The fly factor phenomenon is mediated by interkingdom signaling between bacterial symbionts and their blow fly hosts

We tested the recent hypothesis that the"fly factor"phenomenon(food cur-rently or previously fed on by flies attracts more flies than the same type of food kept inccessible to flies)is mediated by bacterial symbionts deposited with feees or regur-gitated by feeding flies.We allowed laboratory-reared black blow flies,Phormia regina(Meigen),to feed and de fecate on bacterial Luria-Bertani medium solidified with agar,and isolated seven morphologically distinct bacterial colonies.We identified these us-ing matrix-assisted laser desorption/ionization mass spectrometry and sequencing of the 165 rRNA gene.In two-choice laboratory experiments,traps baited with cultures of Pro-teus mirabilis Hauser,Morganella morganii subsp.sibonii Jensen,or Serratia marcescens Bizio,captured significantly more flies than corresponding control jars baited with tryptic soy agar only.A mixture of seven bacterial strains as a trap bait was more attractive to flies than a single bacterial isolate(M.m.siboni).In a field experiment,traps baited with agar cultures of P:mirabilis and M.m siboni in combination captured significantly more flies than lraps baited with either bacterial isolate alone or the agar control.As evident by gas chromatography-mass spectrometry,the odor profiles of bacterial isolates differ,which may explain the additive effect of bacteria to the attractiveness of bacterial trap baits.As"generalist bacteria,"P mirabilis and M.m.sibonii growing on animal protein(beef liver)or plant protein(tofu)are similarly effective in attracting flies.Bacteria-derived airborne semiochemicals appear to mediate foraging by flies and to inform their feeding and oviposition decisions.     

Gender-specific prandial response to dietary restriction and oxidative stress in Drosophila melanogaster

Drosophila melanogaster is ideal for studying lifespan modulated by dietary restriction (DR) and oxidative stress, and also for screening prolongevity compounds. It is critical to measure food intake in the aforementioned studies. Current methods, however, overlook the amount of the food excreted out of the flies as feces or deposited in eggs. Here we describe a feeding method using a radioactive tracer to measure gender-specific food intake, retention and excretion in response to DR and oxidative stress to account for all the ingested food. Flies were fed a full, restricted or paraquat-containing diet. The radioactivity values of the food in fly bodies, feces and eggs were measured separately after a 24-hr feeding. Food intake was calculated as the sum of these measurements. We found that most of the tracer in the ingested food was retained in the fly bodies and < 8% of the tracer was excreted out of the flies as feces and eggs in the case of females during a 24-hr feeding. Under a DR condition, flies increased food intake in volume to compensate for the reduction of calorie content in the diet and also slightly increased excretion. Under an oxidative stress condition, flies reduced both food intake and excretion. Under all the tested dietary conditions, males ingested and excreted 3-5 fold less food than females. This study describes an accurate method to measure food intake and provides a basis to further investigate prandial response to DR and prolongevity interventions in invertebrates.     

Does behaviour play a role in house fly resistance to imidacloprid‐containing baits?

The objective of this research was to examine the role and type of behavioural mechanisms that function in house fly, Musca domestica L. (Diptera: Muscidae), resistance to an imidacloprid‐containing commercial fly bait, QuickBayt^®, using an insecticide‐susceptible and an imidacloprid‐resistant strain. Mortality and feeding behaviour were observed through choice bioassays of three post‐imidacloprid selected house fly generations to determine whether flies would consume the bait in the presence of an alternative food source. Mortality rates in choice containers progressively decreased in post‐selection flies as QuickBayt^® no‐choice selections proceeded. There were no differences between the proportions of flies observed contacting QuickBayt^® and sugar, respectively, a finding that eliminates repellency as a mechanism of stimulus‐dependent behavioural resistance. However, differences in QuickBayt^® consumption and subsequent mortality between choice and no‐choice containers provided strong support for the evolution of consumption irritancy‐ or taste aversion‐related behavioural resistance. The results of this study support the responsible rotation of insecticide bait formulations for house fly control.     

Effect of food deprivation on attractiveness of food sources, containing natural and artificial sugar and protein, to three African fruit flies: Ceratitis cosyra, Ceratitis fasciventris, and Ceratitis capitata

The effects of food deprivation, age, and mating status on the responses of three fruit fly species, Ceratitis cosyra (Walker), Ceratitits fasciventris (Bezzi), and Ceratitits capitata (Wiedemann) (Diptera: Tephritidae) to natural and artificial sugar and protein food sources were investigated. Natural food sources included guava [Psidium guajava L. (Myrtaceae)] juice (a common host fruit for all three fruit fly species) and bird faeces (farm chicken). Artificial food sources included molasses (obtained from a local sugar factory) and a locally produced protein bait (the International Centre of Insect Physiology and Ecology yeast). In all species studied, sugar deprivation of immature (1–2‐day‐old) male and female flies increased their response to food odours, although it did not change their preference for the type of odour (protein or sugar). Protein deprivation of mature (14–17‐day‐old) male and female flies also increased their response to food odours compared to protein‐fed flies. Protein‐deprived females were highly attracted to odours from protein sources in particular. Odours from natural food sources, guava juice, and chicken faeces, were more attractive to food‐deprived flies than were odours from artificial sugar and protein sources. Attraction to food odours increased significantly with increasing age for protein‐deprived females of all species. For males and females of all species, nutritional state was a more important factor than mating status in influencing responses of flies to food odours. Practical implications of these findings are discussed in terms of strategies for fruit fly control using food baits.     

Dietary fatty acids promote sleep through a taste‐independent mechanism

Consumption of foods that are high in fat contribute to obesity and metabolism‐related disorders. Dietary lipids are comprised of triglycerides and fatty acids, and the highly palatable taste of dietary fatty acids promotes food consumption, activates reward centers in mammals and underlies hedonic feeding. Despite the central role of dietary fats in the regulation of food intake and the etiology of metabolic diseases, little is known about how fat consumption regulates sleep. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and metabolic traits, and flies potently regulate sleep in accordance with food availability. To investigate the effects of dietary fats on sleep regulation, we have supplemented fatty acids into the diet of Drosophila and measured their effects on sleep and activity. We found that flies fed a diet of hexanoic acid, a medium‐chain fatty acid that is a by‐product of yeast fermentation, slept more than flies starved on an agar diet. To assess whether dietary fatty acids regulate sleep through the taste system, we assessed sleep in flies with a mutation in the hexanoic acid receptor Ionotropic receptor 56D, which is required for fatty acid taste perception. We found that these flies also sleep more than agar‐fed flies when fed a hexanoic acid diet, suggesting the sleep promoting effect of hexanoic acid is not dependent on sensory perception. Taken together, these findings provide a platform to investigate the molecular and neural basis for fatty acid‐dependent modulation of sleep.     

Is aggregated oviposition by the blow flies Lucilia sericata and Phormia regina (Diptera: Calliphoridae) really pheromone‐mediated?

When female blow flies Lucilia sericata and Phormia regina (Diptera: Calliphoridae) oviposit in aggregations on carrion, even‐aged larval offspring reportedly develop faster, and fewer are parasitized or preyed upon. The benefits of aggregated oviposition equally affect con‐ and heterospecific larvae sharing a resource. The benefits imply that female blow flies engage in coordinated, pheromone‐mediated oviposition behavior. Yet, repeated attempts to identify oviposition pheromones have failed invoking doubt that they exist. Simply by regurgitating and feeding on carrion, flies may produce attractive semiochemicals. If flies were to aggregate in response to feeding flies rather than ovipositing flies, then the semiochemical cue(s) may be associated with the salivary gland. Working with L. sericata and P. regina and using liver as a surrogate oviposition medium, we test the hypotheses, and present data in their support, that (i) gravid or nongravid females ovipositing and/or feeding on liver enhance its attractiveness to gravid and nongravid females; (ii) females respond to semiochemicals from feeding heterospecific females; (iii) females respond equally well to semiochemicals from feeding con‐ and heterospecific females; (iv) macerated head tissues of females applied to liver enhance its attractiveness; and (v) females in direct contact with and feeding on liver, but not when next to yet physically separated from liver, enhance attraction of flies. We conclude that oviposition site‐seeking females do not respond to an oviposition pheromone. Instead, they appear to coopt semiochemicals associated with feeding flies as resource indicators, taking chances that resources are suitable for oviposition, and that ovipositing flies are present.     

House Fly (Musca domestica L.) Attraction to Insect Honeydew

House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.     

High nocturnal CO2 emanation guides the sand fly Phlebotomus papatasi to sugar‐rich plants

Abstract In arid areas, Phlebotomus papatasi obtains essential carbohydrates by feeding on green tissues of plants. There is a great variation in sugar content, metabolic rates and dark respiration between conspecific plants, and also between branches and leaves of the same plant. It is hypothesized that high nocturnal release of CO₂ may guide the sand flies to rich sugar sources. Comparisons of Ph. papatasi feeding on branches of different plant species demonstrate a significant positive correlation between the level of sugar in leaves, the rate of sand fly feeding on them and the size of ingested sugar meals. Mean nocturnal CO₂ emanation of low‐sugar Ricinus communis branches is 26.5 ppm and that of sugar rich branches is 86.3 ppm above room level. Low‐sugar Capparis spinosa branches release 45.0 ppm and the emission from sugar rich branches is 76.0 ppm above room level. Branches with similar emissions, placed behind net partitions, are used in no‐choice orientation experiments: R. communis branches releasing high CO₂ levels are approached by 53.7% of the flies compared with 3.0% of flies that orientate to low CO₂ branches. Capparis spinosa with high CO₂ emission are approached by 9.8% of the flies compared with 0.95% of flies that orientate to low CO₂ branches.     

Gender-specific prandial response to dietary restriction and oxidative stress in Drosophila melanogaster

Drosophila melanogaster is ideal for studying lifespan modulated by dietary restriction (DR) and oxidative stress, and also for screening prolongevity compounds. It is critical to measure food intake in the aforementioned studies. Current methods, however, overlook the amount of the food excreted out of the flies as feces or deposited in eggs. Here we describe a feeding method using a radioactive tracer to measure gender-specific food intake, retention and excretion in response to DR and oxidative stress to account for all the ingested food. Flies were fed a full, restricted or paraquat-containing diet. The radioactivity values of the food in fly bodies, feces and eggs were measured separately after a 24-hr feeding. Food intake was calculated as the sum of these measurements. We found that most of the tracer in the ingested food was retained in the fly bodies and < 8% of the tracer was excreted out of the flies as feces and eggs in the case of females during a 24-hr feeding. Under a DR condition, flies increased food intake in volume to compensate for the reduction of calorie content in the diet and also slightly increased excretion. Under an oxidative stress condition, flies reduced both food intake and excretion. Under all the tested dietary conditions, males ingested and excreted 3-5 fold less food than females. This study describes an accurate method to measure food intake and provides a basis to further investigate prandial response to DR and prolongevity interventions in invertebrates.     

Evaluation of commercial and field‐expedient baited traps for house flies,Musca domestica L. (Diptera: Muscidae)

A comparison of nine commercial baited fly traps on Florida dairy farms demonstrated that Terminator traps collected significantly more (13,323/trap) house flies (Musca domestica L.) than the others tested. Final Flight, Fly Magnet, and FliesBeGone traps collected intermediate numbers of flies (834‐2,166), and relatively few were caught with ISCA, Advantage, Fermone Big Boy, Squeeze & Snap, or OakStump traps (<300). Terminator traps collected about twice as many flies (799.8/trap) as FliesBeGone traps (343.8) when each trap was baited with its respective attractant, but when the attractants were switched between the two trap types, collections were significantly lower (77‐108) than was observed with traps baited with their respective attractant. Solutions of molasses were significantly more attractive to house flies than honey, maple syrup, or jaggery (date palm sugar). Field‐expedient traps constructed from discarded PET water bottles were much less effective than commercial traps, but painting the tops of such traps with black spray paint resulted in a six‐fold increase in trap capture.     

Evaluation of ammonia,human sweat,and bovine blood as attractants for the female canyon fly,Fannia conspicua (Diptera: Muscidae), in southern California

Human sweat, liquid ammonia, and bovine blood are known to be attractive to some hematophagous flies. These materials were evaluated with and without carbon dioxide (CO₂) for their ability to increase capture of female “canyon flies” (Fannia conspicua Malloch) using CDC‐type suction traps (without light). Ammonia acted synergistically with CO₂ to increase trap catch 89.9% over CO₂ alone. There was no synergistic effect of human sweat or bovine blood with CO₂. In the absence of CO₂, none of the three materials increased trap catch of female canyon flies relative to non‐baited traps. Implications for canyon fly control and further trap improvement are discussed.     

Dose‐dependent fate of GFP‐expressing Escherichia coli in the alimentary canal of adult house flies

The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe‐rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose‐dependent survival of bacteria in house flies. Flies were fed four different ‘doses’ of green fluorescent protein (GFP)‐expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post‐ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time‐points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time‐points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose‐fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune‐mediated. Understanding dose‐dependent bacterial survival in flies can help in predicting bacteria transmission potential.     

Cover Caption

Females of the common green blow fly, Lucilia sericata, ovipositing on rat carrion. When females oviposit in aggregations, even‐aged larval offspring develop faster and fewer are preyed upon. These benefits imply that females engage in coordinated, pheromone‐mediated oviposition behavior. Yet, new data show that oviposition site‐seeking females do not respond to pheromones but to semiochemicals associated with gravid or non‐gravid feeding flies, taking chances that resources are suitable for oviposition and that ovipositing flies are present (see pages 651–660). Image: Sean McCann.     

Quantification of Food Intake in Drosophila

Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo^D increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.     

Neural mechanisms of context-dependent processing of CO2 avoidance behavior in fruit flies

The fruit fly, Drosophila melanogaster, innately avoids even low levels of CO₂. CO₂ is part of the so-called Drosophila stress odor produced by stressed flies, but also a byproduct of fermenting fruit, a main food source, making the strong avoidance behavior somewhat surprising. Therefore, we addressed whether feeding states might influence the fly’s behavior and processing of CO₂. In a recent report, we showed that this innate behavior is differentially processed and modified according to the feeding state of the fly. Interestingly, we found that hungry flies require the function of the mushroom body, a higher brain center required for olfactory learning and memory, but thought to be dispensable for innate olfactory behaviors. In addition, we anatomically and functionally characterized a novel bilateral projection neuron connecting the CO₂ sensory input to the mushroom body. This neuron was essential for processing of CO₂ in the starved fly but not in the fed fly. In this Extra View article, we provide evidence for the potential involvement of the neuromodulator dopamine in state-dependent CO₂ avoidance behavior. Taken together, our work demonstrates that CO₂ avoidance behavior is mediated by alternative neural pathways in a context-dependent manner. Furthermore, it shows that the mushroom body is not only involved in processing of learned olfactory behavior, as previously suggested, but also in context-dependent innate olfaction.     

Opposed bands of cilia in the nonfeeding larvae of the serpulid annelid Salmacina tribranchiata

The nonfeeding planktonic larvae of marine invertebrates typically lack larval feeding structures. One puzzling exception to this generalization is the annelid clade Sabellidae, in which nonfeeding larvae possess ciliary bands (specifically, food groove and metatroch) that, to the best of our knowledge, have no function other than in feeding. Nishi and Yamasu (1992b, Bulletin of the College of Sciences, University of the Ryukyus, 54 , 107–121) published a scanning electron micrograph showing that nonfeeding larvae of the serpulid annelid Salmacina dysteri also possess food groove and metatrochal cilia. Here I demonstrate that nonfeeding larvae of Salmacina tribranchiata also bear ciliary bands identifiable as food groove and metatroch by position. High‐speed video of ciliary beat patterns shows that, together with the prototrochal cilia, these bands function in an opposed band system. The presence of feeding structures in nonfeeding annelid larvae is thus more widely distributed than previously recognized. The presence of feeding structures may make evolutionary transitions to planktotrophy more likely, and may underlie an inferred origin of larval feeding in the common ancestor of one of the two major clades of serpulid annelids, Serpulinae.     

Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations

The toxicity of spinosad was determined in one susceptible and five insecticide-resistant laboratory strains of house fly, Musca domestica L. Spinosad was relatively slow-acting, but highly toxic to house flies. In a feeding bioassay, spinosad LC50 at 72 h was 0.51 microg of spinosad per gram of sugar, making it 6.3- and 3.5-fold more toxic to house flies compared with azamethiphos and methomyl, respectively. In topical application bioassay, the LD50 at 48 h of spinosad in susceptible house flies was 40 ng per 20 mg of house fly, making spinosad less toxic than the pyrethroid bioresmethrin synergized by piperonyl butoxide and the organophosphate dimethoate. The insecticide-resistant laboratory strains had resistance factors to spinosad at LC50 in feeding bioassay from 1.5 to 5.5 and at LD50 in topical application bioassay from 2.5 to 4.7, indicating that in house fly cross-resistance to the major insecticide classes will not initially be of major concern for the use of spinosad for house fly control. The toxicity of spinosad was also evaluated against 31 field populations of house flies collected from livestock farms across Denmark. The field populations were 2.2- to 7.5-fold resistant to spinosad at 72 h in feeding bioassay, but based on steep slopes in the bioassay and the limited variation of spinosad toxicity against the various field populations, we consider the field populations to be spinosad-susceptible. We propose a diagnostic dose of 12 microg of spinosad per gram of sugar in feeding bioassay with impregnated sugar for determination of resistant house flies, which is 10x the LC95 of the susceptible strain WHO and approximately = 2x the LD95 of the field populations. Spinosad showed no substantial cross-resistance to the pyrethroid bioresmethrin synergized by piperonyl butoxide, the anticholinesterases dimethoate, azamethiphos, methomyl, and spinosad in house fly field populations.     

Physiological state influences the antennal response of Anastrepha obliqua to male and host volatiles

The sexual and host‐related behaviours of the fruit fly Anastrepha obliqua Macquart (Diptera: Tephritidae) are mediated by volatile compounds. However, whether the physiological state of this species affects its antennal and behavioural responses to semiochemicals is unknown. The effects of age, mating status, diet and the topical application of methoprene, a Juvenile hormone analogue (JHA), on the antennal sensitivity of this tephritid fruit fly species to selected male [(Z)‐3‐nonenol] and host fruit volatiles (ethyl benzoate, ethyl hexanoate, ethyl butyrate and trans‐β‐ocimene) are investigated using electroantennography (EAG). Overall, (Z)‐3‐nonenol and ethyl benzoate elicit the highest EAG responses in both sexes. Flies of both sexes aged 1, 5 and 10 days old show higher EAG responses to the tested compounds compared with flies aged 20 days old. Virgin females and males show higher EAG responses to volatile compounds than mated flies. Females and males fed with sugar plus protein show higher antennal responses to volatiles compared with flies fed sugar or protein alone. Flies of both sexes treated with methoprene show higher antennal responses than flies treated with acetone (control). These results suggest that the peripheral olfactory system in A. obliqua is modulated by the physiological state of the flies.     

Comparative field studies of various traps and attractants of the olive fruit fly,Bactrocera oleae

In the framework of an ongoing testing and refining process of a mass trapping method, using a combination of semiochemicals, for the control of the olive fruit fly,Bactrocera (Dacus) oleae, four trap designs, three trap colors, six different food attractants, and two pheromone formulations were compared under field conditions. No differences were observed between the trap designs and the food attractants tested. Trap color had a significant effect in preference tests only. Traps combining food attractants and pheromones attracted higher numbers of both male and female flies but the differences became statistically significant only in cases of low trap densities. Pheromones enclosed in cyclodextrenes were not as attractive as standard pheromone formulations. The findings of these tests allow a choice among trap types and semiochemicals for cost reduction and convenience, but not for enhancement of the efficacy of the method.