Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach.

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.

Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs.

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.

Molecular response of Musca domestica L. to Mintostachys verticillata essential oil, (4R)+-pulegone and menthone.

Intense applications of synthetic insecticides for the control of adult Musca domestica have led to the insects developing resistance to most of them. In consequence, there is interest in new active ingredients as alternatives to conventional insecticides. Essential oils (EO) are potential tools for controlling M. domestica because of their effectiveness and their minimal environmental effects. In a fumigant assay, M. domestica adults treated with Minthostachys verticillata EO [LC(50)=0.5 mg/dm(3); majority components by SPME-GC: (4R)(+)-pulegone (67.5%), menthone (22.3%) and (4R)(+)-limonene (3.8%)], died within 15 min or less. The terpenes absorbed by the flies and their metabolites, analyzed using SPME fiber, were (4R)(+)-limonene (LC(50)=6.2 mg/dm(3)), menthone (LC(50)=1.9 mg/dm(3)), (4R)(+)-pulegone (LC(50)=1.7 mg/dm(3)) and a new component, menthofuran (LC(50)=0.3 mg/dm(3)), in a relative proportion of 12.4, 6.5, 35.9 and 44.2% respectively. Menthofuran was formed by oxidation of either (4R)(+)-pulegone or menthone mediated by cytochrome P450, as demonstrated by a fumigation assay on flies previously treated with piperonyl butoxide, a P450 inhibitor, which showed a decrease in toxicity of the EO, (4R)(+)-pulegone and of menthone, supporting the participation of the P450 oxidizing system in the formation of menthofuran. The enzymatic reaction of isolated fly microsomes with the EO or the (4R)(+)-pulegone produced menthofuran in both cases. Contrary to expectations, the insect detoxification system contributed to enhance the toxicity of the M. verticillata EO. Consequently, resistant strains overexpressing P450 genes will be more susceptible to either M. verticillata EO or (4R)(+)-pulegone and menthone.

Role of the triad N46, S106 and T107 and the surface charges in the determination of the acidic pH optimum of digestive lysozymes from Musca domestica.

Structures of digestive lysozymes 1 and 2 from housefly (MdL1 and MdL2) show that S106-T107 delimit a polar pocket around E32 (catalytic acid/base) and N46 contributes to the positioning of D50 (catalytic nucleophile), whereas those residues are replaced by V109-A110 and D48 in the non-digestive lysozyme from hen egg-white (HEWL). Further analyses revealed that MdL1 and MdL2 surfaces are less positively charged than HEWL surface. To verify the relevance of these differences to the acidic pH optimum of digestive lysozymes it was determined that pKas of the catalytic residues of the triple mutant MdL2 (N46D-S106V-T107A) are similar to HEWL pKas and higher than those for MdL2. In agreement, triple mutant MdL2 and HEWL exhibits the same pH optimum upon methylumbelliferylchitotrioside. In addition to that, the introduction of six basic residues on MdL1 surface increased by 1unit the pH optimum for the activity upon bacterial walls. Thus, the acidic pH optimum for MdL2 and MdL1 activities upon methylumbelliferylchitotrioside is determined by the presence of N46, S106 and T107 in the environment of their catalytic residues, which favors pKas reduction. Conversely, acidic pH optimum upon bacterial walls is determined by a low concentration of positive charges on the MdL2 and MdL1 surfaces.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut.

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.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae.

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (mu = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (mu = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotriozide (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (ì = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (ì = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica.

Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 angstroms using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2(1) (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 angstroms, beta = 102.97 degrees) and the orthorhombic space group P2(1)2(1)2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 angstroms), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.

7-Coumaryl permethrate and its cis- and trans-isomers as new fluorescent substrates for examining pyrethroid- cleaving enzymes.

New esterase substrates were synthesized using cis-, trans- and cis-trans-permethrinic acid chloride and then used to measure pyrethroid-cleaving enzymes in insects. The new substrates, namely cis-, trans- and cis-trans-7-coumaryl permethrates (7-CP), show a structure very similar to permethrin insecticide and yield fluorescent products on hydrolysis. These substrates were hydrolyzed by a commercial porcine preparation that provided esterase-specific activity, and were stable at different pH values (5.2-7.8). Studies made with house fly, Musca domestica (L.), homogenates showed that these compounds are appropriate for determining pyrethroid hydrolysis activity on individual insects. The measured activity of house fly esterase was 870 relative fluorescence units (RFU) min(-1) with cis-7-CP as substrate, 1117 RFU min(-1) with trans-7-CP as substrate and 1423 RFU min(-1) with cis-trans-7-CP as substrate. The fluorescent substrates for pyrethroid-cleaving esterases described in this paper have advantages over methods already given in the literature. They are substrates with structures very similar to pyrethroids, the cleavage of which can be followed by an increase in fluorescence emission at 440 nm; it takes only about 5 min to measure the reaction, and moreover the high sensitivity of the fluorescence technique allows the quantification of esterase activity on individual insects.

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.

Regional distribution and substrate specificity of digestive enzymes involved in terminal digestion in Musca domestica hind-midguts.

One membrane-bound alpha-glucosidase and two soluble alpha-glucosidases were isolated from homogenates of the hind-midgut, the main digestive region in Musca domestica larvae. The membrane-bound alpha-glucosidase and the low-Mr soluble alpha-glucosidase hydrolyze maltopentaose better than maltose, maltotriose, and maltotetraose, the reverse being true for the high-Mr soluble alpha-glucosidase. A membrane-bound glucoamylase previously described in Musca domestica midgut was shown by gradient centrifugation and dialysis against EDTA to result from the combined action of an amylase and an alpha-glucosidase. The determination of amylase, alpha-glucosidases, soluble and membrane-bound carboxypeptidase A, membrane-bound aminopeptidase and dipeptidase along the tissue and luminal contents of the hind-midgut is described. The data support a proposal concerned with how starch and protein are digested in Musca domestica larval hind-midguts and where and how midgut glycosidases and peptidases are secreted.

Assay of pyrethroid-hydrolysing esterases using (1,R)-cis-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropane carboxylates as substrates.

1. p-Nitrophenyl-, alpha-naphthyl-, and beta-naphthyl-(1,R)-cis-3-(2,2-dibromovinyl)2,2-dimethyl cyclopropane carboxylates are proposed as pyrethroid-like substrates for the detection of pyrethroid esterases (PyEs). 2. The substrates have been used in a spectrophotometric assay of PyEs and in a staining method to locate PyEs on polyacrylamide electrophoresis.