Sulfur fertilization increases defense metabolites and nitrogen but decreases plant resistance against a host-specific insect.

We tested the sulfur-modulated plant resistance hypothesis using potted cabbage (Brassica oleracea var. capitata) plants that were grown without and with increasing levels of sulfur fertilization. Changes in plant chemical traits were assessed and developmental performance of Plutella xylostella, a highly host-specific leaf-chewing insect, was followed. Leaf sulfur concentration gradually increased with growing addition of sulfur in soil; however, there was a generalized saturation response curve, with a plateau phase, for improvements in total leaf nitrogen, defense glucosinolates and insect performance. Plutella xylostella performed better in sulfur-fertilized cabbage probably because of the higher level of nitrogen, despite of the higher content of glucosinolates, which are toxic for many non-specialized insects. Despite the importance of sulfur in plant nutrition and production, especially for Brassica crops, our results showed that sulfur fertilization could decrease plant resistance against insects with high feeding specialization.

Proteolytic activity of gut bacteria isolated from the velvet bean caterpillar Anticarsia gemmatalis.

The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar-Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-α-benzoyl-L-Arg-p-nitroanilide (L-BApNA) and N-α-p-tosyl-L-Arg methyl ester (L-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-tosyl-L-lysine chloromethyl ketone (TLCK)] as well as CaCl2, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.

Effects of aflatoxins on performance and exocrine pancreas of broiler chickens.

The aim of this research was to evaluate, on a weekly basis, the effects of aflatoxins on the activity of digestive enzymes (alpha-amylase, lipase, and trypsin) in the pancreas as well as on the performance and histology of pancreas in broiler chickens over the course of 42 days. One thousand and eighty 1-day-old male Cobb broilers were divided into four treatments with 18 replicates and 15 birds per replicate (i.e., 270 broilers per treatment). Treatments were established according to the amount of aflatoxins added to the diet, as follows: T1 = 0 mg of aflatoxins per kilogram of feed (mg/kg); T2 = 0.7 mg/kg; T3 = 1.7 mg/kg; and T4 = 2.8 mg/kg. Pancreas sample collection was performed from one bird out of each replicate at 7, 14, 21, 28, 35, and 42 days of experiment, which yielded a total of 18 samples per treatment on each collection. Each sample was homogenized in distilled water, frozen in liquid nitrogen, lyophilized, and stored at -20 C until analysis. Performance parameters (body weight, feed consumption, and feed conversion rate) were measured at 21, 35, and 42 days of experiment. At the end of the experiment (42 days), six birds from each treatment were randomly chosen for histologic evaluation of the pancreas. The presence of aflatoxins in the diet induced a negative effect on all performance parameters. The pancreatic activity of lipase and alpha-amylase were significantly increased in treatments T3 and T4, while the specific activity of trypsin was only affected during treatment T4. In addition, several histologic changes were observed in the pancreas of birds receiving aflatoxin-contaminated feed. Aflatoxins present in the feed determined an increase in the activity of pancreatic enzymes in broilers, affecting the digestibility of the diet, thereby leading to losses in performance and productivity.

Cabbage Seasonal Leaf Quality Mediating the Diamondback Moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) Performance.

Seasonal variation in plant quality may be intense enough to generate predictable patterns in insect herbivore populations. In order to explain seasonal oscillations in neotropical populations of the diamondback moth Plutella xylostella (L.), we tested the following: (1) if nutritional quality of cabbage (Brassica oleraceae var. capitata), a primary host plant of diamondback moth, adversely affects the performance of this insect in late spring and early summer, when populations decline and go extinct, and (2) if nutritional features of cabbage change with the seasons. We measured the performance of diamondback moth reared on leaves of cabbages grown during the four seasons of the year. Summer plants proved to be worse for the survival of the immature stages and subsequent adult fecundity, but there were no significant differences between the remaining seasons. Our results support the hypothesis that short-lived plants, grown in different seasons of the year in the tropics, have different nutritional and defensive attributes. We analyzed nutritional quality of cabbage leaves from the four seasons, but only total lipids were reduced in summer plants. Neotropical populations of diamondback moth collapse before plant quality decay in the summer. If the diamondback moth is well adapted to the seasonal deterioration of the habitat, including the reduction in the quality of host plants, it is expected that emigration happens before the mortality increases and natality decreases during the summer.

Enzymatic response of the eucalypt defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a bis-benzamidine proteinase Inhibitor. i.

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.

Altered cysteine proteinase activity in insecticide-resistant strains of the maize weevil: purification and characterization.

Insecticide resistance is usually associated with fitness costs, but such costs may be mitigated by increased energy and amino acid accumulation and mobilization as has been suggested in the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). To address this adaptation, cysteine proteinases (E.C. 3.4.22), one of the main proteinases in weevils, was purified from an insecticide-susceptible and two insecticide-resistant strains of the maize weevil (one with fitness costs, referred as resistant-cost, and the other without it, referred to as resistant no-cost) using thiol-sepharose affinity chromatography. Purification of the cysteine proteinases revealed a single 74,000 Da molecular mass band in the susceptible strain, two bands of 72,000 and 83,000 Da in the resistant cost strain, and two bands of 68,000 and 74,000 Da in the resistant no-cost strain. Purified cysteine proteinases of the three strains behaved differently regarding casein degradation and inhibition; the proteinases least sensitive to inhibition by the specific cysteine proteinase inhibitor E-64 were those from the resistant no-cost strain as indicated by their highest I(50) value. The pH and temperature profile of cysteine proteinase activity differed among strains and although substrate affinity (i.e. K(M)) of the cysteine proteinases was similar, the V(max) value for cysteine-proteinases from the resistant cost strain was 3-fold and 5-fold higher than V(max) values for the resistant no-cost and susceptible strains respectively. Cysteine proteinase activity was highest for the resistant cost strain rather than the resistant no-cost. Therefore enhanced cysteine proteinase activity is unlikely to be playing significant role in mitigating the costs usually associated with insecticide resistance.

Modified alpha-amylase activity among insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais.

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, alpha-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main alpha-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (>or=70-fold purification,

Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais.

Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides--one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin-agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 microM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V(max)-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-alpha-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.

Characterization and identification of proteolytic bacteria from the gut of the velvetbean caterpillar (Lepidoptera: Noctuidae).

The characterization and identification of proteolytic bacteria from the gut of the velvetbean caterpillar (Anticarsia gemmatalis) were the objectives of this study. Twelve aerobic and anaerobic isolates of proteolytic bacteria were obtained from the caterpillar gut in calcium caseinate agar. The number of colony forming units (CFUs) of proteolytic bacteria was higher when the bacteria were extracted from caterpillars reared on artificial diet rather than on soybean leaves (1.73 +/- 0.35 x 10(3) and 0.55 +/- 0.22 x 10(3) CFU/mg gut, respectively). The isolated bacteria were divided into five distinct groups, according to their polymerase chain reaction-restriction fragment-length polymorphism profiles. After molecular analysis, biochemical tests and fatty acid profile determination, the bacteria were identified as Bacillus subtilis, Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii, and Staphylococcus xylosus. Bacterial proteolytic activity was assessed through in vitro colorimetric assays for (general) proteases, serine proteases, and cysteine proteases. The isolated bacteria were able of hydrolyzing all tested substrates, except Staphylococcus xylosus, which did not exhibit serine protease activity. This study provides support for the hypothesis that gut proteases from velvetbean caterpillar are not exclusively secreted by the insect cells but also by their symbiotic gut bacteria. The proteolytic activity from gut symbionts of the velvetbean caterpillar is suggestive of their potential role minimizing the potentially harmful consequences of protease inhibitors from some of this insect host plants, such as soybean, with implications for the management of this insect pest species.

Contribution of gut bacteria to digestion and development of the velvetbean caterpillar, Anticarsia gemmatalis.

Bacteria colonies from gut homogenates of fifth instar velvetbean caterpillars (Lepidoptera: Noctuidae) were subjected to antibiotic sensitivity experiments using discs containing 22 antibiotics. The antibiotic tetracycline provided the best results, followed by chloramphenicol. Tetracycline also provided higher inhibition of colony forming units than chloramphenicol and was therefore provided to the caterpillars in increasing diet concentrations to assess the contribution of gut bacteria to their digestion and development. The activity of proteases (general), serine-proteinases and lipases were significantly suppressed by tetracycline. Concentration-inhibition curves were successfully established for tetracycline and this antibiotic was effective in suppressing them, particularly serine-proteinases, suggesting that gut bacteria may significantly contribute with lipid- and mainly protein-digestion in velvetbean caterpillars. Increased diet concentrations of tetracycline led only to mild increase in insect mortality (ca. 20%), with the surviving insects showing faster development (< or =4 days) and higher pupa weight (<0.04 mg) with increased concentrations of tetracycline. Therefore, the gut bacteria inhibited by tetracycline does not seem to play a crucial role in the survival and development of the velvetbean caterpillar, but may be important in the adaptation of this pest species to hosts rich in protease inhibitors, such as soybean.

Enhanced activity of carbohydrate- and lipid-metabolizing enzymes in insecticide-resistant populations of the maize weevil, Sitophilus zeamais.

Insecticide resistance is frequently associated with fitness disadvantages in the absence of insecticides. However, intense past selection with insecticides may allow the evolution of fitness modifier alleles that mitigate the cost of insecticide resistance and their consequent fitness disadvantages. Populations of Sitophilus zeamais with different levels of susceptibility to insecticides show differences in the accumulation and mobilization of energy reserves. These differences may allow S. zeamais to better withstand toxic compounds without reducing the beetles' reproductive fitness. Enzymatic assays with carbohydrate- and lipid-metabolizing enzymes were, therefore, carried out to test this hypothesis. Activity levels of trehalase, glycogen phosphorylase, lipase, glycosidase and amylase were determined in two insecticide-resistant populations showing (resistant cost) or not showing (resistant no-cost) associated fitness cost, and in an insecticide-susceptible population. Respirometry bioassays were also carried out with these weevil populations. The resistant no-cost population showed significantly higher body mass and respiration rate than the other two populations, which were similar. No significant differences in glycogen phosphorylase and glycosidase were observed among the populations. Among the enzymes studied, trehalase and lipase showed higher activity in the resistant cost population. The results obtained in the assays with amylase also indicate significant differences in activity among the populations, but with higher activity in the resistant no-cost population. The inverse activity trends of lipases and amylases in both resistant populations, one showing fitness disadvantage without insecticide exposure and the other not showing it, may underlay the mitigation of insecticide resistance physiological costs observed in the resistant no-cost population. The higher amylase activity observed in the resistant no-cost population may favor energy storage, preventing potential trade-offs between insecticide resistance mechanisms and basic physiological processes in this population, unlike what seems to take place in the resistant cost population.

Morphology and preliminary enzyme characterization of the salivary glands from the predatory bug Podisus nigrispinus (Heteroptera: Pentatomidae).

Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.

Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis.

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris-HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates L-BApNA and N-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees C when using L-TAME. Maximum enzyme activity against L-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for L-BApNA and 52.5 microM for L-TAME.

Efeitos das associaçöes de morina-ácido nicotínico e quercetina-ácido nicotínico no controle de lipídeos / Effects of the associations of morin-nicotinic acid and quercetin-nicotinic acid in the lipidic control

A aterosclerose pode ser considerada uma doença na circulaçäo coronariana. O presente trabalho estuda a açÄo da quercetina, morina, ácido nicotínico de maneira isolada e em associaçÄo envolvendo quercetina + ácido nicotínico e morina + ácido nicotínico no metabolismo lipídico. Foram dosados colesterol, colesterol-HDL e triacilglicerol após administraçäo de duas doses dos compostos morina, quercetina, morina + ácido nicotínico e quercetina + ácido nicotínico sendo a primeira imediatamente após a administraçäo do triton e a segunda dose, vinte horas depois. Decorridos quarenta e três horas após a administraçäo do triton o sangue foi analisado. Os resultados mostraram que morina + ácido nicotínico e quercetina + ácido nicotínico apresentaram os melhores resultados para colesterol (-83,77 porcento e 74,42 porcento). Para colesterol-HDL os melhores resultados foram com morina e com associaçäo quercetina + ácido nicotínico (+17,99 porcento e +21,96 porcento). Morina + ácido nicotínico mostraram os melhores níveis para triacilgliceróis (-83,77 porcento)