Poplars, Populus spp. (Salicaceae), are important tree species in China. However, insects and diseases are causing economic damage to these trees as larger areas are being planted with poplars. Conventional pesticides are routinely used to control insect and disease pests of poplars, but many of these compounds also negatively affect poplar trees (Tang et al. 2012). Therefore, it is crucial to identify chemistries that can manage pests but minimize the damage to the poplars.

Polyphenol oxidase (PPO), also known as tyrosinase (EC 1.14.18.1), is a copper enzyme that is widely distributed among plastids of microorganisms, plants, insects, and animals (Wang et al. 2005, Yang et al. 2005). It can catalyze both the hydroxylation of monophenols and the oxidation of o-diphenols into o-quinones. It is involved in melanin formation and in immune responses (Meng et al. 2004). Thus, PPO concentration in insects can be used as an index to assess the comparative competency of the insect's immune system (Cai et al. 2001). PPO is also localized in the thylakoid of chloroplasts and other types of plastids in plants where it can be used as an oxidoreductase in photosynthesis (e.g., regulating the rate of harmful photo-oxidation reaction, involvement in electron transfer) (Dai et al. 2007). Furthermore, PPO can promote wound healing and increase the resistance of plants to pathogens.

Clostera anastomosis (L.) (Lepidoptera: Notodontidae) is an important leaf-feeding insect on poplars for which insecticides are widely used for its control. Our objective in this study was to provide a basis for the identification and development of insecticides or allelochemicals that effectively manage this pest insect but have no deleterious effect on the host poplar tree. To this end, we compared the in vitro inhibitory effects of 21 selected chemicals on PPO activity in C. anastomosis larvae and in poplar trees.

Materials and Methods

Clostera anastomosis larvae used in these assays were initially collected from Nanjing (N 31°56′17.00′′, E 118°22′35.98′′) of Jingsu Province, China. They were transported to the laboratory and reared in a room maintained at 25 ± 1°C and 75% relative humidity on a 12L:12D-h photoperiod.

Poplar cuttings (populous × euramericana ‘NanLin895′) were obtained as seedlings from Nanjing Forestry University and grown in a growth chamber at 25 ± 1°C and with a 12L:12D-h photoperiod. They were watered daily and supplied with Hoagland nutrient solution (Afrousheh et al. 2010) each week. They were used in experiments after they had grown to a height of 60–80 cm.

The chemicals 2-tridecanone, quercetin (99%), and tannic acid (>99%) were purchased from Sigma Chemical Co. (St. Louis, MO). Catechol was purchased from Shanghai Qingxi Chemical Technology Co., Ltd. (Shanghai, China). Formulations, active ingredients, and manufacturers of the insecticides used in the tests are shown in Table 1. Allelochemicals and insecticides were dissolved in absolute acetone for experimentation. All other chemicals were of domestic analytical grade and purchased from commercial sources.

Table 1 Insecticides and their active ingredient concentration and manufacturer.

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Enzyme solutions were prepared by first homogenizing 10, fifth-instar larvae in 5 ml of phosphate buffer at 4°C (pH 7.5, 0.1 M) after peritrophic membranes and associated midgut contents were removed. The homogenate was centrifuged at 25,000g for 20 min at 4°C, and the supernatant was used to determine the enzyme activity after being filtered through three layers of cellulose filter paper (grade 3, Whatman, Middlesex, UK). Fresh poplar leaves (0.1 mg) were combined with 0.01 g polyvinylpolypyrrolidone (PVPP) and liquid nitrogen and were then ground. Phosphate buffer (1.5 ml) (pH7.5) was added, and the solution was centrifuged at 25,000g for 20 min at 4°C. The resultant supernatant was used to measure the enzyme activity. All experiments were performed in triplicate.

Diphenolase activity of PPO was assayed using the method described by Vanitha and Umesha (2011), modified by adding 5 mM catethol to the assay mixture and initiating the assay by adding 70 μl of enzyme. Absorbance at 420 nm was monitored for 2 min. The appropriate controls without any enzyme accompanied each assay. Enzyme activity was expressed as _______ [optical density (OD)]/min/mg protein. The method of Bradford (1976), with bovine serum albumin (BSA) as a standard, was used for protein quantification.

Inhibition of PPO activity was determined in assays containing 5 mM catethol and the various insecticides (0.17 mM) or allelochemicals (0.17 mM) serving as inhibitors. All assays, including controls, contained 1.7% acetone, and all were run in triplicate.

Dose-dependent inhibition of the PPO activity was measured with fixed concentrations of 5 mM catethol by adding 30 μl of insecticides or allelochemicals dissolved in acetone at various concentrations to the incubating reaction mixtures. The I₅₀ values, concentrations of inhibitors required to reduce the reaction rate by 50%, were determined by linear regression of the inhibition percent on the log of the inhibitor concentration. All experiments were performed in triplicate.

Results

The inhibition of the diphenolase activity of PPO in C. anastomosis larvae and poplar by insecticides and allelochemicals is shown in Table 2. Three inhibitors (quercetin, phenyl thiourea, and phoxim) had the strongest inhibitory effect on PPO in C. anastomosis larvae, inhibiting more than 60%. Among the tested inhibitors, four organophosphates (triazophos, chlorpyrifos, omethoate, profenofos), four pyrethroids (fenpropathrin, beta-cypermethrin, bifenthrin, lambda-cyhalothrin), and three other insecticides (acetamiprid, fipronil, pyridaben) were moderate inhibitors whereas two allelochemicals (tannic acid and 2-tridecanone), two organophosphates (malathion, isocarbophos), one carbamate (methomyl), and other insecticides (hexaflumuron, imidacloprid) were the least inhibitory. Seven inhibitors (phoxim, omethoate, profenofos, fenpropathrin, phenyl thiourea, pyridaben, and quercetin) had the strongest inhibitory effect on PPO in poplar. For the diphenolase activity of PPO, phenyl thiourea was the most potent inhibitor tested, inhibiting more than 60% of PPO activity at a final concentration of 0.17 mM. Furthermore, five organophosphates (triazophos, malathion, phoxim, omethoate, and profenofos), two pyrethriods (fenpropathrin and beta-cypermethrin), three other insecticides (hexaflumuron, pyridaben, and acetamiprid), and allelochemicals (quercetin) were moderate inhibitors. In addition, one organophosphate insecticide (chlorpyrifos), one carbamate insecticide (methomyl), two pyrethriods (bifenthrin and lambda-cyhalothrin), two allelochemicals (tannic acid and 2-tridecanone), and two other insecticides (fipronil and imidacloprid) were the least inhibitory.

Table 2 Inhibition of polyphenol oxidase by insecticides and allelochemicals in C. anastomosis and poplars.*

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The sensitivity of PPO activity to three inhibitors (quercetin, phenyl thiourea, and phoxim) was also evaluated. Quercetin, phenyl thiourea, and phoxim inhibited the diphenolase activity of PPO in vitro in a dose-dependent manner (Fig.1A, B, Fig. 2A, B, Fig. 3A, B). The I₅₀ values of these three inhibitors for PPO activity in C. anastomosis larvae ranged from 1.84 × 10⁻⁷ M to 1.28 × 10⁻⁴ M and in poplars from 1.51 × 10⁻⁴ M to 3.35 × 10⁻⁴ M (Table 3).

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Table 3 I₅₀ values of inhibitors for the activity of polyphenol oxidase in C. anastomosis larvae and poplars.*

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Discussion

PPO is an important enzyme in insects and plays a key role in the tanning process of insect cuticle. It is also involved in melanin formation and wound healing in insects (Zhou and Jiang. 2004). In plants, PPO catalyzes ortho-dihydroxy phenol into ortho-quinine, which may impact solubility of proteins or amino acids and the nutritional value and may serve as a component of plant defense mechanisms (Ludlum et al. 1991; Mahanil et al. 2008).

In vitro inhibition assays may help clarify modes of action of insecticides and facilitate the development of novel insecticides as well as serve as a reasonable evaluation of pesticides (Liu et al. 2014). However, there were few studies on the in vitro inhibition of polyphenol oxidase in insects. Liu et al. (2004) showed that the I₅₀ value of phenyl thiourea for the activity of PPO in Musca domestica (L.) was 1.5 × 10⁻⁷ M whereas three insecticides (phoxim, methomyl, and imidacloprid) were not significantly inhibitory. Liang et al. (2003) also reported that the I₅₀ values of phenyl thiourea for the activity of PPO from both the resistant and susceptible strains of Plutella xylostella (L.) ranged from 1.18 × 10⁻⁶ M to 1.28 × 10⁻⁶ M with no obvious differences. In addition, Tang et al. (2009) showed that the I₅₀ values of these three inhibitors (quercetin, phenyl thiourea, and phoxim) for PPO activity in Micromelalopha troglodyta (Graeser) were 5.24 × 10⁻⁵ M, 3.34 × 10⁻⁸ M, and 7.25 × 10⁻⁵ M, respectively. In our study, we examined the in vitro inhibition of 21 selected insecticides and allelochemicals on the activity of PPO not only in C. anastomosis larvae but also in poplar. Our results show that quercetin, phenyl thiourea, and phoxim can strongly inhibit the activity of PPO in both C. anastomosis and poplars, but their inhibitory degree differed significantly. The I₅₀ values of these three inhibitors for polyphenol oxidase activity were 14.17, 0.18, and 127.67 μM, respectively, in C. anastomosis while in poplars the values were 0.34, 0.15 and 0.21 mM, respectively. These results suggest that quercetin and phenyl thiourea might offer a degree of control of insect pests while producing little damage to the host poplar plant.

Our finding that phenyl thiourea was the greatest inhibitor of PPO activity in C. anastomosis (I₅₀ = 1.84 × 10⁻⁷ M) was consistent with that of Tang et al. (2009), who reported the I₅₀ value of phenyl thiourea for the PPO activity in M. troglodyta was 3.34 × 10⁻⁸ M. Liu et al. (2004) and Liang et al. (2003) also reported that I₅₀ values of phenyl thiourea for the activity of PPO in M. domestica and P. xylostella were 1.5 × 10⁻⁷ M and 1.18 × 10⁻⁶–1.28 × 10⁻⁶ M, respectively. Furthermore, we found that phenyl thiourea was about 1,000 times more inhibitory for the activity against PPO in poplars (I₅₀ = 1.51 × 10⁻⁴ M) than in C. anastomosis. Perhaps these results will serve as a foundation for the development of effective, selective PPO inhibitors and, thus, novel insecticides.

We also found that some allelochemicals might be potent inhibitors of insects. The I₅₀ value of quercetin for the diphenolase activity of PPO in C. anastomosis was 1.42 × 10⁻⁵ M, which was consistent with the findings of others. Tang et al. (2009), for example, reported that the I₅₀ value of quercetin for the activity of PPO in M. troglodyta was 5.24 × 10⁻⁵ M, and Luo et al. (2005) showed that the I₅₀ value of quercetin for the activity of PPO in Spodoptera exigua (Hübner) was 8.70 × 10⁻⁵ M. Further, our results showed that the I₅₀ value of quercetin for the activity of PPO in poplars was 3.35 × 10⁻⁴ M, which was approximately 23.6 times greater than in C. anastomosis. Therefore, quercetin might have the potential of serving as a synergist for enhancing the toxicity of pesticides in C. anastomosis.

Zhang and Leng (1993) summarized that an appropriate approach to explore novel insecticide development was to explore biological pathways and that PPO may become a target for controlling insect pests. Therefore, the research on PPO requires further investigation.