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Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.

Animals

Effect of experimental prolongation of life span on lipofuscin content and lysosomal enzyme activity in the brain of the housefly, Musca domestica.

The effect of experimental prolongation of life span on the rate of lipofuscin accumulation in the giant neurons of the brain and lysosomal enzyme activity in the head was studied in the male housefly, Musca domestica. The median and the maximum life span of the flies was extended twofold by varying their flight activity. The rate of lipofuscin accumulation was significantly faster in short-lived, high-activity flies as compared to the long-lived, low-activity flies. The maximal content of lipofuscin in the neurons was similar in both groups but was reached at a much older age in the low-activity flies. Age-associated profiles of the activities of the lysosomal enzymes beta-glycerophosphatase and beta-acetylglucosaminidase did not correspond to the mortality characteristics of the housefly populations. The results support the view that lipofuscin represents an integral aspect of aging and may be used as a cellular marker for physiological age whereas lysosomal enzyme activities do not seem to be directly related to the rate of aging.

Acetylglucosaminidase

Soluble cytochrome P-450 from housefly microsomes. Partial purification and characterization of two hemoprotein forms.

Housefly microsomes contain two spectrally different forms of cytochrome P-450 which we have termed P-450 and P-450I. Methods have been developed for the fractionation and chromatographic purification of these two hemoprotein forms. Microsomes are solubilized first with Triton X-100 in the presence of glycerol, dithiothreitol, ethylenediaminetetra-acetic acid, and phenobarbital. Cytochrome P-450 is recovered in a floating pellet after the addition of 25% ammonium sulfate followed by centrifugation, whereas cytochrome P-450I remains in the 25% ammonium sulfate supernatant fluid. Cytochrome P-450 is purified further by Sephadez G-200 and DEAE-Sephadex A-50 column chromatography, which also allows the isolation of cytochrome b5 and NADPH-dependent cytochrome P-450 reductase in good yields and with little cross-contamination. Cytochrome P-450 apparently is free of cytochromes b5 and P-420 as well as of reductase and is obtained in a final yield of approximately 16% with a 6.9-fold purification. Its maximum absorbance is at 45 mn in the CO-difference spectrum and its average extinction coefficient is 103 cm-1 nm-1. Cytochrome P-450I is purified by Sephadex G-25 column chromatography but still contains some cytochromes b5 and P-420 as well as reductase. Its maximum absorbance is at 448.5 nm in the CO-difference spectrum and its extinction coefficient is 83 to 86 cm-1 mM-1. Both cytochromes hydroxylate type I substrates such as aminopyrine. Sufficient amounts of reductase are present in the cytochrome P-450I preparation to sustain activity, but the reductase has to be added to cytochrome P-450 in a reconstituted system for activity. Cytochrome P-450 is fairly stable, whereas cytochrome P-450I can be isolated only when protected by a substrate (phenobarbital). Detergent-solubilized housefly cytochromes P-450 and P-450I seem to correspond to either aggregates or oligomeric proteins. Cytochrome P-450 appears to correspond to a tetramer, each subunit having a molecular weight of 45,000, whereas cytochrome P-450I may correspond to an aggregate of at least 10 subunits. The cytochrome P-450 aggregate is dissociated by 6 M urea, but cytochrome P-450I remains as such.

Amines

Resistant level of houseflies to six kinds of synthetic insecticides in Malaysia.

The resistant level of the houseflies to six kinds of insecticides, DDT, Resmethrin, DDVP, Baytex, Sumithion and Diazinon, was examined on the seven strains collected in Malaysia. It was found that their susceptibility is rather higher than that of the Takatsuki strain which is a standard strain in Japan. However, their susceptibility to Sumithion was the same or slightly lower than that of the Takatsuki strain. The resistant level to five of six kinds of insecticides was the highest in the strain of Cameron Highland. The values were close to Singh's data in 1973, and this means that the resistance of the houseflies to the insecticides is increasing in Malaysia.

Animals

Resistant level of houseflies to seven kinds of synthetic insecticides and effect of synergists to the Malathion resistant strain in Singapore.

The resistant level of the houseflies to the seven kinds of insecticides, Malathion, Sumithion, Baytex, Diazinon, DDVP, DDT and Chrysron, was examined on the 3 strains collected in Singapore. It was found that most housefly colonies in Singapore showed a higher susceptibility to those insecticides than that of the Takatsuki strain of Japan, especially to DDT. However, the resistant level to Malathion was remarkably higher than that of the Takatsuki strain. The synergists, piperonyl butoxide, Safroxane and S-421, mixed with Malathion were effective for lowering the resistant level of the Malathion-resistant strain.

Animals

Levels of resistance to six synthetic insecticides in the Borneo housefly.

The resistant levels of the 4 strains of the housefly collected in Borneo to six insecticides, namely DDT, Chrysron, DDVP, Baytex, Sumithion and Diazinon, were examined. It was found that most housefly population in Borneo showed a higher susceptibility than that of the Takatsuki strain of Japan, especially to DDT.

Animals

Penetration and comparative metabolism of leptophos in susceptible and resistant houseflies.

The metabolism and rate of penetration of leptophos (O-methyl O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate) was determined in a susceptible strain and a strain of housefies which was 50-fold resistant to leptophos. Penetration of leptophos into resistant flies was substantially slower than into susceptible flies but large differences in metabolism, both quantitatively and qualitatively, were not observed. No difference was observed in the sensitivity of flyhead and thorax acetylcholinesterase to leptophos-oxon in vitro, and tolerance to leptophos by the resistant strain is explained in terms of decreased rates or penetration and minor differences in metabolism.

Animals

Problems of housefly (Musca domestica) control due to multiresistance to insesticides.

The development of chemical control of Musca domestica on Danish farms 1945--72 is outlined. It has been strongly influenced by successive development of resistance and failure of control by one insecticide after another. The chlorinated hydrocarbons used as residual sprays failed 1947--51. Organophosphorus compounds (OPC) were widely used from 1953, first as strips impregnated with parathion and residual sprays with diazinon. Resistance to OPC was first found in 1955, diazinon was given up in 1957--59 and parathion strips failed in the early '60's. Trichlorfon paint-on baits were widelyused 1958--64 and serious resistance did not appear until 1967, induced by selective pressure of fenthion and dimethoate used as residual sprays. High resistance to the contact effect of trichlorfon now occurs everywhere in Denmark. However, trichlorfon baits are still able to kill many flies. Residual sprays with fenthion, ronnel and fenitrothion were used to some extent 1960--70, but increased resistance reducing the residual effect developed in 2--3 years. Dimethoate was used on the majority of farms 1965--72. It was very effective the first years and resistance increased slowly until 1971--72, when high to extreme dimethoate-resistance became general on Danish farms. This was associated with high resistance to other OPC for fly control, e.g. fenthion, fenitrothion, bromophos, and tetrachlorvinphos, and to carbamates, with the result that no generally effective residual sprays were available. In 1971--72 frequent treatments with synergized pyrethroids have been tried. However, the method is often expensive, and serious resistance problems have appeared on a few farms. In this situation preventive, sanitary measures to eliminate or reduce fly breeding in manure are becoming decisive again, but difficult to practise due to lack of farm labour. The extreme Danish situation is compared with those in other areas, and probable reasons for differences in resistance and control problems are discussed, as well as possibilities for strategies to reduce resistance development.

Animals

[Residual action of Neguvon, Neocidol, Nuvanol and Dursban on the imaginal and larval stages of the housefly].

It is shown that Dursban, Neocydol and Neguvon preparations, mixed with swine manure at amounts of 500 mg/kg, kill 100 per cent the larvae of the domestic fly up to the 30th, 20th and 10th day, respectively, while the Neguvon and Neocydol preparations in cattle manure, up to the 10th day. Lower concentrations possess a residual effect of a shorter duration. It is also shown that the type of the surfaces and the pH of the mater on which the preparations are placed markedly affect the inctesicide activity and the residual effect of the preparations. The highest activity and the longest residual effect manifest preparations that have been placed on surfaces with a low absorption capacity. The highest effect has been achieved when the preparations are smeared on glass, a lower one, when placed on wood, and the poorest, when laid on mortar.

Animals

Insecticide movement following application to crevices in rooms.

The movement of chlorpyrifos and diazinon from cracks and crevices in empty dormitory rooms with and without forced air movement to houseflies and plates in non-target areas was studied. Aluminum pie plates in rooms with the high concentrations of an insecticide, no forced air movement, and at the earlier sampling interals contained more insecticide. Insecticides were not detectable at 48 hr. Increased housefly movement occurred simultaneously with the initiation of insecticide application in rooms with forced air and 1 min later in rooms with no forced air. Mortality of houseflies exposed during and at 5 hr after insecticide application was greater for males than females in all tests except one; at the high insecticide concentrations; and at successive time intervals through the 24-hr interval after their transfer from the treated rooms to holding rooms. Houseflies in cages near the ceiling, exposed to an insecticide, and forced air movement, exhibited greater mortality than those in cages on the floor, while the reverse was true for houseflies in rooms with no forced air movement.

Air Movements

Comparative studies on the molecular weight of glutathione S-transferases from mammalian livers and an insect.

1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.

Animals

The in vitro conversion of a specific molecular form of glucose 6-phosphate dehydrogenase from Musca domestica L.

A nicotinamide adenine dinucleotide phosphate dependent glucose 6-phosphate dehydrogenase (G6PD), belonging to type I of Kamada and Hori's classification, is present on the zymograms of newly emerged males of Musca domestica. It is capable of undergoing tryptic degradation and being thus transformed into a different active enzymatic form, with some of its catalytic properties unchanged, but with different electrophoretic mobility. We show in this paper that this specific G6PD form of gut origin in M. domestica is not a tissue-specific enzyme, but rather a product of hydrolytic degradation by gut proteinases which act during the process of homogenization. Besides, the G6PD of type I in the housefly is shown to be sensitive to the "storage effect" and to protection by mercaptoethanol, contrary to its hydrolytic gut form which is not sensitive to these processes. In this connection, we discuss the possible reasons for these differences in behavior.

Animals

The characterization of multiple forms of kynurenine formidase in Drosophila melanogaster.

Two enzymic forms of kynurenine formamidase (EC 3.5.1.9) from Drosophila melanogaster were separated and partially purified by pH fractionation, (NH4) 2SO4 fractionation and Sephadex G-75 gel filtration. The enzymes were also separated by DEAE-cellulose ion-exchange chromatography and distinguished by their different rates of thermal inactivation. The multiple forms are termed formamidase I and formamidase II. The molecular weight of formamidase I as measured by Sephadex G-75 chromatography is 60 000 and that of formamidase II is 31 000. The pH optima are broad, ranging between 6.7 and 7.8 for formamidase I and 6.5 and 8.0 for formamidase II. The apparent Km values are 5-10(-3) and 0.83-10(-3) M, resepctively. The possibility that formamidase II is an active subunit of formamidase I is discussed, although neither enzyme will convert to the other when separated and rechromatographed. Eight organisms were tested for the presence or absence of multiple forms of formamidase. Drosophila melanogaster and Drosophila virilis have both enzymes; cow, chicken, yeast and housefly have formamidase I only, and mouse and frog have formamidase II only.

Amidohydrolases

Pathogen transmission in relation to feeding and digestion by haematophagous Arthropods.

The blood feeding habit, especially among opportunist feeders such as tabanids and Stomoxys is known to result in transmission of diseases for which the vectors are not the obligate or alternate hosts. Thus, mechanical transmission of trypanosomes such as T. vivax can occur in cattle herds outside tsetse fly areas where tabanids are actively feeding. In the case of Yaws, mechanical transmission of the spirochaetes by eye flies (Hippelates pallipes) in the West Indies is thought to be most likely. The spirochaetes remain motile in the pharynx and oesophageal diverticula for several hours but are apparently immobilised in the midgut (Kumm & Turner, 1936). There is apparently no development ofspirochaetes in the fly. They have been shown to pass through houseflies, but in mechanical transmission, biochemical transformation or adaptation of the pathogen is not implied. Virus transmission is common among arthropods and transovarial transmission to succeeding generations is frequent in mites and ticks. Although Yellow Fever virus is not transovarially transmitted by its vector Aedes aegypti, the mosquito only becomes infective some time after ingesting an infected blood meal (Chandler, 1955). Thus, metabolic or biochemical changes or adaptations in the virus or in the vector are in some way implicated, as they must also be in transovarially transmitted viruses. However, the causal relationships between virus infectivity and vector physiology are poorly understood. As with virus infections, those disease organisms possessing a cyclical host/vector relationship will possess a variable infectivity potential which is not necessarily related to the abundance of any of the organisms in the cycle. Clearly, feeding behavior and host preferences of the vector are important in determining the rate and extent of disease transmission, and such parameters can be quantified in epidemiological studies. However, a complete understanding of the factors concerned in cyclical disease transmission also depends on a knowledge of the physiology of the organisms involved, and particularly of the interdependence of their physiologies. The subject is vast, and it is proposed to illustrate the problems involved and the progress made, by reference largely to trypanosome transmission by tsetse flies (Glossina spp.).

Adaptation, Physiological