Insecticide resistance status in the whitefly, Bemisia tabaci genetic groups Asia-I, Asia-II-1 and Asia-II-7 on the Indian subcontinent

The present study is a summary of the current level of the insecticide resistance to selected organophosphates, pyrethroids, and neonicotinoids in seven Indian field populations of Bemisia tabaci genetic groups Asia-I, Asia-II-1, and Asia-II-7. Susceptibility of these populations was varied with Asia-II-7 being the most susceptible, while Asia-I and Asia-II-1 populations were showing significant resistance to these insecticides. The variability of the LC50 values was 7x for imidacloprid and thiamethoxam, 5x for monocrotophos and 3x for cypermethrin among the Asia-I, while, they were 7x for cypermethrin, 6x for deltamethrin and 5x for imidacloprid within the Asia-II-1 populations. When compared with the most susceptible, PUSA population (Asia-II-7), a substantial increase in resistant ratios was observed in both the populations of Asia-I and Asia-II-1. Comparative analysis during 2010–13 revealed a decline in susceptibility in Asia-I and Asia-II-1 populations of B. tabaci to the tested organophosphate, pyrethroid, and neonicotinoid insecticides. Evidence of potential control failure was detected using probit analysis estimates for cypermethrin, deltamethrin, monocrotophos and imidacloprid. Our results update resistance status of B. tabaci in India. The implications of insecticide resistance management of B. tabaci on Indian subcontinent are discussed.

Insecticide bioassays. Insecticide bioassays were conducted in 2013 to generate dose response data for the B. tabaci populations (from different geographic locations) against OP, pyrethroid, and neonicotinoid compounds. The results of dose response regressions analyzed by probit analysis are shown in Table 2. The χ 2 analysis showed that dose responses of all the tested populations fitted the log-dose probit mortality model and the linearity was rejected only for cypermethrin against New Delhi and for Nadia populations ( Table 2). Resistance ratios were computed separately for Asia-I and Asia-II-1 populations in comparison to the most susceptible B. tabaci population within the respective genetic groups for each insecticide. In the absence of a characterized susceptible strain, we have also computed resistance ratios for the field populations using the PUSA population (Asia-II-7) as the reference check (as it had significantly lower lethal concentration values for all the tested insecticides).
Pyrethroids. The tested B. tabaci populations exhibited the highest slopes in response to the pyrethroids. The slopes of probit response curves ranged from 1.32 to 2.89 for cypermethrin and 1.33 to 4.81 for deltamethrin.
The LC 50 values for cypermethrin were in the range of 194 to 1362 mg L −1 among the Asia-I populations, and 238 to 701 mg L −1 among the Asia-II-1 populations. There was upto threefold increase in resistance ratio in Khandwa (Asia-I); four and a sevenfold increase in resistance ratio values respectively in Ludhiana and Sriganganagar (Asia-II-1) in comparison to the most susceptible populations within the respective genetic groups. However, the magnitude of resistance was high in comparison to the PUSA with Sriganganagar and Ludhiana populations recording respectively 136 and 78 fold resistance to cypermethrin, while, the Khandwa population Scientific RepoRts | 7:40634 | DOI: 10.1038/srep40634 was showing 70 fold resistance to this pyrethroid. The LC 50 values for deltamethrin ranged from 120 to 760 mg L −1 in the Asia-II-1 populations and 128 to 242 mg L −1 in the Asia-I populations. Ludhiana and Sriganganagar showed respectively 76 and 71 fold resistance to deltamethrin in comparison to the PUSA (Table 2).
Organophosphates. Triazophos, monocrotophos, and chlorpyrifos were the tested OP compounds. The slopes of the response lines ranged from 1.50 to 2.86 for triazophos; 1.35 to 1.87 for the monocrotophos and 1.57 to 2.34 for chlorpyrifos. For triazophos, the LC 50 values ranged from 324 to 525 mg L −1 in Asia-II-1 and 445 to 1429 mg L −1 in Asia-I populations of B. tabaci. A threefold increase in resistance ratio to triazophos was observed in Nadia (Asia-I), while, resistance to triazophos was not significant among the Asia-II-1 populations. The Nadia population was found to be showing 27 fold resistance to triazophos in comparison to the reference PUSA population ( Table 2). Analysis of the dose response to monocrotophos showed that the LC 50 values were ranging from 528 to 2114 mg L −1 and 843 to 3934 mg L −1 respectively, in the Asia-II-1 and Asia-I populations resulting in fourfold resistance in Ludhiana and fivefold resistance in Nadia in comparison to the susceptible checks within the respective genetic groups. However, the Nadia (Asia-I) and Ludhiana (Asia-II-1) populations recorded significantly higher resistance ratios of 44 and 24 in comparison to the PUSA (Asia-II-7). Among the OP compounds, the chlorpyrifos recorded significantly lower LC 50 values of 137 to 201 mg L −1 and 56 to 309 mg L −1 respectively in the Asia-II-1 and Asia-I populations. Comparisons within Asia-I and Asia-II-1 showed a fivefold increase in resistance ratio to chlorpyrifos in Guntur (Asia-I), while no significant increase in resistance ratio was noticed among the Asia-II-1 populations. However, the two Asia-I populations from Guntur and Amravati were showing respectively 25 and 18 fold resistance to chlorpyrifos in comparison to the PUSA population.

Neonicotinoids.
Imidacloprid and thiamethoxam were the tested neonicotinoids. The slopes of the response lines to imidacloprid ranged from 1.37 to 2.23 and 1.52 to 2.11 respectively in Asia-I and Asia-II-1 populations. The LC 50 values were in the range of 178 to 901 mg L −1 and 130 to 956 mg L −1 respectively, for the Asia-II-1 and Asia-I populations. Sriganganagar and Nadia were showing respectively fivefold and sevenfold resistance to imidacloprid in comparison to the most susceptible population within the respective genetic groups (Table 2). But, these two populations were found to be showing respectively 18 and 17 fold resistance to imidacloprid in comparison to the PUSA population. For thiamethoxam, the LC 50 values were ranging from 73 to 194 mg L −1 and 23 to 179 mg L −1 respectively, for the Asia-II-1 and Asia-I populations resulting in upto sevenfold increase in resistance ratio of Guntur (Asia-I) & Amravati (Asia-I) and twofold increase in resistance ratio of Sriganganagar (Asia-II-1) in comparison to the susceptible checks within the respective genetic groups. However, in comparison to PUSA, Sriganganagar, Amravati and Guntur populations showed about a sevenfold increase in resistance ratios to thiamethoxam (Table 2).

Chlorpyrifos
Continued positive and significant correlations between triazophos and imidacloprid (r = 0.911, P < 0.05) within the Asia-I populations of B. tabaci. Additionally, a negative correlation was found between chlorpyrifos and most other evaluated insecticides for both the Asia-I and Asia-II-1 populations (Tables 3 and 4).
Control failure likelihood. The analysis of potential control failure likelihood 54,55 was done by extrapolation of resistance dataset generated in this investigation ( Table 5). The analytical test detected possible cases of control failures for both the pyrethroids for all the field populations of B. tabaci barring PUSA, with the expected mortality (4 to 30% for cypermethrin; < 1 to 14% for deltamethrin) at recommended doses were being significantly lower than the lower confidence limits of their estimated LC 50 values (Table 5). Similarly, this test detected  Table 2. Log-dose (mg L -1 ) probit mortality data of B. tabaci populations tested. a Chi-square test for linearity of the dose-mortality response: ***P < 0.001, **P < 0.01, *P < 0.05. b Degrees of freedom. Resistance ratios (RR) with 95% confidence limits indicating the fold-difference for each population in comparison to the most susceptible population at LC 50 and LC 90 . Confidence limits that include 1.0 indicate no significant difference from the susceptible population (Lethal ratio test-Robertson et al. 85 ). c RR = Asia-I or Asia-II-1 populations divided by most susceptible Asia-II-7 population. d RR = Asia-II-1 populations divided by most susceptible Asia-II-1 population. e RR = Asia-I populations divided by most susceptible Asia-I population.
possible control failure for monocrotophos for all the B. tabaci populations except PUSA with the expected mortality at the recommended field dose (150 mg L −1 ) being 3 to 21% (Table 5). This test detected possible control failure for triazophos only for the Nadia population of B. tabaci, with its expected mortality being significantly lower than the lower confidence limits of LC 50 at the recommended dose (800 mg L −1 ). No cases of possible control failures were detected for chlorpyrifos against the chosen B. tabaci populations. Regarding the neonicotinoids, possible control failure was detected only for imidacloprid, in all the B. tabaci populations except PUSA with the estimated mortalities (< 1 to 22% ) at recommended field dose (35.7 mg L −1 ) were being significantly lower than lower confidence limits of their LC 50 estimates. Whereas, for thiamethoxam, this test detected a possible control failure only for Sriganganagar, Ludhiana, Amravati and Guntur populations ( Table 5).  Table 1). The field populations were brought to the laboratory and maintained in separate chambers of insect proof climate control chambers. The mitochondrial cytochrome oxidase 1 sequence analyses revealed that the field populations from the three geographic locations belonged to the same genetic group throughout the course of the investigation with New Delhi and Sriganganagar belonged to Asia-II-1, while, Guntur population was assigned to Asia-I. Changes in dose-mortality responses to imidacloprid, triazophos, and cypermethrin were estimated in the three field populations of B. tabaci during 2010 to 2013 for examining dynamics of resistance (Tables 6, 7 (Table 6).

Monitoring of resistance in
Although the Sriganganagar population was found to be the least susceptible to cypermethrin (LC 50 = 1362 mg L -1 ) and imidacloprid (LC 50 = 901 mg L -1 ) as per dose-response analysis in 2013, there was only threefold rise in the resistance ratio from the baseline susceptibility of these compounds in 2010 ( Table 7). The New Delhi population had also shown the substantial loss in susceptibility to cypermethrin from 2010 to 2013 as denoted by the sixfold rise in resistance ratio from 2010 to 2013 and this population also showed about the threefold rise in resistance ratios to triazophos and imidacloprid during 2013 compared to the baseline LC 50 estimates generated during 2010 (Table 8).

Discussion
The present study is significant because it gives a summary of the current levels of insecticide resistance expressed by B. tabaci populations belonging to Asia-I, Asia-II-1, and Asia-II-7, drawn across geographical areas of India. Susceptibility of these populations was varied with Asia-II-7 being the most susceptible, while Asia-I and Asia-II-1 populations were showing significant resistance to the selected organophosphate, pyrethroid and neonicotinoid insecticides.   Worldwide, new and novel chemistries have been employed for the control of sucking pests. However, older chemistries are continued to be in use in India, because they are less expensive. The results of our survey in major cotton growing regions in India have also proved this point. The dose response analysis (Table 2) suggests the development of significant resistance to monocrotophos and to a lesser extent to triazophos and chlorpyrifos in Asia-I and Asia-II-1 genetic groups of B. tabaci across geographical areas of India. Compared to an earlier report, there has been a significant increase in the levels of resistance to these OP compounds in the contemporary populations of B. tabaci 12 . Higher intensity of the insecticides use (frequency, dose, space) leads to genetically based resistances in insects over time 56 . Very high levels of resistance to monocrotophos noticed in this study in Indian B. tabaci populations, with a magnitude of resistance recorded being higher than ever before, could be attributed to the large scale use of this OP compound by the Indian farmers 57 . Similarly, high levels of resistance to triazophos noticed in the B. tabaci from Nadia could also be attributed to long term exposure of this B. tabaci population to triazophos. Increased frequency of insecticide usage on vegetable crops has been documented in this region 58 . Varying levels of resistance to triazophos has earlier been recorded in Asian genetic groups of B. tabaci from India (resistance ratio = 3) 10 , Pakistan (resistance ratio = 42) 37 and in MEAM 1 genetic group of B. tabaci from Turkey (resistance ratio = 310) 38 . Incipient resistance to chlorpyrifos observed in this study is comparable to the reports on the occurrence of 14 fold resistance to this compound in Asian genetic groups of B. tabaci from Pakistan 37 .
Low to moderate (resistance ratios ranged from 5 to 45 fold) resistances to cypermethrin have earlier been reported in the B. tabaci populations (which may be belonged to Asia-I considering reports of the predominance of Asia-I in this region 53 ) from southern India 12 . Our data has shown a considerable increase in the level of resistance to cypermethrin in the contemporary B. tabaci Asia-I and Asia-II-1 populations in India compared to the earlier records 12 . Especially, Ludhiana and Sriganganagar locations from northern India recorded a high level of resistance to pyrethroids ( Table 2). It may be pertinent to note that this region has been an endemic area of cotton leaf curl disease vectored by B. tabaci. We hypothesize that regular outbreaks of cotton leaf curl disease and significantly increased usage of insecticides, including pyrethroids for controlling the vector, could have triggered strong selection pressure for resistance development in these B. tabaci populations. The increased use of pyrethroids was found to be one of the factors linked to the recent outbreak of whitefly in cotton belts of Punjab province of India during 2015 13 . Resistance to pyrethroids has been documented in Asia-I 35  Significant resistance to imidacloprid recorded in this study could be attributed to the long term exposure of this compound in the cotton ecosystem of this country. Since the inception of commercial Bt cotton cultivation in India during 2002, every Bt cotton seed has been mandatory treated with a seed dressing formulation of imidacloprid, besides the application of foliar sprays of imidacloprid by farmers for control of sucking pests including whitefly on cotton 7 . Consequently, the imidacloprid seed treatment which had earlier conferred protection against sucking pests upto at least 40 to 45 days after sowing (DAS), was later reported to provide protection for only upto 20-25 DAS 63 . Resistance to neonicotinoids has widely been documented in Asia-I 35  Paired comparisons of the log LC 50 values for the insecticides showed significant positive correlations between OP, Pyrethroid and neonicotinoid compounds and a negative correlation was found between chlorpyrifos and other insecticides evaluated in the B. tabaci Asia-I and Asia-II-1 populations (Table 4). In line with the revelation of several earlier works, we speculate the possibility of cross resistance between imidacloprid, OP and pyrethroid compounds. The concurrent occurrence of high levels of resistance to OPs and pyrethroids had been observed in West Africa, Pakistan, and Turkish B. tabaci populations 17,37,38 . Inconsistency in the neonicotinoid cross-resistance pattern has been reported by Prabhaker et al. 41 and by Horowitz et al. 30 . Earlier reports from china 66 and US 43 have also demonstrated the prevalence of cross resistance between imidacloprid and thiamethoxam in an MEAM genetic group of B. tabaci 34 , while, studies with Cyprus populations of B. tabaci (MEAM 1 genetic group) revealed the absence of cross resistance between these two neonicotinoid compounds 21,67 . Besides target site insensitivity, one or more metabolic resistance mechanisms involving carboxylesterases,  3 . However, there is a limited literature available on the insecticide status of indigenous B. tabaci genetic groups of Asia. This study clearly provided the insecticide resistance/susceptibility status of Asian genetic groups like Asia-I, Asia-II-1, and Asia-II-7 against the selected OP, pyrethroid, and neonicotinoid compounds. As insecticide resistance is regarded by some workers as a major driving force for the selection and establishment of specific B. tabaci genetic groups in a region 19,30,31,51 , there is a need for regular monitoring of insecticide resistance status in diverse B. tabaci genetic groups in India.
Knowledge on the susceptibility level of insect populations from different geographical areas is critical for measuring the trends in temporal and spatial resistance development of B. tabaci 45 . Likewise, our studies have established the decrease in susceptibility levels of three B. tabaci populations to select OP, pyrethroid, neonicotinoid insecticides during 2010 to 2013 (Table 5) and the trend clearly showed the evolution of significant resistances to these insecticides in North Indian field populations of B. tabaci. The recent outbreak of whitefly in the Punjab state of India would appear to be at least partly due to the manifestation of significant resistance development in the field populations of B. tabaci 13 .
The higher values of LC 50 along with the high value of the slopes (Table 2; Figs 2, 3 and 4) may be indicating significant resistance development in Indian field populations of B. tabaci. Chilcuit and Tabashnik 68 proposed that slope was not a good indicator of the genetic variability in susceptible organisms, and further, that genetic variation was not related to the LC 50 values. However, Hussain et al. 69 opined that the higher inter-population variations in the slopes coupled with high level of resistance to the insecticides indicated the possibility of an existence of qualitatively different resistance mechanisms in field strains of H. armigera in Pakistan. Hence, further studies are needed to unravel the biochemical and molecular basis of resistance to these compounds in Indian B. tabaci populations.
The potential for control failure of insecticides was estimated by use of analytical tools as described in Silva et al. 54 and Roditakis et al. 55 . Our results (Table 5) indicate the likelihood of control failures for insecticides such as monocrotophos, imidacloprid, cypermethrin and deltamethrin at the recommended label rates in the selected field populations. Nevertheless, that it was only an estimate and was not based on a rigorous assessment of actual control efficacy of the said chemicals against the field populations. Our results suggest that the field dose of these chemicals have to be higher than the recommended label rate of Central Insecticides Board and Registration Committee, Government of India, to have effective control of B. tabaci at least in these regions. Although insecticide quality is legitimately regulated in India, factors such as poor knowledge on the selection of chemicals by the farmers, use of unscientific tank mixtures and sub-standard application practices exacerbate the problem of control failure of insecticides in field conditions 12,63 . Hence, appropriate field tests are needed to verify the bioefficacy of these chemicals at recommended label rates against these populations of B. tabaci.
Integrated Pest Management (IPM) has been the overriding principle of plant protection in India and greater emphasis is laid on reducing dependency on chemical control in several crop pests. As our results have shown the widespread development of resistance to OP, pyrethroids and neonicotinoids in the B. tabaci genetic groups, Asia-I and Asia-II-1, we emphasize the need for undertaking regular monitoring of insecticide resistance status of different B. tabaci genetic groups across India. The management of this pest in India may be strengthened by taking clues from successful global IPM programmes.   Host plant resistance is a major, often preventative measure for managing B. tabaci. Studies have shown that pubescent varieties are more preferred by B. tabaci as compared with glabrous ones 70,71 . Natural defenses in a wild species of cotton, Gossypium arboretum, including long trichome or presence of inorganic salts with increased concentration of waxes provide protection against whitefly and cotton leaf curl virus 72 . Increasing the area under indigenous varieties of G. arboreum may mitigate the frequent epidemics of whitefly and cotton leaf curl virus especially in Northwestern India.
Rotational scheme of insecticides with different modes of action has been found effective in insecticide resistance management of B. tabaci in Israel. Application of pyriproxyfen in cotton during the first month, followed by an additional treatment with buprofezin (if required), do not markedly alter the susceptibility of B. tabaci to either compounds or no appreciable increase of resistance to the conventional insecticides 73 . Application of insect growth regulators like pyriproxyfen or buprofezin during the early stage of crop growth is found effective in controlling MEAM 1 genetic group of B. tabaci in Arizona, USA, as these insect growth regulator compounds have helped to conserve natural enemies and substantially reduce sprays of broad-spectrum insecticides 74 .
The refuge strategy is mandated by the regulatory authorities worldwide to manage the evolution of resistance in bollworms targeted by Bt cotton. Simulation analysis has shown the effectiveness of this strategy in delaying insecticide resistance in MEAM 1 genetic group of B. tabaci 75 . Although B. tabaci is polyphagous, the cotton refuges have been particularly found more useful in delaying insecticide resistance development in B. tabaci 76 .
Therefore, a comprehensive, integrated pest management and insecticide resistance management strategies, including identification of whitefly resistant Bt hybrids and G. arboreum genotypes, rotation of conventional insecticides with novel molecules including insect growth regulator (IGR) compounds, use of sticky traps and exploitation of native biological control agents will augur the sustainable management of B. tabaci in the Indian subcontinent.

Methods
Whitefly collection, rearing, and maintenance. The field populations of B. tabaci were collected from seven locations across eastern, central, southern and northern regions of India. Geographically, these locations fall under five agro-climatic zones of India (India has 15 agro-climatic zones). Uniform whitefly infestation pattern and easy accessibility encouraged us to select these regions for the collection of B. tabaci populations. To generate adequate information on the use of insecticide on cotton and vegetable fields, Knowledge-Attitude-Practice (KAP) surveys were conducted in 2010 to 2013 in the study sites by following the protocol used by Yadouleton et al. 77 . Briefly, ten farmers in each locality were interviewed by using a semi-structured questionnaire focussing on the insecticide application pattern in the farms. Further, qualitative data were collected through direct observations and group discussions. The descriptions of the collection sites, the period of the collections, genetic group identity of B. tabaci populations and the background information on cropping pattern and insecticide application details are presented in Table 1. The exact locations of the collection sites are presented in Fig. 1.
While collecting, standard procedure was followed by walking in 'Z' mode at a minimum of two-hectare blocks of the crops. Insects were collected using an aspirator during early morning along with infested leaves containing the nymphs and pupae. The insects were transported to the laboratory in ventilated cages containing leaflets inserted into wet sponges. Infested leaflets were kept in cages for the emergence of fresh adults. The taxonomic identity of B. tabaci species complex was confirmed by examining the insects under a light microscope using the keys of Martin et al. 78,79 . These populations had been raised on insecticide-free cotton plants (G. hirsutum.) at temperatures of 27 ± 2 °C, photoperiod of 14:10 h (Light:Dark) and relative humidity of 60-70% in quarantined insect growth chambers. These populations were maintained as large colonies for five generations without insecticide selection prior to the current bioassays. Insecticides. Purity analyzed technical grade insecticides such as triazophos (60.9%), monocrotophos (99%) and chlorpyrifos (60%); cypermethrin (99.3%); imidacloprid (96.4%), thiamethoxam (98%) and deltamethrin (98%) were procured from the insecticide manufacturers. These insecticides were selected, as they represented the OPs, pyrethroids and neonicotinoids concurrently used for control of whitefly in the respective regions where the whitefly populations were collected (Table 1). These compounds also had the label claim for efficacy against whiteflies as per the registered use of pesticides with Central Insecticides Board and Registration Committee, Government of India as on date (http://www.cibrc.nic.in).

Bioassay.
For assessing the insecticide toxicity to B. tabaci, a modified leaf dip bioassay method of Insecticide Resistance Action Committee was followed 82 . The stock solutions of technical grade insecticides were prepared in acetone, with serial dilutions in deionized water containing 0.1 g L -1 of non-ionic wetting agent Triton X-100. Cotton leaves with petiole, collected from the fifteen to twenty-five days old seedlings were immersed in the serially diluted insecticide solutions for 20 sec; then allowed to air dry on paper towel and kept on agar slants (2%) in Petri plates (90 × 15 mm). Leaves dipped in only diluents served as the untreated control. The adults were briefly anesthetized using CO 2 and transferred in batches of 15-20 onto the treated leaves. The plates were sealed with ventilated lids. All such assays were replicated five times for a minimum of five concentrations for each insecticide. All treatments were placed in an insect rearing room with the temperature, photoperiod, and RH conditions as mentioned earlier. As the mortality rate was too low at earlier hours in some of the doses, observations were taken for an extended period 96 h as described by Gorman et al. 16 . The adult insect was considered to be dead if no coordinated movement or deficient response to external stimulus (i.e. when gently probed with a fine paintbrush) was observed under the light microscope. Mortality was estimated by counting the total number of dead and live insects.

Monitoring insecticide susceptibility in B. tabaci populations.
To compare the changes in susceptibility of B. tabaci populations over time, the populations were collected from same cotton fields located in New Delhi, Sriganganagar, and Guntur (the location details and time of collections are furnished in Table 1). The collection, maintenance and genetic group identity of these B. tabaci field populations were done as described in the earlier section. Dose responses were generated for three insecticides viz., triazophos, cypermethrin, and Imidacloprid during 2010, 2012 and 2013. The details of field populations and their genetic group identities are presented in Table 1.
Data analysis. The mortality data were corrected according to Abbott's formula 83 . The LC 50 and LC 90 values, 95% confidence limits, standard errors, the slopes of the regression lines and χ 2 significance tests, were estimated by probit analysis 84 using PoloPlus 2.0 software (LeOra Software, California, United States). The resistance ratios were calculated by the "lethal ratio test" and were considered significant when the confidence limits at 95% did not include the value one as proposed by Robertson et al. 85 . The resistance ratio for each insecticide was calculated with reference to a population of the same genetic group with the lowest LC 50 or LC 90 17,35 . More specifically, resistance ratio = LC 50 or LC 90 of each population was divided by the LC 50 or LC 90 of the most susceptible population within Asia-I or Asia-II-1. In the absence of a characterized susceptible strain, the actual resistance level could be underestimated by mere comparisons within the genetic groups. Therefore, additional resistance ratios were also computed for each insecticide with reference to the most susceptible B. tabaci population (belonging to Asia-II-7 collected from New Delhi and designated as PUSA) to demonstrate the magnitude of resistance development in Indian B. tabaci populations in the present dataset.
Pearson correlation coefficient (r) test was applied to test the significance of pairwise comparison between the different attributes (log LC 50 ). The correlation analysis was conducted using SPSS version 16  , and imidacloprid (c). The dose response lines of the each population were drawn using a probit linear model y = α x + β in which α and β are the slope and intercept, respectively. x is the log-transformed dose (mg L −1 ). y is the percent mortality.  , and imidacloprid (c). The dose response lines of the each population were drawn using a probit linear model y = α x + β in which α and β are the slope and intercept, respectively. x is the log-transformed dose (mg L −1 ). y is the percent mortality.