Larval susceptibility of Aedes aegypti and Culex quinquefasciatus populations to chemical insecticides



Jairo Campos; Carlos F S Andrade

Departamento de Zoologia do Instituto de Biologia da Universidade Estadual de Campinas. Campinas, SP, Brazil





OBJECTIVE: To evaluate the susceptibility to chemical insecticides of Culex quinquefasciatus and Aedes aegypt larvae from areas subjected to control treatments or not.
METHOD: Bioassays for diagnostic concentration and multiple concentration were performed for organophosphate and pyrethroid insecticides according to World Health Organization parameters. The susceptibility was assessed for mosquito larvae collected from an area not subjected to chemical control (Campinas, State of São Paulo, SP) and from other areas (Campo Grande, Mato Grosso do Sul, MS, and Cuiabá, Mato Grosso, MT), in Brazil, subjected to such treatments.
RESULTS: Tests for Culex quinquefasciatus larvae from Campinas, SP, allowed suspicion of resistance to cypermethrin and gave evidence of resistance to cyfluthrin. Larvae of this species collected in Campo Grande, MS, and Campinas, SP, presented resistance to temephos. For the colony from the latter locality, the following resistance rates were established: RR50=6.36 and RR95=4.94, in relation to a standard susceptible strain. Moreover, tests for Aedes aegypti showed similar susceptibility to temephos for a field population from Cuiabá, MT, and a laboratory population.
CONCLUSIONS: The results indicate resistance of Culex quinquefasciatus to organophosphate and pyrethroid insecticides and make evident the need for evaluation and monitoring of the efficiency of insecticides to be used in mosquito control programs.

Keywords: Aedes. Larva. Insecticides. Insecticide resistance. Insecticides organophosphate. Vector control. Mosquito control.




The synanthropic mosquito species Aedes aegypti (Linn.) and Culex quinquefasciatus Say are of great importance in public health, since they are implicated in the transmission of dengue and Bancroft’s filariasis, respectively, in the Americas. The recent epidemics of dengue that have occurred in Brazil (1998 and 2002) and the endemicity of filariasis are once again posing an administrative and public challenge in mosquito control. In Brazil, there have already been indications of resistance in populations of Cx quinquefasciatus.2,9,12 Thus, in Fortaleza (capital of the State of Ceará) resistance to organophosphates has been recorded for Cx. quinquefasciatus.12 This same species has also shown resistance in Rio de Janeiro9 and São Paulo.2,3 According to World Health Organization parameters, the diagnostic concentration of temephos Cx. quinquefasciatus larvae is 0.002 ppm.13 The present work has evaluated the susceptibility of field and laboratory populations of Ae. aegypti and Cx. quinquefasciatus to one organophosphate and two pyrethroids used in mosquito control.



The larvae of Ae. aegypti evaluated came from a 4.5-year-old colony (Aea-Unicamp strain- Department of Zoology) and the F1 of mosquitoes collected from the Campinas university campus in May 2002 (Aea-FEF strain). For this species in Cuiabá, Mato Grosso (MT), an evaluation was made for parental larvae (Aea-Cuiabá strain) that came from eggs collected in traps by the Centro de Controle de Zoonoses, Cuiabá, MT, in 2000. For the larvae of Cx. quinquefasciatus evaluated, the material came from collections made on the campus of the Universidade Federal de Mato Grosso do Sul (UFMS), in Campo Grande, MS, in May 1998 (UFMS-R strain), and from Córrego Fundo, in Cuiabá, MT, in September 2000, (Cuiabá-R strain). For Cx. quinquefasciatus, the collection locations in Campo Grande and Cuiabá presented a history of control using organophosphates. For the evaluations of Cx. quinquefasciatus in Campinas, State of São Paulo (SP), the material utilized came from collections made on the university campus in July 1999 (UNI-R1 strain), and in February 2001 (UNI-R2 strain). Up to that time, no application of any control products had been made on the campus. As a reference standard, use was made of the Rockefeller strain of Ae. aegypti5 and the IAL-S strain (Instituto Adolfo Lutz, São Paulo) of Cx. quinquefasciatus originating from Iguapé, SP, in 1983.3

The insecticides used were the organophosphate temephos (Abate 500E, Larvin 1G and Fersol 1G) and also the pyrethroids cypermethrin (Ciper 250CE) and cyfluthrin (Solfac 5CE). From stock solutions of these products at a concentration of 100 ppm in water, additional dilutions were made in distilled water to arrive at the working concentrations. For the organophosphate temephos, tests were made with diagnostic concentration (DC=0.012; 0.04; and 0.06 ppm i.a.) and multiple concentration (MC=0.00056 to 0.016 ppm i.a.). For the pyrethroids, tests were made with diagnostic concentration for cypermethrin (DC=0.0096) and multiple concentration for cyfluthrin (MC=0.00075 to 0.03) (Table 1). For temephos, the median lethal concentration (LC50) and the LC95 were established using larvae of Ae. aegypti (Aea-Uni and Aea-FEF) and Cx. quinquefasciatus (UNI-R2).

The bioassays were done using larvae of Ae. aegypti and Cx. quinquefasciatus, following parameters previously proposed.4,5,13 Groups of 20 or more larvae in their third and/ or fourth instar were used, in discardable cups with insecticide solutions for three or more repetitions. The final mortality was evaluated 24 hours after contact with the insecticides. The selected data values were submitted to probit analysis using the POLO-PC10 program. When the program verified parallelism in the responses, the resistance rates (RR) were calculated for the lethal concentrations LC50 and LC95 in comparison with the values for standard strains.



The Table 1 presents a list of the biological material utilized and the results from the susceptibility tests. In assays using parental larvae (UNI-R1 strain) of Cx. quinquefasciatus collected in Campinas, SP, low mortality (11%) to a commercial formulation of cypermethrin (DC=0.0096 ppm i.a.) was verified. In the same way, resistance to the diagnostic concentration of pyrethroid cyfluthrin (DC=0.03 ppm i.a.) was found in F2 larvae (UNI-R2 strain). Larvae of Cx. quinquefasciatus presented survivals of 6.3 and 11.9% for temephos (DC=0.04 ppm i.a.) for the UFMS-R strain, and 5% survival (DC=0.012 ppm i.a.) for the Cuiabá-R strain. For larvae from the UNI-R2 colony of this species in Campinas, LC50=0.0076 ppm i.a. and LC95=0.0144 ppm i.a. of temephos were established. On the basis of the results for the standard strain IAL-S, the resistance rates were RR50=6.36 and RR95=4.94.

Evaluations for temephos using the Aea-Cuiabá strain showed tolerance. The tests recently performed using larvae of the Aea-Unicamp (4.5-year-old colony) and Aea-FEF (F1, from the field) strains of Ae. aegypti also indicated tolerance for temephos. When these strains were compared with the Rockefeller strain, the resistance rates were RR50=1.67 and RR95=1.85 for Aea-Unicamp, RR50=1.92 and RR95=1.83 for Aea-FEF. The angular coefficients (s) of the concentration-response lines were 4.8±0.6 for Aea-Unicamp, 5.9±0.7 for Aea-FEF and 5.5±0.6 for Rockefeller (Table 2).



The result from the test using cypermethrin on Cx. quinquefasciatus (UNI-R1) gave rise to the suspicion of resistance to insecticides for this strain, since the cypermethrin lethality records for this species are greater at lower concentrations. In Cuban populations,1 LC50=0.0016 ppm i.a. was found for larvae from a susceptible strain and LC50=0.004 ppm i.a. for a parental strain with resistance. LC50=0.003 and LC90=0.01±0.006 ppm i.a. of this pyrethroid were recorded for a resistant colony of Cx. quinquefasciatus in Rio de Janeiro, in comparison with LC50=0.0008 for the reference strain.9

For the pyrethroid cyfluthrin, few records of tests with mosquitoes are known. RR=4 for LC50 was recorded for larvae of Ae. aegypti from Venezuela when compared with the Rockefeller standard strain,8 thereby indicating increased esterase as a mechanism involved in resistance to pyrethroids for the strain evaluated. Tests of susceptibility to this insecticide performed on Cx. quinquefasciatus from Venezuela have not recorded resistance.11 Our preliminary tests on Cx. quinquefasciatus (UNI-R2) indicated resistance to this product when compared with the Rockefeller strain of Ae. aegypti, since the sensitivity to insecticides of standard strains is generally greater for Cx. quinquefasciatus than for Ae. aegypti (Cutkomp & Subramanyam,7 1986; WHO,13 1992) and in comparison with the LC50 of cyfluthrin and cypermethrin for Ae. aegypti (Campos & Andrade,5 2001) and cypermethrin and other pyrethroids for Cx. quinquefasciatus.1,9

In the same way, the results from evaluations using the UNI-R2 strain of Cx. quinquefasciatus indicated low resistance (RR­­­­­99­­­<5) to temephos, when compared with the susceptible strain IAL-S (LC­­­­­95=0.0029 ppm i.a.). The diagnostic dose of temephos used for Cx. quinquefasciatus in Campo Grande was 20 times greater than what is recommended by WHO,13 and 13.8 times the LC­­95 of IAL-S, and therefore the survival indicates real resistance of the population to the insecticide. On the other hand, for the larvae of Cx. quinquefasciatus in Cuiabá, low resistance to this insecticide was found. When the results from Cuiabá are compared with those obtained for the standard strain of Cx. quinquefasciatus (IAL) in Campinas, a priori we would have RR95@4.

Although the metabolic mechanism involved in resistance to organophosphates has not been identified in the present work, increased esterase has already been recorded in Brazilian populations of Cx. quinquefasciatus,3,12 and we have observed chromosome amplification markers of esterase genes that are associated with resistance to organophosphates in the UNI-R1 and UNI-R2 strains (Campos et al6). Thus, the control strategies for Cx. quinquefasciatus using organophosphates and pyrethroids in Campinas, Campo Grande and Cuiabá must include prior monitoring of their efficiency. The development of resistance in these populations may be associated with indiscriminate use of insecticides in domestic and agricultural environments, as well as the specific treatments in Campo Grande and Cuiabá.

The results from evaluations using temephos on larvae of Ae. aegypti from Cuiabá, MT, did not show resistance when compared with the Rockefeller standard strain. Monitoring of the laboratory and field populations of Ae. aegypti in Campinas did not show four years later the same resistance (DC=0.04 ppm i.a.) recorded in 1998 (Campos & Andrade,5 2001). Because of the small differences in the responses to temephos between the two populations and the overlapping of the confidence intervals, it can be considered that there is no difference between the lethal concentrations for the strains. Thus the susceptibility towards temephos 1G of Ae. aegypti would not be compromised, at least for use as necessary at non-strategic points, like those evaluated in Campo Grande, Campinas and Cuiabá. Evaluations of commercial products are feasible and must become the practice in monitoring.

The results obtained for the areas subjected (Campo Grande and Cuiabá) or not (Campinas) to treatments with insecticides have given evidence of low resistance to these products in Cx. quinquefasciatus and susceptibility in Ae. aegypti. Thus, when field populations are evaluated and compared with standard populations, changes in response to insecticides (LC50 and LC95) of the order of up to threefold in the resistance rates may be considered as tolerance; three to fivefold, low resistance; five to tenfold, moderate; 10 to 20-fold, medium; and greater than 20-fold, high resistance. Such knowledge may assist in adequate planning of resistance management and control strategies to be applied case-by-case to each point, without compromising the efficiency of the products.



To the Universidade Federal de Mato Grosso do Sul , the Universidade Estadual de Campinas and the Centro de Controle de Zoonoses, Cuiabá, MT, for the use of the installations and laboratories; to the veterinarian Gerson Blatt and the biologists Leny Bezerra da Costa and Benedito Oscar Fernandes de Campos of the Centro de Controle de Zoonoses, Cuiabá, MT, for their collaboration in the evaluations of the tests; to the Professor Dr Délsio Natal of the Faculdade de Saúde Pública da Universidade de São Paulo, for making available the IAL strain.



1. Bisset J, Rodríguez M, Soca A. Cross-resistance to malathion in Cuba Culex. quinquefasciatus induced by larval selection with deltamethrin. Med Vet Entomol 1998;12:109-12.        

2. Bracco JE, Dalbon M, Marinotti O, Barata JMS. Resistência a inseticidas organofosforados e carbamatos em população de Culex quinquefasciatus. Rev Saúde Pública 1997;31:182-3.        

3. Bracco JE, Barata, JMS, Marinotti O. Evaluation of insecticide resistance in a population of Culex quinquefasciatus (Diptera: Culicidae) from São Paulo, Brazil. Mem Inst Oswaldo Cruz 1999;94:115-20.        

4. Brown AWA. Insecticide resistance in mosquitoes: a pragmatic review. J Am Mosq Control Assoc 1986;2:123-40.        

5. Campos J, Andrade CFS. Susceptibilidade larval de duas populações de Aedes aegypti a inseticidas químicos. Rev Saúde Pública 2001;35:232-6.        

6. Campos J, Andrade CFS, Recco Pimentel SM. Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae). Mem Inst Oswaldo Cruz 2003;98:383-386.        

7. Cutkomp LK, Subramanyam B. Toxicity of pyrethroids to Aedes aegypti larvae in relation to temperature. J Am Mosq Control Assoc 1986;3:347-9.        

8. Fernández DM, Rodríguez M, Bisset J, Perez E. Identification of esterase resistance mechanisms in Aedes aegypti from Carabobo State, Venezuela in Summaries of Mosquito Vector Control and Biology in Latin America - a Ninth Symposium, St. Louis, MO, February 1999. J Am Mosq Control Assoc 1999;15:421.        

9. González T, Bisset JA, Díaz C, Rodríguez MM, Brandolini MB. Insecticide resistance in a Culex quinquefasciatus strain from Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 1999;94:121-2.        

10. LeOra Software. Polo-PC, probit or logit analysis. Berkeley (CA); 1987.        

11 Reyes-Lugo M, Neus M. Insecticide resistance in Culex quinquefascitus Say 1823 (Diptera: Culicidae) from Zulia State, Venezuela. Rev Cient-Fac Cienc Veter 2000;10:441-7.        

12. Yébakima A, Yp-Tcha MM, Reiter P, Bisset J, Delay B, Chevillon C, Pasteur N. Detoxifying esterases in Culex pipiens quinquefasciatus from the caribbean countries. J Am Mosq Control Assoc 1995;11:363-6.        

13. World Health Organization. Vector resistance to pesticides. Geneva; 1992. (WHO - Technical Report)        



Correspondence to
Carlos F. S. Andrade
Departamento de Zoologia, Instituto de Biologia, Unicamp
13084-971 Campinas, SP, Brasil
E-mail: cfeandra@unicamp.br; jairocag@yahoo.com

Received em 22/8/2002.
Reviewed on 21/3/2003.
Approved on 7/4/2003.



*Based on thesis presented to the Instituto de Bilogia of Unversidade Estadual de Campinas, 2002.

Faculdade de Saúde Pública da Universidade de São Paulo São Paulo - SP - Brazil
E-mail: revsp@org.usp.br