The Joint Action of Destruxins and Botanical Insecticides (Rotenone, Azadirachtin and Paeonolum) Against the Cotton Aphid, Aphis gossypii Glover

The joint action of destruxins and three botanical insecticides, rotenone (Rot), azadirachtin (Aza) and paeonolum (Pae) against the cotton aphid, Aphis gossypii, was bioassayed. In laboratory experiment, several synergistic groups of destruxins with botanical insecticides were found by means of Sun’s Co-toxicity Coefficients (CTC) and Finney’s Synergistic Coefficient (SC). The best synergistic effect was discovered in the ratio group Des/Rot 1/9 with the CTC or SC and LC50 values of 479.93 or 4.8 and 0.06 μg/mL, respectively. The second and third synergistic effects were recorded in the ratio groups Des/Rot 7/3 and 9/1. Although the ratio groups Des/Aza 6/4, Des/Pae 4/6, 3/7 and 2/8 indicated synergism by Sun’s CTC, they were determined as additive actions by Finney’s SC. Additive actions were also found in most of the ratio groups, but antagonism were recorded only in three ratio groups: Des/Pae 9/1, 7/3 and 6/4. In greenhouse tests, the highest mortality was 98.9% with the treatment Des/Rot 1/9 at 0.60 μg/mL, meanwhile, the treatments Des/Pae 4/6 and Des/Aza 6/4 had approximately 88% mortality.


The Joint Action of Destruxins and Rotenone
In total, synergistic or additive interactions were recorded for all ratio groups of destruxin mixed with rotenone ( Table 1). The best synergistic effect was found in the ratio group Des/Rot 1/9 for its CTC or SC values of 479.93 or 4.8 with a LC 50 of 0.06 μg/mL. Similarly synergistic actions were recorded in the ratio groups Des/Rot 7/3 and 9/1 with CTC values of 427.06 and 374. 40 or SC values of 4.3 and 3.7, respectively. The other treatment ratio groups were determined as additive effects according to their CTC and SC values ( Table 1).

The Joint Action of Destruxins and Azadirachtin
The mixtures of destruxins and azadirachtin showed different results depending on their ratios ( Table 2). In accordance with Sun's method, the ratio group Des/Aza 6/4 was determined as synergistic for its CTC value of 191.08, but antagonistic effects were found in the ratio groups Des/Aza 8/2 and 1/9 for their CTC < 50, then, additive effects were recorded in other ratio groups. However, based on Finney's standard, the treatment Des/Aza 6/4 was determined as an additive effect for its SC value >0.4 and <2.7. Additive actions were also found in the other ratio groups as well (Table 2).

The Joint Action of Destruxins and Paeonolum
There were different joint actions between destruxins and paeonolum. According to Sun's standard, synergistic effects were found in the ratio groups Des/Pae 4/6, 3/7 and 2/8 according to their CTC values of 200-250, but antagonistic effects were recorded in the groups Des/Pae 9/1, 7/3 and 6/4 for their CTCs < 50, meanwhile, additive effects were found in other groups. However, based on the Finney's standard, although similarly antagonistic effects were found in the groups Des/Pae 9/1, 7/3 and 6/4, additive effects and no synergisms were recorded in any other group (Table 3).

Discussion
In this study, different joint actions were discovered for the mixtures of destruxins and botanical insecticides. In general, there were synergistic or additive effects in destruxins mixed with rotenone, azadirachtin or paeonolum, except for a few ratio groups. In particular, when destruxins were mixed with rotenone at a ratio 1/9, an extremely low LC 50 value of 0.06 mg/mL was recorded. The level of the synergism is more obvious than for the botanical insecticides mixed with chemical or biological insecticides [27][28][29][30], although it was slightly lower than that for destruxins mixed with matrine described by the authors [31]. In the greenhouse trial, best control effects on the aphids were also found in the mixtures of destruxins with rotenone and matrine. These results suggest that the mixing destruxins with each of these botanical insecticides at an appropriate ratio would result in a mixture that might be used to control cotton aphids.
In practice, using the mixture of the various classes of insecticides is an important part of insecticide application to increase the control effects, to reduce the costs and avoid the insects developing resistance against an individual pesticide. A similar strategy is often found in the area of medicine, for example, in the cocktail therapy was used to treat patients with HIV/AIDS [32]. However, the interaction of different insecticides usually has three results: addition, antagonism and synergism. Whether synergism occurs is influenced by many factors, such as the insect species and class of insecticides used. Biochemically, the different penetration, absorption, detoxication and target proteins of insecticides determine their interaction effects, generally, synergistic actions are preferably found in the mixtures of the insecticides targeting on different proteins or organs and tissues [33].
Destruxins and botanical insecticides have different mechanisms of action. It is generally recognized that destruxins target the immune system of the insects, causing damage to the hemocytes [12], suppressing phagocytic activity [34] and the expression of various antimicrobial peptides [35]. Destruxins are also often found to act more slowly on insects [9]. By contrast, rotenone is known to be an inhibitor of the respiratory chain, preventing the transport of electrons from NADH to CoQ. Azadirachtin has the behavior regulation properties of an antifeedant and deterrent for many insects, and it also disrupts insect growth, although it acts slowly [36]. However, the insecticide mechanism of paeonolum is still unknown. As far as the large difference of action mechanisms are concerned, the certain synergistic action between destruxins and the three botanical insecticides can be accepted. Probably, the synergistic action seen between destruxins and rotenone might be related to their different target systems. In addition, the slower pest-killing speed of destruxins in combination with the quicker pest-killing speed of rotenone might also contribute to this synergistic interaction. However, the antifeedant and deterrent actions of azadirachtin do not prevent aphids from feeding on the plants, because aphids suck the phloem juices of host plants. It might be related to the non-synergistic effects of destruxins mixed with azadirachtin.
Furthermore, the methods for evaluating joint action usually result in different conclusions. In this experiment, we used Sun' CTC and Finney's SC standards to determine if synergistic effects existed in the different insecticide ratio groups. We can give different conclusions for an identical treatment, for example, the ratio groups Des/Pae 4/6, 3/7 and 2/8 were determined to have synergistic effects by Sun's CTC and additive effects by Finney's SC. It seems that Finney's SC has a more severe standard than Sun's CTC to define a synergistic effect, so researchers should select the most suitable methods to evaluate joint action.
In this experiment, we also found that there were large changes in the different ratio groups of destruxins with the three botanical insecticides. The phenomenon was probably related to the impurity of compounds used in the experiment. The 30.4% destruxins contains as active ingredients destruxin A and B, in addition, destruxin E and other destruxin homologues, etc., and many other unknown components. Similarly, 34% azadirachtin includes many homologues and other unknown materials. Of course, rotenone and paeonolum are not pure, although their contents are more than 95%. Multiple components of insecticides probably make understanding the interactions more complicated. However, the trends of synergism between destruxins and botanical insecticides found in this experiment should be useful as a reference. Undoubtedly, the more acute interactions and the mechanisms of synergisms between destruxins and the botanical insecticides should be the focus of future research.

Insects
A population of aphids was collected from local fields and moved onto potted taro Colocasia esculenta (L.) Schoot plants, which were then placed in a greenhouse. Once the aphids had reproduced for two generations, similar non-winged adults were selected for the bioassay.

Bioassays
Insecticides were dissolved in acetone to a concentration of 100,000 μg/mL, and then diluted with 0.05% Tween 80 into stock solutions at a concentration of 10,000 μg/mL. Working solutions were diluted from stock solutions with 0.05% Tween 80. Serial mixtures were prepared with Des/Rot (or Pae, Aza) at a ratio of 10/0, 9/1, 8/2, 7/3, 6/4, 5/5, 4/6, 3/7, 2/8, 9/1 or 0/10. Controls were 0.05% Tween 80 solutions with acetone at the same concentration as the treatment solutions. A leafimmersing method was used as the bioassay, Detached leaves (4 cm × 4 cm) were cut from the potted plants and checked under a stereomicroscope to ensure that approximately 20 similar non-winged adult aphids remained on each leaf. The leaves were then dipped into the insecticide solutions and controls for a total of 5 s. After drying, the leaves were put into Petri dishes (diameter 9 cm) with wet cottonwool balls and cultured at temperature 25.0 °C and photoperiod 14:10 h light:dark. Three leaves were used for each treatment and the experiments were duplicated twice [39].

Statistical Analysis
Mortality was scored 24 h after treatment. Aphids were considered dead if their antenna showed no movement in response to a physical stimulus (touch). Data were corrected for control mortality [40] and analyzed by probit analysis [41] using the software DPS [42]. Sun's Co-toxicity coefficients (CTC) [43] and Finney's synergistic coefficient (SC) [41] were used to determine the joint action of the solutions: if CTC > 150 or SC > 2.7, it shows a synergistic effect, whereas CTC < 50 or SC < 0.4 indicates an antagonistic effect, other CTCs and SCs give additive effects.

Greenhouse Experiments
In greenhouse experiments, the average temperature was 24.0 °C (18.5-35.5 °C) and the photoperiod was 14:10 h light:dark. Assay units were pot-planted C. esculenta approximately 40 cm high with three leaves, on which there were at least 300 non-winged aphids. Based on the results of the laboratory tests, eight treatments were investigated in the greenhouse experiments. In addition, 0.05% Tween 80 was used as a control (Table 4). Insecticide solutions were diluted with 0.05% Tween 80. Each treatment was assigned three potted plants. Each potted plant was sprayed with 30 mL insecticide solution. Before and 24 h after treating, the number of aphids was surveyed and then the number of mortalities was evaluated according to Abbott's equation. Every treatment and control was replicated three times. The data were analyzed using the F-test and Tukey test employing DPS software [42].