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Feminino , Masculino , Dados de Sequência Molecular , Mutação , Replicon , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaAssuntos
Genes , Pigmentos da Retina/genética , Animais , Humanos , Rodopsina/genética , Rodopsina/fisiologiaRESUMO
In vitro mutagenesis and germline transformation were used to create a Drosophila mutant, delta Asn20, lacking the N-linked glycosylation site near the amino terminus of the major rhodopsin (Asn20-Gly-Ser changed to Ile-Gly-Ser). Low opsin protein levels are detected in delta Asn20 photoreceptors. Electroretinogram responses of mutant flies show that the residual rhodopsin found in this mutant is capable of initiating phototransduction. The organization of rhabdomeres, the photoreceptor organelle containing nearly all of the rhodopsin, is aberrant in the delta Asn20 mutant and undergoes age-dependent deterioration. These results establish that an N-linked glycosylation site, and likely glycosylation itself, plays a critical role in the maturation of Drosophila rhodopsin.
Assuntos
Asparagina/metabolismo , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/genética , Sequência de Bases , Drosophila , Eletroforese em Gel de Poliacrilamida , Eletrorretinografia , Proteínas do Olho/análise , Expressão Gênica , Glicosilação , Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestrutura , Rodopsina/genética , Opsinas de Bastonetes , Transdução de SinaisRESUMO
Four well characterized Drosophila rhodopsin (ninaE) mutants possess low levels of rhodopsin in their major class of photoreceptors. The molecular defect present in each strain was determined by isolating and sequencing the mutant genes. Two missense mutants encode proteins which have arginine residues positioned within membrane-spanning domains. The third missense mutant eliminates a proline found near an extracellular domain/membrane-spanning domain interface. Thus, the low levels of rhodopsin protein found in these mutants result directly from changes in protein structure which likely affect the positioning and stability of membrane-spanning domains. The fourth and most severe mutation is a nonsense mutation.
Assuntos
Drosophila/genética , Mutação , Pigmentos da Retina/genética , Rodopsina/genética , Animais , DNA/genética , Genes , Conformação ProteicaRESUMO
Rab6 is a GTP binding protein that regulates vesicular trafficking within the Golgi and post-Golgi compartments. We overexpressed wild-type, a GTPase defective (Q71L), and a guanine nucleotide binding defective (N125I) Rab6 protein in Drosophila photoreceptors to assess the in vivo role of Rab6 in the trafficking of rhodopsin and other proteins. Expression of Drab6(Q71L) greatly reduced the steady state levels of two rhodopsins, Rh1 and Rh3, whereas Drab6(wt) and Drab6(N125I) showed weaker effects. Analysis of a strain carrying Rh1 rhodopsin under a heat shock promoter showed that Drab6(Q71L), but not Drab6(wt) or Drab6(N125I), prevents the maturation of rhodopsin beyond an immature 40 kDa form. Drab6(Q71L) is a GTPase defective mutant, indicating that anterograde transport of rhodopsin requires Rab6 GTPase function. The three Drab6 strains had no effect on the expression of several other photoreceptor proteins. The Drab6(Q71L) photoreceptors show marked histological defects at young ages and degenerate over a two week time span. These results establish that rhodopsin is transported via a Rab6 regulated pathway and that defects in trafficking pathways lead to retinal degeneration.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Drosophila/fisiologia , Rodopsina/metabolismo , Proteínas rab de Ligação ao GTP , Proteínas ras/metabolismo , Animais , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Eletrofisiologia , GTP Fosfo-Hidrolases/fisiologia , Regulação da Expressão Gênica/genética , Complexo de Golgi/fisiologia , Luz , Microscopia Eletrônica , Mutação/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Regiões Promotoras Genéticas/genética , Retina/patologia , Proteínas ras/genéticaRESUMO
The Drosophila mutant, oraJK84, lacks rhabdomeres in the major (R1-6) class of photoreceptors because these rhabdomeres rapidly degenerate in young flies. Genetic analysis reveals that oraJK84 actually contains two mutations (a ninaE and an ort allele) that affect the visual process. The mutation in ort appears to have no effect on photoreceptor structure. The other mutation occurs within the ninaE gene, which encodes the species of rhodopsin found in the R1-6 class of photoreceptors. Our analysis shows that this mutation is responsible for R1-6 rhabdomere degeneration in oraJK84 mutants. We also examined a ninaE mutant, denoted ninaEo117, that produces no ninaE transcript. The morphological phenotype observed in ninaEo117 is similar to that seen in oraJK84 mutants. We conclude that rhodopsin plays a vital role in maintaining photoreceptor structure in Drosophila.
Assuntos
Drosophila/genética , Proteínas do Olho/genética , Genes , Mutação , Células Fotorreceptoras/fisiologia , Animais , Mapeamento Cromossômico , Cruzamentos Genéticos , Drosophila/fisiologia , Feminino , Masculino , Microscopia Eletrônica , Células Fotorreceptoras/ultraestrutura , Rodopsina/análise , Opsinas de BastonetesRESUMO
The Drosophila retinal degeneration C (rdgC) gene is required to prevent light-induced retinal degeneration. Molecular analysis shows that the rdgC transcription unit encodes a novel serine/threonine protein phosphatase. Amino acids 153-393 define a domain that has 30% identity with the catalytic domains of types 1, 2A, and 2B serine/threonine protein phosphatases. A putative regulatory domain is appended that contains multiple potential Ca(2+)-binding sites or "EF hand motifs." Thus, the analysis suggests that the rdgC protein is a novel type of serine/threonine protein phosphatase that is directly regulated by Ca2+. rdgC is expressed in the visual systems of the fly, as well as in the mushroom bodies of the central brain.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Drosophila , Drosophila melanogaster/genética , Fosfoproteínas Fosfatases/genética , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Análise Mutacional de DNA , Genes , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/metabolismo , Conformação Proteica , Mapeamento por Restrição , Alinhamento de Sequência , Distribuição TecidualRESUMO
Rhodopsin is essential for normal photoreceptor development in Drosophila (O'Tousa et al., 1989; Leonard et al., 1992; Kumar and Ready, 1995) and in mice (Humphries et al., 1997). Here we report studies in which a rhodopsin transgene is expressed at restricted stages during the development of Drosophila photoreceptors otherwise lacking rhodopsin. Substantial rescue of normal photoreceptor structure and physiology is effected by rhodopsin expression during the time of the normal onset of rhodopsin synthesis. Expression shortly before or after this critical period does not rescue these deficits. There is a critical developmental period in which rhodopsin plays its key role in photoreceptor morphogenesis.
Assuntos
Proteínas de Drosophila , Proteínas do Olho/fisiologia , Células Fotorreceptoras de Invertebrados/crescimento & desenvolvimento , Rodopsina/fisiologia , Animais , Animais Geneticamente Modificados , Drosophila , Eletrorretinografia , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Microscopia Eletrônica , Morfogênese , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Regiões Promotoras Genéticas , Retina/fisiologia , Rodopsina/genética , TemperaturaRESUMO
Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn(20) is the sole site for glycosylation. The Rh1(N20I) protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1(N20I); therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1(N20I)/NinaA interaction. We further show that Rh1(N20I) interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.
Assuntos
Asparagina , Proteínas de Drosophila , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Oligossacarídeos/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Proteínas de Membrana/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Oligossacarídeos/química , Células Fotorreceptoras de Invertebrados/metabolismo , Estrutura Secundária de Proteína , Rodopsina/genéticaRESUMO
ninaE(D1), a dominant allele of the major Drosophila rhodopsin gene, expresses a rhodopsin that is predominantly recovered in a 80-kD complex that likely represents rhodopsin dimers. By driving either ninaE(D1) or ninaE+ expression from a heat-shock promoter, we show that the 80-kD rhodopsin complex forms immediately after gene activation. In wild type, but not ninaE(D1), rhodopsin monomeric forms are detected at later times. The generation of monomeric forms of wild-type rhodopsin is suppressed in vitamin A-deprived flies or in flies heterozygous for the dominant rhodopsin mutation. We also show that ninaE(D1) expression does not affect the maturation of another Drosophila visual pigment, Rh3. These results are consistent with the view that the ninaE(D1) rhodopsin antagonizes an early posttranslation process that is specific for maturation of the ninaE-encoded rhodopsin.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Olho/genética , Expressão Gênica , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/genética , Animais , Western Blotting , Proteínas de Choque Térmico HSP70/genética , Reação em Cadeia da Polimerase , Rodopsina/metabolismo , Sitios de Sequências RotuladasRESUMO
The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.