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Resistance of Colorado potato beetle (Leptinotarsa decemlineata Say) to commonly used insecticides in Iran
Journal of Asia-Pacific Entomology 17 (2014) 213–220
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Review
Resistance of Colorado potato beetle (Leptinotarsa decemlineata Say) to commonly used insecticides in Iran M. Malekmohammadi ⁎ Department of Plant Protection, Faculty of Agriculture, Bu Ali Sina University, Hamedan, Iran
a r t i c l e
i n f o
Article history: Received 21 August 2013 Revised 19 November 2013 Accepted 17 January 2014 Available online 27 January 2014 Keywords: Iran Colorado potato beetle Insecticide resistance Resistance mechanism
a b s t r a c t The Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), is a major pest of potatoes in Iran and many other parts of the world. Injury is caused when adults and larvae feed on the foliage and stems of potato plants, resulting in poor yields and/or plant death. Adult beetles can also vector plant diseases. Historically, the CPB been controlled using different insecticides, but it is currently resistant to nearly all classes of insecticides and remains a serious pest in many parts of the world. All of the resistance mechanisms reported in insects have been demonstrated in CPB. L. decemlineata invaded Iran in the early 1980s, probably through the importation of infested potatoes. It has caused significant damage to potato crops in affected areas, and it accordingly remains a major threat to Iranian potato production. Regrettably, no IPM programs have been developed for managing CPB infestations in Iran. Furthermore, there are no organized CPB resistance monitoring programs in Iran, and the recommended insecticides for CPB control, endosulfan and phosalon, have not changed in over 22 years. Anecdotal evidence from local farmers suggests a reduction in the efficacy of control of CPB by commonly used insecticides, probably due to the reduced susceptibility to these insecticides. Given the economic significance of L. decemlineata infestations, the increasing prevalence of resistance in this species, the rate of spread of infestations, and the extent of the area infested, there is an urgent need to develop effective and sustainable integrated pest management programs for CPB in Iran. © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Biology of the Colorado potato beetle in Iran . . . . . . . . . Economic threshold for Colorado potato beetle damage . . . . Current control methods for Colorado potato beetle . . . . . . Chemical control and insecticide resistance . . . . . . . . . . Mechanisms of resistance . . . . . . . . . . . . . . . . . . Current status of resistance in CPB to commonly used insecticides Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction The Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), a leaf beetle (Coleoptera: Chrysomelidae) native to the southwestern United States and Mexico, was first collected by Thomas Nuttall in ⁎ Tel.: +98 9183130963; fax: +98 8118251013. E-mail address: [email protected].
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1811 and then described in 1824 by Thomas Say (Jacques, 1988). This beetle is a major pest of potato (Solanum tuberosum) in Iran and many other parts of the world. Injury is caused when adults and larvae feed on the foliage and stems of potato plants, resulting in poor yields and/ or plant death. Adults can also vector plant diseases. This species can also feed, develop, and reproduce on tomato (Lycopersicon esculentum Miller) and eggplant (Solanum melongena L.) and cause significant damage to these crops (Hare, 1990; Phyllis, 2004).
1226-8615/$ – see front matter © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220
Iran is the third-largest potato producer in Asia, after China and India. Potatoes are grown in almost all of the provinces of Iran but production is focused in the western and northwestern provinces, where they are a single-season crop. Potatoes rank third in terms of production, behind rice and wheat. In 2010–2011, the total area under potato cultivation in Iran was around 146,000 ha, with a total production of 4,300,000 t. The average yield was 29,445.5 kg/ha (Anonymous, 2011). Leptinotarsa decemlineata is a key quarantine pest in Iran. It is believed to have invaded the country in the early 1980s, probably on potatoes imported from infested areas. After its initial detection in the northeastern province of Ardabil, in 1984 (Nouri-Ganbalani, 1986), it gradually became established in northwestern Iran. Since then, it has spread westward to the main potato-growing areas. Despite its initial designation as an internal quarantine pest, potatoes originating in infested areas were, for a long time, not subject to a plant health inspection before being transported within Iran. This oversight has resulted in the introduction of L. decemlineata to other provinces of Iran. Currently, it is distributed across nearly all of the principal potato-growing provinces, and continues to expand its range into central and northeastern Iran (Fig. 1). Various insecticides have been used to control CPB, but it has developed resistance to most classes of insecticide. Because of the significant economic impacts of L. decemlineata infestations, the increasing prevalence of insecticide resistance, the high rate of infestation development, and the wide distribution of this species, there is an urgent need to develop an effective and sustainable integrated pest management program for CPB in Iran. Although resistance monitoring is crucial for developing insecticide application strategies, there is no organized CPB resistance monitoring
program in Iran. The recommended insecticides for CPB control in Iran, endosulfan and phosalon, have not changed in over 22 years. For a long time, phosalon and endosulfan proved to be economically viable, effective, and reliable in controlling CPB in Iran. However, recent anecdotal evidence from local farmers indicates a reduction in the efficacy of control of CPB by these insecticides, which may be due to the development of insecticide resistance.
Biology of the Colorado potato beetle in Iran In Iran, the biology of the CPB on potato plants has been studied in the province of Ardabil (Nouri-Ganbalani, 1989). This study observed that overwintered adults emerged and fed on the leaves of potato plants in early May, when mean temperatures were approximately 12 °C. Oviposition occurred primarily from late May through late July. The highest larval densities were observed in late June and early July, coinciding with early blooming of the potato crop. Adults from the first generation emerge from pupation in the soil in late July. Depending on environmental conditions, there may occur multiple generations of L. decemlineata per year. According to Nouri-Ganbalani (1989), the CPB has two overlapping generations per year in Iran. The development periods for the first and second generations were indicated to be about 45 and 52 days, respectively. Given that the emergence of secondgeneration adults coincided with a sudden drop in temperature in autumn and the drying of plant foliage, the majority of them died. As a result, most of the overwintering adults came from the first (summer) generation. As much as 50% of the overwintered population may enter a second diapause.
Fig. 1. The geographical range of Colorado potato beetle in Iran. Top six potato producer provinces: 1: Hamedan, 2: Ardabil, 3: Esfahan, 4: East Azarbaijan, 5: Kordestan, 6: Zanjan (Anonymous, 2011).
M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220 Table 1 Insecticides to which L. decemlineata has already developed resistance (Whalon et al., 2008). Chemical group
Common names
Bacillus thuringiensis subsp. tenebrionis endotoxin Carbamates
Bt
Cyclodiene organochlorines Inorganics Isoflavones Macrocyclic lactones (avermectins) Neonicotinoids
Nereistoxin analogs Organochlorines Organophosphates
Organotins Pyrethroids, pyrethrins
Spinosyns
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(Iran) as 5 and 6.458 late larvae per plant in year 2004 and 2005 respectively. According to Nouri-Ganbalani et al. (2010), at larval densities up to five late-stage larvae per plant chemical control is not economically justified. Current control methods for Colorado potato beetle
Aldicarb, carbaryl, carbofuran, cloethocarb, dioxacarb, oxamyl, propoxur Aldrin, chlordane, dieldrin, endosulfan, endrin, HCH-gamma, toxaphene Hydrogen cyanide Rotenone Abamectin Thiamethoxam, acetamiprid, clothianidin, dinotefuran, imidacloprid, N-desmethylthiamethoxam, N-methylimidaclopridnitenpyram, thiacloprid Cartap DDT, methoxychlor Azamethiphos, azinphosethyl, azinphosmethyl, chlorfenvinphos, malathion, methamidophos, methidathion, monocrotophos, parathion, parathion-methyl, phorate, phosmate, phoxim, quinalphos, tetrachlorvinphos, trichlorfon Azocyclotin Cypermethrin, deltamethrin, esfenvalerate, fenvalerate, permethrin Spinosad
Economic threshold for Colorado potato beetle damage Each beetle consumes approximately 40 cm2 of foliage during the larval stage (Ferro et al., 1985; Logan et al., 1985) and an additional 10 cm2 per day as an adult (Ferro et al., 1985). If left uncontrolled, the pest can cause yield losses of up to 100% by defoliating potato plants prior to tuber formation. On potato crops, CPB is particularly damaging during the tuber formation and filling stages. On the other hand, potato plants are tolerant to some insect damage; they can lose up to 30% of their leaves without suffering significant yield losses. Various studies on the effects of CPB defoliation on potato yields concluded that intense defoliation early or late in the season may have little effect on yield, whereas a moderate mid-season defoliation can cause significant yield losses (Cranshaw and Radcliffe, 1980; Hare, 1980; Shields and Wyman, 1984). As a result, failure to control the first-generation larvae may cause significant yield losses as a result of complete defoliation. The economic threshold for L. decemlineata has been calculated on the basis of either the number of insects per plant (Martel et al., 1986; Senanayake and Holliday, 1990; Mailloux et al., 1991) or the degree of leaf damage due to CPB feeding (Ferro et al., 1983; Connell et al., 1991; Zehnder et al., 1995). Nouri-Ganbalani et al. (2010) determined the economic injury level for the potato cultivar ‘Agria’ in Ardabil
Several different insecticides have been used to control CPB infestations. Although IPM-based approaches exist, most potato growers have traditionally relied on insecticides to prevent damage by L. decemlineata. Some studies have indicated that applications of certain newly developed insecticides with novel modes of action may result in efficient control of CPB (Sirota and Grafius, 1994; Furlong and Groden, 2001; Cutler et al., 2005a,b). The effects of five chitin synthesis inhibitors (diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron) against the second instars of CPB were studied by Karimzadeh et al. (2007), with the finding that lufenuron and hexaflumuron may be particularly effective in future CPB management programs. The inclusion of bioinsecticides in IPM programs is essential for the efficient control of CPB and for minimizing the development of insecticide resistance (Barcic et al., 2006). According to Faridi et al. (2011), rotation of the insecticide spinosad with conventional insecticides can prolong the effectiveness of the former and result in a reduction in the total number of sprays necessary. In another study, the effect of Bacillus thuringiensis var. kurstaki on different larval instars of CPB, and the roles of caffeine and aqueous neem extracts in the enhancement of its action, was evaluated by Javvi et al. (2005). Their study indicated evident synergistic effects of these plant extracts on the efficacy of B. thuringiensis var. kurstaki. A similar effect was reported by Gassemi Kahrizeh et al. (2004), who observed a noticeable synergistic effect of henna extract on the efficacy of this bacterial strain in controlling CPB. In another study, the natural predators and pathogenic agents of CPB in potato fields in Ardabil were identified as follows: the pathogenic fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes); the parasitic mite Linobia sp. (Acari: Hemisarcoptidae); the long-legged spider Phalangium sp. (Opilions: Phalangidae); the predatory bug, Rhinocoris punctiventris (Heteroptera: Reduviidae); and the green lacewing Chrysoperla carnea (Neuroptera: Chrysopidae) (Nouri-Ganbalani et al., 1998). This study revealed that B. bassiana and C. carnea could be successfully incorporated into IPM programs to control L. decemlineata. Chemical control and insecticide resistance Colorado potato beetles experience intense selection pressure for insecticide resistance, because of the significant economic impacts of this pest and the subsequent intensive use of pesticides in infested areas. More than 30 active ingredients are currently registered for use against L. decemlineata around the world (Alyokhin et al., 2008). On the other hand, this species, like many other herbivorous insects, has evolved to resist a variety of defensive phytochemicals, particularly alkaloids. Therefore, it is biochemically well-adapted to develop
Table 2 Toxicity of endosulfan to larvae of a susceptible strain (SS) and field population of Colorado potato beetle, Bostanabaad (ba) with and without synergists (Mohammadi Sharif et al., 2007). Population
na
Synergist
Slope (SE)
LD50 (95% CLb) (μg/insect)
Resistance ratioc
Synergist ratiod
SS
360 366 341 360 360 358
– DEF PBO – DEF PBO
1.9 (0.23) 1.7 (0.27) 2.0 (0.24) 0.9 (0.14) 1.0 (0.12) 0.9 (0.1)
0.1 (0.07–0.12) 0.2 (0.17–0.26) 0.1 (0.09–0.14) 10.9 (7.1–16.3) 3.1 (2.1–4.41) 4.7 (3.1–7.0)
– – – 109 – –
– 0.5 1 – 3.5 2.3
Ba
a b c d
The number of larvae used in each bioassay. CL, confidence interval limit. LD50 of resistant population/LD50 of susceptible strain. LD50 without synergist/LD50 with synergist.
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Table 3 Substrate specificities and kinetic properties of glutathione-S-transferase from the susceptible and resistant strains of CPB (Mohammadi Sharif et al., 2007). Parameter
Susceptible
Resistant (Bostanabaad strain)
Activitya (4)b Kmc Vmaxa
73.2 ± 7.6 154 ± 31.3 66.5 ± 16.3
61.9 ± 12.4 279 ± 54 72.5 ± 6.6
a b c
development of resistance is occurring. Under these circumstances, it is vital that CPB populations under insecticide selection pressure are monitored. However, very little background information is available concerning the insecticide resistance displayed by such populations to insecticides or the mechanisms that may be involved.
Mechanisms of resistance
Mean value (μM/min/mg protein) ± SD. Number of replicates (n). Mean value (μM) ± SD.
insecticide resistance. This tendency, in combination with the aforementioned high selection pressure, has resulted in the development of resistance to a large number of insecticides from various classes (Table 1). Direct correlations between the frequency of insecticide applications and the development of resistance have been reported by many researchers (Hare, 1980; Roush and Miller, 1986; Tisler and Zehnder, 1990). Rather than evolving in isolated areas and spreading geographically, the development of insecticide resistance in CPB is heavily dependent on the patterns of insecticide use and local selection pressure, resulting in repeated and independent occurrences of resistance development. The degree of insecticide resistance in L. decemlineata may vary greatly, regionally or locally, or among different life stages (Silcox et al., 1985; Zehnder, 1986; Zehnder and Gelernter, 1989; Pourmirza, 2005). Heim et al. (1990) indicated that there were differences in CPB susceptibility to insecticides, even among populations originating from the same region. The development of resistance to organochlorine, organophosphorus (OP), carbamate (CB), and pyrethroid classes of insecticides has been documented in several studies (Harris and Svec, 1976; McDonald, 1976; Harris and Svec, 1981; Heim et al., 1990; French et al., 1992; Pap et al., 1997). Other studies have also demonstrated the acquisition of resistance to recently registered products (Zhao et al., 2000; Cutler et al., 2005a). Presently, CPB is resistant to nearly all classes of insecticides and consequently remains a serious pest in many parts of the world (Pedigo, 2004). All of the resistance mechanisms reported in insects have been demonstrated in CPB. The current status of CPB resistance to conventional insecticides in Iran is unclear. However, the continuous use of insecticides over several years, as the major means of CPB control, has fueled concern that the
All of the resistance mechanisms reported in insects have been demonstrated in CPB. Many studies have indicated an increase in the activity or quantity of mixed function oxidases, esterases, and glutathione-Stransferases, in insecticide-resistant CPBs (Zhao et al., 2000; Cutler et al., 2005b). Target site insensitivity has been demonstrated as the major cause of resistance to OPs, CBs, pyrethroids, and cyclodienes (Ffrench-Constant et al., 1993; Anthony et al., 1995; Bass et al., 2004). However, alteration of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) to an insensitive form associated with increased AChE activity has been proven to be an important mechanism conferring resistance to OPs and/or CBs in some insect species (Plapp and Tripathi, 1978; Hama et al., 1980; Fournier et al., 1992; Zhu and Gao, 1999). Other known mechanisms of CPB resistance to insecticides include the increased excretion and/or reduced penetration of certain insecticides. In addition, there is some evidence of behavioral resistance to insecticides. The activity of AChE is commonly used as a biomarker of exposure to different contaminants, including pesticides (Podolska et al., 2008). Kinetic analysis of AChE was used to explain the resistance of some insect strains and the selectivity of some OP insecticides (Guedes et al., 1997; Pradhan and Mishra, 1998; Zhu and Gao, 1998; Ali et al., 2005). Previous studies have determined the process of development of resistance to OPs and CBs in CPB (Argentine et al., 1989a, 1994, 1995; Zhu and Clark, 1995, 1997; Clark, 1997; Zhang et al., 1999; Kim et al., 2006, 2007). Resistance to azinphosmethyl has also been reported in CPB (Argentine et al., 1989b; Wierenga and Hollingworth, 1993). The high level of resistance to azinphosmethyl (136-fold) in a nearly isogenic CPB strain (AZ-R) was postulated to be the result of multiple resistance mechanisms, including reduced penetration, enhanced xenobiotic metabolism, and target site insensitivity (Argentine et al., 1989b; Wierenga and Hollingworth, 1993; Zhu and Clark, 1995; Zhang et al.,
Table 4 Toxicity of phosalone to larvae of a susceptible strain (SS) and field populations of Colorado potato beetle, Aliabad (Aa), Bahar (B), Dehpiaz (Dp), and Yengijeh (Yg) with and without synergists (Malekmohammadi et al., 2010). Population
na
dfb
Synergist
Slope (SE)
LD50 (95% CLc) (μg/insect)
χ2d
SS
260 190 270 210 302 270 230 270 183 270 270 270 190 250 230 230
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
– DEF DEM PBO – DEF DEM PBO – DEF DEM PBO – DEF DEM PBO
1.3 (0.1) 1.6 (0.1) 2.1 (0.2) 1.7 (0.1) 2.8 (0.2) 2.1 (0.2) 2.7 (0.2) 4.7 (0.4) 3.1 (0.2) 2.1 (0.2) 2.7 (0.2) 1.6 (0.2) 3.6 (0.3) 1.6 (0.1) 3.1 (0.3) 4.3 (0.4)
2.4 (2.1–2.7) 1.5 (1.3–1.6) 1.7 (1.5–1.9) 4.2 (3.6–4.8) 548.8 (522.1–578.6) 184.5 (166.1–200) 206 (183.5–225.3) 785.8 (752.9–818.6) 373.9 (345.9–398.3) 330.3 (291.8–361.5) 278.4 (206.4–328.5) 880.4 (840.9–919.9) 610.1 (573.9–642.5) 109.7 (93.4–124.2) 547 (505.7–580.8) 965.7 (925.9–1008.4)
1.2 3.7 4.6 1.8 0.2 1.4 1.2 0.6 0.8 3.1 7.6 1.2 1.1 1.8 0.9 3.6
Aa
B
Dp
a b c d e f
The number of larvae used in each bioassay. Degree of freedom. CL, confidence interval limit. χ2 represents Chi-square goodness-of-fit test. LD50 of resistant population/LD50 of susceptible strain. LD50 without synergist/LD50 with synergist.
Resistance ratioe
Synergist ratiof
1.6 1.4 0.6 226.7 76.2 85.1 324.7 154.5 136.5 115 125.7 252.1 45.3 226 237.7
2.9 2.6 0.7 1.1 1.3 0.4 5.6 1.1 0.6
M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220
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Table 5 Substrate specificities and kinetic properties of AChEs from the SS and the field populations of CPB (Malekmohammadi et al., 2010). Substrate
Kinetic propertya
SS
Aa
B
Dp
Yg
Acetylthiocholine iodide
Kmb Vmc Km Vm Km Vm
10.6 ± 0.21A 20.2 ± 0.51a 7.7 ± 0.51B 13.7 ± 0.61b 7.1 ± 0.44C 2.3 ± 0.52c
15.6 ± 0.32A 11.5 ± 0.42a 9.2 ± 0.52B 8 ± 0.72b 11.8 ± 0.63C 3.7 ± 0.51c
12.6 ± 0.33A 14.9 ± 0.43a 8 ± 0.61B 13 ± 0.91b 9.2 ± 0.51C 4.7 ± 0.21c
18.8 ± 0.44A 10.7 ± 0.32a 13.1 ± 0.62B 7.1 ± 0.52b 13.5 ± 0.42B 3.3 ± 0.51c
12 ± 0.43A 18 ± 0.84a 6.7 ± 0.32B 7.8 ± 0.62b 8.9 ± 0.31C 4.2 ± 0.31c
Propionylthiocholine iodide Butyrylthiocholine iodide
Means in same row and column followed by the same subscript number and letter, respectively, are not statistically different by ANOVA (P N 0.05). a Results are reported as the means ± SD of five replicates (n = 5). b Km; Michaelis constant in μM. c Vmax; maximum velocity in mU/mg protein. One unit of AChE hydrolyzes 1 μmol of ATC per min at 25 °C and pH 7.5.
1999; Kim et al., 2006). Kinetic studies revealed a reduced rate of hydrolysis of acetylthiocholine, increased butyrylcholinesterase activity, and a significantly reduced bimolecular rate constant for azinphosmethyloxon (Argentine et al., 1989b; Wierenga and Hollingworth, 1993; Zhu and Clark, 1995; Zhang et al., 1999; Kim et al., 2006). In the AZ-R strain, AChE was less sensitive to inhibition by azinphosmethyl-oxon and dichlorvos but was more sensitive to inhibition by paraoxon and diisopropyl fluorophosphate, relative to the insecticide-susceptible strain (Zhu and Clark, 1995). Current status of resistance in CPB to commonly used insecticides The continuous use of insecticides over several years, as the dominant measure of control for CPB, has fueled concern that resistance development is occurring. Under these circumstances, it is important that CPB populations under insecticide selection pressure are monitored. To date, very little background information is available concerning either the resistance of such populations to insecticides or of the mechanisms that may be involved. The first study to investigate the presence and diffusion of insecticide resistance in Iranian CPB populations was initiated in 2004. The toxicities of phosalon and endosulfan toward adults of L. decemlineata were evaluated to determine the degree of resistance that was present (Allahyari et al., 2005). This study revealed that CPB was not effectively controlled with the recommended field rates of these insecticides. Mohammadi Sharif et al. (2007) reported resistance to endosulfan, ranging from 18- to 220-fold, in field populations of CPB in the province of East Azarbaijan. Two insecticide synergists, piperonyl butoxide (PBO) and S, S, S-tributylphosphorotrithioate (DEF), decreased this resistance to 2.3- and 3.5-fold, respectively, in the resistant strain, indicating that metabolic detoxification played a minor role in conferring resistance (Table 2). Biochemical assays
indicated that there was no significant difference in glutathione Stransferase activity between the susceptible and resistant strains (Table 3) (Mohammadi Sharif et al., 2007). Mohammadi Sharif et al. (2008) also identified a point mutation, resulting in the replacement of an alanine residue by a serine residue, in the Rdl gene of CPB; this mutation conferred resistance to endosulfan. Insecticide-resistant populations of CPB with insensitive acetylcholinesterase (AChE) have been observed in commercial potato fields in the province of Hamedan, the most important potato-growing region in Iran (Malekmohammadi et al., 2010). Phosalone (OP) has commonly been used for CPB control in this region. Bioassays involving topical application of phosalone to fourth instars of CPB revealed resistance of up to 252-fold in field populations, relative to susceptible strains (SS). Synergism studies indicated that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not play a major role in resistance (Table 4). In our previous study (Malekmohammadi et al., 2010), AChEs from field populations of CPBs presented relatively greater hydrolysis activities on substrates with larger alkyl group substitutions (e.g., butyrylthiocholine iodide vs. acetylthiocholine iodide); they were also less sensitive to inhibition by methoxy-substituted insecticides (e.g., methyl paraoxon) when compared to the susceptible forms of AChEs (Tables 5 and 6). The aforementioned differences in the biochemical parameters of AChE from L. decemlineata may have resulted from differences in the degree of exposure to OP insecticides in the field. Thus, AChEs elicited structure–activity relationships similar to those previously reported for the native form of AChE (Zhu and Clark, 1995); small substrates and inhibitors interacted more efficiently with the native AChE from the susceptible strain, whereas large substrates and inhibitors interacted more efficiently with the native AChE from the resistant populations. These results are indicative of a typical
Table 6 Affinity (Kd), phosphorylation (kp), and bimolecular rate (ki) constants of organophosphorus compounds in inhibition reaction with AChEsa from the SS strain and the field populations of CPB (Malekmohammadi et al., 2010). SS (ratio of ki)b
Aa (ratio of ki)
B (ratio of ki)
Dp (ratio of ki)
Yg (ratio of ki)
Methyl paraoxon Kd (M) kp ki (M−1 min−1)
(6.3 ± 0.3) × 10−6 0.2 ± 0.02 (3.4 ± 0.09) × 104 (1)
(2.79 ± 1.8) × 10−6⁎ 0.2 ± 0.05 (8.1 ± 0.57) × 103⁎ (4.2)
(13.3 ± 0.64) × 10−6⁎ 0.2 ± 0.01 (1.5 ± 0.07) × 104⁎ (2.3)
(1.7 ± 0.05) × 10−4⁎ 0.2 ± 0.06 (1.4 ± 0.03) × 104⁎ (24.2)
(2.0 ± 1.3) × 10−5⁎ 0.2 ± 0.01 (1.0 ± 0.1) × 104⁎ (3.3)
Ethyl paraoxon Kd (M) kp ki (M−1 min−1)
(1.9 ± 1.3) × 10−5 0.2 ± 0.02 (1.2 ± 0.09) × 104 (1)
(1.2 ± 0.1) × 10−6⁎ 0.4 ± 0.03⁎ (3.3 ± 0.3) × 105⁎ (0.04)
(1.96 ± 0.3) × 10−6⁎ 0.3 ± 0.06⁎ (1.3 ± 0.2) × 105⁎ (0.1)
(1.4 ± 0.1) × 10−6⁎ 0.7 ± 0.05⁎ (4.9 ± 0.9) × 105⁎ (0.02)
(3.4 ± 0.09) × 10−6⁎ 0.2 ± 0.02 (6.0 ± 0.6) × 104⁎ (0.2)
Diazoxon Kd (M) kp ki (M−1 min−1)
(4.5 ± 0.7) × 10−5 0.2 ± 0.01 (3.8 ± 0.5) × 103 (1)
(2.1 ± 0.07) × 10−6⁎ 0.23 ± 0.01 (1.1 ± 0.06) × 105⁎ (0.03)
(1.0 ± 0.3) × 10−5⁎ 0.2 ± 0.01 (2.3 ± 0.1) × 104⁎ (0.2)
(7.0 ± 0.4) × 10−6⁎ 0.2 ± 0.02 (3.0 ± 0.2) × 104⁎ (0.1)
(1.1 ± 0.5) × 10−5⁎ 0.2 ± 0.01 (1.9 ± 0.2) × 104⁎ (0.2)
a
Results are reported as means ± SD of 9 replicates (n = 9). Ratios of bimolecular rate constants (ki) were ki of SS strain/ki of field populations. Ratios greater than 1 indicate that field populations are less sensitive to the inhibitory action of OP. ⁎ Indicates significant difference from the SS strain (P b 0.01, t test). b
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M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220
Table 7 Frequencies of resistance alleles in L. decemlineata from susceptible strain vs. resistant populations by PCR-RFLP (restriction fragment length polymorphism) analysis (Malekmohammadi et al., 2012). Population/strain
Sample size
S291G allelic frequencies (heterozygote/homozygote)
Susceptible Bahar Dehpiaz Aliabad Yengijeh
10 15 15 15 15
– 66.6 (5/5) 73.3 (4/7) 53.3 (6/2) 26.6 (3/1)
negative cross-insensitivity of AChE to different OP inhibitors. Another study was carried out to clarify the molecular mechanism of this insensitivity. A serine to glycine change at position 291 (S291G) in the AChE gene was reported previously in an azinphosmethyl-resistant strain of CPB (AZ-R) (Zhu et al., 1996). A study on the three-dimensional structure of AChE from Torpedo californica (Sussman et al., 1991) revealed that S291G could alter the position of the α–E′1 helix and induce conformational changes in both the catalytic and peripheral anionic binding sites. In view of the above findings, we used PCR-RFLP to determine the relative frequency of the S291G mutation in insecticide-resistant field populations of CPB (Malekmohammadi et al., 2012). Sequence analysis revealed that the Ser-Gly (AGT to GGT) mutation known to be associated with azinphosmethyl resistance was present in all of the populations tested, but at a relatively low frequency (Table 7). There was no significant correlation between the mutation frequency and resistance level in these populations (up to 252-fold resistance was observed), indicating that other mutations may also contribute to this variation. In this study, we also used PCR-SSCP to determine sequence variation in the AChE gene (Malekmohammadi et al., 2012). Allelic differences were detected by confirming the presence of a distinctive electrophoretic pattern for each single strand. Nucleotide sequence variation in the different SSCP patterns were verified by direct DNA sequencing. Sixteen amino acid replacements (V44G, E128D, R140G, I143F, T248S, F250S, G251C, I261M, S265I, S291G, G353R, L356R, E366K, S378E, S378R, and E382D) were observed (Table 8). The most frequently observed mutations were R140G, S291G, and S378E. A consistent nonsilent SNP was detected in all resistant populations, involving a substitution of serine for glycine at position 291 (S291G) (Malekmohammadi et al., 2012). Alignment of the AChE gene sequences with experimentally determined AChE sequences in field populations of CPB also revealed some single-base insertion mutations and single-base deletions; these may also contribute to AChE insensitivity.
Conclusion Leptinotarsa decemlineata has caused significant damage to potato crops in affected areas worldwide, and this pest remains a significant threat to Iranian potato production. The need to control this pest has involved the use of different insecticides. Unfortunately, historical records concerning the types and extent of insecticides used against CPB are incomplete and/or difficult to obtain, and there is no organized CPB resistance monitoring program in Iran. The recommendations regarding insecticides to be used for CPB control in Iran have not changed in over 22 years. Previous studies have indicated that there is significant potential for CPB to develop resistance to cyclodienes, OPs, and even carbamates (Allahyari et al., 2005; Mohammadi Sharif et al., 2007, 2008; Malekmohammadi et al., 2010, 2012). The reliance on insecticides can be minimized through the use of appropriate alternative control methods. A combination of cultural and chemical controls may be useful in managing CPB infestations. Moreover, the development of insecticide resistance can be managed by monitoring CPB population densities, crop rotation, growing resistant or tolerant potato varieties, promoting artificial defoliation of potato plants before harvest, accomplishing complete removal of potato tubers from fields, effectively removing volunteer potatoes and weed hosts in and around potato fields, applying insecticides only when necessary, and rotating insecticides after each generation. Annual rotation of potatoes with non-host plants reduces CPB population growth, the proportion of each population treated during a generation, the frequency of insecticide use, and thereby the selection pressure. Control of CPB is hampered by the restricted knowledge of its biology; its complex, diverse, and flexible life history; the paucity of historical data concerning the type and extent of insecticides used; and the indiscriminate use of insecticides. Furthermore, the absence of training programs for growers, for the purpose of providing them with the necessary background information to select appropriate control methods, has resulted in poor participation in IPM programs. All of these factors constitute significant obstacles to the effective management of CPB infestations in Iran. Abbreviations AChE ATC AZ-R BTC CB CPB DEF DEM
acetylcholinesterase acetylthiocholine iodide azinphosmethyl-resistant CPB strain butyrylthiocholine iodide carbamates Colorado potato beetle S,S,S-tributylphosphorotrithioate diethyl maleate
Table 8 Number of nucleotide differences and amino acid substitutions found in the L. decemlineata AChE gene (Malekmohammadi et al., 2012). Fragment 1
Fragment 2
Fragment 3
Strain/population
Nucleotide differences (silent mutation)
Point mutation
Nucleotide differences (silent mutation)
Point mutation
Nucleotide differences (silent mutation)
Point mutation
Susceptible Bahar
– 12 (11)
– V44G, R140G
– 4 (3)
– F250S
– 4 (1)
Dehpiaz
15 (14)
R140G
7 (5)
I143F, T248S
16 (6)
Aliabad
5 (4)
E128D
3 (2)
T248S
2 (–)
Yengijeh
26 (24)
V44D, R140G
3 (3)
–
4 (3)
– S291G E382D G353R G251C I261M S265I S291G L356R E366K S378E S378R S291G S378R S291G
M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220
OP PBO PCR RFLP SNP SS SSCP
organophosphate insecticide piperonyl butoxide polymerase chain reaction restriction fragment length polymorphism single-nucleotide polymorphism susceptible strain single-strand conformation polymorphism.
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Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Review
Resistance of Colorado potato beetle (Leptinotarsa decemlineata Say) to commonly used insecticides in Iran M. Malekmohammadi ⁎ Department of Plant Protection, Faculty of Agriculture, Bu Ali Sina University, Hamedan, Iran
a r t i c l e
i n f o
Article history: Received 21 August 2013 Revised 19 November 2013 Accepted 17 January 2014 Available online 27 January 2014 Keywords: Iran Colorado potato beetle Insecticide resistance Resistance mechanism
a b s t r a c t The Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), is a major pest of potatoes in Iran and many other parts of the world. Injury is caused when adults and larvae feed on the foliage and stems of potato plants, resulting in poor yields and/or plant death. Adult beetles can also vector plant diseases. Historically, the CPB been controlled using different insecticides, but it is currently resistant to nearly all classes of insecticides and remains a serious pest in many parts of the world. All of the resistance mechanisms reported in insects have been demonstrated in CPB. L. decemlineata invaded Iran in the early 1980s, probably through the importation of infested potatoes. It has caused significant damage to potato crops in affected areas, and it accordingly remains a major threat to Iranian potato production. Regrettably, no IPM programs have been developed for managing CPB infestations in Iran. Furthermore, there are no organized CPB resistance monitoring programs in Iran, and the recommended insecticides for CPB control, endosulfan and phosalon, have not changed in over 22 years. Anecdotal evidence from local farmers suggests a reduction in the efficacy of control of CPB by commonly used insecticides, probably due to the reduced susceptibility to these insecticides. Given the economic significance of L. decemlineata infestations, the increasing prevalence of resistance in this species, the rate of spread of infestations, and the extent of the area infested, there is an urgent need to develop effective and sustainable integrated pest management programs for CPB in Iran. © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Biology of the Colorado potato beetle in Iran . . . . . . . . . Economic threshold for Colorado potato beetle damage . . . . Current control methods for Colorado potato beetle . . . . . . Chemical control and insecticide resistance . . . . . . . . . . Mechanisms of resistance . . . . . . . . . . . . . . . . . . Current status of resistance in CPB to commonly used insecticides Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction The Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), a leaf beetle (Coleoptera: Chrysomelidae) native to the southwestern United States and Mexico, was first collected by Thomas Nuttall in ⁎ Tel.: +98 9183130963; fax: +98 8118251013. E-mail address: [email protected].
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1811 and then described in 1824 by Thomas Say (Jacques, 1988). This beetle is a major pest of potato (Solanum tuberosum) in Iran and many other parts of the world. Injury is caused when adults and larvae feed on the foliage and stems of potato plants, resulting in poor yields and/ or plant death. Adults can also vector plant diseases. This species can also feed, develop, and reproduce on tomato (Lycopersicon esculentum Miller) and eggplant (Solanum melongena L.) and cause significant damage to these crops (Hare, 1990; Phyllis, 2004).
1226-8615/$ – see front matter © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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Iran is the third-largest potato producer in Asia, after China and India. Potatoes are grown in almost all of the provinces of Iran but production is focused in the western and northwestern provinces, where they are a single-season crop. Potatoes rank third in terms of production, behind rice and wheat. In 2010–2011, the total area under potato cultivation in Iran was around 146,000 ha, with a total production of 4,300,000 t. The average yield was 29,445.5 kg/ha (Anonymous, 2011). Leptinotarsa decemlineata is a key quarantine pest in Iran. It is believed to have invaded the country in the early 1980s, probably on potatoes imported from infested areas. After its initial detection in the northeastern province of Ardabil, in 1984 (Nouri-Ganbalani, 1986), it gradually became established in northwestern Iran. Since then, it has spread westward to the main potato-growing areas. Despite its initial designation as an internal quarantine pest, potatoes originating in infested areas were, for a long time, not subject to a plant health inspection before being transported within Iran. This oversight has resulted in the introduction of L. decemlineata to other provinces of Iran. Currently, it is distributed across nearly all of the principal potato-growing provinces, and continues to expand its range into central and northeastern Iran (Fig. 1). Various insecticides have been used to control CPB, but it has developed resistance to most classes of insecticide. Because of the significant economic impacts of L. decemlineata infestations, the increasing prevalence of insecticide resistance, the high rate of infestation development, and the wide distribution of this species, there is an urgent need to develop an effective and sustainable integrated pest management program for CPB in Iran. Although resistance monitoring is crucial for developing insecticide application strategies, there is no organized CPB resistance monitoring
program in Iran. The recommended insecticides for CPB control in Iran, endosulfan and phosalon, have not changed in over 22 years. For a long time, phosalon and endosulfan proved to be economically viable, effective, and reliable in controlling CPB in Iran. However, recent anecdotal evidence from local farmers indicates a reduction in the efficacy of control of CPB by these insecticides, which may be due to the development of insecticide resistance.
Biology of the Colorado potato beetle in Iran In Iran, the biology of the CPB on potato plants has been studied in the province of Ardabil (Nouri-Ganbalani, 1989). This study observed that overwintered adults emerged and fed on the leaves of potato plants in early May, when mean temperatures were approximately 12 °C. Oviposition occurred primarily from late May through late July. The highest larval densities were observed in late June and early July, coinciding with early blooming of the potato crop. Adults from the first generation emerge from pupation in the soil in late July. Depending on environmental conditions, there may occur multiple generations of L. decemlineata per year. According to Nouri-Ganbalani (1989), the CPB has two overlapping generations per year in Iran. The development periods for the first and second generations were indicated to be about 45 and 52 days, respectively. Given that the emergence of secondgeneration adults coincided with a sudden drop in temperature in autumn and the drying of plant foliage, the majority of them died. As a result, most of the overwintering adults came from the first (summer) generation. As much as 50% of the overwintered population may enter a second diapause.
Fig. 1. The geographical range of Colorado potato beetle in Iran. Top six potato producer provinces: 1: Hamedan, 2: Ardabil, 3: Esfahan, 4: East Azarbaijan, 5: Kordestan, 6: Zanjan (Anonymous, 2011).
M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220 Table 1 Insecticides to which L. decemlineata has already developed resistance (Whalon et al., 2008). Chemical group
Common names
Bacillus thuringiensis subsp. tenebrionis endotoxin Carbamates
Bt
Cyclodiene organochlorines Inorganics Isoflavones Macrocyclic lactones (avermectins) Neonicotinoids
Nereistoxin analogs Organochlorines Organophosphates
Organotins Pyrethroids, pyrethrins
Spinosyns
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(Iran) as 5 and 6.458 late larvae per plant in year 2004 and 2005 respectively. According to Nouri-Ganbalani et al. (2010), at larval densities up to five late-stage larvae per plant chemical control is not economically justified. Current control methods for Colorado potato beetle
Aldicarb, carbaryl, carbofuran, cloethocarb, dioxacarb, oxamyl, propoxur Aldrin, chlordane, dieldrin, endosulfan, endrin, HCH-gamma, toxaphene Hydrogen cyanide Rotenone Abamectin Thiamethoxam, acetamiprid, clothianidin, dinotefuran, imidacloprid, N-desmethylthiamethoxam, N-methylimidaclopridnitenpyram, thiacloprid Cartap DDT, methoxychlor Azamethiphos, azinphosethyl, azinphosmethyl, chlorfenvinphos, malathion, methamidophos, methidathion, monocrotophos, parathion, parathion-methyl, phorate, phosmate, phoxim, quinalphos, tetrachlorvinphos, trichlorfon Azocyclotin Cypermethrin, deltamethrin, esfenvalerate, fenvalerate, permethrin Spinosad
Economic threshold for Colorado potato beetle damage Each beetle consumes approximately 40 cm2 of foliage during the larval stage (Ferro et al., 1985; Logan et al., 1985) and an additional 10 cm2 per day as an adult (Ferro et al., 1985). If left uncontrolled, the pest can cause yield losses of up to 100% by defoliating potato plants prior to tuber formation. On potato crops, CPB is particularly damaging during the tuber formation and filling stages. On the other hand, potato plants are tolerant to some insect damage; they can lose up to 30% of their leaves without suffering significant yield losses. Various studies on the effects of CPB defoliation on potato yields concluded that intense defoliation early or late in the season may have little effect on yield, whereas a moderate mid-season defoliation can cause significant yield losses (Cranshaw and Radcliffe, 1980; Hare, 1980; Shields and Wyman, 1984). As a result, failure to control the first-generation larvae may cause significant yield losses as a result of complete defoliation. The economic threshold for L. decemlineata has been calculated on the basis of either the number of insects per plant (Martel et al., 1986; Senanayake and Holliday, 1990; Mailloux et al., 1991) or the degree of leaf damage due to CPB feeding (Ferro et al., 1983; Connell et al., 1991; Zehnder et al., 1995). Nouri-Ganbalani et al. (2010) determined the economic injury level for the potato cultivar ‘Agria’ in Ardabil
Several different insecticides have been used to control CPB infestations. Although IPM-based approaches exist, most potato growers have traditionally relied on insecticides to prevent damage by L. decemlineata. Some studies have indicated that applications of certain newly developed insecticides with novel modes of action may result in efficient control of CPB (Sirota and Grafius, 1994; Furlong and Groden, 2001; Cutler et al., 2005a,b). The effects of five chitin synthesis inhibitors (diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron) against the second instars of CPB were studied by Karimzadeh et al. (2007), with the finding that lufenuron and hexaflumuron may be particularly effective in future CPB management programs. The inclusion of bioinsecticides in IPM programs is essential for the efficient control of CPB and for minimizing the development of insecticide resistance (Barcic et al., 2006). According to Faridi et al. (2011), rotation of the insecticide spinosad with conventional insecticides can prolong the effectiveness of the former and result in a reduction in the total number of sprays necessary. In another study, the effect of Bacillus thuringiensis var. kurstaki on different larval instars of CPB, and the roles of caffeine and aqueous neem extracts in the enhancement of its action, was evaluated by Javvi et al. (2005). Their study indicated evident synergistic effects of these plant extracts on the efficacy of B. thuringiensis var. kurstaki. A similar effect was reported by Gassemi Kahrizeh et al. (2004), who observed a noticeable synergistic effect of henna extract on the efficacy of this bacterial strain in controlling CPB. In another study, the natural predators and pathogenic agents of CPB in potato fields in Ardabil were identified as follows: the pathogenic fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes); the parasitic mite Linobia sp. (Acari: Hemisarcoptidae); the long-legged spider Phalangium sp. (Opilions: Phalangidae); the predatory bug, Rhinocoris punctiventris (Heteroptera: Reduviidae); and the green lacewing Chrysoperla carnea (Neuroptera: Chrysopidae) (Nouri-Ganbalani et al., 1998). This study revealed that B. bassiana and C. carnea could be successfully incorporated into IPM programs to control L. decemlineata. Chemical control and insecticide resistance Colorado potato beetles experience intense selection pressure for insecticide resistance, because of the significant economic impacts of this pest and the subsequent intensive use of pesticides in infested areas. More than 30 active ingredients are currently registered for use against L. decemlineata around the world (Alyokhin et al., 2008). On the other hand, this species, like many other herbivorous insects, has evolved to resist a variety of defensive phytochemicals, particularly alkaloids. Therefore, it is biochemically well-adapted to develop
Table 2 Toxicity of endosulfan to larvae of a susceptible strain (SS) and field population of Colorado potato beetle, Bostanabaad (ba) with and without synergists (Mohammadi Sharif et al., 2007). Population
na
Synergist
Slope (SE)
LD50 (95% CLb) (μg/insect)
Resistance ratioc
Synergist ratiod
SS
360 366 341 360 360 358
– DEF PBO – DEF PBO
1.9 (0.23) 1.7 (0.27) 2.0 (0.24) 0.9 (0.14) 1.0 (0.12) 0.9 (0.1)
0.1 (0.07–0.12) 0.2 (0.17–0.26) 0.1 (0.09–0.14) 10.9 (7.1–16.3) 3.1 (2.1–4.41) 4.7 (3.1–7.0)
– – – 109 – –
– 0.5 1 – 3.5 2.3
Ba
a b c d
The number of larvae used in each bioassay. CL, confidence interval limit. LD50 of resistant population/LD50 of susceptible strain. LD50 without synergist/LD50 with synergist.
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Table 3 Substrate specificities and kinetic properties of glutathione-S-transferase from the susceptible and resistant strains of CPB (Mohammadi Sharif et al., 2007). Parameter
Susceptible
Resistant (Bostanabaad strain)
Activitya (4)b Kmc Vmaxa
73.2 ± 7.6 154 ± 31.3 66.5 ± 16.3
61.9 ± 12.4 279 ± 54 72.5 ± 6.6
a b c
development of resistance is occurring. Under these circumstances, it is vital that CPB populations under insecticide selection pressure are monitored. However, very little background information is available concerning the insecticide resistance displayed by such populations to insecticides or the mechanisms that may be involved.
Mechanisms of resistance
Mean value (μM/min/mg protein) ± SD. Number of replicates (n). Mean value (μM) ± SD.
insecticide resistance. This tendency, in combination with the aforementioned high selection pressure, has resulted in the development of resistance to a large number of insecticides from various classes (Table 1). Direct correlations between the frequency of insecticide applications and the development of resistance have been reported by many researchers (Hare, 1980; Roush and Miller, 1986; Tisler and Zehnder, 1990). Rather than evolving in isolated areas and spreading geographically, the development of insecticide resistance in CPB is heavily dependent on the patterns of insecticide use and local selection pressure, resulting in repeated and independent occurrences of resistance development. The degree of insecticide resistance in L. decemlineata may vary greatly, regionally or locally, or among different life stages (Silcox et al., 1985; Zehnder, 1986; Zehnder and Gelernter, 1989; Pourmirza, 2005). Heim et al. (1990) indicated that there were differences in CPB susceptibility to insecticides, even among populations originating from the same region. The development of resistance to organochlorine, organophosphorus (OP), carbamate (CB), and pyrethroid classes of insecticides has been documented in several studies (Harris and Svec, 1976; McDonald, 1976; Harris and Svec, 1981; Heim et al., 1990; French et al., 1992; Pap et al., 1997). Other studies have also demonstrated the acquisition of resistance to recently registered products (Zhao et al., 2000; Cutler et al., 2005a). Presently, CPB is resistant to nearly all classes of insecticides and consequently remains a serious pest in many parts of the world (Pedigo, 2004). All of the resistance mechanisms reported in insects have been demonstrated in CPB. The current status of CPB resistance to conventional insecticides in Iran is unclear. However, the continuous use of insecticides over several years, as the major means of CPB control, has fueled concern that the
All of the resistance mechanisms reported in insects have been demonstrated in CPB. Many studies have indicated an increase in the activity or quantity of mixed function oxidases, esterases, and glutathione-Stransferases, in insecticide-resistant CPBs (Zhao et al., 2000; Cutler et al., 2005b). Target site insensitivity has been demonstrated as the major cause of resistance to OPs, CBs, pyrethroids, and cyclodienes (Ffrench-Constant et al., 1993; Anthony et al., 1995; Bass et al., 2004). However, alteration of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) to an insensitive form associated with increased AChE activity has been proven to be an important mechanism conferring resistance to OPs and/or CBs in some insect species (Plapp and Tripathi, 1978; Hama et al., 1980; Fournier et al., 1992; Zhu and Gao, 1999). Other known mechanisms of CPB resistance to insecticides include the increased excretion and/or reduced penetration of certain insecticides. In addition, there is some evidence of behavioral resistance to insecticides. The activity of AChE is commonly used as a biomarker of exposure to different contaminants, including pesticides (Podolska et al., 2008). Kinetic analysis of AChE was used to explain the resistance of some insect strains and the selectivity of some OP insecticides (Guedes et al., 1997; Pradhan and Mishra, 1998; Zhu and Gao, 1998; Ali et al., 2005). Previous studies have determined the process of development of resistance to OPs and CBs in CPB (Argentine et al., 1989a, 1994, 1995; Zhu and Clark, 1995, 1997; Clark, 1997; Zhang et al., 1999; Kim et al., 2006, 2007). Resistance to azinphosmethyl has also been reported in CPB (Argentine et al., 1989b; Wierenga and Hollingworth, 1993). The high level of resistance to azinphosmethyl (136-fold) in a nearly isogenic CPB strain (AZ-R) was postulated to be the result of multiple resistance mechanisms, including reduced penetration, enhanced xenobiotic metabolism, and target site insensitivity (Argentine et al., 1989b; Wierenga and Hollingworth, 1993; Zhu and Clark, 1995; Zhang et al.,
Table 4 Toxicity of phosalone to larvae of a susceptible strain (SS) and field populations of Colorado potato beetle, Aliabad (Aa), Bahar (B), Dehpiaz (Dp), and Yengijeh (Yg) with and without synergists (Malekmohammadi et al., 2010). Population
na
dfb
Synergist
Slope (SE)
LD50 (95% CLc) (μg/insect)
χ2d
SS
260 190 270 210 302 270 230 270 183 270 270 270 190 250 230 230
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
– DEF DEM PBO – DEF DEM PBO – DEF DEM PBO – DEF DEM PBO
1.3 (0.1) 1.6 (0.1) 2.1 (0.2) 1.7 (0.1) 2.8 (0.2) 2.1 (0.2) 2.7 (0.2) 4.7 (0.4) 3.1 (0.2) 2.1 (0.2) 2.7 (0.2) 1.6 (0.2) 3.6 (0.3) 1.6 (0.1) 3.1 (0.3) 4.3 (0.4)
2.4 (2.1–2.7) 1.5 (1.3–1.6) 1.7 (1.5–1.9) 4.2 (3.6–4.8) 548.8 (522.1–578.6) 184.5 (166.1–200) 206 (183.5–225.3) 785.8 (752.9–818.6) 373.9 (345.9–398.3) 330.3 (291.8–361.5) 278.4 (206.4–328.5) 880.4 (840.9–919.9) 610.1 (573.9–642.5) 109.7 (93.4–124.2) 547 (505.7–580.8) 965.7 (925.9–1008.4)
1.2 3.7 4.6 1.8 0.2 1.4 1.2 0.6 0.8 3.1 7.6 1.2 1.1 1.8 0.9 3.6
Aa
B
Dp
a b c d e f
The number of larvae used in each bioassay. Degree of freedom. CL, confidence interval limit. χ2 represents Chi-square goodness-of-fit test. LD50 of resistant population/LD50 of susceptible strain. LD50 without synergist/LD50 with synergist.
Resistance ratioe
Synergist ratiof
1.6 1.4 0.6 226.7 76.2 85.1 324.7 154.5 136.5 115 125.7 252.1 45.3 226 237.7
2.9 2.6 0.7 1.1 1.3 0.4 5.6 1.1 0.6
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Table 5 Substrate specificities and kinetic properties of AChEs from the SS and the field populations of CPB (Malekmohammadi et al., 2010). Substrate
Kinetic propertya
SS
Aa
B
Dp
Yg
Acetylthiocholine iodide
Kmb Vmc Km Vm Km Vm
10.6 ± 0.21A 20.2 ± 0.51a 7.7 ± 0.51B 13.7 ± 0.61b 7.1 ± 0.44C 2.3 ± 0.52c
15.6 ± 0.32A 11.5 ± 0.42a 9.2 ± 0.52B 8 ± 0.72b 11.8 ± 0.63C 3.7 ± 0.51c
12.6 ± 0.33A 14.9 ± 0.43a 8 ± 0.61B 13 ± 0.91b 9.2 ± 0.51C 4.7 ± 0.21c
18.8 ± 0.44A 10.7 ± 0.32a 13.1 ± 0.62B 7.1 ± 0.52b 13.5 ± 0.42B 3.3 ± 0.51c
12 ± 0.43A 18 ± 0.84a 6.7 ± 0.32B 7.8 ± 0.62b 8.9 ± 0.31C 4.2 ± 0.31c
Propionylthiocholine iodide Butyrylthiocholine iodide
Means in same row and column followed by the same subscript number and letter, respectively, are not statistically different by ANOVA (P N 0.05). a Results are reported as the means ± SD of five replicates (n = 5). b Km; Michaelis constant in μM. c Vmax; maximum velocity in mU/mg protein. One unit of AChE hydrolyzes 1 μmol of ATC per min at 25 °C and pH 7.5.
1999; Kim et al., 2006). Kinetic studies revealed a reduced rate of hydrolysis of acetylthiocholine, increased butyrylcholinesterase activity, and a significantly reduced bimolecular rate constant for azinphosmethyloxon (Argentine et al., 1989b; Wierenga and Hollingworth, 1993; Zhu and Clark, 1995; Zhang et al., 1999; Kim et al., 2006). In the AZ-R strain, AChE was less sensitive to inhibition by azinphosmethyl-oxon and dichlorvos but was more sensitive to inhibition by paraoxon and diisopropyl fluorophosphate, relative to the insecticide-susceptible strain (Zhu and Clark, 1995). Current status of resistance in CPB to commonly used insecticides The continuous use of insecticides over several years, as the dominant measure of control for CPB, has fueled concern that resistance development is occurring. Under these circumstances, it is important that CPB populations under insecticide selection pressure are monitored. To date, very little background information is available concerning either the resistance of such populations to insecticides or of the mechanisms that may be involved. The first study to investigate the presence and diffusion of insecticide resistance in Iranian CPB populations was initiated in 2004. The toxicities of phosalon and endosulfan toward adults of L. decemlineata were evaluated to determine the degree of resistance that was present (Allahyari et al., 2005). This study revealed that CPB was not effectively controlled with the recommended field rates of these insecticides. Mohammadi Sharif et al. (2007) reported resistance to endosulfan, ranging from 18- to 220-fold, in field populations of CPB in the province of East Azarbaijan. Two insecticide synergists, piperonyl butoxide (PBO) and S, S, S-tributylphosphorotrithioate (DEF), decreased this resistance to 2.3- and 3.5-fold, respectively, in the resistant strain, indicating that metabolic detoxification played a minor role in conferring resistance (Table 2). Biochemical assays
indicated that there was no significant difference in glutathione Stransferase activity between the susceptible and resistant strains (Table 3) (Mohammadi Sharif et al., 2007). Mohammadi Sharif et al. (2008) also identified a point mutation, resulting in the replacement of an alanine residue by a serine residue, in the Rdl gene of CPB; this mutation conferred resistance to endosulfan. Insecticide-resistant populations of CPB with insensitive acetylcholinesterase (AChE) have been observed in commercial potato fields in the province of Hamedan, the most important potato-growing region in Iran (Malekmohammadi et al., 2010). Phosalone (OP) has commonly been used for CPB control in this region. Bioassays involving topical application of phosalone to fourth instars of CPB revealed resistance of up to 252-fold in field populations, relative to susceptible strains (SS). Synergism studies indicated that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not play a major role in resistance (Table 4). In our previous study (Malekmohammadi et al., 2010), AChEs from field populations of CPBs presented relatively greater hydrolysis activities on substrates with larger alkyl group substitutions (e.g., butyrylthiocholine iodide vs. acetylthiocholine iodide); they were also less sensitive to inhibition by methoxy-substituted insecticides (e.g., methyl paraoxon) when compared to the susceptible forms of AChEs (Tables 5 and 6). The aforementioned differences in the biochemical parameters of AChE from L. decemlineata may have resulted from differences in the degree of exposure to OP insecticides in the field. Thus, AChEs elicited structure–activity relationships similar to those previously reported for the native form of AChE (Zhu and Clark, 1995); small substrates and inhibitors interacted more efficiently with the native AChE from the susceptible strain, whereas large substrates and inhibitors interacted more efficiently with the native AChE from the resistant populations. These results are indicative of a typical
Table 6 Affinity (Kd), phosphorylation (kp), and bimolecular rate (ki) constants of organophosphorus compounds in inhibition reaction with AChEsa from the SS strain and the field populations of CPB (Malekmohammadi et al., 2010). SS (ratio of ki)b
Aa (ratio of ki)
B (ratio of ki)
Dp (ratio of ki)
Yg (ratio of ki)
Methyl paraoxon Kd (M) kp ki (M−1 min−1)
(6.3 ± 0.3) × 10−6 0.2 ± 0.02 (3.4 ± 0.09) × 104 (1)
(2.79 ± 1.8) × 10−6⁎ 0.2 ± 0.05 (8.1 ± 0.57) × 103⁎ (4.2)
(13.3 ± 0.64) × 10−6⁎ 0.2 ± 0.01 (1.5 ± 0.07) × 104⁎ (2.3)
(1.7 ± 0.05) × 10−4⁎ 0.2 ± 0.06 (1.4 ± 0.03) × 104⁎ (24.2)
(2.0 ± 1.3) × 10−5⁎ 0.2 ± 0.01 (1.0 ± 0.1) × 104⁎ (3.3)
Ethyl paraoxon Kd (M) kp ki (M−1 min−1)
(1.9 ± 1.3) × 10−5 0.2 ± 0.02 (1.2 ± 0.09) × 104 (1)
(1.2 ± 0.1) × 10−6⁎ 0.4 ± 0.03⁎ (3.3 ± 0.3) × 105⁎ (0.04)
(1.96 ± 0.3) × 10−6⁎ 0.3 ± 0.06⁎ (1.3 ± 0.2) × 105⁎ (0.1)
(1.4 ± 0.1) × 10−6⁎ 0.7 ± 0.05⁎ (4.9 ± 0.9) × 105⁎ (0.02)
(3.4 ± 0.09) × 10−6⁎ 0.2 ± 0.02 (6.0 ± 0.6) × 104⁎ (0.2)
Diazoxon Kd (M) kp ki (M−1 min−1)
(4.5 ± 0.7) × 10−5 0.2 ± 0.01 (3.8 ± 0.5) × 103 (1)
(2.1 ± 0.07) × 10−6⁎ 0.23 ± 0.01 (1.1 ± 0.06) × 105⁎ (0.03)
(1.0 ± 0.3) × 10−5⁎ 0.2 ± 0.01 (2.3 ± 0.1) × 104⁎ (0.2)
(7.0 ± 0.4) × 10−6⁎ 0.2 ± 0.02 (3.0 ± 0.2) × 104⁎ (0.1)
(1.1 ± 0.5) × 10−5⁎ 0.2 ± 0.01 (1.9 ± 0.2) × 104⁎ (0.2)
a
Results are reported as means ± SD of 9 replicates (n = 9). Ratios of bimolecular rate constants (ki) were ki of SS strain/ki of field populations. Ratios greater than 1 indicate that field populations are less sensitive to the inhibitory action of OP. ⁎ Indicates significant difference from the SS strain (P b 0.01, t test). b
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Table 7 Frequencies of resistance alleles in L. decemlineata from susceptible strain vs. resistant populations by PCR-RFLP (restriction fragment length polymorphism) analysis (Malekmohammadi et al., 2012). Population/strain
Sample size
S291G allelic frequencies (heterozygote/homozygote)
Susceptible Bahar Dehpiaz Aliabad Yengijeh
10 15 15 15 15
– 66.6 (5/5) 73.3 (4/7) 53.3 (6/2) 26.6 (3/1)
negative cross-insensitivity of AChE to different OP inhibitors. Another study was carried out to clarify the molecular mechanism of this insensitivity. A serine to glycine change at position 291 (S291G) in the AChE gene was reported previously in an azinphosmethyl-resistant strain of CPB (AZ-R) (Zhu et al., 1996). A study on the three-dimensional structure of AChE from Torpedo californica (Sussman et al., 1991) revealed that S291G could alter the position of the α–E′1 helix and induce conformational changes in both the catalytic and peripheral anionic binding sites. In view of the above findings, we used PCR-RFLP to determine the relative frequency of the S291G mutation in insecticide-resistant field populations of CPB (Malekmohammadi et al., 2012). Sequence analysis revealed that the Ser-Gly (AGT to GGT) mutation known to be associated with azinphosmethyl resistance was present in all of the populations tested, but at a relatively low frequency (Table 7). There was no significant correlation between the mutation frequency and resistance level in these populations (up to 252-fold resistance was observed), indicating that other mutations may also contribute to this variation. In this study, we also used PCR-SSCP to determine sequence variation in the AChE gene (Malekmohammadi et al., 2012). Allelic differences were detected by confirming the presence of a distinctive electrophoretic pattern for each single strand. Nucleotide sequence variation in the different SSCP patterns were verified by direct DNA sequencing. Sixteen amino acid replacements (V44G, E128D, R140G, I143F, T248S, F250S, G251C, I261M, S265I, S291G, G353R, L356R, E366K, S378E, S378R, and E382D) were observed (Table 8). The most frequently observed mutations were R140G, S291G, and S378E. A consistent nonsilent SNP was detected in all resistant populations, involving a substitution of serine for glycine at position 291 (S291G) (Malekmohammadi et al., 2012). Alignment of the AChE gene sequences with experimentally determined AChE sequences in field populations of CPB also revealed some single-base insertion mutations and single-base deletions; these may also contribute to AChE insensitivity.
Conclusion Leptinotarsa decemlineata has caused significant damage to potato crops in affected areas worldwide, and this pest remains a significant threat to Iranian potato production. The need to control this pest has involved the use of different insecticides. Unfortunately, historical records concerning the types and extent of insecticides used against CPB are incomplete and/or difficult to obtain, and there is no organized CPB resistance monitoring program in Iran. The recommendations regarding insecticides to be used for CPB control in Iran have not changed in over 22 years. Previous studies have indicated that there is significant potential for CPB to develop resistance to cyclodienes, OPs, and even carbamates (Allahyari et al., 2005; Mohammadi Sharif et al., 2007, 2008; Malekmohammadi et al., 2010, 2012). The reliance on insecticides can be minimized through the use of appropriate alternative control methods. A combination of cultural and chemical controls may be useful in managing CPB infestations. Moreover, the development of insecticide resistance can be managed by monitoring CPB population densities, crop rotation, growing resistant or tolerant potato varieties, promoting artificial defoliation of potato plants before harvest, accomplishing complete removal of potato tubers from fields, effectively removing volunteer potatoes and weed hosts in and around potato fields, applying insecticides only when necessary, and rotating insecticides after each generation. Annual rotation of potatoes with non-host plants reduces CPB population growth, the proportion of each population treated during a generation, the frequency of insecticide use, and thereby the selection pressure. Control of CPB is hampered by the restricted knowledge of its biology; its complex, diverse, and flexible life history; the paucity of historical data concerning the type and extent of insecticides used; and the indiscriminate use of insecticides. Furthermore, the absence of training programs for growers, for the purpose of providing them with the necessary background information to select appropriate control methods, has resulted in poor participation in IPM programs. All of these factors constitute significant obstacles to the effective management of CPB infestations in Iran. Abbreviations AChE ATC AZ-R BTC CB CPB DEF DEM
acetylcholinesterase acetylthiocholine iodide azinphosmethyl-resistant CPB strain butyrylthiocholine iodide carbamates Colorado potato beetle S,S,S-tributylphosphorotrithioate diethyl maleate
Table 8 Number of nucleotide differences and amino acid substitutions found in the L. decemlineata AChE gene (Malekmohammadi et al., 2012). Fragment 1
Fragment 2
Fragment 3
Strain/population
Nucleotide differences (silent mutation)
Point mutation
Nucleotide differences (silent mutation)
Point mutation
Nucleotide differences (silent mutation)
Point mutation
Susceptible Bahar
– 12 (11)
– V44G, R140G
– 4 (3)
– F250S
– 4 (1)
Dehpiaz
15 (14)
R140G
7 (5)
I143F, T248S
16 (6)
Aliabad
5 (4)
E128D
3 (2)
T248S
2 (–)
Yengijeh
26 (24)
V44D, R140G
3 (3)
–
4 (3)
– S291G E382D G353R G251C I261M S265I S291G L356R E366K S378E S378R S291G S378R S291G
M. Malekmohammadi / Journal of Asia-Pacific Entomology 17 (2014) 213–220
OP PBO PCR RFLP SNP SS SSCP
organophosphate insecticide piperonyl butoxide polymerase chain reaction restriction fragment length polymorphism single-nucleotide polymorphism susceptible strain single-strand conformation polymorphism.
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