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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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