Pesticide Biochemistry and Physiology 108 (2014) 86–91
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Effects of a protease inhibitor protein from Xenorhabdus bovienii on physiology of pea aphid (Acyrthosiphon pisum) Danjuan Jin a,b,1, Fanrong Zeng a,⇑, Shuanglin Dong b, Heqing Zhang a a b
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Beijing 100081, PR China Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, PR China
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Article history: Received 31 July 2013 Accepted 31 December 2013 Available online 9 January 2014 Keywords: Protease inhibitor protein Total sugar Aminopeptidase Amino acids Lipid
a b s t r a c t The effects of a protease inhibitor protein from Xenorhabdus bovienii strain xbi1 (Xbpi-1) on biochemistry and physiology of pea aphid (Acyrthosiphon pisum) were studied, and the effects of Xbpi-1 on the activities of aphid aminopeptidase and the concentrations of carbohydrates, free amino acids and lipids in A. pisum were determined. The results showed that the activity of aminopeptidase was reduced 18.30% by Xbpi-1 in vitro assays. In addition, the total sugar, glycogen, and lipid concentrations per aphid fed on an artificial diet with Xbpi-1 were reduced by 39.41%, 24.61% and 26.25%, respectively, compared to the controls. The total free amino acids in the aphids on the diet with Xbpi-1 was significantly decreased by 20.78% compared to the control. The mortalities of A. pisum were significantly higher when the concentrations of Xbpi-1 were 400 and 800 lg/ml in the artificial diet. The results from this study indicated that Xbpi-1 had adverse effects on the physiology and biochemistry of A. pisum. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The pea aphid (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) is an important agricultural pest. The aphid can causes serious losses in vegetable and other agriculture crops by direct feeding and transferring plant viruses in the field [1]. Chemical insecticide has been used to control this insect pest for a long time. However, the use of chemical pesticide on vegetables can cause problems, such as the presence of chemical residues in vegetables, resistance of insect pests to pesticide and environmental pollution. Therefore, there is a need for the development of new strategies to control aphids. Protease inhibitor proteins have the potential to be used in agricultural pest control. Protease or amylase inhibitors can suppress the activities of proteases or amylases of insect pests, and they can delay larval development, cause weight loss, a decline in immunity, reduce fecundity or cause insect death [2–11]. In addition, with the development of transgenic technology in agricultural production, many protease inhibitor genes have been successfully expressed in transgenic plants for controlling insect pests [12–15]. A group of protease inhibitor proteins from Xenorhabdus and Photorhabdus [16–18] is an important resource for the insecticidal proteins. Xbpi-1 (GenBank accession: FJ624423) has been ⇑ Corresponding author. Fax: +86 10 82109714. E-mail address: [email protected] (F. Zeng). Current address: Linan Environmental Monitoring Station Hangzhou, 310000, PR China. 1
0048-3575/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.12.010
demonstrated to have adverse effects on the growth and survival of A. pisum [19]. However, the effects of Xbpi-1 on the biochemistry and physiology of A. pisum is still unclear. The hypothesis for the Xbpi-1 against aphids is that Xbpi-1 may have adverse effects on aphid physiology and biochemical activity such as enzyme activity. The major purpose of this study was to explore the influence of Xbpi-1, which is a protease inhibitor protein that acts against A. pisum, and to determine its effects on the aphid aminopeptidase activity, and the concentrations of carbohydrates, free amino acids and lipids in A. pisum.
2. Materials and methods 2.1. Experimental insects and reagents The experimental insect colony was reared on broad bean at 23 °C (L 16: D 8 photoperiod) in the laboratory. Adult aphids were allowed to settle for 16 h on plants, and 4-day-old nymphs produced by the adults were used for the experiments. The Xbpi-1 protease inhibitor protein used in this study was purified from the supernatant of an Escherichia coli expression system [19], and the protein concentration was determined by the dye-binding method of Bradford using bovine serum albumin (BSA) as the standard [20]. The L-leucine p-nitroanilide (L-Leu-pNA) chemical used in the experiment to measure aphid aminopeptidase activity was purchased from Sigma–Aldrich (St. Louis, United States), and all reagents were of analytical grade.
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2.2. Bioassays
2.4. Analysis of aphid free amino acids
To examine the effect of Xbpi-1 on A. pisum mortality, an artificial diet was supplemented with different concentrations (50, 100, 200, 400 and 800 lg/ml) of Xbpi-1. This diet was a meridic artificial diet with a slight modification according to Dadd et al. [21–23]. The diet included the following ingredients: 20% sucrose, 2% casein hydrolysate, 2% yeast extract, 0.1% ascorbic acid, 0.05% choline chloride, 0.01% nicotinic acid, 0.05% inositol, 0.01% aminobenzoic acid, 0.123% MgSO4, 1.5% K2HPO4, 0.0001% biotin, 0.0005% folic acid, 0.0005% riboflavin, 0.005% Ca pantothenate, 0.0025% thiamin, 0.0002% CuCl2, 0.011% FeCl3, 0.0004% MnCl2, and 0.00085% ZnSO4. The diet was adjusted to pH 7.0 with 0.5 M KOH and sterilized using 0.22 lm MilliporeÒ Express PES Membrane filtration. The 4-day-old aphids were assayed and placed in an incubator at a daytime temperature of 23 °C and night temperature of 21 °C with a 16 L: 8 D photoperiod. Mortality was checked on the 5th day. The experiment was repeated five times.
The 4-day old aphids were reared on an artificial diet (same as above Section 2.2) containing 800 lg/ml Xbpi-1. The control was reared on an artificial diet without Xbpi-1. The aphids were collected at 96 h after rearing the aphids on diet with the treatment or control. The samples were sent to the Analysis Laboratory of Beijing Nutrition Resources Institute and were subjected to an automatic amino acid analyzer. The experiment was repeated three times.
2.3. Aphid aminopeptidase activity Adult aphids (from broad bean) were collected, homogenized in cold 0.15 M NaCl, and centrifuged twice at 14,000 rpm for 10 min at 4 °C. The supernatant was collected and stored at 20 °C until further use. The total protein concentration was determined using the dye-binding method of Bradford with BSA as a standard protein. Leucine aminopeptidase (LAP) activities were assayed using LLeu-pNA as the substrate [24,25]. The substrate solutions (1 mg/ ml) were prepared by dissolving 20 mg of L-Leu-pNA in 1 ml of ethanol followed by adding 19 ml of 50 mM Tris–HCl buffer (pH 8.0). The whole protein extract (40 ll) with 20 lg of Xbpi-1 was added to a well in an assay plate and incubated at 37 °C for 15 min. LAP activity was determined by adding 150 ll of the above L-Leu-pNA substrate solutions. The plate was read at 405 nm with a microplate reader after incubation for 90 min at 37 °C. An endogenous control (whole protein extract with heat treatment) was used. The reading was corrected by subtracting the reading of the endogenous control. The experiment was repeated three times.
2.5. Analysis of aphid sugar, glycogen and lipid levels The 4-day-old aphids were reared on an artificial diet (same as above Section 2.2) containing 800 lg/ml Xbpi-1. The control was reared on an artificial diet without Xbpi-1. The aphids were collected at 48, 72 and 96 h. The amount of total sugars, glycogen and lipid in individual aphids were quantified by the method of Fadamiro et al. [26]. Five pea aphids were crushed with a plastic pestle in a 1.5 ml microcentrifuge tube containing 50 ll of a 2% sodium sulfate solution and placed on ice. The dissolved nutrients were then extracted with 450 ll of a chloroform:methanol (1:2) mixture, and the tubes were then vortexed and centrifuged at 14,000 rpm for 5 min. One hundred microliters of the resulting supernatant was transferred to a 1.5 ml microcentrifuge tube for the sugar assays, and another 100 ll was transferred to a 1.5 ml microcentrifuge tube for the lipid assay. The precipitate in the microcentrifuge tube was used for the glycogen assay. The amount of total sugars in each aphid was measured using the hot anthrone test [26,27]. The anthrone reagent (1 ml) was added to the sugar tube, heated at 90 °C for 10 min and cooled on ice. The absorbance was read at 630 nm to give an estimate of total sugars. To measure glycogen concentration, a previously published method was used [26,27]. Firstly, 1 ml of anthrone reagent was added to the microcentrifuge tube containing the precipitate. After centrifugation, the tube was heated at 90 °C for 10 min and then cooled on ice. The absorbance was read at 630 nm to give an estimate of glycogen. The amount of glycogen was considered to be
Fig. 1. Effect of Xbpi-1, a protease inhibitor protein, on the mortality of Acyrthosiphon pisum. Aphid mortality was calculated after 5 days of feeding. Bars with different letters are significantly different (P < 0.05). The values are represented as the means ± SE.
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Fig. 2. Leucine aminopeptidase activity of the whole protein extract from Acyrthosiphon pisum. Whole protein extract were incubated with Xbpi-1 (20 lg) for 15 min, and the total and specific proteolytic activities were examined. Bars with different letters are significantly different (P < 0.05). The values are represented as the means ± SE.
representative of the 20 aphids because all glycogen in the sample was assumed to precipitate to the bottom of the tube. The amount of lipid was measured by a sulfo-phospho-vanillin reaction [26,28]. Firstly, 100 ll of sulfuric acid was added to the lipid precipitate, and the tube was then heated at 90 °C for 10 min and cooled on ice. After keeping the tube for 5 min at room temperature, 1 ml of vanillin reagent was added to it. The mixture solution was left to react for 15 min at room temperature and read at 525 nm. All the experiments were set up in a completely random design with seven replications. Standard curves were obtained using pure glucose (Sigma–Aldrich, St. Louis, United States) for total sugars and glycogen analysis, and standard curves were obtained using pure cholesterol for lipid analysis.
2.6. Data analysis The statistical software package used for data analysis was the SPSS 16.0 program for Windows (SPSS, Chicago, III). Significant differences in the bioassays were determined by one-way ANOVA and compared by least significant difference (LSD). Other statistical analyses were determined by independent samples t-test. The significance level was checked at P < 0.05. Median lethal concentration (LC50) was obtained by probit analysis.
3. Results 3.1. Bioassay The mortality of A. pisum rearing on the control artificial diet was 12.37%, and the mortalities of A. pisum rearing on the artificial diets containing 50, 100, 200, 400, 800 lg/ml of Xbpi-1 were 16.00%, 17.92%, 25.28%, 39.21% and 80.26%, respectively (Fig. 1). The mortalities of A. pisum were significantly higher when the concentrations of Xbpi-1 were 400 and 800 lg/ml. The LC50 of Xbpi-1 for A. pisum was 396.96 lg/ml.
Table 1 The effects of Xbpi-1, a protease inhibitor protein, on the free amino acids in Acyrthosiphon pisum. Amino acid
Thr Val Met Ile Leu Phe Lys His Arg Pro Ser Glu Gly Ala Asp Tyr Total
Concentration (lg/individual) Control
Xbpi-1
4.29 ± 0.09a 4.51 ± 0.04a 1.75 ± 0.01a 3.17 ± 0.19a 7.42 ± 0.08a 3.59 ± 0.03a 8.54 ± 0.07a 2.41 ± 0.05a 5.93 ± 0.05a 4.79 ± 0.02a 5.28 ± 0.59a 16.19 ± 0.03a 4.41 ± 0.04a 6.38 ± 0.10a 12.09 ± 0.07a 7.33 ± 0.06a 98.07 ± 0.19a
3.36 ± 0.06b 3.51 ± 0.06b 1.36 ± 0.01b 2.40 ± 0.08b 5.62 ± 0.12b 2.75 ± 0.07b 6.70 ± 0.13b 1.88 ± 0.02b 4.60 ± 0.07b 3.56 ± 0.05b 4.62 ± 0.05a 13.31 ± 0.22b 3.41 ± 0.04b 5.07 ± 0.06b 9.70 ± 0.18b 5.84 ± 0.10b 77.69 ± 1.24b
The data in the table are shown as the means ± SE. Means followed by the same letter are not significantly different (P = 0.05).
3.2. Aphid aminopeptidase activity To determine the effects of Xbpi-1on enzyme activity in A. pisum, the activity of aminopeptidase in the whole aphid extract was measured. The results indicated that aminopeptidase activity was reduced by 18.30% compared to the control (Fig. 2). The activity of aminopeptidase in the experimental aphids was significantly reduced by the protease inhibitor protein, Xbpi-1. 3.3. Analysis of aphid free amino acids The effect of Xbpi-1 on the contents of free amino acids in the experimental A. pisum was determined, and 16 free amino acids
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Fig. 3. Effect of Xbpi-1, a protease inhibitor protein, on the total sugar concentration of Acyrthosiphon pisum. The amount (lg ± SE) of total sugars in individual aphids was measured by the hot anthrone test.
Fig. 4. Effect of Xbpi-1, a protease inhibitor protein, on the glycogen concentration of Acyrthosiphon pisum. The amount (lg ± SE) of glycogen in individual aphids was measured by the hot anthrone test.
were found. The result also demonstrated that the total free amino acid level was decreased by 20.78% compared to the control. The concentrations of the 16 free amino acids, except serine, were significantly decreased when aphids fed artificial diet with Xbpi-1 (Table 1).
3.4. Analysis of aphid sugar, glycogen and lipid contents When the aphids were fed on the artificial diet with Xbpi-1, the total sugar concentration was decreased by 39.41% on the 4th day (Fig. 3), and the glycogen concentration was decreased by 18.44% and 24.61% on the 3rd and 4th days, respectively (Fig. 4). The lipid concentration was decreased by 29.26% and 26.25% on the 3rd and 4th days, respectively, when the aphids were fed on the artificial diet with Xbpi-1 (Fig. 5). As compared to the controls, the glycogen and lipid concentrations of the aphids reared on the artificial diet with Xbpi-1 had significant differences on the 3rd day. However,
the total sugar concentration was significantly decreased on the 4th day after feeding on the diet. 4. Discussion In general, the mechanism of the protease inhibitor proteins against insects is that protease inhibitors have adverse effects on protease activity allowing the protease inhibitors to block the activities of proteases in the gut of target insects. Cristofoletti et al. [29] found that the pea aphid has strong aminopeptidase activity in the midgut. Generally, the effect of protease inhibitors on protease activity is determined by measuring the activity of the proteases in vitro with the inhibitor [30,31]. In this study, the effect of Xbpi-1 on the activity of aminopeptidase was determined by measuring the aminopeptidase activity in A. pisum after the aphids fed artificial diets containing Xbpi-1. The result revealed that Xbpi-1 decreased LAP activity of pea aphid by 18.3%. Multiple types of proteases exist in insect guts, and the development and
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Fig. 5. Effect of Xbpi-1, a protease inhibitor protein, on the total lipid concentration of Acyrthosiphon pisum. The amount (lg ± SE) of total lipids in individual aphids was measured by the sulfo-phospho-vanillin reaction.
growth of insects can be affected more quickly if two or more types of proteases are inhibited [32–35]. It is necessary to conduct further research to determine whether Xbpi-1 is a broad spectrum protease inhibitor. In addition, this study showed that the mortalities of A. pisum aphids fed artificial diets containing 200, 400, and 800 lg/ml Xbpi-1 were two, three and four times, respectively, more than the mortality of the controls. The results from this study agreed with those reported by other researchers [19,30,36–38]. Thus, Xbpi-1 may be a potential bio-pesticide for controlling sucking insect pests. This work is the first report on the effects of a novel protease inhibitor protein on carbohydrates, free amino acids and lipids of pea aphid. The major nutritive materials, such as carbohydrates, free amino acids and lipids, are necessary for growth, reproduction and survival of insects [39–45]. Energy metabolism needs glycolysis, fatty acid oxidation, and the tricarboxylic acid cycle. The results from this study showed that Xbpi-1 had adverse effects on the nutrient metabolism of A. pisum. The total sugar, glycogen and lipid concentrations were significantly reduced by rearing the aphids on a diet containing Xbpi-1 after 4 days, and the total free amino acid concentration was also significantly reduced, so Xbpi-1 may affect aphid normal physiological function. The results from this study support our hypothesis that the Xbpi-1 may have the adverse effects on aphid physiology and biochemistry. The protease inhibitor protein, Xbpi-1, inhibited aminopeptidase activity in A. pisum which may lead to the lack of some essential amino acids for the aphids. The Xbpi-1 also reduced the concentrations of the total sugar, glycogen and lipids. These adverse effects may disturb the normal aphid physiological and biochemical function, thus affect aphid growth and ultimately leading to aphid death.
Acknowledgments We thank the editors and reviewers for their comments and suggestions. Thanks are due to Feng-Jiao Liu and Chang-Yan Liu for their technical assistance throughout the experiment. This
work was supported from funds (973 Program 2013CB127600 and the agriculture cooperative research fund 2011-G4).
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Effects of a protease inhibitor protein from Xenorhabdus bovienii on physiology of pea aphid (Acyrthosiphon pisum) Danjuan Jin a,b,1, Fanrong Zeng a,⇑, Shuanglin Dong b, Heqing Zhang a a b
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Beijing 100081, PR China Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, PR China
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Article history: Received 31 July 2013 Accepted 31 December 2013 Available online 9 January 2014 Keywords: Protease inhibitor protein Total sugar Aminopeptidase Amino acids Lipid
a b s t r a c t The effects of a protease inhibitor protein from Xenorhabdus bovienii strain xbi1 (Xbpi-1) on biochemistry and physiology of pea aphid (Acyrthosiphon pisum) were studied, and the effects of Xbpi-1 on the activities of aphid aminopeptidase and the concentrations of carbohydrates, free amino acids and lipids in A. pisum were determined. The results showed that the activity of aminopeptidase was reduced 18.30% by Xbpi-1 in vitro assays. In addition, the total sugar, glycogen, and lipid concentrations per aphid fed on an artificial diet with Xbpi-1 were reduced by 39.41%, 24.61% and 26.25%, respectively, compared to the controls. The total free amino acids in the aphids on the diet with Xbpi-1 was significantly decreased by 20.78% compared to the control. The mortalities of A. pisum were significantly higher when the concentrations of Xbpi-1 were 400 and 800 lg/ml in the artificial diet. The results from this study indicated that Xbpi-1 had adverse effects on the physiology and biochemistry of A. pisum. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The pea aphid (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) is an important agricultural pest. The aphid can causes serious losses in vegetable and other agriculture crops by direct feeding and transferring plant viruses in the field [1]. Chemical insecticide has been used to control this insect pest for a long time. However, the use of chemical pesticide on vegetables can cause problems, such as the presence of chemical residues in vegetables, resistance of insect pests to pesticide and environmental pollution. Therefore, there is a need for the development of new strategies to control aphids. Protease inhibitor proteins have the potential to be used in agricultural pest control. Protease or amylase inhibitors can suppress the activities of proteases or amylases of insect pests, and they can delay larval development, cause weight loss, a decline in immunity, reduce fecundity or cause insect death [2–11]. In addition, with the development of transgenic technology in agricultural production, many protease inhibitor genes have been successfully expressed in transgenic plants for controlling insect pests [12–15]. A group of protease inhibitor proteins from Xenorhabdus and Photorhabdus [16–18] is an important resource for the insecticidal proteins. Xbpi-1 (GenBank accession: FJ624423) has been ⇑ Corresponding author. Fax: +86 10 82109714. E-mail address: [email protected] (F. Zeng). Current address: Linan Environmental Monitoring Station Hangzhou, 310000, PR China. 1
0048-3575/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.12.010
demonstrated to have adverse effects on the growth and survival of A. pisum [19]. However, the effects of Xbpi-1 on the biochemistry and physiology of A. pisum is still unclear. The hypothesis for the Xbpi-1 against aphids is that Xbpi-1 may have adverse effects on aphid physiology and biochemical activity such as enzyme activity. The major purpose of this study was to explore the influence of Xbpi-1, which is a protease inhibitor protein that acts against A. pisum, and to determine its effects on the aphid aminopeptidase activity, and the concentrations of carbohydrates, free amino acids and lipids in A. pisum.
2. Materials and methods 2.1. Experimental insects and reagents The experimental insect colony was reared on broad bean at 23 °C (L 16: D 8 photoperiod) in the laboratory. Adult aphids were allowed to settle for 16 h on plants, and 4-day-old nymphs produced by the adults were used for the experiments. The Xbpi-1 protease inhibitor protein used in this study was purified from the supernatant of an Escherichia coli expression system [19], and the protein concentration was determined by the dye-binding method of Bradford using bovine serum albumin (BSA) as the standard [20]. The L-leucine p-nitroanilide (L-Leu-pNA) chemical used in the experiment to measure aphid aminopeptidase activity was purchased from Sigma–Aldrich (St. Louis, United States), and all reagents were of analytical grade.
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2.2. Bioassays
2.4. Analysis of aphid free amino acids
To examine the effect of Xbpi-1 on A. pisum mortality, an artificial diet was supplemented with different concentrations (50, 100, 200, 400 and 800 lg/ml) of Xbpi-1. This diet was a meridic artificial diet with a slight modification according to Dadd et al. [21–23]. The diet included the following ingredients: 20% sucrose, 2% casein hydrolysate, 2% yeast extract, 0.1% ascorbic acid, 0.05% choline chloride, 0.01% nicotinic acid, 0.05% inositol, 0.01% aminobenzoic acid, 0.123% MgSO4, 1.5% K2HPO4, 0.0001% biotin, 0.0005% folic acid, 0.0005% riboflavin, 0.005% Ca pantothenate, 0.0025% thiamin, 0.0002% CuCl2, 0.011% FeCl3, 0.0004% MnCl2, and 0.00085% ZnSO4. The diet was adjusted to pH 7.0 with 0.5 M KOH and sterilized using 0.22 lm MilliporeÒ Express PES Membrane filtration. The 4-day-old aphids were assayed and placed in an incubator at a daytime temperature of 23 °C and night temperature of 21 °C with a 16 L: 8 D photoperiod. Mortality was checked on the 5th day. The experiment was repeated five times.
The 4-day old aphids were reared on an artificial diet (same as above Section 2.2) containing 800 lg/ml Xbpi-1. The control was reared on an artificial diet without Xbpi-1. The aphids were collected at 96 h after rearing the aphids on diet with the treatment or control. The samples were sent to the Analysis Laboratory of Beijing Nutrition Resources Institute and were subjected to an automatic amino acid analyzer. The experiment was repeated three times.
2.3. Aphid aminopeptidase activity Adult aphids (from broad bean) were collected, homogenized in cold 0.15 M NaCl, and centrifuged twice at 14,000 rpm for 10 min at 4 °C. The supernatant was collected and stored at 20 °C until further use. The total protein concentration was determined using the dye-binding method of Bradford with BSA as a standard protein. Leucine aminopeptidase (LAP) activities were assayed using LLeu-pNA as the substrate [24,25]. The substrate solutions (1 mg/ ml) were prepared by dissolving 20 mg of L-Leu-pNA in 1 ml of ethanol followed by adding 19 ml of 50 mM Tris–HCl buffer (pH 8.0). The whole protein extract (40 ll) with 20 lg of Xbpi-1 was added to a well in an assay plate and incubated at 37 °C for 15 min. LAP activity was determined by adding 150 ll of the above L-Leu-pNA substrate solutions. The plate was read at 405 nm with a microplate reader after incubation for 90 min at 37 °C. An endogenous control (whole protein extract with heat treatment) was used. The reading was corrected by subtracting the reading of the endogenous control. The experiment was repeated three times.
2.5. Analysis of aphid sugar, glycogen and lipid levels The 4-day-old aphids were reared on an artificial diet (same as above Section 2.2) containing 800 lg/ml Xbpi-1. The control was reared on an artificial diet without Xbpi-1. The aphids were collected at 48, 72 and 96 h. The amount of total sugars, glycogen and lipid in individual aphids were quantified by the method of Fadamiro et al. [26]. Five pea aphids were crushed with a plastic pestle in a 1.5 ml microcentrifuge tube containing 50 ll of a 2% sodium sulfate solution and placed on ice. The dissolved nutrients were then extracted with 450 ll of a chloroform:methanol (1:2) mixture, and the tubes were then vortexed and centrifuged at 14,000 rpm for 5 min. One hundred microliters of the resulting supernatant was transferred to a 1.5 ml microcentrifuge tube for the sugar assays, and another 100 ll was transferred to a 1.5 ml microcentrifuge tube for the lipid assay. The precipitate in the microcentrifuge tube was used for the glycogen assay. The amount of total sugars in each aphid was measured using the hot anthrone test [26,27]. The anthrone reagent (1 ml) was added to the sugar tube, heated at 90 °C for 10 min and cooled on ice. The absorbance was read at 630 nm to give an estimate of total sugars. To measure glycogen concentration, a previously published method was used [26,27]. Firstly, 1 ml of anthrone reagent was added to the microcentrifuge tube containing the precipitate. After centrifugation, the tube was heated at 90 °C for 10 min and then cooled on ice. The absorbance was read at 630 nm to give an estimate of glycogen. The amount of glycogen was considered to be
Fig. 1. Effect of Xbpi-1, a protease inhibitor protein, on the mortality of Acyrthosiphon pisum. Aphid mortality was calculated after 5 days of feeding. Bars with different letters are significantly different (P < 0.05). The values are represented as the means ± SE.
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Fig. 2. Leucine aminopeptidase activity of the whole protein extract from Acyrthosiphon pisum. Whole protein extract were incubated with Xbpi-1 (20 lg) for 15 min, and the total and specific proteolytic activities were examined. Bars with different letters are significantly different (P < 0.05). The values are represented as the means ± SE.
representative of the 20 aphids because all glycogen in the sample was assumed to precipitate to the bottom of the tube. The amount of lipid was measured by a sulfo-phospho-vanillin reaction [26,28]. Firstly, 100 ll of sulfuric acid was added to the lipid precipitate, and the tube was then heated at 90 °C for 10 min and cooled on ice. After keeping the tube for 5 min at room temperature, 1 ml of vanillin reagent was added to it. The mixture solution was left to react for 15 min at room temperature and read at 525 nm. All the experiments were set up in a completely random design with seven replications. Standard curves were obtained using pure glucose (Sigma–Aldrich, St. Louis, United States) for total sugars and glycogen analysis, and standard curves were obtained using pure cholesterol for lipid analysis.
2.6. Data analysis The statistical software package used for data analysis was the SPSS 16.0 program for Windows (SPSS, Chicago, III). Significant differences in the bioassays were determined by one-way ANOVA and compared by least significant difference (LSD). Other statistical analyses were determined by independent samples t-test. The significance level was checked at P < 0.05. Median lethal concentration (LC50) was obtained by probit analysis.
3. Results 3.1. Bioassay The mortality of A. pisum rearing on the control artificial diet was 12.37%, and the mortalities of A. pisum rearing on the artificial diets containing 50, 100, 200, 400, 800 lg/ml of Xbpi-1 were 16.00%, 17.92%, 25.28%, 39.21% and 80.26%, respectively (Fig. 1). The mortalities of A. pisum were significantly higher when the concentrations of Xbpi-1 were 400 and 800 lg/ml. The LC50 of Xbpi-1 for A. pisum was 396.96 lg/ml.
Table 1 The effects of Xbpi-1, a protease inhibitor protein, on the free amino acids in Acyrthosiphon pisum. Amino acid
Thr Val Met Ile Leu Phe Lys His Arg Pro Ser Glu Gly Ala Asp Tyr Total
Concentration (lg/individual) Control
Xbpi-1
4.29 ± 0.09a 4.51 ± 0.04a 1.75 ± 0.01a 3.17 ± 0.19a 7.42 ± 0.08a 3.59 ± 0.03a 8.54 ± 0.07a 2.41 ± 0.05a 5.93 ± 0.05a 4.79 ± 0.02a 5.28 ± 0.59a 16.19 ± 0.03a 4.41 ± 0.04a 6.38 ± 0.10a 12.09 ± 0.07a 7.33 ± 0.06a 98.07 ± 0.19a
3.36 ± 0.06b 3.51 ± 0.06b 1.36 ± 0.01b 2.40 ± 0.08b 5.62 ± 0.12b 2.75 ± 0.07b 6.70 ± 0.13b 1.88 ± 0.02b 4.60 ± 0.07b 3.56 ± 0.05b 4.62 ± 0.05a 13.31 ± 0.22b 3.41 ± 0.04b 5.07 ± 0.06b 9.70 ± 0.18b 5.84 ± 0.10b 77.69 ± 1.24b
The data in the table are shown as the means ± SE. Means followed by the same letter are not significantly different (P = 0.05).
3.2. Aphid aminopeptidase activity To determine the effects of Xbpi-1on enzyme activity in A. pisum, the activity of aminopeptidase in the whole aphid extract was measured. The results indicated that aminopeptidase activity was reduced by 18.30% compared to the control (Fig. 2). The activity of aminopeptidase in the experimental aphids was significantly reduced by the protease inhibitor protein, Xbpi-1. 3.3. Analysis of aphid free amino acids The effect of Xbpi-1 on the contents of free amino acids in the experimental A. pisum was determined, and 16 free amino acids
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Fig. 3. Effect of Xbpi-1, a protease inhibitor protein, on the total sugar concentration of Acyrthosiphon pisum. The amount (lg ± SE) of total sugars in individual aphids was measured by the hot anthrone test.
Fig. 4. Effect of Xbpi-1, a protease inhibitor protein, on the glycogen concentration of Acyrthosiphon pisum. The amount (lg ± SE) of glycogen in individual aphids was measured by the hot anthrone test.
were found. The result also demonstrated that the total free amino acid level was decreased by 20.78% compared to the control. The concentrations of the 16 free amino acids, except serine, were significantly decreased when aphids fed artificial diet with Xbpi-1 (Table 1).
3.4. Analysis of aphid sugar, glycogen and lipid contents When the aphids were fed on the artificial diet with Xbpi-1, the total sugar concentration was decreased by 39.41% on the 4th day (Fig. 3), and the glycogen concentration was decreased by 18.44% and 24.61% on the 3rd and 4th days, respectively (Fig. 4). The lipid concentration was decreased by 29.26% and 26.25% on the 3rd and 4th days, respectively, when the aphids were fed on the artificial diet with Xbpi-1 (Fig. 5). As compared to the controls, the glycogen and lipid concentrations of the aphids reared on the artificial diet with Xbpi-1 had significant differences on the 3rd day. However,
the total sugar concentration was significantly decreased on the 4th day after feeding on the diet. 4. Discussion In general, the mechanism of the protease inhibitor proteins against insects is that protease inhibitors have adverse effects on protease activity allowing the protease inhibitors to block the activities of proteases in the gut of target insects. Cristofoletti et al. [29] found that the pea aphid has strong aminopeptidase activity in the midgut. Generally, the effect of protease inhibitors on protease activity is determined by measuring the activity of the proteases in vitro with the inhibitor [30,31]. In this study, the effect of Xbpi-1 on the activity of aminopeptidase was determined by measuring the aminopeptidase activity in A. pisum after the aphids fed artificial diets containing Xbpi-1. The result revealed that Xbpi-1 decreased LAP activity of pea aphid by 18.3%. Multiple types of proteases exist in insect guts, and the development and
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Fig. 5. Effect of Xbpi-1, a protease inhibitor protein, on the total lipid concentration of Acyrthosiphon pisum. The amount (lg ± SE) of total lipids in individual aphids was measured by the sulfo-phospho-vanillin reaction.
growth of insects can be affected more quickly if two or more types of proteases are inhibited [32–35]. It is necessary to conduct further research to determine whether Xbpi-1 is a broad spectrum protease inhibitor. In addition, this study showed that the mortalities of A. pisum aphids fed artificial diets containing 200, 400, and 800 lg/ml Xbpi-1 were two, three and four times, respectively, more than the mortality of the controls. The results from this study agreed with those reported by other researchers [19,30,36–38]. Thus, Xbpi-1 may be a potential bio-pesticide for controlling sucking insect pests. This work is the first report on the effects of a novel protease inhibitor protein on carbohydrates, free amino acids and lipids of pea aphid. The major nutritive materials, such as carbohydrates, free amino acids and lipids, are necessary for growth, reproduction and survival of insects [39–45]. Energy metabolism needs glycolysis, fatty acid oxidation, and the tricarboxylic acid cycle. The results from this study showed that Xbpi-1 had adverse effects on the nutrient metabolism of A. pisum. The total sugar, glycogen and lipid concentrations were significantly reduced by rearing the aphids on a diet containing Xbpi-1 after 4 days, and the total free amino acid concentration was also significantly reduced, so Xbpi-1 may affect aphid normal physiological function. The results from this study support our hypothesis that the Xbpi-1 may have the adverse effects on aphid physiology and biochemistry. The protease inhibitor protein, Xbpi-1, inhibited aminopeptidase activity in A. pisum which may lead to the lack of some essential amino acids for the aphids. The Xbpi-1 also reduced the concentrations of the total sugar, glycogen and lipids. These adverse effects may disturb the normal aphid physiological and biochemical function, thus affect aphid growth and ultimately leading to aphid death.
Acknowledgments We thank the editors and reviewers for their comments and suggestions. Thanks are due to Feng-Jiao Liu and Chang-Yan Liu for their technical assistance throughout the experiment. This
work was supported from funds (973 Program 2013CB127600 and the agriculture cooperative research fund 2011-G4).
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