Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha-casein, and N-benzoyl-DL-arginine napthylamide-(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p-hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman-Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L-Trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.     

Intra-specific variation in female remating in Callosobruchus chinensis and C. maculatus

The effects of mating duration on female remating (exp. 1) and under different male densities (exp. 2) were examined in two strains of the adzuki bean beetle, Callosobruchus chinensis and in one strain of the bruchid beetle, C. maculatus. In experiment 1, the frequency of female remating was markedly different between the two strains of C. chinensis. Females of the jC strain, reared long-term in the laboratory, did not remate after being allowed to mate freely (=monogamy), whereas females of the isC strain, recently established from the field, showed high remating frequencies (=polyandry). In both strains, the frequency of female remating increased after the duration of the first mating was deliberately shortened. The relation between mating duration and remating frequency was significantly different, however, between the two strains. In a closely related species, C. maculatus, which manifests polyandry, this relation was more similar to that of the field-derived (=isC) than to that of the laboratory-derived (=jC) strain of C. chinensis. The reasons for the inter-strain variation observed in the remating frequencies of C. chinensis are also discussed. In experiment 2, the mating duration of the three strains was compared under different male densities. Only the lab-derived strain demonstrated a significantly shorter mating duration when one female was placed together with five males than when paired with one male. The shorter mating duration (approximately 26 s) was similar to that of females allowed to remate in the monogamous strain in experiment 1.     

Resistance to the cowpea weevil (Callosobruchus maculatus) larva in pods of cowpea (Vigna unguiculata)

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7% to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%.Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.     

Vibrations mediate prudent resource exploitation by competing larvae of the bruchid bean weevil Callosobruchus maculatus

Larvae of the bean weevil C. maculatus feeding in a single bean apparently respond to vibrations from each other's chewing: one larva feeds normally, the other is inhibited. If the burrows of the larvae intersect, the inhibited larva dies. If the dominant larva pupates or dies without the burrows intersecting, the inhibited larvae then feeds and matures if enough food remains. Since females add second eggs to the largest beans after most beans carry a single egg, competition is most common in the largest available beans, precisely where inhibited larvae can benefit from avoiding a contest.

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Résumé Lors de la compétition, les larves réagissent l'une par rapport à l'autre, même lorsque leurs galeries sont séparées. L'une est inhibée, tandis que l'autre se comporte comme si elle était seule. Le signal provoquant ces réactions, qui peut traverser du papier métallique, est dû vraisemblablement aux vibrations de mastication des larves; ces dernières perçoivent ces signaux tout au long de leur vie active. La larve la plus âgée ne l'emporte pas obligatoirement, ce qui signifie que le signal indique plus que l'âge.Les conditions expérimentales peuvent inverser les réponses des unes et des autres, bien que la larve la plus âgée puisse souvent mourir de ces conditions anormales de compétition. L'avantage potentiel associé à l'inhibition du futur perdant est mis en évidence en collant ensemble des graines. Quand la larve dominante se nymphose, la larve inhibée recommence à s'alimenter et achève son développement. En ayant eu son alimentation interrompue et en restant dans une galerie superficielle, la larve inhibée évite les affrontements et peut se développer lorsque la larve dominante meurt ou se nymphose, puisque la galerie de la larve inhibée n'avait pas été sectionnée.La stratégie de ponte réduit la fréquence de la compétition en uniformisant la distribution des oeufs; elle augmente les chances de survie des larves vaincues avec l'addition des oeufs sur les graines les plus grosses quand les disponibilités sont limitées. L'étude de l'évolution de ces adaptations a été possible puisque, comme on le sait, C. maculatus est génétiquement polymorphe pour les caractères déterminant le comportement de ponte des femelles et la compétition larvaire.

     

Impact of the egg parasitoid Uscana lariophaga and the larval-pupal parasitoid Dinarmus basalis on Callosobruchus maculatus populations and cowpea losses

In West Africa, Uscana lariophaga Steffan (Hymenoptera: Trichogrammatidae) and Dinarmus basalis (Rondani) (Hymenoptera: Pteromalidae) parasitize the eggs and larvae, respectively, of Callosobruchus maculatus Fab. (Coleoptera: Bruchidae), an important pest of stored cowpea. The impact of the parasitoids on pest populations was evaluated in clay pots similar to the ones used in traditional storage in Niger. At the beginning of the storage period, cowpeas were infested with one density of C. maculatus and with either one or both of the parasitoid species. If the parasitoids we inoculated as single species in stores, both D. basalis and U. lariophaga significantly suppressed the bruchid population, but the former did so more effectively than the latter. A combination of D. basalis and U. lariophaga resulted in the same suppression of bruchid populations as when D. basalis was the only parasitoid. Fifteen weeks after storage, the parasitoids reduced the number of grains damaged significantly by 38–56%. The effect of inoculating single or multiple parasitoid species on C. maculatus populations in an augmentation strategy is discussed.     

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.     

Intraspecific variation in the egg-spacing behavior of the seed beetleCallosobruchus maculatus

Females of Callosobruchus maculatustend to distribute their eggs uniformly among host seeds and, thereby, reduce competition among larvae within seeds. We investigated variability in this behavior by assaying beetles from seven geographic strains on each of three host species. To quantify egg-spacing behavior,we devised an index (U)that estimates the uniformity of the egg distribution but is independent of the number of eggs laid. Egg-spacing behavior was highly variable among beetle strains and legume hosts. Females from an Indian strain frequently achieved the most uniform distribution possible (maximum hyperdispersion), whereas females from two Brazilian strains frequently deposited eggs randomly. Differences among strains were evident throughout the period of oviposition;some strains were sloppy when even egg densities were low. Variation in egg-spacing behavior appears to be influenced by prior host associations and by host size. The most well-developed spacing behavior was observed in a strain associated with the smallest host, which supports only one or two larvae per seed. Realized fecundity also varied significantly among beetle strains. We found no evidence, however, of a predicted tradeoff between the number of eggs laid and the uniformity of the egg dispersion.     

Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Influence of cowpea pod maturity on the oviposition choices and larval survival of a bruchid beetle Callosobruchus maculatus

The active or dispersing morph of Callosobruchus maculatus, a pest of stored cowpeas, is presumed to infest cowpea plants in the field prior to harvest. In oviposition choice tests, both normal and active females preferred pods that were full-size but still green over younger, smaller pods or older, mature pods. Exposed seeds were usually preferred over intact pods of all stages, although females laid more eggs on green pods than on the rough-coated seeds of one cowpea variety. Egg-to-adult survivorship was highest for eggs laid directly on seeds. On both green and mature pods high mortality occurred because newly hatched larvae failed to penetrate a seed after they drilled through the pod wall. However, since 20–50% of eggs laid on green pods do develop into adults, cowpea plants are susceptible to C. maculatus well before pods dehisce to expose the seeds. The oviposition preferences of C. maculatus may be strongly influenced by the surface texture of potential oviposition sites.

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Résumé La forme active de Callosobruchus maculatus, ennemi de Vigna unguiculata stocké, est suspectée d'infester les gousses dans les champs avant la récolte. Lors d'essais sur le choix du lieu de ponte, toutes les femelles, normales et actives, ont préféré les gousses encore vertes mais ayant atteint leur taille définitive aux gousses plus jeunes ou aux gousses plus âgées (jaunes) ou mûres. Les graines étaient presque toujours préférées aux gousses à tous les stades, mais quand les graines étaient rugueuses, elles pondaient de préférence sur les gousses vertes.Le pourcentage le plus élevé d'adultes a été obtenu à partir des oeufs pondus sur les graines. Avec les pontes sur gousses vertes ou mûres, la mortalité élevée se produisait lors de la pénétration dans la gousse. Néanmoins, comme 20 à 50% des oeufs pondus sur des gousses vertes donnent des adultes, V. unguiculata est susceptible d'être attaqué par C. maculatus bien avant la déhiscence des gousses et l'exposition des graines. L'évolution temporelle des propriétés physiques des gousses paraît à l'origine de la relation complexe entre l'âge et l'attractivité des gousses.

     

Effect of experience on host preference inCallosobruchus maculatus (Coleoptera: Bruchidae): Variability among populations

Experiments using naive females established differences in host specificity among geographic strains of the seed beetleCallosobruchus maculatus; some females strongly preferred azuki bean over mung bean, and others failed to discriminate between the two hosts. We examined whether such congenital differences affect the degree to which host preference can be modified by experience. In choice tests, previous exposure to azuki bean increased the proportion of eggs laid on that host, but only in strains with a relatively low host specificity. Under more realistic, no-choice conditions, egg-laying experience affected oviposition rates mostly in strains with a high host specificity, but these experiments could not distinguish between the effects of a female's experience per se and her physiological state (i.e., egg load). Our results indicate that the likelihood of detecting an effect of experience on host choice depends on both the experimental protocol and the source of the test population. In natural populations ofC. maculatus, recent egg-laying experience probably plays little or no role in discrimination between host species but may influence discrimination between conspecific seeds that differ in quality.     

Influence du tégument des graines mûres de Vicia faba sur le développement larvaire de Callosobruchus maculatus

Résumé Des élevages expérimentaux deCallosobruchus maculatus (F.) (Coleoptera, Bruchidae) sur les graines deVicia faba décortiquées ou non, ou sur des pastilles faites des mêmes graines, ont permis de mettre en évidence une barrière empêchant le développement de la bruche.Avec les graines entières, toutes les larves meurent au premier stade enfoncées dans le tégument; alors qu'elles se développent normalement dans les graines décortiquées.Avec des pastilles de broyats de graines non décortiquées, la majorité des larves meurt avant le terme de leur développement; la plus forte mortalité est observée au quatrième stade larvaire. Les larves, qui survivent, accusent une augmentation de leur durée de développement par rapport à celles qui se nourrissent aux dépens des pastilles faites de graines sans tégument.     

Female mating receptivity after injection of male-derived extracts in Callosobruchus maculatus

The effects of male-derived extracts on female receptivity were investigated in Callosobruchus maculatus (Coleoptera: Bruchidae). Injection of aqueous extracts of the male reproductive tract into the abdomen of females reduced receptivity. Aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: Fractions: (I) MW<3 kDa, (II) 3-14 kDa, and (III)>14 kDa. Fraction II reduced female receptivity from 3h after injection, and Fraction III reduced female receptivity from 2 days after injection. On the other hand, no effect on receptivity was found for Fraction I. Furthermore, male reproductive tract organs were divided into accessory gland, testis, and seminal vesicle including the ejaculatory duct. Aqueous extracts of the seminal vesicle reduced receptivity of females immediately following injection, while aqueous extracts of the accessory gland reduced receptivity at the second day. The results suggest that the components of Fraction II existed in the seminal vesicle, and those of Fraction III in the accessory gland. The results of the present and the previous studies in Callosobruchus chinensis, a species closely related to C. maculatus, were compared and are discussed from the viewpoint of the significance of ejaculation in the two species.     

Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: relationship with trypsin inhibitor and vicilin excretion

Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.     

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.     

Relationships between larval density, adult size and egg production in the cowpea seed beetle, Callosobruchus maculatus

ABSTRACT.

• 1 The eggs of the cowpea seed bettle Callosobruchus maculatus (Fab.) are attached to cowpeas. On hatching, the larvae penetrate the testa and remain in the seed until development is complete and adult eclosion has occurred. The adults do not need to feed and were not allowed to do so.
• 2 Strains from Brazil and Nigeria can produce more than twelve adults from a seed bearing numerous eggs, whereas a strain from the Yemen Republic rarely produced more than three.
• 3 In all three strains the mean weight of the adults produced from a single cowpea declined with increasing initial larval density in the seed.
• 4 Egg production by females is positively correlated with their weight at the time of mating, shortly after emergence.
• 5 Lifetime egg production by females of the Brazilian and Nigerian strains was lower if they came from cowpeas with higher initial larval densities. No such relationship could be demonstrated in the Yemen strain.
• 6 The fecundity of one generation of these beetles, at least in some geographical strains, is significantly affected by the number of larvae entering the hosts in which the adults of that generation have developed.

     

Influence des tanins condensés du tégument de fèves (Vicia faba) sur le développement larvaire de Callosobruchus maculatus

Des tanins condensés sont extraits et isolés à partir du tégument de Vicia faba var. Géante Portugaise. Quatre polymères sont quantifiés et identifiés à quatre fractions: A, B, C et D. Ces fractions sont incorporées, en mélange ou séparées à différentes concentrations, dans un milieu nutritif favorable au développement de Callosobruchus maculatus (F.) (Coleoptera, Bruchidae).Les composés phénoliques provoquent la mortalité d'une partie de la population et retardent le développement des survivants. La mortalité observée varie suivant les fractions, leurs doses et le stade de développement de la bruche.La barrière constituée par le tégument des fèves est bien de nature chimique et concerne les tanins condensés.Dans un système de production agricole, l'utilisation des cultivars de fèves riches en tanins faiblement condensés pourrait permettre de réduire les taux d'infestation des graines de V. faba par C. maculatus.     

Synergistic effects of geographical strain,temperature and larval food on insecticide tolerance in Callosobruchus maculatus (F.)

This study investigated the multifactorial interaction of various environmental factors including geographical strain (Brazil, Cameroon and Yemen strains), temperature, dose and larval food (cowpea and mungbean) on the response of Callosobruchus maculatus adult to insecticide. All the main factors, their two‐way interactions and the four‐way interaction had significant effects on C. maculatus response to malathion (an organophosphate insecticide). However, the three‐way interactions were not statistically significant (except strain × food × dose, P = 0.002). The Brazil strain was the most responsive to temperature irrespective of the larval food type. The impact of food type differs from one strain to the other; for instance, the food that imparts higher tolerance in a strain might reduce the tolerance in another. Likewise, the hierarchy of tolerance among the cowpea‐reared strains (Brazil > Cameroon > Yemen) was totally different from that of the mungbean‐reared strains (Cameroon > Yemen > Brazil). The reasons for these differences were discussed in the light of their impact on C. maculatus management. Both the management of C. maculatus and development of resistance could be complex; hence, the states of a variety of environmental factors need to be considered. This is necessary to maximize management success of this bruchid, especially in tropical/subtropical developing countries.     

Phenotypic plasticity in a complex world: interactive effects of food and temperature on fitness components of a seed beetle

Most studies of phenotypic plasticity investigate the effects of an individual environmental factor on organism phenotypes. However, organisms exist in an ecologically complex world where multiple environmental factors can interact to affect growth, development and life histories. Here, using a multifactorial experimental design, we examine the separate and interactive effects of two environmental factors, rearing host species (Vigna radiata, Vigna angularis and Vigna unguiculata) and temperature (20, 25, 30 and 35°C), on growth and life history traits in two populations [Burkina Faso (BF) and South India (SI)] of the seed beetle, Callosobruchus maculatus. The two study populations of beetles responded differently to both rearing host and temperature. We also found a significant interaction between rearing host and temperature for body size, growth rate and female lifetime fecundity but not larval development time or larval survivorship. The interaction was most apparent for growth rate; the variance in growth rate among hosts increased with increasing temperature. However, the details of host differences differed between our two study populations; the degree to which V. unguiculata was a better host than V. angularis or V. radiata increased at higher temperatures for BF beetles, whereas the degree to which V. unguiculata was the worst host increased at higher temperatures for SI beetles. We also found that the heritabilities of body mass, growth rate and fecundity were similar among rearing hosts and temperatures, and that the cross-temperature genetic correlation was not affected by rearing host, suggesting that genetic architecture is generally stable across rearing conditions. The most important finding of our study is that multiple environmental factors can interact to affect organism growth, but the degree of interaction, and thus the degree of complexity of phenotypic plasticity, varies among traits and between populations.