Autosomal sex-associated pyrethroid resistance in a strain of house fly (Diptera: Muscidae) with a male-determining factor on chromosome three

Mechanisms and genetics of resistance to pyrethroid insecticides were investigated in a strain of house fly (ASPR) collected from a cattle ranch in Miyagi, Gunma Prefecture, Japan. Flies were selected in the laboratory with the pyrethroid insecticide permethrin. Both sexes were resistant to pyrethroids; however, females were 22- to 245-fold more resistant than males. Permethrin resistance could be partly suppressed by the monooxygenase inhibitor piperonyl butoxide in females, but not in males. In this strain, sex was determined by a male factor on the third autosome. The relationship of the autosomal male factor to the lower resistance levels observed in the males and the mechanisms of resistance expressed in each sex are discussed.     

Assessing permethrin resistance in the stable fly (Diptera: Muscidae) in Florida by using laboratory selections and field evaluations

Insecticide resistance in the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae),has been demonstrated previously, but mostly with insecticides that are no longer used, such as the organochlorines. Resistance to commonly used pyrethroids has been evaluated twice, but only in the midwestern United States. Stable fly susceptibility to a commonly used pyrethroid, permethrin, was determined in Florida to assess the possibility of resistance development. Diagnostic concentration evaluations of three stable fly field strains demonstrated a maximum of 57 and 21% survival to permethrin residues of 3x and 10x the LC99 of a susceptible strain, respectively. Stable flies from an equine facility with no reported insecticide use demonstrated approximately 20% survival with a 3x diagnostic concentration. Despite a distance of 91-km between field collection sites, survival profiles of field-collected stable fly strains were similar. Although an established stable fly colony collected from a local dairy previously expressed low level resistance to permethrin residues, five generations of laboratory permethrin selection increased resistance 15-fold.     

Genetics of organophosphate resistance in field populations of the house fly (Diptera: Muscidae)

The genetics of resistance to the organophosphate insecticide diazinon were investigated in four populations of the house fly, Musca domestica L., collected in the southern United States. Crosses were made between individual females of lines derived from each population and males of a susceptible strain with three recessive mutants on chromosome II. Individual F1 females were crossed to mutant males, and the progenies were scored for resistance to diazinon and for the presence of mutant phenotypes. A major chromosome II gene for resistance to diazinon was present in all populations at an overall frequency of 83%. Map distances between the resistance gene and the mutant aristapedia and between the mutants aristapedia and stubby wing were highly variable in all populations. Recombination among the visible mutants was usually reduced in resistant progenies relative to susceptible progenies. The data suggest that a single major gene for resistance to diazinon was present on chromosome II in all test populations at variable map positions and is usually associated with a chromosome rearrangement, probably an inversion. The results are similar to those obtained earlier with house fly populations selected for resistance to insecticides in the laboratory; therefore, they seem to be characteristic of field and laboratory populations of the house fly. Overall, the data offer an explanation for previous results suggesting the existence of multiple, closely linked genes for metabolic resistance to insecticides on house fly chromosome II.     

Genetics of house flies. Variability studies with North Dakota, Texas, and Florida populations.

Genetic data were used to compare the structure of native house fly populations collected in North Dakota, Texas, and Florida. Recombination studies with mutant markers on chromosomes 3 and 4 indicated a lack of inversion polymorphism among the three populations in those areas of the genetic map studied. Significant differences were observed among flies from the three regions with regard to the frequency of 1) females that produced only male progeny, and 2) male-determining 3rd chromosomes (IIIm chromosomes). However, the North Dakota and Texas flies were more similar to each other than to the Florida flies since populations from the two former areas possessed a low frequency of both male-producing females and IIIm chromosomes; in contrast, the Florida population was void of females that produced males only and a high percentage if not all Florida males appeared to possess the IIIm male-determining mechanism. Tests for recessive lethal 3rd chromosomes showed that there was no significant difference in the frequency of lethal factors recovered from the North Dakota and Texas flies; the presence of IIIm chromosomes in Florida males precluded the recovery of lethal factors from this population by the method employed. The data suggest that house fly strains to be employed in genetic control programs should 1) originate from target control areas to avoid possible behavioral differences existing among flies from different locales, 2) be initiated with as many flies as possible to provide a background for the maintenance of variability, and 3) be renewed periodically with field-collected material since the genotype may be capable of rapid reorganization in response to laboratory selection pressures.     

Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies, Musca domestica

Two cytochrome P450 alleles, CYP6A5 and CYP6A5v2, were isolated from a pyrethroid-resistant house fly stain, ALHF. The two alleles shared 98% similarity in amino acid sequence. To understand the importance of these two alleles in resistance and examine the expression profile of the two alleles between resistant and susceptible strains, quantitative real-time PCR (qRT-PCR) was performed and compared with the Northern blot analysis. We found that qRT-PCR was an efficient method to characterize the expression profiles between these two sequence-closely-related P450 genes between resistant and susceptible houses flies. One of them, CYP6A5v2, was constitutively overexpressed in ALHF house flies compared with susceptible house fly strains. Moreover, this gene was predominantly expressed in the abdominal tissues of ALHF, in which the primary detoxification organs of insects are located. However, there was no significant difference in the expression of CYP6A5 between ALHF and susceptible house flies. The genetic linkage analysis was conducted to determine the possible link between the constitutively overexpressed CYP6A5v2 and insecticide resistance. CYP6A5v2 was mapped on autosome 5, which is correlated with the linkage of resistance in ALHF. Taken together, the study suggests the importance of CYP6A5v2 in increasing metabolic detoxification of insecticides in ALHF. The distinct expression of CYP6A5 and CYP6A5v2 in resistant and susceptible house flies implies the functional difference of theses two genes in house flies and suggests that they are two recently diverged P450 genes presented in a single organism.     

Commercial and naturally occurring fly parasitoids (Hymenoptera: Pteromalidae) as biological control agents of stable flies and house flies (Diptera: Muscidae) on California dairies

Filth fly parasites reared by commercial insectaries were released on two dairies (MO, DG) in southern California to determine their effect on populations of house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). Spalangia endius Walker, Muscidifurax raptorellus Kogan and Legner, and Muscidifurax zaraptor Kogan and Legner were released on the MO dairy from 1985 to 1987 in varying quantities. Parasitism by Muscidifurax zaraptor on the MO dairy was significantly higher (P less than 0.05) from the field-collected stable fly (4.4%) and house fly (12.5%) pupae, compared with a control dairy (0.1%, stable fly; 1.3%, house fly). Muscidifurax zaraptor, released from April through October during 1987 on the DG dairy (350,000 per month), was not recovered in a significantly higher proportion from either fly species relative to the corresponding control dairy. No specimens of Muscidifurax raptorellus were recovered from the MO dairy. Parasite treatments had no apparent effect on adult populations of either fly species or on overall parasitism rate of field-collected stable fly (16.8%, MO; 17.2%, DG) and house fly (23.3%, MO; 20.9%, DG) pupae. Spalangia spp. were the predominant parasites recovered from field-collected stable fly and house fly pupae on all four dairies. Sentinel house fly pupae placed in fly-breeding sites on both release dairies were parasitized at a significantly higher rate, as compared with sentinel pupae on control dairies. The generic composition of parasites emerging from sentinel house fly pupae was 20.6% Spalangia spp. and 73.2% Muscidifurax spp., whereas in field-collected house fly pupae, Spalangia spp. and Muscidifurax spp. constituted 74.3 and 19.6% of the parasites, respectively.     

Cyromazine resistance in the house fly (Diptera: Muscidae): genetics and cross-resistance to diflubenzuron

Larvae of a house fly, Musca domestica L., strain collected in a chicken house near Pittsburg, Tex, after a control failure with the poultry feedthrough insecticide cyromazine showed 6.5-fold resistance to cyromazine and 10-fold resistance to diflubenzuron. Adults of the strain showed high levels of resistance to carbaryl, DDT, and diazinon; moderate resistance to cypermethrin and permethrin; and low resistance to dieldrin. In contrast, no resistance to cyromazine was observed in eight laboratory house fly strains with resistance to four groups of conventional insecticides. When the genetics of cyromazine resistance was investigated in crosses to susceptible strains with visible mutant markers, results indicated cyromazine resistance was incompletely dominant over susceptibility and the resistance gene was on chromosome V. The same or a closely linked gene conferred resistance to diflubenzuron. A strain containing only chromosome V from the original resistant strain was resistant to cyromazine and diflubenzuron, but not to other insecticides except for low level resistance to DDT and carbaryl. Resistance to the latter insecticides appeared to be due to a linked, but distinct, gene. Therefore, resistance to cyromazine and probably diflubenzuron appears to be genetically distinct from other types of insecticide resistance.     

Autosomal interactions and mechanisms of pyrethroid resistance in house flies, Musca domestica

Five BC? lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.     

Selection for resistance to imidacloprid in the house fly (Diptera: Muscidae)

The house fly, Musca domestica L. (Diptera: Muscidae), continues to be a primary pest of livestock facilities worldwide. This pest also has shown a propensity for pesticide resistance development when under high selection pressures. In this study the house fly strain FDm was created by a 20% contribution from each of five colonies collected from dairies in Florida with known imidacloprid resistance. The FDm strain was used to evaluate the level ofimidacloprid resistance after five selections near the LC70 value of each selected generation. Overall, the mean selection mortality was 72.7, with males being considerably more susceptible than females. The unselected (F0) FDm strain showed considerable susceptibility to imidacloprid after its creation, compared with the five parental strains. Between 9500 and 14,000 virgin house flies were used in each selection. After the fifth and final selection, a 331-fold increase in imidacloprid resistance at the LC70 was observed over the parental FDm strain. In parallel studies, the FDm strain showed increasing tolerance of the commercial imidacloprid product QuickBayt. These results suggest that livestock producers should use caution when choosing pesticides and consider rotating fly baits, as is encouraged with other pesticide treatment regimes on farms.     

Resistance Monitoring and Enzyme Activity in Field-collected Populations of the Spiraea Aphid,Aphis citricola van der Goot

Susceptibility of the spiraea aphid (Aphis citricola van der Goot) to several classes of insecticides revealed regional variation. Four field-collected A. citricola populations have developed resistance to several classes of insecticides, including pyrethroids, organophosphates, and carbamates. Of the 15 insecticides tested four insecticides (deltamethrin, clopyrifos, omethoate and carbaryl) showed the relative resistance ratio (RRR) values of 10 or above, against four field-collected populations, whereas the other 11 insecticides had RRR values above 2. There were no significant differences in esterase activities among the field-collected populations. Esterase activity was generally homogeneous in the Yeasan-collected population, but was more heterogeneous in the populations collected from Andong, Kunwi and Suwon. Glutathione S-transferase (GST) activity in the Andong-and Yeasan-collected populations was higher than that in the other populations. More heterogeneous pattern of esterase activity and higher activity of GST in field populations of A. citricola would be due to multiple resistance mechanism. Fortunately, the levels of resistance in four field-collected populations examined are still increasing. Therefore, careful selective use of insecticides through both continued resistance monitoring and resistance mechanism study can be warranted to extend the effective life of insecticides for controlling A. citricola that was already resistant to various insecticides.     

Impact of exposure length and pupal source on Muscidifurax raptorellus and Nasonia vitripennis (Hymenoptera: Pteromalidae) parasitism in a New York poultry facility

Commercially obtained Nasonia vitripennis Walker and Muscidifurax raptorellus Kogan & Legner were released weekly for 12 wk into a high-rise, caged-layer poultry house. After the release period, parasitoids were sampled using sentinel house fly (Musca domestica L.) pupae that were either laboratory-reared or field-collected as larvae and exposed for 2, 4, 7, and 14 d. Parasitoid-induced mortality was observed in 31% of laboratory colony pupae and in 26% of field-collected pupae, whereas successful parasitism rates of 48 and 51% were observed from these pupal sources, respectively. Parasitism was primarily by M. raptorellus (88%), and Muscidifurax raptor Girault & Sanders (11%), while N. vitripennis accounted for <1%. Percent female progeny ranged from 43%, in M. raptorellus to 76% in N. vitripennis. Parasitoid emergence from 2-d exposed sentinel pupae was the lowest of all treatments. Parasitoid emergence from 7-d exposed sentinel pupae was the highest of all treatments. We found no differences between pupal source, suggesting that when sampling for M. raptor, M. raptorellus, and N. vitripennis, in poultry facilities, pupal source is not a confounding factor.     

Studies on the resistance to various insecticides of a house fly strain (Diptera: Muscidae) selected with azamethiphos.

A colony of azamethiphos-resistant house flies, Musca domestica (L.), was obtained from Denmark and further selected in the laboratory with azamethiphos for four generations. LD50s for various insecticides were determined and compared with those of a susceptible house fly strain. The selected flies showed cross-resistance to all insecticides evaluated. The flies were highly resistant to most organophosphorus, carbamate, and chlorinated hydrocarbon insecticides except prothiophos, p,p'-DDT, and the pyrethroids. We conclude that the main mechanisms responsible for resistance are presumed to be factors other than acetylcholinesterase sensitivity and nerve sensitivity due to knockdown resistance.     

Frequencies of pyrethroid resistance-associated mutations of Vssc1 and CYP6D1 in field populations of Musca domestica L. in Turkey

House flies were collected from 16 different provinces in the Aegean and Mediterranean regions of Turkey, and the frequencies of pyrethroid resistance-associated mutations in Vssc1 and CYP6D1 in these field-collected populations were studied. Although there is no organized resistance management program for house fly control in Turkey, it is known that different groups of insecticides, including pyrethroids, are used. The frequencies of both Vssc1 and CYP6D1 alleles were weighted toward the susceptibles, with Vssc1-susceptible alleles having higher frequencies in both regions (0.75 in Aegean and 0.69 in Mediterranean populations) than CYP6D1-susceptible alleles (0.65 in Aegean and 0.56 in Mediterranean populations). The frequencies of kdr-his alleles were higher than the frequencies of kdr alleles in these populations. While the frequencies of kdr-his alleles were close to each other in the Aegean (0.23) and Mediterranean (0.17) populations, the frequencies of kdr alleles remarkably differed in these two regions, with values of 0.02 and 0.14, respectively. In contrast to Europe, Asia, and the U.S.A., no super-kdr allele was detected in the samples from both regions. We identified six and eight different Vssc1+CYP6D1 genotype classes in the Aegean and Mediterranean regions, respectively. The three most common genotype classes in the regions were susceptible Vssc1 with heterozygous CYP6D1v1 (29%), sus/kdr-his1 with heterozygous CYP6D1v1 (23%), and susceptible Vssc1 with CYP6D1 (22%). The total frequencies of these three most common genotype classes (approximately 75%) obtained in our study were very close to the value obtained in Florida in a previous study, which was related by the similarity of temperature patterns between Florida and the corresponding regions of Turkey. This may reflect the lack of overwintering fitness cost associated with resistance alleles in both climates.     

Resistance of field-collected populations of Culex pipiens pallens (Diptera: Culicidae) to insecticides in the Republic of Korea

Toxicities of 10 insecticides were examined against late third instars of Culex pipiens pallens, the northern house mosquito, using a direct-contact mortality bioassay. Several strains of mosquitoes were tested (insecticide-susceptible KS-CP strain and five geospatially distant field-collected strains (DG-CP, US-CP, BS-CP, GS-CP, and SG-CP)) and identified by polymerase chain reaction. Marked regional variations of insecticide susceptibility were observed. Extremely high to low levels of resistance were measured: bifenthrin, resistance ratio (RR) = 1-521; β-cyfluthrin, RR = 16-397; α-cypermethrin, RR = 9-343; deltamethrin, RR = 1-40; etofenprox, RR = 2-42; permethrin, RR = 3-46; chlorpyrifos, RR = 2-675; fenitrothion, RR = 0.5-364; and fenthion, RR = 2-360. All strains were susceptible to one or more of the insecticides examined. These results indicate that careful selection and rotational use of these insecticides may result in continued satisfactory control against field populations of northern house mosquitoes.     

Meiotic studies in mice carrying the sex reversal (Sxr) factor

A sex reversal factor (Sxr) that causes mice having apparently normal X chromosomes to become phenotypically male is transmitted in an autosomal pattern. The origin of the Sxr factor is still unknown. It seems most likely that it has originated from an autosomal gene mutation or is the result of a translocation of part of the Y chromosome to one of the autosomes. Chromosomes from four XY and six XO mice carrying this sex reversal factor were examined in the diakinesis stage of meiosis. The following unusual observations were noted: (1) in XY males carrying the Sxr factor, the X and Y chromosomes were separated more often than in controls. (2) The Y chromosome tends to be closer to an autosome when the X and Y are separate than when the X and Y are attached. (3) A chromosome fragment was present in 4/226 cells from two XO males and a single cell from an XY, Sxr carrier. Although there is no direct evidence, these observations seem to favor the possibility that the Sxr factor involves a chromosomal rearrangement rather than a single gene mutation.     

Impact of an insecticide resistance strategy for house fly (Diptera: Muscidae) control in intensive animal units in the United Kingdom

A strategy for house fly (Musca domestica L.) control in intensive animal units in the United Kingdom was proposed by the Pesticide Safety Directorate (PSD) of the Ministry of Agriculture, Fisheries and Food in 1993. An advice leaflet was circulated to farmers, and label recommendations for insecticides used to control house flies were altered to prevent their long-term and frequent use. A study was carried out between 1996 and 1998 to gather data on insecticide use and resistance in house fly populations and compared with results from a study carried out in 1990-1992 to assess the impact of the 1993 label recommendations. As in the 1990-1992 study, resistance to methomyl, azamethiphos and pyrethrins + piperonyl butoxide was assessed. Larvicide tests with cyromazine, which had recently been released in the United Kingdom, were also included in this study. Most of the farmers claimed to have received and read the PSD insecticide advice leaflet, and half claimed to have altered insecticide treatments as a result. Comparing results for insecticides used before and after 1993, the proportion of farmers claiming to have used each of the insecticides had decreased. However, there had been no amelioration in resistance to synergised pyrethrins, and the number of house fly populations with reduced response to the insecticide baits had increased between 1990-1992 and 1996-1998. All the house fly populations tested were fully susceptible to cyromazine. There is an urgent need, therefore, to devise new strategies and particularly to minimize the risk of selecting for resistance to cyromazine.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

Thiamethoxam Resistance in the House Fly,Musca domestica L.: Current Status,Resistance Selection,Cross-Resistance Potential and Possible Biochemical Mechanisms

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC₅₀ ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.     

Salivary gland hypertrophy virus of house flies in Denmark: prevalence, host range, and comparison with a Florida isolate

House flies (Musca domestica) infected with Musca domestica salivary gland hypertrophy virus (MdSGHV) were found in fly populations collected from 12 out of 18 Danish livestock farms that were surveyed in 2007 and 2008. Infection rates ranged from 0.5% to 5% and averaged 1.2%. None of the stable flies (Stomoxys calcitrans), rat-tail maggot flies (Eristalis tenax) or yellow dung flies (Scathophaga stercoraria) collected from MdSGHV-positive farms displayed characteristic salivary gland hypertrophy (SGH). In laboratory transmission tests, SGH symptoms were not observed in stable flies, flesh flies (Sarcophaga bullata), black dump flies (Hydrotaea aenescens), or face flies (Musca autumnalis) that were injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), virus injection resulted in suppression of ovarian development similar to that observed in infected house flies, and injection of house flies with homogenates prepared from the salivary glands or ovaries of these species resulted in MdSGHV infection of the challenged house flies. Mortality of virus-injected stable flies was the highest among the five species tested. Virulence of Danish and Florida isolates of MdSGHV was similar with three virus delivery protocols, as a liquid food bait (in sucrose, milk, or blood), sprayed onto the flies in a Potter spray tower, or by immersiion in a crude homogenate of infected house flies. The most effective delivery system was immersion in a homogenate of ten infected flies/ml of water, resulting in 56.2% and 49.6% infection of the house flies challenged with the Danish and Florida strains, respectively.