Ectropis obliqua Prout (Lepidoptera: Geometridae) is a major pest of tea, Camellia sinensis L., in China (Hu et al. 1994, Yang et al. 2016). Its larvae are defoliators that seriously affect the growth of tea plants, as well as tea production and quality. Approximately 4 million hectares of tea are infested by E. obliqua annually (Gao et al. 2007). In order to effectively conduct research on the biology and management of E. obliqua, it is necessary to produce large numbers of healthy specimens in laboratory colonies. An artificial diet is an important component of these efforts (Singh and Moore 1985).

Currently used artificial diets for E. obliqua contain tea powder (Liu et al. 2015, Yuan et al. 2017). Ectropis obliqua fed on a diet (Zhang et al. 2007) that did not contain tea powder exhibited a pupation rate of 76.2%, a larval development duration of 11 d, and an emergence rate of 73.6%, but larval and pupal weights were lower than those feeding on tea leaves or on diets containing tea powder. These lower-performing larvae are not adequate for use in assays and other studies.

Diet supplemented with appropriate amounts of tea powder can yield E. obliqua that grow and develop normally, but the complex composition of tea powder interacts with individual diet additives (Fang et al. 2003, Hiyama et al. 2010). It is necessary to develop a diet to overcome this problem and to identify ingredients to replace tea powder.

Materials and Methods

Insect rearing. Ectropis obliqua larvae were maintained in a ventilated plastic boxes in the environmental chamber maintained at 23 ± 2°C with a photoperiod of 16:8 h (light:dark), and a relative humidity of approximately 75%. Larvae were fed on fresh leaves of tea, C. sinensis cv. Nongkangzao for several generations.

Preliminary optimization of artificial diet. The artificial diet developed by Zhang et al. (2007) was used as the control (Table 1). Artificial diets used to rear Spodoptera litura (F.), Hyphantria cunea (Drury), E. obliqua, and other lepidopter-ans (Gupta et al. 2005, Mukaiyama 1966) were used to develop a recipe for a proposed alternative diet for E. obliqua (Table 1). Ingredients of the general purpose artificial diet containing tea powder are listed in Table 1.

Table 1 Composition and proportion (%) of ingredients of selected artificial diets for Ectropis obliqua.

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Additive concentration assay. The compound (–)-epigallocatechin-3-gallate (EGCG) is the major catechin (phenol) found in green tea (Nagle et al. 2006) and reportedly possesses antimicrobial (Matsumoto et al. 2012), anti-inflammatory, and antioxidative (Cavet et al. 2011) properties in humans. And, according to the content of EGCG in tea, we determined that the concentration of EGCG in an artificial diet for E. obliqua might range from 0.5% to 3% (Wan 2003). We therefore established EGCG concentrations of 0%, 0.5%, 1%, 1.5%, 2%, and 2.5%.

Briefly, the Zhang et al. (2007) diet and the modified diet (Table 1) were prepared with ingredients in proportion to those listed. For each, agar (Group A) was dissolved in an appropriate amount of distilled water and heated to 50–60°C when ingredients from Group B were added, stirred, and boiled for 1 to 2 min. These mixtures were then cooled to 50–60°C when ingredients from Group C were added and mixed until the temperature decreased to 20°C, when Group D ingredients were added and mixed. Each diet was then allowed to solidify as a paste and stored at 4°C. The diet supplemented with EGCG was prepared following the same method, except that EGCG at different concentrations was added with the Group D ingredients.

The prepared artificial diets were cut into thin slices and placed upside down on the inside top of the sterile petri dish. Neonates were transferred to the petri dish. The petri dish was placed directly under the light source so that larvae were phototropically attracted to the diet. Petri dishes containing larvae and diet were placed in an environmental chamber maintained as previously described. Controls consisted of the general-purpose diet, the diet containing tea powder, and the artificial diet with 0% EGCG.

In each experiment, the feeding assays were repeated independently three times; replicates included 10 larvae each. Growth and development were monitored daily. Larval duration, pupation, and pupal weight were recorded. Emergence from pupae also was recorded. Data were subjected to analysis of variance followed by a least significant difference test to separate significantly different treatment means (P < 0.05).

Results and Discussion

No significant differences of pupation rate, larval duration, or emergence rate were observed with the general-purpose diet or the improved artificial diet (Table 2). The effects of diet supplemented with different concentrations of EGCG are shown in Fig. 1. Larvae fed the diet containing 3% EGCG are not included in Table 2 or the figures because almost all larvae died before pupation.

Table 2 Mean ± SD pupation rate, larval duration, pupal weight, and emergence rate of Ectropis obliqua fed an improved artificial diet and the Zhang et al. (2007) artificial diet.

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Pupation increased with an increase of dietary EGCG from 0% to 2.5%.

Pupation increased by more than 35% when the larvae fed on diet supplemented with 2.5% EGCG (Fig. 1A). Duration of larval development was slightly but significantly (P > 0.01) extended with larvae fed on diet supplemented with EGCG; however, there were no significant differences in larval duration among different EGCG additive concentrations. Pupal weight also increased with an increase of EGCG concentration from 0% to 2.5%. Diet supplemented with 2% EGCG was significantly higher than 0% (P < 0.05). Pupal weight increased by more than 41% when the larvae fed on diet supplemented with 2.5% EGCG (Fig. 1C). Emergence rate also increased as dietary EGCG increased from 0% to 2.5%. Emergence on diet supplemented with 0.5–2.5% EGCG was significantly higher than 0% (P < 0.01). Emergence increased by more than 33% when the larvae fed on diet supplemented with 2.5% EGCG (Fig. 1D).

These results demonstrate that EGCG added to the standard diet is beneficial for E. obliqua. The artificial diet supplemented with 2.5% EGCG appears to be the most suitable for E. obliqua (i.e., optimal diet) of the six diets supplemented with EGCG and tested herein. Performance increased with a dietary supplement of EGCG between 0% and 2.5%. When EGCG concentration exceeded 2.5%, performance decreased. This optimal diet (diet + 2.5% EGCG) yielded similar responses as the diet containing tea powder (Table 3). Although the average pupal weight of the improved diet was approximately 20 mg lower than the average pupal weight with the general purpose tea powder diet, significant differences were observed with pupation rate (t = –1.000, P = 0.356), pupal weight (t = –5.113, P = 0.248), and emergence rate (t = –0.701, P = 0.660).

Table 3 Mean ± SD pupation rate, larval duration, pupal weight, and emergence rate of Ectropis obliqua fed an improved artificial diet supplemented with (–)-epigallocatechin-3-gallate (EGCG) and the Zhang et al. (2007) artificial diet.

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In plants, phenols act as a defensive mechanism not only against herbivores, but also against microorganisms and competing plants (War et al. 2012). Artificial feeding experiments have shown that most plant phenols are toxic to many insects (Chan et al. 1978, Kubo et al. 2008).

Consumption of EGCG was positively correlated with epigallocatechin-3-gallate (PO) activity (an immune-related enzyme) of E. obliqua in a range of 0% to 2.5%. EGCG also enhanced PO activity in the mud crab, Scylla paramamosain Estampador (Wang et al. 2017). Therefore, EGCG may increase the activity of the enzyme so that E. obliqua immunity, and therefore growth, is improved. However, when the amount of EGCG concentration reached 3%, the EGCG may exceed the larvae's own detoxification capacity.