Effect of rotenone-induced stress on physiologically active substances in adult Aphis glycines

The aim of this study was to determine the effect of rotenone stress on Aphis glycines Matsumura (Hemiptera: Aphididae) populations in different habitats of Northeast China. The changes in kinase expression activity of endogenous substances (proteins, total sugars, trehalose, cholesterol, and free amino acids), detoxifying enzymes (cytochrome P450 and glutathione S-transferase), and metabolic enzymes (proteases and phosphofructokinases) in specimens from three populations were compared before and after stress with rotenone at median lethal concentration (LC50) and their response mechanisms were analyzed. Following a 24 h treatment with rotenone, the average LC50 rotenone values in A. glycines specimens from field populations A and B, and a laboratory population were 4.39, 4.61, and 4.03 mg/L, respectively. The degree of changes in the kinase expression activity of endogenous substances also differed, which indicated a difference in the response of A. glycines specimens from varying habitats to LC50 rotenone stress. The content of endogenous substances, detoxifying enzymes, and metabolic enzymes, except for that of free amino acids, changed significantly in all populations treated with rotenone at LC50 compared with that in the control (P < 0.05). The decrease in protein and trehalose content, and the obstruction of cholesterol transportation owing to decreased feeding in stressed individuals were the causes of A. glycines death after rotenone treatment. Aphis glycines resistance to rotenone may be related to cytochrome P450 expression.

The authors have declared that no competing interests exist. NO  The aim of this study was to determine the effect of rotenone stress on Aphis glycines 23 populations in different habitats in Northeast China. The changes in kinase expression activity 24 of endogenous substances (proteins, total sugars, trehalose, cholesterol, and free amino acids), 25 detoxifying enzymes (cytochrome P450 and glutathione-S-transferase), and metabolic enzymes 26 (proteases and phosphofructokinases) in specimens from field populations A and B and a 27 laboratory population were compared before and after stress with rotenone at median lethal 28 concentration (LC50) and their response mechanisms were analyzed. Following a 24-h treatment 29 with rotenone at LC50, the LC50 levels for the specimens from the three populations were 4.3859, 30 4.6088, and 4.0305 mg/mL, respectively. The degree of changes in the kinase expression 31 activity of endogenous substances also differed, which indicated a difference in the response of 32 A. glycines from varying habitats to LC50 rotenone stress. The content of endogenous substances, 33 detoxifying enzymes, and metabolic enzymes, except for free amino acids, changed 34 significantly in all populations treated with rotenone at LC50 compared with that in the control 35 (P < 0.05). The decrease in protein and trehalose content and the obstruction of cholesterol 36 transportation due to decreased feeding were one of the causes of A. glycines death after 37 rotenone treatment. A. glycines resistance to rotenone may be related to cytochrome P450 38 expression. 39

43
Aphis glycines (Hemiptera: Aphididae: Aphis) is one of the main pests on soybeans and harms 44 soybean plants by feeding on the leaves and causing undesirable effects such as soybean leaf 45 curling and plant dwarfing [1], which in turn leads to a series of economic problems such as 46 decreased soybean yield and reduced quality [2]. Currently, the prevention and control of A. 47 glycines is primarily based on chemical methods, but the abuse of chemical pesticides has not 48 only caused certain damage to the environment, but also resulted in pesticide-resistant A. The outbreak and damage caused by A. glycines is due to the interaction of multiple factors, 55 including aphids, natural enemies, soybeans, and environmental factors. The initial period of A. 56 glycines infection in the field is generally in the middle of June, and the peak slightly varies 57 with year, generally in early July or end of July. The abundance of A. glycines gradually 58 decreases after reaching the peak, and it disappears in the fields by the end of August to the 59 beginning of September. The abundance of natural enemies gradually peaks with the increase in 60 the abundance of A. glycines. The natural enemies occur approximately 15 days after the 61 occurrence of A. glycines, and they disappear approximately 20 days before the disappearance 62 of soybean aphids. The population of natural enemies peaks at 5-10 days earlier than that of A. 63 glycines. Therefore, prevention and control of aphids can effectively control their spread to the 64 whole field before the peak of aphid population [6]. Currently, several studies have tried to use 65 biodiversity to control pests. Some studies have shown that intercropping and adjacent cropping 66 patterns have a regulatory effect on A. glycines populations, and this approach, along with the 67 natural enemies of A. glycines, can inhibit the growth rate of A. glycines. When soybean and 68 corn were intercropped at ratios of 8:2 and 8:8, the first peak number of A. glycines was lower 69 than that in clear soybean fields [7]. A comprehensive investigation showed that among the 70 soybean and early-maturing potato fields, intercropped at a ratio of 2:2, 6:6, 8:8, 16:16, and 71 32:32, the field intercropped at a ratio of 8:8 offered the best control against A. glycines with a 72 reducing effect on its population density [8]. When potato-soybean and corn-soybean neighbor 73 cropping patterns were adopted, the peak number of A. glycines was lower than that in control 74 fields [9]. These studies prove that the use of biodiversity to control pests is an effective method 75 of pest control. 76 Rotenone, extracted from the roots of leguminous plants, is a broad-spectrum plant insecticide. 77 It causes stomach and contact toxicity, and acts as an antifeedant and a fumigant with control 78 effects on the pests of 137 families in 15 orders [10,11]. Rotenone can inhibit cell respiration; it 79 is an electron transfer inhibitor that blocks electron transfer from nicotinamide adenine 80 dinucleotide to coenzyme Q. Rotenone is a natural compound that can degrade fast with low 81 toxicity. It is a pesticide that can meet the needs of an ecologically aware civilization [10]. 82 The processes of growth, development, metamorphosis, and reproduction of insects are 83 inseparable from the synthesis, decomposition, and transformation of proteins, lipids, In this study, we compared and analyzed the variable differences and trends in the protein, total 90 sugar, trehalose, cholesterol, and free amino acid (FAA) content as well as protease, cropping field were used as field population A and those collected in a potato and soybean 100 neighbor cropping field were used as field population B. The adults of A. glycines for the 101 laboratory population were collected from an artificial climate chamber (with ambient 102 temperature: 24℃; photoperiod 16L:8D; and relative humidity: 60% ± 5%) in the laboratory, 103 and were cultured continuously for more than 3 years. 104 Determination of the effect of 24-h LC50 rotenone treatment 105 on A. glycines 106 We spread 1% agar medium in a 6-cm diameter plastic Petri dish and allowed it to solidify. We Statistical analysis software SPSS23.0 was used for data analyses. Independent sample t-test 137 was adopted to compare the significant difference in physiologically active substances before 138 and after chemical treatment. By using analysis of variance (ANOVA) combined with the least 139 significant difference (LSD) method, multiple comparisons were made to analyze significant 140 differences in physiologically active substances among the three populations, and the level of 141 significance was P = 0.05. 142

Determination of the virulence of rotenone against A. glycines 144
As shown in Table 1, the highest LC50 value of rotenone in A. glycines tissues was from field 145 population B at 4.6088 mg/mL, followed by field population A at 4.3859 mg/mL, and the least 146 was from the laboratory population at 4.0305 mg/mL. 147  19.8%, respectively (Fig 1). As shown in Table 2, the content of protein in A. glycines adults before the stress was the 210 highest in field population A at 0.534 mg/mL and lowest in the laboratory population, at 0.27 211 mg/mL; the difference among the three populations was significant (P < 0.05). The activity of 212 protease in population A was the highest, at 733.89 U/mL, and that in the laboratory population 213 was the lowest, at 481.83 U/mL, with a significant difference (P < 0.05) between either field 214 population (A or B) and laboratory population. The content of FAA was the highest in field 215 population A at 659.30 μmol/L and lowest in the laboratory population at 384.82 μmol/L, with a 216 significant difference (P < 0.05) between either field population (A or B) and laboratory 217

population. 218
The content of total sugar in A. glycines was the highest in field population B at 0.368 mg/mL 219 and lowest in the laboratory population at 0.346 mg/mL, with a significant difference (P < 0.05) 220 between either field population (A or B) and laboratory population. The content of trehalose was 221 the highest in population A at 0.29 mg/mL and lowest in the laboratory population at 0.20 222 mg/mL, with a significant difference (P < 0.05) between either field population (A or B) and 223 laboratory population. The activity of PFK was the highest in field population B at 22.22 U/mL 224 and lowest in the laboratory population at 15.54 U/mL, without significant difference among the 225 three populations. 226 The content of cholesterol in A. glycines was the highest in field population A at 0.0416 mg/mL 232 and lowest in the laboratory population at 0.0360 mg/mL, without significant difference among 233 the three populations. The activity of GST was the highest in field population A at 0.044 U/mL 234 and lowest in the laboratory population at 0.035 U/mL, with no significant difference among the 235 three populations. The content of CYP450 was the highest in field population A at 10.61 ng/mL 236 and lowest in the laboratory population at 7.72 ng/mL, with a significant difference (P < 0.05) 237 between either field population (A or B) and laboratory populations. 238

239
The virulence test results showed that A. glycines individuals from the laboratory population 240 were the most sensitive to rotenone, followed by those from field population A and field 241 population B. The LC50 value of individuals from field populations A and B was 1.09 and 1.14 242 fold higher that of the laboratory population, respectively, indicating that A. glycines adults of 243 these field populations had not developed resistance to rotenone. The results of the rotenone 244 stress test showed that the variable quantity of physiologically active substances in the 245 laboratory population changed the most, indicating that the resistance of soybean aphids in 246 laboratory population to the stress of rotenone was the weakest, followed by that of individuals 247 from field population A and field population B, that is, different populations in different 248 cropping patterns had a difference in resistance to rotenone. The analysis of metabolic 249 substances in A. glycines adults before being stressed with LC50 rotenone in the three 250 populations revealed higher content in the field population than in the laboratory population, 251 indicating that A. glycines in field had developed a corresponding adaptability after long-term 252 interspecific competition along with the climatic stress and other factors. The results of this 253 study showed that A. glycines in field with different cropping patterns had differing 254 susceptibility to pesticides. This result can provide guidance for the use of precise doses in 255 fields, thus reducing cost and pollution at the same time (Fig 4). 256 The results showed that after a 24-h stress with LC50 rotenone, the activity of protease increased 261 significantly (p < 0.05) in A. glycines adults in all the three populations, the content of protein 262 decreased significantly (p < 0.05), and the content of FAA showed no significant difference. 263 This indicated that protein decomposition was accelerating, most amino acids produced after 264 decomposition were involved in the synthesis of new proteins to maintain normal living 265 activities, and a small part of the amino acids continued to be free in the hemolymph to maintain The results showed that after a 24-h stress with rotenone at LC50, the cholesterol content 277 increased significantly (p < 0.05) in A. glycines adults in the three populations. Simultaneously, 278 A. glycines adults' feeding capability was weakened and the possibility of receiving more 279 cholesterol from food was low. Therefore, the most likely reason is that transportation in A. 280 glycines was blocked, which ultimately affected the growth and development of A. glycines [22,281 23, 24, 25]. The activity of GST was weakened significantly (p < 0.05) in the three populations, 282 whereas the content of CYP450 increased but the increase was significant (p < 0.05) only in the 283 laboratory population, indicating that CYP450 in A. glycines played a role in the response to 284 rotenone stress, presumably due to the increased expression of this gene and may be related to 285 the development of resistance [26,27]. Previous studies have shown that the resistance of 286 mosquitoes to insecticides was related to the increase in the expression of CYP450 [28], 287 suggesting that we should pay attention to the changes in this gene when using rotenone in the 288 future. The weakened activity of GST may be caused by increased consumption or by the 289 inhibitory effect of rotenone, but the specific reason for this phenomenon needs further study 290 [29,30,31]. 291 In summary, after being stressed with rotenone at LC50, A. glycines showed some effects on 292 some physiological factors, such as the decrease in protein and trehalose content, and blocking 293 of cholesterol transport, that resulted in their metabolic imbalance, slowed movement, and 294 eventual death. A. glycines of different populations from different cropping patterns showed a 295 difference in resistance and adaptability to rotenone, therefore, their controlling methods should 296 be adjusted according to the cropping patterns to achieve the goal of effective pest control and 297 reduced environmental pollution.