Digestive Proteolytic Activity in Apodiphus Amygdali Germar (Hemiptera: Pentatomidae): Effect of Endogenous Inhibitors S

JournalJournal of Entomological of Entomological and andAcarological Acarological Research Research 2014; 2012; volume volume 46:1868 44:e Digestive proteolytic activity in Apodiphus amygdali Germar (Hemiptera: Pentatomidae): effect of endogenous inhibitors S. Ramzi, A. Zibaee Department of Plant Protection, University of Guilan, Rasht, Iran process in insects. Moreover, understanding the nature of digestive Abstract proteases might be used to develop several inhibitors for providing resistant crop varieties against pests. The digestive proteolytic profile of Apodiphus amygdali was determined by using several substrates and specific inhibitors. Analysis of optimal pH and temperature showed the highest enzymatic activity at the pH range of 6-7 and temperature of 40°C when azocasein was used Introduction as a substrate. By using a negative control, the presence of several specific proteases were determined including tryspin-like, chymotrypsin- Apodiphus amygdali Germar (Hemiptera: Pentatomidae) is a like, elastase, cathepsin B, cathepsin L, amino- and carboxypeptidases polyphagous and univoltine hemipteran distributed throughout in the midgut content of A. amygdali, with the highest and the lowest Europe and the Middle East (Schuh & Slater 1995). It feeds on several activities of cathepsin L and carboxypeptidase, respectively. pH fruit trees, especially plum,only apricot, apple, olive, pear and pistachio as dependency of specific proteases revealed optimal pHs of 9, 8 and 9 for well as non-fruit trees like poplar, pine, plane-tree, elm and willow bark trypsin-, chymotrypsin-like, 6 for cathepsins and 5-6 for carboxy- and (Muhammed & Al-Iraqi, 2010). Nymphs and adults feed on leaves, aminopeptidases, respectively. Specific inhibitors, including phenyl- fruits and flowers by injecting their salivary secretions into the plant methylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, N- tissues to liquefyuse nutrient materials. Feeding on stems and fruits caus- tosyl-L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinyl- es host weakness and attracts other insects to feed on host plants. leucylamido-(4-guanidino)-butane, phenanthroline and ethylendi- Feeding on fruits is the primary damage and causes complete degrada- amidetetraacetic acid, significantly decreased proteolytic activity, indi- tion and yield loss (Schuh & Slater, 1995). cating the presence of different proteases in the midgut of A. amygdali. Digestive proteases are one of the crucial enzymes in the alimenta- Extracted inhibitors from the midgut demonstrated significant inhibi- ry canals of insects since proteins are the key nutrients for growth, tion of specific proteolytic activities of A. amygdali except for cathep- development and reproduction (Nation, 2008). The enzymes are clas- sin B and aminopeptidase. The results indicated that determination of sified based on their activity against protein molecules. In fact, pro- digestive proteolytic activity could be helpful to clarify digestion teases that attack internal bonds are called endopeptidases (proteinas- es) but exopeptidases refers to proteases separating amino acids from N- and C-terminal of proteins, known as aminopeptidases (EC 3.4.11.2) and carboxypeptidases (EC 3.4.11.7) (Terra & Ferreira, Correspondence: Arash Zibaee, Department of Plant Protection, Faculty of 2012). Endopeptidases are divided into different subclasses based on Agricultural Sciences, University of Guilan, Rasht, 416351314, Iran. their pH dependency and catalytic sites. Serine proteases, including Tel.: +98.0131.6690274 - Fax: +98.0131.6690281. trypsin- (EC 3.4.21.4), chymotrypsin-like (EC 3.4.21.1) and elastase E-mail: [email protected] ; [email protected] (EC 3.4.21.36), are active in alkaline pH and they have serine, histi- dine, and aspartic acid residues in their catalytic sites (Terra & Key words: Apodiphus amygdali, digestiveNon-commercial protease, inhibitor. Ferreira, 2012). Meanwhile, these enzymes have different structural features that are associated with their different substrate specificities Acknowledgements: the authors want to thank Dr. Ali Sarafrazi from Iranian (Terra & Ferreira, 2012). Cysteine proteases are active at acidic pH, Institute of Plant Protection for his contribution in identification of collect- including cathepsins B (EC 3.4.22.1) and L (EC 3.4.22.15). Cathepsin ed insects. L (EC 3.4.22.15) is a true endopeptidase that preferentially cleaves Received for publication: 7 August 2013. peptide bonds in P2 against the hydrophobic amino acid residues, but Revision received: 19 November 2013. cathepsin B prefers arginine at the same position (Barrett et al., Accepted for publication: 19 November 2013. 1998). Digestion of proteins is initiated by endopeptidases; due to the activity of endopeptidases, oligopeptidases are attacked from the N- ©Copyright S. Ramzi and A. Zibaee, 2014 and C-terminal by amino- and carboxypeptidases, resulting in dipep- Licensee PAGEPress, Italy tides that are hydrolyzed by dipeptidases (Terra & Ferreira, 2012). Journal of Entomological and Acarological Research 2014; 46:1868 Since synthetic chemicals cause severe negative effects on the envi- doi:10.4081/jear.2014.1868 ronment, non-target organisms and induce resistance in pests, numer- ous studies have been conducted on the use of biocontrol agents and This article is distributed under the terms of the Creative Commons digestive enzyme inhibitors against agricultural pests. Enzymatic Attribution Noncommercial License (by-nc 3.0) which permits any noncom- inhibitors disrupt digestion process of insects and nutrient absorption, mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. thereby decreasing reproduction and therefore pest populations. Protease inhibitors are small proteins that are present in up to 100 plant [Journal of Entomological and Acarological Research 2014; 46:1868] [page 35] Article species and they are effective on serine and cysteine proteases (Nation, After adding 20 mL of the sample, the reaction mixture was incubated 2008). Although this approach may be suitable for decreasing economic at each given temperature for 60 min. Other steps were conducted as losses caused by pests with the least environmental disruption, compre- described above. hensive experiments are required to achieve the final goal. Determination of proteolytic profiles and their properties are the first Specific protease assays and fundamental step in this approach. The objectives of the current study were to determine digestive proteolytic profiles in A. amygdali, the Serine proteases effect of synthetic inhibitors, and to extract endogenous inhibitors. Trypsin-, chymotrypsin- and elastase-like activities (as three subclasses of serine proteases) were assayed using a 1 mM concentration of BApNA (Nabenzoyl-L-arginine-p-nitroanilide, Sigma-Aldrich, 19362), 1 mM SAAPPpNA (N-succinyl-alanine-alanine-proline-pheny- Materials and methods lalanine-p-nitroanilide, Sigma-Adrich, S7388) and 1 mM SAAApNA (N-succinyl-alanine-alanine-alanine-p-nitroanilide, Sigma-Aldrich, Insect rearing S4760) as substrates, respectively. The reaction mixture consisted of Fifth nymphal instars of A. amygdali were collected from elm trees in 35 mL of universal buffer (pH 8, as the recommended pH for serines Shiraz (Fars province, Iran) and transferred to the laboratory. The in the literature), 5 mL of each substrate, and 5 mL of enzyme solu- nymphs were reared on elm leaves at 28±1°C, 70% relative humidity tion. The reaction mixture was incubated at 30°C for a period of 0-10 and a 16L:8D of photoperiod. When adults emerged, they were allowed min before adding 30% TCA to terminate the reaction. The to feed on leaves for 48 h prior to being randomly selected for biochem- absorbance of the resulting mixture was then measured spectropho- ical analysis. tometrically at 405 nm by p-nitroaniline release. To prove the specific proteolytic activity, a negative control for each substrate was pro- Sample preparation vided separately containing all the above components except for Based on the method of Cohen (1993), adults of A. amygdali were enzyme pre-boiled at 100°C for 30 min (Oppert et al., 1997). randomly selected and their midgut removed by dissection in iced saline solution (NaCl, 10 mM, 1.06404.1000, Merck-Chemicals, Cysteine proteases only Darmstadt, Germany). Integument and unneeded organs were removed Cathepsins B and L activities (as three subclasses of cysteine pro- and the midgut was gently separated and rinsed in 1 mL of iced distilled teases) were assayed using a 1 mM concentration of Z-Ala-Arg-Arg 4- water. To obtain appropriate samples, five midguts were placed in one methoxy-b-naphtylamide acetate (Sigma-Aldrich, C8536) and N- Eppendorff tube (Eppendorff, Hamburg, Germany) containing 1 mL of Benzoyl-Phe-Val-Arg-p-nitroanilideuse hydrochloride (Sigma-Aldrich, distilled water. Tissues were ground using a homogenizer and cen- B2133) as substrates, respectively. The reaction mixture consisted of trifuged at 13,000 rpm for 20 min at 4°C (Daika, Rika Kugyo Co., Tokyo, 35 mL of universal buffer (pH 5 as the recommended pH for cysteines Japan). The supernatant was carefully removed, transferred to new in the literature), 5 mL of each substrate and 5 mL of enzyme solution. tubes and stored at –20°C for no more than one week until use in the The reaction mixture was incubated at 30°C for a period from 0-10 min experiments. before adding 30% TCA to terminate the reaction and

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