Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmoLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmooLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.     

The African yam bean seed lectin affects the development of the cowpea weevil but does not affect the development of larvae of the legume pod borer

Artificial feeding assays were used to study the effect of purified galactose-specific lectins from African yam beans (Sphenostylis stenocarpa) on development of larvae of the cowpea weevil, Callosobruchus maculatus (Coleoptera : Bruchidae) and the legume pod-borer, Maruca vitrata (Lepidoptera : Pyrialidae). Inhibition of development of C. maculatus was observed when larvae were fed on artificial cowpea seeds containing 0.2%, 2.0% and 5.0% (wt/wt) of dietary lectin. Larval mortality was between 30% and 88%, while delays in total developmental time ranged between 7 and 13 days. The lectin had no effect on development of larvae of M. vitrala, when tested through topical artificial diet incorporation assays, except at the extremely high dose of 35% dietary level.     

Effects of a chitin binding vicilin from Erythrina velutina seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus)

Erythrina velutina vicilin, EvV, is a dimeric glycoprotein with Mr of 124.6 kDa. EvV was tested for anti-insect activity against bean bruchid larvae. EvV had LD(50) of 0.10% and ED(50) of 0.14% for Z. subfasciatus and LD(50) of 0.26% and ED(50) of 0.19% for C. maculatus. EvV was not digested by bean larvae enzymes until 12 h of incubation, and at 24 h EvV was more resistant to Z. subfasciatus enzymes.     

Effect of a trypsin inhibitor from Dimorphandra mollis seeds on the development of Callosobruchus maculatus

Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil, are pests that damage stored seeds. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (DMTI-II) isolated from Dimorphandra mollis seeds was tested for anti-insect activity against Callosobruchus maculatus larvae. The inhibitor produced ca. 67% mortality to this bruchid when incorporated into an artificial diet at a level of 1%. The doses necessary to cause 50% mortality (LD₅₀) and to reduce weight by 50% (ED₅₀) for DMTI-II were ca. 0.50% and 0.60%, respectively. The action of DMTI-II on C. maculatus larvae may involve the inhibition of trypsin-like activity of larval midgut extracts, the absence of digestion by midgut preparations or with a mixture of pepsin and papain, and its association with a chitin column and chitinous structure in the midgut of this insect.     

The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H+-Pumping Inorganic Pyrophosphatase (Studies Using Ligand Protection from Covalent Inhibitors)

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.     

Isolation of insecticidal lectin-enriched extracts from African yam bean (Sphenostylis stenocarpa) and other legume species

Insecticidal lectins were isolated from 20 resistant Vigna and non-Vigna legumes and tested againstn 3 pests of cowpea namely: Maruca vitrata, Callosobruchus maculatus and Clavigralla tomentosicollis. Crude lectins were separated from seeds using sodium chloride extraction, ammonium sulfate fractionation, and dialysis. SDS-PAGE indicated the molecular size of ca. 30 kDa for the most intense (and presumably active) band. Haemagglutination assays using trypsin-treated rabbit erythrocytes suggested that lectins were among the extracted proteins. Extracts from Lablab purpureus and Sphenostylis stenocarpa both non-Vigna spp., caused greater agglutination than those from the wild Vigna species. Bioassays on all three insect species using the lectin extracts incorporated in either artificial cowpea seeds (5% w/w) or in modified Vanderzant legume pod borer diet (1% w/v) indicated that the non-Vigna extracts were highly toxic to the insects. Mortality after 10 days was >80% in the most toxic extracts. The extract from one of the accessions of Sphenostylis stenocarpa, an edible legume, was singled out for lectin purification and future gene cloning with the view of using it for engineering resistance to cowpea pests.     

在玻璃表面上短时接触硅藻土和多杀菌素对四纹豆象的致死效应和亚致死效应（英文）

四纹豆象Callosobruchus maculatus (F.)是伊朗豇豆种子上的主要贮藏害虫。控制这一害虫时, 用生物杀虫剂比用常规杀虫剂更为合适。本研究评价了室内条件下在玻璃表面上硅藻土和多杀菌素对四纹豆象成虫的致死效应和亚致死效应。结果表明： 硅藻土处理24 h和48 h后, 对四纹豆象成虫的LC50 值分别为 1.47和0.2 g/m²; 多杀菌素处理24 h和48 h后, 对四纹豆象成虫的LC50 值分别为102.9 和68.8 mg ai/L, 说明两种化合物都对四纹豆象成虫具有较高的急性毒性。通过检测生物学参数, 研究了LC₂₀浓度的硅藻土和多杀菌素对四纹豆象的亚致死效应。LC₂₀浓度的硅藻土和多杀菌素使四纹豆象成虫的繁殖力分别比对照降低了71.5%和17.2%, 卵孵化率降低了57.5%和27.8%, 成虫寿命缩短了74.7%和17.1%。接触LC₂₀浓度的硅藻土和多杀菌素使这一害虫的蛹期分别比对照延长了4.8%和2.3%。亚致死效应研究表明, 硅藻土和多杀菌素对四纹豆象的生命参数均产生了负面影响。总之, 致死效应和亚致死效应综合显示, 硅藻土在防治四纹豆象上具有较大的潜力。     

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.     

Deterrent activity of plant lectins on cowpea weevil Callosobruchus maculatus (F.) oviposition

A set of 14 plant lectins was screened in a binary choice bioassay for inhibitory activity on cowpea weevil Callosobruchus maculatus (F.) oviposition. Coating of chickpea seeds (Cicer arietinum L.) with a 0.05% (w/v) solution of plant lectins caused a significant reduction in egg laying. Control experiments with heat inactivated lectin and BSA indicated that the observed deterrent effects are specific and require carbohydrate-binding activity. However, no clear correlation could be established between deterrent activity and sugar-binding specificity/molecular structure of the lectins. Increasing the insect density reduced the inhibitory effect of the lectins confirming that female insects are capable of adjusting their oviposition rates as a function of host availability.     

Alpha-amylase inhibitors from Ficus sp. seeds and their activities towards coleoptera insect pests

Alpha-amylase Inhibitors were isolated from Ficus sp. (Gameleira) seeds by acetone fractionation and Sephadex G-50. Two inhibitors (alpha-PPAI and alpha-ZSAI) were tested against alpha-amylases from coleopteran larvae. alpha-PPAI was active to alpha-amylases of Callosobruchus maculatus (52%) and Zabrotes subfasciatus (53%). alpha-ZSAI was strongly active to Z. subfasciatus (100%) of and Mimosestes mimosae (98%). The alpha-ZSAI is a glycoprotein of approximately 50 kDa with an IC50 value of 0.074 microg microl(-1).     

The effect of arcelin-1 on the structure of the midgut of bruchid larvae and immunolocalization of the arcelin protein

Some wild accessions of the common bean (Phaseolus vulgaris) contain a family of proteins called arcelins, that are toxic to the larvae of certain bruchid species. Among the six allelic variants of arcelin tested so far, arcelin-5 and arcelin-1 confer the highest level of resistance against the Mexican bean weevil, Zabrotes subfasciatus. The same proteins are not toxic to the bean weevil, Acanthoscelides obtectus, which is also a serious pest of cultivated beans. Arcelins belong to the bean lectin family that includes phytohemaggutinins and alpha-amylase inhibitors. Although homologous to lectins, arcelins are themselves only very weak lectins, and their binding properties have not been clearly established. The toxic properties of arcelins may be related to their recognition of and interaction with the glycoproteins and other constituents of the membranes along the digestive tract of insects. Since arcelin-1 was shown to have growth inhibitory effects for the larvae of Z. subfasciatus but not of A. obtectus, we examined the effect of an arcelin-1 containing diet on the structure of the cells that line the intestinal tract of the larvae of these two bruchid species, and used antibodies against arcelin to examine the distribution of arcelin within the cells and tissues. Here we show that dietary arcelin-1 caused an alteration of the gut structure and the penetration of arcelin into the haemolymph in Z. subfasciatus but not in A. obtectus. These results lead us to suggest that arcelins exert their toxic effect by severely damaging the epithelial cells.     

Suitability of five species of stored-product insects as hosts for development and reproduction of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

We investigated the ability of two populations of Anisopteromalus calandrae (Hymenoptera: Pteromalidae), to parasitize and develop on late instars of five different stored-product insects that typically complete their development inside seeds of grain or legume species or other dry commodity. The host species were the cigarette beetle, Lasioderma serricorne (F.); cowpea weevil, Callosobruchus maculatus (F.); rice weevil, Sitophilus oryzae (L.); lesser grain borer, Rhyzopertha dominica (F.); and Angoumois grain moth, Sitotroga cerealella (Olivier). Experiments were conducted in the laboratory in a no-choice design by using petri dishes (15 by 100 mm) as experimental arenas with 20 host larvae. A. calandrae females from populations originating in Georgia (GA) and Oklahoma (OK) were introduced singly into experimental arenas and allowed to sting and oviposit for 24 h. Parasitism by the OK population was greater than that for the GA population across all hosts. However, no or very low parasitism was found on Angoumois grain moth for either population in this experiment. The highest number of parasitoid progeny was recorded on cowpea weevil (15.9) followed by rice weevil (11.5) and cigarette beetle (10.8) for the OK population. A similar trend was observed in the GA population. The highest proportion of female progeny was produced on cowpea weevil (73.0%) by the OK population. Conversely, a higher proportion of female progeny was produced on rice weevil (64.6%) by the GA population than produced by the OK population. Parasitoid adults were significantly larger and heavier when they developed on cowpea weevil irrespective of parasitoid population. The possible application of these results for biological control of stored-product insects is discussed.     

Screening and secretomic analysis of enthomopatogenic Beauveria bassiana isolates in response to cowpea weevil (Callosobruchus maculatus) exoskeleton

The production of cowpea (Vigna unguiculata), an important self-sustained crop in Latin America and Africa, is severely affected by damage by the cowpea weevil Callosobruchus maculatus. The presence of a single larva in stored seeds can lead to losses of almost 40%. Control of C. maculatus currently relies on the inefficient use of chemical insecticides and post-harvest treatments. The use of entomopathogenic fungus became a reliable alternative for coleopteran pest control and has been extensively investigated. Among them, Beauveria bassiana and Metarhizium anisopliae were widely evaluated in order to measure their virulence toward many insects. In this report, we evaluated the insecticidal activity of ten strains of B. bassiana and the most lethal fungi strains were analyzed for proteinaceous secretions by two dimensional electrophoresis and for enzyme activities, including chitinolytic, proteolytic and alpha-amylolytic activities. This study could, in the near future, help to establish novel biotechnological tools to use for cowpea weevil control.     

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.     

Soyacystatin N inhibits proteolysis of wheat alpha-amylase inhibitor and potentiates toxicity against cowpea weevil

Genetic engineering may be used to introduce multiple insect resistance genes with different modes of action into crop plants. We explored the possible interactions of two differing gene products fed in the diet of cowpea weevil, Callosobruchus maculates (F.), a stored grain pest. The soybean cysteine protease inhibitor soyacystatin N (scN) and alpha-amylase inhibitor (alphaAI) from wheat have defensive function against this coleopteran. When artificial seeds containing both scN and alpha(AI) were infested with eggs of C. maculatus, the delays in larval development were longer than was predicted by summing the developmental delays seen when larvae were fed a diet containing the individual proteins, indicating that the effects of scN and alpha(AI) are synergistic. Alpha(AI) was readily hydrolyzed when incubated with insect gut extract. This proteolytic degradation was inhibited by scN, but not by Kunitz inhibitor (a serine protease inhibitor). Thus, degradation of alpha(AI) was due to proteolysis by insect digestive cysteine proteases. These data suggest that C. maculatus uses digestive enzymes not only to function in food protein digestion but also to defend the insects themselves by helping reduce the concentration of a toxic dietary protein.     

Mechanisms of the insecticidal action of TEL (Talisia esculenta lectin) against Callosobruchus maculatus (Coleoptera: Bruchidae)

Plant lectins have insecticidal activity that is probably mediated through their ability to bind carbohydrates. To examine the influence of sugars on the insecticidal activity of a lectin from Talisia esculenta seeds (TEL), the lectin was mixed with mannose, glucose, or mannose plus glucose. Mannose abolished the insecticidal activity. Affinity chromatography showed that TEL bound to midgut proteins of the insect Callosobruchus maculatus. Immunoblotting showed that TEL recognized some proteins, probably glycoproteins, present in the midgut membrane of this insect. The principal proteases responsible for digestive proteolysis in fourth instar larvae of C. maculatus were purified by chromatography on activated thiol-Sepharose. These purified proteases were unable to digest TEL after a 15-h incubation. These results suggest that the insecticidal activity of TEL involves a specific carbohydrate-lectin interaction with glycoconjugates on the surface of digestive tract epithelial cells, as well as binding to assimilatory glycoproteins present in midgut extracts and resistance to enzymatic digestion by cysteine proteinases.     

Presence of the storage seed protein vicilin in internal organs of larval Callosobruchus maculatus (Coleoptera: Bruchidae)

Variant vicilins (7S storage globulins) of cowpea seeds (Vigna unguiculata) are considered as the main resistance factor present in some African genotypes against the bruchid Callosobruchus maculatus. It has been suggested that the toxic properties of vicilins may be related to their recognition and interaction with glycoproteins and other membrane constituents along the digestive tract of the insect. However, the possibility of a systemic effect has not yet been investigated. The objective of this work was to study the fate of 7S storage globulins of V. unguiculata in several organs of larvae of the cowpea weevil C. maculatus. Results demonstrated binding of vicilins to brush border membrane vesicles, suggesting the existence of specific receptors. Vicilins were detected in the haemolymph, in the midgut, and in internal organs, such as fat body and malpighian tubules. There is evidence of accumulation of vicilins in the fat body of both larvae and adults. The absorption of vicilins and their presence in insect tissues parallels classical sequestration of secondary compounds.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.