Vicilin variants and the resistance of cowpea (Vigna unguiculata) seeds to the cowpea weevil (Callosobruchus maculatus)

1. A globulin fraction prepared from the meal of Callosobruchus maculatus-resistant cowpea (Vigna wiguiculata) seeds was shown to be detrimental to this bruchid when incorporated in artificial seeds.2. The performance of C. maculatus was also shown to be strongly hindered by vicilins from resistant seeds when these storage proteins were incorporated in artificial seeds at the level of 2%.3. The purified vicilins from seeds of both resistant and susceptible cowpea varieties were shown to have the same SDS-PAGE pattern but different mobilities in non-denaturing polyacrylamide gel electrophoresis.4. These results and previous ones obtained by us (Silva and Xavier-Filho, 1991; Sales et al., 1992) strongly suggest that the resistance of cowpea seeds from the cultivar TVu 2027 and from others bred from it is associated with the presence of vicilin molecules which are refractory to digestion by bruchid midgut proteinases.     

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.     

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.     

A novel cysteine proteinase inhibitor from seeds of Enterolobium contortisiliquum and its effect on Callosobruchus maculatus larvae

This study focused on the characterization of a novel cysteine proteinase inhibitor from Enterolobium contortisiliquum seeds targeting the inhibition of the growth of Callosobruchus maculatus larvae, an important cosmopolitan pest of the cowpea Vigna unguiculata during storage. The inhibitor was isolated by ion-exchange besides of size exclusion chromatography. EcCI molecular mass is 19,757 Da, composed of two polypeptide chains. It strongly inhibits papain (Ki_app 0.036 nM) and proteinases from the midguts of C. maculatus (80 μg mL^?1, 60% inhibition). The inhibitory activity is reduced by 40% after a heat treatment at 100 °C for 2 h. The protein displayed noxious activity at 0.5% and 1% (w/w) when incorporated in artificial seeds, reducing larval mass in 87% and 92%, respectively. Treatment of C. maculatus larvae with conjugated EcCI-FIT and subsequent biodistribution resulted in high fluorescence intensity in midguts and markedly low intensity in malpighian tubules and fat body. Small amounts of labeled proteins were detected in larvae feces. The detection of high fluorescence in larvae midguts and low fluorescence in their feces indicate the retention of the FITC conjugated EcCI inhibitor in larvae midguts. These results demonstrate the potential of the natural protein from E. contortisiliquum to inhibit the development of C. maculatus.     

Valeur trophique des acides aminés dans les graines de légumineuses et développement larvaire de Callosobruchus maculatus

Interspecific variation of protein amino acids between peanut, horse bean, french bean, lentil, cowpea and chick-pea was undertaken in relation to larval development of C. maculatus. Twenty-one groups of amino acids were identified and quantified. In all seeds, free amino acids exist in low quantities; but they are more numerous than bound amino acids. Essential amino acids for man are sufficient in the six species, except for the sulphur amino acids in all seeds, and for lysine in peanut. Utilization of those pulses, after cooking, in human food complements cereals. Larval development possibilities of C. maculatus upon the used seeds shows up that: (a) with chick-pea and cowpea, the bruchid thrives normally. However, although amino acids are higher in cowpea than in chick-pea, the better performances were observed on the latter; (b) peanut, french bean, horse bean and lentil are unsuitable for larval development of the insect. Although they are rich in amino acids, all larvae die at first in star within these pulses. We conclude that larval development of C. maculatus does not depend only on the amounts of amino acids in the seeds but also on other trophic factors. These could consist of the balance between different nutritive compounds and the presence of allelochemical substances in the tested seeds which bruchid larvae are unable to utilize.     

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).     

The fate of vicilins, 7S storage globulins, in larvae and adult Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)

The fate of vicilins ingested by Callosobruchus maculatus and the physiological importance of these proteins in larvae and adults were investigated. Vicilins were quantified by ELISA in the haemolymph and fat body during larval development (2nd to 4th instars), in pupae and adults, as well as in ovaries and eggs. Western blot analysis demonstrated that the majority of absorbed vicilins were degraded in the fat body. Tracing the fate of vicilins using FITC revealed that the FITC-vicilin complex was present inside cells of the fat body of the larvae and in the fat bodies of both male and female adult C. maculatus. Labelled vicilin was also detected in ovocytes and eggs. Based on the results presented here, we propose that following absorption, vicilins accumulate in the fat body, where they are partially degraded. These peptides are retained throughout the development of the insects and eventually are sequestered by the eggs. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack as these peptides are known to have antimicrobial activity. Quantifications performed on internal organs from larvae of C. maculatus exposed to extremely dry seeds demonstrated that the vicilin concentration in the haemolymph and fat body was significantly higher when compared to larvae fed on control seeds. These results suggest that absorbed vicilins may also be involved in the survival of larvae in dry environments.     

Comparative toxicity of phosphine to developmental stages of three Callosobruchus species infesting stored pulses

Bruchids pose serious threat to stored pulses, and their successful management largely depends on phosphine fumigation. For the first time, the comparative assessment of phosphine toxicity to all the developmental stages of three bruchid species in India was attempted at varied concentrations and exposure times. Based on probit estimates, the egg stages found to be least sensitive to phosphine followed by pupae and larvae, whereas adults were highly sensitive. Among the age groups of eggs, the early age groups (0–2 days old) were less sensitive than later stages (3–6 days old). The bruchid species viz., Callosobruchus maculatus (F.), C. analis (F.), and C. chinensis L. exhibited a variable degree of susceptibility irrespective of life stages and exposure periods tested. However, the collective probit estimates indicated C. maculatus being slightly less sensitive followed by C. analis and C. chinensis. Except in 0–2 day old eggs, the mortality response in 3–6 day old eggs, larvae, pupae, and adults was increased with increasing phospine concentrations and exposure period. The mortality response got even steeper at 48 and 72 h exposures as evident by decreasing LC50 and LC90 values. The information generated on phospine sensitivity in different life stages would serve as baseline data to design insecticide resistance studies in future and also to ascertain stage-specific Concentration vs. time (Ct) products for the recommendation of phosphine doses for successful management of bruchids during any of their life stages under storage conditions.     

Efficacy of artificial seeds in the delivery of bioactive compounds to the seed dwelling larvae of Callosobruchus maculatus (Coleoptera: Bruchidae)

Artificial seeds offer an important method to assay the bioactivity of natural and synthetic compounds against insect larvae that develop within the cotyledons of seeds. Here, the efficacy of artificial seeds as a mechanism to deliver bioactive compounds to larvae of the bruchid beetle, Callosobruchus maculatus, was compared to that of black-eyed beans that had been imbibed with the same bioactive compounds: malachite green or the methanolic extract of neem (Azadirachta indica). Females laid an equivalent number of eggs on control artificial seeds in comparison with black-eyed beans, although egg-to-adult survival on artificial seeds was reduced. Manipulation of the hardness of artificial seeds influenced female oviposition decisions, with more eggs laid on the harder seeds, although seed hardness had no effect on egg-to-adult survival. Incorporation of neem extract or malachite green into the artificial seeds resulted in 100 % larval mortality, while larval mortality on seeds imbibed with neem extract or malachite green was between 50 and 70 %. This suggests incorporation of toxins into artificial seeds, produces a more sensitive assay of compound toxicity in comparison with the method of imbibing seeds and offers a useful method to study of seed–arthropod interactions.     

Assessment of frozen larvae of Callosobruchus maculatus as hosts for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae)

The suitability of frozen host larvae for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae), an ectoparasitoid of Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) and other stored-product insects was investigated. The reproductive potential (number and sex ratio of progeny) of female P. cerealellae was compared on live (fresh) C. maculatus larvae (concealed within cowpea seeds) versus frozen larvae (obtained by freezing infested cowpea seeds at ?20 °C for 48 h) which were subsequently thawed and held at ambient conditions (～25 ± 1 °C, 50 ± 5% RH) for 4, 24, 48, 72, 96, and 120 h before exposure to female parasitoids. No significant differences were recorded in the numbers and sex ratios of the progeny produced by female P. cerealellae on live larvae compared to frozen host larvae that were thawed and held at ambient conditions for up to 96 h, suggesting that live and frozen larvae of C. maculatus are equally suitable for rearing P. cerealellae. However, the data showed that progeny production on frozen hosts gradually declined with thawing duration and was significantly reduced at the thawing duration of 120 h. When live and frozen host larvae were simultaneously presented together to female P. cerealellae at different exposure periods, relatively greater progeny production was recorded on live hosts than on frozen hosts at 12, 24, and 48 h of exposure. This may suggest preference of female P. cerealellae for live versus frozen host larvae. These results are discussed in relation to the life history strategy and host location behavior of P. cerealellae, and may have practical implications in the development of efficient mass rearing systems for the parasitoid.     

The Toxicity of a Lipid Transfer Protein (Cc-LTP1) from Coffea canephora Seeds on the Larval Development of Callosobruchus maculatus (Coleoptera: Bruchidae)

In this work, we analyzed the effects of coffee seed proteins, especially Cc-LTP₁ on the larval development of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), a bruchid pest of beans and the most important insect pest of Vigna unguiculata (L.) Walp. Artificial seed assay, which incorporated the F/0-90 fraction from Coffea canephora seeds, resulted in the reduction of oviposition and caused an inhibition of C. maculatus larval development in a dose-dependent manner. The F/0-90 fraction used at a 4 % concentration resulted in the survival of no larvae. The purified Cc-LTP₁, at a concentration of 0.5 %, also demonstrated effective inhibition of larval development, reducing both females oviposition and the weight and number of larvae. Cc-LTP₁ was also able to inhibit the C. maculatus gut α-amylase activity, and immunolabeling by an anti-LTP serum was observed in the midgut tissues of the C. maculatus larvae. Cc-LTP₁ has shown binding affinity towards microvillar cells, endoplasmic reticulum and mitochondria, as demonstrated by micrographic images taken by a transmission electron microscope. The results from this study indicate that Cc-LTP₁ has insecticidal actions toward C. maculatus and exerts anti-nutritional effects with direct actions on intestinal tissues.     

Impact of αAI-1 Expressed in Genetically Modified Cowpea on Zabrotes subfasciatus (Coleoptera: Chrysomelidae) and Its Parasitoid,Dinarmus basalis (Hymenoptera: Pteromalidae)

Genetically modified (GM) cowpea seeds expressing αAI-1, an α-amylase inhibitor from the common bean, have been shown to be immune against several bruchid species. Effective control of such pests by growing GM cowpea could promote the spread of bruchid species that are αAI-1 tolerant. Consequently, the sustainability of bruchid pest control could be increased by combining GM seeds and hymenopteran parasitoids. However, there are concerns that αAI-1 could interfere with the biological control provided by parasitoids. Here, we assessed the impact of GM cowpea seeds expressing αAI-1 on the αAI-1-tolerant bruchid Zabrotes subfasciatus and its parasitoid Dinarmus basalis. αAI-1 in cowpea seeds did not increase resistance to Z. subfasciatus or affect the mortality rate of Z. subfasciatus larvae. Parasitism of Z. subfasciatus by D. basalis and fitness of D. basalis offspring were not affected by the presence of αAI-1. Thus, αAI-1-expressing cowpeas and parasitoids should be compatible for the control of bruchid pests.     

Response of cowpea bruchid,Callosobruchus maculatus (Fab.) (Coleoptera: Chrysomelidae) to cheese wood,Alstonia boonei De Wild stem bark oil extracted with different solvents

Cheese wood, Alstonia boonei De Wild stem bark was extracted with five different solvents namely: methanol, ethanol, acetone, petroleum ether and n-hexane. The extracts were tested for insecticidal activity on cowpea bruchid, Callosobruchus maculatus (Fab.). Adult mortality and adult emergence of the insects were investigated. Oviposition deterrences using dual-choice and multiple-choice tests were also investigated as well as percentage damage and weight loss at temperature of 28?±?2?°C and 75?±?5% relative humidity. Results showed that at the rate of 2% extract per 20?g of cowpea seeds, methanol, petroleum ether and n-hexane extracts caused 100% mortality of adult cowpea bruchid after four?days of post treatment. When the bruchid had choice of oviposition substrate, they laid significantly (p?<?0.05) fewer eggs on cowpea seeds treated with extracts compared to untreated seeds. There was no adult emergence in seeds treated with methanol, ethanol, petroleum ether and n-hexane extracts and 100% reduction in F₁ progeny was recorded compared with untreated that had 81.86% adults emergence and 0% reduction in F₁ progeny. The results obtained from this study revealed that methanol, ethanol, acetone, petroleum ether and n-hexane extracts of A. boonei stem bark were effective in controlling C. maculatus and could serve as an alternative to synthetic insecticides for the protection of stored cowpeas against bruchids.     

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.     

Lupinus luteus,Vicia sativa and Lathyrus cicera as protein sources for piglets: ileal and total tract apparent digestibility of amino acids and antigenic effects

Twenty-four male piglets, weaned at 28 days of age, were used to measure the total and ileal digestibility and serum immune responses to dietary leguminous seeds. The experimental diets consisted of a control starter (C) and three other diets prepared by replacing 30% of the crude protein content of the C diet by the protein of Lupinus luteus (LL), Vicia sativa (VS) or Lathyrus cicera (LC). The total tract apparent digestibility (TTAD) of energy and crude protein (CP) was lowest (P<0.001) for the LC diet. Similarly TTAD of lysine, methionine and threonine were also lowest for the LC diet. The apparent ileal digestibility (AID) of lysine, methionine and threonine were lowest for the LC diet. AID of lysine and methionine was highest for the LL seeds (0.764 and 0.834, respectively) and lowest (P<0.05) for the LC seeds (only 0.454 and 0.420, respectively). Immunoblots of individual pig serum were made to detect both residual antigenic storage proteins of the seeds of each legume used and IgGs specific to those storage proteins. Antibodies against β-conglutin of L. luteus, vicilin of V. sativa and vicilin of L. cicera were detected 28 days after feeding the diet in the sera of piglets fed on the LL, VS or LC diets, respectively. Conversely, no storage protein was found in the serum of any piglet fed either on LL, VS or LC diets. The presence of antibodies against β-conglutin of lupine, vicilin of V. sativa and vicilin of L. cicera, respectively indicated an immune response in weaned piglets. The absence of residual antigenic proteins may be due to the digestive adaptation of the piglet to the legume-based diets. However, no direct relation between the differences in digestibility coefficients among the legume seeds and their antigenicity was established.     

Rotenoids of Lonchocarpus salvadorensis: Their effectiveness in protecting seeds against bruchid predation

Seeds of the legume Lonchocarpus salvadorensis, having unusually low bruchid predation, contain deguelin (0.29%) rotenone (0.22%), elliptone (0.06%) and α-toxicarol (0.003%). The relation between rotenone content and toxicity towards the generalist bruchid Callosobruchus maculatus is examined and quantified.     

Binding of Vigna unguiculata vicilins to the peritrophic membrane of Tenebrio molitor affects larval development

We investigated the effects of vicilins (7S storage proteins) from Vigna unguiculata (L.) Walp. (Fabaceae), cultivars EPACE‐10 [genotype susceptible to the cowpea weevil, Callosobruchus maculatus (Fabricius)] and IT81D‐1045 [cowpea weevil‐resistant genotype], seeds on Tenebrio molitor L. (Coleoptera: Tenebrionidae) larval development. Toxicity of vicilins was investigated through the incorporation of these proteins in artificial diet offered to the larvae. Binding tests of vicilins to the peritrophic membranes (PM) were carried out by in vitro incubation of PM with solutions of vicilins. Bound proteins were desorbed from PM with 100 mm HCl. Desorbed vicilins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoprobing on Western blotting using an anti‐vicilin cv. EPACE‐10 antibody. The chitin content of the T. molitor PM was evaluated by the Von Wisselingh color test and presence of chitin in the larval PM was confirmed. Bioassays showed that both vicilins from EPACE‐10 and IT81D‐1045 genotypes were toxic to T. molitor larvae, and in vitro binding assays showed that these seed‐storage proteins were capable of binding to the larval PM.     

Parasitism by Pteromalus cerealellae (Hymenoptera: Pteromalidae) on the Cowpea weevil, Callosbruchus maculatus (Coleoptera: Bruchidae): Host density, temperature effects, and host finding ability

Studies on interactions between a larval parasitoid, Pteromalus cerealellae (Boucek) and one of its hosts, Callosobruchus maculatus (F.) were carried out in the laboratory. The number of host larvae parasitized by P. cerealellae increased with host larvae at low densities and tended to a plateau at a density of 25 larvae per female parasitoid. Each parasitoid was able to parasitize more hosts and produced more offspring at 20 and 25 °C than at 30 °C. The number of non-infested seeds mixed with seeds infested with the last instar of C. maculatus did not preclude P. cerealellae from identifying infested seeds and attacking larvae inside them. When infested seeds were tightly packed, several host larvae escaped parasitism. P. cerealellae may be a useful biological control agent in newly harvested cowpea with low C. maculatus infestation, and lowering the temperature of the storage system may enhance the effectiveness of this parasitoid.     

Comparison of life history and genetic properties of cowpea bruchid strains and their response to hypoxia

The cowpea bruchid (Callosobruchus maculatus) is the most important storage pest of grain legumes and comprises geographically distinct strains. Storage under a modified atmosphere with decreased O₂ content represents an alternative to chemical fumigants for pest control of stored grains. In this study, we compared reproduction, development and survival, as well as genome size of bruchid strains from South India (SI), Burkina Faso (BF), Niger (CmNnC) and the United States (OH), reared on mung bean (Vigna radiata). Fecundity and egg-to-adult duration varied significantly among these strains. Notably, strain BF had the highest fecundity, and strain SI displayed the fastest development whereas strain OH was the slowest. Differences in adult lifespan among strains were only detected in unmated but not in the mated group. Genome size of SI females was significantly larger than that of OH females, and for all four strains, the female genomes were larger than those of their corresponding males. Furthermore, we studied effects of exposure to 1% O₂ + 99% N₂ on strains SI and BF. Mortality caused by hypoxia was influenced by not only developmental stage but also by insect strain. Eggs were most sensitive, particularly at the early stage, whereas the 3rd and 4th instar larvae were most tolerant and could survive up to 15 days of low O₂. Strain SI was slightly more resistant than BF in egg and larval stages. Proteolytic activity prior to, during and after hypoxia treatment revealed remarkable metabolic plasticity of cowpea bruchids in response to modified atmosphere.     

Temporal/spatial structure and the dynamical property of laboratory host-parasitoid systems

The effects of spatial structure in terms of local capacity, or the maximum number of larvae surviving competition at resource patches, and temporal structure in terms of the period vulnerable to parasitoid attack in host populations on the persistence of host-parasitoid systems were quantitatively evaluated by laboratory experiments and well-parameterized model analyses. One of two bruchid beetles,Callosobruchus maculatus andC. phaseoli, were used as a host with Heterospilus prosopidis used as the parasitoid.C. maculatus, in which few larvae survive competition to become adults in each bean, andC. phaseoli, in which many larvae become adults in each bean, along with two kinds of beans, the mung and the azuki, were combined to construct four (2×2) resource-herbivorous host-parasitoid systems that differed in local capacity and vulnerable period. The mung-C. maculatus system with the parasitoid was the most persistent, i.e., took the longest time for extinction of either the host or parasitoid to occur. Since this resource-herbivorous host combination exhibited the lowest local capacity and the shortest vulnerable period, these two conditions possibly promoted the persistence of the system. A model incorporating the host population structure supported the observed persistence. Furthermore, the possible contribution of the timing of density-dependent competition of the host on the host-parasitoid persistence is predicted.