RESUMO
Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 °C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila.
Assuntos
Quimotripsina/genética , Quimotripsina/metabolismo , Drosophila melanogaster/enzimologia , Moscas Domésticas/enzimologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Quimotripsina/química , Clonagem Molecular , DNA Complementar/genética , Sistema Digestório/química , Sistema Digestório/enzimologia , Drosophila melanogaster/química , Drosophila melanogaster/genética , Escherichia coli , Moscas Domésticas/química , Moscas Domésticas/genética , Proteínas de Insetos/química , Larva/química , Larva/enzimologia , Larva/genética , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3.
Assuntos
Catepsina D/genética , Catepsina D/metabolismo , Moscas Domésticas/enzimologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lisossomos/enzimologia , Sequência de Aminoácidos , Animais , Catepsina D/química , Bovinos , Sistema Digestório/química , Sistema Digestório/enzimologia , Moscas Domésticas/química , Moscas Domésticas/genética , Proteínas de Insetos/química , Insetos/classificação , Insetos/genética , Lisossomos/química , Lisossomos/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Vertebrados/classificação , Vertebrados/genéticaRESUMO
Spiders are important predators of several agricultural pests and they play an important role as indicators of ecosystem disturb. In Argentina, soybean crop has increased from the introduction of transgenic soybean resistant to glyphosate. This expansion produced an increase in the use of conventional and non-selective pesticides to control soybean pests. The objective of this work was to evaluate the side effects of subletal concentrations of two neurotoxican insecticides with a different mode of action: endosulfan (Glex, 35%, 25 mg/l a.i.) and spinosad (Tracer, 48%, 30 and 3 mg/l a.i) on Araneus pratensis. The insecticides were applied by ingestion of the treated prey (Musca domestica), and the effects on mortality, prey consumption, web building, mating, ootheca construction and fecundity were determined. Spinosad (30 mg/l a.i.) produced higher mortality than endosulfan (25 mg/l a.i.). Tremors and non-coordinated movements were observed in this treatment. The prey consumption was significantly reduced by the two insecticides (approximately 40% lower than control). The spider web building was significantly affected by the two insecticides, but spinosad had a greater effect. Though mating was not affected by both pesticides, abnormal oothecas and dehydrated eggs were observed. This work reports that sublethal concentrations representing approximately from 25 to 2.5% of the maximum field recommended concentrations (105 and 120 mg/l a.i., respectively) showed negative effects on A. pratensis. The consequences of these effects on role of A. pratensis as a natural mortality factor of soybean pests are discussed.
Assuntos
Cadeia Alimentar , Glycine max/parasitologia , Moscas Domésticas/química , Inseticidas/farmacologia , Aranhas/fisiologia , Animais , Argentina , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Endossulfano/farmacologia , Comportamento Alimentar , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Controle de Insetos/métodos , Macrolídeos/farmacologia , Masculino , Oviposição/efeitos dos fármacos , Oviposição/fisiologia , Aranhas/efeitos dos fármacos , Aranhas/crescimento & desenvolvimentoRESUMO
A larval specific high-density lipoprotein (HDL) has been isolated from Musca domestica hemolymph by a combination of density gradient and glycerol gradient ultracentrifugations. The larval lipoprotein has a density of 1.134 g/ml and is formed by at least four apoproteins with molecular weights equal to 26,000, 23,000, 21,000, and 20,000. This lipoprotein contains large amounts of hydrocarbons and phospholipids and minor amounts of diacylglycerols and cholesterol. The larval lipoprotein is completely distinct from lipophorin in regard to apoprotein composition, lipid moiety, physiological pattern, and immunological reactions. Larval lipoprotein is accumulated until the end of the feeding period. During the pupal molt this protein is utilized and is no longer detected after 2 days of pupal stadium. The results obtained imply a possible role of this protein in the puparia and/or pupal cuticle formation. Judging from the properties shown, the Musca domestica larval lipoprotein is a completely new type of insect lipoprotein.