NIH Public Access Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
Published in final edited form as: J Med Entomol. 2014 May ; 51(3): 644–649.
Susceptibility to Chlorpyrifos in Pyrethroid-Resistant Populations of Aedes aegypti (Diptera: Culicidae) from Mexico Beatriz Lopez1, Gustavo Ponce1, Jessica A. Gonzalez1, Selene M. Gutierrez1, Olga K. Villanueva1, Gabriela Gonzalez1, Cristina Bobadilla2, Iram P. Rodriguez1,3, William C. Black IV4, and Adriana E. Flores1,5 1Universidad
Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/n Cd. Universitaria, San Nicolas de los Garza, N.L., 66451 Mexico 2Laboratorio
Estatal de Salud Publica de los Servicios de Salud de Veracruz, Mexico
3Universidad
NIH-PA Author Manuscript
Autonoma de Nuevo Leon, Departamento de Genetica, Hospital Universitario Dr. Jose Eleuterio Gonzalez, Monterrey, Nuevo Leon 64460, Mexico
4Department
of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins,
CO 80523
Abstract
NIH-PA Author Manuscript
Resistance to the organophosphate insecticide chlorpyrifos was evaluated in females from six strains of Aedes aegypti (L) that expressed high levels of cross resistance to eight pyrethroid insecticides. Relative to LC50 and LC90 at 24h of a susceptible New Orleans (NO) three strains were highly resistant to chlorpyrifos (Coatzacoalcos, resistance ratio (RRLC90) =11.97; Pozarica, RRLC90=12.98; and Cosoleacaque, RRLC50= 13.94 and RRLC90=17.57), one strain was moderately resistant (Veracruz, RR=5.92), and two strains were susceptible (Tantoyuca and Martinez de la Torre, RRLC50 and RRLC90 < 5) in CDC bottle bioassays. Furthermore, high levels of α/β-esterase activity in the sample populations were correlated with resistance, suggesting that esterase activity may be a mechanism causing the development of organophosphate resistance in these populations. Overall, the populations in this study were less resistant to chlorpyrifos than to pyrethroids. Rotation of insecticides used in control activities is recommended to delay or minimize the occurrence of high levels of resistance to chlorpyrifos among local populations of Ae. aegypti. The diagnostic dose (DD) and diagnostic time (DT) for chlorpyrifos resistance monitoring was determined to be 85 μg/ bottle and 30min, respectively, using the susceptible NO strain.
Keywords chlorpyrifos; insecticide resistance; Aedes aegypti; α-esterases; β-esterases
5
Corresponding author: Adriana E. Flores, Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/n Ciudad Universitaria, San Nicolas de los Garza, N.L., 66451 Mexico, Tel: (81) 83294000 ext 3683, Fax: (81) 83294110, [email protected].
Lopez et al.
Page 2
NIH-PA Author Manuscript
The mosquito Aedes aegypti (L.) is the primary vector of dengue in Mexico. In addition, Aedes albopictus (Skuse), originally from Asia, represents a secondary dengue vector that was introduced via the international trade in used tires and other goods (e.g., lucky bamboo), and has now spread to North America (including Mexico) and Europe. Generally, denguevector control programs include activities to control both the immature and adult stages of the Ae. aegypti lifecycle. For example, chemical and/or biological larvicides and habitat reduction are widely used in an attempt to maintain mosquito populations below the threshold levels that disrupt dengue virus (DENV) transmission (Reiter and Gubler 1997, WHO 2007).
NIH-PA Author Manuscript
Since 1950, operational vector control programs in Mexico have used a series of insecticides to control Ae. aegypti (Flores et al. 2006). The organochlorine insecticide DDT was used extensively for indoor house spraying from 1950–1960 and was used in some locations as recently as 1998. In recent decades, the chemical control of mosquito immatures has primarily relied on the use of organophosphate insecticides with temephos as the active ingredient. Other organophosphate insecticides incorporating the adulticide malathion were also used for ultra-low volume (ULV) space spraying from 1981–1999. Furthermore, an oilbased formulation of chlorpyrifos was registered for use in Mexico for adult mosquito control and was used in some locations from 1996–1999. However, these practices changed in 2000 due to the Norma Oficial Mexicana NOM-032-SSA2-2002 (DOF 2003), which dictated a national switch to permethrin-based space spraying for the purpose of adult suppression, and this practice remained in place until 2009. Studies of enzymatic mechanisms (Flores et al. 2005, 2006, 2009) and target-site insensitivity (kdr) (Saavedra et al. 2007, 2008; Ponce et al. 2009, Siller et al. 2011) have shown evidence of permethrin resistance in Mexican Ae. aegypti populations. These findings suggest that the widespread use of permethrin has conferred cross-resistance to many other pyrethroid compounds, including those not commonly used in mosquito-control programs in Mexico (Flores et al. 2013).
NIH-PA Author Manuscript
In 2011, a new policy (NOM-032-SSA2-2010: DOF 2011) was implemented that established characteristics for insecticides used in disease-vector control programs in Mexico, such as those used to treat Ae. aegypti. However, these standards do not specify particular agents to be used, stating instead that specific insecticides should be chosen based on proven effectiveness, resistance and safety characteristics related to exposure. Chlorpyrifos is currently one of the insecticides approved for use as an adulticide in spacespraying programs in Mexico. In Mexico, chlorpyrifos is primarily used to control agricultural pests in more than 20 types of crops, including grasses, cereals, fruits and vegetables, among others. Chlorpyrifos is available commercially in various formulations from more than 30 companies (Senasica 2011). Few reports describe the use of chlorpyrifos as a mosquito-control agent in Latin America, although Rawlins and Ragoonansingh (1990) reported high levels of chlorpyrifos resistance in populations of Ae. aegypti in Puerto Rico, St. Lucia, and Trinidad.
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 3
NIH-PA Author Manuscript
In the current study, we tested Ae. aegypti populations that were resistant to eight pyrethroid insecticides to determine their susceptibility to chlorpyrifos and to investigate the enzymatic mechanisms involved in cross-resistance (Flores et al. 2013).
Materials and Methods Collection Sites and Colony-Rearing Conditions Six populations of Ae. aegypti were collected during 2009 in the state of Veracruz on the eastern coast of Mexico (Fig. 1). Each of the sampled populations has been previously reported as resistant to pyrethroids (Flores et al. 2013). Sample populations were named based on the location of their collection sites: Tantoyuca (21°20′54.44″N, 98°13′45.90″W), Poza Rica (20°32′00.00″N, 97°26′59.84″W), Martinez de la Torre (20°03′42.55″N, 97°03′06.97″W), Veracruz (19°10′21.48″N, 96°07′59.93″W), Coatzacoalcos (18°08′16.00″N, 94°26′07.49″W) and Cosoleacaque (18°00′03.16″N, 94°37′46.90″W). The New Orleans (NO) strain was used as a susceptible reference strain.
NIH-PA Author Manuscript
Females from the F1 generation (1–3 d post-emergence, without blood feeding) were used for the bioassays and biochemical tests. Laboratory colonies were established using larvae collected from natural breeding sites and maintained at 25±4°C under a 12-h light-dark cycle. F1 eggs were obtained from field-collected parents, and the eggs were placed in plastic containers with dechlorinated water along with a 50% aqueous solution of powdered liver protein, which served as food source for the subsequent larval stage. Pupae were placed in 250-mL flasks in cages (30×30 cm) until adult females had emerged. Bioassays The Brogdon and McAllister (1988) bottle bioassay was used in bioassays, which involves adding 1 ml of an acetone solution containing technical-grade chlorpyrifos (>98% purity; ChemService, West Chester, PA, USA) to a 250-mL Wheaton® bottle. The bottles were then capped and shaken to ensure uniform coverage and allowed to dry for 1 h at room temperature. Mortality rate was measured following 1, 2, 4 and 24 h of exposure. The dosage (μg/bottle) range was predetermined to yield mortality rates between 0 and 99% after 24 h of exposure. The tested doses varied from 0.003 to 6 μg/bottle, with three replicates per dose and 20–25 females per replicate. Control bottles were coated with acetone alone.
NIH-PA Author Manuscript
LC50, LC90 and Resistance Ratios LC50 and LC90 values with 95% confidence limits were calculated using the IRMA quick calculator software program (http://sourceforge.net/projects/irmaproj/files/) with logistic regression. The mortality rates were corrected using the formula developed by Abbott (1925) when mortality was observed in the control group. LC values without overlapping fiducial limits were considered to be significantly different. Resistance in the field strains was calculated determining the resistance ratio (RR), by dividing the LC50 of the test population by the LC50 of the NO strain. The criteria proposed by Mazarri and Georghiou (1995) were used to classify the RRs as high (>10-fold), medium (between 5 and 10), or low (< 5). The degree of resistance relative to the susceptible NO strain also was assessed at the LC90 level.
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 4
Enzymatic Assays
NIH-PA Author Manuscript
Sixty females from each of the field populations, as well as the reference NO strain, were individually homogenized in 100 μl 0.01 M potassium phosphate buffer, pH 7.2, and resuspended in 2 ml of the same buffer. Next, 100-μl aliquots were transferred to microtiter plates; each individual sample was analyzed in triplicate on each plate. We then quantified the activities of the α- and β-esterases, mixed-function oxidases (MFOs), glutathione-Stransferases (GSTs) and insensitive acetylcholinesterase (iAChE) according to the methods of Brogdon (1989), using the NO strain as reference. Absorbances were measured using a UVM-340 Microplate reader (ASYS Hitech GmbH; Eugendorf, Austria), and triplicate values were averaged. Protein concentrations were determined using the method of Brogdon (1984), and in some cases it was necessary to dilute the homogenates due to variation in the size of the mosquitoes.
NIH-PA Author Manuscript
The data from each of the biochemical assays were evaluated using analysis of variance (ANOVA) and Tukey’s test; a significance level of P ≤ 0.05 was used to compare the means between the field strains and the NO reference strain. We calculated the OD (optical density) ratios by dividing the mean absorbance values for each enzyme obtained from the field strains by the mean absorbance value obtained for the NO strain. The RRLC50, RRLC90, and OD ratio values were subjected to linear regression analysis. The correlation (r) was performed to determine the degree of association between the two variables. Diagnostic Dose (DD) and Diagnostic Time (DD) We used 1–3 d post-emergence females without blood feeding of the susceptible NO strain and chlorpyrifos (>98% purity; Chem Service, West Chester, PA) diluted in acetone. Five doses of chlorpyrifos between 15 and 115 μg/bottle were used to treat 450 Ae. aegypti females, including control females that were treated with acetone alone. Subjects were divided into groups of 20 for each dose, with 4 replicates per dose. For each dose, mortality rate and observations were recorded every 5 min for 80 min.
Results and Discussion Lethal Concentrations and Resistance Ratios
NIH-PA Author Manuscript
The results of the LC50, LC90 and RR analyses from the studied populations are presented in Table 1. The slopes of dose-response regression lines for the field strains were lower than that of the NO strain in all cases, suggesting a higher level of heterogeneity on field strains. The LC50 value for the Martinez de la Torre strain (0.028 μg/bottle) was significantly lower than that observed for the control (0.066 μg/bottle), and the LC50 value in the Veracruz strain (0.056 μg/bottle) was similar to the control value. However, the LC50 values for the remaining populations were significantly larger: Tantoyuca showed an LC50 of 0.117 μg/ bottle, Coatzacoalcos showed a value of 0.273 μg/bottle, and Cosoleacaque showed the highest with an LC50 of 0.920 μg/bottle. With respect to the LC90 values, the control NO strain was significantly more susceptible than all of the field-collected strains. The control strain showed an LC90 value of 0.286 μg/ bottle, followed by Tantoyuca with an LC90 of 0.627 μg/bottle and Martinez de la Torre
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 5
NIH-PA Author Manuscript
with an LC90 of 1.036 μg/bottle; the LC90 values for the Tantoyuca and Martinez de la Torre strains were not significantly different and showed overlapping confidence intervals. The LC90 value for the Veracruz strain (1.7 μg/bottle) was statistically equivalent to that of the Martinez de la Torre strain. The three highly resistant strains Coatzacoalcos (LC90 = 3.425 μg/bottle), Poza Rica (LC90 = 3.714 μg/bottle) and Cosoleacaque (LC90 = 5.027 μg/bottle) showed LC90 values that were not significantly different from one another. The Tantoyuca and Martinez de la Torre strains showed low-level resistance to chlorpyrifos, with resistance ratios of 2.2 and 3.6, respectively. The Veracruz strain showed moderate resistance (RR=5.92), and the remaining three strains were highly resistant to chlorpyrifos (Coatzacoalcos RR=11.97; Poza Rica RR=12.98; and Cosoleacaque RR=17.57). Enzymatic Assays
NIH-PA Author Manuscript
α- and β-esterase levels were significantly higher in the field-sampled populations than in the control strain (Table 2). In particular, α-esterase levels were highest in the strains from Poza Rica and Cosoleacaque (both with a value of 0.874), moderate in the strains from Veracruz and Coatzacoalcos (both with a value of 0.722), and lowest in the Tantoyuca (0.676) and Martinez de la Torre (0.667) strains. Similarly, β-esterase levels were highest in the Poza Rica and Cosoleacaque strains (1.172), moderate in the Veracruz and Coatzacoalcos strains (1.00), and lowest in the Martinez de la Torre (0.827) and Tantoyuca (0.833) strains. Analysis of mixed-function oxidase activity showed mean absorbance values that were significantly different from the control in the Coatzacoalcos (0.225) and Martinez de la Torre (0.289) strains. Glutathione-S-transferase analyses revealed values that were significantly different from the control in the Veracruz (0.073), Martinez de la Torre (0.076), and Coatzacoalcos (0.080) strains. The strain from Poza Rica showed a significantly higher mean absorbance (0.116) than the other studied strains, including the control.
NIH-PA Author Manuscript
When we examined the associations between the RRLC50 and RRLC90 values for chlorpyrifos and the OD ratios based on enzymatic activity of all strains analyzed we found a strong positive correlation between the RRs and α- and β-esterase with values of r = 0.82 and r = 0.91 for RRLC50 and RRLC90, respectively, which were statistically significant (P < 0.05). These results are consistent with earlier findings by Mazzarri and Georghiou (1995), who suggested changes in A4-esterase activity as a possible mechanism for acquiring chlorpyrifos resistance in Ae. aegypti based on three Venezuelan populations. Esterase activity was also linked to chlorpyrifos resistance in Ae. aegypti population in Cuba and Costa Rica (Rodriguez et al. 2007). DD and DT Resistance monitoring is a crucial strategy for anti-insecticide resistance programs. Based on the methods outlined in the Guideline for Evaluating Insecticide Resistance in Vectors Using the CDC Bottle Bioassay (CDC 2011), we provide a diagnostic dose (DD) and a diagnostic time (DT) for chlorpyrifos resistance testing. We observed a saturating dose of 95 μg/bottle 25 min after exposure. Therefore, considering the established criteria, we propose a diagnostic dosage of 85 μg/bottle and with a DT of 30 min (Fig. 2) for chlorpyrifos resistance-monitoring programs. Note that this recommendation is different from that
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 6
published by Bisset et al. (2013), who reported a DD of 90 μg/bottle and a DT of 30 min for the Rockefeller strain.
NIH-PA Author Manuscript
Field-collected populations from Veracruz that showed high RRs to pyrethroids, including δ-phenothrin, permethrin, deltamethrin, cypermethrin, α-cypermethrin, z-cypermethrin, λcyhalothrin and bifenthrin (Flores et al. 2013) have developed tolerance to chlorpyrifos, albeit to a lesser extent than to pyrethroids. This disparity may occur because chlorpyrifos was only used in Mexico from 1996 to 1999. The populations tested in this study and those used by Flores et al. (2013) were collected in 2009, 10 years after the use of the chlorpyrifos was halted in Mexico. Therefore, this phenomenon may be due to cross-resistance, as was described by Mazzarri and Georghiou (1995) for Venezuelan and Cuban populations, as temephos continues to be used for larval mosquito control. However, as previously discussed, this resistance may also be due to the extensive use of chlorpyrifos for controlling agricultural pests.
NIH-PA Author Manuscript
Although only two of the analyzed populations were not resistant to chlorpyrifos and one showed moderate resistance, the levels of resistance to organophosphates were not as high as those observed for pyrethroids in the same populations, suggesting that the rotation of insecticides may significantly delay and/or minimize the development of strong resistance to chlorpyrifos in populations of Ae. aegypti. This strategy was used in Cuba in 1997 and 2001 during DENV epidemics when cypermethrin was applied in rotational schemes with chlorpyrifos and limited the emergence of cypermethrin resistance in Ae. aegypti populations (Montada et al. 2006). It is essential that we consider actions to avoid strong resistance between pyrethroids and alternative adulticides in Mexico. Going forward, strategies must include resistance monitoring, the development of advanced tools for detecting multiple insecticide resistance, and practical tools for efficient vector control, not only in Mexico, but throughout the entire world.
Acknowledgments
NIH-PA Author Manuscript
This study was supported by FOMIX CONACyT, Veracruz through Grant No. 68298 within the project “Estrategia de control del mosquito vector del dengue Aedes aegypti (L.) en base a su resistencia a insecticidas en Veracruz”, CONACyT Ciencia Basica Grant No. 102120 within the project “Mutacion’kdr’ asociada a la resistencia a piretroides en Aedes aegypti (L.) en Mexico” and UANL PAICYT. This work was also funded in part by NIHNIAID U01 AI088647.
References Cited Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol. 1925; 8:265– 267. Bisset JA, Marin R, Rodríguez MM, Severson DW, Ricardo Y, French L, Díaz M, Perez O. Insecticide resistance in two Aedes aegypti (Diptera: Culicidae) strains from Costa Rica. J Med Entomol. 2013; 50:352–361. [PubMed: 23540124] Brogdon WG. Biochemical resistance detection: an alternative to bioassay. Parasitol Today. 1989; 5:56–60. [PubMed: 15463180] Brogdon WG. Mosquito protein microassay. I. Protein determinations from small portions of singlemosquito homogenates. Comp Biochem Physiol B. 1984; 79:457–459. [PubMed: 6509934]
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 7
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Brogdon WG, McAllister JC. Simplification of adult mosquito bioassays through use of time-mortality determinations in bottles. J Am Mosq Cont Assoc. 1998; 14:159–164. CDC (Centers for Disease Control and Prevention). Guideline for evaluating insecticide resistance in vectors using the CDC bottle bioassay: a web-based instruction. 2011. (http://www.cdc.gov/ parasites/education_training/lab/bottlebioassay.html) DOF (Diario Oficial de la Federacion). NOM-032-SSA-2-2002 for epidemiological monitoring. Prevention and control of vector-transmitted diseases. DOF; Mexico: 2003. NOM-032-SSA-2-2002 para la vigilancia epidemiologica. Prevencion y control de enfermedades transmitidas por vector. DOF (Diario Oficial de la Federacion). NOM-032-SSA-2-2002 for epidemiological monitoring. Prevention and control of vector-transmitted diseases. DOF; Mexico: 2011. NOM-EM-003SSA2-2010 para la vigilancia epidemiologica. Prevencion y control de enfermedades transmitidas por vector. Flores AE, Albeldaño VW, Fernandez SI, Badii MH, Loaiza H, Ponce GG, Lozano FS, Brogdon WG, Black WC, Beaty BJ. Elevated α-esterase levels associated with permethrin tolerance in Aedes aegypti (L.) from Baja California, Mexico. Pest Biochem Physiol. 2005; 82:66–78. Flores AE, Grajales JS, Fernandez SI, Ponce GG, Loaiza H, Badii MH, Lozano FS, Brogdon WG, Black WC, Beaty BJ. Mechanisms of insecticide resistance in field populations of Aedes aegypti (L.) from Quintana Roo, Southern Mexico. J Am Mosq Control Assoc. 2006; 22:672–677. [PubMed: 17304936] Flores AE, Reyes G, Fernandez SI, Sanchez RFJ, Ponce GG. Resistance to permethrin in Aedes aegypti (L.) in northern Mexico. Southwest Entomol. 2009; 34:167–177. Flores AE, Ponce G, Silva BG, Gutierrez SM, Bobadilla C, Lopez B, Mercado R, Black WC IV. Wide spread cross resistance to pyrethroids in Aedes aegypti (Diptera: Culicidae) From Veracruz state Mexico. J Econ Entomol. 2013; 106:959–969. [PubMed: 23786088] Mazzarri MB, Georghiou GP. Characterization of resistance to organophosphate, carbamate, and pyrethroid insecticides in field population of Aedes aegypti from Venezuela. J Am Mosq Control Assoc. 1995; 11:315–322. [PubMed: 8551300] Montada D, Zaldivar J, Sanchez F, Figueredo D, Suarez S, Leyva M. Eficacia de los tratamientos intradomiciliarios con los insecticidas cipermetrina, lambdacialotrina y clorpirifos en una cepa de Aedes aegypti. Rev Cubana Med Trop. 2006; 58:130–135. Ponce G, Flores A, Fernández I, Saavedra K, Reyes G, Lozano S, Bond J, Casas M, Ramsey J, García J, Domínguez M, Ranson H, Hemingway J, Eisen L, Black WC IV. Recent rapid rise of a permethrin knock down resistance allele in Aedes aegypti in México. PLoS Negl Trop Dis. 2009; 3:e531. [PubMed: 19829709] Rawlins SC, Ragoonansingh R. Comparative organophosphorous insecticide susceptibility in Caribbean populations of Aedes aegypti and Toxorynchites moctezuma. J Am Mosq Control Assoc. 1990; 6:315–317. [PubMed: 1973450] Reiter, P.; Gubler, DJ. Surveillance and control of urban dengue vectors. In: Gubler, DJ.; Kuno, G., editors. Dengue and dengue hemorrhagic fever. CABI Publishing; Cambridge, MA: 1997. p. 425-462. Rodriguez MM, Bisset JA, Fernandez D. Levels of insecticide resistance and resistance mechanisms in Aedes aegypti from some Latin American countries. J Am Mosq Cont Assoc. 2007; 23:420–429. Saavedra K, Urdaneta L, Rajatileka S, Moulton M, Flores AE, Fernandez I, Bisset J, Rodriguez M, Mccall PJ, Donnelly MJ, Ranson H, Hemingway J, Black WC IV. Mutations in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol. Biol. 2007; 16:785–798. Saavedra K, Strode C, Flores A, Fernandez I, Ranson H, Hemingway J, Black WC IV. QTL mapping of genome regions controlling permethrin resistance in the mosquito Aedes aegypti. Genetics. 2008; 180:1137–1152. [PubMed: 18723882] Senasica (Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria, Mexico). Listado de plaguicidas de uso agrícola. National health, innocuity and agrofood quality service List of agricultural insecticides. 2012. (http://www.senasica.gob.mx/?doc=22993)
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 8
NIH-PA Author Manuscript
Siller Q, Ponce G, Lozano S, Flores AE. Update on the frequency of Ile1016 mutation in voltage-gated channel gene of Aedes aegypti in Mexico. J Am Mosq Control Assoc. 2011; 27:357–362. [PubMed: 22329266] (WHO) World Health Organization. . Scientific working group report on dengue. WHO; Geneva, Switzerland: 2007.
NIH-PA Author Manuscript NIH-PA Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 9
NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 1.
Collection sites of Ae. aegypti populations.
NIH-PA Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 10
NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 2.
Percent of mortality of 1–3-day-old adult female of Ae. aegypti in bottles treated with dilutions of standard grade chlorpyrifos.
NIH-PA Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
NIH-PA Author Manuscript 509 566 426 427
Martinez de la Torre
Veracruz
Coatzacoalcos
Cosoleacaque
0.920 (0.772 – 1.097)f
0.273 (0.207 – 0.360)d
0.056 (0.040 – 0.077)ab
0.028 (0.020 – 0.040)a
0.480 (0.391 – 0.588)e
0.117 (0.098 – 0.139)c
0.066 (0.056 – 0.078)b4
LC50(CI)2
5.027(3.174 – 7.960)d
3.425(1.885 – 6.220)cd
1.695(0.916 – 3.136)c
1.036(0.490 – 2.192)bc
3.714(2.545 – 5.422)c
0.627(0.444 – 0.885)b
0.286(0.204 – 0.402)a
LC90 (CI)2
1.294 (0.159)
0.869 (0.085)
0.643 (0.049)
0.610 (0.056)
1.074 (0.093)
1.306 (0.113)
1.501 (0.157)
Slope±SE
26.48 (5)
13.30 (5)
16.10 (7)
28.96 (6)
10.56 (6)
7.02(6)
9.93(4)
X2(df)
0.001
0.001
0.001
0.001
0.001
0.001
0.001
P-value
13.94
4.14
0.85
0.42
7.27
1.77
1.00
LC50
17.57
11.97
5.92
3.6
12.98
2.2
1.00
LC90
4 Different letter in the columns indicate significant differences in LC50 and LC90 values
Resistance ratios were calculated as the LC50 or 90 field strain/LC50 or 90 New Orleans strain
LC50 and LC90 represent the concentrations (μg/bottle) required to kill 50% and 90% of adult females, respectively; 95% confidence intervals (CI) are shown in parentheses.
Number of adult females assayed
3
2
1
488
Tantoyuca 503
368
New Orleans
Poza Rica
N1
Strain
Resistance ratio3
Lethal concentration (LC50 and LC90) and resistance ratio (RR) values for various Aedes aegypti strains in response to chlorpyrifos
NIH-PA Author Manuscript
Table 1 Lopez et al. Page 11
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
NIH-PA Author Manuscript 0.874d (0.078)
Cosoleacaque
1.172d (0.111)
1.000c (0.075)
1.000c (0.075)
0.827b (0.089)
1.172d (0.111)
0.833b (0.176)
0.367a (0.027)
β-esterases
0.124a (0.054)
0.225c (0.068)
0.210b (0.060)
0.289d (0.108)
0.202b (0.045)
0.178b (0.136)
0.185b (0.058)
MFO
0.011a (0.023)
0.080b (0.029)
0.073b (0.077)
0.076b (0.030)
0.116c (0.033)
0.021a (0.014)
0.025a (0.014)
GST
Different letters in the columns indicate significant differences (P < 0.05) by Tukey test.
0.772c (0.071) 0.772c (0.071)
0.667b (0.061)
Martinez de la Torre
Veracruz
0.874d (0.078)
Poza Rica
Coatzacoalcos
0.265a (0.148) 0.676b (0.090)
New Orleans
Tantoyuca
α–esterases
Strain
0.005a (0.005)
0.011a (0.023)
0.009a (0.008)
0.008a (0.006)
0.008a (0.008)
0.018b (0.029)
0.009a (0.006)
iAChE
Mean absorbance (± SD) values from the biochemical assays carried out on the 6 strains of Ae. aegypti sampled from Veracruz as well as the NO strain
NIH-PA Author Manuscript
Table 2 Lopez et al. Page 12
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
NIH-PA Author Manuscript 3.30 2.52 2.91 2.91 3.30
Martinez de la Torre
Veracruz
Coatzacoalcos
Cosoleacaque 3.19
2.72
2.72
2.25
3.19
2.27
β-esterases
0.67
1.22
1.14
1.56
1.09
0.96
MFO
0.44
3.20
2.92
3.04
4.64
0.84
GST
0.56
1.22
1.00
0.89
0.89
2.00
iAChE
Statistically significant differences are shown in bold according to differences found in the analysis done and shown in table 4.
2.55
Poza Rica
α–esterases
Tantoyuca
Strain
Biochemical assay - OD ratio compared to NO susceptible strain
NIH-PA Author Manuscript
Table 3 Lopez et al. Page 13
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
Published in final edited form as: J Med Entomol. 2014 May ; 51(3): 644–649.
Susceptibility to Chlorpyrifos in Pyrethroid-Resistant Populations of Aedes aegypti (Diptera: Culicidae) from Mexico Beatriz Lopez1, Gustavo Ponce1, Jessica A. Gonzalez1, Selene M. Gutierrez1, Olga K. Villanueva1, Gabriela Gonzalez1, Cristina Bobadilla2, Iram P. Rodriguez1,3, William C. Black IV4, and Adriana E. Flores1,5 1Universidad
Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/n Cd. Universitaria, San Nicolas de los Garza, N.L., 66451 Mexico 2Laboratorio
Estatal de Salud Publica de los Servicios de Salud de Veracruz, Mexico
3Universidad
NIH-PA Author Manuscript
Autonoma de Nuevo Leon, Departamento de Genetica, Hospital Universitario Dr. Jose Eleuterio Gonzalez, Monterrey, Nuevo Leon 64460, Mexico
4Department
of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins,
CO 80523
Abstract
NIH-PA Author Manuscript
Resistance to the organophosphate insecticide chlorpyrifos was evaluated in females from six strains of Aedes aegypti (L) that expressed high levels of cross resistance to eight pyrethroid insecticides. Relative to LC50 and LC90 at 24h of a susceptible New Orleans (NO) three strains were highly resistant to chlorpyrifos (Coatzacoalcos, resistance ratio (RRLC90) =11.97; Pozarica, RRLC90=12.98; and Cosoleacaque, RRLC50= 13.94 and RRLC90=17.57), one strain was moderately resistant (Veracruz, RR=5.92), and two strains were susceptible (Tantoyuca and Martinez de la Torre, RRLC50 and RRLC90 < 5) in CDC bottle bioassays. Furthermore, high levels of α/β-esterase activity in the sample populations were correlated with resistance, suggesting that esterase activity may be a mechanism causing the development of organophosphate resistance in these populations. Overall, the populations in this study were less resistant to chlorpyrifos than to pyrethroids. Rotation of insecticides used in control activities is recommended to delay or minimize the occurrence of high levels of resistance to chlorpyrifos among local populations of Ae. aegypti. The diagnostic dose (DD) and diagnostic time (DT) for chlorpyrifos resistance monitoring was determined to be 85 μg/ bottle and 30min, respectively, using the susceptible NO strain.
Keywords chlorpyrifos; insecticide resistance; Aedes aegypti; α-esterases; β-esterases
5
Corresponding author: Adriana E. Flores, Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/n Ciudad Universitaria, San Nicolas de los Garza, N.L., 66451 Mexico, Tel: (81) 83294000 ext 3683, Fax: (81) 83294110, [email protected].
Lopez et al.
Page 2
NIH-PA Author Manuscript
The mosquito Aedes aegypti (L.) is the primary vector of dengue in Mexico. In addition, Aedes albopictus (Skuse), originally from Asia, represents a secondary dengue vector that was introduced via the international trade in used tires and other goods (e.g., lucky bamboo), and has now spread to North America (including Mexico) and Europe. Generally, denguevector control programs include activities to control both the immature and adult stages of the Ae. aegypti lifecycle. For example, chemical and/or biological larvicides and habitat reduction are widely used in an attempt to maintain mosquito populations below the threshold levels that disrupt dengue virus (DENV) transmission (Reiter and Gubler 1997, WHO 2007).
NIH-PA Author Manuscript
Since 1950, operational vector control programs in Mexico have used a series of insecticides to control Ae. aegypti (Flores et al. 2006). The organochlorine insecticide DDT was used extensively for indoor house spraying from 1950–1960 and was used in some locations as recently as 1998. In recent decades, the chemical control of mosquito immatures has primarily relied on the use of organophosphate insecticides with temephos as the active ingredient. Other organophosphate insecticides incorporating the adulticide malathion were also used for ultra-low volume (ULV) space spraying from 1981–1999. Furthermore, an oilbased formulation of chlorpyrifos was registered for use in Mexico for adult mosquito control and was used in some locations from 1996–1999. However, these practices changed in 2000 due to the Norma Oficial Mexicana NOM-032-SSA2-2002 (DOF 2003), which dictated a national switch to permethrin-based space spraying for the purpose of adult suppression, and this practice remained in place until 2009. Studies of enzymatic mechanisms (Flores et al. 2005, 2006, 2009) and target-site insensitivity (kdr) (Saavedra et al. 2007, 2008; Ponce et al. 2009, Siller et al. 2011) have shown evidence of permethrin resistance in Mexican Ae. aegypti populations. These findings suggest that the widespread use of permethrin has conferred cross-resistance to many other pyrethroid compounds, including those not commonly used in mosquito-control programs in Mexico (Flores et al. 2013).
NIH-PA Author Manuscript
In 2011, a new policy (NOM-032-SSA2-2010: DOF 2011) was implemented that established characteristics for insecticides used in disease-vector control programs in Mexico, such as those used to treat Ae. aegypti. However, these standards do not specify particular agents to be used, stating instead that specific insecticides should be chosen based on proven effectiveness, resistance and safety characteristics related to exposure. Chlorpyrifos is currently one of the insecticides approved for use as an adulticide in spacespraying programs in Mexico. In Mexico, chlorpyrifos is primarily used to control agricultural pests in more than 20 types of crops, including grasses, cereals, fruits and vegetables, among others. Chlorpyrifos is available commercially in various formulations from more than 30 companies (Senasica 2011). Few reports describe the use of chlorpyrifos as a mosquito-control agent in Latin America, although Rawlins and Ragoonansingh (1990) reported high levels of chlorpyrifos resistance in populations of Ae. aegypti in Puerto Rico, St. Lucia, and Trinidad.
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 3
NIH-PA Author Manuscript
In the current study, we tested Ae. aegypti populations that were resistant to eight pyrethroid insecticides to determine their susceptibility to chlorpyrifos and to investigate the enzymatic mechanisms involved in cross-resistance (Flores et al. 2013).
Materials and Methods Collection Sites and Colony-Rearing Conditions Six populations of Ae. aegypti were collected during 2009 in the state of Veracruz on the eastern coast of Mexico (Fig. 1). Each of the sampled populations has been previously reported as resistant to pyrethroids (Flores et al. 2013). Sample populations were named based on the location of their collection sites: Tantoyuca (21°20′54.44″N, 98°13′45.90″W), Poza Rica (20°32′00.00″N, 97°26′59.84″W), Martinez de la Torre (20°03′42.55″N, 97°03′06.97″W), Veracruz (19°10′21.48″N, 96°07′59.93″W), Coatzacoalcos (18°08′16.00″N, 94°26′07.49″W) and Cosoleacaque (18°00′03.16″N, 94°37′46.90″W). The New Orleans (NO) strain was used as a susceptible reference strain.
NIH-PA Author Manuscript
Females from the F1 generation (1–3 d post-emergence, without blood feeding) were used for the bioassays and biochemical tests. Laboratory colonies were established using larvae collected from natural breeding sites and maintained at 25±4°C under a 12-h light-dark cycle. F1 eggs were obtained from field-collected parents, and the eggs were placed in plastic containers with dechlorinated water along with a 50% aqueous solution of powdered liver protein, which served as food source for the subsequent larval stage. Pupae were placed in 250-mL flasks in cages (30×30 cm) until adult females had emerged. Bioassays The Brogdon and McAllister (1988) bottle bioassay was used in bioassays, which involves adding 1 ml of an acetone solution containing technical-grade chlorpyrifos (>98% purity; ChemService, West Chester, PA, USA) to a 250-mL Wheaton® bottle. The bottles were then capped and shaken to ensure uniform coverage and allowed to dry for 1 h at room temperature. Mortality rate was measured following 1, 2, 4 and 24 h of exposure. The dosage (μg/bottle) range was predetermined to yield mortality rates between 0 and 99% after 24 h of exposure. The tested doses varied from 0.003 to 6 μg/bottle, with three replicates per dose and 20–25 females per replicate. Control bottles were coated with acetone alone.
NIH-PA Author Manuscript
LC50, LC90 and Resistance Ratios LC50 and LC90 values with 95% confidence limits were calculated using the IRMA quick calculator software program (http://sourceforge.net/projects/irmaproj/files/) with logistic regression. The mortality rates were corrected using the formula developed by Abbott (1925) when mortality was observed in the control group. LC values without overlapping fiducial limits were considered to be significantly different. Resistance in the field strains was calculated determining the resistance ratio (RR), by dividing the LC50 of the test population by the LC50 of the NO strain. The criteria proposed by Mazarri and Georghiou (1995) were used to classify the RRs as high (>10-fold), medium (between 5 and 10), or low (< 5). The degree of resistance relative to the susceptible NO strain also was assessed at the LC90 level.
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 4
Enzymatic Assays
NIH-PA Author Manuscript
Sixty females from each of the field populations, as well as the reference NO strain, were individually homogenized in 100 μl 0.01 M potassium phosphate buffer, pH 7.2, and resuspended in 2 ml of the same buffer. Next, 100-μl aliquots were transferred to microtiter plates; each individual sample was analyzed in triplicate on each plate. We then quantified the activities of the α- and β-esterases, mixed-function oxidases (MFOs), glutathione-Stransferases (GSTs) and insensitive acetylcholinesterase (iAChE) according to the methods of Brogdon (1989), using the NO strain as reference. Absorbances were measured using a UVM-340 Microplate reader (ASYS Hitech GmbH; Eugendorf, Austria), and triplicate values were averaged. Protein concentrations were determined using the method of Brogdon (1984), and in some cases it was necessary to dilute the homogenates due to variation in the size of the mosquitoes.
NIH-PA Author Manuscript
The data from each of the biochemical assays were evaluated using analysis of variance (ANOVA) and Tukey’s test; a significance level of P ≤ 0.05 was used to compare the means between the field strains and the NO reference strain. We calculated the OD (optical density) ratios by dividing the mean absorbance values for each enzyme obtained from the field strains by the mean absorbance value obtained for the NO strain. The RRLC50, RRLC90, and OD ratio values were subjected to linear regression analysis. The correlation (r) was performed to determine the degree of association between the two variables. Diagnostic Dose (DD) and Diagnostic Time (DD) We used 1–3 d post-emergence females without blood feeding of the susceptible NO strain and chlorpyrifos (>98% purity; Chem Service, West Chester, PA) diluted in acetone. Five doses of chlorpyrifos between 15 and 115 μg/bottle were used to treat 450 Ae. aegypti females, including control females that were treated with acetone alone. Subjects were divided into groups of 20 for each dose, with 4 replicates per dose. For each dose, mortality rate and observations were recorded every 5 min for 80 min.
Results and Discussion Lethal Concentrations and Resistance Ratios
NIH-PA Author Manuscript
The results of the LC50, LC90 and RR analyses from the studied populations are presented in Table 1. The slopes of dose-response regression lines for the field strains were lower than that of the NO strain in all cases, suggesting a higher level of heterogeneity on field strains. The LC50 value for the Martinez de la Torre strain (0.028 μg/bottle) was significantly lower than that observed for the control (0.066 μg/bottle), and the LC50 value in the Veracruz strain (0.056 μg/bottle) was similar to the control value. However, the LC50 values for the remaining populations were significantly larger: Tantoyuca showed an LC50 of 0.117 μg/ bottle, Coatzacoalcos showed a value of 0.273 μg/bottle, and Cosoleacaque showed the highest with an LC50 of 0.920 μg/bottle. With respect to the LC90 values, the control NO strain was significantly more susceptible than all of the field-collected strains. The control strain showed an LC90 value of 0.286 μg/ bottle, followed by Tantoyuca with an LC90 of 0.627 μg/bottle and Martinez de la Torre
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 5
NIH-PA Author Manuscript
with an LC90 of 1.036 μg/bottle; the LC90 values for the Tantoyuca and Martinez de la Torre strains were not significantly different and showed overlapping confidence intervals. The LC90 value for the Veracruz strain (1.7 μg/bottle) was statistically equivalent to that of the Martinez de la Torre strain. The three highly resistant strains Coatzacoalcos (LC90 = 3.425 μg/bottle), Poza Rica (LC90 = 3.714 μg/bottle) and Cosoleacaque (LC90 = 5.027 μg/bottle) showed LC90 values that were not significantly different from one another. The Tantoyuca and Martinez de la Torre strains showed low-level resistance to chlorpyrifos, with resistance ratios of 2.2 and 3.6, respectively. The Veracruz strain showed moderate resistance (RR=5.92), and the remaining three strains were highly resistant to chlorpyrifos (Coatzacoalcos RR=11.97; Poza Rica RR=12.98; and Cosoleacaque RR=17.57). Enzymatic Assays
NIH-PA Author Manuscript
α- and β-esterase levels were significantly higher in the field-sampled populations than in the control strain (Table 2). In particular, α-esterase levels were highest in the strains from Poza Rica and Cosoleacaque (both with a value of 0.874), moderate in the strains from Veracruz and Coatzacoalcos (both with a value of 0.722), and lowest in the Tantoyuca (0.676) and Martinez de la Torre (0.667) strains. Similarly, β-esterase levels were highest in the Poza Rica and Cosoleacaque strains (1.172), moderate in the Veracruz and Coatzacoalcos strains (1.00), and lowest in the Martinez de la Torre (0.827) and Tantoyuca (0.833) strains. Analysis of mixed-function oxidase activity showed mean absorbance values that were significantly different from the control in the Coatzacoalcos (0.225) and Martinez de la Torre (0.289) strains. Glutathione-S-transferase analyses revealed values that were significantly different from the control in the Veracruz (0.073), Martinez de la Torre (0.076), and Coatzacoalcos (0.080) strains. The strain from Poza Rica showed a significantly higher mean absorbance (0.116) than the other studied strains, including the control.
NIH-PA Author Manuscript
When we examined the associations between the RRLC50 and RRLC90 values for chlorpyrifos and the OD ratios based on enzymatic activity of all strains analyzed we found a strong positive correlation between the RRs and α- and β-esterase with values of r = 0.82 and r = 0.91 for RRLC50 and RRLC90, respectively, which were statistically significant (P < 0.05). These results are consistent with earlier findings by Mazzarri and Georghiou (1995), who suggested changes in A4-esterase activity as a possible mechanism for acquiring chlorpyrifos resistance in Ae. aegypti based on three Venezuelan populations. Esterase activity was also linked to chlorpyrifos resistance in Ae. aegypti population in Cuba and Costa Rica (Rodriguez et al. 2007). DD and DT Resistance monitoring is a crucial strategy for anti-insecticide resistance programs. Based on the methods outlined in the Guideline for Evaluating Insecticide Resistance in Vectors Using the CDC Bottle Bioassay (CDC 2011), we provide a diagnostic dose (DD) and a diagnostic time (DT) for chlorpyrifos resistance testing. We observed a saturating dose of 95 μg/bottle 25 min after exposure. Therefore, considering the established criteria, we propose a diagnostic dosage of 85 μg/bottle and with a DT of 30 min (Fig. 2) for chlorpyrifos resistance-monitoring programs. Note that this recommendation is different from that
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 6
published by Bisset et al. (2013), who reported a DD of 90 μg/bottle and a DT of 30 min for the Rockefeller strain.
NIH-PA Author Manuscript
Field-collected populations from Veracruz that showed high RRs to pyrethroids, including δ-phenothrin, permethrin, deltamethrin, cypermethrin, α-cypermethrin, z-cypermethrin, λcyhalothrin and bifenthrin (Flores et al. 2013) have developed tolerance to chlorpyrifos, albeit to a lesser extent than to pyrethroids. This disparity may occur because chlorpyrifos was only used in Mexico from 1996 to 1999. The populations tested in this study and those used by Flores et al. (2013) were collected in 2009, 10 years after the use of the chlorpyrifos was halted in Mexico. Therefore, this phenomenon may be due to cross-resistance, as was described by Mazzarri and Georghiou (1995) for Venezuelan and Cuban populations, as temephos continues to be used for larval mosquito control. However, as previously discussed, this resistance may also be due to the extensive use of chlorpyrifos for controlling agricultural pests.
NIH-PA Author Manuscript
Although only two of the analyzed populations were not resistant to chlorpyrifos and one showed moderate resistance, the levels of resistance to organophosphates were not as high as those observed for pyrethroids in the same populations, suggesting that the rotation of insecticides may significantly delay and/or minimize the development of strong resistance to chlorpyrifos in populations of Ae. aegypti. This strategy was used in Cuba in 1997 and 2001 during DENV epidemics when cypermethrin was applied in rotational schemes with chlorpyrifos and limited the emergence of cypermethrin resistance in Ae. aegypti populations (Montada et al. 2006). It is essential that we consider actions to avoid strong resistance between pyrethroids and alternative adulticides in Mexico. Going forward, strategies must include resistance monitoring, the development of advanced tools for detecting multiple insecticide resistance, and practical tools for efficient vector control, not only in Mexico, but throughout the entire world.
Acknowledgments
NIH-PA Author Manuscript
This study was supported by FOMIX CONACyT, Veracruz through Grant No. 68298 within the project “Estrategia de control del mosquito vector del dengue Aedes aegypti (L.) en base a su resistencia a insecticidas en Veracruz”, CONACyT Ciencia Basica Grant No. 102120 within the project “Mutacion’kdr’ asociada a la resistencia a piretroides en Aedes aegypti (L.) en Mexico” and UANL PAICYT. This work was also funded in part by NIHNIAID U01 AI088647.
References Cited Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol. 1925; 8:265– 267. Bisset JA, Marin R, Rodríguez MM, Severson DW, Ricardo Y, French L, Díaz M, Perez O. Insecticide resistance in two Aedes aegypti (Diptera: Culicidae) strains from Costa Rica. J Med Entomol. 2013; 50:352–361. [PubMed: 23540124] Brogdon WG. Biochemical resistance detection: an alternative to bioassay. Parasitol Today. 1989; 5:56–60. [PubMed: 15463180] Brogdon WG. Mosquito protein microassay. I. Protein determinations from small portions of singlemosquito homogenates. Comp Biochem Physiol B. 1984; 79:457–459. [PubMed: 6509934]
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 7
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Brogdon WG, McAllister JC. Simplification of adult mosquito bioassays through use of time-mortality determinations in bottles. J Am Mosq Cont Assoc. 1998; 14:159–164. CDC (Centers for Disease Control and Prevention). Guideline for evaluating insecticide resistance in vectors using the CDC bottle bioassay: a web-based instruction. 2011. (http://www.cdc.gov/ parasites/education_training/lab/bottlebioassay.html) DOF (Diario Oficial de la Federacion). NOM-032-SSA-2-2002 for epidemiological monitoring. Prevention and control of vector-transmitted diseases. DOF; Mexico: 2003. NOM-032-SSA-2-2002 para la vigilancia epidemiologica. Prevencion y control de enfermedades transmitidas por vector. DOF (Diario Oficial de la Federacion). NOM-032-SSA-2-2002 for epidemiological monitoring. Prevention and control of vector-transmitted diseases. DOF; Mexico: 2011. NOM-EM-003SSA2-2010 para la vigilancia epidemiologica. Prevencion y control de enfermedades transmitidas por vector. Flores AE, Albeldaño VW, Fernandez SI, Badii MH, Loaiza H, Ponce GG, Lozano FS, Brogdon WG, Black WC, Beaty BJ. Elevated α-esterase levels associated with permethrin tolerance in Aedes aegypti (L.) from Baja California, Mexico. Pest Biochem Physiol. 2005; 82:66–78. Flores AE, Grajales JS, Fernandez SI, Ponce GG, Loaiza H, Badii MH, Lozano FS, Brogdon WG, Black WC, Beaty BJ. Mechanisms of insecticide resistance in field populations of Aedes aegypti (L.) from Quintana Roo, Southern Mexico. J Am Mosq Control Assoc. 2006; 22:672–677. [PubMed: 17304936] Flores AE, Reyes G, Fernandez SI, Sanchez RFJ, Ponce GG. Resistance to permethrin in Aedes aegypti (L.) in northern Mexico. Southwest Entomol. 2009; 34:167–177. Flores AE, Ponce G, Silva BG, Gutierrez SM, Bobadilla C, Lopez B, Mercado R, Black WC IV. Wide spread cross resistance to pyrethroids in Aedes aegypti (Diptera: Culicidae) From Veracruz state Mexico. J Econ Entomol. 2013; 106:959–969. [PubMed: 23786088] Mazzarri MB, Georghiou GP. Characterization of resistance to organophosphate, carbamate, and pyrethroid insecticides in field population of Aedes aegypti from Venezuela. J Am Mosq Control Assoc. 1995; 11:315–322. [PubMed: 8551300] Montada D, Zaldivar J, Sanchez F, Figueredo D, Suarez S, Leyva M. Eficacia de los tratamientos intradomiciliarios con los insecticidas cipermetrina, lambdacialotrina y clorpirifos en una cepa de Aedes aegypti. Rev Cubana Med Trop. 2006; 58:130–135. Ponce G, Flores A, Fernández I, Saavedra K, Reyes G, Lozano S, Bond J, Casas M, Ramsey J, García J, Domínguez M, Ranson H, Hemingway J, Eisen L, Black WC IV. Recent rapid rise of a permethrin knock down resistance allele in Aedes aegypti in México. PLoS Negl Trop Dis. 2009; 3:e531. [PubMed: 19829709] Rawlins SC, Ragoonansingh R. Comparative organophosphorous insecticide susceptibility in Caribbean populations of Aedes aegypti and Toxorynchites moctezuma. J Am Mosq Control Assoc. 1990; 6:315–317. [PubMed: 1973450] Reiter, P.; Gubler, DJ. Surveillance and control of urban dengue vectors. In: Gubler, DJ.; Kuno, G., editors. Dengue and dengue hemorrhagic fever. CABI Publishing; Cambridge, MA: 1997. p. 425-462. Rodriguez MM, Bisset JA, Fernandez D. Levels of insecticide resistance and resistance mechanisms in Aedes aegypti from some Latin American countries. J Am Mosq Cont Assoc. 2007; 23:420–429. Saavedra K, Urdaneta L, Rajatileka S, Moulton M, Flores AE, Fernandez I, Bisset J, Rodriguez M, Mccall PJ, Donnelly MJ, Ranson H, Hemingway J, Black WC IV. Mutations in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol. Biol. 2007; 16:785–798. Saavedra K, Strode C, Flores A, Fernandez I, Ranson H, Hemingway J, Black WC IV. QTL mapping of genome regions controlling permethrin resistance in the mosquito Aedes aegypti. Genetics. 2008; 180:1137–1152. [PubMed: 18723882] Senasica (Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria, Mexico). Listado de plaguicidas de uso agrícola. National health, innocuity and agrofood quality service List of agricultural insecticides. 2012. (http://www.senasica.gob.mx/?doc=22993)
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 8
NIH-PA Author Manuscript
Siller Q, Ponce G, Lozano S, Flores AE. Update on the frequency of Ile1016 mutation in voltage-gated channel gene of Aedes aegypti in Mexico. J Am Mosq Control Assoc. 2011; 27:357–362. [PubMed: 22329266] (WHO) World Health Organization. . Scientific working group report on dengue. WHO; Geneva, Switzerland: 2007.
NIH-PA Author Manuscript NIH-PA Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 9
NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 1.
Collection sites of Ae. aegypti populations.
NIH-PA Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
Lopez et al.
Page 10
NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 2.
Percent of mortality of 1–3-day-old adult female of Ae. aegypti in bottles treated with dilutions of standard grade chlorpyrifos.
NIH-PA Author Manuscript J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
NIH-PA Author Manuscript 509 566 426 427
Martinez de la Torre
Veracruz
Coatzacoalcos
Cosoleacaque
0.920 (0.772 – 1.097)f
0.273 (0.207 – 0.360)d
0.056 (0.040 – 0.077)ab
0.028 (0.020 – 0.040)a
0.480 (0.391 – 0.588)e
0.117 (0.098 – 0.139)c
0.066 (0.056 – 0.078)b4
LC50(CI)2
5.027(3.174 – 7.960)d
3.425(1.885 – 6.220)cd
1.695(0.916 – 3.136)c
1.036(0.490 – 2.192)bc
3.714(2.545 – 5.422)c
0.627(0.444 – 0.885)b
0.286(0.204 – 0.402)a
LC90 (CI)2
1.294 (0.159)
0.869 (0.085)
0.643 (0.049)
0.610 (0.056)
1.074 (0.093)
1.306 (0.113)
1.501 (0.157)
Slope±SE
26.48 (5)
13.30 (5)
16.10 (7)
28.96 (6)
10.56 (6)
7.02(6)
9.93(4)
X2(df)
0.001
0.001
0.001
0.001
0.001
0.001
0.001
P-value
13.94
4.14
0.85
0.42
7.27
1.77
1.00
LC50
17.57
11.97
5.92
3.6
12.98
2.2
1.00
LC90
4 Different letter in the columns indicate significant differences in LC50 and LC90 values
Resistance ratios were calculated as the LC50 or 90 field strain/LC50 or 90 New Orleans strain
LC50 and LC90 represent the concentrations (μg/bottle) required to kill 50% and 90% of adult females, respectively; 95% confidence intervals (CI) are shown in parentheses.
Number of adult females assayed
3
2
1
488
Tantoyuca 503
368
New Orleans
Poza Rica
N1
Strain
Resistance ratio3
Lethal concentration (LC50 and LC90) and resistance ratio (RR) values for various Aedes aegypti strains in response to chlorpyrifos
NIH-PA Author Manuscript
Table 1 Lopez et al. Page 11
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
NIH-PA Author Manuscript 0.874d (0.078)
Cosoleacaque
1.172d (0.111)
1.000c (0.075)
1.000c (0.075)
0.827b (0.089)
1.172d (0.111)
0.833b (0.176)
0.367a (0.027)
β-esterases
0.124a (0.054)
0.225c (0.068)
0.210b (0.060)
0.289d (0.108)
0.202b (0.045)
0.178b (0.136)
0.185b (0.058)
MFO
0.011a (0.023)
0.080b (0.029)
0.073b (0.077)
0.076b (0.030)
0.116c (0.033)
0.021a (0.014)
0.025a (0.014)
GST
Different letters in the columns indicate significant differences (P < 0.05) by Tukey test.
0.772c (0.071) 0.772c (0.071)
0.667b (0.061)
Martinez de la Torre
Veracruz
0.874d (0.078)
Poza Rica
Coatzacoalcos
0.265a (0.148) 0.676b (0.090)
New Orleans
Tantoyuca
α–esterases
Strain
0.005a (0.005)
0.011a (0.023)
0.009a (0.008)
0.008a (0.006)
0.008a (0.008)
0.018b (0.029)
0.009a (0.006)
iAChE
Mean absorbance (± SD) values from the biochemical assays carried out on the 6 strains of Ae. aegypti sampled from Veracruz as well as the NO strain
NIH-PA Author Manuscript
Table 2 Lopez et al. Page 12
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
NIH-PA Author Manuscript
NIH-PA Author Manuscript 3.30 2.52 2.91 2.91 3.30
Martinez de la Torre
Veracruz
Coatzacoalcos
Cosoleacaque 3.19
2.72
2.72
2.25
3.19
2.27
β-esterases
0.67
1.22
1.14
1.56
1.09
0.96
MFO
0.44
3.20
2.92
3.04
4.64
0.84
GST
0.56
1.22
1.00
0.89
0.89
2.00
iAChE
Statistically significant differences are shown in bold according to differences found in the analysis done and shown in table 4.
2.55
Poza Rica
α–esterases
Tantoyuca
Strain
Biochemical assay - OD ratio compared to NO susceptible strain
NIH-PA Author Manuscript
Table 3 Lopez et al. Page 13
J Med Entomol. Author manuscript; available in PMC 2014 August 05.
