DOI: 10.5061/dryad.s1rn8pk5r
title: Association between susceptibility of Thrips palmi to spinetoram and frequency of G275E mutation provides basis for molecular diagnostics of field-evolved resistance

description: Background: Spinosyn resistance is an increasing problem in field control
 of targeted pests. While putative mechanisms underlying spinosyn
 resistance have been identified in controlled studies on many species,
 mechanisms underlying field-evolved resistance and the development of a
 molecular diagnostic method for monitoring field resistance have lagged
 behind. Here, we examined levels of resistance of melon thrips, Thrips
 palmi, to spinetoram as well as target site mutations in field populations
 across China to identify potential mechanisms and useful molecular markers
 for diagnostic purposes. Results: LC50 of 16 field-collected populations
 to the spinetoram varied from 0.12 to 759.34 mg L-1. In resistant
 populations, we identified the G275E mutation, which has previously been
 linked to spinosyns resistance, as well as another nonsynonymous mutation,
 F314V, both located in the α6 subunit of the nicotinic acetylcholine
 receptor. There was a strong correlation between levels of spinetoram
 resistance and allele frequency of G275E mutation in field-collected
 populations (r2 = 0.84) and those reared under laboratory conditions for
 two to five generations (r2 = 0.91). LC50 ranged from 0.12 to 0.66 mg L-1
 in populations without G275E mutation, while it ranged from 33.12 to 39.91
 mg L-1 in most populations with a G275E mutation frequency &gt; 90%,
 with the exception of one field-collected population which had an G275E
 frequency of 92% and a very high LC50 value of 759.34 mg L-1, suggesting
 additional mechanisms. Conclusions: Our results indicate that
 field-evolved resistance of T. palmi to spinetoram in China is mainly
 conferred by the G275E mutation, with other mechanisms contributing to a
 higher level of resistance. The frequency of the G275E mutation provides a
 useful diagnostic for quantifying resistance levels in field populations
 of T. palmi.
description: We collected 16 field populations
 of <i>T. palmi</i> across three areas of China, including one
 population from Hainan province in southern China, three populations from
 Beijing and 12 populations from Shouguang and Qingzhou of Shandong
 province, both in northern China (<b>Table 1</b>). These
 populations are referred to as the F0 generation to designate their status
 as being collected and tested directly from the field. Each population was
 collected from a greenhouse or a field with an area about 600
 m<sup>2</sup>, which had been exposed to the same pest control
 practices. Samples representing each population were obtained from at
 least 24 points scattered within the collection area and then taken to
 laboratory in sealed bags. This sampling method reduces the likelihood of
 the collected thrips being close relatives. We used a
 common garden method to examine the potential influence of field
 environments on levels of resistance by comparing changes in resistance
 between field- and laboratory-reared thrips. Nine of the 16
 field-collected populations were reared in the laboratory for two to five
 generations without exposure to any pesticides. For each population, about
 2000 individuals were maintained from generation to generation. These
 populations were labeled with Fx (x representing the number of generations
 of laboratory culture) (<b>Table S1</b>). We also selected one
 population (SGD1-F4s) for increased resistance by exposing thrips to a 70%
 lethal concentration of spinetoram of SGD1-F0 field
 population for four generations. All populations were reared on seedlings
 of cucumber with 3-5 leaves under 25 °C, 40 - 60%
 relative humidity and a photoperiod of
 16L:8D. Twelve field
 populations and six laboratory reared populations were used for both
 bioassays and mutation detection (<b>Table 2</b>).
 Susceptibility of ten of these populations to spinetoram had previously
 been tested in Gao et al. (2019) and Shi et al. (2020) (<b>Table 1
 and 2</b>). Due to haplodiploidy, only diploid females provided
 information on heterozygosity. We used female adults for bioassays (about
 420 individuals) and molecular assays (about 24
 individuals).         The G275E mutation on the
 nAChR α6 subunit gene (<i>TPα6</i>) is one mechanism
 conferring spinetoram resistance in <i>T. palmi</i>
 <sup>22</sup>. We sequenced a fragment of <i>TPα6 </i>from individual thrips to examined the frequency of the mutation within population. Genomic DNA was exacted from individual adults using the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to the manufacturer instructions. As the previous degenerate primer pairs for detecting G275E were designed based on mRNA and are not suitable for DNA amplification, we designed a new species-specific primer pair for amplification of the <i>TPα6</i> region (250bp) including the transmembrane domain 3 (TM3) from genomic DNA (nAChR-α6-F, 5’-CGTAAGCCTTGACTTTTTCT-3’; nAChR-α6-R, 5’-CTGTAGAGACAATTTGTTTGG-3’). Primers were designed using Primer3 web version 4.1.0 (http://primer3.ut.ee/) based on the DNA sequence <sup>29</sup> and mRNA sequences (Accession: AB905366.1 and AB905365.1) <sup>22</sup> of <i>TPα6</i>. These primers allowed for detection of the mutation in each individual and estimation of the frequency of the mutation within a population. The polymerase chain reaction (PCR) volume was 15 µL, which contained 7.5 µL 2x Master Mix (Promega), 0.6 µL the primer pairs, 1.5 µL template DNA and 5.4 µL ddH<sub>2</sub>O. PCR conditions were as follow: initial denaturation for 3 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 52 °C and 30s at 72 °C, and a subsequent final extension for 10 min at 72 °C. Amplified products were purified and sequenced directly with the nAChR-α6-F primer using an ABI 3730xl DNA Analyzer by Tsingke Biotechnology Co. Ltd (Beijing, China).
description: Sequences of individuals from the same population were put in one
 sqd file. These files can be opened by using softwares such as
 SeqMan. <b>Table 1
 </b>Information on the 16 field populations of <i>Thrips
 palmi</i> used in the study Population 
 Collection
 date (Year/month/day) Host
 plant Collection
 location Latitude
 (°N), longitude (°E) HNSY-F0<sup>
 a</sup> 2018/3/8 
 Eggplant 
 Hainan province, Sanya city, Yazhou
 district, Yacheng town, Beiling vallige 109.19, 18.43 
 BJCY-F0<sup>
 a</sup> 2018/07/05 
 Eggplant 
 Beijing city, Chaoyang district,
 Natural movement farm 116.52,
 39.96 BJFS-F0<sup>
 b</sup> 2018/07/09 
 Cucumber 
 Beijing city, Fangshan district,
 Hongke farm 115.86,
 39.72 SGL1-F0<sup>
 b</sup> 2018/7/11 
 Eggplant 
 Shandong province, Shouguang city,
 Jitai town, Lvjia vallige 118.79,
 36.78 SGL2-F0<sup>
 b</sup> 2018/11/20 
 Eggplant 
 Shandong province, Shouguang city,
 Jitai town, Lvjia vallige 118.79,
 36.78 SGD1-F0<sup>
 b</sup> 2018/11/20 
 Cucumber 
 Shandong province, Shouguang city,
 Luocheng street, Dongliu vallige 118.91,
 36.89 SGF1-F0<sup>
 b</sup> 2018/11/20 
 Pepper Shandong
 province, Shouguang city, Luocheng street, Fuqiao vallige
 118.92, 36.89 
 SGZ1-F0<sup>
 b</sup> 2018/11/20 
 Pepper Shandong
 province, Shouguang city, Jitai town, Zhangjiamiaozi vallige
 118.80, 36.86 
 SGNC-F0<sup>
 b</sup> 2018/11/20 
 Pepper Shandong
 province, Shouguang city, Daotian town, Nanci vallige
 119.00, 36.83 
 SGDY-F0<sup>
 b</sup> 2018/11/20 
 Cucumber 
 Shandong province, Shouguang city,
 Daotian town, Dongyi vallige 118.96,
 36.86 SGD2-F0 2019/02/02 
 Cucumber 
 Shandong province, Shouguang city,
 Luocheng street, Dongliu vallige 118.91,
 36.89 SGF2-F0 2019/02/02 
 Pepper Shandong
 province, Shouguang city, Luocheng street, Fuqiao vallige
 118.92, 36.89 
 SGZ2-F0 2019/03/30 
 Pepper Shandong
 province, Shouguang city, Jitai town, Zhangjiamiaozi vallige
 118.80, 36.86 
 SGZ3-F0 2019/03/30 
 Eggplant 
 Shandong province, Shouguang city,
 Jitai town, Zhangjiamiaozi vallige 118.80,
 36.86 SDQZ-F0 2019/7/20 
 Eggplant 
 Shandong province, Qingzhou city,
 Tanfang town, Dongwu village 118.71,36.75 
 BJDX-F0 2019/8/10 
 Eggplant 
 Beijing city,
 Daxing district, sijiyangkun farm 116.31,39.61 
 <sup>a</sup>,
 populations used in Gao et al., 2019; <sup>b</sup>,
 populations used in Shi et al., 2020.