HORN FLY, HAEMATOM IRRITANS (L.), RESISTANCE IN ONTARIO TO

PYRETHROID AND ORGANOPHOSPHOROUS INSECTICIDES IMPREGNATED

IN CA= EAR TAGS

A Thesis

Presented to

The Facuity of Graduate Studies

of

The University of Guelph

by

SARAH M. BUTLER

In partial fulfilment of requirements

for the degree of

Master of Science

August, 1999

@ Sarah M. Butler, 1999

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HORN FLY, HAEMATOBLQ IRRlTANS (L.), RESISTANCE IN ONTARIO TO

PYRJZHROID AND ORGANOPHOSPHOROUS INSECTICIDES IMPREGNATED

IN CA= EAR TAGS

Sarah M. Butler University of Guelph, 1999

Horn fly, Huemcltobiu im5mrs

organophosphorous insecticides impregnatec

a-) 9

d in cati

Advisor: Dr, G-A- Surgeoner

raistance to pyrethroid and

Ne ear tags was monitored by tag

efficacy evaluation in Perth, and by gIass tube bioassay in Perth and Glencœ, Oatario.

Organophosphorous Protector* (20 % diazinon wlw) and ~ctoguard' tags (10 %

teaachlorvinphos w/w) provided no reduction of horn flies as wmpared to a non-treated

herd for both 1997 and 1998 seasons. Pyrethroid stockais tags (8 % cypermethrin wlw)

provided 23.1% reduction of flies in 1997, and ~ovaid* tags (8% fenvalerate w/w)

provided no reduction of horn flies in 1998. ~liminator' tags (11% dkinon, 6%

cypermethrin w/w), provided 71.7% reduction in 1997 and 60.1 96 reduction in 1998.

Resistance ratios at the LC, level for flies nom Perth to fenvalerate and diazinon were

108 - 253 and 2.2, respectively. Resistance ratios for flies fiom Glemm to fenviiierate

and diazinon were 289 and 3.3, respectively. Colorirnetric assays demonstrateci

significantly reduced (10-50%) acetylcholinesterase inhibition in resistant horn flies as

compared to susceptible flies. Chlorfenapyr (AC 303630). a potential new active

ingreclient for tags, was evaluated.

ACKNO'WLEDGEMENTS

I would Iüce to express my gratitude to Dr. Gord Surgeoner for providing the

opportunity to do this project, and for his guidance and assistance throughout. I would

also like to thank Dr. Keith Solomon and Dr. Jock Buchanan-Smith for their input and

involvement as cornmittee members.

This project was funded by the Ontario Canlemen's Association. Their finamiai

assistance and encouragement are gratefuly ackmwledged. Specgl th& to ail the cattk

producers who participated in this study.

The assistance ofDr. Robbin Lindsay and Dr. Steve Scott in setthg up this project

is great1y appreciated. Jamie Heal provideci invaiuable labotatory and cornputer assistance,

Jim Gallivan statistical advice and anaiysis, and Mary-Ellen Latham field work assistance.

Th& are due to Dr. Vic Park at Novartis Animal Health for providing ear tags,

and to George Amette at Cyanamid for providing chlorfenapyr. Special thanks to Dr.

Doug Colwell and technicians at Agriculture and Agri-Food Canada, Lethbridge Research

Centre for preparing assay tubes promptiy and fiequently. Gerry Finley ,O.V.C., and

Angela Martin, Elora Dairy, provided citrateci bovine blood.

Thanks to Mark Chappe1 and Tara George for their invaluable assistance and advice

with the acetycholinesterase assay. Braden Evans, Holy Teneg Akwar and Kier Taylor

provided much appreciated wmpany during the fiequent and long trips to Perth. Many

thanks to Ailison Butler, Laura Van Eerd, Rachael Cheverie, Diane Stanley-Hom, Mark

Hanson, Sandra Lastovic, Jeremy Allison, and my parents for their never ending support

and encouragement throughout this project.

i

.................................... ACKNOWLEDGMENTS i

... ........................................... LISTOFTABLES 111

.......................................... LISTOFFLGU RES iv

................................. 2 LITERATUREREVIEW 3 ...................... 2.1 Biology of Hacmatobia irn'tm (L.) 3

.................................. 2.2 Economic Impact 4 2.2.1 Weight Gain ................................ 4 2.2.2 MiIkPCoduction .............................. 5 2.2.3 Weaning Weights ............................. 5

.................... 2.2.4 Physiologid Effectî on Cattie 6 .......................... 2.2.5 Econornic Thresholds 6

................................. 2.3 Horn FIy Control 7 2.3.1 Chernical Control ............................. 7

........................ 2.3.2 Non-Chernical Control 10 ..................... 2.4 Insecticide Resisauioe by Hom Flies 11

................ 2.4.1 Mechanism of Pyrethroid Resistance 12 2.4.2 Mechankm of Organophosphorous Resistance .......... 13

................. 2.4.3 Rapid Development of Resistaace 13 ...................... 2.4.4 Negative Cross -Resistance 14

.................... 2.5 A Potential. New. Active Ingredient 15

........................... h!lATERIALS AND METHODS 16 .................................... 3.1 ZntfOduction 16

................................. 3.2 Ear Tag Efficacy 17 ................ 3.3 Resistance Ratio LC, G l a s Tube Bioassays 20

.......................... 3.3.1 Fenvalerate Biaassay 22 ........................... 3 .3.2 Diazinon Bioassay 22

3 -4 Chlorfenapyr Glass Tube Bioassay ...................... 23 3.5 Colorimetric Acetylcholinesterase Activity Assay ............ 24

3 .5 . 1 Detennination of Acetykholinesterase Activity ......... 24

RESULTS AND DISCUSSION ............................ 27 4.1 Ear Tag EEicacy 1997 ............................. 27 4.2 Resistance Ratio LC, Glas Tube Biosssay ................ 41

4.2.1 Fenvalerate Bioassay .......................... 41

4.2.2 DiazinonBioassay ........................... 46 4.3 Chlorfenapyr Glass Tube Bioassay ...................... 50 4.4 Colorimetrk Acetyicholinesterase Assay .................. 53

5 CONCLUSIONS ..................................... 55

...................................... 6 REFERENCES 57

LIST OF TABLES

Table 1

Table 2

Table 3

Location, ear tag treatrnent, and description of cattle and pasture shade conditions of f m used in 1997 and 1998, Perth, Ontario. Farm

........ numbers wf~:espond to numbered locations in Figure 1. -18

LC, LG, and resistance ratios ofpyrethroid and organophosphorous- resistant hom fies fiom Perth and Glencoe, ûntario, and susceptible flies from Rockwood, Ontario, detefmined &er 3 hours exposure to federate, 1998,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 42

LC, LC9, and resistance ratios of pyrethroid and organophospborous- resistant hom fies fiom Perth and Glencoe, Ontario, and suscepti'ble flies fiom Rockwood, Ontario, determineci &er 24 hours exposure to diazinon, 1998 ........................................ . .47

iii

LTsr OF FIGURES

Figure 1 Location of fauns at Perth, Ontario for the 1997 and 1998 ear tag .................................. efficacy study. -19

Figure 2 Mean number (+ SE) of hom fies (Huemafo~iu i r r i ~ ) per side ofcattIe (n=lO) at Perth, Ontario, that were non-treated (location 3) or were treated with two Protecto? (2Q96 diazhon wlw) ear tags @cation 2). Significant diffierences (PsO.05) between ~rotectop and the non-treated animais are

................................. indicated by an asterisk (*). 3 1

Figure 3 Mean nimber (+ SE) of hom flies (Haem~lrobia imm) per side ofcattle (n=lO) at Perth, Ontaxio. that were noa-treated (Iocation 3) or were tnated with two ~ c t o g u a r d ~ (LW tetrachlorvinphos wlw) ear tags (location 6). Significant Merences Qa0.05) between Ectoguardo and the non-treated anirnals are indicated by an asterisk (*). ......................... 32

Figure 4 Mean number (+ SE) of horn fiies (Haemutobia imfm) per side ofcattle (n=10) at Perth, Ontario, that were non-treated (iocation 3) or were treated with two Stocka@' (8% cypermethrin w/w) eu tags (location 5). Signincant ciifFerences Ps0.05) between Stockaido and the non-treated animais are indicated by an asterisk (*). ........................ 33

Figure 5 Mean number (+ SE) of horn nies (Huematobia Mtcms) per side of cattle (n=10) at Perth, Ontario, that were non-treated (location 3) or were treated with two ~lirninator~ (1 1% diazinon, 6% cypermethrin wlw) ear tags (iocation 4). Significant merences (Ps0.05) between ~liminat0P and the

............... non-treated animals are indicated by an asterisk (*). -34

Figure 6 Mean number of hom fies (Haemutobia imiftms) per side of cattle (n = 10) at Perth, Ontario, that are non-treated or are tteated with two ~rotector~ (20 % diazinon wfw), two &toguardœ (10 % tewchlo~nphos wfw), two stockaïde (8 % cypermethrin w/w), or two E l i m i n a d (11 % diazinon and 6 % cypemethrin wlw) ear tags, 1997. .......... -35

Figure 7 Mean number (+ SE) of hom flies (Haemattobia i r r i ~ ) per side of cade (n=10) at Perth, Ontario, that were non-treated (location 3) or were treated with two ~rotector* (20% diazhon w/w) ear tags (location 1). Significant diierences (Pr0.05) between Protectorœ and the non-treateà animals are indicated by an astensk (*). ................................. -36

Figure 8 Mean number (+ SE) ofhorn Ses (Haematoobia intant) per side o f d e (n=10) at Perth, Ontarh, that are non-treated (location 3) or are treated with two Ectogud (1% tetrachlominphos w/w) ear tags (iocation 6). Si@cant daféruices (Ps0.05) between -oguardo and the non-treated

........................ anirnals are indicated by an astaisk (*). -37

Figure 9 Mean numba (+ SE) of hom fies (Haematobiu irritclns) per side of cattle @=IO) at Perth, Ontario, that are non-treated (location 3) or are treated with two Bovaidœ (8% fenvafemte w/w) ear tags (iocation 5). Sipinuint ditferences (Ps0.05) between Bovaidœ and the non-treated animals are indicated by an asterisk (*). ................................. - 3 8

Figure 10 Mean nuniber (+ SE) ofhom fies (tiixmaiobiu imrons) per side o f d e @=IO) at Ped~ , -*O, that are non-treated (location 3) or are treated with two Elirninato~ (1 1% diazinon and 6% cypermethiin wlw) ear tags (location 4). SisnifiCant differences @sO.OS) between ~fiminator@' and the non-treated animals are indicated by an asterisk (*). .............. .39

Figure 1 1 Mean number of hom nies (Haentcltobiu ikriiarrs) per side of cattie (n= 10) at Perth, Ontario, that are non-treateû or are treated with two Protectorœ (2û% diazinon wlw), two Ectoguardœ (100h tetrachiorvinphos wlw), two Bovaid. (8% fenvalerate w/w), or two Eliminator@ (1 1% d i d o n and 6% cypermetbrin wfw) ear tags, 1998. ............... -40

Figure 12 Mortality dose-response with 95 % confidence limits for pyrethroid and organophosphorous-cesistant hom flies (Haematobia iet411s)

LC, =O.Os8 fkom Perth, Ontario, and susceptible flies LC,=O. 15xlû3 fiom RockWood, Ontario, exposed to fenvalerate for 3 hours, July

.........-..... ................-...*.. 10/11, 1998. .. -43

Figure 13 Mortality dose-response with 95 1 confidence limits for pyrethroid and organophosphorous-resistant hom flies (Hmmatobia imtMI) ~ ~ ~ = 0 . 4 l x l û ~ fkom Penh, Ontario, and susceptible flies ~C,=0.45~10~ fkom Rockwood, Ontario, exposed to fenderate for 3 hours, August 20121, 1998. ........................... -44

Figure 14 Mortality dose-response with 95 %-confidence limits for pyrethroid ancl organophosphorous-resistant hom flies ( H m ~ o b i a imlmrr) LC,=O. 12 fiom Glencoe, Ontario, and suscepti.%le flies ~C,=0.45~10~ from Rockwood, Ontario, exposed to fenvaierate for 3 hom, August 20/21, 1998 ..................................... . . . . . 45

Figure 15 Mortaiity dose-response with 95% confidence limits for pyrethroid and organophosphorous-resistant horn flies (Hmmatobia im't~~t~) KM = 1-29 from Perth, Ontario, and susceptible flies LC,=0.59 from Rockwood,

... Ontario. exposed to diazinon for 24 hours, August 20/21, 1998. 48

Figure 16 Mortdity doee-response with 95% confidence limits for pyrethroid and organophosphorous-raistant horn flies (Haem4tobt'a irn'tm) LCC = 1.94 fcom Glencae, Ontario, and susceptiile flies LC, = O . S fiom Rockwood, Ontario, expoJed to diazinon for 24 hours, August 20/21, 1998 .......................................... 49

Figure 17 Mortality dose-responre with 95% confidence limits for pyrethroid and organophosphorous-resistant horn flies (Hcumatobia imtmrr) LCm =O.Ol3 nom Perth, Ontario, e x p d to chlorfenapyr for 24 hours, September 4, 1998. Morialiîy of susceptible flies from RockWood, Ontario. was 1 0 % at d l concentrations presented. ........... -51

Figure 18 Mortality dose-response with 95 % confidence limits for pyrethroid and organophosphorous-tesistant horn flies (Hocmatobia irriians) LC,=0.012 fkom Glencoe, Ontario, exposecl to chlodenapyr for 24 hours, September 5, 1998. Mortality of susceptible nies fkom

. . . . Rockwood, Ontario, was 100% at al1 concentrations presented 52

Figure 19 Percent of inhibited acetylcholinesterase activity in pyrethroid and organophoaphorous-resistant horn fly (Haemutobia irn?izns) heads fiom Perth and Glencoe, Ontario, and susceptible horn fiies nom Rockwood, Ontario exposed to wncentratiom of diazinon for 24 hours, August 20

.................................. and21,1998.. 54

1 GENERAL INTRODUCTION

The horn fly, Haematobia imï&ns (L.), is an econornic pest ofcattie caushg major

losses ($730 million US.) to the North Amencan livestock industry annudy @yford a al.

1992). Since its introduction in the early 19808s, the slow-release insecticide- impregnated

ear tag has been the most popular and economical method of control because tags can provide

excelient season-long control with one application (Ahrens and Cocke 1979; Müla et al.

1984; Lanaister et al. 1991; Byfbrd a al. 1992; Surgeoner a al. 1994). Howeva; because

of the widespread and annual use of in&cidal ear tags, resistame to the pyrethroid and

organophosphorous insecticides used in tags rapidly developed in the United States (Sparks

et al. 1985). Faiiure of pyrethroid ear tags was first reported in Ontario, in the GIencoe area

(Surgeoner et al. 1994). Lindsay et al. (1996a) reported organophosphorous eartag fkilure

near Perth, Ontario, and Butler et ai. (1998) observecl hom fly resistance to both

organophosphorous and pyrethroid ear tags for the first time in the same area.

The following objectives were fuifilied to test the hypothesis that ear tag Mures

reported in the Perth area are the result of multiple resistance by hom Dies to

organophosphorous and pyrethroid insecticides used in tags; i ) evaluate the efficacy of

different ear tag formulations (Lindsay et al. 1 W6b) containhg organophosphorous,

pyrethroid, or a combination of both insecticides, and iï) detennine the resistance ratio of

hom Ses to diazinon and fenvalerate by laboratory LC, glass tube bioassays (Cilek and

Knapp 1991; Kunz et al. 1995; Lindsay et al. 1996a,c). The efncacy of a potential, new,

active ingredient for ear tags, chlorfeaapyr (AC 303630) fiom American Cyanamid, was

evaluated for control of organophosphorous and pyrethroid-resistant hom nies by laboratory

LC, glas tube bioassay (Sheppard and Joyce 1998). As weU, colorimetric assays (EIIman

et al. 1961; Bradford 1976) were perfîomed to measure acetylchoiinesterase activity in

organophosphorous resistant and suscepn"b1e horn fües.

Supplernentary experiments were conducted in 1998 on organophosphorous and

pyrethroid resistant hom fies coiiected nom the Glencoe ana in response to cornplaints of

poor horn fly control by producers. Diazinon and fenvderate resistance ratios were

determineci by LC, glass tube bioassay, &cacy of chlorfenapyr was e-valuated byLCw glass

tube bioassay, and colorimetric assays were performed to muisure acetylchohesterase

activity in organophosphorous resistant and susceptible horn Bies.

2.1 Bidogy o f H4ematobia h * f o r r s (L.1

The horn fly, Haematobia mians ( L i i s ) , (Diptera: MuSadae), was introducod

accidentab nom Europe into the United States in 1887 (Rilq 1889 nted in Fuicha 1990).

By the turn ofthe century, horn flies had spread throughout the U.S.A and into Canada

(Kunz and Schmidt 1985).

Both male and f d e adult horn flles an obligate blood-fidhg parades of d e ,

b&o and occasionally horses. Adults fad 20-38 timdday, collsuming up to 14.3 mg of

blood/fly/day (Harris and Frazar 1970). with peak feeding tirne ocanring between l lam and

3pm (Knapp et al. 1992). Hom fies spend most of theiraduit lives on their ho@ kaviag the

host only to oviposit, to move to otha uiimals, or whm animais enter buildings m p et

al. 1992). Adults are nonnally found dong the shouiders and back ofcattle, but take refùge

on the ventrai abdomen durhg rain or on particulady hot, sunny days. Bulls are more

attractive to horn flies than cows, with numbers reachhg 4000 on non-treated b d s and up

to 1500 on non-treated cows by mid-August in the southem U S A Wunz et al. 1984).

H o m fies overwinter as pupae in soil nuu or under manuce pats. Adult horn flies

emerge £tom puparia in late April to June and immediately seek cattle hosts upon which to

feed and mate. Adults fly up to severai kilometres to find a host (Byford a al. 1987a; Mey

et al. 1991). The female horn fly lays up to 400 eggs during her three to seven week Wee

Eggs are laid in batches of 12-20 in fhsh undisturbed cattle faeces. Eggs batch in one to two

days and larme feed within the manun pat for four to ten days before pupating under the pat

or in soi1 beneath the pat. Adults emerge in five to thirteen days to repeat the lifè cycle.

3

Depending on the temperature, the egg-to-egg Iüe cycle of the hom fly takes thme to five

weeks, producing up to four generations in Canada fiom late spring to the fht fiost.

2.2 Economic Impact

The hom fiy causes major economic losses to the North American livestock industry.

In the UNted States, hom flies are estimated to cost the cattle industry $730 d o n anmudly

(Byford et al. 1992). In Canada, an estimatecl S455 mülion in lost production ocnirs smnully

as a result oflivestockectoparasites (Hadieand W.intraub 1985). Losses in productionfkom

hom fly parasitism are maUily due to irritation which d s in: diminished weight gains,

reduced milk production and decreased weaning weights of calves (Palmer and Bay 1981;

Byford et al. 1992; Baron and Lysyk 1995). 0th- -ors that crin c o n t n i to production

losses include anaemia fiom blood loss in seven infestations (Hams and Frazar 1970), as well

as transmission of the nlarial nematode SfephaMlfiGoria stiyesi which causes bide damage

(Steelman 1976).

2.2.1 W&gM Goin

Hom fly parasitism Uinuences behavioural activities of catt1e which can r d t in

diminished feed intake and reduced fad conversion efiiciency(Byford et al. 1992; Baron and

Lysyk 1995). Harvey and Launchbaugh (1982) observeci that non-treated cattie grand lea,

swished theirtails more fkquentIy, and traveiied 0.5 km per day more than treated cattle with

no hom flies. Laake (1946) found thrit an infestation of horn fies caused a reduction in

weight gain o f 14-23 kg live weight in both cows and suclding calves o v a a 5 month p e r i d

H d e (1982) and Derouen a al. (1995) demonstrateci that cettie treated with insecticide

impregnated ear tags gained approxhady 17% (21kg) more thrm non-treated d e over a

16 week period Kinza et ai. (1984) reported a 12% (0.04 kg per day) weight gain

advantage in unuifested yearling stem as compand to infesteci steers o v a a 120 day study.

In addition, hom By control improved steer f d conversion efliciency by 9?% (Kinzer et al.

1984).

2.2.2 Mi IRmcriOn

The eariiest report evaluating the economic importana ofthe horn fly is that ofBeach

and Clark (1904, cited in Palmer and Bay 1981) who reported that cattle treaîed with fly

ointment did not produce mon milk than non-treated cattie. However, Freebom et al.

(1925), Bruce and Decker (1947), and Granette and Hansens (1956, 1957) obsaved

O. 14-1 -79 kg of milk loss per day in d e that were infested with lM0 hom fies. Steelman

(1976) repoaed that hom fly wntrol increased milk production by 37.5 lb during a five week

period-

2-2-3 Wmœng Wight3

Studies on the effêcts of reduced mük production on weanuig weight o f calves have

had variable results. Essig and Pund (1965) found that horn fly i n f ~ o m did not influence

the weight ofweaning calves as a result ofdecreased mük production from cows. In contrast,

Carnpbeii (1976), Kunz et al. (1984), Quisenbeny and Strohbehn (1984), and SteelmaD et ai.

(1991) reported a 5.8 - 7.4 kg increase in cllf weaning weights when wws wae protected

£tom hom fies.

2-2- 4 P h y s Ü ~ b ~ E f f i on (70rmc

Schwinghamrner et al (1986) and Presley et al. (1996) measured physiological and

nutritional responses ofbeefsteers to horn fly uiféstations. Higher heart and respiration rates

as weil as increased rectal temperatures were observeci in s t a n exposexi to X û û fies per

animal as cornpared to d e fkee of fies. Wsta collsumption, urine production, andiirinsry

nitrogen exCretion were increased when steers were a c p o d to >250 fies per animal. Dry

matter digesti'bility and nitrogen digestib'ility did not dWèr from stem fia of Bies, however

nitrogen retention was reduced in gr infksted steers.

2-23 Economr'c Tkrcskodds

The number of hom flies required to cause economic injury to cattle has been studied

by several researchers who found varyïng results. Butkr (1975) reported a threshold of 50

fiies per animal caused economic injury. Knapp et el. (1992) detennined a level of 100 fies

per animai, whereas Schrel'ber et al. (1987) and Hogsette et al. (1991) reported economic

injury at 200 or more flies per animal. W e (1986) proposed a mode1 where two

mechanisms affect cattle productïvity, each having its own thnshold densiq. The first

mechanism is a quantal response to low Unestation levels with a threshold of 12 fies per

animal. Produdivity is reduced by 17-2(r/. due to the e f f â on neurosecxetoxy responses,

grazing and social behaviour, and utilization offorage. The second mechanism is a graded

response when there are more than 230 Bies per animal. As a r d t of the additional stress

caused by fly f&g, the productivity ofthe animaL is reduced as a hear bction offiy

density.

2.3 Horn Fïy Contiol

. . Economic benefits ofhom fly mntrol in Canadian grazing systems are magmized if

fly-fke grazing is maintained for at least 115 dpys (EIaioe 1986). This requires initiation of

a control mame eady in t h e s p ~ g More the fint flics amrge and mahtenance throughout

the summer and into the fidl (Lysyk and Colweli, 1996).

2J.I Ckemicd Gritrol

Direct application of insecticides to the animal is the most common and effective

method ofhom fiy control. Various methods of insecticide appücation, such as sprays, dust

bags, back rubbers, pour-ons, and insecticide-impregnated car tags, are used for controhg

adult horn fies on cade, In addition, insecticidal feed and mineral additives as weli as

insecticidal rumen boluses are available (umently only in the U.S.A.) to control the

development of the larval stages in manure.

Sprays containing pyrethnim extrsct are the earliest recorded mdhod of chernical

control of hom flies (Freebom a al. 1925). In the late 194û'q DDT and other

organochlorines (methoxychior, toxaphene) were remmmended in the U.S.A for hom fly

control (Laake 1946; Sparks et al. 1985). In the 1960's the number of compounds adab l e

for control expanded to include organophosphorous (fenchlorfos, domate , malathion) and

carbarnate (mbaryl) insecticides (Sparks et al. 1985). C d y , both p y d m i d and

organophosphorous insecticides are available as back or wholebody sprays. Although sprays

can provide excellent horn fly reduction, lW? (Ladce 1946; Sparks et al. 1985). the resiiiuai

control lasts hom to days, and thus cattie must be re-sprayed on a dULy to weekEy basis. As

well, some r&ctions apply to lactating diny cows and to the withdrawd period More

slaughter. The high labour requkements plus the stress to cattle in herdhg and handling

offset the benefits of sprays as an economic method of hom fiy control @&y 1996).

Dust bags and back-rubbers are seEtreatment devices that dispaue insecticide to au

animal in either fiee-choice or forced-use situations. Excellent fly control can be achïeved

(Kessler and Bemdt 1971), howeverY much labour is involveci to ensure the deMce is not

empty or rnalfhctioning. Free-choice application can result in the ova-application to some

animals and little to no treatrnent ofothers. Cher-application of insecticide in tum mcreases

the cost of control and potentially lead to residue problems. Subletha1 doses of insecticide

increases the likelihood of resistance development(Ge0rghiou and Taylor 1977). Forcedvse

requires the animal to pass under the dust baglback-rubber to drink, eat, get shelter, or to

enter milking barn. Again, over-application of insecticide can be a problem. As weUy ifthe

animal is anaid ofthe device they may choose not to eat or drink and th& pdonnance wiU

suffer May 1996).

Polywlyl chloride (WC) ear tags containhg organophosphorous, pyrethroid, or a

combination of both insecticides reduce hom fiies by >go?!% to >99?/. for the entire season

(Ahrens and Cocke 1979; Miller et al. 1984; Lancaster et al. 1991; Byford et al. 1992;

Surgeoner et al. 1994). The insecticide, formulateci in the PVC polymer ma* is slows

released f?om the matrix in a continuous but slowly declining Miount and deposited on the

haïr mat of the animal for many months (MiUr et ai. 1983; MwangaIa n al. 1993).

Residuw deposited on body parts of cattie vary in time and space because of

dïerences in insecticide release rate, rate of spread and degradation on hair mat, and

behaviour ofanimals (Beadles et al. 1977; Miller et al. 1983; Mmgaiaetd. 1993). Beause

ear tegs provide exceuent hom fly reduction nequiring one application per season, they have

gained widespread acceptane as on efficient method of c o d (Schmidt et aL 1985; -

McDonaid et al. 1987). Howmr, the continued use ofear tags since t h e introduction in

the early 1980's has lead to hom fly mistance to pyretbroids in most areas of the Unitcd

States (Sheppard 1984; Qwsenberry et ai. 1984; Schmidt et al. 1985; Knapp a 1 1992),

some areas of Canada (&fwaagda and Gall0wa.y 19934b; Surgeonet et ai. 1994; Liadsiiy et

al. 1996b,c), and South Ametica (Sheppard and Toms 1998). Resistance to

organophosphorous insecticides has also been observed in many areas of the Umted States

(Sheppard 1983; Harvey et ai. 1984; Cilek a al. 1991) and some amas ofCanada Gindsay

et ai. 1996a).

Inseaikide feed and minerd additives as well as insecticidal rumen bolunes can destroy

100% of the developing larval caek and Knapp 1991) md/or pupal stages (Moon a ai.

1993) in manure. These fads contain stirofos or one ofthe insect growth regulators @GR)

diflubenzuron or methoprene. Boluses are retained in the animal ' s reticulum and slowly

erode, providïng a constant release of insecticide into the digestive system and manure (C ik

and Knapp 1991). Feed and mineral additives pass through the digestive system to the

manure but do not pmvide a constant insecticide release because some mimais choose not

to eat the additive (Moon et al. 1993). Although additives and boluses can provide 1W/o

mortality of hom fly larvae a d o r pupae in muune, thae may not be a comsponding

reduction in the number of fies on the animal. Newly emaged ad& hom flns fkom a

neighbouring herd can repopulate d e treated with additives or boluses (Byford et PL 19873-

M d e y a al. 1991).

2.3.2 Non-C'hcnÙcal C o d

Because of the rapid development of resistance by the hom fiy to insecticides, non-

chernid control strategies are sought. Fhcher (1990) rewiewed biological control of dung

breechg flies including the hom fly. Nme of twenty-nine na& enemy and cornpetitor

species of Coleoptera, Hymenoptera, and Diptera released in the US.A have established.

However, the impact of biologicai control agents is dependent on several ecologicai fâctors

uicluding seasonal distn'bution and habitat preference. As a r d t , few biocontrol ageats have

been successfùl in wntrohg hom fly populations (Ficher 1990). Also, a biocontrol

program should be area wide, otherwise a nduction of lervae in the manure of a treated site

may not result in a large reduction of adults because of emergmt Ses fiom neighbouring

herds (Byford et al. 1987a; Marley n al. 1991).

Surgeoner et al. (1996) and Tozer and Sutherst (1996) reported traps designed to

brush off and capture adult flia as cattle w a k through provided 84% and >900! reduction

of horn fies on dajr cattle, respectively. However, both studies reporteci producer concem

for cost of the trap and labour involved in encouraging cattle to use the trap. Tozer and

Sutherst (1996) found the trap was unsucced with range cattle, in some cases causing los

of production in forceci use situations.

Increased resistance to horn %es by Brahman cattle breds has bem exarnined.

TugweU et al. (1 969) reported a decrease in hom fly numbers d g as the percentage of

Srahman genetrDcs increased in two year old hcinn. Derouen et ai. (1995) demonstraîed

yearling cattle with 25-50036 Btabman background gained 17% mon weight over three years

as compareci to non-Brahman background, when hom fly wntrol was maintaineci at 68% or

greater reduction. Steelman et ai. (1993) observcd d e with Brahm~ genetic background

atïracted sigaiocantly fewa organophosphorous resistanrflies as c o m p d to d e with no

Brahman background, suggesting that breeding is a potential resïstance management tooL

2.4 Insecticide Rwistuice by Hom Flics

Difnwlty with hom fly contr01 wm first observecl in the late 1950's when DDT no

longer provided efféctive control in some areas of the United States (Bums et al. 1959; Lewis

and Eddy 1961). Resistance to DDT and related compounds such as methoxychlor and

toxaphene was eventually confinned (McDufEe 1960; Hams 1964; Byford et al. 1985).

Burns and Wdson (1963) demonstrated horn fly resistance to fenchlorphos inlouisiana a f k

intensive use of the chernical in back rubbers for three co~lsecutive y-; HMis e$ al. (1966)

colonized the nies h m Louisiana in the laboratory and demonstrated that they were 50 b e s

more tolerant to fenchlorphos than laboratory-rd tlies that had bem colonized four years

earlier. Through the late 1960's and 1970's research on resistance deciined greatly

(Georghiou 1980; Byford et al. 1985). A f k the introduction of insecticide impregnated ear

tags, a rapid rise in hom fly resistance to the active ingrdents used wcis obmed in the

United States (McDonald et ai. 1987; Sheppud et ai. 1989). Demonstrable resistance to

11

stirofos and fenvalerate in ear tags ocaimd in hom fies a f k two seasons of use in Georgia

(Sheppard L983,1984), and with pyrethroids in Louisinna (Quisembeny et ai. 1984). Byford

et al. (1985) and Sparks a al. (1985) demonstratedthat pyrethroid resistanceis characterized

by a broad spectrum of cross &ance to virtually aU commerciaUy adable pyrethroids as

weii as to DDT and methoxychlor. However, the cross-resistmt spectrum of pyrethroid-

resistant hom flies aomially does not extend to the organophosphorous and arbamate

insecticides (Bflord et al. 1985,1988)- Mriisterd, negatbe cross-resîstance ocaus whaàa

resistance to one insecticide clas confers increased toxicity to an unrelated class ofil~~~ectrkide

when compand with a saain not exposed to insecticides (Ciiek and Knapp 1993b).

2.4.1 Mechanlsrn of qLm!klOid Rabtance

The mechanisms of pyrethroid resistance Ui the horn fly have ken investigated

extensively. Studia hdicate that pyrethroid raistance is due to a combination of factors

including knockdown resistance 0, target-site hemitivity (8yford et ai.1985;

McDonald and Schmidt 1987) and enhanced degradation of the insecticide by esterases and

other enzymes (Mord et d.1985; Bull et d.1988; Spprks et ai.1990; Sheppard 1995). Xu

and Buii (1995) detected higher levels of carboxyiesterase activity in pyrethroid resistant hom

flies as compared to susceptible fies. Modifieci behaviour o f the fiy is also involved in the

resistance of hom flies to pyrethroids. Quisenberry a I (1984), Lockwood a ai. (1985) and

Byford et al. (1987b) observeci resistant individuals redistriiuting thanselves to non-treclted

areas of w tagged cattle such es the beUy and the hind legs. Lockwood ct al. (1985)

determineci that behaviod resistance is characterized by a lower sensitivity thnshold

causing hyperirritabîky and repeliency in mistant individuals, and that these symptoms are

increased in the presence of the synergïst pipemnyI butoxïde. Zyzak et al. (1996) conormal

that increased irritability and repellency is seen with the addition ofa synergist in a resistsnt

popuiation exhibithg KDR.

2.42 MechoniSm of ~ a n o p b s p h o ~ Redhnce

The mechanisms of org~ophosphorous resistance in the hom fly hve not been as

thoroughly studied. Reduced sensïîivity of the acetylcholinesterasc enqme to

organophosphorous inhibition is a conmon resistance mechanism in insects (EIams 1983).

Other mechanisms of organophosphonws resisîana have been proposed wbich hclude

sequestration and metaboüc detoxification by ester hydrolysis (Puker a al. 1991).

Unpublished research on susceptiile hom flies by Pmett a al. (1998) indicaie that with aghg

the total esterase activity in adults'increases while sensitnRty to diazinon decreases.

2.4.3 Rapiif DeveIopmenî of R-

The rapid development of resïstance to pyrethroid insecticides fonowing the

introduction of impregnated ear tags has been reviewed (Sparks et al. 1985; Sheppard et ai.

1989; Knapp et al. 1992). The steady release but deciinhg dose ofchernid dispaisod by the

ear tag is a system that rapidly selects for resistance (Brown 1971; Georghiou and Taylor

1977; Sheppard 1987). Initidy the insecticide concentration is sufncient to kül ail but the

homozygous resistant types (McDonald a al. 1987). Although homozygous re&tant types

are rare in a previously non-treated population, because the fiequency of the gaie in an

unselected population would be low, the rrsi*stmt geme is present in heterozygous individds

(Sparks et al. 1985). As the dose of chemicai delivemi fiom the tags dcaeases, some

individuals heterozygous for cesistance can s u ~ v e wMe the homozygous susceptible

individuals are eliminated (McDonald e!t aL 1987). As the hetaozygotes Uaerbreed and the

susceptible individuais continue to be etiminated, the genetks ofthe population quickly M s

to one wntaining a high fiequency ofhomozygous resistnnt UdMduals (Sparks et al. 1985;

McDonald et al. 1987; Sheppard et aL 1989). $ p h et aL (1985) and McDodd et al.

(1987) suspect that resistance is accelerated in many U.S.A. populations because of the

numerous generations (5-20) ofhom fiies exposed to the same errtag throughout one stsson.

2.4.4 N e g d k C~oss -Rcsistrrnce

Enhanced toxicity of chemids to insecticide-cesistant hom fies has b a n reportai in

severai studies. Byford et al. (1988) and Cilek and Knapp (1993rgb) found that the toxicity

of diazinon increased as susceptibility to pennethrin decreased in hom flies. Sheppard aud

Joyce (1998) detennined that chlorfenapyr (AC 303630) is 5 times more toxic to pyrethroid-

resistant horn fies as to susceptible fies. Cilek a al. (1995) obsaved increased activity of

mixed fiinction oxidases thus dowing the pyrethroid-resistant individual to convert more

diazinon to the insecticidal fom. Rotating chernical fâmifïes to d o w for negative cross-

resistance is an important tool in slowing the development ofœIIISeCticide resi*stance (McKenzie

and Byford 1993).

2.5 A Potcatiil, New, Active hgrdient

Chiorfenapyr (AC 303630), an N-substituted haiogenated pyrrole dmloped by

American Cyanamid, is cumently bang testecl as a poteutid new active hgmdient f& control

of hom fies (Sheppard and Joyce 1998; Doscher et al. 19984b). C h l o r f i fùnctions as

a propesticide, converthg to an active toxic fom AC 303268, by Ndealkylation withh the

insect (Black et pl. 1994). The toxic fonn kiiIs the inscct by stoppbg energy production in

the mitochondria as a remit of uncoupliag oxidative phosphoqhtion ('ïregcy et aL 1994).

The mode ofadon of chlorfenapyr is distiactly differcnt firom the insecticides wreatly used

for hom fly cWol.

Prototype chlorfenapyr ear tag fomulatians containhg 3W active ingicdient

provided >95% reduction of hom fiies for 18 weeks, as compared to non-treated d e

@oscher et al. 199%). Bioasays Wormed by Sheppard and Joyce (1998) found

chlorfenapyr to be five times as toxk to pyrethroid-resistuit horn flies as compared to

susceptible fies. The novel mode of action of chlorfenapyr comb'ied with the m e cross-

resistance to pyrethroids demonstrates that chlo&napyr has potential as a new managernent

tool for horn fly control.

3 MATERIALS AND METEODS

3.1 Introduction

In Ontario, horn fly nsistance to insecticides used in ear tags was iht reporteci in

1994 in the Glencne ana (Surgeoner et al. 1994). At that the, the LC, for resistant %es

exposed to fenvalerate was 0.04 @#cm2 compared to an LC, of 0.0002 pg/cm2 for

suscepti'ble flies, thus the resistance ratio was 267 (Surgeoner et al. 1994). In 1996, as part

of an evduation of new ear tags f o d a t e d cig9nst pyrethroid-resi0stant horn fies, M e r

bioassays were performed on Giencoe horn fües to determine the Ievel of resistance ta

fenvalerate and cypermahnn (Lindsay et ai. 19%~). Resïstance ratios wae 249 for

f d e r a t e and 634 for cypermethrin (Liidsay a ai. 1994%). In addition, horn fies rcastcmr

to tetrachlowinphos were obmed in the Perth area (Lindsay a al. 1996a). Mortiüty ofhom

fies coliected fiom tagged animals near Perth and exposed to concentrations of

tetrachlonhphos ranged nom 7.3 - 40% whereas mortality of susceptible flies ranged fiom

92 - 100% when exposed to the same concentrations (Lindsay et aL 1996a). Failwe of both

organophosphorou~ and pyrethroid ear tags was obsaved in the Perth a - in 1997 (Butler

et al. 1998).

The objectives of this research were to monitor the efficacy of organophosphorous

and pyrethroid impregnated ear tags in the Perth ana, to detemùne the resistana ratio of

horn fies to diazinon and fenvdemte by LC, glass tube bioassay (Cüek and Knapp 1991;

Kunz et ai. 1995; Lindsay et al. 1996a,c), and to evduate chlorfenapyr (AC 303630) by giass

tube bioasszy as a potentiai aew active ingredient in tags for horn fly control (Sheppard and

Joyce 1998).

Colorùnetric assays @liman a al. 1961; Bradford 1976) were per5onned to masure

acetyicholinestefa~e h v i t y in organophosphorous resistant and suscepa'ble horn flies.

Supplementary experirnents were conducteci on hom fies collected fkom Giencoe in response

to producers' conams ofhigh hktations. Diazinon and fenderate rrsistance d o s were

detennined by LC, glas tube bioassays. Chlorfenapyr efficacy was evaluated with Oiencoe

organophosphorous and pyrethroid resistant hom fies. As well, acetylcholineserase activiry

was measured in organophosphorous horn fies

3.2 Ear Tag Eniucy

Five separate herds of cattle ofmixed b d s (ca. 30 - 50 anirnals per herd), located

within ten küometres of each otha in the Perth area, were used in thk trial (see Figure L and

Table 1). During early Iune 1997 and late May 1998 animais in each herd were tagged with

either two ~rotector. (20% diazinon w/w Novartis), or two Ecto~ardO (10.h

tetrachlorvinphos w/w; Boehringer Ingeîheim)' or two Stockaida (8% m e t l u i n w/w;

Novartis), or two Eliminator@ (1 1% diazinon and 6% cypermethrin w/w, Novartis) ear tags.

in 1998 the Stockaido treatment was replaced with Bovaido (8% favalerate w/w; N o d s ) .

AU animals within a herd received the same treatment. The fiAh herd was non-treated and

served as a control.

At approximately weekly intarals, the numba of adult horn flies per one side of

animal were counted from ten randornly sdected animals within each h d . Counts for aii

herds were made on the same day between 11:30 am and 3:30 pm. Air temperature, wind

conditions and percent cloud cover were recordeci during each sampiing intenal rad counts

Scale: 1 division = i km

Figure 1 Location of fams at Perth Ontario for the 1 997 and 1 998 ear tag efficacy study.

were not perfonned on unseasonably cool days or when high winds (>25 kmph) or iam were

foitcast-

Hom fly were counted weekly after tag application unil the first frost for both

1997 and 1998 seasons. In 1997, efIicacy was obsaved for twelve weeks pst treatment

fkom June 21 - September 5. For the 1998 season, counts were made one week prior to

treatment Way 22) and efficacy was observed for 17 weeks pst treatment h m May 30 - September 21.

Differ~cesinthenumbaofhomfües onaounalsintheseparatetrratmentherdswere

determineci using analysis of variance (-A) and cornparisons at Pr0.05 were made on

weekly means and on pooled data for the entire season using the Dunnett's test (Nodis

1994). The percent reduction in the number of fies, provided by the different ear tap, was

also calculated for each weekly wunt and o v a t h e e n h scason using the formula: [(Number

of nies on non-treated animals - numba of fies on treated animais)/ numba of fies on non-

treated animals] x 10% (Abbot 1925).

3.3 Resistance Ratio LC, Glas Tube Bioassiys

The level of raistance in horn fües to organophosphorous and pyrethroid insediades

was obtained by detemùning the LC, of resistant and susceptible flies aspirateci into glatis

tubes (surfiice ares of 71-32 cm3 treated with 1 ml per tube of various concentrations of

fenvderate and diazinon (Dunning et ai. 1986; Ciek and Knapp 199 1; Cilek et al. 1995; Kunz

et al'. 1995; Lindsay et al. 19%a,c). The treated glass tubes were prepard by Dr- Doug

Coiwell (Research Scientist) and technicians at Agriculture and Ag@-Food Canada in

Lethbridge, Alberta. Technical grades of fenderate and ofdiazhon were dissolved in

acetone, and aliquots ofthe soiution were placed in the glasstubes et al. 1995; CoIwd

1998). The vials were then roUed on th& side untif the acetone ewprated, 1 a . g a h

of insecticide on the d e r su- munzet o1.1995; Colwell1998)- Once prepued, the tubes

were delivered to Guelph by courïer and then maintainecl in the dsrk at 8 OC. The tubes were

effective up to two weeks after prepadon, thus the bioassay was pafonned as soan as

weather permitted effdve collection of fies. ApproxhteIy 30 minutes prior to use, the

tubes were removed fiom cefngeration and WMMd to ambient ternpaciaue (22 - 2S°C).

Adult hom fies wexe coiiected with a sweep net âom the backs, sides aad undersides

of cattle. Resistant hom fies were collecteci Born uttk in the Perth area (Codedak Farm,

RR. 6 Perth ON) and fiom the Glencoe am (Earl McEachern, RR 4 Glencoe ON).

Susceptible flies were coilected h m cattle near Rockwood ('iün May, RR #3 Rockwood

ON). Captureci hom aies were transferred to a 26 x 26 x 26 cm plexigiw holding cage thn

had a cotton sleeve which allowed for ventilation. Flies were provided with cotton wicks

soaked in citrated bovine blood (Km et ai. 1995). When the cage containeci enough fies

for a test, it was taken immediately to a nearby shaded site where fiies were transfand to the

glas assay tubes. Groups of approximately 20 ullsexed, mixed age fiies were aspirateci into

each tube wunz et al. 1995; Lindsay et al. 1996a,c). Each treatment was replicated twice.

Two non-treated wntrol tubes (1 ml acetone only) were used in each test for cornparison

(Cilek et al. 1995; Kunz a al. 1995; Luidsay et al. 19964~). The starthg time was recorded

as each group of flies was put into a glass tube. When fiiîed, assay tubes wen kept at ambient

temperature (ca 22-2S°C) in the shade. Dead and live fies were counted at the end of the

test period, 3 hours for the fenvalerate test and 24 houn for the diazinon test. Flies were

considered dead athey were not moving. Concentration-response regressions were d y s e û

with the EPA Probit Analysis Program Version 1.5. Resistance ratios were determineci by

dividing the LC, of resistaat nies by the LC, of suscepfiile fies.

3.3.1 Fenvarlc~ofe Biwrsqy

Two sets oftuks with two replications were prepared for each f d e r a t e bioauoy

perfiomed, one set for the resistanî stxain and one set for the suscepti'ble straia.

Concentrations of fenvaIerate for susceptible hom fies ranged f h n 8.1 x log' Cc&m2 to

9.77 x 104 ~(s /cm~, and for resistant fies ranged fkom 3.91 x 10-~ pg/cm2 to 0.128 ~s/d~

Once Ses were aspirateci into the fenvalerate treated bioassaytubes, the tubes wem stoppered

with foam. The number of dead fies exposed to fenvalerate was remrded every h o u for

three hours of exposure. Dead and Live fiies were counted at the end ofthe three hour test

period (Km e$ al. 1995; C o l 4 1998).

3.3.2 Diaahon Bioassay

One set of tubes with two replications was prepared for each diazinon bioassay.

Concentrations of diazinon ranged fiom 0.13 &xn2 to 5.0 &m2. The tubes wae

stoppered with 2 cm (outer diameta) plastic tubing wvered witb cheese doth at one end.

The hose was inserted approxhately 2 cm into the glass tube. This dowed for a cotton plug

soaked in citrateci bovine blood to be placed in the centre of the tubing, providing a blood

meal for the flies. To determine accurate levels ofmortaüty, the numba ofdead and Eveflies

22

was recorded after 24 hours aposun because organophosphorous insecticides Lin more

slowly than do pyrethroids Wunz et al. 1995; Colwelll998).

3.4 Chlorttnapyr Glus Tube Bioamay

Pyrethroid and organophosphorous-resistant hom flies nom Perth and Glencoe, and

susceptible hom fies nom RockWood were exposed to various concentrations of

chlorfenapyr to determine ïts &cacy on fly mortrliry and whether cross nsistance was

occurrîng. The testing was done by g l u tube bioassay. The tubes were prepared by Dr.

Doug Colwell at Agriculture and Agri-Food Canada in Lethbridge, Alberîa Chlorfairpyr

(AC 303630), obtained nom Amencan Cyanamid, was diluted in water and 0.5 ml aüquots

of the solution were placed into each glass tube (surface aru 56.52 cm3 (Colweil 1998).

Tubes were rolled horbntally on a metal trsy placed on a hot plate set to 40°C (Colwell

1998). When the solution had wmpletely evaporated, the tubes were chiüed at 8 OC (Colwell

1998). Bioassay tubes were shipped to Guelph and maintaineci unda the same conditions as

described for the pyrethroid and organophosphorous tests. The initial concentntions testeci

(O. 1 - O. 8 ps/cm3 were too high resuiting in 1 Wh morioüty of nsistant and susceptiiIe flies.

Thus, several attempts were made at preparing concentrations to achieve LC, for the

resistant population (Colwell1998). LC, concentrations have yet to be determineci for the

susceptible population. Final concentrations of chlo~enapyr testeci rangeci fiom 1-25 x 105

&cm2 to 0.2 ps/~rn*~

Simifar procedures for horn fly coliection were used as describeci for the raistance

ratio glass tube bioassays. Flies were provided with a citrated blood meai duriag the 24 hour

23

test period. M e r 24 houn, the number of l in and dead fies was remrded Concentration-

response regressions were analyseci with the EPA Probit Analysis Program Version 1.5.

3.5 Colorimetric Acetykholinesteri~ Activity Assay

Acetylchoünesterase is an enzyme located in the post-synaptic membrane and

fiuctions to hydrolyse the esta bond ofthe neurotransmitter acetylcbobe- Inhibition of tbis

enzyme by the irreversiôle binding of ocgaaophosphorous compouads resuits in the

accumulation ofacetylchohe Pt the synapse.

Inhibition of acetylchoiinesterase can be quantified through the detemination of

acetylcholinesterase activity (Eliman a al. 1961) when standardid to protein concentration

(Bradford 1976). The colorimetric assays, wbich have been adopted to a microplate r d e r

(Galgani and Boquene 199 1). provide an indication of acetyIcholinesterase activity @ h a n

et al. 1961).

3.5.1 Determ0n&n of Acetylckou'nalape Acft0vily

Acetylcholinesterase levels in adult hom fiies were measured using the methd of

Ehan et al. (1 961). in which the hydrolysis ofthe substrate analogue acetylthiocholw iodide

(ATChI) is measured colorirnetridy by the absorbance of2-nitro-S-tbi0berizoate at 405 nxn,

after the r d o n of S,S'-dithiobis-2-nitrobenu,ic acid @TNB) with the i i i thiocholine.

Kinetic reactions were monitored using a microplate reader that determines wan

acetylcholinesterase adivities by linear regrasions of absorbante-thne data @bores a ai.

1988)-

Hom flies were coiiected fiom Perth, Glenca, and Rodouood, aspiratad into the

glass bioassay tubes, and provided with a citrated bovine blood meai as dcscribcd above (p.

22). Pyrethroid and organophosphorou&nsistant Penh and Glencoe, and ~~1scepti'ble

Rockwood hom fies were exposed for 24 hours to concentrations of d i d o n mghg fiom

0.13 &cm2 to 5.0 &cm2. Approximately 20 hom fiies nom each location wae kcpt in non-

treated glas tubes (1 ml acetone only) as wntrols. After 24 hours ~ t p o s w , flics were

wllected and fiozen at -80°C (Xu Md B d 1994). Smples wac Lcpt at -80°C and were

analysed 4 7 and 9 weeks ifta coliection. Ffy hads, which contain 72.9% of totd body

acetylcholinesterase (Xu and Bull 1994). w a e rernoved and hornogenates of 5 hcads were

made in 8 ml of phosphate buff i (pH 7.2). Sampkr were caitrifirged for 20 minutes at

10,000 x g. Thme 30 pl aiiquots of eich sample wae placed in a % wdl niaoplate. DTM)

(30 J )(Sigma D-8130) and 100 3 phosphate buffer @H 7.9) were added to each sample

well and inaibatad for 10 minutes. After incubation 30 pl of ATCM (Sigma A-5751) was

added to each sample weU. Activity was measured using a Bio-Rad (mode1 3550-UV)

microplate reader at 405 rn for 10 minutes. Absohance readings were made at 1 minute

intervais, Sumng each microplate M o r e each reading. Protein concentrations ofthe samplcs

were detennined fiom a standard avve developcd with bovine m m aibumin (Pierce 23209)

as desaibed by Bradford (1 976). Acetylcholinata~se activity was calcufated using the

formula (EUman et al. 1% 1):

Activity= * absmefFx light path x ample volume

AA change in absorbana (OD) &S. weE 13.6 cm2 (absorbame d c i e n t ofthionitrobenzoic at 405 nm) Iight path 0.5547 cm for total volume of 190 pl

The standardized activity was CalcuIated ushg the fomuia @ d o r d 1976):

Standardîzed actîvity = Actikitv Protein concentration

4 RESULTS AND DISCUSSEON

4.1 Ear Tag Efïicacy 1997 and 1-

Weekly mean numbers of hom flies on cattie &Perth, Ontario treated with athatwo

~rotectoe (20% diazhon wlw). or two EctoguardO (lm tetrachlowinphos w/w), or two

Stockaid6 (8% cypemahrin w/w), or two EIuninetd (1 1% d*nnon, 6% cypennethrin

W/W) ear tags for the 1997 season are plotîed with the non-treated means in Figures 2 - 5.

Figure 6 summacÛes weekly mean n u m k n of horn fTies on cattle at Perth, Ontario for

al1 ueatments in 1997. Boch orgawphosphomus t p g ~ (Protector0and -do) provided

poor hom 8y wnuol in 1997. The Protecd (20% diazinon w/w) tags provided a

statistidy signincant reduction of honi flies as compared to the non-treated herd in the week

of June 27 and the week of September 5, 1997 Figure 2). The air temperature inaeased

nom daily highs of 20" C at the end of August to daiiy highs of 25 OC in the fint week of

September 1997, resulting in adult emefgence and thadore increased fSr numbers in

September. The ~rotecto? herd (location 2) may have experienced stati*sticaliy sigdcant

reductions of hom fies compamd to the non-treated herd (Iocation 3) in week 12 because

they had access to a 2 acre woodlot which provided shade and coola temperatures. Low

numbers of fies on control cattle throughout the season codd have r d t e d Born r@ar

manure removal ftom the feed bunk area located just outside the müking barn, thus reducing

the number of oviposition sites. As well, unseasonably dry conditions fbr the summer o f 1997

may have affected moisture content of manure, and thus reduced lame and pupae d v d .

The ~ctoguard' (10% tetrachlorvinphos wlw) tags provided statistically signifiant reduction

of fiies as wmpared to the non-treatcd herd for one week der tag application (F~igure 3).

27

The large increase in fly numbers on Ektogutud0 treated ca#k observed in the nnt week of

September 1997 may have resulted h m i n d addt emergeme because of warm

temperatures, and the low amount of s u e adable at this location (6) comparecl to o h

treatment locations. ûver the entire 1997 season, the ~otector' and Ectoguarde tags

provided no statistical season meui reduction in horn %es u cornpanxi to the non-treated

herd.

nie pyrethroid tag stockaid*@% cypennethrinw/w) provided statistidysigniûcaut

horn fly reduction as comparexi to the non-treated had in the weeks of Jdy 5, and August 24-

September 5,1997 (Figure 4). Cattie wae moved the wak ofhgust 16,1997 to a pasture

that had access to a wooded area ( i d o n 5). Stotisticaiiy signifiant reductions in homfly

numbers as compared to the non-treated herd for August 24 - Septanber 5, 1997 rnay have

been due to the increase in shade provided. Statisticaüy higher numbers of horn flies as

compared to the non-treated herd were o b d on cattle in the wadrs ofJuly 23 and August

8, 1997. Temperatures during the weeks fiom July 23 - Augua 8, 1997 ranged fkom daily

highs of 25-27°C. This increase in temperature may have contn'buted to increaseû

degradation of the pyrethroid on the animal, as well as increased metabolism in the bect.

Over the entire season, stockaidm provided a 23.8% reduction in horn fies as compared to

the non-treated herd.

The ~limu>ator' (1 1% diasinon, 6% cypermethrin wlw) wmb'mtion tag provïded

statisticaiiy signifiant reductions in horn fly numbers as compared to the non-treated &rd for

8 weeks after tag application Figure 5). ûva the enth seasm the ~liminator* tag reduccd

horn Ses by 71.7% as compareci to the non-treatd herd.

Weekly mean numbers of horn flics on cattie at Perth Ontano treated with either two

ProtectoP (20% diazinon w/w), or two Ectoguard0 (IW tetrachiorvinphos w/w), or two

~ o v a i d . (8% favalemte wlw), or two Eliminator@ (1 1% diainon, 6% cypametlnin wlw)

ear tags fot the 1998 sason are plotteci with the non-treated maris in Figures 7 - 10. Figure

11 sumrnarivs weekly mean number of hom flies on cattle at Perth, OMM0 for ali

treatments in 1998. The 1998 season was longer than the 1997 season- 16 weeks versus

12 weeb. respectively. Ullseasonably w a m temperatures in &y 1998 d t e d in ear1y

emergenœ of hom fly adults, -fore application of insecticide irnpregnatad ear tags was

earlier than in 1997.

nie protector (2% diazinon w/w) tags provided strtisticaiiy signiacant reductions

of hom fies as wmpared to the non-treated herd spofadically throughout the season (walr

of June 4,28, July 2.3 1, and August IO, 1998) (Figure 7). niaie statistical reductions may

the r d t of acass to more shade at the Protectop site (location 1). The Ectoguado (10./.

tetrachiorvinphos w/w) tags provided statistical hom fly reduction for 6 weeks post

application (Figure 8). Mer 6 weeks, horn fly numben increased on Ectoguard* ereated

animals to levels that were staîistidy signincantly higher than the non-treated animals. This

difference in fIy numbers may have beai because of mure management around the milking

barn at the wntrol hwd location (3), or besaust less shade was adable at the Ectoguard0

location (6). Over the entire 1998 season, both organophosphorous tags, Protectop a d

~ctoguardo, did not provide a statistically significant reduction in horn fly numkn as

compared to the non-ueated herd.

The pyrethroid tag Bovaidœ (8% fenvaierate w/w) provided statistcaily significant

horn fly reduction fiic 7 weeks post tag applÉltion (Figure 9). Afkr 7 mdo horn tly

numbers on Bovaida treated cattle were not statisticaily signifimtiy difkmt h m the

non-treated herd or were statistidy higher. Over the entire 1998 sauon, the ~ o v a i d ~ tag

did not provide a statistically significant reduction of horn aies as compareû to the non-

treated herd.

The Eliminatorm (1 1% diahon, 6% cypermethrin w/w) tag provided a statisticaily

significant reduction in horn flks fot the weeks of May 30 - August 10, with the exception

of the week of June 4 (Figure 10). Ova the 6 sason the ~liminatof' tag d u c c d fly

numbers by 60.1% as compared to the non-treated M.

Ali tags tested in the P d area in 1997 and 1998 were wasidaed Mures becwse

impregnated tags un d u c e horn fly numbas by to for the entire eason

(Ahrens and Cocke 1979; Miller et al. 1984; Byford a al. 1992; Surgeoncf et al. 1994),

indicating that resistance to both organophosphorous and pyrethroid mseaicides is ooamiag.

Sample Dates 1997

Figure 2 Mean number (+ SE) of horn fies (Haemotobia imi&am) per side of cattie @=IO) at Perth, Ontario, that were non-treated o d o n 3) or were treated with two Protectore (2W diazinon wlw) ea. tags (iocation 2). Signihmt differences (PzO.05) between Protecto? and the non-treaîed animais are indicaîed by an astensk (*).

Sample Oates 1997

Figure 3 Mean number (+ SE) of hom flies (Himaiobia hitans) per side of cattle @=IO) at Perth, Ontario, that were non-treated (iocation 3) or were treated with two Ectoguard0 (Io./. tetrachlorvinphos wh) ear t a p (location 6). Significant Mérrnces (Ps0.05) b e e n Ectoguardo and the non-treated animais an indideci by an asterisic (*).

Sample Dates 1997

Figure 4 Mean numba (+ SE) of hom fiies (Haemutobia irritaPts) per side o f d e @=IO) at Perth, Ontario, that were non-treated Gocation 3) or were treated with two Stockaido (8% cypermethrin wlw) ear tags (location 5). Signifiant dinerences (PrO.05) between Stockaida and the non-treated a n k d s are indicated by an asterisic (*).

Sample Dates 1997

Figure 5 Mean numba (+ SE) of hom fies (Nlematobia in+-) per side of cattle @=IO) at Perth, Ontario, that were non-treated flocation 3) or were treated with two Elimiaotor ( I l% diazinon, 6% cypermethrin wlw) ear tags (iocation 4). SipifIcant différences (Pr0.05) between ~liminnt~P and the non-treated animais are indicated by an asterisic (*).

(o~=u) ap!s rad sa!U uroy 40 raqurnu ueaw

é: 0 P Z ,

e Sample Dates 1998

Figure 7 Mean number (+ SE) of hom fües (Haematobiu Mians) per side of c d e (n=lO) at Perth, Ontario, that were non-treated (location 3) or were treated with two Protaop (2% diainon w/w) ear tags gocation 1). Signincant differences (Ps 0.05) betweai protector@ and the non-treated anirnals are indicated by an asterisk (*).

QI' Sample Dates 1998

Figure 8 Mean number (+ SE) of hom fies (Hmm4fobia imtons) per side of d e (n=lO) at Perth, Ontario, that are non-treated (location 3) or are treated with two Ectoguardm (1W tetrachlocvinphos w/w) ear tags @cation 6). Signiscant differences (Pr0.05) h a n Ectoguardœ and the non-treated anunals are indicated by an a*aisk (9.

Mean number of horn nies per side (n=10) O C cn

O O $ a Pretreatment

May 30

Jun 4

15

2 1

28

Jd 2

10

17

24

3 1

Au@ 10

17

24

3 1

Sep 8

14

2 1 J

~ r l Mean number of hom flies per side (n-10)

May 30

Jun 4

15

21

28

Jul2

I O

17

24

31

Aug 10

17

24

31

14

21

(O ~ = u ) app rad sa!U woy p raquinu ueaw

Fenvalerate LC, values and raistance ratios for pyrethroid and

organophosphorous-resistant honi flies nom Perth and Glenm, Ontario, and susceptible

horn flies nom Rockwood, Ontario, are presented in Table 2. Dose-response cwes for

al1 fenderate bioassays are presented in Figures 12 - 14. Susceptible hom nies collecteâ

for raistance ratios determined August 20121 were nOm a Rockwood daky k d that had

been exposed to a 0.5% pyrethrin barn spray on August 6, 1998 (Tim May, R.R 3,

Rockwood ON). This spray may have selected for flies more tolerant to fenderate and

therefore may have lowered the resistanct ratios.

Table 2 LC,, LC, and resistance ratios of pyrethroid and organophosphorous-rcsistint hom nies from Perth and Gltncot, Ontario, and susceptible flics trom Rockwood, Ontario, determincd afttr 3 hours exposun to fenvalerate, 1998.

Dite Source EN EH Slope Resictance Ratio - -

July 10 Pe fth 0,038 0.32 1,77 (0.026,0.059) (O. 15,2.09) 253

Iuly 1 1 Rockwood 0.15 x 105 0.1 1 x 1W2 1.89 (0.99x104,00.20x10-3) (0.64x10",0.45~10-~)

- -

August 20 Perth 0,041 1 ,O4 1.17 (0.024,0.061) (0.43,7.65) 1 OS

August 2 1 Rockwood 0.38 x lu3 0.18 x. 1c2 2.72 .+ (0.25 x 1@, 0.56 x W3) (O. 13 x IO'*, 0.37 x 10'3

August 21 Glencoe 0.1 1 1.38 1.54 289 (0.025, 1.84) (0.35,8.12)

LC, and LC, values are expressed in &cm2 (95% confidence limits). Resistance ratios are dotedned as RLCdSLC,.

SusceptiMe hom fiy population

Resistant hom fly population - - - - 95% confidence limits

Concentration of fenvale rate pglcm2

Figure l2 Mortality dose-response with 95 % confidence limits for pyrethoid and organophosphorous-resistant hom flies (Haematobiu irnlon~) LCm=0.038 fiom Perth, Ontario, and susceptible flies LCm =O. 1SxlV fiom RockWood, Ontario, e x p d to fenderate for 3 hours, h l y 10/11, 1998.

Susceptibte horn fly population Resistant horn fly population

---- 95% confidence limits

I o5 1 0 ~ 1 o - ~ 1 0-1 1 00 I O'

Concentration of femlerate Clglcd

Figure 13 Mortaiity dose-response with 95% confidence limits for pyretbroid and organophosphorous-resistant hom flies (Hoenxatobia U350 = 0.41x10-' from Perth, ontario, and suscepti'ble flies LC50 =0.45xlU3 fiom Rockwood, Ontario, e x p e d to fenvalate for 3 hours, August 20121, 1998.

SusceptiMe hom fiy population Resistant hom fiy population - - - - 95% confdence limits

Concentration of fenvalerate j@cm2

14 Mortaiity dose-response with 95 % confidence limits for pyrethroid and organophosphorous-resistant hom flies (Haem4tobia imlms) LC,=O. 12 from Glencoe, Ontario, and susceptible flies LC,=0.45xlV fiom Rockwood, Ontario, exposexi to fenvalerate for 3 hours, August 20121, 1998.

4.2.2 LMàzhn Bioassay

Diazinon LC, values and resistance ratios for pymhroid and organopho~phorous-

resistant horn flies fiom Perth and Glencoe, Ontario, and susceptible aies fiom Rochivood,

Ontario, are presented in Table 3. Do~e-response cums for the diazimn bioassays are

plotteci in Figures 15 and 16. The resistanoe ratios obBerved for diazinon raistant dies

were low compared to resistance ratios observeci for tizmderate resistant flies. However,

MIme of both organo~hosphorous tags (protectoi 20% diazinon, and ~ctogupid. 10%

tetrachlorvinphos) indicate that mistance to organophosphorous insecticides is a problem.

-

Table 3 LC,, LC, and resistancc ratios of pyrethroid and organophosphorous-resistant horn nies from Perth and Glencoc, Ontario, and susçeptible flics from Rockwood, Ontario, dttermincd alter 24 houra cxposure to diazinon, 1998.

Date Source LCs LCM Slopt Rtsistanct Ratio - - -

Aupst 20 Perth 1.29 6.27 2.39 (0.77,2.04) (3,43,28.84) 2,2

August 2 1 Rockwood O, 59 3.18 2,48 (0.44,0.74) (2.43,4.69)

August 21 Glencoe 1.94 12,54 1,38 3,3 (1 .25,2,92) (7.26,30.59)

LC, and LC, values are expressed in @cm2 (95% confidence limits). Resistance ratios are determined as RLCdSLCM.

1 0 Susceptible hom fiy population 1 1 . Resistant hom fly population - - - - 95% confidence limits

Concentration of diazinon Iig/m2

Figure 15 Mortality dose-response with 95% confidence limits for pyrethroid and organophosphorous-nsistant horn flies (Haematobia im'tm) Km = 1-29 from Penh, Ontario, and susceptible flies LC,=O.S9 from Roc-, Ontario, exposed to diazinon for 24 hours, August 20/21, 1998.

a SusceptiMe horn ffy population Resistant hom fly population ---- 95% confidence limits

-.

I

.4 r

4

6'

l

r

10-5 1 o4 i o3 1 o - ~ 1 O-' 1 o0 1 01

Concentration of diazinon pg/cm2

Fgve 16 Mortality dose-response with 95% confidence l i m h for pyrethroid and organophosphorous-resistant hom flies (Hmltcofobia im'tum) = 1.94 fiom Glencoe, Ontario, and susceptible aies &=OS9 fkom Rochvood, Ontario, exposed to diazinon for 24 hours, Auguft 20/21, 1998.

4.3 ChlorCenapyr G k Tube B~O(LSEII~

Initial chlorfenapyr (AC 303630) concentrations tested, 0.1 &cm2 - 0.8 fig/cm2

(Sheppard and Joyœ 1998). m l t e d in 100% mortdity of pyrethioid and

organophosphotou~-rais- and suscepbiile ' horn fly strains. Therefore, lower

concentrations were prepared so that a differential mortaiity was observable. The

chlorfenapyr LC, values for resistant Perth and Glencoe horn fly populations were

determined as 0.013 pgfcrn2 and 0.012 pgfcm2, respectnieLy. September 4 and 5, 1998.

Dose-response moctality of pyrethroid and organophosphomus-mistant horn flies fiom

Perth and RockWood, Ontario are presented in Figure-s 17 and 18, mpectively. Mortality

of pyrethroid and organophosphotous-supceptible horn f lks fiom RockWood was 100 % at

al1 levels tested (lowest concentrations tested was 1.25 x 1W2 rcgLcm3. 'IbeJe preliminuy

results suggest that chlocfenapyr resistance wuld develop in orgamphosphorous and

p yrethroid-mistant horn flies because LC,,,s for resistant flies are alrrody higher thPn for

susceptible flies. These results disagree with those of Sheppard ami Joyce (1998) who

observed a 5 fold increased sensitivity to chlorfenapyr in pyrekoii-cesistant horn flies as

compared to a susceptible strain. Sheppard a d Joyœ (1998) predict that high levels of

rnixed function oxidases in pyrethroid resistant horn flies (Sheppard 1995) are responsible

for a rapid conversion of chlorfenap yr to the active toxic form AC 303268.

Hom fly population 95% confidence limits

Concentration of chlorfenapyr p@cm2

Flgure 17 Mortality dose-response with 95% confidence limits for pyrethroid and organophosphorous-resistant horn flies (Haemc~tobia irn'tans) LCm=0.013 fmm Perth, Ontario, exposed to chlorfenapyr for 24 hours, Septernber 4, 1998. Monality of susceptiile flies from Rockwood, Ontario, was 100% at al1 concentrations presented.

I Hom fiy population 95% confidence limits

i o4 103 1 1 O-'

Concentration of chlorfenapyr Crg/cm2

Figure 18 Mortality dose-response with 95 % confidena limits for pyrethroid and organophosphorous-resistant hom flies (Haematobia irritaas) Km =0.012 from Glencoe, Ontario, exposed to chlorfenapyr for 24 houn, September 5, 1998. Morraiity of suscepa%le flies from RockWood, Ontario, was 100 % at al1 concentrations presented.

4.4 Colorimetrîc Acetykholinesterase Asay

The effects of diazinon on pyretïuoid and organophosphorous-resistant and

susceptible hom fly acetylcholinestense activity are presented in Figure 19.

Acety lcholinesterase activity of exposed fly heads k exprwsed as a percent of non-exposed

wntrol values. Mean activity in control aies for the thrœ populations tested (Perth,

Glencoe, and Rockwood, Ontario) mis aot significantly différent at Pr0.05 (Student's t-

test). Mean activity at 100% was 89.8 nmoIehg protcia/mia. PreIiminsry mdts

demonstrate thM the pyrethroid and otganophosph~f~~~-cesistant Perth rid Glencoe

populations had 10 - 50% Iess acetylcholinestcrase inhibition than the susceptible

Rockwood population when expoaed to mmentrations ofdiazinon. The mistant Perth Pnd

GIencoe horn Oies may have a reduced sensitivity to ~atylcholinesteraae inhibition by

diazinon, and for may metabolically degrade diazinon at a higher rate than the suscepti'ble

population (Guerrero et al. 1999). Low fly collection numben in August 1998 at the P d

location lirnited the number of heads available for the wlorimeaic iuxtylcholiaestetase

assays and as a result the assays were based on one collection &te. Further research is

recommended.

Percent inhibited acetylchollnesterase activlty after 24 houn of expsosure

5 CONCLUSIONS

Failure of proteclor' (20% diazinon wlw), ~ctoguard* (10% tetrachlorviaphos

w/w), stockaido (8 96 cypemethrin wlw), BoviÜde (8 % ftnvaierate wlw) and ~lîmiiiotor'

(11 56 diazinon, 6% cypermethrin) tags, and LC, biuassay results indicate that horn flies

in Perth, Ontario have developed multiple resisîanœ to p p t h i d and a&uropbDBphosphorops

iosecticides used in impregnated cpttle ear tags. LC,,, bioessays tevealed thnt ~ie~istance

to fenvalerate (Surgeoner et ai. 19%) has remaïneci high (mistance ratio 289) in horn flics

from Glencoe, Ontario. The resistame ratio detemined for diazioon (3.3) and repotted

lack of control with pyrethroid and organopbosphorous impregnated ear tigs implies that

multiple resistance exists in Glencœ. Resistaace of horn flies to organophosphorous

andor pyrethroid insecticides impregnated in cattle ear tags is a fairly recent development

in Ontario. Surgeoner et al. (1994) and Lindsay et al. (19%) were the fint to document

horn fl y resistance to pyrethroids in Glencoe and organophosphorous insecticides in Perth,

respectively. MultipIe mistance to organophosphorous and pyrethroid insecticides bas not

been documented in Ontario prior to this thesis rrsuuch. However, horn fiy resistance in

Ontario is not as severe a problem as in the USA where resistance ratios have reached

600x in the fieid for pyrethroids and 1ûx for organophosphorous insecticides (Butler

1999).

Effective reduction of horn flies with impregnated tags cumntiy available is no

longer a control option in the Perth and Glencoe areas. Producers in the Perth area may

be able to retum to the ~liminator' (1 1 % diazinon, 6 96 cypermethrin) tag which provided

60.1 - 71.7% season reduction, but must fint use a strategy that has a different active

ingredient (such as the avermectùi pour-on, ~vomsc'). Producers in al MU of Ontario

m u t rotate insecticide mode of action on a yearly b i s a d shodd remove impregiirtcd

ear tags at the end of ihe seamn to prevent M e r resistanœ deveIopment, Also, the irse

of alternative methods of application, e.g. back rubbers, pour-ons and sprays, although

often more costly and labour intensive, may prolong the usefulncss of pyrethids and

organophosphorous insecticides for the control of hom nies in meas where mirbure has

not yet occurred.

Evaluation of a potential new active ingredient for ear tsgs. chlorfenapyr (AC

303630). by glas tube bioassay demonsmted excellent cesistant anci susceptiile fly

mortality (100%) at al1 concentrations suggested by Shepperd and Joyce (1998),

0.1 pg/cm2 to 0.8 fig/cm2. At concentrations ranging from 1.25 x 1C2 pg/cm2 to 0.2

pg/cm2, pyrethroid and organophosphorous-cesistant nies from Perth and Glencoe,

Ontario. were less sensitive to expure than the susceptiile flies nom RockWood,

Ontano. Although the differential mortality observed in the resistant populations may

suggest the potential for resistance development to chlorfenapyr, the high mortality

achieved in both resistant and susceptible horn fly populations îdicate that chlorfeaapyr

could be a useful new tool for horn fly management.

Preliminaty results of colorimetric assays demonstrated higher l a

acety lcholinesterase inhibition (10-50 1) in pyrethroid and orgamphosphorous-resistant

horn tlies than in susceptibles when exposed to concentrations of diazinon.

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