Metabolic regulation of two types of phenol oxidase activity in ontogenesis of house fly

Changes of the tyrosinase activity in ontogenesis of the house fly Musca domestica were shown to be phase-specific and ontogenetic changes of tyrosinase and dihydroxyphenylalanine oxidase activities proved to be coordinated. Ascorbic acid stimulated some ontogenetic stages of the house fly and physiological indices, such as fertility, survival at different stages, and weight of puparia. Also, ascorbic acid modulated the tyrosinase activity.     

Stabilization of mineralized and sclerotized puparial cuticle of muscid flies

Calcium, magnesium and phosphorus are the major mineral elements in puparial exuviae of the face fly, Musca autumnalis, house fly, M. domestica and stable fly, Stomoxys calcitrans, but they are 20–50 times more prevalent in face fly than in the other two species that sclerotize the puparium. Carbon and nitrogen are approx. 5 times more abundant in house fly puparia than in face fly puparia. Face fly puparia contain two and three-fold less total amino acids than the house fly and stable fly, respectively. β-Alanine is a major amino acid in puparial cuticle of the house fly and stable fly, but it is absent in the face fly. There is no significant difference in glucosamine (chitin) content between the three species. Dopamine is the major catechol detected in face fly puparial cuticle while N-β-alanyldopamine (NBAD) is 10 to 15 times more prevalent than other catechols such as dopamine, N-acetyldopamine (NADA), 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in house fly and stable fly puparial cuticles. The latter two species have 75 to nearly 200 times higher levels of extractable catechols than the face fly. At the onset of pupariation, dopamine and NBAD attain nearly equivalent titres in puparial cuticles of face fly and house fly, respectively. Dopamine subsequently decreases more than 40-fold in the face fly as the cuticle becomes stabilized, while NBAD continues to accumulate in the house fly. The house fly covalently incorporates about 150 times more catechols in the puparium than does the face fly. The force required to fracture house fly and stable fly puparia is about three-fold greater than that required to fracture face fly puparia of comparable thickness. However, the face fly puparium attains a strength comparable to those of house fly and stable fly puparia by significantly increasing its thickness. These results demonstrate that dipterans use both catecholamines and minerals for stabilization of puparial cuticle with the house fly and stable fly relying primarily on sclerotization and the face fly on mineralization.     

Expression of cytochrome P-450lpr is developmentally regulated and limited to house fly.

Expression of house fly cytochrome P-450lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450lpr was expressed in both male and female adult house flies. P-4501pr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-4501pr, was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450lpr.     

Larvicidal activity of endectocides against pest flies in the dung of treated cattle

Cattle were treated with topical formulations of endectocides to assess the larvicidal activity of faecal residues against horn fly, Haematobia irritans (L.), house fly, Musca domestica L., and stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae). In laboratory bioassays, doramectin, eprinomectin and ivermectin suppressed horn fly in dung of cattle treated at least 4 weeks previously and suppressed house fly and stable fly in dung of cattle treated 1-5 weeks previously. Moxidectin suppressed horn fly in dung from cattle treated no more than one week previously and did not suppress house fly and stable fly. Results combined for the three species across two experiments suggested that, ranked in descending order of larvicidal activity, doramectin > ivermectin approximately = eprinomectin > moxidectin.     

Immature house fly (Musca domestica) control in breeding sites with a new Brevibacillus laterosporus formulation

A bacterial formulation containing spores of a Brevibacillus laterosporus strain from Sardinia, known to be toxic by ingestion to the house fly (Musca domestica), was assayed in laboratory, outdoor, and field conditions for the control of immature stages of this pest. In all laboratory assays, the bacterial formulation exhibited toxicity against house fly larvae. A concentration of 1 x 10(8) spores of B. laterosporus/g of diet caused 100% mortality of house fly immature stages. The same formulation, applied at a concentration of 1 x 10(8) spores/ml, equivalent to a dose of 2 liters/m(2), caused a reduction in adult emergence from laboratory and natural breeding substrates (outdoor cage experiments) up to 80.3 and 57.8%, respectively. Similarly, this formulation applied in the cow pen of a diary farm at a dose of 2 liters/m(2) produced a significant reduction (30%) in immature fly development. Therefore, the use of this bacterial preparation in microbiological control strategies for the integrated pest management of this species is promising.     

Expression of cytochrome P-450lpr Is developmentally regulated and limited to house fly

Expression of house fly cytochrome P-450_lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450_lpr was expressed in both male and female adult house flies. P-450_lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450_lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450_lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450_lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450_lpr.     

Anti-P450lpr antiserum inhibits specific monooxygenase activities in LPR house fly microsomes.

Monooxygenase activity in microsomes from the LPR strain of house fly (Musca domestica L.) was inhibited by anti-P450lpr, and antiserum specific for house fly cytochrome P450lpr. Anti-P450lpr did not inhibit house fly cytochrome P450 reductase or rat cytochrome P450 monooxygenase assays, consistent with specific inhibition of P450lpr. Anti-P450lpr inhibited the ability of cytochrome P450 reductase to reduce carbon monoxide treated LPR microsomal cytochrome P450, up to 49% of the total, showing that inhibition of cytochrome P450 reduction is the major mechanism of inhibition. Anti-P450lpr inhibited 98% of methoxyresorufin-O-demethylase activity and all the benzo(a)pyrene hydroxylase activity in LPR microsomes, but none of the pentoxyresorufin-O-dealkylase activity. The antiserum partially inhibited ethoxyresorufin-O-dealkylase and ethoxycoumarin-O-dealkylase activity. These results demonstrate that methoxyresourfin-O-demethylase activity and benzo(a)pyrene hydroxylase activity are characteristic substrates for P450lpr activity in the LPR strain of house fly.     

Evaluation of specific activity of preparations of allergens from synanthropic insects

Physical, chemical and immunobiological characteristics of allergens from synanthropic insects were studied by tests for anaphylaxis, indirect degranulation of mast cells test and ELISA. Sera from 20 patients with severe and intermediate atopic asthma with sensiblization to common allergens have been studied. All extracts of allergens from synanthropic insects (german cockroach, oriental cockroach, american cockroach, speckled feeder cockroach, cricket, common house fly, brown house moth, confused flour beetle, rice weevil, grain weevil) have specific activity. Extracts of allergens from common house fly, brown house moth, german cockroach and oriental cockroach had the strongest allergenic activity as measured by ELISA. Obtained allergens can be used for insect allergy diagnostics.     

Evaluation of surveillance methods for monitoring house fly abundance and activity on large commercial dairy operations

Relative house fly, Musca domestica L., activity at three large dairies in central California was monitored during the peak fly activity period from June to August 2005 by using spot cards, fly tapes, bait traps, and Alsynite traps. Counts for all monitoring methods were significantly related at two of three dairies; with spot card counts significantly related to fly tape counts recorded the same week, and both spot card counts and fly tape counts significantly related to bait trap counts 1-2 wk later. Mean fly counts differed significantly between dairies, but a significant interaction between dairies sampled and monitoring methods used demonstrates that between-dairy comparisons are unwise. Estimate precision was determined by the coefficient of variability (CV) (or SE/mean). Using a CV = 0.15 as a desired level of estimate precision and assuming an integrate pest management (IPM) action threshold near the peak house fly activity measured by each monitoring method, house fly monitoring at a large dairy would require 12 spot cards placed in midafternoon shaded fly resting sites near cattle or seven bait traps placed in open areas near cattle. Software (FlySpotter; http://ucanr.org/ sites/FlySpotter/download/) using computer vision technology was developed to count fly spots on a scanned image of a spot card to dramatically reduce time invested in monitoring house flies. Counts provided by the FlySpotter software were highly correlated to visual counts. The use of spot cards for monitoring house flies is recommended for dairy IPM programs.     

LIFE IN ZERO MAGNETIC FIELD. IV. INVESTIGATION OF DEVELOPMENTAL EFFECTS ON FRUITFLY VISION

The fruitfly (Drosophila melanogaster) visual system was investigated electrophysiologically in vivo after exposure to a zero magnetic field (ZMF). Electroretinographic (ERG) recording of fly eye electrical activity was performed on adult insects raised from pupae maintained for 20 hr in zero magnetic field. A flickering excitation regime was applied to excite the visual system, since in this way, a quasistable hyperpolarization component of the electroretinogram can be obtained, containing information from the neural cells, which are the most sensitive to the action of external factors during early ontogenetic stages. Results of the investigation of two D. melanogaster populations, sample and control, were statistically compared.

We found a significant statistical increase of sensitivity in neural cells from the first optic ganglion in the fly population developed from pupae exposed to ZMF.     

Tyrosinase inhibition kinetics of anisic acid

Anisic acid (p-methoxybenzoic acid) was characterized as a tyrosinase inhibitor from ani-seed, a common food spice. It inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase with an IC50 of 0.60 mM. The inhibition of tyrosinase by anisic acid is a reversible reaction with residual enzyme activity. This phenolic acid was found to be a classical noncompetitive inhibitor and the inhibition constant K(I) was obtained as 0.603 mM. Anisic acid also inhibited the hydroxylation of L-tyrosine catalyzed by tyrosinase. The lag phase caused by the monophenolase activity was lengthened and the steady-state activity of the enzyme was decreased by anisic acid.     

Larvicidal potential of the polyol sweeteners erythritol and xylitol in two filth fly species

The house fly, Musca domestica (L.) (Diptera: Muscidae), and the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), are two filth flies responsible for significant economic losses in animal production. Although some chemical control products target adults of both species, differences in mouthpart morphology and behavior necessitates distinct modalities for each. For these reasons, larvicides are an attractive means of chemical control. We assessed the potential of the polyol sweeteners erythritol and xylitol as larvicides to the house fly and stable fly. LC₅₀ values of erythritol against 2^nd instar larvae were 34.94 mg/g media (house fly) and 22.10 mg/g media (stable fly). For xylitol, LC₅₀ values were 74.91 mg/g media (house fly) and 41.58 mg/g media (stable fly). When given a choice, neither species showed a preference for ovipositing in media treated with either sweetener at various concentrations or in media without sweetener. Significantly lower development from egg to adult was observed when the 2^nd instar LC₅₀ equivalent of each sweetener was present in the media compared to controls. Erythritol and xylitol both have larvicidal qualities, however their effective concentrations would necessitate creative product formulation and deployment methods to control all stages of developing flies.     

Atypical Granules in the Eyes of the White Mutant of Drosophila melanogaster Are Lysosome-Related Organelles

In the pigment cells of the white mutant of Drosophila melanogaster, as described earlier, two types of abnormal granules are found by conventional electron microscopy. However, both types of abnormal granules, in addition to those in pigment cell invaginations, are also present in the cytoplasm of the photoreceptor cells. Three enzymes (acid phosphatase, peroxidase, and tyrosinase) are localized within the eyes of wild type and white mutant Drosophila melanogaster by electron microscopy. Peroxidase activity is present in lamellar bodies close to the rhabdomeral microvilli of both fly types. However the organelles containing peroxidase activity are 6-fold more frequent in the wild type than in the mutant. Acid phosphatase is present in lamellar bodies between and at the bases of the rhabdomeral microvilli of the wild type, as well as in ommochrome granules of the photoreceptor cells. In the white mutant, however, acid phosphatase was located in electron lucent vacuoles in the cytoplasm of the receptor cells. These acid phosphatase-positive vacuoles also contained both types of abnormal granules. The latter result indicates that abnormal granules in the receptor cells originate from lysosomal degradation and that targeting of lysosomal enzymes is altered in the white mutant. Due to the tyrosinase activity in the hemolymph of flies, the extracellular spaces are electron dense after DOPA incubation. Since some abnormal granules within the photoreceptor cells are not surrounded by an extracellular space, they can be assumed to originate within the photoreceptor cells.     

Dot-blot test for identification of insecticide-resistant acetylcholinesterase in single insects

A test was developed to detect the presence of insecticide-resistant acetylcholinesterase (AChE) in single insects based on the quasipermanent binding of proteins onto blotting membranes. The method is simple, sensitive, requires inexpensive equipment, and produces a permanent record of results. AChE activity is revealed by the Karnovsky & Roots staining technique in the presence of propoxur, or after exposure of the membrane to paraoxon and rinsing with water. We chose insecticide concentrations that inhibited the sensitive AChE while allowing detectable residual activity of the resistant AChE to remain. By comparing the staining of insecticide-treated and control membranes, susceptible and resistant genotypes for the AChE gene could be distinguished in laboratory strains of mosquitoes (Culex spp. and Anopheles albimanus Wiedemann) and the house fly (Musca domestica L.). Resistant AChE from mosquitoes was less susceptible both to propoxur and paraoxon than the corresponding sensitive AChE, whereas resistant AChE from house fly was less susceptible mainly to paraoxon. The technique worked well for mosquito adults and house fly heads but not for mosquito larvae. Blotted AChE did not show detectable loss of activity during storage of the membranes for 3 wk at 25 degrees C. Storage is an important asset of the technique because transportation of live insect material to the laboratory may not be necessary.     

Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis

Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme-linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL-11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM-7. Both cell lines responded to melanocyte-stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM-7 cells were found to be approximately one-half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM-7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.     

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅰ:水解代谢

水解代谢在家蝇对二氯苯醚菊酯抗性中起重要作用。正常家蝇和抗性家蝇酯酶在对SV₁、SV₂等抑制剂的敏感性和电泳性质上存在着差异。抗性家蝇酯酶水解二氯苯醚菊酯的活性较高,水解乙酸-α-萘酯的活性相对比正常家蝇要低。SV₁及其在体内的代谢产物SV₂在离体和活体情况下对家蝇酯酶都有明显的抑制作用。SV₁和SV₂抑制相同的酯酶电泳条带,但SV₁的抑制作用相对小一些。SV_t对酯酶的抑制是它在家蝇体内对二氯苯醚菊酯增效的机理之一。     

Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures

The activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, is higher in Black skin melanocytes than in melanocytes derived from Caucasian skin. This variation in enzyme activity is not due to differences in tyrosinase abundance or tyrosinase gene activity, but, rather, is due to differences in the catalytic activity of preexisting tyrosinase. In melanocytes, tyrosinase is localized to the membrane of melanosomes and in Caucasian melanocytes the melanosome-bound enzyme is largely inactive. Conversely, in melanosomes of Black melanocytes, tyrosinase has high catalytic activity. Treatment of Caucasian melanocytes with the lysosomotropic compound ammonium chloride or with the ionophores nigericin and monensin results in a rapid and pronounced increase in tyrosinase activity. This increase occurs without any change in tyrosinase abundance, indicating that these compounds are increasing the catalytic activity of preexisting enzyme. Inhibition of the vacuolar proton pump V-ATPase by treatment of Caucasian melanocytes with bafilomycin also increases tyrosinase activity. In contrast to the 10-fold increase in tyrosinase observed in Caucasian melanocytes, neither ammonium chloride, monensin, nigericin, nor bafilomycin is able to increase the already high level of tyrosinase activity present in melanosomes of melanocytes derived from Black skin. Finally, staining of Caucasian melanocytes with the fluorescent weak base acridine orange shows that melanosomes of Caucasian, but not Black, melanocytes are acidic organelles. These data support a model for racial pigmentation that is based on differences in melanosome pH in Black and Caucasian skin types. The models suggests that melanosomes of Caucasian melanocytes are acidic, while those of Black individuals are more neutral. Since tyrosinase is inactive in an acid environment, the enzyme is largely inactive in Caucasian melanosomes but fully active in Black melanosomes.     

Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis

A novel kojic acid derivative containing a trolox moiety, (±)-5-hydroxy-4-oxo-4H-pyran-2-yl methyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (3a), was synthesized. The two biologically active compounds, namely, kojic acid and trolox, were conjugated via an ester bond as they are expected to behave synergistically. The antioxidant activity and the tyrosinase inhibitory activity of this novel kojic acid derivative on melanogenesis were evaluated. Compound 3a exhibited potent tyrosinase inhibitory activity and radical scavenging activity. Limited structure–activity relationship (SAR) investigations indicated that the tyrosinase inhibitory activities may originate from the kojic acid moiety, and the radical scavenging activity may be due to the phenolic hydroxyl group of trolox. Compound 3a also exhibited potent depigmenting activity in a cell-based assay. The limited SAR investigations revealed that the depigmenting activity of 3a may be due to the synergistic activities of kojic acid and its trolox moiety.