RESEARCH ARTICLE
Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications Long Pan, Lili Ren, Fang Chen, Yuqian Feng, Youqing Luo* Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing, 100083, P.R. of China * [email protected]
a11111
OPEN ACCESS Citation: Pan L, Ren L, Chen F, Feng Y, Luo Y (2016) Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications. PLoS ONE 11(5): e0155682. doi:10.1371/journal.pone.0155682 Editor: Youjun Zhang, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, CHINA Received: February 4, 2016 Accepted: May 3, 2016 Published: May 23, 2016 Copyright: © 2016 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by Special Project for Scientific Research of Forestry Commonweal Industry of National Forestry Bureau (201404401). Competing Interests: The authors have declared that no competing interests exist.
Abstract Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P50%
doi:10.1371/journal.pone.0155682.t001
recorded. The pest resistance percentage was calculated as the number of pest-free trees among the total number of trees in the plot. Pest resistance percentage ¼
Number of pest free trees 100 Total number of trees in the plot
Verification of Antifeedant Activity To make the normal diet, feed powder (20 g), distilled water (80 ml), and agar power (1.6g) were mixed and heated to completely dissolve the agar. To make the test diet, G. biloba secondary metabolites were dissolved in 10 ml ethanol before mixing them with the normal diet at different concentrations. All the diets were poured into a rearing box (100ml, d = 5cm) and allowed to solidify. The resulting circular pie (δ = 2mm) was then cut into two equal semicircles for future use. Six other rearing boxes of the same shape was prepared, and a semicircular pie of the normal diet and six semicircular pies containing 4%, 8%, or 16% G. biloba extract, 2% flavonoids, 0.4% ginkgolides, or 0.2% bilogalide were placed in the boxes. Then 30 third-instar H. cunea larvae were put into each box, and the number of larvae choosing to feed on each of the semicircular pies in the boxes was recorded after 24h. These experiments were repeated five times [17–20].
Detoxifying Enzyme Activity Assay Circular diet pies containing 8% G. biloba extracts, 2% ginkgo flavonoids, 0.4% ginkgolide, 0.2% bilobalide, or no extra additive were prepared as described above, to feed 120 third-instar H. cunea larvae in each group. The activities of glutathione transferase, acetylcholinesterase, carboxylesterase and mixed function oxidase were measured at 12, 24, 36, and 48 h for each group of 30 larvae. Third-, fourth-, and fifth-instar H. cunea larvae were fed on a diet containing the above four G. biloba secondary metabolites, and the activities of the above detoxifying were measured at 24 h in each group of enzymes in each group of 30 larvae. These experiments were repeated five times. Glutathione transferase (GSTs) activity was measured using Lee’s method. Briefly, the tissue sample was weighed, and 9 volumes of normal saline were added to produce a 10% tissue homogenate, which was then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was considered as the enzyme solution. The enzyme reaction was measured in a cuvette containing 2.4 mL phosphate buffer (66 mmol/L, pH 7.0), 0.1 mL enzyme solution, 0.3 mL of 50 mmol/L glutathione (GSH), and 0.1 mL of 30 mmol/L 1-chloro-2,4-dinitrobenzene (CDNB), and changes in absorption at 340 nm were recorded. The measurement was repeated five times [21]. Acetylcholinesterase (AChE) activity was measured using a modified Ellman method. Briefly, the tissue sample was weighed, and 9 volumes of normal saline were added to produce a 10% tissue homogenate, which was then centrifuged at 3,500 rpm for 10 min at 4°C. The
PLOS ONE | DOI:10.1371/journal.pone.0155682 May 23, 2016
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Study on insect resistance of Ginkgo biloba
supernatant was collected for enzyme activity measurements. Supernatant (0.1 mL) and ATCh (0.1 mL) (a substrate of AChE) were mixed together and incubated at 30°C for 15min, and then 3.6 mL DTNB was added to terminate the reaction. Absorbance was measured at 412nm. The experiment was repeated five times [22–23]. Carboxylesterase (CarE) activity was measured using Asperen’s method. Briefly, the tissue sample was weighed, and 9 volumes of normal saline were added to produce a 10% tissue homogenate, which was then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected and diluted 50× with phosphate buffer before using for enzyme activity measurement. The enzyme reactions contained 0.3 mL of the diluted supernatant, 0.7 mL of 0.04 mol/L phosphate buffer, and 5 mL substrate, which were mixed and incubated on a shaker for 30 min at 37°C. Then, 1 mL of chromogenic reagent was added, and the reactions were incubated for a further 30 min at room temperature, followed by measurement of absorbance at 600 nm. The experiment was repeated five times. Known concentrations of α-naphthol were used to obtain a standard curve [24]. Mixed function oxidase (MFO) activity was measured using Yu and Nguyen’s method. Tissue samples were weighed and homogenized in 200μl ice-cold homogenization buffer (10% glycerol in 0.1 mol/L phosphate buffer, pH7.5). Then the homogenate was centrifuged at 4°C, 12000r/min for 10min, and the supernatant was centrifuged again. The final supernatant was collected for enzyme activity measurements. Paranitroanisole was added to the supernatant as a substrate for MFO, and the para-nitrophenolate produced was used to calculate MFO activity. The activity of each sample was measured five times [25]. Protein content was measured according to the Bradford’s method using bovine serum albumin as the standard.
Leaf-Disc Assay Ginkgo biloba extracts, ginkgo flavonoids, ginkgolide, and bilobalide were dissolved in 20 mL absolute alcohol:water [1:7 (v/v)] solution in a concentration series (Table 2). Fresh Fraxinus americana leaf discs (1.5 cm diameter) were prepared with a drilling machine from healthy and pesticide-free leaves, and were soaked in the secondary metabolite solutions or alcohol: water [1:7 (v/v)] (as a control) for 10 sec. Four of the treated leaf discs were placed into each empty rearing box (100 ml, d = 5cm) to feed 30 third-instar H. cunea larvae for 24 h. Then, the leaves were removed from the boxes and placed on 1×1 mm graph paper to measure the eaten area by counting the grids. The experiment was repeated five times, and the antifeedant rate was calculated [26]. Antifeedant rate ¼
Eaten area of the control group Eaten area of the test group 100 Eaten area of the control group
Field Antifeedant Test For field antifeedant tests, 2 g of G. biloba extracts, 0.5g of ginkgo flavonoids, 0.1g ginkgolide and 0.05g of bilobalide were respectively dissolved in 10 mL absolute alcohol, mixed with seven volumes of water, and stored in a 100 mL plastic watering can that was properly labeled. Alcohol: water [1:7 (v/v)] was used as the vehicle control. The tests included 183 branches of 24 Salix trees with a diameter of 12–21 cm in the suburban area of Tianjin, China, an area of 0.08 km2 where Salix trees suffered severe damage from H. cunea larvae infestation in August 2015 (Table 3). Photographs were taken to record the insect feeding situation. The five solutions (four secondary metabolites and one control) were sprayed on infested leaf surfaces 3–5 times,
PLOS ONE | DOI:10.1371/journal.pone.0155682 May 23, 2016
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Study on insect resistance of Ginkgo biloba
Table 2. Concentration series for four Gingko biloba secondary metabolites. Secondary metabolite
High(g/L)
Medium(g/L)
Low(g/L)
Control group(g/L)
GBE
50
25
12.5
0
GF
12.5
6.25
3.125
GL
2.5
1.25
0.625
0
BB
1.25
0.625
0.3125
0
0
GBE, Ginkgo biloba extract; GF, gingko flavonoids; GL, ginkgolide; BB, bilobalide. doi:10.1371/journal.pone.0155682.t002
and the larval feeding situation was recorded by photographing again from the same perspective 5 h later. The repelling rate was calculated by comparing the number of larvae on the photographs taken before and after spraying [27]. Repelling rate ¼
Number of larvae before Number of larvae after 100 Number of larvae before
Data Analysis Variance analysis and data processing were performed using SPSS 17.0 or Prism 6.0. One-way ANOVA followed by least significant difference test (LSD, at a level of statistical significance of P = 0.05) were used to analyze the percentage of larva feeding on a given diet or food source, the detoxifying enzyme activities, antifeedant rate, and repelling rate. All of the data expressed as percentages were arcsine transformed and analyzed using SPSS 17.0.
Results Pest Damage to G. biloba Trees in Beijing City The average height of G. biloba trees in the 10 sample plots was 6.1–15.1 m, and the average diameter was 13.3–49.9 cm. Symptoms of pest feeding on the trees are summarized in Table 4. All pest feeding symptoms were confined to the trunk, and no symptoms were observed on leaves. Approximately 93.3% or more trees in the selected plots were free of insect damage in the same year. Only six G. biloba trees in Plots A, C, F, and G were damaged by Holcocerus insularis Staudinger (Lepidoptera, Cossidae) larvae, and only two trees were rated as Class 3 damage. No damage by any insect was found in the other six plots. Table 3. Instars of Hyphantria cunea larvae infesting branches treated with Ginkgo biloba secondary metabolites. Instar
1st
2nd
3rd
4th
5th
Total
GBE
10
11
6
6
6
39
GF
13
9
7
5
5
39
GL
15
10
8
5
6
44
BB
9
9
7
6
5
36
CG
5
5
5
5
5
25
GBE, Ginkgo biloba extract; GF, gingko flavonoids; GL, ginkgolide; BB, bilobalide; CG, control group. doi:10.1371/journal.pone.0155682.t003
PLOS ONE | DOI:10.1371/journal.pone.0155682 May 23, 2016
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Study on insect resistance of Ginkgo biloba
Table 4. Evaluation of pest feeding on Ginkgo biloba in Beijing City. Location
Samplesize
Averageheight (m)
A
30
15.1
B
30
12.1
C
30
D
30
E F
Average diameter(cm)
Class 1
Class 2
Class 3
Proportions without pest(%)
Affected part of tree
Pest species
49.9
28
1
1
93.3
32.7
30
0
0
100.0
Trunk
H. insularis
11.7
31.3
29
1
0
96.7
11.4
30.3
30
0
0
100.0
Trunk
H. insularis
30
10.8
28.7
30
0
0
100.0
30
10.5
28.5
28
1
1
G
30
10.1
27.9
29
1
0
93.3
Trunk
H. insularis
96.7
Trunk
H
30
8.8
24.7
30
0
0
100.0
H. insularis
I
30
8.7
21.0
30
0
0
100.0
J
30
6.1
13.3
30
0
0
100.0
A, Peking University; B, Diaoyutai State Guesthouse; C, Ditan Park; D, Tsinghua University; E, Changping Zhangge village; F, Beijing Forestry University; G, Beijing Botanical Garden; H, Olympic Forest Park; I, Chaoyang District, Sanlitun West Fifth Street; J, Haidian Minzhuang Road. doi:10.1371/journal.pone.0155682.t004
Antifeedant Activity of Different G. biloba Secondary Metabolites against H. cunea Larvae The two curves (Fig 1) show that the proportion of H. cunea larva feeding on the diet containing G. biloba secondary metabolites was significantly lower than the number feeding on the normal diet (F = 36.579; df = 5, 48; P
Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications Long Pan, Lili Ren, Fang Chen, Yuqian Feng, Youqing Luo* Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing, 100083, P.R. of China * [email protected]
a11111
OPEN ACCESS Citation: Pan L, Ren L, Chen F, Feng Y, Luo Y (2016) Antifeedant Activity of Ginkgo biloba Secondary Metabolites against Hyphantria cunea Larvae: Mechanisms and Applications. PLoS ONE 11(5): e0155682. doi:10.1371/journal.pone.0155682 Editor: Youjun Zhang, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, CHINA Received: February 4, 2016 Accepted: May 3, 2016 Published: May 23, 2016 Copyright: © 2016 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by Special Project for Scientific Research of Forestry Commonweal Industry of National Forestry Bureau (201404401). Competing Interests: The authors have declared that no competing interests exist.
Abstract Ginkgo biloba is a typical relic plant that rarely suffers from pest hazards. This study analyzed the pattern of G. biloba pest hazards in Beijing; tested the antifeedant activity of G. biloba extracts, including ginkgo flavonoids, ginkgolide, and bilobalide, against Hyphantria cunea larvae; determined the activities of glutathione transferase (GSTs), acetylcholinesterase (AChE), carboxylesterase (CarE) and mixed-functional oxidase (MFO), in larvae after feeding on these G. biloba secondary metabolites; and screened for effective botanical antifeedants in the field. In this study, no indicators of insect infestation were found for any of the examined leaves of G. biloba; all tested secondary metabolites showed significant antifeedant activity and affected the activity of the four larval detoxifying enzymes. Ginkgolide had the highest antifeedant activity and the most significant effect on the detoxifying enzymes (P50%
doi:10.1371/journal.pone.0155682.t001
recorded. The pest resistance percentage was calculated as the number of pest-free trees among the total number of trees in the plot. Pest resistance percentage ¼
Number of pest free trees 100 Total number of trees in the plot
Verification of Antifeedant Activity To make the normal diet, feed powder (20 g), distilled water (80 ml), and agar power (1.6g) were mixed and heated to completely dissolve the agar. To make the test diet, G. biloba secondary metabolites were dissolved in 10 ml ethanol before mixing them with the normal diet at different concentrations. All the diets were poured into a rearing box (100ml, d = 5cm) and allowed to solidify. The resulting circular pie (δ = 2mm) was then cut into two equal semicircles for future use. Six other rearing boxes of the same shape was prepared, and a semicircular pie of the normal diet and six semicircular pies containing 4%, 8%, or 16% G. biloba extract, 2% flavonoids, 0.4% ginkgolides, or 0.2% bilogalide were placed in the boxes. Then 30 third-instar H. cunea larvae were put into each box, and the number of larvae choosing to feed on each of the semicircular pies in the boxes was recorded after 24h. These experiments were repeated five times [17–20].
Detoxifying Enzyme Activity Assay Circular diet pies containing 8% G. biloba extracts, 2% ginkgo flavonoids, 0.4% ginkgolide, 0.2% bilobalide, or no extra additive were prepared as described above, to feed 120 third-instar H. cunea larvae in each group. The activities of glutathione transferase, acetylcholinesterase, carboxylesterase and mixed function oxidase were measured at 12, 24, 36, and 48 h for each group of 30 larvae. Third-, fourth-, and fifth-instar H. cunea larvae were fed on a diet containing the above four G. biloba secondary metabolites, and the activities of the above detoxifying were measured at 24 h in each group of enzymes in each group of 30 larvae. These experiments were repeated five times. Glutathione transferase (GSTs) activity was measured using Lee’s method. Briefly, the tissue sample was weighed, and 9 volumes of normal saline were added to produce a 10% tissue homogenate, which was then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was considered as the enzyme solution. The enzyme reaction was measured in a cuvette containing 2.4 mL phosphate buffer (66 mmol/L, pH 7.0), 0.1 mL enzyme solution, 0.3 mL of 50 mmol/L glutathione (GSH), and 0.1 mL of 30 mmol/L 1-chloro-2,4-dinitrobenzene (CDNB), and changes in absorption at 340 nm were recorded. The measurement was repeated five times [21]. Acetylcholinesterase (AChE) activity was measured using a modified Ellman method. Briefly, the tissue sample was weighed, and 9 volumes of normal saline were added to produce a 10% tissue homogenate, which was then centrifuged at 3,500 rpm for 10 min at 4°C. The
PLOS ONE | DOI:10.1371/journal.pone.0155682 May 23, 2016
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Study on insect resistance of Ginkgo biloba
supernatant was collected for enzyme activity measurements. Supernatant (0.1 mL) and ATCh (0.1 mL) (a substrate of AChE) were mixed together and incubated at 30°C for 15min, and then 3.6 mL DTNB was added to terminate the reaction. Absorbance was measured at 412nm. The experiment was repeated five times [22–23]. Carboxylesterase (CarE) activity was measured using Asperen’s method. Briefly, the tissue sample was weighed, and 9 volumes of normal saline were added to produce a 10% tissue homogenate, which was then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected and diluted 50× with phosphate buffer before using for enzyme activity measurement. The enzyme reactions contained 0.3 mL of the diluted supernatant, 0.7 mL of 0.04 mol/L phosphate buffer, and 5 mL substrate, which were mixed and incubated on a shaker for 30 min at 37°C. Then, 1 mL of chromogenic reagent was added, and the reactions were incubated for a further 30 min at room temperature, followed by measurement of absorbance at 600 nm. The experiment was repeated five times. Known concentrations of α-naphthol were used to obtain a standard curve [24]. Mixed function oxidase (MFO) activity was measured using Yu and Nguyen’s method. Tissue samples were weighed and homogenized in 200μl ice-cold homogenization buffer (10% glycerol in 0.1 mol/L phosphate buffer, pH7.5). Then the homogenate was centrifuged at 4°C, 12000r/min for 10min, and the supernatant was centrifuged again. The final supernatant was collected for enzyme activity measurements. Paranitroanisole was added to the supernatant as a substrate for MFO, and the para-nitrophenolate produced was used to calculate MFO activity. The activity of each sample was measured five times [25]. Protein content was measured according to the Bradford’s method using bovine serum albumin as the standard.
Leaf-Disc Assay Ginkgo biloba extracts, ginkgo flavonoids, ginkgolide, and bilobalide were dissolved in 20 mL absolute alcohol:water [1:7 (v/v)] solution in a concentration series (Table 2). Fresh Fraxinus americana leaf discs (1.5 cm diameter) were prepared with a drilling machine from healthy and pesticide-free leaves, and were soaked in the secondary metabolite solutions or alcohol: water [1:7 (v/v)] (as a control) for 10 sec. Four of the treated leaf discs were placed into each empty rearing box (100 ml, d = 5cm) to feed 30 third-instar H. cunea larvae for 24 h. Then, the leaves were removed from the boxes and placed on 1×1 mm graph paper to measure the eaten area by counting the grids. The experiment was repeated five times, and the antifeedant rate was calculated [26]. Antifeedant rate ¼
Eaten area of the control group Eaten area of the test group 100 Eaten area of the control group
Field Antifeedant Test For field antifeedant tests, 2 g of G. biloba extracts, 0.5g of ginkgo flavonoids, 0.1g ginkgolide and 0.05g of bilobalide were respectively dissolved in 10 mL absolute alcohol, mixed with seven volumes of water, and stored in a 100 mL plastic watering can that was properly labeled. Alcohol: water [1:7 (v/v)] was used as the vehicle control. The tests included 183 branches of 24 Salix trees with a diameter of 12–21 cm in the suburban area of Tianjin, China, an area of 0.08 km2 where Salix trees suffered severe damage from H. cunea larvae infestation in August 2015 (Table 3). Photographs were taken to record the insect feeding situation. The five solutions (four secondary metabolites and one control) were sprayed on infested leaf surfaces 3–5 times,
PLOS ONE | DOI:10.1371/journal.pone.0155682 May 23, 2016
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Study on insect resistance of Ginkgo biloba
Table 2. Concentration series for four Gingko biloba secondary metabolites. Secondary metabolite
High(g/L)
Medium(g/L)
Low(g/L)
Control group(g/L)
GBE
50
25
12.5
0
GF
12.5
6.25
3.125
GL
2.5
1.25
0.625
0
BB
1.25
0.625
0.3125
0
0
GBE, Ginkgo biloba extract; GF, gingko flavonoids; GL, ginkgolide; BB, bilobalide. doi:10.1371/journal.pone.0155682.t002
and the larval feeding situation was recorded by photographing again from the same perspective 5 h later. The repelling rate was calculated by comparing the number of larvae on the photographs taken before and after spraying [27]. Repelling rate ¼
Number of larvae before Number of larvae after 100 Number of larvae before
Data Analysis Variance analysis and data processing were performed using SPSS 17.0 or Prism 6.0. One-way ANOVA followed by least significant difference test (LSD, at a level of statistical significance of P = 0.05) were used to analyze the percentage of larva feeding on a given diet or food source, the detoxifying enzyme activities, antifeedant rate, and repelling rate. All of the data expressed as percentages were arcsine transformed and analyzed using SPSS 17.0.
Results Pest Damage to G. biloba Trees in Beijing City The average height of G. biloba trees in the 10 sample plots was 6.1–15.1 m, and the average diameter was 13.3–49.9 cm. Symptoms of pest feeding on the trees are summarized in Table 4. All pest feeding symptoms were confined to the trunk, and no symptoms were observed on leaves. Approximately 93.3% or more trees in the selected plots were free of insect damage in the same year. Only six G. biloba trees in Plots A, C, F, and G were damaged by Holcocerus insularis Staudinger (Lepidoptera, Cossidae) larvae, and only two trees were rated as Class 3 damage. No damage by any insect was found in the other six plots. Table 3. Instars of Hyphantria cunea larvae infesting branches treated with Ginkgo biloba secondary metabolites. Instar
1st
2nd
3rd
4th
5th
Total
GBE
10
11
6
6
6
39
GF
13
9
7
5
5
39
GL
15
10
8
5
6
44
BB
9
9
7
6
5
36
CG
5
5
5
5
5
25
GBE, Ginkgo biloba extract; GF, gingko flavonoids; GL, ginkgolide; BB, bilobalide; CG, control group. doi:10.1371/journal.pone.0155682.t003
PLOS ONE | DOI:10.1371/journal.pone.0155682 May 23, 2016
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Study on insect resistance of Ginkgo biloba
Table 4. Evaluation of pest feeding on Ginkgo biloba in Beijing City. Location
Samplesize
Averageheight (m)
A
30
15.1
B
30
12.1
C
30
D
30
E F
Average diameter(cm)
Class 1
Class 2
Class 3
Proportions without pest(%)
Affected part of tree
Pest species
49.9
28
1
1
93.3
32.7
30
0
0
100.0
Trunk
H. insularis
11.7
31.3
29
1
0
96.7
11.4
30.3
30
0
0
100.0
Trunk
H. insularis
30
10.8
28.7
30
0
0
100.0
30
10.5
28.5
28
1
1
G
30
10.1
27.9
29
1
0
93.3
Trunk
H. insularis
96.7
Trunk
H
30
8.8
24.7
30
0
0
100.0
H. insularis
I
30
8.7
21.0
30
0
0
100.0
J
30
6.1
13.3
30
0
0
100.0
A, Peking University; B, Diaoyutai State Guesthouse; C, Ditan Park; D, Tsinghua University; E, Changping Zhangge village; F, Beijing Forestry University; G, Beijing Botanical Garden; H, Olympic Forest Park; I, Chaoyang District, Sanlitun West Fifth Street; J, Haidian Minzhuang Road. doi:10.1371/journal.pone.0155682.t004
Antifeedant Activity of Different G. biloba Secondary Metabolites against H. cunea Larvae The two curves (Fig 1) show that the proportion of H. cunea larva feeding on the diet containing G. biloba secondary metabolites was significantly lower than the number feeding on the normal diet (F = 36.579; df = 5, 48; P
