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Resistance risk assessment to chlorpyrifos and cross-resistance to other insecticides in a field strain of Phenacoccus solenopsis Tinsley
Crop Protection 94 (2017) 38e43
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Resistance risk assessment to chlorpyrifos and cross-resistance to other insecticides in a field strain of Phenacoccus solenopsis Tinsley Muhammad Ismail a, *, Masood Ejaz a, Naeem Abbas b, Sarfraz Ali Shad a, Muhammad Babar Shahzad Afzal c a b c
Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan Department of Entomology, University College of Agriculture and Environmental Sciences, The Islamia University of Bahawalpur, Bahawalpur, Pakistan Citrus Research Institute, Sargodha, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2016 Received in revised form 30 November 2016 Accepted 7 December 2016
The organophosphate insecticide chlorpyrifos is recommended for control of a number of insect pests, including cotton mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae) in Pakistan. This work assessed chlorpyrifos resistance evolution and cross-resistance to other insecticides. After 23 generations of selection, the P. solenopsis strain (Chlor-SEL) had a 26652-fold level of resistance to chlorpyrifos compared to a susceptible strain. Realized heritability (h2) of resistance to chlorpyrifos was 0.04. The Chlor-SEL strain also had a low level of cross resistance to lambda-cyhalothrin (14-fold) and a very low level cross-resistance to nitenpyram and profenofos after 23 generations of selection. The projected rate of resistance development indicated that if 50e90 percent of a P. solenopsis population were selected with chlorpyrifos, a ten-fold increase in the lethal concentration 50 (LC50) would occur in 22e10 generations (h2 ¼ 0.04, Slope ¼ 0.70). At a similar slope, if h2 ¼ 0.14, then only 6-3 generations are required for a ten-fold increase in the LC50 at 50e90 percent selection intensity, respectively. Likewise, if h2 ¼ 0.24, then the same would occur in 4e2 generations. This study showed that P. solenopsis has the ability to become resistant to chlorpyrifos but insect resistance management strategies such as rotation of different group of insecticides are needed to prolong the effectiveness of chlorpyrifos in controlling P. solenopsis. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Cotton Mealybug Invasive pest Organophosphate Resistance evolution
1. Introduction Cotton mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae), is a major insect pest of cotton, vegetables, ornamental and medicinal plants worldwide (Abbas et al., 2010; Afzal et al., 2015d; Fand and Suroshe, 2015; Wang et al., 2009) due to its polyphagous nature. In Pakistan, P. solenopsis caused havoc to cotton production in 11 growing districts of Punjab during 2005 (Saeed et al., 2007) and average cotton yield was reduced by 50% (Muhammad, 2007). Apart from Pakistan, P. solenopsis has also created economic losses for cotton growers in India (Nagrare et al., 2009), United States of America (Fuchs et al., 1991), Republic of China (Wang et al., 2009), Taiwan and Thailand (Hodgson et al., 2008), Australia (Charleston et al., 2010) and Turkey (Kaydan et al., 2013). P. solenopsis damage plants by sucking cell sap from
* Corresponding author. E-mail address: [email protected] (M. Ismail). http://dx.doi.org/10.1016/j.cropro.2016.12.011 0261-2194/© 2016 Elsevier Ltd. All rights reserved.
the phloem and secreting honey dew that results in the development of a sooty mold which affects the photosynthesis process and can cause the premature death of plants (Afzal et al., 2015a; Culik and Gullan, 2005; Wang et al., 2010b). Synthetic chemical insecticides are used for the management of P. solenopsis worldwide, including Pakistan. However, the unnecessary and over use of insecticides in cotton agroecosystem has led to the development of resistance by P. solenopsis (Saddiq et al., 2014, 2015). Resistance in P. solenopsis under laboratory conditions has previously been documented to insecticides including acetamiprid (Afzal et al., 2015a,d), chlorpyrifos (Afzal et al., 2015b), emamectin benzoate (Afzal and Shad, 2015), deltamethrin (Saddiq et al., 2016), and indoxacarb (Afzal et al., 2015e). Extensive use of the organophosphate chlorpyrifos has resulted in resistance reported in a range of pests including P. solenopsis, Tetranychus urticae (Koch), n), and Liriomyza sativae (Blanchard) Laodelphax striatellus (Falle (Afzal et al., 2015b; Askari-Saryazdi et al., 2015; Kumral et al., 2009; Recep and Yorulmaz, 2010; Saddiq et al., 2016; Wang et al., 2010a).
M. Ismail et al. / Crop Protection 94 (2017) 38e43
Frequent use of different insecticides may result in loss of efficacy due to cross-resistance in the insect populations which imposes difficulties in developing successful insecticide resistance management plans (Kranthi et al., 2001; Basit et al., 2011). Crossresistance to other insecticides due to selection of chlorpyrifos resistance has been reported in L. striatellus (Wang et al., 2010a), L. sativae (Askari-Saryazdi et al., 2015), and Sogatella furcifera (Horv ath) (Mu et al., 2016). Studying resistance and crossresistance is useful to limit the development of resistance by employing practices such as insecticide mixtures and rotation of insecticides with different modes of action (Shen and Wu, 1995; Abbas et al., 2015). Risk assessment of insecticide resistance using laboratory or field selection studies can help to avoid or postpone resistance problems in the field (Jutsum et al., 1998; Lai and Su, 2011). Laboratory insecticide selection provides a quick way with fewer variables than those that occur in the field and can reveal the maximum potential of an insect to become resistant (Abbas and Shad, 2015; Sial and Brunner, 2010; Lin et al., 2003; Tabashnik, 1992). In this work, we studied the impact of continuous selection with chlorpyrifos on resistance allele frequencies (h2) under laboratory conditions in P. solenopsis and observed cross-resistance to other insecticides such as profenofos, lambda-cyhalothrin and nitenpyram in the Chlor-SEL strain. The results of this study will be helpful in our understanding of chlorpyrifos resistance and its management in controlling P. solenopsis. 2. Materials and methods 2.1. Insects Approximately 300 insects (nymphs and adults) were randomly selected from ten different areas of a cotton field located in Multan (30.1978 N, 71.4697 E). At the time of collection, the cotton plants were at the reproductive stage. The cotton field of the collection site received heavy amount of sprays from organophosphates, pyrethroids and new chemicals classes during the growing season to control various sucking and chewing pests prior to collecting insects (Afzal et al., 2015b,d; Saddiq et al., 2014, 2015). After collection, the insects were transported in plastic jars (12 24 cm) to the laboratory and were maintained at 27 ± 2 C, 65 ± 5% R.H. and 16:8 h L:D and reared on China rose, Hibiscus rosasinensis L. leaves and tender shoots. All stages of P. solenopsis were kept in plastic jars (12 24 cm) covered with a muslin cloth. The culture was refreshed every 2e3 days with clean fresh leaves along with small twigs. For a reference susceptible strain, a field strain was collected from a cotton field located in Multan district and reared without insecticide exposure for more than one year in the laboratory (Afzal et al., 2015b). 2.2. Insecticides
39
bioassay and each concentration was replicated five times. The five concentrations ranged between 0.625 and 10 mg a.i/ml for the susceptible, 31.25e1000 mg a.i/ml for the field population (Field Pop; G3), and 62.5e8000 mg a.i/ml for the chlorpyrifos selected strain (Chlor-SEL; G5-G25). Fresh leaves were dipped into serial dilutions of insecticides for 10 s and air dried at room temperature. Leaves for control were immersed into water only. Treated dried leaves were placed into petri-dishes (5 cm diameter). Five 2nd instars nymphs were placed in each petri dish so a total of 150 nymphs were used for a single bioassay (including the control). Bioassays were kept under the laboratory conditions as mentioned above. Mortality data were assessed 48 h after exposure to chlorpyrifos, lambda-cyhalothrin and profenofos, and 72 h after exposure to nitenpyram. Nymphs were considered to be dead if there was no leg movement after a gentle touch with fine hairbrush (Afzal et al., 2015d). 2.4. Chlorpyrifos selection The field population was selected with chlorpyrifos for 23 generations (G3-G25) and designated as the Chlor-SEL strain. Selection was carried out with the leaf dip method by exposing 2nd instar nymphs of P. solenopsis to chlorpyrifos (Afzal et al., 2015b). Selection was done at each generation and averages of 300 nymphs were exposed to increasing concentrations (75.26e2601.42 mg a.i/ ml). The selection of chlorpyrifos concentrations was based on the objective of having a sufficient number of nymphs to produce the next generation. Nymphal mortality was assessed after 48 h exposure to chlorpyrifos and the survivors of each selection were reared to obtain the next generation. 2.5. Estimation of realized heritability Realized heritability (h2) was determined according to the method of Falconer et al. (1996) and Tabashnik (1992) by the following equation.
h2 ¼
Selection ResponseðRÞ Selection differentialðSÞ
We estimated R, the difference in mean phenotype and whole parental generation before selection by Falconer (1989):
Selection responseðRÞ ¼
Log final LC50 Log initial LC50 N
The final LC50 was the LC50 value after N number of generations and the initial LC50 was the LC50 value of the field population before selection. Selection differential was calculated as:
Selection differentialðSÞ ¼ i sp Intensity of selection was calculated as follows:
®
Commercial formulations of chlorpyrifos (Lorsban , 40EC; Dow Agro Sciences, Pakistan), lambda-cyhalothrin (Karate® 2.5EC, Syngenta), profenofos (Curacron® 500EC, Syngenta) and nitenpyram (Paranol® 10EC, Kanzo Agro Chemicals) were used for the experiments.
i ¼ 1:583 0:0193336p þ 0 : 0000428p2 þ 3:65194=p where p is the average percent survival of Chlor-SEL strain after N number of selection (Tabashnik and McGaughey, 1994) and sp is the phenotypic standard deviation calculated by:
2.3. Bioassays
sp ¼ ½ðinitial slope final slopeÞ0:51
To assess the toxicities of selected insecticides, a leaf dip bioassay was conducted on 2nd instar nymphs of P. solenopsis (Afzal et al., 2015d). Serial dilutions of insecticide concentrations (mg a.i/ ml) were prepared using chlorpyrifos, lambda-cyhalothrin, profenofos, and nitenpyram. Five concentrations were used for each
To estimate changes in R, S, and h2 during the selection pressure, each parameter was determined for the first and second half (7 generations in one half) of the experiment. The generation G13 was used in both halves of the experiment. The generation (G) needed for a ten-fold increase in LC50 was calculated by:
40
M. Ismail et al. / Crop Protection 94 (2017) 38e43
1
G¼R
1 ¼ h2 S
Resistance ratios (RR) were calculated as: LC50 value of resistant strain/LC50 value of susceptible strain and 95% CI of RR were calculated according to Robertson and Preisler (1992). 2.6. Statistical analysis Data was analyzed by using probit analysis (Finney, 1971) with POLO Plus software (Software, 2002) to determine the LC50 values, confidence intervals (CIs), slopes and their standard errors. Control mortality was corrected using Abbott's formula (Abbott, 1925). 3. Results 3.1. Selection of resistance to chlorpyrifos Selection with chlorpyrifos for 23 generations resulted in an increase in LC50 values (ranging from 75.30 to 9328.24 mg a.i/ml for the Chlor-SEL strain) compared to the susceptible strain. The RR increased from 123-fold to 15292-fold compared to the susceptible strain and 52e124-fold compared to the Field Pop (Table 1). The LC50 of Field pop was previously published by Afzal et al. (2015b). 3.2. Cross-resistance to other insecticides in the Chlor-SEL strain The Chlor-SEL strain of P. solenopsis had low levels of crossresistance to lambda-cyhalothrin (10.5-fold) and profenofos (11.2fold) after 23 generations of selection (G25) compared to the Field Pop (G3). Moderate level of cross-resistance to nitenpyram (30.3fold) was observed in the Chlor-SEL strain (Table 2). 3.3. Realized heritability (h2) The overall mean estimated h2 of chlorpyrifos resistance in P. solenopsis (G3eG25) was 0.04. The estimated h2 of chlorpyrifos resistance was 0.12 and 0.24 in first half and in second half of present study, respectively. The R and S were higher in the first half compared with second half of selection. Therefore, the estimated h2 of chlorpyrifos resistance was higher in the second than in the first half of selection. (see Table 3). 3.4. Projected rate of chlorpyrifos resistance development The projected rate of resistance development is directly proportional to h2 and selection intensity (Fig. 1). For example, if we assume that the slope ¼ 0.70 (the value of mean slope for 23 Chlor-
SEL generations in this study) and h2 ¼ 0.04, then 22-10 generations would be required for a ten-fold increase in the LC50 at a 50e90 percent selection intensity, respectively. However, at a similar slope, if h2 ¼ 0.14, then 6e3 generations would be required for a ten-fold increase in the LC50 at a 50e90 percent selection intensity. Likewise, if h2 ¼ 0.24, then the same would occur in 4e2 generations at 50e90 percent selection intensity (Fig. 1). The projected rate of resistance development is inversely proportional to the slope. For example, if we assume that h2 ¼ 0.04 (heritability of chlorpyrifos resistance estimated in this study) and slope ¼ 0.70, then a ten-fold increase in the LC50 would occur in 84e38 generations at a 50e90 percent selection intensity, respectively. However, at the same h2, if the slope ¼ 1.70, then 53e24 generations would be required for a tenfold increase in the LC50 at 50e90 percent selection intensity, respectively. Likewise, if the slope ¼ 2.70, then the same would occur in 22e10 generations at 50e90 percent selection intensity, respectively (Fig. 2).
4. Discussion In this study, the selection of P. solenopsis with chlorpyrifos for 23 generations resulted in the development of very high resistance (15292-fold). It has recently been documented that P. solenopsis in Pakistan is capable of developing high levels of resistance to different insecticides under laboratory selection pressure (Afzal and Shad, 2015, 2016a; Afzal et al., 2015c,d), suggesting that selection has an obvious effect on the evolution of resistance. Evolution of resistance occurs faster under high selection pressure when susceptible genes are replaced by resistant genes, resulting in a high number of resistant individuals in a population (Ijaz et al., 2016). Realized heritability (h2) provides a standardized approach to evaluate the results of selection by combining the selection strength estimates and resistance development rate (Abbas and Shad, 2015; Tabashnik, 1992). The h2 provide a good way to present selection results with a solid foundation of the theoretical and empirical literature of evolutionary biology (Falconer et al., 1996; Mousseau and Roff, 1987). The lower h2 (0.04) after 23 generations of selection with chlorpyrifos suggests that P. solenopsis strains are less likely to develop resistance to chlorpyrifos when compared with other insecticides, such as acetamiprid, h2 ¼ 0.58 (Afzal et al., 2015a), spinosad, h2 ¼ 0.94 (Afzal et al., 2015c), and indoxacarb, h2 ¼ 1.13 (Afzal and Shad, 2016b). The lower h2 indicates higher environmental variations and lower additive genetic variations for chlorpyrifos resistance in P. solenopsis. The selection response and the selection differential declined as the selection pressure of chlorpyrifos were raised, causing lower h2 in the first compared with the second half of selection. These results showed that the expression level of alleles responsible for chlorpyrifos
Table 1 Resistance development of P. solenopsis to chlorpyrifos after 23 generations of selection. Selection
LC50 (95% Cl) [mg a.i/ml ]
Slope ± SE
Susceptible Field Pop (G3)c Chlor-SEL (G11) Chlor-SEL (G13) Chlor-SEL (G15) Chlor-SEL (G17) Chlor-SEL (G19) Chlor-SEL (G21) Chlor-SEL (G23) Chlor-SEL (G25)
0.61 (0.30e0.87) 75.30 (25.90e123.00) 4631.20 (2220.00e66645.) 3752.90 (2270.10e10768.00) 3848.90 (2406.00e9709.30) 5872.20 (3625.40e16055.00) 4427.10 (2778.80e11328.00) 4530.48 (2874.20e11301.00) 7851.45 (3649.70e179650.00) 9328.24 (4279.10e202142.10)
2.22 1.51 0.90 1.12 1.24 1.26 1.32 1.37 0.84 0.90
a b c
± 0.50 ± 0.36 ± 0.30 ± 0.29 ± 0.30 ± 0.31 ± 0.34 ± 0.33 ± 0.29 ± 0.30
Resistance ratio calculated as LC50 of Chlor-SEL/LC50 of susceptible strain. Resistance ratio calculated as LC50 of Chlor-SEL/LC50 of Field Pop. Published by Afzal et al. (2015b).
c2
df
P
RRa
RRb
4.62 1.74 0.04 0.15 0.59 0.29 0.40 1.14 0.41 1.05
3 3 3 3 3 3 3 3 3 3
0.20 0.63 0.99 0.99 0.90 0.96 0.94 0.77 0.94 0.79
1.00 123.44 (55.42e285.95) 7595.13 (2331.88e25733.91) 6152.30 (2803.37e14056.25) 6309.67 (2980.93e13903.88) 9626.56 (4438.98e21733.87) 7257.62 (3412.95e16067.05) 7427.02 (3529.15e16271.83) 12,871.23 (3736.58e46157.76) 15,292.20 (4311.38e56467.93)
1.00 61.50 (17.24e200.00) 49.84 (20.00e125.00) 51.11 (21.28e125.00) 77.98 (31.25e200.00) 58.79 (24.39e142.86) 60.16 (25.00e142.86) 104.26 (27.78e333.33) 123.88 (32.26e500.00)
M. Ismail et al. / Crop Protection 94 (2017) 38e43
41
Table 2 Cross-resistance to some tested insecticides in Chlor-SEL of P. solenopsis. Strain
Insecticide
LC50 (95% Cl) [mg a.i/ml]
RR
Slope þ SE
Field (G3)
Lambda-cyhalothrin Nitenpyram Profenofos Lambda-cyhalothrin Nitenpyram Profenofos
105.70 (61.84e191.61) 55.84 (11.80e105.93) 171.64 (59.65e288.02) 1111.48 (731.12e1969.40) 1693.64 (1101.77e3623.55) 1922.60 (1166.10e4857.20)
1.00 1.00 1.00 10.52 (5.35e20.83) 30.33 (11.63e76.92) 11.20 (4.67e27.03)
1.31 1.00 1.19 1.30 1.32 1.07
Chlor-SEL (G25)
± ± ± ± ± ±
0.32 0.31 0.31 0.39 0.31 0.29
c2
df
P
0.24 0.03 0.84 0.04 0.61 0.17
3 3 3 3 3 3
0.97 0.99 0.84 0.99 0.89 0.98
Table 3 Estimated realized heritability (h2) of resistance to chlorpyrifos in P. solenopsis. Insecticides
Selected generations
Initial LC50 (mg a.i/ml)
Final LC50 (mg a.i/ml)
R
I
Mean slope
sp
S
h2
Chlorpyrifos Chlorpyrifos Chlorpyrifos
11 (G3-G13) 12 (G13-G25) 23 (G3-G25)
75.30 4359.83 75.30
4359.83 9328.24 9328.24
0.16 0.03 0.10
0.45 0.60 0.54
0.34 0.92 0.70
2.95 1.09 1.42
1.32 0.66 0.76
0.12 0.24 0.04
h2 =0.04 25
22
h2 =0.24
18
20 Generations
h2 =0.14
15 15 10
13 10 6
5
4
4
4
3
3
2
50
60
5
0
70 80 Selection intensity %
3 2 90
Fig. 1. Effect of heritability (h2) on the number of generations of P. solenopsis required for a tenfold increase in LC50 of chlorpyrifos (slope ¼ 0.70) at different selection intensities.
90
84
80
Slope = 0.70
Generations
60
48
44 37
40 22
20
Slope = 2.70
58
53
50 30
Slope = 1.70
70
70
18
15
38 30 13
10
24 10
0 50
60
70
80
90
Selection intensity (%) Fig. 2. Effect of slope on the number of generations of P. solenopsis required for a tenfold increase in LC50 of chlorpyrifos (h2 ¼ 0.04) at different selection intensities.
resistance varied in the entire selection pressure. Random genetic drift could be a likely cause of such observation. This contrasts with the findings of Tabashnik (1992), Sial and Brunner (2010), Lai and Su (2011), Abbas and Shad (2015), Shah et al. (2015a) and Ijaz et al. (2016), where additive genetic variations were present initially and declined after further selection, but agree with the finding of Shah et al. (2015b) and Abbas et al. (2016). The estimation of h2 on the basis of selections (Tabashnik, 1992) has some
important limitations, such as technical difficulties in estimation of parameters and doubt about extrapolation of results to field strains. The technical difficulties generally encountered in estimating h2 from selection experiments were discussed by Falconer et al. (1996). Despite recognized difficulties in extrapolating laboratory results to the field, we used estimates of realized h2 and slope of probit lines in conjunction with varying selection intensities to project the rate of resistance development (Figs. 1 and 2). The phenotypic standard deviation estimated as sp ¼ 1/average slope according to Tabashnik and McGaughey (1994), can provide a better mean slope estimation than the average of final and initial slopes due to different values of the slope in different generations. The projected rate of resistance evolution is directly proportional to h2 and selection intensity. The h2 of insecticide resistance may vary between populations due to a change in allele frequencies and the environment through time (Tabashnik, 1992). However, the predictions made from quantitative genetic theory on the basis of G ¼ R1 give valuable information to develop strategies for managing pesticide resistance which may slow resistance development by declining heritability (Firko and Hayes, 1990; Tabashnik, 1992). The estimation of h2 from a laboratory selection and factors responsible for resistance are necessary to assess insecticideresistance risks in pests (Lai and Su, 2011). The results of selection for chlorpyrifos resistance showed that P. solenopsis populations have the ability to develop resistance to this insecticide and act as an early warning to cotton farmers. If the laboratory estimates of h2 apply to field strains and 90 percent mortality occurs in each generation, a ten-fold increase in its LC50 after 38 generations of exposure to chlorpyrifos (h2 ¼ 0.04) may occur. Furthermore, assuming that the slope ¼ 0.70 and selection mortality ¼ 90 percent, then 10 generations are needed for a tenfold increase in the LC50 whereas 24 and 38 generations are required for the same to happen at slopes of 1.70, and 2.70, respectively. This estimate must be taken with caution due to the limitations discussed above. While cross-resistance generally occurs between the same groups of chemical compounds, it remains hard to predict without proper investigation (Denholm et al., 1998). Cross-resistance among insecticides having different structures and modes of action is extremely unpredictable (Gorman et al., 2010) but crossresistance to unrelated insecticides could be due either to a common mechanism affecting the insecticides or independent mechanisms. Cross-resistance among insecticides from different chemical groups is also possible when an iso-enzyme in an insect acts on different type of insecticides (Abbas et al., 2014). In this
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M. Ismail et al. / Crop Protection 94 (2017) 38e43
study, a moderate level of cross-resistance to nitenpyram and low level of cross-resistance to lambda-cyhalothrin and profenofos were observed in the Chlor-SEL strain of P. solenopsis. Previously, a chlorpyrifos resistant strain of L. striatellus (188-fold) had a 14-fold cross-resistance to dichlorvos and 1.6-fold cross-resistance to thiamethoxam after 25 generations (Wang et al., 2010a). In a codling moth resistant to azinphosmethyl, a negative cross-resistance to chlorpyrifos and methyl parathion was identified (Dunley and Welter, 2000). In conclusion, this study reveals a rapid rate of resistance development to chlorpyrifos in the selected strain of P. solenopsis. Resistance developing under selection pressure in the laboratory showed the likelihood of resistance development in the field if the selection pressure of chlorpyrifos persists for a long period of time. The insecticides (i.e. lambda-cyhalothrin and profenofos) with low cross-resistance can be used in a resistance management program (Abbas et al., 2016; Ijaz et al., 2016) to suppress P. solenopsis. Cultural and biological controls such as rotation of host and non-host plants and by conserving and releasing natural enemies such as Chrysoperla carnea Stephens and Cryptolaemus montrouzieri Mulsant through the use of more selective insecticides should also be incorporated into a resistance management program. In addition, an exploration of the biochemical and genetic mechanisms of resistance to chlorpyrifos and other insecticides is needed. Acknowledgements The authors are highly thankful to Prof. (Rtd.) Dr. Gerald Wilde, Department of Entomology, Kansas State University, USA, for sparing his precious time to check the manuscript for the improvement of English grammar. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cropro.2016.12.011. References Abbas, G., Arif, M.J., Ashfaq, M., Aslam, M., Saeed, S., 2010. Host plants, distribution and overwintering of cotton mealybug (Phenacoccus solenopsis; Hemiptera: Pseudococcidae). Int. J. Agric. Biol. 12, 421e425. 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. Abbas, N., Ijaz, M., Shad, S.A., Binyameen, M., 2016. Assessment of resistance risk to fipronil and cross resistance to other insecticides in the Musca domestica L. (Diptera: Muscidae). Vet. Parasitol. 223, 71e76. Abbas, N., Khan, H.A.A., Shad, S.A., 2014. Resistance of the house fly Musca domestica (Diptera: Muscidae) to lambda-cyhalothrin: mode of inheritance, realized heritability, and cross-resistance to other insecticides. Ecotoxicology 23, 791e801. Abbas, N., Shad, S.A., 2015. Assessment of resistance risk to lambda-cyhalothrin and cross-resistance to four other insecticides in the house fly, Musca domestica L. (Diptera: Muscidae). Parasitol. Res. 114, 2629e2637. Abbott, W., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265e267. Afzal, M.B.S., Abbas, N., Shad, S.A., 2015a. Inheritance, realized heritability and biochemical mechanism of acetamiprid resistance in the cotton mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae). Pestic. Biochem. Physiol. 122, 44e49. Afzal, M.B.S., Ijaz, M., Farooq, Z., Shad, S.A., Abbas, N., 2015b. Genetics and preliminary mechanism of chlorpyrifos resistance in Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae). Pestic. Biochem. Physiol. 119, 42e47. Afzal, M.B.S., Shad, S.A., 2015. Resistance inheritance and mechanism to emamectin benzoate in Phenacoccus solenopsis (Homoptera: Pseudococcidae). Crop Prot. 71, 60e65. Afzal, M.B.S., Shad, S.A., 2016a. Characterization of Phenacoccus solenopsis (Tinsley) (Homoptera: Pseudococcidae) resistance to emamectin benzoate: crossresistance patterns and fitness cost analysis. Neotrop. Entomol. 45, 310e319. Afzal, M.B.S., Shad, S.A., 2016b. Genetic analysis, realized heritability and synergistic suppression of indoxacarb resistance in Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae). Crop Prot. 84, 62e68.
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Cross-resistance relationships between neonicotinoids and pymetrozine in Bemisia tabaci (Hemiptera: Aleyrodidae). Pest Manag. Sci. 66, 1186e1190. Hodgson, C., Abbas, G., Arif, M.J., Saeed, S., Karar, H., 2008. Phenacoccus solenopsis Tinsley (Sternorrhyncha: Coccoidea: Pseudococcidae), an invasive mealybug damaging cotton in Pakistan and India, with a discussion on seasonal morphological variation. Zootaxa 1913, 1e35. Ijaz, M., Afzal, M.B.S., Shad, S.A., 2016. Resistance risk analysis to acetamiprid and other insecticides in Acetamiprid-Selected population of Phenacoccus solenopsis. Phytoparasitica 1e10. Jutsum, A.R., Heaney, S.P., Perrin, B.M., Wege, P.J., 1998. Pesticide resistance: assessment of risk and the development and implementation of effective management strategies. Pestic. Sci. 54, 435e446. Kaydan, M.B., Caliskan, A.F., Ulusoy, M.R., 2013. New record of invasive mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) in Turkey. EPPO Bull. 43, 169e171. 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Shen, J.L., Wu, Y.D., 1995. Insecticide Resistance in Cotton Bollworm and its Management. China Agricultural Press, Beijing, China, pp. 259e328. Sial, A.A., Brunner, J.F., 2010. Assessment of resistance risk in obliquebanded leafroller (Lepidoptera: Tortricidae) to the reduced-risk insecticides chlorantraniliprole and spinetoram. J. Econ. Entomol. 103, 1378e1385. Software, L., 2002. Polo Plus, a User's Guide to Probit or Logic Analysis. LeOra Software, Berkeley, CA. Tabashnik, B.E., 1992. Resistance risk assessment: realized heritability of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae), tobacco budworm (Lepidoptera: Noctuidae), and Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol 85, 1551e1559. Tabashnik, B.E., McGaughey, W.H., 1994. Resistance risk assessment for single and multiple insecticides: responses of Indianmeal moth (Lepidoptera: Pyralidae) to Bacillus thuringiensis. J. Econ. Entomol. 87, 834e841. Wang, L., Zhang, Y., Han, Z., Liu, Y., Fang, J., 2010a. Cross-resistance and possible n). Pest mechanisms of chlorpyrifos resistance in Laodelphax striatellus (Falle Manag. Sci. 66, 1096e1100. Wang, Y., Watson, G.W., Zhang, R., 2010b. The potential distribution of an invasive mealybug Phenacoccus solenopsis and its threat to cotton in Asia. Agric. For. Entomol 12, 403e416. Wang, Y.P., SanAn, W., RunZhi, Z., 2009. Pest risk analysis of a new invasive pest, Phenacoccus solenopsis, to China. Chin. Bull. Entomol. 46, 101e106.
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Resistance risk assessment to chlorpyrifos and cross-resistance to other insecticides in a field strain of Phenacoccus solenopsis Tinsley Muhammad Ismail a, *, Masood Ejaz a, Naeem Abbas b, Sarfraz Ali Shad a, Muhammad Babar Shahzad Afzal c a b c
Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan Department of Entomology, University College of Agriculture and Environmental Sciences, The Islamia University of Bahawalpur, Bahawalpur, Pakistan Citrus Research Institute, Sargodha, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2016 Received in revised form 30 November 2016 Accepted 7 December 2016
The organophosphate insecticide chlorpyrifos is recommended for control of a number of insect pests, including cotton mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae) in Pakistan. This work assessed chlorpyrifos resistance evolution and cross-resistance to other insecticides. After 23 generations of selection, the P. solenopsis strain (Chlor-SEL) had a 26652-fold level of resistance to chlorpyrifos compared to a susceptible strain. Realized heritability (h2) of resistance to chlorpyrifos was 0.04. The Chlor-SEL strain also had a low level of cross resistance to lambda-cyhalothrin (14-fold) and a very low level cross-resistance to nitenpyram and profenofos after 23 generations of selection. The projected rate of resistance development indicated that if 50e90 percent of a P. solenopsis population were selected with chlorpyrifos, a ten-fold increase in the lethal concentration 50 (LC50) would occur in 22e10 generations (h2 ¼ 0.04, Slope ¼ 0.70). At a similar slope, if h2 ¼ 0.14, then only 6-3 generations are required for a ten-fold increase in the LC50 at 50e90 percent selection intensity, respectively. Likewise, if h2 ¼ 0.24, then the same would occur in 4e2 generations. This study showed that P. solenopsis has the ability to become resistant to chlorpyrifos but insect resistance management strategies such as rotation of different group of insecticides are needed to prolong the effectiveness of chlorpyrifos in controlling P. solenopsis. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Cotton Mealybug Invasive pest Organophosphate Resistance evolution
1. Introduction Cotton mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae), is a major insect pest of cotton, vegetables, ornamental and medicinal plants worldwide (Abbas et al., 2010; Afzal et al., 2015d; Fand and Suroshe, 2015; Wang et al., 2009) due to its polyphagous nature. In Pakistan, P. solenopsis caused havoc to cotton production in 11 growing districts of Punjab during 2005 (Saeed et al., 2007) and average cotton yield was reduced by 50% (Muhammad, 2007). Apart from Pakistan, P. solenopsis has also created economic losses for cotton growers in India (Nagrare et al., 2009), United States of America (Fuchs et al., 1991), Republic of China (Wang et al., 2009), Taiwan and Thailand (Hodgson et al., 2008), Australia (Charleston et al., 2010) and Turkey (Kaydan et al., 2013). P. solenopsis damage plants by sucking cell sap from
* Corresponding author. E-mail address: [email protected] (M. Ismail). http://dx.doi.org/10.1016/j.cropro.2016.12.011 0261-2194/© 2016 Elsevier Ltd. All rights reserved.
the phloem and secreting honey dew that results in the development of a sooty mold which affects the photosynthesis process and can cause the premature death of plants (Afzal et al., 2015a; Culik and Gullan, 2005; Wang et al., 2010b). Synthetic chemical insecticides are used for the management of P. solenopsis worldwide, including Pakistan. However, the unnecessary and over use of insecticides in cotton agroecosystem has led to the development of resistance by P. solenopsis (Saddiq et al., 2014, 2015). Resistance in P. solenopsis under laboratory conditions has previously been documented to insecticides including acetamiprid (Afzal et al., 2015a,d), chlorpyrifos (Afzal et al., 2015b), emamectin benzoate (Afzal and Shad, 2015), deltamethrin (Saddiq et al., 2016), and indoxacarb (Afzal et al., 2015e). Extensive use of the organophosphate chlorpyrifos has resulted in resistance reported in a range of pests including P. solenopsis, Tetranychus urticae (Koch), n), and Liriomyza sativae (Blanchard) Laodelphax striatellus (Falle (Afzal et al., 2015b; Askari-Saryazdi et al., 2015; Kumral et al., 2009; Recep and Yorulmaz, 2010; Saddiq et al., 2016; Wang et al., 2010a).
M. Ismail et al. / Crop Protection 94 (2017) 38e43
Frequent use of different insecticides may result in loss of efficacy due to cross-resistance in the insect populations which imposes difficulties in developing successful insecticide resistance management plans (Kranthi et al., 2001; Basit et al., 2011). Crossresistance to other insecticides due to selection of chlorpyrifos resistance has been reported in L. striatellus (Wang et al., 2010a), L. sativae (Askari-Saryazdi et al., 2015), and Sogatella furcifera (Horv ath) (Mu et al., 2016). Studying resistance and crossresistance is useful to limit the development of resistance by employing practices such as insecticide mixtures and rotation of insecticides with different modes of action (Shen and Wu, 1995; Abbas et al., 2015). Risk assessment of insecticide resistance using laboratory or field selection studies can help to avoid or postpone resistance problems in the field (Jutsum et al., 1998; Lai and Su, 2011). Laboratory insecticide selection provides a quick way with fewer variables than those that occur in the field and can reveal the maximum potential of an insect to become resistant (Abbas and Shad, 2015; Sial and Brunner, 2010; Lin et al., 2003; Tabashnik, 1992). In this work, we studied the impact of continuous selection with chlorpyrifos on resistance allele frequencies (h2) under laboratory conditions in P. solenopsis and observed cross-resistance to other insecticides such as profenofos, lambda-cyhalothrin and nitenpyram in the Chlor-SEL strain. The results of this study will be helpful in our understanding of chlorpyrifos resistance and its management in controlling P. solenopsis. 2. Materials and methods 2.1. Insects Approximately 300 insects (nymphs and adults) were randomly selected from ten different areas of a cotton field located in Multan (30.1978 N, 71.4697 E). At the time of collection, the cotton plants were at the reproductive stage. The cotton field of the collection site received heavy amount of sprays from organophosphates, pyrethroids and new chemicals classes during the growing season to control various sucking and chewing pests prior to collecting insects (Afzal et al., 2015b,d; Saddiq et al., 2014, 2015). After collection, the insects were transported in plastic jars (12 24 cm) to the laboratory and were maintained at 27 ± 2 C, 65 ± 5% R.H. and 16:8 h L:D and reared on China rose, Hibiscus rosasinensis L. leaves and tender shoots. All stages of P. solenopsis were kept in plastic jars (12 24 cm) covered with a muslin cloth. The culture was refreshed every 2e3 days with clean fresh leaves along with small twigs. For a reference susceptible strain, a field strain was collected from a cotton field located in Multan district and reared without insecticide exposure for more than one year in the laboratory (Afzal et al., 2015b). 2.2. Insecticides
39
bioassay and each concentration was replicated five times. The five concentrations ranged between 0.625 and 10 mg a.i/ml for the susceptible, 31.25e1000 mg a.i/ml for the field population (Field Pop; G3), and 62.5e8000 mg a.i/ml for the chlorpyrifos selected strain (Chlor-SEL; G5-G25). Fresh leaves were dipped into serial dilutions of insecticides for 10 s and air dried at room temperature. Leaves for control were immersed into water only. Treated dried leaves were placed into petri-dishes (5 cm diameter). Five 2nd instars nymphs were placed in each petri dish so a total of 150 nymphs were used for a single bioassay (including the control). Bioassays were kept under the laboratory conditions as mentioned above. Mortality data were assessed 48 h after exposure to chlorpyrifos, lambda-cyhalothrin and profenofos, and 72 h after exposure to nitenpyram. Nymphs were considered to be dead if there was no leg movement after a gentle touch with fine hairbrush (Afzal et al., 2015d). 2.4. Chlorpyrifos selection The field population was selected with chlorpyrifos for 23 generations (G3-G25) and designated as the Chlor-SEL strain. Selection was carried out with the leaf dip method by exposing 2nd instar nymphs of P. solenopsis to chlorpyrifos (Afzal et al., 2015b). Selection was done at each generation and averages of 300 nymphs were exposed to increasing concentrations (75.26e2601.42 mg a.i/ ml). The selection of chlorpyrifos concentrations was based on the objective of having a sufficient number of nymphs to produce the next generation. Nymphal mortality was assessed after 48 h exposure to chlorpyrifos and the survivors of each selection were reared to obtain the next generation. 2.5. Estimation of realized heritability Realized heritability (h2) was determined according to the method of Falconer et al. (1996) and Tabashnik (1992) by the following equation.
h2 ¼
Selection ResponseðRÞ Selection differentialðSÞ
We estimated R, the difference in mean phenotype and whole parental generation before selection by Falconer (1989):
Selection responseðRÞ ¼
Log final LC50 Log initial LC50 N
The final LC50 was the LC50 value after N number of generations and the initial LC50 was the LC50 value of the field population before selection. Selection differential was calculated as:
Selection differentialðSÞ ¼ i sp Intensity of selection was calculated as follows:
®
Commercial formulations of chlorpyrifos (Lorsban , 40EC; Dow Agro Sciences, Pakistan), lambda-cyhalothrin (Karate® 2.5EC, Syngenta), profenofos (Curacron® 500EC, Syngenta) and nitenpyram (Paranol® 10EC, Kanzo Agro Chemicals) were used for the experiments.
i ¼ 1:583 0:0193336p þ 0 : 0000428p2 þ 3:65194=p where p is the average percent survival of Chlor-SEL strain after N number of selection (Tabashnik and McGaughey, 1994) and sp is the phenotypic standard deviation calculated by:
2.3. Bioassays
sp ¼ ½ðinitial slope final slopeÞ0:51
To assess the toxicities of selected insecticides, a leaf dip bioassay was conducted on 2nd instar nymphs of P. solenopsis (Afzal et al., 2015d). Serial dilutions of insecticide concentrations (mg a.i/ ml) were prepared using chlorpyrifos, lambda-cyhalothrin, profenofos, and nitenpyram. Five concentrations were used for each
To estimate changes in R, S, and h2 during the selection pressure, each parameter was determined for the first and second half (7 generations in one half) of the experiment. The generation G13 was used in both halves of the experiment. The generation (G) needed for a ten-fold increase in LC50 was calculated by:
40
M. Ismail et al. / Crop Protection 94 (2017) 38e43
1
G¼R
1 ¼ h2 S
Resistance ratios (RR) were calculated as: LC50 value of resistant strain/LC50 value of susceptible strain and 95% CI of RR were calculated according to Robertson and Preisler (1992). 2.6. Statistical analysis Data was analyzed by using probit analysis (Finney, 1971) with POLO Plus software (Software, 2002) to determine the LC50 values, confidence intervals (CIs), slopes and their standard errors. Control mortality was corrected using Abbott's formula (Abbott, 1925). 3. Results 3.1. Selection of resistance to chlorpyrifos Selection with chlorpyrifos for 23 generations resulted in an increase in LC50 values (ranging from 75.30 to 9328.24 mg a.i/ml for the Chlor-SEL strain) compared to the susceptible strain. The RR increased from 123-fold to 15292-fold compared to the susceptible strain and 52e124-fold compared to the Field Pop (Table 1). The LC50 of Field pop was previously published by Afzal et al. (2015b). 3.2. Cross-resistance to other insecticides in the Chlor-SEL strain The Chlor-SEL strain of P. solenopsis had low levels of crossresistance to lambda-cyhalothrin (10.5-fold) and profenofos (11.2fold) after 23 generations of selection (G25) compared to the Field Pop (G3). Moderate level of cross-resistance to nitenpyram (30.3fold) was observed in the Chlor-SEL strain (Table 2). 3.3. Realized heritability (h2) The overall mean estimated h2 of chlorpyrifos resistance in P. solenopsis (G3eG25) was 0.04. The estimated h2 of chlorpyrifos resistance was 0.12 and 0.24 in first half and in second half of present study, respectively. The R and S were higher in the first half compared with second half of selection. Therefore, the estimated h2 of chlorpyrifos resistance was higher in the second than in the first half of selection. (see Table 3). 3.4. Projected rate of chlorpyrifos resistance development The projected rate of resistance development is directly proportional to h2 and selection intensity (Fig. 1). For example, if we assume that the slope ¼ 0.70 (the value of mean slope for 23 Chlor-
SEL generations in this study) and h2 ¼ 0.04, then 22-10 generations would be required for a ten-fold increase in the LC50 at a 50e90 percent selection intensity, respectively. However, at a similar slope, if h2 ¼ 0.14, then 6e3 generations would be required for a ten-fold increase in the LC50 at a 50e90 percent selection intensity. Likewise, if h2 ¼ 0.24, then the same would occur in 4e2 generations at 50e90 percent selection intensity (Fig. 1). The projected rate of resistance development is inversely proportional to the slope. For example, if we assume that h2 ¼ 0.04 (heritability of chlorpyrifos resistance estimated in this study) and slope ¼ 0.70, then a ten-fold increase in the LC50 would occur in 84e38 generations at a 50e90 percent selection intensity, respectively. However, at the same h2, if the slope ¼ 1.70, then 53e24 generations would be required for a tenfold increase in the LC50 at 50e90 percent selection intensity, respectively. Likewise, if the slope ¼ 2.70, then the same would occur in 22e10 generations at 50e90 percent selection intensity, respectively (Fig. 2).
4. Discussion In this study, the selection of P. solenopsis with chlorpyrifos for 23 generations resulted in the development of very high resistance (15292-fold). It has recently been documented that P. solenopsis in Pakistan is capable of developing high levels of resistance to different insecticides under laboratory selection pressure (Afzal and Shad, 2015, 2016a; Afzal et al., 2015c,d), suggesting that selection has an obvious effect on the evolution of resistance. Evolution of resistance occurs faster under high selection pressure when susceptible genes are replaced by resistant genes, resulting in a high number of resistant individuals in a population (Ijaz et al., 2016). Realized heritability (h2) provides a standardized approach to evaluate the results of selection by combining the selection strength estimates and resistance development rate (Abbas and Shad, 2015; Tabashnik, 1992). The h2 provide a good way to present selection results with a solid foundation of the theoretical and empirical literature of evolutionary biology (Falconer et al., 1996; Mousseau and Roff, 1987). The lower h2 (0.04) after 23 generations of selection with chlorpyrifos suggests that P. solenopsis strains are less likely to develop resistance to chlorpyrifos when compared with other insecticides, such as acetamiprid, h2 ¼ 0.58 (Afzal et al., 2015a), spinosad, h2 ¼ 0.94 (Afzal et al., 2015c), and indoxacarb, h2 ¼ 1.13 (Afzal and Shad, 2016b). The lower h2 indicates higher environmental variations and lower additive genetic variations for chlorpyrifos resistance in P. solenopsis. The selection response and the selection differential declined as the selection pressure of chlorpyrifos were raised, causing lower h2 in the first compared with the second half of selection. These results showed that the expression level of alleles responsible for chlorpyrifos
Table 1 Resistance development of P. solenopsis to chlorpyrifos after 23 generations of selection. Selection
LC50 (95% Cl) [mg a.i/ml ]
Slope ± SE
Susceptible Field Pop (G3)c Chlor-SEL (G11) Chlor-SEL (G13) Chlor-SEL (G15) Chlor-SEL (G17) Chlor-SEL (G19) Chlor-SEL (G21) Chlor-SEL (G23) Chlor-SEL (G25)
0.61 (0.30e0.87) 75.30 (25.90e123.00) 4631.20 (2220.00e66645.) 3752.90 (2270.10e10768.00) 3848.90 (2406.00e9709.30) 5872.20 (3625.40e16055.00) 4427.10 (2778.80e11328.00) 4530.48 (2874.20e11301.00) 7851.45 (3649.70e179650.00) 9328.24 (4279.10e202142.10)
2.22 1.51 0.90 1.12 1.24 1.26 1.32 1.37 0.84 0.90
a b c
± 0.50 ± 0.36 ± 0.30 ± 0.29 ± 0.30 ± 0.31 ± 0.34 ± 0.33 ± 0.29 ± 0.30
Resistance ratio calculated as LC50 of Chlor-SEL/LC50 of susceptible strain. Resistance ratio calculated as LC50 of Chlor-SEL/LC50 of Field Pop. Published by Afzal et al. (2015b).
c2
df
P
RRa
RRb
4.62 1.74 0.04 0.15 0.59 0.29 0.40 1.14 0.41 1.05
3 3 3 3 3 3 3 3 3 3
0.20 0.63 0.99 0.99 0.90 0.96 0.94 0.77 0.94 0.79
1.00 123.44 (55.42e285.95) 7595.13 (2331.88e25733.91) 6152.30 (2803.37e14056.25) 6309.67 (2980.93e13903.88) 9626.56 (4438.98e21733.87) 7257.62 (3412.95e16067.05) 7427.02 (3529.15e16271.83) 12,871.23 (3736.58e46157.76) 15,292.20 (4311.38e56467.93)
1.00 61.50 (17.24e200.00) 49.84 (20.00e125.00) 51.11 (21.28e125.00) 77.98 (31.25e200.00) 58.79 (24.39e142.86) 60.16 (25.00e142.86) 104.26 (27.78e333.33) 123.88 (32.26e500.00)
M. Ismail et al. / Crop Protection 94 (2017) 38e43
41
Table 2 Cross-resistance to some tested insecticides in Chlor-SEL of P. solenopsis. Strain
Insecticide
LC50 (95% Cl) [mg a.i/ml]
RR
Slope þ SE
Field (G3)
Lambda-cyhalothrin Nitenpyram Profenofos Lambda-cyhalothrin Nitenpyram Profenofos
105.70 (61.84e191.61) 55.84 (11.80e105.93) 171.64 (59.65e288.02) 1111.48 (731.12e1969.40) 1693.64 (1101.77e3623.55) 1922.60 (1166.10e4857.20)
1.00 1.00 1.00 10.52 (5.35e20.83) 30.33 (11.63e76.92) 11.20 (4.67e27.03)
1.31 1.00 1.19 1.30 1.32 1.07
Chlor-SEL (G25)
± ± ± ± ± ±
0.32 0.31 0.31 0.39 0.31 0.29
c2
df
P
0.24 0.03 0.84 0.04 0.61 0.17
3 3 3 3 3 3
0.97 0.99 0.84 0.99 0.89 0.98
Table 3 Estimated realized heritability (h2) of resistance to chlorpyrifos in P. solenopsis. Insecticides
Selected generations
Initial LC50 (mg a.i/ml)
Final LC50 (mg a.i/ml)
R
I
Mean slope
sp
S
h2
Chlorpyrifos Chlorpyrifos Chlorpyrifos
11 (G3-G13) 12 (G13-G25) 23 (G3-G25)
75.30 4359.83 75.30
4359.83 9328.24 9328.24
0.16 0.03 0.10
0.45 0.60 0.54
0.34 0.92 0.70
2.95 1.09 1.42
1.32 0.66 0.76
0.12 0.24 0.04
h2 =0.04 25
22
h2 =0.24
18
20 Generations
h2 =0.14
15 15 10
13 10 6
5
4
4
4
3
3
2
50
60
5
0
70 80 Selection intensity %
3 2 90
Fig. 1. Effect of heritability (h2) on the number of generations of P. solenopsis required for a tenfold increase in LC50 of chlorpyrifos (slope ¼ 0.70) at different selection intensities.
90
84
80
Slope = 0.70
Generations
60
48
44 37
40 22
20
Slope = 2.70
58
53
50 30
Slope = 1.70
70
70
18
15
38 30 13
10
24 10
0 50
60
70
80
90
Selection intensity (%) Fig. 2. Effect of slope on the number of generations of P. solenopsis required for a tenfold increase in LC50 of chlorpyrifos (h2 ¼ 0.04) at different selection intensities.
resistance varied in the entire selection pressure. Random genetic drift could be a likely cause of such observation. This contrasts with the findings of Tabashnik (1992), Sial and Brunner (2010), Lai and Su (2011), Abbas and Shad (2015), Shah et al. (2015a) and Ijaz et al. (2016), where additive genetic variations were present initially and declined after further selection, but agree with the finding of Shah et al. (2015b) and Abbas et al. (2016). The estimation of h2 on the basis of selections (Tabashnik, 1992) has some
important limitations, such as technical difficulties in estimation of parameters and doubt about extrapolation of results to field strains. The technical difficulties generally encountered in estimating h2 from selection experiments were discussed by Falconer et al. (1996). Despite recognized difficulties in extrapolating laboratory results to the field, we used estimates of realized h2 and slope of probit lines in conjunction with varying selection intensities to project the rate of resistance development (Figs. 1 and 2). The phenotypic standard deviation estimated as sp ¼ 1/average slope according to Tabashnik and McGaughey (1994), can provide a better mean slope estimation than the average of final and initial slopes due to different values of the slope in different generations. The projected rate of resistance evolution is directly proportional to h2 and selection intensity. The h2 of insecticide resistance may vary between populations due to a change in allele frequencies and the environment through time (Tabashnik, 1992). However, the predictions made from quantitative genetic theory on the basis of G ¼ R1 give valuable information to develop strategies for managing pesticide resistance which may slow resistance development by declining heritability (Firko and Hayes, 1990; Tabashnik, 1992). The estimation of h2 from a laboratory selection and factors responsible for resistance are necessary to assess insecticideresistance risks in pests (Lai and Su, 2011). The results of selection for chlorpyrifos resistance showed that P. solenopsis populations have the ability to develop resistance to this insecticide and act as an early warning to cotton farmers. If the laboratory estimates of h2 apply to field strains and 90 percent mortality occurs in each generation, a ten-fold increase in its LC50 after 38 generations of exposure to chlorpyrifos (h2 ¼ 0.04) may occur. Furthermore, assuming that the slope ¼ 0.70 and selection mortality ¼ 90 percent, then 10 generations are needed for a tenfold increase in the LC50 whereas 24 and 38 generations are required for the same to happen at slopes of 1.70, and 2.70, respectively. This estimate must be taken with caution due to the limitations discussed above. While cross-resistance generally occurs between the same groups of chemical compounds, it remains hard to predict without proper investigation (Denholm et al., 1998). Cross-resistance among insecticides having different structures and modes of action is extremely unpredictable (Gorman et al., 2010) but crossresistance to unrelated insecticides could be due either to a common mechanism affecting the insecticides or independent mechanisms. Cross-resistance among insecticides from different chemical groups is also possible when an iso-enzyme in an insect acts on different type of insecticides (Abbas et al., 2014). In this
42
M. Ismail et al. / Crop Protection 94 (2017) 38e43
study, a moderate level of cross-resistance to nitenpyram and low level of cross-resistance to lambda-cyhalothrin and profenofos were observed in the Chlor-SEL strain of P. solenopsis. Previously, a chlorpyrifos resistant strain of L. striatellus (188-fold) had a 14-fold cross-resistance to dichlorvos and 1.6-fold cross-resistance to thiamethoxam after 25 generations (Wang et al., 2010a). In a codling moth resistant to azinphosmethyl, a negative cross-resistance to chlorpyrifos and methyl parathion was identified (Dunley and Welter, 2000). In conclusion, this study reveals a rapid rate of resistance development to chlorpyrifos in the selected strain of P. solenopsis. Resistance developing under selection pressure in the laboratory showed the likelihood of resistance development in the field if the selection pressure of chlorpyrifos persists for a long period of time. The insecticides (i.e. lambda-cyhalothrin and profenofos) with low cross-resistance can be used in a resistance management program (Abbas et al., 2016; Ijaz et al., 2016) to suppress P. solenopsis. Cultural and biological controls such as rotation of host and non-host plants and by conserving and releasing natural enemies such as Chrysoperla carnea Stephens and Cryptolaemus montrouzieri Mulsant through the use of more selective insecticides should also be incorporated into a resistance management program. In addition, an exploration of the biochemical and genetic mechanisms of resistance to chlorpyrifos and other insecticides is needed. Acknowledgements The authors are highly thankful to Prof. (Rtd.) Dr. Gerald Wilde, Department of Entomology, Kansas State University, USA, for sparing his precious time to check the manuscript for the improvement of English grammar. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cropro.2016.12.011. References Abbas, G., Arif, M.J., Ashfaq, M., Aslam, M., Saeed, S., 2010. Host plants, distribution and overwintering of cotton mealybug (Phenacoccus solenopsis; Hemiptera: Pseudococcidae). Int. J. Agric. Biol. 12, 421e425. 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. Abbas, N., Ijaz, M., Shad, S.A., Binyameen, M., 2016. Assessment of resistance risk to fipronil and cross resistance to other insecticides in the Musca domestica L. (Diptera: Muscidae). Vet. Parasitol. 223, 71e76. Abbas, N., Khan, H.A.A., Shad, S.A., 2014. Resistance of the house fly Musca domestica (Diptera: Muscidae) to lambda-cyhalothrin: mode of inheritance, realized heritability, and cross-resistance to other insecticides. Ecotoxicology 23, 791e801. Abbas, N., Shad, S.A., 2015. Assessment of resistance risk to lambda-cyhalothrin and cross-resistance to four other insecticides in the house fly, Musca domestica L. (Diptera: Muscidae). Parasitol. Res. 114, 2629e2637. Abbott, W., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265e267. Afzal, M.B.S., Abbas, N., Shad, S.A., 2015a. Inheritance, realized heritability and biochemical mechanism of acetamiprid resistance in the cotton mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae). Pestic. Biochem. Physiol. 122, 44e49. Afzal, M.B.S., Ijaz, M., Farooq, Z., Shad, S.A., Abbas, N., 2015b. Genetics and preliminary mechanism of chlorpyrifos resistance in Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae). Pestic. Biochem. Physiol. 119, 42e47. Afzal, M.B.S., Shad, S.A., 2015. Resistance inheritance and mechanism to emamectin benzoate in Phenacoccus solenopsis (Homoptera: Pseudococcidae). Crop Prot. 71, 60e65. Afzal, M.B.S., Shad, S.A., 2016a. Characterization of Phenacoccus solenopsis (Tinsley) (Homoptera: Pseudococcidae) resistance to emamectin benzoate: crossresistance patterns and fitness cost analysis. Neotrop. Entomol. 45, 310e319. Afzal, M.B.S., Shad, S.A., 2016b. Genetic analysis, realized heritability and synergistic suppression of indoxacarb resistance in Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae). Crop Prot. 84, 62e68.
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