Screening of Juvenile Hormone Disruptors from Myzus persicae using Yeast β-galactosidase Assay

Introduction

Peach aphids, Myzus persicae (Sulzer 1776; Hemiptera: Aphididae), mediate and transmit over 100 types of plant viruses (Blackman and Eastop, 2000), causing damage to both horticultural plants and economically important crops. They reduce the value of agricultural products by interfering with plant growth (Powell et al., 2005; Goggin et al., 2017), and they are one of the most economically important pests in the world (van Emden and Harrington, 2007). Although various methods have already been adopted for the control of peach aphids, the demand for environmentally friendly pesticides has been increasing (Briassoulis et al., 2001; Vontas et al., 2012). Additionally, owing to their short life cycle and high reproductive potential (Kim et al., 2014), aphids are not effectively controlled by current neonicotinoid-based insecticides, which contribute to environmental pollution (Foster et al., 2008; Puinean et al., 2010). Therefore, it is necessary to identify alternative means to prevent infections by M. persicae in order to reduce the use of chemical pesticides.

Insect growth regulators (IGR) that control the processes of insect metamorphosis and ecdysis are gaining attention, because of their low environmental toxicity, as eco-friendly pest control alternatives. Studies on various juvenile hormones and their receptors, which are a type of IGR, have been conducted (Smith, 1995; Palli and Retnakaran, 2001; Lee et al., 2015). Notably, the juvenile hormone receptor Methoprene-tolerant (Met), a transcription factor of the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) domain, has been identified in Drosophila melanogaster (Ashok et al., 1998; Wilson and Ashok, 1998). In order to activate the bHLH-PAS domain, bHLH-PAS proteins, known as Taiman, βFTZ-F1 interacting steroid receptor co-activator (FISC), or steroid receptor co-activator (SRC), and the juvenile hormone receptor bind to each other in the presence of a juvenile hormone (JH) to regulate the juvenile hormone-dependent genes (Lee et al., 2015).

In this study, plant extracts, with the potential to control peach aphids, were evaluated based on a screening system, using the transformant produced through a yeast two-hybrid assay with the juvenile hormone receptor of the peach aphid (Met) and the partner protein of the juvenile hormone receptor (SRC). Extracts with juvenile hormone disruptor (JHD) activity which could potentially be used to develop eco-friendly pest control agents were selected and sorted by plant groups.

Materials and Methods

Results and Discussion

JHD activities per plant groups

We tested 3648 plant extracts (from 167 families, including 1628 species), directly adding each extract to the yeast culture to investigate whether the pyriproxyfen-mediated Met-SRC binding could be disrupted by plant extracts in the two-hybrid yeast assay (Table 1). We selected the threshold for high JHD activity based on the results of a previous study (Oh et al., 2017). We set the high JHD value (JHD > 0.7) based on the JHD value (JHD > 0.5) indicated in the previous study (Oh et al., 2017). Among the plant extracts evaluated, in our study, 320 plant extracts were found to disrupt the pyriproxyfen-mediated binding of M. persicae to Met-SRC. We summarized the high JHD value (JHD > 0.7) based on the JHD value (JHD > 0.5) indicated in the previous study (Oh et al., 2017). Based on this, among 320 plant extracts, 272 (angiosperms) and 46 (gymnosperms) extracts were identified (Supplementary Table 1). The 20 most active extracts were identified and selected, and the extract from Camellia (Theaceae) showed the highest JHD activity (Fig. 1). Furthermore, most of the plants whose extracts showed superior JHD activity corresponded to gymnosperms and angiosperms (Fig. 2).

Table 1.

Summary of the screening of the juvenile hormone disruptor (JHD) activity of plant extracts

Fig. 1.

Plant species with high juvenile hormone disruptor (JHD) activity among plant extracts evaluated as treatments against M. persicae. Values and error bars indicate means ± SD, respectively.

Fig. 2.

Average juvenile hormone disruptor (JHD) activity values per plant groups. Values and error bars indicate means ± SD, respectively (Numbers in parentheses indicate plant extract).

Confirmation of JHD activity per plant group

Extracts from the four major vascular plant groups, namely the Pteridophyta, Lycopodiophyta, gymnosperms, and angiosperms, had significant effects on M. persicae two-hybrid yeast assay. We found that the JHD activity was particularly elevated in gymnosperms, with a mean value significantly higher than that of the Pteridophyta, Lycopodiophyta, and angiosperms, respectively (Fig. 2). Furthermore, after determining the average JHD activity for Pteridophyta, Lycopodiophyta, and gymnosperm extracts, excluding less than 5 extracts, the top 10 plants were selected and identified from more than five extracts of angiosperms (Fig. 3). High JHD activity was evaluated from the extracts of two gymnosperm families, namely Cupressaceae, which contained the species Platycladus orientalis, and Pinaceae, which included pines (Fig. 3A), as well as for the Lycopodiophyta family Selaginellaceae (Fig. 3D), and Dryopteridaceae (Pteridophyta; Fig. 3C). In addition, JHD activity was also measured in angiosperm, namely Magnoliaceae and Alismataceae (Fig. 3B). The JHD activity of both Cupressaceae and Pinaceae was higher than that of other plant taxa and higher than the average value for all plant extracts. Also, this study confirmed that the effect of JHD on the M. persicae-specific JHD assay system differs according to plant group. We found that the JHD activity was particularly concentrated in the gymnosperms (Oh et al., 2017); the mean JHD activity of the gymnosperm extracts was significantly higher than that of the Lycopodiophyta, Pteridophyta, and angiosperms, respectively.

Fig. 3.

Average juvenile hormone disruptor (JHD) activity values per plant family, subdivided in plant group. (A) gymnosperms; (B) angiosperms; (C) Pteridophyta; (D) Lycopodiophyta. Values and error bars indicate means ± SD, respectively. (Numbers in parentheses indicate plant extract)

In addition, camellidin II from the camellia flower, which was shown to have high JHD activity in this study, is known to be an antifeedant for Eurema hecabe larvae (Numata et al., 1987). Secondary metabolites which are produced by plants, such as camellidin II, are known to react against insects that harm plants and to elicit the production of substances similar to the hormones of those insects (Toong et al., 1988; Dinan, 2001). Accordingly, plant extracts containing secondary metabolites can potentially be developed as novel insecticides that are safe and environment-friendly. Notably, the present screening method with plant extracts can rapidly identify candidates for new insecticide substances. Therefore, our results indicate that the discovered JHD compounds could be used to develop novel insecticides.

Acknowledgments

This research was supported by a grant (PJ 014845) from National Institute of Agricultural Sciences in 2020 and National Research Foundation of Korea Research Initiative Program (2019R1A2C1003571).

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