Effects of Honeysuckle Varieties on Protective and Detoxifying Enzyme Activities in Heterolocha Jinyinhuaphaga Chu (Lepidoptera: Geometridae) Larvae

Investigating the effects of various host plants on protective and detoxifying enzyme activities in insects could provide insights into the adaptation mechanisms of insects to host plants. In the present study, we measured superoxide dismutase (SOD), peroxidase (POD), catalase(CAT), carboxylesterase(CarE), acetylcholinesterase (AchE), and glutathione S-transferase (GST) activity levels in Heterolocha jinyinhuaphaga Chu (Lepidoptera: Geometridae) larvae fed on four honeysuckle varieties (wild variety, Jiufeng 1, Xiangshui 1, and Xiangshui 2). The results showed that levels of SOD, POD, CAT, CarE, AchE, and GST activities in H. jinyinhuaphaga larvae fed on the four honeysuckle varieties differed. The enzyme activity levels were the highest when larvae were fed on the wild variety, followed by Jiufeng 1 and Xiangshui 2, and the lowest when fed on Xiangshui 1. Furthermore, the enzyme activity levels increased with an increase in larval age. According to the results of two - way analysis of variance, the interaction between host plants and larval age had no significant effect on SOD, POD, CAT, CarE, AchE, and GST activities in H. jinyinhuaphaga larvae (p ˃0.05).

Keywords: Heterolocha jinyinhuaphaga; Enzyme activity; Protective enzyme; Detoxifying enzyme; Host plant

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Introduction

Herbivorous insects feed on suitable host plants during their normal growth and development, and they can digest and absorb nutrients from the host plants. Such insects can also break down and excrete the toxic substances. However, host plants may produce a wide variety of secondary metabolites, which provide defense against insect herbivory (Li et al. [16]; Huang et al. [10]). Studies have shown that nutrient composition and secondary metabolites may vary across different varieties of host plants (Abdel-Aal et al. [1]; Zheng et al. [44]; Wang et al. [31]; Chen et al. [8]). Feeding on a variety of host plants can result in varying physiological and biochemical responses among herbivorous insects, which in turn significantly influence their growth and development, such as fecundity, longevity, and survival (Slansky and Scriber [27]; Awmack and Leather [3]; Hwang et al. [12]; Saeed et al. [25]; Lytan and Firake [20]; Carrasco et al. [5]). Typically, herbivorous insects select the most nutritious plants as their host plants, and a proper diet invariably results in the rapid development of insects; however, plant secondary metabolites function as defensive mechanisms against feeding by insects. Therefore, herbivorous insects diet depend on their adaptation mechanisms to nutrients and secondary compounds in the host plants. During the long-term coevolution with host plants, insects have developed corresponding adaptation mechanisms in relation to physiological functions, such as the secretion of protective and detoxifying enzymes, which are generally considered to be one of the most important forms of insect adaptation to plant diets.

The protective enzymes in insects include superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), which can eliminate free radicals, maintain free radicals at low levels, and prevent poisoning of insects by free radicals (Acar et al. [2]; Krishnan and Kodrik [14]). Carboxylesterase (CarE), acetylcholinesterase (AchE), and glutathione S-transferase (GST) are the main detoxifying enzymes in insects, which are involved in the detoxification of xenobiotics (Tiwari et al. [28]; Yang et al. [38]). According to previous studies, detoxifying and protective enzyme activities among insects vary with the types of host plants (Lindroth [17]; Wang et al. [29]; Yin et al. [40]; Saha et al. [26]; Rigsby et al. [24]; Yang et al. [39]). For example, a previous study revealed that, SOD activity levels in Hyphantria cunea larvae fed on plum leaves were the highest, and those fed on walnut leaves were the lowest, CAT activity levels were the highest when larvae fed on plum leaves, and those fed on apple leaves were the lowest (Yanar et al. [37]). GST and cytochrome P450 monooxygenases (CYP450) levels in Helopeltis theivora Waterhouse increase considerably when the insect pest feeds on Mikania micrantha Kunth (Asteraceae) and Psidium guajava L.(Myrtaceae) when compared to those feeding on Camellia sinensis (Theaceae) (Saha et al. [26]). Furthermore, protective and detoxifying enzyme activities among insects were also different when feeding on different varieties of the same host plant species(Liu et al. [18]). Therefore, investigating the effects of various host plants on the protective and detoxifying enzyme activities in insects could provide insight into the adaptation mechanisms of herbivorous insects to host plants.

The honeysuckle geometrid, Heterolocha jinyinhuaphaga Chu (Lepidoptera: Geometridae) is a major pest of the honeysuckle plant (Lonicera japonica Thunb). The insect produces up to three generations annually in China (Xiang et al. [32]), and its life cycle consists of egg, larva, pupa, and adult stages. The moth can mate on the first night after emergence and females oviposit during 6–8 h after mating. Larvae feed on honeysuckle leaves after hatching and often cause extensive damage to plants, which results in substantial economic losses in many regions of China (e.g., Shandong, Henan, and Anhui provinces) (Xiang et al. [32]). The numbers of larvae in the first - generation peaks in mid-May, followed by the second generation that peaks in mid to late July, and the third generation that peaks in late September. Overwintering of pupae begins in early October.

Several studies have focused on investigating the biological characteristics and control methods of H. jinyinhuaphaga (Ni et al. [21]; Xiang et al. [32], [33], [34], [35]). However, no studies have investigated the roles of protective and detoxifying enzymes in H. jinyinhuaphaga. In the present study, we first evaluated the potential effects of four honeysuckle varieties (wild variety, Jiufeng 1, Xiangshui 1, and Xiangshui 2) on the activities of three protective enzymes (SOD, POD, and CAT) and three detoxifying enzymes (CarE, AchE, and GST) in H. jinyinhuaphaga larvae.

Materials and Methods

Insect Rearing

Heterolocha jinyinhuaphaga larvae were collected from honeysuckle plantations of Jiufeng 1 variety in May in Chuzhou city, Anhui province, China, and reared with honeysuckle leaves (Jiufeng 1 variety) in the laboratory at a temperature of 25 ± 1 °C, relative humidity of 70± %, and photoperiod of 14:10 h (light : dark cycle). Larvae were reared together before the third instar, and then reared individually after the third instar to avoid cannibalism. Male and female pupae were placed separately in round glass jars (1000 mL) under the same conditions, as previously described. The newly emerged male and female moths were collected daily, maintained in round glass jars (1000 mL) and fed with 5% sugar solution to facilitate mating and oviposition.

Determination of Secondary Metabolites Contents in Host Plant

Honeysuckle leaves were collected from the same plant parts of different honeysuckle varieties (3 years old), they were then dried for about 96 h to constant weight at 50 °C in an oven. The dried honeysuckle leaves were ground into a powder using a pulverizer.

The total flavonoid contents of the leaves were determined by ultraviolet spectroscopy method (Li et al. [15]), the standard solution was prepared using rutin and the optical density (OD) value was determined at the maximum absorption wavelength to obtain the standard curve. The chlorogenic acid contents were determined by ultraviolet spectroscopy method (Huang et al. [11]), the standard solution was prepared using chlorogenic acid and the optical density (OD) value was determined at the maximum absorption wavelength to obtain the standard curve. The tannin contents were determined by Folin-Denis method (Folin and Denis [9]), tannin was used as a standard, and tannin contents were calculated using the tannin standard curve.

Determination of Host Plant Effects on Protective and Detoxifying Enzyme Activities in H. jin...

Newly hatched H. jinyinhuaphaga larvae were placed individually in round glass jars (500 mL), and reared with leaves of different varieties of honeysuckle (Jiufeng 1, Xiangshui 1, Xiangshui 2, and a wild variety). Leave bases of host plants were wrapped with wet cotton to keep the leaves fresh, and then the jars were covered with pieces of gauze and maintained under the same conditions as previously described. The leaves were replaced daily at 9 o'clock until the fifth instar larval stage was reached. Larval instar stages were identified based on the larval head capsule widths. A total of 100 larvae were reared simultaneously for each honeysuckle variety.

Preparation of Enzyme Solutions

Ten fifth instar H. jinyinhuaphaga larvae fed on each honeysuckle variety were placed in 5 mL of phosphate buffer, homogenized in an ice bath, and the homogenate was centrifuged for 20 min (12,000 r/min, 4 ℃). The supernatants were each placed in centrifuge tubes and used as enzyme sources. The phosphate buffer dosages for SOD, POD, CAT, CarE, AchE, and GST were 0.1 mol/L (pH 7.6), 0.2 mmol/L(pH 7.0), 0.05 mmol/L (pH 7.0), 0.1 mol/L (pH 7.6), 0.1 mol/L (pH 8.0), and 0.1 mol/L (pH 8.0), respectively.

Determination of Superoxide Dismutase Activity

SOD activity was determined using the photochemical nitro blue tetrazolium (NBT) reduction method (Zhu et al. [46]). Reaction mixtures (3 mL) which comprised 0.7 mL phosphate buffer (0.1 mol/L, pH 7.6), 1.2 mL methionine (Met,13 mmol/L), 0.4 mL nitroblue- tetrazolium (75 µmol/L), 0.1 mL ethylenediaminetetraacetic acid (0.1 mmol/L), 0.3 mL enzyme solution, and 0.3 mL riboflavin (20 µmol/L) were placed in test tubes and allowed to react. The reaction was terminated in the dark after the mixtures reacted for 15 min under illumination of 4 000 lx at 25 ℃, and optical density (OD) values were immediately measured at a wavelength of 560 nm. For the control group, similar amounts of distilled water were added to replace enzyme solutions. Each treatment was repeated six times. The required enzyme amount caused 50% inhibition of NBT by 1 mg of enzyme protein in 1 mL of reaction solution was defined as one unit of enzyme activity (U), and enzyme activity was expressed as U/mg. SOD activity was calculated according to the following formula :SOD activity (U/mg) = (A0-A)×V/[A0 × 50%×W×V0 ] (Liu et al. [18]).

Where A0 is the OD value for the control group, A is the OD value for the sample, V is the total volume of reaction mixture (mL), V0 is the enzyme volume (mL), and W is the protein concentration in enzyme solution (mg/mL).

Determination of Peroxidase Activity

POD activity was determined based on a previously described colorimetry method using guaiacol (Zhu et al. [46]). Reaction mixtures (3 mL), which comprised 1 mL of phosphate buffer (0.2 mmol/L, pH 7.0), 1 mL hydrogen peroxide (H2O2 , 30%), 0.9 mL guaiacol (30 mmol/L), and 0.1 mL enzyme solution, were placed in test tubes and allowed to react. The OD values were measured at a wavelength of 470 nm after the reaction mixtures reacted for 5 min. For the control group, similar amounts of distilled water were added to replace enzyme solutions. Each treatment was repeated six times. The OD value changes per minute caused by 1 mg of enzyme was defined as one unit of enzyme activity (U), and enzyme activity was expressed as U/mg. POD activity was calculated according to the following formula:POD activity (U/mg) = ΔOD470 / [t×W×(V0 / V)].

Where ΔOD470 is the change in absorbance value with reaction time, t is the reaction time (min), V is volume of enzyme solution (mL), V0 is the volume of the reaction mixture (mL), and W is the protein concentration in enzyme solution (mg/mL).

Determination of Catalase Activity

Reaction mixtures (3 mL) which comprised 1 mL phosphate buffer (0.05 mol/L, pH 7.0), 0.2 mL H2O2 (30%), 1.7 mL distilled water, and 0.1 mL enzyme solution, were placed in test tubes and allowed to react (Zhang et al. [43]). The OD values of the reaction mixtures were measured immediately at a wavelength of 240 nm. Two minutes later, the OD values were measured again. For the control group, similar amounts of distilled water were used to replace enzyme solutions. Each treatment was repeated six times. The OD value changes per minute caused by 1 mg of enzyme was defined as one unit of enzyme activity (U), and enzyme activity was expressed as U/mg. CAT activity was calculated according to the following formula:CAT activity (U/mg) = ΔOD240 /[t×W×(V0 / V)].

Where ΔOD240 is the change in absorbance value with reaction time, t is the reaction time (min), V is the total volume of enzyme solution (mL), V0 is the volume of the reaction mixture (mL), and W is the protein concentration in enzyme solution (mg/mL).

Determination of Carboxylesterase Activity

CarE activity was determined according to a previously described method (Liu et al. [18]). Enzyme solutions (10 µL), 400 µL α-napthyl acetate (300 µmol/L), 30 µL physostigmine (10 µmol/L), and 560 µL of phosphate buffer (0.1 mol/L, pH 7.6) were placed in test tubes. After shaking the reaction mixtures by orbital shaker for 30 min at 30 ℃, 100 µL of chromogenic reagent (1% of Fast blue B salt : 5% of sodium dodecyl sulfate solution = 2:5) was added to the reaction mixtures to terminate the reaction. OD values for the reaction mixtures were measured at a wavelength of 600 nm after 15 min. A standard curve of α-naphthol was generated and used to calculate the production. One micromole of production catalyzed by 1 mg of enzyme per minute was defined one unit of enzyme activity (U), and enzyme activity was expressed as U/mg. For the control group, similar amounts of distilled water were added to replace enzyme solutions. Each treatment was repeated six times. CarE activity was calculated according to following formula:CarE activity (U/mg) = P/[t×W×(V0 / V)] (Liu et al. [18]).

Where P is the production quantity (µmol), t is the reaction time (min), V is the volume of enzyme solution (mL), V0 is the volume of the reaction mixture (mL), and W is the protein concentration in enzyme solution (mg/mL).

Determination of Acetylcholinesterase Activity

AchE activity was determined according to a previously described method (Yin et al. [40]). Enzyme solutions (50 µL), 2.8 mL phosphate buffer (0.1 mol/L, pH 8.0), 50 µL acetylcholine iodide (0.075 mol/L), 0.1 mL 2-nitrobenzoic acid (0.01 mol/L) were placed in test tubes. After heating the reaction mixtures in a water bath (25 ℃) for 15 min, 0.1 mL physostigmine (1 × 10− 3 mol/L) was added. Subsequently, the changes in OD values for the reaction mixtures within the first 10 min were measured at a wavelength of 410 nm. For the control group, similar amounts of distilled water were added to replace enzyme solutions. A standard curve of glutathione was generated and used to calculate the production. A total of 1 µmol of production catalyzed by 1 mg of enzyme per minute was defined as one unit of enzyme activity (U), and enzyme activity was expressed as U/mg. Each treatment was repeated six times. AchE activity was calculated according to the following formula:AchE activity (U/mg)= [(A-A0) /(A1-A2)]×C/(t× W).

Where A is the OD value of the sample, t is the reaction time (min), A0 is the OD value of the control, A1 is the OD value of the standard sample, A2 is the OD value of the blank, C is the dosage of the standard sample (µmol/mL), W is the protein concentration in enzyme solution (mg/mL).

Determination of Glutathione S-transferase Activity

GST activity was determined according to a previously described method (Yin et al. [40]). Enzyme solutions (100 µL), 2.9 mL phosphate buffer (0.1 mol/L, pH 8.0), 0.7 mL reduced glutathione (0.03 mol/L), 50 µL 1-chloro-2, 4-dinitrobenzene (CDNB, 0.1 mol/L) were placed in test tubes. After full oscillation, the reaction mixtures were placed in a water bath (25 ℃) for 5 min. Afterward, the changes in OD values for the reaction mixtures within the first 5 min were measured at a wavelength of 340 nm. For the control group, similar amounts of distilled water were added to replace enzyme solutions. Each treatment was repeated six times. The log (GST) drop value per minute was obtained by deducting the non-enzymatic reaction (sample OD - non-enzymatic OD). A decrease in log (GST) by 0.001 per minute caused by 1 mg of enzyme was defined as one unit of enzyme activity (U), and GST activity was expressed as U/mg. GST activity was calculated according to the following formula:GST activity (U/mg) =[ (Δ OD340×V)/(ε×L)] / W (Liu et al. [18]).

Where ΔOD340 is the variation in OD values per minute (ΔOD340/min), V is the reaction mixture volume (mL), ε is the extinction coefficient (9.6 mmol/cm), L is the optical path of the colorimetric cup, and W is the protein concentration in enzyme solution (mg/mL).

Measurement of Protein Content

Protein content was determined using the Coomassie brilliant blue G-250 method (Bradford [4]), where samples (0.1 mL) were placed in centrifuge tubes and 5. 0 mL Coomassie brilliant blue G-250 solution was added. OD values were measured at a wavelength of 595 nm after 10 min of reaction. Bovine serum albumin was used as a protein standard, and protein contents in original enzyme solutions were calculated using the bovine serum albumin standard curve.

Statistical Analysis

Data were analyzed using SPSS Statistics v11.5 (SPSS Inc., Chicago, IL, USA). Comparisons of means between different host varieties and larval ages were analyzed using one-way and two-way analysis of variance (ANOVA), where differences among means were compared using Tukey's multiple comparison test at a P < 0.05 significance level.

Results

Secondary Metabolites Contents in Host Plant

Secondary metabolites contents differed among honeysuckle varieties (Table 1). The wild variety had the highest total flavonoid, chlorogenic acid, and tannin contents, i.e.,7.65%, 3.12%, and 0.94%), respectively, while Xiangshui 1 had the lowest total flavonoid, chlorogenic acid, and tannin contents (6.44%, 2.68%, and 0.73%, respectively).

Table 1 Major secondary metablites contents of leaves from different honeysuckle varieties

Variety	Total flavonoid content (%)	Chlorogenic acid content (%)	Tannin content (%)
Jiufeng 1	7.22 ± 1.09a	2.89 ± 0.14a	0.86 ± 0.01a
Xiangshui 1	6.44 ± 0.58a	2.68 ± 0.29a	0.73 ± 0.02a
Xiangshui 2	6.73 ± 0.48a	2.71 ± 0.44a	0.79 ± 0.01a
Wild variety	7.65 ± 1.08a	3.12 ± 0.28a	0.94 ± 0.03a

The data in the table represent the mean ± SE. Letters after the results are based on multiple comparison tests, where the different lower case letters in the same column indicate a significant difference at P < 0.05.

Superoxide Dismutase Activity

SOD activity levels in H. jinyinhuaphaga larvae differed fed on the various honeysuckle varieties (Fig. 1-A). SOD activity levels were the highest when the first instar larvae were fed on wild variety (48.32 U/mg), followed by Jiufeng 1 (46.25 U/mg), and Xiangshui 2 (25.73 U/mg), and the lowest when larvae were fed on Xiangshui 1 (24.27U/mg). No significant differences were observed in SOD activity levels between larvae fed on the wild variety and Jiufeng 1, and those fed on Xiangshui 1 and Xiangshui 2; however, significant differences were observed in SOD activity levels among larvae fed on other varieties (df1 = 3, df2 = 8, F = 270.277, p < 0.0001). SOD activity increased with an increase in larval age, and SOD activity levels in the fifth instar larvae fed on wild variety, Jiufeng 1, Xiangshui 2 and Xiangshui 1 were 58.77 U/mg, 54.48 U/mg, 35.55 U/mg, and 33.31 U/mg respectively, and increased by 21.63%, 17.79%, 38.17%, and 37.25% when compared to those in the first instar larvae. No significant differences were observed in SOD activity between the fifth instar larvae fed on Xiangshui 1 and Xiangshui 2; however, significant differences were observed in SOD activities among fifth instar larvae fed on other varieties (df1 = 3, df2 = 8, F = 158.446, p < 0.0001).

Graph: Fig. 1 Protective and detoxifying enzyme activities in H. jinyinhuaphaga larvae fed on four honeysuckle varieties (wild variety, Jiufeng 1, Xiangshui 1, and Xiangshui 2). (The letters above the columns represent the results of the Tukey's multiple comparison test for enzyme activities among the same larval instars fed on the various honeysuckle varieties; the different lowercase letters represent statistically significant differences at P<0.05)

According to the results of two-way ANOVA, the interaction between host plant and larval age had no significant effect on SOD activity in H. jinyinhuaphaga larvae (F = 0.02, df1 = 2, df2 = 4, p ˃ 0.05).

Peroxidase Activity

POD activity levels in H. jinyinhuaphaga larvae differed fed on the various honeysuckle varieties (Fig. 1-B). POD activity levels were the highest when the first instar larvae were fed on the wild variety (30.78 U/mg), followed by Jiufeng 1 (26.19 U/mg), and Xiangshui 2 (24.27 U/mg), and the lowest when larvae were fed on Xiangshui 1 (18.43 U/mg). When the larvae were fed on Jiufeng 1 and Xiangshui 2, there were not significant differences in POD activity levels between them; however, significant differences were observed in POD activities among larvae fed on other varieties (df1 = 3, df2 = 8, F = 14.776, p = 0.001). With the increasing of larval age, POD activity increased. POD activity levels in the fifth instar larvae fed on wild variety, Jiufeng 1, Xiangshui 2 and Xiangshui 1 were 38.84 U/mg, 34.72 U/mg, 30.45 U/mg, and 28.81 U/mg respectively, and increased by 26.19%, 32.57%, 25.46%, and 56.32% than those in the first instar larvae. There were not significant differences in the POD activity between fifth instar larvae fed on Xiangshui 1 and Xiangshui 2; however, significant differences were observed in POD activities among fifth instar larvae fed on other varieties (df1 = 3, df2 = 8, F = 17.253, p = 0.001).

Two - way ANOVA showed that the interaction between host plant and larval age had no significant effect on POD activity in H. jinyinhuaphaga larvae (F = 0.08, df1 = 2, df2 = 4, p ˃ 0.05).

Catalase Activity

CAT activity levels in H. jinyinhuaphaga larvae differed fed on the various honeysuckle varieties (Fig. 1-C). When the first instar larvae were fed on the wild variety, CAT activity levels in them were the highest (44.82 U/mg), followed by Jiufeng 1 (43.81 U/mg), Xiangshui 2 (38.47 U/mg), and Xiangshui 1 (35.92 U/mg). There were not significant differences in CAT activity between larvae fed on the wild variety and Jiufeng 1, and those fed on Xiangshui 1 and Xiangshui 2; however, significant differences were observed in CAT activities among larvae fed on other varieties (df1 = 3, df2 = 8, F = 17.812, p = 0.001). With an increase in larval age, CAT activity increased. In the fifth instar larvae fed on wild variety, Jiufeng 1, Xiangshui 2 and Xiangshui 1, CAT activity levels were 61.46 U/mg, 55.71U/mg, 50.83 U/mg, and 48.47 U/mg respectively, and increased by 37.12%, 27.16%, 32.13%, and 34.94% than those in the first instar larvae. No significant differences were observed in CAT activity between the fifth instar larvae fed on Xiangshui 1 and Xiangshui 2; however, there were significant differences in CAT activities among the fifth instar larvae fed on other varieties (df1 = 3, df2 = 8, F = 34.457, p < 0.0001).

Two - way ANOVA showed that the interaction between host plant and larval age had no significant effect on CAT activity in H. jinyinhuaphaga larvae (F = 0.001, df1 = 2, df2 = 4, p ˃0.05).

Carboxylesterase Activity

CarE activity levels in H. jinyinhuaphaga larvae differed fed on the various honeysuckle varieties (Fig. 1-D). In the first instar larvae fed on the wild variety, CarE activity levels were the highest (18.69 U/mg), followed by Jiufeng 1(18.35 U/mg), Xiangshui 2 (16.33U/mg), and Xiangshui 1 (14.32 U/mg). Between the larvae fed on the wild variety and Jiufeng 1, and those fed on Xiangshui 1 and Xiangshui 2, there were not significant differences in CarE activity; however, there were significant differences in CarE activities among larvae fed on other varieties (df1 = 3, df2 = 8, F = 6.161, p = 0.018). CarE activity increased with larval age increase, and CarE activity levels in the fifth instar larvae were 26.37 U/mg, 24.63U/mg, 23.83 U/mg, and 22.44 U/mg when fed on wild variety, Jiufeng 1, Xiangshui 2 and Xiangshui 1 respectively, and increased by 41.09%, 34.22%, 45.93%, and 56.70% than those in the first instar larvae. Except the fifth instar larvae fed on the wild variety and Jiufeng 1, and those fed on Xiangshui 1 and Xiangshui 2, there were significant differences in CarE activities among the fifth instar larvae fed on other varieties (df1 = 3, df2 = 8, F = 5.053, p = 0.03).

The results of two -way ANOVA showed that the interaction between host plant and larval age had no significant effect on the CarE activity in H. jinyinhuaphaga larvae (F = 0.10, df1 = 2, df2 = 4, p ˃ 0.05).

Acetylcholinesterase Activity

AchE activity levels in H. jinyinhuaphaga larvae were different fed on the various honeysuckle varieties (Fig. 1-E). The first instar larvae fed on the wild variety had the highest AchE activity levels (16.68 U/mg), followed by Jiufeng 1 (14.71 U/mg), Xiangshui 2 (13.54 U/mg), and Xiangshui 1 (12.22 U/mg). There were significant differences in AchE activity between larvae fed on the wild variety and Xiangshui 1, and those fed on the wild variety and Xiangshui 2 (df1 = 3, df2 = 8, F = 6.741, p = 0.014). AchE activity increased with larval age increase, their activity levels in the fifth instar larvae fed on wild variety, Jiufeng 1, Xiangshui 2 and Xiangshui 1 were 23.63 U/mg, 22.34 U/mg, 20.76 U/mg, and 19.23 U/mg respectively, which increased by 41.67%, 51.87%, 53.32%, and 57.36% than those in the first instar larvae. Significant differences were observed in AchE activity between the fifth instar larvae fed on the wild variety and Xiangshui 1, and those fed on Jiufeng 1 and Xiangshui 1 (df1 = 3, df2 = 8, F = 4.881, p = 0.032); however, there were no significant differences in AchE activities among the fifth instar larvae fed on other varieties.

The results of two -way ANOVA showed that the interaction between host plant and larval age had no significant effect on AchE activity in H. jinyinhuaphaga larvae (F = 0.053, df1 = 2, df2 = 4, p ˃ 0.05).

Glutathione S-transferase Activity

H. jinyinhuaphaga larvae had different GST activity levels fed on the various honeysuckle varieties (Fig. 1-F). In the first instar larvae, GST activity levels were the highest fed on the wild variety (55.83 U/mg), then were the second fed on Jiufeng 1 (53.79 U/mg), followed by Xiangshui 2 (52.53 U/mg) and Xiangshui 1(50.68 U/mg). Except larvae fed on the wild variety and Xianghui 1, no significant differences were observed in GST activities among larvae fed on other varieties (F = 3.246, df1 = 3, df2 = 8, p = 0.081). With the increasing of larval age, GST activity increased, and reached the highest levels in the fifth instar larvae fed on wild variety, Jiufeng 1, Xiangshui 2 and Xiangshui 1, which were 68.39 U/mg, 65.63 U/mg, 63.14 U/mg, and 61.46 U/mg respectively. GST activity levels in the fifth instar larvae increased by 22.49%, 22.01%, 20.19%, and 21.27% when compared to those in the first instar larvae. There were significant differences in GST activity between the fifth instar larva fed on the wild variety and the other three varieties (df1 = 3, df2 = 8, F = 6.63, p = 0.015); however, no significant differences were observed in GST activities among the fifth instar larvae fed on the other three varieties.

The results of two -way ANOVA showed that the interaction between host plant and larval age had no significant effect on GST activity in H. jinyinhuaphaga larvae (F = 0.042, df1 = 2, df2 = 4, p ˃ 0.05).

Discussion

The selection of host plants by herbivorous insects is closely associated with their enzyme activities (Karasov et al. [13]). Secondary metabolites in host plants could influence the protective and detoxifying enzyme activities in herbivorous insects (Chen et al. [6]; Zeng et al. [41]). For example, low dosages of tannin could induce an increase of GST activity in Helicoverpa armigera (Hübner) (Chen et al. [6]). SOD, CAT, and POD activities in Apolygus lucorum increased fed on artificial diet containing different dosages of tannin at 24 ~ 72 h, then they decreased (Luo and Cui [19]). GST activities in adipose body of Spodoptera litura larvae increased significantly fed on artificial diet containing flavone (Wang et al. [30]). GST and CYP450 activities in the gut of Hyphantria cunea were significantly induced fed on artificial diet containing different dosages of chlorogenic acid, and reached the highest levels at dosages of 1.00% (Pan et al. [22]).

Secondary metabolites in host plants activate or inhibit protective and detoxifying enzyme activities in herbivorous insects depending on the types of host plant species fed on by the insects, which in turn, influence secondary metabolism, and causes insects to develop various adaptation mechanisms to host plants (Saha et al. [26]; Razmjou et al. [23]). The adaptation mechanisms of enzyme systems to secondary metabolism influence host range expansion of herbivorous insects. The results of the present study have demonstrated that SOD, POD, CAT, CarE, AchE, and GST activities in H. jinyinhuaphaga larvae fed on the four honeysuckle varieties (wild variety, Jiufeng 1, Xiangshui 1, and Xiangshui 2) varied considerably. Enzyme activity levels were the highest when larvae were fed on the wild variety, followed by Jiufeng 1 and Xiangshui 2, and the lowest when larvae were fed on Xiangshui 1. A previous study (Xiang et al. [33]) investigated the feeding preferences of H. jinyinhuaphaga larvae using the four honeysuckle varieties used in the present study, and the results revealed that the feeding preferences of the insect larvae were consistent with the enzyme activities observed in the present study. The observation suggests that the wild variety is the most preferred host plant among the four host pant varieties, and H. jinyinhuaphaga larvae have developed associated adaptation mechanisms to overcome defense compounds in the host plant. However, the protective and detoxifying enzyme activities in other insect species increase when they feed on resistant host plant varieties. For example, SOD and CAT activities increased significantly in Sogatella furcifera fed on a resistant Oryza sativa variety (N22) and highly resistant Oryza sativa variety (Ptb33) (Chen et al. [7]). A few studies have revealed that enzyme activities among herbivorous insects feeding on different host plants vary. For example, SOD activity in Bemisia tabaci was the lowest when it fed on Solanum lycopersicum; POD activity was the lowest when it fed on Abutilon theophrasti; CarE and GSTs activities were the lowest when it fed on Cucumis sativus (Zhou et al. [45]). CarE activity in adult Anoplophora glabripennis was the highest when it fed on Acer negundo, and GST activity was the highest when it fed on Betula platyphylla (Yan et al. [36]). The findings suggest that different insect species may have developed their own adaptation mechanisms to host plants, and that various host plants contain different types of secondary metabolites, which results in the variations in enzymes activity (Zhang et al. [42]).

Overall, SOD, POD, CAT, CarE, AchE, and GST activities in H. jinyinhuaphaga larvae fed on the four varieties of honeysuckle (wild variety, Jiufeng 1, Xiangshui 1, and Xiangshui 2) increased with an increase in larval age, and reached the highest levels in the fifth instar larvae. According to the results, enzyme activities in the larvae were enhanced to promote a gradual adaptation to secondary metabolites in host plants, and the adaptability of larvae to host plants could ensure population growth and insect species survival. In addition, the interaction between host plants and larval age had no significant effect on SOD, POD, CAT, CarE, AchE, and GST activities in H. jinyinhuaphaga larvae.

In conclusion, the present study revealed various adaptation mechanisms of H. jinyinhuaphaga larvae to different honeysuckle varieties, and provides insights into the roles of protective and detoxifying enzymes in enhancing tolerance of H. jinyinhuaphaga larvae to secondary metabolites in host plants, thereby presenting a pest management strategy through the cultivation of resistant host plant varieties. However, secondary metabolites among host plant species may vary; therefore, further studies should be conducted to determine the relationship between enzyme activity and secondary metabolites in host plants.

Acknowledgements

We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. This study was supported by the Academic Funding Project for the Top-Notch Personnel of College Subject Specialty (Grant No. gxbjZD2020089), and the Scientific Research Foundation of Chuzhou University (Grant No.2019qd02).

Author Contributions

Yuyong Xiang contributed with the experimental design, data analysis, manuscript writing, and manuscript review. Hehui Niu and Baoling Jin contributed with the experimental study. Yuanchang Zhang contributed with the data analysis. Peifeng Yin contributed with the experimental design.

Funding Information

This study was supported by the Academic Funding Project for the Top-Notch Personnel of College Subject Specialty (Grant No. gxbjZD2020089), and the Scientific Research Foundation of Chuzhou University (Grant No.2019qd02).

Declarations

Competing Interests

The authors declare that they have no conflict of interest relevant to the content of this article.

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By Yuyong Xiang; Hehui Niu; Baoling Jin; Yuanchang Zhang and Peifeng Yin

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