182 ISJ 14: 182-189, 2017 ISSN 1824-307X SHORT COMMUNICATION Effect of Teucrium polium (Lamiaceae) essential oil on digestive enzyme activities and energy reserves of Ephestia kuehniella (Lepidoptera: Pyralidae) M Shahriari1, N Sahebzadeh1, A Zibaee2 1Department of Plant Protection, Faculty of Agriculture, University of Zabol, Zabol, Iran 2Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Accepted May 3, 2017 Abstract In the current study, the effects of Teucrium polium L. (Lamiaceae) essential oil (Tp EO) and α- pinene as a major component of this essential oil, were tested against Ephestia kuehniella Z. (Lep.: Pyralidae) for larvicidal activity, digestive enzymes and non-enzymatic (amount of macromolecules) responses. Concentrations of 4.91 and 10.66 µl/ml were determined as LC50 values for Tp EO and α- pinene, respectively. The activities of α-amylase, triacylglycerol lipase, general protease, serine proteases (trypsin and chymotrypsin-like), carboxy- and aminopeptidases significantly decreased when the larvae fed on the diet supplemented with Tp EO and α-pinene but significant increases were observed in elastase-like proteinase. The amount of protein, glycogen and triglyceride decreased in the treated larvae by both treatments. The results showed that the digestive enzymes and non enzymatic activities of the treated larvae were decreased compared to the control. Therefore, Tp EO and α-pinene are suggested as botanicals for controlling flour moth larvae. Key Words: digestive enzymes; essential oil; flour moth; macromolecules; Teucrium polium; α-pinene Introduction In view of enzymology, digestive enzymes (such as amylases, lipases, and proteases) are commonly found in the midgut of insects. To provide metabolites and energy, these enzymes convert the food complexes into micro molecules (Klowden, 2007). α-amylase catalyzes the endo-hydrolysis of long chains such as starch and glycogen (Terra and Ferriera, 2012). This activity converts them to maltose and other oligosaccharides, which are easily absorbed by the epithelial cells of insect gut (Rahimi and Bandani, 2014). The outer links of triacylglycerols are hydrolyzed by triacylglycerol lipases (TAG-lipases). These enzymes preferentially release fatty acids (preferably unsaturated fatty acids) which are activated by calcium ions (Terra and Ferriera, 2012). Insect proteases are the diverse groups of hydrolytic enzymes, which break peptide bonds in dietary proteins (Terra and Ferriera, 2012; Subala and Shivakumar, 2015). Insects receive their required energy from ingested macromolecules such as carbohydrates, lipids and proteins. In insects, the most important lipid and ___________________________________________________________________________ Corresponding author: Najmeh Sahebzadeh Department of Plant Protection Faculty of Agriculture University of Zabol, Zabol, Iran E mail: najmeh.sahebzadeh@gmail.com carbohydrate reserves are triglyceride and glycogen (or trehalose), respectively (Klowden, 2007; Nation, 2008). Proteins are composed of amino acids, associated to molecular transformation, cuticular scleritization, and visual pigment synthesis, and even engaged as neural transmitters (Klowden, 2007; Ramzi et al., 2014). The flour moth, Ephestia kuehniella (Lep.: Pyralidae) is a polyphagous and dangerous pest on the stored products that has been observed in cereals flour. The larvae cause high levels of damage by feeding and producing frass on the stored products (Jallouli et al., 2013). Currently, synthetic insecticides such as methyl bromide are most often used to control the stored products pests. However, concerns over the environmental pollution, residue levels, toxicity to non-target organisms and pesticide resistance are associated with conventional application of these insecticides, which compel us to attention to new compounds for pest control (Rajendran and Sriranjini, 2008; Muthusamy and Shivakumar, 2017). To manage the stored product pests, the application of botanical insecticides is considered as an appropriate alternative to synthetic pesticides (Rajendran and Sriranjini, 2008; Karaborklu et al., 2011). Plant secondary metabolites play insecticidal activities which could potentially act in anti-herbivore defenses against 183 agricultural pests (Isman, 2006). Lamiaceae plants are quite promising to develop as novel biopesticides for the control of pest (Kim et al., 2013). Teucrium polium is a member of this family already showing strong insecticidal activity on Musca domestica (Dip.: Muscidae), Callosobruchus maculatus (Col.: Chrysomelidae), and Tribolium castaneum (Col.: Tenebrionidae) (Bigham et al., 2010; Hydarzade and Moravvej, 2012; Khani and Hydarian, 2014). The major constituents of its essential oil are α-pinene (12.52 %), linalool (10.63 %) and caryophyllene oxide (9.69 %) (Moghtader, 2009). A comprehensive understanding of plant essential oils (EOs), with their active metabolites, and their interactions on physiology of the insect pests is necessary before using these compounds against the stored grain pests. Accordingly, we examined the toxicity and biochemical properties of T. polium essential oil (Tp EO) and α-pinene with a specific focus on digestive enzymes including α- amylase, triacylglycerol lipase, general protease, serine proteases (trypsin and chymotrypsin-like), carboxy- and aminopeptidases, as well as elastase- like proteinase of E. kuehniella. It is well documented that these enzymes alter during physiological process in insects. In addition, the effects of Tp EO and α-pinene on amount of protein, glycogen, and triglyceride were also considered on the treated fourth instar larvae of E. kuehniella under laboratory conditions. Materials and Methods Insect rearing The larvae of E. kuehniella were collected from the infected wheat flour in Zabol, Sistan and Baluchestan province, Iran. The insects were reared in rectangular plastic boxes (17×9×9 cm3). The insects were reared in the laboratory under controlled (25 ± 1 °C, 65 ± 5 % R.H., and 15L:9D) on an artificial diet (43 gr wheat flour, 6 gr yeast, and 20 ml of glycerin) (Lima et al., 2001). Essential oil preparation Seeds of T. polium were ground and 40 g of powdered seeds was incubated at 500 ml of distilled water for 4 h in 100 ºC. Water of obtained sample was taken by sodium sulfate and kept at 4 ºC prior to the experiment tests (Negahban et al., 2007). α-pinene was purchased from Sigma- Aldrich (Spain) and were applied without further purification. Bioassay According to Shahriari et al. (2017), forth instar larvae of E. kuehniella were randomly selected and were exposed to 500 mg of artificial diet containing 2, 3, 4.5, 6.7 and 10 µl/ml of Tp EO, and 4, 5.6, 8, 11.3 and 16 µl/ml of α-pinene, respectively. Control larvae were fed on the diet containing acetone as solvent for dilution of serial concentrations of Tp EO and α-pinene. Larval mortality was recorded after 24 h. The experiments were carried out using 180 larvae with 3 replicates in each concentration (total numbers were 360 larvae for both Tp EO and α- pinene concentrations). Larval gut extraction The treated larvae of E. kuehniella were randomly and individually selected and their midguts were dissected under a stereomicroscope in ice- cold distilled saline solution (NaCl, 10 mM). The midguts were rinsed in ice-cold distilled water and placed in a pre-cooled homogenizer. Then the prepared midguts were grounded prior to be centrifuged at 13,000 rpm (20 min at 4 oC). The supernatants were stored at -20 oC for determination of digestive enzyme activities (Ramzi et al., 2013). Effects of Tp EO and α-pinene on digestive enzymes α-amylase assay was carried out as described by Bernfeld (1955). Ten microliter of gut extraction and 20 μl of 1 % soluble starch as substrate were incorporated using phosphate buffer (50 μl, 20 mM, pH 7.1), mixed thoroughly and incubated for 30 min at 35 °C. Eighty microliter dinitrosalicylic acid (to stop the reaction) was added and heated in boiling water for 10 min and the absorbance of this reaction was read at 545 nm. One unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35 °C (Ramzi et al., 2014). The method described by Tsujita et al. (1989) was used to assay TAG-lipase activity. Twenty microliter of homogenated guts were incubated at 37°C with 100 μl of Tris-HCl buffer (10 mM, pH 8) and 40 μl of p-nitrophenyl butyrate (27 mM) as substrate. The absorbance was read at 405 nm. One unit of enzyme released 1.0 nmol of p- nitrophenol per min at pH 7.2 and 37 °C when p- nitrophenyl butyrate was used as substrate. Standard curve of p-nitrophenol was used to calculate the specific activity of this enzyme. General proteolytic activity was measured using azocasein (2 %) based on a method described by Elpidina et al. (2001). The reaction mixture consisted of 20 μl of the enzyme solution, 100 μl of Tris-HCl buffer (pH 8) and 40 μl of azocasein (2 %) as substrate. The reaction mixture was incubated at 37°C for 60 min before adding 150 μl of 10 % trichloroacetic acid (TCA) to terminate the reaction. Precipitation was achieved by cooling at 4 °C for 120 min and it was centrifuged at 13,000 rpm for 10 min. An equal volume of NaOH (2 M) was added to the supernatant and the absorbance was recorded at 440 nm. The blank consisted of all mentioned portions except the enzyme solution of guts. Trypsin, chymotrypsin and elastase-like activities were assayed using a concentration 1 mM of BApNA (Nabenzoyl-L-arginine-p-nitroanilide), 1 mM N-succinyl alanine alanine proline phenylalanine pnitroanilide (SAAPPpNA) and 1 mM SAAApNA (N- succinyl-alanine-alanine-alanine-p-nitroanilide) as substrates, respectively. The reaction mixture included 35 μl of Tris-HCl buffer (20 mM, pH 8 as literature recommended pH for serines in lepidopteran species), 5 μl of BApNA, SAAPPpNA and SAAApNA substrates individually and 5 μl of enzyme solution. This mixture was incubated at 30 °C (10 min), and its absorbance was then read at 405 nm. To prove the specific proteolytic activity, a negative control was provided for each substrate separately containing all mentioned components 184 besides enzyme pre-boiled at 100 °C for 30 min (Oppert et al., 1997). Activities of the two exopeptidases in the midgut of E. kuehniella were obtained using Hippuryl-L-Arginine and Hippuryl-L- Phenilalanine for carboxy- and aminopeptidases, respectively. The reaction mixture consisted 35 μl of Tris-HCl buffer (20 mM, pH 7), 5 μl of each mentioned substrate and 5 μl of enzyme solution. Following by incubation (30 °C, 10 min), the absorbance was read at 340 nm. To prove the specific proteolytic activity, a negative control was provided as mentioned previously (Oppert et al., 1997). Effects of TP EO and α-pinene on macromolecules The amount of protein was measured using Lowry et al. (1951) procedure. Twenty microliter of homogenized sample was added to 100 µl of reagent, and incubation was made for 30 min at 25 °C. The absorbance was read at 545 nm (Recommended by Ziest Chem. Co., Tehran-Iran) (Ramzi et al., 2014). To determine triacylglyceride content, a diagnostic kit (Pars Azmoon Co., Tehran, Iran) was used to measure the amount of triacylglyceride in the fourth instar larvae. The reagent solution contained 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). Samples (10 μl) were incubated with 10 μl of distilled water and 70 μl of reagent for 20 min at 25 °C. The following equation was used to calculate the amount of triacylglyceride: mg/dl = ×0.01126. Optical densities (ODs) of samples and reagent as standard were read at 545 nm (Fossati and Prencipe, 1982). For estimation of glycogen, fat bodies of 20 larvae were cut and immersed to 1 mL of 30 % KOH w/Na2SO4. Tubes containing the samples were covered with aluminium foil (to avoid evaporation) and were boiled for 30 min. Tubes were shaken and cooled in ice. Then, 2 mL of 95 % EtOH was added to precipitate glycogen from digested solution. The samples were again shaken and incubated in ice for 30 min. Tubes were centrifuged 13,000 rpm for 30 min. Supernatant was removed and pellets (glycogen) were re-dissolved in 1 mL of distilled water before being shaken. Glycogen standard (0, 25, 50, 75 and 100 mg/mL) was prepared before adding phenol 5 %. The samples were incubated on ice bath for 30 min. Finally, the absorbance of standards and samples were read at 490 nm (Chun and Yin, 1998). Data analysis For estimation of LC50 and corresponding 95 % CI values, POLO-Plus software was used. Biochemical data were estimated in a complete randomized design, and were compared by one- way analysis of variance (ANOVA) followed by Tukey’s test. Differences between samples were statistically considered at a probability less than 5 % and marked in the tables and figures. Results and Discussion Bioassay In this study, the lethal concentrations (LC50) values were observed in 4.91 µl/ml and 10.66 µl/ml for Tp EO and α-pinene, respectively (Table 1). By increasing of concentration, the mortality of larvae was increased. Similarly, α-pinene was evaluated against T. castaneum larvae, at 24 and 48 h post- treatment, and LC50 of this compound was determined as 12.85 and 8.39 µl/ml, respectively (Shahriari et al., 2016). Bigham et al. (2010) found a significant oral toxic activity of T. polium on M. domestica (LC50 = 80ppm). LC50 values were showed contact toxicity of T. polium 1263.09 and 1469.72 μl/m2 on adult males and females of C. maculatus, respectively (Hydarzade and Moravvej, 2012). Plant essential oils (EOs) are aromatic compounds and complex mixture of monoterpenes. It is often observed that complex essential oil compounds are more efficacious than the pure combination (Don-Pedro, 1996; Bakkali et al., 2008; Kumrungsee et al., 2014). In our study, the oral toxicity of Tp EO was higher than α-pinene on E. kuehniella. Similarly, Don-Pedro (1996), Ho et al. (1997), Hori (1998), and Kim et al. (2010, 2013) demonstrated that essential oil from different plant species (anise, rosemary, citrus, origanum and cumin) were more effective than the their pure secondary metabolites (trans-anethole, linalool, limonene, carvacrol and p-cymene) on different pests such as C. maculatus, Myzus persicae S. (Homoptera: Aphididae), T. castaneum and Sitophilus oryzae (Col.: Curculionidae). Table 1 Dose-response parameters of fourth instar larvae of Ephestia kuehniella exposed to Teucrium polium essential oil and α-pinene Treatment LC50(µl/ml) X 2 (df) Slope±SE Tp EO 4.91 (4.16-5.87) 2.05 (3) 3.16 ± 0.504 α-pinene 10.66 (8.61-14.91) 2.37 (3) 2.28 ± 0.526 Tukey test, p < 0.05 185 Table 2 Activity of digestive enzymes in fourth instar larvae of Ephestia kuehniella at 24, 48 and 72 h post treatment with LC50 value of Teucrium polium essential oil and α-pinene Digestive enzyme Time (h) Control Tp EO α-pinene F- Value Pr α-amylase (U/mg protein) 24 0.658±0.0049a 0.144±0.0024c 0.240±0.0053b 3794.32 0.0001 48 0.763±0.0085a 0.355±0.0034b 0.370±0.0090b 992.23 0.0001 72 0.762±0.0008a 0.290±0.0049c 0.417±0.0017b 6250.88 0.0001 TAG lipase (U/mg protein) 24 0.562±0.0045a 0.352±0.0045b 0.346±0.0092b 340.02 0.0001 48 0.563±0.0046a 0.506±0.0058b 0.338±0.0052c 676.9 0.0001 72 0.589±0.0053a 0.456±0.0035b 0.328±0.0035c 658.24 0.0001 General protease (U/mg protein) 24 0.118±0.0046a 0.059±0.0043b 0.053±0.0050b 59.38 0.0001 48 0.120±0.0043a 0.088±0.0017b 0.074±0.0023b 50.89 0.0002 72 0.123±0.0033a 0.106±0.0038b 0.094±0.0026b 19.07 0.0023 Trypsin-like (U/mg protein) 24 0.166±0.0032a 0.063±0.0031b 0.033±0.0038c 409.32 0.0001 48 0.165±0.0035a 0.067±0.0037b 0.058±0.0031b 371.03 0.0001 72 0.163±0.0040a 0.073±0.0030b 0.059±0.0030b 275.65 0.0001 Chymotrypsin-like (U/mg protein) 24 0.092±0.0029a 0.049±0.0013c 0.080±0.0038b 58.59 0.0001 48 0.094±0.0034a 0.078±0.0003b 0.087±0.0025ab 7.33 0.0245 72 0.092±0.0035a 0.069±0.0008b 0.086±0.0012a 29.64 0.0008 Elastase-like (U/mg protein) 24 0.103±0.0024b 0.117±0.0029a 0.119±0.0036a 7.73 0.0219 48 0.099±0.0029b 0.125±0.0032a 0.130±0.0040a 22.44 0.0016 72 0.096±0.0030c 0.164±0.0034a 0.132±0.0043b 60.91 0.0001 Carboxypeptidase (U/mg protein) 24 0.297±0.0031a 0.202±0.0040b 0.189±0.0031b 17.22 0.0033 48 0.300±0.0041a 0.218±0.0018b 0.199±0.0058c 159.55 0.0001 72 0.300±0.0041a 0.226±0.0040c 0.260±0.0030b 94.55 0.0001 Aminopeptidase (U/mg protein) 24 0.105±0.0031a 0.079±0.0029b 0.108±0.0031a 26.59 0.0010 48 0.106±0.0041a 0.073±0.0011b 0.068±0.0028b 57.86 0.0001 72 0.108±0.0041a 0.072±0.0080b 0.073±0.0027b 75.66 0.0001 Statistical differences have been done within each time intervals and marked by different letters in each row (Tukey test, p < 0.05). Effects of Tp EO and α-pinene on digestive enzymes The lower activity of α-amylases was found in the larvae fed on the diet containing LC50 concentrations of Tp EO and α-pinene compared to the control (Table 2). Similarly, Bigham et al. (2010) reported that Tp EO decreased the activity of α- amylase in the treated M. domestica larvae versus control. The reduced activity of α-amylase by plant- based compounds could imply their cytotoxic effect on the midgut epithelial cells those synthesize insect α-amylase (Franco et al., 2002; Senthil-Nathan et al., 2006; Zibaee and Bandani, 2010). This result is consistent with previous studies which were demonstrated the reduction of α-amylase activities in pests such as E. kuehniella (Shahriari and Sahebzadeh, 2017), Ectomyelois ceratoniae (Lep.: Pyralidae) (Ramzi et al. 2013, 2014), Glyphodes pyloalis (Lep.: Crambidae) (Khosravi et al., 2011; Yazdani et al., 2013), and Pieris rapae (Lep.: Pieridae) (Hashminia et al., 2011) after treatment with botanical toxins. Midgut TAG-lipase has a primary role to obtain dietary lipid as well as strong antiviral activity. We found a significant reduction in TAG-lipase activity in the larvae of E. kuehniella fed on the artificial diet containing Tp EO and α-pinene (Table 2). Yazdani et al. (2013) reported that LC50 concentration of some 186 Fig. 1 Effects of essential oil of Teucrium polium essential oil and α-pinene on amounts of storage macromolecules (total protein, triacylglyceride, and glycogen) in the fourth instar larvae of E. kuehniella. Statistical differences have been done within each time intervals and marked by different letters (Tukey test, p < 0.05). essential oils such as increased the activity of TAG- lipase in the treated G. pyloalis larvae versus control. Khosravi et al. (2011) found similar resus while Senthil-Nathan et al. (2006) reported the lower activity of TAG-lipase in Cnaphalocrocis medinalis (Lep.: Pyralidae) midgut after treatment with Azadirachta indica (Melliaceae) extract. Great utilization of exogenous lipids could increase the 187 activity of TAG-lipase in insect midgut (Yazdani et al., 2013). Decreased activity of TAG-lipase by botanical insecticide probably resulted to disturbance of digestion and absorption processes (Senthil- Nathan et al., 2006; Zibaee and Bandani, 2010). Treatment of E. kuehniella larvae with LC50 concentrations of Tp EO and α-pinene showed the lower general proteolytic activity (Table 2). Senthil- Nathan et al. (2006) found a significant reduction in protease activity of C. medinalis fed on a diet containing Bt toxins and botanical insecticides including neem seed and leaf extract of Vitex negundo (Verbenaceae). Digestive proteolytic activity increased in the larvae of G. pyloalis treated by Lavandula angustifolia (Lamiaceae) (Yazdani et al., 2013). The digestion of proteins in the first stage in insects mostly occurs under the effect of serine proteases (Terra and Ferreira, 2012). In our study, Tp EO and α-pinene decreased the activities of trypsin and chymotrypsin-like protease but a significant addition was observed on elastase activity of the treated larvae (Table 2). Similar to our results, Mojarab-Mahboubkar and Jalali-Sendi (2016) demonstrated that activity of serine proteases decreased by Artemisia annua methanolic extract except for elastase. Zibaee et al. (2014) reported that activity of serine proteases decreased in the larvae of Pieris brassicae (Lep.: Pieridae) treated with 0.5, 1 and 2 % concentrations of Polygonum persicaria (Polygonaceae) Agglutinin. Senthil-Nathan et al. (2006), Zibaee and Bandani (2010), and Ramzi et al. (2013) reported that plant- based compounds may affect the construction of some kinds of proteases and inhibition them to digestion ingestion proteins. It was shown that activities of two exopeptidases including amino and carboxy- peptidases of treated E. kuehniella larvae with LC50 concentration of Tp EO and α-pinene significantly decreased in comparison with the control larvae (Table 2). Ramzi et al. (2013, 2014) and Zibaee et al. (2014) found no significant differences in the activities of aminopeptidase between control and treated larvae, while carboxypeptidas activity significantly decreased 48 h post-treatment by lectins extracted from plant source. Exopeptidases hydrolyze single amino acids either from the N- terminus or from the C-terminus (aminopeptidases and carboxypeptidases, respectively) at the end of a polypeptide chain (Kanost and Clem, 2012). The potential mechanisms of EO's and plant secondary metabolites on exopeptidase activities are still lacking. Effects of Tp EO and α-pinene on macromolecules In this study, amounts of protein, glycogen, and triglyceride in E. kuehniella larvae treated by Tp EO and α-pinene decreased significantly versus control (Fig. 1). Several studies have been demonstrated that synthetic and botanical insecticides could alter the amount of carbohydrates, lipids and proteins in insects. The significant reductions in amount of protein in G. pyloalis treated with lethal and sublethal doses of L. angustifolia EO have been reported by Yazdani et al. (2013). In a similar study, Zibaee et al. (2011) showed that the amount of protein, glycogen (trehalose) and lipid as non- enzymatic components in hemolymph and fat bodies of Eurygaster integriceps (Hemiptera: Scutelleridae) reduced after exposure to pyriproxifen. Citrullus colocynthis (Cucurbitaceae) agglutinin decreased the amount of glycogen and triglyceride levels in E. ceratoniae (Ramzi et al., 2014). The reductions occurring by Tp EO and α- pinene in E. kuehniella might be due to accelerating in glycogenolysis at larval fat bodies, transport glycogen from fat body to hemolymph as a response to energy depletion when larvae were exposed to toxins (Zibaee et al., 2011). Lipid depletion after toxin treatments could be occurred because of alteration in its synthesis patterns (Klowden, 2007), hormonal dysfunction for controlling lipid metabolism (Steel, 1980) and the utilization of this metabolic reserve to generate insect energy demand (Sak et al., 2006). 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