Soluble and particle-bound trehalase in extracts from larvae, pupae, and adults of Musca domestica

Trehalase activity was measured in tissue homogenates and extracts from the larval, pupal, and adult stages of Musca domestica, the common housefly. The tissue homogenates were separated into soluble and particlebound fractions by differential centrifugation, and the trehalase activities of the fractions were measured. The trehalase specific activity (units of enzyme/mg protein) in homogenates from adult insects was nearly twenty times greater than activity in homogenates of larvae. Homogenates of pupae showed intermediate values. In both the adults and larvae the enzyme activity was approximately evenly distributed between soluble and particle-bound forms, whereas 95 per cent of the trehalase activity in the extract of pupae was in the soluble fraction. The results show that the form and amount of trehalase present during housefly development is adjusted to accommodate the enzyme's physiological rôle of splitting trehalose to glucose for the insect's use as an energy source.     

In vitro glucosylation of 1-naphthol by insects

Glucosylation in insects was investigated using tobacco hornworms (Manduca sexta) as the primary test insect and 1-naphthol-¹⁴C as the substrate. Of 6 common co-factors tested, only UDPG was utilized by the conjugating enzyme system. Neither the hornworm nor housefly enzymes could form the glucuronic acid derivative of 1-naphthol using UDPGA. Centrifugal fractionation of the hornworm homogenates showed that the glucosyltransferase activity was in the 105,000 g soluble fraction. In the housefly, the enzyme activity was associated with the 15,000 g pellet and to a lesser extent with the 105,000 g pellet. In vitro inhibition of the glucosyltransferase by sulphoxide, piperonyl butoxide, and other insecticide synergists was demonstrated.     

The separation of multiple forms of housefly 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) dehydrochlorinase from glutathione S-aryltransferase by electrofocusing and electrophoresis

Electrophoresis in a sucrose gradient at pH values between 5 and 8 separated housefly DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] dehydrochlorinase into two major fractions. GSH S-aryltransferase under similar conditions migrated as a single peak of activity. Separation of housefly homogenates or partially purified enzyme preparations by electrofocusing in a natural pH gradient also showed the presence of multiple forms of DDT dehydrochlorinase.     

tRNA methyltransferases during embryogenesis in Musca domestica

Activity and diversity of the tRNA methyltransferases were examined during embryogenesis of the housefly, Musca domestica. A rapid rise in the activity of the tRNA methyltransferases was observed during the first 3 hr of embryogenesis. Activity increased slowly until the tenth hour of embryogenesis and then declined until hatching at 12 hr. The greatest diversity of tRNA methyltransferases, as indicated by extent of methylation, existed at 6 hr of embryogenesis; the least diversity was observed in 1-hr embryos, while 12-hr embryos showed intermediate levels. Inhibition of embryonic tRNA methyltransferases at high concentrations of enzyme was observed in all extracts examined.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium

S-Adenosylmethionine:homocysteine methyltransferase activity was monitored during embryogenesis of the housefly, Musca domestica. A rapid decrease in the activity of S-adenosylmethionine:homocysteine methyltransferase was observed during the first 3 hr of embryogenesis. Activity continued to decline less rapidly until hatching at 12 hr. An inverse relationship was found to exist between the activities of S-adenosylmethionine:homocysteine methyltransferase and the tRNA methyltransferases during Musca embryogenesis.     

Soluble acid phosphatases from the posterior reproductive system of the female housefly, Musca domestica

When the kinetics of the acid phosphatase enzyme system from the posterior reproductive tract of the female housefly, Musca domestica, were studied, enzyme activity was zero order for 2 hr and was temperature-dependent. Orthophosphate release was linearily related to enzyme concentration, and pH optima between pH 3·3 to 3·7, 4·2 to 4·8, and 5·2 to 5·7 were observed. Magnesium and calcium ions were enzyme activators; arsenate, phosphate, fluoride, and hydroxymalonate ions were inhibitors. Sodium azide had little effect. The similarity of activity exhibited on the test substrates indicated that the soluble enzymes corresponded to the non-specific phosphomonoesterases.Disk electrophoresis showed that at least 6 proteins had acid phosphatase activity and were pH-dependent. Also, electrophoresis of extracts of the various structures of the reproductive tract showed that there was a tissue specificity in the distribution of the acid phosphatase isoenzymes.     

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica)

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR?DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.     

Low speed preparation of microsomes: a comparative study

Microsomal fractions were isolated from hepatic tissue, housefly abdomen and southern armyworm midgut, either by centrifugation of post-mitochondrial supernatant for 1 h at 105 000×g or by calcium sedimentation and low speed centrifugation. A comparison of the two methods of isolation were made on the basis of: NADPH oxidase activity, O-demethylation of p-nitroanisole, spectral parameters of cytochrome P-450, and integrity of ultrastructural constituents. The method of isolation did not effect these parameters appreciably for microsomes isolated from hepatic tissue. Calcium sedimentation of insect microsomes resulted in a reduction of enzyme activity and an apparent decrease in cytochrome P-450. Ultrastructural integrity did not appear to be influenced by the method of sedimentation.     

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.     

Comparative study of the interaction of acetylcholinesterases of human erythrocytes and the heads of houseflies with phosphorylated alkylchloroformoximes

Studies have been made on the interaction of four types of phosphorylated alkylchloroformoximes, i.e. analogues of an insecticide-acaricide valexon, with acetylcholinesterases from human erythrocytes and from the heads of the housefly Musca domestica. Antiacetylcholinesterase activity of the drugs depended both on the structure of the organophosphorus compounds, and the origin of the enzyme, indicating the existence of differences in the active surface of these acetylcholinesterases. Incorporation of one or two chloride atoms into alkylchloroformoxime group of the cleaved part of the organophosphorus compounds increased anticholinesterase activity with respect to both enzymes. Diethyl derivatives of these drugs exhibited higher specificity with respect to housefly enzyme as compared to human acetylcholinesterase.     

Spectral and catalytic properties of cytochrome P-450 from a diazinon-resistant housefly strain

Microsomes from the diazinon-resistant Rutgers strain of housefly contain amounts of cytochrome P-450 that are larger than those reported for rat liver, but the specific activity expressed as nmole of cytochrome P-450 per mg protein is much lower. The hemoprotein shows that spectral changes type I, II and IV are essentially in the low-spin form as judged by the n-octylamine and ethyl isocyanide difference spectra, and is unstable at pH below 6.5 and above 8.0. Cytochrome P-420 is also produced with time when CO-difference spectra are recorded. This is accelerated at pH above 8.0. The presence of contaminating amounts of cytochrome P-420, due to denaturation during spectral analysis or to the method used to isolate the microsomes, makes questionable the practice of characterizing the hemoprotein on the basis of the 455 nm peak in the ethyl isocyanide spectra, since a 434 nm peak is produced with concomitant decrease of the 455 nm peak. Microsomes hydroxylate naphthalene, aminopyrine and aniline, but the activity when expressed as nmole of product per nmole of cytochrome P-450 is the same or lower than that reported for other resistant housefly strains.     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

溴氰菊酯对不同品系家蝇脑突触体膜蛋白磷酸化及ATP酶活性的影响

以敏感品系家蝇和溴氰菊酯抗性品系家蝇(Musca domestica L.)为材料,研究和比较了神经毒剂溴氰菊酯对其脑突触体蛋白磷酸化作用的影响。结果表明,浓度为10^-5 mol/L溴氰菊酯抑制了敏感品系家蝇脑突触体蛋白磷酸化作用,而对抗性品系家蝇脑突触体蛋白磷酸化作用无明显影响。若反应体系中加入2.5×10^-6 mol/L的cAMP显著激活了敏感品系家蝇脑突触体蛋白磷酸化水平,但是当0.6 mmol/Lca²⁺或0.6 mmol/L Ca²⁺加10^-5 mol/L钙调蛋白时明显增强了抗性品系家蝇脑突触体蛋白磷酸化水平,甚至超过了其对敏感品系的作用水平。此外,还发现不同浓度的溴氰菊酯可抑制突触膜上的Na/K-ATP酶和Ca-ATP酶活力,浓度越高抑制作用也越大,并且敏感品系家蝇对溴氰菊酯的敏感度要高于抗性品系。     

Housefly Population Density Correlates with Shigellosis among Children in Mirzapur,Bangladesh: A Time Series Analysis

Background

Shigella infections are a public health problem in developing and transitional countries because of high transmissibility, severity of clinical disease, widespread antibiotic resistance and lack of a licensed vaccine. Whereas Shigellae are known to be transmitted primarily by direct fecal-oral contact and less commonly by contaminated food and water, the role of the housefly Musca domestica as a mechanical vector of transmission is less appreciated. We sought to assess the contribution of houseflies to Shigella-associated moderate-to-severe diarrhea (MSD) among children less than five years old in Mirzapur, Bangladesh, a site where shigellosis is hyperendemic, and to model the potential impact of a housefly control intervention.

Methods

Stool samples from 843 children presenting to Kumudini Hospital during 2009–2010 with new episodes of MSD (diarrhea accompanied by dehydration, dysentery or hospitalization) were analyzed. Housefly density was measured twice weekly in six randomly selected sentinel households. Poisson time series regression was performed and autoregression-adjusted attributable fractions (AFs) were calculated using the Bruzzi method, with standard errors via jackknife procedure.

Findings

Dramatic springtime peaks in housefly density in 2009 and 2010 were followed one to two months later by peaks of Shigella-associated MSD among toddlers and pre-school children. Poisson time series regression showed that housefly density was associated with Shigella cases at three lags (six weeks) (Incidence Rate Ratio = 1.39 [95% CI: 1.23 to 1.58] for each log increase in fly count), an association that was not confounded by ambient air temperature. Autocorrelation-adjusted AF calculations showed that a housefly control intervention could have prevented approximately 37% of the Shigella cases over the study period.

Interpretation

Houseflies may play an important role in the seasonal transmission of Shigella in some developing country ecologies. Interventions to control houseflies should be evaluated as possible additions to the public health arsenal to diminish Shigella (and perhaps other causes of) diarrheal infection.     

Alkaline phosphatase in the integument of Ceratitis capitata: Developmental profile and functional properties

Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine-O-phosphate. Tyrosine-O-phosphate is the preferred substrate for the integumental enzyme form with a K_m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine-O-phosphate.     

家蝇幼虫组织匀浆液的抗病毒活性

以家蝇Musca domesticaL.幼虫为材料,研究了蝇蛆组织匀浆液的抗病毒活性。用鸡胚培养检测了其抗流感病毒的活性,乙型肝炎病毒表面抗原诊断试剂盒检测了其对乙型肝炎病毒表面抗原的破坏作用。结果表明:15μL的家蝇幼虫组织匀浆液对流感病毒的抑制率为72.41%,而30,50和80μL剂量组对病毒的杀灭率都达到80%以上;蝇蛆组织匀浆液对乙型肝炎病毒表面抗原的破坏率可达到87.5%。说明家蝇幼虫体内存在着抗病毒活性物质。这为开发天然的抗病毒药物提供了新的途径。     

Proteinase Enzyme System of Lactic Streptococci II. Role of Membrane Proteinase in Cellular Function

The effect of several environmental conditions on the structure and activity of a membrane-associated proteinase from Streptococcus lactis was investigated. The activity of the enzyme varied with pH. Before storage at 3 C, maximal activity occurred at pH 6.0, but was minimal at this pH after storage. At all pH values tested, the enzyme was inactivated after storage. After storage at 3 C, the enzyme showed gross structural alterations with a concomitant loss of activity. Gel filtration and sedimentation velocity data indicated that inactivation of the enzyme was the result of aggregation to higher molecular weight forms. p-Hydroxymercuribenzoate prevented inactivation of the enzyme during storage by preventing aggregation. Activity was correlated with disaggregation of polymer forms of the enzyme to an active monomer. The storage-inactivated enzyme could be reactivated by treatment of the enzyme with cysteine, glutathione, or ferrous ion. Glutathione enabled stored cells to produce acid at their original rate when subcultured in milk. This was attributed to the effect of glutathione on the membrane proteinase. The data suggested that the biological activity of stored cells may be dependent upon the activity of the membrane proteinase.     

The impact of different plant extracts on biological parameters of Housefly [Musca domestica (Diptera: Muscidae)]: Implications for management

Housefly is a significant domestic pest, which causes nuisance. The use of insecticides is discouraged to manage housefly; therefore, alternative management strategies are inevitable. The current study investigated the impact of different plant extracts, i.e., Moringa oleifera (moringa), Allium sativum (garlic) and Piper nigrum (black pepper) on biological parameters of house fly. Two different concentrations (i.e., 25 and 50%) of the extracts were blended in larval diet made through mixing of wheat bran, yeast and dried milk powder. The results indicated significant differences for larval duration. Maximum larval duration was recorded for garlic followed by black pepper and moringa, respectively. In case of pupal duration, non-significant differences were observed among plant extracts. Increase rate of oviposition was noticed with moringa at 25% concentration, while decreased oviposition rate was noted for garlic with 50% concentration. Egg hatching percentage remained non-significant for the botanical extracts. The highest survival was observed with moringa, while garlic resulted in the lowest survival. The highest repellency was noticed for garlic followed by black pepper, whereas moringa resulted in the lowest repellency after 30, 60 and 90 min. Prolonged developmental time was observed for bot concentrations of garlic, whereas moringa noted the shortest developmental time. Thus moringa was found to be a promoter of housefly development. Minimum adult emergence was found with both concentrations of garlic followed by 50% concentration of black pepper. The 50% concentration of black pepper promoted the population of adult males, while both concentrations of moringa and 25% concentration of black pepper encouraged the population of female adults. Study outcomes depicted that tested botanical extracts had significant potential for disturbing biological parameters of housefly. The garlic extracts can potentially be used to manage housefly. However, further investigations on the larval and adult mortality are needed.