Insecticide Resistance and Detoxification Enzymes Activity in Nilaparvata lugens Stål Against Neonicotinoids

The Nilaparvata lugens (Stål) is one of the most destructive pests of rice crops in Asian. To assess the resistance of imidacloprid, thiamethoxam, clothianidin, and nitenpyram, N. lugens exposed to each pesticide up to 15 generations. Results showed that the resistance of N. lugens increased significantly against imidacloprid, thiamethoxam, clothianidin, and nitenpyram (neonicotinoids) under selection pressure. There was a 118.07-fold increase in resistance against imidacloprid, 90.37-fold against thiamethoxam, 217.81-fold against clothianidin, and 34.09-fold against nitenpyram in 15 generation as compared to F0. Based on fold increase, imidacloprid and clothianidin subjected for enzymatic analysis and results showed that enzyme activity involves resistance development against neonicotinoids. Cytochrome P450, esterase, and GST had significantly higher activity as the generation passes under the selection pressure of imidacloprid and clothianidin. There was a significant correlation existed between GST, and esterase activity, when compared to LC50 of imidacloprid. GST, esterase and P450 showed a significant correlation with LC50 of clothianidin. The results showed that detoxification enzymes play an important role in insecticide detoxification. When the mixture of imidacloprid and clothianidin tested results showed that the mortality exerted was similar to control when imidacloprid and clothianidin resistant populations were exposed.


Introduction
The brown planthopper (Nilaparvata lugens) is an important and notorious pest in rice agroecosystem in many parts of Asia. N. lugens is a monophagous insect pest, which feeds only on rice (O. sativa) or wild rice species. Moreover, this pest is a destructive cropland insect pest in temperate parts of Asia, especially frequently erupting in China Y. Wang & M. Wang, 2007). It causes hopper burn and other direct damages on rice crops by sucking from the phloem and transmitting plant viral diseases, including the Rice Grassy Stunt Virus (RGSV) and Rice Ragged Stunt Virus (RRSV) to the rice plant (Maholidy, 2019).
During the 1950s, DDT (dichlorodiphenyltrichloroethane) and BHC (benzoate hexachloride) used on a large area (Zhu & Cheng, 2013) along with different insecticides related to organophosphates, carbamates, pyrethroids, neonicotinoids, and insect growth regulators have been used since 1970s to up till now for the management of N. lugens Matsumura et al., 2008). However, thiamethoxam, chlorpyrifos, nitenpyram, and pymetrozine are more trusted pesticides for the management of N. lugens in China Zhang et al., 2014). Due to the high fecundity of N. lugens, synthetic pesticides were used widely for its management (Su et al., 2013). This intense application of insecticides in paddy fields, induced a rapid insecticide resistance in N. lugens but also play an important role in the failure of different chemical insecticides (Nagata, 1985;Zhang et al., 2014). N. lugens already developed resistance against organochlorines, organophosphates, carbamates, and pyrethroids, which are for major insecticidal groups (Matsumura et al., 2009;). Comparatively, low-level resistance found in N. lugens against buprofezin (insect growth regulator) (Li, 2007;Zhuang et al., 2004). Many previous studies reported the resistance of N. lugens against different insecticides. In 2005 an outbreak of N. lugens was reported due to imidacloprid resistance . Intergroup cross resistance is also a cause of failure of whole insecticide group (Ausborn et al., 2005) There is a need for strict monitoring of insecticide resistance in N. lugens for better control (Min et al., 2014).
Neonicotinoids are an important group of pesticides all over the world due to their high efficacy and versatile range against different insect pests. Insects started to develop resistance against neonicotinoid insecticides (Jeschke et al., 2011). The first reported resistance was in 1996 when low resistance observed in whitefly against imidacloprid (Cahill et al., 1996). Up till now, according to SciFinder® 2014, American Chemical Society published more than 500 publications for neonicotinoid resistance and more than 300 cases of imidacloprid resistance reported Arthropod Pesticide Resistance Database (APRD) (Bass et al., 2015;University Michigan State, 2014). Most reported resistant insects are green peach aphid, Myzus persicae, the cotton aphid, Aphis gossypii, the rice brown planthopper, Nilaparvata lugens, houseflies, Musca domestica, Colorado potato beetle, Leptinotarsa decemlineata and glasshouse whitefly, Trialeurodes vaporariorum (Bass et al., 2015).
In whole life, insects deal with many toxic substances that can be natural as well as many agrochemicals like insecticides. This detoxification process is a key defensive mechanism for the insects for resistance or tolerance (Wu et al., 2011). Enzymatic activities can play an important role in understanding the susceptibility and tolerance or resistance against insecticide. The alternation in the level of detoxification enzymes is a common mechanism in response to the exposure of any stress of insects (Baker et al., 1998). Detoxification enzymes are typically involved in the metabolic process to act on the target site where toxic substance attacks in the insect body (Lin et al., 2015). The activity of enzymes in the detoxification process was observed higher in resistant species of insects as compared to susceptible ones, which is an important mechanism to confer the insecticide detoxification process (Denholm, 1992;Lu & Gao, 2009). Previously reported literature stated that Glutathione-S-transferase (GST), carboxylesterases (CarEs), acetylcholinesterase (AChE), and others are the basic detoxification enzymes that involve in the metabolic detoxification of xenobiotic (Hafeez et al., 2019;Hu et al., 2014;. The present study aimed to assess the current status of insecticide resistance in N. lugens against neonicotinoids insecticides (imidacloprid, nitenpyram, thiamethoxam, and clothianidin). This study also aimed to determine how different enzymes act for the detoxification of these insecticides and their role in insecticide resistance under pesticide selection pressure.

Insects
The population of N. lugens collected from different locations of the paddy fields of Huazhong Agricultural University. This population raised under laboratory conditions for several generations without exposure to any insecticide. Population reared more than ten generations under laboratory conditions (27±1 °C, 70-80% humidity and a 16:8 h L:D cycle) were used for experimentation. For experimentation, fourth instar nymphs of N. lugens used. The insecticide selection process, the toxicity of all the insecticides evaluated of all the generations, and a median lethal concentration (LC 50 ) and lethal concentration (LC 90 ) determined. This median lethal concentration used for a successive generation as a selected concentration.

Bioassay
The topical application method (Khan et al., 2018) used in this experiment for toxicity determination bioassay. Progressive concentrations applied to the fourth instar nymphs of N. lugens. The test insects were placed in a glass tube and then immobilised by using CO 2 . 0.5 μl of test concentration applied on the ventral side of N. lugens with the help of a micro-applicator (Burkard, England). For each concentration, twenty individuals used in one replication, and four replications set up. Treated insects kept in glass jars containing rice seedlings and a jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 5; mixture of nutrient water. These jars kept in climatic chambers with a temperature of 27±1 °C with a 16:8 h L:D photoperiod. Mortality data was taken after 24 hours.

Resistance Selection
Resistance selection performed by utilising the leaf dip method . Almost 20 rice seedlings were washed and assembled and kept into a 500 mL plastic jar after treatment with test concentration. Around 3000, 4 th instar nymphs treated from each generation during the resistance selection procedure. After five days of treatment, survivors were collected and moved to a pesticide-free jar with new rice seedlings for the successive generation. Two pesticides, one against which N. lugens was most susceptible, and one against which N. lugens was more resistant was taken and subjected for determination of detoxification enzyme activity.

Pesticide Additive Bioassay
Due to the high resistance ratio, imidacloprid and clothianidin were selected and subjected to synergistic bioassay. The LC 50 imidacloprid (2.74 mg a.i L -1 ) and clothianidin (0.64 mg a.i. L -1 ) computed from susceptible strains (F 0 ) of N. lugens were mixed in 1:1 ratio. A 100 ml solution formed. The pesticides were applied to susceptible N. lugens population, imidacloprid resistant strain and clothianidin resistant strain by the method mentioned above. The enzymatic analysis was also done at this stage to assess the enzymatic activity.

P450 Activity
The activity of cytochrome P450 assessed by ethoxycoumarin-O-dethylase (ECOD). 4 th instar nymphs of N. lugens, which exposed to pesticides, were grinded, and 0.3 g sample homogenised in 900 μl mixture of 1.0 mM dithiothreitol (DTT), 1.0 mM PMSF, 1.0 mM EDTA and 10% glycerol (0.1M, pH 7.5). The mixing was done on Ice and then centrifuged at 4 o C, 10,000 RPM for 15 min. Enzyme reaction system includes 125 μl of enzyme solution, Sodium phosphate buffer 365 μl (0.1M, pH 7.5), NADPH 10 μl (10 mM) and 7-EC 5 μl of (40 mM). After mixing, the mixture incubated at 30 °C for 15 min. 15% TCA (trichloroacetic acid) added (150 μl) to sack the reaction. The whole mixture was centrifuged at 10000 RPM for 2 min and collected. 40 μl of 1.6 mM (pH 10.5) glycine-NaOH buffer was added. The optical density (OD) set at 34 °C with the help of Spark 10M multimode Microplate Reader (TECAN, CA, Switzerland) at an excitation wavelength of 358 nm (Slit 5 nm) and an emission wavelength of 465 nm. The enzyme source was diluted 200 times for protein analysis.

GST Activity
Insects were treated topically with micro-applicator, and after 24 hours alive, N. lugens nymphs were transferred into centrifuge tubes and immediately frozen by liquid nitrogen (-80 °C). The activity of GST enzyme was assessed by following the procedure as described by Khan (2019). GST determination Kit (SR No. A004) bought from Nanjing Jiancheng Bioengineering Institute, China. The kit was mixed, as instructed by the manufacturer. Each sample ground in 0.2M phosphate buffer (pH = 7.0). Bio-Rad spectrophotometer used for the analysis of absorbance of light at 412 nm wavelength. Moreover, the protein level sample also determined by using the Bradford assay kit purchased from Beyotime, Shanghai, China, by using BSA (Bovine Serum Albumin) as a standard.

Carboxylesterases (CarE) Activity
The activity of CarE was assessed by following the procedure as described by Nawaz (2018). For the assessment of esterase activity, 200 μl of assay mixture shifted into a 96-well-plate. This mixture was consisting of 2 μl of 0.2M alpha naphthyl acetate and 10 μl of enzyme solution diluted in 0.2M (pH 7.2) sodium phosphate buffer. The incubation of the solution done for 15 min at 37 °C, and the reaction halted by supplementing of colorimetric reagent fast blue RR salt. After this, at 600 nm wavelength, the absorbance was measured with the help of a Bio-Rad spectrophotometer.

Statistical Analysis
PoloPlus ( One-way ANOVA was used to assess the mean difference in enzymatic activity analysis with three replications via the Tuckey's test at P = 0.05 and the correlation between LC 50 and enzyme activity also computed by SPSS. Graphical work is done with the help of Sigmaplot 12.0.

Mortality and Resistance Development
Results (Table 1) showed that the LC 50 of imidacloprid at F 0 was 2.74 mg a.i. L -1 and LC 50 of F 15 computed 323.17 mg a.i. L -1 , due to selection pressure after 15 generations from F 0 and F 15 , the resistance ratio increased up to 118.07-fold. Thiamethoxam also showed 1.01 mg a.i. L -1 LC 50 , while this LC 50 reached up to 91.54 mg a.i. L -1 after F 15 . The increase in resistance was about 90.37-fold. The LC 50 of F0 on N. lugnes treated with clothianidin was 0.64 mg a.i. L -1 , which reaches up to 139.18 mg a.i. L -1 after F 15 under pesticide selection pressure. The resistance increased up to 217.81-fold as compared to LC 50 . The LC 50 of susceptible strain exposed to nitenpyram was 0.41 mg a.i. L -1 , which increased up to 34.09-fold and showed LC 50 of 13.94 mg a.i. L -1 after 15 generations under selection pressures. The increase in LC 50 of different pesticides in different generations also can be seen in Figure 1. Note. G = generations; CL = Critical limit; RR = Resistance Ratio. jas.ccsenet.

Pesticide Additive Bioassay
For testing the additive effect of these pesticides, the mixture of imidacloprid and clothianidin tested against F 15 resistant strain. Results in (Figure 3) showed that the imidacloprid resistance strain (IRS) and clothianidin resistant strain (CRS) exerted 68.33±5.69% and 65.00±5.69% mortality. The exerted mortality has no significant difference as compared to that in control (susceptible strain). These results showed that the resistant strains still showed high susceptibility to different pesticide combinations. The enzymatic analysis showed that P450, esterase and GST enzyme activity in CRS and IRS was non-significantly varying with control, which revealed that the combination of pesticide has an additive effect on resistant populations of N. lugens (Figure 4).  Vol. 12, No. 5; imidacloprid. Results showed that due to continuous exposure of imidacloprid, thiamethoxam, clothianidin and nitenpyram for 15 generations, the resistance increased 118. 07-fold, 90.37-fold, 217.81-fold and 34.09-fold, respectively. Gorman et al. (2008) studied the resistance development in N. lugens in 2005 and 2006 and reported a 100-fold increase in imidacloprid resistance in N. lugens. Jin et al. (2019) exposed N. lugens to clothianidin for 13 generations and reported the increase of resistance up to 174.33-fold. (Sun et al., 2018) reported 26.5-fold increase in resistance due to selection pressure under thiamethoxam. The resistance levels of N. lugens against thiamethoxam (RR 25.9-159.2) rapidly increased in the period 2012-2014, a phenomenon similar to what had observed in the paddy field populations of N. lugens in China from 2011 to 2012 (Zhang et al., 2014) with moderate to high levels of resistance in Korea observed in this pest (Min et al., 2014). Zhang et al. (2017) reported a 33.9-fold increase in resistance when N. lugens was exposed to nitenpyram. These all results fortified the findings of the current study that under selection pressure, the N. lugens developed resistance against neonicotinoids. The current study also showed that nitenpyram is more effective as compared to other insecticides because of less resistance development.  confirmed these results that N. lugens developed less resistance against nitenpyram.
The main reason for the rapid resistance development in N. lugens against neonicotinoids is not only its extensive and intensive use but also the cross-resistance between neonicotinoid insecticides (Matsumura et al., 2014;Wang et al., 2009). Neonicotinoid cross-resistance was reported in Bemisia tabaci (Feng et al., 2010), Leptinotarsa decemlineata (Mota-Sanchez et al., 2006) and Trialeurodes vaporariorum (Longhurst et al., 2013). The cross-resistance occurred when two different insecticides are hitting the same target side and due to over and over exposure, the mutation occurs which induced resistance against other insecticides (Bass et al., 2015).
The study of resistance mechanisms in insects considered a prerequisite for developing resistance management strategies Xu et al., 2014). Gene regulation is often the main cause of insecticide resistance, which increased the cytochrome P450, GST and esterase enzyme activity for detoxification (Heckel, 2012). Increased activity of these detoxification enzymes is the most common mechanism of resistance (Heckel, 2012). In this study, the results showed that for detoxification of imidacloprid P450, esterase and GST enzymes played important role. The activity of all the enzymes increases as the generation increased. We found that the activity of the detoxification enzymes (esterase and GST) were significantly correlated with the LC50 values of imidacloprid, indicating that these detoxification enzymes may be involved in the observed resistance of N. lugens to imidacloprid. Previous studies showed that for the detoxification of imidacloprid, esterase enzymes showed high activity as compared to GST enzymes . Wen et al. (2009) reported that resistant N. lugens strain against imidacloprid after 25 generations have a high activity of esterase enzymes as compared to the parent generations. Zhang et al. (2016) also reported that among all the populations collected from different provinces of China, most resistance populations against imidacloprid showed the higher activity of detoxification enzymes. There have been a few reports that esterase appeared to be responsible for neonicotinoid insecticide resistance. However, previous studies have not demonstrated that glutathione-S-transferase plays a role in neonicotinoid insecticide resistance in insect species. Current study also showed that esterase and GST enzymes play important role to detoxify clothianidin. Previous studies have reported that GST and esterase play a significant role in the detoxifying strategies of pests (Han et al., 1998;Heckel, 2012;Hemingway et al., 2002;Vontas et al., 2000;Zhang et al., 2012). Current study showed that when mixture of imidacloprid and clothianidin tested the resulted mortality was more than 60% and enzymatic activity was not significantly different. Many researchers also reported that mixture of different pesticides can enhance the toxicity which can also be an effective method to deal with resistance. LC 50 : LC 50 ratio mixture can produce the synergistic or antagonistic effect (Khan et al., 2013). If different pesticides mixed, their toxicity is also enhanced to control the resistant strain of P. xylostella (Attique et al., 2006).
From the present study, it can be concluded that continuous exposure to a single insecticide for a long time can be a reason of resistance induction. In the present study, due to the long term exposure of neonicotinoid insecticides, N. lugens induced resistance. Resistance against any pesticide is due to the detoxification of enzymes. Esterase and GST enzymes are more important in detoxification of neonicotinoid insecticides as compared to P450. The combination of different pesticides can also play an important role do deal with resistant strains of insects.