Imidacloprid resistance of melon thrips, Thrips palmi, is conferred by CYP450-mediated detoxification

Abstract

The nicotinic acetylcholine receptor β1 subunit gene of the most imidacloprid-susceptible (OK) strain and the most imidacloprid-resistant (TS7) strain of Thrips palmi encoded a susceptible amino acid, Arg, at amino acid position 81. The OK and TS7 strains showed LC₅₀ values of 9.4 mg/L and 115.0 mg/L, respectively. The synergist, piperonyl butoxide, decreased the resistance ratio to one-third in the TS7 strain. These results suggest that the imidacloprid resistance in T. palmi is conferred mainly by cytochrome P450-mediated detoxification.

Introduction

Melon thrips, Thrips palmi Karny, is the most destructive pest affecting widely diverse ornamental and vegetable crops, particularly plants of the families Solanaceae, Cucurbitaceae, and Leguminosae.¹⁾ Adults and nymphs of T. palmi suck cell fluids from leaves, stems, flowers, and the surfaces of fruits, thereby causing silvery scars and leaf chlorosis.¹⁾ Moreover, T. palmi is known as a vector of plant viruses of the genus Tospovirus, including Tomato spotted wilt virus.¹⁾ In Japan, chemical control using insecticides has been applied to control T. palmi infestations since their first appearance in Miyazaki Prefecture in 1978.^2,3) However, the effectiveness of insecticides of various groups, including imidacloprid, against T. palmi has been undermined because of its extremely strong capability of developing insecticide resistance.

Imidacloprid, a nicotinic acetylcholine receptor (nAChR) agonist with high insecticidal potency and low mammalian toxicity, became commercially available in Japan in 1991. Imidacloprid blocks the nicotinergic neuronal pathway, thereby leading to the accumulation of acetylcholine (an important neurotransmitter), to paralysis, and eventually to death.⁴⁾ Actually, nAChR comprises five subunits, each containing four transmembrane domains and extracellular N-terminal domains that include the acetylcholine binding site. The acetylcholine binding site, located at the interface of two subunits, may comprise six loops.⁵⁾

Cytochrome P450s (CYP450s) are an important degradation system involved in the metabolism of xenobiotics and endogenous compounds in insects.^6–8) CYP450-mediated insecticide resistance is most commonly attributable to increased detoxification resulting from a change in the catalytic activity and/or a change in the level of expression.⁹⁾ The involvement of CYP450s in imidacloprid resistance has been reported in some insects, such as Nilaparvata lugens,^10,11) Musca domestica,¹²⁾ Bemisia tabaci,¹³⁾ and Aedes aegypti.¹⁴⁾ Consequently, increased metabolic detoxification by CYP450s is regarded as an important mechanism of imidacloprid resistance.

Target-insensitive imidacloprid resistance mechanisms have been characterized in Myzus persicae,¹⁵⁾ Aphis gossypii,¹⁶⁾ and N. lugens.¹⁷⁾ Laboratory-selected imidacloprid-resistant N. lugens had a single point mutation within the extracellular, agonist-binding domain of two nAChR α subunits (Y151S).¹⁷⁾ However, no evidence to date indicates that the Y151S mutation is responsible for imidacloprid resistance in the field,^10,18) suggesting that imidacloprid resistance in N. lugens occurs exclusively via CYP450-mediated detoxification. In contrast, imidacloprid resistance in the field has been linked with a single point mutation in the β1 subunit loop D region of nAChR (R81T) in M. persicae¹⁵⁾ and A. gossypii.¹⁶⁾

No mechanism conferring resistance to imidacloprid in T. palmi has been reported to date. For this study, we cloned DNA fragments encoding the nAChR β1 subunit (TPβ1) gene and examined the involvement of the R81T mutation in imidacloprid resistance in T. palmi. Furthermore, we examined the involvement of degradation enzymes in imidacloprid resistance using the respective synergists for enzymes such as CYP450s, glutathione S-transferases (GSTs), and carboxylesterases (CEs).

Materials and Methods

1. Insects and chemicals

Table 1 presents descriptions of the T. palmi strains used for this study. Insects collected on eggplants were maintained at 25°C on fava bean Vicia faba sprouts under a long photoperiod (16L : 8D).

Table 1. Thrips palmi strains used in this study and their LC₅₀ values against imidacloprid

Strain	n	LC50 (mg/L) (95% CLa))	RRb)	Location and year
OK	333	9.4 (8.6–10.3)	1.0	Akaiwa City, Okayama, 1994
KC	217	20.3 (19.3–21.3)	2.2	Aki City, Kochi, 2008
OS	282	52.4 (49.8–55.1)	5.6	Habikino City, Osaka, 2010
NR	251	26.2 (18.6–34.0)	2.8	Shiki County, Nara, 2013
TS1	316	22.2 (16.8–28.0)	2.4	Kaiyo Town, Tokushima, 2011
TS4	260	87.5 (63.4–130.0)	9.3	Kaiyo Town, Tokushima, 2011
TS5	248	24.0 (14.0–35.4)	2.6	Kaiyo Town, Tokushima, 2011
TS6	340	22.3 (16.1–28.9)	2.4	Kaiyo Town, Tokushima, 2011
TS7	221	115.0 (77.5–216.2)	12.2	Kaiyo Town, Tokushima, 2011
TS8	346	30.1 (28.9–31.3)	3.2	Kaiyo Town, Tokushima, 2011

^a) CL: confidence limit. ^b) Resistance ratio (RR): LC₅₀ of each strain/ LC₅₀ of the OK strain.

Imidacloprid (Admire FL 20%) and diethyl maleate (DEM) were purchased from Bayer CropScience and Nacalai Tesque, Inc., respectively. Piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) were purchased from Wako Pure Chemical Industries, Ltd.

2. Bioassay

The leaf-dipping bioassay method was used.¹⁹⁾ Fava bean leaves (about 3.5 cm×2 cm) were dipped in five concentrations of imidacloprid containing 0.1% of the spreading agent (Gramin; Sankyo Agro Co., Ltd.) for 2 min. For the control test, fava bean leaves were dipped in distilled water containing the spreading agent. The treated leaves were allowed to air-dry at room temperature. Then 20 females were introduced into a plastic dish (3.7 cm diameter; 1.0 cm height) containing air-dried leaves. They were kept at 25°C. A bioassay was conducted with at least three replicates. Mortality was recorded at 48 hr after treatment. Lack of response of an insect to a prodding using the tip of a brush was judged as death. The LC₅₀ value was estimated for each strain using probit analysis.²⁰⁾

3. Synergism test

For this study, PBO, DEM, and DEF, the respective inhibitors of CYP450, GST, and hydrolytic enzymes including CE, were used as synergists. The fava bean leaves were dipped for 2 min in five concentrations of imidacloprid containing 0.1% of the spreading agent, synergists (PBO, 0.295 mM; DEM, 0.581 mM; DEF, 0.318 mM), and 0.1% acetone. The synergist concentrations were the same with those reported in Zhang et al.²¹⁾ The synergist concentrations caused no mortality for T. palmi. No other synergist concentrations were tested in this study. For the control test, fava bean leaves were dipped in distilled water containing 0.1% of the spreading agent and 0.1% acetone. The dried leaves were used for bioassay, as described above.

4. RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from ca. 100 adults of each strain using Sepasol RNA I Super G (Nacalai Tesque, Inc.). cDNA was constructed from 1 µg of total RNA using ReverTra Ace (Toyobo Co., Ltd.).

A DNA fragment encoding the TPβ1 gene was amplified from the most susceptible (OK) strain and the most resistant (TS7) strain using the primers 5′-caacgtgaatgaaaagaacca-3′ and 5′-gtagaaggtgatgtccgtctc-3′ to compare the nucleotide sequences corresponding to the R81T mutation. The primers were designed based on the nucleotide sequence of the nAChR β1 subunit of Frankliniella occidentalis²²⁾ (GenBank/EMBL/DDBJ accession no. AB748926). Amplified fragments were cloned into pGEM-T Easy Vector (Promega Corp.) and were then sequenced.

A DNA fragment encoding the TPβ1 gene was amplified from each strain using the primers, 5′-caacgtgaatgaaaagaacca-3′ and 5′-caggaaacagctatgacgtagaaggtgatgtccgtctc-3′ to examine the presence of insects containing the R81T mutation using direct sequencing. The latter primer was designed to contain the M13 reverse primer sequence as underlined.

The PCR conditions were 1 cycle of 3 min at 94°C followed by 40 cycles of 15 sec at 94°C, 30 sec at 53°C, and 1 min 20 sec at 72°C and a final extension of 72°C for 7 min. Quick Taq HS DyeMix (Toyobo Co., Ltd.) was used for PCR amplification in this study.

5. Nucleotide sequencing

The plasmid DNA used for nucleotide sequencing was purified using a Plasmid Mini Extraction Kit (Bioneer Corp.). The nucleotide sequence was determined using a dye terminator cycle sequencing kit (Applied Biosystems) and a DNA sequencer (3130xl; Applied Biosystems). Nucleotide and deduced amino acid sequences were analyzed using Genetyx ver. 11 (Genetyx Corp.). More than three independently isolated clones were sequenced.

Direct sequencing of DNA fragments encoding TPβ1 amplified from cDNA as described above was conducted using the primer 5′-cttccagtagtccgacaagtcc-3′ or the M13 reverse primer.

Results and Discussion

1. Resistance levels of T. palmi strains against imidacloprid

The LC₅₀ values of 10 strains examined in this study were 9.4 mg/L (OK strain) to 115.0 mg/L (TS7 strain) (Table 1). The resistance level of the most resistant TS7 strain was estimated to be 12.2-fold higher than that of the most susceptible OK strain.

2. Nucleotide sequence analyses of the TPβ1 gene

The R81T mutation at the top of the loop D region of the nAChR β1 subunit was shown to be involved in imidacloprid resistance in M. persicae¹⁵⁾ and A. gossypii.¹⁶⁾ The DNA fragments encoding the TPβ1 gene were amplified using RT-PCR reactions from the OK and TS7 strains and were then cloned and sequenced (Fig. 1). The cloned DNA fragments covered 151 amino acid residues (amino acid positions 72 to 222) out of 418 in the nAChR β1 subunit of F. occidentalis.²²⁾ The deduced amino acid sequence of the fragments displayed homology (98%) with that of the nAChR β1 subunit in F. occidentalis,²²⁾ suggesting that the amplified DNA fragments encode the nAChR β1 subunit. The cloned DNA fragments from both strains showed the same nucleotide sequences and encoded a susceptible amino acid, Arg, at the R81T site (numbered according to nAChR β1 subunit in M. persicae) (Fig. 1).

Fig. 1. ‌Nucleotide and deduced amino acid sequences of the nAChR β1 subunit gene from the most msusceptible (OK) strain of Thrips palmi (GenBank/EMBL/DDBJ accession no. LC016859). The six loops (lpD, lpA, lpE, lpB, lpF, and lpC) of the N-terminal extracellular domain are underlined. The amino acid position 81 (numbering according to nAChR β1 subunit of Myzus persicae) is boxed.

To ascertain further whether the R81T mutation is involved in imidacloprid resistance in T. palmi, direct sequencing of the amplified TPβ1 gene fragments was conducted using 10 strains with different sensitivities to imidacloprid. The agriculturally recommended concentration of imidacloprid to control T. palmi in eggplants is 50 mg/L in Japan. Therefore, some strains, such as OS, TS4, and TS7, might be defined as imidacloprid-resistant strains (Table 1). Nevertheless, the frequencies for the TPβ1 gene encoding a susceptible amino acid, Arg, at the R81T site might be 100% for all strains examined (data not shown). These results suggest that R81T is not responsible for the imidacloprid resistance in T. palmi.

3. Involvement of CYP450-mediated detoxification in imidacloprid resistance

Increased metabolic detoxification of the insecticides has been recognized as another important mechanism involved in insecticide resistance.^6–8) In insecticide detoxification, the involvement of three major groups of enzymes, CYP450s, GSTs, and CEs, has been reported.⁸⁾ Bioassays with enzyme inhibitors showed the involvement of CYP450s in imidacloprid resistance in N. lugens,^10,11) M. domestica,¹²⁾ B. tabaci,¹³⁾ and A. aegypti.¹⁴⁾ In addition to CYP450s, the involvement of CEs and, to a lesser degree, GSTs in imidacloprid resistance was reported in A. aegypti.¹⁴⁾ The involvement of CEs in imidacloprid resistance was also shown in B. tabaci.²³⁾

The LC₅₀ value of the TS7 strain against imidacloprid decreased to one-third (i.e., synergistic ratio of 3.0) with PBO treatment (Table 2). However, PBO treatment did not revert the resistance level of the TS7 strain to that of the OK strain. No such decrease in resistance level was observed in the OK strain (Table 2). No synergism effect was observed for DEM or DEF in either strain (Table 2). The synergism effects by PBO observed in the TS7 strain suggest the association of CYP450s with imidacloprid resistance in T. palmi. Consequently, the results of this study suggest that the resistance to imidacloprid in T. palmi is conferred mainly by CYP450-mediated detoxification rather than target insensitivity caused by the R81T mutation in TPβ1. PBO is known to block nonspecific esterases in some insect species.^24–26) Nonspecific esterases might also be involved in imidacloprid resistance in T. palmi.

Table 2. Synergistic effects of PBO, DEM, and DEF on Thrips palmi strains with different sensitivities to imidacloprid

Treatment	OK			TS7
	n	LC50 (mg/L) (95% CLa))	SRb)	n	LC50 (mg/L) (95% CL)	SR
Imidacloprid	333	9.4 (8.6–10.3)	1.0	221	115.0 (77.5–216.2)	1.0
Imidacloprid+PBOc)	254	15.9 (8.3–23.8)	0.6	314	38.4 (22.5–61.6)	3.0
Imidacloprid+DEMd)	222	17.8 (9.4–26.4)	0.5	298	106.0 (78.0–161.5)	1.1
Imidacloprid+DEFe)	241	12.7 (3.5–23.1)	0.7	238	136.6 (85.9–321.3)	0.8

^a) CL: confidence limit. ^b) Synergist ratio (SR): LC₅₀ of imidacloprid alone/ LC₅₀ of imidacloprid+synergist. ^c) PBO: piperonyl butoxide. ^d) DEM: Diethyl maleate. ^e) DEF: S,S,S-tributyl phosphorotrithioate.

Some specific CYP450 enzymes such as the human CYP3A4,²⁷⁾ D. melanogaster CYP6G1,²⁸⁾ B. tabaci CYP6CM1vQ,²⁹⁾ and N. lugens CYP6AY1,¹¹⁾ have been shown to metabolize imidacloprid. Cloning of the CYP450 gene and verification of its involvement in imidacloprid resistance in T. palmi remain subjects for future investigations.

Acknowledgment

This study was supported in part by the Ohara Foundation for Agricultural Research. The authors thank M. Ito (Kochi Prefecture, Japan), Y. Narai (Shimane Prefecture, Japan), A. Nakano (Tokushima Prefecture, Japan), T. Kaneda (Tokushima Prefecture, Japan), M. Shibao (Osaka Prefecture, Japan), Y. Kunimoto (Nara Prefecture, Japan), and T. Murai (Utsunomiya University, Japan) for their assistance with insect collection.

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