J Arthropod-Borne Dis, December 2018, 12(4): 370–377 O Guntay et al.: Evaluation of Pyrethroid … 370 http://jad.tums.ac.ir Published Online: December 25, 2018 Original Article Evaluation of Pyrethroid Susceptibility in Culex pipiens of Northern Izmir Province, Turkey Onur Guntay 1, Mehmet Salih Yikilmaz 1, Huseyin Ozaydin 1, *Savas Izzetoglu 1, Asli Suner 2 1Section of Molecular Biology, Department of Biology, Faculty of Science, Ege University, Turkey 2Department of Biostatistics and Medical Informatics, Faculty of Medicine, Ege University, Turkey (Received 6 Mar 2018; accepted 21 Oct 2018) Abstract Background: Mosquitoes, being a nuisance species, are considered as one of the most important species in public health control programs due to their role as a vector in mosquito-borne diseases observed in humans and animals. We evaluated the susceptibility status of Culex pipiens collected from northern Izmir, Turkey in 2011-16. Methods: Mosquito larvae, collected from three different locations in northern İzmir, were reared in the laboratory. Adult susceptibility bioassays were performed using the WHO insecticide-impregnated papers including deltamethrin 0.05%, permethrin 0.75%, α-cypermethrin 0.05% and cyfluthrin 0.15%. In addition, adult bioassays were performed after the pre-exposure to piperonyl butoxide (PBO) to determine the contribution of P450 detoxification enzymes to the phenotypic resistance. Results: In all of the three populations, high levels of resistance were observed (mortalities<63%) to all of the four pyrethroids. Different pyrethroids but with the same mode of action can exhibit significantly different phenotypic re- sistance in a single population. PBO bioassays also showed that P450 detoxification enzymes can have diverse effects on different pyrethroids. Conclusion: Using just one chemical in a class of insecticide can be misleading for resistance studies. Keywords: Bioassay, Insecticide resistance, Pyrethroids, PBO, Culex pipiens Introduction More than 48 mosquito species have been identified in Turkey including Culex pipiens, is one of the major nuisances (1). Culex species are important vectors for lymphatic filariasis and many other viral diseases such as West Nile (2, 3). Since the 1950s, the control of pests has gained momentum with the inclusion of chem- ical compounds in the integrated pest manage- ment. Pyrethroids, which are in the recommend- ed insecticides list of WHO for the adult mos- quito control (4), are widely used in both in- door and outdoor in Turkey. Long-term and frequent applications of the same insecticide may select resistant individu- als and this selection pressure may eventually lead to resistant populations becoming estab- lished (4-6). Hence, monitoring of the re- sistance/susceptibility status of mosquito pop- ulations to insecticides is very important for the success of the vector-control programs. The most common form of resistance to py- rethroids is called knock-down resistance (KDR), linked to a single nucleotide substitution in the Vssc gene encoding the voltage-sensitive sodi- um channel protein and involves reduced target- site sensitivity to pyrethroids (7, 8). The second common mechanism involved in pyrethroid re- sistance involves detoxification enzymes be- longing to the three major enzyme families; namely, cytochrome P450 monooxygenases (CYPs), glutathione-S-transferases (GSTs) and carboxyl/ cholinesterases (CCEs). These en- zymes cause metabolic detoxification of pyre- throids before they reach their target site (9-11). A few studies have reported the occurrence, distribution and some mechanisms involved in *Corresponding author: Dr Savas Izzetoglu, E-mail: savas.izzetoglu@ege.edu.tr J Arthropod-Borne Dis, December 2018, 12(4): 370–377 O Guntay et al.: Evaluation of Pyrethroid … 371 http://jad.tums.ac.ir Published Online: December 25, 2018 insecticide resistance among certain mosquito species throughout Turkey and in neighboring countries such as Greece and Iran (12-22). Yet, we have a very limited knowledge of the in- secticide resistance status of mosquito popula- tions in Turkey. In this study, our aims were to determine resistance/susceptibility status of three natural populations of Cx. pipiens in northern Izmir, Turkey and to understand to role of P450 de- toxification enzymes in different pyrethroids (deltamethrin, permethrin, α-cypermethrin and cyfluthrin). Materials and Methods Mosquito samples Mosquito larvae were collected from three different field locations: Maltepe Village, Mene- men (N38°37’06”, E26°53’25”) in 2011, Sasalı, Çiğli (N38°28’23”, E26°56’51”) and Ege Uni- versity Botanical Garden, Bornova (N38°27’33”, E27°14’01”) in 2016. Sites were located in the same climatic area with a slight difference (the average winter and summer temperatures are 7.9 °C and 26.3 °C, respectively and the average annual rainfall is 687mm). Larvae were trans- ported to the laboratory and reared under con- ditions at a temperature of 25 °C and 70% (±5%) RH with a 12:12h light: dark photoperiod. Preparation for morphological identification Species identification based on morphology was performed by selecting 4th stage larvae from the three abovementioned populations. For the preparations, the method (23) has been used with slight modifications. Culex pipiens larvae are yellowish-brown in color and have long-me- dium thickness siphon (24). These character- istics were used for identification. Species de- terminations based on morphological keys (25- 27) were completed using stereomicroscope with an installed Argenit camera (CAMERA M5 CMOS). Susceptibility bioassays Adult bioassays were performed following WHO guidelines (WHO/VBC/81.80) using the WHO susceptibility test kits. All specimens used in the bioassay were at fixed ages (3–5d old) and non-blood fed female adults. Mosqui- toes were exposed to filter papers impregnated separately with 0.05% deltamethrin (Batch No: DE 499, Expiry Date: Apr 2018), 0.75% per- methrin (Batch No: PE 406, Expiry Date: Apr 2018), 0.05% α-cypermethrin (Batch No: AL 237, Expiry Date: Apr 2018), 0.15% cyfluthrin (Batch No: CY 123, Expiry Date: Apr 2018) and control (Batch No: PY 249, Expiry Date: Mar 2018) for 1h. In order to evaluate the P450 detoxification enzyme activity, 4% PBO-im- pregnated papers (Batch No: PB 019, Expiry Date: Mar 2018) were pre-exposed for 1h be- fore deltamethrin and permethrin applications. This test was not carried out for α-Cyperme- thrin and Cyfluthrin applications. We have called this test as synergist bioassay test within this manuscript. Oil-impregnated papers were used for controls. All of the impregnated papers were obtained from WHO Vector Control Research Unit at Universiti Sains Malaysia. During the experiments, knock-down (KD) rates were rec- orded every 15min (28). After completing the exposure time, mosquitoes were gently trans- ferred into plastic cups and provided with 10% sucrose solution for 24h. Experimental mor- tality was recorded after 24h recovery period. For each test session, 4 batches of 20–25 mosquitoes and 2 batches of 20–25 mosqui- toes were exposed to insecticide-impregnated and oil-impregnated papers, respectively (28). For each insecticide, three replicates were car- ried out and the results were pooled to obtain the mean value of each test. Temperature and humidity were maintained at 27 °C (±1), 80% (±10) RH throughout the bioassay and recov- ery period (28). Data analysis Statistical analyzes of the bioassay results were performed using Kruskal-Wallis and Mann- Whitney U tests with the IBM SPSS ver. 21.0 (Chicago, IL, USA). The normal distribution of the data was examined using Shapiro-Wilk J Arthropod-Borne Dis, December 2018, 12(4): 370–377 O Guntay et al.: Evaluation of Pyrethroid … 372 http://jad.tums.ac.ir Published Online: December 25, 2018 normality test. For all the hypotheses testing, an alpha of 0.05 was used as the cutoff for significance. Results Morphological identification Some characteristics used for genus and spe- cies identification are shown in Fig. 1. Before applying bioassays, we identified that all pop- ulations collected were Cx. pipiens. Bioassays We have first analyzed the KD rates at the end of a 1h-application of each pyrethroid in each population. The pairwise comparisons of KD rates within the populations were tested for significance. Out of 18 pairwise comparisons (3 populations, 4 different pyrethroid applica- tions, therefore 6 pairwise comparisons within each population), 7 were found to be significant (*P< 0.05). These significant results were ob- tained from the comparisons between the KD rates of Permethrin and Deltamethrin, Perme- thrin and Cyfluthrin, and α-Cypermethrin and Cyfluthrin applications in both Çiğli and Bor- nova populations. Moreover, one more signif- icant result was also revealed from the pairwise comparison between KD rates of Deltamethrin and Cyfluthrin applications in Menemen pop- ulation. The knock-down rates at 1h observed in all the populations of the study were sum- marized in Fig. 2. All of the Cx. pipiens populations showed different levels of resistance to all tested in- secticides with a mortality rate ranging between 0% and 63%. Overall, the lowest mortality rates were obtained with permethrin (ranged between 0% and 21%) and the highest mortality rates with cyfluthrin (ranged between 8% and 63%) in all populations. Mortality rates resulted from α-cypermethrin and deltamethrin applications ranged between 0% and 25% and between 0% and 38%, respectively. All the results obtained from susceptibility tests carried out in all of the three populations were summarized in Table 1. Mortalities observed in cyfluthrin applica- tions were significantly different than the mor- tality rates obtained from the other three pyre- throids used in the study in both Menemen and Bornova populations (*P< 0.05). For Çiğli pop- ulation, we have found statistically significant difference (*P< 0.05) between mortality rates of cyfluthrin and permethrin applications as well as that of between cyfluthrin and α-Cyperme- thrin applications. However, there was no sig- nificant difference (P> 0.05) between mortal- ity rates of cyfluthrin and deltamethrin appli- cations. In addition, there was no significant difference between mortality rates of permethrin and α-Cypermethrin applications in Çiğli pop- ulation. Furthermore, the pairwise comparison of mortality rates of Permethrin, α-Cyperme- thrin and deltamethrin applications in Borno- va and Menemen populations, separately, did not reveal a significant difference. The synergist bioassay test carried out in Menemen and Bornova populations in order for the assessment of the contribution of P450 detoxification enzymes did not restore suscep- tibility to permethrin and deltamethrin. How- ever, in both populations, pre-exposure to PBO significantly increased the effect of the pyre- troids applied. In Menemen population, the mor- tality rates significantly (*P< 0.05) increased from 1.24% (deltamethrin) to 23.96% (PBO+ deltamethrin) (estimated P= 0.000) and from 0.96% (permethrin) to 4.26% (PBO+ perme- thrin) (estimated P= 0.014). Similarly, in Bor- nova population, mortality rates significantly (*P< 0.05) increased from 16.98% (deltame- thrin) to 55.96% (PBO+deltamethrin) (estimat- ed P= 0.000) and from 8.15% (permethrin) to 27.95% (PBO+permethrin) (estimated P= 0.000). This bioassay was not carried out in Çiğli pop- ulation. The comparative results of the syner- gist bioassays were summarized in Table 2. J Arthropod-Borne Dis, December 2018, 12(4): 370–377 O Guntay et al.: Evaluation of Pyrethroid … 373 http://jad.tums.ac.ir Published Online: December 25, 2018 Table 1. Mortality rate results (Izmir, Turkey, 2017) of permethrin (0.75%), α-cypermethrin (0.05%), deltamethrin (0.05%) and cyfluthrin (0.15%) tests obtained at 24h after the application in each of the tested Culex pipiens populations were presented below (95% CI) Populations % Mortality (range) Permethrin α-Cypermethrin Deltamethrin Cyfluthrin Menemen 0.96 (0–4) 1.83 (0–9) 1.24 (0–7) 17.97 (8–29) Çiğli 2.52 (0–7) 2.59 (0–8) 13.12 (3–22) 27.42 (14–46) Bornova 8.15 (0–21) 11.39 (0–25) 16.98 (0–38) 46.27 (36–63) Table 2. Susceptibility test results of Culex pipiens with and without pre-exposure to PBO in northern Izmir, Turkey, 2017 (95% CI) Populations Insecticide tested % Mortality (range) Menemen Deltamethrin 1.24 (0–7) PBO+Deltamethrin 23.96 (8-35) Permethrin 0.96 (0–4) PBO+Permethrin 4.26 (0–9) Bornova Deltamethrin 16.98 (0–38) PBO+Deltamethrin 55.96 (37–68) Permethrin 8.15 (0–21) PBO+Permethrin 27.95 (16–50) Fig. 1. Some characteristics used in morphological identification. a) The last abdominal segment, anal segment and siphon of 4th stage larvae. b) Pecten teeth on siphon (shown by) and setae on siphon (all labeled with asterisk,). c) Setae on the head; labeled as 4C-7C. d) Combs on the last abdominal segment as shown by the arrow J Arthropod-Borne Dis, December 2018, 12(4): 370–377 O Guntay et al.: Evaluation of Pyrethroid … 374 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 2. Knock-down rates of Culex pipiens at 1h of each tested population (A-C) in northern Izmir, Turkey, 2017 Discussion Culex pipiens is the most common mosquito in Turkey and mosquito control is mainly di- rected against larval stages besides, many py- rethroids are currently registered and used for adult control (15). Many pesticides commonly used in agricultural areas and public health cause mosquito populations to become resistant. The insecticides with same mode of action used in these different areas cause cross-resistance (29, 30). Resistance was expected in these populations due to frequent use of agricultural pesticides in the vicinity of the settlement areas where pop- ulations were collected. Nevertheless, some mor- tality rates for permethrin, deltamethrin and α- cypermethrin were 0%, and therefore the detec- tion of such a high resistance was surprising. Resistance to permethrin and deltamethrin was also detected in Cx. pipiens populations col- lected from similar regions in 2012 and 2013, but such low mortality rates have not been de- termined (18). In our study, the overall mortal- ity rate for deltamethrin was 17%. In contrast, value of this species for deltamethrin in north- western and southeastern Iran reported as 91% and 93% respectively (21, 22). Nevertheless, in Tehran, mortality rate for deltamethrin in same species was reported as 18% (20). In Greece, the lowest mortality rate was reported 64% in 13 different populations of the same species (19). Different mortality rates can occur within different pyrethroids with the same mode of ac- tion (31). However, in the same population, the difference between cyfluthrin and other pyre- throid mortality means was not expected to be as high as 38% (Table 1). This may be related to the chemical structure of the pyrethroid used. The presence of the α-cyano group in the struc- ture of the pyrethroid has an effect on the re- sistance mechanism(s) (32). This difference can also be explained by the reflection of different kdr mutations in phenotypic resistance in dif- ferent ways (33). A possible third explanation is the different detoxification enzymes involved in metabolic resistance mechanisms. CYPs are major mechanism of insecticide metabolic re- sistance (34). Previous years, different results have been obtained about the role of GSTs and CCEs in pyrethroid resistance (10, 35, 36). Under different pyrethroid pressures, metabolic resistance mechanisms can change and pheno- typic resistance can reflect differently. Under low pyrethroid selection pressure, metabolic re- sistance is mainly mediated by CYPs, but under high pyrethroid selection pressure, high level of metabolic resistance is related to CYPs and J Arthropod-Borne Dis, December 2018, 12(4): 370–377 O Guntay et al.: Evaluation of Pyrethroid … 375 http://jad.tums.ac.ir Published Online: December 25, 2018 CCEs (11). In this study, we used PBO, the CYPs in- hibitor (37, 38) to demonstrate the effects of CYPs in metabolic resistance of deltamethrin and permethrin. Our results showed that CYPs indeed play a role in the metabolic resistance of both deltamethrin and permethrin (Table 2). In Menemen and Bornova populations, syner- gistic value between permethrin and PBO+ permethrin was 3.3% and 19.8% respectively. Synergistic results between same pyrethroid and PBO reported in two different population of same species in Marin County, California as 55.5% and 12.8% respectively (39). Interestingly, in both populations with pre- exposure PBO, the effect of CYPs was higher in deltamethrin than in permethrin. Besides the detoxification enzymes, the physical properties and chemical structures of the insecticide used may also play a role together in metabolic re- sistance mechanisms. Conclusion Culex pipiens mosquitoes collected from the three different localities in northern Izmir have high levels of resistance to permethrin, deltame- thrin, α-cypermethrin and cyfluthrin. In addi- tions, P450 detoxification enzymes have an ef- fect on phenotypic resistance. However, the ma- jor mechanism is due to the kdr resistance. Fur- ther studies are needed to explain these mech- anisms responsible for pyrethroid resistance in Cx. pipiens. 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