Effect of plant a-amylase inhibitors on sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), alpha-amylase activity

Plant-insect interaction is a dynamic system, subjected to continual variation and change. In order to reduce insect attack, plants developed different defence mechanisms including chemical and physical barriers such as the induction of defensive proteins, volatiles that attract predators of the insect herbivores and secondary metabolites. Proteinaceous inhibitors of alpha-amylase and proteases are widely distributed in cereals, legumes and some other plants. Because of the possible importance of these inhibitors in plant physiology and animal nutrition, extensive research has been conducted on their properties and biological effects. Sunn pest like other insect pests of wheat lives on a polysaccharide-rich diet and depends to a large extent on effectiveness of their alpha-amylases for survival, a-amylase (1-4-alpha-D-glucan glucanohydrolase) hydrolyses starch, and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic linkages and has a major role in the utilization of polysaccharides. The enzyme inhibitors act on key insect gut digestive hydrolyses, alpha-amylase. Several kinds of a-amylase inhibitors present in seeds and vegetative organs of plant, act to regulate number of phytophagous insects. Therefore, the aim of the current study is to study cereal proteinaceous inhibitors of insect digestive enzymes and their potential use as resistance factors against Sunn pest. The proteinaceous inhibitors from different cereal species including barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were extracted and tested in in vitro condition against Sunn pest alpha-amylase. Extraction was made with NaCl (0.15 M) at room temperature and further purification was done by ammonium sulphate precipitation. It was found that fractions obtained from barley had more inhibitory effect on amylase activity of Sunn pest than fractions obtained from wheat. Knowledge gained through these studies can be used to select resistant plant against insect pest.     

Inhibitory effect of proteinaceous seed extract of three Iranian wheat cultivars on Eurygaster integriceps (Sunn pest) digestive enzymes

Abstract

To investigate interaction between proteinaceous extracts of three Iranian wheat cultivars and digestive enzymes of Sunn pest, a population of adult insects was collected in summer from a wheat field located in Borkhar Region, Isfahan, Iran. Seed proteins were extracted by 0.15?M NaCl solution and partially purified using ammonium sulphate. Spectrophotometric assays were implemented to determine enzyme activities. Results indicated cultivar- and dose-dependent efficacy of seed protein extracts. Inhibition percentages of α-amylase, α-glucosidase, β-glucosidase and proteolytic activities at concentration of 32?μg/mL and saturation percentage of 70% ammonium sulphate were 54, 37, 25 and 59% by Hamoon, 16, 28, 18 and 19% by Karkheh and 15, 16, 15 and 23% by Dena, respectively. Zymography also confirmed the effect of inhibitors on α-amylase activity. Among wheat cultivars, Hamoon has the highest biological activity against Sunn pest major digestive enzymes and could contribute towards the development of new insect pest control strategies.     

Esterase activity in summer population of sunn pest, Eurygaster integriceps Put. (Hemiptera: Scutelleridae)

A key constraint on increasing wheat production in Iran and some neighbouring countries is Sunn pest which cause severe damage to vegetative growth stage of plant in the early season. It also feeds on wheat grain in late growth stage of plants thus damaged wheat grains loose their bakery properties. Because of injecting protease enzymes into the grain during feeding, enzymes degrade gluten proteins and cause rapid relaxation of dough which results in the production of bread with poor volume and texture. Organophosphorus insecticides are the main pesticides used to control the insect pest. However, suitable reduction in pest population has not been achieved partly due to resistance to pesticides. Esterase plays crucial roles in insect physiology and detoxifies a broad range of xenobiotics including insecticides. Enhanced esterase activity is a major mechanism if insecticide resistance and has been detected in a number of insects. To evaluate esterase activity adult bugs were collected from wheat field in Karaj area of Iran and transferred to the laboratory. For biochemical assay, two adult bugs (either males or females) were homogenized in 500 microl Na-phosphate buffer pH 7.2. The homogenates were centrifuged at 14000 g for 10 minutes at 4 degrees C. The supernatants as the enzyme source were pooled and stored at -20 degrees C for later use. For enzyme assay, 300 microl of supernatant was mixed with equal volume of substrates (30 mM alpha-naphthyl acetate or 30 mM beta-naphthyl acetate) and incubated at 30 degrees C for 30 minutes. Then, 50 microl of fast blue solution (150 mg fast blue B in 15 ml distilled water plus 35 ml 5% SDS) was added and esterase activity was determined in a spectrophotometer at 595 nm. Data showed that there are no differences in esterase activity between male and female. However, There was significant differences between hydrolysis of substrates, alpha-naphthyl acetate and beta-naphthyl acetate. Insect esterase hydrolyzes alpha-naphthyl acetate much more than beta-naphthyl acetate.     

Comparison of α- and β-mannosidase activity in the three cereal pests,Haplothrips tritici Kurdjumov (Thysanoptera: Phlaeothripidae), Rhopalosiphum padi L. (Homoptera: Aphididae) and Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

The Sunn pest, Eurygaster integriceps, the bird cherry-oat aphid, Rhopalosiphum padi, and wheat thrips, Haplothrips tritici are the major pests of wheat and other cereals in a wide area of the world. All these three insect species could produce damage to the wheat to some extent. Therefore, the purpose of the present study was to determine α- and β-mannosidase of the three mentioned insect pests. These insects were collected from the wheat farm and their guts (the Sunn pest and the aphid) and salivary glands of Sunn pest were removed. However, regarding tiny body of thrips, the whole body used in order to extract the enzymes. The enzymes, including α- and β-mannosidase activity, were measured by the hydrolysis of p-nitrophenyl-α-d-mannopyranoside (pNPαGal) and p-nitrophenyl-β-d-mannopyranoside (pNPβGal), respectively, using phosphate citrate buffer (pH 5.0). Mannosidases were not active in all three tested insect species, and also there were significant differences in activities of the two enzymes in three species. The greatest activity of α-mannosidases was observed in the Sunn pest salivary glands, E. integriceps, and the least activity was found in Sunn pest midgut with no activity. However, the activity of β-mannosidase was established in Sunn pest midgut, but there was no activity in the aphid midgut, R. padi. Activities of these two enzymes were modest in the thrips, H. tritici. The greatest amount of β-mannosidases in the Sunn pest midgut makes sense, since the Sunn pest is the main pest in the wheat farm that can feed on wheat grains. In the wheat grains, the highest amount of glycoproteins and glycolipids are present. Thus, it has been known that these enzymes (α- and β-mannosidases) are active on digestion of carbohydrates.     

Comparison of the effects of cereal and legume proteinaceous seed extracts on α-amylase activity and development of the Sunn pest

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is a significant limiting factor in the production of wheat and barley in many areas of the world. In the current study, the effect of semi-purified proteinaceous extracts of seeds on digestive enzymes, and the growth and development of the Sunn pest were studied. The results showed that the purified α-amylase inhibitor from Triticum aestivum (type І) and rice semi-purified seed extract did not significantly affect the Sunn pest α-amylase activity. However, bean and cowpea seed extracts significantly affected α-amylase activity in vitro. For example, the bean seed extract at concentrations of 0.125 and 2.0 mg · mL^− 1 inhibited α-amylase activity of the pest by 15% and 45%, respectively, while the cowpea seed extract, at the same concentrations, inhibited α-amylase activity of the pest by 9% and 40%, respectively. Further, incorporation of the seed extracts into the insect diet showed that the rice seed extract did not affect insect development time, while bean and cowpea seed extracts at high concentrations (e.g., 3.0%) significantly affected nymphal development time and survivability (P > 0.05). These results show that semi-purified seed extracts affect α-amylase activity, developmental time, and survivability but not the adult weight of the Sunn pest.     

Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage.     

Digestive alpha-amylases from Tecia solanivora larvae (Lepidoptera: Gelechiidae): response to pH, temperature and plant amylase inhibitors

The biochemical properties of the digestive alpha-amylase from Tecia solanivora larvae, an important and invasive insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive alpha-amylases with isoelectric points 5.30, 5.70 and 5.98, respectively, which were separated using native and isoelectric focusing gels. The alpha-amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzymes are stable when heated to 50 degrees C and were inhibited by proteinaceous inhibitors from Phaseolus coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The inhibitors present in an amaranth hybrid inhibited 80% of the activity at pH 9.0. The results show that the alpha-amylase inhibitor from amaranth seeds may be a better candidate to make genetically-modified potatoes resistant to this insect than inhibitors from common bean seeds.     

Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases.

Insect pests and pathogens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects feed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, beta-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive alpha-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be improved through the use of transgenic technology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six different alpha-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, gamma-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to different modes of inhibition and different specificity profiles against diverse alpha-amylases. Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant's own alpha-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitinase activity. The area has benefited from the recent determination of many structures of alpha-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of alpha-amylase inhibition. The continuing discovery of new classes of alpha-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect alpha-amylases, plant alpha-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed.     

First combined resistance to Hessian fly and Sunn pest identified in synthetic hexaploid wheat

Hessian fly, Mayetiola destructor (Say), and Sunn pest, Eurygaster integriceps (Puton), are the two most damaging insect pests of wheat in North Africa, West and Central Asia. Host plant resistance is the most environmental friendly, cost-effective and practical means of controlling insect pests. Twenty synthetic hexaploid wheat lines selected as resistant to Syrian Sunn pest in 2010 were screened for resistance to Moroccan Hessian fly biotype in 2016. The Hessian fly screening was carried out in standard greenhouse flats using a randomized complete block design with three replications, with susceptible and resistant checks in every test flat. The results showed that three synthetic hexaploid wheat lines exhibited resistance to both Moroccan Hessian fly biotype and Syrian Sunn pest. This is the first record of combined resistance to these two pests in wheat. Mapping populations using these sources of resistance are being developed using double haploid techniques for subsequent genetic characterization and identification of linked molecular markers for marker assisted selection.     

The elm leaf beetle α-amylase and its activity relationship with insect feeding

The elm leaf beetle, Xanthogalerucella luteola (Coleoptera: Chrysomelidae) is the most serious pest of elm trees. This pest causes severe damage to elm trees during its growth stages and as a result, in the middle of summer, leaves become skeletised and start to fall down. In this work, biochemical characterisation of digestive α-amylase of this insect and its relationship with insect feeding was investigated. The insect gut was isolated and its α-amylase was extracted and starch (1%) was used as a substrate for the enzyme. Results showed that the enzyme’s optimum temperature and pH was 35?°C and 5.5, respectively. Some ions such as NaCl decreased the enzyme activity whilst MgCl₂ and CaCl₂ increased the enzyme activity. Gut content electrophoresis showed that only one α-amylase is active in this insect species. There was a correlation between the amount of leaf eaten by the insect and the amount of the enzyme activity.     

A review of research on Sunn Pest {Eurygaster integriceps Puton (Hemiptera: Scutelleridae)} management published 2004–2016

Wheat is an important food crop that provides over 40% of the per capita dietary supply of calories and proteins in many developing countries. Wheat production has a crucial role in food security and the global economy. With the world's population estimated to reach 9.6 billion by 2050, the demand for wheat is expected to increase 60%. Sunn Pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is one of the major constraints to wheat production in Central and West Asia, Eastern Europe and North Africa. The economic impact of Sunn Pest is around 42 million USD for the region and that is only the cost of the chemicals used for its management. Application of chemical insecticides has been the main strategy for management of Sunn Pest. However, emergence of resistance in Sunn Pest populations against most pesticides and increased awareness of their adverse impacts on the environment have prompted investigation of alternative approaches. This review provides information on the most current literatures on Sunn Pest management with emphasis on practices such as application of chemical insecticides, insect growth regulators (IGRs) and anti-juvenile hormones, use of Sunn Pest egg parasitoids, entomopathogenic nematodes, entomopathogenic fungi, use of digestive enzyme inhibitors and development of resistant wheat varieties.     

The effect of cereal seed extracts on amylase activity of the rose sawfly,Arge rosae Linnaeus (Hymenoptera: Argidae)

The sawfly, Arge rosae Linnaeus (Hymenoptera: Argidae) is a serious pest of rose plants in a vast area of the world including Iran. Pesticides are applied in order to control in areas where infestation is high. So, there is a need for more environmentally benign alternatives to control this insect pest in urban areas. Therefore, the aim of the current study was to study the effect of cereal seed proteinaceous extracts including wheat and triticale on the insect α-amylase. Wheat and triticale seed proteins as well as fourth instars larvae α-amylase were extracted. The results showed that there are two different α-amylase isoenzymes in the insect gut, one major band and the other minor band, which had optimal activity at alkaline pH (pH 8.0). The effect of five concentrations including 0.42, 0.21, 0.105, 0.05 and 0.025?mg?ml^?1 of each seed extract was tested on α-amylase activity of the larvae gut. At the highest concentration (0.42?mg?ml^?1), wheat seed extract caused 74% inhibition of the enzyme activity while triticale seed extract inhibited 62% enzyme activity. Experiments proved that seed proteinaceous extract affected the enzyme activity in a pH-dependant manner.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Gut compartments and ovary bacterial symbionts of the Sunn pest

The Sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae), is the severe pest of cereals, especially of wheat in many parts of the world. Many insect species, including the Sunn pest that feed solely on nutritionally restricted diets, harbor symbiotic microorganisms. In the current study, we isolated and identified the Sunn pest bacterial symbionts of gut fractions and ovary. The phylogenetic analysis indicated that Sunn pest gut bacterial symbionts are polyphyletic and contained a taxonomic diversity belonging to three different phyla, including Firmicutes, Tenericutes, and Proteobacteria. Firmicutes was represented by Enterococcus, Proteobacteria by Pantoea and Acetobacteraceae, and Tenericutes by Spiroplasma. We isolated and identified Enterococcus, Acetobacteraceae, Spiroplasma and Pantoea from Sunn pest different gut compartments, and Pantoea from ovaries. There was considerable overlap between recognized symbionts from the 2nd and 3rd midgut sections (Acetobacteraceae), the 4th midgut section and hindgut (Spiroplasma), and 4th midgut section and ovary (Pantoea). Niche heterogeneity within a microbial habitat of gut fractions resulted in colonizing and adaptation of various communities of symbionts in each fraction. The Sunn pest gut compartments and ovary symbionts have been demonstrated to be of multiple evolutionary origins. This diversity may be of great importance to the Sunn pest fitness and survival in various overwintering niches.     

Purification and characterization of alpha-amylase from the infective juveniles of the nematode Heterorhabditis bacteriophora

Glycogen content and alpha-amylase activity were estimated in the infective juveniles (IJs) of Heterorhabditis bacteriophora at different times of storage. The glycogen content declined from 5.8 to 2.5 ng/IJ during storage for 40 days at 27 degrees C. The change in glycogen content coincided with the change of alpha-amylase activity during storage. alpha-Amylase was purified from IJs at zero time of storage by ion exchange chromatography and gel filtration. Ion exchange chromatography resolved alpha-amylase into three isoenzymes. The major isoenzyme alpha-amylase I had the highest specific activity and was purified to homogeneity. A molecular mass of 46-47 kDa was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. The Km values were 6.5 and 9.6 mg/ml using starch and glycogen as substrates, respectively. alpha-Amylase I showed optimum activity at pH 7.0 and had an optimum temperature of 40 degrees C. The enzyme was unstable at temperatures above 40 degrees C. The enzyme activity was severely inhibited by EDTA, p-CMB and iodoacetic acid, but potentiated by CaCl2 and NaCl. These results are discussed and compared with previously reported alpha-amylases in the insect hosts of the parasite.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

An investigation of the action of porcine pancreatic alpha-amylase on native and gelatinised starches

The action of pancreatic alpha-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine alpha-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat/Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100 degrees C, kcat/Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat/Km is not a simple one. The value of kcat/Km rises to reach a maximum at a pre-treatment temperature of 75 degrees C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although alpha-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.     

Identification and biochemical characterisation of α-amylase in the alimentary tract of Mediterranean fruit fly,Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Mediterranean fruit fly (Medfly), Ceratitis capitata, is an important pest of many fruit crops in temperate and subtropical regions worldwide. α-Amylases are hydrolytic enzymes involved in carbohydrate metabolism in insects. There is no report about α-amylase activity in C. capitata in literature. So, the aim of the current study was biochemical characterisation of α-amylase in the alimentary canal of the pest to gain a better understanding of digestive physiology of the insect. α-Amylase of Medfly was extracted and characterised using starch as the substrate. The results showed the presence of α-amylase activity in the gut of the insect for carbohydrate digestion. Optimum activity of the enzyme occurs at pH 8.0 and 40?°C. The most effective activator of the enzyme was determined in treatment with 20?mM CaCl₂. Na⁺, K⁺ and Mg²⁺ ions also activated the enzyme. Native PAGE of α-amylase showed two isoenzymes suggesting the importance of α-amylase in the carbohydrate digestion in the insect. Understanding of the digestive physiology and α-amylase activity of Medfly is important when new management strategies for this economically important pest are devised.     

The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat,primarily from Israel and Golan

Background  

Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature). Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences) were used to detect natural selection.