Presence and partial characterisation of a major proteolytic enzyme in the larval gut of Callosobruchus maculatus

Protease activity in the gut of larvae of the bruchid beetle, Callosobruchus maculatus (a storage pest of cowpea seeds), has been investigated to help clarify nutritional mechanisms in view of reports that these insects carry out little or no proteolysis (Applebaum, 1964). Larval gut homogenates showed protease activity against a variety of different protein substrates, but did not hydrolyse a synthetic trypsin substrate. The proteolytic activity had a pH optimum of 5.4. It was not inhibited by serine protease inhibitors, but was inhibited by reagents reactive against-SH groups. Protein trypsin inhibitors from legume seeds which are not hosts to C. maculatus (soybean, limabean) were not effective inhibitors of the larval proteolytic activity but a cowpea protease inhibitor preparation and aprotinin partially inhibited proteolysis. The latter two inhibitors also inhibited the plant thiol protease papain. It is suggested that C. maculatus has replaced the normal insect proteases with an enzyme similar to plant proteases to evade the antimetabolic effects of trypsin/chymotrypsin inhibitors in seeds. Besides trypsin/chymotrypsin inhibitors, cowpea seeds also contain proteins which inhibit papain; these inhibitors were purified and were shown to be effective inhibitors of C. maculatus larval protease.

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Mise en évidence et caractérisation partielle d'une enzyme protéolytique importante du tube digestif des larves de Callosobruchus maculatus

Résumé Callosobruchus maculatus attaque les graines stockées particulièrement de Vigna unguiculata. L'activité protéolytique du tube digestif des larves a été examinée. Aucune hydrolyse n'a été observée contre la N-benzoyl arginine p-nitroanilide (substrat de la trypsine synthétique), mais la protéolyse a été mise en évidence en utilisant la viciline de V. unguiculata comme substrat naturel ou la myoglobine comme substrat artificiel, le p.H optimum est de 5,4. Les inhibiteurs chimiques des protéases de la sérine n'ont pas altéré l'activité enzymatique, mais les réactifs du groupe SH l'ont inhibée. Nous suggérons que cette protéase est une thiol protéase, c'est à dire avec un groupe actif semblable à celui de la papaïne, protéase végétale. En accord avec cette hypothèse, les inhibiteurs de protéine efficaces seulement contre la trypsine, c'est à dire les inhibiteurs de la trypsine de Glycine max et de la trypsine de Phaseolus lunatus n'affectent pas cette protéase larvaire, tandis que les protéines avec une faible action inhibitrice contre la papaïne (préparation inhibitrice de la protéase de V. unguiculata, aprotinine) inhibent partiellement la protéolyse par des extraits de tube digestif larvaire.Des inhibiteurs protéiques de papaïne extraits de graines de V. unguiculata sont des inhibiteurs très efficaces de la protéase larvaire. On peut penser que C. maculatus contient une protéase semblable aux protéases végétales, plutôt qu'aux protéases classiques, d'insectes, ce qui permet d'éviter les effets antimétaboliques directs des inhibiteurs de protéase (inhibiteurs de trypsine et de chymotrypsine) trouvés en quantité relativement importantes dans les graines de légumineuses.

     

Response of Callosobruchus maculatus (F.) to varying temperature and relative humidity under laboratory conditions

The effect of temperature and relative humidity were determined on the development of Callosobruchus maculatus (F.) on stored bean (Vigna unguiculata) seeds exposed to five temperatures (20, 25, 30, 35 and 40 °C) and six relative humidity levels (40, 50, 60, 70, 80 and 90%). Oviposition and total adult progeny responded in a curvilinear pattern to temperature and relative humidity while developmental period presented a linear response. Egg laying (117.33 ± 3.21) and adult emergence (35.00 ± 1.70) were least at 20 °C and 90% R.H. but those showed the optimal values at 30 °C and 70% R.H. The implication of these findings is that the growth attributes of C. maculatus relate with the weather variables studied in a curvilinear manner and thus quadratic equations generated could be used for the prediction of optimum temperature and relative humidity in a given area for the management of C. maculatus in stored beans.     

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.     

Fumigant toxicity of essential oil from Tanacetum balsamita L. (Compositae) against adults and eggs of Callosobruchus maculatus F. (Coleoptera: Bruchidae)

The essential oil of Tanacetum balsamita L. was tested against Callosobruchus maculatus F. at the rates of 5.12, 7.23, 10.19, 14.36 and 20.24?μl/l?air for adults and 5.3, 8.5, 10.8, 13.7 and 17.4?μl/l?air for eggs. All experiments were conducted at 27?±?1?°C and 65?±?5% relative humidity. Mortality was counted after 24, 48 and 72?h of exposure and all adults (dead and alive) were taken out of the vials and the vials were left at the same conditions for further 35?days to assess progeny production. For mortality of eggs, unhatched eggs were counted after 7?days. The adults treated with oil were more susceptible than eggs. At highest dose (20.24?μl/l?air) mortality of adults after 72?h, received 75.2%, but this amount for egg at 17.4?μl/l?air after 72?h was 49.06%. The lowest LC₅₀ value on 72?h was observed in adults (1.96?μl/l?air). Also, the lowest LC₉₅ value on 72?h was observed in adults (42.86?μl/l?air). The percentage of reduced progeny on 72?h was 50.92% at 20.24?μl/l?air. Our results suggest that T. balsamita can be used as an alternative to synthetic insecticides but higher dose may be needed.     

Physicochemical properties of thiol proteinase inhibitor isolated from goat pancreas

Thiol proteinase inhibitors are crucial to proper functioning of all living tissues consequent to their cathepsin regulatory and myriad important biologic properties. Equilibrium denaturation of dimeric goat pancreas thiol proteinase inhibitor (PTPI), a cystatin superfamily variant has been studied by monitoring changes in the protein's spectroscopic and functional characteristics. Denaturation of PTPI in guanidine hydrochloride and urea resulted in altered intrinsic fluorescence emission spectrum, diminished negative circular dichroism, and loss of its papain inhibitory potential. Native like spectroscopic properties and inhibitory activity are only partially restored when denaturant is diluted from guanidine hydrochloride unfolded samples demonstrating that process is partially reversible. Coincidence of transition curves and dependence of transition midpoint (3.2M) on protein concentration in guanidine hydrochloride‐induced denaturation are consistent with a two‐state model involving a native like dimer and denatured monomer. On the contrary, urea‐induced unfolding of PTPI is a multiphasic process with indiscernible intermediates. The studies demonstrate that functional conformation and stability are governed by both ionic and hydrophobic interactions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 708–717, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Insecticidal activity of Poppy (Papaver somniferum L.) seed oil against cowpea weevil (Callosobruchus maculatus F.) in stored cowpea

In this study, the insecticidal effect of Poppy (Papaver somniferum) seed oil was investigated against Callosobruchus maculatus (Bruchidae) in cowpea, at different concentrations and exposure time. The Poppy seed oil at 2, 4, 6, 8 and 10?ml/kg was tested against adults of C. maculatus, and the mortality was counted after 24, 48 and 72?h of exposure. All tests were conducted at 27–30?°C and 65?±?5% r.h. The experiments were carried out based on the factorial experiment by randomized complete design with four replications. Twenty-five insects with 0–24?h old were used for each replication. The effect of Poppy seed oil on the reduction of emergence of insects in next generation (F₁) was also assayed. The results of experiments indicate the significant differences between concentrations and exposure time (p?<?0.0001), and the increase of concentrations and exposure time increased mortality. The amount of mortality at high concentrations was remarkable, and highest mortality rate (96.91%) was recorded at 10?ml/kg, after 72?h of exposure. The application of oil significantly reduced F₁ progeny production and even at lowest concentration (2?ml/kg), the amount of F₁ production was reduced more than 70%, compared with control treatment. Complete (100%) reduction in progeny production was recorded at the rates of 8 and 10?ml/kg. The results of seed germination assay showed no significant differences between control and treated seed, and no harmful effect was observed on the seed germination. These results proved that Poppy seed oil can be used as a controlling agent of storage pests, especially C. maculatus, although more detailed studies are necessary.     

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.     

Effects of search experience in a resource-heterogeneous environment on the oviposition decisions of the seed beetle, Callosobruchus maculatus (F.)

Abstract.  1. This study investigates how female seed beetles, Callosobruchus maculatus , distribute their eggs on various-sized seeds when the size of seed was varied during the egg-laying period.
2. Beetles were allowed to lay eggs on one of three arrays of 64 adzuki beans ( Vigna angularis ). Each array contained four size classes of seed, ranging from small (5.0–5.5 mm diameter) to large (6.5–7.0 mm), but differed in how they were distributed within the environment. In the most heterogeneous condition (the 64-patch design), the four sizes were interspersed, while in the least heterogeneous condition (the four-patch design) they were grouped into four separate blocks. Thus, a beetle exploring the 64-patch design would frequently encounter all four seed sizes, whereas a beetle exploring the four-patch design would only rarely encounter a change in bean size.
3. Beetles experiencing greater seed size heterogeneity were more likely to lay eggs on larger seeds, whereas those in the blocked condition were more likely to oviposit on small seeds. Beetle responses to seed size heterogeneity suggest that the degree of preference for large seeds depends on a female's recent experience.
4. Female beetles exhibited size discrimination throughout their egg-laying process; however, there was a trade-off between seed size and egg discrimination (i.e. avoiding those seeds already containing developing eggs) in response to the change in fitness gained from either laying on larger seeds or lower egg-load seeds during the egg-laying process.
5. Our model provides the first evidence that evolving seed size discrimination ability is adaptive for the seed beetle with egg-discrimination ability.     

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。     

四纹豆象发育起点温度及有效积温

在 2 1～ 36℃范围内设置 6个温度对四纹豆象卵、幼虫、蛹的发育速率进行了测定 ,结果表明 :卵在 2 1～ 36℃、幼虫在 2 1～ 30℃和蛹在 2 1～ 33℃之间的发育速率与温度均呈直线关系 ;用直线回归法所得卵、幼虫、蛹的发育起点温度分别为 15.82 ,15.18,16 .0℃ ,有效积温分别为 6 2 .54,151.0 6 ,10 2 .76日度     

The function of multiple mating in oviposition and egg maturation in the seed beetle Callosobruchus maculatus

Abstract.  To comprehend the significance of multiple mating in female Callosobruchus maculatus , the material-benefits hypothesis is tested, as well as the potential function and mechanism of additional mating for females. The results show that longevity and lifetime fecundity are significantly higher in doubly-mated females, whereas females subject to an interrupted second copulation (which transfers no ejaculate) show resemblance with singly-mated females in lifetime fecundity and daily fecundity, supporting the material-benefits hypothesis. Female lifetime fecundity increases with ejaculate size in doubly-, but not singly-mated females. Doubly-mated females exhibit an immediate increase in fecundity on the day of remating. Moreover, dissection of ovaries after remating shows that the number of unlaid eggs is lower in doubly-mated females in comparison with singly-mated females, whereas the total number of unlaid and laid eggs shows no significant difference. This suggests that the second mating increases the oviposition rate, but not the egg maturation rate in female C. maculatus . Thus, ejaculate serves as a stimulus for oviposition, at least on the remating day. However, because doubly-mated females lay more eggs in total than singly-mated females, doubly-mated females appear to mature more eggs than the singly-mated counterparts. This is attributed to the 'indirect-driven' hypothesis, which states that the reduction in the number of mature eggs in the oviducts brought about by oviposition stimulates the maturation of oocytes in ovarioles to replenish the number of mature eggs.     

Partial purification and characterization of an alkaline proteinase from the rabbit testis

A proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α?N?benzoyl?DL?arginine β?naphthylamide (Bz-Arg-NNap), α-N-benzoyl-L-arginine p-nitroanilde (Bz-Arg-NPhNO₂), and α-N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS-polyacrylamide gel electrophoresis corresponded to a M_t 48,000. The same value was established by the gel filtration over Sephadex G-75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz-Arg-OEt. However, CaCl₂, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.     

Partial characterization of digestive tract proteinases from western corn rootworm larvae,Diabrotica virgifera

We have partially characterized the proteolytic activity within luminal contents of the digestive tracts of larvae of the western corn rootworm, Diabrotic virgifera LeConte. At least 15 proteinases were detected based on chromatographic behavior on ion exchange high-performance liquid chromatography and their mobility on sodium dodecyl sulfate gels containing gelatin. Inhibitors of proteolytic activity indicated that these enzymes are primarily sulfhydryl proteinases. Native polycrylamide gel electrophoresis revealed that a single proteinaceous inhibitor, egg white cystatin, was capable of abolishing a substantial part of the proteolytic acitivity. Our data suggest, accordingly, that gene transfer experiments utilizing the cystatin gene may generate lines of maize which have increased resistance to western corn rootworm larvae. © 1992 Wiley-Liss, Inc.     

Acute toxicity of kaolin and essential oils from Mentha pulegium and Zingiber officinale against different stages of Callosobruchus maculatus under laboratory conditions

Naturally derived compounds such as essential oils and natural mineral are relatively cheap, non-toxic to food grains and environmentally friendly and would be suitable alternatives for currently used chemical insecticides if they have high insecticidal effectiveness. In the present study, acute toxicity of kaolin and essential oils from Mentha pulegium and Zingiber officinale were assessed on different stages of Callosobruchus maculatus at 28?±?2?°C, 65?±?5% R. H and dark condition. The calculated LC₅₀ values on the egg, larvae and adult stages of C. maculatus were 1.15, 2.33 and 2.18?μl/ml air for Z. officinale and 0.07, 0.11 and 0.09?μl/ml air for M. pulegium, respectively. The result showed that M. pulegium was more effective essential oil against different stages of C. maculatus compared with the Z. officinale, and also the egg and adult stages of C. maculatus were more susceptible against essential oils compared with larval stage. The LC₅₀ values of kaolin were 0.71 and 0.18?mg/cm² on egg and adult of C. maculatus, respectively. The combination of tested essential oils with kaolin increased mortality of C. maculatus adults compared with their application alone. It was found that tested essential oils and kaolin had high potential in controlling different stages of C. maculatus.     

Consequences of inbreeding for the cowpea seed beetle, Callosobruchus maculatus (F.)(Coleoptera: Bruchidae)

Inbreeding is said to reduce vigour and fitness. It may also determine how a population responds to selection. Local populations of Callosobruchus maculatus , the cowpea seed beetle, are established annually from small numbers of founders and the species has been distributed to many parts of the world where isolated populations may have been founded by very small numbers of individuals. After more than 20 generations of inbreeding, inbred lines have been shown to diverge from a common ancestral stock in similar directions with respect of some variables such as developmental speed, but haphazardly in respect of other parameters such as male weight. The respective roles of drift and of selection as effective evolutionary forces in inbred lines are discussed in the light of these results. It is argued that some intraspecific differences in C. maculatus may be explained as a product of periodic inbreeding, but that the process does not impair the ability to adapt to local conditions so contributing to the status of the species as a pest of international importance.     

The fecundity and oviposition behavior of a South Indian strain of Callosobruchus maculatus

The fecundity of the South India strain of Callosobruchus maculatus (Fab.) (Bruchidae: Coleoptera) is 73 eggs for females developing in 31 days and increases to 94 eggs for females emerging after 41 days. Fed females lay 45–55 more eggs which is the egg-equivalent of the reserves unfed females had to sacrifice for activity. Competition as a larva did not reduce a female's fecundity. Oviposition is inhibited when females are given 25 or fewer beans for oviposition and it is reversed if females find fresh beans. Oviposition is generally released by beans carrying a below average number of eggs, thus, the eggs are almost uniformly distributed over the beans. Eggs are preferentially placed on the largest egg-free beans as expected if oviposition is released when a beetle goes from a smaller to a larger bean. The cues resulating host preferences, cowpea > mung pigeonpea chickpea, are secondary to the cues producing hyperdispersion and the selection of larger beans. We present a model for the oviposition behavior that accounts for the observed responses to species, size, and egg load of oviposition sites.

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Résumé La fécondité de femelles isolées avec un mâle, sans aliment ni eau, et laissées sans perturbation avec 50 graines au moins sert de fécondité standard. Elle augmente avec le temps de développement de l'uf à l'adulte de 73 ufs (32 j) à 94 ufs (44 j). La fécondité potentielle, obtenue avec des femelles alimentées sur sucre est de 125 ufs. Des femelles maintenues avec moins de 25 graines réduisent leur fécondité.Plus de la moitié des femelles distribuent leurs ufs uniformément jusqu'à une densité de 1,5 uf. L'uniformité peut provenir de l'induction de réactions de ponte par les graines portant moins d'ufs que l'effectif moyen. L'hyperdistribution au dessus des densités de 3 ufs/graine révèle l'aptitude à distinguer les graines avec 3 ufs des graines avec 4 ufs. Il y a moins d'écarts par rapport à l'uniformité (erreurs) avec des graines à surface rugueuse. Des femelles avec 10 graines font moins d'erreurs que des femelles avec 50 ou 100 graines.Les ufs sont ajoutés aux plus grosses graines sans ufs; ainsi, plus il y a de graines qui portent des ufs, plus le poids des graines avec ufs diminue. Un tel schéma est prévisible si la ponte est induite par des graines plus grosses que les graines rencontrées précédemment. Les femelles présentent un ordre de préférence clair quand elles sont en rapport avec leurs hôtes classiques: Vigna unguiculata > V. radiata > Cajanus cajan > Cicer arietinum Les caractères spécifiques de l'hôte, plutôt que la taille ou la texture de la surface servent à la distinction entre les graines d'un même hôte.Ces résultats expérimentaux peuvent être utilisés pour la modélisation du comportement de ponte d'une femelle.