An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Thermostable cellulase production of Aspergillus fumigatus Z5 under solid-state fermentation and its application in degradation of agricultural wastes

A lignocellulosic decomposing fungus Z5 was isolated and identified as Aspergillus fumigatus, its capacity to produce cellulase was assessed under solid-state fermentation (SSF) using lignocellulosic materials as substrates. Cultivation conditions of A. fumigatus Z5 for cellulase production were optimized, results showed that for carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase), the best condition was 50 °C, 80% initial moisture, initial pH 4.0 and 7% initial inoculum, the average activity of CMCase activity, FPase activity reached 526.3 and 144.6 U g⁻¹ dry weight (dw) respectively, much higher than most of previous reports of this genus. Optimal temperature and pH for the CMCase activity of the crude enzyme were found to be 50 °C and 5.0, respectively. Zymogram analysis showed that eight kinds of CMCase were secreted by A. fumigatus Z5 when cellulose-containing materials were supplied in the culture. The crude enzyme secreted by the strain was further applied to hydrolyze pretreated corn stover and the enzymatic hydrolysate was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of bio-ethanol was 0.112 g g⁻¹ dry substrate (gDS), suggesting that it is a promising fungus in the bio-ethanol production process.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

Regulation of environmental factors on the expression of a solid-state specific lipase (Lip1) with Rhizopus chinensis by western blot and indirect Elisa

To identify which solid-state typical environmental factors are involved in the induction of a solid-state special lipase (Lip1), western blot and Elisa based on Lip1 antibody were used. A low water activity played a significant role in the induction of Lip1, as evidenced by the increased expression level (20–46 μg/g dry cell) along with the decrease of water activity (0.927–0.969). Physical barrier against hyphal extension was found to be another required factor, since the expression of Lip1 was significantly enhanced by 3-fold using a membrane with smaller pore size (0.45 and 0.22 μm) covered on top of surface culture.     

Differences in penetration routes and establishment rates of four entomopathogenic nematode species into four white grub species

We compared the penetration of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), S. glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and H. bacteriophora (GPS11 strain) into third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis. When larvae were exposed to nematodes for 6-72 h larval mortality and nematode establishment rate and occasionally speed of kill often showed the same pattern within nematode-white grub combinations. But no two nematodes or white grub species had the same pattern for these observations for all white grub or nematode species, respectively. Mortality, establishment, and speed of kill followed a similar pattern for H. zealandica, S. glaseri, and S. scarabaei, but there was no clear relationship for H. bacteriophora. Significant nematode establishment was only observed after at least 48 h exposure in most nematode-white grub combinations. Faster establishment was observed only for H. zealandica in A. orientalis and R. majalis (after 24 h) and for S. scarabaei in P. japonica and R. majalis (after 12 h). Nematode establishment after 72 h in the different scarab species was generally low for S. glaseri (<1.5%) and H. bacteriophora (<3%), higher for H. zealandica (2-5%), and the highest for S. scarabaei (1-14%). However, in another experiment establishment was generally higher after 96h exposure. Nematode penetration sites were determined by comparing nematode establishment in larvae with mouth, anus, mouth+anus, or none sealed with glue. The trends for each nematode species were very similar in the different white grub species. H. zealandica and H. bacteriophora showed excellent cuticular penetration ability but may also penetrate through mouth and/or anus. S. glaseri also penetrated through the cuticle but lower establishment in larvae with mouth or mouth+anus sealed suggested that the mouth is an important penetration site. S. scarabaei showed a preference for the mouth as a penetration site, but it showed some cuticular penetration ability and may also use the anus as a penetration site. The methodology used cannot exclude that cuticular penetration also included penetration through the spiracles. To fully understand the effect of nematode and white grub species on nematode virulence, future studies will have to compare host immune response to the penetrating IJs and the role of the symbiotic bacteria in these interactions.     

Efficient mosquitocidal toxin production by Bacillus sphaericus using cheese whey permeate under both submerged and solid state fermentations

Whey permeate (WP) was used efficiently for production of mosquitocidal toxin by Bacillus sphaericus 2362 (B. sphaericus 2362) and the Egyptian isolate, B. sphaericus 14N1 (B. sphaericus 14N1) under both submerged and solid state fermentation conditions. Under submerged fermentation, high mosquitocidal activity was produced by B. sphaericus 2362 and B. sphaericus 14N1 at 50-100% and 25-70% WP, respectively. Initial pH of WP was a critical factor for toxin production by both tested organisms. The highest toxicity was obtained at initial pH 7. Egyptian isolate, B. sphaericus 14N1 was tested for growth and toxin production under solid state fermentation conditions (SSF) by using WP as moistening agent instead of distilled water. The optimum conditions for production of B. sphaericus 14N1 on wheat bran-WP medium were 10 g wheat bran/250 ml flask moistened with 10-70% WP at 50% moisture content, inoculum size ranged between 17.2 × 10⁷ and 34.4 × 10⁷ and 6 days incubation under static conditions at 30 °C. Preliminary pilot-scale production of B. sphaericus 14N1 under SSF conditions in trays proved that wheat bran-WP medium was efficient and economic for industrial production of mosquitocidal toxin by B. sphaericus.     

Cost-effective production of Bacillus thuringiensis biopesticides by solid-state fermentation using wastewater sludge: effects of heavy metals

This study demonstrated the feasibility to produce Bacillus thuringiensis subsp. kurstaki (Btk) based biopesticides using wastewater sludge as raw materials under solid-state fermentation (SSF). More than 10¹⁰ CFU/g viable cells of Btk were obtained using sludge or its mixture with agricultural wastes. This study well considered the effect of heavy metals on Btk growth and their changes of chemical speciation caused by SSF. The IC₅₀ (concentration causing 50% inhibition in total cell biomass) for Pb(II), Cu(II), Cd(II) and Cr(III) on Btk were determined to be 227, 82, 15 and 263 mg/L, respectively. Exposure to 150 mg/L of Cu(II) severely reduced the amount and size of toxin crystals, which decreased the endotoxin synthesis and entomotoxicity potency of Btk cells. Using Tessier’s sequential extraction procedure, the exchangeable heavy metals in sludge were shown to be transformed into residual fractions after SSF, and thus significantly reduced their bioavailability and potential environmental risks.     

Invertase production by Aspergillus niger in submerged and solid-state fermentation

Three Aspergillus nigerstrains were grown in submerged and solid state fermentation systems with sucrose at 100 g l^–1. Average measurements of all strains, liquid vs solid were: final biomass (g l^–1), 11 ± 0.3 vs 34 ± 5; maximal enzyme titres (U l^–1) 1180 ± 138 vs 3663 ± 732; enzyme productivity (U l^–1h^–1) 20 ± 2 vs 87 ± 33 and enzyme yields (U/gX) 128 ± 24 vs 138 ± 72. Hence, better productivity in solid-state was due to a better mould growth.     

Differences between the pathogenic processes induced by Steinernema and Heterorhabditis (Nemata: Rhabditida) in Pseudaletia unipuncta (Insecta: Lepidoptera)

Larvae of Pseudaletia unipuncta are moderately susceptible to infections caused by entomopathogenic nematodes, being a desirable host to study pathogenic processes caused by Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri and their associated bacteria. The ability of the infective stage of these nematodes to invade hosts is quite different. S. carpocapsae invades the highest number of insects and presents the highest penetration rate, followed by H. bacteriophora. Regression analysis between the number of insects parasitized and the number of IJs counted per insect, over time, showed a high correlation for S. carpocapsae whereas for H. bacteriophora it was low. Dose-response was most evident at a concentration below 100 IJs per insect on H. bacteriophora, whereas on S. carpocapsae it was found for doses ranging from 100 to 2,000 IJs. Student's t test analysis of dose-response showed parallel, yet unequal, slopes for both strains of H. bacteriophora, whereas distinct regressions were obtained for S. carpocapsae and S. glaseri, thus, evidencing each species develop a distinct pathogenic process. Insects injected with Photorhabdus luminescens died within 50 h after injection, whereas those treated with X. nematophila died much later. Moreover, the mortality in insects exposed to H. bacteriophora complex and injected with P. luminescens was close, but insects injected with bacteria died faster. Insect mortality in treatments with complexes S. carpocapsae and S. glaseri was significantly higher than that which was observed in insects injected with symbiotic bacteria.     

Non-irradiation-derived reactive oxygen species (ROS) and cancer: therapeutic implications

Summary. Owing to their chemical reactivity, radicals have cytocidal properties. Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalysed reactions. Although these developments are currently still in their infancy, they nevertheless deserve consideration. There are now numerous examples known of conventional anti-cancer drugs that may at least in part exert cytotoxicity by induction of radical formation. Some drugs, such as arsenic trioxide and 2-methoxy-estradiol, were shown to induce programmed cell death due to radical formation. Enzyme-catalysed radical formation has the advantage that cytotoxic products are produced continuously over an extended period of time in the vicinity of tumour cells. Up to now the enzymatic formation of toxic metabolites has nearly exclusively been investigated using bovine serum amine oxidase (BSAO), and spermine as substrate. The metabolites of this reaction, hydrogen peroxide and aldehydes are cytotoxic. The combination of BSAO and spermine is not only able to prevent tumour cell growth, but prevents also tumour growth, particularly well if the enzyme has been conjugated with a biocompatible gel. Since the tumour cells release substrates of BSAO, the administration of spermine is not required. Combination with cytotoxic drugs, and elevation of temperature improves the cytocidal effect of spermine metabolites. The fact that multidrug resistant cells are more sensitive to spermine metabolites than their wild type counterparts makes this new approach especially attractive, since the development of multidrug resistance is one of the major problems of conventional cancer therapy.     

Inter- and intraspecific variation in defensive compounds produced by five neotropical stink bug species (Hemiptera: Pentatomidae)

The differences in composition of defensive secretions between nymphs, adult males and adult females of Chinavia impicticornis (=Acrosternum impicticorne), Chinavia ubica (=Acrosternum ubicum), Euschistus heros, Dichelops melacanthus and Piezodorus guildinii (Hemiptera, Pentatomidae) were analysed within and between species using compositional log-ratio statistics and canonical variates analysis. Differences in composition between nymphs, males and females were found for all species, as well as when all species were pooled. In particular, tetradecanal appears to be a predominantly nymphal compound in D. melacanthus, E. heros and P. guildinii. In the two Chinavia species 4-oxo-(E)-2-hexenal and an unknown compound were more dominant in nymphs. The interspecific analysis revealed a good separation of defensive compounds according to their taxonomic relationship. Thus, the two Chinavia species grouped together, with (E)-2-decenal and (E)-2-hexenyl acetate, contributing to this separation. The other three species also differed from each other, with (E)-2-octenal associated to D. melacanthus, (E)-2-hexenal to P. guildinii and (E,E)-2,4-decadienal and tetradecanal to E. heros. The pooled analysis of stage ignoring species revealed tetradecanal and 4-oxo-(E)-2-decenal (tentative identification) strongly associated to nymphs. Thus, there are predictable differences between stages, and many of the differences are conserved between species. Consideration of these differences could prove to be important in understanding stink bug-natural enemy interactions, and in optimising biocontrol efforts.     

Zinc induces distinct changes in the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in the roots of two Brassica species with different sensitivity to zinc stress

Background and Aims Zinc (Zn) is an essential micronutrient naturally present in soils, but anthropogenic activities can lead to accumulation in the environment and resulting damage to plants. Heavy metals such as Zn can induce oxidative stress and the generation of reactive oxygen and nitrogen species (ROS and RNS), which can reduce growth and yield in crop plants. This study assesses the interplay of these two families of molecules in order to evaluate the responses in roots of two Brassica species under high concentrations of Zn.Methods Nine-day-old hydroponically grown Brassica juncea (Indian mustard) and B. napus (oilseed rape) seedlings were treated with ZnSO₄ (0, 50, 150 and 300 µm) for 7 d. Stress intensity was assessed through analyses of cell wall damage and cell viability. Biochemical and cellular techniques were used to measure key components of the metabolism of ROS and RNS including lipid peroxidation, enzymatic antioxidants, protein nitration and content of superoxide radical (O2·−), nitric oxide (NO) and peroxynitrite (ONOO⁻).Key Results Analysis of morphological root damage and alterations of microelement homeostasis indicate that B. juncea is more tolerant to Zn stress than B. napus. ROS and RNS parameters suggest that the oxidative components are predominant compared with the nitrosative components in the root system of both species.Conclusions The results indicate a clear relationship between ROS and RNS metabolism as a mechanism of response against stress caused by an excess of Zn. The oxidative stress components seem to be more dominant than the elements of the nitrosative stress in the root system of these two Brassica species.     

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35–60 °C and pH 4.0–6.0, with a maximum activity at 50 °C and pH 4.0–5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 °C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0–C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.     

An Endoreticulatus species from Ocinara lida (Lepidoptera: Bombycidae) in Taiwan

A microsporidium from the Ficus pest, Ocinara lida, in Taiwan is characterized. The taxonomic position of this species was preliminarily determined by sequencing small subunit rRNA gene (SSUrRNA). Analysis of the SSUrRNA sequence indicated that this isolate from O. lida is a member of the genus Endoreticulatus and belongs to the genetic grouping containing other lepidopteran Endoreticulatus species we have analyzed phylogenetically. The taxonomic position of this isolate was also confirmed by the ultrastructural characteristics of this isolate. The congruence between SSUrRNA sequence analysis and ultrastructural characteristics shows that this isolate is more closely related to Endoreticulatus bombycis than to Endoreticulatus schubergi Zw?lfer.     

Hygrophorones A-G: fungicidal cyclopentenones from Hygrophorus species (Basidiomycetes)

Twenty new 5-(hydroxyalkyl)-2-cyclopentenone derivatives (hygrophorones) could be isolated from Hygrophorus latitabundus, H. olivaceoalbus, H. persoonii, and H. pustulatus. Their fungicidal activity was exemplarily tested. The hygrophorones have structural similarities to the antibiotic pentenomycin. Chemically, hygrophorones are 2-cyclopentenones with hydroxy or acetoxy substituents at C-4 and/or C-5. An odd-numbered 1' oxidized alkyl chain (C(11), C(13), C(15), or C(17)) is attached at C-5. In addition, from H. persoonii the new gamma-butyrolactone derivative [5-(E)-2-hydroxytetradexylidene-5H-furan-2-one] could be isolated. Some hygrophorones are responsible for the color reaction of the stipes of these fungi upon treatment with potassium hydroxide solution. Structural elucidations are based on 1D ((1)H, (13)C) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectroscopic analyses as well as HR-FT-ICR-MS investigations.     

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC-MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772-2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a beta-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O(9)-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O(9)-esters may be further diversified by O(7)- and/or O(3')-acylation.