A amylase activity of nymphal stages of sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

Wheat production in Iran has changed substantially over the past one or two decades with development of higher-yielding cultivars and improved methods of planting. Sunn pest, Eurygaster integriceps (Heteroptera: Pentatomidae), is the most important cereal pest in Iran. 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. alpha-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 recent increase in study of insect digestive enzymes seems to make sense in the realization that the gut is the major interface between the insect and its environment. Hence, an understanding of digestive enzyme function is essential when developing methods of insect control such as the use of enzyme inhibitor's and transgenic plants to control phytophagous insects. The aim of the current study is to identify and characterize alpha-amylase activity in order to gain a better understanding of its digestive physiology, which hopefully will lead to new strategies of the insect control. In order to analyze a-amylase activity adult and different nymphal stages were collected from wheat field from Karaj area and midgut complex from these individuals were dissected under a light microscope in ice-cold saline buffer (0.006M NaCl). After homogenization in buffer, homogenate was centrifuged at 15000 g for 20 min at 4 degrees C. The supernatant was pooled and stored at -20 degrees C for subsequent analysis. alpha-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure using soluble starch as substrate (starch 1%). Our result showed that enzyme activities in different nymphal stages (first, second, third, fourth and fifth stadium) were 0.19, 0.78, 1.21, 1.23, 1.25 units/mg protein, respectively.     

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.     

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.     

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.     

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye)

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

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.     

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.     

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.     

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.     

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.     

Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.     

一株新的a-淀粉酶抑制剂生产菌株ZG0656及其产物的发酵、分离、性质与应用

从土壤中分离并筛选得到了一株a-淀粉酶抑制剂生产菌, 编号ZG0656。根据形态特征、培养特征、生理生化特征、细胞壁化学组成特征和16S rDNA全序列相似性比较分析等多相分类方法, 确认菌株ZG0656为天蓝黄链霉菌的新变种, 命名为天蓝黄链霉菌南开变种(Streptomyces coelicoflavus var. nankaiensis)。该菌经10 L发酵罐水平发酵, 发酵液中可积累一定量的a-淀粉酶抑制剂。采用浓缩, 树脂吸附, 凝胶过滤, 减压干燥等方法得到a-淀粉酶抑制剂混合物。该a-淀粉酶抑制剂为含氮的拟低聚糖类物质, 能强烈抑制哺乳动物来源的a-淀粉酶, 对餐后高血糖的形成有明显改善作用, 可用于制备治疗糖尿病、肥胖症的药物或功能性食品。     

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.     

Inhibition of amylases from different origins by albumins from the wheat kernel.

The amylase activity of water extracts from 18 insect species, from 23 marine species and from 17 different species of birds and mammals was determined quantitatively. The inhibition of amylase in these extracts by three albumin fractions from the mature wheat kernel, which had been separated according to their molecular weights (60 000, 24 000 and 12 500 D), was determined as well. The inhibition activity of the three albumin fractions toward amylases extracted from a number of cereal species or from immature and germinating wheat kernel was also tested. The extracts from insects that are destructive of wheat grain and stored wheat products showed much higher amylase activities as compared to the other insect species that do not attack wheat and wheat products. On the basis of the effectiveness with which the three albumin fractions inhibit their activities, the amylase preparations tested were divided into susceptible, partially susceptible and resistent. Susceptible amylases, inhibited by any of the three albumin fractions, were found mainly in insects that attack wheat and in marine species. Partially susceptible amylases, inhibited by only one or two of the three albumin fractions, were present in a few avain and mammalian species including man. Resistent amylases were largely distributed in cereal, avian and mammalian species as well as in insect species that do not usually attack wheat grain or wheat flour products. At no stage of development, wheat alpha-amylase was inhibited by the albumin fractions from the mature kernel. The 12 500 dalton albumin fraction was the most effective in inhibiting insect amylases, but it was inactive toward avian and mammalian amylases. The 24 000 dalton albumin fraction was the most effective in inhibiting amylases from marine avian and mammalian species and inhibited as much as 33 amylases over 66 different amylases tested. It is suggested that protein inhibitors of amylase contributed to natural selection of polyploid wheats by giving some insect resistence to such wheats, even though some insect species were able to overcome this biochemical defense toa large degree by producing higher amylase activities.     

Biology,host preferences and fitness of <Emphasis Type="Italic">Oulema melanopus</Emphasis> (Coleoptera: Chrysomelidae), a recent invasive pest in Western Canada

The developmental parameters and fitness of a recent invasive insect pest of cereals in western Canada, the cereal leaf beetle, Oulema melanopus (L.) (Coleoptera: Chrysomelidae), were compared on live plants and excised leaves of commercial cultivars of potential cereal hosts including wheat (winter and spring), oats, barley, corn, rye and triticale. Host preference and utilization within the fundamental host range of O. melanopus differed. The biological parameters differed significantly when the larvae were reared on excised tissues versus on live host plants. In both studies, wheat (winter and spring), oat (cv. Morgan) and barley were the most preferred hosts in terms of development, survivorship, adult weights and fecundity. Prolonged development with low fitness gains was noted on corn. Although the development on hosts such as rye and triticale was prolonged and adult fitness was low, the survivorship was high on these hosts. Hence, these crops can act as secondary hosts for the beetle in its new eco-region. Larvae fed foliage of Waldern, a local oat cultivar, had increased developmental time and lower survivorship compared with other cereal hosts. Despite differences in fitness gains among hosts, similar numbers of eggs were laid on all hosts. Hence, the oviposition choice of O. melanopus may not be driven by fitness gains alone and it may indicate adaptive strategy to hosts in newer environments.     

Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca2+ situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca2+-modulated kinetics of the AMY2/BASI interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors.     

Rice bifunctional alpha-amylase/subtilisin inhibitor: cloning and characterization of the recombinant inhibitor expressed in Escherichia coli

The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.     

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.     

Inhibitory specificity and insecticidal selectivity of alpha-amylase inhibitor from Phaseolus vulgaris

The primary structure and proteolytic processing of the alpha-amylase isoinhibitor alpha AI-1 from common bean (Phaseolus vulgaris cv. Magna) was determined by protein chemistry techniques. The inhibitory specificity of alphaAI-1 was screened with a panel of the digestive alpha-amylases from 30 species of insects, mites, gastropod, annelid worm, nematode and fungal phytopathogens with a focus on agricultural pests and important model species. This in vitro analysis showed a selective inhibition of alpha-amylases from three orders of insect (Coleoptera, Hymenoptera and Diptera) and an inhibition of alpha-amylases of the annelid worm. The inhibitory potential of alphaAI-1 against several alpha-amylases was found to be modulated by pH. To understand how alphaAI-1 discriminates among closely related alpha-amylases, the sequences of the alpha-amylases sensitive, respectively, insensitive to alphaAI-1 were compared, and the critical determinants were localized on the spatial alpha-amylase model. Based on the in vitro analysis of the inhibitory specificity of alphaAI-1, the in vivo activity of the ingested alphaAI-1 was demonstrated by suppression of the development of the insect larvae that expressed the sensitive digestive alpha-amylases. The first comprehensive mapping of alphaAI-1 specificity significantly broadens the spectrum of targets that can be regulated by alpha-amylase inhibitors of plant origin, and points to potential application of these protein insecticides in plant biotechnologies.