Cypermethrin resistance and reproductive types in onion thrips, Thrips tabaci (Thysanoptera: Thripidae)

Abstract

Cypermethrin resistance and reproductive types were examined for T. tabaci strains. Some arrhenotokous and thelytokous strains encoded the sodium channel mutation (T929I) involved in cypermethrin resistance. However, the resistance levels varied to some degree among the strains. A cytochrome P450 inhibitor, piperonyl butoxide, showed different synergistic effects on the strains examined. These results suggest that fundamental and additional levels of cypermethrin resistance in T. tabaci are conferred respectively by reduced sensitivity of the sodium channel and by cytochrome P450-mediated detoxification.

Introduction

Onion thrips, Thrips tabaci Lindeman, is a destructive agricultural pest worldwide.¹⁾ T. tabaci is also known as a vector of Tomato spotted wilt virus (TSWV),²⁾ and Iris yellow spot virus (IYSV).³⁾ The pyrethroid resistance of T. tabaci has been reported in some countries such as Japan, the USA, Canada, and Australia.^4–8)

The mechanisms of pyrethroid resistance have been characterized in many insect pests including T. tabaci. Nerve insensitivity associated with amino acid mutations in the sodium channel is known as a major mechanism of pyrethroid resistance. Reportedly, T. tabaci strains that showed high levels of resistance had two amino acid mutations (M918T and L1014F) heterozygously.⁹⁾ One moderately resistant strain was homozygous for amino acid mutation at the T929I site.⁹⁾

Cytochrome P450 (CYP450), an important degradation system for the metabolism of xenobiotics and endogenous compounds in insects,¹⁰⁾ is another important mechanism of pyrethroid resistance. In thrips, the involvement of CYP450 in cypermethrin resistance was reported for Thrips palmi Karny.¹¹⁾

Actually, T. tabaci has two distinct reproductive types: thelytoky and arrhenotoky. Thelytoky, an original reproductive type in Japan, shows complete parthenogenetic reproduction.¹²⁾ Arrhenotoky is a form of parthenogenesis in which unfertilized eggs develop into haploid males.¹²⁾ In Japan, arrhenotoky first appeared in Shimane Prefecture in 1990.¹³⁾ The reproductive types of T. tabaci were examined previously for pyrethroid resistance.¹⁴⁾ All insects with M918T and L1014F were judged as thelytokous. Most insects encoding T929I exhibited arrhenotoky. Takezawa¹⁴⁾ determined reproductive types using nucleotide sequences encoding the mitochondrial cytochrome oxidase I (COI). However, Sogo et al.¹⁶⁾ showed that some arrhenotokous insects were phylogenetically clustered incorrectly into the clade of thelytoky according to the method using COI sequences. Therefore, the relation between reproductive type and pyrethroid resistance must be re-examined. In this study, reproductive types of field-collected strains were determined based on progeny production of the adult females. Furthermore, nucleotide sequences corresponding to the three mutation sites in the sodium channel gene were examined for insects of different reproductive types.

Materials and Methods

1. Insects

In all, nine T. tabaci strains were collected in the field at three locations (Kochi, KOC; Tokushima, TOK; Kagawa, KAG) (Table 1). The TTU laboratory strain was obtained from T. Murai (Utsunomiya University, Japan). Insects were maintained with germinated fava bean (Vicia faba L.) at 23°C under a long photoperiod (16L:8D).

Table 1. Thrips tabaci strains used in this study

Strain	Location	Host plant	Year
TTU	Utsunomiya City, Tochigi Pref.	Allium cepa	2005
KOC50	Hataeda, Nankoku City, Kochi Pref.	Allium fistulosum	2011
KOC2	Hondo, Shimanto-cho, Takaoka-gun, Kochi Pref.	Asparagus officinalis	2012
KOC2442	Hataeda, Nankoku City, Kochi Pref.	Allium fistulosum	2011
KOC16	Hataeda, Nankoku City, Kochi Pref.	Allium fistulosum	2011
TOK12	Hegawa, Mugi-cho, Kaifu-gun, Tokushima Pref.	Allium fistulosum	2012
TOK6	Hegawa, Mugi-cho, Kaifu-gun, Tokushima Pref.	Allium fistulosum	2012
TOK401	Mugi-cho, Kaifu-gun, Tokushima Pref.	Allium fistulosum	2011
KAG1	Kita, Ayagawa-cho, Ayauta-gun, Kagawa Pref.	Allium cepa	2015
KAG2-1	Shichika, Manno-cho, Nakatado-gun, Kagawa Pref.	Asparagus officinalis	2013

2. Bioassay

The leaf-dipping bioassay method¹⁷⁾ was used for this study with a slight modification. Briefly, kidney bean leaves (3×3 cm) were dipped for 3 min in more than five concentrations of cypermethrin (Agrosrin 6.0% E.C.; Sumitomo Chemical Co., Ltd.) containing the spreading agent (0.02%, Agrura; Agro Kanesho Co., Ltd.). For the control test, kidney bean leaves were dipped in distilled water containing the spreading agent. The treated leaves were allowed to air-dry at room temperature and were inserted into plastic vials (10 mL) that had been coated in advance with the same concentrations of cypermethrin. Ten female adults were introduced into plastic vials and were kept at 23°C. Each bioassay was replicated three times. Mortality was recorded at 24 hr after treatment. Insects showing a lack of response to prodding using a brush tip were judged as dead. The LC₅₀ value was estimated for each strain using probit analysis.¹⁸⁾

3. Synergism test with piperonyl butoxide (PBO)

PBO (Wako Pure Chemical Industries, Ltd.) was used as the synergist in this study. Kidney bean leaves were dipped for 3 min in more than five concentrations of cypermethrin containing the spreading agent (0.02%), PBO (0.295 mM), and acetone (0.1%). The PBO concentration caused no mortality for T. tabaci (data not shown). For the control test, kidney bean leaves were dipped in distilled water containing the spreading agent (0.02%), PBO (0.295 mM), and acetone (0.1%). The treated leaves were allowed to air-dry and were inserted into the insecticide-treated plastic vials as described above.

4. Genomic DNA extraction

Genomic DNA was extracted from 10–30 adult females using a Wizard Genomic DNA Purification Kit (Promega Corp.) according to the manufacturer’s instructions.

5. PCR amplification of sodium channel gene sequences

The genomic DNA fragments corresponding to domains IIS4-IIS6 of the sodium channel gene were amplified using PCR with the primers Tt-Na-5′-3 (5′-tgagtccgaagttctatttt-3′) and Tt-Na-3′-5 (5′-ggtccgagatctgattcgtc-3′). The primers were designed based on the nucleotide sequence of the sodium channel gene from T. tabaci (GenBank/EMBL/DDBJ accession No. LC164017). The PCR conditions were 1 cycle of 3 min at 94°C, 40 cycles of 15 sec at 94°C, 30 sec at 60°C, and 1 min at 72°C; and final extension of 72°C for 7 min. Amplified DNA fragments were sequenced directly using the primer Tt-Na-direct-seq4 (5′-gcgaacgtttgctttgatcc-3′).

6. Determination of reproductive types

Females of the laboratory and field-collected strains were reared individually and were allowed to oviposit on fava beans for more than 14 days. When males were included among the progeny, the reproductive type was judged as arrhenotokous. Females producing only daughters were selected. Then the obtained daughters were allowed to lay eggs. The productive type was regarded as thelytokous if only females were obtained from the eggs.

The reproductive type was also examined based on the nucleotide sequences of the COI. A part of the COI sequences was amplified by PCR using the primers UEA3 (5′-tatagcattcccacgaataaataa-3′)¹⁹⁾ and Tt448 (5′-atgagaaattagtccaaatcctgg-3′).¹⁵⁾ The PCR conditions were 1 cycle of 3 min at 94°C, 40 cycles of 15 sec at 94°C, 30 sec at 50°C, and 1 min at 72°C; and final extension of 72°C for 7 min. Amplified DNA fragments were sequenced directly using the primer, Tt-COI-direct-5′-2 (5′-gtctgatcagtttattttaacagcc-3′).

7. Nucleotide sequencing

Nucleotide sequencing was conducted using a dye terminator cycle sequencing kit (Applied Biosystems) and a DNA sequencer (3130xl; Applied Biosystems).

Results

1. Reproductive type determination

Reproductive types of the examined strains are presented in Table 2. The TTU, KOC50, KOC2, TOK12, TOK6, KAG1, and KAG2-1 strains were judged as thelytokous according to progeny production of the adult females. The remaining three strains (KOC2442, KOC16, and TOK401) were regarded as arrhenotokous. Based on the COI sequences, KOC16 was classified as thelytokous.

Table 2. Toxicity of cypermethrin and synergistic effect of PBO on Thrips tabaci

Strain	Reproductive typea		T929I	Treatment	n	LC50 (mg/L)	95% CLd	RRe	SRf
	Phenotype	COI
TTU	TH	TH	SS	cypermethrin	163	0.287	0.235–0.350	22	1.00
				cypermethrin+PBOg	158	0.226	0.180–0.282	14	1.27
KOC50	TH	TH	SSc	cypermethrin	246	0.013	0.012–0.015	1	1.00
				cypermethrin+PBO	216	0.016	0.014–0.018	1	0.81
KOC2	TH	TH	RRb	cypermethrin	151	94.885	81.762–107.776	7,299	1.00
				cypermethrin+PBO	162	66.461	59.116–74.860	4,154	1.43
KOC2442	AR	AR	RR	cypermethrin	244	112.905	87.978–137.379	8,685	1.00
				cypermethrin+PBO	191	63.567	55.610–74.170	3,973	1.78
KOC16	AR	TH	RR	cypermethrin	195	148.968	134.568–164.627	11,459	1.00
				cypermethrin+PBO	196	48.149	41.014–55.102	3,009	3.09
TOK12	TH	TH	SS	cypermethrin	166	0.033	0.029–0.041	3	1.00
				cypermethrin+PBO	192	0.028	0.023–0.040	2	1.18
TOK6	TH	TH	RR	cypermethrin	205	146.781	124.486–158.835	11,291	1.00
				cypermethrin+PBO	236	81.195	65.485–88.063	5,075	1.81
TOK401	AR	AR	RR	cypermethrin	221	393.060	336.079–482.295	30,235	1.00
				cypermethrin+PBO	237	105.119	92.759–120.763	6,570	3.74
KAG1	TH	TH	SS	cypermethrin	196	0.054	0.009–0.077	4	1.00
				cypermethrin+PBO	227	0.049	0.031–0.060	3	1.10
KAG2-1	TH	TH	RR	cypermethrin	236	70.982	57.813–81.006	5,460	1.00
				cypermethrin+PBO	202	52.995	48.428–57.151	3,312	1.34

^a AR: arrhenotoky, TH: thelytoky. ^b RR: Resistant homozygote for the T929I site. ^c SS: Susceptible homozygote for the T929I site. ^d CL: confidence limit. ^e Resistance ratio (RR): cypermethrin LC₅₀ of each strain/cypermethrin LC₅₀ of KOC50 strain or cypermethrin+PBO LC₅₀ of each strain/cypermethrin+PBO LC₅₀ of KOC50 strain. ^f Synergist ratio (SR): LC₅₀ of cypermethrin alone/LC₅₀ of cypermethrin+PBO. ^g PBO: piperonyl butoxide.

2. Bioassay

The LC₅₀ values of 10 strains examined were 0.013 mg/L (KOC50)–393.06 mg/L (TOK401) (Table 2). The resistance level of the TOK401 strain was estimated as 30, 235-fold higher than that of the KOC50 strain (Table 2).

3. Nucleotide sequence analyses of the sodium channel gene

The DNA fragments corresponding to domains IIS4–IIS6 of the sodium channel gene from 10 strains were amplified by PCR. Amplified DNA fragments were sequenced directly. According to the signal appearance at the T929I site on the sequence chromatograms, the KOC2, KOC2442, KOC16, TOK6, TOK401, and KAG2-1 strains were inferred as resistant homozygotes for the mutation site (Table 2). The remaining four strains (TTU, KOC50, TOK12, and KAG1) were regarded as susceptible homozygotes (Table 2). No strain with both M918T and L1014F was found in this study.

4. Synergistic analysis with PBO

The synergistic ratios of PBO for 10 strains were 0.81 (KOC50)–3.74 (TOK401) (Table 2).

Discussion

Reproductive type determination of T. tabaci based on progeny production of the adult females takes a considerable amount of time. A time-saving method for discrimination of the reproductive types using the COI sequences was reported by Takeuchi and Toda.¹⁵⁾ However, some insects exhibiting arrhenotoky were classified incorrectly as showing thelytoky according to the COI-based method.¹⁶⁾ In fact, in this study, the reproductive type of one strain (KOC16) was predicted incorrectly based on COI-based method (Table 2). Consequently, reproductive types determined by progeny production of the adult females were used in this study.

The LC₅₀ values of the KOC2, KOC2442, KOC16, TOK6, TOK401, and KAG2-1 strains were 70.982 mg/L (KAG2-1)–393.06 mg/L (TOK401) (Table 2). The agriculturally recommended concentration of cypermethrin for T. tabaci in onions is 30 mg/L in Japan. Direct sequencing showed that the six strains had T929I. One strain showing a moderate level of resistance to cypermethrin contained T929I.⁹⁾ These results suggest that the six strains with T929I are cypermethrin resistant. However, the resistance level of the TOK401 strain was estimated as 5.5-fold higher than that of the KAG2-1 strain, suggesting the involvement of other resistance mechanisms.

The involvement of CYP450 in cypermethrin resistance was reported for T. palmi.¹¹⁾ To examine the involvement of CYP450 at various levels of resistance of the resistant T. tabaci strains, a synergism test using PBO was executed. PBO caused 3.09-fold and 3.74-fold decreases in the resistance ratio for the KOC16 and TOK401 strains, respectively. The synergistic effects of PBO in the resistance were limited for the KOC2, KOC2442, TOK6, and KAG2-1 strains. These results suggest that CYP450 is involved differentially in the cypermethrin resistance of T. tabaci strains. PBO is also known to block nonspecific esterases in some insect species.^20–22) The involvement of nonspecific esterases in the cypermethrin resistance of T. tabaci must also be examined.

Takezawa¹⁴⁾ reported that most strains encoding T929I are arrhenotokous. In the present study, T929I was encoded not only in arrhenotoky but also in thelytoky, suggesting that both reproductive types have the potential to encode the mutation. To date, all insects encoding M918T and L1014F show thelytoky.^9,14) No insect encoding both mutations has been obtained in Shikoku Island to date (this study by Aizawa is unpublished). The genetic potential of arrhenotokous strains to encode both mutations heterozygously must be examined in future studies.

Acknowledgment

This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) as part of Joint Research Program implemented at the Institute of Plant Science and Resources, Okayama University in Japan.

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