Effects of Pyriproxyfen on Intermediary Metabolism of Rice Striped ...

Effects of Pyriproxyfen on Intermediary Metabolism of Rice Striped Stem Borer, Chilo suppressalis Walker (Lepidoptera: Crambidae) Seyyedeh Kimia Mirhaghparast, Arash Zibaee & Hassan Hoda

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences ISSN 0369-8211 Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-014-0436-2

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Author's personal copy Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-014-0436-2

RESEARCH ARTICLE

Effects of Pyriproxyfen on Intermediary Metabolism of Rice Striped Stem Borer, Chilo suppressalis Walker (Lepidoptera: Crambidae) Seyyedeh Kimia Mirhaghparast • Arash Zibaee Hassan Hoda

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Received: 4 May 2014 / Revised: 26 July 2014 / Accepted: 15 September 2014 Ó The National Academy of Sciences, India 2014

Abstract Effects of Pyriproxyfen were determined on intermediary metabolism in the fifth larval instars of Chilo suppressalis via assessments of various enzymatic and nonenzymatic compounds. Bioassay of larvae revealed LC10–50 values of 18, 72 and 190 lg/ml of pyriproxyfen. Results on alanine aminotransferase revealed lower activities in treated larvae versus control for all time intervals while activity of aspartate aminotransferase showed higher activities in treated larvae by 18 and 72 lg/ml of pyriproxyfen. Activities of c-Glutamyl transferase and aldolase in treated larvae were significantly higher than those of control larvae. Treating of larvae by different concentrations of pyriproxyfen significantly increased lactate dehydrogenase activity for all time intervals although activity of the enzyme after 1 h was not significantly different among control and treated larvae. Activities of acid and alkaline phosphatases in treated larvae were higher than those of control. Amount of high and low density lipoproteins in treated larvae was higher than those of control except for time interval of 3 h of HDL in which control larvae revealed higher amount than 72 and 190 lg/ml treated larvae. Increased activity of the enzyme was noticed in treated larvae versus control in case of general esterases. Similar results were observed in case of glutathione S-transferase. Amount of triacylglyceride, glycogen and protein in control larvae was higher than those of treated larvae for all time intervals indicating their depletion due to S. K. Mirhaghparast A. Zibaee (&) Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran e-mail: [email protected]; [email protected] H. Hoda Biological Control Department, Iranian Institute of Plant Protection, Amol, Iran

energetic costs of pyriproxyfen treatment. These results clearly indicated negative effects of pyriproxyfen on intermediary metabolism of larvae that might lead to desirable mortality in pest population. Keywords Chilo suppressalis Pyriproxyfen Intermediary metabolism Rice striped stem borer

Introduction Rice striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), is the major pest of rice in Latin America and Asia particularly northern areas of Iran [1]. Larval feeding on inner parts of the stems causes severe damage to rice production. Utilizing broad-spectrum insecticides such as diazinon, padan and fenitrothione is the main way to decrease population outbreaks of C. suppressalis in a given area. Although broad-spectrum insecticides have provided somehow stable management against rice striped stem borer, public issues over health and environmental effects of these insecticides have been increased by reporting various disorders on non-target organisms [1]. Moreover, resistance of C. suppressalis to diazinon has been reported in four different populations of northern Iran [1]. Insect growth regulators (IGRs) have shown many alternatives of organophosphorous insecticides due to their selective and special mode of action [2]. Synthetic IGRs mimic natural hormones of insects and disrupt major physiological processes in them [3]. There are three main groups of IGRs namely chitin synthesis inhibitors, ecdysone agonists (20E) and juvenile hormone analogues [4]. Chitin synthesis inhibitors decrease synthetic amount of chitin and induce its malformations [5]. Ecdysone agonists

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Author's personal copy S. K. Mirhaghparast et al.

imitate biological activity of insect’s natural molting hormone, 20-hydroxyecdysone (20E), in a competitive manner with ecdysteroids [6]. Pyriproxyfen is a juvenile hormone analogue showing low toxicity against mammals [2]. It is a pyridine-based insecticide that competes with natural juvenile hormone on its binding receptors and imitates action of juvenile hormone leading to maintain immature state of insects [2]. Pyriproxyfen was initially used against public health pests [2] but recently it was considered to be effective against agricultural pests such of Diptera, Homoptera and Lepidoptera [2]. Intermediary metabolism shows various pathways in which digested foods such as carbohydrates, lipids and proteins are processed to generate energy [7]. Several enzymatic and non-enzymatic components are involved in energy production for insects. As described earlier, insect growth regulators (IGRs) interfere in physiology of insects and cause destructive effects on growth, development and reproduction. Although the first target of juvenile hormone analogues is endocrine system but many biochemical and physiological components are changed in several metabolic pathways. For example, larvae of Bombyx mori have shown an increase in hemolymph compounds such as glucose, urea, uric acid, cholesterol, total protein, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, 24 h after being treated by pyriproxyfen [8]. Zibaee et al. [9] have shown that pyriproxyfen leads to changes in enzymatic and non-enzymatic compounds in adults of Eurygaster integriceps Puton (Hemiptera: Scutelleridae). The aim of the current study is to demonstrate effects of pyriproxyfen on larvae of C. suppressalis by evaluating the involved enzymatic and non-enzymatic compounds in detoxification and intermediary metabolism.

Material and Methods Insect Rearing Eggs of C. suppressalis were collected from rice fields of Amol (north Iran) and reared on seedlings of Hashemi variety in the laboratory conditions of 28 ± 1 °C, 70 ± 5 % of relative humidity (RH) and 16 h light:8 h dark (LD 16:8). Hatched larvae were reared up to fifth larval instars. Fresh stems were provided to the larvae for feeding. Bioassay Five concentrations of Pyriproxyfen (50, 100, 300, 500 and 1,000 lg/ml) were topically exposed on fifth instar larvae to evaluate LC10, LC30 and LC50 values along with a control treated by acetone (N = 180). Larvae were treated

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topically by 2 ll of each concentration on dorsal cuticle using a microapplicator. Mortality was recorded at 72 h and the LC values were calculated using POLO-PC software. Determined LC10, LC30 and LC50 values were treated on 1,200 larvae for biochemical experiments. Sample Preparation for Enzymatic Assay Treated larvae were randomly selected and their hemolymph and fat bodies were collected separately at 1, 3, 6, 12, 24 h post-treatment by pyriproxyfen. The fat bodies were stored in distilled water but the hemolymph samples were combined with 250 ll of anticoagulant solution (0.01 M ethylenediamine tetra-acetic acid, 0.1 M glucose, 0.0062 M NaCl, 0.026 M citric acid, pH 4.6) as described by Azambuja et al. [10]. Samples of fat body were homogenized by a glass homogenizer and centrifuged at 28,0009g for 15 min at 4 °C. Samples for hemolymph were directly centrifuged at 28,0009g for 15 min at 4 °C without homogenizing. Assay of Alanine (EC 2.6.1.1) and Aspartate (EC 2.6.1.1) Aminotransferases Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using procedure of Thomas [11]. This assay was done by AST and ALT kit (Biochem Co., Iran). The reagents AST and ALT were mixed (4:1) and samples were added and absorption was read at 340 nm. Assay of c-Glutamyl Transferase The method described by Szasz [12] was used to measure c-glutamyl transferase activity in control and treated larvae (ZiestChem Diagnostic Co., Tehran-Iran). Reaction mixture consisted of 50 ll of buffer reagent and 20 ll of substrate reagent (L-c-glutamyl-3-carboxy-4-nitrinilide). Absorbance was read at 405 nm after incubation for 3 min. Assay of Aldolase Activity of aldolase was assayed according to Pinto et al. [13]. As the instruction of manufacturer (ZiestChem Diagnostics Co., Tehran-Iran), 50 ll of buffer reagent, 25 ll of substrate reagent (Fructose-1,6 di-phosphate), 10 ll of cofactor reagent (NADH) and 20 ll of sample were incubated for 5 min prior to the reading of absorbance at 340 nm. Assay of Lactate Dehydrogenase (EC 1.1.1.27) For evaluating the lactate dehydrogenase (LDH) King‘s method [14] was used. To standardize volumes, 0.2 ml of

Author's personal copy Pyriproxyfen and Intermediary Metabolism of C. suppresslais

NAD? solution was taken in a test tube and 0.2 ml of water in control test tube. Then, 1 ml of the buffered substrate and 0.01 ml of the sample were added. Test tube was incubated for 15 min at 37 °C. One milliliter of color reagent (2,4-dinitrophenyl hydrazine) was added prior to incubation for 15 min. The contents were cooled in room temperature, 10 ml of 0.4 N NaOH was added to make the solution strongly alkaline. Finally, the intensity of color was measured at 340 nm.

Protein Determination

Assay of Acid (EC 3.1.3.2) and Alkaline (EC 3.1.3.1) Phosphatase

Triacylglyceride Determination

The enzyme assays were carried out as described by Bessey et al. [15]. The buffered substrate (phosphate buffer, 0.02 m, pH 7.2) was incubated with the sample for 30 min. Alkali was added to stop the reaction and adjust the pH to determine the concentration of the product. Finally, absorbance was read at 405 nm. Determination of HDL and LDL The method of Schaefer and McNamara [16] was used to determine the amount of HDL and LDL in control and treated larvae by using diagnostic kit of ZiestChem Co, Tehran-Iran. In case of HDL, 50 ll of precipitant reagent (PTA-Magnesium) and 20 ll of samples were incubated for 15 min. The reaction mixture was centrifuged at 28,0009g for 2 min. The absorbance was read at 492 nm after 60 min. For LDL evaluation, 50 ll of buffer reagent was incubated with 20 ll of enzyme reagent (Containing H2O2 and aminoantipyrine) and 10 ll of sample for 5 min. The absorbance was read at 545 nm. Determination of EST Activity Esterase activity was determined by the method described in Han et al. [17]. Twenty microliters of a-naphthyl acetate, b-naphthyl acetate (10 mM) and 50 ll fast blue RR salt (1 mM) was put in each tube. The reaction was initiated by addition of 20 ll of enzyme solution, incubated for 1 min and then optical density (OD) was recorded at 450 nm using microplate reader. Determination of GST Activity For glutathione S-transferase activity, the method reported by Oppenorth [18] was adopted. Twenty microliters of CDNB (20 mM) and DCNB (40 mM) were separately pipetted into microplate wells, and then 100 ll of enzyme solution was added. The OD value was recorded at 340 nm after 1 min incubation.

Protein concentration was assayed according to the method described by Lowry et al. [19]. The method recruits reaction of Cu2?, produced by the oxidation of peptide bonds with FolinCiocalteu reagent. In the assay, 20 ll of homogenized sample was added to 100 ll of reagent, and incubation was made for 30 min. The absorbance was read at 545 nm (Recommended by Ziest Chem. Co., Tehran-Iran).

A diagnostic kit from PARS-AZMOONÒ Co. was used to measure the amount of triacylglyceride. Reagent solution contained phosphate buffer (50 mM, pH 7.2), 4-chlorophenol (4 mM), Adnosine Triphosphate (2 mM), Mg2? (15 mM), glycerokinase 0.4 kU/L), peroxidase (2 kU/L), lipoprotein lipase (2 kU/L), 4-aminoantipyrine (0.5 mM) and glycerol-3phosphate-oxidase (0.5 kU/L). Samples (10 ll) were incubated with 10 ll distilled water and 70 ll of reagent for 20 min at 25 °C [20]. ODs of samples and reagent as standard were read at 545 nm. The following equation was used to calculate the amount of triacylglyceride: mg/dl ¼

OD of sample 0:01126 OD of standard

Glycogen Determination Fat bodies of 30 larvae were taken out and immersed in 1 ml of 30 % KOH w/Na2SO4. Tubes which contained the samples were covered with foil (to avoid evaporation) and were boiled for 20–30 min. Tubes were shaked and cooled in ice. Two ml of 95 % ethanol solution was added to precipitate glycogen from digested solution. The samples were again shaked and incubated in ice for 30 min. Tubes were centrifuged at 28,0009g for 30 min. Supernatant was removed and pellets (glycogen) were re-dissolved in 1 ml of distilled water before being shaked. Glycogen standard (0, 25, 50, 75 and 100 mg/ml) was prepared before adding 5 % phenol. Incubation was performed on ice bath for 30 min. Standards and samples were read at 490 nm and distilled water was used as blank [21]. Statistical Analysis All data were compared by one-way analysis of variance (ANOVA) followed by Tukey’s test when significant differences were found at p B 0.05 and marked in figures with letters. Results and Discussion Bioassay of fifth instar larvae by different concentrations of pyriproxyfen revealed LC10, LC30 and LC50 values of 18,

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Fig. 1 Effect of pyriproxyfen on activities aminotransferases in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

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especially the concentration of 72 lg/ml of pyriproxyfen (Fig. 1). In case of c-glutamyl transferase, enzymatic activities of treated larvae were significantly higher than those of control in all time intervals (Fig. 1). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the two main transaminases in hemolymph and fat bodies of insects that have crucial roles in transformation of amino acids or keto acid [11, 22]. AST catalyze the conversion of aspartate and a-ketoglutarate to oxaloacetate and glutamate or vice versa during the krebs cycle and transamination. ALT is a transaminase that involves in two parts of the alanine cycle during proline metabolism [7, 19]. c-glutamyl transferase (c-GT) is another transaminase similar to AST and ALT that plays a crucial role in cglutamyl cycle for synthesis and degradation of glutathione and xenobiotic compounds although it has a key role in

72 and 190 lg/ml for pyriproxyfen. These concentrations were topically exposed to larvae providing any alterations in intermediary metabolism at various time intervals. In fact, results of the current study revealed interference of pyriproxyfen in intermediary metabolism of C. suppressalis larvae in various time intervals by alteration in activities of involved enzymes and the amount of nonenzymatic components. Activities of ALT in all time intervals were the highest in the control larvae versus treatment although no statistical differences were observed between control and treated larvae by concentration of 18 lg/ml after 1 and 3 h post-treatment (Fig. 1). AST activities of control larvae at time intervals of 3–12 showed lower activities than those of treated ones (Fig. 1). In other time intervals, AST activities in control and treated larvae by 18 lg/ml of pyriproxyfen were lower than others Fig. 2 Effect of pyriproxyfen on activities of aldolase and lactate dehydrogenase in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

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transamination by moving the c-glutamyl moiety of glutathione to a receptor leading to produce glutamate [23]. The present results showed two different activities of ALT and AST in control and treated larvae by pyriproxyfen. Higher activity of ALT in control larvae indicates efficiency of amino acid transformation to be used in proline metabolism for both energy demand and tissue repair. Since AST involved in providing oxaloacetate for krebs cycle, its activity could be attributed to necessity for energy demand via krebs cycle or tissue repair. In case of c-GT, higher activity in treated larvae indicates synthesis and degradation of glutathione and xenobiotic compounds like pyriproxyfen. Currently, Ramzi et al. [24] found similar results on Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae) fed on the artificial diet containing 2 % of Citrullus colocynthis L. agglutinin. Also, Zibaee et al. [9] reported that treating adults of Eurygaster integriceps

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Fig. 3 Effect of pyriproxyfen on activities of acid and alkaline phosphatases in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

Puton (Hemiptera: Scutelleridae) by pyriproxyfen caused high levels of AST and ALT after 24 and 48 h post-treatment. Etebari et al [8] showed lower activity of AST in Bombyx mori L. (Lepidoptera: Bombycidae) after 24 h of post-treatment by pyriproxyfen although the enzymatic activity sharply increased after 48 and 120 h. Sharifi et al. [25] reported lower activity of ALT 24 and 48 h posttreatment of 30 lg/ml pyriproxyfen on Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) larvae. In case of synthetic organophosphorous insecticides, Ender et al. [26] reported that diet containing high level of methyl parathion significantly increased ALT activity in Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. Treating C. suppressalis larvae by different concentrations of pyriproxyfen caused higher activity of aldolase and LDH in treated larvae versus control (Fig. 2). The only difference was found in case of LDH in time interval of 1 h

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activity of LDH increased in the treated adults of E. integriceps by pyriproxyfen. Acid phosphatase (ACP) and alkaline phosphatase (ALP) are the hydrolytic enzymes that remove phosphate groups from various types of molecules such as nucleotides, proteins and alkaloids in acid and alkali conditions, respectively [9] in addition their involvement in lipid digestion in the midgut [27]. Hence, these enzymes could affect physiological mechanisms of insects via digestion and positive transportation of nutrients in the midgut, hemolymph and fat body. Both ACP and ALP activities in treated larvae by pyriproxyfen were higher than those of control except for ACP activity of treated larvae by 190 lg/ml after 3 h that showed lower activity of control (Fig. 3). In accordance, amounts of HDL and LDL in the

in which activity of LDH in control larvae was higher than that of treated larvae by 72 and 190 lg/ml of pyriproxyfen (Fig. 2). Aldolase is an important enzyme for initial steps of glycolysis that breaks down some sugars to produce energy [13]. Also, pyruvate is the final product of glycolysis which is converted to lactate by lactate dehydrogenase (LDH) in anaerobic conditions and shortage in oxygen supplement. Moreover, LDH is an indicator showing exposure of organic tissues to chemical stresses when any elevation is obtained in its activity [9]. In the current study, it was found that activity of both enzymes elevated in the treated larvae by pyriproxyfen versus control. These findings could point out tissue damage caused by pyriproxyfen and energy demands via glycolysis. Although there is no report in case of aldolase but Zibaee et al. [9] found that

Fig. 4 Effect of pyriproxyfen on amounts of HDL and LDL in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

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Fig. 5 Effect of pyriproxyfen on activities of general esterases in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

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Time (Hour) treated larvae were found to be higher than those of control. Amount of HDL in control larvae was lower than those of treated larvae in all time intervals except for 3 h post-treatment. The control value was significantly higher than that of 190 lg/ml of pyriproxyfen (Fig. 4). Similar results were found in amount of LDL except for time interval of 6 h (Fig. 4). HDL and LDL are the two lipid carriers in the hemolymph [7]. HDL is responsible for transferring the mono and diacylglycerols from the midgut to fat bodies, while LDL transfers them to tissues to produce energy [19]. These findings could be explained by elevating energy demands in body which leads to a shift from glycolysis to b-oxidation of lipids to produce efficient energy to recovery. This is in accordance with lower amount of triacylglycerols that indicate energy demands vis metabolism of lipids. Besides components involved in intermediary metabolism of C. suppressalis larvae, activities of two main

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detoxifying enzymes were also evaluated. Treating of C. suppressalis larvae by different concentrations of pyriproxyfen caused statistical increase of general esterase activities by using both substrates except for b-naphtyl acetate assessment in 1 h post-treatment by pyriproxyfen (Fig. 5). Although similar results were observed in case of glutathione S-transferase activity but activity of the enzyme in the larvae treated by 190 lg/ml of pyriproxyfen were significantly lower than the control when CDNB was used as substrate (Fig. 6). Esterase (EST) is an important detoxifying enzyme which hydrolyses esoteric bonds in synthetic chemicals [28]. Glutathione S-transferases (GST) are the cytosolic enzymes catalyzing conjugation of electrophile molecules with reduced glutathione (GSH) to increase water solubility of toxic substances and to decrease their toxicity [29]. Meanwhile, GSTS have crucial role in the metabolism of synthetic insecticides and plant allelochemicals [30]. Treating of C. suppressalis larvae by


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Fig. 6 Effect of pyriproxyfen on activities of glutathione Stransferases in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

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Time (Hour) different concentrations of pyriproxyfen significantly increased activities of ESTs and GSTs in all time intervals. These results showed involvement of these enzymes in possible degradation of pyriproxyfen in hemolymph. Moreover, it could be concluded that although these enzymes detoxify pyriproxyfen but it still affects intermediary metabolism of the larvae. Amounts of triacylglyceride, glycogen and protein, as storage macromolecules in fat bodies, in control larvae were higher than in treated larvae for all time intervals although lower statistical differences were found in amount of protein among control and treated larvae (Fig. 7). Insects store their additional ingested food as three main macromolecules, protein, glycogen and triacylglyceride, in their fat bodies. These macromolecules are involved in energy production via intermediary metabolism including glycolysis, krebs cycle, b-oxidation, proline metabolism and electron transfer cycle. Except for protein that showed

various results, amount of glycogen and triacylglyceride significantly decreased in treated larvae by pyriproxyfen in all time intervals. Etebari et al. [8], Zibaee et al. [9], and Ramzi et al. [24] reported similar results on decrease in amount of storage macromolecules in insects exposed to insecticidal compounds. These results in addition to findings of engaged enzymatic components indicate energy demands of treated larvae leading to depletion of storage macromolecules.

Conclusion Results of the current study clearly depicted significant alterations in enzymatic and non-enzymatic components involved in intermediary metabolism of C. suppressalis larvae treated by pyriproxyfen. Moreover, there are a few studies on aldolase, c-GT, HDL and LDL followed

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Fig. 7 Effect of pyriproxyfen on amounts (mg/ml) of storage macromolecules in C. suppressalis larvae. Statistical differences have been marked by letters in each time intervals (Tukey test, p B 0.05)

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treatment by chemicals. These alterations were caused due to pyriproxyfen treatment nevertheless activities of two detoxifying enzymes, ESTs and GSTs, were also elevated in the treated larvae. Upcoming results of the current study could verify appropriate effects of pyriproxyfen against C. suppressalis showing its application as an alternative for currently used insecticides. Acknowledgments The research work has been supported by University of Guilan.

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