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 > 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.

We collected 16 field populations of T. palmi 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 (Table 1). 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 m², 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) (Table S1). 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 (Table 2). Susceptibility of ten of these populations to spinetoram had previously been tested in Gao et al. (2019) and Shi et al. (2020) (Table 1 and 2). 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 (TPα6) is one mechanism conferring spinetoram resistance in T. palmi ²². We sequenced a fragment of TPα6 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 TPα6 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 ²⁹ and mRNA sequences (Accession: AB905366.1 and AB905365.1) ²² of TPα6. 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 ddH₂O. 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).

Sequences of individuals from the same population were put in one sqd file. These files can be opened by using softwares such as SeqMan.

Table 1 Information on the 16 field populations of Thrips palmi used in the study

^a, populations used in Gao et al., 2019; ^b, populations used in Shi et al., 2020.