ISJ 13: 122-133, 2016 ISJ 13: 122-133, 2016 ISSN 1824-307X RESEARCH REPORT Chemical and organic fertilizers affect physiological performance and antioxidant activities in Myzus persicae (Hemiptera: Aphididae) M Mardani-Talaee1, A Zibaee2, G Nouri-Ganblani1, J Razmjou1 1Department of Plant Protection, Faculty of Agricultural Sciences, University of Mohaghegh Ardabili, Iran 2Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Accepted April 6, 2016 Abstract Myzus persicae is a widespread and polyphagous insect that causes severe damages to hundreds of host plants. In the current study, zinc sulfate and vermicompost as chemical and organic fertilizers, were added into cultural soil of Capsicum annuum to determine their effects on physiology and antioxidant activities of M. persicae. The aphids reared on zinc sulfate-treated culture showed the highest activities of general protease, trypsin, cathepsins, carboxypeptidase and lipase but activities of chymotrypsin and aminopeptidase were the highest in vermicompost-treated culture. Although activities of α-amylase in the fertilizer-treated cultures were higher than control but activities of α- and β-glucosideases showed the highest values in zinc sulfate and vermicompost treatments, respectively. Aspartate aminotransferase and γ-glutamyl transferase showed the highest activity in the aphids reared on the vermicompost-treated culture but alanine aminotransferase activity got the lowest value in fertilizer-treated cultures. Activities of aldolase and lactate dehydrogenase in the fertilizer-treated aphids were higher than those of control and vermicompost-treated aphids, but alkaline phosphatase showed the lower activity although activity of acid phosphatase decreased in vermicompost- treated aphids compared to other treatments. Activities of antioxidant enzymes were found to be the highest in the aphids fed on vermicompost-treated culture including glucose-6-phosphate dehydrogenase, superoxide dismutase, peroxidase and ascorbate oxidase but catalase in vermicompost treatment had lower activity than control and zinc-sulfate treatments. Also, malondialdehyde and RSSR/RSH ratio demonstrated higher values in the aphids fed on zinc sulfate- and vermicompost-treated plants than control, respectively. Finally, the amounts of glycogen and triglyceride revealed the highest values in zinc sulfate-treated plants compared to other treatments. These results indicated significant effects of fertilizers on physiology and antioxidant activity of M. persicae which are important to be considered in integrated pest management programs. Key Words: Myzus persicae; fertilizer; physiological performance; antioxidant indices   Introduction The green peach aphid, Myzus persicae (Hemiptera: Aphididae), is one of the most important insect pests worldwide because of its severe damages to many crop plants, vegetables and fruit trees as well as transmission of plant pathogenic viruses such as potato leaf roll virus (PLRV), potato virus Y (PVY), pepper mottle virus (Pep MoV), tobacco etch virus (TEV) and cucumber mosaic virus (CMV) (Hill, 1983; Robert et al., 2000). Although population outbreaks of M. persicae are ___________________________________________________________________________ Corresponding author: Arash Zibaee Department of Plant Protection Faculty of Agricultural Sciences University of Guilan, Rasht, Iran E-mail: Arash.zibaee@gmx.com, arash.zibaee@guilan.ac.ir somehow suppressed by synthetic insecticides but strong selection pressure of chemical spraying induces resistance to the most registered insecticides so other control methods need to be investigated for appropriate control (Bolandandam et al., 2004). Although fertilizers are recommended to increase crop yields but they can affect pest populations leading to to use control procedures (Patriquin et al., 1995; Arancon et al., 2006; Edwards et al., 2009). In fact, fertilizers can fluctuate amounts of defensive chemical components in plants which finally change ecological fitness and physiological performance of herbivorous insects (Edwards et al., 2009; Mardani- Talaee et al., 2016). Numerous studies have reported the effects of macronutrient chemical 123   mailto:Arash.zibaee@gmx.com mailto:arash.zibaee@guilan.ac.ir fertilizers (such as N, P and K) on population dynamics of insect pests (Lu et al., 2007). Since fertilizers may result in higher growth rate and population increase of herbivorous insects through improving nutritional quality of host plants (Edwards et al., 2009). Moreover, fertilizers residue have raised a great concern of consumers in recent years because of their chemical constituent. So, organic fertilizers may be more appropriate because of their least effects on environment or residual contaminations. In case, vermicompost is an organic fertilizer produced through the interactions between earthworms and microorganisms in a mesophilic process from organic wastes. It reduces pH and C:N ratio in soil, stabilizes the organic matter and makes nutrients readily available to plants (Yardim et al., 2006). Soil amendment with vermicompost have reduced population growth rates in some herbivores such as Manduca quinquemaculata, Acalymma vittatum, Diabrotrica undecimpunctata (Yardim et al., 2006), Leptinotarsa decemlineata (Mardani-Talaee et al., 2015), Pseudococcus sp., Teranychus urticae, M. persicae (Arancon et al., 2002, 2006; Edwards et al., 2009; Mardani-Talaee et al., 2016), and Aphis gossypii (Razmjou et al., 2011). In a previous study, we found that vermicompost increased levels of phenolic compounds in the leaves of bell pepper and thereby decreased life table parameters of M. persicae (Mardani-Talaee et al., 2016). On the other hand, our results demonstrated an induced resistance in bell pepper cultured in vermisompost-treated culture (Mardani-Talaee et al., 2016). In case, physiological parameters of treated and non-treated aphids by fertilizers must be determined to better understanding of observed changes. So, the current study was conducted to compare potential changes in physiological processes of M. persicae induced by fertilizers. In details, a chemical fertilizer (zinc sulfate) and vermicompost (30 %) were separately added into cultural soil of C. annum to find their effects on digestion, intermediary metabolism and antioxidant activities of M. persicae under greenhouse conditions. These findings will increase our understanding on beneficial or detrimental effects of fertilizers to better management of M. persicae in greenhouses. Materials and Methods Plant sources, Insect colony and fertilizer treatments The sandy loam soil collected from a fallow potato field in Ardabil plain (38°14´ N and longitude 48°19´ E) was used to grow C. annuum (cv. California Wonder). Seeds of bell pepper, C. annuum were grown in an aphid-free greenhouse set at 25 ± 5 oC, 60 ± 5 % of relative hunidity and a photoperiod of 14:10 (L:D) h. The aphids were collected from a tomato field from Meshkin-Shahr (Ardabil province), Iran and transferred to cultured C. annuum at six-leaf stage. To maintain a suitable aphid colony, some aphids were transferred from infested plants to new young plants every week. Experimental treatments here were; i) bell pepper grown in field-collected soil as control treatment; ii) bell pepper grown in the field-collected soil sprayed on 4 - 6 expanded leaves stages of C. annuum with zinc sulfate (Provided by Iranian Soil and Water Research Institute in Karaj) at the concentration of 0.001 % (50 mL per plant). iii) bell pepper grown in the same soil amended with 30% of vermicompost (Obtained from cattle manure of Pars Koud Company, Gorgan, Iran) which contained 1.8 % N, 3.9 mg/kg P, pH of 7.3 and EC of 2.2 ds/m before planting. Sample preparation for biochemical assays Adults of M. persicae from control and fertilized cultures were randomly selected and homogenized in ice cold NaCl solution (0.15 M) in proportion of 20 aphids per ml of saline solution. The samples were then centrifuged at 20000 g for 5 min at 4 ºC (Dubovskiy et al., 2008). Supernatants were collected and stored at -20 ºC to onset of the biochemical experiments (< a week). Determination of digestive enzyme activities General proteolytic activity General proteolytic activity in control and treated aphids by fertilizers were determined based on the method of Elpidina et al. (2001) using Table 1 Effects of fertilizers on activities of digestive proteases in M. persicae adults Statistic (Mean±SE U/mg protein) Treatments General Protease1 Trypsin Chymotrypsin Elastease CathepsinL CathepsinB Aminopeptidases Carboxypeptidases Control 0.062±0.016 b 4.568±0.336 b 2.818±0.759 c 38.51±1.950 c 2.819±0.459 b 3.885±0.374 c 0.134±0.017 c 1.123±0.125 a Vermicompost (30%) 0.040±0.008 b 2.829±0.154 b 12.143±0.432 a 60.15±1.890 b 14.97±1.430 b 6.000±1.150 b 5.150±0.797 a 0.657±0.302 b Zinc sulfate 0.224±0.027 a 14.842±0.996 a 5.25±0.231 b 128.82±0.682 a 23.25±1.280 a 13.280±1.080 a 0.550±0.057 b 1.092±0.0519 a 1Activity unit of all enzymes were U per mg protein except for general protease as OD/min. The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. 124   Azocasein (2 %) as substrate. Briefly, 40 µL of azocasein solution, 80 µL of universal buffer [20 mM, (Frugoni, 1957; Mardani-Talaee and Zibaee, 2015)] and 20 µL of sample were incubated for 60 min at 37 ºC. Then, proteolysis was stopped by adding 80 µL of 30 % trichloroacetic acid (TCA) and precipitation was achieved by cooling at 4 °C for 5 min prior to centrifugation at 20,000g for 10 min. Finally, an equal volume of NaOH (2 N) was added and absorbance was read at 450 nm (Microplate reader, Awareness Statfax, 3200, USA). Specific proteolytic activity Based on Rahbe et al. (2003), activities of trypsin, chymotrypsin, cathepsin B, cathepsin L, amino- and carboxypeptidase were determined in control and fertilized-treated aphids. Trypsin and chymotrypsin activities were assayed using 1 mM of BApNA (Nabenzoyl- DL-arginine-p-nitroanilide) and SAAPPpNA (N-succinyl-alanine- alanine-proline- phenylalanine-p-nitroanilide), as substrates, respectively. Substrates (20 µL) were separately added into 50 µL of universal buffer (20 mM, pH 8) and incubation was initiated by adding 10 µL of enzyme solution for 10 min at 30 ºC. The reaction was terminated by adding 100 µL of TCA (30 %) and absorbance was read at 405 nm. Cathepsin B and L activities were determined using Z-Ala-Arg- Arg 4-metjoxy-bnaphtylamide acetate (1 mM) and N-benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride (1 mM), respectively. Substrates (20 µL) were separately added into 50 µL of universal buffer (20 mM, pH 5) and incubation was initiated by adding 10 µL of enzyme solution for 10 min at 30 ºC. The reaction was terminated by adding 100 µL of TCA (30 %) and absorbance was read at 405 nm. Activities of exopeptidases were determined using hippuryl-L-arginine and hippuryl-L-phenylalanine as the specific substrates (1 mM) of carboxy- and aminopeptidases, respectively. Substrates (20 µL) were separately added into 50 µL of universal buffer (20 mM, pH 7) and incubation was initiated by adding 10 µL of enzyme solution for 10 min at 30 ºC. The reaction was terminated by adding 100 µL of TCA (30%) and absorbance was read at 340 nm. α-Amylase assay Activity of α-amylase was determined based on the method of Bernfeld (1955) using starch (1 %) and dinitrosalicylic acid (DNS). Briefly, 40 µL of starch solution (1 %), 80 µL of universal buffer (pH 7) and 20 µL of enzyme sample were incubated for 30 min at 35 ºC. Then, 100 µL of DNS was added and the tubes containing reaction mixture were additionally incubated for 10 min at boiling water. Finally, 100 µL of the reaction mixture was poured into wells of microplate and absorbance was read at 540 nm. α- and β-glucosidase assay According the method of Silva and Terra (1995), α- and β-glucosidase activities were assayed by adding 20 µL of p-nitrophenol α- glucopyranoside for α-glucosidase (5 mM) and p- nitrophenol β -glucopyranoside for β-glocusidase (5 mM) (Separately) in 50 µL of universal buffer (pH 7). The incubation was initiated by adding 10 µL of the enzyme solution for 10 min prior to read absorbance at 405 nm. Lipase assay Activity of lipase was determined using the method of Tsujita et al. (1989) in control and fertilized-treated aphids. Ten microliter of sample and 20 μL of p-nitrophenyl butyrate (27 mM) as substrate were added into 50 µL of universal buffer (pH 7), mixed thoroughly and incubated at 37˚C. After 1 min, 100 μL of NaOH (1 M) was added and absorbance was read at 405 nm. Determination of intermediary metabolism in M. persicae Assay of alanine (EC 2.6.1.1) and aspartate (EC 2.6.1.1) aminotranferases This is a chlorometric assay using 2,4- dinitrophenyl hydrazine to synthesize pyruvate hydrazine by combination of pyruvate with 2,4- dintitrophenyl puruvate (Thomas, 1998). Based on a kit provided by Biochem Company (Tehran, Iran), reagent A (for AST) and reagent B (for ALT) were incubated separately with reagent D for 5 min. Then, 10 µL of the enzyme solution was added and incubation continued for 60 min. At the end, reagent C was added and absorbance was read at 340 nm. Specific activity was calculated by dividing absorbance with protein content in sample. Assay of γ-Glutamyl transferase The assay was done by the method of Szasz (1976) using a kit provided by ZiestChem Diagnostic Company (Tehran-Iran). The reaction mixture consisted 50 μL of buffer reagent, 20 μL of substrate reagent (L-γ-glutamyl-3-carboxy-4-nitrianilide) and 10 μL of the sample. Incubation was prolonged for 3 min and absorbance was read at 405 nm (ZiestChemDiagnostic Co., Tehran-Iran). Specific activity was calculated by dividing absorbance with protein content in sample. Assay of aldolase As the instruction of manufacturer, ZiestChem Diagnostics Company (Tehran-Iran), 50 µL of buffer reagent, 25 µL of substrate reagent (Fructose-1,6 di-phosphate), 10 µL of cofactor reagent (NADH) and 20 µL of sample were incubated for 5 min prior to read absorbance at 340 nm (Pinto et al., 1969). Assay of lactate dehydrogenase The method of King (1965) was used to evaluate activity of lactate dehydrogenase (LDH). To standardize volumes, 0.2 ml of NAD+ solution was added to the test tubes and 0.2 ml of water was added to control test tubes, each containing 1 ml of the buffered substrate and 0.01 ml of the sample was also added to the test tubes. Test tube samples were incubated for exactly 15 min at 37 °C and then arrested by adding 1 ml of color reagent (2,4- dinitrophenyl hydrazine) to each tube and the incubation continued for an additional 15 min. Then, the contents were cooled at room temperature, 10 ml of 0.4 N NaOH was added to each tube to make the solutions strongly alkaline. At exactly 60 s after adding of alkali to each tube, the intensity of color was measured at 340 nm. 125   Table 2 Effect of fertilizers on activities of α- amylase, glucosidases and lipase in M. persicae adults Statistic (Mean±SE U/mg protein) Treatments α-amylase α- glucosidases ß-glucosidases Lipase Control 5.870±0.142 b 43.49±2.560 b 6.500±1.480 c 79.790±1.640 b Vermicompost (30%) 7.120±0.194 a 29.32±1.894 c 25.320±9.120 a 51.19±1.059 c Zinc sulfate 7.346±0.759 a 65.88±3.730 a 15.723±0.512 b 169.94 ±2.271 a The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. Assay of acid (EC 3.1.3.2) and alkaline (EC 3.1.3.1) phosphatases The assays were carried out as described by Bessey et al. (1946). The buffered substrate (phosphate buffer, 0.02 M, pH 7.2) was incubated with samples for 30 min. Alkali was added to stop the reaction and to adjust pH for determination of product concentration. The spectral absorbance of p-nitrophenolate was maximal at 340 nm. The molar absorbance of p-nitrophenolate at 400 nm is approximately double that of p-nitrophenyl phosphate at 310 nm. On converting the p- nitrophenolate into p-nitrophenol by acidification, the absorption maximum shifted to approximately 320 nm with no detectable absorption at 405 nm. Determination of antioxidant activities in M. persicae Catalase (CAT) activity was measured based on the method of (Wang et al., 2001). Briefly, 100 μL of sample was added into 500 μL of hydrogen peroxide (1 %), incubated at 28 °С for 10 min and the activity was determined as the ΔA at 240 nm/min/mg protein. Superoxide dismutase (SOD) activity was determined based on McCord and Fridovich (1969) as the reduced rate of NBT (Nitro blue tetrazolium) by the superoxide anion due to xanthine oxidation by xanthine oxidase (McCord and Fridovich, 1969). Sample (100 μL) was added into 500 μL of the reaction solution containing 70 μM of NBT; 125 μM of xanthine; both dissolved in PBS and the xanthine oxidase solution [(100 μL; 10 mg of bovine albumin; 100 μL of xanthine oxidase (5.87 units/mL); dissolved in 2 mL of PBS]. The reaction mixture was incubated in darkness for 20 min at 28 °С prior to calculate the activity as differences in absorbance between a sample containing the mixture and a clean reagent mixture at ΔA 560 nm/min/mg protein. Peroxidase activity was assayed based on by the method of Addy and Goodman (1972). The reaction mixture consisted 500 µL of buffered pyrogallol [0.05 M pyrogallol in 0.1 M phosphate buffer (pH 7.0)] and 500 µL of H2O2 (1 %) prior to adding 100 µL of enzyme extract. Changes in absorbance was measured at 430 nm for every 30 seconds in 2 minutes. The peroxidase activity was calculated using an extinction coefficient of oxidized pyrogallol (4.5 litres/mol). Ascorbate peroxidase assay was carried out according to Asada (1984). The reaction mixture consisted 100 µL of sample and 250 µL of 67 mM potassium phosphate buffer (pH 7) containing 2.5 mM ascorbic acid and 200 µL of 30 mM H2O2. Absorbance was monitored at 290 nm for 5 min. MDA (malondialdehyde) concentration was determined by considering the process of lipid peroxidation due to formation of MDA. Based on Bar-Or et al. (2001), Briefly, 100 μL of 20 % trichloroacetic acid was mixed with 100 μL of the sample prior to be centrifuged at 15,000g for 10 min at 4 °C. The obtained supernatant was mixed with 100 μL of 0.8 % TBA reagent, and the mixture was incubated at 100 °C for 60 min prior to read absorbance at 535 nm. The MDA concentration is reported as amount of MDA produced per mg protein using a molar extinction coefficient of 1.56×105 M-1 cm-1. Activity of glucose-6-phosphate dehydrogenase was measured based on Balinsky and Bernstein (1963). The solution containing 100 μL Tris-HCl buffer (100 mM, pH 8.2), 0.2 mM NADP and 0.1M of MgCl2 was taken in a cuvette along with 100 μL of water and suitable aliquots of enzyme extract. The reaction was initiated by adding 100 of 6 mM glucose-6-phosphate and OD increase was measured at 340 nm. The activity of the enzyme is expressed as μmol/min/mg protein. The concentrations of oxidized (RSSR) and reduced (RSH) thiols were determined based on the method of Khramtsov et al. (1997). Initially, RSSRs were decomposed for 20 min by 1 M of hydrochloric acid to form RSH prior to neutralize the mixture pH by sodium hydroxide. Then, fifty microliters of the homogenate was mixed with 500 μL of 0.1 % DNTB solution in PBS, and the mixture was incubated for 10 min at 37 °С. The absorbances of RSH and RSH and RSSR were measured at 405 nm. The concentration of RSSR was calculated as the difference between the final concentration of reduced thiols after reduction by hydrochloric acid (RSH and RSSR) and the initial concentration of one RSH in the sample. The results are presented as the ratio of RSSR to RSH. 126   Table 3 Effects of fertilizers on activities of transaminases in M. persicae adults Statistic (Mean±SE U/mg protein) Treatments AST ALT γ-GT Control 0.121±0.020 b 0.182±0.022 a 0.256±0.040 b Vermicompost (30%) 0.242±0.044 a 0.137±0.027 b 0.680±0.247 a Zinc sulfate 0.147±0.018 b 0.167±0.023 ab 0.389±0.088 b AST, aspartate amino transferase; ALT, alanine amino transferase; γ-GT, γ-glutamyl transferase. The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. Determination of storage macromolecules in M. persicae Triglyceride determination The amount of triglyceride was measured using a diagnostic kit provided by Pars Azmoon Company (Tehran, Iran) in M. persicae adults. Briefly, 10 µL of sample was incubated with 70 µL of reagent solution containing phosphate buffer (50 mM, pH 7.2), 4-chlorophenol (4 mM), adenosine 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-3- phosphate-oxidase (0.5 kU/L) for 20 min at 25 °C (Fossati and Prencipe, 1982). Then, absorbance of sample mixture and reagent was read at 546 nm. The following equation was used to calculate the amount of triacylglyceride: mg/dl = Glycogen determination Bodies of 10 aphids were immersed in 1 mL of 30 % KOH with Na2SO4; covered with foil (to avoid evaporation) and put in boiling water for 30 min. Tubes then were shaken and cooled in ice. Two milliliters of 95 % EtOH was added and the tubes were shaken again and incubated in ice for 30 min. Followed by centrifugation at 22,000g for 30 min, supernatant was removed and the pellets (glycogen) were re-dissolved in 1 mL of distilled water before being re-shaken. Incubation continued after adding 5 % phenol for 30 min prior to read absorbance at 492 nm. Standard of glycogen was prepared as 0, 25, 50, 75 and 100 mg/mL to calculate amount of glycogen in sample (Chun and Yin, 1998). Protein determination The method of Lowry et al. (1951) was used to determine protein concentration in the control and treated aphids (ZiestChem. Co., Tehran, Iran). Statistical analysis Normality of data was tested by Kolmogorov- Smirnov method prior to analysis by one-way analysis of variance (ANOVA) followed by comparison of the means with Tukey post hoc Honestly Significant Difference (HSD) test at α = 0.05. Results Effects of fertilizers on digestive enzymes Statistical differences were found in activities of general protease, serine proteases, cysteine proteases and exopeptidases (amino- and carboxy- peptidases) in M. persicae fed on control and fertilizer-treated C. annum (Table 1). Activities of general protease (F = 27.14; df = 2, 8; p < 0.01), trypsin (F = 111.95; df = 2, 8; p < 0.01), elastase (F = 149.79; df = 2, 8; p < 0.01), cathepsin L (F = 80.84; df = 2, 8; p < 0.01), cathepsin B (F = 27.88; df = 2, 8; p < 0.01) and carboxypeptidases (F = 1.86; df = 5, 17; p < 0.01) were the highest in the aphids fed on the zinc sulfate treated-plants while the highest activities of chymotrypsin (F = 30.69; df = 2, 8; p < 0.01) and aminopeptidase (F = 36.37; df = 2, 8; p < 0.01) were found in vermocompost treatment (Table 1). For all proteases, the lowest activities were observed in control treatments except for carboxypeptidase (Table 1). The highest activities of α-amylase (F = 9.75; df = 2, 8; p < 0.01), α- glucosidase (F = 9.30; df = 2, 8; p < 0.01) and lipase (F = 416.68; df = 2, 8; p < 0.01) were found in the aphids fed on C. annum treated by zinc sulfate although no significant difference was observed in α-amylase of the aphids fed on both zinc sulfate- and vermicompost-treated plants (Table 2). Aphids fed on control and vermicompost- treated C. annum showed the highest and the lowest ß-glucosidase (F = 3.10; df = 2, 8; p < 0.01) activity, respectively (Table 2). The lowest activities of α-amylase and ß-glucosidase were obtained in control but the aphids fed on vermicompost-treated C. annum showed the lowest activities of α- glucosidase and lipase (Table 2). Effects of fertilizers on intermediary metabolism of M. persicae Significant differences were found in activities of aspartate amino transferase (AST), alanine amino transferase (ALT) and γ-glutamyl transferase (γ-GT) of M. persicae fed on different fertilizer 127   Table 4 Effects of fertilizers on activities of aldolase, alkaline and acid phosphatase and lactate dehydrogenase in M. persicae adults Statistic (Mean±SE U/mg protein) Treatments Aldolase ALP ACP LDH Control 0.424±0.099 b 0.104±0.002 a 0.337±0.033 a 0.259±0.049 b Vermicompost (30%) 0.773±0.030 a 0.039±0.026 b 0.150±0.012 b 0.796±0.097 a Zinc sulfate 0.931±0.034 a 0.068±0.017 b 0.353±0.027 a 0.671±0.166 a ALP, alanine phosphatase; ACP, acid phosphatase; LDH, lactate dehydrogenase. The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. treatments (Table 3). AST (F = 4.73; df = 2, 8; p < 0.01) and γ-GT (F = 2.13; df = 2, 8; p < 0.01) showed the highest activities in M. persicae fed on vermicompost-treated C. annum while control and zinc sulfate treatments caused the lowest enzymatic activities (Table 3). In case of ALT, the aphids fed on both fertilizer treatments showed lower activity than control (F = 0. 94; df = 2, 8; p < 0.01) (Table 3). Aldolase (F = 1.90; df = 2, 8; p < 0.01) and LDH (F = 6.06; df = 2, 8; p < 0.01) had the highest activities in the aphids fed on fertilizer-treated C. annum compared to control while activity of ALP decreased in these treatments (F = 3.36; df = 2, 8; p < 0.01) (Table 4). Finally, activity of ACP (F = 20.54; df = 2, 8; p < 0.01) was the highest in M. persicae fed on control and zinc sulfate-treated C. annum (Table 3). Effects of fertilizers on antioxidant enzymes Both control and zinc-sulfate treatments caused the highest activity of catalase in M. persicae but the aphids fed on vermicompost-treated C. annum had the lowest enzymatic activity (Table 5; F = 4.10; df = 2, 8; p < 0.01). Activities of peroxidase (F = 4.90; df = 2, 8; p < 0.01), superoxide dismutase (F = 5.33; df = 2, 8; p < 0.01), ascorbate oxidase (F = 6.52; df = 2, 8; p < 0.01) and glucose-6-phosphate dehydrogenase (F = 8.90; df = 2, 8; p < 0.01) were also the highest in the aphids fed on vermicompost- treated C. annum (Table 5). Significant differences were found in the amounts of malondialdehyde (MDA) and oxidized to reduced thiols (RSSR/RSH ratio) in control and fertilizer treatments (Table 6). Adults of M. persicae fed on zinc sulfate-treated and control plants showed the highest and the lowest values of MDA, respectively (Table 6; F = 7.82; df = 2, 8; p < 0.01) while the highest RSSR/RSH ratio was found in the aphids fed on vermicompost- treated C. annum (Table 6; F= 1.84; df = 2, 8; p < 0.01). Effects of fertilizers on storage macromolecules in M. persicae Significant differences were found in triacyl- glyceride (TAG) and glycogen contents of M. persicae reared on different fertilizer treatments (Table 7). M. persicae fed on C. annum treated by zinc sulfate had the highest amount of triglyceride (F = 4.31; df = 2, 8; p < 0.01) and glycogen (F = 65.46; df = 2, 8; p < 0.01) compared to other treatments (Table 7). Discussion According to the results obtained here, zinc sulfate and vermicompost fertilizers significantly affected physiological performance and antioxidant activities of M. persicae. Researches have demonstrated that fertilizer treatment affect fecundity, life table parameters and physiological performance of herbivorous insects (Edwards et al., 2009; Razmjou et al, 2011; Mardani-Talaee et al., 2015) because soil amendment with fertilizers increases level of organic matters and soil biological interactions led to fertility and relative host plant resistance to pest damages (Luong and Heong, 2005). Also, organic fertilizers, e.g., vermicompost, may increase amounts of phenolic compounds in host plants which definitely affect biological performance of insects, the phenomenon has been observed in M. persicae (Mardani-Talaee et al., 2016). Adults of M. persicae fed on zinc sulfate-treated C. annum showed the highest activities of digestive enzymes while the aphids on control and vermicompost treatments had the lowest enzymatic activities except for chymotrypsin, aminopaptidase and β-glucosidase. These results imply on suitability of C. annum reared on zinc sulfate cultural soil. The suitability may be created due to higher amounts of nutrients or lower levels of plant secondary metabolites which prevent growth and development of insect via repellency or inhibitory mechanisms on digestive enzymes (Terra and Ferreira, 2005; Nation, 2008). Our previous studies revealed the effect of vermicompost to increase amounts of secondary compounds in M. persicae and Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) (Mardani-Talaee et al., 2015, 2016). In details, Mardani-Talaee et al. (2015) reported the higher contents of flavonoids, anthocyanins and phenolic compounds in potatoes cultured in the soils containing 30 % of vermicompost. Similar findings were obtained in C. annum cultured in vermicompost compared to control 128   Table 5 Effects of fertilizers on activities of antioxidant enzymes in M. persicae adults Statistic (Mean±SE U/mg protein) Treatments Catalase Ascorbate oxidase GPDH SOD PO Control 0.328±0.081 a 0.778±0.549 c 0.136±0.026 b 0.080±0.002 b 0.003± 0.001 b Vermicompost (30%) 0.148±0.049 b 7.176±0.463 a 0.447±0.0216 a 1.860±0.310 a 0.013± 0.005 a Zinc sulfate 0.119 ± 0.021 a 3.704±0.106 b 0.286±0.085 ab 0.834±0.029 ab 0.002± 0.001 b GPDH, glucose-6-phosphate dehydrogenase; SOD, superoxide dismutase; PO, peroxidase. The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. and zinc sulfate treatments (Mardani-Talaee et al., 2016). Moreover, Stevenson et al. (1993) found that phenolic compounds were responsible for development retardation of Spodoptera litura (Lepidoptera: Noctuidae) reared on wild ground nut. Haukioja et al. (2002) reported changes of consumption rate in Epirrita autumnata (Lepidoptera: Geometridae) due to presence of phenolic compounds. Finally, Edwards et al. (2009) highlighted the role of phenolic substances to alleviate feeding performance in sap sucking insects. Insects are depend on several processes involved in intermediary metabolism to gain their required energy for biological activities such as flight, reproduction and etc. Intermediary metabolism relies on activities of transaminases to process amino acids for energetic demands, tissue construction and lipid oxidation to provide energy and metabolic water, besides processing of glucose via glycolysis and krebs cycle (Nation, 2008). ALT and AST are the two important enzymes in transaminase mechanisms of insects that catalyze alanine cycle in proline metabolism and facilitate conversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate, respectively (Nation, 2008). These two enzymes are involved in proline metabolism and providing some components for krebs cycle. Although γ-GT is a transaminase but it transfers γ-glutamyl moiety of glutathione to a receptor for glutamate formation so it is important in γ-glutamyl cycle to synthesize and degrade glutathione and xenobiotic compounds (Tate and Meister, 1985). In our study, activities of AST and γ- GT significantly increased in the aphids fed on vermicompost-treated C. annum compared to control and zinc sulfate treatments while activity of ALT decreased in both fertilizer treatments compared to control. The lower activity of ALT in fertilized-treated aphids may be attributed to non- dependence of energy production via proline metabolism or protein shortage in hemolymph and fat bodies because of disordered protein digestion. The second reason seems to be more important because ALT is crucial to convert amino acids to each other and keto-acid. Meanwhile, increased activities of AST and γ-GT may be due to energy demand via Krebs cycle by aspartate and α- ketoglutarate conversions to oxalate and glutamate as well as detoxification of secondary metabolites entered into hemolymph. Aldolase is an isomerase which make sugars available in initial steps of glycolysis (Pinto et al., 1969). LDH involves in pyruvate conversion to lactate in anaerobic conditions and utilizes electrons to provide NADH from NAD+ (Nation, 2008). Also, the enzyme is considered as an index of tissue damage in clinical chemistry (Kaplan and Pesce, 1996). The higher activities of aldolase and LDH in the aphids fed on fertilized-treated plants refer to glycolysis cycle for energy demand from one hand and potential tissue damages due to increased levels of plants’ secondary metabolites. Mardani- Talaee et al. (2015) reported higher activity of LDH in L. decemlineata adults fed on potatoes cultured in vermicompost (30 %) with regard to increased levels of secondary metabolites in the treated plants. Acid and alkaline phosphatases are the two hydrolytic enzymes that detach phosphate groups from different molecules such as nucleotids, proteins and alkaloinds in acid and alkali media. The higher activities of these enzymes indicate digestion efficacy and nutrient absorption in midgut and appropriate transfer of dietary nutrients to fat bodies (Senthil-Nathan et al., 2006). Lower activities of these enzymes mainly ALP in fertilized-treated aphids indicate disturbance of dietary utilization of ingested food because of disordered performance of digestive enzymes. On the other hand, it can be concluded that impaired digestive process led to lower availability of nutrients in aphids mainly storage in fat bodies which affect activities of given phosphatases. Antioxidant system is recruited to protect animal cells against ROS (Reactive Oxygen Species) by decrease level of lipid peroxidation, DNA and protein damage (Felton and Duffey, 1991; Dubovskiy et al., 2008). Several enzymes are involved in antioxidant defense system including 129   Table 6 Effects of fertilizers on RSSR/RSH Ratio and malondialdehyde (MDA) in M. persicae asults Statistic (Mean±SE) Treatments MDA (nM/mg protein) RSSR/RSH Ratio Control 0.674±0.157 b 0.977±0.132 b Vermicompost (30%) 3.699±1.22 ab 1.626±0.773 a Zinc sulfate 4.783±0.467 a 0.5200±0.0560 b The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. ascorbate peroxidases (APX), superoxide dismutases (SOD), catalases (CAT), peroxidases (POX), glutathione-S-transferase (GST), glucose-6- phosphate dehydrogenase and nonenzyme antioxidants such as ascorbic acid, thiols, and α- tocopherol (Fridovich, 1978; Dubovskiy et al., 2008). Functionally, SOD converts superoxide radical О2 - into H2O2; CAT and POX convert H2O2 into H2O; GST removes the products of lipid peroxidation or hydroperoxides from cells; thiols are important to protect cells against hydroxyl radical (OH-); nitroxyl radical (NO−) and superoxide radical О2 - (Udupi and Rice-Evans, 1992; Dubovsky et al., 2008). Besides, thiol ratio (RSSR/RSH) is an important index to highlight the lower amounts of reduced SH-groups (RSH) and the higher amounts of oxidated SH- groups (RSSR) due to oxidative stress (Dubovsky et al., 2008). Meanwhile, increased amount of MDA is an evidence of oxidative stress in an organism (Dubovsky et al., 2008). Peroxidase, SOD, ascorbate oxidase and GPDH had the highest activity in the aphids fed on vermicompost treated plants but activities of ascorbat oxidase and catalase were the highest in control and zinc sulfate treatments. Although the highest amount of MDA was found in Zinc sulfate treatment but the aphids fed on vermicompost-treated plants showed the highest value of RSSR/RSH ratio. Enhanced activity of SOD in M. persicae reared on vermicompost indicated the higher amount of H2O2. Meanwhile, CAT is known to be inhibited by accumulation of superoxide anions in oxidative stressed organisms so the lower activity in vermicompost treatments may be attributed to possible destruction of aphid tissues mainly gut due to increased amounts of secondary metabolites (Kono and Fridovich, 1982; Pardini et al., 1988). In glycolysis, glycerol-3-phosphate shuttle transfers the reducing equivalents from cytoplasmic pool of NADPH to the mitochondrial membrane (Nation, 2008). Then, NADPH is re-oxidized by transferring electrons across the mitochondrial membrane rather than NADH itself (Nation, 2008). Moreover, Ascorbate peroxidase enzymatically removes H2O2 with the concurrent oxidation of ascorbate (Asada, 1992). NADPH is the final reducing equivalent in GSH-GSSG system to equilibrate chemical constitutions between dehydroascorbate and ascorbate. So, the higher activity of ascorbate oxidase should be compensated with increase in glucose-6-phosphate dehydrogenase activity. Our results on the highest activities of both enzymes in the aphids fed on vermicompost-treated plants indicate more reliance of aphids to provide energy from glycolysis and to maintain the balance between produced oxidative reagents. Malondialdehyde (MDA) is an organic compound which naturally exist in biological media indicating oxidative stress. MDA is produced due to lipid peroxidation of polyunsaturated fatty acids. In fact, reactive oxygen species produced in cells degrade polyunsaturated lipids and produce MDA which is a reactive electrophile species leading to toxic stress in cells (Wang et al., 2001; Dubovsky et al., 2008). In our study, adults of M. persicae fed on Zinc sulfate and vermicompost showed higher amounts of MDA compared to control. It can be concluded that oxidative stress in fertilizer-treated aphids caused a high level of lipid peroxidation which is accompanied with the lower amounts of storage triglyceride mainly in vermicompost. Our results demonstrated the highest RSSR/RSH ratio in the aphids fed on vermicompost-treated C. annum. Alteration of balance between oxidized thiols in an organism indicates the higher activity of radical oxidative species. Wang et al. (2001) believe higher ratio of oxidized to reduced thiols is accompanied with increase in lipid peroxidation processes led to higher amount of MDA. Their conclusion is corresponds with our findings on the aphids fed on vermicompost-treated plants with the highest RSSR/RSH ratio and MDA concentration. Insects are highly dependent on their stored nutrients to survive in environment (Nation, 2008). Dietary food is transferred to fat bodies and stored as three macromolecules, protein, glycogen and triglyceride (Nation, 2008). Amounts of the two measured macromolecule, glycogen and triglyceride, were the highest in the aphids fed on Zinc sulfate-treated plants compared to vermicompost and control. In fact, C. annum reared in soil containing zinc sulfate is an appropriate host 130   https://en.wikipedia.org/wiki/Organic_compound https://en.wikipedia.org/wiki/Organic_compound https://en.wikipedia.org/wiki/Oxidative_stress Table 7 Effect of fertilizers on storage macromolecules in M. persicae adults Statistic (Mean±SE mg/mL) Treatments TAG Glycogen Control 1.136±0.029 ab 0.034±0.025 b Vermicompost (30%) 0.032±0.032 b 0.042±0.009 b Zinc sulfate 4.783±0.015 a 0.510±0.001 a The means followed by different letters in a column are significantly different [p < 0.01, Tukeys (HSD)]. plant for M. persicae because of nutrient quality and lack of feeding inhibitors mainly secondary metabolites. In other two treatments, mainly vermicompost, shortage of nutrients due to impaired digestive process led to utilize storage glycogen and triglyceride to support energetic mechanisms via glycolysis or β-oxidation of lipids. The conclusion can be supported by altered activities of intermediary enzymes highlighted earlier. In summary, significant differences were observed in digestion, intermediary metabolism and antioxidant activities of M. persicae fed on C. annum cultured in control and fertilizer-treated soils. The physiological findings here and life table parameters reported in our previous study imply on negative effect of vermicompost on ecological fitness of M. persicae. So, it is recommended to use vermicompost as a culture substrate in greenhouses infested by M. persicae to suppress population outbreaks and to alleviate production costs due to unnecessary control procedures. Acknowledgment The research was supported by a grant of University of Guilan, Ministry of Science, Research and Technology. References Addy SK, Goodman RN. Polyphenol oxidase and peroxidase in apple leaves inoculated with a virulent or an avirulent strain for Ervinia amylovora. Ind. Phytopathol. 25: 575-579, 1972. Arancon NQ, Edwards CA, Yardim EN, Oliver T, Byrnem RJ, Keeney G. Suppression of two- spotted spidermite (Tetranychus urticae), mealy bugs (Pseudococcus sp.) and aphid (Myzus persicae) populations and damage by vermicomposts. Crop. Prot. 26: 29-39, 2006. Arancon N, Edwards CA, Yardim F, Lee S. Management of plant parasitic nematodes by use of vermicomposts. Proc. Brighton. Crop. Prot. Con. Pest. Dis. 2: 705-710, 2002. Asada K. Chloroplasts: formation of active oxygen species and its scavenging. Meth. Enzymol. 105: 422-429, 1984. Balinsky D, Bernstein RE. The purification and properties of glucose-6- phosphate dehydrogenase from human erythrocytes. Biochem. Biophys. Acta 67: 313-315, 1963. Bar-Or D, Rael LT, Lau EP, Rao NKR, Thomas GW, Winkler JV, et al. An analog of the human albumin N-terminus (Asp- Ala-His-Lys) prevents formation of copper-induced reactive oxygen species. Biochem. Biophys. Res. Commun. 284: 856-862, 2001. Bernfeld P. Amylases, α and β. Meth. Enzymol. 1: 149-158, 1955. Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164: 321-329, 1946. Bolandandam J, Barker H, Fenton B. Differences in potato leafroll transmitting ability of individual genotypes of Scottish Myzus persicae with different susceptibilities to Lambda- cyhalothrin insecticide. Proc. 15th Intl. Plant Protect. Cong., Beijing, China, 2004. Bourgaund F, Gravot A, Milesi S, Gontier E. Production of plant secondary metabolite: A historical perspective. Plant. Sci. 161: 839-851, 2010. Chun Y, Yin ZD. Glycogen assay for diagnosis of female genital Chlamydia trachomatis infection. J. Clin. Microbiol. 36: 1081-1082, 1998. Dubovskiy IM, Martemyanov VV, Vorontsova YL, Rantala MJ, Gryzanova EV, Glupov VV. Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae). Comp. Biochem. Physiol. 148C: 1-5, 2008. Edwards CA, Arancon NQ, Vasko-Bennett M, Askar A, Keeney G, Little B. Suppression of green peach aphid (Myzus persicae) (Sulz.), citrus mealy bug (Planococcus citri) (Risso), and two spotted spider mite (Tetranychus urticae) (Koch.) attacks on tomatoes and cucumbers by aqueous extracts from vermicomposts. Crop. Prot. 28: 1-14, 2009. Elpidina EN, Vinokurov KS, Gromenko VA, Rudenskaya YA, Dunaevsky YE, Zhuzhikov DP. Compartmentalization of proteinases and 131   amylases in Nauphoeta cinerea midgut. Arch. Insect Biochem. Physiol. 48: 206-216, 2001 Felton GW, Summers CB. Antioxidant systems in insects. Arch. Insect. Biochem. Physiol. 29: 187-197, 1995. Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28: 2077-2080, 1982. Fridovich I. The biology of oxygen radicals. Sci. 201: 875-879, 1978. Frugoni JAC. Tampone universale di Britton e Robinson a forza ionica costante. Gazz. Chim. Ital. 87: 403-407, 1957. Haukioja E, Ossipov V, Lempa K. The interactive effects of leaf maturation and phenolics on consumption and growth of a geanetrid moth. Entomol. Exp. Appl. 104: 125-136, 2002. Hill DS. Agricultural insect pests of the tropics and their control, 2nd ed., Cambridge University Press, New York, USA, 1983. Kaplan LA, Pesce AJ. Clinical chemistry, theory analysis and correlation. St. Louise, MO: Mosby-Year Book, 1996. Khramtsov VV, Yelinova VI, Glazachev YuI, Reznikov VA, Zimmer G, Weiner L, et al. Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide labl. J. Biochem. Biophys. Meths. 35: 115-128, 1997. King J. The dehydrogenases or oxidoreductases. Lactate dehydrogenase, pp. 83-93. In: Van Nostrand D (ed.), Practical clinical enzymology, Elsevier, London, 1965. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. Lu ZX, Yu XP, Heong KL, Hu C. Effect of nitrogen fertilizer on herbivores and its stimulation to major insect pests in rice. Rice. Sci. 14: 56-66, 2007. Luong MC, Heong KL. Effects of organic fertilizers on insect pest and diseases of rice. Omonrice 13: 26-33, 2005. Mardani-Talaee M, Nouri-Ganblani G, Razmjou J, Hassanpour M, Naseri B, Asgharzadeh A. Effects of chemical, organic and bio-fertilizers on some secondary metabolites in the leaves of bell pepper (Capsicum annuum) and their impact on life table parameters of Myzus persicae (Hemiptera: Aphididae). J. Econ. Entomol. 1-10, 2016 [In press] Mardani-Talaee M, Zibaee A, Nouri-Ganbalani G, Rahimi V, Tajmiri P. Effect of vermicompost on nutrition and intermediary metabolism of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Arch. Phytopathol. Plant Prot. 8: 623-645, 2015. McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythro-cuprein (hemocuprein). J. Biol. Chem. 244: 6049-6055, 1969. Nation JL. Insect physiology and biochemistry, 2nd ed., CRC press, London, 2008. Pardini RS, Pritsos CA, Bowen SM, Ahmad S, Blomquist GJ. Adaptations to plant pro-oxidants in a phytophagous insect model: enzymatic protection from oxidative stress. In: Simic MG, Taylor KA, Ward JF, Sonntag C (eds), Oxygen radicals in biology and medicine, Plenum Press, New York, pp 725-728, 1988. Patriquin D G, Baines D, Abboud A. Diseases, pests and soil fertility. In: Cook HF, Lee HC (eds), Soil management in sustainable agriculture. Wye College Press, Wye, UK, pp 161-174, 1995. Pinto PVA, Kaplan A, Dreal PA. Aldolase: I. Colorimetric determination. Clin. Chem. 15: 349-360, 1969. Rahbe´ Y, Deraison C, Bonade´-Bottino M, Girard C, Nardon C, Jouanin L. Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape. Plant Sci. 164: 441-450, 2003. Razmjou J, Mohammadi M, Hassanpour M. Effect of vermicompost and cucumber cultivar on population growth attributes of the melon aphid (Hemiptera: Aphididae). J. Econ. Entomol. 104: 1379-1383, 2011. Robert Y, Woodford JA, Ducray-Bourdin DG. Some epidemiological approaches to the control of aphid-borne virus diseases in seed potato crops in northern Europe. Virus. Res. 71: 33-47, 2000. Senthil Nathan S, Chunga PG, Muruganb K. Combined effect of biopesticides on the digestive enzymatic profiles of Cnaphalocrocis medinalis (Guenee) (the rice leaffolder) (Insecta: Lepidoptera: Pyralidae). Ecotoxicol. Environ. Saf. 64: 382-389, 2006. Silva CP, Terra WRM. An α-glucosidase from perimicrovillar membranes of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) midgut cells: Purification and properties. Insect Biochem. Mol. Biol. 25: 487-494, 1995. Stevenson PC, Anderson JC, Blaney WM, Simmonds MSJ. Developmental inhibition of Spodoptera litura (Fab.) larvae by a novel caffeoylquinic acid from the wild ground, Arachis paraguariensis (Chhod et Hassl.). J. Chem. Ecol. 19: 2917-2933, 1993. Szasz G. Reaction-rate method for gamma- glutamyltransferase activity in serum. Clin. Chem. 22: 2051-2055, 1976. Tate SS, Meister A. Gamma-glutamyl transpeptidase from kidney. Meth. Enzymol. 113: 400-419, 1985. Terra WR, Ferreira C. Biochemistry of digestion. In: Lawrence IG, Kostas I, Sarjeet SG (eds), Comprehensive molecular insect science. Vol. 3. Oxford: Elsevier, pp 171-224, 2005. Thomas L. Clinical laboratory diagnostic, pp 89-94, 1th ed., THBooks Verlasgesellschaft, Frankfurt, 1998. Tsujita T, Ninomiya H, Okuda H. p-nitrophenyl butyrate hydrolyzing activity of hormone- sensitive lipase from bovine adipose tissue. J. Lipid Res. 30: 997-1004, 1989. Udupi V, Rice-Evans C. Thiol compounds as protective agents in erythrocyte under oxidative stress. Free Rad. Res. Comm. 16: 315-323, 1992. Wang Y, Oberley LW, Murhammer DW. Evidence of oxidative stress following the viral infection of 132   two Lepidopteran insect cell lines. Free Rad. Biol. Med. 31: 1448-1455, 2001. Yardim EN, Arancon NQ, Oliver TJ, Byrne RJ. Suppression of tomato hornworm (Manduca quinquemaculata) and cucumber beetles (Acalymna vittatum and Diabrotica unidecimpunctata) populations and damage by vermicomposts. Pedobiologia 50: 23-29, 2006. 133