Assessment of the importance of α-amylase inhibitor-2 in bruchid resistance of wild common bean

Both α-amylase inhibitor-2 (αAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1–5 were generated by backcrossing wild common bean accessions with a cultivated variety. Whereas seeds of a wild accession (G12953) containing both αAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing αAI-2 accumulated high levels of this protein in seeds. The expression of αAI-2 in these lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive α-amylase. However, although the seed content of αAI-2 in these transgenic lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that αAI-2 alone does not provide a high level of resistance to Z. subfasciatus. However, αAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of αAI-1, and it may prove beneficial in genetic engineering of insect resistance in legumes.     

QUES, a new Phaseolus vulgaris genotype resistant to common bean weevils, contains the Arcelin-8 allele coding for new lectin-related variants

In common bean (Phaseolus vulgaris L.), the most abundant seed proteins are the storage protein phaseolin and the family of closely related APA proteins (arcelin, phytohemagglutinin and α-amylase inhibitor). High variation in APA protein composition has been described and the presence of arcelin (Arc) has been associated with bean resistance against two bruchid beetles, the bean weevil (Acanthoscelides obtectus Say) and the Mexican bean weevil (Zabrotes subfasciatus Bohemian). So far, seven Arc variants have been identified, all in wild accessions, however, only those containing Arc-4 were reported to be resistant to both species. Although many efforts have been made, a successful breeding of this genetic trait into cultivated genotypes has not yet been achieved. Here, we describe a newly collected wild accession (named QUES) and demonstrate its resistance to both A. obtectus and Z. subfasciatus. Immunological and proteomic analyses of QUES seed protein composition indicated the presence of new Arc and arcelin-like (ARL) polypeptides of about 30 and 27 kDa, respectively. Sequencing of cDNAs coding for QUES APA proteins confirmed that this accession contains new APA variants, here referred to as Arc-8 and ARL-8. Moreover, bioinformatic analysis showed the two proteins are closely related to APA components present in the G12949 wild bean accession, which contains the Arc-4 variant. The presence of these new APA components, combined with the observations that they are poorly digested and remain very abundant in A. obtectus feces, so-called frass, suggest that the QUES APA locus is involved in the bruchid resistance. Moreover, molecular analysis indicated a lower complexity of the locus compared to that of G12949, suggesting that QUES should be considered a valuable source of resistance for further breeding purposes.     

Linkage disequilibrium at the APA insecticidal seed protein locus of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Background  

An interesting seed protein family with a role in preventing insect herbivory is the multi-gene, APA family encoding the α-amylase inhibitor, phytohemagglutinin and arcelin proteins of common bean (Phaseolus vulgaris). Variability for this gene family exists and has been exploited to breed for insect resistance. For example, the arcelin locus has been successfully transferred from wild to cultivated common bean genotypes to provide resistance against the bruchid species Zabrotes subfasciatus although the process has been hampered by a lack of genetic tools for and understanding about the locus. In this study, we analyzed linkage disequilibrium (LD) between microsatellite markers at the APA locus and bruchid resistance in a germplasm survey of 105 resistant and susceptible genotypes and compared this with LD in other parts of the genome.     

Biochemical properties of Turkish common beans and their resistance against bean weevil <Emphasis Type="Italic">Acanthoscelides obtectus</Emphasis> (Coleoptera: Bruchidae)

Beans (Phaseolus vulgaris L.) with highly nutritional values are cultivated worldwide. Bean seeds are commonly exposed to bruchid attacks throughout the storage. Acanthoscelides obtectus (Say), also known as the bean weevil, is one of the most important insect pests and causes significant economic losses each year in warehouses. Chemical and alternative methods are commonly used to control A. obtectus. However, alternative control methods are getting popular because of negative impacts of chemicals on environment and human health. Identification and development of natural resistant bean genotypes may constitute a good alternative in fighting against bruchid pests. In this study, seed testa thickness and biochemical properties of 13 commonly grown Turkish bean genotypes were investigated, their resistance against damage caused by A. obtectus was determined, and finally the correlations among all these parameters were investigated. The highest ash and oil content was observed in Yakutiye-98 genotype while the highest protein and fiber ratio was observed in Noyanbey-98 and Zülbiye genotypes, respectively. The highest moisture ratio was observed in Karaca?ehir-90 genotype. Akda?, Akman-98, Noyanbey-98 and K?r?kkale genotypes were found to be more resistant against A. obtectus than the other genotypes and the lowest infection rates were detected in these genotypes. Consequently, Akda?, Akman-98, Noyanbey-98 and K?r?kkale genotypes which were resistant to A. obtectus can be recommended to farmers for cultivation in Turkey.     

Bruchid resistance of common bean lines having an altered seed protein composition

 Arcelin seed proteins of common bean (Phaseolus vulgaris L.) are toxic to one of the most damaging pests of bean seeds, Zabrotes subfasciatus (Boheman), but they appear to have little effect on another important bean pest, Acanthoscelides obtectus (Say), when introduced into standard cultivars by backcrossing. With the goal of increasing arcelin concentration to improve resistance, we modified seed-protein composition by introducing a null allele for the major seed protein, phaseolin, into lines (SMARC1, 2 and 4) or three phytohemagglutinin types (SMPHA lines). These lines were tested for resistance to both insects by measuring percentage insect emergence (%E) and days-to-adult emergence (DAE). For SMARC lines, arcelin type was the most important factor in resistance levels, with SMARC1 lines being most resistant, SMARC2 lines intermediate, and SMARC4 lines the least resistant to both bruchids. Additionally, the absence of phaseolin was a significant factor in the resistance of SMARC lines to A. obtectus. SMARC1 lines without phaseolin had half the percentage insect emergence of lines with phaseolin. SMARC1 lines with an altered seed composition had the highest levels of resistance to both bruchids of any large-seeded line reported to-date. Received: 2 April 1997 / Accepted: 20 May 1997     

Genetic mapping of microsatellite markers around the arcelin bruchid resistance locus in common bean

The deployment in common beans (Phaseolus vulgaris L.) of arcelin-based bruchid resistance could help reduce post-harvest storage losses to the Mexican bean weevil [(Zabrotes subfasciatus (Boheman)]. Arcelin is a member of the arcelin-phytohemagglutinin-α-amylase inhibitor (APA) family of seed proteins, which has been extensively studied but not widely used in bean breeding programs. The purpose of this study was to evaluate microsatellite markers for genetic analysis of arcelin-based bruchid resistance and to determine the orientation of markers and the rate of recombination around the APA locus. A total of 10 previously developed microsatellites and 22 newly developed markers based on a sequenced BAC from the APA locus were screened for polymorphism and of these 15 were mapped with an F₂ population of 157 individuals resulting from a susceptible × resistant cross of SEQ1006 × RAZ106 that segregated for both the arcelin 1 allele and resistance to the bruchid, Z. subfasciatus. Microsatellites derived from APA gene sequences were linked within 0.8 cM of each other and were placed relative to the rest of the b04 linkage group. In a comparison of genetic to physical distance on the BAC sequence, recombination was found to be moderate with a ratio of 125 kb/cM, but repressed within the APA locus itself. Several markers were predicted to be very effective for genetic studies or marker-assisted selection, based on their significant associations with bruchid resistance and on low adult insect emergence and positions flanking the arcelin and phytohemagglutinin genes.     

Molecular Cloning and Expression of an α-Amylase Inhibitor from Rye with Potential for Controlling Insect Pests

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new -amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of -amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic -amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.     

Effects of Phaseolus vulgaris (Fabaceae) seed coat on the embryonic and larval development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetles infest various seeds. The seed coat is the first protective barrier against bruchid infestation. Although non-host seed coats often impair the oviposition, eclosion and survival of the bruchid Callosobruchus maculatus larvae, morphological and biochemical aspects of this phenomenon remain unclear. Here we show that Phaseolus vulgaris (non-host) seed coat reduced C. maculatus female oviposition about 48%, increased 83% the seed penetration time, reduced larval mass and survival about 62 % and 40 % respectively. Interestingly, we found no visible effect on the major events of insect embryogenesis, namely the formation of the cellular blastoderm, germ band extension/retraction, embryo segmentation, appendage formation and dorsal closure. Larvae fed on P. vulgaris seed coat have greater FITC fluorescence signal in the midgut than in the feces, as opposed to what is observed in control larvae fed on Vigna unguiculata. Cysteine protease, α-amylase and α-glucosidase activities were reduced in larvae fed on P. vulgaris natural seed coat. Taken together, our results suggest that although P. vulgaris seed coat does not interfere with C. maculatus embryonic development, food digestion was clearly compromised, impacting larval fitness (e.g. body mass and survivability).     

Mesoamerican Origin and Pre- and Post-Columbian Expansions of the Ranges of Acanthoscelides obtectus Say,a Cosmopolitan Insect Pest of the Common Bean

An unprecedented global transfer of agricultural resources followed the discovery of the New World; one consequence of this process was that staple food plants of Neotropical origin, such as the common bean (Phaseolus vulgaris), soon expanded their ranges overseas. Yet many pests and diseases were also transported. Acanthoscelides obtectus is a cosmopolitan seed predator associated with P. vulgaris. Codispersal within the host seed seems to be an important determinant of the ability of A. obtectus to expand its range over long distances. We examined the phylogeographic structure of A. obtectus by (a) sampling three mitochondrial gene sequences (12s rRNA, 16s rRNA, and the gene that encodes cytochrome c oxidase subunit I (COI)) throughout most of the species’ range and (b) exploring its late evolutionary history. Our findings indicate a Mesoamerican origin for the current genealogical lineages of A. obtectus. Each of the two major centers of genetic diversity of P. vulgaris (the Andes and Mesoamerica) contains a highly differentiated lineage of the bean beetle. Brazil has two additional, closely related lineages, both of which predate the Andean lineage and have the Mesoamerican lineage as their ancestor. The cosmopolitan distribution of A. obtectus has resulted from recent expansions of the two Brazilian lineages. We present additional evidence for both pre-Columbian and post-Columbian range expansions as likely events that shaped the current distribution of A. obtectus worldwide.     

Comparative value of four arcelin variants in the development of dry bean lines resistant to the Mexican bean weevil

Wild Phaseolus vulgaris L. accessions containing arcelin codominant alleles 1 through 5 were reconfirmed and characterized for resistance to the Mexican bean weevil, Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae). Accession G 02771 (arcelin 5) had the highest level of antibiosis resistance, followed by G 12952 (arcelin 4), G 12882 (arcelin 1) and G 12866 (arcelin 2). Arcelin 3 accessions conferred the lowest levels of resistance. As the presence of arcelin is inherited as a single dominant gene, a backcross breeding program has been used to transfer resistance to the Mexican bean weevil from wild beans to bean cultivars using serological techniques to detect the presence of arcelin and replicated insect feeding tests to measure resistance levels. Progeny containing arcelin 1 showed resistance equal or superior to that of the resistant check. Arcelin 2-deerived lines had intermediate levels of resistance while no resistant progenies were obtained from crosses with arcelin 3 and 4 sources. Results are discussed in relation to the deployment of arcelin alleles in bean cultivars.

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Valeurs comparées de 5 types d'arcéline dans l'obtention de lignées de Phaseolus vulgaris résistantes à Zabrotes subfasciatus

Résumé La résistance à Zabrotes subfasciatus est associée à la présence d'arcéline, une nouvelle protéine des graines, découverte chez quelques populations de Phaseolus vulgaris. 5 types d'arcéline, hérités comme allèles codominants ont été décrits dans la littérature. Nous avons reprécisé les différentes populations contenant différents types d'arcéline et caractérisé leurs résistances à Z. subfasciatus. La population G 02771, correspondant à l'arcéline 5, présente la résistance la plus élevée par antibiose, suivie de G 12952 (arcéline 4), G 12882 (arcéline 1) et G 12866 (arcéline 2). Les populations contenant l'arcéline 3 présentent le moins de résistance à Z. subfasciatus.Un programme de croisements en retour associé à des tests sérologiques pour déceler la présence d'arcéline chez les descendants jeunes et des expériences répétées d'alimentation par les insectes vec BC2F₃ a été réalisé pour transférer la résistance de populations naturelles à des cultivars de haricots. Les lignées, provenant de croisements avec des populations sauvages avec de l'arcéline 1, ont été fortement résistantes à Z. subfasciatus. Les lignées contenant de l'arcéline 2 ont été considérées comme ayant une résistance intermédiaire. Les lignées avec arcélines 3 et 4 étaient sensibles. Les raisons de l'échec du transfert de la résistance élevée des parents contenant de l'arcéline 4, sont inconnues. On a constaté que la concentration de l'arcéline dans les lignées contenant cet allèle était très faible, tandis que la concentration en arcéline 1 restait remarquablement élevée. Les recherches sont poursuivies pour déterminer les raisons de l'absence de transfert de l'arcéline 4 chez les descendants contenant cet allèle. Quoi qu'il en soit, les caractéristiques agronomiques et les qualités des lignées résistantes (codées RAZ) ont été évaluées en vue d'une diffusion pour les programmes nationaux de recherche des pays de basses altitudes intertropicaux ou Zabrotes subfasciatus fait des dégâts importants.

     

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

Plant α-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 α-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 α-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 α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Comparative resistance of Phaseolus vulgaris beans to Callosobruchus chinensis and Acanthoscelides obtectus (Coleoptera: Bruchidae): The differential digestion of soluble heteropolysaccharide

At a concentration of 1 %, a soluble heteropolysaccharide fraction derived from Phaseolus vulgaris beans prevents development of Callosobruchus chinensis but not of Acanthoscelides obtectus. At a concentration of 2 %, it partly inhibits A. obtectus. The differential response could provide a basis for selecting resistant bean varieties. The heteropolysaccharide contains arabinose, xylose, rhamnose, glucose and galactose in approximate molar proportions of 9 : 2 : 1 : 1 : 4. Its biological activity resides mainly in its integral basic structure and only to a small extent in its constituent sugars. The insensitivity of A. obtectus is due to its effectively digesting a large portion of the heteropolysaccharide, releasing most of the contained arabinose, at the larval mid-gut pH of 6.7–6.8. C. chinensis hydrolyses much less of it at its mid-gut pH of 6.4–6.6, whereby the biological activity of the polymer is retained. In vitro digestion of the heteropolysaccharide by A. obtectus enzyme results in a residue which is comparatively inactive on this beetle in vivo, but is still partly toxic for C. chinensis.

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Résumé A la concentration de 1 %, l'extrait de Phaseolus contenant l'heteropolysaccharide soluble, empêche le développement de Callosobruchus chinensis, mais non celui d'Acanthoscelides obtectus. A une concentration de 2 %, il inhibe partiellement ce dernier. Cette différence dans la réponse pourrait servir de base, en vue de la sélection de variétées de pois résistants.L'heteropolysaccharide contient de l'arabinose, du xylose, du rhamnose, du glucose et du galactose dans les proportions respectives approchées en moles 9 : 2 : 1 : 1 : 4. Son activité biologique réside principalement dans l'intégrité de sa structure fondamentale, et seulement à un faible degré dans ses sucres constitutifs. L'insensibilité de Acanthoscelides obtectus est due à ce qu'il digère effectivement une grande portion de l'heteropolysaccharide, libérant la plupart de l'arabinose contenu, cela à PH 6.7–6.8 de l'intestin moyen de la larve, Callosobruchus chinensis en hydrolyse beaucoup moins au PH 6.4–6.6 de son intestin moyen, à la suite de quoi l'activité biologique du polymère est conservée. In vitro la digestion de l'heteropolysaccharide par les enzymes d'Acanthoscelides obtectus engendre un résidu qui est comparativement inactif sur cette espèce in vivo mais est encore partiellement toxique pour Callosobruchus chinensis.

     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

Effects of Temperature on Resistance in Phaseolus vulgaris Genotypes and on Development of Meloidogyne Species

Phaseolus vulgaris lines with heat-stable resistance to Meloidogyne spp. may be needed to manage root-knot nematodes in tropical regions. Resistance expression before and during the process of nematode penetration and development in resistant genotypes were studied at pre- and postinoculation temperatures of 24 °C and 24 °C, 24 °C and 28 °C, 28 °C and 24 °C, and 28 °C and 28 °C. Resistance was effective at all temperature regimes examined, with fewer nematodes in roots of a resistant line compared with a susceptible line. Preinoculation temperature did not modify resistance expression to later infections by root-knot nematodes. However, postinoculation temperatures affected development of Meloidogyne spp. in both the resistant and susceptible bean lines tested. The more rapid development of nematodes to adults at the higher postinoculation temperature of 28 °C in both bean lines suggests direct temperature effects on nematode development instead of on resistance expression of either of two gene systems. Also, resistance was stable at 30 °C and 32 °C.     

Expression of the Arcelin 1 gene from Phaseolus vulgaris L. in cowpea seeds (Vigna unguiculata L.) confers bruchid resistance

Persistent decrease in the productivity of cowpea (Vigna unguiculata L.) has been partly due to attack by bruchids including Zabrotes subfasciatus and Callosobruchus maculatus. Resistance to these insects in Phaseolus vulgaris L. has been shown to be associated with arcelins, a family of seed proteins encoded by a multigenic family of lectins on the APA locus. In this work, we report the construction of an expression vector containing Arc1 gene isolated from P. vulgaris and introduced into cowpea as a strategy to confer resistance to insect attack. Following transformation and selection, feeding experiments in which C. maculatus and Z. subfasciatus were fed with transgenic (L3 and L5) and non-transgenic (control) grains showed that introduced gene protected the transgenic line. Significant differences (p < .05 and p < .01) were found in the number of eggs laid, the number of emerging insects and the loss of grain mass in L3, compared with control, for both insects. Similar observations were made in L5 with the exception of the number of laid eggs. The strategy here described may form the basis for the development of a cowpea variety tolerant to bruchids in a crop cultivated by farmers throughout Latin America and Africa.     

Predicting evolution of insect resistance to transgenic crops in within-field refuge configurations,based on larval movement

The selection pressure imposed by the widespread use of transgenic technologies can lead to the evolution of insect resistance, and the availability of refuge areas that allow susceptible homozygous insects to survive is a key factor in delaying the evolution of resistance in agricultural landscapes. Different strategies to exploit refuge areas exist, but several insect-related ecological traits may directly affect the efficiency of refuges in slowing the development of resistance. Insect larval movement is one such trait that may affect the management of resistance, depending on the refuge strategy adopted. We developed a computational model to simulate how larval movement would affect the spatio-temporal dynamics of the evolution of resistance of insect pests to Bt crops, under different refuge configurations. In order to test the model, we used population data for Spodoptera frugiperda, one of the main target pests for control with Bt toxins. Simulations were run for spatial arrangements composed of three refuge configurations (seed mixture, blocks, and strips), with sizes ranging from 20% to 50% for two types of resistance (incomplete and complete) and three rates of larval movement (proportion of larvae moving per time step), equal to 0, 0.1 and 0.5. We demonstrated that with a seed mixture, in most cases the higher the rate of larval movement, the higher the proportion of resistant insects in the population in an area, regardless of the type of resistance tested. Strip configurations showed the opposite trend. In a block configuration, the number of resistant larvae was highest at an intermediate dispersal rate (0.1). We concluded that larval movement is an important variable affecting the evolution of resistance to Bt crops, but its effect depends on the type of resistance and the configuration and size of the refuge.     

Molecular basis for the specific binding of different α-amylase inhibitors from Phaseolus vulgaris seeds to the active site of α-amylase

In search of a possible mechanism of inhibition which might be responsible for the different specificities of the three isoforms of the bean (Phaseolus vulgaris) α-amylase inhibitor α-AI1, α-AI2 and α-AIL (EC 3.2.1.1), the two isoforms α-AI2 and α-AIL were modelled from the atomic co-ordinates of α-AI1 in the α-AI1/PPA complex and docking experiments were performed with pig pancreatic α-amylase (PPA) and the modelled amylase from Zabrotes subfasciatus (ZSA). The modelled α-AI2 penetrates without any steric hindrance in the substrate cleft of both enzymes but the possible hydrogen bonds between PPA and α-AI2 seem too few to maintain the stability of the complex. α-AIL, which differs from α-AI1 and α-AI2 by the absence of post-translational proteolytic cleavage and the occurrence of two additional loops of fifteen and six residues, creates steric clashes with PPA and ZSA that prevent its penetration into the substrate cleft of the enzyme. Docking experiments explain at the molecular level the specificity of α-amylase inhibitor isoforms towards enzymes of different origins. In addition, they explain why, according to its unprocessed and more bulky character, α-AIL was previously shown to be inactive on all α-amylases assayed. In fact, this last isoform is now considered as an evolutionary intermediate between phytohaemagglutinins, arcelins and α-amylase inhibitors.     

Development, fecundity and egg dispersion of Zabrotes subfasciatus

Zabrotes subfasciatus Boh. (Coleoptera: Bruchidae) is the smallest of the bruchids commonly infesting stored legume seeds, yet its wild and cultivated hosts, Phaseolus lunatus and P. vulgaris, have large seeds. It is demonstrated that the maximum fecundity of females is around 55 eggs which are aggregated onto some of the available hosts. About 80% of the eggs normally hatch and development at 27°C and 70% relative humidity takes around 34 days. The sex ratio of emerging adults is slightly biased towards males. About 75% of the larvae in a seed produce adults at low and moderate initial densities and up to 20 adults can emerge from a single seed. Adult weight is not influenced by the initial larval density in the seed but there is a strong correlation between the weight of females at emergence and their fecundity. These results are considered in the light of existing knowledge of Z. subfasciatus, much of which is apparently contradictory or inconsistent. Many of these difficulties are resolved and it is demonstrated that the behaviour and bionomics are well adapted to the normal situation in which the beetle is found and that the differences between this species and other bruchids are explicable in this context.     

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety “Red Kidney.”