Journal of the Ameican Mosquito Control Association, 16(3):223-228,200,0 Copyright @ 2000 by the American Mosquito Control Association, Inc.

METHOPRENE TOLERANCE IN AEDES NIGROMACULIS IN FRESNO COUNTY CALIFORNIA

A. J. CORNEL,' M. A. STANICH,I D. FARLEX' E S. MULLIGAN III3.rNo G. BYDEa

ABSTRACT. Methoprene, a juvenile hormone analogue, has been used for at least 20 years as the primary insecticide to control the pasture mosquito Aedes nigrornaculis in Fresno County, California. First reports of apparent methoprene control failures were noted in a pasture west of Fresno in September 1998. Insufficient control was noted in 12 different pastures the following season from April to September 1999. In September of 1999, field trials were conducted to better ascertain the level of control. Results based on pupal counts from different methoprene formulations and rates of application indicated that in some pastures low levels of control were achieved with Altosid@ (Liquid Larvicide) and Altosid XR-G. Control with Altosid Pellets was reported at 52-99Vo.

KEY WORDS Aedes nigromaculis, methoprene tolerance, California, insecticide resistance, mosquito control

INTRODUCTION lations of either have been made from collections in California (Hardy and Reeves 1990). Aedes nigromaculis (Ludlow) is a floodwater, Aedes nigromaculis llias a notorious reputation multivoltine mosquito species with a range from for rapidly developing resistance to insecticides. southern Canada, through central and western Unit- Resistance was detected only 3 years after the in- ed States,to Mexico (Carpenterand LaCasse1955). troduction of DDT (Bohart and Murray 1950). By The lst collections of Ae. nigromaczlls were made 1952, some populations had developed resistance by Aitken in 1937 (Bohart and Washino 1978). to other chlorinated hydrocarbons such as lindane, Since then, collection records of this mosquito have aldrin, and toxaphene (Schaefer and Wilder 1970). demonstratedits associationwith irrigated pastures, In the 1950s, organophosphates became the main alfalfa fields, almond orchards, and wetlands. In pesticides used, and the lst reports of resistance to Fresno County, Ae. nigromacufts numbers peak in parathion were recorded in Ae. nigromaculis in suilrner from July to September (average maxi- 1958 in Kings and Tilare counties (Lewallen and mum temperatures of 32-4O"C and minimum of Nicholson 1959). Because of cross reactivity with 20"C), and by mid-October, they produce overwin- other organophosphates and carbamates by the tering eggs. During this period of peak activity, 1970s, there were few chemicals available to con- completion of the aquatic cycle takes between 5 trol Ae. nigromaculis, and some attempts to control and 7 days, but reports of 4 days have been noted it in the Central Valley had stopped. Methoprene in hot conditions (Bohart and Washino 1978). proved to be a highly effective control agentof Ae. Aede s nig romaculis, an opportunistic bloodfeed- nigromaculis (Schaefer and Dupras 1973, Schaefer er, will bite humans readily. If not controlled, their et al. 1975) and is still in extensive use today. numbers can rapidly reach nuisance levels in areas Three formulations containing methoprene are neighboring breeding sites.In Fresnoand surround- used to control Ae. nigromaculis in California. ing counties, pasturesare generally flooded in 10- They are Altosid@ Pellets (Pellets), Altosid XR-G 14-day cycles, resulting in new hatches every (XR-G), and Altosid liquid formulation (ALL). The flooding cycle. Becauseof its nuisance status,Ae. formulation used is essentially driven by the type nigromaculis control constitutes an important and of breeding site and the cost of application. After costly componentof severalmosquito control agen- initial field trial evaluations of methoprene showed cies in California. Additionally, laboratory studies 10O7o control in Central Valley pastures (Schaefer have demonstrated that Ae. nigromnculis is capable and Wilder 1973), records show that ALL has been of transmitting western equine encephalomyelitis used effectively in pastures since 1974 in parts of and St. Louis encephalitisviruses, although no iso- Fresno County (particularly west of Fresno). In the last few floodings of a west-Fresno pasture in September of 1998, the lst signs of ALL control rDepartment of Entomology, University of California, failures appeared. High numbers of flying and host- Davis, Mosquito Control ResearchLaboratory, Keamey seeking adult Ae. nigromaculis remained after the Agricultural Center,9240 S. Riverbend Avenue, Parlier, field was sprayed. The following year, more careful cA 93648. observation and attention were given to applica- 2Fresno Mosquito and Vector Control District, PO Box tions of ALL in these and other pastures during the 2, Fresno,CA^93707. numbers of adults 3Consolidated Mosquito Abatement District, PO Box Ae. nigromaczlis season. High 278. Selma. CA 93662. remained after treatment. and control failures were a Kings Mosquito Abatement District, PO Box 907, noticed in 12 additional pastures. This provided op- Hanford, CA 93232. portunities to examine other factors that could be

223 224 JounNer-or run AntenrceNMosqurro Coxtnol AssoctarroN

Galilornia

Fig. l. Location of field site. Numbers refer to pasture or site number. Hatched areas represent urban and residential areas of Fresno. responsible for control failures such as incorrect designated with a site number. The site not included pesticide application, flooding regime anomalies, in the flgure was 38.4 km southwest of downtown and methoprene formulation problems. All of these Fresno in Kings County and is referred to as Kings other factors failed to explain the unusually high 4004. Only I pasture where previous methoprene numbers of adults remaining after treatment, and failures were noted covered a large enough area in the possibility of resistance to methoprene was con- a single flooding for simultaneous application and sidered for the lst time. evaluation of all 3 methoprene formulations (site To avoid reliance on anecdotal information and l1). This site was divided into 4 areas. To one, ALL to ascertain more specifically the percent emer- (Zoecon division of Wellmark International. Dallas. gence that was occurring in these and other pas- TX;5Vo active ingredient (AI), S-methoprene) was tures, a fleld evaluation was done in the final month applied, XR-G (Zoecon; l.S%o AI, S-methoprene) of the 1999 Ae. nigromaculis season in Fresno and in a 2nd, Pellets (Zoeconi 4.25Vo AI,S-methoprene) Kings counties. in a 3rd, and a 4th area was left untreated as a control. For comparative purposes, ALL was ap- plied in 6 other pastures, 3 of which were pastures MATERIALS AND METHODS of previous methoprene failures (sites 2, 10, and Study site and methoprene application.' Small l4). The 3 other pastures had shown no evidence pasture sizes, lack of control of flooding and drying of failure (sites 12, 13 and 15). In addition to pas- regimes, time constraints, sensitivity associated ture 11, XR-G was applied in 3 pastures (sites 1, with methoprene failures, and spread of methopre- 6, and Kings 4004), and Pellets were applied in 3 ne resistant adults limited the design of the field pastures (sites 2, 3, and 4). Simultaneous applica- study. The study site (7,702.4 ha) is presented in tion of ALL and Pellets was accomplished at site Fig. 1; it shows all but I pasture. Each pasture was 2. In addition to a portion of site 11, site 9 was SEPTEMBER2000 METHopRENE Tor-rneNcs tN Aeoes t'trcnouecuus used as a control to determine natural mortality. All Climatic conditions were recorded at a weather methoprene formulations were applied either by station located in Fresno. hand with a granule bag or Whirly Bird@ seeder or applied with a Herd@ seeder mounted on an all- RESULTS terrain vehicle. Altosid Liquid Larvicide sand was prepared by using a formulation of 118.3 ml of Results based on field-collected pupal death and ALL adhered to 4.536 kg of l6-mesh sand (RMC adult emergence after applications of 3 Altosid Lonestar@, Pleasanton, CA) and a drying agent (Hi- methoprene formulations revealed unexpectedly Sil 233 Harwick@, Pico Rivera, CA). The larvicide high emergence rates from various dispersed pas- was applied at a rate of 623 g of methoprene to 90 tures and expected control in others. kg of sand glanule mixture when larvae were in Sites 2 and 11: During the study period (l Sep- late 3rd and early 4th stages. XR-G and Pellets tember-2 October 1999), daily maximum and min- were applied I day prior to pasture flooding to imum temperatures ranged from 27.79 to 35.57"C maximize effectiveness. Actual methoprene appli- (mean, 32.2"C) and 11.11 to 19.45"C (mean, cation rates were determined for each pasture using 14.5'C), respectively. Precipitation (0.254 mm) oc- the following formula: curred on I day. Percent control obtained for each of the metho- w.-w. prene 11 is presented in : APPlication Rate formulations at sites 2 and A Table l. Percent mortality was not corrected for natural death, as more pupae died in the control The weight of material before application is W,, W, plots than in the ALL and XR-G treatment plots at is the weight after application, and A is the area of site ll, and the water dried up before pupae could application. Actual application rates are provided in develop in site 2. Essentially, no control of Ae. ni- Tables I and 2. gromaculis was achieved with either ALL or XR- Methoprene failures had been observed in 3 ad- G at sites 2 and ll. Some control, albeit at unac- ditional sites (5, 7, and 8), but these were not in- ceptably low levels, was obtained with Pellets at cluded because they were not flooded at the time site ll (53Vo and 4lVo) and 2 (85.12Va). of this study. Interestingly, higher mortalities were consistently Sampling : Ae de s ni g romaculis immattres devel- observed in early collections ofpupae from site 11. op synchronously, allowing for simultaneous col- This could be due to a bias in the early collec- lections of pupae. Pupae were only collected from tions-the sick and lethargic pupae were more eas- treated and control pastures, ensuring that the lar- ily collected on the Lst day, whereas the 2nd and vae received sufficient exposure to methoprene. 3rd day favored the collection of healthy and more Whenever possible, 2 or more pupal collections, 24 resistant pupae. Another explanation could be that h apart, were made from each pasture so that mor- not all of the eggs hatched at once, and those that tality rates could be calculated based on more than hatched 1st, developing into the early pupae, were I day's collections. Additionally, pupal samples exposed to slightly higher concentrations of meth- were taken from several remaining pools of water oprene than those hatching and developing later. In from each pasture to avoid bias caused by uneven mosquitoes, males often develop more quickly and methoprene application across pastures. Samples pupate earlier than females, and a sex difference in were transported in 5-liter buckets to the laboratory susceptibility to methoprene may be possible. where the pupae and the water were transferred to Methoprene has been noted to slow down larval BioQuip@ mosquito breeders or Pyrex@ storage and pupal development, and thus most pupae were dishes. A maximum of 100 pupae was placed in present a day later in the plots treated with Pellets. each breeding container and maintained at room Pellets are designed to provide 30 days of control, temperature (*21'C). A pad of cotton wool soaked and a 2nd collection of pupae was obtained from a in lOVo sucrose solution was placed on the top of subsequent flooding (17 days later) in the portion each adult container. Pupae were allowed to com- of site 11 that had been originally treated with Pel- plete development (24 h after field collection), and lets. An ll.9%o drop in mortality was recorded in counts of dead pupae, partially emerged adults, Ae. nigromaculis from the 2nd flooding when com- dead adults, and pupal cases were made. Percent pared with mortality from the lst flooding. monality was then calculated based on the follow- Other pastures.' Pupal mortality rates obtained ing formula: percentage mortality : (dead pupae + with methoprene treatrnents in all sites are sum- dead adults + total mosquitoes collected) X 100. maized in Table 2. Site 9 was used as a control, Corrected percentage of mortality rates were cal- recording 98.25Vo emergence in the 571 pupae col- culated using Abbott's (1925) formula. lected. This control was used in Abbott's (1925) In all pastures that were used in this study, re- formula to calculate the corrected mortalities for the maining pupae were treated with larvicidal oil. other treated pastures. Adults were killed with adulticide a day after emer- Prior to the study, increased ALL failure was no- gence to prevent the spread of resistant mosquitoes ticed in a pasture in Kings County, and XR-G as a and reduce nuisance problems. more potent formulation was then applied. In the Jounn.qr oF THE AMERTCANMoseuno CoNrnol AssocrenoN Vol. 16,No.3

Table l. Pupal mortality evaluations of Aedes nigromaculis treated with various Altosid@ methoprene formulations __:-"tt ":9"rly:n2l1:l :ures in Fresno County, California. Treatmentl Area Date Percent (g active treated pupae mortality Average ingredient/ha) (ha) collected (n) mortality Control (no treatment) 15 Sep 10.85(258) 16 Sep l r.35(141) 11.03 Ar,L' (12.33) 1.54 15 Sep ro.28(2s3) 16 Sep 3.36(r49) 7.71 Ar,L3(11.21) o.26 17 Sep 2.98(168) xR-G' (290.85) 0.89 15 Sep 23.26(86) 16 Sep 2 (1s0) 17 Sep r.69 (59) 8.14 Pellets2 (395.37) o;77 15 Sep 60 (5) 16 Sep 84.56(149) 17 Sep 20.ss(146) 53 I Oct 4r.r (146) Pellets3 (333.44) 17 Sep 8s.r2 (r2r)

'ALL, Altosid liquid fomulation; XR-G, Altosid XR-G; Pellets, Altosid Pellets. , Site 11 data. 3 Site 2 data. lst day's collection of 160 pupae, 96.22Vo of pupae As in site 11, earlier pupal collections showed and adults either drowned or died in another way. higher mortality rates. For example, from Pellet A day later the pasture was revisited, and a sample treatments at site 3, the percentage of control of 1st- of 25 pupae was brought back from which only day pupae was 9O.64Vo (n : 2O3), whereas 2nd- 44Vo dred. The Kings pasture was treated a few day control was only 68.5Vo (n : 254). This dif- days before the trial in Fresno County began, and ference of 22.l4vo was double that observed at site it was from our experience in this pasture that we I 1. Higher mortality rates for earlier collections attempted to continue evaluating methoprene con- also occurred in XR-G-treated plots. Control at site trol from more than I day's pupal collection when- I of lst-day pupal collections was 37.l7vo (n : ever possible. Unfortunately, all of the All-treated 374) a;nd 16.76%o(n : 149) for 2nd-day collections. pastures other than pasture 11 dried up too rapidly Tllis ZOVo difference is similar to that seen at the for more than I day of pupal collection. site 11 XR-G-treated area.

Table 2. Pupal mortality evaluations of Aedes nigromaculis treated with various Altosid@ formulations in several pastures in Central California, September, 1999.

Treatment Percent Corrected (g active Area treated mortality mortality ingredient/ha)' (ha) \n) (vo) l1 12.33 ALL t.54 7.7r (4O2) 6.O7 2 tt.zt Ar,L 0.26 2.98(r68) 1.25 10 10.65ALL 0.52 0 (6) Na l4 1t.2t At L o.4 1.81(386) o.06 122 tr.zt ALL 3.O4 87.(X (s4) 86.81 732 tt.zt Ar-L 0.81 97.37(76) 97.32 152 13.67 ALL 2.O3 99.06 (841) 99.O4 1l 2.90.85XR-G 0.89 8.r4 (295\ 6.5 1 312.71XR-G 2.9 31.33(553) 30.11 6 403.5 XR-G 1.03 8.34(s1) 6.7 Kings 4OO4 168.12XR-G 3.24 90 (r85) 89.82 l1 396.37 Pellets 1.46 53 (30O) 52.16 J 571.62Pellets o.7 79.57 (4s7) 79.21 4 314.39Pellets 1.08 99.18(488) 99.17 2 333.44 Pellets o.26 85.12 (r2r) 84.85 'ALL, Altosid liquid fomulation; XR-G, Altosid XR-G; Pellets,Altosid Pellets. 'Pasturesnoted to still have effective ALL control prior to study. SeP,rEnspn 2000 METHoPRENEToLERANCE w Atons Nrcnouecuus

Generally, very low control was achieved with between the islands and the mainland. However, in ALL and XR-G methoprene applications in pas- California, the pastures are not isolated habitats and tures in which previous failures had occurred. An form a continuum across the Central Valley. Hence, unintentional application of XR-G at a rate higher one would expect the Ae. nigromaculis to be a sin- than the recommended rate (26.9 kg/ha) still did not gle panmictic population. In California, methoprene produce acceptablecontrol levels at site 6. Much tolerance appears to have been selected in a large higher control with ALL was achieved in the 3 pas- population by repetitive treatment. Dispersal stud- tures where methoprene still appeared to kill Ae. ies have shown that Ae. nigromaculds mosquitoes nigromaculis prior to the study (sites 12, 13, and are capable of flying at least 11 km (Husbands and l5). At site 15 (<1 mile from site ll), the same Rosay 1952), and genetic exchange should occur ALL sand and drying agent mixture that was used from one pasture to the next. Interestingly, the pres- at site 11 was used 16 days after application at site ent study shows that pastureswith susceptiblepop- 11. This fell within the recommended period of 20 ulations are dispersed among pastures with treat- days of shelf life of methoprene (ALL) sand mix- ment-resistant populations. This may suggest that ture. All other ALL treatrnents were with material development and spread of methoprene resistance that had not necessarilycome from the same mix- is still at its early stages. Methoprene failures were ture but that had originated from the same liquid originally noticed at site I during the past summer. methoprene batch. Numerous pastures southwest of this pasture, es- sentially downwind, developed resistance. Howev- DISCUSSION er, that some levels of tolerance were noticed more than 30 km south in Kings 4OO4may indicate that In the early 1970s, the question of why insecti- methoprene resistance is developing independently cide resistance always appears to start in alkaline in several locations. pastures was raised. Schaefer (1972) suggestedthat Selection for increased tolerance to methoprene it had nothing to do with the affect of alkaline soil has been achieved in the laboratory after many gen- and water on the insecticides. Rather, it was be- erations with Culex tarsalis Coq. (Georghion 1974) cause the pastures in question were on soils with and Culex pipiens pipiens L. (Brown and Brown poor drainageand could not be usedfor crops. Con- 1974). The mechanismsof resistancein thesemos- stant irigation to create a sufficient gr.\zing area quitoes were not investigated, but cytochrome P450 problem. pastures created a mosquito These were or carboxylesterase mediated detoxification mech- thus targeted for mosquito control. If one assumes anisms(Brown and Brown l9T4,Feyereisen1998) a very conservative of estimate flooding every 18 have been postulated.Additionally, methoprenere- days, each pasture has to be treated 5 times during sistant strains of Drosophila melanogasterMeigen the peak Ae. nigromncurr.r season. The repetitive have been selected in the laboratory by P-element chemical treatrnent that has been conducted since mutagenesis (T\rrner and Wilson 1995), and recent 1974 (25 years) created a strong selection pressure molecular genetic studies on these strains have pro- on mosquito populations that favored chemical-tol- vided considerable insights into a possible insen- erant individuals. In addition, those pastures that involving are closest to urban areas are treated more exten- sitive target site resistance mechanism the gene (Ashok sively than pastures further away because of their Met et al. 1998, Wilson and Ashok proximity to more people. In the sites in this study, 1998). The Met gene appears to be a transcription it appearsthat the highest levels of methoprene fail- factor gene coding for a family of bHLH-PAS pro- juvenile ures occurred in pasturesclosest to residential areas teins, and the role that this protein has in juvenile (sites 1, 3, and l1). All the resistantand susceptible hormone and hormone mimic metabolism pastures occur in similar sandy loam soil types. is presently under investigation. Comparisons of the Hence, different soil substratesaffecting methoprene homolog Met gene between methoprene susceptible silica binding properties between pastures is unlike- and resistantpopulations of Ae. nigromaculls could ly. also prove insightful. The mechanismsof resistanceand spreadof tol- The high methoprene failure rates observed in erance to methoprene in Ae. nigromaculis needs ad- this study and the lack of evidence of alternative ditional study. Resistance to methoprene has arisen explanations suggest that Ae. nigromaculis has de- in a mosquito population in isolated barrier islands veloped increased tolerance to methoprene. It had (Captiva and Lover's Key Islands) off the west been hoped that an endogenouscompound similar coast of Florida. In Florida, the salt marsh mosquito in molecular structure or physiological action Aedes taeniorhynchus (Wied.) on Captiva island would be less likely to induce resistance.To deter- and Lover's Key island was found to be 14.9- and mine quantitatively what the levels of methoprene 14.8-fold more tolerant of methoprene, respective- resistance or tolerance levels are, bioassays com- ly, than was a mainland (Flamingo strain) popula- paring methoprene mortality dose responsesof sus- tion (Dame et al. 1998).Ae. taeniorlrynchusappears ceptible, resistant, and if possible, methoprene-na- to have developed increased tolerance in isolated ive populations are currently being conducted. island habitats where little to no gene flow occurs Offspring from host-seeking Ae. nigromaculis fe- 228 JoURNAL oF THE AMERTcIN Mosqurro CoNrnol AssocrATIoN Vol. 16.No.3 males that were collected this past season are being Feyereisen R. 1998. Juvenile hormone resistance: no PA- used for these bioassays. Saran. Proc Natl Acad Sci USA 95:2725--2726. Georghiou GP 19'14. Assessment of potential of Culex tarsalis for development of resistance to carbamate in- ACKNOWLEDGEMENTS secticides and insect growth regulators. Proc Calif Mosq Control Assoc 42:117-118. We are thankful to Wellmark International for Hardy JL, Reeves WC. 1990. Experimental studies on in- supplying the methoprene to conduct the field trials. fection in vectors. In: Reeves WC, ed. Epidemiology thankful for and control of mosquito-bome arboviruses in Califor- We are also assistancefrom Tim Phil- nia, 1943-1987. Sacramento, CA: Mosquito Vector lips (Fresno County Mosquito and Vector Control Control Association. p 145-253. District) and Henry Doll (Consolidated Mosquito Husbands RC, Rosay B. 1952. A cooperative ecological Abatement District) for applying the methoprene study of mosquitoes of irrigated pastures. Proc Calif and collecting pupae for analysis. Mosq Control Assoc 2O:l'126. Lewallen LL, Nicholson LM. 1959. Parathion-resistant Aedes nigromaculis in California. Mosq News 19:12- REFERENCES CITED 14. Schaefer CH. 1972. Mosquito control crisis in the central Abbott WS. 1925. A method for computing the effective- valley: where do we go from here? Proc Calif Mosq ness of an insecticide. J Econ Entomol 18l.265-267. Control As soc 4O:.76-8O. Ashok M, Turne C, Wilson TG. juvenile hor- 1998. Insect Schaefer CH, Dupras EF Jr. 1973. Insect developmental gene mone resistance homology with the bHLH-PAS inhibitors. 4. Persistence of ZR-515 in water. J Econ family of transcriptional regulators. Proc NatI Acad Sci Entomol 66:923-925. USA 95:2761-2766. Schaefer CH, Wilder WH. 1970. Insecticide resistance and Bohart RM, Murray WD. 1950. DDT resistance in Aedes cross-resistance in Aedes nigromaculis. J Econ Entomol nigromaculis larvae. Proc Calif Mosq Control Assoc 63:1224-1226. 18:.22-23. Schaefer CH, Wilder WH. 1973. Insect developmental in- Bohart RM, Washino RK. 1978. 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