RESUMO
Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl- (NKCC) and K+:Cl- (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.
Assuntos
Moscas Domésticas/metabolismo , Proteínas de Insetos/metabolismo , Animais , Transporte Biológico , Trato Gastrointestinal/metabolismo , Moscas Domésticas/enzimologia , Moscas Domésticas/genética , Moscas Domésticas/crescimento & desenvolvimento , Proteínas de Insetos/genética , Filogenia , Proteoma/metabolismo , RNA-Seq , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Membro 4 da Família 12 de Carreador de Soluto/genética , Membro 4 da Família 12 de Carreador de Soluto/metabolismo , Água/metabolismoRESUMO
To disclose the molecular mechanisms involved in luminal midgut buffering of M.â¯domestica, we used RNA-seq analyses from triplicate samples of seven sections along the midgut to evaluate the expression levels of genes coding for selected manually curated protein sequences. Channels, pumps and transporters were confirmed as being apical by proteomics of purified microvillar membranes. Midgut pH determinations with a microsensor and a pH indicator were carried out in larvae in different diets with or without added compounds to evaluate the role of proteins in buffering. The data suggested that acidification occurs at middle midgut by the action of H+ V-ATPase with protons produced by carbonic anhydrase, followed by chloride ions transported by a K+Cl- symporter. K+ ions are recovered by an apical K+ channel and K+ homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is also affected by a Na+/H+ exchanger and a multidrug resistance protein. Posterior midgut alkalization results from the action of a NH3 transporter and H+-coupled peptide transporter, mainly in a diet rich in free peptides. A working model was proposed for the midgut luminal acidification and alkalization, as well as for mucosal protection against acid by a neutralized mucus layer.
Assuntos
Transporte Biológico/genética , Moscas Domésticas/genética , Proteínas de Insetos/genética , Larva/genética , Ácidos/química , Ácidos/farmacologia , Álcalis/química , Álcalis/farmacologia , Animais , Sistema Digestório/metabolismo , Moscas Domésticas/metabolismo , Concentração de Íons de Hidrogênio , Larva/efeitos dos fármacos , Larva/metabolismo , Proteômica , RNA-Seq , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Until now there is no molecular model of starch digestion and absorption of the resulting glucose molecules along the larval midgut of Musca domestica. For addressing to this, we used RNA-seq analyses from seven sections of the midgut and carcass to evaluate the expression level of the genes coding for amylases, maltases and sugar transporters (SP). An amylase related protein (Amyrel) and two amylase sequences, one soluble and one with a predicted GPI-anchor, were identified. Three highly expressed maltase genes were correlated with biochemically characterized maltases: one soluble, other glycocalyx-associated, and another membrane-bound. SPs were checked as being apical or basal by proteomics of microvillar preparations and those up-regulated by starch were identified by real time PCR. From the 9 SP sequences with high expression in midgut, two are putative sugar sensors (MdSP4 and MdSP5), one is probably a trehalose transporter (MdSP8), whereas MdSP1-3, MdSP6, and MdSP9 are supposed to transport glucose into cells, and MdSP7 from cells to hemolymph. MdSP1, MdSP7, and MdSP9 are up-regulated by starch. Based on the data, starch is at first digested by amylase and maltases at anterior midgut, with the resulting glucose units absorbed at middle midgut. At this region, low pH, lysozyme, and cathepsin D open the ingested bacteria and fungi cells, freeing sugars and glycogen. This and the remaining dietary starch are digested by amylase and maltases at the end of middle midgut and up to the middle part of the posterior midgut, with resulting sugars being absorbed along the posterior midgut.
Assuntos
Glucose/metabolismo , Moscas Domésticas/metabolismo , Amido/metabolismo , Animais , Sistema Digestório/enzimologia , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/metabolismo , Expressão Gênica , Moscas Domésticas/genética , Moscas Domésticas/crescimento & desenvolvimento , Larva/enzimologia , Larva/genética , Larva/metabolismo , Proteômica , Análise de Sequência de RNARESUMO
Most insects have a gut lined with a peritrophic membrane (PM) consisting of chitin and proteins, mainly peritrophins that have chitin-binding domains. The PM is proposed to originate from mucus-forming mucins (Mf-mucins), which acquired a chitin-binding domain that interlocked with chitin, replacing mucus in function. We evaluated the expression of Mf-mucins and peritrophins by RNA-sequencing (RNA-seq) throughout the midgut of four distantly related insects. Mf-mucins were identified as proteins with high o-glycosylation and a series of uninterrupted Pro/Thr/Ser residues. The results demonstrate that the mucus layer is widespread in insects, and suggest that insect Mf-mucins are derived from those found in other animals by the loss of the cysteine knot and von Willebrand domains. The data also support a role of Mf-mucins in protecting the middle midgut of Musca domestica against acidic buffers. Mf-mucins may also produce a jelly-like material associated with the PM that immobilizes digestive enzymes in Spodoptera frugiperda. Peritrophins with a domain similar to Mf-mucins may be close to the ancestor of peritrophins. Expression data of peritrophins and chitin synthase genes throughout the midgut of M. domestica, S. frugiperda and Tenebrio molitor indicated that peritrophins were incorporated along the PM, according to their preferential sites of formation. Finally, the data support the view that mucus has functions distinct from the PM.
Assuntos
Proteínas de Insetos/genética , Mucinas/metabolismo , Animais , Sistema Digestório/metabolismo , Gafanhotos/genética , Gafanhotos/metabolismo , Moscas Domésticas/genética , Moscas Domésticas/metabolismo , Proteínas de Insetos/metabolismo , Análise de Sequência de RNA , Spodoptera/genética , Spodoptera/metabolismo , Tenebrio/genética , Tenebrio/metabolismoRESUMO
Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.
Assuntos
Moscas Domésticas/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica , Moscas Domésticas/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Masculino , Dados de Sequência Molecular , Oogênese , Regiões Promotoras GenéticasRESUMO
During Musca domestica vitellogenesis a protein is preferentially synthesized by the female fat body and accumulates in the haemolymph but not in the ovaries. This protein, designated nonvitellogenic female protein (NVFP), was purified and shown to be a hexamer with an M(r) = 430 kDa, and subunits of M(r) = 70 kDa. The hexamer dissociates into subunits when the pH is elevated from 7.0 to 9.0. Two cDNA clones, F0 and F2, were isolated and analysed. The 2.2 kb F2 clone has an open reading frame that encodes a conceptual translation product that has similarity to the Drosophila melanogaster LSP-2 hexamerin. Recombinant protein from the F2-cDNA is recognized by a specific anti-NVFP serum. The temporal pattern of mRNA expression of the gene represented by the F2 clone follows that determined for the synthesis of NVFP. The data support the conclusion that NVFP is an hexamerin specific to the adult stage of Musca domestica.
Assuntos
Moscas Domésticas/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Clonagem Molecular , DNA Complementar , Escherichia coli , Feminino , Moscas Domésticas/metabolismo , Proteínas de Insetos/imunologia , Proteínas de Insetos/isolamento & purificação , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Homologia de Sequência de Aminoácidos , VitelogêneseRESUMO
We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 +/ 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage.
Assuntos
Ferritinas/sangue , Moscas Domésticas/metabolismo , Animais , Ferritinas/isolamento & purificação , Hemolinfa , Larva , Pupa , CoelhosRESUMO
The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al., 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al., 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).
Assuntos
Catepsinas/metabolismo , Moscas Domésticas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Feminino , Cinética , Dados de Sequência Molecular , ÓvuloRESUMO
1. Lysozyme is absent from tissues other than the midgut in the drug-feeding larvae of Musca domestica (Diptera, Cyclorrhapha, Muscidae) and in the fruit-feeding larvae of Anastrepha fraterculus (Diptera, Cyclorrhapha, Tephritidae), whereas in the detritus-feeding larvae of Trichosia pubescens (Diptera, Nematocera, Sciaridae) lysozyme is only found in the hemolymph and in the fat body. 2. A. fraterculus larvae have a midgut region with a luminal pH of 3.4, and display a pepstatin-inhibited acid proteolytic activity which has a spec. act. (7.2 U/mg protein) similar to that of M. domestica. 3. The midgut lysozyme from M. domestica and A. fraterculus is more active (high ionic strength) at pH 3.5 than at pH 6.0, the contrary being true for a midgut chitinase. 4. The results suggest that the adaptations to digest bacteria in insects are similar to those in vertebrate foregut fermenters, and that these characteristics were probably present in the Cyclorrhapha ancestor, but not in the Diptera ancestor.
Assuntos
Dípteros/metabolismo , Muramidase/metabolismo , Animais , Bactérias/metabolismo , Bacteriólise , Evolução Biológica , Quitinases/metabolismo , Moscas Domésticas/metabolismo , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Larva/metabolismoRESUMO
Two distinct fractions of Musca domestica arylphorin were isolated by affinity chromatography on Concanavalin A-Sepharose column. The results show that in the hexameric arylphorin that do not bind to the lectin there is no Concanavalin A binding subunit and in the majority of the hexamers that bind to the lectin there is only one subunit with Concanavalin A binding site. The results indicate that the carbohydrate moiety of the arylphorin is not involved in its specific uptake by the fat bodies and integument.