J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 243 http://jad.tums.ac.ir Published Online: September 30, 2019 Original Article Investigation of Susceptibility Levels of Culex pipiens L. (Diptera: Culicidae) Populations to Synthetic Pyrethroids in Antalya Province of Turkey *Onder Ser1; Huseyin Cetin2 1Malaria Control Unit, Antalya Provincial Directorate of Health, Antalya, Turkey 2Department of Biology, Faculty of Science, Akdeniz University, Antalya, Turkey (Received 6 June 2018; accepted 26 June 2019) Abstract Background: Culex pipiens L. (Diptera: Culicidae) is an important vector of several pathogens. This mosquito is widely distributed throughout the world. We aimed to determine the susceptibility levels of Cx. pipiens populations to some synthetic pyrethroid insecticides in Antalya, Turkey. Methods: The immature stages of mosquitoes were collected from eight locations in Alanya, Döşemealtı, Kemer, Kumluca, and Manavgat districts of Antalya between Apr and Oct of 2017. Adult susceptibility tests were carried out according to a modified version of the Centers for Disease Control and Prevention bottle bioassay. In the tests, the World Health Organization recommended diagnostic doses; permethrin (0.75%), etofenprox (0.5%), deltamethrin (0.05%) and lambda-cyhalothrin (0.05%) were used. Results: As a result of the susceptibility tests, deltamethrin was the least effective insecticide and it caused 58.78– 97.56% mortalities on Cx. pipiens populations while permethrin was the most effective substance that caused 100% mortality on all populations. While all of the tested populations were found susceptible to permethrin, and possible resistant or resistant to deltamethrin. Etofenprox and lambda-cyhalothrin led to 91.54–100% and 93.1–100% mortali- ties, respectively. Conclusion: The possible resistance or resistance to deltamethrin in all the areas is caused by the widespread use of this chemical against pests in agriculture and public health applications for long-term. Moreover, a concordance was found between resistance levels and the intensity of pesticide application in agriculture and public health, and organic and chemical pollution levels in the sampled habitats. Keywords: Antalya; Culex pipiens; Mosquito; Resistance; Synthetic pyrethroid Introduction There are over 3500 species of mosquitoes (Diptera: Culicidae) in the world, and more than 50 of them have been documented in Turkey (1, 2). Culex pipiens L. is an important vector of several disease-causing pathogens, such as filarial nematode (Wuchereria bancrofti), West Nile virus (WNV), Rift Valley fever virus, and St. Louis encephalitis virus. This mosquito species is widely distributed throughout the world (3, 4). Culex pipiens is the predominant species or it is intensively encountered in mosquito fauna studies conducted in different provinces of Turkey (1, 5-8). Culex pipiens was also seen as a dominant mosquito species in studies carried out in Antalya Province (9, 10). This mosquito is the possible vector of the WNV in Turkey (11, 12). In 2010, 47 WNV infections were detected and 10 patients died from the WNV infection (12). One of the most effective manners of controlling mosquito-borne diseases trans- mission is to control of their vectors. Despite the use of various methods to control mos- quitoes; the application of insecticides con- tinue to be the most preferred method due to easy accessibility, fast and effective results in a short time. However, excessive and uncon- scious use of insecticides lead to various prob- *Corresponding author: Dr Onder Ser, E-mail: onderser62@hotmail.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 244 http://jad.tums.ac.ir Published Online: September 30, 2019 lems in terms of environment and human health and also cause to development of resistance by mosquitoes. Parallel to resistance develop- ment, control of mosquito populations are be- coming more difficult and it may be an in- crease in the incidence of mosquito-borne dis- eases (13-15). Therefore, it is essential to monitor the insecticide resistance and sus- ceptibility in field mosquito populations to ensure the sustainability of mosquito control programs (4). Synthetic pyrethroids (SP) were produced to increase the chemical stability and biolog- ical activity of natural pyrethrins, which have insecticidal effects. Natural pyrethrins are ob- tained from the extraction of dried flower heads of Chrysanthemum spp. These contain a mix- ture of insecticidal action esters. SP are ne- urotoxic effect to insects and their primary action site is the voltage-gated sodium chan- nels (16, 17). The mode of action of these insecticides is similar to the organic chlorine insecticide, dichloro diphenyl trichloroethane (DDT). These insecticides act the voltage- gated sodium channels on the membranes of the nerve cells and cause over-stimulation due to the longer opening of channels (18, 19). Traditionally, SP are classified into two groups namely type I and type II, according to their chemical structure and toxicology. Type I pyrethroids do not contain the α-cyano group, whereas type II pyrethroids include the α-cyano group on the phenoxybenzyl moiety (19). SP have killer, knock-down and repellent effects on insects. In addition, these insec- ticides can be used with synergistic com- pounds to increase their activity (16). SP have several advantages, as compared with other insecticides in terms of cost, safety (less toxic to mammals), repellency, and duration of re- sidual action (20). SP are broad-spectrum in- secticides, effective against a number of insect pests (21). Currently, these insecticides are widely used, in agriculture, public health, vete- rinary medicine and as household pesticides (20, 21). Using them as larvicides are limited due to their toxicity against non-target aquatic organisms. Nowadays, SP are used on all cer- tified long-lasting insecticidal nets and indoor residual spraying programmes for control of major vector-borne diseases worldwide (20). We aimed to determine the susceptibility levels of Cx. pipiens populations collected from different districts of Antalya, important tourism and agricultural center of Turkey, to some synthetic pyrethroid insecticides com- monly used against mosquito adults. Materials and Methods Mosquitoes The immature stages (egg raft, larva, and pupa) of mosquitoes were collected from aquat- ic habitats in Alanya (Çıplaklı and Süleyman- lar), Döşemealtı (Ilıca and Killik), Kemer (Tekirova), Kumluca (Naranciye and solid waste storage area) and Manavgat (Çakış) districts of Antalya between Apr and Oct of 2017 (Fig. 1). Global positioning system (GPS) coordinates of the sampling areas are shown in Table 1. The immature stages collected from breeding sites were transported to the Depart- ment of Biology, Faculty of Science, Akdeniz University and reared to adults under stan- dard conditions (at a temperature of 25±2 °C, 60±10% relative humidity and 12h light: 12h dark photoperiod in an insectary). Larval feeding was done by using fish food. A pad of cotton soaked in 10% sucruse solution was provided for adult mosquitoes feeding. The species identification was made using the mor- phological characters according to the iden- tification keys (16, 22, 23). Insecticides In the susceptibility tests, synthetic pyre- throid active substances (permethrin, etofen- prox, deltamethrin, and lambda-cyhalothrin) were purchased from Tagros Chem. India Ltd. and used at the diagnostic doses recommended by the World Health Organization (WHO) (20). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 245 http://jad.tums.ac.ir Published Online: September 30, 2019 Bioassays Adult susceptibility tests were carried out according to a modified version of the Centers for Disease Control and Prevention (CDC) bottle bioassay (24). The WHO susceptibili- ty assay and the CDC bottle test are the most frequently used methods for detection of in- secticide resistance in mosquito populations (25). The results obtained from WHO and CDC test methods support each other. Similar re- sults were found between the two methods in terms of mortality rates in the studies conduct- ed to determine the susceptibility levels to in- secticides in malaria vectors (26, 27). Both methods have several advantages and disad- vantages. For example, WHO test kits are expensive and may not be easily attainable, but CDC bottle test method is simple, fast and cost-effective (25-27). However, the licensing of insecticides used in indoor residual spra- ying applications in Turkey is carried out ac- cording to doses recommended by the WHO. In addition, the doses used in the CDC bottle test method are considerably lower than the doses used in the WHO susceptibility tests. Therefore, the diagnostic doses and time re- commended in the WHO (2013) test procedures (20) were used in our study. For this pur- pose, WHO recommended stock solutions (0.75% permethrin, 0.5% etofenprox, 0.05% deltamethrin and 0.05% lambda-cyhalothrin) were prepared by dissolving the active sub- stances in acetone. For each synthetic pyre- troid, 1.836ml of stock solution was applied to the inner surface of the glass jars having an inner surface area of about 500cm2 and solution spreads in the jar. In this way, an equal amount of the active substance to the amount of per square centimeter insecticide on the WHO tube test method papers was applied to the glass surfaces (20, 25, 28). Af- ter the solvent was evaporated (2h waiting period), tests were performed with 20–40 non- blood fed, 3–5 day-old adult female mos- quitoes. These individuals were recorded with knock-down rates at 5min intervals for 1h. After the 1h exposure period, mosquitoes were transferred to clean jars for recovery. The num- ber of dead mosquitoes in both the treated and the control jars was recorded 24h post-ex- posure. According to the WHO recommen- dations, mosquitoes are categorized as dead if they are immobile or unable to stand on or fly in a coordinated manner. Each bioassay was conducted at least using four replicates. In each bioassay, one control group was used. Only acetone is applied to the jars where the cont- rol groups are located and the inside surfaces are dried. Statistical analysis Corrected mortality rate was calculated using Abbott's formula when mortality rate in the control group was between 5–20% (29). Values of times for 50% knockdown (KDT50) and KDT95 were calculated by Stat- Plus probit analysis program. Insecticide resistance status of populations was assessed according to WHO (2013) (20) criteria, where mortality range 98–100% was susceptible, 90–97% possible resistant, and < 90% resistant. Results Knock-down effect of synthetic pyrethroids The KDT50 and KDT95 values of active substances are presented in Table 2. At the end of 1h, while the knock-down rates for permethrin were 100% in all populations, the knock-down rates for etofenprox, deltamethrin and lambda-cyhalothrin were between 88.21– 100%, 31.31–99.58% and 94.27–100%, res- pectively. According to KDT50 values, per- methrin showed the highest knock-down effect on Cx. pipiens, except for the Döşemealtı-Ilıca population. Although different results were obtained for other active substances, delta- methrin showed the highest KDT50 values and the lowest knockdown effect at the five populations. KDT50 values of permethrin were http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 246 http://jad.tums.ac.ir Published Online: September 30, 2019 6.00–12.10min, of etofenprox, were 6.57–27.61 min, of deltamethrin were 11.26–122.53min and of lambda-cyhalothrin were 5.57–20.66 min. The lowest KDT50 value in all popu- lations was 5.57min at lambda-cyhalothrin in the Döşemealtı-Ilıca population, while the highest KDT50 value was 122.53min at delta- methrin in the Kumluca-solid waste storage area population. When KDT95 values were compared, deltamethrin showed the lowest knock-down effect except for the Döşemealtı- Killik population while permethrin showed the highest knock-down effect on all populations. KDT95 values of permethrin were 12.96–29.59 min, of etofenprox were 17.36–85.36min, of deltamethrin were 40.83–744.30min and of lambda-cyhalothrin were 14.82–69.66min in all populations. The lowest KDT95 value of all populations was 12.96min at the permeth- rin in the Döşemealtı-Ilıca population, while the highest KDT95 value was 744.30min at the deltamethrin in the Kumluca-solid waste storage area population. Mortality rates As a result of the susceptibility tests, del- tamethrin was the least effective insecticide and it caused 58.78–97.56% mortalities on Cx. pipiens while permethrin was the most effective substance that caused 100% mortal- ity on all populations (Fig. 2). According to WHO criteria, while all of the tested popula- tions were susceptible to permethrin, none of the populations were susceptible to deltame- thrin, four of the eight populations were re- sistant to deltamethrin and the remains were possible resistant (Table 2). In addition, the lowest mortality rate among all tested popu- lations was obtained from deltamethrin at 58.78% in the Kumluca-solid waste storage area population. Etofenprox and lambda- cyhalothrin led to 91.54–100% and 93.10– 100% mortality in the eight tested popula- tions, respectively (Fig. 2). Populations col- lected from two sampling sites for each of etofenprox and lambda-cyhalothrin were de- termined as possible resistant and the other six populations were susceptible (Table 2). Table 1. Location and global positioning system (GPS) coordinates of Cx. pipiens collection sites No District Location GPS Coordinate 1 Alanya Çıplaklı 36º33'39.9" N 32º02'44.2" E 2 Alanya Süleymanlar 36º40'42.299" N 31º59'04.256" E 3 Döşemealtı Ilıca 37º09'12.868" N 30º37'54.912" E 4 Döşemealtı Killik 37º12'51.987" N 30º39'52.588" E 5 Kemer Tekirova 36º31'04.634" N 30º32'18.758" E 6 Kumluca Narenciye 36º21'59.7" N 30º17'21.3" E 7 Kumluca Solid waste storage area 36º25'10.538" N 30º18'30.969" E 8 Manavgat Çakış 36º54'46.150" N 31º09'47.825" E Fig. 1. Culex pipiens collection sites in various districts of Antalya Province, Turkey in 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 247 http://jad.tums.ac.ir Published Online: September 30, 2019 Table 2. Knock-down (KD) rates, mortality rates, KDT50 and KDT95 values and susceptibility status of Cx. pipiens populations from Antalya, Turkey to four synthetic pyrethroids Populations Parameters Permethrin (0.75%) Etofenprox (0.05%) Deltamethri n (0.05%) Lambda- cyhalothrin (0.05%) Control Alanya- Çıplaklı N 165 172 190 167 171 %KD±SE (after 60min) 100 88.21±5.40 80.03±5.09 94.27±1.47 0 KDT50±SE (min) 12.10±0.82 26.02±0.82 26.85±1.24 20.66±0.73 KDT95±SE (min) 27.27±2.32 85.36±5.74 188.76±27.6 9 69.66±4.25 χ2 24.4440 2.0809 1.0577 3.1637 P-level 0.0065 0.9957 0.9998 0.9773 %Mortality±SE (after 24h) 100 91.54± 2.51 80.30±3.01 93.10±2.08 0.63±0.63 Susceptibility status S PR R PR Alanya- Süleymanlar N 115 112 131 121 102 %KD±SE (after 60min) 100 94.34±2.67 92.96±4.32 100 0 KDT50±SE (min) 11.81±0.68 27.61±1.43 22.89±0.82 14.79±0.48 KDT95±SE (min) 29.59±2.04 81.18±9.09 86.54±6.32 33.66±1.39 χ2 12.6888 17.5235 5.6160 4.5321 P-level 0.2416 0.0636 0.8464 0.9202 %Mortality±SE (after 24h) 100 91.95±3.37 90.23±2.45 99.19±0.81 0.96±0.96 Susceptibility status S PR PR S Döşemealtı- Ilıca N 118 110 124 138 114 %KD±SE (after 60min) 100 100 65.98±7.32 100 0 KDT50±SE (min) 6.00±0.30 6.57±0.37 47.19±1.41 5.57±0.36 KDT95±SE (min) 12.96±0.84 17.36±1.06 124.14±10.9 4 14.82±1.00 χ2 0.0344 0.0390 11.7727 0.0646 P-level 0.9999 0.9999 0.3006 0.9999 %Mortality±SE (after 24h) 100 100 76.51±4.92 100 5.15±3.16 Susceptibility status S S R S Döşemealtı- Killik N 158 154 196 169 144 %KD±SE (after 60min) 100 98.33±1.67 99.58±0.42 100 0 KDT50±SE (min) 9.89±0.41 15.85±0.58 13.09±0.55 12.03±0.46 KDT95±SE (min) 24.78±1.23 46.07±2.25 40.83±2.10 30.61±1.41 χ2 4.7670 4.6889 1.7015 0.8662 P-level 0.9062 0.9110 0.9982 0.9999 %Mortality±SE (after 24h) 100 99.17±0.83 97.56±0.86 100 0 Susceptibility status S S PR S Kemer- Tekirova N 118 121 139 115 109 %KD±SE (after 60min) 100 100 92.00±8.00 100 0 KDT50±SE (min) 6.37±0.35 8.96±0.44 11.26±0.70 12.01±0.50 KDT95±SE (min) 15.95±1.00 25.89±1.39 60.06±4.72 34.32±1.68 χ2 0.2756 1.1492 0.9450 1.2361 P-level 0.9999 0.9997 0.9999 0.9995 %Mortality±SE (after 24h) 100 100 91.70±4.39 100 1.71±1.05 Susceptibility status S S PR S Kumluca- Narenciye N 149 147 171 155 148 %KD±SE (after 60min) 100 99.28±0.72 93.10±3.01 96.14±1.76 0 KDT50±SE (min) 11.35±0.43 16.84±0.75 19.69±0.70 14.67±0.58 KDT95±SE (min) 26.91±1.24 41.77±2.47 65.40±3.85 45.94±2.37 χ2 5.8699 11.9181 1.8928 2.4029 P-level 0.8261 0.2906 0.9971 0.9922 %Mortality±SE (after 24h) 100 98.55±1.45 90.07±2.86 96.94±1.08 0 Susceptibility status S S PR PR http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 248 http://jad.tums.ac.ir Published Online: September 30, 2019 Kumluca- Solid waste storage area N 137 134 138 115 138 %KD±SE (after 60min) 100 100 31.31±2.52 100 0 KDT50±SE (min) 8.99±0.34 11.93±0.72 122.53±20.6 6 9.88±0.37 KDT95±SE (min) 18.51±0.92 20.67±1.78 744.30±310. 93 20.81±0.99 χ2 2.3809 32.7119 2.9895 0.7357 P-level 0.9925 0.0003 0.9817 0.9999 %Mortality±SE (after 24h) 100 100 58.78±5.34 100 2.48±1.78 Susceptibility status S S R S Manavgat- Çakış N 137 120 133 114 119 %KD±SE (after 60min) 100 100 84.38±4.61 100 0 KDT50±SE (min) 8.96±0.39 12.40±0.48 24.82±0.87 13.85±0.47 KDT95±SE (min) 21.94±1.13 32.58±1.52 95.55±7.47 32.08±1.36 χ2 0.0921 1.8646 4.6029 7.4133 P-level 0.9999 0.9973 0.9161 0.6859 %Mortality±SE (after 24h) 100 100 80.67±5.76 100 7.93±4.78 Susceptibility status S S R S N: Number of individuals tested, PR: Possible Resistant, R: Resistant, S: Susceptible, SE: Standard Error Swsa: Solid waste storage area Fig. 2. Mortality rates of Cx. pipiens populations to diagnostic concentrations of synthetic pyrethroids Table 1. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 249 http://jad.tums.ac.ir Published Online: September 30, 2019 Discussion Knock-down rates SP are rapid-acting insecticides, which have a knock-down effect (20). The target site of these insecticides is voltage-gated sodium channels of nerve cell membranes in insect body (30, 31). Mutations in genes encoding the amino acid sequence in these channels cause a reduction in the sensitivity of the channels to the binding of pyrethroid insec- ticides. Alterations in the target site that lead to resistance to insecticides are usually referred to as knockdown resistance or kdr (32). In the case of kdr in a insect population, the rates of knock-down obtained from susceptibility tests are an important parameter for early detec- tion of resistance to the insecticide (20). From this point of view, there is currently no re- sistance development against permethrin in all studied populations due to 100% knock- down rates at the end of 1h and low KDT50 and KDT95 values. Permethrin is a highly ac- tive substance with rapid knockdown effect against a variety of insects (33). However, intensive use of permethrin may cause deve- lopment of resistance in mosquito populations. Extensive use of permethrin and DDT is in- volved in the selection of resistance against these insecticides in field populations of Anopheles (An.) gambiae in Burkina Faso (34). While mosquitoes collected from cotton- growing and urban areas were resistant to permethrin and DDT, mosquitoes collected from areas with limited insecticide selection pressure (rice fields and control areas) were susceptible. Mosquitoes in all tested areas were susceptible to deltamethrin with high morta- lity rates. In addition, mosquitoes collected from cotton-growing and urban areas showed high- er KDT50 and KDT95 values with permethrin and DDT than deltamethrin. Resistance to per- methrin and DDT may explain extensive use of these insecticides in the cotton-growing area and domestic use of same insecticides as bomb spray or coils in the urban areas. The appearance of 31.31–99.58% knock- down rates at the end of 1h and high values of KDT50 (11.26–122.53min) and KDT95 (40.83–744.30min) of deltamethrin in this study indicate resistance development in all studied populations against this active substance. Sus- ceptibility to deltamethrin of field-collected populations and colonized strains of An. cu- licifacies were investigated in India (35). Al- though there was 100% mortality in all the strains of An. culicifacies exposed to diagnostic concentration of deltamethrin for 1h, knock- down bioassays revealed more than two-fold higher values of KDT50 and KDT90 in An. cu- licifacies from Rameshwaram Island (both in field-collected and colonized strain), than pop- ulations from other areas. Results indicated the development of incipient resistance to delta- methrin in this strain of An. culicifacies. In- secticide resistance levels were evaluated in nine populations of An. gambiae sampled in three areas in the east of Tanzania (36). These areas are represented as an agriculture area, an urban area and a low pollution area depen- ding on urbanization, agriculture activity, and usage of insecticides for vector control. For adult mosquitoes, resistance ratio fifty (RR50) was obtained by dividing the KDT50 of each population to the KDT50 of the susceptible reference strain. Adult susceptibility tests re- vealed that populations from urban and agri- culture areas demonstrated the moderate re- sistance levels to deltamethrin with mean RR50 of 3.1 fold and 5.6 fold, respectively. Mor- tality rates after 1h exposure to WHO diag- nostic dose of deltamethrin were between 84 and 100% in all populations. A significant correlation was between deltamethrin resistance and agriculture activity. The presence of 88.21% and 94.34% knock-down rates at the end of 1h, KDT50 values of 26.02 and 27.71min and KDT95 values of 85.36 and 81.18min respectively in the Alanya-Çıplaklı and Alanya-Süleymanlar http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 250 http://jad.tums.ac.ir Published Online: September 30, 2019 populations for etofenprox indicate that these populations may develop resistance to eto- fenprox. The appearance of 94.27% and 96.14% knock-down rates at the end of 1h, the KDT50 values of 20.66 and 14.67min and the KDT95 values of 69.66 and 45.94min res- pectively in the Alanya-Çıplaklı and Kumlu- ca-Narenciye populations for lambda-cyhalo- thrin indicate that these populations may de- velop resistance to lambda-cyhalothrin. The knock-down rates, KDT50 and KDT95 values obtained from susceptibility tests are compatible with the mortality rates at the end of 24h. Mortality rates in the populations with high KDT50 and KDT95 values with low knock-down rates against etofenprox, deltame- thrin and lambda-cyhalothrin were determined to be resistance or possible resistant levels. Mortality rates In this study, the possible resistance or resistance to deltamethrin in all the sampling areas is caused by the widespread use of this active substance against the pests of agricul- ture and public health for many years. Insec- ticide resistance in mosquitoes collected from areas where pesticide use is high against ag- riculture and/or public health pest is higher levels than in areas where pesticide use is less (37-40). Kasap et al. (41) researched re- sistance to some insecticides of An. sacha- rovi collected in five malaria-endemic prov- inces of Turkey. According to results of sus- ceptibility tests, Adana, Adıyaman, and An- talya populations were resistant to most in- secticides tested, while Aydın and Muğla pop- ulations were susceptible to most of the insec- ticides. Intensive insecticide usage against ag- ricultural pests and mosquitoes has selected resistance in An. sacharovi to a number of com- pounds in these areas. The resistance levels to two types of insecticides of four major vec- tor species (An. gambiae, Cx. quinquefasciatus, Aedes (Ae.) aegypti, and Ae. albopictus) were assessed in Mayotte, a small island in the In- dian Ocean (42). Adult bioassays revealed that while field populations of An. gambiae and two Aedes species were found susceptible to deltamethrin, field population of Cx. quinquefasciatus was found stronge resistance to deltamethrin (only 10% mortality after 24h and strong decrease of knock-down effect: RR50= 4.2, RR95= 4.9). Culex quinquefasciatus is the major vector of the Bancroftian filariasis, which has been plaguing Mayotte for many years. Since the 1950s, intense vector control efforts had been carried out by using DDT and followed syn- thetic pyrethroids against this species. These important selective pressures certainly explain the strong resistance to deltamethrin observed in the Cx. quinquefasciatus population. In- secticide resistance status of Cx. quinque- fasciatus populations from four areas was studied in Benin (43). Adult tests showed high frequency of resistance in mosquitoes to per- methrin (ranging from 4% to 24% mortality) and deltamethrin (24% to 48% mortality) in the four research areas. SP have been exten- sively used in agriculture since 1980s parti- cularly in cotton and vegetable fields in Benin. Moreover, the massive free campaign of bed nets impregnated with permethrin and delta- methrin as the major control strategy against Wuchereria bancrofti transmitted by Cx. quinquefasciatus. These cases may cause the resistance of Cx. quinquefasciatus to SP. In our study, the highest level of deltame- thrin resistance with the lowest mortality rates was detected in mosquitoes collected from the Kumluca-solid waste storage area. This situ- ation is thought to have been caused by the application of residual spraying, thermal and cold fogging by using the SP regularly against the vectors such as mosquitoes, sand flies and house flies by the Antalya Metropolitan Mu- nicipality vector control teams in this area. In addition, Kumluca is a district where green- house activities and the use of pesticides re- lated to greenhouses is intense in Antalya Prov- ince. Therefore, the greenhouse wastes on the solid waste storage area contain intense pes- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 251 http://jad.tums.ac.ir Published Online: September 30, 2019 ticide residues and the leaking water from these wastes constitutes a breeding site for mosqui- toes. Moreover, there are intensively empty packages of various chemicals such as pesti- cides, fertilizers, and detergents containing res- idues in the solid waste storage area and ag- ricultural pesticides are used in fields and gar- dens near to this area. The resistance levels to deltamethrin of mosquito populations are in agreement with pollution level of the sampling areas and pes- ticide application frequency in these areas. Alanya-Çıplaklı, Manavgat-Çakış, Döşemealtı- Ilıca, and Kumluca-solid waste storage area populations were resistant to deltamethrin with mortality rates lower than 90%. The collec- tion areas of these four populations are habi- tats where pollutants such as organic and chem- ical wastes are concentrated. In addition, vec- tor control applications are regularly carried out by the Antalya Metropolitan Municipal in these four areas and pesticides are used in the fields and fruit gardens near these areas. Pes- ticides used in agricultural and public health applications are effective for improving re- sistance of mosquitoes to insecticides. Besides, various anthropogenic or natural xenobiotics in the breeding sites of mosquitoes can also contribute to develop resistance of mosquitoes to insecticides, especially by altering the ex- pression of genes encoding detoxification en- zymes and/or cuticular proteins (44). Aedes ae- gypti larvae can be improved resistance to var- ious insecticides in different classes by chang- es in the expression of genes responsible for detoxification of heavy metal (copper), poly- cyclic aromatic hydrocarbons (fluoranthene and benzo[a]pyrene) or herbicides (glyphosate and atrazine) (45-47). Similarly, the expression of genes encoding cuticular proteins was changed in Ae. aegypti larvae exposed to various pol- lutants and pesticides from different classes (48, 49). Alanya-Süleymanlar, Döşemealtı-Killik, Kemer-Tekirova and Kumluca-Narenciye pop- ulations were possible resistant to deltame- thrin with mortality rates in the range of 90– 97%. The habitats of these populations seem to be cleaner in terms of organic and chemi- cal pollutants when compared to the habitats of the other four populations. In addition, pes- ticide applications against pests of agricultur- al and public health are carried out at lower intensity in these sampling areas. In this study, the permethrin in all sampled locations was the most effective of the four active substances tested, leading to 100% mor- tality on the mosquito populations. In Turkey, use of permethrin as plant protection product has been terminated by Republic of Turkey Ministry of Food, Agriculture and Livestock since 01 Jan 2011. Therefore, this active sub- stance has not been used against pests of ag- ricultural in Antalya province for the last six years. In addition, permethrin has been used in limited quantities in cold fogging applica- tions against adult mosquitoes by the Antalya Metropolitan Municipality in 2013 and 2015 years. This active substance has not been used against adult mosquitoes in the last two years (50). As a result of the susceptibility tests, etofenprox was caused 91.54% to 100% mor- talities in all populations. While Alanya- Çıplaklı (91.54% mortality) and Alanya-Süley- manlar (91.95% mortality) populations were determined to be possible resistant to eto- fenprox, the other six populations were found susceptible. Etofenprox is used in low amounts against pests of agriculture and public health in Antalya Province. This case is consistent with the susceptibility to etofenprox of the six tested populations. The possible resistance to etofenprox of the Alanya-Çıplaklı and Alan- ya-Süleymanlar populations may be due to the local use of this active substance in these sam- pling areas or development cross-resistance to other insecticides from the used synthetic pyrethroid group in fruit growing (grape, avo- cado, lemon, orange, medlar, banana etc.) at outdoor area of Alanya District. Because of tests performed with lambda- cyhalothrin, it was observed mortality in the http://jad.tums.ac.ir/ https://www.tarim.gov.tr/Sayfalar/EN/AnaSayfa.aspx https://www.tarim.gov.tr/Sayfalar/EN/AnaSayfa.aspx J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 252 http://jad.tums.ac.ir Published Online: September 30, 2019 range of 93.1–100% in all populations. While Alanya-Çıplaklı (93.1% mortality) and Kumlu- ca-Narenciye (96.94% mortality) populations were possible resistant to this active substance, the other six populations were susceptible. Lambda-cyhalothrin is used in low amounts against pests of agriculture and public health in Antalya Province. This case is consistent with the susceptibility to this active substance of the six tested populations. The possible re- sistance to lambda-cyhalothrin of the Alan- ya-Çıplaklı and Kumluca-Narenciye popula- tions may be due to the local use of this ac- tive substance in these sampling areas or de- velopment cross-resistance to other insecticides from the used synthetic pyrethroid group in these sampling areas. In our literature review, Cx. pipiens has developed resistance to SP at different levels in a variety of researches conducted in Tur- key and worldwide. Culex pipiens populations collected from Aksu district of Antalya were resistant to permethrin (0.75%) and delta- methrin (0.05%) with 78.3% and 75.8% mor- tality rates, respectively (3). Similarly, Cx. pipiens populations collected from Aksu Dis- trict of Antalya were resistant to permethrin (0.75%) and deltamethrin (0.05%) with 74% and 62% mortality rates, respectively (12). Seasonal dynamics of insecticide resistance were investigated in field populations of Cx. pipiens from Çanakkale, Balıkesir, İzmir, Aydın, Muğla, and Denizli provinces in western Turkey (4). In the results of bioassays, all populations showed seasonally different levels of resistance to permethrin and deltamethrin. The resistance status to four insecticides was examined in thirteen populations of Cx. pipiens collected from five regions of Greece (39). Adult bioassays showed that while one popu- lation was resistant to deltamethrin with 64% mortality rate, three populations were possible resistant with mortalities of 92%, 90% and 87%, and other nine populations were sus- ceptible. Salim-Abadi et al. (51) evaluated the susceptibility status of Cx. pipiens col-lected from the capital city of Tehran, Iran. Field population was resistant to all tested SP (lambda-cyhalothrin 0.05%, deltamethrin 0.05% and cyfluthrin 0.15%). The irritability levels of different groups of insecticides on laboratory strain and field population of Cx. pipiens complex were investigated in Tehran, Iran (52). Permethrin (0.75%) and deltamethrin (0.05%) were moderately irritable against both field population and laboratory strain of Cx. pipiens complex. While cyfluthrin (0.15%) was moderately irritable for field population, it was hypo-irritable for laboratory strain. Lambda- cyhalothrin (0.05%) was hypo-irritable against both field population and laboratory strain. Whereas etofenprox (0.5%) was hypo-irritable for field population, it was non-irritable for laboratory strain. Ghorbani et al. (53) assessed the susceptibility status to 12 adulticides and two larvicides recommended by WHO of Cx. pipiens collected from Sari County in the north of Iran. The susceptibility tests showed that Cx. pipiens was resistant to all tested in- secticides. Nevertheless, the resistance level was lower to SP compared to the others. The mortality rates after exposure to etofenprox (0.5%), cyfluthrin (0.15%), permethrin (0.75%), deltamethrin (0.05%) and lambda-cyhalothrin (0.05%) were 76.47%, 72.09%, 70.73%, 39.08% and 33.33% respectively. Insecticide resistance may vary among regions, provinc- es, districts, and even smaller localities with- in a country. These differences in insecticide resistance include many factors such as the species of mosquito, life stage, physiological status, even the various symbioses or patho- gens found in the body of mosquitoes, the climatic characteristics of the study area, al- titude, vegetation cover, acreage, socioeconom- ic structure, agriculture and animal husband- ry activities, pesticides used in this area, doses, frequency, and methods of application of pes- ticides, agricultural chemicals, urban and in- dustrial pollutants (44). In order to prevent and/or delay to the de- velopment of resistance to insecticides in mos- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 253 http://jad.tums.ac.ir Published Online: September 30, 2019 quitoes, integrated control programs should be implemented in which chemical use is kept in a minimum level, with emphasis on phys- ical, cultural and biological control methods (15, 54). If chemical use is needed; products with high selective toxicity, low toxicity to non-target organisms, less persistence in the environment and no resistance developed in target organism should be preferred and these products should be used at the doses indicat- ed on the label. In addition, whether mosqui- toes have resistance or not should be regularly monitored against the products used in com- bat (55). In the case of resistance detection, various tests can be performed to determine the mechanism. For this purpose, synergist tests can be used to determine the presence of re- sistance related to the detoxification enzymes, biochemical enzyme assays to determine the metabolic resistance, and molecular biological tests to determine the target site resistance (20). According to the results obtained from re- sistance tests, insecticide resistance maps should be prepared. These maps should be used in the selection of insecticide and re- sistance management (56). Insecticide appli- cations should be made in more limited areas where mosquitoes are heavily infested or mos- quito-borne disease risk is high rather than large areas (55). Since larval and adult control must be performed simultaneously to have an effect on mosquito populations, unrelated classes of insecticides with different modes of action should be used for each life stage of mos- quitoes (57). It may be beneficial to apply a mosaic approach by using products of dif- ferent insecticide classes and mode of action in neighboring areas (32, 57). The insec- ticides with same modes of action should not be used in an area for a long time. Instead, insecticides of different classes with unre- lated modes of action should be used in ro- tation (32, 54, 57, 58). Addition of synergistic substances to products may increase the sus- ceptibility of mosquitoes to insecticides (58). Synergists are compounds that do not have insecticidal activity by themselves. However, when they are mixed with insecticides of a certain class, significantly increase their effect by inhibiting an enzyme that detoxifies the insecticide in the insect body (20, 59). Syner- gists include piperonyl butoxide (PBO), which inhibits oxidase activity, S.S.S tributlyphos- phorotrithioate (DEF), which inhibits esterase activity, ethacrynic acid (EA), diethyl maleate (DM), and chlorfenethol (CF), which inhibit glutathione transferase activity (24). PBO is used as a synergist in insecticide formulations against the public health pests in Turkey. However, there is no standard for synergist ratios to be used in formulations. In addition, the development of more efficient and environ- ment-friendly new compounds with different modes of action, such as herbal, microbial and synthetic origin, as an alternative to the existing insecticides will contribute to preventing or delaying the development of resistance to in- secticides in mosquitoes (55, 58, 60). Conclusion In our study, all Cx. pipiens populations were found to be possible resistant or re- sistant to deltamethrin. This situation is thought to be due to the widespread use of this active substance in agriculture and pub- lic health applications for many years. The resistance levels to deltamethrin of the mos- quito populations are related to pesticide ap- plication frequency in these areas and the pollution level of the sampling area. Insecticides are used intensively to control mosquito populations at present. However, mosquitoes develop resistance to almost every kind of insecticides used in combat. This case causes serious concerns. One of the effective ways, in order to prevent and/or delay to the development of resistance to insecticides in mosquitoes, is to minimize usage of insecti- cide. To achieve this, integrated control pro- grams should be implemented. In addition, http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2019, 13(3): 243–258 O Ser and H Cetin: Investigation of … 254 http://jad.tums.ac.ir Published Online: September 30, 2019 the resistance status of mosquito populations against insecticides used in combat should be regularly monitored and insecticide re- sistance maps should be prepared. The results of this study will contribute to the planning of the resistance management and selection of insecticides that will be used by the mosquito control agencies and insti- tutions in Antalya, Turkey. However, it needs new studies that will be used for other mos- quito species from more localities and dif- ferent active substances. It is also thought to be useful to conduct further studies to deter- mine resistance mechanism in mosquito pop- ulations in areas where resistance is detected. Acknowledgements The authors declare that this study received no financial support. 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