Comparisons of antifeedancy and spatial repellency of three natural product repellents agains horn flies

Transcription

1 United Sttes Deprtment of Agriculture From the SelectedWorks of Dvid B Tylor 14 Comprisons of ntifeedncy nd sptil repellency of three nturl product repellents gins horn flies Junwei J. Zhu, USDA-ARS Agroecosystem Mngement Reserch Unit Gry J. Brewer, University of Nersk-Lincoln Dvid J. Boxler, University of Nersk-Lincoln Kristin A. Friesen, USDA-ARS Agroecosystem Mngement Reserch Unit Dvid B. Tylor, USDA-ARS Agroecosystem Mngement Reserch Unit Aville t:

2 Reserch Article Received: 18 Septemer 14 Revised: Novemer 14 Accepted rticle pulished: 1 Decemer 14 Pulished online in Wiley Online Lirry: (wileyonlinelirry.com) DOI 1.2/ps.396 Comprisons of ntifeedncy nd sptil repellency of three nturl product repellents ginst horn flies, Hemtoi irritns (Dipter: Muscide) Junwei J Zhu, * GryJBrewer, Dvid J Boxler, Kristin Friesen nd Dvid B Tylor Astrct BACKGROUND: Horn flies re mong the most importnt iting fly pests of cttle in the United Sttes. Horn fly mngement is lrgely dependent upon pesticides, which ultimtely leds to the rpid development of insecticide resistnce. Alterntive control strtegies, including repellents, hve shown promising results in reducing fly iting. In the present study, we exmined the efficcy nd longevity of recently identified nturl product repellents ginst horn flies. RESULTS: Ctnip oil, gerniol nd C891 cids reduced horn fly feeding in lortory iossy nd lso exhiited sptil repellency in the olfctometer. Residul ctivity ws oserved for up to 3 dys in lortory ssys; however, 24 h of residul effectiveness ws oserved from the two repellents when pplied on cttle in the field. The limited residul effectiveness ws correlted with the high voltility of the mjor ctive repellent compounds. CONCLUSION: All three nturl product repellents effectively repel iting horn flies, exhiiting oth feeding deterrence nd sptil repellency. They my e used for developing n effective push-pull strtegy with slow relese mtrix tht cn prolong their effectiveness for horn fly mngement. 14 Society of Chemicl Industry Keywords: Hemtoi irritns; repellent efficcy; ctnip oil; gerniol; ftty cids 1 INTRODUCTION Horn flies, Hemtoi irritns, re mong the most importnt pests of pstured cttle in the United Sttes. 1 The species ws introduced into North Americ from Europe etween 1884 nd ,3 Both sexes feed on cttle, cusing nnoynce, ltertion of grzing ehvior, reduction in feed conversion efficiency nd reduced milk production nd weight gin. 4 8 Kunz et l. 9 reported tht the dmge cused y horn flies costs US cttle producers $US 1 illion nnully. Furthermore, horn flies hve een implicted in the trnsmission of Stphylococcus ureus mstitis nd cuse ovine tet tresi. 1,11 Horn fly mngement in pstures is lrgely dependent upon chemicl control methods, such s pour-ons, insecticideimpregnted er tgs, ck-ruers nd feed-through. However, the low costs of generic insecticide formultions hve enled producers to use them frequently, which hs led to widespred resistnce Additionlly, some of them re not leled for use on lctting diry cttle either in conventionl or in orgnic frming systems. In Nersk, Prolte/Lintox-HD ( spryle phosmet pesticide) hs een suggested for horn fly control on psture cttle owing to the very low repellency oserved. Although insecticides remin the min horn fly mngement tool, lterntive mngement methods re under development. Nturl product repellents hve een used for hundreds of yers to protect humns nd their nimls from rthropod ttck. 16 Recently, severl reports hve demonstrted the repellency of plnt oils nd ftty cids, including essentil oils of ctnip (Nepet ctri) nd gernium, s well s short-chin-length ftty cids, ginst iting fly The present pper reports the efficcy nd longevity of repellent nd ntifeeding ctivity of three nturl products in lortory iossys nd on cttle under field conditions. 2 MATERIALS AND METHODS 2.1 Horn flies Lortory iossys were conducted using n insecticide susceptile lortory strin of horn flies mintined t the USDA-ARS Knipling-Bushlnd US Livestock Insects Reserch Lortory in Correspondence to: Junwei J Zhu, USDA-ARS Agroecosystem Mngement Reserch Unit, USDA-ARS, University of Nersk, Est Cmpus, Lincoln, NE 68583, USA. E-mil: jerry.zhu@rs.usd.gov USDA-ARS Agroecosystem Mngement Reserch Unit, USDA-ARS, University of Nersk, Est Cmpus, Lincoln, NE, USA Deprtment of Entomology, University of Nersk Lincoln, Lincoln, NE, USA Pest Mng Sci (15) 14 Society of Chemicl Industry

3 JJ Zhu et l. Kerrville, Texs. Horn flies were mintined t 23 ± 2 C with vrile humidity (3 5%) nd light:drk photoperiod of 12:12 h. Adult horn flies were fed citrted ovine lood (3.7 g sodium citrte L 1 ) twice dily y soking feminine hygiene pds (Styfree ; McNeil-PPC Inc., Skillmn, NJ) in lood nd plcing them inside the cges. 2.2 Nturl product repellents nd insecticides Ctnip oil ws purchsed from Brmle Berry Inc. (Bellinghm, Wshington, DC). The chemicl composition ws determined y gs chromtogrphy mss spectrometry (GC-MS) nlysis to e 85% (Z,E)- nd (E,Z)-nepetlctone. Gerniol ws purchsed from FASST Products (Rockville Centre, NY), nd the oil contined >9% (2E)-3,7-dimethyloct-2,6-dien-1-ol. C891 cids, 1:1:1 mixture of octnoic, nonoic nd decnoic cids, ws purchsed from Strtcor Inc. (Richmond, CA). Prolte/Lntox-HD contining 11.75% phosmet ws purchsed from Vlley Vet Supply (Mrysville, KS). The two nepetlctones were ccumulted nd purified (>95%) from the purchsed ctnip essentil oil, following the method descried in Peterson. 21 Synthetic stndrds of (2E)-3,7-dimethyloct-2,6-dien-1-ol, octnoic cid, nonoic cid nd decnoic cid were purchsed from Sigm-Aldrich (St Louis, MO) with purity of >98%. 2.3 Lortory feeding iossy The lortory iossy for testing ntifeeding ctivity used six-cell pprtus similr to tht descried y Klun et l. 22 (K&D module), with modifictions for horn flies. 23 Test flies (3 4 dys old) were strved for 24 h prior to testing. Three doses of repellents (.2, 2 nd mg) were dissolved in 3 μl of hexne (high-purity solvent; Burdick & Jckson, Muskegon, MI) nd then evenly pplied to the outer lyer cut from snitry pd (4 5 cm). After the solvent evported (2 3 min), the repellent-impregnted lyer ws plced on top of lood-soked snitry pd in the K&D module. A control smple ws treted with 3 μlofhexneonly. Test flies were trnsferred into ech of the six testing cells (3 5 flies cell 1 )yusingglss/ruertuepooter.after4h,horn flies were nesthetized with CO 2 nd checked for feeding sttus y squshing their domen nd exmining for the presence of lood. Flies in the repellent iossy were exposed to rndomized tretments (repellent cndidtes t vrious dosges) until t lest ten replictes were completed (fresh flies nd lyers were used for ech test). During the experiments, we recorded the time to knockdown t the mg dosge, which ws defined while flies were ded or lying on the floor of the ox unle to fly. Results of the feeding iossy were nlyzed y logistic regression. LS mens were seprted y t-tests (Proc Genmod, SAS 9.3; SAS Institute, Inc., Cry, NC). Antifeedncy is reported s cktrnsformed LS mens with 95% confidence limits. 2.4 Sptil repellent ssy in the single-cge olfctometer A single-cge, dul-port glss olfctometer ws used to ssess sptil repellency of the three repellents ginst horn flies. 23 Horn flies (3 4 dys old) were strved for 24 h prior to testing. Flies were relesed into the olfctometer individully nd given 3 min to respond; their presence in the repellent-treted or control port (>1 cm inside the port) ws recorded. Normlly, one set of twelve tests (three repellent products t four doses) ws performed ech dy. Repellents were first dissolved in hexne to mke solutions t concentrtions of.1,.1, 1 nd 1 μg μl 1.Aquntityof1μL of repellent solution ws pplied to piece of filter pper (cut s smll tringle, 2 cm of ech side). For the control, 1 μlofhexne ws pplied. The filter pper ws ir dried, fixed to n insect pin nd plced in the middle of test port of the olfctometer. Within ech set of tests, the order of ports, repellents or control ws rndomized. All three ports (including the relesing port) were clened with cetone followed y hexne efore ech test. A test consisted of the sequentil introduction of five flies into the olfctometer. Ech test ws replicted times. New sets of five flies were used for ech replicte. Responses were recorded s the percentge of flies inside the tretment or control ports. After checking the homogeneity of vrince nd normlity of dt, they were nlyzed using Student s t-test. Log trnsformtion ws done when necessry. Results with P <.5 were considered to e sttisticlly significnt. 2.5 Electrontennl responses to repellents Electrontennogrms (EAGs) were recorded s indicted in Zhu et l. 23 Three dosges (1, 1 nd μg) of ech repellent dissolved in redistilled HPLC-grde hexne (1 μl) were prepred. The prepred solutions were pplied to filter pper strips ( cm, Whtmn No. 1; Whtmn Interntionl Ltd, Midstone, Kent, UK). Air-dried filter pper strips were inserted into 15 cm long Psteur pipettes. A 5 ml puff (Auto-Puffer; SYNTECH, Kirchzrten, Germny) contining odornt compound ws lown through the pipette nd directed cross the ntenne to elicit n EAG response. Control puffs (hexne only) of ir were pplied fter ech puff of test stimulus. The EAG response for ech stimulus ws recorded s the men mplitude of ech of the six replicted mesurements. The sequence of exposure of ech stimulus to ech ntenn ws rndom. The significnces of differences of horn fly reltive EAG responses (solute EAGs controls) were determined y multiwy ANOVA followed y Scheffe test (PASW Sttistics 18, SPSS Inc.,Chicgo,IL),ndStudent st-test ws used for comprisons of EAG recordings etween mle nd femle ntenne. 2.6 Longevity of repellency of the three nturl product repellents in the lortory nd field Antifeednt longevity tests were first conducted under lortory conditions. The three nturl products were tested t mg dose. Smples were prepred s in Section 2.5. Repellent-impregnted lyers were ged y hnging from metl stnd plced inside fume hood (Air Sentry, New York, NY) with continuous ventiltion t 27 m min 1. Antifeedncy ws tested on horn flies (3 4 dys old) strved for 24 h on smples ged for (freshly mde), 24, 48, 72 nd 96 h. Ech ge clss ws replicted minimum of 1 times. Repellency ws tested on cttle under field conditions during the summers of 11 nd 12 t the University of Nersk, West Centrl Reserch nd Extension Center, North Pltte, Nersk. Tests were conducted using criteri specified y the Americn Society for Testing nd Mterils (ASTM, 19) nd protocols pproved y the Institutionl Animl Cre nd Use Committee of the University of Nersk (IACUC Protocol No C). Test cttle were restrined in chute, nd 25 ml of repellent (15% ctnip oil, or 3% gerniol in light minerl oil, or C891 cid formultion) ws spryed evenly over the entire ody, except for the fce re, using compressed-ir hnd gun (J.E. ADAMS Industries Ltd, Cedr Rpids, IA) with 241 kp ir pressure. This dose ws estimted to e equivlent to pproximtely.1 ml cm 2. Control cttle were treted with 25 ml of minerl oil. Prolte/Lntox-HD spryle solution ws diluted s per lel instructions (1: in wileyonlinelirry.com/journl/ps 14 Society of Chemicl Industry Pest Mng Sci (15)

4 Repellency of three nturl products ginst horn flies Men % of ntifeedncy mg 2 mg mg Ctnip oil C891 cids Gerniol Figure 1. Comprisons of ntifeedncy from three nturl product repellents t 4 h ginst horn flies t three dosges (.2, 2 nd mg) in lortory iossys compred ginst control. There were no significnt differences mong repellent tretments (P >.5), nd only slight differences mong dosges (P <.5). wter). Five nimls were used for ech tretment. Cttle were plced in pens seprted y > m y tretment. Tretment pens were >5 m downwind from the control pen. Horn fly density on nimls ws recorded y digitl photogrphy t 1, 3, 6 nd 24 h fter tretment. The numer of flies in ech imge ws counted using GIMP Imge Editor (v.2.8; nd douled to express the totl numer of flies per niml. The numer of flies per niml ws nlyzed reltive to tretment nd time intervl with ANOVA. Mens were seprted with Tukey s HSD when the overll F-vlue ws significnt. 2.7 Relese rtes of the three repellents The relese rtes of the repellents were estimted y mesuring the sorption rtes of their mjor compositionl compounds [(Z,E)- nd (E,Z)-nepetlctone for ctnip oil, (2E)-3,7-dimethyloct-2,6-dien-1-ol for gerniol nd octnoic cid, nonnoic cid nd decnoic cid for C891 cids] with solid-phse microextrction (SPME) fiers ( μm of polydimethylsiloxne for ctnip oil nd gerniol nd 7 μm of polydimethylsiloxne/croxen for C891 cids; Supelco, Bellefonte, PA). Cttle hide (Turkey Creek Furs & Recycling, Crete, NE) ws cut into 1 cm 1 cm smples, nd 1 ml of repellent solution ws pplied to ech smple. Repellent-treted hide smples were plced outdoors in n open field, seprted y 1 m mong tretments in July. SPME fiers were plced 2 3 cm from the hide smples for collecting relesed voltiles (five hide smples per tretment) for 5 min. Collections were conducted t 1, 3 4, 6 7 nd h fter repellent pplictions. The reltive concentrtions of the compositionl compounds were nlyzed with n Agilent GC system equipped with DB-FFAP column (3 m.25 mm i.d.; Agilent Technologies Inc., Plo Alto, CA, USA). Helium ws used s the crrier gs, nd the flow rte ws mintined t 2.5 ml min 1. Smples were injected under the splitless mode. The temperture progrm for GC nlyses ws 5 C for 3 min, rising y 1 Cmin 1 to 23 C. The quntities of the compositionl compounds were ssessed y the externl stndrd method. Synthetic stndrds were weighed nd dissolved in hexne. Clirtion curves to determine linerity were otined for ech stndrd t 5, 1, 5, nd 5 ng μl 1 with three replictes per concentrtion. Linerity ws ssumed when the regression coefficient provided n R 2 vlue of >.98. The quntities of the compositionl compounds were otined y integrting the res of the stndrds peks nd clculting the concentrtions sed on the stndrd curves. The mount of compositionl compounds sored on SPME fiers s function of time since ppliction ws nlyzed with ANOVA (α =.5), followed y Tukey s HSD if the overll F-vlue ws significnt. 3 RESULTS 3.1 Antifeednt ssy Differences were oserved mong the three repellents nd the control (F = 389.2; df = 3, 125; P <.5) nd mong the three repellent doses (F = 7.7; df = 2, 125; P <.5). The interction term ws non-significnt (F = 8.9; df = 6, 125; P =.18). Fewer thn 15% of the flies fed when exposed to.2 or 2 mg of repellentm wheres 93.3 ± 1.9% of the control flies fed (Fig. 1). When exposed to mg of repellent, 2% or less of the flies fed. Among the repellents, A Percentge responses B Percentge responses C Percentge responses 6 * ** * 6 6 Tretment Control * ** * * * ** ** * *.1 g 1 g 1 g Figure 2. Behviorl responses of horn flies to the four dosges of three nturl product repellents (A, ctnip oil; B, C891 cids; C, gerniol) tested in single-cge olfctometer. Asterisks inside the columns indicte significnt differences etween the tretments nd control (P <.5). Pest Mng Sci (15) 14 Society of Chemicl Industry wileyonlinelirry.com/journl/ps

5 JJ Zhu et l. horn fly feeding did not differ significntly etween ctnip oil nd gerniol (Z =.24; P =.81), ut the ntifeedncy ws slightly higher with C891 (Z = 9.7 for ctnip nd Z = 9.6 for gerniol; P <.5). Horn fly knockdown time t mg dosge ws 2.58 ±.28 min for ctnip oil, 9.38 ± 1.68 min for gerniol nd 13. ±.3 min for C891 cids respectively. 3.2 Sptil repellency test During the course of the lortory ntifeednt iossy, we oserved tht horn flies plced in the repellent-treted cells tended to fly wy from the treted surfce, which indicted sptil repellency. The single-cge olfctometer demonstrted sptil repellency for ll three repellents (Fig. 2). Horn flies were strongly repelled from treted ports with repellent with dosges of 1 μg (t = ; P <.5). However, t the lowest dose tested (.1 μg), only gerniol repelled flies (t = 2.44; P <.5) (Fig. 2C). Overll, >% of horn flies tested in the olfctometer responded to either tretments or the control. 3.3 Antennl response Olfctory sensill of horn flies responded strongly to ech of the repellents (Fig. 3), compred with the controls (222 ± 13 μv for femles nd 159 ± 5 μv for mles, solute responses). Responses were similr for the three repellents t ech of the doses tested. However, significnt differences were oserved mong doses nd A 7 Femle A 1 μg 1 μg μg 6 5 A Reltive EAGS (μv) 3 B B B B B A B Ctnip oil Gerniol C891 Acids B 7 Mle 6 1 μg 1 μg μg 5 Reltive EAGS (μv) 3 c Ctnip oil Gerniol C891 Acids Figure 3. Reltive electrontennl responses of femle (A) nd mle (B) horn flies to three dosges of the nturl product repellents. Vlues re mens ± SE; different letters on top of the rs denote significnt differences (P <.5). wileyonlinelirry.com/journl/ps 14 Society of Chemicl Industry Pest Mng Sci (15)

6 Repellency of three nturl products ginst horn flies 6 h 24 h 48 h 72 h 96 h Control feeding 94% ± 2 9 c 7 Percentge feeding Ctnip oil Gerniol C891 cids Repellents pplied t mg Figure 4. Longevity of ntifeedncy effects of three nturl product repellents ginst horn flies t dosge of mg pplied to sheets ged for vrile periods in lortory iossys. Different letters on top of the rs indicte significnt differences (P <.5). etween the sexes. For femle horn flies, significntly higher EAG responses were elicited to the higher dosges (1 nd μg) of gerniol nd C891 cids (t = , P <.1), compred with those of mles. Among femles, responses were highest when responding to the intermedite 1 μg dose(f = ; df = 2, 15; P <.1). Antenne of mles responded strongly to thelowestdose(f = ; df = 2, 15; P <.1). 3.4 Effectiveness nd longevity of ntifeedncy Under the lortory conditions, the three repellents retined more thn 9% ntifeedncy during the first 24 h (Fig. 4). The efficcy of ntifeedncy of ctnip oil nd gerniol decresed significntly fter 6 h (F = ; df = 4, 45; P <.5), ut they still retined more thn 5% of their ntifeeding ctivity t 72 h. All three repellents lost more thn 7% of their ntifeeding ctivity fter 4 dys. Residul ctivity ws similr mong the three repellents during the course of the 4 dy tril. Averge feeding of control flies ws 94 ± 2%. During the field trils, ll three repellents strongly repelled horn flies. A significntly lower numer of flies per niml ws oserved up to 6 h fter ppliction (F = ; df = 4, 35; P <.1) (Fig. 5). Gerniol nd C891 cids retined repellency up to 24 h (F = ; df = 4, 35; P <.1). Prolte ws not repellent. Overll, the numer of horn flies on ll of the nimls, including the controls, decresed 2 dys fter tretment. 3.5 Relese rtes of the repellent compounds Differences were found from ll three repellents, ut only significnt in two repellents (ctnip oil nd gerniol: F = 7.51; df = 4, P <.1 nd F = 5.95; df = 4,, P <.1). Gerniol hd the highest relese rte with more thn decreses found t 6 h, compred with the other two repellents with losses. Almost zero sorption ws found fter 48 h from ctnip oil nd gerniol. 4 DISCUSSION AND CONCLUSION In the United Sttes, for horn fly control, insecticides re often pplied t lest 2 3 times per month to mnge them on pstured cttle. 6 Such frequent nd high-dose uses of insecticides rpidly led to the development of insecticide-resistnt flies. Resistnce to the two most commonly used insecticide groups, orgnophosphtes nd pyrethroids, hve een reported. 8 Therefore, novel, pproprite control strtegies tht minimize resistnce re needed. 6,8 Among the lterntive pest controls, iopesticides, including repellents, re of growing importnce, with glol mrket vlue of $US 3 illion nnully. 24 These products cn effectively control rthropod pests y cting s repellents or feeding deterrents of iting insects. The results from this study on the three nturl product feeding deterrents my provide useful informtion towrds n lterntive method for control of flies on psture cttle. Ctnip, Nepet ctri (Lmiles: Lmicee), is herceous mint ntive to Eursi nd North Afric, well known for its pseudonrcotic effects in cts. 25,26 Topicl ppliction of ctnip oil on humn skin cn prevent iting y severl mosquito species Recently, ctnip oil hs lso een reported s n effective ntifeednt ginst severl species of muscoid flies. 17 Gerniol is primry component of rose oil, plmros oil nd citronell oil (the Merck Index). Brnrd nd Xue 29 demonstrted its repellency ginst severl mosquitoes. Gerniol t 3% tested Pest Mng Sci (15) 14 Society of Chemicl Industry wileyonlinelirry.com/journl/ps

7 JJ Zhu et l. 25 Control Prolte Ctnip oil Gerniol C891 cids Men numer of horn flies per niml h 3 h 6 h 24 h Hours fter ppliction Figure 5. Numer of horn flies per niml fter tretments with repellents nd n insecticide t different hours fter ppliction. Different letters on top of the rs indicte significnt differences within time period (P <.5). on cttle s horn fly repellent effectively reduced fly density under the economic threshold ( per niml; Wtson W, privte communiction). Short-chin ftty cids (C 8,C 9 nd C 1 ) tested in lortory trils showed strong ntifeedncy ginst horn flies. 17 The present study using the modified K&D module to test these three nturl product repellents demonstrted strong ntifeedncy ginst strved horn flies, with over 9% efficcy even from the lowest dose (.2 mg) tested (pproximtely 8 μgcm 2 ). An verge of 9% ntifeedncy ws otined y the three test products during the first 4 6 h of testing; however, the effect declined to out 5% from dy 2 to dy 3, proly owing to voltiliztion. Asorption rtes of mjor compositionl compounds (ng min 1 ) Ctnip oil C891 cids Gerniol c c 25.8 c c c c 1 h 3 h 6 h 24 h 48 h Time fter ppliction Figure 6. SPME sorption rtes of their mjor compositionl compounds from three topiclly pplied repellents on cttle hides mesured t 1, 3, 6, 24 nd 48 hours. Different letters on top of the SE rs indicte significnt differences (P <.5). wileyonlinelirry.com/journl/ps 14 Society of Chemicl Industry Pest Mng Sci (15)

8 Repellency of three nturl products ginst horn flies In generl, the definition of nturl product repellent is sustnce found in nture tht elicits n voiding rection. Repellents cn e further chrcterized s contct repellents (ntifeednts in most iting insects) nd sptil repellents. All three repellents tested in the present study demonstrted strong ntifeedncy in the lortory feeding ssy. In ddition, tested flies were lso oserved eing driven wy from memrnes treted with repellents, which indicted sptil repellency. Our single-cge olfctometer ssy showed tht s little s 1 μg of ctnip oil or C891 ftty cids repelled horn flies. The strong electrontennl responses elicited from horn fly olfctory sensill to the three repellents re evidence of physiologicl response to the repellents s well. Among the three repellents, the vpor pressures of their mjor constituent compounds [nepetlctones, (E)-3,7-dimethyl-2,6-octdien-1-ol nd three short-chin ftty cids] re ll less thn 1. mmhg (25 C), which indictes tht they exist solely in vpor in the tmosphere. A reduction in EAG responses from oth sexes of horn flies from the repellents t highest dose my e induced y dpttion in the olfctory receptors, which hs een commonly found in moth sex pheromone communiction. 3 However, the highest EAG responses to 1 μgof ll three repellents oserved mong femle horn flies nd differences in EAG responses etween the two sexes to higher dosges of gerniol nd C891 cids remin unexplined. In the present study, ll three repellents seem to operte in the vpor phse. Repellents with high vpor pressure my offer protection t low concentrtions, ut ecuse of rpid voltiliztion, they my only provide limited residul ctivity. The results from SPME sorption nlyses supported the notion tht decreses in repellency re correlted with their fst evportion in the field. However, the numer of horn flies in the field ws reltively low y dy 2. It is uncertin tht the decrese in horn fly popultion in the field ws due to our testing or to other cuses. Ctnip oil hs een reported to hve contct nd fumignt toxicity ginst iting flies. 31 Gerniol nd C891 cids lso possess low toxicity. 18,19,32 Our lortory ntifeedncy ssys ( mg tretment) demonstrted tht the tested horn flies were knocked down in less thn 1 min from ctnip oil nd gerniol, which indicte their toxic effects s well. During the course of field trils, commonly used orgnophosphorous insecticide (Prolte/Lintox-HD) on eef/diry cttle ginst horn flies ws lso tested, nd no repellency ws oserved. However, owing to the windy conditions in the field, no ded horn flies were found round the treted cttle, even with reltively high lndings on the cttle. All three nturl products demonstrted strong ntifeedncy nd repellency ginst horn flies, ut with reltively short effective periods. More efforts need to e focused on prolonging residul repellent ctivity if these compounds re to e used for prcticl pplictions. Reifenrth et l. 33 suggested mixing severl repellents together for extended longevity. They discovered tht evportion rtes re slightly less nd tht repellency decyed less rpidly from mixing repellents reltive to individul compounds t the sme dose. More studies re under wy to evlute the extended longevity nd effectiveness of lends of the three repellents ginst horn flies in the field. Push-pull strtegies hve een proposed for confined livestock systems employing on-niml repellents nd ttrctnt-ited trps. 34,35 A modified push-pull methodology employing repellents on the mjority of the nimls nd few insecticidetreted trp nimls my e more effective. Interestingly, Prolte/Lintox-HD insecticide did not exhiit repellency, mking its use on trp nimls vile option. The reltively strong toxicity of gerniol nd ctnip oil my further help to kill the flies, while flies contct residues left on treted cttle hide fter the loss of sptil repellency. ACKNOWLEDGEMENTS We express our deep grtitude to B Voelker, B Mills, Dennis Berkeile nd T Weinhold for their technicl help with this study. We lso thnk Dr K Lohmeyer, USDA-ARS, Kerrville, Texs, for providing the horn flies for the lortory iossys. This work ws done in coopertion with the Institute of Agriculture nd Nturl Resources, University of Nersk Lincoln, nd supported prtly y Regionl Project 13. This study ws prtly supported y funding from USDA-NIFA ( ) nd USDA-CSREE ( ). REFERENCES 1 Byford RL, Crig ME nd Crosy BL, A review of ectoprsites nd their effect on cttle production. JAnimSci7: (1992). 2 Osorn H, Insects ffecting domestic nimls. 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