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Yearly changes of insecticide susceptiblity and possible insecticide resistance mechanisms of Anopheles maculipennis Meigen (Diptera: Culicidae) in Turkey
Acta Tropica 126 (2013) 280–285
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Yearly changes of insecticide susceptiblity and possible insecticide resistance mechanisms of Anopheles maculipennis Meigen (Diptera: Culicidae) in Turkey M.M. Akiner a,∗ , S.S. Caglar b , F.M. Simsek c a
Department of Biology, Faculty of Science and Arts, Recep Tayyip Erdogan University, Fener, Rize, Turkey Department of Biology, Faculty of Science, Hacettepe University, 06800 Beytepe, Ankara, Turkey c Department of Biology, Faculty of Science and Arts, Adnan Menderes University, 09010 Aydın, Turkey b
a r t i c l e
i n f o
Article history: Received 6 September 2012 Received in revised form 17 February 2013 Accepted 24 February 2013 Available online 15 March 2013 Keywords: Anopheles maculipennis Insecticide resistance DDT Malathion Permethrin Deltamethrin
a b s t r a c t To evaluate the adulticide susceptibility and yearly changes of Anopheles maculipennis Meigen (Diptera: Culicidae) in Thrace, five mosquito populations were evaluated against the resistance status of four different adulticides. Three biochemical resistance mechanisms and yearly changes of activities were investigated. All the strains were highly resistant to DDT, and all the strains were placed in the resistance surveillance category for malathion, permethrin and deltamethrin in 2007. Although DDT mortality rates had increased from 2007 to 2008 except in the Seremkoy strain, malathion, permethrin and deltamethrin mortality rates have decreased in all of the tested strains. High rates of increase were determined for nonspecific esterases (NSEs) activity by using the substrate p-NPA and these results showed correlation with malathion mortality rates. All the strains showed high level of glutathione S transferases (GSTs), and their activity level had significantly increased from 2007 to 2008. Different insecticide susceptibility statuses were observed between localities, and high DDT resistance was observed although DDT was banned in the 1980s. Biochemical assay results suggest that NSEs and GSTs could play a role insecticide resistance in all tested strains. Malathion susceptibility has decreased in all the tested strains and NSE’s activity is possibly the main enzymatic mechanism related to the insecticide resistance. DDT resistance is at a high degree in all the strains and GST’s activity is probably related to this situation. GST’s activity could play an important role for permethrin and deltamethrin susceptibility but needs to be confirmed for molecular studies. Our results provide important data on insecticide susceptibility and change over time for the Anopheles maculipennis populations in Turkey. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Malaria remains a serious problem in the Middle East. Anopheles maculipennis complex is the vector of malaria in Europe and Middle East historically (Becker et al., 2003). This complex comprises 12 palearctic members and three species of this complex, and An. atroparvus, An. labranchiae, and An. sacharovi, are known to be efficient current and historical vectors of malaria in the palearctic region (Bruce-Chwatt and de Zulueta, 1980; Jetten and Takken, 1994; Linton et al., 2002; Sedaghat et al., 2003a,b). An. sacharovi is the main vector in Turkey and has widespread
∗ Corresponding author at: Recep Tayyip Erdogan University, Faculty of Science and Arts, Department of Biology, Zoology Section, 53100 Fener, Rize, Turkey. Tel.: +90 464 2236126x1835; fax: +90 464 2234019. E-mail address: [email protected] (M.M. Akiner). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.02.020
distribution in many parts of our country, but is generally found in mixed populations with An. maculipennis and An. melanoon in Thrace. Although development of resistance to chemical insecticides has been reported by many of the authors, chemical insecticides are still being heavily used in control operations in many areas of the World (Bonning et al., 1991; Chandre et al., 1999; Chavillon et al., 2001). Ramsdale et al. (1980) had described carbamate and organophosphate resistance in An. sacharovi populations from Cukurova plain, Osmancık and Blacksea region in Turkey. Kasap et al. (1999) had described carbamate, organophosphate (OP), and pyrethroid (PY) resistance in An. sacharovi, Culex tritaeniorhyncus, and Aedes caspius. Kasap et al. (2000) had identified OP, PY, carbamate, and organochlorine resistance in five different An. sacharovi populations of southern Turkey. Akiner et al. (2009) had described OP and PY resistance in Culex pipiens larvae and they have also found organochlorine, OP, and PY resistance in adult samples. However,
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Fig. 1. Location and GPS coordinates of the collected strains.
all of the aforementioned studies were conducted by using classical bioassays and can only provide clues as to molecular and biochemical mechanisms. Furthermore, there are few studies which focus on biochemical mechanisms of insecticide resistance in Turkish Anopheles species. Hemingway et al. (1985) had indicated that DDT resistance may be related to the qualitative or quantitative changes of DDT dehydrochlorinase, but did not find any changes in nonspecific esterases, multifunction oxidases and glutathion S transferases (GST) activity levels in An. sacharovi populations from the Cukurova plain. Luleyap and Kasap (2000) had reported that AChE, GSTs, and non-specific esterases activity levels were higher in the resistant group than in the susceptible groups. Studies about insecticide resistance of Anopheles and Culex populations in Turkey is not up-to-date and also these previous studies are narrow scoped and they did not cover An. maculipennis that is also the most abundant species in many parts of our country. Thus, new studies are necessary for finding out current resistance status of Anopheles and Culex populations. The objective of this study was to evaluate resistance, yearly changes and enzyme activity levels responsible for the insecticide resistance in the An. maculipennis populations from the Thracian part of Turkey.
2. Materials and methods 2.1. Strains of An. maculipennis The study sites’ locations, GPS coordinates, and a brief description of the localities are provided in Fig. 1. Larvae and adult samples of An. maculipennis were collected from five different localities in the Thrace region of Turkey. We especially chose rice field areas near the border between Turkey, Greece, and Bulgaria (Avariz, Su Akacagi, Tatarkoy, Seremkoy), an area where human trafficking occurs. The chosing criteria for the last collection site is population density and different agricultural usage types (corn and bean). All the adult samples were transferred to a −80 ◦ C deep freezer until the date of biochemical analysis. Insecticide usage over the previous 5 years at the sampling sites is listed in Table 1 and Fig. 1. 2.2. Bioassays The adult mosquitoes were assayed for susceptibility by diagnostic tests (WHO, 1981). Susceptibility was determined against DDT, permethrin, deltamethrin, and malathion. One diagnostic
Table 1 Anopheles maculipennis strains used to survey insecticide resistance and the use of insecticides in the last 5 years for control and use of insecticides agriculturally for over 10 years. Insecticides Last 5 years
Over 10 years
Adulticides
Agricultural
Deltamethrin Cypermethrin Permethrin D-phenothrin Cyfluthrin Pyrethrum Thiamethoxam Endosulfan Carbaryl Malathion Chlorpyriphos ethyl Deltamethrin Alpha cypermethrin
Avariz
Tatarkoy
Derekoy
X X
X X
X X X
X
Seremkoy
Su Akacagi
X X X X X X X X X
X X X X X
X X X
X X X
X
X
X X X X
X X X
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Table 2 Percentage mortality of Anopheles maculipennis adults from five different areas exposed to diagnostic doses of DDT, malathion, permethrin, and deltamethrin in 2007 and 2008. Insecticides
DDT 4%
Permethrin 0.75%
Deltamethrin 0.05%
Strain/Year
2007
2008
2007
2008
2007
2008
2007
2008
19 47.5 22 55 20 50 26 65 24 60
20 50 25 62.5 21 52.5 24 60 27 67.5
38 95 36 90 37 92.5 36 90 36 90
35 87.5 33 82.5 33 82.5 31 77.5 32 80
37 92.5 34 85 36 90 38 95 37 92.5
34 85 33 82.5 34 85 35 87.5 35 87.5
38 95 37 92.5 37 92.5 36 90 38 95
37 92.5 36 90 34 85 37 92.5 35 87.5
Avarız Tatarkoy Derekoy Seremkoy Su Akacagi
No. Dead Mortality (%) No. Dead Mortality (%) No. Dead Mortality (%) No. Dead Mortality (%) No. Dead Mortality (%)
Malathion 5%
Note: Experiments were performed using 2 replicates and each replicate contained 20 female mosquitoes.
concentration was used for all insecticides and a 1-h application period was used for permethrin, deltamethrin, and malathion, while a 2-h application period was used for DDT. Mortality was recorded after the 24 h resting period. Insecticide resistance status was evaluated by using the classification determined by the WHO (WHO, 2006), in which 98–100% mortality indicates susceptibility, 80–97% mortality suggests possible resistance requiring confirmation, and <80% mortality suggests resistance. 2.3. Biochemical analysis Non-specific esterases (NSEs), mixed function oxidases (MFOs), and glutathione S-transferases (GSTs) levels were measured individually in the adult females as described by the WHO (WHO, 1998) for possible biochemical insecticide resistance mechanisms. Standard flat-bottom microtiter plates (Nunch maxisorp® , Nunch A/S, Roskilde, Denmark) were used for individual testing and absorbance was read spectrophotometrically with an ELISA reader (Power Wave® XS, Biotek Instruments USA). Biochemical analyses for individual mosquitoes were performed with two replicates of the homogenate. A total of 553 (253 in 2007, 300 in 2008) adult females from five localities (Avariz (55, 60), Tatarkoy (54, 60), Derekoy (24, 60), Seremkoy (60, 60), Su Akacagi (60, 60)) were analyzed. Each An. maculipennis specimen was homogenized on ice in 200 l of 50 mM sodium phosphate buffer, pH 7.2, and the homogenate was centrifuged at 10,000 × g for 10 min at +4 ◦ C. The supernatant was used as the source of enzymes. Two 20 l replicates of the supernatant were transferred to the microtiter plate for alpha and beta naphthyl acetate (␣,  NA) and MFOs assays. GSTs, p-nitrophenyl acetate (p-NPA), and protein assays were conducted with two 10 l replicates of supernatant. NSEs activity was measured by using three substrates: ␣-naphthyl acetate, -naphthyl acetate, and p-nitrophenyl acetate (p-NPA). For the ␣NA and NA assay, 200 l of working solution was added (0.02 M phosphate buffer including 30 mM ␣ NA and  NA) for each, separately. After 30 min of incubation, 50 l of Fast Blue B Salt solution was added to stop the reaction. The final concentration of the final volume was measured at 570 nm endpoint for ␣- and -naphthyl acetate. For the p-NPA assay, 200 l of p-NPA working solution (20 ml 0.05 M phosphate buffer including 100 l 0.02 M p-NPA) was added to two replicates. The plate was read at 405 nm kinetically (WHO, 1998). MFOs activities were measured via the amount of heme (Brogdon et al., 1998). 200 l working solution (0.01 g 3,3 ,5,5 tetramethyl benzidine (TMBZ) + 5 ml methanol + 15 ml sodium acetate buffer) was added to each replicate. 25 l of 3% hydrogen peroxide was added and the mixture was incubated for 5 min at room temperature. Absorbance was read at 620 nm.
GSTs assays were measured by using a 200 l GSH/CDNB working solution (100 l of 0.060 g reduced glutathione in 20 ml 0.1 M Na phosphate buffer +0.013 g CDNB in 1 ml ethanol) added to each replicate well. The absorbance was read at 340 nm kinetically (WHO, 1998). Total protein amount in 10 l of supernatant was measured by using the Bradford assay in order to calculate enzyme activity for each sample (Bradford, 1976). Then 300 l of Bradford dye reagent was added to each replicate and the endpoint absorbance was read at 595 nm. Protein values were calculated by using a standard curve of absorbance of bovine serum albumin. 2.4. Statistical analysis Mean enzyme activity values were compared between populations and years by the Kruskal–Wallis test by means of Statistica 8® . 3. Results 3.1. Bioassay The results of the diagnostic bioassays carried out with DDT, malathion, permethrin and deltamethrin are shown in Table 2. As a result of the diagnostic tests, all the populations were resistant to DDT (mortality rates under 80%) and showed low mortality with this insecticide. Mortality rates decreased in all of the strains except in the Seremkoy strain (Table 2). All the strains were placed in the surveillance category (80–97%) for malathion in 2007. Mortality rates had decreased from 2007 to 2008 and went below 90%, approaching the resistance category. The mortality rate of the Seremkoy population was placed in the resistance category (under 80%) in 2008. All the populations were determined to be in the surveillance category (80–97%) except for the Su akacagi population in 2007 for permethrin. The highest change of mortality rates was also determined in the same strain (15%) from 2007 to 2008. Deltamethrin mortality rates were placed from 85 to %100 and decreased from 2007 to 2008 and were determined to be in the surveillance category (Table 2). 3.2. Biochemical analysis A total of 553 (253 in 2007, 300 in 2008) adult females from five localities (Avariz, Tatarkoy, Derekoy, Seremkoy, and Su Akacagi) were analyzed with the biochemical test for three different possible resistance mechanisms: elevated NSEs activity, elevated MFOs, and elevated GSTs activity (Table 3). NSEs activities of samples were detected from all of the field sites from 2007 to 2008 with artificial ester substrates; ␣, , NA
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Table 3 Non-specific esterases (NSEs), mixed function oxidases (MFOs), and glutathione S transferases (GSTs) activity in Anopheles maculipennis tested strains (mean ± S.D.) in 2007 and 2008. p-NPA
␣ Esterases
 Esterases
mole pNPA/min/mg protein
nmole ␣ naphthol/min/mg protein
nmole  naphthol/min/mg protein
Strains
2007
Avariz Tatarkoy Derekoy Seremkoy Su Akacagi
0.230 0.261 0.456 0.652 0.745
2008 ± ± ± ± ±
0.245 0.207 0.235 0.472 0.377
0.781 0.774 0.658 1.254 1.235
2007 ± ± ± ± ±
0.528 0.483 0.505 0.878 0.636
0.00024 0.00025 0.00028 0.00023 0.00013
2008 ± ± ± ± ±
0.00008 0.00008 0.00004 0.00005 0.00002
0.00025 0.00026 0.00035 0.00030 0.00023
MFOs
2007
Avariz Tatarkoy Derekoy Seremkoy Su Akacagi
0.0027 0.0037 0.00095 0.00023 0.0008
0.00004 0.00007 0.0001 0.00006 0.00004
2008
0.00025 0.00026 0.00030 0.00022 0.00012
± ± ± ± ±
0.0001 0.00008 0.00004 0.00005 0.00002
0.00025 0.00026 0.00033 0.00029 0.00022
± ± ± ± ±
0.00004 0.00008 0.00009 0.00007 0.00004
GSTs mole CDNB/min/mg protein
nmole product/min/mg protein Strains
2007 ± ± ± ± ±
2008 ± ± ± ± ±
0.0021 0.0034 0.0004 0.0001 0.0007
0.0016 0.0014 0.00108 0.00040 0.0014
and p-NPA. With p-NPA, the enzyme activity fluctuated in all the tested populations and increased from 2007 to 2008. The highest activity was found in the Su Akacagi strain and the lowest activity was found in the Avariz strain for 2007 samples. The highest activity was observed in the Seremkoy strain and the lowest activity was observed in the Derekoy strain for the 2008 samples (Table 3). Although increased activity was detected from 2007 to 2008, there were no significant differences between 2007 and 2008 (p > 0.05). ␣ and  esterase activities had fluctuated between all of the strains but nearly the same range was observed for Avariz, Tatarkoy and Seremkoy strains in 2007 and 2008. ␣ and  esterases activities increased from 2007 to 2008. ␣ esterase activity showed significant differences (p < 0.05) between 2007 to 2008, while  esterase did not show the same trend in the Derekoy strain. The Su Akacagi strain had showed low level activity for ␣ and  esterase in 2007, and activity levels increased nearly two-fold in 2008 and were significantly different from 2007 levels (Table 3). The MFOs levels in the 2007 samples ranged from 0.00023 ± 0.0001 (Seremkoy) to 0.0037 ± 0.0034 (Tatarkoy) nmole product min/mg protein. In 2008, MFOs levels ranged from 0.0004 ± 0.0002 (Seremkoy) to 0.0016 ± 0.0015 (Avariz) nmole product min/mg protein. The Avariz and Tatarkoy samples’ activity levels decreased from 2007 to 2008, while other strains’ activity levels increased. Although increasing and decreasing were observed in all the strains, statistically significant differences were found only in Seremkoy strain (p < 0.05) (Table 3). The GSTs levels ranged from 0.414 ± 0.14 (Su Akacagi) to 0.737 ± 0.18 (Derekoy) mole CDNB/min/mg protein in 2007. In 2008, the GSTs levels had increased and the highest increasing rate was found for the Su Akacagi strain (Table 3). All the strains in 2008 had GSTs levels which were significantly different from that of the 2007 samples except in the Derekoy strain. 4. Discussion 4.1. DDT and pyrethroid resistance Herein we reported that DDT resistance decreased and pyrethroid resistance increased to varying degrees. Many of the authors have indicated that DDT resistance is related to the kdr mechanism, elevated oxidases and DDT dehydrochlorinase activity of GSTs for different Anopheles, Aedes and Culex species (El-Sayed et al., 1989, Prapanthadara et al., 1995; Brengues et al., 2003; Lumjuan et al., 2005). High and increased NSE’s and GST’s activity
2007 ± ± ± ± ±
0.0015 0.0008 0.0006 0.0002 0.0012
0.586 0.693 0.737 0.447 0.414
2008 ± ± ± ± ±
0.31 0.23 0.18 0.17 0.14
0.795 0.774 0.766 0.736 0.736
± ± ± ± ±
0.17 0.27 0.32 0.28 0.25
may be related to DDT resistance, but are not fully explained by this situation. Hemingway et al. (1985) had indicated that DDT resistance is related to the DDT dehydrochlorinase activity in An. sacharovi. Luleyap and Kasap (2000) had reported high GSTs activity in An. sacharovi populations and indicated that DDT and pyrethroid resistance are related to high GST’s activity. Herein we identified the same situation and increasing GST’s and NSE’s levels for all the strains. This situation and high DDT resistance led to the cross resistance for pyrethroid groups. The decreasing of the DDT resistance from 2007 to 2008 is related to the fact that there is no usage of this insecticide, however, DDT resistance is still high. All the strains were placed in the surveillance category for permethrin and deltamethrin, but permethrin mortality rates were lower than those of deltamethrin. In addition, 2008 mortality rates for permethrin were close to the resistant category. Permethrin has been used in all areas over last five years except in Su Akacagi, but deltamethrin is used only in Seremkoy. There is no information about the residents’ personal usage. This type of usage and amount are not described exactly, but its availability to customers in all the markets indicate intensive use of permethrin and deltamethrin. Deltamethrin was used for pest control only in the Seremkoy area. Seremkoy mortality rate had increased from 2007 to 2008 against deltamethrin, though the increase was very little. In contrast to this situation, deltamethrin and alpha cypermethrin were used in all the tested areas for agricultural purposes for over 10 years. High GST’s levels and their increasing trend may be explained to permethrin and deltamethrin resistance and decreasing mortality levels in all the tested areas. In addition to this situation, Derekoy and Seremkoy MFOs’ levels increased from 2007 to 2008 and mortality rates decreased for permethrin and deltamethrin except Seremkoy’s 2008 samples. Increased MFOs; levels suggest that they possibly contributed to the pyrethroid resistance for these populations. MFOs activity change is reported to affect insecticide metabolization by many of the authors, but elevated NSE’s and GST’s levels have also been reported to confer pyrethroid resistance (Berge et al., 1998; Rodriguez et al., 2002; Vontas et al., 2001). High DDT resistance and pyrethroid resistance increasing trend for these strains may related to the kdr resistance and correlation for GST, MFO levels. Insecticide resistance conferred by the kdr gene and metabolic resistance due to amplification of the oxidative detoxifying enzymes (glutathione s transferases and oxidases) and co-factors have been widely reported in An. gambiae s.l. due to selection pressure as a result of extensive use of pyrethroid insecticides for malaria control and pest control in
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agriculture (Miller, 1988; Elissa et al., 1993; Awolola et al., 2007). Kdr mutation described for Iranian An. maculipennis and Turkish An. sacharovi strains after extensive use of DDT and pyrethroids. Iranian An. maculipennis’ samples showed high DDT resistance and moleculer studies revealed that this resistance is related to the kdr mutation (Djadid et al., 2012). Turkish An. sacharovi species have shown that high GST and low level of MFO levels, as well as kdr type mutation are also present and are similar to the An. gambiae mutation type (Lüleyap et al., 2002). Our study has revealed that the different biochemical mechanisms are responsible for the DDT and pythroid resistance, and there is a need for confirmation of molecular basis of resistance mechanisms and correlation between the molecular and biochemical mechanisms. DDT and Pyrethroid usage profiles in these areas have supported the kdr-like resistance mechanisms, which is as per results of other studies. Hemingway and Ranson (2000), Hemingway et al. (2004) had reported that esterases play an important role in organophosphates, carbamate and pyrethroid resistance and besides this, GSTs can provide resistance to these insecticides. Our data are also consistent with those previously reported for areas where insecticides are used agriculturally in Turkey (Kasap et al., 2000; Luleyap and Kasap, 2000). Etang et al. (2007) had reported the same situation of elevated MFO and NSE activity for Pitao An. gambiae samples where pyrethroid, organophosphate, and carbamate insecticides were widely used for cotton. Diabate et al. (2002) had reported the same situation and indicated that agricultural and urban areas insecticide usage played an important role in pyrethroids resistance in An. gambiae s.l. 4.2. Malathion resistance Our results indicated that all of the 2007 strains were placed in the surveillance category, but mortality rates decreased from 2007 to 2008. 2008 bioassay results had shown that three strains were placed in the border resistance category (Tatarkoy, Derekoy, Su Akacagi) and one strain was placed in the resistance category (Seremkoy). One of the major biochemical mechanisms responsible for the development of malathion resistance in mosquitoes is the enhanced activity of the carboxylesterase (Raymond et al., 1987; Bisset et al., 1990). We have used three different substrates for determination of NSEs activity. In mosquitoes, esterases related to the resistance to insecticides can be activated with ␣ and  naphtyl acetate substrates. We used substrates as well as paranitrophenyl acetate because some of the esterases such as malathion carboxylesterases act preferentially on this substrate (Etang et al., 2007). The p-NPA assays had indicated that esterase activity increased from 2007 to 2008 and determined a nearly two to threefold increase for all the tested strains. These results correlated with the malathion bioassays. According to the results, malathion resistance may be explained with an increased amount of malathion carboxylesterase, but a detailed study is needed to determine the genetic basis of this situation. Malathion has not been used directly for mosquito or household pest control in all of the tested areas over the last 5 years (Table 1). Therefore, malathion resistance is not fully explained by the insecticide usage profile, i.e. direct use for mosquito control. However, this insecticide and other organophosphates had been used for agricultural purposes, and malathion resistance can be related to agricultural insecticide application. Kasap et al. (2000) had reported the same situation and indicated that the selection of insecticide resistance in An. sacharovi populations from Adana was a consequence of agricultural pesticide usage. 5. Conclusion Understanding resistance and mechanisms can be a guide to control mosquito populations. All the collection points were
located at the border of Turkey-Greece and Turkey-Bulgaria in this study. The main occupation of the residents in these areas is rice farming and residents extremely suffer from the mosquito problem. Therefore, these residents use agricultural insecticides and commercial hand sprays for mosquito control. These regions are also considered as a zone of intense human trafficking areas. Therefore, these areas are likely to experience epidemics and monitoring efforts need to be established in these areas. Insecticide resistance and cross resistance status are possible obstacles for mosquito control and possible epidemics that relate to the human trafficing. More detailed studies are needed to establish new and effective insecticide usage profiles to avoid insecticide resistance and to arrest possible epidemics. Acknowledgements This work is supported by Tubitak (TBAG Grant number 105T531) colloborative project with GSRT. We are thankful to Elcin Eksi and Aysun Demet Gulsoy and Jennifer Kalvenas (Department of Biological Sciences, The Middle East Technical University (METU)) for reviewing the work in English. References Akiner, M.M., Simsek, F.M., Caglar, S.S., 2009. Insecticide resistance of Culex pipiens (Diptera: Culicidae) in Turkey. J. Pest. Sci. 34 (4), 259–264. Awolola, T.S., Oduola, A.O., Oyewole, I.O., Obansa, J.B., Amajoh, C.N., Koekemoerd, L.L., Coetzeed, M., 2007. Dynamics of knockdown pyrethroid insecticide resistance alleles in a field population of Anopheles gambiae s.s. in southwestern Nigeria. J. Vect. Borne Dis. 44, 181–188. Becker, N., Petric, D., Zgomba, M., Boase, C., Dahl, C., Lane, J., Kaiser, A., 2003. Mosquitoes and Their Control. Kluwer Academic, Plenum publishers, USA498. Berge, J.B., Feyereisen, R., Amichot, M., 1998. Cytochrome P450 monooxygenases and insecticide resistance in insects. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 353, 1701–1705. Bisset, J.A., Rodríguez, M.M., Díaz, C., Ortiz, E., Marquetti, M.C., 1990. The mechanims of organophosphate and carbamate resistance in Culex quinquefasciatus (Diptera: Culicidae) from Cuba. Bull. Entomol. Res. 80, 160–168. Bonning, B.C., Hemingway, J., Romi, R., Majori, G., 1991. Interaction of insecticide resistance genes in field populations of Culex pipiens (Diptera:Culicidae) from Italy in response to changing insecticide selection pressure. Bull. Entomol. Res. 81, 5–10. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7 (72), 248–254. Brengues, C., Hawkes, N.J., Chandre, F., McCarroll, L., Duchon, S., Guillet, P., Manguin, S., Morgan, J.C., Hemingway, J., 2003. Pyrethroid and DDT cross resistance in Aedes aegypti is correlated with novel mutations in the voltage gated sodium channel gene. Med. Vet. Entomol. 17, 87–94. Brogdon, W.G., McAllister, J.C., Vulule, J., 1998. Heme peroxydase activity measured in single mosquitoes identifies individuals expressing the elevated oxidase mechanism for insecticide resistance. J. Am. Mosq. Contr. Assoc. 13, 233–237. Bruce-Chwatt, L., de Zulueta, J., 1980. The Rise and Fall of Malaria in Europe. A Historico-Epidemiological Study. University Press, Oxford240. Chandre, F., Frederic, D., Sylvie, M., Cecile, B., Carnavale, P., Guillet, P., 1999. Pyrethroid cross resistance spectrum among populations of Anopheles gambiae s.s. from Cote d’Ivore. J. Am. Mosq. Control Assoc. 15, 53–59. Chavillon, C., Bernard, C., Marquine, M., Pasteur, N., 2001. Resistance to Bacillus sphaericus in Cx. pipiens (Diptera: Culicidae): interaction between recessive mutant and evolution in southern France. J. Med. Entomol. 38, 657–664. Diabate, A., Baldet, T., Chandre, F., Akogbeto, M., Guiduemde, T.R., Darriet, F., Brengues, C., Guillet, P., Hemingway, J., Small, G.J., Hougard, J.M., 2002. The role of agriculture use of insecticides in resistance to pyrethroids in An. gambiae s.l. in Burkina Faso. Am. J. Trop. Med. Hyg. 67, 617–622. Djadid, N.D., Forouzesh, F., Zakeri, S., 2012. Identification of knock-down (kdr) mutation in VGSC gene, related to pyrethroids resistance in An. sacharovi and An. maculipennis, Archive of SID, www. SID. ir Elissa, N., Mouchet, J., Riviere, F., Meunier, J.Y., Yao, K., 1993. Resistance of Anopheles gambiae s.s. to pyrethroids in Cote D’ Ivoire. Ann. Soc. Belg. Med. Trop. 73, 291–294. El-Sayed, S., Hemingway, J., Lane, R.P., 1989. Susceptibility baselines for DDT metabolism and related enzyme systems in the sandfly Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Bull. Entomol. Res. 79, 679–684. Etang, J., Manga, L., Toto, J.C., et al., 2007. Spectrum of metabolic based resistance to DDT and pyrethroids in Anopheles gambiae s.l. populations from Cameroon. J. Vector Ecol. 32, 123–133. Hemingway, J., Hawkes, N.J., McCarroll, L., Ranson, H., 2004. The molecular basis of insecticide resistance in mosquitoes. Insect Biochem. Mol. Biol. 34, 653–665.
M.M. Akiner et al. / Acta Tropica 126 (2013) 280–285 Hemingway, J., Malcolm, C.A., Kissoon, K.E., Boddington, R.G., Curtis, C.F., Hill, N., 1985. The biochemistry of insecticide resistance in Anopheles sacharovi: comparative study with a range of insecticide susceptible and resistant Anopheles and Culex species. Pestic. Biochem. Physiol. 24, 68–76. Hemingway, J., Ranson, H., 2000. Insecticide resistance in insect vector of human disease. Annu. Rev. Entomol. 45, 375–391. Jetten, T.H., Takken, W., 1994. Anophelism without malaria in Europe – a review of the ecology and distribution of the genus Anopheles in Europe, Wageningen Agricultural University Papers, pp. 94–95. Kasap, H., Luleyap, U., Alptekin, D., Kasap, M., 1999. Use of insecticides in Cukurova and development of resistance in mosquitoes. Act. Par. Turcica 23 (3), 267–272. Kasap, H., Kasap, M., Alptekin, D., Luleyap, U., Herath, P.R.C., 2000. Insecticide resistance in Anopheles sacharovi Favre in southern Turkey. Bull. WHO 78 (5), 687–692. Linton, Y.M., Smith, L., Harbach, R.E., 2002. Molecular confirmation of sympatric populations of Anopheles messeae and Anopheles atroparvus overwintering in Kent, southeast England. Eur. Mosq. Bull. 13, 8–16. Luleyap, U., Kasap, H., 2000. Insecticide resistance in malaria vector An. sacharovi. Turk. J. Biol. 24, 437–460. Lüleyap, H.U., Alptekin, D., Kasap, H., Kasap, M., 2002. Detection of knockdown resistance mutations in Anopheles sacharovi (Diptera: Culicidae) and genetic distance with Anopheles gambiae (Diptera: Culicidae) using cDNA sequencing of the voltage-gated sodium channel gene. J. Med. Entomol. 39, 870–874. Lumjuan, N., McCarrroll, L., Prapandhatara, L.A., Hemingway, J., Ranson, H., 2005. Elevated activity of an epsilon class GST confers DDT resistance in the dengue vector, Aedes aegypti. Insect Biochem. Mol. Biol. 35, 861–871. Miller, T.A., 1988. Mechanisms of resistance to pyrethroid insecticides. Parasitol. Today 4, S8–S13. Ramsdale, C.D., Herath, P.R., Davidson, G., 1980. Recent developments of insecticide resistance in some Turkish Anophelines. J. Trop. Med. Hyg. 83 (1), 11–19.
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Yearly changes of insecticide susceptiblity and possible insecticide resistance mechanisms of Anopheles maculipennis Meigen (Diptera: Culicidae) in Turkey M.M. Akiner a,∗ , S.S. Caglar b , F.M. Simsek c a
Department of Biology, Faculty of Science and Arts, Recep Tayyip Erdogan University, Fener, Rize, Turkey Department of Biology, Faculty of Science, Hacettepe University, 06800 Beytepe, Ankara, Turkey c Department of Biology, Faculty of Science and Arts, Adnan Menderes University, 09010 Aydın, Turkey b
a r t i c l e
i n f o
Article history: Received 6 September 2012 Received in revised form 17 February 2013 Accepted 24 February 2013 Available online 15 March 2013 Keywords: Anopheles maculipennis Insecticide resistance DDT Malathion Permethrin Deltamethrin
a b s t r a c t To evaluate the adulticide susceptibility and yearly changes of Anopheles maculipennis Meigen (Diptera: Culicidae) in Thrace, five mosquito populations were evaluated against the resistance status of four different adulticides. Three biochemical resistance mechanisms and yearly changes of activities were investigated. All the strains were highly resistant to DDT, and all the strains were placed in the resistance surveillance category for malathion, permethrin and deltamethrin in 2007. Although DDT mortality rates had increased from 2007 to 2008 except in the Seremkoy strain, malathion, permethrin and deltamethrin mortality rates have decreased in all of the tested strains. High rates of increase were determined for nonspecific esterases (NSEs) activity by using the substrate p-NPA and these results showed correlation with malathion mortality rates. All the strains showed high level of glutathione S transferases (GSTs), and their activity level had significantly increased from 2007 to 2008. Different insecticide susceptibility statuses were observed between localities, and high DDT resistance was observed although DDT was banned in the 1980s. Biochemical assay results suggest that NSEs and GSTs could play a role insecticide resistance in all tested strains. Malathion susceptibility has decreased in all the tested strains and NSE’s activity is possibly the main enzymatic mechanism related to the insecticide resistance. DDT resistance is at a high degree in all the strains and GST’s activity is probably related to this situation. GST’s activity could play an important role for permethrin and deltamethrin susceptibility but needs to be confirmed for molecular studies. Our results provide important data on insecticide susceptibility and change over time for the Anopheles maculipennis populations in Turkey. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Malaria remains a serious problem in the Middle East. Anopheles maculipennis complex is the vector of malaria in Europe and Middle East historically (Becker et al., 2003). This complex comprises 12 palearctic members and three species of this complex, and An. atroparvus, An. labranchiae, and An. sacharovi, are known to be efficient current and historical vectors of malaria in the palearctic region (Bruce-Chwatt and de Zulueta, 1980; Jetten and Takken, 1994; Linton et al., 2002; Sedaghat et al., 2003a,b). An. sacharovi is the main vector in Turkey and has widespread
∗ Corresponding author at: Recep Tayyip Erdogan University, Faculty of Science and Arts, Department of Biology, Zoology Section, 53100 Fener, Rize, Turkey. Tel.: +90 464 2236126x1835; fax: +90 464 2234019. E-mail address: [email protected] (M.M. Akiner). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.02.020
distribution in many parts of our country, but is generally found in mixed populations with An. maculipennis and An. melanoon in Thrace. Although development of resistance to chemical insecticides has been reported by many of the authors, chemical insecticides are still being heavily used in control operations in many areas of the World (Bonning et al., 1991; Chandre et al., 1999; Chavillon et al., 2001). Ramsdale et al. (1980) had described carbamate and organophosphate resistance in An. sacharovi populations from Cukurova plain, Osmancık and Blacksea region in Turkey. Kasap et al. (1999) had described carbamate, organophosphate (OP), and pyrethroid (PY) resistance in An. sacharovi, Culex tritaeniorhyncus, and Aedes caspius. Kasap et al. (2000) had identified OP, PY, carbamate, and organochlorine resistance in five different An. sacharovi populations of southern Turkey. Akiner et al. (2009) had described OP and PY resistance in Culex pipiens larvae and they have also found organochlorine, OP, and PY resistance in adult samples. However,
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Fig. 1. Location and GPS coordinates of the collected strains.
all of the aforementioned studies were conducted by using classical bioassays and can only provide clues as to molecular and biochemical mechanisms. Furthermore, there are few studies which focus on biochemical mechanisms of insecticide resistance in Turkish Anopheles species. Hemingway et al. (1985) had indicated that DDT resistance may be related to the qualitative or quantitative changes of DDT dehydrochlorinase, but did not find any changes in nonspecific esterases, multifunction oxidases and glutathion S transferases (GST) activity levels in An. sacharovi populations from the Cukurova plain. Luleyap and Kasap (2000) had reported that AChE, GSTs, and non-specific esterases activity levels were higher in the resistant group than in the susceptible groups. Studies about insecticide resistance of Anopheles and Culex populations in Turkey is not up-to-date and also these previous studies are narrow scoped and they did not cover An. maculipennis that is also the most abundant species in many parts of our country. Thus, new studies are necessary for finding out current resistance status of Anopheles and Culex populations. The objective of this study was to evaluate resistance, yearly changes and enzyme activity levels responsible for the insecticide resistance in the An. maculipennis populations from the Thracian part of Turkey.
2. Materials and methods 2.1. Strains of An. maculipennis The study sites’ locations, GPS coordinates, and a brief description of the localities are provided in Fig. 1. Larvae and adult samples of An. maculipennis were collected from five different localities in the Thrace region of Turkey. We especially chose rice field areas near the border between Turkey, Greece, and Bulgaria (Avariz, Su Akacagi, Tatarkoy, Seremkoy), an area where human trafficking occurs. The chosing criteria for the last collection site is population density and different agricultural usage types (corn and bean). All the adult samples were transferred to a −80 ◦ C deep freezer until the date of biochemical analysis. Insecticide usage over the previous 5 years at the sampling sites is listed in Table 1 and Fig. 1. 2.2. Bioassays The adult mosquitoes were assayed for susceptibility by diagnostic tests (WHO, 1981). Susceptibility was determined against DDT, permethrin, deltamethrin, and malathion. One diagnostic
Table 1 Anopheles maculipennis strains used to survey insecticide resistance and the use of insecticides in the last 5 years for control and use of insecticides agriculturally for over 10 years. Insecticides Last 5 years
Over 10 years
Adulticides
Agricultural
Deltamethrin Cypermethrin Permethrin D-phenothrin Cyfluthrin Pyrethrum Thiamethoxam Endosulfan Carbaryl Malathion Chlorpyriphos ethyl Deltamethrin Alpha cypermethrin
Avariz
Tatarkoy
Derekoy
X X
X X
X X X
X
Seremkoy
Su Akacagi
X X X X X X X X X
X X X X X
X X X
X X X
X
X
X X X X
X X X
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Table 2 Percentage mortality of Anopheles maculipennis adults from five different areas exposed to diagnostic doses of DDT, malathion, permethrin, and deltamethrin in 2007 and 2008. Insecticides
DDT 4%
Permethrin 0.75%
Deltamethrin 0.05%
Strain/Year
2007
2008
2007
2008
2007
2008
2007
2008
19 47.5 22 55 20 50 26 65 24 60
20 50 25 62.5 21 52.5 24 60 27 67.5
38 95 36 90 37 92.5 36 90 36 90
35 87.5 33 82.5 33 82.5 31 77.5 32 80
37 92.5 34 85 36 90 38 95 37 92.5
34 85 33 82.5 34 85 35 87.5 35 87.5
38 95 37 92.5 37 92.5 36 90 38 95
37 92.5 36 90 34 85 37 92.5 35 87.5
Avarız Tatarkoy Derekoy Seremkoy Su Akacagi
No. Dead Mortality (%) No. Dead Mortality (%) No. Dead Mortality (%) No. Dead Mortality (%) No. Dead Mortality (%)
Malathion 5%
Note: Experiments were performed using 2 replicates and each replicate contained 20 female mosquitoes.
concentration was used for all insecticides and a 1-h application period was used for permethrin, deltamethrin, and malathion, while a 2-h application period was used for DDT. Mortality was recorded after the 24 h resting period. Insecticide resistance status was evaluated by using the classification determined by the WHO (WHO, 2006), in which 98–100% mortality indicates susceptibility, 80–97% mortality suggests possible resistance requiring confirmation, and <80% mortality suggests resistance. 2.3. Biochemical analysis Non-specific esterases (NSEs), mixed function oxidases (MFOs), and glutathione S-transferases (GSTs) levels were measured individually in the adult females as described by the WHO (WHO, 1998) for possible biochemical insecticide resistance mechanisms. Standard flat-bottom microtiter plates (Nunch maxisorp® , Nunch A/S, Roskilde, Denmark) were used for individual testing and absorbance was read spectrophotometrically with an ELISA reader (Power Wave® XS, Biotek Instruments USA). Biochemical analyses for individual mosquitoes were performed with two replicates of the homogenate. A total of 553 (253 in 2007, 300 in 2008) adult females from five localities (Avariz (55, 60), Tatarkoy (54, 60), Derekoy (24, 60), Seremkoy (60, 60), Su Akacagi (60, 60)) were analyzed. Each An. maculipennis specimen was homogenized on ice in 200 l of 50 mM sodium phosphate buffer, pH 7.2, and the homogenate was centrifuged at 10,000 × g for 10 min at +4 ◦ C. The supernatant was used as the source of enzymes. Two 20 l replicates of the supernatant were transferred to the microtiter plate for alpha and beta naphthyl acetate (␣,  NA) and MFOs assays. GSTs, p-nitrophenyl acetate (p-NPA), and protein assays were conducted with two 10 l replicates of supernatant. NSEs activity was measured by using three substrates: ␣-naphthyl acetate, -naphthyl acetate, and p-nitrophenyl acetate (p-NPA). For the ␣NA and NA assay, 200 l of working solution was added (0.02 M phosphate buffer including 30 mM ␣ NA and  NA) for each, separately. After 30 min of incubation, 50 l of Fast Blue B Salt solution was added to stop the reaction. The final concentration of the final volume was measured at 570 nm endpoint for ␣- and -naphthyl acetate. For the p-NPA assay, 200 l of p-NPA working solution (20 ml 0.05 M phosphate buffer including 100 l 0.02 M p-NPA) was added to two replicates. The plate was read at 405 nm kinetically (WHO, 1998). MFOs activities were measured via the amount of heme (Brogdon et al., 1998). 200 l working solution (0.01 g 3,3 ,5,5 tetramethyl benzidine (TMBZ) + 5 ml methanol + 15 ml sodium acetate buffer) was added to each replicate. 25 l of 3% hydrogen peroxide was added and the mixture was incubated for 5 min at room temperature. Absorbance was read at 620 nm.
GSTs assays were measured by using a 200 l GSH/CDNB working solution (100 l of 0.060 g reduced glutathione in 20 ml 0.1 M Na phosphate buffer +0.013 g CDNB in 1 ml ethanol) added to each replicate well. The absorbance was read at 340 nm kinetically (WHO, 1998). Total protein amount in 10 l of supernatant was measured by using the Bradford assay in order to calculate enzyme activity for each sample (Bradford, 1976). Then 300 l of Bradford dye reagent was added to each replicate and the endpoint absorbance was read at 595 nm. Protein values were calculated by using a standard curve of absorbance of bovine serum albumin. 2.4. Statistical analysis Mean enzyme activity values were compared between populations and years by the Kruskal–Wallis test by means of Statistica 8® . 3. Results 3.1. Bioassay The results of the diagnostic bioassays carried out with DDT, malathion, permethrin and deltamethrin are shown in Table 2. As a result of the diagnostic tests, all the populations were resistant to DDT (mortality rates under 80%) and showed low mortality with this insecticide. Mortality rates decreased in all of the strains except in the Seremkoy strain (Table 2). All the strains were placed in the surveillance category (80–97%) for malathion in 2007. Mortality rates had decreased from 2007 to 2008 and went below 90%, approaching the resistance category. The mortality rate of the Seremkoy population was placed in the resistance category (under 80%) in 2008. All the populations were determined to be in the surveillance category (80–97%) except for the Su akacagi population in 2007 for permethrin. The highest change of mortality rates was also determined in the same strain (15%) from 2007 to 2008. Deltamethrin mortality rates were placed from 85 to %100 and decreased from 2007 to 2008 and were determined to be in the surveillance category (Table 2). 3.2. Biochemical analysis A total of 553 (253 in 2007, 300 in 2008) adult females from five localities (Avariz, Tatarkoy, Derekoy, Seremkoy, and Su Akacagi) were analyzed with the biochemical test for three different possible resistance mechanisms: elevated NSEs activity, elevated MFOs, and elevated GSTs activity (Table 3). NSEs activities of samples were detected from all of the field sites from 2007 to 2008 with artificial ester substrates; ␣, , NA
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Table 3 Non-specific esterases (NSEs), mixed function oxidases (MFOs), and glutathione S transferases (GSTs) activity in Anopheles maculipennis tested strains (mean ± S.D.) in 2007 and 2008. p-NPA
␣ Esterases
 Esterases
mole pNPA/min/mg protein
nmole ␣ naphthol/min/mg protein
nmole  naphthol/min/mg protein
Strains
2007
Avariz Tatarkoy Derekoy Seremkoy Su Akacagi
0.230 0.261 0.456 0.652 0.745
2008 ± ± ± ± ±
0.245 0.207 0.235 0.472 0.377
0.781 0.774 0.658 1.254 1.235
2007 ± ± ± ± ±
0.528 0.483 0.505 0.878 0.636
0.00024 0.00025 0.00028 0.00023 0.00013
2008 ± ± ± ± ±
0.00008 0.00008 0.00004 0.00005 0.00002
0.00025 0.00026 0.00035 0.00030 0.00023
MFOs
2007
Avariz Tatarkoy Derekoy Seremkoy Su Akacagi
0.0027 0.0037 0.00095 0.00023 0.0008
0.00004 0.00007 0.0001 0.00006 0.00004
2008
0.00025 0.00026 0.00030 0.00022 0.00012
± ± ± ± ±
0.0001 0.00008 0.00004 0.00005 0.00002
0.00025 0.00026 0.00033 0.00029 0.00022
± ± ± ± ±
0.00004 0.00008 0.00009 0.00007 0.00004
GSTs mole CDNB/min/mg protein
nmole product/min/mg protein Strains
2007 ± ± ± ± ±
2008 ± ± ± ± ±
0.0021 0.0034 0.0004 0.0001 0.0007
0.0016 0.0014 0.00108 0.00040 0.0014
and p-NPA. With p-NPA, the enzyme activity fluctuated in all the tested populations and increased from 2007 to 2008. The highest activity was found in the Su Akacagi strain and the lowest activity was found in the Avariz strain for 2007 samples. The highest activity was observed in the Seremkoy strain and the lowest activity was observed in the Derekoy strain for the 2008 samples (Table 3). Although increased activity was detected from 2007 to 2008, there were no significant differences between 2007 and 2008 (p > 0.05). ␣ and  esterase activities had fluctuated between all of the strains but nearly the same range was observed for Avariz, Tatarkoy and Seremkoy strains in 2007 and 2008. ␣ and  esterases activities increased from 2007 to 2008. ␣ esterase activity showed significant differences (p < 0.05) between 2007 to 2008, while  esterase did not show the same trend in the Derekoy strain. The Su Akacagi strain had showed low level activity for ␣ and  esterase in 2007, and activity levels increased nearly two-fold in 2008 and were significantly different from 2007 levels (Table 3). The MFOs levels in the 2007 samples ranged from 0.00023 ± 0.0001 (Seremkoy) to 0.0037 ± 0.0034 (Tatarkoy) nmole product min/mg protein. In 2008, MFOs levels ranged from 0.0004 ± 0.0002 (Seremkoy) to 0.0016 ± 0.0015 (Avariz) nmole product min/mg protein. The Avariz and Tatarkoy samples’ activity levels decreased from 2007 to 2008, while other strains’ activity levels increased. Although increasing and decreasing were observed in all the strains, statistically significant differences were found only in Seremkoy strain (p < 0.05) (Table 3). The GSTs levels ranged from 0.414 ± 0.14 (Su Akacagi) to 0.737 ± 0.18 (Derekoy) mole CDNB/min/mg protein in 2007. In 2008, the GSTs levels had increased and the highest increasing rate was found for the Su Akacagi strain (Table 3). All the strains in 2008 had GSTs levels which were significantly different from that of the 2007 samples except in the Derekoy strain. 4. Discussion 4.1. DDT and pyrethroid resistance Herein we reported that DDT resistance decreased and pyrethroid resistance increased to varying degrees. Many of the authors have indicated that DDT resistance is related to the kdr mechanism, elevated oxidases and DDT dehydrochlorinase activity of GSTs for different Anopheles, Aedes and Culex species (El-Sayed et al., 1989, Prapanthadara et al., 1995; Brengues et al., 2003; Lumjuan et al., 2005). High and increased NSE’s and GST’s activity
2007 ± ± ± ± ±
0.0015 0.0008 0.0006 0.0002 0.0012
0.586 0.693 0.737 0.447 0.414
2008 ± ± ± ± ±
0.31 0.23 0.18 0.17 0.14
0.795 0.774 0.766 0.736 0.736
± ± ± ± ±
0.17 0.27 0.32 0.28 0.25
may be related to DDT resistance, but are not fully explained by this situation. Hemingway et al. (1985) had indicated that DDT resistance is related to the DDT dehydrochlorinase activity in An. sacharovi. Luleyap and Kasap (2000) had reported high GSTs activity in An. sacharovi populations and indicated that DDT and pyrethroid resistance are related to high GST’s activity. Herein we identified the same situation and increasing GST’s and NSE’s levels for all the strains. This situation and high DDT resistance led to the cross resistance for pyrethroid groups. The decreasing of the DDT resistance from 2007 to 2008 is related to the fact that there is no usage of this insecticide, however, DDT resistance is still high. All the strains were placed in the surveillance category for permethrin and deltamethrin, but permethrin mortality rates were lower than those of deltamethrin. In addition, 2008 mortality rates for permethrin were close to the resistant category. Permethrin has been used in all areas over last five years except in Su Akacagi, but deltamethrin is used only in Seremkoy. There is no information about the residents’ personal usage. This type of usage and amount are not described exactly, but its availability to customers in all the markets indicate intensive use of permethrin and deltamethrin. Deltamethrin was used for pest control only in the Seremkoy area. Seremkoy mortality rate had increased from 2007 to 2008 against deltamethrin, though the increase was very little. In contrast to this situation, deltamethrin and alpha cypermethrin were used in all the tested areas for agricultural purposes for over 10 years. High GST’s levels and their increasing trend may be explained to permethrin and deltamethrin resistance and decreasing mortality levels in all the tested areas. In addition to this situation, Derekoy and Seremkoy MFOs’ levels increased from 2007 to 2008 and mortality rates decreased for permethrin and deltamethrin except Seremkoy’s 2008 samples. Increased MFOs; levels suggest that they possibly contributed to the pyrethroid resistance for these populations. MFOs activity change is reported to affect insecticide metabolization by many of the authors, but elevated NSE’s and GST’s levels have also been reported to confer pyrethroid resistance (Berge et al., 1998; Rodriguez et al., 2002; Vontas et al., 2001). High DDT resistance and pyrethroid resistance increasing trend for these strains may related to the kdr resistance and correlation for GST, MFO levels. Insecticide resistance conferred by the kdr gene and metabolic resistance due to amplification of the oxidative detoxifying enzymes (glutathione s transferases and oxidases) and co-factors have been widely reported in An. gambiae s.l. due to selection pressure as a result of extensive use of pyrethroid insecticides for malaria control and pest control in
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agriculture (Miller, 1988; Elissa et al., 1993; Awolola et al., 2007). Kdr mutation described for Iranian An. maculipennis and Turkish An. sacharovi strains after extensive use of DDT and pyrethroids. Iranian An. maculipennis’ samples showed high DDT resistance and moleculer studies revealed that this resistance is related to the kdr mutation (Djadid et al., 2012). Turkish An. sacharovi species have shown that high GST and low level of MFO levels, as well as kdr type mutation are also present and are similar to the An. gambiae mutation type (Lüleyap et al., 2002). Our study has revealed that the different biochemical mechanisms are responsible for the DDT and pythroid resistance, and there is a need for confirmation of molecular basis of resistance mechanisms and correlation between the molecular and biochemical mechanisms. DDT and Pyrethroid usage profiles in these areas have supported the kdr-like resistance mechanisms, which is as per results of other studies. Hemingway and Ranson (2000), Hemingway et al. (2004) had reported that esterases play an important role in organophosphates, carbamate and pyrethroid resistance and besides this, GSTs can provide resistance to these insecticides. Our data are also consistent with those previously reported for areas where insecticides are used agriculturally in Turkey (Kasap et al., 2000; Luleyap and Kasap, 2000). Etang et al. (2007) had reported the same situation of elevated MFO and NSE activity for Pitao An. gambiae samples where pyrethroid, organophosphate, and carbamate insecticides were widely used for cotton. Diabate et al. (2002) had reported the same situation and indicated that agricultural and urban areas insecticide usage played an important role in pyrethroids resistance in An. gambiae s.l. 4.2. Malathion resistance Our results indicated that all of the 2007 strains were placed in the surveillance category, but mortality rates decreased from 2007 to 2008. 2008 bioassay results had shown that three strains were placed in the border resistance category (Tatarkoy, Derekoy, Su Akacagi) and one strain was placed in the resistance category (Seremkoy). One of the major biochemical mechanisms responsible for the development of malathion resistance in mosquitoes is the enhanced activity of the carboxylesterase (Raymond et al., 1987; Bisset et al., 1990). We have used three different substrates for determination of NSEs activity. In mosquitoes, esterases related to the resistance to insecticides can be activated with ␣ and  naphtyl acetate substrates. We used substrates as well as paranitrophenyl acetate because some of the esterases such as malathion carboxylesterases act preferentially on this substrate (Etang et al., 2007). The p-NPA assays had indicated that esterase activity increased from 2007 to 2008 and determined a nearly two to threefold increase for all the tested strains. These results correlated with the malathion bioassays. According to the results, malathion resistance may be explained with an increased amount of malathion carboxylesterase, but a detailed study is needed to determine the genetic basis of this situation. Malathion has not been used directly for mosquito or household pest control in all of the tested areas over the last 5 years (Table 1). Therefore, malathion resistance is not fully explained by the insecticide usage profile, i.e. direct use for mosquito control. However, this insecticide and other organophosphates had been used for agricultural purposes, and malathion resistance can be related to agricultural insecticide application. Kasap et al. (2000) had reported the same situation and indicated that the selection of insecticide resistance in An. sacharovi populations from Adana was a consequence of agricultural pesticide usage. 5. Conclusion Understanding resistance and mechanisms can be a guide to control mosquito populations. All the collection points were
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