RESUMO
Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (ß-pdh-I and ß-pdh-II). Phylogenetically, ß-pdh-I is more closely related to insect pdf than to ß-pdh-II, and we hypothesized that ß-PDH-I may represent a canonical clock output signal. Accordingly, ß-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf(01)) as a heterologous system, we show that ß-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but ß-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and ß-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but ß-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.
Assuntos
Relógios Biológicos/fisiologia , Braquiúros/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Neuropeptídeos/metabolismo , Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Comportamento Animal/fisiologia , Braquiúros/anatomia & histologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/classificação , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Duplicação Gênica , Dados de Sequência Molecular , Neurônios/metabolismo , Neuropeptídeos/classificação , Neuropeptídeos/genética , Peptídeos/classificação , Peptídeos/genética , Fenótipo , Filogenia , Alinhamento de SequênciaRESUMO
Lipins constitute a novel family of Mg(2+)-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Monoéster Fosfórico Hidrolases/metabolismo , Isoformas de Proteínas/metabolismo , Frações Subcelulares/enzimologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/classificação , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/fisiologia , Feminino , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/classificação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ovário/citologia , Ovário/metabolismo , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/genética , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Alinhamento de Sequência , Distribuição Tecidual , Triglicerídeos/metabolismoRESUMO
A number of insects produce acoustic signals during courtship. Genes involved in the control of the courtship song are particularly interesting from an evolutionary viewpoint because interspecific variation in this signal is potentially important as a reproductive isolation mechanism and, as a consequence, in the speciation process. The cacophony gene was identified by a mutation affecting the "lovesong" in Drosophila melanogaster. Phlebotomine sandflies (Diptera: Psychodidae) also produce acoustic stimuli during courtship and therefore cacophony can be used as an interesting molecular marker in evolutionary studies in these important disease vectors. In this paper we have studied the molecular evolution of the IVS6 region of cacophony in sandflies. We compared the level of divergence in the exon sequences encoding this conserved domain in Drosophila and Phlebotomines. We also analysed the high level of variation in an intron that is present in sandflies but that was lost in Drosophila during evolution. The available cacophony sequences were also used for a phylogenetic analysis of some species of the Neotropical genus Lutzomyia.