Comparative pharmacokinetic analysis of sporoderm-broken and sporoderm-removed Ganoderma lucidum spore in rat by using a sensitive plasma UPLC-QqQ-MS method.

Previous studies have found that removing the sporoderm significantly enhanced antitumor and immunoregulatory activities of Ganoderma lucidum spore (GLS) compared with breaking the sporoderm. However, the pharmacokinetics of sporoderm-removed GLS (RGLS) and sporoderm-broken GLS (BGLS) remain elusive. To compare the pharmacokinetic differences between the two products, we developed a UPLC-QqQ MS method for determining nine representative triterpenoid concentrations. Chloramphenicol was used as an internal standard. The samples were separated on a reversed-phase column using acetonitrile-0.1% formic acid and water-0.1% formic acid as mobile phases. Nine triterpenoids were analyzed using multiple reaction monitoring mode. The results showed that the area under the concentration-time curve from dosing to time t of all nine components was increased in RGLS compared with BGLS. And the time to the maximum concentration in BGLS was delayed compared with that of RGLS. These indicated that the absorption of RGLS was better than that of BGLS, and the sporoderm might hinder the absorption of the active components. These results increase our understanding of the bioavailability of BGLS and RGLS and indicate that increased bioavailability is one of the main reasons for the enhanced efficacy of RGLS.

Differential Analysis of Korean and Chinese Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Spore Powder by Infrared Spectroscopy with Stoichiometry.

To investigate the differences between Korean Ganoderma lucidum spore powder (KP), broken-spo-roderm KP (BSKP), Chinese traditional G. lucidum spore powder (CP), and broken-sporoderm CP (BSCP), they were identified by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), second derivative infrared spectroscopy (SD-IR), dual-index sequence analysis (DISA) and X-ray diffraction (XRD). SEM showed that there were no significant differences in microstructure between the two kinds of spore powders. FT-IR spectra showed that the four spore powders appeared with characteristic peaks of 3400, 3006, 2925, 1745, 1535, 1454, 1249, 1074, 1049, and 896 cm-1, respectively, they were contained the characteristic peaks of total triterpenes, polysaccharides and fatty acids. DISA showed that the same species of spore powders, the overall similarity of before and broken the sporoderm was high with minor differences and there were no differences between the different kinds of spore powders. Similarity analysis showed that the four spore powders were in high agreement and were no differences. The polysaccharide, total triterpene, spore oil and protein content of the four spore powders were determined separately. The results showed that the active ingredients content of the batch of KP were lower than that of CP, that of BSKP were lower than that of BSCP, while the active ingredients content of both broken-sporoderm spore powders were higher than that of before broken-sporoderm. It is inferred that the structure of the main chemical and component of KP is the same as that of CP. This study provides a reference for the future development and application of G. lucidum.

Modifications of Ganoderma lucidum spores into digestive-tissue highly adherent porous carriers with selective affinity to hydrophilic or hydrophobic drugs.

Ganoderma lucidum spores (GLSs) have been suggested to provide optimal structures for transporting orally bioavailable drugs. However, the double-layer wall and cavities of GLSs are naturally closed. This study aimed to modify GLSs into porous carriers by opening the layers and internal cavity with iturin A (IA) followed by potassium hydroxide (KOH) or hydrochloric acid (HCl). The (IA + KOH)- and (IA + HCl)-treated GLS carriers exhibited a high loading rate of 301.50 ± 2.33 and 268.18 ± 7.72 mg/g for the hydrophilic methylene blue (MB) and hydrophobic rifampicin (RF), respectively. The mechanisms underlying the modification involved the enhancement of the specific surface area with IA and the exposure of hydrophilic groups or hydrophobic groups of the GLSs with KOH or HCl. The sustained 48-h molecule-release profiles of the MB- and RF-loaded GLS carriers were best fitted using a first-order kinetics model in simulated gastric (or intestinal) fluid compared with other models. In mice, the designed GLS carriers had high adhesion capacities onto the mucosa of the digestive tract and long retention times (120 h), and even promoted the secretion of mucus and expression of several key intestinal barrier proteins. This study provided a new method to modify GLSs into oral carriers with selective drug affinity, high loading capacity, sustained drug release, and high adhesion to the digestive tract.

Modeling and optimization of the protocol of complex chromatography separation of cyclooxygenase-2 inhibitors from Ganoderma lucidum spore.

INTRODUCTION: The spores of the medicinal fungus Ganoderma lucidum possess hepatoprotective properties. The main components, triterpenes, are particularly beneficial, making the screening and preparation of active triterpenes from Ganoderma lucidum significant. OBJECTIVES: We aimed to screen and verify cyclooxygenase-2 inhibitors from G. lucidum spores, establish a rapid online hyphenated technique for the preparation of active ingredients, and analyze the structures of the active ingredients. METHODS: Ultrafiltration LC combined with an enzyme inhibition assay and molecular docking was employed to screen and evaluate cyclooxygenase-2 ligands, which were prepared by pressurized liquid extraction coupled online with countercurrent chromatography and semi-preparative LC. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Six cyclooxygenase-2 inhibitors, namely, ganoderic acids I, C2 , G, B, and A and ganoderenic acid A, were screened and evaluated. They were prepared using the online hyphenated technique, following which their structures were identified. CONCLUSION: This study provides opportunities for the discovery and development of new therapeutic drugs from other natural resources, as the present instrumental setup achieved efficient and systematic extraction and isolation of natural products compared with reference separation methods, thus exhibiting significant potential for industrial applications.

Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

The Function and Structure of the Microsporidia Polar Tube.

Microsporidia are obligate intracellular pathogens that were initially identified about 160 years ago. Current phylogenetic analysis suggests that they are grouped with Cryptomycota as a basal branch or sister group to the fungi. Microsporidia are found worldwide and can infect a wide range of animals from invertebrates to vertebrates, including humans. They are responsible for a variety of diseases once thought to be restricted to immunocompromised patients but also occur in immunocompetent individuals. The small oval spore containing a coiled polar filament, which is part of the extrusion and invasion apparatus that transfers the infective sporoplasm to a new host, is a defining characteristic of all microsporidia. When the spore becomes activated, the polar filament uncoils and undergoes a rapid transition into a hollow tube that will transport the sporoplasm into a new cell. The polar tube has the ability to increase its diameter from approximately 100 nm to over 600 nm to accommodate the passage of an intact sporoplasm and penetrate the plasmalemma of the new host cell. During this process, various polar tube proteins appear to be involved in polar tube attachment to host cell and can interact with host proteins. These various interactions act to promote host cell infection.

Assessment of a potential bioproduct for controlling Cerotoma arcuata tingomariana (coleoptera: Chrysomelidae).

AIMS: The leaf-feeding pest Cerotoma arcuata tingomariana (Bechyné) (Coleoptera: Chrysomelidae) produces huge economic losses in different crops. This study aimed to produce conidia by semisolid-state fermentation and to establish the insecticidal activity of two formulation prototypes based on a native Beauveria bassiana isolate for controlling this pest. METHODS AND RESULTS: A novel fabric-based semisolid-state fermentation strategy for quick and large-scale conidia production was performed and characterized. Conidia were formulated as an emulsifiable concentrate (EC) and a water-dispersible granulate (WG). Afterwards, the mortality of C. a. tingomariana adults was assessed. A conidia concentration of 2.9 × 109 conidia cm-2 was obtained after 9 days-course fermentation and a yield of 33.4 g kg-1 dry-substrate. CONCLUSIONS: The polyester fabric-based fermentation is an efficient technique for producing and collecting B. bassiana spores. Regarding LC90 , the potency analysis showed that the EC was 21-fold more potent than the non-formulated conidia, and ~ 2.6-fold more potent than the WG. SIGNIFICANCE AND IMPACT OF STUDY: A high throughput fermentation based on polyester fabric as support for B. bassiana conidia production and subsequent formulation as an EC comprises a promising strategy for obtaining a bioproduct to control adults of C. a. tingomariana and other Chrysomelidae pests.

Water-based Fe3O4 magnetic fluid-coated Aspergillus niger spores for treating liquid contaminated with Cr(VI).

The use of magnetic biosorbents for the remediation of heavy metals has attracted increasing attention due to their ease of separation and reusability. We developed a method for preparing superparamagnetic biosorbent materials using water-based magnetic fluids. Water-based magnetic fluid-spores (WMFSs) were obtained by combining water-based magnetic fluid (WMF) with Aspergillus niger spores at ratios of 0.6:1 (WMFS1), 0.8:1 (WMFS2), 1:1 (WMFS3), 1.2:1 (WMFS4), and 1.4:1 (WMFS5). A magnetic composite material was prepared from magnetic nanoparticles and spores in a ratio of 1:1 as a control. The adsorption efficiency and separation effect of WMFS3 were significantly better than those of the magnetic composite material. The morphology and structure of WMFS3 were characterized by performing transmission electron microscopy. The results showed that Fe3O4 magnetic particles were uniformly coated on the spore surface. The superparamagnetism of WMFS3 was tested using a vibrating sample magnetometer. At pH 2.0, the maximum adsorption capacity of WMFS3 for Cr(VI) was 105 mg/g; in the pH range of 2.0-3.0, the adsorption equilibrium time of WMFS3 was 60 min. Thus, the adsorption process conformed to the pseudo-second-order kinetic model and Freundlich isotherm. Thermodynamic studies showed that the process was spontaneous and endothermic. The adsorption mechanisms of WMF3 for Cr(VI) included electrostatic, reduction, and complexation adsorption. This biosorbent material showed excellent adsorption performance for Cr(VI) and is promising for wastewater resource applications.

Structure identification of a polysaccharide in mushroom Lingzhi spore and its immunomodulatory activity.

Ganoderma lucidum spore serves as a well-known immunomodulatory functional food in Asia. The polysaccharides in G. lucidum spore are responsible for the claimed immunomodulatory activity. However, the structural information of polysaccharides remains unclear. In this work, the leading water-soluble polysaccharide in G. lucidum spore (GLSP-I) with a molecular weight of 128.0 kDa was isolated and purified. The monosaccharide composition analysed by gas chromatography indicated that GLSP-I was a glucan. Three side chains, including Glc-(1 â†’ 3)-Glc-(1 â†’ 3)-Glc-(1 â†’ 6)-Glc, Glc-(1 â†’ 6)-Glc-(1 â†’ 6)-Glc-(1 â†’ 6)-Glc and Glc-(1 â†’ 3)-Glc-(1 â†’ 3)-Glc-(1 â†’ 3)-Glc-(1 â†’ 3)-Glc, were identified by UPLC-MS/MS. The structural characteristics were further identified by NMR spectra. The results indicated that the backbone of GLSP-I was (1 â†’ 3)-ß-D-glucan, with side chains linking at O-6. The proposed structure was drawn as below. The immunomodulatory activity assay indicated that GLSP-I could activate macrophages in a dose-dependent manner.

Regulatory function of the novel transcription factor CxrC in Penicillium oxalicum.

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

Antifungal effects of lycorine on Botrytis cinerea and possible mechanisms.

Botrytis cinerea cause postharvest diseases on fruit and lead economic losses. Application of environment-friendly natural compounds is an alternative for synthetic fungicides to control postharvest disease. Lycorine is an indolizidine alkaloid which is widely used for human drug design, however, application of lycorine in controlling postharvest disease and the underlying mechanisms have not been reported. In this study, the effects of lycorine on mycelium growth, spore germination, disease development in apple fruit, cell viability, cell membrane integrity, cell wall deposition, and expression of mitogen-activated protein kinase (MAPK) and GTPase of B. cinerea were investigated. Our results showed that lycorine was effective in controlling postharvest gray mold caused by B. cinerea on apple fruit. In the in vitro tests, lycorine strongly inhibited spore germination and mycelium spreading in culture medium. Investigation via fluorescein diacetate and propidium iodide staining suggested that lycorine could damage the membrane integrity and impair cell viability of B. cinerea. Furthermore, the expression levels of several MAPK and GTPase coding genes were reduced upon the lycorine treatment. Taken together, lycorine is an effective and promising way to control postharvest disease caused by B. cinerea.

Structure and chain conformation of bioactive ß-D-glucan purified from water extracts of Ganoderma lucidum unbroken spores.

Two polysaccharide fractions (GLSB50 and GLSB70) with total sugar content of 82.07 wt% and 53.79 wt%, respectively, were obtained from the water extracts of unbroken Ganoderma lucidum spores by sequential ethanol precipitation treatment. Compared with GLSB70, GLSB50 exhibited better activity on stimulation of humoral immune responses in immunosuppressed mice. A novel ß-D-glucan (GLSB50A-III-1) with weight average molecular weight (Mw) of 1.93 × 105 g/mol was purified from GLSB50 through chromatography separation. The exponent α value of Mark-Houwink-Sakurada equation was calculated to be 0.13, indicating that GLSB50A-III-1 presented globular spheres conformation in aqueous solution. Structural analysis showed that GLSB50A-III-1 mainly consisted of (1 â†’ 3), (1 â†’ 4), (1 â†’ 6)-linked ß-d-glucose residues in the backbone, with two single ß-D-Glcp attached at O-6 of ß-(1 â†’ 3) and ß-(1 â†’ 4)-linked residues separately as side chains. The repeat unit of GLSB50A-III-1 was deduced as follows.

Association of airborne particulate matter with pollen, fungal spores, and allergic symptoms in an arid urbanized area.

Studies focused on the seasonal distribution of pollen and spores in semiarid cities are scarce. At these sites, climate change potentiates the emission and transport of fine (PM10) to ultrafine particles (PM2.5), easily attached to pollen surfaces, causing allergen's release. This study examines the potential correlation of seasonal variations of pollen, fungal spores, PM10, and meteorological parameters with allergic reactions of 150 people living in a Sonoran desert city. We collected PM10, airborne pollen, and spores during a year. We also studied topsoil and road dust samples as potential PM-emission sources. We obtained dust-mineralogy, chemistry, and particle size attached to pollen by X-ray diffraction and scanning electron microscope. Results show that seasonal high PM-loading in the urban atmosphere coincides with aeroallergens promoting micro- to nanoparticles' attachment to pollen's surface. A collapsed membrane was observed in several samples after individual grains show the following maximum wall coverage: Poaceae 28%, Asteraceae 40%, Chenopodiaceae-Amaranthacea 29%, Fabaceae 18%. Most of the particles covering pollen's surface have a geogenic origin mixed with metals linked to traffic (bromide, chlorine, and antimony). Mineralogical, granulometric analysis, and main wind-direction show that two local soil-types are the main contributors to PM. A high frequency of positive sensitization to pollen with high particle loading was detected. These results suggest that climate-driven dust emissions may alter pollen and spore surfaces' physicochemical characteristics with the further consequences in their allergenic potential.

The putative polysaccharide synthase AfCps1 regulates Aspergillus fumigatus morphogenesis and conidia immune response in mouse bone marrow-derived macrophages.

Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections with high mortality in immunosuppressed individuals. Morphogenesis, including hyphal growth, conidiation, and cell wall biosynthesis is crucial in A. fumigatus pathogenesis. Based on a previous random insertional mutagenesis library, we identified the putative polysaccharide synthase gene Afcps1 and its para-log Afcps2. Homologs of the cps gene are commonly found in the genomes of most fungal and some bacterial pathogens. Afcps1/cpsA is important in sporulation, cell wall composition, and virulence. However, the precise regulation patterns of cell wall integrity by Afcps1/cpsA and further effects on the immune response are poorly understood. Specifically, our in-depth study revealed that Afcps1 affects cell-wall stability, showing an increased resistance of ΔAfcps1 to the chitinmicrofibril destabilizing compound calcofluor white (CFW) and susceptibility of ΔAfcps1 to the ß-(1,3)-glucan synthase inhibitor echinocandin caspofungin (CS). Additionally, deletion of Afcps2 had a normal sporulation phenotype but caused hypersensitivity to Na+ stress, CFW, and Congo red (CR). Specifically, quantitative analysis of cell wall composition using high-performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD) analysis revealed that depletion of Afcps1 reduced cell wall glucan and chitin contents, which was consistent with the down-regulation of expression of the corresponding biosynthesis genes. Moreover, an elevated immune response stimulated by conidia of the ΔAfcps1 mutant in marrow-derived macrophages (BMMs) during phagocytosis was observed. Thus, our study provided new insights into the function of polysaccharide synthase Cps1, which is necessary for the maintenance of cell wall stability and the adaptation of conidia to the immune response of macrophages in A. fumigatus.

Prophylactic effects of sporoderm-removed Ganoderma lucidum spores in a rat model of streptozotocin-induced sporadic Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (G. lucidum, Lingzhi), also known as "immortality mushroom" has been broadly used to improve health and longevity for thousands of years in Asia. G. lucidum and its spores have been used to promote health, based on its broad pharmacological and therapeutic activity. This species is recorded in Chinese traditional formula as a nootropic and has been suggested to improve cognitive dysfunction in Alzheimer's disease. However, little is known about the nootropic effects and molecular mechanism of action of G. lucidum spores. AIM OF THE STUDY: The present study investigated the protective effects of sporoderm-deficient Ganoderma lucidum spores (RGLS) against learning and memory impairments and its mechanism of action. MATERIALS AND METHODS: In the Morris water maze, the effects of RGLS on learning and memory impairments were evaluated in a rat model of sporadic Alzheimer's disease that was induced by an intracerebroventricular injection of streptozotocin (STZ). Changes in amyloid ß (Aß) expression, Tau expression and phosphorylation, brain-derived neurotrophic factor (BDNF), and the BDNF receptor tropomyosin-related kinase B (TrkB) in the hippocampus were evaluated by Western blot. RESULTS: Treatment with RGLS (360 and 720 mg/kg) significantly enhanced memory in the rat model of STZ-induced sporadic Alzheimer's disease and reversed the STZ-induced increases in Aß expression and Tau protein expression and phosphorylation at Ser199, Ser202, and Ser396. The STZ-induced decreases in neurotrophic factors, including BDNF, TrkB and TrkB phosphorylation at Tyr816, were reversed by treatment with RGLS. CONCLUSION: These findings indicate that RGLS prevented learning and memory impairments in the present rat model of STZ-induced sporadic Alzheimer's disease, and these effects depended on a decrease in Aß expression and Tau hyperphosphorylation and the modulation of BDNF-TrkB signaling in the hippocampus.

Structural and immunological studies on the polysaccharide from spores of a medicinal entomogenous fungus Paecilomyces cicadae.

A neutral branched heteropolysaccharide (Pc0-1) was purified from the spores of Paecilomyces cicadae, which parasitized in the bamboo cicada (Platylomia pieli Kato). The structure of Pc0-1 was analyzed by HPLC, IR, methylation and NMR spectroscopy. The results reveal that Pc0-1, with an average molecular weight of 18 × 103 kDa, consists of glucose, galactose, mannose and arabinose in the molar ratio of 8:5:4:1. Some of the glucose residues have methyl modification at O-6 position. The Pc0-1 polysaccharide has a core structure containing 1,2-linked α-d-Manp residues as the backbone and branches at the O-3 and O-6 of the α-d-Manp residues. The inner part of the side-chains is comprised of 1,4-linked α-d-Glcp and 1,4-linked 6-O-Me-α-d-Glcp residues. 1,2-linked ß-Galf and minor 1,4-linked Arap and 1,3 or 4-linked Arap residues were occasionally linked at the outside of the side-chains. The side-chains have a single terminal residue of α-d-Glcp, α-Manp, ß-Galf or minor Arap (minor). Studies on the bioactivity of Pc0-1 on the macrophages show it exhibit moderate immunostimulating activity through increasing the production of nitric oxide (NO) and enhancing the secretion of major inflammatory cytokines by macrophages, such as TNF-α, IL-1ß, IL-6, in RAW 264.7 cells. We examined the effect of Pc0-1 on induced NO and cytokine production in macrophages using anti-PRR antibodies to investigate the membrane receptor for the polysaccharide. The results show that Pc0-1 mainly activates macrophages through their mannose receptor (MR). TLR4 and TLR2 also participated in the recognition of Pc0-1.

Identification of toxic mold species through Raman spectroscopy of fungal conidia.

We use a 785 nm shifted excitation Raman difference (SERDS) technique to measure the Raman spectra of the conidia of 10 mold species of especial toxicological, medical, and industrial importance, including Stachybotrys chartarum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and others. We find that both the pure Raman and fluorescence signals support the hypothesis that for an excitation wavelength of 785 nm the Raman signal originates from the melanin pigments bound within the cell wall of the conidium. In addition, the major features of the pure Raman spectra group into profiles that we hypothesize may be due to differences in the complex melanin biosynthesis pathways. We then combine the Raman spectral data with neural network models to predict species classification with an accuracy above 99%. Finally, the Raman spectral data of all species investigated is made freely available for download and use.

Cordycepin, a metabolite of Cordyceps militaris, reduces immune-related gene expression in insects.

Hypocrealean entomopathogenic fungi (EPF) (Sordariomycetes, Ascomycota) are natural regulators of insect populations in terrestrial environments. Their obligately-killing life-cycle means that there is likely to be strong selection pressure for traits that allow them to evade the effects of the host immune system. In this study, we quantified the effects of cordycepin (3'-deoxyadenosine), a secondary metabolite produced by Cordyceps militaris (Hypocreales, Cordycipitaceae), on insect susceptibility to EPF infection and on insect immune gene expression. Application of the immune stimulant curdlan (20 µg ml-1, linear beta-1,3-glucan, a constituent of fungal cell walls) to Drosophila melanogaster S2r+ cells resulted in a significant increase in the expression of the immune effector gene metchnikowin compared to a DMSO-only control, but there was no significant increase when curdlan was co-applied with 25 µg ml-1 cordycepin dissolved in DMSO. Injection of cordycepin into larvae of Galleria mellonella (Lepidoptera: Pyralidae) resulted in dose-dependent mortality (LC50 of cordycepin = 2.1 mg per insect 6 days after treatment). Incubating conidia of C. militaris and Beauveria bassiana (Hypocreales, Cordycipitaceae; an EPF that does not synthesize cordycepin) with 3.0 mg ml-1 cordycepin had no effect on the numbers of conidia germinating in vitro. Co-injection of G. mellonella with a low concentration of cordycepin (3.0 mg ml-1) plus 10 or 100 conidia per insect of C. militaris or B. bassiana caused a significant decrease in insect median survival time compared to injection with the EPF on their own. Analysis of predicted vs. observed mortalities indicated a synergistic interaction between cordycepin and the EPF. The injection of C. militaris and B. bassiana into G. mellonella resulted in increased expression of the insect immune effector genes lysozyme, IMPI and gallerimycin at 72 h post injection, but this did not occur when the EPF were co-injected with 3.0 mg ml-1 cordycepin. In addition, we observed increased expression of IMPI and lysozyme at 48 h after injection with C. militaris, B. bassiana and sham injection (indicating a wounding response), but this was also prevented by application of cordycepin. These results suggest that cordycepin has potential to act as a suppressor of the immune response during fungal infection of insect hosts.

Chitin increases drying survival of encapsulated Metarhizium pemphigi blastospores for Ixodes ricinus control.

Ticks, like Ixodes ricinus, have negative impacts on human and animal health in Germany and worldwide, with almost no specific scientifically proven biological control agent commercially available. Biological control agents containing entomopathogenic fungi present many advantages over chemical acaricides but usually high doses of aerial conidia (1013-1014 conidia/ha) are required to control arthropod pests in the field. A suitable formulation containing nutrients not only makes sensitive blastospores applicable but also functions as a microfermenter to multiply the biomass and thus significantly reduce the required application dosage. For this approach, Metarhizium pemphigi X1c blastospores were encapsulated in calcium alginate beads with granular corn starch or chitin powder as nutrients to ensure formation of aerial conidia on the surface and were then dried. The highest concentration was obtained with moist beads containing chitin (4.68 ±â€¯0.71∙107 conidia∙bead-1). The highest drying survival was also obtained with chitin as the additive (14.7 ±â€¯2.18%). Newly formed aerial conidia of all formulations showed high virulence and caused 100% mortality of I. ricinus nymphs. Altogether, this study paves the way for a lower dose and cost-effective application of blastospores for the control of above ground arthropod pests.

Identification and characterization a novel polar tube protein (NbPTP6) from the microsporidian Nosema bombycis.

BACKGROUND: Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50-500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. RESULTS: A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. CONCLUSIONS: Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.