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Selection of bifenthrin resistance in cotton mealybug Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae): Cross-resistance, realized heritability and possible resistance mechanism
Crop Protection 87 (2016) 55e59
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Selection of bifenthrin resistance in cotton mealybug Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae): Cross-resistance, realized heritability and possible resistance mechanism Muhammad Mudassir Mansoor a, **, Muhammad Babar Shahzad Afzal b, *, Esteban Basoalto c, Abu Bakar Muhammad Raza a, Ansa Banazeer d a
Department of Entomology, University College of Agriculture, University of Sargodha, Sargodha, Pakistan Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan n y Sanidad Vegetal, Facultad de Ciencias Agrarias, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile Instituto de Produccio d Department of Entomology, Muhammad Nawaz Sharif University of Agriculture, Multan, Pakistan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2016 Received in revised form 21 April 2016 Accepted 29 April 2016 Available online 1 June 2016
Cotton mealybug Phenacoccus solenopsis Tinsley is an important pest of cotton in Pakistan, and its management is difficult due to the development of insecticide resistance. This research was conducted to characterize the bifenthrin resistance in populations of P. solenopsis and different parameters such as cross-resistance, realized heritability and possible resistance mechanisms were studied to improve the management of this important pest. A field-collected population was selected with bifenthrin in the laboratory for 14 generations and developed a resistance of 178-fold. The realized heritability of bifenthrin resistance was 0.54 in the selected population. The toxicity of bifenthrin was synergized by the addition of either piperonylbutoxide (PBO) or S,S,S tributylphosphorotrithioate (DEF) which suggests a general metabolic resistance due to possible involvement of mono-oxygenases or esterases. However, the resistant population did not develop a significant cross-resistance to either buprofezin, chlorpyrifos or lambda-cyhalothrin. These data suggest that alternative insecticide-based management programs can be developed for this pest in the short-term, but resistance management strategies which can reduce the sole reliance on insecticides are still needed. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Mealybug Insect resistance Bifenthrin Synergist
1. Introduction Insecticides are commonly used to improve agricultural productivity in developing countries (Karunamoorthi et al., 2012), and are known as effective tools for numerous pests of agricultural importance (Sayyed and Crickmore, 2007; Ishtiaq and Saleem, 2011; Afzal et al., 2015a). Insecticide use improves productivity by minimizing the potential of insect pests to create infestations, but also on the other hand increases production costs and can lead to the development of resistance in insect pest species (Metcalf, 1989; Siqueira et al., 2001). Insects build up resistance to insecticides through uninterrupted utilization as well as by using the natural
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M.M. Mansoor), Shahzad.babar35@ gmail.com (M.B.S. Afzal). http://dx.doi.org/10.1016/j.cropro.2016.04.026 0261-2194/© 2016 Elsevier Ltd. All rights reserved.
phenomenon of cross-resistance that develops because of previous exposure to different insecticides (Basit et al., 2011, 2013). In heavily sprayed crops like cotton, a suitable resistance management plan is required having potential to fulfill the pest management demand while decreasing the selection pressure on special target sites within the insect (Young et al., 2003). Multiple kinds of chewing and sucking pests attack the cotton crop during the season, creating damage to crop productivity and yield (Afzal et al., 2015a). Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae) is an important polyphagous pest and has caused significant yield losses to cotton crops for growers in some regions of Pakistan and other countries in Asia and in the United State of America (Abbas et al., 2005; Mahmood et al., 2011; Kumashiro et al., 2001; Nagrare et al., 2009; Wang et al., 2009; Hodgson et al., 2008). More than 40% losses of the cotton crop have been reported from Pakistan during 2007 as a result of P. solenopsis infestation (Pakistan cotton statistics, 2007). Management of P. solenopsis in Pakistan and many other countries has
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relied on broad-spectrum synthetic pyrethroid insecticides (Saddiq et al., 2014). Bifenthrin is commonly recommended for sucking pests in cotton including P. solenopsis (Saeed et al., 2007). Farmers have applied this insecticide in cotton fields to suppress the P. solenopsis populations but excessive applications of bifenthrin eventually resulted in resistance development. Bifenthrin is an insecticide among synthetic pyrethroids having stomach as well as contact activity (Thomson, 1998). It mainly acts upon sodium channels of insect nervous systems, causing these channels to open for a long time, thus increasing cell permeability and ultimately causing the death of insects (Brown, 2005). Some basic knowledge about the extent of the resistance and the potential for cross-resistance to other classes of insecticides is essential to develop insecticide resistance management strategies (Roush and Croft, 1986). Also, characterizing the genetic heritability and the potential mechanisms of insecticide resistance are important in order to develop alternative programs. These data can guide pest managers to develop programs that allow reversion of existing resistances and to select alternative tools that can act independently of the current resistance mechanisms. We studied resistance to bifenthrin in a field population of P. solenopsis after its selection in the laboratory. To understand the possible mechanism underlying bifenthrin resistance, the involvement of metabolic enzymes was evaluated by using synergists. According to authors' best knowledge; it is the first report of bifenthrin resistance in P. solenopsis. 2. Materials and methods 2.1. Insecticides and synergists Four commercial insecticides including bifenthrin (Talstar 10 EC, FMC, Pakistan), buprofezin (Fuzin 25 WP, 4B Group, Pakistan), lambda-cyhalothrin (Karate 2.5 EC, Syngenta, Pakistan) and chlorpyrifos (Lorsban 40 EC, Arysta Life Sciences, Pakistan) were used in this study. Two synergists including an esterase specific inhibitor, S,S,S tributylphosphorotrithioate (DEF; Sigma Ltd, UK) and a cytochrome P-450 monooxygenase inhibitor, piperonylbutoxide (PBO; Sigma Ltd, Poole, UK) were used for the synergism tests. 2.2. Insect collection and rearing About 500 nymphs and adults were collected from a cotton field in Multan District. This field had a known history of frequent insecticide applications using a number of compounds each season. The cotton fields in that area receive heavy applications of different insecticides mainly of organophosphates (e.g., profenofos, chlorpyrifos, methamidophos), pyrethroids (e.g., bifenthrin, deltamethrin, cypermethrin, lambda-cyhalothrin) and some new chemistry insecticides (e.g., acetamiprid, imidacloprid, buprofezin) for the control of different cotton pests including P. solenopsis. The field collected population was brought in the laboratory and insects were reared by maintaining standard laboratory conditions specifically at 16:8 h (L: D), 27 ± 1 C and 65 ± 5% R.H. This population was designated as Field Pop. Fresh tender shoots and leaves of China rose, Hisbiscus rosa-sinensis Linneaus were used as food of P. solenopsis and food was renewed after every 2 days. Transparent plastic jars (24 10 cm) were used to rear the insect. An insecticide-susceptible strain (Lab-PK) of P. solenopsis was reared in the Pak Arab Biological Control Laboratory (Fatima Sugar Research and Development Center, Muzaffargarh) for more than three years after its collection from cotton field of the Central Cotton Research Institute, Multan Pakistan. The population which was selected from the field population after performing base-line bioassays was called Bifen-Sel.
2.3. Bioassays Bioassays were performed on Field Pop, Bifen-Sel and Lab-PK. Aqueous solutions of insecticides were prepared and serially diluted. Four concentrations of each insecticide were used in each insecticide bioassay. Three-day-old second instar nymphs were used in the bioassays. Fresh leaves of H. rosasinensis were used for bioassay according to the Leaf-dip method (Ahmad et al., 2007; Afzal et al., 2015a). The leaves after dipping for 10 s in insecticide solution were placed in Petri dishes (5 cm in diameter) with moist filter papers (Afzal et al., 2015a) and insects were exposed to insecticide treated leaves. A total of 40 nymphs, 10 nymphs per replication, for each insecticide concentration were used. For control, 20 nymphs with water-treated leaves were kept in Petri dishes. Numbers of insects tested in single bioassay including control were 180. Mortality data of bioassays was obtained after 48 h of treatment with bifenthrin, lambda-cyhalothrin, and chlorpyrifos, while for buprofezin, mortality data was taken after 96 h exposure. The nymphs were touched individually by using camel hair brush to check mortality and non-moving nymphs were considered dead. 2.4. Selection Nymphs were selected at every generation from G1 to G14 with bifenthrin. The concentrations for selection were determined by performing bioassay of bifenthrin on Field Pop. This bioassay provided different lethal concentrations that were then used for selection. Leaf dip method with same protocol as mentioned above was used for selection of population. About 100 to 200 nymphs were selected per generation (Table 1). Mortality was judged 48 h after insecticide exposure. 2.5. Synergism analysis PBO and DEF concentrations were tested to recognize the uppermost non-lethal dose. Acetone (analytical reagent; Fisher Scientific, Lough borough, UK) was mixed with PBO or DEF and mixed in serial solutions having insecticide concentrations. The nonlethal concentrations, 5 mg/ml PBO and 10 mg/ml DEF, were used for Bifen-Sel (G15), while a concentration (1 mg/ml) of both synergists was used for Lab-PK. For the control acetone was used alone. Bioassays were performed according to method described above. 2.6. Data analysis of bioassays The toxicological data was analyzed with POLO software (LeOra Software, 2005) by probit analysis (Finney, 1971) to determine the LC50 (median lethal concentration) values, their standard errors, slopes, and confidence intervals (CIs). Synergism ratio (SR) for synergism bioassays was evaluated as follows: SR ¼ LC50 of insect population exposed to insecticide/LC50 of population exposed to insecticide with synergist. 2.7. Realized heritability estimation Realized heritability (h2) was estimated by using the method of Falconer et al. (1996) and Tabashnik (1992) by the following equation. h2 ¼ Selection response/Selection differential Selection response was calculated as: Selection response ¼ (Log final LC50 Log Initial LC50)/N
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Table 1 Selection history of Phenacoccus solenopsis with bifenthrin. Generation (G)
Concentration (mg/ml)
Number of insects exposed
Number of insects died
% Mortality
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14
3.766 3.766 7.496 14.921 17.56 22.351 35.143 35.143 35.143 35.143 35.143 35.143 35.143 35.143
200 200 200 200 200 200 200 200 200 200 100 100 100 100
176 154 137 121 103 89 74 61 53 42 28 11 8 8
88 77 68.5 60.5 51.5 44.5 37 30.5 26.5 21 28 11 8 8
3.2. Bifenthrin resistance selection and its cross-resistance with other insecticides
Selection differential was calculated as: Selection differential ¼ i s p Hence i is the intensity of selection, calculated according to Falconer (1989) and the sp is the phenotypic standard deviation calculated as:
sp ¼ [(initial slope e final slope) 0.5]1
Selection with bifenthrin increased the resistance from 3.28 (Go) to 178.42-fold (G15) in the population of P. solenopsis (Table 2). Bifenthrin selected resistance in P. solenopsis induced very low cross-resistance to buprofezin (1.53-fold), to chlorpyrifos (1.62fold) and to lambda-cyhalothrin (1.98-fold) at G15 when compared with Field Pop (Table 2). 3.3. Estimation of realized heritability
3. Results 3.1. Toxicity comparison of various insecticides to field pop and LabPK populations For the Field Pop at Go, the toxicity of bifenthrin was significantly higher compared to buprofezin and chlorpyrifos, but it was significantly lower than that of lambda-cyhalothrin (95% CI did not overlap). The toxicities of buprofezin and chlorpyrifos remained statistically similar (95% CI overlap) when these insecticides were tested on Field Pop Go. For the Lab-PK population, the toxicity of bifenthrin was similar (95% CI overlap) to buprofezin, but higher (95% CI did not overlap) than that of chlorpyrifos. The toxicity of lambda-cyhalothrin was significantly higher (95% CI did not overlap) when compared to all other insecticides tested at Lab-PK (Table 2).
After 14 generations of consecutive selection with bifenthrin, the LC50 value of bifenthrin increased from 7.49 to 406.8 mg/ml and slop decreased from 3.46 to 2.16 in the Bifen-sel. Realized heritability (h2) for bifenthrin resistance was 0.54 in P. solenopsis. The numbers of generations estimated to acquire a ten-fold increase in bifenthrin resistance were eight (Reciprocal of R, Table 3). 3.4. PBO and DEF synergism for bifenthrin Lab-PK and Bifen-Sel (G15) populations of P. solenopsis were tested with PBO and DEF to see whether they synergize the toxicity of bifenthrin or not. These synergists decreased the resistance ratio of the Bifen-Sel (G15) from 178.42 to 75.97 and 45.94, respectively (Table 4). Based on the 95% CI comparison of LC50 values, the results indicate that both PBO and DEF have synergistic effect with SR ¼ 2.83 and 3.17, respectively in Bifen-sel. In the Lab-PK, both synergists did not increase the insecticidal activity of bifenthrin
Table 2 Response of Phenacoccus solenopsis populations to different insecticides. Population
Insecticides
LC50 [95% CI] (mg/ml)
Slope ± SE
Field Pop (Go) Field Pop (Go) Field Pop (Go) Field Pop (Go) Lab-PK Lab-PK Lab-PK Lab-PK Bifen-Sel (G15) Bifen-Sel (G15) Bifen-Sel (G15) Bifen-Sel (G15)
Bifenthrin Buprofezin Chlorpyrifos Lambda-cyhalothrin Bifenthrin Buprofezin Chlorpyrifos Lambda-cyhalothrin Bifenthrin Buprofezin Chlorpyrifos Lambda-cyhalothrin
7.49 20.70 25.01 2.76 2.28 1.83 4.73 0.87 406.82 31.70 40.41 5.47
3.46 2.75 2.38 3.44 3.57 3.53 3.89 3.84 2.16 2.39 2.02 3.44
a b
LC50 of Field Pop (Go) or Bifen-sel (G15)/LC50 of Lab-PK. LC50 of Bifen-sel (G15)/LC50 of Field Pop (Go).
(5.76e9.003) (16.18e25.19) (19.41e30.94) (2.05e3.34) (1.73e2.74) (1.21e2.27) (3.32e5.75) (0.56e1.07) (323.22e540.54) (25.304e39.24) (31.63e52.80) (4.59e6.45)
± ± ± ± ± ± ± ± ± ± ± ±
0.60 0.41 0.36 0.63 0.64 0.76 0.84 0.90 0.35 0.35 0.33 0.45
c2
df
P
n
RRa
RRb
4.62 2.72 0.17 0.13 0.07 1.04 0.54 0.39 0.10 1.71 0.16 2.39
3 3 3 3 3 3 3 3 3 3 3 3
0.20 0.43 0.98 0.98 0.99 0.79 0.90 0.94 0.99 0.63 0.98 0.49
180 180 180 180 180 180 180 180 180 180 180 180
3.28 11.31 5.28 3.17 1.00 1.00 1.00 1.00 178.42 17.32 8.54 6.28
54.32 1.53 1.62 1.98
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Table 3 Realized heritability determination. Estimate of mean response per generation
Estimate of mean selection differential per generation
Insecticide
Initiala LC50 (log)
Finala LC50 (log)
Rb
pc
Id
Initial slope
Final slope
spe
Sf
h2g
Bifenthrin
7.49 (0.87)
406.82 (2.61)
0.12
60.00
0.64
3.46
2.16
0.36
0.23
0.54
a b c d e f g
Initial (Go) and final (G15) LC50 values were calculated in mg/mL. Selection response. Average percentage survival of Bifen-sel (G15) after selection. Intensity of selection according to Falconer (1989). Phenotypic deviation. Selection differential. Realized heritability.
Table 4 Synergism tests for bifenthrin resistance in Phenacoccus solenopsis. Population
Treatment
Lab-PK
Bifenthrin Bifenthrin Bifenthrin Bifen Bifenthrin Bifenthrin
Bifen-Sel (G15)
a
þ PBO þ DEF þ PBO þ DEF
LC50 [95% CI] (mg/ml)
Slope ± SE
2.28 1.89 2.79 406.82 143.60 128.18
3.57 2.96 3.12 2.16 2.99 3.15
(1.73e2.74) (1.22e2.40) (2.18e3.37) (323.22e540.54) (111.55e173.92) (98.15e155.22)
± ± ± ± ± ±
0.64 0.59 0.49 0.35 0.46 0.52
c2
df
P
n
RRa
SR
0.07 0.55 0.701 0.1 1.03 1.08
3 3 3 3 3 3
0.99 0.9 0.87 0.99 0.79 0.78
180 180 180 180 180 180
1 1 1 178.42 75.97 45.94
1 1.21 0.81 1 2.83 3.17
LC50 of Bifen-Sel (G15)/LC50 of Lab-PK.
(95% CI overlap) (Table 4). 4. Discussion Synthetic insecticides have been the key tool used in Pakistan since the 1970s against a number of chewing and sucking insect pests of cotton. This continuous reliance on a variety of toxicants has clearly resulted in a reduced product efficacy due to the selection for insecticide resistance in many insect pests (Ahmad et al., 2007; Basit et al., 2011; Ishtiaq and Saleem, 2011; Khan et al., 2014; Afzal and Shad, 2015). Among these synthetic insecticides, bifenthrin, a synthetic pyrethroid has been excessively used by farmers in cotton fields since the introduction of P. solenopsis in Pakistan in the past few years. Our current research has shown that P. solenopsis can develop a high level of resistance to bifenthrin under a regime of continuous selection in the laboratory. In our study, selection of P. solenopsis with bifenthrin for 14 generations amplified the level of resistance to 178.42-fold when compared with Lab-PK and 54.31-fold when compared with Field Pop at Go. Previously, P. solenopsis has also been reported to develop significant levels of resistance following selection in the laboratory to various insecticides such as acetamiprid (315-fold; Afzal et al., 2015a), indoxacarb (2223-fold; Afzal et al., 2015b) and deltamethrin (100-fold; Saddiq et al., 2015). High level of bifenthrin resistance development in selected population of P. solenopsis indicated that frequency of resistant alleles might be higher in field population. Studying resistance and cross-resistance is useful to limit the development of resistance by employing practices such as insecticides mixtures and rotation having different modes of action (Shen and Wu, 1995; Abbas et al., 2015). In our study, selection of P. solenopsis with bifenthrin induced negligible cross-resistance to buprofezin, chlorpyrifos and lambda-cyhalothrin when compared with Field Pop (Table 2). Cross-resistance is a valuable tool to identify insecticide resistance mechanisms (Shad et al., 2010). Cross-resistance can result from non-specific enzymes, such as microsomal oxidases, mutation at an insecticidal target site and some factors such as delayed cuticular penetration (Basit et al., 2011). In this study, very low cross-resistance between bifenthrin and buprofezin was expected as these insecticides are from different classes which exhibit different modes of action. It suggests
that these insecticides can be used in rotational pattern in the field. Similarly, in our study negligible cross-resistance of bifenthrin with lambda-cyhalothrin (pyrethroid) and chlorpyrifos (organophosphate) has practical implications in delaying the development of bifenthrin resistance in P. solenopsis in the field, since both have different mode of actions. Pyrethroids and organophosphates act on the functions of voltage-gated sodium channels and acetylcholinesterase (AChE), respectively (Sayyed et al., 2010). It suggests that bifenthrin can be rotated with lambda-cyhalothrin or alternatively with chlorpyrifos, in an insecticide resistance management (IRM) program of P. solenopsis. The realized heritability of bifenthrin resistance was determined in order to find the genetic variation in the P. solenopsis population. A high level of genetic variation (h2 ¼ 0.54) for the bifenthrin resistance was observed. It shows great additive genetic distinction thus causing significant increase in LC50 values between field collected at Go and Bifen-SEL at G15 for bifenthrin. This indicates that the field collected population of P. solenopsis had a higher frequency of resistance allele (s) than the laboratory population. This finding is consistent with the basic fact that population was obtained from the fields (Multan) where intensive use of several insecticides including pyrethroids and organophosphates for the insect pest control has been reported (Sayyed and Crickmore, 2007; Mansoor et al., 2015). The cotton fields in the area (Multan) receive heavy applications of different insecticides mainly of organophosphates (e.g., profenofos, chlorpyrifos, methamidophos), pyrethroids (e.g., bifenthrin, deltamethrin, cypermethrin, lambda-cyhalothrin) and some new chemistry insecticides (e.g., acetamiprid, imidacloprid, buprofezin) for the control of different cotton pests including P. solenopsis. The resistance development in insect species is mainly the consequence of metabolic activity of different detoxification enzymes (Kang et al., 2006). In order to detect the mechanism pathway responsible for resistance development the synergistic effect of PBO and DEF with bifenthrin was checked for Bifen-sel and Lab-PK populations. PBO and DEF produced a considerable effect on toxicity of bifenthrin in Bifen-Sel but not in Lab PK. Cytochrome P450 mono-oxygenases and esterases seem prominent in bifenthrin resistance development because both PBO and DEF enhanced the toxicity of bifenthrin in the selected population. Resistance to
M.M. Mansoor et al. / Crop Protection 87 (2016) 55e59
bifenthrin was also previously reported due to involvement of monooxygenases (Ahmad et al., 2007) or esterases (Gunning et al., 1999). The current study also confirms that possible connection of P-450 mono-oxygenases and esterases in bifenthrin resistance buildup. Similar results have been reported in deltamethrin resistant Chrysoperla carnea (Sayyed et al., 2010), cypermethrin resistant Amsacta albistriga, (Muthusamy and Shivakumar, 2015), bifenthrin and b-cyfluthrin resistant Amyelois transitella (Demkovich et al., 2015), lambda-cyhalothrin resistant Ceratitis capitata (Arouri et al., 2015), and emamectin benzoate resistant P. solenopsis (Afzal and Shad, 2015). In short, the P. solenopsis exhibits extraordinary potential to develop high levels of resistance under continuous selection pressure of bifenthrin and this could be at least partially due to increased detoxification by the P-450 mono-oxygenases and esterases. This study offers management fundamentals of insecticide resistance in P. solenopsis. Furthermore, the study of genetics of resistance to bifenthrin in the P. solenopsis could be important for resistance progress management. Biological control agents such as C. carnea as well as cultural practices should be incorporated with IRM for the effective control of P. solenopsis. References Abbas, G., Arif, M., Saeed, S., 2005. Systematic status of new species of genus Phenacoccus Cockerell (Pseudococcidae), a serious pest of cotton Gossypium hirsutum L. Pak. Pak. Entomol. 27, 83e84. Abbas, N., Crickmore, N., Shad, S.A., 2015. Efficacy of insecticide mixtures against a resistant strain of house fly (Diptera: Muscidae) collected from a poultry farm. Int. J. Trop. Insect Sci. 35, 48e53. Afzal, M.B.S., Shad, S.A., 2015. Resistance inheritance and mechanism to emamectin benzoate in Phenacoccus solenopsis (Homoptera: Pseudococcidae). Crop Prot. 7, 60e65. Afzal, M.B.S., Shad, S.A., Abbas, N., Ayyaz, M., Walker, W.B., 2015a. Cross-resistance, the stability of acetamiprid resistance and its effect on the biological parameters of cotton mealybug, Phenacoccus solenopsis (Homoptera: Pseudococcidae), in Pakistan. Pest Manage. Sci. 71, 151e158. Afzal, M.B.S., Shad, S.A., Basoalto, E., Ejaz, M., Serrao, J.E., 2015b. Characterization of indoxacarb resistance in Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae): cross-resistance, stability and fitness cost. J. Asia-Pacific Entomol. 18, 779e785. Ahmad, M., Sayyed, A.H., Crickmore, N., Saleem, M.A., 2007. Genetics and mechanism of resistance to deltamethrin in a field population of Spodoptera litura (Lepidoptera: noctuidae). Pest Manage. Sci. 63, 1002e1010. ~ era, P., Arouri, R., Goff, G.L., Hemden, H., Navarro-Llopis, V., M’saad, M., Castan Feyereisen, R., Hern andez-Crespo, P., Ortego, F., 2015. Resistance to lambdacyhalothrin in Spanish field populations of Ceratitis capitata and metabolic resistance mediated by P450 in a resistant strain. Pest Manage. Sci. 71, 1281e1291. Basit, M., Sayyed, A.H., Saleem, M.A., Saeed, S., 2011. Cross-resistance, inheritance and stability of resistance to acetamiprid in cotton whitefly, Bemisia tabaci Genn (Hemiptera: aleyrodidae). Crop Prot. 30, 705e712. Basit, M., Saeed, S., Saleem, M.A., Denholm, I., Shah, M., 2013. Detection of resistance, cross-resistance, and stability of resistance to new chemistry insecticides in Bemisia tabaci (Homoptera: aleyrodidae). J. Econ. Entomol. 106, 1414e1422. Brown, A.E., 2005. Mode of Action of Insecticide and Related Pest Control Chemicals for Production Agriculture, Ornamentals and Turf, vol. 301. Department of entomology, College park. MD 20742, pp. 405e3913. Demkovich, M., Siegel, J.P., Higbee, B.S., Berenbaum, M.R., 2015. Mechanism of resistance acquisition and potential associated fitness costs in Amyelois transitella (Lepidoptera: Pyralidae) exposed to pyrethroid insecticide. Environ. Entomol. 44, 855e863. Falconer, D.S., 1989. Introduction to Quantitative Genetics. Longman, London. Falconer, D.S., Mackay, T.F., Frankham, R., 1996. Introduction to Quantitative Genetics, fourth ed., vol. 12, p. 280 Trends Genet. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, UK, p. 333. Gunning, R.V., Moores, G.D., Devonshire, A.L., 1999. Esterase inhibitors synergize the toxicity of pyrethroids in Australian Helicoverpa armigera (Hubner) (Lepidoptera: noctuidae). Pestic. Biochem. Physiol. 63, 50e62. Hodgson, C., Abbas, G., Arif, M.J., Saeed, S., Karar, H., 2008. Phenacoccus solenopsis Tinsley (Sternorrhyncha: coccoidea: Pseudococcidae), an invasive mealybug
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Selection of bifenthrin resistance in cotton mealybug Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae): Cross-resistance, realized heritability and possible resistance mechanism Muhammad Mudassir Mansoor a, **, Muhammad Babar Shahzad Afzal b, *, Esteban Basoalto c, Abu Bakar Muhammad Raza a, Ansa Banazeer d a
Department of Entomology, University College of Agriculture, University of Sargodha, Sargodha, Pakistan Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan n y Sanidad Vegetal, Facultad de Ciencias Agrarias, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile Instituto de Produccio d Department of Entomology, Muhammad Nawaz Sharif University of Agriculture, Multan, Pakistan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2016 Received in revised form 21 April 2016 Accepted 29 April 2016 Available online 1 June 2016
Cotton mealybug Phenacoccus solenopsis Tinsley is an important pest of cotton in Pakistan, and its management is difficult due to the development of insecticide resistance. This research was conducted to characterize the bifenthrin resistance in populations of P. solenopsis and different parameters such as cross-resistance, realized heritability and possible resistance mechanisms were studied to improve the management of this important pest. A field-collected population was selected with bifenthrin in the laboratory for 14 generations and developed a resistance of 178-fold. The realized heritability of bifenthrin resistance was 0.54 in the selected population. The toxicity of bifenthrin was synergized by the addition of either piperonylbutoxide (PBO) or S,S,S tributylphosphorotrithioate (DEF) which suggests a general metabolic resistance due to possible involvement of mono-oxygenases or esterases. However, the resistant population did not develop a significant cross-resistance to either buprofezin, chlorpyrifos or lambda-cyhalothrin. These data suggest that alternative insecticide-based management programs can be developed for this pest in the short-term, but resistance management strategies which can reduce the sole reliance on insecticides are still needed. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Mealybug Insect resistance Bifenthrin Synergist
1. Introduction Insecticides are commonly used to improve agricultural productivity in developing countries (Karunamoorthi et al., 2012), and are known as effective tools for numerous pests of agricultural importance (Sayyed and Crickmore, 2007; Ishtiaq and Saleem, 2011; Afzal et al., 2015a). Insecticide use improves productivity by minimizing the potential of insect pests to create infestations, but also on the other hand increases production costs and can lead to the development of resistance in insect pest species (Metcalf, 1989; Siqueira et al., 2001). Insects build up resistance to insecticides through uninterrupted utilization as well as by using the natural
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M.M. Mansoor), Shahzad.babar35@ gmail.com (M.B.S. Afzal). http://dx.doi.org/10.1016/j.cropro.2016.04.026 0261-2194/© 2016 Elsevier Ltd. All rights reserved.
phenomenon of cross-resistance that develops because of previous exposure to different insecticides (Basit et al., 2011, 2013). In heavily sprayed crops like cotton, a suitable resistance management plan is required having potential to fulfill the pest management demand while decreasing the selection pressure on special target sites within the insect (Young et al., 2003). Multiple kinds of chewing and sucking pests attack the cotton crop during the season, creating damage to crop productivity and yield (Afzal et al., 2015a). Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae) is an important polyphagous pest and has caused significant yield losses to cotton crops for growers in some regions of Pakistan and other countries in Asia and in the United State of America (Abbas et al., 2005; Mahmood et al., 2011; Kumashiro et al., 2001; Nagrare et al., 2009; Wang et al., 2009; Hodgson et al., 2008). More than 40% losses of the cotton crop have been reported from Pakistan during 2007 as a result of P. solenopsis infestation (Pakistan cotton statistics, 2007). Management of P. solenopsis in Pakistan and many other countries has
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relied on broad-spectrum synthetic pyrethroid insecticides (Saddiq et al., 2014). Bifenthrin is commonly recommended for sucking pests in cotton including P. solenopsis (Saeed et al., 2007). Farmers have applied this insecticide in cotton fields to suppress the P. solenopsis populations but excessive applications of bifenthrin eventually resulted in resistance development. Bifenthrin is an insecticide among synthetic pyrethroids having stomach as well as contact activity (Thomson, 1998). It mainly acts upon sodium channels of insect nervous systems, causing these channels to open for a long time, thus increasing cell permeability and ultimately causing the death of insects (Brown, 2005). Some basic knowledge about the extent of the resistance and the potential for cross-resistance to other classes of insecticides is essential to develop insecticide resistance management strategies (Roush and Croft, 1986). Also, characterizing the genetic heritability and the potential mechanisms of insecticide resistance are important in order to develop alternative programs. These data can guide pest managers to develop programs that allow reversion of existing resistances and to select alternative tools that can act independently of the current resistance mechanisms. We studied resistance to bifenthrin in a field population of P. solenopsis after its selection in the laboratory. To understand the possible mechanism underlying bifenthrin resistance, the involvement of metabolic enzymes was evaluated by using synergists. According to authors' best knowledge; it is the first report of bifenthrin resistance in P. solenopsis. 2. Materials and methods 2.1. Insecticides and synergists Four commercial insecticides including bifenthrin (Talstar 10 EC, FMC, Pakistan), buprofezin (Fuzin 25 WP, 4B Group, Pakistan), lambda-cyhalothrin (Karate 2.5 EC, Syngenta, Pakistan) and chlorpyrifos (Lorsban 40 EC, Arysta Life Sciences, Pakistan) were used in this study. Two synergists including an esterase specific inhibitor, S,S,S tributylphosphorotrithioate (DEF; Sigma Ltd, UK) and a cytochrome P-450 monooxygenase inhibitor, piperonylbutoxide (PBO; Sigma Ltd, Poole, UK) were used for the synergism tests. 2.2. Insect collection and rearing About 500 nymphs and adults were collected from a cotton field in Multan District. This field had a known history of frequent insecticide applications using a number of compounds each season. The cotton fields in that area receive heavy applications of different insecticides mainly of organophosphates (e.g., profenofos, chlorpyrifos, methamidophos), pyrethroids (e.g., bifenthrin, deltamethrin, cypermethrin, lambda-cyhalothrin) and some new chemistry insecticides (e.g., acetamiprid, imidacloprid, buprofezin) for the control of different cotton pests including P. solenopsis. The field collected population was brought in the laboratory and insects were reared by maintaining standard laboratory conditions specifically at 16:8 h (L: D), 27 ± 1 C and 65 ± 5% R.H. This population was designated as Field Pop. Fresh tender shoots and leaves of China rose, Hisbiscus rosa-sinensis Linneaus were used as food of P. solenopsis and food was renewed after every 2 days. Transparent plastic jars (24 10 cm) were used to rear the insect. An insecticide-susceptible strain (Lab-PK) of P. solenopsis was reared in the Pak Arab Biological Control Laboratory (Fatima Sugar Research and Development Center, Muzaffargarh) for more than three years after its collection from cotton field of the Central Cotton Research Institute, Multan Pakistan. The population which was selected from the field population after performing base-line bioassays was called Bifen-Sel.
2.3. Bioassays Bioassays were performed on Field Pop, Bifen-Sel and Lab-PK. Aqueous solutions of insecticides were prepared and serially diluted. Four concentrations of each insecticide were used in each insecticide bioassay. Three-day-old second instar nymphs were used in the bioassays. Fresh leaves of H. rosasinensis were used for bioassay according to the Leaf-dip method (Ahmad et al., 2007; Afzal et al., 2015a). The leaves after dipping for 10 s in insecticide solution were placed in Petri dishes (5 cm in diameter) with moist filter papers (Afzal et al., 2015a) and insects were exposed to insecticide treated leaves. A total of 40 nymphs, 10 nymphs per replication, for each insecticide concentration were used. For control, 20 nymphs with water-treated leaves were kept in Petri dishes. Numbers of insects tested in single bioassay including control were 180. Mortality data of bioassays was obtained after 48 h of treatment with bifenthrin, lambda-cyhalothrin, and chlorpyrifos, while for buprofezin, mortality data was taken after 96 h exposure. The nymphs were touched individually by using camel hair brush to check mortality and non-moving nymphs were considered dead. 2.4. Selection Nymphs were selected at every generation from G1 to G14 with bifenthrin. The concentrations for selection were determined by performing bioassay of bifenthrin on Field Pop. This bioassay provided different lethal concentrations that were then used for selection. Leaf dip method with same protocol as mentioned above was used for selection of population. About 100 to 200 nymphs were selected per generation (Table 1). Mortality was judged 48 h after insecticide exposure. 2.5. Synergism analysis PBO and DEF concentrations were tested to recognize the uppermost non-lethal dose. Acetone (analytical reagent; Fisher Scientific, Lough borough, UK) was mixed with PBO or DEF and mixed in serial solutions having insecticide concentrations. The nonlethal concentrations, 5 mg/ml PBO and 10 mg/ml DEF, were used for Bifen-Sel (G15), while a concentration (1 mg/ml) of both synergists was used for Lab-PK. For the control acetone was used alone. Bioassays were performed according to method described above. 2.6. Data analysis of bioassays The toxicological data was analyzed with POLO software (LeOra Software, 2005) by probit analysis (Finney, 1971) to determine the LC50 (median lethal concentration) values, their standard errors, slopes, and confidence intervals (CIs). Synergism ratio (SR) for synergism bioassays was evaluated as follows: SR ¼ LC50 of insect population exposed to insecticide/LC50 of population exposed to insecticide with synergist. 2.7. Realized heritability estimation Realized heritability (h2) was estimated by using the method of Falconer et al. (1996) and Tabashnik (1992) by the following equation. h2 ¼ Selection response/Selection differential Selection response was calculated as: Selection response ¼ (Log final LC50 Log Initial LC50)/N
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57
Table 1 Selection history of Phenacoccus solenopsis with bifenthrin. Generation (G)
Concentration (mg/ml)
Number of insects exposed
Number of insects died
% Mortality
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14
3.766 3.766 7.496 14.921 17.56 22.351 35.143 35.143 35.143 35.143 35.143 35.143 35.143 35.143
200 200 200 200 200 200 200 200 200 200 100 100 100 100
176 154 137 121 103 89 74 61 53 42 28 11 8 8
88 77 68.5 60.5 51.5 44.5 37 30.5 26.5 21 28 11 8 8
3.2. Bifenthrin resistance selection and its cross-resistance with other insecticides
Selection differential was calculated as: Selection differential ¼ i s p Hence i is the intensity of selection, calculated according to Falconer (1989) and the sp is the phenotypic standard deviation calculated as:
sp ¼ [(initial slope e final slope) 0.5]1
Selection with bifenthrin increased the resistance from 3.28 (Go) to 178.42-fold (G15) in the population of P. solenopsis (Table 2). Bifenthrin selected resistance in P. solenopsis induced very low cross-resistance to buprofezin (1.53-fold), to chlorpyrifos (1.62fold) and to lambda-cyhalothrin (1.98-fold) at G15 when compared with Field Pop (Table 2). 3.3. Estimation of realized heritability
3. Results 3.1. Toxicity comparison of various insecticides to field pop and LabPK populations For the Field Pop at Go, the toxicity of bifenthrin was significantly higher compared to buprofezin and chlorpyrifos, but it was significantly lower than that of lambda-cyhalothrin (95% CI did not overlap). The toxicities of buprofezin and chlorpyrifos remained statistically similar (95% CI overlap) when these insecticides were tested on Field Pop Go. For the Lab-PK population, the toxicity of bifenthrin was similar (95% CI overlap) to buprofezin, but higher (95% CI did not overlap) than that of chlorpyrifos. The toxicity of lambda-cyhalothrin was significantly higher (95% CI did not overlap) when compared to all other insecticides tested at Lab-PK (Table 2).
After 14 generations of consecutive selection with bifenthrin, the LC50 value of bifenthrin increased from 7.49 to 406.8 mg/ml and slop decreased from 3.46 to 2.16 in the Bifen-sel. Realized heritability (h2) for bifenthrin resistance was 0.54 in P. solenopsis. The numbers of generations estimated to acquire a ten-fold increase in bifenthrin resistance were eight (Reciprocal of R, Table 3). 3.4. PBO and DEF synergism for bifenthrin Lab-PK and Bifen-Sel (G15) populations of P. solenopsis were tested with PBO and DEF to see whether they synergize the toxicity of bifenthrin or not. These synergists decreased the resistance ratio of the Bifen-Sel (G15) from 178.42 to 75.97 and 45.94, respectively (Table 4). Based on the 95% CI comparison of LC50 values, the results indicate that both PBO and DEF have synergistic effect with SR ¼ 2.83 and 3.17, respectively in Bifen-sel. In the Lab-PK, both synergists did not increase the insecticidal activity of bifenthrin
Table 2 Response of Phenacoccus solenopsis populations to different insecticides. Population
Insecticides
LC50 [95% CI] (mg/ml)
Slope ± SE
Field Pop (Go) Field Pop (Go) Field Pop (Go) Field Pop (Go) Lab-PK Lab-PK Lab-PK Lab-PK Bifen-Sel (G15) Bifen-Sel (G15) Bifen-Sel (G15) Bifen-Sel (G15)
Bifenthrin Buprofezin Chlorpyrifos Lambda-cyhalothrin Bifenthrin Buprofezin Chlorpyrifos Lambda-cyhalothrin Bifenthrin Buprofezin Chlorpyrifos Lambda-cyhalothrin
7.49 20.70 25.01 2.76 2.28 1.83 4.73 0.87 406.82 31.70 40.41 5.47
3.46 2.75 2.38 3.44 3.57 3.53 3.89 3.84 2.16 2.39 2.02 3.44
a b
LC50 of Field Pop (Go) or Bifen-sel (G15)/LC50 of Lab-PK. LC50 of Bifen-sel (G15)/LC50 of Field Pop (Go).
(5.76e9.003) (16.18e25.19) (19.41e30.94) (2.05e3.34) (1.73e2.74) (1.21e2.27) (3.32e5.75) (0.56e1.07) (323.22e540.54) (25.304e39.24) (31.63e52.80) (4.59e6.45)
± ± ± ± ± ± ± ± ± ± ± ±
0.60 0.41 0.36 0.63 0.64 0.76 0.84 0.90 0.35 0.35 0.33 0.45
c2
df
P
n
RRa
RRb
4.62 2.72 0.17 0.13 0.07 1.04 0.54 0.39 0.10 1.71 0.16 2.39
3 3 3 3 3 3 3 3 3 3 3 3
0.20 0.43 0.98 0.98 0.99 0.79 0.90 0.94 0.99 0.63 0.98 0.49
180 180 180 180 180 180 180 180 180 180 180 180
3.28 11.31 5.28 3.17 1.00 1.00 1.00 1.00 178.42 17.32 8.54 6.28
54.32 1.53 1.62 1.98
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Table 3 Realized heritability determination. Estimate of mean response per generation
Estimate of mean selection differential per generation
Insecticide
Initiala LC50 (log)
Finala LC50 (log)
Rb
pc
Id
Initial slope
Final slope
spe
Sf
h2g
Bifenthrin
7.49 (0.87)
406.82 (2.61)
0.12
60.00
0.64
3.46
2.16
0.36
0.23
0.54
a b c d e f g
Initial (Go) and final (G15) LC50 values were calculated in mg/mL. Selection response. Average percentage survival of Bifen-sel (G15) after selection. Intensity of selection according to Falconer (1989). Phenotypic deviation. Selection differential. Realized heritability.
Table 4 Synergism tests for bifenthrin resistance in Phenacoccus solenopsis. Population
Treatment
Lab-PK
Bifenthrin Bifenthrin Bifenthrin Bifen Bifenthrin Bifenthrin
Bifen-Sel (G15)
a
þ PBO þ DEF þ PBO þ DEF
LC50 [95% CI] (mg/ml)
Slope ± SE
2.28 1.89 2.79 406.82 143.60 128.18
3.57 2.96 3.12 2.16 2.99 3.15
(1.73e2.74) (1.22e2.40) (2.18e3.37) (323.22e540.54) (111.55e173.92) (98.15e155.22)
± ± ± ± ± ±
0.64 0.59 0.49 0.35 0.46 0.52
c2
df
P
n
RRa
SR
0.07 0.55 0.701 0.1 1.03 1.08
3 3 3 3 3 3
0.99 0.9 0.87 0.99 0.79 0.78
180 180 180 180 180 180
1 1 1 178.42 75.97 45.94
1 1.21 0.81 1 2.83 3.17
LC50 of Bifen-Sel (G15)/LC50 of Lab-PK.
(95% CI overlap) (Table 4). 4. Discussion Synthetic insecticides have been the key tool used in Pakistan since the 1970s against a number of chewing and sucking insect pests of cotton. This continuous reliance on a variety of toxicants has clearly resulted in a reduced product efficacy due to the selection for insecticide resistance in many insect pests (Ahmad et al., 2007; Basit et al., 2011; Ishtiaq and Saleem, 2011; Khan et al., 2014; Afzal and Shad, 2015). Among these synthetic insecticides, bifenthrin, a synthetic pyrethroid has been excessively used by farmers in cotton fields since the introduction of P. solenopsis in Pakistan in the past few years. Our current research has shown that P. solenopsis can develop a high level of resistance to bifenthrin under a regime of continuous selection in the laboratory. In our study, selection of P. solenopsis with bifenthrin for 14 generations amplified the level of resistance to 178.42-fold when compared with Lab-PK and 54.31-fold when compared with Field Pop at Go. Previously, P. solenopsis has also been reported to develop significant levels of resistance following selection in the laboratory to various insecticides such as acetamiprid (315-fold; Afzal et al., 2015a), indoxacarb (2223-fold; Afzal et al., 2015b) and deltamethrin (100-fold; Saddiq et al., 2015). High level of bifenthrin resistance development in selected population of P. solenopsis indicated that frequency of resistant alleles might be higher in field population. Studying resistance and cross-resistance is useful to limit the development of resistance by employing practices such as insecticides mixtures and rotation having different modes of action (Shen and Wu, 1995; Abbas et al., 2015). In our study, selection of P. solenopsis with bifenthrin induced negligible cross-resistance to buprofezin, chlorpyrifos and lambda-cyhalothrin when compared with Field Pop (Table 2). Cross-resistance is a valuable tool to identify insecticide resistance mechanisms (Shad et al., 2010). Cross-resistance can result from non-specific enzymes, such as microsomal oxidases, mutation at an insecticidal target site and some factors such as delayed cuticular penetration (Basit et al., 2011). In this study, very low cross-resistance between bifenthrin and buprofezin was expected as these insecticides are from different classes which exhibit different modes of action. It suggests
that these insecticides can be used in rotational pattern in the field. Similarly, in our study negligible cross-resistance of bifenthrin with lambda-cyhalothrin (pyrethroid) and chlorpyrifos (organophosphate) has practical implications in delaying the development of bifenthrin resistance in P. solenopsis in the field, since both have different mode of actions. Pyrethroids and organophosphates act on the functions of voltage-gated sodium channels and acetylcholinesterase (AChE), respectively (Sayyed et al., 2010). It suggests that bifenthrin can be rotated with lambda-cyhalothrin or alternatively with chlorpyrifos, in an insecticide resistance management (IRM) program of P. solenopsis. The realized heritability of bifenthrin resistance was determined in order to find the genetic variation in the P. solenopsis population. A high level of genetic variation (h2 ¼ 0.54) for the bifenthrin resistance was observed. It shows great additive genetic distinction thus causing significant increase in LC50 values between field collected at Go and Bifen-SEL at G15 for bifenthrin. This indicates that the field collected population of P. solenopsis had a higher frequency of resistance allele (s) than the laboratory population. This finding is consistent with the basic fact that population was obtained from the fields (Multan) where intensive use of several insecticides including pyrethroids and organophosphates for the insect pest control has been reported (Sayyed and Crickmore, 2007; Mansoor et al., 2015). The cotton fields in the area (Multan) receive heavy applications of different insecticides mainly of organophosphates (e.g., profenofos, chlorpyrifos, methamidophos), pyrethroids (e.g., bifenthrin, deltamethrin, cypermethrin, lambda-cyhalothrin) and some new chemistry insecticides (e.g., acetamiprid, imidacloprid, buprofezin) for the control of different cotton pests including P. solenopsis. The resistance development in insect species is mainly the consequence of metabolic activity of different detoxification enzymes (Kang et al., 2006). In order to detect the mechanism pathway responsible for resistance development the synergistic effect of PBO and DEF with bifenthrin was checked for Bifen-sel and Lab-PK populations. PBO and DEF produced a considerable effect on toxicity of bifenthrin in Bifen-Sel but not in Lab PK. Cytochrome P450 mono-oxygenases and esterases seem prominent in bifenthrin resistance development because both PBO and DEF enhanced the toxicity of bifenthrin in the selected population. Resistance to
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bifenthrin was also previously reported due to involvement of monooxygenases (Ahmad et al., 2007) or esterases (Gunning et al., 1999). The current study also confirms that possible connection of P-450 mono-oxygenases and esterases in bifenthrin resistance buildup. Similar results have been reported in deltamethrin resistant Chrysoperla carnea (Sayyed et al., 2010), cypermethrin resistant Amsacta albistriga, (Muthusamy and Shivakumar, 2015), bifenthrin and b-cyfluthrin resistant Amyelois transitella (Demkovich et al., 2015), lambda-cyhalothrin resistant Ceratitis capitata (Arouri et al., 2015), and emamectin benzoate resistant P. solenopsis (Afzal and Shad, 2015). In short, the P. solenopsis exhibits extraordinary potential to develop high levels of resistance under continuous selection pressure of bifenthrin and this could be at least partially due to increased detoxification by the P-450 mono-oxygenases and esterases. This study offers management fundamentals of insecticide resistance in P. solenopsis. Furthermore, the study of genetics of resistance to bifenthrin in the P. solenopsis could be important for resistance progress management. Biological control agents such as C. carnea as well as cultural practices should be incorporated with IRM for the effective control of P. solenopsis. References Abbas, G., Arif, M., Saeed, S., 2005. Systematic status of new species of genus Phenacoccus Cockerell (Pseudococcidae), a serious pest of cotton Gossypium hirsutum L. Pak. Pak. Entomol. 27, 83e84. Abbas, N., Crickmore, N., Shad, S.A., 2015. Efficacy of insecticide mixtures against a resistant strain of house fly (Diptera: Muscidae) collected from a poultry farm. Int. J. Trop. Insect Sci. 35, 48e53. Afzal, M.B.S., Shad, S.A., 2015. Resistance inheritance and mechanism to emamectin benzoate in Phenacoccus solenopsis (Homoptera: Pseudococcidae). Crop Prot. 7, 60e65. Afzal, M.B.S., Shad, S.A., Abbas, N., Ayyaz, M., Walker, W.B., 2015a. Cross-resistance, the stability of acetamiprid resistance and its effect on the biological parameters of cotton mealybug, Phenacoccus solenopsis (Homoptera: Pseudococcidae), in Pakistan. Pest Manage. Sci. 71, 151e158. Afzal, M.B.S., Shad, S.A., Basoalto, E., Ejaz, M., Serrao, J.E., 2015b. Characterization of indoxacarb resistance in Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae): cross-resistance, stability and fitness cost. J. Asia-Pacific Entomol. 18, 779e785. Ahmad, M., Sayyed, A.H., Crickmore, N., Saleem, M.A., 2007. Genetics and mechanism of resistance to deltamethrin in a field population of Spodoptera litura (Lepidoptera: noctuidae). Pest Manage. Sci. 63, 1002e1010. ~ era, P., Arouri, R., Goff, G.L., Hemden, H., Navarro-Llopis, V., M’saad, M., Castan Feyereisen, R., Hern andez-Crespo, P., Ortego, F., 2015. Resistance to lambdacyhalothrin in Spanish field populations of Ceratitis capitata and metabolic resistance mediated by P450 in a resistant strain. Pest Manage. Sci. 71, 1281e1291. Basit, M., Sayyed, A.H., Saleem, M.A., Saeed, S., 2011. Cross-resistance, inheritance and stability of resistance to acetamiprid in cotton whitefly, Bemisia tabaci Genn (Hemiptera: aleyrodidae). Crop Prot. 30, 705e712. Basit, M., Saeed, S., Saleem, M.A., Denholm, I., Shah, M., 2013. Detection of resistance, cross-resistance, and stability of resistance to new chemistry insecticides in Bemisia tabaci (Homoptera: aleyrodidae). J. Econ. Entomol. 106, 1414e1422. Brown, A.E., 2005. Mode of Action of Insecticide and Related Pest Control Chemicals for Production Agriculture, Ornamentals and Turf, vol. 301. Department of entomology, College park. MD 20742, pp. 405e3913. Demkovich, M., Siegel, J.P., Higbee, B.S., Berenbaum, M.R., 2015. Mechanism of resistance acquisition and potential associated fitness costs in Amyelois transitella (Lepidoptera: Pyralidae) exposed to pyrethroid insecticide. Environ. Entomol. 44, 855e863. Falconer, D.S., 1989. Introduction to Quantitative Genetics. Longman, London. Falconer, D.S., Mackay, T.F., Frankham, R., 1996. Introduction to Quantitative Genetics, fourth ed., vol. 12, p. 280 Trends Genet. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, UK, p. 333. Gunning, R.V., Moores, G.D., Devonshire, A.L., 1999. Esterase inhibitors synergize the toxicity of pyrethroids in Australian Helicoverpa armigera (Hubner) (Lepidoptera: noctuidae). Pestic. Biochem. Physiol. 63, 50e62. Hodgson, C., Abbas, G., Arif, M.J., Saeed, S., Karar, H., 2008. Phenacoccus solenopsis Tinsley (Sternorrhyncha: coccoidea: Pseudococcidae), an invasive mealybug
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