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Abstract ［Objectives］ This study was conducted to expand the insect resistance spectrum of tea saponin, and its control effect on Cylas formicarius and the potential as an insecticide for pest control were explored.
［Methods］ The olfactory avoidance rate of C. formicarius to tea saponin aqueous solution was determined by Ytype olfactometer; the feeding avoidance rate of C. formicarius to tea saponin was determined by the selective method; the antifeedant rate of C. formicarius to tea saponin was determined by nonselective method; and the development duration and mortality of C. formicarius under the influence of tea saponin were determined by artificial feeding method.
［Results］ C. formicarius had no significant olfactory tendency to every concentration of tea saponin, and the olfactory avoidance rate of 20.0% tea saponin aqueous solution was only 9.14%. Tea saponin had a feeding avoidance effect on C. formicarius, and the avoidance rate increased with the increase of tea saponin concentration. At 6 h, the feeding avoidance rates of 0.5%, 1.0%, 5.0%, 10.0% and 20.0% tea saponin on C. formicarius were 58.14%, 77.77%, 88.23%, 95.00% and 97.65 %, respectively; and the feeding avoidance effect at 6 h was significant, and the feeding avoidance rate was higher than that of 1 h. Tea saponin had a significant antifeedant effect on C. formicarius, and the longer the feeding time, the higher the antifeedant rate. At 72 h, the antifeedant rates of 0.5%, 1.0%, 5.0%, 10.0 % and 20.0% tea saponin to C. formicarius were 63.01%, 67.54%, 97.14 %, 96.42% and 98.57%, respectively. The larval development duration of C. formicarius was shortened with the increase of tea saponin concentration, and the larval death occurred. The development duration of larvae under the influence of 1.0% tea saponin was the shortest, which was 4.01 d shorter than that of the control, and the mortality was the highest, which was 26.65%.
［Conclusions］ Tea saponin had neither olfactory avoidance effect nor olfactory attracting effect to C. formicarius, but had obvious feeding avoidance effect and strong antifeedant effect. Tea saponin can shorten the development duration of the larvae of C. formicarius and cause the death of the larvae.
Key words Tea saponin; Cylas formicarius; Avoidance effect; Antifeedant effect; Artificial feeding method; Developmental duration
Received: August 13, 2019Accepted: October 16, 2019
Supported by National Modern Agricultural Industry Technology System Guangxi Industry Potato Innovation Team (nycytxgxcxtd1101); Guangxi Science and Technology Planning Project (GK AB16380046, GK AB18221101). Rujun PAN (1992-), male, P. R. China, master, devoted to research about pesticide toxicology and application.
*Corresponding author. Email: liyun_ren@163.com.
Cylas formicarius is commonly found in tropical and subtropical sweet potato growing areas in the world. It is widely distributed in southern producing areas in China and is an important pest in import and export quarantine at home and abroad［1］. In addition to adults eating potato vines, leaves, young shoots and potato tubers, the larvae also feed on rough vines and potato tubers, forming tunnels inside potato tubers and inducing the production of terpenoids and phenols in the bored parts, which makes the taste bitter and the quality so poor that it cannot be eaten or fed［2-3］.The pest occurs 6-8 generations per year, and there are overlapping generations［4］. The damage rate of sweet potato in most of the affected areas is as high as 60%-100%［5］, and the yield loss of sweet potato is 5%-20%, and even more than 30% in the worst affected areas［6］. Tea saponin is a pentacyclic triterpenoid saponin substance［7］. It is a new type of natural nonionic surfactant with excellent performance［8］, which is very rich in plants of Camellia in Theacease［9］. Due to its high efficiency of foaming, emulsifying, dispersing, wetting, etc.［10］, it also has direct pesticidal activity, and the application field of tea saponin has expanded to the pesticide industry. Studying the prevention and treatment of tea saponin on C. formicarius is of great significance to the healthy development of sweet potato industry. At present, the registered 30% tea saponin aqueous solution has a good prevention and control effect. The average control effect on Empoasca pirisuga Matumura at 7 d after spraying is as high as 90.5%［11］. Chen et al.［12］ found in the field control of Plutella xylostella that the overall control effect achieved by applying 233 ml of 30% tea saponin solution per 0.067 hm2 was 89.86%, and there was no significant difference from the application of 80 ml of 48% chlorpyrifos EC per 0.067 hm2. In addition, 30% tea saponin aqueous solution has a strong poisoning effect on geometrid moth eggs, and has strong avoidance and antifeedant effects on its larvae［13］. Although a number of experiments have confirmed that tea saponin has pesticidal activity, it has not been determined whether it has an inhibitory effect on C. formicarius. The olfactory tendency and feeding behavior of C. formicarius under the action of tea saponin were determined biologically, and the effects of tea saponin on the developmental duration and survival of the pest was determined by artificial feeding method. The study provides a theoretical basis for the development of tea saponin pesticides for C. formicarius. Materials and Methods
Test materials
The tea saponin powder (purity≥98%) was using weighed with an electronic balance (Sartorius BSA224SCW, Sartorius, Germany), and a tea saponin aqueous solution was prepared according to 20.0 g of tea saponin powder per 100.0 ml of distilled water. The obtained solution was then diluted to 10.0%, 5.0%, 1.0% and 0.5% (g/v) sequentially, for later use.
The sweet potato variety was Hainan Jidanhuang, which was purchased from Dakanghui Supermarket in Nanning, Guangxi. The tubers were brought back to the laboratory, for rinsing with clean water and then drying for use.
C. formicarius was collected from the sweet potato field in the farm of Guangxi University, and then reproduced in an artificial climate chamber (RPX250C, Shanghai Fuma Laboratory Instrument Co., Ltd.) with sweet potato at (28±1) ℃ and (65%±3%) RH for 2 to 3 generations. The adults which had been feathered for 5-8 d were used for the test.
Artificial feed formula［14］: Ascorbic acid ( 1.8 g), stigmasterol (720.0 mg), methylparaben (675.0 mg), potassium sorbate (675.0 mg), yeast extract (9.0 g), choline chloride (450.0 mg), agar powder (40.0 g), mixed vitamin B (45.0 mg), cellulose (14.4 g), inositol (360.0) Mg), mixed salt (2.7 g), glucose (40.0 g), casein (21.6 g), absolute ethanol (20.0 ml), tetracycline hydrochloride (200.0 mg), distilled water (9 000 ml), and sweet potato powder (80.0 g), in which sweet potato powder was prepared by cutting fresh sweet potato into slices (60 ℃, 4 h), drying and then grinding into powder.
Preparation of artificial feed: Agar powder was added into distilled water, followed by boiling and then cooling to 55 ℃. Sweet potato powder, glucose, casein, cellulose, mixed salt, yeast extract and ascorbic acid were added, and the mixture was stirred well for more than 2 min. Mixed vitamin B, choline chloride, inositol, stigmasterol, methylparaben, potassium sorbate and tetracycline hydrochloride (the 0.5% and 1.0% tea saponin treatment groups were additionally added with 4.6 g and 9.2 g of tea saponin powder, respectively) were added to anhydrous ethanol, stirred until dissolved. The two mixtures were mixed and stirred for more than 2 min, giving the artificial feed. Finally, the artificial feed was filled into culture tubes (0.5 ml centrifuge tubes with venting holes) and cooled for use.
Determination of the olfactory tendency of C. formicarius to tea saponin
Ytype olfactometer connection sequence: A drying tower (32.0 cm high, 4.0 cm diameter) wasconnected to a glass rotor flowmeter (LZB3, Changzhou Xinwang Instrument Co., Ltd.; air: 20 ℃, 101. 3 kPa, 0.04-0.40 L/min, a sample vial (height 10.0 cm, diameter 6.5 cm), a Ytype tube (branch tube length 15.0 cm, base tube length 1. 0 cm, tube diameter 3.0 cm), and a pump (vacuum pump VLC650324V, governor TAA0224V, Chengdu Qihai Electromechanical Equipment Manufacturing Co., Ltd.), sequentially. The power supply of the pump was turned on, the flow rate of the flow meter was adjusted to 0.4 L/min, and the olfactometer system was continuously pumped for 10 min to stabilize the flow rate in the system to a specified value. 3.0 ml of the tea saponin aqueous solutions with concentrations of 0.5%, 1.0%, 5.0%, 1.0% and 20.0% were sequentially added into the sample vial in the order of concentration from low to high. Each concentration was treated as one treatment, and the same volume of clear water was placed in the control bottle as a control. The test was carried out in an indoor shading environment［T: ( 27±2) ℃, RH: (65%±5%)］, and 10 healthy C. formicarius were introduced from the straight arm end of the Ytype tube, and the power supply of the pump was turned on. The test was started after the gas flow was stabilized. After 10 min, the numbers in the treatment arm and the control arm were observed and recorded, and each concentration was determined repeatedly for 6 times. Determination of the feeding selectivity of C. formicarius to tea saponin
A selection test was carried out. Cylindrical potato pieces (length 3.0 cm, diameter 1.5 cm) were made with a puncher and immersed in the series of tea saponin aqueous solutions at concentrations of 0.5%, 1.0 %, 5.0 %, 10.0% and 20.0 %, respectively. They were fished out 20 s later, airdried and placed at one side of a culture dish (diameter 7.5 cm), while the potato pieced immersed in clear water were placed at another side as the control. Then, 10 starving adult C. formicarius were added into the culture dish. The culture dish was placed in an incubator［T: ( 28±1) ℃, RH: (65%±3%)］. The numbers of C. formicarius on the treatment potato pieces and the control potato pieces were recorded at 1 and 6 h, respectively, and each concentration was determined repeatedly for 6 times.
Determination of antifeedant effect of C. formicarius on tea saponin
A nonselective method was adopted. Cylindrical potato pieces (length 3.0 cm, diameter 1.5 cm) made with a puncher were immersed in the series of tea saponin aqueous solutions at concentrations of 0.5%, 1.0 %, 5.0 %, 10.0% and 20.0%, respectively. They were fished out 20 s later, airdried and placed in a culture dish (diameter 7.5 cm) according to one potato piece per dish. Then, 10 adults starved for 10 h were added into the culture dish. The culture dish was placed in an incubator［T: (28±1) ℃, RH: (65%±3%)］. A potato piece of the same size immersed in clear water and treated by the same method was placed in a culture dish to serve as a control. The numbers of C. formicarius on the treatment potato piece and the control potato piece were recorded at 12, 24, 48 and 72 h, respectively, and each concentration was determined repeatedly for 6 times.
Effects of tea saponin on the development duration and mortality of sweet potato larvae
40 females and 10 males were placed in a culture bottle (height 9.0 cm, diameter 9.0 cm) containing sweet potato pieces that were not damaged by C. formicarius, and allowed to mate and lay eggs. The eggs under the epidermis were taken out and transferred to culture tubes (0.5 ml centrifuge tube) containing artificial feeds containing 0.5% and 1.0% tea saponin and containing no saponin, respectively. One egg was put into one tube (a small hole was poked in the surface of the feed matrix with a needle, and the egg was inserted into it), and the tubes were numbered one by one and placed in an incubator［T: ( 28±1) ℃, RH: (65%± 3%)］ in medium culture. After the transfer of the eggs, the development of C. formicarius in each stage was observed in every tube every day, and the development duration of egg, larva and pupa and the dead number were recorded. Each treatment was set with 20 tubes and done in triplicate, and there were 180 tubes in total. Statistically analysis
Data were expressed as "mean±standard error (SE)" and analyzed using SPSS 20.0 software. Data significance was tested using oneway ANOVA and Duncan multiple comparison method. The survival of the test larvae was analyzed using the survival function life table.
Avoidance rate (%) =Control group-Treatment group/Control group+Treatment group×100
Antifeedant rate (%) =Feeding holes of control group-Feeding hole of treatment group/Feeding holes of control group
Cumulative mortality (%) = Number of died pests/Total number of pests×100
Corrected mortality (%) = Motality of treatment group-Mortality of control group/1-Mortality of control group×100
Agricultural Biotechnology2019
Results and Analysis
Effect of tea saponin on the olfactory tendency of C. formicarius
It can be seen from Fig. 1 that the olfactory avoidance rate of 0.5% tea saponin was 0.00%, and the difference in the number of pests between the treatment arm and the control arm was not significant (P>0.05, the same below), indicating that tea saponin at the concentration had no significant effect on C. formicarius; and 1.0%, 5.0% and 10.0% tea saponin had negative olfactory avoidance rates, indicating that tea saponin at above concentrations had an attracting effect on C. formicarius, but the differences in the number of pests between the treatment arm and the control arm were not significant. 20.0% tea saponin showed an olfactory avoidance rate of 9.14%, but the difference in the number of pests between the treatment arm and the control arm was also not significant, indicating that tea saponin at the concentration had an olfactory avoidance effect on C. formicarius, which was very limited. It could be seen that tea saponin had neither obvious olfactory avoidance effect nor attracting effect on C. formicarius.
The feeding avoidance effect of tea saponin on C. formicarius
It can be seen from Fig. 2Fig. 3 that tea saponin had a feeding avoidance effect on C. formicarius, which increased with the increase of tea saponin concentration. At 1 h, 0.5% and 1% tea saponin had lower feeding avoidance effects, and 5.0%, 10.0%, and 20.0% tea saponin had higher feeding avoidance effects of 59.88 %, 80.00 %, and 86.67%, respectively, showing significant differences from the control group in the number of pests (P<0.05, the same below). At 6 h, The feeding avoidance effect of tea saponin on C. formicarius was more obvious than that at 1 h, and increased with the increase of tea saponin concentration. 0.5%, 1.0%, 5.0%, 10.0%, and 20.0% tea saponin had strong feeding avoidance effects, and showed the feeding avoidance rates of 58.4%, 77.77%, 88.23%, 95.00% and 97.65%, respectively, and there was a significant difference between each treatment group and the control group in the number of pests. Antifeedant effect of tea saponin on C. formicarius
Each concentration of tea saponin had an antifeedant effect on C. formicarius, but no acute toxicity had been observed. As can be seen from Fig. 4, the antifeedant rates of 0.5% and 1.0% tea saponin at 12 h were 36.26% and 37.50%, respectively, and the differences in the number of feeding holes from the control group were significant; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 85.00%, 81.25% and 92.5 %, respectively, and the differences in the number of feeding holes from the control group were significant. At 24 h, the antifeedant rates of C. formicarius to 0.5% and 1.0% tea saponin were 56.55 % and 62.50 %, respectively, and their feeding holes were significantly different from the control group; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 85.00%, 81.25% and 92.50%, respectively, and the differences in the number of feeding holes were also significant from the control group. At 48 h, the antifeedant rates of C. formicarius to 0.5% and 1.0% tea saponin were 66.95% and 66.27%, respectively, and their feeding holes were significantly different from the control group; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 96.39%, 95.48% and 98.19%, respectively, and the differences in the number of feeding holes were significant from the control group as well. At 72 h, the antifeedant rates of C. formicarius to 0.5% and 1.0% tea saponin were 63.01% and 67.54%, respectively, and the differences in the number of feeding holes from the control group were significant; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 97.14%, 96.42% and 98.57%, respectively, and the differences in the number of feeding holes from the control group were significant. It can be seen from Fig. 4 that the antifeedant rates of C. formicarius to tea saponin at different concentrations gradually increased with time. The antifeedant rate of 0.5% and 1.0% tea saponin tended to be stable within 24-72 h and were in the range of 56.55%-67.54%, which was at the middle level compared with other three concentrations, which meant that C. formicarius was relatively balanced with 0.5% and 1.0% tea saponin in feeding and refusing food. At this time, it could be ensured that C. formicarius could ingest tea saponin to the maximum extent, which helps to study the longterm effects of tea saponin on C. formicarius.
Effects of tea saponin on the development duration of the egg, larva and pupa stages of C. formicarius and its survival It can be seen from Table 1 that the egg, larva and pupa stages of C. formicarius were shortened with the increase of the concentration of tea saponin, and the larval mortality was the highest. The egg duration under the influence of 0.5% tea saponin was 7.29 d, which was 1.44 d shorter than the control group, and the difference was significant. The egg duration under the influence of 1.0% tea saponin was 6.90 d, which was shorted by 1.83 d compared with the control group, which was different. The larval duration under the influence of tea saponin was 22.88 d, which was shortened by 0.62 d compared with the control group, which was significant, and the corrected mortality was 13.30%. Under the influence of 1.0% tea saponin, the larval duration was 22.07 d, which was shortened by 1.43 d compared with the control group, which was significant. And the number of died larvae was significantly different from that of the control group and the 0.5% tea saponin group, and the corrected mortality was 53.33%. The pupal duration under the influence of 0.5% tea saponin was 6. 44 d, which was 0.68 d shorter than the control group, which was significant, and the number of died pupae was significantly different from that of the control group and the 0.5% tea saponin group. The pupal duration under the influence of 1.0% tea saponin was 5.69 d, which was shortened by 0.75 d compared with the control group, which was significant, and by 0.07 d compared with the 0.5% tea saponin group, and the number of died pupae was significantly different from that of the control group. From the trend of survivorship curve (Fig. 5), tea saponin had great effects on the hatching period of C. formicarius eggs and the survival of larvae from late larva stage to pupa stage, and the effects of tea saponin got more obvious with the increase of the concentration. In the test insects after emergence, the survival rate of the CK was 42.0%, and the survival rates of 0.5% and 1.0% tea saponin treatments were 35.0% and 22.00%, respectively.
Discussion
In this study, tea saponin aqueous solution had neither olfactory avoidance effect nor olfactory attracting effect on C. formicarius. It might be because tea saponin is a kind of nonvolatile substance at normal temperature［15］, and the amount wafted through the air is very small and not enough to cause the strong behavior trend of C. formicarius, which needs further verification. However, tea saponin had obvious feeding avoidance effect on C. formicarius, and the feeding avoidance rate increased with the increase of the tea saponin concentration and increased with time. The avoidance behavior of the test pests was more obvious at 6 h of feeding than at 1 h, indicating that C. formicarius was more sensitive to tea saponin at longer feeding time. Compared with the test pests that were hungry at 1 h (that had contact tea saponin for 1 h in the nonselective condition), the test pests in the nonstarved state at 6 h (that had contact tea saponin for 6 h in the nonselective condition) made a better choice for food, so the choice of tea saponincontaining food was also reduced. In addition, tea saponin had a strong antifeedant effect on C. formicarius, and the antifeedant rate increased with time, but no acute toxicity was observed. This is also reflected in the antifeedant activity against cabbage caterpillar. Cai et al.［16］ fed cabbage caterpillar larvae at the 5th instar with cabbage mustard leaf disks treated with 500 mg/L tea saponin aqueous solution by the leaf disk feeding method, and the selective antifeedant rate after 12 h reached 100%, and in the method of irrigating the roots of host plants, the antifeedant rate of cabbage caterpillar reached 63.95 % at 24 h. Under the influence of tea saponin, the development period of C. formicarius larvae was reduced, and it was speculated that tea saponin might have antijuvenile hormone activity. In this study, although tea saponin did not cause significant damage to the eggs of C. formicarius, it greatly shortened the development time of eggs, which might be due to that the maturity enhancement effect of tea saponin promoted embryo development. When the C. formicarius entered the larva stage, a 59.2% mortality occurred. The possible causes are as follows: early maturing embryos hatched unhealthy larvae, leading to rapid death; and after stronglyresistant larvae ingested tea saponin, the maturity enhancement effect of which caused abnormal larva development, leading to early development and pupation or the occurrence of molting disorder leading to death. After entering the pupa stage, although C. formicarius had strong resistance during this period, the influence of the larva stage caused pupa diapause, which further resulted in death. The pest resistance of this type of insect hormone is difficult to find in routine experiments, but its possibility of existence cannot be ruled out. Moreover, the advantages of plantderived agents lie in the diversification of their action modes, and the potential pestresistant action mode should be paid attention to. It has been reported that tea saponin significantly prolongs the development duration of P. xylostella［17］, and studies have shown that tea saponin has strong toxic activity against thirdinstar Ectropis obliqua larvae, secondinstar P. xylostella larvae, Aphis craccivora［18］ and fourth and fifthinstar Spodoptera exigua larvae［19］. These results are similar to the results of this study, verifying the pesticidal activity of tea saponin. The next step is to conduct an indepth study on its action mechanism. Conclusions
Tea saponin had neither olfactory avoidance effect nor olfactory attraction effect on C. formicarius, but obvious feeding avoidance effect and strong antifeedant effect. It had no acute toxicity. Under the influence of tea saponin, the developmental duration of C. formicarius larvae was shortened, and larvae might die in the development period.
References
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［Methods］ The olfactory avoidance rate of C. formicarius to tea saponin aqueous solution was determined by Ytype olfactometer; the feeding avoidance rate of C. formicarius to tea saponin was determined by the selective method; the antifeedant rate of C. formicarius to tea saponin was determined by nonselective method; and the development duration and mortality of C. formicarius under the influence of tea saponin were determined by artificial feeding method.
［Results］ C. formicarius had no significant olfactory tendency to every concentration of tea saponin, and the olfactory avoidance rate of 20.0% tea saponin aqueous solution was only 9.14%. Tea saponin had a feeding avoidance effect on C. formicarius, and the avoidance rate increased with the increase of tea saponin concentration. At 6 h, the feeding avoidance rates of 0.5%, 1.0%, 5.0%, 10.0% and 20.0% tea saponin on C. formicarius were 58.14%, 77.77%, 88.23%, 95.00% and 97.65 %, respectively; and the feeding avoidance effect at 6 h was significant, and the feeding avoidance rate was higher than that of 1 h. Tea saponin had a significant antifeedant effect on C. formicarius, and the longer the feeding time, the higher the antifeedant rate. At 72 h, the antifeedant rates of 0.5%, 1.0%, 5.0%, 10.0 % and 20.0% tea saponin to C. formicarius were 63.01%, 67.54%, 97.14 %, 96.42% and 98.57%, respectively. The larval development duration of C. formicarius was shortened with the increase of tea saponin concentration, and the larval death occurred. The development duration of larvae under the influence of 1.0% tea saponin was the shortest, which was 4.01 d shorter than that of the control, and the mortality was the highest, which was 26.65%.
［Conclusions］ Tea saponin had neither olfactory avoidance effect nor olfactory attracting effect to C. formicarius, but had obvious feeding avoidance effect and strong antifeedant effect. Tea saponin can shorten the development duration of the larvae of C. formicarius and cause the death of the larvae.
Key words Tea saponin; Cylas formicarius; Avoidance effect; Antifeedant effect; Artificial feeding method; Developmental duration
Received: August 13, 2019Accepted: October 16, 2019
Supported by National Modern Agricultural Industry Technology System Guangxi Industry Potato Innovation Team (nycytxgxcxtd1101); Guangxi Science and Technology Planning Project (GK AB16380046, GK AB18221101). Rujun PAN (1992-), male, P. R. China, master, devoted to research about pesticide toxicology and application.
*Corresponding author. Email: liyun_ren@163.com.
Cylas formicarius is commonly found in tropical and subtropical sweet potato growing areas in the world. It is widely distributed in southern producing areas in China and is an important pest in import and export quarantine at home and abroad［1］. In addition to adults eating potato vines, leaves, young shoots and potato tubers, the larvae also feed on rough vines and potato tubers, forming tunnels inside potato tubers and inducing the production of terpenoids and phenols in the bored parts, which makes the taste bitter and the quality so poor that it cannot be eaten or fed［2-3］.The pest occurs 6-8 generations per year, and there are overlapping generations［4］. The damage rate of sweet potato in most of the affected areas is as high as 60%-100%［5］, and the yield loss of sweet potato is 5%-20%, and even more than 30% in the worst affected areas［6］. Tea saponin is a pentacyclic triterpenoid saponin substance［7］. It is a new type of natural nonionic surfactant with excellent performance［8］, which is very rich in plants of Camellia in Theacease［9］. Due to its high efficiency of foaming, emulsifying, dispersing, wetting, etc.［10］, it also has direct pesticidal activity, and the application field of tea saponin has expanded to the pesticide industry. Studying the prevention and treatment of tea saponin on C. formicarius is of great significance to the healthy development of sweet potato industry. At present, the registered 30% tea saponin aqueous solution has a good prevention and control effect. The average control effect on Empoasca pirisuga Matumura at 7 d after spraying is as high as 90.5%［11］. Chen et al.［12］ found in the field control of Plutella xylostella that the overall control effect achieved by applying 233 ml of 30% tea saponin solution per 0.067 hm2 was 89.86%, and there was no significant difference from the application of 80 ml of 48% chlorpyrifos EC per 0.067 hm2. In addition, 30% tea saponin aqueous solution has a strong poisoning effect on geometrid moth eggs, and has strong avoidance and antifeedant effects on its larvae［13］. Although a number of experiments have confirmed that tea saponin has pesticidal activity, it has not been determined whether it has an inhibitory effect on C. formicarius. The olfactory tendency and feeding behavior of C. formicarius under the action of tea saponin were determined biologically, and the effects of tea saponin on the developmental duration and survival of the pest was determined by artificial feeding method. The study provides a theoretical basis for the development of tea saponin pesticides for C. formicarius. Materials and Methods
Test materials
The tea saponin powder (purity≥98%) was using weighed with an electronic balance (Sartorius BSA224SCW, Sartorius, Germany), and a tea saponin aqueous solution was prepared according to 20.0 g of tea saponin powder per 100.0 ml of distilled water. The obtained solution was then diluted to 10.0%, 5.0%, 1.0% and 0.5% (g/v) sequentially, for later use.
The sweet potato variety was Hainan Jidanhuang, which was purchased from Dakanghui Supermarket in Nanning, Guangxi. The tubers were brought back to the laboratory, for rinsing with clean water and then drying for use.
C. formicarius was collected from the sweet potato field in the farm of Guangxi University, and then reproduced in an artificial climate chamber (RPX250C, Shanghai Fuma Laboratory Instrument Co., Ltd.) with sweet potato at (28±1) ℃ and (65%±3%) RH for 2 to 3 generations. The adults which had been feathered for 5-8 d were used for the test.
Artificial feed formula［14］: Ascorbic acid ( 1.8 g), stigmasterol (720.0 mg), methylparaben (675.0 mg), potassium sorbate (675.0 mg), yeast extract (9.0 g), choline chloride (450.0 mg), agar powder (40.0 g), mixed vitamin B (45.0 mg), cellulose (14.4 g), inositol (360.0) Mg), mixed salt (2.7 g), glucose (40.0 g), casein (21.6 g), absolute ethanol (20.0 ml), tetracycline hydrochloride (200.0 mg), distilled water (9 000 ml), and sweet potato powder (80.0 g), in which sweet potato powder was prepared by cutting fresh sweet potato into slices (60 ℃, 4 h), drying and then grinding into powder.
Preparation of artificial feed: Agar powder was added into distilled water, followed by boiling and then cooling to 55 ℃. Sweet potato powder, glucose, casein, cellulose, mixed salt, yeast extract and ascorbic acid were added, and the mixture was stirred well for more than 2 min. Mixed vitamin B, choline chloride, inositol, stigmasterol, methylparaben, potassium sorbate and tetracycline hydrochloride (the 0.5% and 1.0% tea saponin treatment groups were additionally added with 4.6 g and 9.2 g of tea saponin powder, respectively) were added to anhydrous ethanol, stirred until dissolved. The two mixtures were mixed and stirred for more than 2 min, giving the artificial feed. Finally, the artificial feed was filled into culture tubes (0.5 ml centrifuge tubes with venting holes) and cooled for use.
Determination of the olfactory tendency of C. formicarius to tea saponin
Ytype olfactometer connection sequence: A drying tower (32.0 cm high, 4.0 cm diameter) wasconnected to a glass rotor flowmeter (LZB3, Changzhou Xinwang Instrument Co., Ltd.; air: 20 ℃, 101. 3 kPa, 0.04-0.40 L/min, a sample vial (height 10.0 cm, diameter 6.5 cm), a Ytype tube (branch tube length 15.0 cm, base tube length 1. 0 cm, tube diameter 3.0 cm), and a pump (vacuum pump VLC650324V, governor TAA0224V, Chengdu Qihai Electromechanical Equipment Manufacturing Co., Ltd.), sequentially. The power supply of the pump was turned on, the flow rate of the flow meter was adjusted to 0.4 L/min, and the olfactometer system was continuously pumped for 10 min to stabilize the flow rate in the system to a specified value. 3.0 ml of the tea saponin aqueous solutions with concentrations of 0.5%, 1.0%, 5.0%, 1.0% and 20.0% were sequentially added into the sample vial in the order of concentration from low to high. Each concentration was treated as one treatment, and the same volume of clear water was placed in the control bottle as a control. The test was carried out in an indoor shading environment［T: ( 27±2) ℃, RH: (65%±5%)］, and 10 healthy C. formicarius were introduced from the straight arm end of the Ytype tube, and the power supply of the pump was turned on. The test was started after the gas flow was stabilized. After 10 min, the numbers in the treatment arm and the control arm were observed and recorded, and each concentration was determined repeatedly for 6 times. Determination of the feeding selectivity of C. formicarius to tea saponin
A selection test was carried out. Cylindrical potato pieces (length 3.0 cm, diameter 1.5 cm) were made with a puncher and immersed in the series of tea saponin aqueous solutions at concentrations of 0.5%, 1.0 %, 5.0 %, 10.0% and 20.0 %, respectively. They were fished out 20 s later, airdried and placed at one side of a culture dish (diameter 7.5 cm), while the potato pieced immersed in clear water were placed at another side as the control. Then, 10 starving adult C. formicarius were added into the culture dish. The culture dish was placed in an incubator［T: ( 28±1) ℃, RH: (65%±3%)］. The numbers of C. formicarius on the treatment potato pieces and the control potato pieces were recorded at 1 and 6 h, respectively, and each concentration was determined repeatedly for 6 times.
Determination of antifeedant effect of C. formicarius on tea saponin
A nonselective method was adopted. Cylindrical potato pieces (length 3.0 cm, diameter 1.5 cm) made with a puncher were immersed in the series of tea saponin aqueous solutions at concentrations of 0.5%, 1.0 %, 5.0 %, 10.0% and 20.0%, respectively. They were fished out 20 s later, airdried and placed in a culture dish (diameter 7.5 cm) according to one potato piece per dish. Then, 10 adults starved for 10 h were added into the culture dish. The culture dish was placed in an incubator［T: (28±1) ℃, RH: (65%±3%)］. A potato piece of the same size immersed in clear water and treated by the same method was placed in a culture dish to serve as a control. The numbers of C. formicarius on the treatment potato piece and the control potato piece were recorded at 12, 24, 48 and 72 h, respectively, and each concentration was determined repeatedly for 6 times.
Effects of tea saponin on the development duration and mortality of sweet potato larvae
40 females and 10 males were placed in a culture bottle (height 9.0 cm, diameter 9.0 cm) containing sweet potato pieces that were not damaged by C. formicarius, and allowed to mate and lay eggs. The eggs under the epidermis were taken out and transferred to culture tubes (0.5 ml centrifuge tube) containing artificial feeds containing 0.5% and 1.0% tea saponin and containing no saponin, respectively. One egg was put into one tube (a small hole was poked in the surface of the feed matrix with a needle, and the egg was inserted into it), and the tubes were numbered one by one and placed in an incubator［T: ( 28±1) ℃, RH: (65%± 3%)］ in medium culture. After the transfer of the eggs, the development of C. formicarius in each stage was observed in every tube every day, and the development duration of egg, larva and pupa and the dead number were recorded. Each treatment was set with 20 tubes and done in triplicate, and there were 180 tubes in total. Statistically analysis
Data were expressed as "mean±standard error (SE)" and analyzed using SPSS 20.0 software. Data significance was tested using oneway ANOVA and Duncan multiple comparison method. The survival of the test larvae was analyzed using the survival function life table.
Avoidance rate (%) =Control group-Treatment group/Control group+Treatment group×100
Antifeedant rate (%) =Feeding holes of control group-Feeding hole of treatment group/Feeding holes of control group
Cumulative mortality (%) = Number of died pests/Total number of pests×100
Corrected mortality (%) = Motality of treatment group-Mortality of control group/1-Mortality of control group×100
Agricultural Biotechnology2019
Results and Analysis
Effect of tea saponin on the olfactory tendency of C. formicarius
It can be seen from Fig. 1 that the olfactory avoidance rate of 0.5% tea saponin was 0.00%, and the difference in the number of pests between the treatment arm and the control arm was not significant (P>0.05, the same below), indicating that tea saponin at the concentration had no significant effect on C. formicarius; and 1.0%, 5.0% and 10.0% tea saponin had negative olfactory avoidance rates, indicating that tea saponin at above concentrations had an attracting effect on C. formicarius, but the differences in the number of pests between the treatment arm and the control arm were not significant. 20.0% tea saponin showed an olfactory avoidance rate of 9.14%, but the difference in the number of pests between the treatment arm and the control arm was also not significant, indicating that tea saponin at the concentration had an olfactory avoidance effect on C. formicarius, which was very limited. It could be seen that tea saponin had neither obvious olfactory avoidance effect nor attracting effect on C. formicarius.
The feeding avoidance effect of tea saponin on C. formicarius
It can be seen from Fig. 2Fig. 3 that tea saponin had a feeding avoidance effect on C. formicarius, which increased with the increase of tea saponin concentration. At 1 h, 0.5% and 1% tea saponin had lower feeding avoidance effects, and 5.0%, 10.0%, and 20.0% tea saponin had higher feeding avoidance effects of 59.88 %, 80.00 %, and 86.67%, respectively, showing significant differences from the control group in the number of pests (P<0.05, the same below). At 6 h, The feeding avoidance effect of tea saponin on C. formicarius was more obvious than that at 1 h, and increased with the increase of tea saponin concentration. 0.5%, 1.0%, 5.0%, 10.0%, and 20.0% tea saponin had strong feeding avoidance effects, and showed the feeding avoidance rates of 58.4%, 77.77%, 88.23%, 95.00% and 97.65%, respectively, and there was a significant difference between each treatment group and the control group in the number of pests. Antifeedant effect of tea saponin on C. formicarius
Each concentration of tea saponin had an antifeedant effect on C. formicarius, but no acute toxicity had been observed. As can be seen from Fig. 4, the antifeedant rates of 0.5% and 1.0% tea saponin at 12 h were 36.26% and 37.50%, respectively, and the differences in the number of feeding holes from the control group were significant; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 85.00%, 81.25% and 92.5 %, respectively, and the differences in the number of feeding holes from the control group were significant. At 24 h, the antifeedant rates of C. formicarius to 0.5% and 1.0% tea saponin were 56.55 % and 62.50 %, respectively, and their feeding holes were significantly different from the control group; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 85.00%, 81.25% and 92.50%, respectively, and the differences in the number of feeding holes were also significant from the control group. At 48 h, the antifeedant rates of C. formicarius to 0.5% and 1.0% tea saponin were 66.95% and 66.27%, respectively, and their feeding holes were significantly different from the control group; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 96.39%, 95.48% and 98.19%, respectively, and the differences in the number of feeding holes were significant from the control group as well. At 72 h, the antifeedant rates of C. formicarius to 0.5% and 1.0% tea saponin were 63.01% and 67.54%, respectively, and the differences in the number of feeding holes from the control group were significant; and the antifeedant rates of 5.0%, 10.0% and 20.0% tea saponin were 97.14%, 96.42% and 98.57%, respectively, and the differences in the number of feeding holes from the control group were significant. It can be seen from Fig. 4 that the antifeedant rates of C. formicarius to tea saponin at different concentrations gradually increased with time. The antifeedant rate of 0.5% and 1.0% tea saponin tended to be stable within 24-72 h and were in the range of 56.55%-67.54%, which was at the middle level compared with other three concentrations, which meant that C. formicarius was relatively balanced with 0.5% and 1.0% tea saponin in feeding and refusing food. At this time, it could be ensured that C. formicarius could ingest tea saponin to the maximum extent, which helps to study the longterm effects of tea saponin on C. formicarius.
Effects of tea saponin on the development duration of the egg, larva and pupa stages of C. formicarius and its survival It can be seen from Table 1 that the egg, larva and pupa stages of C. formicarius were shortened with the increase of the concentration of tea saponin, and the larval mortality was the highest. The egg duration under the influence of 0.5% tea saponin was 7.29 d, which was 1.44 d shorter than the control group, and the difference was significant. The egg duration under the influence of 1.0% tea saponin was 6.90 d, which was shorted by 1.83 d compared with the control group, which was different. The larval duration under the influence of tea saponin was 22.88 d, which was shortened by 0.62 d compared with the control group, which was significant, and the corrected mortality was 13.30%. Under the influence of 1.0% tea saponin, the larval duration was 22.07 d, which was shortened by 1.43 d compared with the control group, which was significant. And the number of died larvae was significantly different from that of the control group and the 0.5% tea saponin group, and the corrected mortality was 53.33%. The pupal duration under the influence of 0.5% tea saponin was 6. 44 d, which was 0.68 d shorter than the control group, which was significant, and the number of died pupae was significantly different from that of the control group and the 0.5% tea saponin group. The pupal duration under the influence of 1.0% tea saponin was 5.69 d, which was shortened by 0.75 d compared with the control group, which was significant, and by 0.07 d compared with the 0.5% tea saponin group, and the number of died pupae was significantly different from that of the control group. From the trend of survivorship curve (Fig. 5), tea saponin had great effects on the hatching period of C. formicarius eggs and the survival of larvae from late larva stage to pupa stage, and the effects of tea saponin got more obvious with the increase of the concentration. In the test insects after emergence, the survival rate of the CK was 42.0%, and the survival rates of 0.5% and 1.0% tea saponin treatments were 35.0% and 22.00%, respectively.
Discussion
In this study, tea saponin aqueous solution had neither olfactory avoidance effect nor olfactory attracting effect on C. formicarius. It might be because tea saponin is a kind of nonvolatile substance at normal temperature［15］, and the amount wafted through the air is very small and not enough to cause the strong behavior trend of C. formicarius, which needs further verification. However, tea saponin had obvious feeding avoidance effect on C. formicarius, and the feeding avoidance rate increased with the increase of the tea saponin concentration and increased with time. The avoidance behavior of the test pests was more obvious at 6 h of feeding than at 1 h, indicating that C. formicarius was more sensitive to tea saponin at longer feeding time. Compared with the test pests that were hungry at 1 h (that had contact tea saponin for 1 h in the nonselective condition), the test pests in the nonstarved state at 6 h (that had contact tea saponin for 6 h in the nonselective condition) made a better choice for food, so the choice of tea saponincontaining food was also reduced. In addition, tea saponin had a strong antifeedant effect on C. formicarius, and the antifeedant rate increased with time, but no acute toxicity was observed. This is also reflected in the antifeedant activity against cabbage caterpillar. Cai et al.［16］ fed cabbage caterpillar larvae at the 5th instar with cabbage mustard leaf disks treated with 500 mg/L tea saponin aqueous solution by the leaf disk feeding method, and the selective antifeedant rate after 12 h reached 100%, and in the method of irrigating the roots of host plants, the antifeedant rate of cabbage caterpillar reached 63.95 % at 24 h. Under the influence of tea saponin, the development period of C. formicarius larvae was reduced, and it was speculated that tea saponin might have antijuvenile hormone activity. In this study, although tea saponin did not cause significant damage to the eggs of C. formicarius, it greatly shortened the development time of eggs, which might be due to that the maturity enhancement effect of tea saponin promoted embryo development. When the C. formicarius entered the larva stage, a 59.2% mortality occurred. The possible causes are as follows: early maturing embryos hatched unhealthy larvae, leading to rapid death; and after stronglyresistant larvae ingested tea saponin, the maturity enhancement effect of which caused abnormal larva development, leading to early development and pupation or the occurrence of molting disorder leading to death. After entering the pupa stage, although C. formicarius had strong resistance during this period, the influence of the larva stage caused pupa diapause, which further resulted in death. The pest resistance of this type of insect hormone is difficult to find in routine experiments, but its possibility of existence cannot be ruled out. Moreover, the advantages of plantderived agents lie in the diversification of their action modes, and the potential pestresistant action mode should be paid attention to. It has been reported that tea saponin significantly prolongs the development duration of P. xylostella［17］, and studies have shown that tea saponin has strong toxic activity against thirdinstar Ectropis obliqua larvae, secondinstar P. xylostella larvae, Aphis craccivora［18］ and fourth and fifthinstar Spodoptera exigua larvae［19］. These results are similar to the results of this study, verifying the pesticidal activity of tea saponin. The next step is to conduct an indepth study on its action mechanism. Conclusions
Tea saponin had neither olfactory avoidance effect nor olfactory attraction effect on C. formicarius, but obvious feeding avoidance effect and strong antifeedant effect. It had no acute toxicity. Under the influence of tea saponin, the developmental duration of C. formicarius larvae was shortened, and larvae might die in the development period.
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