Establishment of Toxicity and Susceptibility Baseline of Broflanilide for Aphis gossypii Glove

Simple Summary The control of Aphis. gossypii has relied on the application of insecticides, but the resistance to insecticides has become a key factor in the successful management of A. gossypii. It is a critical measure to introduce a novel insecticide with a different action mode in the resistance management. We found that broflanilide has high insecticidal activity against A. gossypii. Broflanilide susceptibility was determined in field populations of A. gossypii from main cotton planting areas of China. Meanwhile, the susceptible baseline of the cotton aphid to broflanilide was established. These results suggested that the cotton aphid possessed high susceptibility to broflanilide. The susceptible baseline provides a comparative basis for the future resistance monitoring in the management of cotton aphids. Abstract The Aphis gossypii is an important pest that can damage cotton plants and can cause a huge economic loss worldwide. Chemical control is a main method to manage this pest, but the cotton aphid resistance to insecticides has become a severe problem in the management of the cotton aphid. It is important to introduce a novel insecticide for rotational application with other insecticides. Broflanilide, as a meta-diamide insecticide with a special mode of action, showed high efficiency against lepidopterous larvae. However, we found that broflanilide possessed high insecticidal activity against the sap-sucking pest A. gossypii. The susceptibility of A. gossypii to broflanilide from 20 field populations in main cotton planting areas of China in 2021 was determined by the leaf-dipping method. LC50 values of broflanilide to A. gossypii ranged from 0.20 μg mL−1 to 1.48 μg mL−1. The susceptible baseline of A. gossypii to broflanilide was established with the LC50 value of 0.41 μg mL−1 and might be used to calculate the resistance ratio (RR) of cotton aphid population in broflanilide resistance monitoring. The RR value of field populations in China was from 0.49 to 3.61 in 2021. It suggested that the broflanilide may be a potential agent in the resistance management of A. gossypii to insecticides. These results are significantly useful for the rational chemical control of cotton aphids.

The cotton aphid, A. gossypii Glover (Hemiptera: Aphididae), is a sap-sucking pest that threatens a wide range of crops, including pepper, tomato, eggplant, watermelon, cucumber, squash, pumpkin, citrus fruit, potato, and cotton [14]. It can damage the hosts by direct sap-feeding plant tissue nutrition and indirectly through the transmission of plant pathogens [15]. The control of cotton aphids is largely dependent on the use of insecticides. The cotton aphid has evolved high level of resistance to many insecticides, including organophosphates [10,16], carbamates [17], pyrethroids [11], and neonicotinoids [18,19], because of their continuous use for a long time.
Organophosphates and carbamates were widely used to control cotton aphids in 1960s-1980s in China, but the cotton aphid evolved 23 and 148 fold resistance to two organophosphate insecticides, parathion and demeton, in North China in 1964 [20]. The resistance of cotton aphids to omethoate increased to 60-80 times in four regions of Shandong province in 2004 compared with 22-37 times in 1985 [17]. The cotton aphid resistance to omethoate in six areas of Xinjiang reached 2137-9501 fold in 2018 [21]. In 1999, the cotton aphid evolved 18-34 times resistance to a carbamate insecticide, carbosulfan, in Shandong province [17]; however, the resistance of the cotton aphid from Xinjiang reached 148.0 times in 2018 [21]. Pyrethroid insecticides were used for the control of cotton aphids since the mid-1980s, but the resistance of the cotton aphid to pyrethroids was rapidly detected [20]. In 1985, the cotton aphid developed 3228 and 241 fold resistance to deltamethrin and fenvalerate [20]; by 2013, the field populations of the cotton aphid evolved more than 1000 times resistance to beta-cypermethrin in China [11]. In 2018, the cotton aphid developed 353-4932 times resistance to beta-cypermethrin in cotton fields of Xinjiang [21]. At present, neonicotinoid insecticides are widely applied for the control of cotton aphids; however, A. gossypii also evolved high resistance to neonicotinoids including imidacloprid, acetamiprid, and thiamethoxam, and the resistance ratio reached more than 471.2-fold in Shandong and Shanxi provinces and the field population in Xinjiang developed 1095 times resistance [21,22].
The insecticide resistance has become a critical problem for the successful management of cotton aphids. The application of the insecticides with new structural types is one of the important measures for the control of A. gossypii and resistance management in fields. The broflanilide displayed a high efficiency against 20 field populations of the cotton aphid in 2021. We established the susceptible baseline of A. gossypii to broflanilide as a reference for future resistance monitoring.

Insects
The field populations of A. gossypii were collected from 20 main cotton planting areas of China from July to September in 2021. More than 2000 apterous aphids were randomly collected according to a five-point sampling method from 20-30 cotton plants at each sample site to ensure that the samples were representative. The information and geographic distribution of the field populations collecting information are shown in Figure 1 and Table 1. Neonicotinoids (imidacloprid, acetamiprid, and thiamethoxam), pyrethroids (beta-cypermethrin and deltamethrin), and a sulfoximine insecticide (sulfoxaflor) have been used in these regions for the control of cotton aphid. These cotton aphid populations were transferred to the laboratory and reared on cotton seedlings (Gossypium hirsutum L. var. Xinmian No. 1) without pesticide exposure. All field populations were reared in insectaria under the controlled conditions with 22 ± 1 • C, 60-70% of relative humidity, and 16:8 h (L:D) of photoperiod. The aphid populations were raised for at least 3 generations in the insectaria and used for subsequent experiments.

Toxicity bioassays
The toxicity of broflanilide to the cotton aphid was determined by the leaf-dipping method as previously described [23] with slight modifications. We used a two-step dilution method. First, the stock solution of broflanilide (5000 μg mL −1 ) was prepared with acetone for easy dilution with water. The desired concentrations (0, 0.1, 0.1, 0.2, 0.4, 0.8, 1.6, 6.4, 10.0 μg mL −1 ) were obtained by diluting the above stock solution with the distilled water with 0.05% triton-X 100 before bioassay. Fresh cotton leaves were cut into 21 mm-

Toxicity Bioassays
The toxicity of broflanilide to the cotton aphid was determined by the leaf-dipping method as previously described [23] with slight modifications. We used a two-step dilution method. First, the stock solution of broflanilide (5000 µg mL −1 ) was prepared with acetone for easy dilution with water. The desired concentrations (0, 0.1, 0.1, 0.2, 0.4, 0.8, 1.6, 6.4, 10.0 µg mL −1 ) were obtained by diluting the above stock solution with the distilled water with 0.05% triton-X 100 before bioassay. Fresh cotton leaves were cut into 21 mm-diameter leaf discs with punch, and then these leaf discs were immersed in the above diluted solutions for 15 s. The leaf discs only treated with 0.05% (v/v) Triton-X 100 water were used as the corresponding control. The treated leaf discs were placed indoors to dry, and then the dried leaf discs were put into 12-well cell plates that contained 2.5 mL of 1.85% (w/v) agar. Healthy apterous adult aphids were gently transferred into 12-well cell plates from cotton seedlings using a soft small brush, and then the plate was sealed with Chinese art paper to prevent aphids from escaping, three replicates per concentration and at least 25 aphids in each well. The 12-well cell plates were placed under the same condition as the aphid culture. The number of live and dead aphids was scored after 72 h exposure. The aphid was considered dead if it could not move by the touch of a soft small brush.

Data Analysis
Probit analysis was used to calculate the slope of the regress curve, LC 50, and LC 90 , and 95% confidence limits by POLO Plus 2.0 software [24], and the Chi-square (χ 2 ) values and degrees of freedom (df ) were obtained from this software. The p-value was calculated by the CHIDIST function of Excel 2019 using Chi-square values and degrees of freedom.
The bioassay data of all aphid populations were pooled for the establishment of the susceptible baseline of A. gossypii to broflanilide, and the susceptible baseline was used to calculate the resistance ratio by LC 50 of field population/susceptible baseline.  Table 2). The slope ranged from 1.24 ± 0.12 to 6.59 ± 1.10 for field populations of A. gossypii (Table 2). It suggested that there was higher susceptible consistency among individuals of cotton aphid population. The ALE10 population was the most susceptible to broflanilide with the LC 50 value of 0.20 µg mL −1 , and the population with the largest LC 50 value was from KC, LC 50 value of 1.48 µg mL −1 . The difference of LC 50 values between ALE10 and KC populations was 7.4 times.

Susceptible Baseline of Aphis gossypii to Broflanilide
The curve of dose-mortality that was used to calculate the susceptible baseline of A. gossypii to broflanilide showed an S-shaped distribution (Figure 2A). The toxicity regression analysis showed the R 2 = 0.96 (p < 0.001) and slope =1.86 ( Figure 2B), which indicated a high linear relationship between concentration logarithm and mortality probability value.

Susceptible Baseline of Aphis gossypii to Broflanilide
The curve of dose-mortality that was used to calculate the susceptible baseline of A. gossypii to broflanilide showed an S-shaped distribution (Figure 2A). The toxicity regression analysis showed the R 2 = 0.96 (p < 0.001) and slope =1.86 ( Figure 2B), which indicated a high linear relationship between concentration logarithm and mortality probability value. We established that the susceptible baseline of A. gossypii to broflanilide for the LC50 value of 0.41 μg mL −1 , and the LC90 was 1.63 μg mL −1 and slope was 2.12 ± 0.08 ( Table 2). The susceptible baseline was used to calculate the resistance ratio (RR) of cotton aphid populations to broflanilide. All field populations in 2021 were susceptible to broflanilide, and the RR ranged from 0.49 to 3.61.

Discussion
The application of chemical insecticides is an indispensable measure in the practice of cotton pest management. The resistance of cotton aphids to traditional insecticides (organophosphorus, carbamate, pyrethroid, and neonicotinoid insecticides) has become a constraint in the control of cotton aphids [10,13,19]. It is an essential way for pest management to rotate application among different action modes of insecticides. Broflanilide has a novel mode of action, classified as a new member of group 30 (mode of action: GABAgated chloride channel allosteric modulator) [1, 25]. As an antagonist of GABAR, broflanilide exhibited high biological activity against various insect species such as Helicoverpa armigera (Lepidoptera: Noctuidae), Spodoptera exigua (Lepidoptera: Noctuidae) [26], Spodoptera Litura (Lepidoptera: Noctuidae) [27], and Anopheles arabiensis (Diptera: Cu- We established that the susceptible baseline of A. gossypii to broflanilide for the LC 50 value of 0.41 µg mL −1 , and the LC 90 was 1.63 µg mL −1 and slope was 2.12 ± 0.08 ( Table 2). The susceptible baseline was used to calculate the resistance ratio (RR) of cotton aphid populations to broflanilide. All field populations in 2021 were susceptible to broflanilide, and the RR ranged from 0.49 to 3.61.

Discussion
The application of chemical insecticides is an indispensable measure in the practice of cotton pest management. The resistance of cotton aphids to traditional insecticides (organophosphorus, carbamate, pyrethroid, and neonicotinoid insecticides) has become a constraint in the control of cotton aphids [10,13,19]. It is an essential way for pest management to rotate application among different action modes of insecticides.
The susceptible baseline of broflanilide against A. gossypii was established by pooling the bioassay data of all field populations with the LC 50 of 0.41 µg mL −1 . According to research reports, LC 50 values of the susceptible baseline for A. gossypii were 0.50 µg mL −1 for beta-cypermethrin and 1.1 µg mL −1 for deltamethrin [11]. LC 50 values of sulfoxaflor and imidacloprid against the susceptible strain were 0.64 µg mL −1 and 0.32 µg mL −1 , respectively [31,33]. Shi et al. (2022) also used the mean LC 50 value (0.149 µg mL −1 ) of 16 field populations as the susceptible baseline of A. gossypii to afidopyropen [34]. Our results demonstrated that A. gossypii has a high susceptibility to broflanilide, which is similar to the susceptible baseline of the insecticides mentioned above.
A. gossypii has developed high resistance to some commonly used insecticides, such as pyrethroids and neonicotinoids [10][11][12]. Detoxifying enzymes including esterases and cytochrome P450 monooxygenases (P450) and point mutations in sodium channels have been demonstrated to contribute A. gossypii resistance to pyrethroids [11,35]. Both the enhancement of P450 activity and target mutations in the nicotinic acetylcholine receptors were involved in A. gossypii resistance to neonicotinoids [19,36]. Broflanilide, as a noncompetitive antagonist of targeting on GABAR, displayed high efficiency to field populations of A. gossypii, although A. gossypii has developed high resistance to pyrethroids and neonicotinoids in cotton fields. Similarly, A. arabiensis was susceptible to brofilanilide, but it possessed high resistance to pyrethroids [28]. In addition, the P. xylostella has developed 1143-fold resistance to abamectin [37], and H. armigera evolved 20.36 and 39.12 fold resistance to chlorantraniliprole and benzoate, but broflanilide still showed high insecticidal activities against their larvae [26]. This indicated that broflanilide did not exhibit the cross-resistance with other insecticides mentioned above.

Conclusions
Broflanilide exhibited good biological activity against A. gossypii, which provides a potential alternative for the control of cotton aphids. These results are useful for the chemical control of A. gossypii in cotton fields.