Antioxidant activities of protein hydrolysates obtained from the housefly larvae.

The housefly is an important resource insect and the housefly larvae are ideal source of food additives. The housefly larvae protein hydrolysates were obtained by enzymatic hydrolysis by alcalase and neutral proteinase. Their antioxidant activities were investigated, including the superoxide and hydroxyl radicalscavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, reducing power and metal chelating activity. The antioxidant activities of both hydrolysates increased with their increasing concentrations. The alcalase hydrolysate (AH) showed higher scavenging activities against hydroxyl radical and superoxide anion radical at low concentrations and higher metal-chelating activity than the neutral proteinase hydrolysate (NPH). The NPH exhibited higher scavenging activity against DPPH free radical and higher reducing power than the AH. Both hydrolysates showed more than 50% superoxide anion radical-scavenging activity at 10 µg/mL. These results indicate that both housefly larvae protein hydrolysates display high antioxidant activities and they could serve as potential natural antioxidant food additives.

Stage selection and restricted oviposition period improves cryopreservation of dipteran embryos.

Embryos of two dipteran species (Musca domestica and Lucilia sericata) were assessed for an effective sampling time that would result in the highest post-cryopreservation hatch rate, with a primary goal to define species-specific egg collection periods and the effects of manual stage selection on post cryopreservation yield. The effects of the time taken to collect eggs on, (a) the proportion of embryos reaching a specific developmental stage between 17 and 20 h of development, and (b) the post-cryopreservation hatch rate were assessed. Permeabilization treatment applied at any stage of embryonic development did not significantly reduce embryo viability. Eggs collected over longer durations significantly reduced the number of embryos available in a specific developmental stage amenable to cryopreservation. Hatch percentage after cryopreservation of the embryos of M. domestica collected over a 60 min period was 10.7 ± 8.7% compared to 31 ± 5% for the eggs collected for just 15 min. Similarly, percent hatch in L. sericata resulted in 17.0 ± 3.9 and <2% for 15 and 60 min samples, respectively. Significantly higher hatching rates were obtained for cryopreservation after manual selection of specific embryonic developmental stages from the dechorionated samples. Post-cryopreservation hatching rate for stage-selected M. domestica embryos was 86.5 ± 5.5% compared to 33.3 ± 4.5% for embryos staged only by an overall visual confirmation. In the case of L. sericata, the hatching percentage was 79.0 ± 11.1 for stage-selected embryos compared to 17.0 ± 3.9% without individual selection.

The influence of housefly Musca domestica embryo age on viability in water and at low temperatures.

The sensitivity of housefly Musca domestica L. (Diptera: Muscidae) embryos to storage at low temperatures (5 and 10 °C on moist sponges in Petri dishes) and in water at 26 °C was investigated to develop suitable protocols for the storage and transport of housefly eggs. The youngest embryos (aged 0-3 h) were the most sensitive to storage at 5 °C, with 45% survival after storage for 24 h. Storage of embryos aged 3-12 h at 5 °C for 24 h had no negative effect; longer storage resulted in significantly decreased larval survival (30-34% after 48-72 h, compared with 61% in the control group) and reduced hatching rates (83% after 72 h storage). No negative effects were observed when embryos aged 0-9 h were stored at 10 °C for 24 h, but this temperature did not completely inhibit development and eggs began to hatch if stored for longer than 24 h. All age groups of embryos showed high mortality after storage in water at 26 °C for 24 h, with the youngest embryos being least resistant to submersion.

Isolamento e seleção de fungos para controle de larvas de terceiro instar de Musca domestica / Isolation and selection of fungi in order to control Musca domestica third-instar larvae

O controle biológico no Brasil vem crescendo devido aos problemas gerados pelo uso indiscriminado de inseticidas químicos. A Musca domestica representa o maior problema em granjas avícolas devido às condições favoráveis para seu crescimento populacional. Sendo assim, foram realizadas capturas de dípteros muscoides em um aviário na região de Montes Claros, MG, usando armadilhas contendo isca química e captura por busca direta e, destas moscas, foram isolados e identificados fungos residentes nestes insetos. Os fungos isolados foram duas espécies do gênero Aspergillus sp., um do gênero Memnoniella sp., Scopulariopsis sp., Paecilomyces sp e um fungo da família Moniliaceae. Foram também requeridos junto ao CENARGEM/EMBRAPA as espécies de fungos entomopatogênicos Beauveria bassiana CG 470 e CG 472; Metarhizium anisopliae CG 34 e CG 312 e o Paecilomyces sp. CG 301. As espécies selecionadas para os bioensaios foram um Aspergillus sp., Memnoniella sp. e os Metarhizium anisopliae CG 34 e CG 312 por terem boa esporulação. Os fungos Aspergillus sp. e o Memnoniella sp. não apresentaram capacidade entomopatogênica, os fungos M. anisopliae CG 34 e CG 312 foram bastante eficientes em controlar a emergência dos adultos de M. domestica, mostrando-se bons agentes de controle biológico.

Use of biological control measures in Brazil has been increasing because of the problems generated from the indiscriminate use of chemical insecticides. Musca domestica represents the biggest problem to poultry farms due to the favorable conditions for its population growth. The present study was therefore conducted, beginning with captures of muscoid diptera on a poultry farm in the Montes Claros region, state of Minas Gerais, Brazil, using traps containing chemical bait and as well as capture by direct search, and the fungi resident on these flies was isolated and identified. The isolated fungi were two species of Aspergillus sp., one each from the genera Memnoniella sp., Scopulariopsis sp., Paecilomyces sp., and one fungi of the family Moniliaceae. Also, the entomopathogenic species of fungi Beauveria bassiana CG 470 and CG 472, Metarhizium anisopliae CG 34 and CG 312, and Paecilomyces sp. CG 301 were requested from CENARGEM/EMBRAPA. The species selected for the bioassays were Aspergillus sp., Memnoniella sp. and Metarhizium anisopliae CG 34 and CG 312. The fungi Aspergillus sp. and the Memnoniella sp. did not present entomopathogenic capacity; the fungi Metarhizium anisopliae CG 34 and CG 312 were sufficiently efficient in controlling the emergence of the adults of Musca domestica, showing themselves good agents of biological control.

[How many neuroblasts build mushroom bodies in Lucilia caesar L. and Musca domestica L. (Diptera, Brachycera Cyclorrhapha)?].

The green-bottle fly Lucilia caesar and the housefly Musca domestica differ greatly in the number of neuroblasts producing mushroom bodies. Four neuroblasts were found in each mushroom body of Lucilia pupae, and its calyx has a quadruple structure. In the housefly, the number of mushroom body neuroblasts rises up 20 in each brain hemisphere. This leads to a more complicated calyx structure. The neuroblast number observed in Lucilia and Musca is compared with that found in other Diptera.

Toxic effects of acrylamide on survival, development and haemocytes of Musca domestica.

The influence of acrylamide, a potentially toxic substance present in some types of food, on survival, postembryonic development and haemocytes, insect's blood cells, of the housefly was examined. Larvae were reared on media contaminated with acrylamide at concentrations of 82 microg/g, 164 microg/g or 246 microg/g. The length of larval and pupal stages as well as the survival of larvae and pupae was examined. To study the effects of acrylamide on haemocytes, the analysis of their index and morphology was performed in the third instar larva. The obtained data showed that the survival of larvae exposed to 82 microg/g and 164 microg/g concentrations of acrylamide decreased by 50% and 85%, respectively, whereas 246 microg/g concentration was lethal. In both groups of flies, larval and pupal stages were significantly lengthened by about 1.5 day in comparison with control. Moreover, acrylamide increased the number of prohaemocytes and intermediate cells while the number of plasmatocytes and granulocytes decreased. The size of plasmatocytes decreased in acrylamide-treated larvae when compared with these cells of control flies. The reduced survival of animals is probably due to affecting haemocytes involved in immune responses in insects. Moreover, the housefly's blood cells showed to be sensitive to toxin, which suggests their usefulness to test toxicity of substances present in food products.

Evolutionary and functional analysis of the tailless enhancer in Musca domestica and Drosophila melanogaster.

To further understand the evolutionary dynamics of the regulatory interactions underlying development, we expand on our previous analysis of hunchback and compare the structure and function of the tailless enhancer between Musca domestica and Drosophila melanogaster. Our analysis shows that although the expression patterns and functional protein domains of tll are conserved between Musca and Drosophila, the enhancer sequences are unalignable. Upon closer investigation, we find that these highly diverged enhancer sequences encode the same regulatory information necessary for Bicoid, Dorsal, and the terminal system to drive tll expression. The binding sites for these transcription factors differ in the sequence, number, spacing, and position between the Drosophila and Musca tll enhancers, and we were unable to establish homology between binding sites from each species. This implies that the Musca and Drosophila Bcd-binding sites have evolved de novo in the 100 million years since these species diverged. However, in transgenic Drosophila embryos the Musca tll enhancer is able to drive the same expression pattern as endogenous Drosophila tll. Therefore, during the rapid evolution of enhancer sequences individual binding sites are continually lost and gained, but the transcriptional output is maintained by compensatory mutations in cis and in trans.

Preliminary studies on the zinc-induced metallothionein protein with antibacterial activity in housefly larvae, Musca domestica.

Three isoforms of metallothionein protein induced with Zinc were isolated and purified from housefly larvae, Musca domestica, by gel filtration on Sephadex G-75, G-25 and anion exchange on DEAE-52 chromatography. Among them, one was found to possess antibacterial activity, and was further characterized by SDS-polyacrylamide gel electrophoresis, sulphydryl group determination, enzyme hydrolysis, and spectra property. Our results showed that the novel protein has the characteristics of heat-stable, low-molecular weight (6 kDa), rich-cysteine (approximately 12 cysteine residues in one molecule), metal affinity, and antibacterial activity. This paper was the first to report that metallothionein had antibacterial activity. We expect that this characteristic would give some help to investigate definite physiological functions of metallothionein.

Sex determination in Drosophila melanogaster and Musca domestica converges at the level of the terminal regulator doublesex.

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins' 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex ( dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX(M)) and female-specific (MdDSX(F)) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX(F) expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX(M) expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.

Cryopreservation of Musca domestica (Diptera: Muscidae) embryos.

Prior studies on cryopreserving embryos of several non-drosophilid flies established that two Drosophila melanogaster embryo cryopreservation protocols were not directly suitable for use with these species. This paper describes our work on developing a protocol for cryopreservation of embryos of the housefly, Musca domestica. Significant progress was made when permeabilization of the vitelline membrane was optimized, a vitrification solution containing ethylene glycol, polyethylene glycol, and trehalose was formulated, and when cooling and recovery of the cryopreservation protocol included a step which passed the embryos through liquid nitrogen vapor. More than 70% of housefly embryos withstand treatments of dechorionation, permeabilization, loading with cryoprotectant, and dehydration in vitrification solution, but the cooling, warming, and poststorage rearing steps still cause a considerable reduction in survival. About 53% of the vitrified M. domestica embryos hatched into larvae. Relative to the percentage of the control adult emergence, about 13% of the embryos stored in liquid nitrogen developed into fertile adults. Hatching of the F(1) progeny of adults having been cryopreserved as embryos was similar to control levels.

Sex-lethal, the master sex-determining gene in Drosophila, is not sex-specifically regulated in Musca domestica.

Sex-lethal (Sxl) is the master switch gene for somatic sex determination in Drosophila melanogaster. In XX animals, Sxl becomes activated and imposes female development; in X(Y) animals, Sxl remains inactive and male development ensues. A switch gene for sex determination, called F, has also been identified in the housefly, Musca domestica. An active F dictates female development, while male development ensues when F is inactive. To test if the switch functions of Sxl and F are founded on a common molecular basis, we isolated the homologous Sxl gene in the housefly. Though highly conserved in sequence, Musca-Sxl is not sex-specifically regulated: the same transcripts and protein isoforms are expressed in both male and female animals throughout development. Musca-Sxl is apparently not controlled by the primary sex-determining signal and, thus, is unlikely to correspond to the F gene. Ectopic expression of Musca-SXL protein in Drosophila does not exert any noticeable effects on the known target genes of endogenous Sxl. Instead, forced overexpression of the transgene eventually results in lethality of both XY and XX animals and in developmental abnormalities in some escaper XY animals. Similar results were obtained with the Sxl homologue of Ceratitis capitata (Saccone, G., Peluso, I., Artiaco, D. , Giodano, E., Bopp, D. and Polito, L. C. (1998) Development 125, 1495-1500) suggesting that, in these non-drosophilid species, Sxl performs a function different from that in sex determination.

The Y-chromosomal and autosomal male-determining M factors of Musca domestica are equivalent.

In Musca domestica, male sex is determined by a dominant factor, M, located either on the Y, the X or on an autosome. M prevents the activity of the female-determining gene F. In the absence of M, F becomes active and dictates female development. The various M factors may represent translocated copies of an ancestral Y-chromosomal M. Double mutants and germ line chimeras show that MY, MI, MII, MIII and MV perform equivalent functions. When brought into the female germ line, they predetermine male development of the offspring. This maternal effect is overruled by the dominant female-determining factor FD. MI and MII are weak M factors, as demonstrated by the presence of yolk proteins in MI/+ males and by the occurrence of some intersexes among the offspring that developed from transplanted MI/+ and MII/+ pole cells. The arrhenogenic mutation Ag has its focus in the female germ line and its temperature-sensitive period during oogenesis. We propose that MI and Ag represent allelic M factors that are affected in their expression. Analysis of mosaic gonads showed that in M. domestica the sex of the germ line is determined by inductive signals from the surrounding soma. We present a model to account for the observed phenomena.

An eye color gene for the detection of transgenic non-drosophilid insects.

A genetic marker for identifying transgenic Musca domestica by changes in eye color is described. The Drosophila melanogaster tryptophan oxygenase gene, vermilion (v), was tested for its ability to genetically complement the mutant tryptophan oxygenase gene in houseflies homozygous for green (ge). The v cDNA, placed under the control of the hsp82 promoter of D. pseudoobscura was transiently expressed in M. domestica embryos homozygous for the tryptophan oxygenase gene, ge, resulting in the rescue of adult eye color. The use of a gene from D. melanogaster to complement an eye color mutant in Musca provides the opportunity to develop a gene vector system for M. domestica and a select group of other non-drosophilid insects in which homologous mutations exist.

Using electroporation and a slot cuvette to deliver plasmid DNA to insect embryos.

Microinjection is the method used almost exclusively to deliver DNA constructs to insect embryos while electroporation is commonly used for DNA delivery to bacteria, cell cultures and certain plant tissues. This communication describes a method using an easily constructed slot cuvette and the electroporation technique for transfer of DNA to insect embryos for possible use in developing methods for germline transformation. This method eliminates time-consuming individual embryo manipulation and thus far has been found to be adaptable for use on several types of insect embryos. Using this method, we show successful transfer of plasmid DNA to embryos of the corn earworm moth, Helicoverpa zea, and the house fly, Musca domestica.

Genetic control of sex determination in the germ line and soma of the housefly, Musca domestica.

In Musca domestica, sex in the soma is cell autonomously determined by the male-determiner M, or by the female-determiner FD. Transplanted pole cells (precursors of the germ line) show that sex determination of germ cells is non-autonomous genotypically male pole cells form functional eggs in female hosts, and genotypically female pole cells form functional sperm in male hosts. When M/+ cells undergo oogenesis, a male-determining maternal effect predetermines offspring without M, i.e. of female genotype, to develop as fertile males. FD is epistatic to M in the female germ line, as it is in the soma, overruling the masculinizing effect of M. The results suggest that maternal F product is needed for activation of the zygotic F gene.

Comparative studies of larval and pupal phenoloxidase of the housefly, Musca domestica L.

1. Larval and pupal phenoloxidase of the housefly, Musca domestica L. resembled each other in some enzymatic properties, such as optimal pH, pH stability, optimal temperature and thermal stability. 2. The pI values of both enzymes were 4.85, as determined by isoelectric focusing. The molecular weights of two enzymes had a similar value (320,000 and 330,000 for larval and pupal phenoloxidase, respectively) and the molecular weights of these subunits had the same value of 60,000. 3. The aminio acid compositions of two enzymes were very similar. N-terminal amino acid sequences of larval and pupal enzymes were Glu-Glu-Ala-Thr-Val-Val-Pro-Asp-Gly- and Ala-Thr-Val-Val-Pro-Asp-Gly-Tyr-Phe-Met-, respectively, and the residues 3-9 (larval phenoloxidase) were in agreement with the residues 1-7 (pupal phenoloxidase).

The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase.

Antisera to purified house fly NADPH-cytochrome P450 reductase were used to select cDNA clones from an expression library of abdomens of phenobarbital-treated house flies. A partial cDNA of 1841 bp containing a TAG termination codon, a consensus polyadenylation site and 269 bp of 3' untranslated sequence was obtained. Sequencing of a genomic clone coupled with mRNA sequencing yielded the complete coding sequence including the starting ATG. The resulting open reading frame of 2013 nucleotides codes for a protein of 671 residues. The native reductase apoprotein has a molecular weight of 76,366 and the deduced molecular weight of the holoenzyme (i.e. with 1 mol of FAD and FMN) is 77,608. The sequence of the house fly P450 reductase protein is highly similar to that of rabbit liver, the overall amino acid positional identity is 54.5% and the overall identity among eukaryotic P450 reductases is about 25%. The P450 reductase gene of 19-23 kb was located on chromosome III, as shown by comparison of RFLP-patterns of the P450 reductase gene in two house fly strains and their hybrids.

Multiple conductances of neuronal nicotinic acetylcholine receptors.

In the presence of acetylcholine, cationic channels with three different conductances were recorded from neurones of the dissociated housefly (Musca domestica). Large conductance (80 pS) channels, resembling those that are abundant in reconstitution studies with a 65 kDa alpha-bungarotoxin affinity purified polypeptide, were detected in situ. The two larger conductance channels (80 pS; 32 pS) exhibited open and closed times that were best fitted by multiple exponential functions, indicating the presence of at least two open states. A third conductance (20 pS) showed brief, sparse openings and was least frequently observed. The 32 pS channel was the most common.

Segmentation gene expression in the housefly Musca domestica.

Drosophila and Musca both belong to the group of higher dipteran flies and show morphologically a very similar early development. However, these two species are evolutionary separated by at least 100 million years. This presents the opportunity for a comparative analysis of segmentation gene expression across a large evolutionary distance in a very similar embryonic background. We have analysed in detail the early expression of the maternal gene bicoid, the gap genes hunchback, Krüppel, knirps and tailless, the pair-rule gene hairy, the segment-polarity gene engrailed and the homoeotic gene Ultrabithorax. We show that the primary expression domains of these genes are conserved, while some secondary expression aspects have diverged. Most notable is the finding of hunchback expression in 11-13 stripes shortly before gastrulation, as well as a delayed expression of terminal domains of various genes. We conclude that the early developmental gene hierarchy, as it has been defined in Drosophila, is evolutionary conserved in Musca domestica.

Evidence for an inactive plasma membrane-associated precursor of active cytoplasmic ornithine decarboxylase in developing embryos of Musca domestica.

During embryonic development of Musca domestica inactive ornithine decarboxylase protein appears in the embryos at 6 h postoviposition, increases in concentration and reaches a maximum level at 9 h postoviposition. The inactive enzyme is associated with the plasma membrane and appears to be the precursor for active ornithine decarboxylase, which is associated with the cytosolic fraction just prior to hatching. Both ornithine decarboxylase protein and enzymatic activity disappear during the early larval stage of this insect.