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Keywords:

  • fluralaner;
  • A1443;
  • GABA;
  • ligand-gated ion channel;
  • parasiticide;
  • receptor

Abstract

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

BACKGROUND

Fluralaner (A1443) is an isoxazoline ectoparasiticide that is a novel antagonist of γ-aminobutyric acid (GABA) receptors (GABARs), with a potency comparable to that of fipronil, a phenylpyrazole ectoparasiticide. To clarify the biological effectiveness of fluralaner against fipronil-resistant pests, differences in the actions of fluralaner and fipronil on GABARs that possess resistance to dieldrin (rdl)-type mutations were evaluated.

RESULTS

Fipronil had neither pest control nor GABAR-antagonistic activities against two-spotted spider mites (Tetranychus urticae) that had two different rdl-type amino acids (A301 [RIGHTWARDS ARROW] H and T350 [RIGHTWARDS ARROW] A: Drosophila melanogaster GABAR numbering) and against small brown planthoppers (Laodelphax striatellus) that had a novel rdl-type (A283 [RIGHTWARDS ARROW] N) mutation in GABARs. In contrast, fluralaner showed not only high pest control activities against these pests, but also excellent antagonistic activities for these rdl-type GABARs.

CONCLUSION

The findings indicate that rdl-type fipronil-resistant pests do not show cross-resistance to fluralaner owing to the differential actions of fluralaner and fipronil on the GABAR. © 2014 Society of Chemical Industry


1 INTRODUCTION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

Phenylpyrazole insecticides such as fipronil and cyclodiene insecticides such as dieldrin act as antagonists against insect ionotropic γ-aminobutyric acid (GABA) receptors (GABARs), and have been extensively used for pest control in agriculture and animal health.[1-3] The binding sites of these insecticides in GABARs have been suggested to reside within the intrinsic channel, on the basis of data obtained using site-directed mutagenesis, radioligand binding and electrophysiological expriments.[4, 5] Of the GABARs, those from insects that show resistance to dieldrin have been well studied.[6, 7] These insects have a resistance to dieldrin (rdl)-type point mutation (alanine to another amino acid) around the 300th position in the amino acid sequences of their subunits; the one derived from Drosophila melanogaster contains a mutation of alanine to serine at position 301 (A301 [RIGHTWARDS ARROW] S).[3] The Drosophila GABAR (DmGABAR) with this mutation shows low sensitivity not only to dieldrin but also to fipronil.[7]

Recently, a novel rdl-type mutation was identified in the fipronil-resistant populations of the small brown planthopper, Laodelphax striatellus.[8] According to the report, fipronil-resistant L. striatellus had an rdl-type mutation (A283 [RIGHTWARDS ARROW] N). Furthermore, Le Goff et al. reported that another rdl-type GABAR of D. simulans (DsGABAR) with two mutations (A302 [RIGHTWARDS ARROW] S and T350 [RIGHTWARDS ARROW] M) showed less sensitivity to fipronil.[9] Dermauw et al. reported that fipronil showed no miticidal activity against the two-spotted spider mite Tetranychus urticae. They found that substitutions at this conserved position of the two rdl-type mutations at A301 and T350 (DmGABAR numbering) were present in T. urticae GABAR (TuGABAR). The TuGABAR had two different rdl-type amino acids (A301 [RIGHTWARDS ARROW] S/H and T350 [RIGHTWARDS ARROW] A) in its TM2 and TM3 domains (Fig. 1). They proposed that the double different amino acids in TuGABAR caused low sensitivity to fipronil.[10]

image

Figure 1. Alignment of the amino acid sequences in transmembrane domain 1 and 2 (TM1 and TM2 respectively) of GABA receptor subunits of D. melanogaster (DmGABAR), M. domestica (MdGABAR), S. furcifera (SfGABAR), L. striatellus (LsGABAR) and T. urticae (TuGABAR). The A301S/G/N and T350M point mutations (D. melanogaster GABAR numbering) associated with dieldrin and/or fipronil resistance in insects are marked with an arrow, while residues at equivalent positions in T. urticae are indicated in red.

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Fluralaner (A1443) is a novel isoxazoline ectoparasiticide that acts as a specific blocker of GABARs.[11] Fluralaner has excellent acaricidal activity against ticks and spider mites. To examine differences in the actions of fluralaner and fipronil, biological assays against T. urticae and L. striatellus were performed. It is reported here that fipronil shows low miticidal activity against T. urticae owing to its low antagonist activity against TuGABAR, whereas fluralaner shows excellent inhibitory activity against TuGABAR, leading to strong miticidal activity. Fluralaner also shows high insecticidal activity against the fipronil-resistant mutant of L. striatellus. These results are the first form of biological evidence of the differential mechanisms of action of fluralaner and fipronil in terms of interaction with their target sites.

2 MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

2.1 Chemicals and plasmids

GABA was purchased from Sigma-Aldrich (St Louis, MO). Fluralaner was synthesised according to the procedure described in its patent.[12] Fipronil was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). The pGEMHE plasmid was kindly supplied by Dr Kubo (National Institute for Physiological Sciences, Aichi, Japan). The vector pcDNA3 was inserted with Musca domestica GABAR (MdGABAR),[11] which was kindly supplied by Prof. Ozoe (Department of Life Science and Biotechnology, Shimane University, Shimane, Japan).

2.2 Animals

The laboratory strains of T. urticae and L. striatellus maintained at Biological Research Laboratories, Nissan Chemical Industries, Ltd (Saitama, Japan), were used for assays. A population of L. striatellus that was resistant to fipronil was kindly supplied by Dr Matsumura (NARO Kyusyu Okinawa Agricultural Research Center, Kumamoto, Japan). Mature female African clawed frogs (Xenopus laevis) were purchased from Japan SLC, Inc. (Shizuoka, Japan).

2.3 Miticidal test

The leaf spray method[13] was used to determine the miticidal activity of fluralaner and fipronil solutions against T. urticae. The solutions were prepared by diluting wettable powder containing 100 g kg−1 of each active ingredient with water containing the non-ionic surfactant Admix (3 mL L−1; Nissan Chemical Industries, Ltd, Tokyo, Japan). Mortality was assessed 3 days after the treatment. The test was carried out in two replicates.

2.4 Insecticidal test

The rice-stem dipping method[14] was used to determine the insecticidal activities of fluralaner and fipronil solutions against L. striatellus. The solutions were prepared by diluting emulsifiable concentrate containing 100 g L−1 of each active ingredient with water containing the non-ionic surfactant Admix (3 mL L−1). Mortality was recorded 6 days after treatment. The test was carried out in two replicates.

2.5 Isolation of the cDNA clone encoding the GABAR subunit of fipronil-resistant L. striatellus GABAR

Total RNA was extracted from adult L. striatellus using the RNeasy kit (Qiagen, Valencia, CA). The PrimeScript II 1st Strand cDNA Synthesis kit (Takara Bio Inc., Shiga, Japan) was used to synthesize cDNA. The primers LsGABAR-F (5′-ACGAAGTGTCACTGCCACAG) and LsGABAR-R (5′-TGTATCCGTTTCGCCATGTA) were used to amplify the GABAR subunit gene fragment (accession number AB253526).

2.6 Isolation of the cDNA encoding the TuGABAR subunit

Total RNA was isolated from adult T. urticae using the RNeasy kit (Qiagen). PrimeScript II 1st Strand cDNA Synthesis kit was used to synthesize the cDNA. The primers TuGABAR-F (5′-TTGGCTGTGTGCTTTTCTCCTT) and TuGABAR-R (5′-GCATTTTCCAGCTTCTGCTT) were used to amplify the full-length cDNA encoding the TuGABAR subunit (accession number AB567686). The PCR product was cloned and subcloned into the Sma I-Hind III site of the plasmid pGEMHE.

2.7 Preparation of the cDNA encoding the M. domestica GABAR (MdGABAR) subunit that contains the fipronil-resistant L. striatellus-type mutation (A299 [RIGHTWARDS ARROW] N)

The cDNA that encodes A299 [RIGHTWARDS ARROW] N mutant of the MdGABAR subunit was prepared from the cDNA of the wild type (bd variant: DDBJ accession number AB177547) using a KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan). The primers MdGABAR-A299N-F (5′-AATTTAGGTGTCACCACTGTGTTGACCA) and MdGABAR-A299N-R (5′-TACACGGGCTGGTGTAGCATTACGA) were used for the mutagenesis. The PCR product was cloned and subcloned into the EcoRI site of the plasmid pGEMHE using the In-Fusion HD Cloning kit (Takara Bio). The primers MdGABAR-IF-F (5′-CCGGGGATCCGAATTCCACCACCATGAGTGATTC) and MdGABAR-IF-R (5′-TTGCTCTAGAGAATTCGGTGACACTATAGAATAGGG) were used with the In-Fusion cloning technology.

2.8 Preparation of cRNA

The TuGABAR and MdGABAR-A299 [RIGHTWARDS ARROW] N templates for in vitro transcription were obtained using the restriction enzyme NheI. Capped RNA transcripts were synthesized using T7 polymerase (mMESSAGE mMACHINE T7 Ultra kit; Ambion, Austin, TX). cRNA samples were stored at −80 °C until use.

2.9 Expression of TuGABAR and MdGABAR-A299N in Xenopus oocytes

Mature female X. laevis was anesthetized by immersing them in 0.1% tricaine for 30 min at room temperature. Ovary lobes were isolated and treated with collagenase type 1A (2 mg mL−1; Sigma-Aldrich;) in Ca2+ ion-free standard oocyte saline (SOS) (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.6) for 90–120 min at 25 °C. Each oocyte (stages V to VI) was injected with 10–50 ng of TuGABAR or MdGABAR-A299 [RIGHTWARDS ARROW] N cRNA dissolved in nuclease-free water. The oocytes were incubated at 16 °C in complete SOS medium (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 1.8 mM CaCl2, 50 µg mL−1 gentamycin, 100 U mL−1 penicillin, 100 µg mL−1 streptomycin, 2.5 mM sodium pyruvate, pH 7.6) for 3–6 days after cRNA injection, and then used for electrophysiological recording.

2.10 Electrophysiology

Electrophysiological experiments were performed at a holding potential of −80 mV, using a two-electrode voltage clamp set-up (TEV 200A; Dagan Corporation, Minneapolis, MN). Micropipettes were prepared from glass capillaries (1.2 × 90 mm; A-M Systems, Sequim, WA), using a pipette puller (PE-2; Narishige, Tokyo, Japan), and filled with 2 M of KCl to have a resistance of 0.2–2 MΩ in the medium. The electrical signals were digitized by Powerlab 4/16 (ADInstruments, Colorado Springs, CO). Experiments were carried out at room temperature. Oocytes were placed in a recording chamber perfused with SOS solution by gravity flow at 8–10 mL min−1. GABA dissolved in SOS medium was applied to each oocyte for 20 s at intervals of 2 min. Fluralaner and fipronil were first dissolved in dimethyl sulfoxide (DMSO) and diluted with SOS medium to a final DMSO concentration of 0.1%, which did not affect the GABA response in oocytes. Fluralaner or fipronil was perfused in the chamber containing an oocyte after three successive control applications of GABA and during the remainder of the experiments. GABA (EC50) was repeatedly applied with a test compound for 20 s at 2 min intervals during the perfusion of the test compound. The minimum response was regarded as the extent of inhibition (5 min after the first application of a test compound). IC50 values were determined from the mean of at least three replications using probit analysis.

3 RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES

Fluralaner exhibited excellent miticidal activity against T. urticae, whereas fipronil showed almost no activity even at the highest dose (500 mg L−1) (Table 1). To evaluate the differential actions of fluralaner and fipronil, the authors compared the antagonist activities of the two insecticides against homo-oligomeric TuGABAR having the different rdl-type amino acids A301 [RIGHTWARDS ARROW] H and T350 [RIGHTWARDS ARROW] A (Fig. 1) by expressing it in Xenopus oocytes. The properties of the TuGABAR ion channel have previously been reported.[15] The application of GABA to Xenopus oocytes expressing TuGABAR elicited inward currents. In the present experiment, the average maximum current induced by GABA was 300 nA, and the concentration of GABA that evoked half the maximum response (EC50) was 135 µM (n = 5).

Table 1. Miticidal activities of fluralaner and fipronil against the two-spotted spider mite Tetranychus urticae
Concentration (mg L−1)Mortality (%)
FluralanerFipronil
500NT5
100NT10
50100NT
25100NT
12.5100NT
6.3100NT
3.1100NT

Figure 2A shows that 30 µM of fipronil slightly blocked currents induced by GABA (100 µM) in TuGABAR. Figure 2B shows that 0.3 µM of fluralaner nearly completely blocked these currents. The antagonistic activity of fipronil against TuGABAR was very low, even at the highest concentration (30 µM), whereas fluralaner had high inhibitory activity, with a 50% inhibitory concentration (IC50) of 12 nM (Fig. 2C). The authors previously reported that the IC50 value of fluralaner for MdGABAR was 5.23 nM.[11] Thus, fluralaner exhibited similar levels of inhibitory activity against the GABARs of the spider mite and the housefly. The IC50 values of fipronil for several insect GABARs have been reported to be between 10 and 50 nM;[9, 16, 17] however, fipronil showed very low antagonistic activity against TuGABAR (Fig. 2C). These findings indicate that the low miticidal activity of fipronil against T. urticae is due to its low inhibitory activity against TuGABAR, whereas fluralaner has high antagonistic activity against TuGABAR, which leads to high miticidal activity (Table 1).

image

Figure 2. Inhibitory activities of fluralaner and fipronil to GABA (EC50)-induced currents in TuGABAR. (A), (B) Blockade of GABA response by 30 µM of fipronil (A) and 0.3 µM of fluralaner (B). More than 80% of GABA currents were blocked by 0.1 µM of fluralaner (n = 5), but 30 µM of fipronil (n = 4) blocked GABA currents by only 27%. (C) Concentration–inhibition curves for the effects of fluralaner and fipronil. Closed circles and closed squares indicate fluralaner and fipronil respectively.

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In 2010, a novel rdl-type mutation was identified from a fipronil-resistant population of the white-backed planthopper Sogatella furcifera.[18] According to the report, the fipronil-resistant S. furcifera had an rdl-type mutation (A284 [RIGHTWARDS ARROW] N). In addition, the same group reported that a fipronil-resistant population of L. striatellus had the same rdl-type mutation (A283 [RIGHTWARDS ARROW] N).[8] To examine the activity of fluralaner against GABAR with this mutation, its insecticidal activity against fipronil-resistant L. striatellus and its antagonistic activity against MdGABAR having an A299 [RIGHTWARDS ARROW] N mutation were determined. Prior to this experiment, it was confirmed that the GABAR subunit of the fipronil-resistant strain of L. striatellus contained the A283 [RIGHTWARDS ARROW] N mutation, as reported by Nakao[8] (data not shown). The 50% lethal concentrations (LC50 values) of fipronil for the wild-type strain and the resistant strain were 0.29 and >500 mg L−1 respectively (Table 2). In contrast, fluralaner showed high insecticidal activities against not only the wild-type strain but also the resistant strain, with LC50 values of 7.6 and 10 mg L−1 respectively. In electrophysiological experiments on the MdGABAR-A299 [RIGHTWARDS ARROW] N mutant, the average maximum current induced by GABA was 900 nA, and the EC50 of GABA responses was 450 µM. Figure 3 shows that the antagonistic activity of fipronil against the MdGABAR-A299 [RIGHTWARDS ARROW] N mutant was very poor, even at the highest concentration (30 µM), whereas fluralaner had high inhibitory activity, with an IC50 of 51 nM. These findings indicate that the low insecticidal activity of fipronil against the resistant strain is due to its low inhibitory activity against GABAR with the rdl-type mutation, whereas fluralaner has high antagonistic activity against GABAR with the rdl-type mutation, leading to high insecticidal activity.

Table 2. Comparison of insecticidal activities of fluralaner and fipronil against sensitive and resistant strains of the small brown planthopper Laodelphax striatellus
StrainLD50 (mg L−1)
FluralanerFipronil
Susceptible7.60.29
Resistant10>500
image

Figure 3. Concentration–inhibition curves for the effects of fluralaner and fipronil against A299 [RIGHTWARDS ARROW] N MdGABAR. Closed circles and closed squares indicate fluralaner and fipronil respectively.

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The authors have previously reported that a strain of M. domestica that possesses an rdl-type mutation (A299 [RIGHTWARDS ARROW] S) in its GABAR is less sensitive to fipronil, and that the strain exhibited no cross-resistance to fluralaner.[11] This finding indicates that the rdl-type mutation does not reduce the potency of fluralaner. In a binding assay using a membrane fraction prepared from housefly heads, fluralaner and fipronil inhibited the binding of 4′-ethynyl-4-n-[2,3-[3]H2]propylbicycloorthobenzoate ([[3]H]EBOB).[11] Recently, García-Reynaga et al. showed that [3H]fluralaner bound to housefly head membranes is poorly displaced by fipronil and other non-competitive antagonists acting at the EBOB-binding site, but that it is displaced by avermectin at a lower concentration (IC50 = 3 nM) than fipronil.[19] These findings suggest that the binding site of fluralaner is distinct from those of EBOB and fipronil. Accordingly, fluralaner might inhibit the binding of EBOB to the binding site by an allosteric mechanism.

This study presents the differential biological actions of the novel GABA receptor antagonists fluralaner and fipronil. The results and previously published information indicate that the binding sites of fluralaner and fipronil in GABAR are located independently, suggesting that fipronil-resistant pests exhibit no cross-resistance to fluralaner.

REFERENCES

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MATERIALS AND METHODS
  5. 3 RESULTS AND DISCUSSION
  6. REFERENCES
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