Detection of IgE antibodies to a wide range of insect species in subjects with suspected inhalant allergies to insects

Sera obtained from subjects diagnosed on the basis of case history and skin tests as having inhalant allergies to insects, were tested for the presence of IgE antibodies to antigens from the house fly Musca domestica, a blowfly Calliphora stygia, the common clothes moth Tineola bisselliella, warehouse moth Ephestia cautella, cockroach Blattella germanica, carpet beetle Anthrenus verbasci and silverfish Ctenolepisma longicaudata. Approximately one-third of the sera reacted with extracts from all seven species, over half the sera reacted with four of the extracts and only 3 sera proved negative to all of the extracts. Twenty-six of the sera also contained IgE antibodies that reacted with the house dust mite Dermatophagoides farinae. Tests with a further eleven species of flies from the order Diptera and with extracts of the grain borer Rhyzopertha dominica, locust Chortoicetes terminifera and Bogong moth Agrotis infusa revealed strong IgE antibody responses to all of the species. Electroblotting studies revealed up to 15 IgE-binding components in extracts from 11 different insect species including 6 species of flies. Some IgE-binding insect components with similar electrophoretic mobilities indicated the possible presence of common allergens in extracts from different species. In particular, blots of each of the fly extracts showed a dense IgE-binding component of MW approximately 37,000 daltons. 'Pan allergy' to insects may occur in subjects who have been sensitized to one or a few insects and allergenic similarities may extend to at least some other noninsect members of the phylum Arthropoda.     

Persistence of hemoglobin allergenicity and antigenicity during metamorphosis of Chironomidae (Insecta: Diptera)

Chironomid hemoglobins are potent allergens. The allergenic and antigenic activities of these hemoglobins are studied with the help of RAST, RAST inhibition and double immunodiffusion. Human as well as rabbit antisera were used. It was shown that hemoglobins are the main antigenic/allergenic components in extracts of Camptochironomus tentans larvae. Furthermore, immunological cross-reactivity among larvae, pupae and adult midges of this species are shown to be due to the existence of hemoglobin antigenic determinants in all developmental stages of this insect.     

Comparison by electroblotting of IgE-binding components in extracts of house dust mite bodies and spent mite culture

Eight Dermatophagoides pteronyssinus extracts (three culture extracts, four mite body extracts, and the World Health Organization International Standard [IS]) were investigated by side-by-side sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by electrotransfer to nitrocellulose, and by probing with a pooled serum from mite-allergic subjects. Representative body and mite culture extracts were compared by probing with individual sera, and both types of extract were also compared by RAST-inhibition studies. Extracts from the same source differed in the molecular weight (MW) of some of their IgE-binding components. In general, most IgE-binding components in culture extracts and the IS were in the 14 to 35 kd MW region, whereas extracts from mite bodies and one culture extract contained more IgE-binding components of higher MW (35 to 110 kd). Comparison of representative mite body and culture extracts by use of 22 separate sera resolved 26 and 19 IgE-binding components in the two extracts, respectively. Patterns of RAST inhibition produced by both types of extracts when they were used either as the inhibitor or as the allergosorbent demonstrated qualitative differences between the two types of extracts. These results demonstrate that mite extracts may differ considerably in their allergenic composition and emphasize the need for standardization of mite allergenic extracts and the reexamination of the suitability of the D. pteronyssinus IS.     

Identification and characterization of allergens in two species of tuna fish.

BACKGROUND: In countries where fish consumption is high, allergenic reactions to fish are common among patients diagnosed with food hypersensitivity. For tuna fish, allergenic proteins are not known. In addition, it is not known how the tuna fish extracts should be processed to obtain optimal in vitro diagnostic performance and to preserve labile antigens. OBJECTIVE: The aim of this study was to characterize IgE-binding components of Yellowfin and Albacore tuna fish. METHODS: Various tuna fish extract preparations were fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose and analyzed by using tuna positive patients with different Western blot profiles. The functional activity of the extracts was evaluated by basophil histamine release. RESULTS: Immunoblot analysis showed the majority of patients responding to Yellowfin tuna extract. Inhibition studies using immunoblot analysis and histamine release testing showed a specific protein with a molecular weight of approximately 46 kD that is present in Yellowfin tuna, but absent in Albacore. Only defatted, lyophilized tuna fish extracts were able to induce histamine release from sensitized basophils although IgE-binding components were detected in fresh raw, fresh cooked, and canned tuna fish preparations. CONCLUSION: These studies indicate that patient sera may contain different tuna fish species IgE specific antibodies directed against unique species specific allergens present in Yellowfin and Albacore tuna fish. Possibly, extracts should contain specific allergenic components from both Albacore and Yellowfin to cover the epitope heterogeneity observed in sera from patients developing IgE antibodies against tuna fish.     

Oviposition preferences of two forensically important blow fly species, Chrysomya megacephala and C. rufifacies (Diptera: Calliphoridae), and implications for postmortem interval estimation

Necrophagous blow fly species (Diptera: Calliphoridae) are the most important agents for estimating the postmortem interval (PMI) in forensic entomology. Nevertheless, the oviposition preferences of blow flies may cause a bias of PMI estimations because of a delay or acceleration of egg laying. Chrysomya megacephala (F.) and C. rufifacies (Macquart) are two predominant necrophagous blow fly species in Taiwan. Their larvae undergo rather intense competition, and the latter one can prey on the former under natural conditions. To understand the oviposition preferences of these two species, a dual-choice device was used to test the choice of oviposition sites by females. Results showed when pork liver with and without larvae of C. rufifacies was provided, C. megacephala preferred to lay eggs on the liver without larvae. However, C. megacephala showed no preference when pork liver with and without conspecific larvae or larvae of Hemipyrellia ligurriens (Wiedemann) was provided. These results indicate that females of C. megacephala try to avoid laying eggs around larvae of facultatively predaceous species of C. rufifacies. However, C. rufifacies showed significant oviposition preference for pork liver with larvae of C. megacephala or conspecific ones when compared with pork liver with no larvae. These results probably imply that conspecific larvae or larvae of C. megacephala may potentially be alternative food resources for C. rufifacies, so that its females prefer to lay eggs in their presence. When considering the size of the oviposition media, pork livers of a relatively small size were obviously unfavorable to both species. This may be because females need to find sufficient resources to meet the food demands of their larvae. In another experiment, neither blow fly species showed an oviposition preference for pork livers of different stages of decay. In addition, the oviposition preferences of both species to those media with larvae were greatly disturbed in a dark environment. If we removed the larvae that had previously fed on the pork liver and let the females choose, no oviposition preference was observed; but both species still showed a preference for the larger media in the dark. This suggests that female blow flies can use visual cues to recognize larvae on the media and decide on their oviposition site. Our studies point out the effects of some biotic and abiotic factors which were previously overlooked, and remind us to reevaluate these effects on oviposition, especially when using insect developmental data to estimate PMIs.     

Identification of cross-reactive proteins amongst different Curvularia species

BACKGROUND: Curvularia lunata is an important inhalant allergen. The present study was undertaken to investigate the shared IgG- and IgE-binding components among seven Curvularia species prevalent in the aerospora. METHODS: Seven different Curvularia species were grown in a semisynthetic medium for 13 days. The extracts were analyzed by SDS-PAGE, immunoblot and ELISA/immunoblot inhibition using sera from C. lunata-positive patients and anti-C. lunata rabbit serum. RESULTS: Different Curvularia species showed 11-19 protein bands on SDS-PAGE. Proteins of 12, 20, 31, 45, 53, 78 and 97 kD were present in all the species. Eight out of 98 nasobronchial patients exhibited positive skin tests to C. lunata and to at least five Curvularia species. ELISA using these sera showed IgE binding with Curvularia species. Immunoblot using pooled anti-C. lunata sera from patients showed 5-12 allergenic proteins. Proteins of 12, 31, 45, 53 and 78 kD showed IgE binding in Curvularia species. Antibodies against C. lunata detected 6-14 antigenic proteins on immunoblot. Proteins of 31, 45 and 53 kD showed IgG binding in all the species. Proteins of 31 and 53 kD showed complete IgE/IgG binding inhibition. IgE/IgG ELISA inhibition showed dose-dependent inhibition in Curvularia species. C. lunata extract required 0.17 and 0.11 microg of protein for 50% IgE and IgG inhibition, respectively. C. clavata and C. pallescens required 10 times more protein to exhibit the same inhibition and other species required similar protein levels as those required by C. lunata. CONCLUSIONS: A high degree of cross-reactivity was observed between C. lunata and the six other Curvularia species tested. C. lunata and C. senegalensis shared maximum allergenic and antigenic components.     

The spectrum of low molecular weight house dust mite (Dermatophagoides pteronyssinus) allergens with emphasis on Der p II

Analysis by protein blotting of sera from 96 different house dust mite-allergic subjects revealed previously unrecognized complexity of low molecular weight (MW) (less than 20 kD) IgE-binding proteins in extracts of whole bodies of Dermatophagoides pteronyssinus. Of 11 different IgE-binding components of MW less than 20 kD identified, two (MW approximately 16 kD and approximately 15 kD), showed both a high frequency (88% and 49% respectively) and a high intensity of IgE-binding. The approximately 16 kD component, identified as allergen Der p II, showed the highest frequency of IgE antibody reactivity of any of the major D. pteronyssinus allergens including Der p I and Der p III.     

Shared allergenic activity in Asian (Blattella asahinai), German (Blattella germanica), American (Periplaneta americana), and Oriental (Blatta orientalis) cockroach species

Whole-body extracts of the feral and peridomestic Asian cockroach (Blattella asahinai) and the three domestic cockroach species, German (Blattella germanica), American (Periplaneta americana), and Oriental (Blatta orientalis), were compared allergenically using an IgE serum pool from 4 German cockroach sensitive individuals. In crossover radioallergosorbent inhibition analysis, the Asian cockroach shared allergenic activity primarily with the German cockroach polymer and to a lesser extent with either the American or Oriental cockroach polymers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thin-layer isoelectric focusing analysis of the extracts showed similar although varying intensities of Coomassie blue stained banding patterns among five extracts analyzed. Electroblotting analysis with 12.5% SDS-PAGE of the whole-body German cockroach extract and IgE serum from individuals sensitive to German cockroach revealed eight allergenic components with apparent molecular weights of 92, 80, 67, 48, 36, 27, 25 and 18 kD. Five components could be identified in the whole-body extract of the Asian cockroach corresponding to apparent molecular weights of 92, 67, 48, 40, and 32 kD. Analysis of individual serum by immunoblot analysis with each of the cockroach extracts showed considerable heterogenicity in the IgE-binding pattern. Although the Asian cockroach demonstrated considerable cross-reacting allergenic components to German and relatively fewer cross-reacting allergenic components to either the Oriental or American, it is too early to establish genus- or species-specific cockroach allergens. It is important to point out that German cockroach sensitive individuals should be made aware of the potential exposure of Asian cockroach aeroallergens both indoors and outdoors in areas with high infestations of Asian cockroaches.     

Occupational sensitivity to Tenebrio molitor Linnaeus (yellow mealworm)

Tenebrio molitor is an abundant stored-grain pest in the northern United States. We evaluated an individual with work-related symptoms of rhinoconjunctivitis on exposure to this insect. Prick skin tests with extracts prepared from the larval, pupal, and adult-life stages were positive for the patient and for another individual with allergy to a closely related species of beetle, Alphitobius diaperinus. Specific IgE antibodies to the extracts were demonstrated by RAST. RAST inhibition demonstrated immunologic cross-reactivity between the life stages of T. molitor and also between T. molitor and A. diaperinus, as well as slight cross-reactivity with blowfly. The proteins in the extracts of each life stage were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 15 protein bands were detected in each of the extracts, although the patterns of separation were different for each life stage. After immunoblotting and autoradiography, six different IgE-binding proteins were identified in the larval extract, five in the pupal extract, and seven in the adult extract, with similar IgE-binding patterns noted for the larval and adult extracts. We conclude that this patient developed IgE-mediated sensitivity to T. molitor antigens as the result of occupational exposure. This study confirms the fact that beetles of the Tenebrionid family are potentially significant allergens for workers exposed to grains or grain products.     

Effects of Psychodiella sergenti (Apicomplexa, Eugregarinorida) on its natural host Phlebotomus sergenti (Diptera, Psychodidae)

Phlebotomine sand flies (Diptera, Psychodidae) are important vectors of human pathogens. Moreover, they possess monoxenous parasites, including gregarines of the genus Psychodiella Votypka, Lantova, and Volf, which can negatively affect laboratory-reared colonies, and have been considered as potential candidates in biological control. In this study, effects of the gregarine Psychodiella sergenti Lantova, Volf, and Votypka on its natural host Phlebotomus sergenti Parrot were evaluated. The gregarines increased the mortality of immature sand fly stages, and this effect was even more apparent when the infected larvae were reared in more dense conditions. Similarly, the gregarines negatively affected the survival of adult males and females. However, no impact was observed on the mortality of blood-fed females, the proportion of females that laid eggs, and the number of eggs oviposited. The 10-times higher infection dose (50 versus five gregarine oocysts per one sand fly egg) led to -10 times more gamonts in fourth-instar larvae and two or three times more gamonts in females and males, respectively. Our study clearly shows that Ps. sergenti is harmful to its natural host under laboratory conditions. However, its potential for use in biological control is questionable as a result of several factors, including this parasite's strict host specificity.     

Purification and partial characterization ofa 67-kD cross-react ive allergen from Imperata cylindrica pollen extract

BACKGROUND: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85-16 kD. OBJECTIVES: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. METHODOLOGY: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. RESULTS: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. CONCLUSION: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.     

Allergenic cross-reactivity between the nematode Anisakis simplex and the dust mites Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, and Dermatophagoides pteronyssinus

Background: The nematode Anisakis simplex is a common parasite on fish and other seafood. It is considered to be a food allergen and to induce IgE-mediated reactions. Allergenic cross-reactivity between A. simplex and other nematodes has been reported, as has cross-reactivity with arthropods: red mosquito larvae and German cockroach. We have here studied the allergenic relationship between A. simplex and four different dust-mite species.
Methods: Serum samples collected from 69 farmers allergic to dust mites were analyzed for IgE to A. simplex by CAP FEIA. Allergenic cross-reactivity between A. simplex and dust mites was studied in two of the sera by CAP FEIA and immunoblotting inhibition.
Results: We found that 14/69 farmers had detectable levels of IgE antibodies to A. simplex . The IgE response in CAP FEIA to A. simplex was inhibited to various degrees in the two studied sera by extracts of the dust mites Acarus siro , Lepidoglyphus destructor , Tyrophagus putrescentiae , and Dermatophagoides pteronyssinus . In the reverse inhibition experiment, extract of A. simplex inhibited the response in both sera to A. siro and T. putrescentiae , but not to L. destructor . The IgE binding to D. pteronyssinus was inhibited in one of the two sera. In blotting inhibition experiments, the IgE binding to several allergens in A. simplex was inhibited by each of the four mite extracts, especially by A. siro and T. putrescentiae , which completely inhibited the IgE binding to several allergens.
Conclusions: The results show allergenic cross-reactivity between several allergens in A. simplex and four dust-mite species. The clinical significance of this cross-reactivity remains to be evaluated.     

Molecular cloning and characterization of hazel pollen protein (70 kD) as a luminal binding protein (BiP): a novel cross-reactive plant allergen

BACKGROUND: Tree pollen contains many allergens showing cross-reactivity to proteins from pollen, seeds, and fruits of different plant species. Amongst Fagales, responsible for several allergenic responses, hazel provides the best material to study pollen as well as food allergens in one species. The aim of this study was to identify and characterize the physiological function of an allergen from hazel pollen and to determine possible cross-reactivity to proteins from hazelnut. METHODS: Monoclonal antibodies (mAbs) against hazel pollen crude extract were produced. On the basis of IgE binding, demonstrated by sera from patients allergic to hazel pollen, one mAb indicating the best correlation has been selected, and the putative allergen was purified by preparative gel electrophoresis. Isoforms were investigated by two-dimensional PAGE, and for molecular identification a hazel pollen cDNA library was constructed. In situ localization of the allergen during pollen development was performed by immunofluorescence labelling. RESULTS: Immunological staining of crude hazel pollen extract with specific IgE and mAb revealed a 70-kD protein. Immunoblot studies with mAb showed cross-reactive proteins of 70-72 kD in different plant tissues and species. After protein purification, the IgE-binding reactivity of the allergen has been reconfirmed, and two isoforms were detected. Molecular cloning identified the allergen as a luminal binding protein (BiP) of the Hsp70 family with 88-92% sequence identity in various plants. Further immunocytological studies indicated involvement of BiP during pollen development. CONCLUSIONS: Chaperons like BiP play an important role in protein synthesis and in the protection of cellular structures during stress-related processes. Because of their highly conserved protein sequences, we propose that such allergens could be responsible for at least a part of the allergenic cross-reactivity between proteins from different pollens and plant foods.     

Dewatered sewage biosolids provide a productive larval habitat for stable flies and house flies (Diptera: Muscidae)

Species diversity and seasonal abundance of muscoid flies (Diptera: Muscidae) developing in biosolid cake (dewatered biosolids) stored at a wastewater treatment facility in northeastern Kansas were evaluated. Emergence traps were deployed 19 May through 20 October 2009 (22 wk) and 27 May through 18 November 2010 (25 wk). In total, 11,349 muscoid flies were collected emerging from the biosolid cake. Stable flies (Stomoxys calcitrans (L.)) and house flies (Musca domestica (L.)), represented 80 and 18% of the muscoid flies, respectively. An estimated 550 stable flies and 220 house flies per square-meter of surface area developed in the biosolid cake annually producing 450,000 stable flies and 175,000 house flies. Stable fly emergence was seasonally bimodal with a primary peak in mid-July and a secondary peak in late August. House fly emergence peaked with the first stable fly emergence peak and then declined gradually for the remainder of the year. House flies tended to emerge from the biosolid cake sooner after its deposition than did stable flies. In addition, house fly emergence was concentrated around midsummer whereas stable fly emergence began earlier in the spring and continued later into the fall. Biosolid age and temperature were the most important parameters affecting emergence for house flies and stable flies, whereas precipitation was not important for either species. This study highlights the importance of biosolid cake as a larval developmental habitat for stable flies and house flies.     

Identification and characterization of Parietaria judaica allergens

Allergens of Parietaria judaica pollen extract have been identified and characterized biochemically. Two main allergenic components, A1 and A2, have been found by crossed-radioimmunoelectrophoresis and demonstrated to be spread in a wide range of pH. Immunoblotting studies revealed that at least eight SDS-denatured polypeptides show IgE-binding activity. The one exhibiting the highest allergenic activity, named Pj10 (MW 10,000 daltons) was found in all the fractions when the pollen extract was fractionated by chromatofocusing. Bidimensional electrophoretic analysis suggested that Pj10 either can form homopolymeric proteins of different molecular weights or can be associated to a number of proteins by disulfide bridges. Furthermore, Pj10 is the main molecular structure with IgE-binding activity in the two allergenic components A1 and A2 defined by immunological criteria.     

Allergenicity and cross-reactivity of cat and dog allergenic extracts

This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophore-tic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. Binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized. If these observations confirmed that CAT-1 is a major allergen in cat-sensitive subjects, they also showed a different profile of reactivity in patients sensitive to cats only and those sensitive to both cats and dogs, which could have diagnostic and therapeutic implications.     

The first documented forensic entomology case in Thailand

The forensic entomology case described herein is the first such case documented in Thailand. A mummified corpse of a 32-yr-old man was discovered in a forested habitat, with the larvae of six species of flies (Diptera) found in association with the corpse at the time of its discovery, i.e., those of Hydrotaea (=Ophyra) spinigera Stein (family Muscidae), Piophila casei (L.) (family Piophilidae), Megaselia scalaris (Loew) (family Phoridae), Sagus sp. (family Stratiomyidae), and larvae of two unidenitified flesh fly species (family Sarcophagidae). The presence and age of the larval specimens of P. casei, M. scalaris, and H. spinigera gave entomological evidence that the postmortem interval for the corpse was 3-6 mo. This report also documets some of the forensically important fly species that occur in Thailand.     

Persistence of Escherichia coli in immature house fly and stable fly (Diptera: Muscidae) in relation to larval growth and survival

The persistence of Escherichia coli in artificially fed larvae was examined for up to 48 h after ingestion by house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). The rate of change in the E. coli load was similar for both species for up to 5 h after ingestion. Up to 48 h after ingestion, abundance of E. coli declined in immature house flies but remained constant in immature stable flies. When different E. coli concentrations were fed to larvae, the abundance of E. coli increased in stable fly larvae regardless of the initial concentration. The E. coli load in house fly larvae increased when larvae were fed a low concentration of bacteria, but it declined when larvae were fed a high concentration of bacteria. Survival of house fly and stable fly larvae averaged 62 and 25%, respectively, when reared on pure E. coli cultures. These observations suggest that house fly larvae digest E. coli and use it as a food source but stable fly larvae do not.     

Laboratory evaluation of novaluron as a development site treatment for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae)

A granular formulation of novaluron (Novaluron 0.2G, 0.2% [AI]), a newer benzoylphenyl urea insecticide, was evaluated for its efficacy in controlling the larval stage of horn flies, Haematobia irritans (L.); house flies, Musca domestica L.; and stable flies, Stomoxys calcitrans (L.), in cow manure. Various rates and insecticide placement locations (top, middle, and bottom of manure) were evaluated in this study and all combinations of these variables reduced adult emergence of all three species when compared with the untreated controls. The presence of deformed pupae indicated that novaluron had an insect growth regulator effect on the developing fly larvae. Top, middle, or bottom application rates of 0.125, 0.195, 0.25, and 0.375 g novaluron onto manure samples, reduced adult horn fly emergence by > 90%. Middle and bottom application rates of 0.195, 0.25, and 0.375 g novaluron reduced adult house fly emergence >93%. All rates and placement combinations resulted in >98% reduction of adult stable fly emergence. The level of control efficacy observed against these three fly species along with the ease of use of a granular formulation, make this product an ideal candidate for use in an integrated livestock pest management program.