THE INFLUENCE OF DIETARY α‐SOLANINE ON THE WAXMOTH Galleria mellonella L

Plant allelochemicals are nonnutritional chemicals that interfere with the biology of herbivores. We posed the hypothesis that ingestion of a glycoalkaloid allelochemical, α‐solanine, impairs biological parameters of greater wax moths Galleria mellonella. To test this idea, we reared wax moths on artificial diets with 0.015, 0.15, or 1.5 mg/100 g diet of α‐solanine. Addition of α‐solanine to the diet affected survival of seventh‐instar larvae, pupae, and adults; and female fecundity and fertility. The diet containing the highest α‐solanine concentration led to decreased survivorship, fecundity, and fertility. The diets supplemented with α‐solanine led to increased malondialdehyde and protein carbonyl contents in midgut and fat body and the effect was dose‐dependent. Dietary α‐solanine led to increased midgut glutathione S‐transferase activity and to decreased fat body glutathione S‐transferase activitiy. We infer from these findings that α‐solanine influences life history parameters and antioxidative enzyme activities in the midgut and fat body of G. mellonella.     

Purification of fat body glutathione S‐transferase from the desert locust Schistocerca gregaria: investigation of flavonoid inhibitory effects on enzyme activity

Focus on the development of botanical insecticides such as polyphenols may represent an alternative method to chemical control. In the present study, total glutathione concentration and its related antioxidant enzymes in foregut, midgut, hindgut and fat body homogenates of the desert locust Schistocerca gregaria are examined. Glutathione S‐transferase (GST) activity exhibits a significantly higher value in fat bodies compared with other tissues. A simple and reproducible procedure for the purification of S. gregaria fat body GST is established and the purified enzyme is shown to be homogenous. The purified GST displays a typical Michaelis behaviour with respect to its substrates. Characterization of the GST, including optimum pH, substrate specificity and inhibitor effects, is carried out. The ability of some flavonoids to inhibit S. gregaria fat body GST activity is examined. High‐performance liquid chromatography analysis indicates that the major components in Glycyrrhiza glabra roots are 18α‐glycyrrhetinic acid, quercetin and rutin, and the major components in Hibiscus sabdariffa calyx are cyanidin 3‐O‐glucoside chloride and delphinidin. Quercetin and delphinidin chloride exhibit strong GST inhibition and the inhibition type is determined for both. Rutin shows a smaller inhibitory effect, whereas 18α‐glycyrrhetinic acid and cyanidin have no effect. Inhibition of S. gregaria fat body GST activity would be expected to prevent, or at least delay, the development of resistance to chemical pesticides. Among the examined levels of the antioxidant enzymes, total glutathione concentration and its related enzymes in foregut, midgut, hindgut and fat body crude homogenates of S. gregaria GST activity exhibit a significantly higher value in fat bodies compared with other tissues. Some flavonoids that are detected in H. sabdariffa calyx and G. glabra root extracts are the most effective inhibitors of the purified S. gregaria fat body GST activity. Inhibition of S. gregaria fat body GST activity by quercetin and delphinidin (major compounds detected by HPLC) would be expected to prevent, or at least delay, the development of resistance to chemical pesticides.     

THE EFFECTS OF XANTHOTOXIN ON THE BIOLOGY AND BIOCHEMISTRY OF Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE)

The effects of a dietary plant allelochemical, xanthotoxin (XA), on survivorship, development, male and female adult longevity, fecundity, and hatchability of the greater wax moth Galleria mellonella L. were investigated. Oxidative stress indicators, the lipid peroxidation product, malondialdehyde (MDA), and protein oxidation products, protein carbonyl (PCO) contents, and activities of a detoxification enzyme glutathione S‐transferase (GST) activity were determined in wax moth adults. The insect was reared from first‐instar larvae on an artificial diets containing XA at 0.001, 0.005, or 0.1% to adult stage in laboratory conditions. Relative to the controls, the diets containing XA concentrations led to decreased survivorship in seventh instar, pupal, and adult stages. Compared to control diet (77.7%), the highest dietary XA concentration decreased survivorship to adulthood to 11.0%. The highest XA concentration (0.1%) reduced female longevity from 10.4 to 5.7 days and decreased egg numbers from 95.0 to 33.5 and hatchability from 82.7 to 35.6%. The lowest XA concentration (0.001%) led to about a sixfold increase in MDA content. XA at high concentrations (0.005 and 0.1%) increased MDA (by threefold) and protein carbonyl (by twofold) contents decreased GST activity. The highest dietary XA concentration decreased GST activity from 0.28 ± 0.025 to 0.16 ± 0.005 μmol/mg protein/min. We infer from these findings that XA‐induced oxidative stress led to decreased biological fitness.     

Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae)

Bacillus thuringiensis is one of the most widely used sources of biorational pesticides, as well as a key source of genes for transgenic expression to provide pest resistance in plants. In this study the effect of Bacillus thuringiensis ssp. galleriae (Bt) infection on the activity of superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), concentrations of oxidated and reduced thiols (RSSR/RSH) and malondialdehyde (MDA) was tested in the midgut of Galleria mellonella larvae. We found that Bt infection resulted in increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio the first day after inoculation. However, catalase activity decreased on the first and following days after bacterial infection by Bt. Our results confirm the hypothesis that Bt infection increases the level of oxidative stress in the larval midgut. In light of this study, it seems possible that oxidative damage contributes to cell death in the midgut during bacteriosis.     

摄入异烟肼对家蚕幼虫体内谷胱甘肽氧化还原循环和谷胱甘肽S-转移酶活性的影响

为了建立家蚕Bombyx mori的药物筛选和毒性评价模型, 以剂量为2 000 mg/kg的抗结核模药异烟肼饲喂家蚕5龄第3天幼虫后检测其中肠和脂肪体的抗氧化解毒相关代谢的变化。结果表明： 雌蚕中肠组织中, 总谷胱甘肽(GSH+2GSSG)、 还原型谷胱甘肽(reduced glutathione, GSH)和氧化型谷胱甘肽(oxidized glutathione, GSSG)含量均呈现迅速上升再缓慢下降趋势; 谷胱甘肽S 转移酶(glutathione S-transferase, GST)活性升高到较大值后逐渐降低; GSH/GSSG的比值下降表明, 在72 min后中肠组织向氧化态转移。脂肪体组织中, 总谷胱甘肽、 GSH和GSSG含量变化均呈现迅速下降再迅速上升的趋势; GST活性达到最大值后逐渐降低后趋于平稳; GSH/GSSG比值升高表明, 在72 min后脂肪体组织向还原态转移。无论雌蚕还是雄蚕, 总谷胱甘肽、 GSH和GSSG含量以及GST活性均是脂肪体高于中肠。雌蚕的总谷胱甘肽含量、 GSH和GSSG含量高于雄蚕, 但雄蚕的GST活性高于雌性。结果说明, 摄入异烟肼引起了家蚕幼虫体内谷胱甘肽氧化还原状态的改变和酶活性的变化, 在这个过程中脂肪体起主要解毒代谢作用。     

Dose‐response effects of the antioxidant α‐lipoic acid in the liver and brain of pompano Trachinotus marginatus (Pisces,Carangidae)

This study evaluated the effect of different doses of the antioxidant α‐lipoic acid (LA) administered by intraperitoneal injection on the detoxifying capacity (activity of glutathione‐S‐transferase, GST) and oxidative damage (lipids and proteins) in the pompano, Trachinotus marginatus. The plasma glucose levels showed that there were no differences between the treatments (P > 0.05). In the brain, GST activity was significantly higher (P < 0.05) in fish injected with 40 mg LA kg^?1 when compared with the control group. In the muscle, GST activity was not influenced by LA treatment (P > 0.05). In the liver, fish injected with 20 mg LA kg^?1 showed higher GST activity than the control group (P < 0.05); however, higher doses (40 and 60 mg LA kg^?1) led to a reduction of GST activity in the liver, which was comparable to that observed in the control group (P > 0.05). The two highest LA doses (40 and 60 mg kg^?1) had opposite effects, depending on the tissue examined: LA was an antioxidant in the brain, reducing lipid peroxidation (P < 0.05), and a pro‐oxidant in the liver, augmenting oxidative lipid damage (P < 0.05). The latter effect was accompanied by an increase in the free iron concentration in the liver at higher LA doses. These results indicate the need to thoroughly evaluate the antioxidant effects on aquatic organisms, since at some doses and/or in some organs their beneficial effects can be lost.     

蛋白质沉淀剂对棉铃虫谷胱甘肽S-转移酶的部分纯化

通过用聚乙烯亚胺（PEI）、硫酸铵、聚乙二醇（PEG）沉淀技术和GSH-Sepharose 4B亲和柱对棉铃虫Helicoverpa armigera (Hübner)幼虫中谷胱甘肽S-转移酶进行了部分纯化研究。结果表明PEG10000和PEG20000的纯化效果优于硫酸铵的沉淀效果。通过PEI沉淀去核酸后,再用硫酸铵沉淀,中肠和脂肪体GST活性分布在70%～75％和60%～65％沉淀段,比活力分别为1 081.49和596.41 nmol/(min·mg),纯化倍数分别为2.53和2.2。在6种PEG中,PEG10000和PEG20000的纯化效果较好。在中肠和脂肪体中PEG10000沉淀的GST活性峰分别在40%～45％和30%～40％,GST比活力分别为795.11和1 080.18 nmol/(min·mg),纯化倍数分别是2.4和3.97。PEG20000沉淀中肠和脂肪体GST的活性峰分别在25%～40％和25%～45％,比活力分别是767.57和945.96 nmol/(min·mg),纯化倍数分别是2.81和3.05。用GSH-Sepharose 4B纯化中肠GST,GST比活力达到5 888.44 nmol/(min·mg),纯化倍数达到107.38。     

Host–pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella. Infection with this fungus triggered systemic immune response in G. mellonella; nevertheless, the infection was lethal if spores entered the insect hemocel. We observed melanin deposition in the insect cuticle and walls of air bags, while the invading fungus interrupted tissue continuity. We have shown colonization of muscles, air bags, and finally colonization and complete destruction of the fat body—the main organ responsible for the synthesis of defense molecules in response to infection. This destruction was probably not caused by simple fungal growth, because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans. This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism. Finally, we were unable to reduce the extremely high virulence of B. bassiana against G. mellonella by priming of larvae with thermally inactivated fungal spores.     

Antioxidative responses in Galleria mellonella larvae infected with the entomopathogenic nematode Heterorhabditis sp. beicherriana

The effects of a new species Heterorhabditis sp. beicherriana on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) content of the host were studied. Last-instar larvae of Galleria mellonella were used as host insects and were experimentally infected with H. beicherriana at 0, 20, 40, 80 infective juveniles (IJs) per insect. At 0, 8, 16, 24, 32 and 40 h after infection, activities of SOD, POD, CAT and MDA content were determined in extracts from infected and control insects. We found that H. beicherriana infection resulted in gradually increased activities of SOD, POD and CAT the first day and decreased activities of these enzymes thereafter. However, MDA content in the insects of both control and IJ-inoculated groups stayed at a similar level at 24 h post-infection, but a significant decrease of MDA content in inoculated groups was recorded after 32 h of the infection, which is 8 hours later than the activities of SOD, POD and CAT were significantly increased. Our results suggest that H. beicherriana infection increases the level of oxidative stress and antioxidative responses in the larval G. mellonella, and it seems that oxidative damage contributes to cell death in this host.     

Penicillin-induced oxidative stress: effects on antioxidative response of midgut tissues in instars of Galleria mellonella

Penicillin and other antibiotics are routinely incorporated in insect culture media. Although culturing insects in the presence of antibiotics is a decades-old practice, antibiotics can exert deleterious influences on insects. In this article, we test the hypothesis that one of the effects of dietary penicillin is to increase oxidative stress on insects. The effects of penicillin on midgut concentrations of the oxidative stress indicator malondialdehyde (MDA) and on midgut antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], glutathione S-transferase [GST], and glutathione peroxidase [GPx]) and transaminases (alanine aminotransferase and aspartate aminotransferase) activities in greater wax moth, Galleria mellonella (L.), were investigated. The insects were reared from first instars on artificial diets containing 0.001, 0.01, 0.1, or 1.0 g penicillin per 100 g of diets. MDA content was significantly increased in the midgut tissues of each larval instar reared in the presence of high penicillin concentrations. Activities of antioxidant and transaminase enzymes did not show a consistent pattern with respect to penicillin concentrations in diet or age of larvae. Despite the increased penicillin-induced oxidative stress in gut tissue, antioxidant and transaminase enzymes did not correlate with oxidative stress level or between each other in larvae of other age stages except for the seventh instar. We found a significant negative correlation of MDA content with SOD and GST activities in seventh instars. SOD activity was also negatively correlated with CAT activity in seventh instars. These results suggest that exposure to dietary penicillin resulted in impaired enzymatic antioxidant defense capacity and metabolic functions in wax moth larval midgut tissues and that the resulting oxidative stress impacts midgut digestive physiology.     

The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella

Larvae of the wax moth, Galleria mellonella (L.), were reared from first instar on a diet supplemented with 156, 620, 1,250, or 2,500 ppm boric acid (BA). The content of malondialdehyde (MDA, an oxidative stress indicator), and activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] were determined in the fat body and hemolymph in the 7th instar larvae and newly emerged pupae. Relative to control larvae, MDA was significantly increased in larval hemolymph, larval and pupal fat body, but decreased in the pupal hemolymph. Insects reared on diets with 156- and 620-ppm BA doses yielded increased SOD activity but 1,250- and 2,500-ppm doses resulted in decreased SOD activity in larval hemolymph. SOD activity was significantly increased but CAT was decreased in the larval fat body. High dietary BA treatments led to significantly decreased GST activity. However, they increased GPx activity in larval hemolymph. Dietary BA also affected larval survival. The 1,250- and 2,500-ppm concentrations led to significantly increased larval and pupal mortality and prolonged development. In contrast, the lowest BA concentration increased longevity and shortened development. We infer that BA toxicity is related, at least in part, to oxidative stress management.     

Optimization of Glycoalkaloid Analysis for Use in Industrial Potato Fruit Juice Downstreaming

Annually, within the European Union about 1.7 million tons of starch is produced by processing over 8 million tons of potato tubers, Solanum tuberosum. In recent years, the potato protein content has gained tremendous industrial interest, since these proteins have excellent nutritional value. As naturally occurring, secondary plant metabolites steroidal potato glycoalkaloids are formed in potatoes. The two major glycoalkaloids in potatoes are α‐solanine and α‐chaconine. Because of the significant toxicity of the glycoalkaloids for human and for animal nutrition it was essential to develop efficient extraction processes. The need for an easy, fast, sensitive and reliable glycoalkaloid assay at the very beginning of the production chain is obvious. In this study an efficient analytical assay for potato glycoalkaloids from powdery protein samples under industrially relevant conditions is described: sample extraction, analyte pre‐purification, and final HPLC analysis. An acetic acid extraction/homogenization process was used for glycoalkaloid extraction from potato protein samples. The extracts were purified by means of solid phase extraction cartridges using the different washing steps developed in this study. The final determination was performed through an HPLC method using a Reprosil‐Pur NH₂ column. The limit of detection was 5 μg/mL for α‐solanine and α‐chaconine, respectively, corresponding to concentrations of 20 ppm in potato protein powder.     

Complex of digestive proteinases of <Emphasis Type="Italic">Galleria mellonella</Emphasis> caterpillars. Composition,properties, and limited proteolysis of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> endotoxins

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.     

Ingestion of the anti‐bacterial agent,gemifloxacin mesylate,leads to increased gst activity and peroxidation products in hemolymph of Galleria mellonella l. (lepidoptera: pyralidae)

Gemifloxacin mesylate (GEM) is a synthetic, fourth‐generation fluoroquinolone antibacterial antibiotic that has a broad spectrum of activity against bacteria. GEM inhibits DNA synthesis by inhibiting DNA gyrase and topoisomerase IV activities. Recent research into insect nutrition and mass‐rearing programs, in which antibiotics are incorporated into the culture media to maintain diet quality, raised a question of whether clinical antibiotics influence the health or biological performance of the insects that ingest these compounds. Because some antibiotics are pro‐oxidant compounds, we addressed the question with experiments designed to assess the effects of GEM (mesylate salt) on oxidative stress indicators, using Galleria mellonella larvae. The insects were reared from first‐instar larvae to adulthood on artificial diets amended with GEM at 0.001, 0.01, 0.1, or 1.0%. Feeding on the 1% diets led to significantly increased hemolymph contents of the lipid peroxidation product, malondialdehyde and protein oxidation products, protein carbonyl. All GEM concentrations led to increased hemolymph glutathione S‐transferase activity. We inferred that although it was not directly lethal to G. mellonella larvae, dietary exposure to GEM exerts measurable oxidative damage, possibly on insects generally. Long‐term, multigenerational effects remain unknown.     

DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE,AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α‐glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane‐bound isoforms, being more abundant as a membrane‐bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion‐exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α‐amylases and α‐galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α‐amylases acting on maize seed starch granules.     

Effect of Terminalia chebula aqueous extract on oxidative stress and antioxidant status in the liver and kidney of young and aged rats

We evaluated the preventive effects of Terminalia chebula (T. chebula) aqueous extract on oxidative and antioxidative status in liver and kidney of aged rats compared to young albino rats. The concentrations of malondialdehyde (MDA), lipofuscin (LF), protein carbonyls (PCO), activities of xantione oxidase (XO), manganese‐superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione‐S‐transferase (GST), and glucose‐6‐phosphate dehydrogenase (G6PDH), levels of glutathione (GSH), vitamin C and vitamin E were used as biomarkers. In the liver and kidney of aged animals, enhanced oxidative stress was accompanied by compromised antioxidant defences. Administration of aqueous extract of T. cheubla effectively modulated oxidative stress and enhanced antioxidant status in the liver and kidney of aged rats. The results of the present study demonstrate that aqueous extract of T. cheubla inhibits the development of age‐induced damages by protecting against oxidative stress. Copyright © 2009 John Wiley & Sons, Ltd.     

Biochemical characterization of α-amylase of the Sunn pest, Eurygaster integriceps

The α‐amylase in the midgut and salivary glands of Eurygaster integriceps was isolated and characterized. The specific activity of α‐amylase in the midgut was 1.77 U/mg protein and in the salivary glands was 1.65 U/mg protein. Sodium dodecylsulfate electrophoresis showed that both midgut and salivary glands contain isozymes. Only a trace amount of α‐amylase activity was detected in the first nymphal stage (0.19 U/mg protein), whereas α‐amylase activity was highest in the third nymphal stage (1.21 U/mg protein). The results show that α‐amylase activity in the immature stages increase constantly to the third instar stage. There was no significant difference in enzyme activity between the third, fourth and fifth nymphal stages and adults. The optimum pH and temperature for the enzyme activity was determined to be 6.5 and 35°C, respectively. The enzyme activity was inhibited by addition of ethylenediaminetetraacetic acid, urea, sodium dodecylsulfate and Mg²⁺, but NaCl and KCl enhanced enzyme activity.     

Host plant defenses of black (Solanum nigrum L.) and red nightshade (Solanum villosum Mill.) against specialist Solanaceae herbivore Leptinotarsa decemlineata (Say)

Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in‐vitro glutathione S‐transferase (GST) inhibition activity, we used insecticide‐resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat‐body homogenate. In‐vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis‐hex‐3‐enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata.