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.     

Campylobacter jejuni in Musca domestica: An examination of survival and transmission potential in light of the innate immune responses of the house flies

The house fly, Musca domestica, has been implicated as a vector of Campylobacter spp., a major cause of human disease. Little is known whether house flies serve as biological amplifying hosts or mechanical vectors for Campylobacter jejuni. We investigated the period after C. jejuni had been ingested by house flies in which viable C. jejuni colonies could be isolated from whole bodies, the vomitus and the excreta of adult M. domestica and evaluated the activation of innate immune responses of house flies to ingested C. jejuni over time. C. jejuni could be cultured from infected houseflies soon after ingestion but no countable C. jejuni colonies were observed > 24 h postingestion. We detected viable C. jejuni in house fly vomitus and excreta up to 4 h after ingestion, but no viable bacteria were detected ≥ 8 h. Suppression subtractive hybridization identified pathogen‐induced gene expression in the intestinal tracts of adult house flies 4–24 h after ingesting C. jejuni. We measured the expression of immune regulatory (thor, JNK, and spheroide) and effector (cecropin, diptericin, attacin, defensing, and lysozyme) genes in C. jejuni‐infected and ‐uninfected house flies using quantitative real time PCR. Some house fly factor, or combination of factors, eliminates C. jejuni within 24 h postingestion. Because C. jejuni is not amplified within the body of the housefly, this insect likely serves as a mechanical vector rather than as a true biological, amplifying vector for C. jejuni, and adds to our understanding of insect–pathogen interactions.     

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.     

Vibrio cholerae laboratory infection of the adult house fly Musca domestica

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)‐labelled, non‐O1/non‐O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ～10⁷ colony‐forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time‐point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose‐dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.     

The Survival of Ingested <Emphasis Type="Italic">Serratia marcescens</Emphasis> in Houseflies (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) After Electrocution with Electric Fly Killers

Electric fly killers (EFKs) are commonly used to control flying insects that enter food establishments. For establishment of the incidence of pathogen-bearing insects in food establishments, insect samples obtained from EFK trays could be used. The principal difficulty with this approach is that the survival time of microorganisms on or within insect corpses after electrocution is unknown. This study determined the survival of Serratia marcescens (as a representative of the enteric bacteria) within houseflies following their electrocution by a commercial EFK. S. marcescens was successfully ingested by houseflies and survived on and within the corpses after electrocution for up to 5 weeks. Maximal levels of bacteria were recovered 24 h postelectrocution. The study also demonstrates the ability of ingested S. marcescens to out-compete resident microbial flora within houseflies. The findings are intended to pave the way for further research to determine the incidence of pathogen-laden flying insects in food establishments. Received: 30 April 2002 / Accepted: 3 July 2002     

Identification of Potential Probiotics in the Midgut of Mulberry Silkworm,Bombyx mori Through Metagenomic Approach

Microorganisms play an important role in the growth and development of numerous insect species. The mulberry silkworm, Bombyx mori (Lepidoptera), harbors several bacteria in its midgut aiding the metabolic processes; however, the variability of bacterial spp. present in the midgut and their role(s) in the growth and development of the silkworm are poorly understood. The present work compares the diversity of midgut bacterial communities in silkworms of variable voltinism (Pure Mysore, PM: multivoltine; CSR2: bivoltine and PM × CSR2: crossbreed) through metagenomics. The predominance of Enterococcus (30.30%) followed by Bacillus (16.96%) was observed in PM, whereas Lactobacillus (56.56%) followed by Enterococcus (10.58%) was seen only in CSR2. Interestingly, crossbreed midgut harbored diverse bacterial communities (36.21% Lactobacillus, 25.94% Bacillus, 8.1% Enterococcus, and 18.37% uncultured bacteria). Metagenomic profiles indicate variability in the gut bacterial population in different kinds of silkworms influencing the physiological activities accordingly. The dominant bacteria, particularly lactobacilli, bacilli, and enterococci could be further explored for identifying the potential probiotic consortia based on a literature survey and potential involvement in nutrient absorption, disease/stress tolerance, and improved economic traits.

     

Kinetics of Colonization of Adult Queensland Fruit Flies (Bactrocera tryoni) by Dinitrogen-Fixing Alimentary Tract Bacteria

The average total population of bacteria remained constant in the alimentary tracts of adult laboratory-raised Queensland fruit flies (Bactrocera tryoni) although the insects had ingested large numbers of live bacteria as part of their diet. The mean number of bacteria (about 13 million) present in the gut of the insects from 12 to 55 days after emergence was not significantly modified when, at 5 days after emergence, the flies were fed antibiotic-resistant bacteria belonging to two species commonly isolated from the gut of field-collected B. tryoni. Flies were fed one marked dinitrogen-fixing strain each of either Klebsiella oxytoca or Enterobacter cloacae, and the gastrointestinal tracts of fed flies were shown to be colonized within 7 days by antibiotic-resistant isolates of K. oxytoca but not E. cloacae. The composition of the microbial population also appeared to be stable in that the distribution and frequency of bacterial taxa among individual flies exhibited similar patterns whether or not the flies had been bacteria fed. Isolates of either E. cloacae or K. oxytoca, constituting 70% of the total numbers, were usually dominant, with oxidative species including pseudomonads forming the balance of the population. Antibiotic-resistant bacteria could be spread from one cage of flies to the adjacent surfaces of a second cage within a few days and had reached a control group several meters distant by 3 weeks. Restriction of marked bacteria to the population of one in five flies sampled from the control group over the next 30 days suggested that the bacterial population in the gut of the insect was susceptible to alteration in the first week after emergence but that thereafter it entered a steady state and was less likely to be perturbed by the introduction of newly encountered strains. All populations sampled, including controls, included at least one isolate of the dinitrogen-fixing family Enterobacteriaceae; many were distinct from the marked strains fed to the flies. Nitrogenase activity detected by the acetylene reduction assay was associated with flies fed dinitrogen-fixing bacteria as well as with control groups given either no supplement or free access to a yeast hydrolysate preparation. Nitrogen fixed from the atmosphere may supplement the nutrition of the alimentary tract microbial population of B. tryoni. Transmission electron microscopy showed that the principal site of bacterial colonization in the abdominal alimentary tract was the lumen of the midgut inside the peritrophic membrane. No intracellular symbionts were seen in the gut tissues nor were bacteria found attached to the cuticular folds of the hindgut. The ultrastructure of the gut resembled that of other fly genera except that the intercellular spaces between rectal epithelial cells were more extensive, suggesting a role for unspecialized epithelium in water and solute uptake in B. tryoni.     

Vector potential of houseflies for the bacterium Aeromonas caviae

Houseflies, Musca domestica Linnaeus (Diptera: Muscidae), have been implicated as vectors or transporters of numerous gastrointestinal pathogens encountered during feeding and ovipositing on faeces. The putative enteropathogen Aeromonas caviae (Proteobacteria: Aeromonadaceae) may be present in faeces of humans and livestock. Recently A. caviae was detected in houseflies by PCR and isolated by culture methods. In this study, we assessed the vector potential of houseflies for A. caviae relative to multiplication and persistence of the bacterium in the fly and to contamination of other flies and food materials. In experimentally fed houseflies, the number of bacteria increased up to 2 days post-ingestion (d PI) and then decreased significantly 3 d PI. A large number of bacteria was detected in the vomitus and faeces of infected flies at 2-3 d PI. The bacteria persisted in flies for up to 8 d PI, but numbers were low. Experimentally infected flies transmitted A. caviae to chicken meat, and transmissibility was directly correlated with exposure time. Flies contaminated the meat for up to 7 d PI; however, a significant decrease in contamination was observed 2-3 d PI. In the fly-to-fly transmission experiments, the transmission of A. caviae was observed and was apparently mediated by flies sharing food. These results support houseflies as potential vectors for A. caviae because the bacterium multiplied, persisted in flies for up to 8 d PI, and could be transmitted to human food items.     

Three metabolites from an entomopathogenic bacterium,Xenorhabdus nematophila,inhibit larval development of Spodoptera exigua (Lepidoptera: Noctuidae) by inhibiting a digestive enzyme,phospholipase A2

Abstract An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune‐associated phospholipase A₂ (PLA₂), which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline‐tyrosine and acetylated phenylalanine‐glycine‐valine, which are known to inhibit enzyme activity of PLA₂ extracted from hemocyte and fat body. This study tested their effects on digestive PLA₂ of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 μg/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA₂ extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA₂ of S. exigua midgut may explain some of their oral toxic effects.     

Studying the “fly factor” phenomenon and its underlying mechanisms in house flies Musca domestica

The “fly factor” was first discovered >60 years ago and describes the phenomenon that food currently or previously fed on by flies attracts more foraging flies than the same type and amount of food kept inaccessible to flies. Since then, there has been little progress made to understanding this phenomenon. Our objectives were (i) to demonstrate the existence of the fly factor in house flies, Musca domestica and (ii) to study underlying mechanisms that may cause or contribute to the fly factor. In 2‐choice laboratory bioassays, we obtained unambiguous evidence for a fly factor phenomenon in house flies, in that we demonstrated that feeding flies are more attractive to foraging flies than are nonfeeding flies, and that fed‐on food is more attractive to foraging flies than is “clean” food. Of the potential mechanisms (fly excreta, metabolic output parameters [elevated temperature, relative humidity, carbon dioxide]), causing the fly factor, fly feces, and regurgitate do attract foraging flies but none of the metabolic output parameters of feeding flies does. Even though feeding flies produce significantly more CO₂ than nonfeeding flies, elevated levels of CO₂ have no behavior‐modifying effect on flies. Preferential attraction of house flies to fly feces and regurgitate indicates that the flies sense airborne semiochemicals emanating from these sources. Hypothesizing that these semiochemicals are microbe‐produced, future studies will aim at isolating and mass producing these microbes to accumulate semiochemicals for identification.     

Transport of peroxidase through the midgut epithelium of Glossina M. Morsitans (diptera, glossinidae)

The peritrophic membrane (pm) of teneral female tsetse flies, Glossina morsitans morsitans, did not extend to the full length of the midgut 1-12 hr after emergence. The ingested blood did not reach the posterior part of the midgut (p-part), and the crop still contained food 12 hr after feeding. In these flies, the p-part contained the remains of the larval gut, the meconium, and bacteria. Ferritin molecules fed to tsetse females together with human serum were only found in the endoperitrophic space of the gut. This electron-dense tracer did not penetrate and cross the pm. On the other hand, ingested peroxidase passed the pm, and was transported through intercellular clefts, the basal labyrinth and the basal lamina to the hemolymph. This uptake was observed in the anterior part and to a smaller extent in the middle part of the midgut within 2 hr after feeding. Peroxidase was incorporated from the hemolymph into fat body cells, where it was found 2 hr and later after feeding. Pinocytosis of the tracer molecules, as an additional intracellular pathway to the intercellular route of transport, could not be demonstrated.     

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography‐tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α‐amylase) or other reactions (β‐1,3‐glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C‐Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase‐2 (McCHS‐2), chitinase (McCHI), and β‐N‐acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS‐2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS‐2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.     

Rapid bacterial growth in the hindgut of a marine deposit feeder

Antibiotic-insensitive mutants of natural sedimentary bacteria from an intertidal site were selected on gradient plates. Two of these strains, anAeromonas sp. andVibrio alginolyticus, were mixed with natural sediments from the field and fed toAbarenicola vagabunda, an intertidal lugworm characteristic of sandy beaches in the Pacific Northwest. Digestive removal was apparent in the midgut, 97% efficiency being seen forAeromonas sp. Both strains showed rapid growth in the hindgut, increasing between 2 and 3 orders of magnitude in abundance between the midgut and rectum of the polychaete, corresponding with a doubling time of about 50 min for each strain. Direct epifluorescence counts of natural bacteria in guts of animals freshly collected from the field suggest a mean doubling time that is only slightly greater (66 min) for all ingested bacteria that survive midgut digestion. These bacterial growth rates exceed by orders of magnitude the greatest rates reported for ambient marine sediments and suggest that hindgut bacterial growth, though of little immediate importance in the energetics of the animals, may strongly influence both population dynamics of marine bacteria and diagenesis of sedimentary organic matter.     

Time and site of assembly of the peritrophic membrane of the mosquito Aedes aegypti

Summary We determined the time and site of secretion of the precursors of the peritrophic membrane (PM) in Aedes aegypti and when the structure is assembled. The fine structure of the developing membrane of blood-feed females was described, and the pattern of secretion of injected tritiated glucosamine analyzed autoradiographically. Immediately following blood feeding, ingested red cells rapidly become compressed, such that the surrounding plasma is extruded to the margin of the midgut contents. Thereby, ingested fluids form a narrow margin separating the blood mass from the midgut epithelium. By electron microscopy, the PM first becomes evident at about 4 to 8 h after blood is ingested, and the membrane attains mature texture by 12 h. The compacted mass of ingested erythrocytes seems to serve as a template for the forming structure. In contrast, tritiated glucosamine, injected into freshly engorged mosquitoes, begins to concentrate on the midgut microvilli by 2 h after feeding. By 8 h the label assumes the layered appearance that characterizes the fine structure of the mature membrane. In contrast to the prevailing concept that the PM of mosquitoes first assumes texture anteriorly immediately after blood is ingested, we find that this potential barrier to pathogen development forms no earlier than 4 h after feeding and that it is formed from precursors secreted along the entire length of the epithelium overlying the food mass.     

Killing of Flies in Electrocuting Insect Traps Releases Bacteria and Viruses

Electrocuting insect traps (EIT) are popular devices frequently used by homeowners and food handlers attempting to localize the control of flying insects, including the ubiquitous house fly (Musca domestica L.). The traps contain a visual attractant and a high-voltage metal grid. Upon contact with the grids, the insects are disintegrated by the high voltage. As part of a systematic evaluation of EITs and their role in infectious disease spread, we quantitated spread of bacteria and a bacterial virus during electrocution of house flies. We loaded flies with Serratia marcescens or with the Escherichia coli phage ΦX174 and placed sprayed or fed flies into a room containing an EIT. While flies were being electrocuted, liberated particles and bacteria were assayed via agar plates or via air filtration samplers. Sprayed flies released one of every 10,000 of the added bacteria or viruses, and fed flies released one of every 1,000,000 of the consumed bacteria or viruses. Results of our studies suggest EITs could play a role in the spread of infectious disease agents, but the potential is influenced by the insect's route of contamination. Received: 26 February 2000 / Accepted: 2 May 2000     

Biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Col., Coccinellidae) by chitinolytic phylloplane bacteria Alcaligenes paradoxus entrapped in alginate beads

The chitinase secreting strain KPM‐012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM‐012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM‐012A‐entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for 1 week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.     

Insecticide,sugar, and diet effects on feeding and mortality in Rhagoletis indifferens (Dipt., Tephritidae)

The effects of spinosad bait and various insecticides, the presence of sugar in insecticides, and diet on feeding responses and mortality in western cherry fruit fly, Rhagoletis indifferens Curran (Dipt., Tephritidae), were determined. Numbers of feeding events on insecticides with sugar were greater than on insecticides alone, but there was only a small effect of diet on feeding responses to insecticides with sugar. Feeding durations on imidacloprid, thiamethoxam and acetamiprid with sugar were shorter than on sugar water and spinosad bait, as the neonicotinoids paralysed flies quickly. Flies that fed on sugar only (nitrogen‐starved) suffered higher mortalities when exposed to spinosad, thiamethoxam and azinphos‐methyl than to imidacloprid, acetamiprid and indoxacarb, and mortality in between these two groups of treatments when exposed to spinosad bait. Mortalities were greater when sugar was added to insecticides, and were higher in nitrogen‐starved than fully‐fed (yeast extract + sugar fed) flies. Flies that fed once on thiamethoxam were killed more quickly than those that fed once on spinosad bait and spinosad. Results suggest that thiamethoxam is comparable to spinosad in its effects on mortality, and that using it with sugar in bait may also have similar results as using spinosad bait or spinosad. One benefit of using thiamethoxam with sugar may be that it kills flies more quickly, before they can oviposit, than spinosad bait, although whether a fly will feed on it may depend on how much sugar or nitrogenous food it has eaten.     

Effects of prococene II on the nutritional physiology of last instar Heliothis zea larvae

When precocene II was fed to last instar larvae of Heliothis zea, it caused significant reductions in the calculated rate of growth, rate of nutrient assimilation, and conversion of ingested and digested food to body mass. No change in the rate of food consumption occurred but respiration was significantly higher. Transport of the nutrient [1-¹⁴C] linoleic acid across midgut tissue was hindered when larvae were fed precocence II. There was also a precocene-induced change in the apical (luminal) morphology of midgut cells, including absence of the glycocalyx and loss of the microvillar absorptive surface. The influence of precocene II on the midgut physiology and metabolic processes in last instar larvae of H. zea may account for the observed reduced growth and delayed development. © 1992 wiley-Liss, Inc.     

Identification of Acetobacter liquefaciens as Causal Agent of Pink-Disease of Pineapple Fruit

Two bacterial strains causing pink-disease of pineapple were identified as Acetobacter liquefaciens and compared with 8 other Acetobacter liquefaciens, 10 Gluconobacter oxydans and 7 Frateuria aurantia strains. The similarieties and differences between these bacteria are discussed.