Effects of azadirachtin on post-embryonic development, energy reserves and α-amylase activity of Plodia interpunctella Hübner (Lepidoptera: Pyralidae)

The effects of azadirachtin on the fourth instar larvae of Plodia interpunctella (Lepidoptera) were investigated. When incorporated into the diet at 2 and 4 ppm, azadirachtin provoked larval weight loss, developmental delay and high larval and pupal mortality. Spectrophotometric assays showed that azadirachtin caused a severe reduction in protein, glycogen and lipid contents 7 days after the beginning of the treatment. In addition, α-amylase activity was reduced in larvaefed azadirachtin.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

BACTERIAL EXPRESSION OF THE BIFUNCTIONAL α-AMYLASE/SUBTILISIN INHIBITOR FROM BARLEY

The bifunctional α-amylase/subtilisin Inhibitor (BASI) is an endogenous inhibitor of the high pl cereal α-amylases encoded by the amyl genes. Evaluation of the potential role of this protein in malting and brewing would be greatly assisted by the availability of large quantities of the protein. We have produced the protein by expression of the barley gene in bacteria. The barley gene was cloned into a pMAL vector and expressed as a fusion protein. The purified fusion protein was successfully cleaved with a specific protease to release the native BASI protein. The BASI produced by bacterial expression will be a useful source of the protein for studies of interactions with barley α-amylases and studies of the influence of this protein on malting and brewing.     

Effects of different food commodities on larval development and α-amylase activity of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

The present work was undertaken to study the influence of four commodities (wheat flour, dates, sorghum and barley) on Plodia interpunctella post-embryonic development. Larval weight, larval mortality, pupation and adult emergence were recorded. The study also aimed to find out the effect of these commodities on protein and glycogen production as well as on α-amylase activity. Results indicated that the weight of fourth instar larvae placed on dates increased gradually. Percentage mortality was low. Pupation and adult emergence were delayed. In contrast, the weight of larvae placed on wheat flour, sorghum or barley remained low. Pupation and adult emergence occurred sooner than among those placed on dates and the percentage mortality was highest for larvae placed on barley. Results also showed that protein content and α-amylase activity were lower for larvae placed on dates than for those placed on other commodities. The biochemical composition of different commodities showed that dates are a rich source of glucose, while their protein and starch contents were very low as compared to the other commodities. In contrast, wheat flour, sorghum and barley contained large amounts of starch and protein and low amounts of glucose. Thus, the reduction in α-amylase activity was probably due to the high levels of glucose in dates.     

Control of wheat α-amylase using inhibitors from cereals

A survey of 46 varieties of cereals and related species (including 27 different species from the Poaceae) indicated the presence of a strong inhibitor of wheat α-amylase in all seven Hordeum species tested. Rye contained a lower level of inhibitor activity, but the other species contained insignificant amounts of wheat α-amylase inhibitor activity. The partially purified barley inhibitor was most effective in inhibiting wheat α-amylase activity at high pH. The addition of chromosome 2 of barley to wheat (Chinese Spring addition line 2H) resulted in an apparent increase in the molecular weight of the α-amylase produced during germination. This was probably due to the formation of a complex between the inhibitor encoded by the asi gene on chromosome 2 of barley and wheat α-amylase 2. Breeding of wheat with the barley inhibitor gene may reduce the impact of the high α-amylase levels that result from pre-harvest sprouting in wheat.     

Immediate and delayed effects of short exposures to phosphine on adults and larvae of the khapra beetle,Trogoderma granarium

Adults and larvae of the khapra beetle, Trogoderma granarium Everts were exposed to 3000 ppm of phosphine through the Phosphine Tolerance Test. In a first series of bioassays, observations were taken every 2 min and the exposed individuals were classified either as walking normally or as being immobilized (knocked down), i.e., not walking normally. In the second series of bioassays all individuals were exposed for 90 min to phosphine. For both bioassays delayed mortality was noted after a 7 and 14-day post exposure interval. Larvae were found to be more tolerant than adults, as the time required for the individuals to be immobilized was up to 20 min, which was almost twice as long as the time required for the immobilization of the adults. There were high levels of adult mortality 7 days later, and complete (100%) mortality 14 days after the exposure. In contrast, larval mortality was low, for both post-exposures. Adults were 100% immobilized after the termination of the 90-min exposure interval at 3000 ppm of phosphine, while the percentage of the active larvae was extremely low. Regarding the 7 days post exposure interval the percentage of larval immobilization was higher than that of adults, but this was reversed seven days later. Interestingly, development of the larvae was delayed compared to the control, after the 90 min exposure, in contrast to 20 min exposure which did not cause any delayed effect. Our work provides some first data for the evaluation of the influence of short exposures to phosphine on adults and larvae of T. granarium, which may be very useful in creating an effective initial quantification plan for the control of this species.     

Solubilization of Collagen in Restructured Beef with Collagenases and α-Amylase

Three microbial collagenases were evaluated for their ability to degrade collagen structure in a restructured beef product. Collagenase preparations with and without α-amylase were added to flaked meat, and their effect on collagen solubility was determined by quantitating the percent soluble hydroxyproline following cooking of product slices. Results demonstrated statistically significant increases in collagen solubility above that of a control product (no added enzymes) for purified Clostridium histolyticum and partially purified Vibrio B-30 collagenases alone and for purified Achromobacter iophagus and partially purified Clostridium histolyticum collagenases with the addition of α-amylase. Thus, the use of collagenase may prove valuable in the production of quality-enhanced meat products.     

Microbial control using bacteria of the almond moth,Cadra (Ephestia) cautella Walker (Lepidoptera: Pyralidae)

Almond moth, Cadra (Ephestia) cautella (Lepidoptera: Pyralidae) is one of the most serious pests of dried fruits and other stored products. Almond moth is polyphagous pest that is widespread in Turkey and all over the world. Almond moth larvae cause serious damages on trees, warehouse and threshing floor of drying fig. Though various cultural, chemical and biological methods are used to control this pest, it is still effective in Turkey and various part of the world. In order to find a significant microbial control agent against this pest, we isolated 13 bacterial isolates and identified these isolates based on morphological, physiological, biochemical and molecular characteristics. According to these characteristics, isolates were identified as Serratia marcescens (Eca1 and Eca3), Serratia sp. (Eca11), Bacillus thuringiensis (Eca2, Eca4, Eca6, Eca7, Eca8, Eca9, Eca10, Eca12, Eca13) and Bacillus axarquiensis (Eca5). The insecticidal activities of these isolates were performed against three insect species from Lepidoptera group which cause serious damage in warehouses. The highest insecticidal activity is 57% for Eca9 isolate on the 3rd instar larvae of C. cautella, 100% for Eca9 isolate on the 3rd instar larvae of Plodia interpunctella and 100% for Eca10 and Eca3 isolate on the 3rd instar larvae of E. kuehniella. Results indicate that Eca9, Eca3 and Eca10 isolates may be valuable as potential biological control agents for the control of warehouse pests.     

α-Amylase reactive extrusion enhances the protein digestibility of saponin-free quinoa flour while preserving its total phenolic content

The original cellular structure and the presence of starch are known to reduce quinoa protein digestibility. Here, we aimed at optimizing an integrated single-step process exploiting the synergisms of amylolysis (<0.42% thermostable α-amylase, starch basis) and extrusion (at different temperature profiles) to enhance protein digestibility in saponin-free quinoa flour while minimizing polyphenol losses. In vitro protein digestion rate (velocity of substrate depletion) and extension (percentage of digested substrate at the end of the reaction) were significantly enhanced with every extrusion treatment, reaching up to four-fold faster protein digestion rate and up to 47% reduction of residual non-digested protein at 100 °C (last barrel temperature) and 0.36 g/100 g α-amylase concentration compared to native flour. Generally, reactive extrusion lowered the content of extractable (free) polyphenols and non-extractable proanthocyanins, but this effect was minimized at lower extrusion temperatures and higher α-amylase concentration. Contrarily, the more thermoresistant hydrolysable bound polyphenols increased in all cases, especially at harsh extrusion conditions.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.     

Digestion and protein metabolism of Trogoderma granarium (Coleoptera: Dermestidae) fed on different barley varieties

The Khapra beetle, Trogoderma granarium Everts, is one of the most important pests of grains which rapidly infests durable stored products in hot and dry conditions. In the current study, effects of omit ten varieties of barley (Abidar, Bahman, Line20, Line22, Line30, Lisivi, Lokht11, Makuyi, Sahand and Sahraa) were determined on digestive enzymes and protein metabolsim of T. granarium larvae under laboratory conditions as 33 ± 1 °C, 65 ± 5% RH and photoperiod of 14:10 (L: D) h. The highest and the lowest activities of α-amylase were observed in T. granarium larvae fed on Line30 and Lisivi varieties, respectively but the glucosidases showed the highest activities followed by feeding on Lokht 11. Also, the latter variety caused the highest lipase activity in the larvae while Abidar, Line20, Lisuei and Sahand imposed the lowest lipase activity. The highest activities of serine proteases including trypsin, chymotrypsin and elastase were detected in the larvae fed on Line 30 but the two exopeptidases, amino- and carboxypeptidases, showed the highest activities when the larvae fed on Lisuei. All the three aminotransferses had the highest activities in the larvae fed on Lokht 11 as the related individuals contained the maximum amount of protein compared to other ones. Results of the current study highlighted Line30 as the most suitable variety to be digested by serine proteases and α-amylase but Lokht11 is appropriately digested by glucosidases and lipase which emboss their susceptibilities to T. granarium. Our findings on digestion and protein metabolism imply on susceptibility of varieties Line30 and Lokht11 to T. granarium larvae.     

芸豆提取物α-淀粉酶抑制剂对SD大鼠的减肥效果研究

探讨从云南白芸豆所提取的α-淀粉酶抑制剂对SD大鼠降脂减肥作用。按中国卫生部减肥功能检验规范方法第2003年版[1]进行相应实验。采用高(352mg/kg)、中(141mg/kg)、低(70mg/kg)3个剂量组的芸豆提取物α-淀粉酶抑制剂连续灌胃45d后均对降低SD大鼠体重、睾丸周围脂肪垫质量及大鼠血液总胆固醇有显著效果(P<0.05)。芸豆α-淀粉酶抑制剂对降低肥胖大鼠体重、内脏脂肪及总胆固醇有显著作用。     

环斑猛猎蝽对斜纹夜蛾低龄幼虫的捕食功能反应

为挖掘环斑猛猎蝽(Sphedanolestes impressicollis)对斜纹夜蛾(Spodoptera litura)的捕食潜力，在室内条件下研究了环斑猛猎蝽4龄若虫对斜纹夜蛾1龄、2龄和3龄幼虫的捕食功能、寻找效应、环斑猛猎蝽不同密度对捕食斜纹夜蛾的干扰效应以及捕食空间大小对环斑猛猎蝽捕食功能的影响。结果显示，4龄环斑猛猎蝽若虫对低龄斜纹夜蛾幼虫的捕食功能反应与Holling II模型相符，对1~3龄斜纹夜蛾幼虫的瞬时攻击率分别为0.979、0.999和0.164，处理时间分别为0.013、0.020和0.018 d，最大日捕食量分别为74.5、51.2和6.1头。4龄环斑猛猎蝽若虫的寻找效应与斜纹夜蛾低龄幼虫密度呈负相关，对斜纹夜蛾低龄幼虫的捕食作用率随自身密度的增加而下降。在捕食1龄斜纹夜蛾幼虫时，环斑猛猎蝽4龄若虫自身密度对其捕食能力干扰最大。因此4龄环斑猛猎蝽若虫对低龄斜纹夜蛾幼虫具有一定的捕食能力，可用于烟田斜纹夜蛾幼虫的绿色防控。      

Effects of spinosad and spinetoram on larval mortality,adult emergence,progeny production and mating in Cadra cautella (Walk.) (Lepidoptera: Pyralidae)

The bacterial formulations, spinosad and spinetoram, were evaluated for their efficacy in suppressing development and mating success in Cadra cautella (Walk.) (Lepidoptera: Pyralidae), the almond moth. A dilution series of spinosad and spinetoram was sprayed on rice flour. Rice flour samples sprayed with water served as the control. Late instar C. cautella larvae were introduced onto spinosad-, spinetoram-, or water-treated rice flour. The first experiment tested the effects of spinosad and spinetoram on larval mortality, as well as emergence of adults and progeny at different insecticide concentrations. In the second experiment, the mating success of C. cautella adults that had emerged from larvae exposed to spinosad was tested inside a cubicle. Both spinosad and spinetoram increased larval mortality, whereas both compounds reduced adult emergence and progeny production. Natural mating was reduced in the presence of the synthetic sex pheromone (Z,E)-9,12-tetradecadienyl acetate. However, exposure of C. cautella larvae to spinosad did not alter mating in adult progeny. Spinosad was more effective than spinetoram at suppressing C. cautella development. The study concludes that both spinosad and spinetoram suppress the development of immatures of C. cautella to the adult stage as well as mating. Thus, the both compounds can be used to protect stored grains from infestation by C. cautella.     

Identification,biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation

Common bean is a legume of significant socioeconomic importance and is cultivated worldwide. This crop is affected by several pests and diseases, which cause considerable economic losses and reduce yield. In recent years, several studies have demonstrated the role of proteins and peptides with activity against a wide range of insects and pathogens. The objective of this work was to identify defense proteins, such as antimicrobial peptides, protease and amylases inhibitors in common bean genotypes and evaluate the relationship of these proteins with Phaseolus vulgaris seed resistance to Callosobruchus maculatus infestation. Nineteen common bean genotypes were subjected to protein extraction, pH 5.4, and precipitation with ammonium sulfate at 70% saturation. The obtained extracts were separated by tricine gel electrophoresis. Experiments were carried out with natural seeds of common beans and artificial seeds (Vigna unguiculata seeds covered with seed coats of common beans) to evaluate the rate of oviposition and development of the insect species Callosobruchus maculatus. Lipid-transfer proteins were identified in nine genotypes whereas defensins were present in all genotypes. The inhibitory activity of α-amylases and trypsin and fungal development were determined in crude extracts (50 μg mL⁻¹). The results also indicated that the extracts from all bean genotypes inhibited the activity of human salivary α-amylase and C. maculatus larval α-amylase. Except for the extracts of four genotypes, all other extracts inhibited trypsin activity. None of the extracts from the evaluated bean genotypes inhibited the growth of tested fungi. Natural seeds from all genotypes did not inhibit insect oviposition, however, the larvae did not survive after feeding on these seeds. Artificial seeds containing seed coat flour of all genotypes inhibited the oviposition of C. maculatus, indicating that the seed coat was also repellent to insect.     

Extraction optimisation,antioxidant activity and inhibition on α-amylase and pancreatic lipase of polyphenols from the seeds of Syzygium cumini

The objectives of this study were to determine the optimal extraction conditions of polyphenols from Syzygium cumini seeds by response surface methodology and investigate their antioxidant activity and inhibition on α-amylase and pancreatic lipase. As results, the optimal extraction conditions in the ultrasonic extraction process which maximised total polyphenols content, minimised the IC₅₀ values of α-amylase and pancreatic lipase were determined as follows: extraction time 60 min, ethanol concentration 63% and solvent/solid ratio 44 mL g⁻¹. The main phenolic compounds in partially purified fraction of Syzygium cumini seeds were catechin, epicatechin, kaempferol, gallic, 5-caffeoylquinic, caffeic and ferulic acids. In addition, the partially purified fraction inhibited 87.66 ± 5.55 and 86.61 ± 3.15% of α-amylase and pancreatic lipase, respectively. The results suggested that Syzygium cumini seeds could be explored as a natural antioxidant and could be used as a source of highly antidiabetic and anti-obesity bioactive compounds.     

Two-sex life table analysis and digestive physiology of Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae) on different barley cultivars

The life table parameters and digestive enzymatic activity of Sitotroga cerealella Olivier (Lepidoptera: Gelechiidae), an important insect pest of grains, were studied under laboratory conditions (25 ± 1 °C, relative humidity of 65 ± 5% and a photoperiod of 16: 8 (L: D) h). Seeds of ten barley cultivars including Bahman, CB-84-10, Fajr30, Makuyi, Nosrat, Yousof, 12A₁, 13A₁, 18A₁ and 19A₁ were used as host diet. S. cerealella showed the shortest developmental time and the highest survival and fecundity after feeding on cultivar 19A₁. The longest developmental time and the lowest fecundity were on cultivars Bahman and Fajr30, respectively. The gross and net reproductive rates and intrinsic rate of increase were the highest when S. cerealella was reared on cultivar 19A₁. However, a corresponding decrease in these parameters was observed on cultivar Fajr30. Fourth instar larvae fed on cultivar 19A₁ showed higher α-amylase activity than those fed on other barley cultivars. However, S. cerealella larvae exhibited no significant difference in proteolytic activity among barley cultivars. Correlation analyses showed that high correlations existed between the life table parameters and α-amylase activity on one side and particle size index on the other. It is suggested that nutritional quality, especially seed hardness, may be the major factor responsible for the susceptibility of barley cultivars to S. cerealella.     

Contributions of diet quality and diapause duration to the termination of larval diapause in khapra beetle,Trogoderma granarium (Coleoptera: Dermestidae)

Larvae of khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae) have an unusual diapause during which they may sporadically feed for several years, but not complete development. Diapausing larvae have an enhanced tolerance to starvation, insecticides and extreme temperatures. Thus, knowledge of factors that terminate larval diapause may aid in the control of this pest. In the current study, we assessed the effect of diet quality (five mixtures of fresh and spent diet in a replacement series) on diapause termination for diapausing larvae from laboratory cultures aged 3, 10, or 14 months. Larvae (n = 10 replicates for each combination of diet quality and culture, 10 larvae/replicate) were held at 30 °C and observed at different intervals over the course of 188 days to record larval mortality and larval pupation (= diapause termination). Larvae were least able to survive on diets of lower quality; i.e., 88% mortality after 188 days on 0% fresh diet versus 8% mortality on 100% fresh diet (averaged across cultures). Larvae were most likely to terminate diapause on diets of higher quality; i.e., 87% termination after 188 days on 100% fresh diet versus 0% termination on 0% fresh diet (averaged across cultures). Diapausing larvae from older cultures were least likely to terminate diapause and least likely to survive. On 100% fresh diet, diapause termination of larvae from cultures aged 3, 10 and 14 months was 93, 91, and 78%, respectively. On 0% fresh diet, mortality of larvae from cultures aged 3, 10 and 14 months was 67, 97, and 100%, respectively. These results indicate that diapausing larvae can accumulate the nutrients required to terminate diapause and complete development when provided with an opportunity to do so. However, the window of opportunity continues to shrink the longer the larvae remain in diapause.     

The effect of modified atmospheres,an alternative to methyl bromide,on the susceptibility of immature stages of angoumois grain moth Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae)

The angoumois grain moth, Sitotroga cerealella is recognized as a common pest of grains world-wide. This investigation aimed at determining the most effective CO₂ concentration in air against the immature stages of the pest in laboratory tests. The sensitivity of newly-laid eggs (<24 h old), 4th instar larvae and 3-day-old pupae to 4 modified atmospheres (MAs) containing 30%, 45%, 65% and 75% CO₂ in air was investigated at 27 °C and exposure periods between 2 h and 264 h. The percentage mortality of the newly-laid eggs, as well as the reduction of adult emergence from 4th instar larvae and 3-day-old pupae tended to increase with the increase of CO₂ concentrations in air and exposure period. According to LT₉₅ values, the order of sensitivity of the three developmental stages of S. cerealella to the four MAs tested was as follows: eggs > pupae > larvae.     

Efficacy of alpha-cypermethrin and thiamethoxam against Trogoderma granarium Everts (Coleoptera: Dermestidae) and Tenebrio molitor L. (Coleoptera: Tenebrionidae) on concrete

Laboratory bioassays were conducted to evaluate alpha-cypermethrin and thiamethoxam for the control of adults, small larvae and large larvae of the khapra beetle Trogoderma granarium, and the yellow mealworm beetle Tenebrio molitor, on concrete. Factors such as dose (0.025 and 0.1 mg alpha-cypermethrin or thiamethoxam/cm²), exposure interval (1, 3 and 7 d), and formulation (alpha-cypermethrin SC and thiamethoxam WG) were evaluated. Apart from immediate assessment at end of exposure, an assessment of delayed mortality was performed with the survivors of the 7-d exposure by removing them from the treated substrate and keeping them on untreated surfaces for 7 more days. After the 7-d exposure, more T. granarium adults were dead on dishes treated with alpha-cypermethrin than with thiamethoxam. Small larvae were generally less susceptible than adults. After 7 d, small larval mortality reached 64.4% for alpha-cypermethrin, while for thiamethoxam it was <6%. Large T. granarium larvae were more tolerant than the small ones. Delayed mortality of T. granarium adults was generally high for both insecticides and doses, and ranged between 43.3 and 63.3% of those that were still alive immediately after the 7-d treatment. For both larval categories, delayed mortality was higher for larvae that had been previously exposed to alpha-cypermethrin, than with thiamethoxam. For T. molitor, after the 7-d exposure, significantly more adults were dead on dishes treated with alpha-cypermethrin than with thiamethoxam. For small larvae mortality was 38.9% at the lowest thiamethoxam dose, but in the other cases ranged between 88.9 and 95.6%. In the case of large larvae, the overall mortality was low in all tested combinations. Regarding delayed mortality of this species, it remained at low levels, for both adults and small larvae. Our results indicate that T. molitor was more susceptible than T. granarium in both insecticides tested, but alpha-cypermethrin was more effective than thiamethoxam.