Baseline Susceptibility and Laboratory Selection of Resistance to Bt Cry1Ab Protein of Chinese Populations of Yellow Peach Moth, Conogethes punctiferalis (Guenée)

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.


Introduction
Bacillus thuringiensis (Berliner), a ubiquitous Gram-positive and endospore-forming soil bacterium, produce insecticidal crystal proteins during the sporulation phase of its growth cycle [1,2]. It has been using as an alternative to chemical insecticides to control the critical agricultural pests in the orders Lepidoptera, Coleoptera, and Diptera [3][4][5].
Since 1996, transgenic maize expressing Bt insecticidal proteins has been commercialized and used successfully to manage corn borer problems. It is widely grown in the key maize growing regions such as the USA, Brazil, Argentina, Canada, South Africa, and the Philippines and the estimated acreage reached 58.9 million hectares in 2018 [6].

Baseline Susceptibility of YPM to Bt Cry1Ab Protein
Fourteen YPM larvae populations were collected in the non-Bt maize fields from China's different geographical areas in 2019 and 2020 (Table 1). The largest populations were collected in the Shandong, Henan, Hebei, and Anhui provinces from the Huang-Huai-Hai summer maize production region which is the China's second largest maizegrowing region (Figure 1). The susceptibility of YPM larvae to Cry1Ab for fourteen field and lab populations was presented in Table 2. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC 50 values ranged from 0.35 to 2.38 ng/cm 2 for the populations over the two years of the collection, while the LC 99 values ranged between 40.13 and 409.65 ng/cm 2 . The population from Luohe had the lowest LC 50 value (0.35 ng/cm 2 ); it was not significantly different from Luoyang (0.55 ng/cm 2 ) and Jiyuan population (0.65 ng/cm 2 ), all from Henan province. Tangshan population produced the highest LC 50 value (2.38 ng/cm 2 ), followed by Handan (2.19 ng/cm 2 ), Suzhou (2.14 ng/cm 2 ), and Linyi (1.72 ng/cm 2 ) populations.

Baseline Susceptibility of YPM to Bt Cry1Ab Protein
Fourteen YPM larvae populations were collected in the non-Bt maize fields from China's different geographical areas in 2019 and 2020 (Table 1). The largest populations were collected in the Shandong, Henan, Hebei, and Anhui provinces from the Huang Huai-Hai summer maize production region which is the China's second largest maize growing region (Figure 1). The susceptibility of YPM larvae to Cry1Ab for fourteen field and lab populations was presented in Table 2. Diet-overlay bioassay results showed tha all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC5 values ranged from 0.35 to 2.38 ng/cm 2 for the populations over the two years of the col lection, while the LC99 values ranged between 40.13 and 409.65 ng/cm 2 . The population from Luohe had the lowest LC50 value (0.35 ng/cm 2 ); it was not significantly different from Luoyang (0.55 ng/cm 2 ) and Jiyuan population (0.65 ng/cm 2 ), all from Henan province Tangshan population produced the highest LC50 value (2.38 ng/cm 2 ), followed by Handan (2.19 ng/cm 2 ), Suzhou (2.14 ng/cm 2 ), and Linyi (1.72 ng/cm 2 ) populations.  Table 1.   Table 1.

YPM Larval Growth Inhibition to Bt Cry1Ab Protein
For the larval growth inhibition, YPM larvae showed high susceptibility to Cry1Ab in all tested populations ( Table 3). The larval growth inhibition recorded >50% in the Cry1Ab lowest concentration (0.004 ng/cm 2 ) and above 99% larval growth inhibition was observed in the concentration of 13.68 ng/cm 2 and 68.42 ng/cm 2 , respectively.

Development of Diagnostic Concentration
After pooling the six populations collected in 2019 in the F2 generation, the LC 99 for Cry1Ab was 43.33 ng/cm 2 , with a lower and upper limit 24.28 ng/cm 2 and 92.73 ng/cm 2 , respectively. The upper limit of 92.73 ng/cm 2 exhibited larval mortality >99% for all eight populations collected in 2020 (Table 4). However, both LC 99 (24.28 ng/cm 2 ) and the lower limit of 43.33 ng/cm 2 produced larval mortality <99% for eight populations. Based on this, the upper limit of the LC 99 estimates could be potentially suitable as the diagnostic concentration for future monitoring work of Cry1Ab resistance in the YPM population.

Laboratory Selection
Large-scale diet-overlay bioassays similar to those described above were conducted for the selection experiments. As mentioned in the methodology, the laboratory strain was steadily selected with increasing doses of Cry1Ab from 1.08 to 6.16 ng/cm 2 for the 14th generation (Table 5). Resistance ratio increased 2.26-fold in 3rd generation, 3.30-fold in 6th generation, 4.76-fold in 9th generation, 5.42-fold in 11th generation, 6.08-fold in 13th generation, and after the 14th generation of selection, the resistance ratio increased by 7.10-fold, respectively.

Discussion
Before the broad commercial deployment of Bt maize, the establishment of baseline susceptibility of target pests to Bt protein is the first step for generating an effective resistance monitoring and management program [22]. In the present study, we conducted the diet overlay bioassays to verify the baseline susceptibility of YPM to Cry1Ab from fourteen diverse geographical populations of China. In general, Cry1Ab proteins have observed high toxicity against the species of genus Ostrinia [25,[41][42][43]. A previous report documents that YPM is highly susceptible to Cry1Ab proteins than other tested proteins [44]. Zhang et al., 2010 [45] concluded that high genetic similarity among 11 populations but Shandong populations showed lower genetic diversity than others and biggest genetic distance was identified between Sichuan and Shandong populations. Here the LC 50 of Shandong populations were in general higher than those from inner land provinces (although not significant).
The natural variation in baseline susceptibility associate with various factors such as population vigor, the source of the Bt proteins, bioassay method, environmental condition, time of exposure, and artificial diet [46][47][48][49]. Several studies have shown minor to moderate variation in the Crambidae family to Cry1Ab among various countries. The Cry1Ab susceptibility varied less than 10-fold among ten different O. furnacalis populations across China [25]. Alcantara [50]. However, the ranges of LC 50 among populations in response to Cry1Ac protein were 16-fold for Helicoverpa zea in the USA [51]. H. armigera population from China displayed a 100-fold high level of natural variability in response to Cry1Ac protein [52]. In this study, the LC 50 values among fourteen different populations ranged from 0.35 to 2.38 ng/cm 2 with 7-fold variations, while the LC 99 values ranged between 40.13 and 409.65 ng/cm 2 with 10-fold variations. These findings collectively indicated that the Crambidae family exhibits relatively little variability in response to the Cry1A protein compare with those of the Noctuidae.
The high sensitivity growth inhibition may be considered as an additional indicator for diagnostic concentration response in future monitoring work [53]. In our study, severe larval growth inhibition of YPM larvae byCry1Ab was detected in the different geographical populations ( Table 3). The results showed that >50% larval growth inhibition in the Cry1Ab lowest concentration (0.004 ng/cm 2 ) and >99% larval growth inhibition in the concentration of 13.68 ng/cm 2 and 68.42 ng/cm 2 , respectively. Accordingly, when increasing the Cry1Ab concentration, YPM larval weight also reduced after one week of treatment. Marçon et al. (1999) documented that high larval stunting was showed in the low concentration of Cry1Ab is common among tested species, including Ostrinia spp. Furthermore, in Vietnam, the Ostrinia population exhibited >90% larval stunting in a concentration of above 0.82 ng/cm 2 [42]. In addition, high larval growth inhibition was observed to Bt Cry1A protein in the noctuid in different countries [52,[54][55][56][57].
To monitor insect resistance's evolution to Bt proteins, the diagnostic dose's development and estimation are necessary steps in the insect resistance monitoring program. In some countries, the LC 99 values have been applied to develop a diagnostic dose for monitoring the evolution of insect resistance to Cry1 proteins [23,53,58]. In this study, we collected six populations in 2019 to designate the diagnostic concentration of YPM to Cry1Ab. The LC 99 estimates for Cry1Ab were 43.33 ng/cm 2 , with a lower and upper limit of 24.28 ng/cm 2 and 92.73 ng/cm 2 , respectively. The upper limit of the LC 99 (92.73 ng/cm 2 ) was selected as the candidate diagnostic concentration because of exhibited consistent larval mortality above 99% when treated on eight representative YPM populations in China (Table 4). A study from 2020, the LC 99 estimate of Cry1Ab was 93 ng/cm 2 , killed >99% of O. furnacalis populations, and was identified as the diagnostic dose for monitoring susceptibility of O. furnacalis in China [59]. These observations suggested that the development of effective diagnostic concentration could be successfully applied to control both YPM and O. furnacalis in future monitoring work.
The establishment of Bt resistance to target pests is threatening to the continuous success of Bt crops. Several studies found O. furnacalis population has been evolved resistant to Bt Cry1 protein after several generations in the laboratory selection [60][61][62]. Besides, both a laboratory and field study observed that some noctuid could evolve resistance to Cry1Ab [63,64]. We attempted the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Resistance ratio increased 2.26-fold in 3rd generation, 3.30-fold in 6th generation, 4.76-fold in 9th generation, 5.42-fold in 11th generation, 6.08-fold in 13th generation, and after the 14th generation of selection, the resistance ratio increased by 7.10-fold, respectively (Table 5). Although the resistant proportion was generally low, YPM potentially could evolve resistance to Cry1Ab. The possible evolution of resistance to target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.

Conclusions
We report here on the baseline susceptibility of distinct geographical populations of YPM to Cry1Ab, which is crucial to establish an effective resistance monitoring program for YPM in China. Our findings collectively pointed that the Crambidae family exhibits relatively little variability in response to the Cry1A protein compare with those of the Noctuidae. Additionally, the upper limit of the LC 99 (92.73 ng/cm 2 ) was verified as the candidate diagnostic concentration. Presently, a resistant strain of YPM is being developed in the laboratory and can be potentially used for testing the efficacy of the diagnostic technique. The diagnostic concentration validation study should compare the YPM resistant population and the field-collected population to future monitoring programs.

Insects Collection
Fourteen YPM field populations were collected from the maize fields in seven provinces, including Shandong, Hebei, Henan, Shaanxi, Beijing, and Anhui provinces across the Huang-Huai-Hai summer maize region and Sichuan Province from Southeast Hilly Maize Region in China. Approximately 200-350 larvae (4th-5th instar) were collected from maize ears at each location in autumn before maize harvest. Only the Chengdu population was collected about 65 larvae because of less population in that collected area. Collection sites were chosen based upon their infestation history and were made in 2019 and 2020 (Figure 1).

Laboratory Rearing of Field-Collected YPM
Field collected larvae were reared on the freshly prepared artificial diet until pupation [65]. The pupae were transferred to mating cages for adult emergence. Inside the adult rearing cages, the apple substrate wrapped with gauze was placed for oviposition. A cotton swab soaked in 15% honey solution was provided for the adults and renewed daily. The YPM was maintained in the rearing room at 27 ± 1 • C, 60-70% relative humidity (RH), and a 16:8 h (L:D) photoperiod. Newly hatched neonate larvae (<24 h) from the F1 generation were used for diet-overlay bioassays and diagnostic concentration assays. The Jiyuan and Chengdu populations were raised to F2 and F3 generation as not enough for further experiment. YPM laboratory strain was collected in a maize field where Bt spraying is not practiced at Langfang, Hebei Province, China. The collected larvae were reared using the same method mentioned above for eighteen generations without exposure to Bt protein.

Baseline Susceptibility Bioassay
Trypsin-activated Cry1Ab protein was purchased from Envirologix (Portland, OR, USA). Cry1Ab protein's insecticidal activity against YPM larvae was assessed in concentrationresponse bioassays by applying Cry1Ab preparations on the artificial diet's surface. Seven protein concentrations ranged from 0.004 to 68.42 ng/cm 2 (protein/diet). All bioassays were performed by transferring neonate larvae (<24 h) with a fine brush to contaminated diet surfaces in a 24-well plate. The plates were sealed with a membrane (Cat# 3M-9733, Minnesota Mining, and Manufacturing Company, Saint Paul, MN, USA) perforated with a sharp pin on each cell to provide aeration. Plates were maintained in the rearing room at 27 ± 1 • C, 60-70% RH, and a 16:8 h (L:D) photoperiod. Three replicates of 24 larvae per concentration (n = 72) were applied for each Bt protein dose, and double-distilled water (no Bt) was applied for the control. The larval mortality and larval weight were scored one week after treatment. The larvae that failed to move when prodded with a fine pin were considered dead.

Establishment of Diagnostic Concentrations of Bt Cry1Ab Protein
Diagnostic concentrations were established with the pooled population from six locations during 2019 field collections. A pooled analysis using the logistic option in the PROBIT procedure was performed across populations to estimate LC 99 and its lower and upper limit, which was considered the diagnostic concentration for future resistance monitoring purposes. To evaluate the LC 99 , lower limit, and the upper limit for use as the diagnostic concentration, eight YPM populations were collected in Shandong, Hebei, Henan, Beijing, Anhui, and Sichuan provinces in 2020. About 240 neonate larvae from each population were tested with each of the three candidates' diagnostic concentrations of Cry1Ab using the diet overlay bioassay described above.

Laboratory Selection
Laboratory strains of YPM originated from the maize field at Langfang experimental station of Chinese Academy of Agricultural Sciences, Hebei Province, China, and reared on the artificial diet in the laboratory for eighteen generations without exposure to Bt proteins. Diet overlay bioassay was conducted to evaluate this strain's susceptibility to Cry1Ab before conducting a selection experiment, and the LC 50 estimate was 1.08 ng/cm 2 (toxin/diet). The population was initially treated with LC 50 value of Cry1Ab for ten days and transferred to an untreated artificial diet to complete its life cycle. It was selected steadily with increasing the concentrations of Cry1Ab (2.05 ng/cm 2 ) for 6th generation, (3.12 ng/cm 2 ) for 9th generation, and (4.32 ng/cm 2 ) for 11th generation and then maintained at (6.16 ng/cm 2 ) for 14th generation, respectively. The resistance ratio was calculated LC 50 value of the Cry1Ab resistance strain divided by LC 50 value of the Cry1Ab susceptible strain.

Statistical Analysis
Fifty percent lethal concentration (LC 50 ) with 95% fiducial limits (FL) and LC 99 were determined using the PoloPlus (v 1.0, LeOra Software, Parma, MO, USA) for Cry1Ab protein and the slope for all bioassays by probit analysis. The difference between LC 50 values and LC 99 values in diet-overlay bioassays was considered significantly different if their 95% fiducial limits did not overlap. The mortality percentage for each concentration was analyzed by one-way ANOVA, followed by post hoc Tukey HSD test. Statistical data analysis was based on the software package SPSS v.20 (IBM Corporation, Armonk, NY, USA). The percentage of larval growth inhibition was calculated using the formula below based on [40]: