278 JoURNALoF THE AupRrcau Moseurro CoNtnor, AssocretroN Vor,.8,No.3 LETHAL EFFECTS OF IVERMECTIN ON ANOPHEZES QUADNIMACULATUS J. W. JONES, M. V. MEISCH, C. L. MEEK1 lNo W. S. BIVIN' Department of Entomology,Uniuersity of Arknnsas,Fayetteuille, AR 72701 ABSTRACT. Female Anophelesquadrimorulatus adults were blood fed on 15 mixed breed dogs 4 h after the dogswere given oral dosagesof ivermectin. Dogs were divided into 5 treatment groups of 3 dogs each, at 10, 500, 1,000, 2,500 ptg/kg, and.untreated. Additionally, An. quadrimaculatus werc fed on lambskin-membranescontaining blood drawn from one dog in each treatment group. Mosquitoes wele allowed to feed on the dogs or the lambskin-membranesand were observedfor death at 24 and 48 h post-feeding.Greater than g0% mortality was recordedin all ivermectin treatment groups except at the 24 h post-feedingperiod with the l\pe/ke dog dose blood fed through the lambskin-membrane (65.4Vo mortality). The highest 2 dosagesproduced 100% mosquito mortality at 48 h post-feedingfrom either a dog or the in uitro systemusing a lambskin-membrane. INTRODUCTION MATERIALS AND METIIODS Avermectins have a wide range of activity Adults of An. quadrirnaculatuswere obtained against a large number of helminths and arthro- from resting stations on the University of Ar- pods. Many publications describe the effect of kansas Rice Researchand Extension Center avermectins on helminths and arthropods par- approximately 13 km east of Stuttgart, AR (Ar- asitic on mammals, but few studies report the kansas County). Female mosquitoeswere man- effect of these compoundson mosquitoes. ually separatedfrom malesby chilling collection Pampiglioneet al. (1985)reported 100% mor- tubes in a freezer for 40 sec and then placing tality in Arwpheles stephensi Liston 36 h after them on a chill table. Aliquots of 40 females feeding on mice given 2.8 mg/kg of ivermectin, weretransferred by mouth aspirator to each236- a semisyntheticavermectin. When Aedesaegypti ml cardboard container (Fonda Group Inc., (Linn.) and.Culex quinquefasciatusSay were fed Union, NJ).3 Each container was subsequently on mice given 28 mg/kg of ivermectin they ex- covered with nylon mesh screening. Seventy hibited abort 50% mortality within 72 h of feed- containers of mosquitoeswere placed in an in- ing. The LD56 for Cx. quinquefasciotusfed on sulatedchest for ground transportation to Baton ivermectin-treated mice was 82 mg/kg. Focks et Rouge,LA. Moistened paper toweling was added al. (1991) examined the effects of ivermectin on to the chest to sustain a high humidity level for the reproductive rate of.Ae. aegypti. After Ae. mosquito survival during transportation. aegyptifemales were fed on rabbits injected with Fifteen mixed breed dogs,housed at the Lou- ivermectin at 10 mg/kg, the mosquitoes exhib- isiana State University School of Veterinary ited reduced survival and egg production when Medicine, were divided into 5 groups of 3 dogs compared with females fed on control rabbits. with one group acting as a control (untreated). Ivermectin levels of 3.4 and 4.3 ng/ml in human Dogswere approximately 1 year old and weighed blood caused 50% of Ae. aegypti and Aedes aL- 19.0+ 3.6 kg. Ivermectinawas diluted with For- bopictus (Skuse) eggsto be infertile (Tesh and mulation Bu and was administered orally with a Guzman 1990).They reported that the LDsosfor 3 cc syringe atl0 pg/kg (: the prophylactic dog Ae. aegypti,Ae. albopictusand Cr. quinquefascia- heartworm dosage),500 pg/kg, J",000pg/kg and tus fed ivermectin in human blood were 126,208 2,500pg/kg. The control dogswere given 3 cc of and 698 ng/ml, respectively. Formulation B. Four hours after administering We evaluatedthe effects of canine blood con- ivermectin, the dogs were immobilized using 1 taining selecteddosages of ivermectin on freshly ml pentobarbital/2.25 kg. Blood samples were fed. Anophelcs quadrimaculatus Say females. drawn from each dog, centrifuged and the su- Adult survival was the primary factor consid- ered. " Mention of a product doesnot imply a recommen- dation for use or endotsementfor saleby the Univer- l Department of Entomology, Louisiana Agricul- sity of Arkansas or Louisiana State University. a tural Experiment Station, Louisiana State Agricul- Ivermectin and Formulation B were supplied by tural Center, Baton Rouge,LA 70803. Merck, Sharp and Dohme Research Laboratories, 2Division of Laboratory Animal Medicine, School Rahway NJ. 6 of Veterinary Medicine, Louisiana State University, Formulation B is a solvent consisting of 60Vopro- Baton Rouge,LA 70803. pylene glycol and 40% glycerol formal. SEPTEMBER1992 Errncrs oF IvERMECTIN oN Arv. 1vADRTMACULATUI 279 pernatant placed in 3 cc EDTA tubes. Four Table 2. Concentrationsof ivermectin in blood containers, each holding 40 mosquitoes, were sampledprior to and subsequentto Arwpheles taped to each dog with the nylon screening of Etad r i mar ulah-zsfeedin gr the container in contact with 4 previously shorn Concentration areasalong one sideofeach dog (front shoulder, of ivermectin rib cage,hip and inside the hind leg). Mosquitoes (ng/ml)'time were allowedto feedthrough the nylon screening post-treat- Dosage for 30 min. Previous trials had indicated that 30 ment Animal rate min would be sufficient for the mosquitoes to no. fus/ke) 4h 4.5h feed to repletion. After this period, the con- tainers were removed,and the mosquitoes,while 110 39 37 still in the original containers, were provided 2r0 27 26 310 45 37 cotton balls soaked in lIVo sugar water as a Mean dl ild carbohydrate source.Blood sampleswere taken 4 500 191 157 again and handled as before.Blood sampleswere 5 500 161 146 packedin ice and shippedto Merck Laboratories 6 500 205 t75 for ivermectin concentration analysis. Mosqui- Mean 186 159 toes were transported to the University of Ar- 7 1,000 394 363 kansas Medical-Veterinary Entomology Labo- 8 1,000 369 305 ratory in Fayetteville, AR to continue the study. 9 1,000 96 180 Mean Mortality readingswere taken 24 and48 h post- 286 282 10 2,500 629 554 feeding. Data were subjected to analysis ofvar- 11 2,500 601 494 iance and means were separated by Duncan's 12 2,500 r34 101 new multiple-range test (Dowdy and Wearden Mean 456 383 1991). 13 Control 00 A 50-ml aliquot of blood was taken from one 14 Control 00 dog in eachtreatment and control group,packed 15 Control 00 in ice and transported to Fayetteville to feed on ] Initial blood sampleswere taken 4 h after dogs An. quadrimaculatus via a natural lambskin- weretreated. membrane,i.e., condom. Membrane feeding was 2Blood samplesanalyzed by high-pressureliquid initiated to ensure that substanceson the hair chromatography. or skin of the dog had not influenced the out- come of the test. The mosquitoeswere collected at the samelocation near Stuttgart, transported water bath and then placed on the top of the to Fayetteville, and handled in the laboratory as cardboard containers for mosquitoes to feed. previously describedfor those mosquitoesused Four containers,each containing 40 mosquitoes, in the dog-host test. The procedure was begun were used for each treatment. Mosquitoes fed 72h after blood was drawn from the dogs.Con- on the condoms through the nylon screen as domscontaining blood wereheated to 38'C in a discussedpreviously. Mortality was determined at 24 and 48 h, and data were analyzed as previously described. Table1. Percentmortality of Anopheles quadrimaculatusfed on bloodcontaining tte.-ecttn* RESULTS AND DISCUSSION (hours) Posttreatment Mortality rates exceeded90% for mosquitoes Lambskin-mem- fed on dogs at all rates of ivermectin Dogs at 24 h branes post-feeding (Table 1). Mortality in mosquitoes Dosage rates fed on the lambskin-membranes 0'e/ke) 24h 48 h 24h 48h also exceeded 90Voat all rates at 24 h post-feeding,except the 10 90.5Aa 98.6Aa 65.4Bb 90.8Aa l0 pg/kg treatment (65.4%. However at 48 h 500 93.2Aa 99.5Aa 92.6Aa 9Z.3Aa post-feeding, mortality at that particular 1,000 94.4Aa 100.0Aa gg.2Aa 100.0Aa treat- 2,500 96.9Aa 100.0Aa 100.0Aa 100.0Aa ment level increasedto 90.8% and was not sig- Control 1.0Bb 4.3Ab 1.6Bc 3.4Ab nificantly different (P > 0.05) from the other 3 t dosages.The mortality recorded at all dosages Means in the samerow followedby the same upper exceeded90% in both dog-fed case letter are not sigrrificantly different (P > 0.0S and the lambskin- level) by DMRT. membrane-fed groups after 48 h post-feeding. 2 Means in the sarnecolumn followed bv the same The 2 highest dosage rates resulted in 100% lower case letter are not significantly diffirent (p = mortality; whereas,the control mortality never 0.05 level) by DMRT. exceeded,4.37o. 280 JoURNALoF THEAurnrcer Moseurro CorrRor, AssocrnrroN VoL.8,No. 3 We prepared individual oviposition cups for as an anthelminthic has increaseddramatically mosquitoesto measurefecundity as well as mor- in canines and it is likely that ivermectin is tality but the high rate of mortality precluded often imbibed during blood meal acquisitionsby any measurementof fecundity. Resultsfrom this female mosquitoes. Its effect on mosquito fe- study demonstratedmuch higher ivermectin ac- cundity as well as survival is an area requiring tivity against a mosquito speciesthan previously further evaluation. reported. The blood ivermectin concentrationsrecorded ACKNOWLEDGMENTS in the 10 prg/kggroup (Table 2) are much higher than Tesh and Guzman (1990)recorded as caus- We are grateful to Richard Endris of Merck, ing egg infertility in culicine mosquitoes. The Inc. for his assistancein the project and to David blood ivermectin concentrationsat all other dos- Fink, Pierre DeMontigny and JessicaCha of Merck Laboratories for ages exceededthe LDro recorded by Tesh and conducting the blood sample analysis. Guzman (1990) for Ae. aegypti. All concentra- tions producedvery high mortality in An.