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.     

Identification of lactic acid bacteria and yeast from boza

Boza is a low-alcohol beverage produced from the fermentation of barley, oats, millet, maize, wheat or rice. The number of lactic acid bacteria isolated from three boza samples ranged from 9 × 10⁶ to 5 × 10⁷ CFU/mL. Carbohydrate fermentation reactions and PCR with species-specific primers classified the isolates as Lactobacillus paracasei subsp. paracasei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus rhamnosus and Lactobacillus fermentum. No filamentous fungi were isolated. Yeasts were isolated from two of the three boza samples, with cell numbers ranging from 1.3 × 10² to 1.8 × 10³ CFU/mL. Results obtained from sequencing of the D1/D2 rDNA region identified the yeasts as Candida diversa, Candida inconspicua, Candida pararugosa, Issatchenkia orientalis, Pichia fermentans, Pichia guillliermondii, Pichia norvegensis, Rhodotorula mucilaginosa and Torulaspora delbrueckii. C. inconspicua has been isolated from human sputum and tongue and is an opportunistic pathogen. R. mucilaginosa is also an opportunistic pathogen implicated in fungaemia, endocarditis and meningitis. P. norvegensis has been associated with septicaemia in humans. Saccharomyces cerevisiae, commonly associated with fermented beverages, has not been detected in any of the boza samples, despite enrichment.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen–thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5 ± 5.4 × 10⁶ sperm (range, 6.8–22 × 10⁶) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 × 10⁶ sperm, with 70 ± 5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17β and progesterone were determined in most queens on the day of AI and again 30–40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P = 0.58); overall 33% (5/15) of the queens became pregnant. For frozen–thawed semen, AI was consistently done 28 h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P = 0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen–thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H₂O₂. The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H₂O₂ in the linear range from 2.6 × 10^− 6 mol/L to 8.8 × 10^− 3 mol/L with a detection limit of 6.4 × 10^− 7 mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Variations in hemocyte counts in the mussel,Mytilus edulis: Similar reaction patterns occur in disappearance and return of molluscan hemocytes and vertebrate leukocytes

It was asked whether variations in hemocyte counts in a mussel can be explained by mechanisms known to govern the leukocyte number in vertebrates. Hemolymph of 25 freshly collected Mytilus edulis contained (4.2 ± 1.75) × 10⁶ cells/mL including basophilic and eosinophilic granulocytes and 6.6 ± 5.5% hyalinocytes (15 animals). After 12 or 30 days under optimal laboratory conditions, hemocytes in circulation decreased to less than 1 × 10⁶/mL, the lowest number observed being 5 × 10⁵ cells/mL. Within 2 min of a stressful stimulus, cell numbers doubled or increased by a factor of 3 or 4. After stressing mussels by keeping them out of water for 1 h, cell counts were as high as 1.2 × 10⁷ cells/mL. The quick rate of increase in cell counts is not due to hemocyte proliferation. In mussels, returned to optimal water conditions, cell numbers dropped following an exponential decay curve (y = 5.6865 · (0.9936^X). Not all hemocyte types decreased in number to the same extent. After a strong decrease in the total cell count induced by injection of LPS, the remaining hemocyte population contained a larger percentage of basophils. This indicated the disappearance of eosinophilic cells from the circulation. Stress situations caused their return. Hemocytopenia or stress-induced hemocytosis in M. edulis, both in conjunction with changes in the percentage of granulocytes present, resembles margination/demargination processes in mammals where the concentration of circulating leukocyte subsets depends on the expression of adhesive receptor–ligand molecules on the surface of specific leukocyte types and vascular endothelial cells. In Mytilus edulis, variations in the concentration of distinct cell groups excluded heart activity to explain cell fluctuations. Furthermore, in this mussel, where hemocyte proliferation is not the reason for rapid hemocytosis, cell divisions were nevertheless demonstrated; they seem to be important in maintaining hemocyte homeostasis as 10–20% of cells in circulation possess the capacity to proliferate. They belong to the group of basophilic granulocytes.     

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10⁸ CFU ml^?1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10⁵ spores ml^?1; While the blue mold decay was completely inhibited at concentrations of 5 × 10⁸ CFU ml^?1, at challenged with P. expansum spores suspension of 5 × 10⁴ spores ml^?1_. These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10⁵ CFU per wound to 2.24 × 10⁷ CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.     

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): Purification,primary structure,and inhibitory properties

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K_a = 1.1 × 10⁹ M^?1 and 2.5 × 10⁹ M^?1, respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K_a = 4.5 × 10⁸ M^?1 and 1.2 × 10¹⁰ M^?1). Weak interaction with human plasmin (K_a = 1.2 × 10⁷ M^?1) was also revealed.     

Effects of dietary Bacillus subtilis and inulin supplementation on performance,eggshell quality,intestinal morphology and microflora composition of laying hens in the late phase of production

Eighty Lohmann White laying hens were used to investigate the effect of dietary inclusion of Bacillus subtilis and inulin, individually or in combination, on egg production, eggshell quality, tibia traits, Ca retention, and small intestine morphology and microflora composition from 64 to 75 weeks of age. Hens were randomly distributed into 4 treatment groups, with 5 replicates per treatment and 4 hens per replicate. Treatment groups were fed basal diet (control), basal diet plus 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g), basal diet plus 1 g/kg inulin, or basal diet plus a synbiotic combination of 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g) and 1 g/kg inulin. Dietary supplementation of B. subtilis, inulin or synbiotic improved (P<0.05) feed conversion, egg performance, eggshell quality and calcium retention compared with the control. B. subtilis and synbiotic groups exhibited the highest (P<0.05) increase in egg production and egg weight. Inulin and synbiotic groups exhibited the highest (P<0.05) increase in eggshell thickness and eggshell calcium content, and the lowest (P<0.05) eggshell deformations. Unmarketable eggs were 8.4% (P<0.05) of the total eggs produced by the control group compared to 3.5%, 1.7%, and 1.5% for the B. subtilis, inulin and synbiotic groups, respectively. Tibia density, ash, and Ca content increased (P<0.05) by inulin and synbiotic inclusions, compared with the control. B. subtilis, inulin, and their synbiotic combination increased (P<0.05) villus height and crypt depth in all intestinal segments, compared with the control. B. subtilis and inulin modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of bifidobacteria and lactobacilli, compared with the control. Colonization of the beneficial microflora along with increasing the villi–crypts absorptive area were directly associated with the improvements in performance and eggshell quality. It can be concluded that egg production and eggshell quality of laying hens can be improved (P<0.05) in the late phase of production by dietary inclusion of B. subtilis and inulin.     

Semi-interpenetrating polymer networks (semi-IPNs) for entrapment of laccase and their use in Acid Orange 52 decolorization

Laccase enzyme (L) from Trametes versicolor was entrapped in three hydrogel structures namely poly(acrylamide-N-isopropylacrylamide), P(AAm-NIPA), and semi-interpenetrating networks of poly(acrylamide)/alginate, P(AAm)/Alg, and poly(acrylamide-N-isopropylacrylamide)/alginate, P(AAm-NIPA)/Alg. The optimum temperatures for free and all immobilized systems were found to be 40 °C. For free and immobilized laccase systems of P(AAm-NIPA)-L, P(AAm)/Alg-L and P(AAm-NIPA)/Alg-L, K_m values were found to be 6.7 × 10^?3, 8.8 × 10^?2, 5.5 × 10^?2 and 1.8 × 10^?2 mM; V_max values were calculated as 1.8 × 10^?3, 2.5 × 10^?2, 1.5 × 10^?2 and 6.1 × 10^?3 mM min^?1, respectively. For free and the same immobilized systems, the enzymes retained 42%, 91%, 79% and 86% of their initial activities at the end of 56 days of storage. After using the mentioned immobilized systems repeatedly 10 times, they retained 77%, 71% and 84% of their original activities, respectively. For free and the same immobilized systems, decolorization of Acid Orange 52 (AO52) in 6 h were found to be 63%, 50%, 48% and 66%, respectively. Addition of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, into the assay medium increased these values up to 73%, 73%, 74% and 75%, respectively.     

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

Background aimsWe evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte–colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m² once daily, intravenously on day 1). We also investigated the relationship between the number of SSC^lo CD45^dim CD34^hi cells, SSC^lo ALDH^br cells and engraftment.MethodsThirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n% = 14), Hodgkin's lymphoma (n% = 7), non-Hodgkin's lymphoma (n% = 2), acute myloid leukemia (n% = 2), chronic lymphocytic leukemia (n% = 1) and germ cell testis tumor (n% = 1).ResultsNumbers of SSC^lo CD45^dim CD34^hi cells and SSC^lo ALDH^br cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC^lo CD45^dim CD34^hi cell dose or SSC^lo ALDH^br cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC^lo CD45^dim CD34^hi cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 × 10⁶/kg for platelet recovery. The optimal thresholds for SSC^lo ALDH^br cells were 6.53 × 10⁶/kg for neutrophil recovery and 8.72 × 10⁶/kg platelet recovery.ConclusionsAccording to our data, numbers of SSC^lo ALDH^br cells are in very good agreement with numbers of SSC^lo CD45^dim CD34^hi cells and can be a predictor of stem cell mobilization.     

Synthesis,X-ray crystal structure,DNA/protein binding and cytotoxicity studies of five α-aminophosphonate N-derivatives

Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal structures of 1–5 are monoclinic or triclinic system with the space group P 21/c, P − 1, P − 1, P2(1)/c and P − 1, respectively. All P atoms of 1–5 have tetrahedral geometries involving two O-ethyl groups, one C_α atom, and a double bond O atom. The binding interaction of five new α-aminophosphonate N-derivatives (1–5) with calf thymus(CT)-DNA have been investigated by UV–visible and fluorescence emission spectrometry. The apparent binding constant (Kapp) values follows the order: 1 (3.38 × 10⁵ M⁻¹) > 2 (3.04 × 10⁵ M⁻¹) > 4 (2.52 × 10⁵ M⁻¹) > 5 (2.32 × 10⁵ M⁻¹) > 3 (2.10 × 10⁵ M⁻¹), suggesting moderate intercalative binding mode between the compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the compounds 1–5 showed that the quenching mechanism might be a static quenching procedure. For the compounds 1–5, the number of binding sites were about one for BSA and the binding constants follow the order: 1 (2.72 × 10⁴ M⁻¹) > 2 (2.27 × 10⁴ M⁻¹) > 4 (2.08 × 10⁴ M⁻¹) > 5 (1.79 × 10⁴ M⁻¹) > 3 (1.17 × 10⁴ M⁻¹). Moreover, the DNA cleavage abilities of 1 exhibit remarkable changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have been examined by MTT and shown antitumor effect on the tested cells.     

Spore production in solid-state fermentation of rice by Clonostachys rosea,a biopesticide for gray mold of strawberries

Gray mold caused by Botrytis cinerea is an important disease of strawberry. Clonostachys rosea is a mycoparasite of B. cinerea that reduces fruit losses when used as a biocontrol agent. Since spore production by C. rosea has not been optimized, we investigated factors affecting sporulation under aseptic conditions on white rice grains. The greatest spore production in glass flasks, 3.4 × 10⁹ spores/g-dry-matter (gDM), occurred with an initial moisture content of 46% (w/w wet basis), inoculated with 1 × 10⁶ spores/gDM and hand shaken every 15 days. However, a lower inoculum density (9 × 10³ spores/gDM) and no shaking also gave acceptable sporulation. In plastic bags 1.1 × 10⁸ spores/gDM were produced in 15 days, suggesting that larger scale production may be feasible: with this spore content, 24 m² of incubator space would produce sufficient spores for the continued treatment of 1 ha of strawberry plants.     

Immunotherapeutic effects of chitin in comparison with chitosan against Leishmania major infection

Chitin and chitosan microparticles (MPs) are important immune system stimulators. The aim of this study was to evaluate the protective effects of these compounds in comparison with each other against Leishmania infection in BALB/c mice infected with Leishmania major (L. major).Female BALB/c mice were injected subcutaneously with 2 × 10⁵ promastigotes. Chitin and/or chitosan MPs (< 40 μm) were subcutaneously injected in the BALB/c mice with two-day intervals until two weeks. Mice in all groups were sacrificed at 12 weeks post-infection. Enumeration of viable parasites was performed using limiting dilution assay. Furthermore, the animals (5 mice/group) were sacrificed two weeks post-infection. The lymph node cells were isolated and the effects of the chitinous MPs on the proliferation and production of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) were determined. The mean sizes of lesions were significantly smaller in chitin (0.6 ± 0.12 mm) and chitosan treated groups (1.2 ± 0.8 mm) than in the control group (6.2 ± 1.7 mm) (P < 0.05). The parasite load in the lymph nodes of the treated mice was significantly lower than that in the lymph nodes of controls (1.31 × 10⁶ vs 8.24 × 10⁷ parasite/lymph node [P = 0.032] and 7.49 × 10⁶ vs 8.24 × 10⁷ parasite/lymph node [P = 0.05] for chitin and chitosan MPs treatment, respectively). We found that chitinous MPs induced cell proliferation and that chitin but not chitosan increased TNF-α and IL-10 production. Chitin appears that it has more effect than chitosan against leishmaniasis. The current study revealed that chitinous MPs had significant activity against L. major and could be considered as new therapeutic modality in leishmaniasis.     

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

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.     

Evaluation of new fermentation and formulation strategies for a high endophytic establishment of Beauveria bassiana in oilseed rape plants

Since several years it is known that strains of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) are able to colonize plants as a true endophyte. However, so far no integrated bioprocess engineering approaches have been published where fermentation and formulation strategies are combined to optimize colonization of oilseed rape plant tissues. We therefore aimed at investigating whether and how blastospore (BS) formation can be shifted to resilient submerged conidiospores (SCS) by introducing osmotic stress in different growth phases.When 50 g/L NaCl was added after 48 h to a culture of B. bassiana a yield of 1.4 ± 0.1 × 10¹⁰ SCS/g sucrose in shake flasks and 1.8 ± 0.1 × 10¹⁰ SCS/g sucrose in a stirred tank reactor were obtained. In a bioreactor, 24 h after the addition of NaCl, the formation of BS slowed down, the respiratory quotient decreased and a shift from BS to SCS set in.Following these steps, different formulation strategies, namely encapsulation, film coating and liquid formulation were evaluated. B. bassiana grew out of beads as well as on commercial fungicide-coated seeds. Due to the complete suppression of fungal growth on non-sterile soil, the most suitable option was a foliar application. A liquid formulation consisting of 0.1% Triton X-114, 1% molasses, 1% titanium dioxide and 10⁶ spores/mL was applied on leaf tips. After 14 days, the endophyte was detected by PCR and microscopic analysis in the leaves.Further research should focus on formation of SCS and protection of plants colonized by B. bassiana against herbivorous insects.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana

Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5 × 10⁶ or 1 × 10² promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12 weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4 wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5 × 10⁶ promastigotes developed signs of LCL at 12 wpi while the mice inoculated with 1 × 10² remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5 × 10⁶) compared with subclinical ones (1 × 10²). High TGF-β expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12 wpi.     

Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and the relationship with bloom occurrences in Kesennuma Bay,Japan

While cyst germination may be an important factor for the initiation of harmful/toxic blooms, assessments of the fluctuation in phytoplankton cyst germination, from bottom sediments to water columns, are rare in situ due to lack of technology that can detect germinated cells in natural bottom sediments. This study introduces a simple mesocosm method, modeled after previous in situ methods, to measure the germination of plankton resting stage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellates resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A. pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was conducted in addition to the typical samplings of seawater and bottom sediments to detect the dinoflagellates vegetative cells and resting cysts. Bloom occurrences of A. fundyense were observed June 2014 and February 2015 with maximum cell densities reaching 3.6 × 10⁶ cells m⁻² and 1.4 × 10⁷ cells m⁻², respectively. The maximum germination fluxes of A. fundyense cysts occurred in April 2014 and December 2014 and were 9.3 × 10³ cells m⁻² day⁻¹ and 1.4 × 10⁴ cells m⁻² day⁻¹, respectively. For A. pacificum, the highest cell density was 7.3 × 10⁷ cells m⁻² during the month of August, and the maximum germination fluxes occurred in July and August, reaching 5.8 × 10² cells m⁻² day⁻¹. Thus, this study revealed the seasonal dynamics of A. fundyense and A. pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred one to two months after peak germination, which strongly suggests that both the formation of the initial population by cyst germination and its continuous growth in the water column most likely contributed to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.     

Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and insect larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28 ± 0.15) × 10⁶ U ml⁻¹ and (1.3 ± 0.2) × 10⁶ U ml⁻¹ respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7 ± 0.2) × 10⁶ U ml⁻¹ and (3.5 ± 0.4) × 10⁶ U ml⁻¹ at 2 dpi in Rachiplusia nu larvae and (1.1 ± 0.2) × 10⁶ U ml⁻¹ and (1.0 ± 0.15) × 10⁶ U ml⁻¹ at 5 dpi in Spodoptera frugiperda larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.