The endophytic fungal entomopathogens Beauveria bassiana and Purpureocillium lilacinum enhance the growth of cultivated cotton (Gossypium hirsutum) and negatively affect survival of the cotton bollworm (Helicoverpa zea)

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum, were assessed on the growth of cultivated cotton (Gossypium hirsutum) and development of the cotton bollworm (Helicoverpa zea). In two replicate greenhouse trials, cotton plants were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia and evaluated for effects on both plant dry biomass, number of nodes and number of developing flowers (squares). We similarly treated cotton plants and evaluated H. zea performance using no-choice in planta assays starting at the 2nd larval instar. Treatment with both fungal endophytes resulted in a significant increases in plant dry biomass (ANOVA, P = 0.024). Plant developmental stage and number of squares were also significantly enhanced in the endophyte treated plants (ANOVA, P = 0.005 and P = 0.027, respectively). The survivorship of H. zea was significantly different among the endophyte treatment groups (Kaplan–Meier, P = 0.02), where insects feeding on control plants exhibited higher survival than insects on the endophyte treated plants. There were no significant endophyte treatment effects on larval or pupal weights of H. zea individuals. There was no endophyte effect on days to pupation among treatments, but there was a marginal effect on days to eclosion (Kaplan–Meier, P = 0.07). Overall, our results demonstrate (i) the positive plant growth enhancing effects of the target endophyes on cultivated cotton under greenhouse conditions and (ii) the negative effects of endophytic P. lilacinum and B. bassiana on H. zea survivorship and development using whole plant assays.     

Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae

Two fungal cyclooligomer depsipeptide synthetases (CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC 7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8±1.4 mg/l) and bassianolide (21.7±0.1 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding d-hydroxyisovaleric acid (d-Hiv) and the corresponding l-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of d-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins. The total titer of beauvericin and its congeners (beauvericins A–C) was increased to 61.7±3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC 7159. Supplement of l-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8±2.1 mg/l. Using this yeast system, we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules.     

Production of isomaltulose using Erwinia sp. D12 cells: Culture medium optimization and cell immobilization in alginate

Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. An experimental Design and Response Surface Methodology were applied for the optimization of the nutrient concentration in the culture medium for enzyme production in shaken flasks at 200 rpm and 30 °C. A higher production of glucosyltransferase (7.47 Uml⁻¹) was observed in the culture medium containing sugar cane molasses (160 gl⁻¹), bacteriological peptone (20 gl⁻¹) and yeast extract Prodex Lac SD^® (15 gl⁻¹) after 8 h, at 30 °C. The highest production of glucosyltransferase in the 6.6-l bioreactor (14.6 Uml⁻¹) was obtained in the optimized culture medium after 10 h at 26 °C. When Erwinia sp. D12 cells were immobilized in sodium alginate, it was verified that sodium alginate solution A could be substituted by a cheaper one, sodium alginate solution B. Using a 40% cell suspension and 2% sodium alginate solution B for cell immobilization in a packed-bed reactor, 64.1% conversion of sucrose to isomaltulose was obtained. The packed-bed reactor with immobilized cells plus glutaraldehyde and polyethylenimine solutions remained in a pseudo-steady-state for 180 h.     

Fructanolytic and saccharolytic enzymes of Treponema zioleckii strain kT

Enzymes in the newly described rumen bacterium, Treponema zioleckii strain kT, capable of digesting Timothy grass fructan, inulin, and sucrose were identified and characterized. Two specific endolevanases and one non-specific β-fructofuranosidase were found in a cell-free extract. The molecular weight of the endolevanases were estimated to be 60 and 36 kDa, whereas that of β-fructofuranosidase, 87 kDa. The former of the specific enzymes was associated with the outer membrane, while the latter and the non-specific β-fructofuranosidase, with the periplasm or cytosol. The K_m and V_max for Timothy grass fructan degradation by endolevanase were 0.27% and 15.75 μM fructose equivalents × mg protein^?1 × min^?1, those for sucrose and inulin digestion by β-fructofuranosidase were 1.35 × 10^?3 M and 1.73 μM hexoses × mg protein^?1 × min^?1 and 1.77% and 1.83 μM hexoses × mg protein^?1 × min^?1, respectively.     

Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L⁻¹ to 3.16 g L⁻¹, with a molecular weight range of 1.40×10⁶–1.83×10⁶ Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10⁶ U mL⁻¹), the production of HA was substantially increased from 5.96 g L⁻¹ to 19.38 g L⁻¹. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10³–1.42×10⁶ Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.     

Production,characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

The production, characterization and antioxidant activities in vitro of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3 were investigated. For EPS production, the preferable culture conditions were 24 °C and pH 8 for 60 h with sucrose and yeast extract as the carbon and nitrogen sources, respectively. Notably, sucrose concentration was the prominent factor, and the maximum yield of EPS (22.82 g/L) was obtained at a sucrose concentration of 160 g/L. The crude EPS was purified by chromatography of DEAE-52 and Sephadex G-100, affording EPS-1 and EPS-2 with molecular weights of 1.22 × 10⁶ and 8.69 × 10⁵ Da, respectively. They were composed of mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. In addition, both crude and purified EPS showed strong scavenging activities on superoxide and hydroxyl radicals, and their antioxidant activities decreased in the order of crude EPS > EPS-2 > EPS-1.     

Characterization of methylated azopyridine as a potential electron transfer mediator for electroenzymatic systems

N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron transfer mediator for oxidation reactions catalyzed by NAD⁺- and pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity with MAZP was defined as (2.2 ± 0.1) × 10⁵ M⁻¹ s⁻¹, whereas the bimolecular rate constant of reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was determined to be (4.7 ± 0.1) × 10⁴ M⁻¹ s⁻¹. The use of MAZP for the regeneration of the cofactors was investigated by applying the electrochemical oxidation of the mediator. The total turnover numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD⁺-dependent alcohol dehydrogenase depended on the concentration of the substrate and the duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable, and the turnover number of the enzyme reached a value of 2.3 × 10³. In addition, oxidation of 1,2-propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded enantioselectively to yield l-lactic acid.     

Laboratory and semi-field evaluation of Beauveria bassiana (Ascomycota: Hypocreales) against the lettuce aphid,Nasonovia ribisnigri (Hemiptera: Aphididae)

The lettuce aphid, Nasonovia ribisnigri (Mosley), is an economically important pest of lettuce worldwide. The entomopathogenic fungus Beauveria bassiana strain GHA has recently been reported as a potential biocontrol candidate for use against the lettuce aphid. This study provides information on the mortality inflicted by B. bassiana when applied against different life stages of the lettuce aphid under laboratory conditions and how fungus infection affects the aphid fecundity. In addition, temporal changes in persistence of fungus inoculum applied to foliage of young lettuce plants under semi-field conditions was analysed. Immature life stages were generally the least susceptible to fungal infection and the susceptibility of all stages was dose-dependent, with the highest mortality occurring at the highest dose. B. bassiana significantly affected the rate of nymph production by the lettuce aphid, with the highest effect seen when the alatoid fourth instar of N. ribisnigri was inoculated with B. bassiana. The persistence of B. bassiana conidia on lettuce foliage was not influenced by leaf position. Within 5 days, the cumulative percentage decline in the conidial population was 38% which declined further to 92% and 99% on day 11 and 20 post-spraying, respectively. In accordance, the infectivity to second instar lettuce aphid nymphs of B. bassiana conidia deposited on leaves declined according to an exponential decay model predicting an intercept of 0.59 ± 0.03 (S.E), a reduction in aphid mortality at a rate of 11% with each increasing day after fungal application and a fungus half-life of 6.34 ± 0.69 days.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Enhancement of polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by optimization of medium compositions and feeding of sucrose

A strategy that optimization of medium compositions for maximum biomass followed by feeding of sucrose for maximum polysaccharide synthesis was developed for enhancing polysaccharide production in suspension culture of protocorm-like bodies (PLBs) of Dendrobium huoshanense C.Z. Tang et S.J. Cheng. In growth stage, the original half-strength MS medium was optimized with carbon sources, nitrogen sources and metal ion combinations. The effects of different carbon sources on PLBs growth were remarkable and sucrose at 35 g l⁻¹ was the most suitable. Sole nitrate nitrogen of 30 mmol l⁻¹ was the best for PLBs growth. Metal ions (Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) showed different influences on PLBs growth. The optimal concentration of Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ was 4.5 mmol l⁻¹, 0.1 mmol l⁻¹, 0.5 mmol l⁻¹ and 0.06 mmol l⁻¹, respectively. In the optimized medium (sucrose, nitrate, Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ concentration as described above, the other component concentration seen in half-strength MS), 33.9 g DW l⁻¹ PLBs were harvested after 30 days of culture and biomass increase was improved 245% as compared with that in the original medium. In production stage, polysaccharide synthesis was significantly improved by the feeding sucrose. The maximum polysaccharide production (22 g l⁻¹) was obtained in the case of 50 g l⁻¹ sucrose feeding at day 30 of culture, which was about 109-fold higher than that in the original medium without feeding of sucrose.     

Purification and biochemical characterization of angiotensin I-converting enzyme (ACE) from ostrich lung: The effect of 2,2,2-trifluoroethanol on ACE conformation and activity

This work reports the purification and biochemical characterization of angiotensin I-converting enzyme (ACE) from ostrich (Struthio camelus) lung. The molecular weight of the purified enzyme was approximately evaluated to be 200 kDa and the maximum enzyme activity was observed at pH 7.5. The enzyme activity was increased by detergents of Triton X-100 (0.01%), cetyltrimethylammonium bromide (CTAB) (0.1 and 1 mM) and sodium dodecyl sulfate (SDS) (0.1 mM), while decreased by Triton X-100 (1% and 10%) and SDS (1 mM and 10 mM). The secondary and tertiary structure and activity of ACE in the absence and presence of trifluoroethanol (TFE) were investigated using circular dichroism, fluorescence quenching and UV–visible spectroscopy, respectively. Our results revealed that TFE stabilizes ACE at low concentrations, while acts as a denaturant at higher concentration (20%). The K_m, K_cat and K_cat/K_m values of ostrich ACE towards FAPGG were 0.8 × 10^?4 M, 59,240 min^?1 and 74 × 10⁷ min^?1 M^?1, respectively. The values of IC₅₀ and K_i for captopril were determined to be 36.5 nM and 16.6 nM, respectively. In conclusion, ostrich lung ACE is a new enzyme which could be employed as a candidate for studying ACE structure and its natural or synthetic inhibitors.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products

The aim of this research was to identify a low cost medium based on commercial products and by-products that provided maximum Bacillus subtilis CPA-8 growth and maintained biocontrol efficacy. Low cost media combining economical nitrogen and carbon sources such as yeast extract, peptone, soy products, sucrose, maltose and molasses were tested. Tests were carried out in 250-ml flasks containing 50 ml of each tested medium. Maximum cell growth (>3 × 10⁹ CFU ml^?1) was obtained in defatted soy flour 44% combined with sucrose or molasses media. Second, CPA-8 production was scaled up in a 5-l fermenter and CPA-8 population dynamics, pH and oxygen consumption in the optimized medium (defatted soy flour 44% – molasses) was recorded. In these tests, there was a 5-h lag phase before growth, after which exponential growth occurred and maximum production was 3 × 10⁹ CFU ml^?1 after 20 h. Fruit trials with cells and cell free supernatants from CPA-8 grown in optimized medium maintained biocontrol efficacy against Monilinia fructicola on peaches, resulting in disease reductions up to 95%. CPA-8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results show that B. subtilis CPA-8 can be produced in a low cost medium combining inexpensive nitrogen and carbon sources (40 g l^?1 defatted soy flour 44%, 5 g l^?1 molasses plus mineral trace supplements) in shake flasks and a laboratory fermenter (5 l). The results could be used to provide a reliable basis for scaling up the fermentation process to an industrial level.     

Efficacy assessment of Pichia guilliermondii strain Z1, a new biocontrol agent,against citrus blue mould in Morocco under the influence of temperature and relative humidity

The interactions of Penicillium italicum, which causes blue mould, and antagonistic yeast Pichia guilliermondii strain Z1 were examined in controlled environments, to determine the influence of relative humidity (RH) (45%, 75%, 85%, 98%, and 100%) and temperature (T) (5, 10, 15, 20, and 25 °C). All main effects and interactions were significant (P ? 0.05), with the exception of interactions RH×T and strain Z1 (BCA)×RH×T. In the pathogen control, the lesion diameter of blue mould developed under all environmental conditions but was the largest at a RH range between 98% and 100%, independent of the temperature. The efficacy of strain Z1 appeared to be independent of the environment and reduced disease incidence by more than 85% in all environmental conditions. Rapid colonization of the antagonistic yeast strain Z1 on citrus wounded sites was recorded during the first week at 5 °C. Colonization then stabilized at ±6.9 × 10⁶ CFU/ml for 30 days. This indicates that P. guilliermondii is able to adapt itself and colonize the wound sites prior to the arrival of the pathogen, displaying greater efficiency than when colonizing wounds after pathogen. The antagonist was capable of growing in low concentrations of orange juice (0.1–5%), with greatest growth at 5%. Applying strain Z1 (1 × 10⁸ CFU/ml) as a formulated product significantly reduced the incidence of infected fruits and the percentage of infected wounds relative to the pathogen control. Disease control with formulated product (45%) was slightly lower than that obtained with thiabendazole (20%) or strain Z1 culturable cells (25%). These results suggest that strain Z1 may be a useful BCA for control of blue mould under varying environmental conditions, and control may be enhanced by combining with other eco-friendly post-harvest treatments or improved formulation.     

Microbial biological control potential of three strains of Beauveria bassiana s. l. against greenhouse shore fly Scatella tenuicosta: Assessment of virulence,mass production capacity,and effects on shore fly reproduction

The microbial biological control potential of three strains of Beauveria bassiana sensu lato originally isolated from the shore fly Scatella tenuicosta (Diptera: Ephydridae) was assessed in a series of laboratory bioassays. Comparisons were made to two commercially-available strains of B. bassiana. Two of the shore fly strains proved 27–67 times more virulent than the commercial strains in terms of LC₅₀ (14–17 vs. 458–942 conidia/mm²) and killed shore flies more rapidly. B. bassiana s. l. strain ST1 exhibited a mass production capacity comparable to the commercial B. bassiana stain GHA, producing 2.8 × 10¹² conidia/kg barley-based solid substrate in ventilated mushroom spawn bags. The shore fly strains of Beauveria sporulated on a higher percentage of killed adult shore flies and produced substantially greater numbers of conidia per cadaver than the commercial strains, indicating that these pathogens are well adapted to this host. Female shore flies treated with strain ST1 survived for only 5 days, with longevity being reduced by 8–10 days compared to control insects. This reduction in survival had a large impact on total lifetime egg production, reducing it by 78–88%, depending on the time of treatment relative to the pre-oviposition period. However, fungal growth within infected female shore flies had no effect on egg production or egg viability until the day before the flies succumbed to mycosis (day 4 post-inoculation). As a consequence, the intrinsic rate of shore fly population increase and population doubling time were little affected by fungal infection (0.4357 vs. 0.4152 and 1.6 vs. 1.7 days for control vs. Beauveria-treated populations, respectively). These findings underscore the challenges involved with use of slow-acting pathogens for control of highly fecund greenhouse pests and the fundamental necessity of integrating these agents into integrated pest management systems.     

Alginate embedding and subsequent sporulation of in vitro-produced Conidiobolus thromboides hyphae using a pressurised air-extrusion method

Conidiobolus thromboides is an entomophthoralean fungus with potential as a biological control agent of aphids. However, its application in biological control is limited due to its formulation requirements. The objective of this study was to develop and optimise a novel air-extrusion method to embed C. thromboides hyphae at high density in alginate pellets. An orthogonal experimental design was used to investigate selected combinations of parameters known to affect hyphal density within pellets. The diameter of pellets produced, and the calculated density of hyphae within them, ranged from 0.18 ± 0.09 to 3.17 ± 0.06 mm and from 0.02 to 350.56 mg/mm³ respectively. These data were used to predict the optimal parameter combination to deliver the greatest density of hyphae of C. thromboides per pellet: 1% sodium alginate, a 1:2 ratio of hyphae to sodium alginate, an orifice diameter of 0.232 mm and an air pressure of 0.05 MPa. Pellets made under the optimal conditions predicted produced a mean total of 4.3 ± 0.6 × 10⁵ conidia per pellet at 100% relative humidity which was significantly greater than the mean total number of conidia produced from infected aphid cadavers of comparable size (9.35 ± 0.85 × 10⁴) (p < 0.001). In conclusion, air-extrusion embedding appears to be a promising method for formulating in vitro-produced hyphae of C. thromboides for use in biological control.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L⁻¹ and 36.3 g L⁻¹ succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L⁻¹ succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol⁻¹ hexose.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.