Linkage relationships of several insecticide resistance factors in the house fly (Musca domestica L.)

Summary Strains of the house fly resistant to parathion, malathion, Isolan, and DDT were crossed with a susceptible strain which carried the recessive marker, stubby-wing (stw). F₁ populations were composed of normal-winged insecticide-resistant flies. When the F_1's were backcrossed to the stw parent, the resultant normal-winged progeny were resistant and the stw progeny were susceptible. Thus the major factors for resistance in the strains studied were all located on the same chromosome.Similar cross-over ratios were observed with parathion and malathion-resistant strains and populations of resistant stw flies were established. Low ali-esterase, characteristic of the parent resistant strains, was also present in the resistant stw strains.No crossing-over occurred between stw and DDT-resistance. Resistance to DDT, present in the malathion-resistant strain, was not introduced with malathion-resistance into the stw strain. Therefore, the factors for DDT and malathion resistance, although linked, are genetically distinct.

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Zusammenfassung Stubenfliegenstämme, die Resistenzfaktoren gegen Parathion, Malathion, Isolan^R (1-isopropyl-3-methyl-5-pyrazolyl dimethylcarbamate) und DDT tragen, wurden mit einem anfälligen Stamm gekreuzt, der das rezessive Markierungsgen, stummelflügelig (stw), aufweist. Die F₁-Populationen jeder Kreuzung bestanden aus normalflügeligen, insektizidresistenten Fliegen. Wenn die F₁-Fliegen mit den stw-Eltern rückgekreuzt wurden, waren die normalflügeligen Nachkommen resistent gegen alle Insektizide und die stw-Nachkommen anfällig. Also sind die Hauptresistenzfaktoren der untersuchten Stämme alle in dem gleichen Chromosom lokalisiert.Für die Faktoren, welche die Resistenz gene Parathion und Malathion kontrollieren, wurden ähnliche crossing over-Verhältnisse beobachtet, und es wurden Populationen von stw-Fliegen aufgebaut, die resistent gegen diese Insektizide sind. Niedrige Ali-Esterase-Aktivität, die für die parathion- und malathionresistenten Ausgangsstämme charakteristisch ist, war auch in den resistenten stw-Stämmen vorhanden, so daß das Gen für niedrige Ali-Esterase-Aktivität identisch oder eng mit den Resistenzgenen verbunden ist.Eine unzulängliche Trennung zwischen anfälligen und isolanresistenten Fliegen schloß die Möglichkeit der crossing over-Messung des Faktors für Isolanresistenz aus. Zwischen stw und DDT-Resistenz wurde kein crossing over beobachtet. Hohe Kreuzungsresistenz gegen DDT, die in einem malathionresistenten Stamm vorhanden ist, wurde mit dem Faktor für Malathionresistenz nicht in den stw-Stamm eingeführt. Die Faktoren für die Resistenz gegen DDT und Malathion sind demnach genetisch unterschiedlich.

     

Acetylcholine in the choline-deficient housefly, Musca domestica

The distribution of choline metabolites has been studied in the nervous system of housefly larvae reared on diets containing various amounts of choline or on diets containing either β-methylcholine (β-MCh) or N-dimethylethylcholine (DMECh). Adult houseflies were obtained from larvae reared on diets containing at least 4·6 times the level of choline in the basic diet or on diets containing the two choline analogues. The distribution of choline metabolites has been studied in the heads and thoraces plus abdomina of these adults.

Acetylcholine was found to be concentrated in the larval nervous system where it was synthesized preferentially to other choline metabolites when the amount of choline available to the insect was severely restricted. The acetylcholine content of the adult was between six and nine times that of the larval nervous system and approximately 70 per cent was concentrated in the head. The amount of acetylcholine present in flies obtained from larvae fed on diets containing the lowest amount of choline which allowed adults to develop was 230 pmole/insect. Flies obtained from larvae fed on diets with added β-MCh contained 150 pmole of acetylcholine/insect but no detectable acetyl-βMCh. Flies obtained from larvae fed on diets with added DMECh contained 43 pmole of acetylcholine and 127 pmole of acetyl-DMECh/insect.

It is concluded that the two choline analogues spare the choline requirement of the housefly by two different mechanisms. β-MCh displaces choline from the lipids of non-nervous tissue making more available for uptake into the nervous system where sufficient acetylcholine is synthesized for adult development. DMECh is a complete replacement for choline; the acetyl-DMECh acting as a neurotransmitter in place of acetylcholine.     

Evaluation of Beauveria bassiana applications against adult house fly, Musca domestica, in commercial caged-layer poultry facilities in New York state

Applications of a commercially produced Beauveria bassiana product, balEnce, were compared with pyrethrin treatments for the control of adult house flies in New York high-rise, caged-layer poultry facilities. An integrated fly management program, which included the release of house fly pupal hymenopteran parasitoids, was used at all facilities. Adult house fly populations were lower in B. bassiana-treated facilities during the spray and post-spray periods, as recorded on spot cards. Concurrently, the numbers of house fly larvae recovered in B. bassiana-treated facilities were less than one-half that of the pyrethrin-treated facilities. House fly pupal parasitism levels were low, but similar under both treatment regimes. The numbers of adult and larval Carcinops pumilio, a predatory beetle, recovered from B. bassiana-treated facilities were 43 and 66% greater than from the pyrethrin-treated facilities, respectively.     

The contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin.

Most of the photoreceptors of the fly compound eye have high sensitivity in the ultraviolet (UV) as well as in the visible spectral range. This UV sensitivity arises from a photostable pigment that acts as a sensitizer for rhodopsin. Because the sensitizing pigment cannot be bleached, the classical determination of the photosensitivity spectrum from measurements of the difference spectrum of the pigment cannot be applied. We therefore used a new method to determine the photosensitivity spectra of rhodopsin and metarhodopsin in the UV spectral range. The method is based on the fact that the invertebrate visual pigment is a bistable one, in which rhodopsin and metarhodopsin are photointerconvertible. The pigment changes were measured by a fast electrical potential, called the M potential, which arises from activation of metarhodopsin. We first established the use of the M potential as a reliable measure of the visual pigment changes in the fly. We then calculated the photosensitivity spectrum of rhodopsin and metarhodopsin by using two kinds of experimentally measured spectra: the relaxation and the photoequilibrium spectra. The relaxation spectrum represents the wavelength dependence of the rate of approach of the pigment molecules to photoequilibrium. This spectrum is the weighted sum of the photosensitivity spectra of rhodopsin and metarhodopsin. The photoequilibrium spectrum measures the fraction of metarhodopsin (or rhodopsin) in photoequilibrium which is reached in the steady state for application of various wavelengths of light. By using this method we found that, although the photosensitivity spectra of rhodopsin and metarhodopsin are very different in the visible, they show strict coincidence in the UV region. This observation indicates that the photostable pigment acts as a sensitizer for both rhodopsin as well as metarhodopsin.     

Functional Expression of House Fly (Musca domestica) Cytochrome P450 CYP6D1 in Yeast (Saccharomyces cerevisiae)

Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides. In house fly microsomes CYP6D1 requires cytochrome b₅ for the metabolism of some substrates, such as benzo[a]pyrene, but does not require cytochrome b₅ for the metabolism of other substrates such as methoxyresorufin. To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed. Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into the yeast microsomes. CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase. However, CYP6D1 expression did not result in measurable benzo[a]pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b₅, or that there is insufficient cytochrome b₅ in the yeast microsomes to support this CYP6D1-mediated activity. Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function.     

Map position of Aldox (Aldehyde-oxidase) and Adh (Alcohol dehydrogenase) loci on autosome II of Musca domestica L.

The Aldox and Adh structural loci of Musca domestica L. belong to autosome II. They code for the enzymes aldehyde oxidase and alcohol dehydrogenase. Both these enzymes have allelic variants with specific electrophoretic mobility, which, on cellogel, are seen as single bands. The Aldox and Adh loci encompass a large map interval, which includes the morphological markers ar, cm, and car. The recombination frequencies between these five loci indicate the alignment Aldox-ar-cm-car-Adh.

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Localisation chez Musca domestica L. des gènes Aldox et Adh codant les enzymes ald\;ehyde oxydase (AO) et alcool déshydrogénase (ADH)

Résumé Chez la mouche, les enzymes AO et ADH, observées par électrophorèse sur cellogel, présentent toutes 2 des formes alléliques exprimées par des bandes ayant une mobilité anodique propre.Les gènes structuraux Aldox et Adh, codant ces formes, sont liés entre eux et situés sur le chromosome 2. Ils se recombinent avec une fréquence élevée d'interchange; ils sont donc séparés par un intervalle important dans lequel sont compris les caractères visibles ar, cm, car. La fréquence des recombinaisons entre caractères visibles et gènes enzymatiques indique l'ordre suivant sur ce segment du deuxième chromosome de la mouche: Aldox, ar, cm, car, Adh.

     

Fine structure of the Malpighian tubules in the housefly, Musca domestica

The epithelium of the Malpighian tubules in the housefly is comprised of four distinct cellular types. Type I cells are characterized by the presence of intimate associations between infoldings of basal plasma membrane and mitochondria. On the luminal surface, cytoplasm is extended into microvilli which contain mitochondria. Membrane-bound vacuoles in the cytoplasm seem to progressively accumulate granular material. Type II cells have dilated canaliculi. Microvilli lack mitochondria. The Type III cell has not been reported previously in Malpighian tubules. It has very well-developed granular endoplasmic reticulum which contains intracisternal bundles of tubules. Cytoplasm contains numerous electron dense bodies. Type IV cells occur in the common duct region of the Malpighian tubules. Mitochondria do not extend into the microvilli.     

Synergism of organophosphates in Musca domestica and Chrysomya putoria

A series of symmetrical trisubstituted phosphorus compounds was tested as synergists for malathion and certain other organophosphorus insecticides against resistant and normal strains of Chrysomya putoria and Musca domestica. All the compounds synergised the insecticides more effectively against resistant than normal strains. This was especially true with malathion, notably with the C. putoria strain the resistance of which is highly specific for malathion. A 5:1 mixture of the best synergists virtually abolished its resistance. Among the synergists tested, the two best were common to the C. putoria and the two different resistant strains of M. domestica; these two synergists were also outstanding against Culex tarsalis larvae, as reported by other authors.In addition to the synergistic action on malathion, the better compounds were also effective in reducing resistance to other phosphorus insecticides (Dicapthon, parathion, coumaphos) though not quite so effectively. It is concluded that their action is not entirely specific for suppressing carboxyesterase detoxication.

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Synergismus von organophosphaten bei Musca domestica und tChrysomya putoria

Zusammenfassung Eine Reihe symmetrisch dreifach substituierter Phosphorverbindungen wurde als Synergisten für Malathion und gewisse andere Organophosphat-Insektizide gegenüber resistenten und normalen Stämmen von Chrysomya putoria und Musca domestica geprüft. Alle diese Verbindungen verstärkten die Insektizide gegen resistente Stämme wirkungsvoller als gegen normale. Das galt besonders für Malathion, vor allem bei dem C. putoria-Stamm, dessen Resistenz gegenüber Malathion hoch spezifisch ist. Ein 5:1-Gemisch der besten Synergisten hob seine Resistenz praktisch auf. Unter den geprüften Synergisten waren die zwei besten C. putoria und den beiden verschiedenen resistenten Stämmen von M. domestica gemeinsam; diese beiden Synergisten wirkten auch gegenüber Culex tarsalis-Larven ausgezeichnet, wie andere Autoren berichteten.Neben der synergistischen Wirksamkeit für Malathion reduzierten die besseren Verbindungen auch die Resistenz anderer Phosphor-Insektizide (Dicapthon, Parathion, Coumaphos), wenn auch nicht ganz so vollkommen. Es wird geschlossen, daß ihre Wirkungsweise für die Unterdrückung der Carboxyesterase-Entgiftung nicht ganz spezifisch ist.

     

The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe

Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion.     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.     

(Z)-3-nonenol, (Z,Z)-3,6-nonadienol and (S,S)-(-)-epianastrephin: male produced pheromones of the Mexican fruit fly

(Z)-3-nonenol, (Z,Z)-3,6-nonadienol and (S,S)-(-)-epianastrephin proved active as male-produced pheromones which elicited behavioral responses from virgin female Mexican fruit flies (Anastrepha ludens) (Diptera: Tephritidae) in laboratory bioassays. All three chemicals elicited attraction and/or locomotor arrest when tested individually. When tested together, (Z)-3-nonenol inhibited the behavioral effect of (Z,Z)-3,6-nonadienol but either of the alcohols synergized the effect of (S,S)-(-)-epianastrephin. Quantities of the pheromones per male abdomen were: (Z)-3-nonenol-100 ng; (Z,Z)-3,6-nonadienol-40 ng; and total epianastrephin (R,R and S,S enantiomers)-700 ng. Relative to the quantities in abdominal extract, males released these chemicals during sexual display in a blend containing a higher proportion of the alcohols.

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Résumé Le (Z)-3-nonènol, le (Z,Z)-3,6-nonadiènol et le (S,S)-(-)-épianastréphine produits par les mâles de la mouche mexicaine du fruit (Anastrepha ludens) (Diptera: Tephritidae) ont attirés les femelles lors de tests conduits en laboratoire. Le pouvoir attractif du (Z)-3-nonènol est faible comparé à celui des deux autres phéromones. Evalués ensemble, le (Z)-3-nonènol inhibe l'attractivité du (Z,Z)-3,6-nonadiènol mais chacun des deux alcools synergise l'effet attractif de la (S,S)-(-)-épianastréphine. Les quantités recouvrées par mâle dans un extrait abdominal étaient respectivement de 100, 40 et 700 ng pour le (Z)-3-nonènol, le (Z,Z)-3,6-nonadiènol, et l'épianastréphine totale (R,R et S,S) énantiomères. Comparées à ces valeurs, le mélange élange émis par les mâles lors de leur cour sexuelle est plus riche in alcools. Le rapport (Z)-3-nonènol (Z,Z)-3,6-nonadiènol est cependant identique dans l'extrait abdominal et dans le mélange émis par les mâles.

     

Field Studies of Entomophthora (Zygomycetes: Entomophthorales)—Induced Behavioral Fever in Musca domestica (Diptera: Muscidae) in Denmark

House flies were collected over 3 days (three to five times per day) from specific sites on a dairy farm with a range of high to low temperatures. Flies were held individually to determine whether the distribution of fungus-infected (Entomophthora muscae and E. schizophorae) house flies differed according to the stage of infection and temperature. All but 2 of 396 infected flies (99.5%) had E. muscae. More E. muscae-infected flies collected from cool areas were in later stages of infection (i.e., dying 0–2 days after capture), whereas flies collected on sun-exposed surfaces tended to be in earlier stages of infection (i.e., dying 6–8 days after capture). Most flies died 3–5 days after capture and were consequently in the middle stages of infection. A mark and release experiment was conducted to determine whether E. schizophorae-inoculated flies frequented surfaces with higher temperatures than did uninfected control flies. About 3000 yellow-marked house flies inoculated with E. schizophorae and 3000 blue-marked control flies were released in an enclosed swine farrowing barn. Significantly more inoculated flies were recorded on the heat lamps than flies in the control group. The results suggest that behavioral fever occurs in the field for flies infected with both E. muscae and E. schizophorae and that flies can cure themselves of infection through the use of artificial heat sources.     

A complex glutathione transferase gene family in the housefly Musca domestica

In most metazoans, the glutathione S-transferases (GST) are encoded by gene families, and are used to detoxify xenobiotics. We describe the structure of genomic loci coding for the GSTs in the housefly that have been implicated, by both genetic and biochemical means, in mediating insecticide resistance. In earlier work, we showed that one of the theta-class enzymes, MdGST-3, is overproduced in resistant flies and degrades certain insecticides. We used a fragment from a cDNA clone of MdGST-3 as a probe to screen a housefly genomic DNA bank in phage λ. This probe detected multiple gst loci. Genes for GSTs were found in five different, nonoverlapping λ clones, three of which carry multiple, closely linked gsts. Multiple genes for both MdGST-3 and MdGST-4 were found; some of which have introns in their 5′ untranslated regions. In adults, the only MdGST-3 enzymes that are expressed are encoded by the intron-free genes. A new theta-class GST (called MdGST-5) was also discovered. Fusion genes comprising 5′ MdGST-3 sequences and either MdGST-4 or MdGST-5 sequences in their 3′ halves were encountered at three separate loci. The genes described here are found in both the ancestral sensitive strain and the insecticide-resistant strains. Received: 28 June 1996 / Accepted: 23 April 1997     

Absorption properties of a photostable pigment (P456) in rhabdomere 7 of the fly

Summary One photoreceptor type (R7y) in the compound eye of fly (Musca) contains, besides the visual pigment, a photostable pigment (most probably a carotene) with maximal absorption in the blue spectral range. The extinction and dichroism due to this pigment are determined, taking into account waveguide properties, birefringence, anomalous dispersion and possible twisting of the rhabdomeres. The concentration of this pigment, if it is a carotene, is rather high: there are 7–10 molecules per rhodopsin molecule.