Applied Entomology and Zoology https://doi.org/10.1007/s13355-019-00616-2 ORIGINAL RESEARCH PAPER Tebufenozide resistance in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae): establishment of a molecular diagnostic method based on EcR mutation and its application for feld‑monitoring Miwa Uchibori‑Asano1 · Toru Uchiyama2 · Akiya Jouraku1 · Akihito Ozawa2 · Gaku Akiduki3 · Kohji Yamamura4 · Tetsuro Shinoda1 Received: 27 August 2018 / Accepted: 7 March 2019 © The Author(s) 2019 Abstract The smaller tea tortrix, Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae), is one of the main insect pests of tea, Camellia sinensis Kuntz, in Japan. Recently, A. honmai has developed a high resistance to diacylhydrazine analog insect growth regulators, such as tebufenozide, in Shizuoka Prefecture. Previously, we identifed a point mutation (A415V) in the ecdysone receptor gene (EcR), a candidate factor responsible for tebufenozide resistance. In this study, we have developed a molecular method of diagnosis to detect the EcR A415V mutation by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). This method was confrmed to be successfully applicable to larvae reared in the laboratory and adults collected by pheromone traps in the feld. The appearance ratio of the resistant allele in the A. honmai populations from various Japanese districts examined by the method revealed a high correlation with the magnitude of tebufenozide resistance. These results verifed that the A415V mutation is the principal factor responsible for tebufenozide resistance and the PCR–RFLP method may be used as a reliable and convenient tool for monitoring tebufenozide resistance in the feld. Keywords Adoxophyes honmai · Insecticide resistance · Tebufenozide · Ecdysone receptor · Molecular diagnosis Introduction The smaller tea tortrix, Adoxophyes honmai Yasuda (Lepi- doptera: Tortricidae), is a polyphagous insect pest infest- Electronic supplementary material The online version of this ing many agricultural crops, including tea, citrus, rose, and article (https ://doi.org/10.1007/s1335 5-019-00616 -2) contains grape [9]. Adoxophyes honmai has 4–5 generations per year supplementary material, which is available to authorized users. in the tea felds of Shizuoka Prefecture, which produces the * Tetsuro Shinoda largest quantity of tea in Japan [20]. Larvae prefer feeding [email protected] on leaf buds and young leaves to old leaves [12]. Therefore, 1 outbreaks of this pest cause severe damage to tea leaves, Institute of Agrobiological Sciences, National Agriculture developmental delay of tea shoots, reductions in tea yield, and Food Research Organization (NARO), Tsukuba, Ibaraki 305-8634, Japan and decreased quality in tea manufacturing. 2 Various insecticides have been used for the control of A. Tea Research Center, Shizuoka Research Institute of Agriculture and Forestry, Kurasawa, Kikugawa, honmai in Shizuoka Prefecture to date. However, A. hon- Shizuoka 439-0002, Japan mai has already developed resistance to many insecticides, 3 Kyushu Okinawa Agricultural Research Center, National including carbamates [13], organophosphates, synthetic Agriculture and Food Research Organization (NARO), pyrethroids, and benzoylurea analog insect growth regula- Kumamoto 861-1192, Japan tors (IGRs) [7]. In addition, the development of high levels 4 Institute for Agro-Environmental Sciences, National of resistance to diacylhydrazine (DAH) analog IGRs and Agriculture and Food Research Organization (NARO), diamide insecticides has been recently reported [19, 20]. Tsukuba, Ibaraki 305-8604, Japan Vol.:(0123456789)1 3 Applied Entomology and Zoology DAH analog IGRs, including tebufenozide, chromafe- Here, we have developed a molecular diagnostic method nozide, and methoxyfenozide are nonsteroidal ecdysteroid targeting the A415V mutation in EcR. Using this method, agonists. They exhibit insecticidal activity by binding to we elucidated the relationship between the A415V mutation the target protein, ecdysone receptor, and promoting aber- and the magnitude of tebufenozide resistance in various A. rant ecdysis in the larvae of lepidopteran insect pests [11, honmai feld populations. Accordingly, we revealed that the 24, 25]. Tebufenozide has been used as a popular insecti- A415V mutation is indeed a principal factor in tebufenozide cide for the control of major lepidopteran pests, A. honmai resistance and this method is useful for feld-monitoring of and the oriental tea tortrix Homona magnanima Diakonof tebufenozide-resistance in A. honmai. (Lepidoptera: Tortricidae), in the tea felds of Shizuoka Prefecture since 1993 [7]. However, A. honmai quickly developed resistance to tebufenozide, where the resistant Materials and methods ratio of a population collected in the Makinohara region of Shizuoka Prefecture in 2004 was 57.7-fold compared to Insects a susceptible strain, and reached 1,528-fold in 2008 [19]. In addition to A. honmai, the development of tebufe- Larval or adult A. honmai were collected in the tea felds nozide resistance has been reported in feld populations of Shizuoka, Mie, Kyoto, Saitama, Ibaraki, Kanagawa, of the apple pandemis leafroller moth Pandemis pyrusana Kagoshima, and Fukuoka Prefectures, Japan (Table 1). Egg Kearfott (Lepidoptera: Tortricidae) [3], the codling moth masses were obtained from more than 20 females for each Cydia pomonella L. (Lepidoptera: Tortricidae) [5], and the regional population and maintained on an artifcial diet oblique-banded leafroller moth Choristoneura rosaceana (Insecta LFS or Silk Mate 2S, Nihon-Nosan Kogyo, Yoko- Harris (Lepidoptera: Tortricidae) [3, 15], and in labora- hama, Japan) at 25 °C under a 16:8 h light:dark photoperiod tory-selected strains of the diamondback moth Plutella [20]. F2 or F3 generation larvae were used for bioassays for xylostella L. (Lepidoptera: Plutellidae) [2], the beet army- insecticide sensitivity and molecular diagnoses unless oth- worm Spodoptera exigua Hübner (Lepidoptera: Noctui- erwise mentioned. dae) [6], and the greenheaded leafroller Planotortrix octo Male adults were collected using pheromone traps (SE Dugdale (Lepidoptera: Tortridae) [10, 23]. However, the trap, Sankei chemical, Kagoshima, Japan) equipped with mechanisms of tebufenozide resistance remain largely pressure-sensitive adhesive sheets and synthetic sex phero- unknown in these species. mones of A. honmai (Shin-Etsu Chemical, Tokyo, Japan or Genetic studies have revealed that tebufenozide resist- Sumitomo Chemical, Tokyo, Japan). The traps were set on ance in A. honmai is incompletely dominant and is associ- plucking surface of tea plants using agricultural poles in the ated with multiple autosomal genes [21]. More recently, feld for 1–7 days. by next-generation sequencing analyses, we have identifed two types of candidate genes that are putatively responsi- Extraction of genomic DNA ble for the tebufenozide resistance: (1) ecdysone receptor gene (EcR) that encodes the target molecule of tebufe- For molecular diagnostics, abdomens of adults or larvae nozide, and (2) cytochrome P450 monooxygenase genes (4th–5th instar) were excised, individually placed into 0.2 ml (CYP9As) that encode metabolic enzymes [18]. In resistant PCR tubes containing 100 µl of an alkaline solution (50 mM strains, a single nucleotide polymorphism (SNP) causing NaOH, 0.2 mM EDTA), lightly crushed with a toothpick, an amino acid substitution of alanine (A) at position 415 to and heated at 95 °C for 10 min. Supernatants (30 µl) were valine (V) was found in the ligand binding domain of EcR. transferred to clean 0.2 ml PCR tubes containing 50 µl of These amino acids adjoin an amino acid residue critical 0.2 M Tris–HCl (pH 8.0), and mixed by pipetting. Extracts for DAH binding. Moreover, biochemical analyses using were diluted 100 times with distilled water (DW) and used a recombinant EcR protein showed that the A415V muta- as templates for PCR. tion resulted in a 5-fold decrease in the binding afnity for tebufenozide. Meanwhile, the expression level of CYP9A PCR‑restriction fragment length polymorphism genes were high in resistant strains and moderately corre- (PCR–RFLP) lated with the magnitude of tebufenozide-resistance. Thus, both the desensitization of target protein (EcR) and the Previously, we have identifed a candidate SNP marker enhancement of detoxifcation activity by overexpressed for tebufenozide-resistance in A. honmai [18]. This SNP CYP9As are relevant to the resistant mechanism [18]. locates on exon 6 of the genomic DNA of EcR (Fig. 1a), However, it remains unclear to what extent the EcR muta- which corresponds to C1244T in the EcR B1 isoform tion contributes to tebufenozide resistance in A. honmai cDNA (Accession Nos. LC209228 for resistant type in the feld. and LC209229 for susceptible type) and causes A415V 1 3 Applied Entomology and Zoology Table 1 Collection data for Adoxophyes honmai in Japan and developmental stages used for PCR-restriction fragment length polymorphism analyses Collection Collection site (town, city) Prefecture Latitude/longitude Collection year Collection stage for PCR–RFLPa number 1 Yui, Shimada Shizuoka N34.81, E138.19 2015 Larvae (30) 2 Kasaume, Iwata Shizuoka N34.78, E137.86 2015 Larvae (28) 3 Kamiuchida, Kakegawa Shizuoka N34.76, E138.04 2015 Larvae (30) 4 Higashihagima, Makinohara Shizuoka N34.77, E138.14 2016 Larvae (30) and adults (513) 5 Kurasawa, Kikugawa Shizuoka N34.78, E138.14 2015 Larvae (30) 6 Nunohikihara, Makinohara Shizuoka N34.77, E138.15 2016 Larvae (30) and adults (288) 7 Jina,