Resistance to deltamethrin and fenitrothion in dubas bug, Ommatissus lybicus de Bergevin (Homoptera: Tropiduchidae) and possible biochemical mechanisms

Environmental pollution, ill-effects on human health, insecticide resistance development and insect pest resurgence are some serious problems that may arise due to excessive chemical spraying for pest control. Despite of heavy aerial and surface insecticide spraying, incomplete control of Ommatissus lybicus de Bergevin 1930 (Homoptera: Tropiduchidae) is reported in Oman every year, which requires investigation of insecticides resistance in pest. Fifteen populations of O. lybicus, collected from diverse vicinities were exposed along with a deltamethrin-selected (DEL-SEL) and lab-susceptible (LAB-SUS) strain to deltamethrin and fenitrothion insecticides in bioassay tests for estimation of their resistance status. All the field populations of O. lybicus, exhibited minor (RR = 3–5-folds) to low (RR = 5–10-folds) levels of resistance to deltamethrin, however, two out fifteen populations collected from Al-Hajir and Sint were found susceptible against fenitrothion (RR < 3-folds). Enzyme assays were conducted to detect the activities of cytochrome p-450-reductase (CPR), glutathione s-transferase (GST) and acetylcholinesterase (AChE) in the field collected, DEL-SEL and LAB-SUS strains of O. lybicus. Results revealed significantly increased activities of all enzymes in the field collected as well as DEL-SEL strains of O. lybicus when compared with LAB-SUS strains.


Results
Reference line susceptibility. The mortality scores of the laboratory reared nymphs of O. lybicus LAB-SUS strain were used as reference values for estimation of resistance and the corresponding LD 50 values were used to calculate the resistance ratios (RRs) in the field collected populations. Both insecticides (deltamethrin and fenitrothion) showed highest toxicities to the LAB-SUS reference strain by displaying lowest LD 50 values of 0.108 and 0.009 ppm, respectively. In comparison with the reference strain, neither the 95% CI values of field populations overlapped, nor the resistance ratios (RRs) contained 1 (Tables 1 and 2).
Deltamethrin resistance in O. lybicus field populations. The entire field populations showed some resistance (minor to low levels) against deltamethrin by displaying resistance ratios between 3-10-folds (Table 1). Highest resistance ratio (8.16-fold) was determined in the insects collected from Baa' d with LD 50 value of 0.881 ppm and exhibited a low level of resistance against deltamethrin. The insects collected from Al-Ruddah, Al-Ghabbi, Al-Mudhairib, Ghiyazah and Hail Al-Khanabisha also exhibited low levels of resistance against deltamethrin with resistance ratios ranging from 5.08 to 8.16-folds. The O. lybicus strain selected with deltamethrin also displayed a low level of resistance (RR = 7.36-folds) when tested with deltamethrin, with LD 50 value of 0.795 ppm. Other populations collected from Miss, Al-Kharma, Sayyah, Al-hajir, Al-Hajar, Al-Waqba, Sint, Falaj Al-Maragha and Al-Wasil exhibited minor levels of resistance against deltamethrin and displayed resistance ratios between 3.22 to 4.70-folds. Comparison of the toxicity values of deltamethrin against field collected populations and LAB-SUS strain demonstrates that no field strain was found susceptible against deltamethrin. fenitrothion resistance in O. lybicus field populations. Five out of fifteen O. lybicus field populations unveiled low levels of resistance (RR = 5-10-folds), whereas, eight populations displayed minor levels (RR = 3-5folds), when the representative toxicity values were compared with those of the LAB-SUS strain against fenitrothion (Table 2). Highest level of resistance (7.22-folds) was determined in the insects collected from Baa' d with LD 50 value of 0.065 ppm. The same strain also exhibited a low level of resistance against deltamethrin. Insects collected from Hail Al-Khanabisha, Al-Ruddah, Al-Ghabbi and Ghiyazah also displayed low levels of resistance against fenitrothion with resistance ratios ranging between 5.11 and 5.89-folds. Minor levels of resistance (RR = 3-5-folds) were estimated in the insects collected from Al-Hajir, Miss, Al-Mudhairib, Al-Kharma, Sayyah, Falaj Al-Maragha, Al-Wasil and Al-Waqba. Insects collected from Al-Hajar and Sint were found susceptible by displaying low toxicity scores (RR < 3-folds) and LD 50 values of 0.025 and 0.019 ppm, respectively. Table 1. Toxicity of deltamethrin in various field populations of Ommatissus lybicus under laboratory conditions. *n = number of test insects, **LD 50 = median lethal concentration (ppm), ***CI = confidence intervals, ****RR = resistance ratio, and + = significantly different from the LAB-SUS (Reference) strain (95% CIs of RR didn't include 1).

Discussion
In Sultanate of Oman, O. lybicus is reported to pose serious challenges for devising effective management strategies. Spraying of insecticides is the major pest control option which is carried out through area wide aerial applications using helicopters 10 fitted with ULV spray equipment and through high pressure water pumps for ground and surface applications 12 . Emulsifiable concentrate (EC) and ultra-low volume formulations of organophosphorus and pyrethroid insecticides are more often used for ground and aerial applications, respectively 12,46,47 . Serious pest infestations in date palm orchards even after heavy aerial and surface spraying confer a challenge for the pest exterminators to devise suitable and environment-friendly pest management strategies 23,27,47,48 . Over and repeated use of pesticides may cause side effects to the non-target organisms and pose health risks to consumers as well as other organisms by contributing in environmental pollution [1][2][3][4][5] . Additionally, the injudicious pesticide spraying may result in insecticide resistance development and resurgence of the target and non-target pests. In order to avoid the unnecessary spraying of chemicals, it is necessary to investigate the status of insecticide resistance and the underlying mechanism for its mitigation. We have reported previously that the field collected populations did not develop any moderate or higher level of resistance against deltamethrin and fenitrothion, but only one strain collected from Wadi Qari was reported to possess intermediate resistance level against fenitrothion 18 . The research presented in this study, however, does not report any field strain to possess intermediate or higher level of resistance against the tested insecticides. A possible reason for the slow development of resistance can be less number of generations in a year and a higher frequency of mutations is crucial for pesticide resistance development 49 .
Some field and laboratory studies were performed to appraise and evaluate the toxicity of certain chemicals against O. lybicus with the objective to pick the effective one for its management 21,23 . Pyrethroids (deltamethrin, bifenthrin and cypermethrin) were mostly reported as effective insecticides in controlling O. lybicus in the field studies 25,28 . However, only one report has surveyed and reported minor and low levels of resistance against deltamethrin and fenitrothion in O. lybicus 18 . We could not find any report describing the underlying mechanism of resistance development in O. lybicus. Elevated and amplified levels of mixed function oxidases in certain pest insects, are reported in literature to aid in resistance development to carbamates, pyrethroids, organochlorines and organophosphates 1,31-37 .
In our study, we also performed enzyme activity assays to estimate the activities of detoxifying enzymes viz., CPR, GST and AChE in the field collected, DEL-SEL and LAB-SUS strains of O. lybicus to determine their possible role in resistance development. The analyses revealed significant increased activities of all assayed enzymes in the field collected as well as DEL-SEL strains of O. lybicus when compared with LAB-SUS strains (DF = 16, P = 0.000). The involvement of CPR in resistance mechanism can be determined because a higher CPR activity was observed in the insects collected from Baa' d, Al-Hajir, Al-Ruddah and Al-Ghabbi. These populations were found resistant against both insecticides i-e deltamethrin (RR = 8.16-folds) and fenitrothion (RR = 7.22-folds). Lowest enzyme (CPR) activities were observed in insects collected from Al-Waqba and Al-Wasil after the LAB-SUS strain and displayed enzyme activity ratios of 1.11 and 1.50-folds, respectively. These populations exhibited minor levels of resistance against both insecticides. The insects from DEL-SEL population displayed highest GST activity whereas, amongst the field collected populations, insects collected from Sint and Al-Mudhairib showed highest GST activities. In our bioassay experiments, insects collected from these locations also exhibited minor to low levels of resistance. Insects collected from Miss, Al-Ruddah, Al-Hajir, Al-Ghabbi, Al-Wasil, Sayyah and Al-Waqba showed moderately higher levels of GST activities and were found with minor to low levels of resistance in bioassay experiments. Role of GST in resistance to certain insecticides including organophosphates, organochlorines and pyrethroids is also documented and described in many insects including mosquitoes and aphids 30,36,[41][42][43][44] .
All the field collected populations and the DEL-SEL strain exhibited raised levels of AChE activity as compared to the LAB-SUS strain. The AChE activities and role of insensitive and altered AChE in resistance to organophosphates and carbamates have been described in previous studies [30][31][32][33]50  Conclusively, the study reveals minor to low levels of resistance development in the field populations of O. lybicus. As a consequence of selection pressure under frequent and heavy spray regimes, the pest may develop even higher levels of resistance due to enhanced activities of detoxifying enzymes. Discontinuation of spraying with deltamethrin and fenitrothion and use of alternate chemical pesticides in rotation can help to mitigate the resistance development in O. lybicus. Further studies are also recommended to investigate more efficient and eco-friendly pesticides for inclusion in pest management program in Omani date palm orchards. Less reliance on conventional pesticides and use of biorational insecticides along with some alternate pest management strategies will help in insecticide resistance mitigation and reducing the environmental pollution.  (Table 4, Fig. 1). The insects were collected from the date palm orchards having medium to high pest infestation and were shifted to the mesh cages (80 × 60 × 50 cm) containing potted date palm off-shoots, maintained under the laboratory conditions of 27 ± 2 °C, 70 ± 5% RH and 12L:12D photoperiod. A lab strain was used as a reference strain (LAB-SUS) which was collected in 2013 from unsprayed date palms and was maintained without exposing to any insecticide under lab conditions. However, some batches of insects from the LAB-SUS strain were used to select successively by exposing to sub-lethal doses of deltamethrin till seven generations 18 . The 13th generation of the same DEL-SEL strain was again tested for its resistance status in our current experiments. experimental procedure. The residue film bioassay protocols were adapted to observe the toxicity of the selected insecticides (deltamethrin and fenitrothion) against different field collected populations of O. lybicus 51,52 . A measured volume (1 ml/petri plate) of each dilution (from range of dilutions determined through pre-bioassays and demonstrating 1-99% mortality of the test insect) was applied to the sterilized petri plates and the lid covers. A complete and even application of pesticide surface-residues were obtained by a continu- www.nature.com/scientificreports/ ous rotation of petri plates which were then set underneath the fume hood for evaporation. The petri plates in control treatment received acetone (without insecticide). Batches of each strain containing counted number of healthy and homogeneous 3rd instar nymphs were released in the pesticide-treated petri plates. After specified interval (48 h) of insect releases in the treated plates, mortality scores (dead insects) were noted separately and analyzed for comparison. The insects with least movement gestures (appendages) upon physical stimulations with the camel hairbrush were considered alive, and vice versa. The bioassays were performed with all necessary precautions and cares to diminish the experimental error. The data for mortality were corrected by using the abbot's formula 53 .

Materials and methods
where, n is number of insects, T is treated, and Co is Control. The enzyme analyses were performed to detect the activities of CPR, GST and AChE in 3 rd instar nymphs of field collected, DEL-SEL and LAB-SUS strains of O. lybicus. The protocols provided by the manufacturer were strictly followed while performing the assays. Briefly, for CPR activity, whole body tissues (fresh samples) weighing 5 mg from each strain were homogenized in 500 µL of ice-cold CPR Assay Buffer after washing with Phosphate-buffered saline (PBS). After incubating for five minutes on ice, samples were centrifuged (1,500 × g) at 4 °C. The supernatant from each sample was transferred to pre-chilled microfuge tube and further centrifuged (12,000 × g) at 4 °C for 5 min. The supernatant was again transferred to pre-chilled tubes and stored on ice until CPR activity assay was performed. A standard curve was obtained from standard dilutions (0, 2, 4, 6,  where 'B' is the amount of G6P (nmol) consumed (calculated through the standard Curve), '∆T' is the reaction time (minutes) and 'P' is the amount of protein sample (mg) added in the reaction well. The whole-body tissues (3-5 mg) were used and rinsed with PBS (pH 7.4) for performing the GST activity assays. The tissues were then homogenized in 50 ml cold buffer (pH 7.0, containing 2 mM EDTA) and were centrifuged at 10,000 × g for 15 min (4 °C). The resultant supernatant was used in GST activity assays. The background wells were set up by adding 170 µL of Assay Buffer and 20 µL Glutathione. The positive control wells contained 150 µL of Assay buffer, 20 µL of Glutathione and 20 µL of reconstituted GST (control). In sample wells, we added 150 µL of Assay Buffer, 20 µL of Glutathione and 20 µL of samples. The reaction was initiated by adding 10 µL of CDNB (1-chloro-2,4-dinitrobenzene) to each well. The absorbance was measured once every minute at 340 nm at 5 time points. The GST activity was calculated as where '∆A 340 ′ is the change in absorbance per minute and was determined as In order to perform the AChE activity assays, supernatant was obtained from homogenization of wholebody tissues (3-5 mg) in 0.1 M phosphate buffer (pH 7.5) and 5 min centrifugation at 14,000 rpm. Briefly, 2 mg reagent was mixed in 200 μL Assay Buffer to prepare the Working Reagent. A volume (200 μL) of each, water and calibrator, were added separately to the wells, along with samples (10 μL) in separate wells. The reaction was initiated by transferring freshly prepared Working Reagent (190 μL) to all sample wells and the plats were tapped briefly to mix well. First absorbance was measured at OD = 412 nm at 2 min and second at 10 min in the plate reader. AChE activity was calculated as follows, where 'OD 10 ′ and 'OD 2 ′ are the OD 412nm values of samples at 10 min and 2 min, respectively. 'OD CAL ' and 'OD H2O ' are the OD 412nm values of Calibrator and water at 10 min. The number '200′ is the equivalent activity of the calibrator under assay conditions. Data analyses. The toxic dose values (LD 50 ) of deltamethrin for test populations, resistance ratios over susceptible (Reference) strain and their confidence interval values were estimated by performing the Probit Analysis 54 of the recorded mortalities in each experiment using Polo Plus software version 1.0 (LeOra software LLC). Treatments with non-overlapping confidence intervals were considered significantly different for their toxicities (LD 50 ). Higher slope numbers (> 0.90) described lower dissimilarity in physical appearances of the treated insects from each field strain 55 . The resistance ratios (RRs) were calculated through the division of median lethal dose of the respective (field/selected) strain and median lethal dose the reference strain. Categories for resistance were determined by following the scale described by Jin and his co-workers 56 i-e susceptible (RR < 3 folds), minor resistance (3 ≤ RR ≥ 5 folds), low resistance (5 ≤ RR ≥ 10 folds), intermediate resistance (10 ≤ RR ≥ 40 folds), high resistance (40 ≤ RR ≥ 160 folds) and extremely high resistance (RR > 160 folds). The values of RRs were measured significantly different only when 95% CI (confidence interval) did not include 1 54 . The results obtained in enzyme activity studies were analyzed for variance and the means were compared by Tukey's HSD test through Minitab 18 software.

ethical statement
Any author did not perform any experiment with humans as participants. All the standard protocols for performing the bioassay and enzyme activity experiments with the insect O. lybicus belonging to phylum Arthropoda, were performed after approval from the Plant Protection Research Center, Ministry of Agriculture and Fisheries, Oman.