Male House Flies Research Articles

Low sex drive and choosy females: fungal infections are a reproductive downfall for male house flies

Abstract Many entomopathogenic fungi cause infections that kill their insect host. Little is understood about changes in the reproductive investment that occurs during an infection by a lethal disease over the waning life of an insect. Life-history theory suggests the host will respond by investing resources into fighting the disease or increasing reproduction. Here, we investigate how the reproductive life of adult house flies, Musca domestica, is impacted by its host-specific fungal pathogen, Entomophthora muscae. Specifically, we test how the week-long infection alters the mating behavior of virgin adult male house flies. We find that the pathogen significantly decreases male libido, an effect which grows stronger over the course of the infection. Furthermore, females were significantly less likely to choose an infected male, reducing male mating success. Additionally, we assessed sperm viability to understand the reproductive costs for monandrous females to mate with infected males. Analyses revealed that sperm quality decreases as early as 3 days post-infection. These results show that E. muscae, which can have a prevalence near 100% in wild populations, causes severe lifetime reproductive costs to male house flies. Understanding how host–pathogen interactions affect host life history is crucial for elucidating all the negative effects pathogen virulence exerts on hosts.

Sex-specific Feeding Behavior of Adult House Flies, Musca domestica L. (Diptera: Muscidae).

House flies, Musca domestica L., (Diptera: Muscidae) mechanically vector diverse disease-causing microorganisms while foraging for food in agricultural and urban habitats. Although flies are diverse feeders, nutrient composition of food is important for both fly longevity and reproduction, especially for anautogenous females who require protein for egg production. We investigated whether fly sex and/or mating status influenced their preference for foods with varying macronutrient composition. Presumably mated or unmated male and female flies were separated by sex and offered four food, each in 10% solution offered on cotton wicks: sugar (carbohydrate-rich), fat-free milk (protein-rich, moderate carbohydrate), egg-yolk (protein and lipid-rich), and water (no macronutrients). Foods were colored with nontoxic dyes, which were rotated between replicates. After 4h exposure, flies were dissected to determine the type of food(s) ingested. The interaction of house fly sex and food type significantly influenced food preference, where females preferred milk (protein and carbohydrate-rich food), and males preferred mainly sugar (carbohydrate-rich). Furthermore, 32.8% of females and 10.6% of males foraged on multiple foods. While interaction of sex and mating status had no effect on food preference, milk preference was significantly higher in presumably mated than unmated females. We also tested whether food color influenced fly feeding preference, and found that color was most significant when flies were offered one food type, but negligible when multiple food types were present. This study suggests that bait-based fly control strategies should consider sex-specific preferences for various food attractants if aiming to target and control both male and female house flies.

Pathogenic fungus uses volatiles to entice male flies into fatal matings with infected female cadavers.

To ensure dispersal, many parasites and pathogens behaviourally manipulate infected hosts. Other pathogens and certain insect-pollinated flowers use sexual mimicry and release deceptive mating signals. However, it is unusual for pathogens to rely on both behavioural host manipulation and sexual mimicry. Here, we show that the host-specific and behaviourally manipulating pathogenic fungus, Entomophthora muscae, generates a chemical blend of volatile sesquiterpenes and alters the profile of natural host cuticular hydrocarbons in infected female housefly (Musca domestica) cadavers. Healthy male houseflies respond to the fungal compounds and are enticed into mating with female cadavers. This is advantageous for the fungus as close proximity between host individuals leads to an increased probability of infection. The fungus exploits the willingness of male flies to mate and benefits from altering the behaviour of uninfected male host flies. The altered cuticular hydrocarbons and emitted volatiles thus underlie the evolution of an extended phenotypic trait.

Temperature-dependent effects of house fly proto-Y chromosomes on gene expression could be responsible for fitness differences that maintain polygenic sex determination.

Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Evolutionary turnover in a sex determination pathway may occur via selection on alleles that are genetically linked to a new master sex determining locus on a newly formed proto-sex chromosome. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto-sex chromosomes, are informative models to study the evolution of sex determination and sex chromosomes. House flies are such a model system, with male determining loci possible on all six chromosomes and a female-determiner on one of the chromosomes as well. The two most common male-determining proto-Y chromosomes form latitudinal clines on multiple continents, suggesting that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. Temperature-dependent fitness effects could be manifested through temperature-dependent gene expression differences across proto-Y chromosome genotypes. These gene expression differences may be the result of cis regulatory variants that affect the expression of genes on the proto-sex chromosomes, or trans effects of the proto-Y chromosomes on genes elswhere in the genome. We used RNA-seq to identify genes whose expression depends on proto-Y chromosome genotype and temperature in adult male house flies. We found no evidence for ecologically meaningful temperature-dependent expression differences of sex determining genes between male genotypes, but we were probably not sampling an appropriate developmental time-point to identify such effects. In contrast, we identified many other genes whose expression depends on the interaction between proto-Y chromosome genotype and temperature, including genes that encode proteins involved in reproduction, metabolism, lifespan, stress response, and immunity. Notably, genes with genotype-by-temperature interactions on expression were not enriched on the proto-sex chromosomes. Moreover, there was no evidence that temperature-dependent expression is driven by chromosome-wide cis-regulatory divergence between the proto-Y and proto-X alleles. Therefore, if temperature-dependent gene expression is responsible for differences in phenotypes and fitness of proto-Y genotypes across house fly populations, these effects are driven by a small number of temperature-dependent alleles on the proto-Y chromosomes that may have trans effects on the expression of genes on other chromosomes.

Temperature-dependent effects of house fly proto-Y chromosomes on gene expression

Code, commands, and data to perform analyses of house fly RNA-seq data. House fly has a stable polygenic sex determination system. The male determining factor (Mdmd) is commonly found on the Y chromosome (Y^M) or the third chromosome (III^M). These proto-Y chromosomes are clinally distributed, with Y^M found most commonly in northern latitudes and III^M most commonly found at southern latitudes, hinting at possible genotype-by-temperature interactions that maintain the polymorphism. If this distribution is maintained by temperature-dependent selection pressures, we expect the fitness of III^M and Y^M flies to vary across developmental temperatures. These temperature-dependent effects could be driven by differential gene expression in III^M and Y^M males at different temperatures. Here, we performed RNA-seq experiments to study how genotype-by-temperature interactions affect gene expression in male houseflies carrying different proto-Y chromosomes. We raised a Y^M strain known as IsoCS and a III^M strain known as CSrab at 18°C and 29°C for two generations. We dissected 5 heads and 15-20 pairs of testes for each of three replicates of each genotype-by-temperature combination. We carried out RNA-seq on these tissues. Data are available from NCBI Gene Expression Omnibus accession GSE136188.

Cytochrome P450 monooxygenases-mediated sex-differential spinosad resistance in house flies Musca domestica (Diptera: Muscidae)

Females developed notably higher resistance than males in a spinosad-resistant house fly strain, however, resistance factors responsible for this phenomenon are poorly understood. In this study, the potential role of cytochrome P450 monooxygenases involved in the sex-differential spinosad resistance in house flies was investigated, using a susceptible strain (SSS) and a spinosad resistant near-isogenic line (N-SRS). Combination of the synergism of spinosad by PBO and increased cytochrome P450 monooxygenase activity in the N-SRS strain implied that cytochrome P450 monooxygenases contributed to spinosad resistance in house flies. Transcriptional levels of eight P450 genes related to insecticide resistance in two genders of the SSS and N-SRS strain were separately evaluated by quantitative real-time PCR. Notably, compared with the corresponding gender of susceptible SSS house flies, CYP4G2 and CYP6A5v2 were overexpressed in resistant N-SRS females, while the expression of these two P450 genes was significantly decreased in resistant N-SRS males. Furthermore, by measuring the expression of CYP4G2 and CYP6A5v2 in female and male house fly populations with different spinosad resistance levels, which were generated from a series of genetic crosses, the genetic linkage between spinosad resistance and P450 gene expression was analyzed. It was found that with increased spinosad resistance, CYP4G2 and CYP6A5v2 were up-regulated in females, while both of them were down-regulated in males, and this suggested their involvement in the female-linked spinosad resistance of house flies. Taken together, our results provide valuable insight into the involvement of cytochrome P450 monooxygenases in the sex-differential spinosad resistance in house flies.

Abundance and Accumulation ofEscherichia coliandSalmonellaTyphimurium Procured by Male and Female House Flies (Diptera: Muscidae) Exposed to Cattle Manure

Abundance and Accumulation of<i>Escherichia coli</i>and<i>Salmonella</i>Typhimurium Procured by Male and Female House Flies (Diptera: Muscidae) Exposed to Cattle Manure

Comparison of sucrose intake and production of elimination spots among adult Musca domestica, Musca autumnalis, Phormia regina and Protophormia terraenovae

ObjectiveTo compare the differences in intake and excretion between Musca domestica and other three species from families Muscidae and Calliphoridae which may help explaining the significance of house fly in the transmission of pathogens. MethodsThe four adult species were supplied with two concentrations of sucrose via modified capillary feeder assay system. The two sucrose concentrations were applied to one adult male/each experiment and the elimination spots were counted. Using 0.25 mol/L sucrose + 0.25% bromophenol blue, one active non-starved male/cup was observed carefully for 1 h to record its behavior. As a growing medium used in bacterial transmission experiments, undiluted trypticase soy broth was used to feed 3-day-old females and males of Musca domestica following two different diets upon emergence and the frequency of elimination spots was estimated. ResultsThe two Musca species have half the weight of the two Phormia species. Comparing the volume of intake per hour, house fly took as much as the other species, all of which were larger. House fly produced twice, or more, the number of elimination spots/h than the other three species. Feeding the flies a sugar liquid diet resulted in producing more fecal spots than regurgitation spots. The male house flies produced less elimination spots/h when fed with trypticase soy broth than with the two sucrose solutions. ConclusionsHouse flies eliminated more than the other examined fly species and most of these elimination events were defecation which implicates the fecal route for pathogen transmission by this important vector.

The evolving puzzle of autosomal versus Y-linked male determination in Musca domestica.

Sex determination is one of the most rapidly evolving developmental pathways, but the factors responsible for this fast evolution are not well resolved. The house fly, Musca domestica, is an ideal model for studying sex determination because house fly sex determination is polygenic and varies considerably between populations. Male house flies possess a male-determining locus, the M factor, which can be located on the Y or X chromosome or any of the five autosomes. There can be a single M or multiple M factors present in an individual male, in heterozygous or homozygous condition. Males with multiple copies of M skew the sex ratio toward the production of males. Potentially in response to these male-biased sex ratios, an allele of the gene transformer, Md-traD, promotes female development in the presence of one or multiple M factors. There have been many studies to determine the linkage and frequency of these male determining factors and the frequency of Md-traD chromosomes in populations from around the world. This review provides a summary of the information available to date regarding the patterns of distribution of autosomal, X-linked and Y-linked M factors, the relative frequencies of the linkage of M, the changes in frequencies found in field populations, and the fitness of males with autosomal M factors vs. Y-linked M. We evaluate this natural variation in the house fly sex determination pathway in light of models of the evolution of sex determination.

Fungi in housefly (Musca domestica L.) as a disease risk indicator—A case study in South Africa

Houseflies are the commonest insects which have increasingly overcrowded human dwellings, particularly in rural areas and constitute a health hazard. In the environment they move back and forth by feeding and breeding on food commodities and filth. This may lead to the spread of diseases and also mycotoxin-producing fungi. Thus frequent exposure to the activity of such houseflies will have an impact on the welfare of humans. The study investigated the natural occurrence of fungal contamination in housefly samples captured from different households and pit toilets from a rural community in South Africa. Fungal contamination data were based on the prevalence, contamination level and morphological characteristics of the different identified species. A total of 497 fungal isolates of 15 genera including Aspergillus (37%), Fusarium (17%), Penicillium (21%), Alternaria (1.4%), Chrysosporium (2%), Cladosporium (0.2%), Curvularia (0.4%), Epicoccum (1%), Eupenicillium (1%), Moniliella (9%), Mucor (2%), Nigrospora (1%), Rhizopus (2%), Scopulariopsis (2%) and Yeasts (3%) were identified from the external surfaces of both female and male houseflies. The range of fungal contamination per total fungal count isolated from female and male houseflies were recorded with mean fungal load of 4.1×106, 8.4×106, 4.4×106, 3.3×105, 9.8×106, 2.2×104, 5.6×104, 2.9×106, 5.2×106, 4.7×106, 4.5×107, 4.6×106, 2.3×106, 4.9×107 and 6.4×106CFU/ml, respectively. However, the range from The most dominant fungal isolates of the female housefly samples were Aspergillus flavus, Fusarium verticillioides, Penicillium verrucosum and Moniliella suaveolens, while A. flavus, Aspergillus parasiticus, F. verticillioides, Fusarium proliferatum and Penicillium aurantiogriseum were most prevalent in male samples. The study proves that housefly is a vector for fungal spores. Therefore, it is crucial to implement housefly-control measures to curb the spread of diseases.

Identification of Volatile Compounds From a Food-Grade Vinegar Attractive to House Flies (Diptera: Muscidae)

A commercial vinegar product (ChinKiang) was found to be highly attractive to adult house flies, Musca domestica L. Field experiments on a Nebraska dairy demonstrated that traps baited with vinegar and brown sugar captured more house flies than those baited with other house fly attractants. Solid phase microextraction was used in the field to collect volatiles from the vinegar bait. Seven compounds were identified as, acetic acid, furfural, butanoic acid, isovaleric acid, hexanoic acid, 2-phenylethanol, and p-cresol. Electroantennograms showed that the seven vinegar components elicited significant responses from antennae of female and male house flies. Bioassays indicated that the vinegar blend of the seven volatile components were more attractive than any of the individual components. Field evaluations demonstrated that traps baited with the synthetic seven component blend caught as many flies as those baited with vinegar. This is the first detailed report of house fly attractants from vinegar. The vinegar volatile compounds identified in this study will be useful for the development of less objectionable alternatives to the fetid, manure mimicking volatiles currently used in commercial fly bait systems, especially those designed for use in indoor environments.

Spinosad resistance in female Musca domestica L. from a field‐derived population

Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC(50), whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression.

Adult House Fly (Diptera: Muscidae) Activity and Age of Females Near Varying Levels of (&lt;I&gt;Z&lt;/I&gt;)-9-Tricosene on a Southern California Dairy

The number of adult male and female house flies, Musca domestica L. (Diptera: Muscidae), near varying levels of (Z)-9-tricosene alone (5, 50, or 100 micdrol) or combined (50 microl) with sugar was determined using conical screened traps on a dairy in southern California. Overall, significantly more males than females were collected in the traps. Significantly more flies (male and female) were collected in traps with (Z)-9-tricosene. There were no significant differences among doses of (Z)-9-tricosene alone, but numbers of both sexes were significantly higher in traps baited with (Z)-9-tricosene and sugar compared with the 5- and 50-microl doses without sugar. The age of female flies collected in traps was determined by pterin analysis. Mean female ages ranged from 94.7 to 99.6 degree-days (6.3-6.8 d of age) and did not differ significantly among treatments. Dissections of a subset of females from each treatment determined that collected females were primarily nongravid (86.3%). Proportions of gravid females that were collected did not differ among treatments.

A microsatellite marker linkage map of the housefly,Musca domestica: evidence for male recombination

We present the first molecular marker linkage map for Musca domestica containing 35 microsatellite plus six visible markers. We report the development of 33 new microsatellite markers of which 19 are included in the linkage map. Two hundred and thirty-six F2 individuals were genotyped from three crosses yielding a linkage map consisting of five linkage groups that represent the five autosomes of the housefly. The map covers a total of 229.6 cM with an average marker spacing of 4.4 cM spanning approximately 80.2% of the genome. We found up to 29% recombination in male houseflies in contrast to most previous studies. The linkage map will add to genetic studies of the housefly.

Hormones and Sex-Specific Transcription Factors Jointly Control Yolk Protein Synthesis inMusca domestica

In the housefly Musca domestica, synthesis of yolk proteins (YPs) depends on the level of circulating ecdysteroid hormones. In female houseflies, the ecdysterone concentration in the hemolymph oscillates and, at high levels, is followed by expression of YP. In male houseflies, the ecdysterone titre is constantly low and no YP is produced. In some strains, which are mutant in key components of the sex-determining pathway, males express YP even though their ecdysterone titre is not significantly elevated. However, we find that these males express a substantial amount of the female variant of the Musca doublesex homologue, Md-dsx. The dsx gene is known to sex-specifically control transcription of yp genes in the fat body of Drosophila melanogaster. Our data suggest that Md-dsx also contributes to the regulation of YP expression in the housefly by modulating the responsiveness of YP-producing cells to hormonal stimuli.

Changes in the frequency of YMversus IIIMin the housefly,Musca domesticaL., under field and laboratory conditions

In the housefly, Musca domestica L., sex is usually determined by a dominant factor, M, located on the Y chromosome. However, there are 'autosomal male' (AM) populations in which the M factor is located on one or more of the five autosomes (I-V) or on X. We examined changes in the frequency of AM and YM males in North Carolina populations of houseflies after 4 years in the laboratory (NC Lab 02:06) and after 4 or 5 years in the field (NC 2006 and NC 2007). In 2002, 77.7% of the male houseflies were III/III;XYM, 20% were IIIM/III;XX, and 2.3% were IIIM/III;XYM. After 4 years in the laboratory, IIIM/III males disappeared and 17.4% of the males were XMYM. Conversely, 4 years later, the field population was relatively unchanged from 2002. Thus, there was a strong selection against IIIM/III males in the laboratory, but not in the field. Field-collected flies from 2007 indicated a slight increase in the frequency of XYM males and a slight decrease in the frequency of IIIM/III males (relative to 2002 and 2006), suggesting that the relative frequency of XYM and IIIM/III can vary slightly over time in field populations. The detection of XMYM males in 2007 offered the opportunity to evaluate the frequency of the female-determining FD factor, which was found to be present in both the laboratory and field populations, but frequencies varied greatly. The present study represents the first report of FD in houseflies from North America. The significance of these results, relative to observed clines in AM versus YM males, is discussed.

Crossmodal Visual Input for Odor Tracking during Fly Flight

Flies generate robust and high-performance olfactory and visual behaviors. Adult fruit flies can distinguish small differences in odor concentration across antennae separated by less than 1 mm [1], and a single olfactory sensory neuron is sufficient for near-normal gradient tracking in larvae [2]. During flight a male housefly chasing a female executes a corrective turn within 40 ms after a course deviation by its target [3]. The challenges imposed by flying apparently benefit from the tight integration of unimodal sensory cues. Crossmodal interactions reduce the discrimination threshold for unimodal memory retrieval by enhancing stimulus salience [4], and dynamic crossmodal processing is required for odor search during free flight because animals fail to locate an odor source in the absence of rich visual feedback [5]. The visual requirements for odor localization are unknown. We tethered a hungry fly in a magnetic field, allowing it to yaw freely, presented odor plumes, and examined how visual cues influence odor tracking. We show that flies are unable to use a small-field object or landmark to assist plume tracking, whereas odor activates wide-field optomotor course control to enable accurate orientation toward an attractive food odor.

Effect of caloric restriction on life span of the housefly, Musca domestica.

Caloric restriction (CR) has been found to extend the life spans of a wide variety of species, transcending phylogenetic boundaries. The objective of this study was to test the generality of this phenomenon, using the male housefly as an insect model in which food intake can be quantified precisely. Sucrose was found to promote a longer life span than diets additionally containing proteins and lipids. Flies were fed sucrose or a more complex diet ad libitum (AL), or in amounts ranging from 50% to 100% of the average amount consumed by young flies. CR shortened rather than prolonged the life span of houseflies, particularly flies fed sucrose only. The rate of oxygen consumption was not affected by caloric restriction or by the exclusion of proteins and lipids from the diet, and the reproductive activity of male flies remained unchanged by sucrose feeding. Thus, it is unlikely that the life-shortening effects of CR can be explained either in terms of an adaptive response in metabolic rate or use of a suboptimal food source. Results of this study contradict the widely held view that CR has a life-extending effect in all species.

Continuous recording of excretory water loss from Musca domestica using a flow-through humidity meter: hormonal control of diuresis

Water loss from adult male houseflies was continuously recorded using a flow-through humidity meter, which enabled losses to be apportioned between the sum of cuticular and respiratory transpiration, salivation and excretion. Transpiration accounted for >95% of water lost from sham-injected flies, compared with excretion (3.0%) and salivation (2.4%). In contrast, excretion accounted for 40% of water lost from flies injected with ≥3 μl of saline, whereas salivary losses were unchanged. Saline injections (1–5 μl) expanded the abdomen in the dorsal–ventral plane, and this expansion was positively correlated with the magnitude of the ensuing diuresis, suggesting the signal for diuretic hormone release originates from stretch receptors in abdominal tergal–sternal muscles. The effects of decapitation, severing the ventral nerve cord within the neck or ligaturing the neck, showed the head was needed to initiate and maintain diuresis, but was neither the source of diuretic hormone nor did it control the discharge of urine from the anus. These findings indicate the head is part of the neural–endocrine pathway between abdominal stretch receptors and sites for diuretic hormone release from the thoracic-abdominal ganglion mass. Evidence is presented for Musdo-K having a hormonal role in the control of diuresis, although other neuropeptides may also be implicated.

MALE HOUSEFLIES CLEARLY SEE BETTER

![Figure][1] When a male housefly sets his sights on a female, there's a lot at stake. In a high-speed chase, he's got to get his gal, or loose his chance of genetic immortality. The stakes are high enough for male flies to have developed a specialised eye structure, the `lovespot', that is