Microplate assay analysis of the distribution of organophosphate and carbamate resistance in Guatemalan Anopheles albimanus

Bulletin of the World Health Organization, 66 (3): 339-346 (1988) Microplate assay analysis of the distribution of organophosphate and carbamate resistance in Guatemalan Anopheles albimanus W. G. BROGDON,1 R. F. BEACH,2 J. M. STEWART,3 & L. CASTANAZA4 Simple microplate assay methods for determining the frequency of insecticide re- sistance in single mosquitos were used to study the distribution and localization of organophosphate and carbamate resistance in field populations of Anopheles albimanus Weidemann in Guatemala, where such resistance, caused by heavy use of agricultural pesticides, has long been assumed to be widespread. Areas of complete susceptibility to organophosphates and carbamates were observed, as well as areas where the resistant phenotypes represented up to 98% of the population. Overall, the resistance levels were lower and more localized than expected. Two mechanisms of resistance were identified by the microassay methods. These were the elevated esterase (nonspecific esterase) and insensitive acetylcholinesterase mechanisms which were selected independently, the for- mer (documentedfor the first time in Central American anophelines) being predominant. These methods represent a promising new technology for the detection and assessment of resistance and will facilitate improved control strategy decisions. The malaria vector, Anopheles albimanus, from the coastal areas of Central America has shown re- sistance to nearly all known insecticides through the development of a variety of resistance mechanisms. Davidson was the first to review organochlorine mul- tiresistance in this vector; resistance to both DDT and 1 Research Entomologist, Malaria Branch, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333, USA. Requests for reprints should be sent to this author. 2 Research Entomologist, Medical Entomology Research and Training Unit-Guatemala, Centers for Disease Control, Atlanta, GA 30333, USA. 3 Research Biologist, Medical Entomology Research and Training Unit-Guatemala, Centers for Disease Control, Atlanta, GA 30333, USA. 4 Entomological Technician, Centre for Tropical Disease Research, Universidad del Valle de Guatemala, Guatemala City, Guatemala. dieldrin in the same mosquito populations appeared in Central America in the late 1950s (1). Subsequently, resistance to organophosphates and carbamates appeared, for which one mechanism has been des- cribed (2-7). Synthetic pyrethroid resistance has also recently been reported in Guatemala (8). This rise and spread of resistance in Central America has been attributed to the rapidly increasing use of agricultural pesticides (9). Most studies of resistance in A. albimanus, particularly concerning the biochemical mechanisms of resistance, have been evaluated in laboratories on colonized, selected mosquito strains because of the lack of a methodology for ascertaining resistance mechanisms in the field. As a result, the practical (operational) field impact of these studies on Central American A. albimanus has been minimal. For 4886 -339- 340 W. G. BROGDON ET AL. example, there have been no studies of the spatial distribution of resistance mechanisms or levels, and the mechanisms that were identified in the laboratory have not been confirmed in the field. For decades, field studies on resistance problems in situ have relied on the WHO bioassay kit for determining resistance in mosquitos. Although much has been learned from this detection system, it has several inherent limitations (9). Only one insecticide can be tested per insect and, without a known dis- criminating dosage, large numbers of insects are needed to generate probit lines. Using discriminating dosages allows detection of resistance with smaller numbers of insects but not determination of the resistance level or mechanism. Moreover, discrimi- nating dosages that could be applied to all mosquitos do not exist. Also, false positives may occur because of deteriorating filter-papers or precedural variables, such as temperature or humidity. Finally, the bio- assay methods are ineffective in detecting resistance phenotypes at low frequencies. For these reasons, we proposed the development of microplate assay methods for detecting resistance in single mosquitosa based upon methods we had devel- oped (10) and were using in resistance studies.b Since then, workers in several laboratories have developed or are developing similar biochemical and new immunological methods for evaluating resistance (11-15). Recently, these methods have been intro- duced into field studies of resistance in the anopheline vectors of malaria in Haiti and Sri Lanka (16). This paper describes the results of a microplate assay method that was used to detect and assess the spatial distribution of organophosphate and carba- mate insecticide resistance in Guatemalan A. albi- manus in various parts of the country. Other studies on the relationship between the microplate assay and the WHO bioassay, on the temperature effects in microplate assays, and on longitudinal studies of resistance foci are under way. MATERIALS AND METHODS Mosquito collections The spatial distribution of resistance was deter- mined on specimens ofA. albimanus that were collec- ted throughout the country from selected locations. The mosquitos were collected in three ways. (1) Human-biting collections were made in the early a BROGDON, W. G. A proposed new method under development for field detection and evaluation of insecticide resistance. Un- published WHO document VBC/84.859. b BROGDON, W. G. New methods for biochemical field studies of insecticide resistance. Paper presented at the annual meeting of the American Mosquito Control Association, Sacramento, CA, 1982. evening in sites that were distant from corrals since A. albimanus, a zoophilic species, rarely bites man in close proximity to cattle. (2) In the corrals, mosquitos that had virtually all been blood-fed were collected from their resting sites in vegetation or other struc- tures (17). (3) Mosquitos were also collected using ultraviolet light updraft traps (18). The collections decreased in size in the order: corral captures > human-biting captures > UV-trap captures. The live mosquitos were immobilized using a dry- ice chest and transferred to labelled vials that were returned to the laboratory on dry ice for storage at -70 OC. Collections of more than 200 A. albimanus females were obtained from each study site. Microassays and experimental design Elevated esterase microplate assay. Individual mosquitos were homogenized in 100 1l of0.05 mol/l potassium phosphate buffer, pH 6.8, and diluted to 1 ml with buffer. Aliquots of 100 Id were used for each assay replicate. The microplate assay procedure of Brogdon & Dickinson was used for enzyme assays (10). To each 100 Id of homogenate were added 100 ,sl of ,3-naphthyl acetate (56 mg/10 ml 2-pro- panol/90 ml buffer) using a 96-tip transfer platec and the preparation was incubated at ambient tem- perature for 10 minutes (in Guatemala City, the laboratory temperature was 25 OC). A 100 Id aliquot of dianisidine (100 mg/100 yd water) was then added. Absorbances were read at 550 nm in an enzyme immunoassay readerd or evaluated visually. Insensitive acetylcholinesterase microplate assay. Individual mosquitos were homogenized in 100 i1 of 0.05 mol/l potassium phosphate buffer, pH 6.8, and diluted to 1 ml with buffer. Although the pH opti- mum for the original acetylcholinesterase assay of Brogdon & Dickinson was 7.4, the use of a buffer with a pH of 6.8 did not affect the detection efficiency and allowed both the elevated esterase and the insen- sitive acetylcholinesterase microassays to be more easily run with replication on the same mosquitos (10). To each assay well were added 100 gd acetyl- thiocholine iodide (75 mg/100 ml buffer) containing 0.1 mmol/l propoxur and 100 A1 Ellman's reagent(DTNB, 13 mg/100 ml buffer). Absorbance (at 414 nm) was measured at 30 min using the microplate reader. Reaction kinetics may also be monitored using the same system (19). Protein microplate assay. The significance of size variations between mosquitos for the interpretation - Vaccupette, Research Products International Corp., Mount Prospect, IL, USA. Use oftrade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services. d Minireader II, Dynatech Laboratories, Alexandria, VA, USA. INSECTICIDE RESISTANCE IN GUATEMALAN ANOPHELESALBIMANUS 341 of results was evaluated using a microplate protein assay (20, 21). To 100 I1 aliquots of mosquito homo- genate were added 200 1l of diluted dye reagent.e Absorbance (at 600 nm) was noted using the micro- plate reader, and values were compared with a stan- dard curve. For each mosquito, three replicates were assayed (for the elevated esterase, insensitive acetylcholin- esterase, and protein). The microtitration plates were organized so that thirty-two mosquitos (three repli- cates) could be microassayed using one of the three types of assays on a single plate. Thus, three plates were used for running the three assays on the same thirty-two mosquitos, and the transfer plates could be used to maximum advantage. Resistancejfrequencies. Estimates of the percentage resistance were made from comparative bioassay/ microassay data collected from Guatemalan-A. albi- manus. The resistance thresholds in microplate assays of Guatemalan A. albimanus are (at absorbance 550) >0.9 in the elevated esterase assays and (at absorb- ance 410) >0.3 in the insensitive acetylcholin- esterase assays. Bio-Rad Laboratories: Richmond, CA, USA. PETEN OEIC(i) BELIZE MEXICO HIGHLAND NDURAS OAS1 1 A1 i ~ 1 LSALVADOR PACIFIC OCEAN Fig. 1. Map showing the 21 collection sites in Guatemala; at least 200 mosquitos were collected from each location (total collection, 9417 mosquitos). Table 1 gives the name of the department for each site number. RESULTS Approximately 1100 mosquitos were microassayed for the elevated esterase and insensitive acetylcholin- esterase mechanisms. The protein levels were suf- ficiently consistent and no corrections for mosquito size variation were needed. Data were organized according to the Guatemalan administrative depart- ments in which the study sites were located (Table 1, Fig. 1). Neither of the two resistance mechanisms was detected in non-agricultural areas and where there had been little or no use of organophosphate or carbamate insecticides for malaria control. Both mechanisms were detectable but uncommon in areas where agriculture was relatively light (Fig. 2, 3); higher frequencies of both resistance mechanisms were observed throughout the heavily agricultural areas near the Pacific coast (Fig. 4, 5). Frequency distributions for elevated esterase absorbance values from the Pacific coastal areas revealed three absorbance peaks (Fig. 4) which cor- respond to susceptible or resistant homozygotes and Table 1. Locations of the 21 study sites by administrative department and intensity of agriculture in the area Site number/ Intensity of department Location agriculture 1, 2/San Marcos Pacific coast/Mexican border: Heavy Ocos, Hacienda la Zarca 3, 4/Retalhuleu Pacific coast: Heavy Colonia la Felicidad, Hacienda la Agricola 5, 6, 7, 8/Escuintla Pacific coast: Heavy Aldea San Pedro Nahualate, Aldea Santa Marta el Mar, Aldea Empalizada, Barrio el Mangelar 9, 10/Santa Rosa Pacific coast: Heavy Aldea el Chapeton, Aldea la Avellana 11, 12,13/Jutiapa Salvadoran border: Light Parcelamiento Montufar, Aldea el Toro, Aldea las Moritas 14/Guatemala Foothills near Guatemala City: Light Finca Rancho Grande 15, 16, 17/lzabal Atlantic coast: Light Hacienda las Vegas, Aldea el Relleno, Aldea San Francisco II 18/Alta Verapaz Northem rain forest: None Aldea Chajmaic 19, 20, 21 /Pet6n Northern rain forest: None Finca Esquipulas, Aldea el Quetzal, Aldea Los Angeles W. G. BROGDON ET AL. TL~~~~~~~~~EE 1 0 2=03 0.4 05 0.6 0.7210 1.1 1.2 13 14 .5 168 !7 1.8 1. 1 ABSORBANCE 550 30 - 20 fI IT1 1-1 1 1 1 1 1 SITES 15, 18, 17 0.3 0.4 0.5 0.6 0.7 0.8 0.9 *1. 112 1A1.541.ABSORBANCE 550 30 [10 0 0 0 l 1 1.1 1 1 1 1 20 H , , ,._ __ ABSORBANCE 550 60 0.1 0.2 0.3 0.4 0.5 0.5 0.7 0.5 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 ABSORBANCE 550 Fig. 2. Elevated esterase microassay data from four Guatemalan departments with little or no agricultural spraying. Frequencies (at absorbance 550) were based on four replicates per mosquito (n - sample size; r - micro- assay resistance level determined by the described criteria). heterozygotes, although certain sites did not show all three. The elevated esterase data from the Pacific coast were pooled, and the resulting frequency distri- bution was plotted (Fig. 6); the peaks (at absorbance 550) at 0.7, 0.9, and 1.1 correspond to the pink, lavender, and purple hues observed in the assays. The frequency distribution for insensitive acetyl- cholinesterase absorbance values from mosquitos in Escuintla (Fig. 5) shows a second peak in frequency at 0.4 (heterozygotes); the susceptible homozygote peak is at 0.2. The distribution of these resistance mechanisms has been found to be geographically localized. For example, as shown in Fig. 7, a site near Lake Ama- titlan showed a higher frequency of elevated esterase resistance than the area with the most resistance on 3 Fig. 3. Insensitive acetylcholinesterase microassay data from four Guatemalan departments with little or no agri- cultural spraying. Frequencies (at absorbance 410) were based on three replicates and one control replicate per mosquito (n = sample size; r - microassay resistance level determined by the described criteria). LHmT 1 --lLI- 1-1 ESCUINT -LL ABSORBANCE 550 E---- I ~~~I I ISr8 0-SANTfA ROSA 20 SIES 10-101 r-53% U, x U.3 V.'4 U.5 U.{ U.7f U. U.9 1.UI1.1 . 1.3I 1.c 1.E 1. fO 1.0 .. .U ABSORBANCE 550 Ii1111111111 liiSAN MARCOS '°TT : I 'I-4 I I0 n0 57 lx 10U. r - 49% 30. . . . . . . . . 0.1 0.2 S0.3 OA 0.5 0.5 07-01 01 1 011 112 13 114 15 111 ABSORBANCE55 I I I I RETALHULEU I I I>1 1 1 1 1 1 IS 1---1 1 1 1 1I _LI a_ I III".31 z20 - IISI I S TES3,4 l 0.1 0.2 0.3 04 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1A 1.5 1.6 1.7 1.8 1.9 2.0 ABSORBANCE 550 Fig. 4. Elevated esterase microassay from four Guate- malan departments with heavy agricultural spraying. Frequencies (at absorbance 550) were based on four replicates per mosquito (n - sample size; r - microassay resistance level determined by the described criteria). 0 z W e U. 0 z U. 0 z > ~~~~~~IJtI.PYA!A I ~ L1III1isTSsB...L40 SITES 1.13 ll ll lI30ll ISITE5 0 01 02 03 0. 3. . .>n0 aU I I10 T i 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1. 1.8 .9 tO . . . . . . . 1-7 tR. .I 342 6 .W 2.uW INSECTICIDE RESISTANCE IN GUATEMALAN ANOPHELESALBIMANUS Iii i g 9 IL7 1-i I.--. 30 I l 1-1 1111 1 1 1 1 .A-- 03 OA 0.7 I. 1.1 1.2 1.J 1.4 1.5 1.6 1.7 1.9 1.9 20 2.1 2.2 m1-I M I11.1 I I1>.!,..L.1 0 . . _ _ _ _ 91%~~~~~~~n ao 10____:a1iSil l l lL__~~~~~~~~~~~r.0 0.5 1.0 1.1 1.2 1.3 1.4 1.6 1.6 1.7 1. 1 30 2.1 2.2 ABSoRNC NO Fig. 5. Insensitive acetylcholinesterase microassay data from four Guatemalan departments with heavy agricul- tural spraying. Frequencies (at absorbance 410) were based on three replicates and one control replicate per mosquito (n - sample size; r - microassay resistance level determined by the described criteria). the Pacific coast, San Marcos, which might be ac- counted for by intense, indiscriminate insecticide spraying of surface waters by the owners of lakeside vacation villas. Foci for insensitive acetylcholin- esterase resistance were identified in Escuintla. Two sites, Puerto San Jose and Santa Marta el Mar, showed much higher frequencies of insensitive acetylcholinesterase than the other sites in Escuintla (Fig. 8) or elsewhere in Guatemala. Investigations revealed very large (33 km2) cotton fields adjoining the breeding sites where insecticide was applied by frequent aerial spraying during the season. -A3 --W-r-T--l..I | | | m~-rnr SMSOE Fig. 6. Pooled elevated esterase microassay data for the Pacific coastal areas. Frequencies (at absorbance 550) were based on four replicates per mosquito (n = sample size; r = microassay resistance level determined by the described criteria). Fig. 7. Comparison of the frequencies and levels of elevated esterase microassay absorbances from two Guatemalan A. albimanus collection sites. Frequencies (at absorbance 550) were based on four replicates per mosquito (n = sample size; r= microassay resistance level determined by the described criteria). T IBUIATE ESC6UINTLA 20 -- - - r''-so 10F o e 11 0.4 U -c ABSOFBANCE 410 PUERTiO S'AN JOS:11111 HEISICUII iI nTLA on * A |-X_ I X .W I _ e 10 k / 1 1 ABSORBANCE 410 Fig. 8. Comparison of frequencies and levels of insensi- tive acetylcholinesterase microassay absorbances from two Guatemalan A. albimanus study sites in Escuintla. Frequencies (at absorbance 410) were based on three test replicates and one control replicate per mosquito (n = sample size; r = microassay resistance level deter- mined by the described criteria). 343 O.3 0.4 0.5 0.6 QL7 0.9A_ EAN410 ABOOORA""41 344 W. G. BROGDON ET AL. Certain sites showed interesting differences in frequencies of the two resistance mechanisms. For example, in Escuintla, resistant homozygotes were missing from the elevated esterase microassay fre- quency distribution, but the insensitive acetylcholin- esterase mechanism was present at relatively high frequency. In San Marcos, the insensitive acetyl- cholinesterase mechanism was at relatively low frequency but resistant homozygotes occurred at relatively high frequency. DISCUSSION Until the present study, the only organophosphate/ carbamate resistance mechanism known in Central American A. albimanus was related to insensitive acetylcholinesterase (22). An elevated esterase mechanism similar to that detected in Guatemala has recently been reported in HaitianA. albimanus, but in no other anophelines. However, we have observed this mechanism in the field in A. crucians in Haiti and A. pseudopunctipennis in Guatemala (Brogdon & Beach, unpublished data). Others have found that antibodies raised to Culex resistance esterases did not react with esterases in a number of anophelines, but the epitope(s) involved in that study are unknown and may not be broadly diagnostic for resistance esterases (23). It appears that both the heterozygotes and the homozygous susceptible and resistant mosquitos were detected using the elevated esterase assay. How- ever, great caution must be exercised in analysis of isolated data showing only one genotype, since multiple copies of resistance genes may complicate analysis of otherwise simple dominant gene resist- ance (24, 25). Few homozygous resistant genotypes were detected in the insensitive acetylcholinesterase assays, which is to be expected with such low levels of resistance. Both resistance mechanisms studied in Guatemala appear to be closely associated with heavily agricul- tural areas, but these are also the areas of highest mosquito densities and heaviest use of insecticides by the public health departments. Further studies will be necessary to delineate the relative roles played by agricultural and public health pesticide application in modulating the resistance patterns observed at par- ticular locations at particular times. Selection for resistance for public health spraying has been impli- cated in field studies of A. albimanus in Haiti and A. culicifacies in Sri Lanka (16). The most important issue of operational signifi- cance raised by the Guatemalan data is that resistance distribution is heterogeneous. First, the two mechan- isms (elevated esterase and insensitive acetylcholin- esterase), aside from their association with agricul- tural areas, appear to be selected independently. Areas where one mechanism is common do not nec- essarily have high levels of the other, as shown by data from San Marcos and Puerto San Jose. Also, resistance due to either mechanism may be highly localized and examples are Tiquisate vs. Puerto San Jose, Lake Amatitlan vs. San Marcos. Finally, the patterns of local insecticide use are associated with resistance foci, as illustrated by the data from Lake Amatidan and Puerto San Jose. Recognition of these differences in resistance dis- tribution was facilitated through use ofour microplate assay methods (10), which through further studies could help resolve resistance problems in the field! The ability to spot low frequency resistance geno- types for specific mechanisms will make possible the earlier detection of resistance and precise studies of resistance microepidemiology. Most importantly, these methods will ultimately make resistance man- agement techniques available to malaria control personnel in the field. f See footnote a, page 340. ACKNOWLEDGEMENTS This study was supported, in part, by the World Health Organization. RESUMME ANALYSE PAR TITRAGE SUR MICROPLAQUE DE LA DISTRIBUTION DE LA RESISTANCE AUX ORGANOPHOSPHORtS ET AUX CARBAMATES CHEZ ANOPHELES ALBIMANUS AU GUATEMALA Afin d'etudier la distribution et la localisation de la rdsistance aux organophosphores et aux carbamates chez des populations sauvages d'Anopheles albimanus Weide- mann au Guatemala, oti l'on soupconne cette resistance, due INSECTICIDE RESISTANCE IN GUATEMALAN ANOPHELESALBIMANUS 345 a un usage intensif de pesticides agricoles, d'etre largement r6pandue, on a utilist des methodes simples de titrage sur microplaque permettant de determiner la frequence de la r6sistance aux insecticides chez des moustiques isoles. D'autres etudes sur la relation entre les r6sultats du titrage sur microplaque et ceux du titrage biologique OMS sont 6galement en cours, ainsi que des etudes longitudinales sur les foyers de resistance. On a observe aussi bien des zones de sensibilite totale aux organophosphores et aux carba- mates que des zones oti les ph6notypes resistants repr6sen- taient jusqu'a 98% de la population anophelienne. Dans l'ensemble, la r6sistance dtait plus localis&e et de niveau plus faible que l'on ne pensait. Grace aux methodes de microtitrage, on a identifie deux mecanismes de resistance: l'un faisait intervenir une ele- vation des est6rases (esterases non specifiques) et l'autre une insensibilit6 de l'acetylcholinesterase, le premier m6ca- nisme (observ6 pour la premiere fois chez des anophelines d'Amerique centrale) 6tant predominant. Aucun de ces micanismes n'a d6t d6cele dans les regions non agricoles et ot l'on n'emploie que tres peu les organophosphores ou les carbamates pour la lutte antipaludique. Les deux m6ca- nismes 6taient observables, mais rares, dans les r6gions relativement peu agricoles; en revanche, on observait une fr6quence elevee des deux m6canismes de rdsistance dans toute la r6gion d'agriculture intensive le long de la c6te pacifique. Les deux m6canismes de r6sistance sont donc etroitement associ6s aux zones d'agriculture intensive, qui sont egale- ment des zones de tres forte densite de moustiques et d'emploi intensif d'insecticides en sant6 publique. D'autres etudes seront necessaires pour determiner le r6le relatif de l'application de pesticides dans l'agriculture et en santd publique en ce qui concerne la modulation des niveaux de resistance observes en un lieu donne et a un moment donne. La selection de la resistance sous l'effet de pulverisations a but sanitaire a ete observee lors d'etudes sur le terrain d'A. albinanus en Haiti et d'A. culicifacies a Sri Lanka. Les donnees obtenues au Guatemala sont d'un interet particulier sur le plan operationnel car elles montrent que la distribution de la resistance est heterogene. Tout d'abord, les deux mecanismes de resistance (elevation des esterases et insensibilite de l'acetylcholinesterase), a part leur association avec les zones agricoles, semblent selectionnes independamment. Lorsque l'un d'eux est fr&quent dans une region, l'autre ne l'est pas necessairement, comme il ressort de leur incidence relative dans deux regions du pays. De plus, la resistance due a l'un ou l'autre mecanisme peut etre extremement localisee, comme le montrent plusieurs exemples dans chaque cas. Enfin, on observe une associa- tion entre les types locaux d'emploi d'insecticides et les foyers de resistance. Le titrage portant sur l'elevation des esterases permet de deceler les moustiques heterozygotes et homozygotes pour la sensibilite ou la resistance aux insecticides. n faut toute- fois etre tres prudent lors de l'analyse de donn6es isolees ne revelant qu'un genotype car la presence de copies multiples des genes de resistance peut compliquer l'analyse, la resis- tance 6tant par ailleurs de type dominant simple. Les titrages portant sur l'insensibilite de l'acetylcholinesterase n'ont montre que peu de genotypes homozygotes resistants, ce qui etait previsible avec un niveau de resistance aussi bas. Ces methodes constituent une nouvelle technologie pro- metteuse pour la d6tection et 1'evaluation de la resistance et devraient faciliter les decisions en matiere de strategie de lutte. REFERENCES 1. DAVIDSON, G. DDT-resistance and dieldrin-resistance in Anopheles albimanus. Bulletin of the World Health Organization, 28: 25-33 (1963). 2. ARIARATNAM, V. & GEORGHIOU, G. P. 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