Insecticide Resistance in Rice Planthoppers - American Chemical

Rice is a major crop in Asia. However, production of rice has been seriously damaged by three rice planthoppers: the brown planthopper (BPH), Nilaparv...
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Chapter 2

Insecticide Resistance in Rice Planthoppers

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Toshifumi Nakao* Agrochemical Research Center, Mitsui Chemicals Agro, Inc., Mobara, Chiba 297-0017, Japan *E-mail: [email protected].

Rice is a major crop in Asia. However, production of rice has been seriously damaged by three rice planthoppers: the brown planthopper (BPH), Nilaparvata lugens (Stål); the white backed planthopper (WBPH), Sogatella furcifera (Horváth); and the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). To control rice planthoppers, various insecticides such as organochlorines, organophosphates, carbamates, pyrethroids, and buprofezin were used. However, rice planthoppers have developed resistance to these insecticides. In 1991, imidacloprid was introduced to control planthoppers. Imidacloprid suppressed the rice planthopper populations and was heavily used in Asia. However, populations of imidacloprid-resistant rice planthoppers have appeared. Since 2005, outbreaks of rice planthoppers have occurred in Asia. After introduction of imidacloprid, fipronil and ethiprole were commercialized to control rice planthoppers. But rice planthoppers have developed resistance to fipronil and ethiprole. In this chapter, insecticide resistance and its mechanisms in rice planthoppers are reviewed.

Introduction Rice is a major crop in Asia. However, production of rice has been seriously damaged by three rice planthoppers: the brown planthopper (BPH), Nilaparvata lugens (Stål); the white backed planthopper (WBPH), Sogatella furcifera (Horváth); and the small brown planthopper (SBPH), Laodelphax striatellus (Fallén).

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BPH attacks only rice plants, by sucking and transmitting viruses such as, rice ragged stunt virus and rice grassy stunt virus. WBPH causes sucking damage on rice plants. Recently, WBPH has been reported to cause southern rice blackstreaked dwarf virus disease. SBPH infests not only rice plants but also other crops, such as wheat, barely, and rye, and transmits viruses, such as rice stripe virus (1, 2). In Asia, BPH is found in Bangladesh, Brunei, Burma (Myanmar), China, regions of Hong Kong, India, Indonesia, Japan, Cambodia, Korea, Laos, Malaysia, Nepal, Pakistan, the Philippines, Singapore, Sri Lanka, regions of Taiwan, Thailand, and Vietnam (3). SBPH is found in Bangladesh, Cambodia, China, regions of Hong Kong, India, Indonesia, Japan, Korea, Laos, Malaysia, Myanmar, Nepal, Pakistan, the Philippines, Ryukyu Islands, Sri Lanka, regions of Taiwan, Thailand, and Vietnam (3). WBPH is observed in Bangladesh, China, India, Indonesia, Japan, Korea, Malaysia, the Philippines, Taiwan, Thailand, and Vietnam BPH and WBPH live in tropical regions of Southeast Asia. They emigrate from tropical regions of Southeast Asia into northern subtropical and temperate regions from May to July. But they do not overwinter in temperature regions, such as Japan and Korea. SBPH undergoes diapauses and overwinters in temperature regions. Furthermore, overseas migration of SBPH from China to western Japan was reported (4). To control planthoppers, various insecticides such as organochlorines, organophosphates, carbamates, pyrethroids, insect growth regulators, neonicotinoids, phenylpyrazoles, and pymetrozine, were used (5, 6). However, rice planthoppers have developed resistance to these insecticides. For example, the field collected populations of BPH in China from 2012 to 2014 had developed high levels of resistance to neonicotinoid insecticide imidacloprid (resistance ratio, RR = 233.3-2029) and buprofezin (RR = 147.0-1222). Furthermore, these populations of BPH showed low to moderate resistance to carbamte insecticide isoprocarb (RR = 17.1-70.2) and organophosphate insecticide chlorpyrifos (RR = 7.4-30.7) (7). There are sevelal insecticide resistant mechanisms, such as, target-site mutation, metabolic detoxification, penetration resistance, and excretion. In this chapter, insecticide resistance in rice planthoppers is reviewed.

Resistance to Organochlorines, Organophosphates, Carbamates, Pyrethroids, Buprofezin, and Pymetrozine Organochlorines, such as BHC (benzene hexachloride) and DDT (dichloro-diphenyl-trichloroethane), were the first insecticide used to control BPH (5). According to the Insecticide Resistance Action Committee (IRAC), BHC belongs to IRAC group 2A which acts as a GABA-gated chloride channel blocker. DDT belongs to IRAC group 3B which acts as modulators against sodium channels. Rice planthoppers developed resistance to BHC and DDT (8, 9). 24 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Organophospahtes and carbamates belong to IRAC group 1 which acts as acethylcholinesterase (AChE) inhibitors. As organophospahtes and carbamates were widely used to control rice planthoppers, rice planthoppers developed resistance to these insecticides relatively fast (5, 8, 9). The main factor contributing to resistance of the BPH to organophospahtes and carbamates was suggested to be detoxification as a result of high esterase activity, which was caused by high expression of amplified carboxyesterase gene, Ni-EST1 (10–12). Comparison of amino acid sequences of the AChEs between the susceptible and resistant BPH strains revealed a point mutation, G185S. The G185S mutation was suggested to change the affinity of AChE for its substrates and inhibitors. Thus, G185S mutation was likely responsible for the insensitivity of the AChE to methamidophos in the resistant strain (13). Pyrethroids belong to IRAC group 3A and act as sodium channel modulators. Use of pyrethroids to control BPH was limited to only a few countries (5). Selection of a laboratory colony of the BPH with the pyrethroids permethrin and λ-cyhalothrin increased its resistance to both insecticides. Biochemical analysis and synergistic studies with metabolic inhibitors indicated that elevated glutathione S-transferases (GSTs) with a predominant peroxidase activity conferred resistance to both pyrethroids (14, 15). Etofenprox is a non-ester pyrethroid insecticide. Although the susceptibility of BPH to etofenprox remained a susceptible level of resistance in China (7), the successive selection by etofenprox for 16 generations in the laboratory resulted in a high level resistance and it was suggested that high expression of a P450 gene CYP6FU1 was associated with resistance (16). Buprofezin belongs to IRAC group 16 and acts as an inhibitor of chitin biosynthesis. Buprofezin was used from 1984 to control BPH, but the sensitivity of BPH to buprofezin began to decrease after 10 years (5). Most populations of WBPH in eastern China in 2010 and 2011 developed moderate resistance to buprofezin (up to 25-fold) (17). Pymetrozine is the first and only substance from the azomethyne pyridine (5). Pymetrozine belongs to IRAC group 9 and acts as a chordotonal organ TRPV channel modulator. Pymetrozine resistance of the nine field populations of the BPH collected from China in 2012 was at a moderate level. Resistance ratio of the nine populations ranged from 34.9 to 46.8-fold (18),

Resistance to Imidacloprid As mentioned above, rice planthoppers have developed resistance to organochlorines, organophosphates, carbamates, pyrethroids, and buprofezin. In 1991, imidacloprid was introduced to control planthoppers. Imidacloprid belongs to IRAC group 4A and acts as a nicotinic acetylcholine receptor (nAChR) agonist. Imidacloprid suppressed the rice planthopper populations and was heavily used in Asia. However, imidacloprid-resistant BPH was first observed in Thailand and appeared in Asian counties, such as, Vietnam, China, and Japan (19). Since 2005, outbreaks of BPH have occurred in East Asian countries (5). 25 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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The main resistant factor that confers imidacloprid resistance in BPH was suggested to be detoxification caused by increased P450 monooxygenase activity (20, 21). Imidacloprid-resistant BPH strain, which originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain. Expression levels of CYP6AY1 mRNA was found to be highest in imidacloprid-resiatant BPH strain. By expressing CYP6AY1 in Escherichia coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently. When CYP6AY1 mRNA levels in imidacloprid-resistant strain were reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found (22). In contrast, overexpression of CYP6ER1 was associated with field-evolved resistance to imidacloprid in BPH populations in five countries in South and East Asia (23). RNA interference of CYP6ER1 and transgenic expression of CYP6ER1 in Drosophila melanogaster both suggested that the expression of CYP6ER1 was sufficient to confer imidacloprid resistance (24, 25). It was suggested that four P450 genes (CYP6AY1, CYP6ER1, CYP6CS1 and CYP6CW1) conferred imidacloprid resistance to BPH and that CYP6ER1 was important at all stages of resistance development (25). Although target site mutation Y151S in two nAChR subunits from laboratory selected imidacloprid-resistant BPH was reported (26), this mutation has never been observed in any field collected populations (5). Continuous selection of a filed collected strain of BPH with imidacloprid in the laboratory resulted in a substantial increase in resistance and it was suggested that reduction in mRNA and protein expression of a nAChR α8 subunit was associated with resistance (27). SBPH also has developed resistance to imidacloprid in China, and immigration of SBPH into Japan from China caused imidacloprid resistance in Japan (4, 28). The main factor that confers imidacloprid resistance in SBPH was suggested to be detoxification caused by increased P450 monooxygenase activity of CYP353D1v2. Expression level of CYP353D1v2 of imidacloprid-resistant SBPH was significantly different to that of the susceptible strain. Strong correlation was found between CYP353D1v2 expression levels and imidacloprid treatments. Additionally, depression of the expression of CYP353D1v2 by RNA interference could significantly enhance the sensitivity of SBPH to imidacloprid (29).

Resistance to Fipronil Followed by introduction of imidacloprid, fipronil was commercialized to control rice planthoppers. Fipronil belongs to IRAC group 2B and acts as an RDL GABA receptor blocker (Figure 1). Fipronil was effective against rice palnthoppers and became one of the main alternatives after rice palnthoppers developed resistance to imidacloprid (5, 19). However, fipronil-resistant rice palnthoppers have been reported. 26 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Figure 1. Structures of phenylpyrazoles, fipronil and ethiprole.

Figure 2. RDL GABA receptor subunit and mutations that confer fipronil resistance against planthoppers. SBPH has developed resistance to fipronil in Japan (19, 30). Sequence analysis of the Rdl genes from a fipronil-resistant SBPH population collected in Fukuoka Prefecture in Japan during 2009 identified an A2′N mutation (index number for M2 membrane spanning region) (Figure 2) in the heterozygous state (31). A membrane potential assay was carried out using Drosophila S2 cells expressing the wild-type and A2′N mutant SBPH Rdl genes, either individually or together. The EC50 value of GABA for the wild-type homomers was 0.72 μM. Expression of A2′N mutant receptor homomers decreased the sensitivity 27 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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to GABA. The EC50 value of GABA for the A2′N mutant receptor homomers was 11.0 μM (Figure 3). By contrast, the EC50 value for GABA in cells that expressed the wild-type and A2′N mutant Rdl genes was 1.4 μM (Figure 3), thereby indicating that co-expression of wild-type and A2′N mutant RDL GABA receptor subunits restored the sensitivity to GABA. The A2′N mutation abolished the inhibitory activity of fipronil in cells expressing the A2′N mutant Rdl gene with or without the wild-type Rdl gene (Figure 4) (31). A two-electrode voltage clamp study showed that the A2′N mutant from Musca domestica GABA receptors expressed in Xenopus oocytes decreased the sensitivity to fipronil profoundly (32). A fipronil-resistant SBPH population selected in a laboratory showed 112.1-fold resistance to fipronil and 24.5-fold resistance to ethiprole. A2′N mutation was suggested to play an important role in conferring both fipronil and ethiprole resistance (33, 34). To predict the appearance of fipronil-resistant SBPH, a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay has been developed (35). One hudred and fourteen individuals of SBPH were collected from 4 regions in Gunma Prefecture in Japan in 2014 and more than 80 % of SBPH individuals were diagnosed as insects carrying A2′N mutation in the Rdl gene using the PCR-RFLP assay (36). Wei et al. (2016) has developed the ASPCR assay to detect the A2′N mutation in the SBPH Rdl gene, and the mutation frequencies of 19.2% and 3.6% had appeared in Lujiang and Gaochun populations in China in 2016, respectively (34).

Figure 3. Concentration–response curves for GABA in the SBPH RDL GABA receptors. Data are expressed as percentages of the maximal response to GABA for homo-oligomeric wild-type SBPH RDL GABA receptor homomers (open circle), homo-oligomeric A2′N mutant SBPH RDL GABA receptors (open square), and RDL GABA receptors on cells co-transfected with wild-type and A2′N mutant SBPH Rdl genes (open triangle). Vertical bars represent SEM for three independent experiments done in duplicate. (Reproduced with permission from ref. (31). Copyright 2011 Oxford University Press). 28 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Figure 4. Concentration–response curves for fipronil in the SBPH RDL GABA receptors. Data are expressed as percentage inhibition of the response to EC80 concentrations of GABA in the absence of fipronil with homo-oligomeric wild-type SBPH RDL GABA receptors (open circle), homo-oligomeric A2′N mutant SBPH RDL GABA receptors (open square), and RDL GABA receptors on cells co-transfected with wild-type and A2′N mutant SBPH Rdl genes (open triangle). Vertical bars represent SEM for three independent experiments done in duplicate. (Reproduced with permission from ref. (31). Copyright 2011 Oxford University Press). WBPH has also developed fipronil resistance (19, 37). Biochemical studies indicated that the esterase and P450 monooxygenase might be related to fipronil resistance, but the existence of other more important factors have been suggested (37). From a fipronil-resistant WBPH population collected in Japan in 2007, the A2′N mutation was found in the heterozygous state (Figure 2) (38). In addition, a novel R340Q mutation was associated with the A2′N mutation in the cytoplasmic loop M3–M4 (Figure 2) (39). A membrane potential assay showed that the EC50 value for GABA with the wild-type receptor homomer was 0.25 μM. The EC50 values for GABA with A2′N and A2′N · R340Q mutant receptor homomers were 5.63 and 6.03 μM, respectively (Figures 5A and 6A, respectively) (39). As is the case for SBPH RDL GABA receptors, the decreased sensitivity of the A2′N and A2′N · R340Q mutant WBPH RDL GABA receptors was recovered by coexpression of the wild-type receptor (Figures 5A and 6A, respectively) (39). The IC50 value for fipronil with the wild-type receptor homomer was 79 nM (Figure 5B) (39). The RDL GABA receptors were inhibited by up to 40% with 3 μM fipronil in cells that expressed the wild-type and A2′N mutant genes (Figure 5B). By contrast, A2′N · R340Q double-mutant gene abolished the inhibitory activity of fipronil in cells that expressed the wild-type and A2′N · R340Q double-mutant genes (Figure 6B) (39). These results suggest that the A2′N · R340Q double mutation confers a higher level of resistance to fipronil than the A2′N mutation in the heterozygous state (Figures 5B and 6B, respectively) (39). 29 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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A rapid PCR-RFLP assay was also developed to detect the fipronil-resistant WBPH carrying the A2′N mutation in the Rdl gene (35). The PCR-RFLP assays for SBPH and WBPH are useful for monitoring fipronil resistance in planthoppers that carry the A2′N mutation in the Rdl gene, and they may facilitate the management of fipronil-resistant planthoppers.

Figure 5. Influence of the A2′N mutation on the GABA and fipronil sensitivities in WBPH RDL GABA receptors. (A) Concentration–response curves for GABA. Data are expressed as percentages of the maximal response to GABA with wild-type WBPH RDL GABA receptor homomers (open circle), A2′N mutant WBPH RDL GABA receptor homomers (closed square), and RDL GABA receptors on cells co-transfected with wild-type and A2′N mutant WBPHRdl genes (open square). Vertical bars represent SEM for three independent experiments done in duplicate.(B) Concentration–response curves for the inhibitory effects of fipronil on wild-type WBPH RDL GABA receptor homomers (open circle), A2′N mutant WBPH RDL GABA receptor homomers (closed square), and RDL GABA receptors in cells co-transfected with wild-type and A2′N mutant WBPH Rdl genes (open square). The inhibitory effects of fipronil on the response to EC80 concentrations of GABA are shown as the percentage difference relative to control cells in the absence of fipronil. Vertical bars represent SEM for three independent experiments done in duplicate. (Reproduced with permission from ref. (39). Copyright 2013 the Pesticide Science Society of Japan). 30 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Figure 6. Influence of the A2′N• R340Q double mutation on the GABA and fipronil sensitivities of the WBPH Rdl GABA receptors. (A) Concentration–response curves for GABA. Data are expressed as percentages of the maximal response to GABA with A2′N•R340Q mutant WBPH RDL GABA receptor homomers (closed square) and RDL GABA receptors on cells co-transfected with wild-type and A2′N•R340Q mutant WBPH Rdl genes (open square). For comparison, the concentration–response curve for GABA with wild-type WBPH RDL GABA receptor homomers is also represented by a line. Vertical bars represent SEM for three independent experiments done in duplicate. (B) Concentration–response curves for the inhibitory effects of fipronil on A2′N•R340Q mutant WBPH RDL GABA receptor homomers (closed square) and RDL GABA receptors on cells co-transfected with wild-type and A2′N•R340Q mutant WBPH Rdl genes (open square). For comparison, a concentration–response curve for fipronil with wild-type WBPH RDL GABA receptor homomers is also represented by a line. The inhibitory effects of fipronil on the response to EC80 concentrations of GABA are shown as the percentage different relative to control cells in the absence of fipronil. Vertical bars represent SEM for three independent experiments done in duplicate. (Reproduced with permission from ref. (39). Copyright 2013 the Pesticide Science Society of Japan)

In contrast to SBPH and WBPH, fipronil-resistant BPH has not been observed until recently (30). BPH populations collected from six field populations in China in 2009 showed fipronil resistance with a 23.8-to 43.3-fold resistance ratio (40). Fipronil-resistant BPH was selected in laboratory. As the generation increased, 31 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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frequency of A2′S mutation (Figure 1) in the Rdl gene increased and R0′Q · A2′S double mutation (Figure 1) appeared. R0′Q · A2′S double mutation caused a much higher level of fipronil resistance (41). In field populations from China, Vietnam and Thailand, A2′S mutation was detected. Furthermore R0′Q · A2′S double mutation was observed in field populations from China and Vietnam, although the frequency of the double mutation was low. These results showed that fipronil resistance and related target mutations were widely distributed in field populations, but the low frequency indicated that the target mutations were not the dominant mechanism for fipronil resistance in the field populations (41).

Resistance to Ethiprole Ethiprole, the structure of which is similar to fipronil, also belongs to IRAC group 2B and acts as an RDL GABA receptor blocker (Figure 1). Unfortunately, resistance to ethiprole in BPH has been reported in China, Thailand, Vietnam, and India (23, 40, 42). An ethiprole-resistant population collected from Thailand developed 308.5-fold resistance to ethiprole and further selection with ethiprole for nine generations led to 453.1-fold resistance. Synergism and biochemical studies have suggested that the esterase and P450 monooxygenase activities caused the ethiprole resistance, although involvement of mutations in GABA receptor was also speculated (42). Garrood et al. (43) showed a casual cause of A2′S mutation on the GABA receptor in ethiprole resistance. Two ethiprole-resistant populations collected from Thailand and India showed 406- and 331-fold resistance, respectively. In contrast, fipronil resistance ratios of BPH populations collected from Thailand and India were 32-fold and 3-fold, respectively (43). After selection of these strains with ethiprole, ethiprole resistance ratios of these strains increased to >14,000-fold and fipronil resistance ratios of these strains reached 860-fold. The ethiprole-selected population from India showed 100% homozygous for A2′S mutation. Thus, major factor of mechanism of ethiprole resistance in BPH seems to A2′S mutation. SBPH also has developed ethiprole resistance (44). The resistance ratio of an ethiprole-resistant population of SBPH collected in China in 2013 was 107-fold and increased to 180-fold after selection with ethiprole. P450 monooxygenase genes, CYP4dE1 and CYP6CW3v2 were overexpressed in the resistant strain. When mRNA levels of CYP4dE1 and CYP6CW3v2 in ethiprole-resistant strain were reduced by RNA interference, ethiprole susceptibility was recovered, suggesting that CYP4dE1 and CYP6CW3v2 play an important role in ethiprole resistance in SBPH (44). Although approximately 25% of ethiprole-resistant SBPH carried the A2′N mutation, mutation might not be the major mechanism of observed ethiprole resistance. It is possible that frequency of A2′N mutation increase if selection continues (44).

32 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Effects of A2′ Mutations in Drosophila melanogaster RDL GABA Receptors on Inhibitory Activities of Phenylpyrazoles We examined the effects of A2′ mutations in Drosophila melanogaster RDL GABA receptors on inhibitory activities of fipronil and ethiprole. Three different concentrations of GABA, EC50, EC80, and EC95 of GABA, were used, because a concentration-dependent effect of GABA on the insensitivity to fipronil in the A2′S mutant Oulema oryzae RDL GABA receptor was suggested (45). The membrane potential assay showed that there was no difference in fipronil sensitivity between the wild-type and A2′S mutant receptor homomers when EC50 and EC80 of GABA were applied (Figures 7A and 7B, Table 1). However, an approximately 14-fold reduction in the potency of fipronil was observed with the A2′S mutant receptor homomers when EC95 GABA was applied. By contrast, the fipronil sensitivity of the wild-type receptor homomers was not affected by the application of EC95 of GABA (Figures 7A and 7B, Table 1), suggesting that inhibitory activity of fipronil was affected by GABA concentration in A2′S mutant receptor.

Table 1. Comparison of Inhibitory Activities of Fipronil and Ethiprolea Fipronil

Wildtype

A2′S

A2′N

A2′G

Ethiprole

GABA concentration

IC50 (nM)

pIC50

IC50 (nM)

pIC50

EC50= 1.0 μM

69.8

7.156±0.054

55.7

7.254±0.041

EC80= 2.0 μM

33.7

7.472±0.061

53.9

7.268±0.033

EC95= 5.0μM

34.4

7.464±0.058

327.4

6.485±0.048

EC50= 1.4 μM

60.4

7.219±0.052

465.5

6.332

EC80= 3.0μM

42.3

7.373±0.036

3565.0

5.448

EC95= 8.0 μM

822.0

6.085±0.056

>10000

10000

10000

10000

10000

10000

10000