327 ISJ 15: 327-337, 2018 ISSN 1824-307X RESEARCH REPORT Biochemical basis of conventional and novel mode of action insecticides resistance in field population of Diaphorina citri collected from Southern Punjab, Pakistan A Naeem, S Freed* Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan Accepted August 19, 2018 Abstract Diaphorina citri Kuwayama is a phloem-feeding pest of citrus worldwide and vectors the putative causal agent of citrus greening, “Candidatus Liberibacter asiaticus” (CLas). Baseline toxicity and activities of esterases (EST), and glutathione-S-transferases (GST) sensitivity to bifenthrin, chlorpyrifos, thiamethoxam, acetamiprid, nitenpyram, chlorfenapyr and imidacloprid were established with field populations of D. citri. The highest calculated toxicity was observed in thiamethoxam treated D. citri with LC50 of 4.6 μg/mL and minimum in chlorpyrifos and imidacloprid with LC50 values of 96.9 and 91.9 μg/mL, respectively. The EST activity recorded in D. citri treated with different insecticides ranged from 11.8 to 52.1 µmol/min/mg of protein, while GST activity ranged from 10.9 to 51.3 µmol/min/mg of protein which subsequently linked with insecticide resistance. The present results provide a specific baseline to study levels of detoxifying enzymes against commonly used insecticides in D. citri that’s indirectly correlated with level of resistance. Key Words: Insecticides; HLB; Esterase; GST; resistance; citrus psylla; detoxification Introduction Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), Asian citrus psyllid (Burckhardt and Ouvrard, 2012), a worldwide pest of citrus (Sapindales: Rutaceae) (Halbert and Manjunath, 2004; Bové, 2006) and primarily important due to its role as fastidious vector of Candidatus (Ca.) Liberibacter (L.) africanus, Ca. L. asiaticus., and Ca. L. americanus associated with “citrus greening” also known as Asian huanglongbing (HLB) (Pelz-Stelinski et al., 2010; Boina and Bloomquist, 2015). Citrus trees infected by this disease produce small, discolored, lopsided and misshapen fruit over a shortened life span (Bové, 2006; Gottwald et al., 2012; Grafton- Cardwell et al., 2013) and infected trees can cause 33% yield loss of citrus fruits (Salcedo et al., 2016). HLB management programs include the removal of infected trees, use of disease-free nursery stock and psyllid vector control by pesticide sprays (Belasque et al., 2010; Tiwari et al., 2010; Grafton- Cardwell et al., 2013). ___________________________________________________________________________ Corresponding author: Shoaib Freed Faculty of Agricultural Sciences and Technology Bahauddin Zakariya University Multan, Pakistan E-mail: sfareed@bzu.edu.pk The injudicious use of different insecticides puts D. citri under pressure and the insects use different mechanism (increased detoxification, reduced penetration, alterations of target sites and behavioral adaptations) for evolving resistance (Brattsten et al., 1986; Chen et al., 2006; Sétamou et al., 2010) that may be found among D. citri populations. The enzymatic mechanism involves increased activities of esterases (EST) and glutathione-S-transferases (GST) (Hemingway et al., 2004; Tiwari et al., 2011a,b,c). A positive correlation between level of insecticide resistance and detoxifying enzymes has already been documented in several insect pests (Scott, 1999; Maymo et al., 2002; Srigiriraju et al., 2009). The insecticide resistance management involves reducing the frequency of resistance alleles within a field population so that the efficacy of a specific insecticide can be preserved (Georghiou, 1994). EST are detoxifying enzymes produced through biotransformation of xenobiotics and directly linked to insecticides resistance (Devorshak and Roe, 1998) EST are known to detoxify organophosphate insecticides by hydrolyzing the ester moiety (Oakeshott et al., 2005) and are studied in several insects like Culex quinquefasciatus (Diptera: Culicidae) (Sahgal et al., 1994), Blattella germanica (Blattodea: Ectobiidae) (Anspaugh et al., 1994), and 328 Table 1 List of Insecticides used for bioassays Trichoplusia ni (Lepidoptera: Noctuidae) (Ishaaya and Casida, 1980). Similarly, increased level of GST is correlated with insecticide resistance (Li et al., 2007). The enhanced oxidative and hydrolytic enzyme activities of GST expression have been associated with organophosphate and synthetic pyrethroid resistance in Musca domestica (Diptera: Muscidae) (Clark et al., 1984; Tang et al., 1990); Helicoverpa armigera (Lepidoptera: Noctuidae) (Srinivas et al., 2004), and Spodoptera litura (Lepidoptera: Noctuidae) (Armes et al., 1997; Kranthi et al., 2001). The understanding of baseline activity of enzymes is necessary to suggest better management tactics and to minimize insecticide resistance in D. citri (Boina et al., 2011; Tiwari et al., 2011a). The present study intended to examine the activities of EST and GST after application of conventional and noval mode of action insecticides in field populations of D. citri so that better management program involving insecticides can be devised. Materials and methods Insects The adult D. citri were collected from citrus orchard of Khanewal, Punjab, Pakistan through an aspirator and released on small citrus plants in Plexiglas cages 40×40×40 cm under laboratory conditions. Insects were reared in Laboratory of Insect Microbiology, Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan until specific numbers were obtained to be used in bioassays. The rearing conditions were maintained at 27 ± 2 ºC and 60-65% relative humidity with a (12: 12h) light: dark photoperiod. Insecticides Seven insecticides belonging to different toxicological groups were chosen on the basis of their current use in field (Table 1). Insecticidal Bioassays The leaf dip method was used to determine the toxicity of different insecticides against D. citri. Serial dilutions of insecticides with selected concentrations (through hit and trial method) were formed for bioassay with distilled water (Naeem et al., 2016). Fresh shoots/flushes of citrus were exposed for about 10 sec with different insecticide dilutions and were allowed to dry for 1 h at room temperature, while distilled water was used to treat control insects instead of insecticide dilution. In each treatment fifteen (5-7 days old) adult psyllids were released on treated citrus flushes within plastic jars (9×6×6 cm) covered with net from two sides for good aeration. Each treatment was replicated three times under similar conditions as described earlier. The mortality was recorded after 48, 72 and 96 h for conventional, neonicotinoid and pyrrole insecticides, respectively. The treatment samples for further biochemical assay were collected after 24, 48 and 72 h during bioassay. Biochemical assays Subsequent to the insecticidal bioassay, collected alive adult D. citri of different treatment levels and control were processed for further biochemical assay. Firstly, three to five psyllids were taken from each treatment and crushed by using the 0.15 M NaCl in the Eppendorf tube. For centrifugation the final volume was made 400 μl. The samples were spun at 3000 rpm for 10 mins at 5 ± 1 °C using the centrifuge machine (Z216MK, Hermle Labortechnik, Germany) to obtain the supernatants. The supernatants (D. citri homogenate) were further used to determine EST and GST activities. Determining the activities of EST and GST Determination of Protein Contents The protein contents were determined in D. citri homogenates through Bradford (1976) procedure, using bovine serum albumin as standard at 25 °C with a spectrophotometer (UV3000, O.R.I. Germany). Insecticide Trade name and manufacturer Target site of Action Group of Insecticide chlorpyrifos (Lorsban®, 400 EC; Dow Agro Sciences) Acetyl cholinesterase inhibitors Organophosphates bifenthrin (Talstar®,10 EC; FMC) Sodium channel modulators Pyrethrins imidacloprid (Confidor®, 20 SL; Bayer Crop Sciences) Agonists of nicotinic acetylcholine receptor Neonicotinoids acetamiprid (Mospilan®, 20 SP; Arysta Life Sciences) Agonists of nicotinic acetylcholine receptor Neonicotinoids thiamethoxam (Actara®, 25W G; Syngenta) Agonists of nicotinic acetylcholine receptor Neonicotinoids nitenpyram (Paranol®, 10 EC; Kanzo, Agrochemicals) Agonists of nicotinic acetylcholine receptor Neonicotinoids chlorfenapyr (Pirate®, 360 SC; BASF Chemicals and Polymers) Uncouplers of oxidative phosphorylation via disruption of the proton gradient Pyrroles 329 Table 2 Toxicity of conventional and novel mode of action insecticides against D. citri Insecticide LC50 (μgmL−1) (FD) Slope χ2 df Pa nb chlorpyrifos 91.88 (62.67-144.85) 1.35 ± 0.25 2.722 4 0.61 270 bifenthrin 40.14 (23.05-80.21) 0.95 ± 0.23 0.231 4 0.99 270 imidacloprid 96.91 (49.17-487.05) 0.70 ± 0.22 1.218 4 0.88 270 acetamiprid 67.42 (43.13-135.74) 1.18 ± 0.26 0.024 4 1.00 270 thiamethoxam 4.64 (3.15- 6.61) 1.25 ± 0.22 1.195 4 0.88 270 nitenpyram 6.10 (4.25- 9.67) 1.25 ± 0.23 1.599 4 0.81 270 chlorfenapyr 23.99 (16.58-37.89) 1.21 ± 0.22 0.363 4 0.99 270 aP-values are based on Chi-square goodness of fit test. P values > 0.05 suggest goodness of fit of the model bNumber of D. citri used in the bioassay, including the control EST activity EST activity was measured by using 1 mM 4- Nitrophenyl acetate as a substrate (Damayanthi and Karunaratne, 2010). In each replicate, 90 µl of phosphate buffer (PBS, pH 6.5) and 100 µl of 0.6 M aNa (or bNa) was added to 10 µl of D. citri homogenate. After 30 min incubation, 100 µl of Fast Garnett BC solution was added to stop the reaction. The concentration of the final product was determined at 405 nm as an endpoint calculated from standard curves of a- and b-Naphtol, respectively. GST activity The GST activity was determined according to Caballero et al. (2008). For this purpose, 1- CDNB (chloro-2, 4-dinitrobenzene) (1 mM) was used as a substrate in ultraviolet (UV) semi-micro cuvettes (4 ml) with 5 mM reduced glutathione, 0.1 M Tris buffer pH 8 and 30 µl of D. citri homogenates. Enzyme activity was determined by monitoring continuous changes in absorbance at 340 nm for 4 min at 25 °C with a spectrophotometer. Statistical analysis The mortality data was evaluated by probit analysis (Finney, 1971) using the software package EPA probit analysis version 1.5. The enzymatic activities were analyzed through split plot design. Significance was accepted at α = 0.05. The enzymatic activities in D. citri homogenates were expressed by plotting graphs using Graph PrismPad 6.02. Results Effect of insecticides on D. citri The toxicity of two groups of insecticides (conventional and novel mode of action) was evaluated against D. citri and their LC50 are given in Table 2. The highest percentage mortalities by chlorpyrifos, bifenthrin, imidacloprid, acetamiprid, thiamethoxam, nitenpyram and chlorfenapyr were 75.0, 66.0, 62.0, 59.0, 82.0, 73.0 and 71.0, respectively at their highest concentrations μgmL−1 (Table 3). However, thiamethoxam with lowest LC50 value of 4.6 (3.2-6.6) μgmL−1 proved to be most potential insecticide against D. citri as compared to imidacloprid with LC50 value of 96.9 (49.2-487.1) μgmL−1, chlorpyrifos 91.9 (62.7-144.8) μgmL−1, acetamiprid 67.4 (43.1-135.7) μgmL−1, bifenthrin 40.1 (23.0-80.2) μgmL−1, chlorfenapyr 23.9 (16.6- 37.9) μgmL−1 and nitenpyram 6.10 (4.2-9.7) μgmL−1, respectively. EST activity Activity of EST in D. citri after the exposure to conventional insecticides In case of conventional insecticides bifenthrin showed maximum inhibition of EST i.e., (3.3 ± 0.7) µmol/min/mg of protein (F= 278.0, df= 5, P= 0.0000) observed after 48 h of treatment, while chlorpyrifos (34.6 ± 2.3) µmol/min/mg of protein (F= 2118.7, df= 5, P= 0.0000) expressed highest EST activity at 200 μgmL−1 after 24 h (Fig. 1). Activity of EST in D. citri after the exposure to novel mode of action insecticides The maximum inhibition of EST was expressed in chlorfenapyr treated D. citri (2.8 ± 0.4) µmol/min/mg of protein (F= 391.6, df= 5, P= 0.0000), while maximum EST activity was recorded in nitenpyram (52.1 ± 5.8) µmol/min/mg of protein (F= 45.2, df= 5, P= 0.0000) followed by imidacloprid, chlorfenapyr, thiamethoxam and acetamiprid. In case of imidacloprid maximum activity (24.4 ± 1.9) µmol/min/mg of protein (F= 703.2, df= 5, P= 0.0000) was expressed in 24 h treatment as compared to 48 h and 72 h. In case of acetamiprid highest activity (11.8 ± 1.5) µmol/min/mg of protein (F= 368.5, df= 5, P= 0.0000) was observed after 48 h and lowest in 72 h post treatment. On the other hand, in case of nitenpyram, chlorfenapyr and thiamethoxam highest activity (12.9 ± 1.5) µmol/min/mg of protein (F= 110.7, df= 5, P= 0.0000) was expressed in 24 h than 48 h and 72 h samples (Fig. 1). 330 Table 3 Percent mortalities of D. citri against conventional and novel mode of action insecticides Insecticides Concentration (μgmL−1) %Mortality F-value P-value LSD chlorpyrifos 200 75.5 ± 8.4 A 24 0.000 23.9 100.0 44.4 ± 1.9 B 50.0 40.0 ± 3.3 BC 25.0 26.6± 3.3 BCD 12.5 17.7 ± 1.9 CD control 4.4 ± 1.9 D bifenthrin 100 66.6 ± 3.3 A 41.1 0.000 16.7 50 57.7 ± 3.8 AB 25 46.6 ± 3.3 B 12.5 28.8 ± 1.9 C 6.2 24.4 ± 1.9 C Control 6.6 ± 1.9 D imidacloprid 150 62.2 ± 1.9 A 26.8 0.000 18.3 75 44.4 ± 3.8 AB 37.5 37.7 ± 3.8 B 18.7 28.8 ± 1.9 B 9.4 28.8 ± 5.1 B Control 2.2 ± 1.9 C acetamiprid 100 59.9 ± 3.3 A 28.5 0.000 18.3 50 46.6 ± 5.7 AB 25 33.3 ± 3.3 BC 12.5 22.2 ± 1.9 CD 6.2 15.5 ± 1.9 CD Control 4.4 ± 1.9 D thiamethoxam 20 82.2 ± 3.8 A 45.4 0.000 20.2 10 66.6 ± 5.7 AB 5 46.6 ± 3.3 BC 2.5 31.1 ± 3.8 C 1.2 28.8 ± 1.9 C Control 2.2 ± 1.9 D nitenpyram 14 73.3 ± 8.4 A 31.9 0.000 21.1 7 46.6 ± 1.9 B 3.5 40.0 ± 3.3 BC 1.7 22.2 ± 3.3 CD 0.8 17.7 ± 1.9 DE Control 2.2 ± 1.9 D chlorfenapyr 60 71.1 ± 5.1 A 50.3 0.000 16.7 30 53.3 ± 3.3 B 15 39.9 ± 3.3 BC 7.5 26.6 ± 0.0 CD 3.7 17.7 ± 1.9 DE Control 2.2 ± 1.9 E GST activity Activity of GST in D. citri after the exposure of conventional insecticides In case of conventional insecticides, maximum inhibition of GST i.e., (5.3 ± 0.9 µmol/min/mg) of protein was due to chlorpyrifos at 200 μgmL−1 after 72 h of treatment (F= 2196.4, df= 5, P= 0.0000), while bifenthrin expressed highest GST activity (47.6 ± 3.6 µmol/min/mg of protein) (F= 122.1, df= 5, P= 0.0000) at 100 μgmL−1, 24 h post treatment (Fig. 2). Activity of GST in D. citri after exposure of novel mode of action insecticides Chlorfenapyr expressed maximum GST inhibition (2.7 ± 1.6 µmol/min/mg of protein) (F= 220.1, df= 5, P= 0.0000) followed by thiamethoxam and acetamiprid. In contrast, nitenpyram expressed maximum GST activity. The GST activities of novel mode of action insecticides were as follows: imidacloprid (10.9 ± 2.5 µmol/min/mg of protein), acetamiprid (24.2 ± 1.9 µmol/min/mg of protein) (F= 38.6, df= 5, P= 0.0000), nitenpyram 331 Fig. 1 EST specific activity expressed as mean in bars followed by the same letter within columns indicate no significant difference (P≤ 0.05) in a Tukey’s test after the exposure of different insecticides in D. citri. The line in the bars represents the mean standard error 332 Fig. 2 GST specific activity expressed as mean in bars followed by the same letter within columns indicate no significant difference (P≤ 0.05) in a Tukey’s test after the exposure of different insecticides in D. citri. The line in the bars represents the mean standard error 333 Fig. 3 Linear regression analysis showing the relation between percent mortality and EST enzymatic expression with respect to different hours (P≤ 0.05) (51.3 ± 1.7 µmol/min/mg of protein) (F= 95.7, df= 5, P= 0.0000), thiamethoxam (16.9 ± 2.6 µmol/min/mg of protein) and chlorfenapyr (14.0 ± 1.6 µmol/min/mg of protein) after 24 h of treatment (Fig. 2). Relation between percent mortality of D. citri and enzymatic expression The relation between percent mortality and EST (Fig. 3) and GST (Fig. 4) was expressed by drawing regression lines activity in D. citri. The difference between the slopes showed significant values. Discussion Asian HLB has grown into a great risk to the world citrus industry (Bové, 2006; Hall et al., 2013) as the vector D. citri has developed resistance to a wide range of insecticides (Naeem et al., 2016; Chen and Stelinski, 2017). Detoxification enzymes like EST and GST are described as the defense mechanism against foreign toxic compounds (Li and Liu, 2007) and play important roles in maintaining the normal physiological activities in the insect’s body (Chen et al., 2017). For example, due to neurotoxic action of insecticides, insects release the 334 Fig. 4 Linear regression analysis showing the relation between percent mortality and GST enzymatic expression with respect to different hours (P≤ 0.05) detoxifying enzymes which correlate with insecticide resistance (Bilal et al., 2018). The biochemical mechanisms of insecticide resistance in D. citri have received significant recent attention (Liu et al., 2016; Chen et al., 2018). Tiwari et al. (2011a) reported high levels of detoxifying enzymes activity at higher toxicity level of insecticide (thiamethoxam, imidaclorpid, bifenthrin, chlorpyrifos and fenpropathrin) in D. citri populations. Similarly, our result showed higher enzymatic function at higher toxicity level of insecticides as compared to control D. citri population. Our results suggested that D. citri showed maximum EST (52.1 ± 5.8 µmol/min/mg of protein) after 24 h of exposure to nitenpyram. In relation with percent mortality 73.3% regression showed highly significant correlation with EST activity after 24 h of exposure (F= 124.3, P= 0.0004) However, our results indicated that acetamiprid (11.8 ± 1.5) 335 µmol/min/mg of protein induced reduced activity of EST whereas, imidacloprid (10.9 ± 2.5) µmol/min/mg of protein induced a reduction in activity of GST. Results declared some inner connection between enzyme activity, time, percent mortality and concentrations of insecticide. For example, in case of EST activity for acetamiprid, chlorfenpyr and thiamethoxam slope was positively related with percent mortalities at specific time i.e., 24 h, 48 h and 72 h, respectively as compared to other insecticides. Its means that increase in EST enzyme release was directly linked with percent mortality, which might be a reason for resistance development at specific time of exposure of insecticide. Whereas, GST had significantly negative slope between percent mortality and enzyme activity for all insecticides with respect to time. Suppression of detoxification enzymes indicates that these enzymes play no role in the detoxification of tested compounds and may increase the susceptibility of insect pests to these insecticides (Abd-Elaziz and El-Siad, 2009). Previous studies revealed that GST enzymes contribute to pyrethroid resistance (Grant and Matsumura, 1989; Tiwari et al., 2011a, c). Our results revealed that bifenthrin expressed (47.6 ± 3.6) µmol/min/mg of protein GST activity which coincides with previous study. In contrast, bifenthrin revealed very low EST activity i.e., 3.3 ± 0.7 µmol/min/mg of protein. Current research showed that in vivo, metabolism of imidacloprid changed to the main metabolite 4/5-hydroxyimidacloprid, a product of oxidative degradation, in honeybee Apis mellifera (Suchail et al., 2004). Similarly, the main metabolite of imidacloprid i.e., 5- hydroxyimidacloprid was detected in imidacloprid- resistant strains of Bemisia tabaci (Rauch and Nauen, 2003). Parallel to previous results of our studies, imidacloprid showed EST and GST activities i.e., 24.4 ± 1.9 and 10.9 ± 2.5 µmol/min/mg of protein, which subsequently linked with imidacloprid resistance. In future, there is crucial need to reduce P450 activity which increase the susceptibility to imidacloprid through silencing of CYP4 genes in adult D. citri (Killiny et al., 2014). Insecticide resistance management becomes a serious threat due to insecticide failures in the field. By monitoring detoxifying enzymes of currently used insecticides we practiced a strategy to obtain optimal resistance management. Use of enzyme inhibitors, such as piperonyl butoxide, diethyl maleate and triphenyl phosphate, for cytochrome P450, GST and carboxylesterase, respectively may be useful in cases where increased enzymatic detoxification is contributing to resistance (Terriere, 1984; Ishaaya, 1993). Biochemical assays explain that the insensitivity of different insecticides is likely the biochemical basis of conventional and novel mode of action insecticides resistance in D. citri. A proactive approach has been taken to characterize the molecular and biochemical response of D. citri to insecticides for optimization of rotation schedules (Tiwari et al., 2011a; Coy et al., 2016). 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