Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

Isolation,characterization, and properties of factor F1 from mitochondrial preparations of housefly (Musca domestica L.) thorax

A water-soluble Mg²⁺-dependent ATPase (coupling factor F₁) was isolated from the mitochondria of housefly thorax. It comprised about 14% of the proteins from a crude preparation. The F₁ preparation was nearly homogeneous as assessed by gel electrophoresis, isoelectric focusing, and electron microscopy. It was composed of five subunits with the following apparent molecular weights: α, 68,000; β, 61,000; γ, 38,000; δ, 27,000; and ?, 17,500. The isoelectric pH (pI) of this protein was 7.3. F₁ had a pH optimum of 8.2 and a temperature optimum between 37 and 45°C. The enzyme was fairly stable at 25°C. Nearly complete loss of activity was noticed at 0°C, while at 0 or 25°C, glycerol (20%) partially stabilized the enzyme activity against such inactivation. The K_m value of the enzyme with respect to ATP was 0.4 mM. The activity was stimulated by low concentrations of 2,4-dinitrophenol. The enzyme was inhibited by azide, p-hydroxymercuribenzoate, and guanidine hydrochloride. Oligomycin and the pesticides pyrethrin, cyhexatin, and DDT have no effect on the enzyme activity. However, all of these chemicals inhibited intact Mg²⁺- ATPase. The results are discussed in the light of differential responses of soluble and intact ATPase to these pesticides.     

The genetic basis of insecticide resistance in the house fly: Evidence that a single locus plays a major role in metabolic resistance to insecticides

Genetic evidence indicates that insecticide resistance in insects is controlled by relatively few genes. In the house fly, Musca domestica L., major resistance genes include one for decreased uptake of insecticides, three for changes at the target sites of insecticide action, and a single gene for metabolic resistance to multiple types of insecticides. The latter gene, which is located on chromosome II, interacts with minor genes located on other chromosomes. The product of the major gene for metabolic resistance appears to be a receptor protein which recognizes and binds xenobiotics, including insecticides and plant defense substances, and then induces synthesis of appropriate detoxifying enzymes.     

Genetic studies on glutathione-dependent reactions in resistant strains of the house fly,Musca domestica L

Genetic studies of glutathione-dependent reactions were conducted with a diazinon-resistant house fly strain in which resistance is controlled primarily by genes on chromsome II. The resistant strain was crossed with a susceptible strain which had mutant markers on chromosomes II, III, and V, and the F₁ was backcrossed to the susceptible strain. Glutathione transferase activities of the resultant eight phenotypes were measured using 3,4-dichloronitrobenzene, methyl iodide, and γ-benzene hexachloride as substrates. High levels of all these activities are controlled by gene(s) on chromosome II. Further analysis was made by introducing diazinon resistance into a susceptible strain via genetic crossing-over. Intermediate activity levels for 3,4-dichloronitrobenzene and methyl iodide conjugations were introduced along with intermediate levels of resistance. Assays of individual flies of the synthesized strain revealed they were heterogeneous for glutathione-dependent activities, consisting of individuals with low, intermediate, and high transferase activity. Based on these results, high levels of the glutathione-dependent enzymes are not a major biochemical mechanism responsible for diazinon resistance. It was also demonstrated that glutathione S-aryltransferase and S-alkyltransferase in the house fly, as measured with 3,4-dichloronitrobenzene and methyl iodide, are inseparable genetically and may, therefore, be the same enzyme.     

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental‐maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl’s reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal‐placental tissues in buffalo throughout pregnancy.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

Delta-aminolevulinic acid synthetase in the house fly and its possible role in oxidative resistance to insecticides

δ-Aminolevulinic acid synthetase (ALA synthetase EC 2.3.1.37) is the initial and rate-limiting enzyme in the biosynthetic pathway leading to heme and cytochrome formation in animals. The occurrence of ALA synthetase in house fly mitochondria was established and its possible relationship to oxidative resistance to insecticides was investigated.Levels of ALA synthetase in five house fly strains were measured and compared with levels of microsomal oxidases and cytochrome P-450 in the same strains. ALA synthetase was elevated in those strains with elevated levels of microsomal oxidases and cytochrome P-450 and was highest in the strain with the highest levels of microsomal oxidases and P-450. A possible regulatory role for ALA synthetase in relation to oxidative resistance to insecticides in the house fly is discussed.     

Electrical conductivity in the precambrian lithosphere of western canada

The subcrustal lithosphere underlying the southern Archean Churchill Province (ACP) in western Canada is at least one order of magnitude more electrically conductive than the lithosphere beneath adjacent Paleoproterozoic crust. The measured electrical properties of the lithosphere underlying most of the Paleoproterozoic crust can be explained by the conductivity of olivine. Mantle xenolith and geological mapping evidence indicate that the lithosphere beneath the southern ACP was substantially modified as a result of being trapped between two nearly synchronous Paleoproterozoic subduction zones. Tectonically induced metasomatism thus may have enhanced the subcrustal lithosphere conductivity of the southern ACP.