Phaseolus Vulgaris Seeds Research Articles

The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply.

Phaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain etal., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on sucrose non-fermenting related kinase 1 (SnRK1) activity during this process. Bean fruits removed from the plant at 20 days after flowering (DAF) demonstrated active remobilisation of nutrients from the pod to the seeds. After 5 days, the pod dry weight was reduced by 50%. The process was characterized by a rapid degradation of starch, with the greatest decrease observed on day 1 after the fruits were removed. The pod nutrients were insufficient for the needs of all the seeds, and only some seeds continued their development. Those seeds exhibited a transient reduction in sucrose levels on day 1 after the fruits were removed. However, the normal level of sucrose was recovered, and the rate of starch synthesis was identical to that of a seed developed under normal conditions. Removing the fruits from the plant had no effect on the activity of SnRK1 in the pods, whereas in the seeds, the activity was increased by 35%. Simultaneously, a large reduction in seed sucrose levels was observed. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. At 20–25 DAF, cotyledons actively accumulate storage materials. It is possible that the increase in SnRK1 activity observed in seeds developed in fruits that have been removed from the plant is part of the mechanism required for nutrient remobilisation under conditions of stress.

Application of the flour of four leguminous crops for the control of tenebrionidae beetle (Tribolium castaneum - herbst)

Provender used to breed animals is constituted mainly of cereal flour. However, due to poor post-harvest technologies, more than 35% of annual crop yield is often lost during storage as a consequence of insect attack. While synthetic insecticides constitute an efficient method for reducing these losses; current excessive usage runs the risk of increasing the food insecurity. The use of entomotoxic proteins of leguminous plant for grain storage has been advocated as an alternative method with limited or no effect on the quality and safety of the grains or their products. This study was carried out as an evidence of bioactivity of four flours of seeds of Phaseolus vulgaris , Pisum sativum and two varieties of Glycine max (SJ320 and DOCKO) against adults of Tribolium castaneum Herbst which is a very resistant stored product pest to chemicals in Northern Cameroon. The growth of ten couples of T. castaneum in different formulations made by the mixture of sorghum flours with leguminous seed flours is evaluated 50 days after application. The research carried out reveals that the population of T. castaneum is important in the soybean formulations. In the pots of bean and pea flours, the average numbers of living pests were respectively 21.17 ±10.5 and 32.38 ±18.3 against 65.99 ±28.1 and 95.2 ±36.7 for the pots containing soybean SJ320 and DOCKO flours, respectively. In treatments PP20 and BB20, the number of nymphs 82.4±6.7, 86.6±2.7, respectively and the larvae V 22.2±9.9, 68.8±8.9 in that order decreases comparatively to the control 143.6±7.4, 142±12.5, respectively . The incorporation of these promising flours of pea and bean at a rate of 20% during manufacturer of provender used to feed animals can contribute to their better cereal food conservation and can reduce the residues of chemical pesticides in the environment. The formulation with 20% of black beans or pea flours in sorghum flour ensures an effective control of T. castaneum. The development of a bio insecticide made of flour of pea or black bean is to be considered seriously since their residues are not poisonous to consumers. Cereals which are important and indispensable for the production of animal feed can be preserved from insect attack by the use of natural products, which will contribute to diminish the toxicity of chemical preservatives used in feed, environmental pollution and the phenomenon of resistance of insects to chemical pesticides.

Proteomic changes in the roots of germinating Phaseolus vulgaris seeds in response to chilling stress and post-stress recovery

Plants respond to different environmental cues in a complex way, entailing changes at the cellular and physiological levels. An important step to understand the molecular foundation of stress response in plants is the analysis of stress-responsive proteins. In this work we attempted to investigate and compare changes in the abundance of proteins in the roots of bean (Phaseolus vulgaris L.) germinating under long continuous chilling conditions (10°C, 16 days), exposed to short rapid chilling during germination (10°C, 24h), as well as subjected to recovery from stress (25°C, 24h). The results we obtained indicate that germination under continuous chilling causes alterations in the accumulation of the proteins involved in stress response, energy production, translation, vesicle transport, secondary metabolism and protein degradation. The subsequent recovery influences the accumulation of the proteins implicated in calcium-dependent signal transduction pathways, secondary metabolism and those promoting cell division and expansion. Subjecting the germinating bean seeds to short rapid chilling stress resulted in a transient changes in the relative content of the proteins taking part in energy production, DNA repair, RNA processing and translation. Short stress triggers also the mechanisms of protection against oxidative stress and promotes expression of anti-stress proteins. Subjecting bean seeds to the subsequent recovery influences the abundance of the proteins involved in energy metabolism, protection against stress and production of phytohormones. The exposure to long and short chilling did not result in the alterations of any proteins common to both treatments. The same situation was observed with respect to the recovery after stresses. Bean response to chilling is therefore strongly correlated with the manner and length of exposure to low temperature, which causes divergent proteomic alterations in the roots.

Correlation of protein content to flatulent galactooligosaccharides and exogenous amino acids in seeds of Phaseolus vulgaris L.

In order to estimate the possible correlations among constituents of <em>Phaseolus vulgaris </em>seeds, the contents of protein, exogenous amino acids and flatulent galactooligosaccharides (raffinose and stachyose) were analyzed in 16 Polish bean cultivars for dry seeds. A negative correlation coefficient (r =-0.9490) was found between protein and methionine contents. High positive correlations among exogenous amino acids, such as lysine and isoleucine, valine and isoleucine, lysine and tyrosine, were observed indicating a chance of selecting far more than one at a time. The small-seeded bean cultivars contained higher contents of protein and galactooligosaccharides than the large-seeded ones.

The "white kidney bean incident" in Japan.

Lectin poisoning occurred in Japan in 2006 after a TV broadcast that introduced a new diet of eating staple foods with powdered toasted white kidney beans, seeds of Phaseolus vulgaris. Although the method is based on the action of a heat-stable α-amylase inhibitor in the beans, phaseolamin, more than 1,000 viewers who tried the method suffered from acute intestinal symptoms and 100 people were hospitalized. Lectins in the white kidney beans were suspected to be the cause of the trouble. We were asked to investigate the lectin activity remaining in the beans after the heat treatment recommended on the TV program. The test suggested that the heat treatment was insufficient to inactivate the lectin activity, which, combined with our ignorance of carbohydrate signaling in the intestine, was the cause of the problem.

Isolation and characterization of plant growth promoting rhizobacteria and their application in plant growth

Five major genera of bacteria namely Pseudomonas, Bacillus, Klebsiella, Azotobacter, and Enterobacter has been isolated from the rhizosphere of leguminous plants. Screening of these bacterial isolates revealed different plant growth promoting properties. Phosphate solubilizers (44.82%), Siderophore producers (54.02%), IAA producers (51.72%) and Ammonia producers (82.75%) were obtained among rhizopheric bacteria isolated. Wide variation in the anti fungal properties with large number of bacteria active against Alternaria sp. and Rhizoctonia sp. Possibility of ten bacterial strains as PGPRs was explored by carrying out controlled study of exposure of seeds of Phaseolus vulgaris with culture of bacterial strains. Increase in dry mass of root and shoot were observed.

Analysis of rhizobial strains nodulating Phaseolus vulgaris from Hispaniola Island, a geographic bridge between Meso and South America and the first historical link with Europe

Hispaniola Island was the first stopover in the travels of Columbus between America and Spain, and played a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis of recA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Island showed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512, which were initially named as Rhizobium etli and have been recently reclassified into Rhizobium phaseoli after the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America than previously thought, and since the proposal of the American origin of R. etli was based on the analysis of several strains that are currently known to be R. phaseoli, it can be concluded that both species have an American origin coevolving with their host in its distribution centres. The analysis of the symbiovar phaseoli nodC gene alleles carried by different species isolated in American and European countries suggested a Mesoamerican origin of the α allele and an Andean origin of the γ allele, which is supported by the dominance of this latter allele in Europe where mostly Andean cultivars of common beans have been traditionally cultivated.

Isolation, purification and biochemical characterization of dipeptidyl peptidase-III from germinated Vigna radiata seeds

Genome analysis of plants indicated majority of putative protease genes that need characterization at enzymatic and molecular level. Proteases execute important role in seed development but knowledge of dipeptidylpeptidases in seeds is limited. A dipeptidylpeptidase-III that cleaves Arg-Arg from Arg-Arg-4mβNA was purified from germinated Mung bean seeds. Screening of cereals and legumes for DPP-III revealed highest activity in Phaseolus vulgaris and Vigna radiata (variety P.9531) seeds germinated for 48h. Homogenate (10%) was acid precipitated upto pH 4.2 and then subjected to 0–55% ammonium sulphate precipitation followed by successive chromatographies on cation exchanger, gel filtration and anion exchanger. Enzyme purity was confirmed by native polyacrylamide gel electrophoresis and in situ gel assays. DPP-III is a heterodimer of ∼60kDa with two subunits of 32.8kDa and 27kDa on SDS-PAGE. DPP-III worked optimally at pH 8.0 and at 37°C with pH stability between 7.5 to 9.5. Arg-Arg-4mβNA was preferred substrate and none of endopeptidase and monopeptidase substrates were hydrolysed. Inhibitors studies revealed it as thiol protease with involvement of metals at active site. The enzyme might be involved in plant's development by generating/deactivating bioactive peptides. Further studies on functional characterization and molecular structure are in progress in our laboratory.

Bioremediation of Four Food Industrial Effluents

Some effluents ((Whey Effluent (WhE); Orange Effluent (OE); Carrot Effluent (CE) and Chocolate Effluent (ChE)) were bioremediated using some allochthonous microorganisms (Lactobacillus delbrueckii subsp. bulgaricus, Saccharomyces cerevisiae Y-1347 and Dekkera bruxellensis). The highest biodegradable efficiency of the Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD) and Total Organic Nitrogen (TON) of the effluents under investigation was noticed when using the allochthonous microorganisms together with the autochthonous one. Saccharomyces cerevisiae Y-1347 proved to be the best utilizer of Whey (WhE) organic and nitrogenous compounds with the reduction of BOD, COD and TON by 12.36, 20.09 and 68.42%, respectively. Dekkera bruxellensis proved to be the organism of choice on using Orange Effluent (OE) where BOD, COD and TON were reduced by 18, 20 and 53.39%, respectively. Lactobacillus delbrueckii subsp. bulgaricus proved to be the best utilizer of the Carrot Effluent (CE) constituents by reducing BOD, COD and TON by 24.27, 19.33 and 63.63%, respectively. Dekkera bruxellensis proved to be the best utilizer of the Chocolate Effluent (ChE) constituents by improving its quality and reducing BOD, COD and TON by 18.36 and 15.86 and 73.07%, respectively. A successful trial was made to use the treated effluents in the irrigation of Lens culinaris and Phaseolus vulgaris seeds for germination.

Endoreduplication intensity as a marker of seed developmental stage in the Fabaceae

Flow cytometry (FCM) can be used to study cell cycle activity in developing, mature and germinating seeds. It provides information about a seed's physiological state and therefore can be used by seed growers for assessing optimal harvest times and presowing treatments. Because an augmented proportion of 4C nuclei usually is indicative of high mitotic activity, the 4C/2C ratio is commonly used to follow the progress of seed development and germination. However, its usefulness for polysomatic (i.e., containing cells with different DNA content) seeds is questioned. Changes in cell cycle/endoreduplication activity in developing seeds of five members of the Fabaceae were studied to determine a more suitable marker of seed developmental stages for polysomatic species based on FCM measurements. Seeds of Phaseolus vulgaris, Medicago sativa, Pisum sativum, Vicia sativa, and Vicia faba var. minor were collected 20, 30, 40, 50, and 60 days after flowering (DAF), embryos were isolated and the proportion of nuclei with different DNA contents in the embryo axis and cotyledon was established. The ratios 4C/2C and (Σ>2C)/2C were calculated. Dried seeds were subjected to laboratory germination tests following international seed testing association (ISTA) rules. Additionally, the absolute nuclear DNA content was estimated in the leaves of the studied species. During seed development nuclei with DNA contents from 2C to 128C were detected; the endopolyploidy pattern depended on the species, seed organ and developmental stage. The cell cycle/endoreduplication parameters correlated negatively with genome size. The (Σ>2C)/2C ratio in the cotyledons reflected the seed developmental stage and corresponded with seed germinability. Therefore, this ratio is recommended as a marker in polysomatic seed research and production instead of the 4C/2C ratio, which does not consider the occurrence of endopolyploid cells.

Allelopathic effects of aqueous extract of Brassica napus on germination of seeds of Phaseolus vulgaris

Secondary compounds produced by plants are considered an alternative method of weed suppression but can cause negative effects on crops in succession, especially in a no-tillage system, due to the degradation of crop residues with allelopathic potential. The objective of this work was to analyze the influence of foliar aqueous extracts of Brassica napus on the germination and initial development of seedlings of Phaseolus vulgaris. The extract was prepared as a stock 10 % weight/volume solution, and diluted into treatments of relative concentrations of 100 % (i.e. 10 % w/v stock), 75 %, 50 %, 25 % and 0 % (untreated control consisting of distilled water), in a completely randomized design. The seeds of Phaseolus vulgaris were moistened with the differing concentrated extracts and kept in a germination chamber at 25 °C, with a photoperiod of 12 h for nine days. The variables evaluated were: percentage germinating, first count of germination and germination velocity index, as well the root and hypocotyl length, and fresh and dry mass of the seedlings. The aqueous leaf extracts of Brassica napus did not influence the germination of Phaseolus vulgaris seeds, but did induce the growth of abnormal seedlings by inhibition of secondary roots and reduced prominence of the primary root.

Effects of cryopreservation of Phaseolus vulgaris L. seeds on early stages of germination

In this work, we studied the effects of cryopreservation on various parameters of early stages of germination of Phaseolus vulgaris seeds (0, 7 and 14days). Percentages of germination, fresh mass of different plant parts, levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and protein were determined. No phenotypic changes were observed visually in seedlings recovered from cryopreserved seeds. However, several significant effects of seed liquid nitrogen exposure were recorded at the biochemical level. There was a significant negative effect of cryopreservation on shoot protein content, which decreased from 3.11mgg(-1) fresh weight for non-cryopreserved controls to 0.44mgg(-1) fresh shoot weight for cryopreserved seeds. On the other hand, cryopreservation significantly increased levels of other aldehydes than malondialdehyde in shoots at day 7, from 56.47μmolg(-1) for non-cryopreserved controls to 253.19μmolg(-1) fresh shoot weight for cryopreserved samples. Liquid nitrogen exposure significantly reduced phenolics contents (free, cell-wall linked, total) in roots at day 7 after onset of germination. In general, roots were more affected by cryostorage compared with other plant parts, while leaves were the least affected. The effects of seed cryopreservation seem to decline progressively along with seedling growth.

Low Soil Phosphorus Availability Increases Acid Phosphatases Activities and Affects P Partitioning in Nodules, Seeds and Rhizosphere of Phaseolus vulgaris

The effect of phosphorus (P) deficiency on phosphatases activities in N2-fixing legumes has been widely studied in hydroponic culture. However, the response of acid phosphatase (APase) and phytase in rhizosphere, nodules and seeds of Phaseolus vulgaris to low soil’s P-availability is not yet fully understood. In this study, six genotypes of N2-fixing P. vulgaris were grown under contrasting soil P-availabilities; i.e., low (4.3 mg P kg−1) and sufficient (16.7 mg P kg−1) in the Haouz region of Morocco. At flowering and maturity stages, plants were harvested and analyzed for their phosphatases activities, growth and P content. Results show that, low P decreased nodulation, growth, P uptake and N accumulation in all the genotypes, but to a greater extent in the sensitive recombinant inbreed line 147. In addition, while seed P content was slightly reduced under low P soil; a higher P was noticed in the Flamingo and Contender large seeded-beans (6.15 to 7.11 mg g−1). In these latter genotypes, high APase and phytase activities in seeds and nodules were associated with a significant decline in rhizosphere’s available P. APase activity was mainly stimulated in nodules, whereas phytase activity was highly induced in seeds (77%). In conclusion, the variations of APase and phytase activities in nodules and seeds depend on genotype and can greatly influence the internal utilization of P, which might result in low P soil tolerance in N2-fixing legumes.

Purification of a novel α-amylase inhibitor from local Himalayan bean (Phaseolus vulgaris) seeds with activity towards bruchid pests and human salivary amylase.

Six bean (Phaseolus vulgaris L.) cultivars of Himalayan region were analysed for α- amylase inhibitor activity. The α-amylase inhibitor from seeds of screened bean cultivar KR-9, showing maximum inhibitory activity was purified using ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-100) and ion exchange chromatography (DEAE-Sephadex). The inhibitor was purified to homogeneity as judged by native-PAGE with 14.22 fold purification and 71.66% recovery. Purified inhibitor consisted of three subunits of molecular weight 15,488, 18,620 and 26,302 daltons, respectively as determined by SDS-PAGE. It was found to be heat stable up to 30°C-40°C and had two pH optima of 5.0 and 6.9. Nature of inhibition was found to be of non-competitive type. The purified inhibitor was found to be effective against α-amylases extracted from larvae of Callosobruchus chinensis, Tribolium castaneum and gut enzyme of Spodoptera littoralis. Larvae of Tribolium castaneum fed on flour mixed with purified inhibitor for 5days showed 100% larval mortality. Purified α-amylase inhibitor was also found to inhibit human salivary α-amylase, suggesting its potential in prevention and therapy of obesity and use as drug design targets for treatment of diabetes. The gene encoding the inhibitor may be used to develop transgenic plants resistant against insect pests.

Bioremoval of chemically different synthetic dyes by Aeromonas hydrophila in simulated wastewater containing dyeing auxiliaries

Aeromonas hydrophila, isolated from acclimated textile effluent after selective enrichment, was assessed for its dye biodegradability potential using chemically different dyes. The results revealed that the bacterium has a broad spectrum dye-degrading activity since it was able to efficiently decolorize the azo (Solophenyl red 3BL and Polar brilliant red B), triphenylmethane (Basic fuchsin and Crystal violet) and azine (Safranin and Nigrosine) dyes tested. Decolorization of azo dyes was more effective under microaerophilic conditions, whereas aerobic conditions were a prerequisite for the efficient decolorization of triphenylmethane and azine dyes by this isolate. UV/visible spectra of decolorized cultures indicated that color removal was the result of biodegradation rather than adsorption. Color reduction percentages obtained after 24 h of incubation at 30°C were: 95 (Solophenyl red); 92 (Polar brilliant red B); 93 (Basic fuchsin); 75 (Crystal violet); 70 (Safranin) and 42 (Nigrosine). Optimal decolorization activity was obtained at an initial medium pH range of 6–9, and various concentrations of dyes (5–50 mg/L) and dyeing auxiliaries (sodium chloride, 0–20 g/L; SDS, 0–5 mg/L). The addition of carbon sources (especially glucose) significantly enhanced color reduction by this bacterium. A phytotoxicity bioassay using seeds of Phaseolus vulgaris confirmed the detoxification of dyes after treatment with A. hydrophila. The results indicate the potential of this isolate for use in biotechnological applications in the treatment of dye wastewater containing chemically different dyes

Antifungal Activity of PvD1 Defensin Involves Plasma Membrane Permeabilization, Inhibition of Medium Acidification, and Induction of ROS in Fungi Cells

In recent years, studies have demonstrated the function of many antimicrobial peptides against an extensive number of microorganisms that have been isolated from different plant species and that have been used as models for the study of various cellular processes linked to these peptides' activities. Recently, a new defensin from Phaseolus vulgaris (L.) seeds, named PvD(1,) was isolated and characterized. PvD(1) was purified through anion exchange and phase-reverse chromatography. PvD(1)'s antifungal activity was tested. A SYTOX Green uptake assay revealed that the defensin PvD(1) is capable of causing membrane permeabilization in the filamentous fungi Fusarium oxysporum, Fusarium solani, and Fusarium laterithium and in yeast strains Candida parapsilosis, Pichia membranifaciens, Candida tropicalis, Candida albicans, Kluyveromyces marxiannus, and Saccharomyces cerevisiae at a concentration of 100 μg/ml. Ultrastructural analysis of C. albicans and C. guilliermondii cells treated with this defensin revealed disorganization of both cytoplasmic content and the plasma membrane. PvD(1) is also able to inhibit glucose-stimulated acidification of the medium by yeast cells and filamentous fungi, as well as to induce the production of reactive oxygen species and nitric oxide in C. albicans and F. oxysporum cells.

Stereoisomers of Castasterone, 3-Epicastasterone and 2,3-Diepicastasterone, in Immature Seeds of Phaseolus vulgaris

A capillary GC-MS analysis revealed that two stereoisomers of castasterone are contained in immature seeds of Phaseolus vulgaris. 400 MHz proton NMR analysis of the stereoisomers determined they are A ring epimers of castasterone, 3-epicastasterone and 2,3-diepicastasterone. In rice lamina inclination assay, 3-epicastasterone and 2,3-diepicastasterone showed reduced biological activity than that of castasterone. Together with our previous finding that 2-epicastasterone exhibits a less biological activity than CS, this result indicates that epimerization of hydroxyl at C-2 or/and C-3 is/are inactive processes to reduce biological activity of CS after exerting as a bioactive brassinosteroid in P. vulgaris.

Screening for antidiabetic activity and phytochemical constituents of common bean (Phaseolus vulgaris L.) seeds

Seeds of Phaseolus vulgaris were given individually at different doses to different batches of rats (both normal and hyperglicemic rats) after an overnight fast. Seeds contain the bioactive components – alkaloids, flavonoids, fiber, proteins, tannins, terpenoids, saponins, quercetin, anthocyanin and catechin. The blood glucose levels were measured at 0, 1, 2, 3, 4, 5 and 6 h after the treatment. Most active doses were further studied to dose-dependent (300, 200 and 100 g/kg bw) antihyperglycemic effects alone and in combination with glibenclamide (0.20, 0.10 and 0.05 g/kg bw). Seeds of P. vulgaris at a dosage of 300 g/kg bw is showing maximal blood glucose lowering effect in diabetic rats after third hour. The antihyperglycemic activity of P. vulgaris seeds was compared with the treatment of glibenclamide, an oral hypoglycemic agent. The combination of seeds of most dose (300 mg/kg bw) and higher dose of glibenclamide (0.20 g/kg bw) showed safer and potent hypoglycemic as well as antihyperglycemic activities without creating severe hypoglycemia in normal rats. Key words: Phaseolus vulgaris, seeds, diabetes mellitus, antihyperglycemic activity, glucose-infused diabetes, glibenclamide.

An antifungal defensin from Phaseolus vulgaris cv. ‘Cloud Bean’

An antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3 kDa was purified from dried seeds of Phaseolus vulgaris ‘Cloud Bean’. The isolation procedure entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography an Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Although the antifungal peptide was unadsorbed on DEAE-cellulose, it was adsorbed on both Affi-gel blue gel and SP-Sepharose. The antifungal peptide exerted antifungal activity against Mycosphaerella arachidicola with an IC50 value of 1.8 μM. It was also active against Fusarium oxysporum with an IC50 value of 2.2 μM. It had no inhibitory effect on HIV-1 reverse transcriptase when tested up to 100 μM. Proliferation of L1210 mouse leukemia cells and MBL2 lymphoma cells was inhibited by the antifungal peptide with an IC50 of 10 μM and 40 μM, respectively.

Host‐mediated induction of α‐amylases by larvae of the Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is irreversible and observed from the initiation of the feeding period

Larvae of Zabrotes subfasciatus secrete alpha-amylases that are insensitive to the alpha-amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non alpha-amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible alpha-amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible alpha-amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days (age when the larvae stopped feeding), we could detect higher activity of the inducible alpha-amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible alpha-amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible alpha-amylase isoforms. Incubations of brush border membrane vesicles with the alpha-amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes.