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Diptera: Ceratopogonidae) on Animals Journal of the American Mosquito Control Association,9(3):256-259, l9g3 Copyright @ 1993 by the American Mosquito Control Association, Inc. IN VITRO ASSAY FOR PERMETHRIN PERSISTENCE AND INTERFERENCE WITH BLOODFEEDING OF CULrc OI DES (DIPTERA: CERATOPOGONIDAE) ON ANIMALS BRADLEY A. MULLENS Department of Entomology, University of California, Riverside, CA 92521 ABSTRACT. Permethrin (650/oAI) wasapplied to the dorsum of 3 goats(3-4 ml/animal). Hair clippings were taken before treatment and,4-6, 18-20,39-4 I and 67-69 days after treatment. Culicoidesvariipennis fed upward through a thin layer ofhair and through a membrane on an artificial feeder containing cattle blood. Midge knockdown was usually complete after the 45 min exposureperiod, but many bloodfed first. Belly hair residuesdid not statistically reduce engorgementbeyond zf-6 days. Head hair residues reduced engorgementby up to 98o/oat 18-20 days, but did not significantly reduce engorgementat 67- 69 days. Back hair residuessignificantly reduced engorgementfor the entire period (99-1000/othrough day 3941 and 660/oby day 67-69). Partial engorgementwas higher in the treatments. INTRODUCTION h-old, Culicoides variipennis reared in the labo- ratory (Joneset al. 1969). Approximately 200- Efficacy testing of insecticidal or repellent 300 adult midges (mixed sexes;approximately compounds on bloodfeedingDiptera commonly half females)from the same cohort were lightly is done with human volunteers, who can note anesthetizedwith CO, and added to each of a the number of bites (Harbach et al. 1990). The number of clean 23 ml (0.75 oz) plastic cups. A information can then be extrapolatedto estimate piece of sheer nylon stocking (15 denier) was whether treatments might reduce diseaseagent stretchedtightly acrossthe top ofeach cup (sur- transmission.This method obviously is not pos- face area 19.6 cm') and affixed with a small rubber sible in assessingtreatment eftcacy on animals. band. Approximately 50-70 mg of hair from the Typically the residual effectsofinsecticides ap- goat then was arranged loosely and evenly on the plied to animals are assessedby direct obser- top of the nylon. Blood feeders (Lillie Glass- vation of feeding insects in the field (Foil et al. blowers, Inc., 3431 Lake Drive, Smyrna, GA 1990). This may risk bias due to the proximity 30082) were used to warm cattle blood (EDTA of a human observer,but, more significantly, ob- added to prevent clotting) to 37oC above a servation generally is not possible for many of stretched parafilm membrane (Hunt and Mc- the smaller crepuscularor nocturnal species.An Kinnon 1990). Each cup then was raised im- alternative method is to expose insects directly mediately to contact the membrane. The midges to animals or treated substratesunder controlled would move upward to the feeding surface and conditions.Holbrook (1986),for example,took probe successfully nylon hair clippings from ear-tagged(permethrin-treat- through the mesh, hair and membrane to obtain a blood meal. A period ed) and untreatedcattle, all held under field con- of 45 min was allowed for bloodfeeding.A pre- ditions, and showed that the treated hair was treatment batch of hair similarly was clipped from toxic to Culicoidesvariipennis (Coquillett) adults the upper side ofeach goat and used as control for at least 70 days after treatment. hair throughout the tests. The present studies were initiated after it was Three castratedmale goats(47 .7-70.5 kg body found that free-ranging desert bighorn sheep in weight) used in the test were housed in individ- southern California were subject to high lamb ual, contiguous, chain-link fence enclosures.They mortality influenced by hemorrhagic diseasevi- were outside during the day under shade cloth nrses,presumably transmittedby Culicoidesspp. and were inside at night. When outside, they (Mullens and Dada 1992). Persistent pesticides receivedapproximately l-3 hours of direct sun,/ might be usedto treat field-capturedanimals and day. The tests were done in the fall, with mean protect against Culicoides attack (Holbrook 1986). maximum daytime temperatures generally 25- 35t. A permethrin formulation containing 650lo AI, packaged in plastic MATERIALS AND METHODS l-ml ampules, was se- lected for the tests and applied at either 3 or 4 Domestic goats,which have a hair coat similar mVanimal (depending on body weight). This high to that of bighorn sheep,were used as a model. concentration formulation, used commercially Hairwas clipped from the upper side of one goat. for ectoparasitecontrol on dogs,was intended to Preliminary laboratory experimentsused 36-72- enhancepersistence in the field. The amount re- 256 Srrrnrranen1993 PrrurlnrnnrN Errscrs oN C uttco tozs ENconcnvENt quired per animal could easily be carried in a Table l. Engorgementof Culicoides researcher'spocket and applied quickly to sheep variipennis allowed to feed on cattle blood captured for study in remote bighorn sheephab- (parafilm membrane, artificial blood feeder) itats, thus minimizing stressto the animal. On through hair from different body areas of day 0 the permethrin was applied to one goal permethrin-treated goats. Treatment along the dorsal midline. After 2 days with no applied to dorsal midline. apparent ill effects, the other 2 goats were treated Days o/oreduc- similarly. after tion in en- On day 6 (,1-6 days post-treatment) hair clip- treat- Body o/oengorge- gorge- pings were taken from near the ventral midline, ment area ment (SD)t ment2 the head around the base of the ears, and the dorsal midline (in that order, cleansingthe clip- 4-6 Control3 82.2(r.3)a persthoroughly with soapand hot water between Belly 40.2(r.l)b t.t animals).This wasdone becausesome Culicoides Head 5.2(7.8)c 93.7 spp.have preferredbody regionsfor feeding(e.g., Back o.2 (o.7)c 99.8 Schmidtmannet al. 1980,Braverman I 988)and l8-20 Control 41.3(1.0)a we wanted to determine how the permethrin Belly l5.a (3.4)ab 62.7 might become distributed around the body. Care Head 1.0(3.1)bc 97.6 was taken not to remove hair from exactly the Back 0.1 (0.3)c 99.8 same spot on different dates.Hair sampleswere 394r Control 70.6(0.9)a placedin separateclean plastic petri dishesiden- Belly 55.8(1.3)a 2r.o tified by animal and body region. Head 29.4(0.8)b 58.4 In the laboratory, three glass feeders were ar- Back 0 (0)c 100.0 Adult C. variipenniswere placec rangedin series. 67-69 Control 65.7(0.1)a cups as above, and a set of in clean, labelled Belly 79.6(O.1)a 0.0 (untreated hair from each goat) was run controls Head 45.9(1.6)ab 30.1 a baselinefeeding rate for that cohort to establish Back 22.6(7.1)b 6s.6 of midges. After the 45-min feeding period, the ' were removed, and new membranes and Mean (standard deviation) of arcsin VFioportion- cups Means within a Treated hair from transformed data, backtransformed for table- blood were used in the feeders. column for a given feedingday followed by the sameletter are the samebody region of eachanimal quickly was not significantly different (P > 0.05) using Tukey's test. - o/o applied to the top ofthe nylon, and the cupswere : [(o/0engorgement in control engorgementin treat- brought into contact with the membranes. After mentV% engorgementin controll x 100. r control hair from upper side of animal. the feeding period, the procedure was repeated Pretreatment with hair from another body region. After each feeding period we observed the midges to determine-the number knocked down throughout the test. There were significant dif- : : (midgesonthebottomofthecontainerincapable ferencesamong days (F 9.21; df 3,6; P < of directed movement) or alive (crawling,usually 0.0 I ), with days I 8-20 having a significantlylow- on the sides or top oi the container). ihe con- er engorgementrate using Tukey's test to sepa- tainers then were frozen for l-24 hours and the rate means. There was no difference among an- : : midgessexed and countedunder a dissectingmi- imals (F O.147;df 2,6; P > 0.05)' so control croriop". Midges with visible blood in thi gut data were pooled for each date to obtain an es- were counted is engorged. If the blood occupied timate of control engorgement. This was used to > 3 abdominal segments, the midge *as conlid- calculate the reduction in engorgementfor midges ered fully e.tgotged; less was scoied as partial feeding through treated hair from each body re- engorgement.Hair *u. clipped and the proce- gion for that test date (Table l). duies-were repeated on dayi 18-20, 3941 and One-wayANOVAswereconductedonthedata 67-69. The pircentage engorgementwas trans- for each test date, using Tukey's test to separate formed to aicsin Vprodiifi for analysis of means(Table l).Partialdataforonegoat(treated variance (ANOVAj and-t-tests to compare dif- head and back hair) were omitted from the day ferent animals, test dates and body regions. 67-69 analysis due to complete lack of feeding by those midges, possibly due to inadequate membrane stretching. We did not have enough AND DISCUSSION RESULTS adult midges from the same cohort to repeat the A two-way ANOVA first was done to test for feeding attempts on those hair samples for that differencesamong datesand animals for untreat- date. Residue on back hair consistently resulted ed (control) hair. In no casewere more than 6 in completeknockdown ofthe midgesby the end midges knocked down in any control cup ofthe45-minfeedingperiod. Italsosignificantly 258 Jounwel or rne Ar'reRrcANMoseurro Covrnor Assocrerror Vor. 9, No. 3 reduced engorgement(=99.80/0 reduction rela- assay to quantifu permethrin resistance in Hae- tive to controls through days 3941), though re- matobia irritans (Linn.) in the laboratory, placing duction had declined to 65.60/oby days 6749.
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