Field survey of reproductive modes and sodium channel mutations associated with pyrethroid resistance in Thrips tabaci

Abstract

Using PCR-Restriction Fragment Length Polymorphism (RFLP) with mitochondrial cytochrome c oxidase subunit I sequences, we examined the reproductive modes of female adults of Thrips tabaci collected at 54 sites across Japan. Results showed the presence of heteroplasmic insects harboring mitochondria associated with arrhenotoky and thelytoky. Using the insects, we also applied PCR-RFLP to examine the genotypes for the amino acid mutation (T929I) site involved in pyrethroid resistance. Findings showed the presence of thelytokous heterozygotes under the circumstance that most arrhenotokous insects are resistant homozygotes, and many thelytokous insects are susceptible homozygotes. These results suggest that, in the field, genetic exchange occurs between insects through of both reproductive modes. A survey of the genotypes for the other amino acid mutations using nucleotide sequencing showed a decline of insects with an M918T and L1014F pair and an increase of insects with M918L. These results suggest the evolutional progression of amino acid mutations associated with pyrethroid resistance in T. tabaci.

Introduction

Thrips tabaci Lindeman (Thysanoptera: Thripidae) has been recognized as a destructive agricultural pest species worldwide.¹⁾ The thrips directly suck cell fluids from leaves, stems, flowers, and the surfaces of fruits of widely various crops, thereby causing silvery scarring and leaf chlorosis, which reduce their commercial value considerably. Moreover, they transmit Tomato spotted wilt virus (TSWV)²⁾ and Iris yellow spot virus (IYSV)³⁾ in a persistent manner, widening the scale and scope of economic damage.

Three reproductive modes have been reported in T. tabaci: arrhenotoky, thelytoky, and duterotoky.^4,5) Arrhenotoky is a form of parthenogenesis by which unfertilized eggs develop into haploid males.⁴⁾ With thelytoky, only females develop from unfertilized eggs.⁴⁾ Duterotoky, by which females and males appear from unfertilized eggs, has been reported in the United States,⁵⁾ but not in Japan.^6,7)

Thrips tabaci has developed resistance to various insecticides including pyrethroids. Pyrethroid resistance of T. tabaci is conferred mainly by target insensitivity of the sodium channel caused by amino acid mutations. Six amino acid mutations have been correlated with the resistance: M918T, M918L, T929I, V1010A, L1014F, and K1774N.^6–11) Thelytokous insects with M918T and L1014F exhibit a high level of resistance to a pyrethroid.⁸⁾ Arrhenotokous insects with T929I show a moderate level of pyrethroid resistance.⁸⁾ Jouraku et al.¹⁰⁾ reported that T929I is linked with K1774N and that insects with both mutations show a high degree of pyrethroid resistance. Recently, the presence of thelytokous insects with M918L was reported in Japan.¹⁰⁾ However, the distribution of insects with amino acid mutations and their reproductive modes described above have not been investigated extensively in the field.

Earlier, we showed that all arrhenotokous insects were homozygous for the T929I site, whereas only some thelytokous insects were homozygous for the mutation site.⁷⁾ Arrhenotokous and thelytokous insects sometimes inhabit the same site⁷⁾ and mate irrespective of the reproductive mode of their counterpart.¹²⁾ Therefore, genetic exchange might occur through hybridization between insects of both reproductive modes in the field. In fact, genetic exchange between both reproductive modes has been shown to occur with low frequency.^13,14) Nevertheless, insects heterozygous for the T929I site were not observed in our earlier study.⁷⁾

For this study, we examine genotypes for the sodium channel mutation sites and reproductive modes using larger numbers of female adults of T. tabaci collected from various regions of Japan. Based on these data, the possibility of genetic exchange between arrhenotoky and thelytoky and the evolutional progression of the sodium channel mutations are discussed.

Materials and methods

1. Insect collection and genomic DNA extraction

Thrips tabaci individuals were collected in the field from 2021 through 2023 (Table 1). Collected insects were stored in 99.5% ethanol or at −80°C until DNA extraction.

Table 1. Genotypes for the T929I site in female adults of Thrips tabaci examined in this study.

Study site	n	Reproductive mode									Host plant	Location (Latitude and longitude of municipality)	Month, Year	Note
		Arrhenotoky			Thelytoky			Unknown
		RR	RS	SS	RR	RS	SS	RR	RS	SS
Hokkaido 1	271	176	7	1	3	6	56	2	11	9	A. cepa	Naganuma town, Hokkaido pref (N43°00′ E141°41′)	July, 2021
Hokkaido 2	76	0	0	0	0	0	76	0	0	0	A. cepa	Kunneppu town, Hokkaido pref (N43°43′ E143°45′)	July, 2023
Akita 1	31	31	0	0	0	0	0	0	0	0	A. fistulosum	Noshiro city, Akita pref (N40°12′ E140°01′)	September, 2021
Akita 2	76	66	1	0	1	0	1	2	4	1	A. fistulosum	Happo town, Akita pref (N40°19′ E140°02′)	September, 2021
Iwate 1	39	29	0	0	0	0	10	0	0	0	A. fistulosum	Morioka city, Iwate pref (N39°70′ E141°15′)	September, 2022
Iwate 2	7	0	0	0	0	0	7	0	0	0	A. fistulosum	Ohta, Hanamaki city, Iwate pref (N39°38′ E141°11′)	September, 2022
Iwate 3	28	0	0	0	0	0	28	0	0	0	A. fistulosum	Shiba town, Iwate pref (N39°33′ E141°09′)	September, 2022
Iwate 4	28	0	0	0	0	0	28	0	0	0	A. fistulosum	Nishiwaga town, Iwate pref (N39°19′ E140°46′)	September, 2022
Iwate 5	35	0	0	0	0	0	35	0	0	0	A. fistulosum	Ohata, Hanamaki city, Iwate pref	September, 2022
Yamagata 1	31	29	0	0	2	0	0	0	0	0	A. fistulosum	Sagae city, Yamagata pref (N38°22′ E140°16′)	July, 2022
Yamagata 2	28	26	0	0	2	0	0	0	0	0	A. fistulosum	Shonai town, Yamagata pref (N38°50′ E139°54′)	August, 2022
Miyagi 1	64	0	0	0	0	1	63	0	0	0	A. cepa	Shibata town, Miyagi pref (N38°03′ E140°45′)	July, 2022
Miyagi 2	116	31	0	0	0	0	85	0	0	0	A. fistulosum, A. cepa	Natori city, Miyagi pref (N38°10′ E140°53′)	July, 2022
Tochigi 1	67	59	0	0	0	1	7	0	0	0	Allium fistulosum	Kamikomoriya, Utsunomiya city, Tochigi pref (N36°33′ E139°52′)	June, 2021
Tochigi 2	67	57	0	0	0	0	9	0	1	0	A. fistulosum	Mooka city, Tochigi pref (N36°44′ E140°01′)	June, 2021
Tochigi 3	44	2	0	0	0	0	42	0	0	0	A. fistulosum	Minemachi, Utsunomiya city, Tochigi pref	May, 2022
Chiba 1	45	43	0	0	0	0	2	0	0	0	A. fistulosum	Arai, Yokoshibahikari town, Chiba pref (N35°39′ E140°30′)	June, 2021
Chiba 2	95	95	0	0	0	0	0	0	0	0	A. fistulosum	Haratkata, Yokoshibahikari town, Chiba pref	June, 2021
Chiba 3	42	32	1	0	0	0	9	0	0	0	A. fistulosum	Oamishirosato city, Chiba pref (N35°31′ E140°19′)	June, 2021
Chiba 4	35	35	0	0	0	0	0	0	0	0	A. fistulosum	Kokubun, Ichikawa city, Chiba pref (N35°71′ E139°92′)	August, 2021
Chiba 5	33	33	0	0	0	0	0	0	0	0	A. fistulosum	Kitakokubun, Ichikawa city, Chiba pref	August, 2021
Chiba 6	49	45	0	0	0	1	3	0	0	0	A. cepa	Arai, Yokoshibahikari town, Chiba Pref	June, 2022
Chiba 7	16	11	0	0	0	0	5	0	0	0	A. cepa	Haratkata, Yokoshibahikari town, Chiba Pref	June, 2022
Yamanashi 1	38	37	1	0	0	0	0	0	0	0	A. fistulosum	Iwamori, Kai city, Yamanashi pref (N35°39′ E138°30′)	July, 2022
Yamanashi 2	54	11	28	6	5	1	3	0	0	0	A. fistulosum	Shimoimai, Kai city, Yamanashi pref	July, 2022
Nagano 1	45	43	1	0	1	0	0	0	0	0	A. fistulosum	Yamagata village, Nagano pref (N36°10′ E137°52′)	October, 2022
Nagano 2	48	47	0	0	0	0	1	0	0	0	A. fistulosum	Matsumoto city, Nagano pref (N36°14′ E137°58′)	October, 2022
Kyoto 1	18	0	0	0	2	1	15	0	0	0	Daucus carota subsp. Sativus	Kyotamba town, Kyoto Pref (N35°10′ E135°25′)	December, 2020 and January, 2021
Kyoto 2	14	11	1	0	2	0	0	0	0	0	A. fistulosum	Takeda, Fushimi-ku, Kyoto city, Kyoto pref (N35°01′ E135°76′)	June, 2022
Kyoto 3	9	6	0	0	2	0	1	0	0	0	A. fistulosum	Mukaijima, Fushimi-ku, Kyoto city, Kyoto pref	June, 2022
Osaka	15	0	0	0	0	0	15	0	0	0	A. officinalis	Habikino city, Osaka pref (N34°55′ E135°57′)	July, 2023
Wakayama 1	8	5	0	0	0	0	3	0	0	0	Allium cepa	Gobo city, Wakayama pref (N33°89′ E135°15′)	April, 2022
Wakayama 2	49	25	2	0	0	1	21	0	0	0	A. cepa	Nishikawahara, Kinokawa city, Wakayama pref (N34°26′ E135°36′)	April, 2022
Wakayama 3	53	12	0	1	5	2	32	1	0	0	A. cepa	Kirihata, Kinokawa city, Wakayama pref	April, 2022
Wakayama 4	51	10	0	0	0	0	41	0	0	0	A. cepa	Kokawa, Kinokawa city, Wakayama pref	April, 2022
Hyogo 1	53	0	0	0	1	0	52	0	0	0	A. fistulosum	Kasai city, Hyogo pref (N34°55′ E134°50′)	May, 2022	No pesticide use
Hyogo 2	64	0	0	0	0	0	64	0	0	0	A. cepa	Awaji city, Hyogo pref (N34°26′ E134°54′)	May, 2022
Okayama	51	0	0	0	0	0	51	0	0	0	A. cepa, B. oleracea var. capitata	Okayama city, Okayama pref (N34°65′ E133°91′)	May, 2022	No pesticide use
Hiroshima 1	7	0	0	0	7	0	0	0	0	0	Asparagus officinalis	Funocho, Miyoshi city, Hiroshima pref (N34°48′ E132°51′)	June, 2022
Hiroshima 2	20	0	0	0	0	0	20	0	0	0	A. officinalis	Miwacho, Miyoshi city, Hiroshima pref	June, 2022
Hiroshima 3	37	0	0	0	7	0	30	0	0	0	A. officinalis	Kawashiri, Sera town, Hiroshima pref (N34°35′ E133°03′)	June, 2022
Hiroshima 4	13	0	0	0	0	0	13	0	0	0	A. officinalis	Uzuto, Sera town, Hiroshima pref	June, 2022
Hiroshima 5	14	0	0	0	4	0	10	0	0	0	Solanum melongena	Higashihiroshima city, Hiroshima pref (N34°25′ E132°44′)	July, 2022
Hiroshima 6	7	0	0	0	0	1	6	0	0	0	Eustoma grandiflorum	Higashihiroshima city, Hiroshima pref	July, 2022
Hiroshima 7	14	0	0	0	1	0	13	0	0	0	A. officinalis	Miyoshi city, Hiroshima pref	July, 2022
Shimane 1	51	5	0	0	0	0	46	0	0	0	A. cepa	Hikawa town, Izumo city, Shimane pref (N35°22′ E132°45′)	May, 2022	No pesticide use
Shimane 2	51	1	0	0	0	0	50	0	0	0	A. cepa	Izumo city, Shimane pref	May, 2022	No pesticide use
Nagasaki 1	21	0	0	0	0	0	21	0	0	0	A. officinalis	Isahaya city, Nagasaki pref (N32°84′ E130°05′)	July, 2022
Nagasaki 2	10	0	0	0	3	0	7	0	0	0	A. officinalis	Shimabara city, Nagasaki pref (N32°78′ E130°37′)	October, 2022
Nagasaki 3	38	0	0	0	28	1	9	0	0	0	A. officinalis	Higashisonogi town, Nagasaki pref (N33°02′ E129°55′)	October, 2022
Nagasaki 4	9	0	0	0	0	0	9	0	0	0	A. officinalis	Nagasaki city, Nagasaki pref (N32°75′ E129°87′)	August, 2022
Kumamoto 1	32	0	0	0	0	0	32	0	0	0	A. fistulosum	Yatsushiro city, Kumamoto pref (N32°50′ E130°60′)	May, 2022	No pesticide use
Kumamoto 2	15	2	0	0	0	0	13	0	0	0	A. fistulosum, Allium sativum	Koshi city, Kumamoto pref (N32°53′ E130°77′)	May, 2022	No pesticide use
Miyazaki	45	3	0	0	0	1	41	0	0	0	Brassica oleracea var. capitata	Miyazaki city, Miyazaki pref (N31°90′ E131°42′)	May, 2022	No pesticide use

Genomic DNA was extracted from a single female adult using MightyPrep reagent for DNA (Takara Bio Inc., Kusatsu, Japan). Briefly, a single insect introduced into 10 µL of the reagent was incubated at 95°C for 10 min and then at room temperature for 2 min. After centrifugation at 15,000×g for 2 min, the supernatant was recovered as a DNA sample. For subsequent PCR amplification, 0.5 µL of the supernatant was used.

2. Determination of reproductive mode

The reproductive mode was examined based on the nucleotide sequences of the cytochrome c oxidase subunit I (COI) gene. A part of the COI sequence was amplified by PCR using the primers UEA3 (5′-tatagcattcccacgaataaataa-3′)¹⁵⁾ and Tt448 (5′-atgagaaattagtccaaatcctgg-3′).¹⁶⁾ 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, with final extension of 72°C for 7 min. EmeraldAmp MAX PCR Master Mix (Takara Bio Inc.) was used for PCR amplification. Digestion of the amplified DNA fragments with EcoO109I produces restriction fragments with lengths of 350 bp and 140 bp for arrhenotoky and restriction fragments with lengths of 260 bp, 140 bp, and 90 bp for thelytoky. The digested DNA fragments were confirmed by 1.5% or 2% agarose gel electrophoresis. We applied this method for reproductive mode determination of our cultured arrhenotokous (KOC16, KOC2442, TOK401, KAG4-5, and KTF-SPRR) and thelytokous (KAG1, KOC50, KOC2, and KOC2-2) strains¹¹⁾ and found that the method correctly judged their reproductive modes, except for arrhenotokous KOC16 strain with thelytokous-associated COI sequence.¹⁷⁾ An arrhenotokous strain (KYT-M1) with thelytokous-associated COI sequence was also reported in another study.¹¹⁾ Nevertheless, the frequency of such insects is very low, and this method is useful as an initial screen for the reproductive modes of T. tabaci.

DNA fragments amplified from two insects collected in Hokkaido prefecture showing restriction patterns of both reproductive modes were cloned into pMD20 vector (Takara Bio Inc.) according to the manufacturer’s recommendations. Then they were sequenced.

3. Genotyping of sodium channel mutation sites

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′).⁶⁾ 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, with final extension of 72°C for 7 min. EmeraldAmp MAX PCR Master Mix was used for PCR amplification. Digestion of the amplified DNA fragments with MboI produces restriction fragments with lengths of 519 bp and 216 bp for susceptible homozygotes and restriction fragments with lengths of 287 bp, 232 bp, and 216 bp for resistant homozygotes. All the described restriction fragments (519 bp, 287 bp, 232 bp, and 216 bp) are produced in heterozygotes. The digested DNA fragments were confirmed using 1.5% or 2% agarose gel electrophoresis.

A certain number of heterozygotes and susceptible homozygotes for the T929I site were subjected to genotyping for the amino acid mutation (M918T, M918L, V1010A, and L1014F) sites. For this genotyping, the amplified sodium channel DNA fragments were sequenced directly using the Tt-Na-direct-seq4 (5′-gcgaacgtttgctttgatcc-3′) primer, as described by Aizawa et al.⁶⁾

4. Nucleotide sequencing

Nucleotide sequencing was conducted using a dye terminator cycle sequencing kit (ver. 3.1; Applied Biosystems, Waltham, MA, USA) and a DNA sequencer (3500 Genetic Analyzer; Applied Biosystems). Nucleotide and deduced amino acid sequences were analyzed using software (Genetyx ver. 13; Genetyx Corp., Tokyo, Japan).

Results

1. Determination of reproductive mode

In all, 2,277 female adults were collected at 54 sites from northern to southern Japan and were examined for their reproductive modes. Results showed that 1,068 and 1,178 insects, respectively, were classifiable as arrhenotoky and thelytoky (Table 1). Our PCR-RFLP method failed to indicate the reproductive mode of the remaining 31 insects because restriction patterns of both reproductive modes were observed (The restriction patterns of three out of 31 insects shown in lanes 1, 2, and 7 in Fig. 1). Those insects were detected at four study sites: Tochigi 2, Hokkaido 1, Akita 2, and Wakayama 3. Of the 298 insects collected at the study sites with no pesticide application (Kumamoto 1, Kumamoto 2, Shimane 1, Shimane 2, Miyazaki, Okayama, and Hyogo 1), 11 and 287 were, respectively, classifiable as arrhenotoky and thelytoky.

Fig. 1. Restriction patterns of the COI gene fragments amplified from Thrips tabaci. After the COI gene fragments amplified by PCR were digested with EcoO109I, they were size fractionated on 2% agarose gel. Restriction patterns for arrhenotoky and thelytoky were observed, respectively, in lanes 3, 4, and 5 and in lanes 6, 8, and 9. In lanes 1, 2, and 7, restriction patterns for both reproductive modes were observed.

To assess the possibility of heteroplasmy in 31 insects showing restriction patterns of both reproductive modes, the COI fragments amplified from two insects collected at Hokkaido 1 were cloned and sequenced. For one insect, 11 clones were examined. Among the 11 clones, 10 showed arrhenotokous-associated sequences, whereas the remaining clone was thelytokous-associated (data not shown). For another insect, six examined clones were also classified into two sequences: arrhenotokous-associated (three clones) and thelytokous-associated (three clones). These results suggest the presence of heteroplasmic insects having mitochondria associated with both reproductive modes in T. tabaci.

2. Genotyping of sodium channel mutation sites

The results of genotyping for the T929I site are presented in Table 1. Insects with T929I were detected throughout Japan. Most of the arrhenotokous female adults were resistant homozygotes for the T929I site. In arrhenotoky, susceptible homozygotes (eight insects) and heterozygotes (42 insects) were, respectively, detected at three study sites (Hokkaido 1, Wakayama 3, and Yamanashi 2) and at eight study sites (Chiba 3, Hokkaido 1, Akita 2, Wakayama 2, Kyoto 2, Yamanashi 1, Yamanashi 2, and Nagano 1). On the other hand, many of the thelytokous female adults were susceptible homozygotes for the T929I site. Regarding thelytoky, a total of 17 heterozygous insects were detected at 11 study sites: Tochigi 1, Hokkaido 1, Kyoto 1, Wakayama 2, Wakayama 3, Miyazaki, Chiba 6, Miyagi 1, Yamanashi 2, Hiroshima 6, and Nagasaki 3. Regarding the heterozygous insects, the results of genotyping using PCR-RFLP were confirmed by nucleotide sequencing, except for one insect collected at Yamanashi 2 (data not shown).

The results of genotyping for mutation sites other than the T929I site are presented in Table 2. Thelytokous insects with a pair of M918T and L1014F were detected at a very low frequency (two insects) at only one study site (Hokkaido 2). They encoded the mutations heterozygously as reported by Toda and Morishita⁸⁾ (data not shown). On the other hand, insects heterozygously encoding M918L were found at three study sites (Hokkaido 1, Wakayama 1, and Hokkaido 2). Among them, three arrhenotokous insects collected at Hokkaido 1 were heterozygous for both M918L and T929I sites. If the reproductive mode predicted by the PCR-RFLP method was correct, then this report is the first of insects with M918L in arrhenotoky. The linkage examination between T929I and M918L and other amino acid mutations remained in the future. No insect with V1010A was detected in this study (data not shown).

Table 2. Genotypes for the M918L, M918T, and L1014F sites in heterozygotes and susceptible homozygotes for the T929I site in Thrips tabaci.

Study site	Thelytoky										Arrhenotoky										Unknown
	n	M918L			M918T			L1014F			n	M918L			M918T			L1014F			n	M918L			M918T			L1014F
		RR	RS	SS	RR	RS	SS	RR	RS	SS		RR	RS	SS	RR	RS	SS	RR	RS	SS		RR	RS	SS	RR	RS	SS	RR	RS	SS
Hokkaido 1	59	0	55	4	0	0	59	0	0	59	8	0	3	5	0	0	8	0	0	8	20	0	10	10	0	0	20	0	0	20
Hokkaido 2	76	0	7	69	0	2	74	0	2	74	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Akita 1	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Akita 2	1	0	0	1	0	0	1	0	0	1	1	0	0	1	0	0	1	0	0	1	5	0	0	5	0	0	5	0	0	5
Iwate 1	10	0	0	10	0	0	10	0	0	10	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Iwate 2	7	0	0	7	0	0	7	0	0	7	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Iwate 3	12	0	0	12	0	0	12	0	0	12	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Iwate 4	12	0	0	12	0	0	12	0	0	12	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Iwate 5	11	0	0	11	0	0	11	0	0	11	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Yamagata 1	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Yamagata 2	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Miyagi 1	17	0	0	17	0	0	17	0	0	17	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Miyagi 2	31	0	0	31	0	0	31	0	0	31	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Tochigi 1	8	0	0	8	0	0	8	0	0	8	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Tochigi 2	9	0	0	9	0	0	9	0	0	9	—	—	—	—	—	—	—	—	—	—	1	0	0	1	0	0	1	0	0	1
Tochigi 3	13	0	0	13	0	0	13	0	0	13	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Chiba 1	2	0	0	2	0	0	2	0	0	2	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Chiba 2	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Chiba 3	8	0	0	8	0	0	8	0	0	8	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—
Chiba 4	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Chiba 5	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Chiba 6	4	0	0	4	0	0	4	0	0	4	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Chiba 7	5	0	0	5	0	0	5	0	0	5	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Yamanashi 1	—	—	—	—	—	—	—	—	—	—	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—
Yamanashi 2	—	—	—	—	—	—	—	—	—	—	29	0	0	29	0	0	29	0	0	29	—	—	—	—	—	—	—	—	—	—
Nagano 1	—	—	—	—	—	—	—	—	—	—	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—
Nagano 2	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Kyoto 1	16	0	0	0	0	0	16	0	0	16	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Kyoto 2	—	—	—	—	—	—	—	—	—	—	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—
Kyoto 3	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Osaka	14	0	0	14	0	0	14	0	0	14	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Wakayama 1	3	0	1	2	0	0	3	0	0	3	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Wakayama 2	22	0	0	22	0	0	22	0	0	22	2	0	0	2	0	0	2	0	0	2	—	—	—	—	—	—	—	—	—	—
Wakayama 3	34	0	0	34	0	0	34	0	0	34	1	0	0	1	0	0	1	0	0	1	—	—	—	—	—	—	—	—	—	—
Wakayama 4	40	0	0	40	0	0	40	0	0	40	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hyogo 1	14	0	0	14	0	0	14	0	0	14	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hyogo 2	7	0	0	7	0	0	7	0	0	7	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Okayama	16	0	0	16	0	0	16	0	0	16	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 1	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 2	6	0	0	6	0	0	6	0	0	6	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 3	16	0	0	16	0	0	16	0	0	16	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 4	4	0	0	4	0	0	4	0	0	4	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 5	10	0	0	10	0	0	10	0	0	10	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 6	7	0	0	7	0	0	7	0	0	7	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Hiroshima 7	13	0	0	13	0	0	13	0	0	13	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Shimane 1	46	0	0	46	0	0	46	0	0	46	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Shimane 2	15	0	0	15	0	0	15	0	0	15	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Nagasaki 1	15	0	0	15	0	0	15	0	0	15	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Nagasaki 2	7	0	0	7	0	0	7	0	0	7	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Nagasaki 3	10	0	0	10	0	0	10	0	0	10	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Nagasaki 4	8	0	0	8	0	0	8	0	0	8	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Kumamoto 1	14	0	0	14	0	0	14	0	0	14	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Kumamoto 2	13	0	0	13	0	0	13	0	0	13	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—
Miyazaki	16	0	0	16	0	0	16	0	0	16	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—	—

Discussion

In recent years, decreased susceptibility to multiple insecticides has been reported for T. tabaci.^18–20) However, decreased susceptibility to organophosphates, pyrethroids, neonicotinoids, and diamides has been reported only in the arrhenotokous populations.^19–22) Arrhenotokous and thelytokous strains with T929I exhibited a similar level of resistance to a pyrethroid, but biotic performances such as longevity and fecundity were markedly inferior in the latter.^6,7) Little or no information related to the fitness costs of resistance to organophosphates, neonicotinoids, and diamides in different reproductive modes of T. tabaci has been available. However, considering the ratio of arrhenotokous and thelytokous insects at study sites with and without pesticide applications, the fitness costs to the insecticides in arrhenotokous insects might be lower than those in thelytokous insects. Future studies must examine details of the relation between insecticide resistance and fitness costs.

Heteroplasmy, the existence of mitochondrial gene variants within a single cell or individual, might occur through some mechanisms, including paternal leakage.^23,24) From the present study, we found heteroplasmic insects exhibiting the presence of mitochondria associated with both reproductive modes. Gawande et al.²⁵⁾ reported that heteroplasmic insects might have resulted from the introgression of mitochondria of arrhenotokous males into thelytokous females through paternal leakage during mating. If their inference is correct, then the reproductive mode of heteroplasmic insects must be thelytoky. The reproductive modes of the heteroplasmic insects identified in this study were unclear. The establishment of heteroplasmic strains and examination of their reproductive modes remain as objectives for future investigations.

Most of the arrhenotokous insects were resistant homozygotes for the T929I site, whereas many thelytokous insects were susceptible homozygotes (Table 1). They coexisted at 26 study sites out of 54. The present study detected, for the first time reported, thelytokous insects heterozygously encoding T929I at 11 study sites across 10 prefectures. Reportedly, arrhenotokous males mated not only with arrhenotokous females but also with thelytokous females.¹²⁾ Gene transfer from arrhenotokous males to thelytokous females has been shown to occur with low frequency.^13,14) Consequently, the thelytokous insects heterozygously encoding T929I might have resulted through mating between arrhenotokous insects with T929I and thelytokous insects without the mutation. In an earlier study, we demonstrated that T. tabaci is divisible into three groups based on the COI sequence: diploid thelytoky, triploid thelytoky, and diploid arrhenotoky.¹¹⁾ In addition, our principal component analysis using whole genome resequencing data demonstrated that diploid and triploid thelytokous groups are further classifiable into two based on the sodium channel mutations harbored by respective group members (strains).¹¹⁾ Based on the results of subsequent admixture analysis, we inferred that a thelytokous group without T929I might be a recipient of gene flow from arrhenotokous males.¹¹⁾ Mating experiments must be conducted to clarify the genetic background of arrhenotokous and thelytokous insects that enable genetic exchange between different reproductive modes.

It is of particular interest that arrhenotokous insects without T929I were detected in this study. In Japan, thelytokous insects were dominant.^26,27) In the late 1990s to early 2000s, T. tabaci began to damage a wider range of agricultural crops^28–32) and exhibited decreased insecticide susceptibility.^28,32,33) Arrhenotokous insects began to be observed frequently during the period.⁸⁾ All arrhenotokous strains examined had T929I.^7,34) Based on the observations, it has been speculated that arrhenotokous insects with T929I invaded from overseas.^35,36) However, the discovery of arrhenotokous insects without T929I in this study suggests that arrhenotokous insects susceptible to pyrethroids had invaded together with the resistant insects of the same reproductive mode but they have been eliminated through selection with pyrethroids.

Thelytokous insects with a pair of M918T and L1014F were observed with a certain frequency in 2001–2010.^8,34) However, those insects have not been observed in the past few years.^6,10) From this study, we confirmed the presence of insects with a pair of M918T and L1014F, but their frequency was very low. We also confirmed the increase of insects with M918L. These results suggest the evolutional progression of amino acid mutations associated with pyrethroid resistance. Surveys to elucidate the contribution of these mutations to pyrethroid resistance and fitness costs and their future distribution are expected to be valuable for more effective management of T. tabaci.

Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (21H02192). We would like to thank M. Morishita (Sumitomo Chemical), T. Okamoto (Wakayama pref.), K. Etoh (Wakayama pref.), Y. Ogawa (Nagasaki pref.), Y. Yoshimura (Nagasaki pref.), T. Anazawa (Yamanashi pref.), F. Arai (Yamanashi pref.), Y. Murakami (Yamanashi pref.), T. Ono (Miyagi pref.), T. Sekine (Miyagi pref.), K. Tani (Hyogo pref.), J. Yase (Hyogo pref.), M. Hareyama (Iwate pref.), S. Tokumaru (Kyoto pref.), N. Sawamura (Shimane pref.), S. Toda (NARO), S. Hoshino (Hiroshima pref.), M. Shibao (Osaka pref.), K. Shirotsuka (Osaka pref.), S. Higuchi (Kumamoto pref.), S. Kitabayashi (Nagano pref.), N. Yamagishi (Nagano pref.), S. Ito (Yamagata pref.), K. Nagai (JPPA), A. Matsuura (Miyazaki pref.), K. Takehara (Miyazaki pref.), A. Iwasaki (Hokkaido pref.), Y. Takezawa (Hokkaido pref.), T. Sasaki (Hokkaido pref.), and Z. Nakai (Chiba pref.) for insect collection.

Declaration of interest

none

References

• 1) L. A. Mound: Biological diversity. In “Thrips as crop pests,” ed. by T. Lewis, CAB International, Wallingford, pp. 197–215, 1997.
• 2) I. Zawirska: Untersuchungen uber zwei biologische Typen von Thrips tabaci Lind. (Thysanoptera, Thripidae) in der VR Polen. Arch. Phytopathol. Pflanzenschutz 12, 411–422 (1976).
• 3) M. Doi, S. Zen, M. Okuda, H. Nakamura, K. Kato and K. Hanada: Leaf necrosis disease of lisianthus (Eustoma grandiflorum) caused by Iris yellow spot virus. Ann. Phytopathol. Soc. Jpn. 69, 181–188 (2003) (in Japanese with English summary).
• 4) G. Moritz: Structure, growth and development. In “Thrips as crop pests,” ed. by T. Lewis, CAB International, Wallingford, pp. 15–64, 1997.
• 5) B. A. Nault, A. M. Shelton, J. L. Gangloff-Kaufmann, M. E. Clark, J. L. Wereen, J. C. Cabrera-La Rosa and G. G. Kennedy: Reproductive modes in onion thrips (Thysanoptera: Thripidae) populations from New York onion fields. Environ. Entomol. 35, 1264–1271 (2006).
• 6) M. Aizawa, T. Watanabe, A. Kumano, T. Miyatake and S. Sonoda: Cypermethrin resistance and reproductive types in onion thrips, Thrips tabaci (Thysanoptera: Thripidae). J. Pestic. Sci. 41, 167–170 (2016).
• 7) M. Aizawa, T. Watanabe, A. Kumano, K. Tamagaki and S. Sonoda: Biotic performances of thelytokous and arrhenotokous strains of Thrips tabaci (Thysanoptera: Thripidae) showing resistance to cypermethrin. Appl. Entomol. Zool. 53, 11–17 (2018).
• 8) S. Toda and M. Morishita: Identification of three point mutations on the sodium channel gene in pyrethroid-resistant Thrips tabaci (Thysanoptera: Thripidae). J. Econ. Entomol. 102, 2296–2300 (2009).
• 9) M. Wu, H. Gotoh, T. Waters, D. B. Walsh and L. C. Lavine: Identification of an alternative knockdown resistance (kdr)-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene. Pest Manag. Sci. 70, 977–981 (2014).
• 10) A. Jouraku, S. Kuwazaki, H. Iida, I. Ohta, H. Kusano, M. Takagi, T. Yokoyama, N. Kubota, M. Shibao, K. Shirotsuka, A. Iwasaki, Y. Takezawa and M. Takeda: T929I and K1774N mutation pair and M918L single mutation identified in the voltage-gated sodium channel gene of pyrethroid-resistant Thrips tabaci (Thysanoptera: Thripidae) in Japan. Pestic. Biochem. Physiol. 158, 77–87 (2019).
• 11) A. Jouraku, Y. Tomizawa, K. Watanabe, K. Yamada, S. Kuwazaki, M. Aizawa, S. Toda and S. Sonoda: Evolutionary origin and distribution of amino acid mutations associated with resistance to sodium channel modulators in onion thrips, Thrips tabaci. Sci. Rep. 14, 3792 (2024).
• 12) K. D. Király, M. Ladányi and J. Fail: Reproductive isolation in the cryptic species complex of a key pest: Analysis of mating and rejection behaviour of onion thrips (Thrips tabaci Lindeman). Biology (Basel) 11, 396 (2022).
• 13) X.-W. Li, P. Wang, J. Fail and A. M. Shelton: Detection of gene flow from sexual to asexual lineages in Thrips tabaci (Thysanoptera: Thripidae). PLoS One 10, e0138353 (2015).
• 14) A. L. Jacobson, B. A. Nault, E. L. Vargo and G. G. Kennedy: Restricted gene flow among lineages of Thrips tabaci supports genetic divergence among cryptic species groups. PLoS One 11, e0163882 (2016).
• 15) D.-X. Zhang and G. M. Hewitt: Nuclear integrations: challenges for mitochondrial DNA markers. Trends Ecol. Evol. 11, 247–251 (1996).
• 16) R. Takeuchi and S. Toda: Discrimination of two reproductive forms of Thrips tabaci by PCR-RFLP, and distribution of arrhenotokous T. tabaci in Tottori Prefecture. Jpn. J. Appl. Entomol. Zool. 55, 254–257 (2011) (in Japanese with English summary).
• 17) K. Sogo, K. Miura, M. Aizawa, T. Watanabe and R. Stouthamer: Genetic structure in relation to reproductive mode in Thrips tabaci (Insects: Thysanoptera). Appl. Entomol. Zool. 50, 73–77 (2015).
• 18) M. Takeda: Current status of Thrips tabaci about its host plants, reproductive forms, insecticide resistance and virus transmission by the questionnaire survey in Japan. Plant Prot. 68, 248–254 (2014) (in Japanese).
• 19) K. Sogo, T. Watanabe, M. Ito, K. Takechi and K. Miura: Distribution and insecticide sensitivity of reproductive types of onion thrips (Thrips tabaci Lindeman) living in Shikoku. Plant Prot. 67, 666–671 (2013) (in Japanese).
• 20) N. Sasayama, H. Taguchi, C. Tanaka and M. Nishino: Emergence of arrhenotoky and sensitivity to pesticides in onion thrips (Thrips tabaci Lindeman) in Mie Prefecture. Kansai Byochugai Kenkyukaiho 62, 161–163 (2020) (in Japanese with English summary).
• 21) N. Haruyama and K. Matsumoto: Effect of insecticide on six species of thrips occurring on horticultural crops in Tochigi prefecture. Annu. Rept. Kanto-Tosan Pl. Prot. Soc. 60, 99–101 (2013) (in Japanese).
• 22) M. Doi, Y. Tsuchida, Y. Katai and A. Tatara: Toxicity of insecticides to onion thrips Thrips tabaci Lindeman on allium in western region Shizuoka prefecture. Kansai Byochugai Kenkyukaiho 56, 111–113 (2014) (in Japanese).
• 23) M. D. S. Nunes, M. Dolezal and C. Schlötterer: Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster. Mol. Ecol. 22, 2106–2117 (2013).
• 24) E. Rodríguez-Pena, P. Verísimo, L. Fernández, A. González-Tizón, C. Bárcena and A. Martínez-Lage: High incidence of heteroplasmy in the mtDNA of a natural population of the spider crab Maja brachydactyla. PLoS One 15, e0230243 (2020).
• 25) S. J. Gawande, S. Anandhan, A. A. Ingle, A. Jacobson and R. Asokan: Heteroplasmy due to coexistence of mtCOI haplotypes from different lineages of the Thrips tabaci cryptic species group. Bull. Entomol. Res. 107, 534–542 (2017).
• 26) K. Imai, S. Onogi and T. Tomioka: Thrips tabaci Lindeman. In “Pest thrips in Japan,” ed. by K. Umeya, I. Kudo and M. Miyazaki, Zenkoku Noson Kyoiku Kyokai, pp. 283–292, 1988 (in Japanese).
• 27) K. Sakimura: The life and seasonal histories of Thrips tabaci Lind. in the vicinity of Tokyo Japan. Oyo Dobutsugaku Zasshi 9, 1–24 (1937) (in Japanese).
• 28) M. Morishita and H. Oue: Damage of persimmon fruit by onion thrips, Thrips tabaci (Lindeman) (Thysanoptera: Thripidae), and its chemical control. Kansai Byochugai Kenkyukaiho 43, 43–44 (2001) (in Japanese).
• 29) M. Tsuchiya: Occurrence of blemished fruits caused by Thrips tabaci on greenhouse Satsuma mandarins in Shizuoka Prefecture. Annu. Rep. Kanto-Tosan Pl. Prot. Soc. 48, 153–155 (2001) (in Japanese with English summary).
• 30) K. Fujikawa and T. Muta: Occurrence and chemical susceptibility of onion thrips (Thrips tabaci Lindeman) in greenhouses of Satsuma mandarin in Kagoshima Prefecture. Plant Prot. 57, 61–64 (2003) (in Japanese).
• 31) E. Matsumoto: Occurrence and management of Thrips tabaci on green asparagus. Japan Agric. Tech. 48, 34–38 (2004) (in Japanese).
• 32) H. Oida and T. Mihira: Damage from Thrips tabaci on florist crops and its management. Noko To Engei 56, 178–181 (2001) (in Japanese).
• 33) T. Murai: Current status of the onion thrips, Thrips tabaci, as a pest thrips in Japan. Agrochemicals Japan 84, 7–10 (2004) (in Japanese).
• 34) Y. Takezawa: Monitoring synthetic-pyrethroid insecticide resistant Thrips tabaci using genetic diagnosis in Sorachi and Kamikawa districts of Hokkaido. Annu. Rept. Plant Prot. North. Jpn. 63, 184–188 (2012) (in Japanese).
• 35) T. Murai: Parthenogenetic reproduction in Thrips tabaci and Frankliniella intonsa (Insecta: Thysanoptera). In “Advances in invertebrate reproduction 5,” ed. by M Hoshi and O. Yamashita, Elsevier Science, Amsterdam, pp. 357–362, 1990.
• 36) S. Toda and T. Murai: Phylogenetic analysis based on mitochondrial COI gene sequences in Thrips tabaci Lindeman (Thysanptera: Thripidae) in relation to reproductive forms and geographic distribution. Appl. Entomol. Zool. 42, 309–316 (2007).