Introduction

Brevicoryne brassicae L. (Hemiptera: Polygonaceae) is one of the most serious pests of cruciferous vegetables like late cabbage worldwide (Gabrys and Pawluk, 1999). Cabbage aphids often absorb the juice from foliage and stems of plants as a source of nutrition through their piercing-sucking mouthparts (Ellis et al., 1998). Due to strong reproductive capacity (parthenogenesis), short life cycle, and overlapping generations, cabbage aphids usually densely gather on leaves of vegetables to seriously affect the crop yield and quality (Pontoppidan et al., 2003). Meanwhile, aphids can transmit plant viruses through their oral needles, causing significant crop yield reductions (Blanc et al., 2011). Spraying chemical pesticides like pyrethroid insecticides (especially deltamethrin) is the main method used in modern agricultural practices to control aphid populations, and chemical pesticides must be used widely and frequently due to the high population density of cabbage aphids reached in a short time (Dhaked et al., 2016). However, prolonged use of chemical pesticides results in a variety of adverse impacts, such as enhanced pesticide-resistance of aphids (Gul et al., 2019) and pesticide residues threatening the ecological environment and food safety (Nauen, 2014). Therefore, it is extremely urgent to find low toxicity and environmentally friendly pesticides.

On the one hand, the extraction and separation of new active compounds from plants has become an important approach for the development of new pesticides (Lv et al., 2012). On the other hand, the development of botanical pesticides is another new way to solve the above problems (Nukenine et al., 2010). Compared with traditional chemical pesticides, botanical pesticides are characterized by low residues, high selectivity and lack of development of resistance. Botanical products are anew type of environment-friendly pesticides (Huang et al., 2011).

Abrus precatorius L. is a poisonous legume of the Fabaceae family, native to India with the common name of rosary bean. This species is now distributed in the subtropical regions around the world, and mainly found in Yunnan, Guangdong, Guangxi, and Fujian in China (Garaniya and Bapodra, 2014). A. precatorius has been studied and used in medicine and food in China and abroad widely due to its high medicinal and economic values, high toxicity, detox, and sedative properties (Verma et al., 2011). The ethanol extract of A. precatorius leaves has anti-inflammatory and analgesic effects on rats (Asija et al., 2015). Compounds isolated from cotyledons have anti-cancer activity and have provided an alternative option for breast cancer treatment (Sofi et al., 2018). In addition, the anti-diabetic effect of the chloroform-methanol extract from seeds is assessed in rabbits with alloxan diabetes mellitus (Monago and Alumanah, 2005). However, there are few reports on the prevention and control of agricultural pests with A. precatorius at present. As the main production area of A. precatorius medicinal materials and being one of the countries with the highest demand for pesticides in the world, it is necessary to better promote the development and utilization of this unique medicinal plant resource and deeply examine the prospects for the agricultural applications of A. precatorius in China.

The growth and development of insects are influenced by the activity and content of various enzymes (Ramsey et al., 2010). In addition, the defense and self-protection responses to unhealthy growth environments and exogenous substances are also regulated by enzymes (Yang et al., 2013). Acetylcholinesterase (AChE) is an important hydrolase in the insect nervous system that blocks nerve impulses by catalyzing the hydrolysis of the neurotransmitter acethylcholine (Yeom et al., 2012). Glutathione S-transferases (GSTs) are also important enzymes that develop metabolic resistance to insecticides and are involved in the detoxification mechanisms of many molecules (Enayati et al. 2005). In plant-insect interactions, these metabolic enzyme activities will change after feeding on non-hosts thus reflect the effect of exogenous toxic and harmful substances in insects, ultimately elucidating the insecticidal mechanism of botanical insecticides by determining the impact of active compounds (Liu et al. 2018).

In this study, the insecticidal activities and action mechanism of three organic solvent (methanol, ethanol and petroleum ether) extracts of Abrus precatorius L. against cabbage aphids were investigated by detecting changes in acetylcholinesterase (AChE) and glutathione S-transferase (GSTs) activities at the physiological and biochemical levels. It was shown that the petroleum ether extract of A. precatorius was the optimal one among the three extracts, as the changes of AChE and GSTs activities in cabbage aphids treated with it were strongly dose- and time-dependent. After 24h treatment of petroleum ether extract with different concentrations, the cabbage aphids displayed significant inhibitory effects on the enzyme activities. After treatment with the LC50 dose, the activities of AChE and GSTs in the insects were further inhibited with the prolongation of treatment time. The obtained results can contribute to the development of new botanical aphicides, the more effective control of aphid damage, and the reduced pesticide pollution. These results will also provide references for the efficient use of A. precatorius as a botanical insecticide resource.

Materials and Methods

Insects and plant material

Specimens of Brevicoryne brassicae were collected from the Agricultural Cropping Garden of Anhui Agricultural University and transferred to cabbage seedlings cultivated in the greenhouse without pesticide contact during the growing season. Insects were used for testing after four generations of indoor rearing. The feeding conditions included a growth photoperiod of 16:8h light: dark, a constant temperature of 25 ±1°C, and constant humidity of 70-80%. Apterous adult aphids with similar individual development were selected for testing. Abrusprecatorius was purchased from the traditional Chinese medicine market in Bozhou, Anhui Province, China.

Reagents

Methanol, ethanol, petroleum ether and acetone were all pure analytical grade. Sterile double distilled water was used. Normal saline0.86%, a total protein (TP) determination kit (Coomassie Brilliant Blue), and AChE and GSTs assay kits were purchased from Nanjing Jiancheng Materials Biological Engineering Research Institute.

Extract of A. precatorius

Seeds of A. precatorius with bright color, full-development and of similar sizes were quickly washed with water, drained and then dried at room temperature. The seeds were crushed and passed through 40 mesh sieves. Using the cold dipping method, A. precatorius (100g) was placed into a 1.5liter round-bottomed flask and eight times the volume of organic solvent was added. The mixture was incubated in the dark for 72h. After three days, vacuum filtration was performed using a Buchner funnel. The solution was immersed in an eight-fold volume of organic solvent for three days, and this operation was repeated three times in total. Afterwards, the three filtrates were collected. The extracts were concentrated by vacuum decompression using a rotary evaporator and then concentrated into a paste. Three kinds of organic solvent extraction pastes were weighed to 1g, and acetone was added to dissolve the pastes. Then, a 0.1% Tween-80 aqueous solution was added to prepare solutions with 50, 25, 12.50, 6.25, 3.15, and 1.56mg·ml^-1 of active ingredient, and stored at 4°C.

Contact toxicity bioassay

The contact activity of the three organic solvent extracts of A. precatorius against cabbage aphids was determined by the microdrip method (Zhong et al., 2005). A microsyringe was used to dispense ~0.5μl of the extract solution onto the front chest plate of the cabbage aphids. After dripping, the insects were transferred to the cabbage leaves with a moisturizing treatment. Then, the treated cabbage leaves and insects were placed in a disposable petri dish with wet filter paper. Each concentration was repeated three times, and each treatment was repeated with 30 cabbage aphids. The control group was treated with acetone and 0.1% Tween-80 aqueous solution, and each treatment was repeated three times. After 24, 48 and 72h, the death of cabbage aphids was evaluated. The abdomen and legs of the aphids were touched with a brush, and aphids were considered dead if they did not move. The corrected mortality was calculated as:

Corrected mortality (%) = (M₁) / (M₀) ×100

where M₁: treatment mortality, the blank control mortality, and M₀= 1: the blank control mortality.

Determination of enzyme activities

To study the changes in AChE and GSTs activities in cabbage aphids treated with petroleum ether extract from A. precatorius, the concentration gradients of 0.79, 1.45, 2.23, 3.23 and 4.57mg·ml^-1 were set at the concentration of the LC₁₀, LC₂₀, LC₃₀, LC₄₀ and LC₅₀ levels 24h after treatment. After treatment for 12, 24, 36, 48 and 60h at the LC₅₀ level, the enzyme solution was prepared separately. After the treatment, a 0.1g sample of insects was weighed and rinsed in pre-cooled physiological saline three times to remove impurities. The insects were then dried and ground to powder with liquid N. and in a mortar (pre-cooled). The powder was placed into centrifuge tube and 0.9ml of normal saline (pre-cooled) was added. At this time, a 10% homogenate of the cabbage aphids was centrifuged for 10min (4°C, 3000rpm) and the supernatant was collected as the enzyme solution to be tested. The enzyme extraction step was performed in an ice bath. The physiological saline and homogenizer were pre-cooled before each experiment to prevent enzyme inactivation.

For AChE, 50μl of the enzyme solution was tested, and four groups were established according to the test requirements: blank tubes, standard tubes, control tubes and determination tubes were used. Then, strictly following the instructions of the AChE assay kit, the samples to be determined were added to the enzyme label plate (96 holes, 100μl per hole). The OD value (412nm) of each hole was measured using a microplate reader, and each hole was measured three times with three replicates per sample. The results were calculated using the average values by referring to the kit instructions.

For GSTs, 100μl of the enzyme solution was used to determine the activities of the following four groups according to the test requirements: blank tube, standard tube, control tube and determination tube. Then, strictly following the instructions of the GSTs assay kit according to the corresponding group relationship, the determined enzyme solution was placed into an enzyme label plate (96 holes, 100μl per hole) and the same exact procedure as for AChE was followed.

For total protein, we used 50μl of the enzyme solution to be tested and diluted the enzyme solution to 2% with physiological saline (pre-cooled). Three groups were established according to the test requirements: homogenate determination tube, blank tube, and protein standard tube. Then, strictly following the instructions of the total protein (TP) assay kit according to the corresponding group relationship, the samples to be determined were added to the enzyme label plate (96 holes, 100μl per hole). The OD value (595nm) of each hole was measured using a microplate reader, and each hole was measured three times with three replicates per sample. The results were calculated using the average values by referring to the kit instructions.

Statistical analysis

The test data was systematically counted (Abbott, 1925) in Excel. Data processing software DPS was used to calculate the mean values and standard errors of insecticidal activities and enzyme activities (Tang and Zhang, 2012), to analyze data variance, and to calculate the virulence regression equation and lethal concentration were also counted using the DPS program. Duncan's new repolarization method was used to test and compare the differences between the treatments (p<0.05).

Results

Contact toxicity of A. precatorius extracts against B. brassicae

As shown in Table I, all three organic solvent extracts of A. precatorius had contact activities against cabbage aphids. The contact effects of the three organic solvent extracts were related to the extract concentration. After being treated with 25mg·ml^-1 for 72h, the corrected mortality rates of the methanol extract, ethanol extract and petroleum ether extract were 47.81, 66.9 and 99.07%, respectively.

Table I shows the treatment concentrations of the three types of solvent extracts of A. precatorius and the mortality rate of cabbage aphids treated for 24, 48 and 72h. The regression equation and LC₅₀ values of the three extracts against cabbage aphids were obtained by linear regression analysis of their toxicities. As shown in Table II, longer contact time resulted in the lower LC₅₀. In other words, the best contact effect was achieved with the increase in extract concentration and the prolongation of treatment time. The LC₅₀ values of three organic solvent extracts of A. precatorius after treatment for 72h were 33.04, 8.28 and 2.18mg·ml^-1 respectively. Based on the observed mortality rates and LC₅₀ values, the best contact effect was achieved with the petroleum ether extract followed by the ethanol extract, and then methanol extract.

The experimental concentrations of 0.79, 1.45, 2.23, 3.23 and 4.57 mg·ml^-1 were used to determine the changes in AChE activity in cabbage aphids treated with five concentrations of petroleum ether extract of A. precatorius for 24h. The changes in AChE activity are shown in Figure 1. The extract affected the activity of AChE in cabbage aphids at LC₁₀, LC₂₀, LC₃₀, LC₄₀ and LC₅₀ concentrations. With the increase in treatment concentrations, the activity of AChE in cabbage aphids showed a significant (p<0.05) downward trend compared with the control. Therefore, the petroleum ether extract of A. precatorius had a significant inhibitory effect on the AChE of cabbage aphids.

The lethal concentration of A. precatorius extract for 24h of contact treatment against cabbage aphids was 4.57mg·ml^-1. The activity of AChE in cabbage aphids was assayed under different treatment times at 12, 24, 36, 48 and 60h. Figure 2 shows that the AChE activity in cabbage aphids in the treatment group was obviously inhibited compared with the control. The highest inhibitory rate of AChE activity in cabbage aphids was 56.43% after 24h of treatment (Figure 2). After 48h of treatment, the AChE activity was gradually increased, but was generally lower than that in the control group. Therefore, the effect of petroleum ether extract of A. precatorius on AChE activity in cabbage aphids was time dependent.

Effects of petroleum ether extract of A. precatorius on the GSTs activity of B. brassicae

The experimental concentrations of 0.79, 1.45, 2.23, 3.23 and 4.57mg·ml^-1 were used to determine the changes in GSTs activity in cabbage aphids treated with five concentrations of petroleum ether extract of A. precatorius for 24h. The changes in GSTs activity are shown in Figure 3. The activity of GSTs in the body was affected when cabbage aphids were exposed to the petroleum ether extract of A. precatorius. Compared with the control, the extract significantly inhibited the activity of GSTs in cabbage aphids. The activity of GSTs at the concentration of LC₁₀ and LC₂₀ was first activated and significantly higher than that of the control group (p<0.05). It began to decrease at LC₃₀ until the LC₄₀ and LC₅₀ levels were significantly lower than in the control group (p<0.05). With the increase in extract concentrations, the activity of GSTs was inhibited more significantly.

The lethal concentration of A. precatorius extract was 4.57mg·ml^-1 for contact treatment of cabbage aphids for 24h. The GSTs activity (Figure 4) was assayed under different treatment times at 12, 24, 36, 48 and 60h. The petroleum ether extract of A. precatorius had obvious effects on the activity of GSTs in cabbage aphids. Compared with the control group, the GSTs activity was significantly inhibited after treatment with the extract, and this effect was time dependent. The highest inhibition rate of GSTs activity was 66.20% at 36h. With the prolongation of treatment time, the GSTs activity was gradually increased, but it was inhibited compared with the control group.

Discussion

The insecticidal activities of methanol, ethanol and petroleum ether extracts of A. precatorius and the effects of petroleum ether extract of A. precatorius on the activities of AChE and GSTs in cabbage aphids were studied. The results showed that these extracts had contact effects against cabbage aphids. However, the petroleum ether extract had the highest contact activity against cabbage aphids. Compared with other botanical aphidicides, the extract of A. precatorius showed stronger insecticidal activity against aphids. For example, the essential oil of Cinnamomum camphora seeds also showed insecticidal activity against cotton aphids with an LD50 value of 14.68ng/aphid (Jiang et al., 2016). Three extracts of Illicium verum fruit were reported to have had significant contact activity against M. persicae adults in the following order: ethyl acetate extract > petroleum ether extract > methyl alcohol extract (Zhou et al., 2016).

The secondary metabolites of insecticidal plants occur in various forms, and their modes of action against target pests are also different; the reaction mechanisms of pests against plant secondary metabolites are also varied (Ramsey et al., 2010). Insect pests use different biologically active detoxification enzymes and substance metabolism systems in the body to jointly resist plant secondary metabolites (Li et al., 2007). AChE is a very important enzyme in insect neurotransmission and therefore an important target for insecticides. When the activity of AChE is inhibited in vivo, insects die because of excessive stimulation (Yeom et al., 2012). GSTs are important detoxifying enzymes involved in the metabolism of endogenous and exogenous substances in insects. The GSTs in insects can be increased or decreased due to the action of toxic and harmful exogenous substances (Enayati et al., 2005). Therefore, examination of the indicators of detoxification of metabolic enzymes can be used to quantify the effects of botanical pesticides.

The results of the enzyme activity assay showed that the petroleum ether extract of A. precatorius had an obvious dose and time dependent effect on the activity of AChE and GSTs in cabbage aphids. The petroleum ether extract of A. precatorius had significant concentration dependent effects on the activities of AChE and GSTs in these aphids. The activity of AChE in cabbage aphids was inhibited by both high and low concentrations of petroleum ether extract of A. precatorius 24h after treatment. Therefore, it is speculated that the petroleum ether extract of A. precatorius had a neurotoxic effect. In the treatment with higher concentrations, both enzymes were inhibited, and these effects became more obvious with the increase in concentration. These effects may have been related to the decline in physiological function after insect poisoning, which finally led to the death of the insects (Li et al., 2007).

In addition, the petroleum ether extract of A. precatorius showed a strong time-dependent effect on the activities of AChE and GSTs in cabbage aphids. Compared with the control group, under the treatment with the LC₅₀ of the petroleum ether extract of A. precatorius, the activities of the two enzymes in cabbage aphids were first inhibited and then recovered with the prolongation of the treatment time. Generally, the activities were inhibited. The decrease in the GSTs activity may have inhibited the normal detoxification metabolism of cabbage aphids and the accumulation of toxic substances may have caused death of the insects after treatment (Vanhaelen et al., 2001).

The petroleum ether extract of A. precatorius contains different plant secondary substances, which strongly inhibit the activities of AChE and GSTs after entering the bodies of cabbage aphids. This may have affected their detoxification metabolism, causing the accumulation of toxic substances. At the same time, the extract may have also seriously interfered with the normal physiological and biochemical properties of the insects and accelerating their death (Francis et al., 2005). The experimental results showed that the petroleum ether extract of A. precatorius may be an effective inhibitor of AChE and GSTs in cabbage aphids. For the practical application of A. precatorius extract as a botanical pesticide to control cabbage aphids, metabolic enzyme inhibitors can be considered to improve pesticide efficacy and speed of action. A. precatorius is a unique plant in China, is abundant, and is one of the traditional Chinese herbal medicines. Therefore, it is necessary to further explore the insecticidal activity of this plant. In addition, it is possible to develop a new class of safe, highly efficient botanical insecticides. The findings of the current study can contribute to the development of new botanical aphicides to effectively control aphid damage and reduce pesticide pollution into the environment.

Acknowledgments

The authors thank DBMediting for professional English language editing services. This work was supported by the National Key Research and Development Program of China (Grant NO. 2017YFD0201203), Key Project for Academic and Technical Leader Candidate of Anhui Province, China (Grant No. 2019H238) and Key Program of Anhui Province Tobacco Corporation, China (Grant No. 20170551024).

EFFECTS OF Abrus precatorius L. EXTRACTS ON ACETYLCHOLINESTERASE AND GLUTATHIONE S-TRANSFERASE ACTIVITIES OF Brevicoryne brassicae L. (Hemiptera: Aphididae)

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