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Immature House Fly (Musca domestica) Control in BreedingSites With a New Brevibacillus laterosporus FormulationAuthor(s) Luca Ruiu Alberto Satta Ignazio FlorisSource Environmental Entomology 37(2)505-509 2008Published By Entomological Society of AmericaDOI httpdxdoiorg1016030046-225X(2008)37[505IHFMDC]20CO2URL httpwwwbiooneorgdoifull1016030046-225X28200829375B5053AIHFMDC5D20CO3B2

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BIOLOGICAL CONTROLETHMICROBIALS

Immature House Fly (Musca domestica) Control in Breeding SitesWith a New Brevibacillus laterosporus Formulation

LUCA RUIU ALBERTO SATTA AND IGNAZIO FLORIS1

Dipartimento di Protezione delle Piante Sezione di Entomologia AgrariaUniversita di Sassari via E De Nicola 07100 Sassari Italy

Environ Entomol 37(2) 505ETH509 (2008)

ABSTRACT A bacterial formulation containing spores of a Brevibacillus laterosporus strain fromSardinia known to be toxic by ingestion to the house szligy (Musca domestica) was assayed in laboratoryoutdoor and THORNeld conditions for the control of immature stages of this pest In all laboratory assaysthe bacterial formulation exhibited toxicity against house szligy larvae A concentration of 1 108 sporesofB laterosporusg of diet caused 100 mortality of house szligy immature stages The same formulationapplied at a concentration of 1 108 sporesml equivalent to a dose of 2 litersm2 caused a reductionin adult emergence from laboratory and natural breeding substrates (outdoor cage experiments) upto 803 and 578 respectively Similarly this formulation applied in the cow pen of a diary farm ata dose of 2 litersm2 produced a signiTHORNcant reduction (30) in immature szligy development Thereforethe use of this bacterial preparation in microbiological control strategies for the integrated pestmanagement of this species is promising

KEY WORDS microbial insecticide biological management entomopathogenic bacteriumtreatments Diptera

The house szligy Musca domestica L (Diptera Musci-dae) is the most common szligy pest of livestock Thisinsect is of particular medical and veterinary interestbecause of its importance as a nuisance and in theepidemiology of enteric diseases (Cohen et al 1991)Because of its high reproductive potential the houseszligy control is almost always necessary in diary farmswhere integrated pest management (IPM) programsinvolve several methods such as source reduction andkilling or excluding adults with barriers (Drummondet al 1988)

Because the house szligy completes its life cycle veryquickly in summer wet manure should be removedfrequently on livestock farms In addition insecticidalformulations for control of house szligy larvae can beeither sprayed directly into infested breeding mediaor included in animal feeds as additives that passthrough the intestinal tract and release insecticidalresidues in the feces (Moon 2002) House szligy adultmanagement is mainly based on the use of variouschemical insecticidal formulations but traps andsugar- and pheromone-based insecticidal baits arealso used

Because the use of chemical insecticides is associ-ated with ecological risks and increasing house szligyresistance alternative house szligy control strategies in-cluding the use of biological control organisms(Hogsette 1999) have been studied Many beneTHORNcial

organisms (eg predators parasites and natural com-petitors) commonly occur in the breeding media ofmuscid szligies resulting in their natural biological con-trol (Rueda and Axtell 1985) Moreover certain spe-cies of parasitoid wasps especially Pteromalidae canalso be released to augment their natural populations(Rutz and Axtell 1981 Petersen et al 1983)

Biological control can also be based on the use ofmicrobial agents including entomopathogenic bacte-ria (Glare and OOtildeCallaghan 2000) The successful mi-crobiological control of many injurious insects usingBacillus thuringiensis (Berliner) suggests that the de-velopment of an appropriate formulation of B thurin-giensisor of another related bacterial species effectiveagainst the house szligy might be beneTHORNcial The toxicityof B thuringiensis variety thuringiensis strains againstthe house szligy shown in the past (Hall and Arakawa1959 Briggs 1960) was mainly caused by the presenceof the -exotoxin they produced In fact various in-secticides based on B thuringiensis variety thuringien-sis had been developed and used in Europe for houseszligy control until the prohibition of the use of -exo-toxin in agriculture (Glare and OOtildeCallaghan 2000)More recently several studies have shown the toxicityof -endotoxins from various B thuringiensis strainsagainst M domestica (Indrasith et al 1992 Hodgmanet al 1993 Lonc et al 1997 Johnson et al 1998 Zhonget al 2000)

In a recent laboratory study performed in Sardinia(Italy) a new strain of the entomopathogenic bacte-1 Corresponding author e-mail iszligorisunissit

0046-225X080505ETH0509$04000 2008 Entomological Society of America

riumBrevibacillus laterosporus (Laubach) was discov-ered It showed high toxicity against both house szligylarvae and adults having lethal and sublethal effects onthis pest (Ruiu et al 2006)B laterosporus is an aerobicspore-forming bacterium characterized by a canoe-shaped lamellar body attached to one side of its sporeIt is classiTHORNed as a nonpathogenic agent to humans andits potential use for insect biological control espe-cially against mosquitoes (Favret and Yousten 1985Orlova et al 1998) has been considered recently(Oliveira et al 2004)

To our knowledge there is no information availableon the use of B laterosporus formulations for the con-trol of Diptera in THORNeld conditions The aim of this studywas to evaluate the efTHORNcacy of a formulation based onthe new B laterosporus strain isolated in Sardiniaagainst the house szligy in laboratory assays outdoor cageexperiments and THORNeld trials performed in a diary farm

Materials and Methods

House Fly The rearing of the houseszligies (M do-mestica) used in the laboratory bioassays and in theoutdoor cage experiment was set up with pupae fromthe Entomology Institute of the University of Milan(Italy) and was performed at 25 1C in a laboratoryof the Department of Plant Protection of the Univer-sity of Sassari Adults were reared in Plexiglas cages(30 by 30 by 30 cm) where water and a milk powder-sucrose mixture (11) were provided ad libitum asfood Larvae were grown on an artiTHORNcial diet made ofwheat bran (34) milk powder (1) benzoic acid(01) and water (649) (wtwt) (Ruiu et al 2006)Brevibacillus laterosporus Formulation A formu-

lation of the Sardinian strain of B laterosporus wasproduced by e-nema (Raisdorf Germany) Bacteriawere grown in a 500-ml reactor and the whole sporu-lated culture was transformed into a water suspensionand subjected to gamma-irradiation to kill all sporesThe THORNnal suspension which had a concentration of109 spores of B laterosporusg was diluted withsterilized milliQ water to obtain the different concen-trations used in the bioassays Spore counts were rou-tinely performed under a microscope using a Thomachamber (E Hartnack Berlin Germany) after a briefsonication of the suspension to break any clumpsLaboratory Bioassays Preliminary laboratory ex-

periments were conducted to study the effects ofdifferent bacterial concentrations and to assay somemethods for the incorporation of the formulation intolarval breeding media

In the THORNrst experiment the suspension of B lateros-porus was diluted with sterilized milliQ water beforebeing mixed with the diet to obtain the following THORNnalconcentrations 2 108 1 108 and 05 108 sporesgof diet Groups of 10 THORNrst-instar larvae ofM domesticawere kept in petri dishes (35 cm diameter) and fed25 g of the above described larval diets containing theB laterosporus formulation Larvae were incubated at25 1C and 99 of relative humidity Control szligieswere reared on a diet with no B laterosporus (Ruiu etal 2006) The experiment was replicated four times

with separate szligy cohorts Larval mortality was as-sessed after 5 d and pupae were saved to record adultemergence

To simulate a treatment on the surface of a naturalbreeding substrate in a second experiment the for-mulation was simply sprinkled on the surface insteadof being mixed homogeneously with the diet Thebacterial concentration chosen for the treatments wasbased on the results of the previous experiment whereconcentrations higher than 1 108 sporesg of dietcaused 100 immature mortality One thousand houseszligy eggs estimated with a graduated pipette tube(Moreland and McLeod 1956) were placed into a cy-lindrical jar (15 cm diameter and 20 cm high) con-taining 1 kg of the rearing larval diet and kept in thedark at 25 1C The next day when larvae were inthe THORNrst instar 35 ml of the bacterial formulation at aconcentration of 1 108 sporesml corresponding toa dose of 2 litersm2 were sprinkled on the surface ofthe larval rearing substrate The controls were simi-larly treated using an equivalent quantity of waterinstead This experiment was replicated four timesAfter a week pupae were collected and emergingadults were surveyedOutdoor Cage Experiment This trial was con-

ducted outdoors inside cages (50 by 30 by 60 cm)consisting of a polystyrene container (50 by 30 by 10cm) at the bottom and a frame (50 cm high) coveredwith gauze After THORNlling the container with 10 kg offresh cattle manure the frame was assembled On theTHORNrst day 100 1-wk-old szligies (50 males and 50 females)obtained from the laboratory rearing were collectedby an aspirator and released into each cage In theseconditions szligies were allowed to feed and lay eggs onthe manure

An aliquot of 300 ml of the B laterosporus formu-lation at a concentration of 1 108 sporesml wasapplied to the surface of the manure by a sprinklingcan The dose of this treatment corresponded to 2litersm2 The control was similarly sprinkled with thesame quantity of water

Every 3 d the cages were opened to allow the szligiesto escape the treatments were repeated and 100 newszligies were released inside each cage again as previ-ously described A total of four successive 3-d treat-ments were carried out Three days after the lasttreatment all cages were opened and the frames werethrown away After that each container was coveredby a polystyrene cap and two openings (5 cm diam-eter) were made on a lateral face of the container Thisallowed the emerging szligies to go toward the light andto be collected in plastic transparent cylinders andcounted during the following days Four weeks laterthe containers were opened to verify the presence ofszligies not previously counted in the plastic cylinders

During this experiment the cages were always pro-tected from the wind and the rain maximum air tem-perature varied between 20 and 31C and minimumair temperature varied between 14 and 20C

The experiment was replicated three timesField Experiment Field experiments were con-

ducted in a central-western area of Sardinia (Arborea

506 ENVIRONMENTAL ENTOMOLOGY Vol 37 no 2

Province of Oristano) where dairy farms are abun-dant and the economy is mainly based on milk pro-duction and agriculture The diary farm selected forexperiments contained 100 head and had a cowshedof 2000 m2 of which only a small part was undercover The farms had a dunghill from which manurewas periodically removed In preliminary observa-tions the cow pen and the dunghill were identiTHORNed asthe main developmental sites for immature house szligies

Immature house szligy density on the farm was mon-itored from the beginning of July until the end ofAugust 2003 using modiTHORNed sampling methods of Loo-mis et al (1968) and Skoda et al (1996) A manufac-tured 05-liter standard metal core sampler was used totake eight random subsamples of manure which weremixed together tomakea larger sampleof4 litersEach4-liter sample was replicated three times and newsamples were collected every week The number ofadults that emerged from these manure samples wasused to monitor the abundance of immature szligies

To allow immature house szligy development manurewas incubated in cylindrical plastic containers (20 cmdiameter by 25 cm high) that had an opening on theirpolystyrene cap to permit the emerging szligies to gotoward the light and to be collected in a plastic trans-parent cylinder Plastic containers were left in thefarm and transparent cylinders were inspected for szligyidentiTHORNcation and counting Three weeks later con-tainers were opened to verify the presence of szligies notpreviously counted in the transparent cylinders

Areas (three treated and three control) of 9 m2

within the cow pen were randomized over six plotsthat were previously selected for homogeneity of thebreeding sites Samples of 4 liters of manure werecollected weekly from each area as described abovefor szligy monitoring

A total of THORNve applications using a B laterosporusformulation at a concentration of 1 108 sporesmland a dose of 2 litersm2 was applied at the followingdates 28 July and 1 5 9 and 13 August 2003 Theformulation was distributed on the surface of the cowpen by a sprinkling can The control areas were sim-ilarly treated using only water at the same doseStatistical AnalysisData on larvae (THORNrst experiment

on immature stages) in preliminary laboratory bioas-says were subjected to analysis of variance (ANOVAone factor design concentration) followed by a leastsigniTHORNcant difference (LSD) test to separate treat-ment means

The use of t-tests served to compare data means oftreated and control groups of the preliminary labora-tory bioassays of larvae (second experiment) the out-door cage experiment and the treatments against im-mature stages in the diary farm

In all experiments data met the assumption of ho-mogeneity of variance All statistical analyses wereperformed using Statgraphics Plus software (Stat-graphics Plus 2001)

Results

Laboratory Tests The toxicity of the diets contain-ing B laterosporus formulation against the house szligylarvae was concentration-dependent (Table 1) In theTHORNrst experiment the bacterial concentration signiTHORN-cantly inszliguenced the mortality percentages for larval(F312 8192 P 00001) and larval pupal (F312 1047P 00001) stages Despite the natural mortalityoccurred in the control (10 for larval stage and 20for larval pupal stage) which is related to the rear-ing diet (Floris et al 2004) a concentration of 1 108

sporesg of diet was high enough to cause a highlysigniTHORNcant 100 mortality of immature stages andconsequently no adult emergence

In the second experiment (Table 2) B laterosporustreatment on the surface of the laboratory breedingsubstrate signiTHORNcantly increased larval (t 15516df 6 P 0001) and pupal (t 4037 df 6 P0007) mortality and signiTHORNcantly reduced the numberof pupated insects (t 15516 df 6 P 0001) andemerged adults (t 14212 df 6 P 0001) Con-sequently total mortality percentage (t 14212df 6 P 0001) signiTHORNcantly increased reaching avalue of 803Outdoor Cage Experiment Repeated superTHORNcial

treatments using the B laterosporus formulation at aconcentration of 1 108 sporesml on manure causeda signiTHORNcant reduction in immature development abil-

Table 1 Percentage (mean SEM) of larval and pupal mor-tality of housefly after rearing first-instar larvae on a diet containingdifferent concentrations of a B laterosporus formulation

B laterosporus concentration(sporesg of diet)

Larval mortalitya

()Total mortalityb

()

20 108 1000 000a 100 000a10 108 925 479a 100 000a05 108 650 645b 875 629bControl 100 408c 200 408c

Means within the same column followed by different letters aresigniTHORNcantly different (one-way ANOVA followed by LSD test P 001)a Recorded after 5 dbDifference between the no larvae at the beginning of the exper-

iment and the emerged adults expressed in percentage

Table 2 Number (mean SEM) of flies that developed from eggs on substrate treated with a B laterosporus formulation at theconcentration of 1 108 sporesml and a corresponding dose of 2 litersm2 (n 1000)

TreatmentTotal pupae

obtainedLarval mortality

()Emerging

adultsPupal mortality

()Total mortality

()

Treated 2733 325 727 33 1970 288 287 42 803 29Control 8258 146 174 15 7528 264 89 25 247 26

Means within each column are signiTHORNcantly different (t-test)

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 507

ity The mean number SEM of adults that emergedfrom manure was 947 207 in the treated groupversus 2250 356 in the control group This corre-sponded to a general reduction in immature develop-ment of 578 The mean number of emerged adultsin the treated and control groups were statisticallydifferent from each other (t 31678 df 4 P 0034)Field Experiments Results on the effects of the B

laterosporus treatments in the cow pen of the dairyfarm are summarized in Table 3 No signiTHORNcant dif-ferences in immature stage abundance were noticedbefore treatmentsbetween the treatedand thecontrolarea (t 0141 df 4 P 0894) Successivelytreatments with the bacterial formulation signiTHORNcantlyreduced the immature szligy density in the treated areacompared with the control area (t 3317 df 4 P0029) Finally B laterosporus treatments caused adecrease of 30 in immature house szligy density

Discussion

The B laterosporus formulation containing gamma-irradiated spores of a new Sardinian strain exhibited atoxicity against house szligy larvae that was comparableto that caused by spore suspensions prepared in lab-oratory (Ruiu et al 2006) The bacterial formulationwhen used at a concentration of 1 108 sporesliterand a dose corresponding to 2 litersm2 was able to killlarvae and reduce adult emergence from laboratorylarval medium The same treatment also caused a sig-niTHORNcant reduction in szligy emergence from manure inoutdoor cage experiments that reproduced a naturalszligy habitat Similarly the B laterosporus formulationwas able to produce a signiTHORNcant reduction in imma-ture szligy development when repeated treatments werecarried out in the cow pen of a diary farm Theseresults are in line with those reported in the past byHarvey and Brethour (1960) who mixed formulationscontaining spores of B thuringiensis variety thurin-giensis with cattle feces Although a complete inhibi-tion of larval development at a higher dose was ob-tained this effect was likely to be related to theproduction of -exotoxin by living bacteria reproduc-ing in manure (Jespersen and Keiding 1990 Glare and

OOtildeCallaghan 2000) In contrast in our case spores oftheB laterosporus strain were previously killed so thatmortality was caused only by the presence of toxins inthe spore and the canoe-shaped parasporal body(CSPB) structure (Ruiu et al 2007a)

The newB laterosporus spore formulation was moreeffective when sprinkled on a synthetic medium (lab-oratory larval diet) than on a natural breeding sub-strate (manure) Similarly a greater treatment effec-tiveness in artiTHORNcial media than manure was alsoobserved by Jespersen and Keiding (1990) Moreoverbecause a liquid formulation sprinkled on the surfacehas some difTHORNculty reaching the larvae in the manuremass the efTHORNcacy of the treatments was probablylimited to the superTHORNcial layers where the presence oflarvae was noted Enhancement of these effects mightbe achieved with a more uniform mixing of spores andmanure such as that done by Borgatti and Guyer(1963) feeding B thuringiensis to animals which al-lowed spores to go through their digestive tract and bereleased with feces Similar effects of B thuringiensisfor microbial house szligy control have more recentlybeen obtained by Labib and Rady (2001)

Everything considered the efTHORNcacy of the treat-ments on the manure against the house szligy is encour-aging and provides an important contribution to theprospects of house szligy control in natural breedingplaces In addition the sublethal effects of this micro-bial agent on immature (ie development time) andadult (ie fecundity longevity) house szligies should betaken into account to better evaluate its potential(Ruiu et al 2006)

For these reasons the method assayed in this re-search represents a THORNrst approach to a successful useof B laterosporus for the control of house szligy in diaryfarms However the observed reduction in the effec-tivenessofB laterosporus fromlaboratory toTHORNeld trialssuggests that more studies covering a much largertreated area are needed to THORNnd more suitable methodsof application of this type of formulation In additiona feed additive containingB laterosporus could also bestudied as proposed for B thuringiensis variety thu-ringiensis to control immature szligy development onmanure (Dunn 1960 Smirnoff and MacLeod 1961)

Finally in relation to its compatibility with naturallyoccurring biological control agents such as hymenop-teran parasitoids (Ruiu et al 2007b) the new B lat-erosporus formulation tested in this study has a po-tential application in biological control and integratedpest management programs of this important pest

Acknowledgments

This study was THORNnancially supported by the Italian Min-istero Universita Ricerca ScientiTHORNca (Research Program Bio-tecnologie innovative per il controllo di insetti nocivi medi-ante lOtildeimpiego di agenti microbiologici coordinator ProfIgnazio Floris University of Sassari Italy) and the Fondazi-one Banco di Sardegna We thank AHD Francesconi forassistance

Table 3 Number (mean SEM) of emerged houseflies fromstandard 4-liter manure samples in treated and control areasbefore and after treatment with a B laterosporus formulation at aconcentration of 1 108 sporesml and a dose of 2 litersm2

Treatment

No emerged houseszligies

Beforetreatmenta

Aftertreatmentb

Treated 1270 87a 783 125bControl 1287 184a 1100 108a

Means within each column followed by different letters are sig-niTHORNcantly different (t-test)a Values were calculated as means of the monitoring data of a 4-wk

period before treatmentb Values were calculated as means of the monitoring data of a 4-wk

period after treatment

508 ENVIRONMENTAL ENTOMOLOGY Vol 37 no 2

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Briggs J D 1960 Reduction of adult house-szligy emergenceby the effects of Bacillus spp on the development ofimmature forms J Insect Pathol 2 418ETH432

Cohen D M Green C Block R Slepon R Ambar S SWasserman and M M Lavine 1991 Reduction oftransmission of shigellosis by control of houseszligies (Muscadomestica) Lancet 337 993ETH997

Drummond R O J E George and S E Kunz 1988 Con-trol of arthropod pests of livestock a review of technol-ogy CRC Boca Raton FL

Dunn P H 1960 Control of house szligies in bovine faeces bya feed additive containing Bacillus thuringiensis var thu-ringiensis Berliner J Insect Pathol 213ETH16

Favret E M and A A Yousten 1985 Insecticidal activityof Bacillus laterosporus J Invertebr Pathol 45 195ETH203

Floris I L Ruiu and A Satta 2004 Inszliguenza della qualitadi mosche domestiche allevate in laboratorio sugli effettidi Bacillus thuringiensis Berliner Proceedings of the XIXCongresso Nazionale Italiano di Entomologia 10ETH15 June2002 Catania Italy

Glare T R and M OrsquoCallaghan 2000 Bacillus thuringien-sisbiology ecology and safety Wiley Chichester UnitedKingdom

Hall IM andKYArakawa 1959 Thesusceptibilityof thehouse szligy Musca domestica Linnaeus to Bacillus thurin-giensis var thuringiensis Berliner J Insect Pathol 1 351ETH355

Harvey T L and J R Brethour 1960 Feed additives forcontrol of house szligy larvae in livestock feces J EconEntomol 53 774ETH776

Hodgman T C Y Ziniu SMing T Sawyer CM Nichollsand D J Ellar 1993 Characterization of a Bacillus thu-ringiensis strain which is toxic to the house szligy Muscadomestica FEMS Microbiol Lett 11417ETH22

Hogsette J A 1999 Management of ectoparasites with bi-ological control organisms Internat J Parasitol 29 147ETH151

Indrasith L S N Suzuki KOgiwara S Asano andHHori1992 Activated insecticidal crystal proteins fromBacillusthuringiensis serovars killed adult house szligies Lett ApplMicrobiol 14 174ETH177

Jespersen J B and J Keiding 1990 The effect of Bacillusthuringiensis var thuringiensis onMusca domestica L lar-vae resistant to insecticide pp 215ETH229 InD A Rutz andR S Patterson (eds) Biocontrol of arthropods affectinglivestock and poultry Westview Press Boulder CO

Johnson C A H Bishop and C L Turner 1998 Isolationand activity of strains of Bacillus thuringiensis toxic tolarvae of the House szligy (Diptera Muscidae) and TropicalBlowszligies (DipteraCalliphoridae) J Invertebr Pathol71 138ETH144

Labib I M and M Rady 2001 Application of Bacillusthuringiensis in poultry houses as a biological controlagent against the house szligy Musca domestica sorbens JEgypt Soc Parasitol 31531ETH544

Lonc E M Lecadet T M Lachowicz and E Panek 1997Description of Bacillus thuringiensis wratislaviensis (H-47) a new serotype originating from Wroclaw (Poland)and other Bt soil isolates from the same area Lett ApplMicrobiol 24 467ETH473

Loomis E C A S Deal and W R Bowen 1968 Therelative effectiveness of cumaphos as a poultry feed ad-ditive to control synanthropic szligy larvae in manure JEcon Entomol 61 904ETH908

Moon R D 2002 Muscid szligies pp 279ETH301 In G Mullenand L Durden (eds) Medical and veterinary entomol-ogy Academic London United Kingdom

Moreland C R and W S McLeod 1956 House szligy egg-measuring techniques J Econ Entomol 49 49ETH51

Oliveira E J L Rabinovitch R G Monnerat L K Passosand V Zahner 2004 Molecular characterization of Bre-vibacillus laterosporus and its potential use in biologicalcontrol Appl Environ Microbiol 70 6657ETH6664

Orlova M V T A Smirnova L A Ganushkina V YYacubovich andRRAzizbekyan 1998 Insecticidal ac-tivity of Bacillus laterosporus Appl Environ Microbiol642723ETH2725

Petersen J J J A Meyer D A Stage and P B Morgan1983 Evaluation of sequential releases of Spalangia en-dius (HymenopteraPteromalidae) for control of houseszligies and stable szligies (DipteraMuscidae) associated withconTHORNned livestock in Eastern Nebraska J Econ Entomol76 283ETH286

Rueda L M and R C Axtell 1985 Guide to commonspecies of pupal parasites (Hymenoptera Pteromalidae)of the house szligy and other muscoid szligies associated withpoultry and livestock manure North Carolina Agricul-tural Research Service North Carolina State UniversityRaleigh NC

Ruiu L G Delrio D J Ellar I Floris B Paglietti SRubino and A Satta 2006 Lethal and sublethal effectsof Brevibacillus laterosporus on the house szligy (Musca do-mestica) Entomol Exp Appl 118 137ETH144

Ruiu L I Floris A Satta and D J Ellar 2007a Toxicityof a Brevibacillus laterosporus strain lacking parasporalcrystals against Musca domestica and Aedes aegypti BiolControl 43136ETH143

Ruiu L A Satta and I Floris 2007b Susceptibility of thehouse szligy pupal parasitoid Muscidifurax raptor (Hyme-noptera Pteromalidae) to the entomopathogenic bacte-ria Bacillus thuringiensis and Brevibacillus laterosporusBiol Control 43 188ETH194

Rutz D A and R C Axtell 1981 House szligy (Musca do-mestica) control in broiler-breeder poultry houses bypupal parasites (Hymenoptera Pteromalidae) indige-nous parasite species and release of Muscidifurax raptorEnviron Entomol 10343ETH345

Skoda R S G D Thomas and J B Campbell 1996 Com-parison of core sampling and pupal traps for monitoringimmature stable szligies and house szligies (Diptera Muscidae)in beef feedlot pens J Econ Entomol 89 428ETH434

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Statgraphics Plus 2001 Statgraphics Plus userOtildes guide stan-dard ed Version 4 Manugistics Rockville MD

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Received for publication 31 May 2007 accepted 21 Septem-ber 2007

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 509

riumBrevibacillus laterosporus (Laubach) was discov-ered It showed high toxicity against both house szligylarvae and adults having lethal and sublethal effects onthis pest (Ruiu et al 2006)B laterosporus is an aerobicspore-forming bacterium characterized by a canoe-shaped lamellar body attached to one side of its sporeIt is classiTHORNed as a nonpathogenic agent to humans andits potential use for insect biological control espe-cially against mosquitoes (Favret and Yousten 1985Orlova et al 1998) has been considered recently(Oliveira et al 2004)

To our knowledge there is no information availableon the use of B laterosporus formulations for the con-trol of Diptera in THORNeld conditions The aim of this studywas to evaluate the efTHORNcacy of a formulation based onthe new B laterosporus strain isolated in Sardiniaagainst the house szligy in laboratory assays outdoor cageexperiments and THORNeld trials performed in a diary farm

Materials and Methods

House Fly The rearing of the houseszligies (M do-mestica) used in the laboratory bioassays and in theoutdoor cage experiment was set up with pupae fromthe Entomology Institute of the University of Milan(Italy) and was performed at 25 1C in a laboratoryof the Department of Plant Protection of the Univer-sity of Sassari Adults were reared in Plexiglas cages(30 by 30 by 30 cm) where water and a milk powder-sucrose mixture (11) were provided ad libitum asfood Larvae were grown on an artiTHORNcial diet made ofwheat bran (34) milk powder (1) benzoic acid(01) and water (649) (wtwt) (Ruiu et al 2006)Brevibacillus laterosporus Formulation A formu-

lation of the Sardinian strain of B laterosporus wasproduced by e-nema (Raisdorf Germany) Bacteriawere grown in a 500-ml reactor and the whole sporu-lated culture was transformed into a water suspensionand subjected to gamma-irradiation to kill all sporesThe THORNnal suspension which had a concentration of109 spores of B laterosporusg was diluted withsterilized milliQ water to obtain the different concen-trations used in the bioassays Spore counts were rou-tinely performed under a microscope using a Thomachamber (E Hartnack Berlin Germany) after a briefsonication of the suspension to break any clumpsLaboratory Bioassays Preliminary laboratory ex-

periments were conducted to study the effects ofdifferent bacterial concentrations and to assay somemethods for the incorporation of the formulation intolarval breeding media

In the THORNrst experiment the suspension of B lateros-porus was diluted with sterilized milliQ water beforebeing mixed with the diet to obtain the following THORNnalconcentrations 2 108 1 108 and 05 108 sporesgof diet Groups of 10 THORNrst-instar larvae ofM domesticawere kept in petri dishes (35 cm diameter) and fed25 g of the above described larval diets containing theB laterosporus formulation Larvae were incubated at25 1C and 99 of relative humidity Control szligieswere reared on a diet with no B laterosporus (Ruiu etal 2006) The experiment was replicated four times

with separate szligy cohorts Larval mortality was as-sessed after 5 d and pupae were saved to record adultemergence

To simulate a treatment on the surface of a naturalbreeding substrate in a second experiment the for-mulation was simply sprinkled on the surface insteadof being mixed homogeneously with the diet Thebacterial concentration chosen for the treatments wasbased on the results of the previous experiment whereconcentrations higher than 1 108 sporesg of dietcaused 100 immature mortality One thousand houseszligy eggs estimated with a graduated pipette tube(Moreland and McLeod 1956) were placed into a cy-lindrical jar (15 cm diameter and 20 cm high) con-taining 1 kg of the rearing larval diet and kept in thedark at 25 1C The next day when larvae were inthe THORNrst instar 35 ml of the bacterial formulation at aconcentration of 1 108 sporesml corresponding toa dose of 2 litersm2 were sprinkled on the surface ofthe larval rearing substrate The controls were simi-larly treated using an equivalent quantity of waterinstead This experiment was replicated four timesAfter a week pupae were collected and emergingadults were surveyedOutdoor Cage Experiment This trial was con-

ducted outdoors inside cages (50 by 30 by 60 cm)consisting of a polystyrene container (50 by 30 by 10cm) at the bottom and a frame (50 cm high) coveredwith gauze After THORNlling the container with 10 kg offresh cattle manure the frame was assembled On theTHORNrst day 100 1-wk-old szligies (50 males and 50 females)obtained from the laboratory rearing were collectedby an aspirator and released into each cage In theseconditions szligies were allowed to feed and lay eggs onthe manure

An aliquot of 300 ml of the B laterosporus formu-lation at a concentration of 1 108 sporesml wasapplied to the surface of the manure by a sprinklingcan The dose of this treatment corresponded to 2litersm2 The control was similarly sprinkled with thesame quantity of water

Every 3 d the cages were opened to allow the szligiesto escape the treatments were repeated and 100 newszligies were released inside each cage again as previ-ously described A total of four successive 3-d treat-ments were carried out Three days after the lasttreatment all cages were opened and the frames werethrown away After that each container was coveredby a polystyrene cap and two openings (5 cm diam-eter) were made on a lateral face of the container Thisallowed the emerging szligies to go toward the light andto be collected in plastic transparent cylinders andcounted during the following days Four weeks laterthe containers were opened to verify the presence ofszligies not previously counted in the plastic cylinders

During this experiment the cages were always pro-tected from the wind and the rain maximum air tem-perature varied between 20 and 31C and minimumair temperature varied between 14 and 20C

The experiment was replicated three timesField Experiment Field experiments were con-

ducted in a central-western area of Sardinia (Arborea

506 ENVIRONMENTAL ENTOMOLOGY Vol 37 no 2

Province of Oristano) where dairy farms are abun-dant and the economy is mainly based on milk pro-duction and agriculture The diary farm selected forexperiments contained 100 head and had a cowshedof 2000 m2 of which only a small part was undercover The farms had a dunghill from which manurewas periodically removed In preliminary observa-tions the cow pen and the dunghill were identiTHORNed asthe main developmental sites for immature house szligies

Immature house szligy density on the farm was mon-itored from the beginning of July until the end ofAugust 2003 using modiTHORNed sampling methods of Loo-mis et al (1968) and Skoda et al (1996) A manufac-tured 05-liter standard metal core sampler was used totake eight random subsamples of manure which weremixed together tomakea larger sampleof4 litersEach4-liter sample was replicated three times and newsamples were collected every week The number ofadults that emerged from these manure samples wasused to monitor the abundance of immature szligies

To allow immature house szligy development manurewas incubated in cylindrical plastic containers (20 cmdiameter by 25 cm high) that had an opening on theirpolystyrene cap to permit the emerging szligies to gotoward the light and to be collected in a plastic trans-parent cylinder Plastic containers were left in thefarm and transparent cylinders were inspected for szligyidentiTHORNcation and counting Three weeks later con-tainers were opened to verify the presence of szligies notpreviously counted in the transparent cylinders

Areas (three treated and three control) of 9 m2

within the cow pen were randomized over six plotsthat were previously selected for homogeneity of thebreeding sites Samples of 4 liters of manure werecollected weekly from each area as described abovefor szligy monitoring

A total of THORNve applications using a B laterosporusformulation at a concentration of 1 108 sporesmland a dose of 2 litersm2 was applied at the followingdates 28 July and 1 5 9 and 13 August 2003 Theformulation was distributed on the surface of the cowpen by a sprinkling can The control areas were sim-ilarly treated using only water at the same doseStatistical AnalysisData on larvae (THORNrst experiment

on immature stages) in preliminary laboratory bioas-says were subjected to analysis of variance (ANOVAone factor design concentration) followed by a leastsigniTHORNcant difference (LSD) test to separate treat-ment means

The use of t-tests served to compare data means oftreated and control groups of the preliminary labora-tory bioassays of larvae (second experiment) the out-door cage experiment and the treatments against im-mature stages in the diary farm

In all experiments data met the assumption of ho-mogeneity of variance All statistical analyses wereperformed using Statgraphics Plus software (Stat-graphics Plus 2001)

Results

Laboratory Tests The toxicity of the diets contain-ing B laterosporus formulation against the house szligylarvae was concentration-dependent (Table 1) In theTHORNrst experiment the bacterial concentration signiTHORN-cantly inszliguenced the mortality percentages for larval(F312 8192 P 00001) and larval pupal (F312 1047P 00001) stages Despite the natural mortalityoccurred in the control (10 for larval stage and 20for larval pupal stage) which is related to the rear-ing diet (Floris et al 2004) a concentration of 1 108

sporesg of diet was high enough to cause a highlysigniTHORNcant 100 mortality of immature stages andconsequently no adult emergence

In the second experiment (Table 2) B laterosporustreatment on the surface of the laboratory breedingsubstrate signiTHORNcantly increased larval (t 15516df 6 P 0001) and pupal (t 4037 df 6 P0007) mortality and signiTHORNcantly reduced the numberof pupated insects (t 15516 df 6 P 0001) andemerged adults (t 14212 df 6 P 0001) Con-sequently total mortality percentage (t 14212df 6 P 0001) signiTHORNcantly increased reaching avalue of 803Outdoor Cage Experiment Repeated superTHORNcial

treatments using the B laterosporus formulation at aconcentration of 1 108 sporesml on manure causeda signiTHORNcant reduction in immature development abil-

Table 1 Percentage (mean SEM) of larval and pupal mor-tality of housefly after rearing first-instar larvae on a diet containingdifferent concentrations of a B laterosporus formulation

B laterosporus concentration(sporesg of diet)

Larval mortalitya

()Total mortalityb

()

20 108 1000 000a 100 000a10 108 925 479a 100 000a05 108 650 645b 875 629bControl 100 408c 200 408c

Means within the same column followed by different letters aresigniTHORNcantly different (one-way ANOVA followed by LSD test P 001)a Recorded after 5 dbDifference between the no larvae at the beginning of the exper-

iment and the emerged adults expressed in percentage

Table 2 Number (mean SEM) of flies that developed from eggs on substrate treated with a B laterosporus formulation at theconcentration of 1 108 sporesml and a corresponding dose of 2 litersm2 (n 1000)

TreatmentTotal pupae

obtainedLarval mortality

()Emerging

adultsPupal mortality

()Total mortality

()

Treated 2733 325 727 33 1970 288 287 42 803 29Control 8258 146 174 15 7528 264 89 25 247 26

Means within each column are signiTHORNcantly different (t-test)

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 507

ity The mean number SEM of adults that emergedfrom manure was 947 207 in the treated groupversus 2250 356 in the control group This corre-sponded to a general reduction in immature develop-ment of 578 The mean number of emerged adultsin the treated and control groups were statisticallydifferent from each other (t 31678 df 4 P 0034)Field Experiments Results on the effects of the B

laterosporus treatments in the cow pen of the dairyfarm are summarized in Table 3 No signiTHORNcant dif-ferences in immature stage abundance were noticedbefore treatmentsbetween the treatedand thecontrolarea (t 0141 df 4 P 0894) Successivelytreatments with the bacterial formulation signiTHORNcantlyreduced the immature szligy density in the treated areacompared with the control area (t 3317 df 4 P0029) Finally B laterosporus treatments caused adecrease of 30 in immature house szligy density

Discussion

The B laterosporus formulation containing gamma-irradiated spores of a new Sardinian strain exhibited atoxicity against house szligy larvae that was comparableto that caused by spore suspensions prepared in lab-oratory (Ruiu et al 2006) The bacterial formulationwhen used at a concentration of 1 108 sporesliterand a dose corresponding to 2 litersm2 was able to killlarvae and reduce adult emergence from laboratorylarval medium The same treatment also caused a sig-niTHORNcant reduction in szligy emergence from manure inoutdoor cage experiments that reproduced a naturalszligy habitat Similarly the B laterosporus formulationwas able to produce a signiTHORNcant reduction in imma-ture szligy development when repeated treatments werecarried out in the cow pen of a diary farm Theseresults are in line with those reported in the past byHarvey and Brethour (1960) who mixed formulationscontaining spores of B thuringiensis variety thurin-giensis with cattle feces Although a complete inhibi-tion of larval development at a higher dose was ob-tained this effect was likely to be related to theproduction of -exotoxin by living bacteria reproduc-ing in manure (Jespersen and Keiding 1990 Glare and

OOtildeCallaghan 2000) In contrast in our case spores oftheB laterosporus strain were previously killed so thatmortality was caused only by the presence of toxins inthe spore and the canoe-shaped parasporal body(CSPB) structure (Ruiu et al 2007a)

The newB laterosporus spore formulation was moreeffective when sprinkled on a synthetic medium (lab-oratory larval diet) than on a natural breeding sub-strate (manure) Similarly a greater treatment effec-tiveness in artiTHORNcial media than manure was alsoobserved by Jespersen and Keiding (1990) Moreoverbecause a liquid formulation sprinkled on the surfacehas some difTHORNculty reaching the larvae in the manuremass the efTHORNcacy of the treatments was probablylimited to the superTHORNcial layers where the presence oflarvae was noted Enhancement of these effects mightbe achieved with a more uniform mixing of spores andmanure such as that done by Borgatti and Guyer(1963) feeding B thuringiensis to animals which al-lowed spores to go through their digestive tract and bereleased with feces Similar effects of B thuringiensisfor microbial house szligy control have more recentlybeen obtained by Labib and Rady (2001)

Everything considered the efTHORNcacy of the treat-ments on the manure against the house szligy is encour-aging and provides an important contribution to theprospects of house szligy control in natural breedingplaces In addition the sublethal effects of this micro-bial agent on immature (ie development time) andadult (ie fecundity longevity) house szligies should betaken into account to better evaluate its potential(Ruiu et al 2006)

For these reasons the method assayed in this re-search represents a THORNrst approach to a successful useof B laterosporus for the control of house szligy in diaryfarms However the observed reduction in the effec-tivenessofB laterosporus fromlaboratory toTHORNeld trialssuggests that more studies covering a much largertreated area are needed to THORNnd more suitable methodsof application of this type of formulation In additiona feed additive containingB laterosporus could also bestudied as proposed for B thuringiensis variety thu-ringiensis to control immature szligy development onmanure (Dunn 1960 Smirnoff and MacLeod 1961)

Finally in relation to its compatibility with naturallyoccurring biological control agents such as hymenop-teran parasitoids (Ruiu et al 2007b) the new B lat-erosporus formulation tested in this study has a po-tential application in biological control and integratedpest management programs of this important pest

Acknowledgments

This study was THORNnancially supported by the Italian Min-istero Universita Ricerca ScientiTHORNca (Research Program Bio-tecnologie innovative per il controllo di insetti nocivi medi-ante lOtildeimpiego di agenti microbiologici coordinator ProfIgnazio Floris University of Sassari Italy) and the Fondazi-one Banco di Sardegna We thank AHD Francesconi forassistance

Table 3 Number (mean SEM) of emerged houseflies fromstandard 4-liter manure samples in treated and control areasbefore and after treatment with a B laterosporus formulation at aconcentration of 1 108 sporesml and a dose of 2 litersm2

Treatment

No emerged houseszligies

Beforetreatmenta

Aftertreatmentb

Treated 1270 87a 783 125bControl 1287 184a 1100 108a

Means within each column followed by different letters are sig-niTHORNcantly different (t-test)a Values were calculated as means of the monitoring data of a 4-wk

period before treatmentb Values were calculated as means of the monitoring data of a 4-wk

period after treatment

508 ENVIRONMENTAL ENTOMOLOGY Vol 37 no 2

References Cited

Borgatti A L and G E Guyer 1963 The effectiveness ofcommercial formulations of Bacillus thuringiensis Ber-liner on house szligy larvae J Insect Pathol 5 377ETH384

Briggs J D 1960 Reduction of adult house-szligy emergenceby the effects of Bacillus spp on the development ofimmature forms J Insect Pathol 2 418ETH432

Cohen D M Green C Block R Slepon R Ambar S SWasserman and M M Lavine 1991 Reduction oftransmission of shigellosis by control of houseszligies (Muscadomestica) Lancet 337 993ETH997

Drummond R O J E George and S E Kunz 1988 Con-trol of arthropod pests of livestock a review of technol-ogy CRC Boca Raton FL

Dunn P H 1960 Control of house szligies in bovine faeces bya feed additive containing Bacillus thuringiensis var thu-ringiensis Berliner J Insect Pathol 213ETH16

Favret E M and A A Yousten 1985 Insecticidal activityof Bacillus laterosporus J Invertebr Pathol 45 195ETH203

Floris I L Ruiu and A Satta 2004 Inszliguenza della qualitadi mosche domestiche allevate in laboratorio sugli effettidi Bacillus thuringiensis Berliner Proceedings of the XIXCongresso Nazionale Italiano di Entomologia 10ETH15 June2002 Catania Italy

Glare T R and M OrsquoCallaghan 2000 Bacillus thuringien-sisbiology ecology and safety Wiley Chichester UnitedKingdom

Hall IM andKYArakawa 1959 Thesusceptibilityof thehouse szligy Musca domestica Linnaeus to Bacillus thurin-giensis var thuringiensis Berliner J Insect Pathol 1 351ETH355

Harvey T L and J R Brethour 1960 Feed additives forcontrol of house szligy larvae in livestock feces J EconEntomol 53 774ETH776

Hodgman T C Y Ziniu SMing T Sawyer CM Nichollsand D J Ellar 1993 Characterization of a Bacillus thu-ringiensis strain which is toxic to the house szligy Muscadomestica FEMS Microbiol Lett 11417ETH22

Hogsette J A 1999 Management of ectoparasites with bi-ological control organisms Internat J Parasitol 29 147ETH151

Indrasith L S N Suzuki KOgiwara S Asano andHHori1992 Activated insecticidal crystal proteins fromBacillusthuringiensis serovars killed adult house szligies Lett ApplMicrobiol 14 174ETH177

Jespersen J B and J Keiding 1990 The effect of Bacillusthuringiensis var thuringiensis onMusca domestica L lar-vae resistant to insecticide pp 215ETH229 InD A Rutz andR S Patterson (eds) Biocontrol of arthropods affectinglivestock and poultry Westview Press Boulder CO

Johnson C A H Bishop and C L Turner 1998 Isolationand activity of strains of Bacillus thuringiensis toxic tolarvae of the House szligy (Diptera Muscidae) and TropicalBlowszligies (DipteraCalliphoridae) J Invertebr Pathol71 138ETH144

Labib I M and M Rady 2001 Application of Bacillusthuringiensis in poultry houses as a biological controlagent against the house szligy Musca domestica sorbens JEgypt Soc Parasitol 31531ETH544

Lonc E M Lecadet T M Lachowicz and E Panek 1997Description of Bacillus thuringiensis wratislaviensis (H-47) a new serotype originating from Wroclaw (Poland)and other Bt soil isolates from the same area Lett ApplMicrobiol 24 467ETH473

Loomis E C A S Deal and W R Bowen 1968 Therelative effectiveness of cumaphos as a poultry feed ad-ditive to control synanthropic szligy larvae in manure JEcon Entomol 61 904ETH908

Moon R D 2002 Muscid szligies pp 279ETH301 In G Mullenand L Durden (eds) Medical and veterinary entomol-ogy Academic London United Kingdom

Moreland C R and W S McLeod 1956 House szligy egg-measuring techniques J Econ Entomol 49 49ETH51

Oliveira E J L Rabinovitch R G Monnerat L K Passosand V Zahner 2004 Molecular characterization of Bre-vibacillus laterosporus and its potential use in biologicalcontrol Appl Environ Microbiol 70 6657ETH6664

Orlova M V T A Smirnova L A Ganushkina V YYacubovich andRRAzizbekyan 1998 Insecticidal ac-tivity of Bacillus laterosporus Appl Environ Microbiol642723ETH2725

Petersen J J J A Meyer D A Stage and P B Morgan1983 Evaluation of sequential releases of Spalangia en-dius (HymenopteraPteromalidae) for control of houseszligies and stable szligies (DipteraMuscidae) associated withconTHORNned livestock in Eastern Nebraska J Econ Entomol76 283ETH286

Rueda L M and R C Axtell 1985 Guide to commonspecies of pupal parasites (Hymenoptera Pteromalidae)of the house szligy and other muscoid szligies associated withpoultry and livestock manure North Carolina Agricul-tural Research Service North Carolina State UniversityRaleigh NC

Ruiu L G Delrio D J Ellar I Floris B Paglietti SRubino and A Satta 2006 Lethal and sublethal effectsof Brevibacillus laterosporus on the house szligy (Musca do-mestica) Entomol Exp Appl 118 137ETH144

Ruiu L I Floris A Satta and D J Ellar 2007a Toxicityof a Brevibacillus laterosporus strain lacking parasporalcrystals against Musca domestica and Aedes aegypti BiolControl 43136ETH143

Ruiu L A Satta and I Floris 2007b Susceptibility of thehouse szligy pupal parasitoid Muscidifurax raptor (Hyme-noptera Pteromalidae) to the entomopathogenic bacte-ria Bacillus thuringiensis and Brevibacillus laterosporusBiol Control 43 188ETH194

Rutz D A and R C Axtell 1981 House szligy (Musca do-mestica) control in broiler-breeder poultry houses bypupal parasites (Hymenoptera Pteromalidae) indige-nous parasite species and release of Muscidifurax raptorEnviron Entomol 10343ETH345

Skoda R S G D Thomas and J B Campbell 1996 Com-parison of core sampling and pupal traps for monitoringimmature stable szligies and house szligies (Diptera Muscidae)in beef feedlot pens J Econ Entomol 89 428ETH434

Smirnoff W A and C F MacLeod 1961 Study of thesurvival ofBacillus thuringiensis var thuringiensisBerlinerin the digestive tracts and in faces of a small mammalbirds J Insect Pathol 3266ETH270

Statgraphics Plus 2001 Statgraphics Plus userOtildes guide stan-dard ed Version 4 Manugistics Rockville MD

Zhong C D J Ellar A Bishop C Johnson S Lin and E RHart 2000 Characterization of a Bacillus thuringiensisdelta-endotoxin which is toxic to insects in three ordersJ Invertebr Pathol 76 131ETH139

Received for publication 31 May 2007 accepted 21 Septem-ber 2007

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 509

Province of Oristano) where dairy farms are abun-dant and the economy is mainly based on milk pro-duction and agriculture The diary farm selected forexperiments contained 100 head and had a cowshedof 2000 m2 of which only a small part was undercover The farms had a dunghill from which manurewas periodically removed In preliminary observa-tions the cow pen and the dunghill were identiTHORNed asthe main developmental sites for immature house szligies

Immature house szligy density on the farm was mon-itored from the beginning of July until the end ofAugust 2003 using modiTHORNed sampling methods of Loo-mis et al (1968) and Skoda et al (1996) A manufac-tured 05-liter standard metal core sampler was used totake eight random subsamples of manure which weremixed together tomakea larger sampleof4 litersEach4-liter sample was replicated three times and newsamples were collected every week The number ofadults that emerged from these manure samples wasused to monitor the abundance of immature szligies

To allow immature house szligy development manurewas incubated in cylindrical plastic containers (20 cmdiameter by 25 cm high) that had an opening on theirpolystyrene cap to permit the emerging szligies to gotoward the light and to be collected in a plastic trans-parent cylinder Plastic containers were left in thefarm and transparent cylinders were inspected for szligyidentiTHORNcation and counting Three weeks later con-tainers were opened to verify the presence of szligies notpreviously counted in the transparent cylinders

Areas (three treated and three control) of 9 m2

within the cow pen were randomized over six plotsthat were previously selected for homogeneity of thebreeding sites Samples of 4 liters of manure werecollected weekly from each area as described abovefor szligy monitoring

A total of THORNve applications using a B laterosporusformulation at a concentration of 1 108 sporesmland a dose of 2 litersm2 was applied at the followingdates 28 July and 1 5 9 and 13 August 2003 Theformulation was distributed on the surface of the cowpen by a sprinkling can The control areas were sim-ilarly treated using only water at the same doseStatistical AnalysisData on larvae (THORNrst experiment

on immature stages) in preliminary laboratory bioas-says were subjected to analysis of variance (ANOVAone factor design concentration) followed by a leastsigniTHORNcant difference (LSD) test to separate treat-ment means

The use of t-tests served to compare data means oftreated and control groups of the preliminary labora-tory bioassays of larvae (second experiment) the out-door cage experiment and the treatments against im-mature stages in the diary farm

In all experiments data met the assumption of ho-mogeneity of variance All statistical analyses wereperformed using Statgraphics Plus software (Stat-graphics Plus 2001)

Results

Laboratory Tests The toxicity of the diets contain-ing B laterosporus formulation against the house szligylarvae was concentration-dependent (Table 1) In theTHORNrst experiment the bacterial concentration signiTHORN-cantly inszliguenced the mortality percentages for larval(F312 8192 P 00001) and larval pupal (F312 1047P 00001) stages Despite the natural mortalityoccurred in the control (10 for larval stage and 20for larval pupal stage) which is related to the rear-ing diet (Floris et al 2004) a concentration of 1 108

sporesg of diet was high enough to cause a highlysigniTHORNcant 100 mortality of immature stages andconsequently no adult emergence

In the second experiment (Table 2) B laterosporustreatment on the surface of the laboratory breedingsubstrate signiTHORNcantly increased larval (t 15516df 6 P 0001) and pupal (t 4037 df 6 P0007) mortality and signiTHORNcantly reduced the numberof pupated insects (t 15516 df 6 P 0001) andemerged adults (t 14212 df 6 P 0001) Con-sequently total mortality percentage (t 14212df 6 P 0001) signiTHORNcantly increased reaching avalue of 803Outdoor Cage Experiment Repeated superTHORNcial

treatments using the B laterosporus formulation at aconcentration of 1 108 sporesml on manure causeda signiTHORNcant reduction in immature development abil-

Table 1 Percentage (mean SEM) of larval and pupal mor-tality of housefly after rearing first-instar larvae on a diet containingdifferent concentrations of a B laterosporus formulation

B laterosporus concentration(sporesg of diet)

Larval mortalitya

()Total mortalityb

()

20 108 1000 000a 100 000a10 108 925 479a 100 000a05 108 650 645b 875 629bControl 100 408c 200 408c

Means within the same column followed by different letters aresigniTHORNcantly different (one-way ANOVA followed by LSD test P 001)a Recorded after 5 dbDifference between the no larvae at the beginning of the exper-

iment and the emerged adults expressed in percentage

Table 2 Number (mean SEM) of flies that developed from eggs on substrate treated with a B laterosporus formulation at theconcentration of 1 108 sporesml and a corresponding dose of 2 litersm2 (n 1000)

TreatmentTotal pupae

obtainedLarval mortality

()Emerging

adultsPupal mortality

()Total mortality

()

Treated 2733 325 727 33 1970 288 287 42 803 29Control 8258 146 174 15 7528 264 89 25 247 26

Means within each column are signiTHORNcantly different (t-test)

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 507

ity The mean number SEM of adults that emergedfrom manure was 947 207 in the treated groupversus 2250 356 in the control group This corre-sponded to a general reduction in immature develop-ment of 578 The mean number of emerged adultsin the treated and control groups were statisticallydifferent from each other (t 31678 df 4 P 0034)Field Experiments Results on the effects of the B

laterosporus treatments in the cow pen of the dairyfarm are summarized in Table 3 No signiTHORNcant dif-ferences in immature stage abundance were noticedbefore treatmentsbetween the treatedand thecontrolarea (t 0141 df 4 P 0894) Successivelytreatments with the bacterial formulation signiTHORNcantlyreduced the immature szligy density in the treated areacompared with the control area (t 3317 df 4 P0029) Finally B laterosporus treatments caused adecrease of 30 in immature house szligy density

Discussion

The B laterosporus formulation containing gamma-irradiated spores of a new Sardinian strain exhibited atoxicity against house szligy larvae that was comparableto that caused by spore suspensions prepared in lab-oratory (Ruiu et al 2006) The bacterial formulationwhen used at a concentration of 1 108 sporesliterand a dose corresponding to 2 litersm2 was able to killlarvae and reduce adult emergence from laboratorylarval medium The same treatment also caused a sig-niTHORNcant reduction in szligy emergence from manure inoutdoor cage experiments that reproduced a naturalszligy habitat Similarly the B laterosporus formulationwas able to produce a signiTHORNcant reduction in imma-ture szligy development when repeated treatments werecarried out in the cow pen of a diary farm Theseresults are in line with those reported in the past byHarvey and Brethour (1960) who mixed formulationscontaining spores of B thuringiensis variety thurin-giensis with cattle feces Although a complete inhibi-tion of larval development at a higher dose was ob-tained this effect was likely to be related to theproduction of -exotoxin by living bacteria reproduc-ing in manure (Jespersen and Keiding 1990 Glare and

OOtildeCallaghan 2000) In contrast in our case spores oftheB laterosporus strain were previously killed so thatmortality was caused only by the presence of toxins inthe spore and the canoe-shaped parasporal body(CSPB) structure (Ruiu et al 2007a)

The newB laterosporus spore formulation was moreeffective when sprinkled on a synthetic medium (lab-oratory larval diet) than on a natural breeding sub-strate (manure) Similarly a greater treatment effec-tiveness in artiTHORNcial media than manure was alsoobserved by Jespersen and Keiding (1990) Moreoverbecause a liquid formulation sprinkled on the surfacehas some difTHORNculty reaching the larvae in the manuremass the efTHORNcacy of the treatments was probablylimited to the superTHORNcial layers where the presence oflarvae was noted Enhancement of these effects mightbe achieved with a more uniform mixing of spores andmanure such as that done by Borgatti and Guyer(1963) feeding B thuringiensis to animals which al-lowed spores to go through their digestive tract and bereleased with feces Similar effects of B thuringiensisfor microbial house szligy control have more recentlybeen obtained by Labib and Rady (2001)

Everything considered the efTHORNcacy of the treat-ments on the manure against the house szligy is encour-aging and provides an important contribution to theprospects of house szligy control in natural breedingplaces In addition the sublethal effects of this micro-bial agent on immature (ie development time) andadult (ie fecundity longevity) house szligies should betaken into account to better evaluate its potential(Ruiu et al 2006)

For these reasons the method assayed in this re-search represents a THORNrst approach to a successful useof B laterosporus for the control of house szligy in diaryfarms However the observed reduction in the effec-tivenessofB laterosporus fromlaboratory toTHORNeld trialssuggests that more studies covering a much largertreated area are needed to THORNnd more suitable methodsof application of this type of formulation In additiona feed additive containingB laterosporus could also bestudied as proposed for B thuringiensis variety thu-ringiensis to control immature szligy development onmanure (Dunn 1960 Smirnoff and MacLeod 1961)

Finally in relation to its compatibility with naturallyoccurring biological control agents such as hymenop-teran parasitoids (Ruiu et al 2007b) the new B lat-erosporus formulation tested in this study has a po-tential application in biological control and integratedpest management programs of this important pest

Acknowledgments

This study was THORNnancially supported by the Italian Min-istero Universita Ricerca ScientiTHORNca (Research Program Bio-tecnologie innovative per il controllo di insetti nocivi medi-ante lOtildeimpiego di agenti microbiologici coordinator ProfIgnazio Floris University of Sassari Italy) and the Fondazi-one Banco di Sardegna We thank AHD Francesconi forassistance

Table 3 Number (mean SEM) of emerged houseflies fromstandard 4-liter manure samples in treated and control areasbefore and after treatment with a B laterosporus formulation at aconcentration of 1 108 sporesml and a dose of 2 litersm2

Treatment

No emerged houseszligies

Beforetreatmenta

Aftertreatmentb

Treated 1270 87a 783 125bControl 1287 184a 1100 108a

Means within each column followed by different letters are sig-niTHORNcantly different (t-test)a Values were calculated as means of the monitoring data of a 4-wk

period before treatmentb Values were calculated as means of the monitoring data of a 4-wk

period after treatment

508 ENVIRONMENTAL ENTOMOLOGY Vol 37 no 2

References Cited

Borgatti A L and G E Guyer 1963 The effectiveness ofcommercial formulations of Bacillus thuringiensis Ber-liner on house szligy larvae J Insect Pathol 5 377ETH384

Briggs J D 1960 Reduction of adult house-szligy emergenceby the effects of Bacillus spp on the development ofimmature forms J Insect Pathol 2 418ETH432

Cohen D M Green C Block R Slepon R Ambar S SWasserman and M M Lavine 1991 Reduction oftransmission of shigellosis by control of houseszligies (Muscadomestica) Lancet 337 993ETH997

Drummond R O J E George and S E Kunz 1988 Con-trol of arthropod pests of livestock a review of technol-ogy CRC Boca Raton FL

Dunn P H 1960 Control of house szligies in bovine faeces bya feed additive containing Bacillus thuringiensis var thu-ringiensis Berliner J Insect Pathol 213ETH16

Favret E M and A A Yousten 1985 Insecticidal activityof Bacillus laterosporus J Invertebr Pathol 45 195ETH203

Floris I L Ruiu and A Satta 2004 Inszliguenza della qualitadi mosche domestiche allevate in laboratorio sugli effettidi Bacillus thuringiensis Berliner Proceedings of the XIXCongresso Nazionale Italiano di Entomologia 10ETH15 June2002 Catania Italy

Glare T R and M OrsquoCallaghan 2000 Bacillus thuringien-sisbiology ecology and safety Wiley Chichester UnitedKingdom

Hall IM andKYArakawa 1959 Thesusceptibilityof thehouse szligy Musca domestica Linnaeus to Bacillus thurin-giensis var thuringiensis Berliner J Insect Pathol 1 351ETH355

Harvey T L and J R Brethour 1960 Feed additives forcontrol of house szligy larvae in livestock feces J EconEntomol 53 774ETH776

Hodgman T C Y Ziniu SMing T Sawyer CM Nichollsand D J Ellar 1993 Characterization of a Bacillus thu-ringiensis strain which is toxic to the house szligy Muscadomestica FEMS Microbiol Lett 11417ETH22

Hogsette J A 1999 Management of ectoparasites with bi-ological control organisms Internat J Parasitol 29 147ETH151

Indrasith L S N Suzuki KOgiwara S Asano andHHori1992 Activated insecticidal crystal proteins fromBacillusthuringiensis serovars killed adult house szligies Lett ApplMicrobiol 14 174ETH177

Jespersen J B and J Keiding 1990 The effect of Bacillusthuringiensis var thuringiensis onMusca domestica L lar-vae resistant to insecticide pp 215ETH229 InD A Rutz andR S Patterson (eds) Biocontrol of arthropods affectinglivestock and poultry Westview Press Boulder CO

Johnson C A H Bishop and C L Turner 1998 Isolationand activity of strains of Bacillus thuringiensis toxic tolarvae of the House szligy (Diptera Muscidae) and TropicalBlowszligies (DipteraCalliphoridae) J Invertebr Pathol71 138ETH144

Labib I M and M Rady 2001 Application of Bacillusthuringiensis in poultry houses as a biological controlagent against the house szligy Musca domestica sorbens JEgypt Soc Parasitol 31531ETH544

Lonc E M Lecadet T M Lachowicz and E Panek 1997Description of Bacillus thuringiensis wratislaviensis (H-47) a new serotype originating from Wroclaw (Poland)and other Bt soil isolates from the same area Lett ApplMicrobiol 24 467ETH473

Loomis E C A S Deal and W R Bowen 1968 Therelative effectiveness of cumaphos as a poultry feed ad-ditive to control synanthropic szligy larvae in manure JEcon Entomol 61 904ETH908

Moon R D 2002 Muscid szligies pp 279ETH301 In G Mullenand L Durden (eds) Medical and veterinary entomol-ogy Academic London United Kingdom

Moreland C R and W S McLeod 1956 House szligy egg-measuring techniques J Econ Entomol 49 49ETH51

Oliveira E J L Rabinovitch R G Monnerat L K Passosand V Zahner 2004 Molecular characterization of Bre-vibacillus laterosporus and its potential use in biologicalcontrol Appl Environ Microbiol 70 6657ETH6664

Orlova M V T A Smirnova L A Ganushkina V YYacubovich andRRAzizbekyan 1998 Insecticidal ac-tivity of Bacillus laterosporus Appl Environ Microbiol642723ETH2725

Petersen J J J A Meyer D A Stage and P B Morgan1983 Evaluation of sequential releases of Spalangia en-dius (HymenopteraPteromalidae) for control of houseszligies and stable szligies (DipteraMuscidae) associated withconTHORNned livestock in Eastern Nebraska J Econ Entomol76 283ETH286

Rueda L M and R C Axtell 1985 Guide to commonspecies of pupal parasites (Hymenoptera Pteromalidae)of the house szligy and other muscoid szligies associated withpoultry and livestock manure North Carolina Agricul-tural Research Service North Carolina State UniversityRaleigh NC

Ruiu L G Delrio D J Ellar I Floris B Paglietti SRubino and A Satta 2006 Lethal and sublethal effectsof Brevibacillus laterosporus on the house szligy (Musca do-mestica) Entomol Exp Appl 118 137ETH144

Ruiu L I Floris A Satta and D J Ellar 2007a Toxicityof a Brevibacillus laterosporus strain lacking parasporalcrystals against Musca domestica and Aedes aegypti BiolControl 43136ETH143

Ruiu L A Satta and I Floris 2007b Susceptibility of thehouse szligy pupal parasitoid Muscidifurax raptor (Hyme-noptera Pteromalidae) to the entomopathogenic bacte-ria Bacillus thuringiensis and Brevibacillus laterosporusBiol Control 43 188ETH194

Rutz D A and R C Axtell 1981 House szligy (Musca do-mestica) control in broiler-breeder poultry houses bypupal parasites (Hymenoptera Pteromalidae) indige-nous parasite species and release of Muscidifurax raptorEnviron Entomol 10343ETH345

Skoda R S G D Thomas and J B Campbell 1996 Com-parison of core sampling and pupal traps for monitoringimmature stable szligies and house szligies (Diptera Muscidae)in beef feedlot pens J Econ Entomol 89 428ETH434

Smirnoff W A and C F MacLeod 1961 Study of thesurvival ofBacillus thuringiensis var thuringiensisBerlinerin the digestive tracts and in faces of a small mammalbirds J Insect Pathol 3266ETH270

Statgraphics Plus 2001 Statgraphics Plus userOtildes guide stan-dard ed Version 4 Manugistics Rockville MD

Zhong C D J Ellar A Bishop C Johnson S Lin and E RHart 2000 Characterization of a Bacillus thuringiensisdelta-endotoxin which is toxic to insects in three ordersJ Invertebr Pathol 76 131ETH139

Received for publication 31 May 2007 accepted 21 Septem-ber 2007

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 509

ity The mean number SEM of adults that emergedfrom manure was 947 207 in the treated groupversus 2250 356 in the control group This corre-sponded to a general reduction in immature develop-ment of 578 The mean number of emerged adultsin the treated and control groups were statisticallydifferent from each other (t 31678 df 4 P 0034)Field Experiments Results on the effects of the B

laterosporus treatments in the cow pen of the dairyfarm are summarized in Table 3 No signiTHORNcant dif-ferences in immature stage abundance were noticedbefore treatmentsbetween the treatedand thecontrolarea (t 0141 df 4 P 0894) Successivelytreatments with the bacterial formulation signiTHORNcantlyreduced the immature szligy density in the treated areacompared with the control area (t 3317 df 4 P0029) Finally B laterosporus treatments caused adecrease of 30 in immature house szligy density

Discussion

The B laterosporus formulation containing gamma-irradiated spores of a new Sardinian strain exhibited atoxicity against house szligy larvae that was comparableto that caused by spore suspensions prepared in lab-oratory (Ruiu et al 2006) The bacterial formulationwhen used at a concentration of 1 108 sporesliterand a dose corresponding to 2 litersm2 was able to killlarvae and reduce adult emergence from laboratorylarval medium The same treatment also caused a sig-niTHORNcant reduction in szligy emergence from manure inoutdoor cage experiments that reproduced a naturalszligy habitat Similarly the B laterosporus formulationwas able to produce a signiTHORNcant reduction in imma-ture szligy development when repeated treatments werecarried out in the cow pen of a diary farm Theseresults are in line with those reported in the past byHarvey and Brethour (1960) who mixed formulationscontaining spores of B thuringiensis variety thurin-giensis with cattle feces Although a complete inhibi-tion of larval development at a higher dose was ob-tained this effect was likely to be related to theproduction of -exotoxin by living bacteria reproduc-ing in manure (Jespersen and Keiding 1990 Glare and

OOtildeCallaghan 2000) In contrast in our case spores oftheB laterosporus strain were previously killed so thatmortality was caused only by the presence of toxins inthe spore and the canoe-shaped parasporal body(CSPB) structure (Ruiu et al 2007a)

The newB laterosporus spore formulation was moreeffective when sprinkled on a synthetic medium (lab-oratory larval diet) than on a natural breeding sub-strate (manure) Similarly a greater treatment effec-tiveness in artiTHORNcial media than manure was alsoobserved by Jespersen and Keiding (1990) Moreoverbecause a liquid formulation sprinkled on the surfacehas some difTHORNculty reaching the larvae in the manuremass the efTHORNcacy of the treatments was probablylimited to the superTHORNcial layers where the presence oflarvae was noted Enhancement of these effects mightbe achieved with a more uniform mixing of spores andmanure such as that done by Borgatti and Guyer(1963) feeding B thuringiensis to animals which al-lowed spores to go through their digestive tract and bereleased with feces Similar effects of B thuringiensisfor microbial house szligy control have more recentlybeen obtained by Labib and Rady (2001)

Everything considered the efTHORNcacy of the treat-ments on the manure against the house szligy is encour-aging and provides an important contribution to theprospects of house szligy control in natural breedingplaces In addition the sublethal effects of this micro-bial agent on immature (ie development time) andadult (ie fecundity longevity) house szligies should betaken into account to better evaluate its potential(Ruiu et al 2006)

For these reasons the method assayed in this re-search represents a THORNrst approach to a successful useof B laterosporus for the control of house szligy in diaryfarms However the observed reduction in the effec-tivenessofB laterosporus fromlaboratory toTHORNeld trialssuggests that more studies covering a much largertreated area are needed to THORNnd more suitable methodsof application of this type of formulation In additiona feed additive containingB laterosporus could also bestudied as proposed for B thuringiensis variety thu-ringiensis to control immature szligy development onmanure (Dunn 1960 Smirnoff and MacLeod 1961)

Finally in relation to its compatibility with naturallyoccurring biological control agents such as hymenop-teran parasitoids (Ruiu et al 2007b) the new B lat-erosporus formulation tested in this study has a po-tential application in biological control and integratedpest management programs of this important pest

Acknowledgments

This study was THORNnancially supported by the Italian Min-istero Universita Ricerca ScientiTHORNca (Research Program Bio-tecnologie innovative per il controllo di insetti nocivi medi-ante lOtildeimpiego di agenti microbiologici coordinator ProfIgnazio Floris University of Sassari Italy) and the Fondazi-one Banco di Sardegna We thank AHD Francesconi forassistance

Table 3 Number (mean SEM) of emerged houseflies fromstandard 4-liter manure samples in treated and control areasbefore and after treatment with a B laterosporus formulation at aconcentration of 1 108 sporesml and a dose of 2 litersm2

Treatment

No emerged houseszligies

Beforetreatmenta

Aftertreatmentb

Treated 1270 87a 783 125bControl 1287 184a 1100 108a

Means within each column followed by different letters are sig-niTHORNcantly different (t-test)a Values were calculated as means of the monitoring data of a 4-wk

period before treatmentb Values were calculated as means of the monitoring data of a 4-wk

period after treatment

508 ENVIRONMENTAL ENTOMOLOGY Vol 37 no 2

References Cited

Borgatti A L and G E Guyer 1963 The effectiveness ofcommercial formulations of Bacillus thuringiensis Ber-liner on house szligy larvae J Insect Pathol 5 377ETH384

Briggs J D 1960 Reduction of adult house-szligy emergenceby the effects of Bacillus spp on the development ofimmature forms J Insect Pathol 2 418ETH432

Cohen D M Green C Block R Slepon R Ambar S SWasserman and M M Lavine 1991 Reduction oftransmission of shigellosis by control of houseszligies (Muscadomestica) Lancet 337 993ETH997

Drummond R O J E George and S E Kunz 1988 Con-trol of arthropod pests of livestock a review of technol-ogy CRC Boca Raton FL

Dunn P H 1960 Control of house szligies in bovine faeces bya feed additive containing Bacillus thuringiensis var thu-ringiensis Berliner J Insect Pathol 213ETH16

Favret E M and A A Yousten 1985 Insecticidal activityof Bacillus laterosporus J Invertebr Pathol 45 195ETH203

Floris I L Ruiu and A Satta 2004 Inszliguenza della qualitadi mosche domestiche allevate in laboratorio sugli effettidi Bacillus thuringiensis Berliner Proceedings of the XIXCongresso Nazionale Italiano di Entomologia 10ETH15 June2002 Catania Italy

Glare T R and M OrsquoCallaghan 2000 Bacillus thuringien-sisbiology ecology and safety Wiley Chichester UnitedKingdom

Hall IM andKYArakawa 1959 Thesusceptibilityof thehouse szligy Musca domestica Linnaeus to Bacillus thurin-giensis var thuringiensis Berliner J Insect Pathol 1 351ETH355

Harvey T L and J R Brethour 1960 Feed additives forcontrol of house szligy larvae in livestock feces J EconEntomol 53 774ETH776

Hodgman T C Y Ziniu SMing T Sawyer CM Nichollsand D J Ellar 1993 Characterization of a Bacillus thu-ringiensis strain which is toxic to the house szligy Muscadomestica FEMS Microbiol Lett 11417ETH22

Hogsette J A 1999 Management of ectoparasites with bi-ological control organisms Internat J Parasitol 29 147ETH151

Indrasith L S N Suzuki KOgiwara S Asano andHHori1992 Activated insecticidal crystal proteins fromBacillusthuringiensis serovars killed adult house szligies Lett ApplMicrobiol 14 174ETH177

Jespersen J B and J Keiding 1990 The effect of Bacillusthuringiensis var thuringiensis onMusca domestica L lar-vae resistant to insecticide pp 215ETH229 InD A Rutz andR S Patterson (eds) Biocontrol of arthropods affectinglivestock and poultry Westview Press Boulder CO

Johnson C A H Bishop and C L Turner 1998 Isolationand activity of strains of Bacillus thuringiensis toxic tolarvae of the House szligy (Diptera Muscidae) and TropicalBlowszligies (DipteraCalliphoridae) J Invertebr Pathol71 138ETH144

Labib I M and M Rady 2001 Application of Bacillusthuringiensis in poultry houses as a biological controlagent against the house szligy Musca domestica sorbens JEgypt Soc Parasitol 31531ETH544

Lonc E M Lecadet T M Lachowicz and E Panek 1997Description of Bacillus thuringiensis wratislaviensis (H-47) a new serotype originating from Wroclaw (Poland)and other Bt soil isolates from the same area Lett ApplMicrobiol 24 467ETH473

Loomis E C A S Deal and W R Bowen 1968 Therelative effectiveness of cumaphos as a poultry feed ad-ditive to control synanthropic szligy larvae in manure JEcon Entomol 61 904ETH908

Moon R D 2002 Muscid szligies pp 279ETH301 In G Mullenand L Durden (eds) Medical and veterinary entomol-ogy Academic London United Kingdom

Moreland C R and W S McLeod 1956 House szligy egg-measuring techniques J Econ Entomol 49 49ETH51

Oliveira E J L Rabinovitch R G Monnerat L K Passosand V Zahner 2004 Molecular characterization of Bre-vibacillus laterosporus and its potential use in biologicalcontrol Appl Environ Microbiol 70 6657ETH6664

Orlova M V T A Smirnova L A Ganushkina V YYacubovich andRRAzizbekyan 1998 Insecticidal ac-tivity of Bacillus laterosporus Appl Environ Microbiol642723ETH2725

Petersen J J J A Meyer D A Stage and P B Morgan1983 Evaluation of sequential releases of Spalangia en-dius (HymenopteraPteromalidae) for control of houseszligies and stable szligies (DipteraMuscidae) associated withconTHORNned livestock in Eastern Nebraska J Econ Entomol76 283ETH286

Rueda L M and R C Axtell 1985 Guide to commonspecies of pupal parasites (Hymenoptera Pteromalidae)of the house szligy and other muscoid szligies associated withpoultry and livestock manure North Carolina Agricul-tural Research Service North Carolina State UniversityRaleigh NC

Ruiu L G Delrio D J Ellar I Floris B Paglietti SRubino and A Satta 2006 Lethal and sublethal effectsof Brevibacillus laterosporus on the house szligy (Musca do-mestica) Entomol Exp Appl 118 137ETH144

Ruiu L I Floris A Satta and D J Ellar 2007a Toxicityof a Brevibacillus laterosporus strain lacking parasporalcrystals against Musca domestica and Aedes aegypti BiolControl 43136ETH143

Ruiu L A Satta and I Floris 2007b Susceptibility of thehouse szligy pupal parasitoid Muscidifurax raptor (Hyme-noptera Pteromalidae) to the entomopathogenic bacte-ria Bacillus thuringiensis and Brevibacillus laterosporusBiol Control 43 188ETH194

Rutz D A and R C Axtell 1981 House szligy (Musca do-mestica) control in broiler-breeder poultry houses bypupal parasites (Hymenoptera Pteromalidae) indige-nous parasite species and release of Muscidifurax raptorEnviron Entomol 10343ETH345

Skoda R S G D Thomas and J B Campbell 1996 Com-parison of core sampling and pupal traps for monitoringimmature stable szligies and house szligies (Diptera Muscidae)in beef feedlot pens J Econ Entomol 89 428ETH434

Smirnoff W A and C F MacLeod 1961 Study of thesurvival ofBacillus thuringiensis var thuringiensisBerlinerin the digestive tracts and in faces of a small mammalbirds J Insect Pathol 3266ETH270

Statgraphics Plus 2001 Statgraphics Plus userOtildes guide stan-dard ed Version 4 Manugistics Rockville MD

Zhong C D J Ellar A Bishop C Johnson S Lin and E RHart 2000 Characterization of a Bacillus thuringiensisdelta-endotoxin which is toxic to insects in three ordersJ Invertebr Pathol 76 131ETH139

Received for publication 31 May 2007 accepted 21 Septem-ber 2007

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 509

References Cited

Borgatti A L and G E Guyer 1963 The effectiveness ofcommercial formulations of Bacillus thuringiensis Ber-liner on house szligy larvae J Insect Pathol 5 377ETH384

Briggs J D 1960 Reduction of adult house-szligy emergenceby the effects of Bacillus spp on the development ofimmature forms J Insect Pathol 2 418ETH432

Cohen D M Green C Block R Slepon R Ambar S SWasserman and M M Lavine 1991 Reduction oftransmission of shigellosis by control of houseszligies (Muscadomestica) Lancet 337 993ETH997

Drummond R O J E George and S E Kunz 1988 Con-trol of arthropod pests of livestock a review of technol-ogy CRC Boca Raton FL

Dunn P H 1960 Control of house szligies in bovine faeces bya feed additive containing Bacillus thuringiensis var thu-ringiensis Berliner J Insect Pathol 213ETH16

Favret E M and A A Yousten 1985 Insecticidal activityof Bacillus laterosporus J Invertebr Pathol 45 195ETH203

Floris I L Ruiu and A Satta 2004 Inszliguenza della qualitadi mosche domestiche allevate in laboratorio sugli effettidi Bacillus thuringiensis Berliner Proceedings of the XIXCongresso Nazionale Italiano di Entomologia 10ETH15 June2002 Catania Italy

Glare T R and M OrsquoCallaghan 2000 Bacillus thuringien-sisbiology ecology and safety Wiley Chichester UnitedKingdom

Hall IM andKYArakawa 1959 Thesusceptibilityof thehouse szligy Musca domestica Linnaeus to Bacillus thurin-giensis var thuringiensis Berliner J Insect Pathol 1 351ETH355

Harvey T L and J R Brethour 1960 Feed additives forcontrol of house szligy larvae in livestock feces J EconEntomol 53 774ETH776

Hodgman T C Y Ziniu SMing T Sawyer CM Nichollsand D J Ellar 1993 Characterization of a Bacillus thu-ringiensis strain which is toxic to the house szligy Muscadomestica FEMS Microbiol Lett 11417ETH22

Hogsette J A 1999 Management of ectoparasites with bi-ological control organisms Internat J Parasitol 29 147ETH151

Indrasith L S N Suzuki KOgiwara S Asano andHHori1992 Activated insecticidal crystal proteins fromBacillusthuringiensis serovars killed adult house szligies Lett ApplMicrobiol 14 174ETH177

Jespersen J B and J Keiding 1990 The effect of Bacillusthuringiensis var thuringiensis onMusca domestica L lar-vae resistant to insecticide pp 215ETH229 InD A Rutz andR S Patterson (eds) Biocontrol of arthropods affectinglivestock and poultry Westview Press Boulder CO

Johnson C A H Bishop and C L Turner 1998 Isolationand activity of strains of Bacillus thuringiensis toxic tolarvae of the House szligy (Diptera Muscidae) and TropicalBlowszligies (DipteraCalliphoridae) J Invertebr Pathol71 138ETH144

Labib I M and M Rady 2001 Application of Bacillusthuringiensis in poultry houses as a biological controlagent against the house szligy Musca domestica sorbens JEgypt Soc Parasitol 31531ETH544

Lonc E M Lecadet T M Lachowicz and E Panek 1997Description of Bacillus thuringiensis wratislaviensis (H-47) a new serotype originating from Wroclaw (Poland)and other Bt soil isolates from the same area Lett ApplMicrobiol 24 467ETH473

Loomis E C A S Deal and W R Bowen 1968 Therelative effectiveness of cumaphos as a poultry feed ad-ditive to control synanthropic szligy larvae in manure JEcon Entomol 61 904ETH908

Moon R D 2002 Muscid szligies pp 279ETH301 In G Mullenand L Durden (eds) Medical and veterinary entomol-ogy Academic London United Kingdom

Moreland C R and W S McLeod 1956 House szligy egg-measuring techniques J Econ Entomol 49 49ETH51

Oliveira E J L Rabinovitch R G Monnerat L K Passosand V Zahner 2004 Molecular characterization of Bre-vibacillus laterosporus and its potential use in biologicalcontrol Appl Environ Microbiol 70 6657ETH6664

Orlova M V T A Smirnova L A Ganushkina V YYacubovich andRRAzizbekyan 1998 Insecticidal ac-tivity of Bacillus laterosporus Appl Environ Microbiol642723ETH2725

Petersen J J J A Meyer D A Stage and P B Morgan1983 Evaluation of sequential releases of Spalangia en-dius (HymenopteraPteromalidae) for control of houseszligies and stable szligies (DipteraMuscidae) associated withconTHORNned livestock in Eastern Nebraska J Econ Entomol76 283ETH286

Rueda L M and R C Axtell 1985 Guide to commonspecies of pupal parasites (Hymenoptera Pteromalidae)of the house szligy and other muscoid szligies associated withpoultry and livestock manure North Carolina Agricul-tural Research Service North Carolina State UniversityRaleigh NC

Ruiu L G Delrio D J Ellar I Floris B Paglietti SRubino and A Satta 2006 Lethal and sublethal effectsof Brevibacillus laterosporus on the house szligy (Musca do-mestica) Entomol Exp Appl 118 137ETH144

Ruiu L I Floris A Satta and D J Ellar 2007a Toxicityof a Brevibacillus laterosporus strain lacking parasporalcrystals against Musca domestica and Aedes aegypti BiolControl 43136ETH143

Ruiu L A Satta and I Floris 2007b Susceptibility of thehouse szligy pupal parasitoid Muscidifurax raptor (Hyme-noptera Pteromalidae) to the entomopathogenic bacte-ria Bacillus thuringiensis and Brevibacillus laterosporusBiol Control 43 188ETH194

Rutz D A and R C Axtell 1981 House szligy (Musca do-mestica) control in broiler-breeder poultry houses bypupal parasites (Hymenoptera Pteromalidae) indige-nous parasite species and release of Muscidifurax raptorEnviron Entomol 10343ETH345

Skoda R S G D Thomas and J B Campbell 1996 Com-parison of core sampling and pupal traps for monitoringimmature stable szligies and house szligies (Diptera Muscidae)in beef feedlot pens J Econ Entomol 89 428ETH434

Smirnoff W A and C F MacLeod 1961 Study of thesurvival ofBacillus thuringiensis var thuringiensisBerlinerin the digestive tracts and in faces of a small mammalbirds J Insect Pathol 3266ETH270

Statgraphics Plus 2001 Statgraphics Plus userOtildes guide stan-dard ed Version 4 Manugistics Rockville MD

Zhong C D J Ellar A Bishop C Johnson S Lin and E RHart 2000 Characterization of a Bacillus thuringiensisdelta-endotoxin which is toxic to insects in three ordersJ Invertebr Pathol 76 131ETH139

Received for publication 31 May 2007 accepted 21 Septem-ber 2007

April 2008 RUIU ET AL BIOLOGICAL CONTROL MICROBIALS 509