Background

The ability of mosquitoes to resist to insecticides is a serious threat to disease prevention. Monitoring of the sensitivity of culicidae to insecticides is essential for guidelines of any vector control program to ensure timely control of the situation when the insecticide is no longer effective. Culex pipiens mosquito has been strongly suspected as the most likely vector in the transmission of West Nile virus outbreaks that have affected Tunisia in 1997, 2003, 2007, 2010, 2011 and 2012 (Triki et al., 2001; Hachfi et al., 2010; Bouatef et al., 2012; Riabi et al., 2014). With the exception of the work of Ben Cheikh et al. (1995; 1998) and Tabbabi et al. (2017), no other studies have been published on the susceptibility of culicidae to temephos (organophosphates (OP)). This work, carried out in central Tunisia, aimed to determine the levels of resistance of Culex pipiens to temephos, insecticide usually used in mosquito control (Gambarra et al., 2010).

1 Material and Methods

1.1 Period and study area

This study was carried out from 2002 to 2005 during the period of activity of the mosquitoes in five localities of central Tunisia (Figure 1).

1.2 Tested strains

Five populations of Culex pipiens were collected from central Tunisia and used for different bioassays. The identification of larvae was determined using the key of Brunhes et al. (2000). Three others strains were used us references: S-Lab as susceptible strain, SA2 and SA5 (Berticat et al., 2002) as resistant strains characterized by overproduced esterases A2-B2 and A5-B5, respectively.

1.3 Insecticides

Two technical grade insecticides were used for bioassay: the organophosphates temephos (9l%o; American Cyanamid, Princeton, NJ), and the carbamate propoxur (997o; Mobay). Two synergists were used to help detect detoxification enzymes involved in resistance: S, S, S {ributyl phosphorothioate (DEF), an esterase inhibitor, and piperonyl butoxide (pb), an inhibitor of mixed function oxidases.

1.4 Larval susceptibility tests

The sensitivity of Culex pipiens was studied according to the recommended experimental protocol standardized by the WHO (Raymond et al., 1986). Five insecticide concentrations were prepared with 3 replicates per concentration. LC₅₀s and CL₉₅s, corresponding to 50 and 95% mortality, were determined graphically by the linear relationship between the decimal logarithm of the insecticide concentrations and the mortality percentages transformed into probit values (Raymond et al., 1993). The differentiation between sensitive and resistant strains is based on the resistance rate. Resistance ratios were calculated at the median lethal concentration (LC50) and LC95 by comparing the estimated lethal concentration values of the field populations with those of the susceptible S-Lab strain.

1.5 Biochemical detection of esterases

Phenotype of the resistance associated to esterase genes were detected by esterase starch gel electrophoresis technique (Pasteur et al., 1988).

2 Results

As showed in Table 1, sample # 4 was susceptible to temephos insecticide with RR₅₀ of 0.51. The resistance level was high for samples # 5 (>50-fold), and low, not exceeding 4-fold in the other resistant samples. LC₉₅ showed two susceptible samples (# 2, and 4). The CYP450 enzyme was found related to temephos resistance in three samples. In fact, the addition of Pb to temephos bioassays completely suppressed the resistance in samples # 1 (RR₅₀=0.84, p>0.05, RSR=3.0) and 5 (RR₅₀=1.5, p>0.05, RSR=37.1). The temephos resistance level of sample # 3 decreased slightly (RR=1.9, p<0.05, RSR=1.7). In contrast, any effect was observed for DEF synergist indicating the non-involvement of esterases in the resistance to temephos insecticide (Table 1).

Mortalities due to propoxur (< 25%) were recorded in two resistant samples: # 3, and 5. The important mortalities (>75%) were recorded in the most susceptible samples: # 4.

One or several esterases were detected in all the studied samples: C1, A1, A2-B2, A4-B4 and/or A5-B5, and B12. The frequencies of these esterases were 42% in the sample having the highest resistance to temephos divided in two esterases, A1 (31%) and C1 (11%), and 11% in the sample having the lowest resistance presented by A1-B1/A2-B2 esterases.

3 Discussion

Temephos insecticide has been used for more than thirty years as a larvicide due to its low cost and proven efficiency. Resistance of Culex mosquitoes to temephos is however occurring in many countries including Tunisia, North Africa (Ben Cheikh et al., 1998). The continuation of the extensive use of this insecticide needs monitoring its evolution in resistant populations.

The biochemical assays detected elevated esterases in all studied samples with different frequencies to be involved the recorded resistance to temephos. However, synergists bioassays did not suggested any esterase activity. In fact, the effect of synergist employed in the toxicological tests (DEF) does not always result in the inhibition of esterases and GSTs. Wirth and Georghiou (1999) found a correlation between both synergist and biochemical tests in the revelation of esterases to reported temephos resistance in Aedes aegypti. The CYP450 enzyme was found related to temephos resistance in this study proving previous studies of Ben Cheikh et al. (1998) and Bisset et al. (2000).

The insensitive AChE 1 could be responsible partly in the resistance to temephos insecticide (OP). The AChE 1 resistant phenotypes were present with a frequency ranged from 0.11 to 0.72. It well known that insensitive acetylcholinesterase (AChE 1) is the most recognized OP target site resistance mechanism. Previous studies have been associated mutations on the gene encoding this enzyme with OP resistance in Culex pipiens and other mosquitoes (Weill et al., 2002; 2004; Nabeshima et al., 2004; Alout et al., 2007).

Acknowledgements

This work was kindly supported by the Ministry of Higher Education and Scientific Research of Tunisia by funds allocated to the Research Unit (Génétique 02/UR/08-03) and by DHMPE of the Minister of Public Health of Tunisia. We are very grateful to S. Ouanes, for technical assistance, A. Ben Haj Ayed and I. Mkada for help in bioassays, S. Saïdi, Tunisian hygienist technicians for help in mosquito collecting, and M. Nedhif and M. Rebhi for their kind interest and help.

References

Alout H., Berthomieu A., Hadjivassilis A., and Weill M., 2007, A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culex pipiens mosquitoes from Cyprus, Insect Biochem Mol Biol, 37: 41-47

https://doi.org/10.1016/j.ibmb.2006.10.001

Ben Cheikh H., Haouas-Ben Ali Z., Marquine M., and Pasteur N., 1998, Resistance to organophosphorus and pyrethroid insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia, J. Med. Entomol, 35: 251-260

https://doi.org/10.1093/jmedent/35.3.251

Ben Cheikh H., Marrakchi M., and Pasteur N, 1995, Mise en évidence d’une très forte résistance au chlorpyrifos et à la perméthrine chez les moustiques Culex pipiens de Tunisie, Arch. Institut Pasteur Tunis, 72: 7-12

Berticat C., Boquien G., Raymond M., and Chevillon C., 2002, Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes, Genet. Res, 83: 189-196

https://doi.org/10.1017/S0016672304006792

Bisset J.A., Rodriguez M.M., Diaz C., and Soca A., 2000, Course of insecticide resistance in Culex quinquefasciatus (Diptera: Culicidae) in a region of La Habana, Rev. Cubana Med. Trop, 52: 180-185

Bouatef S., Hogga N., Ben Dhifallah I., Triki H., Ben Alya Bouafif N. and Achour, N., 2012, Monitoring and current situation of meningitis andmeningo-encephalitis to West Nile virus in Tunisia, Tun. Rev. Infect, 6: 181-182

Brunhnes J., Rhaim A., Geoffroy B., Angel G., and Hervy J.P., 2000, Les moustiques de l’Afrique méditerranèenne: logiciel d’identification et d’enseignement. Paris (FRA) ; Tunis : IRD ; IPT, 2000, 1 CD-ROM (Didactiques), ISBN 2-7099-1446-8

Gambarra W.P.T., 2010, Tecnologias de georreferenciamento e genética molecular aplicados à avaliação da resistência de Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) ao Temephos. [Master´s thesis] Campina Grande, Brazil: Centro de Ciências e Tecnologias, Universidade Estadual de Paraíba

Hachfi W., Bougmiza I., Bellazreg F., Bahri O., Kaabia N., Bahri F., and Letaief A., 2010, Second epidemic of West Nile virus meningoencephalitis in Tunisia, Med. Mal. Infect, 40: 456-461

https://doi.org/10.1016/j.medmal.2009.12.005

Nabeshima T., Mori A., Kozaki T., Iwata Y., and Hidoh O., 2004, An amino acid substitution attributable to insecticide-insensitivity of acetylcholinesterase in a Japanese encephalitis vector mosquito, Culex tritaeniorhynchus, Biochem Biophys Res Commun, 313: 794-801

https://doi.org/10.1016/j.bbrc.2003.11.141

Pasteur N., Pasteur G., Catalan J., Bonhomme F., and Britton-Davidian J., 1988, Practical isozyme genetics. Ellis Horwood, Chichester, United Kingdom

Raymond M., Fournier D., Bride J.M., Cuany A., Bergé J., Magnin M., and Pasteur N., 1986, Identification of resistance mechanisms in Culex pipiens (Diptera: Culicidae) from southern France: insensitive acetylchlinesterase and detoxifying oxidases, J. Econ. Entomol, 79: 1452-1458

https://doi.org/10.1093/jee/79.6.1452

Raymond M., Prato G., and Ratsira D., 1993, PROBIT, Analysis of mortality assays displaying quantal response. Praxeme (Licence No. L93019), Saint Georges d’Orques, France

Riabi S., Gaaloul I., Mastouri M., Hassine M., and Aouni M., 2014, An outbreak of WestNile virus infection in the region of Monastir Tunisia, 2003. Pathog. Global Health, 108: 148-157

https://doi.org/10.1179/2047773214Y.0000000137

Tabbabi A., Ben Jha I., Lamari A., and Ben Cheikh H., 2017, Insecticide Resistance Development in Culex pipiens Population under Selection Pressures with Temephos, The Journal of Middle East and North Africa Sciences, 3(5): 16-19

Triki H., Murri S., Le Guenno B., Bahri O., Hili K., Sidhom M., and Dellagi K., 2001, West Nile viral meningo-encephalitis in Tunisia, Medecine Tropicale, 61: 487-490

Weill M., Fort P., Berthomieu A., Dubois M.P., and Pasteur N., 2002, A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila, Proc Biol Sci, 269: 2007-2016

https://doi.org/10.1098/rspb.2002.2122

Weill M., Malcolm C., Chandre F., Mogensen K., and Berthomieu A., 2004, The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors, Insect Mol Biol, 13: 1-7

https://doi.org/10.1111/j.1365-2583.2004.00452.x

Wirth M.C., and Georghiou G.P., 1999, Selection and characterization of temephos resistance in a population of Aedes aegypti from Tortola, British Virgin Islands, J. Am. Mosq. Control Assoc, 15: 315-320