Overexpression of cytochrome P450 CYP6CM1 gene in Bemisia tabaci (Gennadius) in the cotton fields of the Çukurova Region, Turkey

In Cukurova region, neonicotinoid group insecticides are widely used in cotton cultivation areas for controlling B. tabaci; as a result of this use, there is a problem of resistance in these areas. In this study, cytochrome P450 CYP6CM1 relative expression levels, which are the indicators of imidacloprid resistance, were analyzed. Five different B. tabaci populations were collected from Adana city cotton cultivation areas in 2018 and the indicators were observed. On the other hand, GST gene relative expression profile, which is an efficient factor in metabolic resistance, cytochrome P450, carboxylesterase (AcHE) and Glutathion S transferase (GST) enzyme activities were determined. The highest cyp6cm1 and GST genes relative activities were determined in Meletmez population; the activity level was 18, 2 and 9 times higher than susceptible population. On the other hand, the highest and lowest cytochrome P450 enzyme activity was observed in Meletmez and Solaklı populations, respectively. According to the analyses, it can be concluded that there is extensive imidacloprid and neonicotinoid resistance of B. tabaci populations in Çukurova Region cotton cultivation areas. As a result, excessive gene expression and high-level enzyme activities indicate that there is a resistance in the studied area based on enforced monooxygenases activity. Research Article Article History Received : 16.08.2019 Accepted : 21.01.2020


INTRODUCTION
In Turkey, cotton is a highly important plant as it is a resource of economic contribution; the plant is used as a raw material in various textile and industrial products (Şekeroğlu et al., 1998). Approximately a total of 520.000 hectare cotton area was cultivated in 2018 in Turkey. 100.000 hectare cotton area was cultivated in Adana, Mersin and Hatay regions, which shows that the region is a very important cotton resource (Anonymus, 2019). White fly Bemisia tabaci is a polyphagous pest that is highly harmful especially for vegetable and cotton fields; the insect was reported for the first time in 1928 (Kaygısız, 1976;Ulusoy et al., 1996). Bemisia tabaci especially sucks on the leaves of cotton plants and causes honey-like formations; besides, it causes secondary infections such as fumaginate, which are formed by saprophytic fungus. On the other hand, they are carriers of more than 100 seriously harmful viruses (Moya et al., 2001;Hsieh et al., 2006). All these cause important yield and quality loss in cotton cultivation. The most effective and common Bemisia tabaci biotypes are Bemisia tabaci B and Q (De Barro et al., 2011).
The use of insecticides has become more common (neonicotinoids) as they are easy to use and economic for controlling Bemisia tabaci. Besides, different levels of resistances to insecticides (organophosphate, carbamate, pyrethroid and neonicotinoids) are reported in various countries (Ahmad et al., 2003;Wang et al., 2007;Sparks et al., 2015).Neonicotinoids are efficiently used in controlling coleoptera, diptera and lepidoptera families, especially aphid and white fly; on the other hand, they have high market share. Because of the common and unconscious use, different levels of resistances are reported in the Mediterranean Region and in different parts of the world ( (Nauen et al., 2002Jeschke et al., 2011, Ulusoy et. al., 2018, Satar et al., 2018. It is reported that in Adana cotton cultivation areas, there is 100 times higher resistance to imidacloprid in B. tabaci at a specific location when compared to the susceptible population (Satar et al., 2018). It is reported that there are 200 times higher imidacloprid and thiametoxam resistance in Aphis gossypii in different cotton cultivation areas of the same region (Ulusoy et. al., 2018).Resistance mechanism results from the decrease in sensitivity level of organisms against xenobiotics because of the reinforced metabolic enzyme activities and mutations in target proteins (Nauen, 2007). Various enzymes such as Esterase, Glutathione S Transferase (GSTs) and Cytochrome P450, which are included in multi-gene family, are efficient in the xenobiotic detoxification process of living organisms. This activity is closely related with the transcription level of genes at cellular extent (Field et al., 1999;Bass & Field, 2011). In the previous studies, it is reported that there is an increase in P450 (Cyp6cm1) and glutathione S-transferase (GST) enzyme genes relative activities in B. tabaci populations that are resistant to neonicotinoid and organic phosphate insecticides (Puinean et al., 2010;Qiong et al., 2012;Liang et al., 2014;Yang et al., 2014). P450 (encoded as CYP genes) enzyme group is a super family with multi-gene structure; they actively take part in a wide range of processes such as metabolization of internal and external components, growth, development, nutrition and detoxification of xenobiotics (Scott et al., 1999;Feyereisen, 2005).Reinforced p450 activities in imidacloprid resistant B. tabaci individuals are observed through different methods. Firstly, it is proven those imidacloprid individuals' resistance decreases when they are exposed to p450 inhibitors (Nauen et al., 2002). Secondly, it is determined that there is a close correlation between resistance and monoxigenase activity; this is proven with 7ethoxycoumarin-O-deethylase enzyme reactions (Rauch & Nauen, 2003). Finally, it is presented through in-vivo studies, 14 C tests that there are 5hdroxy-imidacloprid end-products as a result of oxidative degradation in imidacloprid resistant species; it is observed that p450 efficiency is important in resistance (Rauch & Nauen, 2003).
In their study, Karunker et al., 2008 determined that in DNA based resistance scanning methods, CYP6CM1 gene regulation level is one of the main indicators of imidacloprid resistant B. tabaci individuals. On the other hand, during imidacloprid resistant B. tabaci individuals RNA interference studies, it is observed that as a result of the P450 CYP6CM1 gene regulation decrease or suspension processes, B.tabaci B and Q biotypes detoxification processes declines and death rate increases (Li et al, 2014). In addition to these, it is proven that E. coli bacteria recombinant enzymes with B. tabaci CYP6CM1 gene clones quickly detoxify imidacloprid molecules (Karunker et al., 2009;Roditakis et al., 2011). Çukurova is one of the most important areas in Mediterranean Region, Turkey as it is a land of polyculture; unfortunately, insecticides are unconsciously and commonly used, which is an important problem for the region. It is reported that there is neonicotinoid resistance in A. gossypii and Bemisia tabaci in cotton cultivation areas of the region (Ulusoy et al., 2018, Satar et al., 2018. The imidacloprid resistance in Bemisia tabaci is carried out only on the basis of the samples collected from cotton cultivation areas, which is why it is limited and there is no other study about the resistance.  B. tabaci populations total nucleic acid were extracted by using a Thermo Scientific purification kit. After that, the total DNA quality and quantities were measured by nano-drops. Acording to Bel-Kadhi, 2008; Bt-23F, 5'-CGGAGCTTGCGCCTTAGTC-3' and Bt-23R, 5'-GGCTTTATCATAGCTCTCGT-3' RAPD primers were used for determining B. tabaci biotypes.
In the classical PCR process, there were one cycle of 5 min at 94 ⁰C, 40 cycles of 1 min at 94 ⁰C, anneling 1 min at 55 ⁰C, 1 min at 72 ⁰C and final extension 10 min 72 ⁰C.
Determining Carboxylesterase activity 30 individuals of B. tabaci were homogenized in Tris HCl buffer (0.05 M, pH 7.5), the homogenate was centrifuged as 10000 g at 4 °C for 25 min, and the supernatant was used as an enzyme source. 125 μl subtrate mixture (4 mg Fast Blue RR salt ve 10 mg α-NA (α-naftil asetat), pH 6.0 Na-P buffer) and 75 μl enzyme were added to the microplate cells and the reaction was started. Reading was carried out in a microplate reader at 450 nm and 27 °C for 7 min(Multiscan Go ). The control cells were read without adding the homogenate. The enzyme readings were made with 3 replications and the activity levels were determined by comparison to the controls (Wang et al., 2009).
Determining glutathione S transferase activity The method by Ulusoy et al., 2018 was modified and used to determine activity. 50 B. tabaci individuals were homogenized in a 500 μl Tris HCL buffer (0.05 M, pH: 7.5) by a homogenizer, the homogenate was centrifuged at 11000 g at 4 ⁰C for 7 min, and the supernatant was used as an enzyme source. 100 μl 0.4 mM 1-chloro-2,4-dinitrobenzene (CDNB), 100 μl 4 mM reduced glutathione (GSH) and 100 μl enzyme were added to the microplate cells and the reaction was started. Reading was carried out in a microplate reader at 340 nm and 25 ºC for 10 min. The control cells were read without adding the homogenate. The enzyme readings were made with 3 replications and the activity levels were determined by comparison to the controls. Changes in absorbance per minute were converted into nM CDNB conjugated/min/mg protein using the extinction coefficient of the resulting 2.4dinitrophenyl-glutatione (Habig et al., 1974).
Determining cytochrome p450 monooxygenase activity Cytochrome p450 activity was measured based on the method describe by Ulusoy et al., 2018. 50 B. tabaci individuals were homogenized in a Na -phosphate buffer (0.1 M pH: 7.5), the homogenate was centrifuged as 10000 g at 4 ºC for 10 min, and the supernatant was used as an enzyme source. 90 μl of enzyme and 100 μl 2 mM of p-nitroanisole (substrate) were mixed and left for 3-4 min at 34 ⁰C. Then, 10 μl 9.6 mM of nicotinamide adenine dinucleotide phosphate (NADPH) was added to this mixture, the resulting mixture was put into the microplate cells, and measurements were made at 40 s intervals for 20 min at 405 nm and 25 ⁰C. The control cells were read without adding the homogenate. The enzyme readings were made with 3 replications and the activity levels were determined by comparison to the controls. The specific enzyme activity was determined using the extinction coefficient of p-nitrophenol (Kranthi, 2005).
All enzymes protein quantities were calculated according to Bradford (1976) with OD values determined.

B.tabaci total RNA extraction and Real-Time
Polymerase Chain Reaction (RT-PCR) B. tabaci individuals were frozen at -80 °C, 100 of the individuals were homogenized, and their total RNAs were extracted by using a Thermo Scientific RNA purification kit. After the extraction, the total RNA amounts were measured by nano-drops, and equal concentrations of 75 ng/μl each were diluted by a TE buffer.
In the classical PCR experiment, the primers were tested and proven to work by using a Thermo Scientific One-Step RT-PCR kit. In the classical PCR process, there were one cycle of 15 min at 50 ⁰C, one cycle of 2 min at 95 ⁰C, 40 cycles of 20 s at 95 ⁰C, waiting time of 30 s at 60 ⁰C and another 1min at 72 ⁰C.
The real-time PCR process was carried out with 3 replications for each gene in each population by using a Thermo Scientific One-Step qRT-PCR kit, the Ct values were determined, and these values are taken into analysis by taking the average of 3 replications. In order to confirm the specificity of the real-time reactions, a melting curve cycle was applied. The same temperature table was used in the RT-PCR process. The melting curve program included a cycle of 30 s at 95 ⁰C, a cycle of 10 s at 60 ⁰C and a waiting time of 10 s at 60 ⁰C.
The relative activity levels of the CYP6CM1 and GST genes were determined by making ΔΔCt calculations based on the reference population by taking the average of the obtained Ct values (Livak & Schmittgen, 2001).

RESULT and DISCUSSION
Since its discovery in 1991, imidacloprid resistance in B. tabaci was firstly observed in Southern Spain and it has rapidly increased since that time (Cahill et al., 1996, Sparks & Nauen, 2015. In Turkey, the first imidacloprid resistances in B. tabaci and A. gossypii pests were observed in 2009and 2016(Satar et al., 2018, Ulusoy et al., 2018 (table 2). At the end of PCR reactions of the study, which are made for B. tabaci biotype discrimination, it was determined that the population used in the study is B biotype (figure 2). When CYP6CM1 expression levels of the samples were observed, it was seen that there was a higher change (when compared to the sensitive population) varying from 7 to 18.2 times (Figure 3). It is found that the highest expression level was in Meletmez population (18.2 times higher than the sensitive population) (Figure 3). Karunker et al., 2008, collected B and Q type B. tabaci from different parts of the world. At the end of the analysis process, they reported that only imidacloprid resistant populations have higher CYP6CM1 gene expression when compared to susceptible population (17 times). On the other hand, it is proven that through imidacloprid resistance B. tabaci RNA interference studies, it is possible to stop or slow down P450 CYP6CM1 gene regulation, which decreases imidacloprid resistance (Li et al., 2014).
When the previous studies in the same region were analyzed, it was seen that there are reports showing that while there is 230 times imidacloprid resistance in A. gossypii (in six different locations) there is up to 95 times imidacloprid resistance in B. tabaci (in one location) (Table 1). In line with these results, the high level of CYP6CM1 expression (18 times) in the study population indicates that imidacloprid resistance in cotton fields is high. Populations in the study have high p450 enzyme activities, which is in parallel with the CYP6CM1 expression levels. The highest level of p450 enzyme activity was observed in Meletmez population (3.24 M min -1 mg -1 protein) which was in line with the CYP6CM1 gene expression level (Table 2).  , 1982;Hemmingway&Georghiou, 1984;Rauch & Nauen, 2003;Yang et al, 2014). Different types of insecticides are commonly used in Çukurova Region which is a poly-culture agricultural production area (vegetables, fruits, cotton etc.). Although different agricultural products are cultivated in the region, agricultural lands are almost within one another. As B. tabaci is a polyphagous species and frequently exposed to insecticides with different mechanisms such as organophosphate, carbamate, pyrethroid and neonicotinoid, it is unavoidable for it to have high carboxylesterase and GST activities.
As a result of the study, it can be said that, there was a common imidacloprid resistance in B. tabaci populations in Adana cotton cultivation areas in 2018; the previous studies and CYP6CM1 gene overexpression criteria support this finding. On the other hand, it is possible to claim that there is a metabolic resistance in the populations resulting from the common use of insecticide. According to these findings, it is important to review and manage the insecticide control of B. tabaci in Çukurova region cotton cultivation areas; it is also crucial to develop more efficient and different control strategies in order to cope with the resistance in the region.

Statement of Conflict of Interest
Authors have declared no conflict of interest.