Cellulase enhances endophytism of encapsulated Metarhizium brunneum in potato plants

The recent discovery that entomopathogenic fungi can grow endophytically in plant tissues has spurred research into novel plant protection measures. However, current applications of fungi aiming at endophytism mostly lack targeted formulation strategies resulting in low efficacy. Here, we aimed at enhancing Metarhizium brunneum CB15 endophytism in potato plants by (i) improvement of fungal growth from beads and (ii) cellulase formation or addition to encapsulated mycelium. We found that beads supplemented with cellulose alone or in addition with inactivated baker's yeast exhibited cellulase activity and increased mycelial growth by 12.6 % and 13.6 %, respectively. Higher enzymatic activity achieved by cellulase co-encapsulation promoted a shift from mycelial growth to spore formation with maximum numbers of 2.5 × 10⁸ ± 6.1 × 10⁷ per bead. This correlated with improved endophytism in potato plants by 61.2 % compared to non-supplemented beads. Our study provides first evidence that customized formulations of fungal entomopathogens with enzymes can improve endophytism and this may increase efficacy in plant protection strategies against herbivorous pests.     

Complement component C1q and its receptor are involved in the interaction of human sperm with zona-free hamster eggs

C1q is a component of the classical complement pathway that can react with the Fc-fragment of immunoglobulins and with other proteins, such as fibronectin, laminin, and a specific C1q receptor present on several cell types. Given its role in many adhesion systems, mainly related to phagocytosis, we tested the effects of C1q on the interaction between human spermatozoa and zona-free hamster eggs. The presence of C1q in the medium used for gamete coincubation resulted in promotion of sperm-oolemma adhesion and an inhibition of penetration. The number of adherent sperm per egg at 5 micrograms/ml concentration was 90 +/- 35 vs. 29 +/- 7 for the control (P less than 0.001). At 1 microgram/ml, the lower concentration at which C1q had an effect, the number of penetrating sperm/egg was 0.6 vs. 1.7 for the control without C1q (P less than 0.01), and the percent of penetrated eggs was 28% vs. 85%. At 50 micrograms/ml, the percent of penetrated eggs was 7%, with a penetration index of 0.07. The addition of C1q to the medium resulted in sperm agglutination, which varied between sperm donors. The presence of C1q receptors, as detected by anti-C1qR monoclonal antibodies (Mabs), was demonstrated both on zona-free hamster eggs by immunobead rosetting and on human spermatozoa by immunobead binding and indirect immunofluorescence. Mabs directed against different epitopes of C1qR had different effects on gamete interaction, with a partial inhibition of penetration mediated by some of them. The binding of C1q to antibody-free human spermatozoa was also demonstrated both by means of indirect immunofluorescence and utilizing 125I-C1q.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hamster oocyte penetrability during preovulatory maturation

In vitro fertilization techniques were used to analyze the penetrability of preovulatory hamster oocytes. The zonas of granulosa cell-free primary (GV) oocytes were penetrated in vitro in 2-3 h as readily as those of ovulated secondary oocytes (80% vs. 88%), whether inseminated separately or as mixed oocyte groups. In fact, a significantly higher (P less than 0.05) mean number of perivitelline spermatozoa was present in immature (3.6) compared with secondary (1.9) oocytes, primarily reflecting a lack of the zona block to polyspermy in the immature population. By contrast, when granulosa cells remained around GV oocytes, zona penetration was low and more were penetrated, with more spermatozoa incorporated into the vitellus as a function of increasing time of oocyte recovery after hCG. We conclude, contrary to previous reports, that the zona pellucida of the hamster GV oocyte is readily penetrable by spermatozoa in vitro. However, the resumption of meiosis brings an increase in the penetrability of the granulosa cell vestment as well as the capacity for cortical granule exocytosis and the ability to decondense and transform the fertilizing sperm nucleus. The fact that the zona pellucida of the immature oocyte has proved to be penetrable in vitro and/or in vivo in all the mammals studied in this respect is discussed with particular reference to the situation in man.     

Influence of fruit traits on the infestation of Dacus persicus in two fruit morphs of Calotropis procera

The larvae of Dacus (Leptoxyda) persicus (aak fruit fly) are key predispersal seed predators in Calotropis procera (Asclepiadaceae). Based on fruit characteristics, two morphs are distinguishable in C. procera viz., the soft-fruited morph (SF morph) and the hard-fruited morph (HF morph). The work reported here examined whether the fruit characteristics influenced the infestation by the aak fruit fly and, if so, what mechanism(s) were operative. Fruits in the SF morph were significantly more acceptable to the aak fruit fly than those of the HF morph irrespective of their size class and availability or fly population density. A general ranking of fruit acceptability for oviposition by the aak fruit fly within the fruit size class was: size class III ≥ size class II > size class I and IV. The negative relationship between fruit infestation and pericarp toughness, which is suggestive of trade-offs between the fly’s oviposition obligation and energy/time (predation risk) constraint, was found to correlate with the requirement of greater force to puncture the pericarp in the hard fruits. Lower penetrability of the pericarp in the hard fruits appeared to be primarily due to the thickness of pericarp and secondarily on account of the thickened walls of endocarpic–mesocarpic cells in the inner pericarpic layer. The present data point to the existence of two fruit morphs in C. procera differing in the acceptability of fruits for oviposition by the aak fruit fly primarily on account of toughness and internal structure of the pericarp.     

Skin absorption and human risk assessment

A common practice is to assume that percutaneous absorption does not significantly contribute to total bioavailability and therefore, absorption through other routes is more important to human risk assessment. The skin can represent a significant barrier to absorption, but some substances are absorbed to a significant extent. Since there is a potential for percutaneous penetration that is not consistent between species or substances, the assessment of the potential contribution of total body burden from dermal exposures should be considered. This review briefly discusses some theories, practices, and factors that affect percutaneous absorption with an emphasis on how percutaneous absorption evaluations apply to human risk assessment.     

Effect of several factors on the mechanical properties of pressure-sensitive adhesives used in transdermal therapeutic systems

The effects of coating thickness, type of adhesive, and type and concentration of enhancer on the mechanical properties of two acrylic pressure-sensitive adhesives (PSAs) were investigated using a 2(4) factorial design and an optimization technique. Sixteen formulations containing 0% or 10% of either caprylic acid or methyl laurate in two different PSAs, namely Duro-Tak 87-2196 and Duro-Tak 87-2097, were prepared. The adhesive properties of these laminates were evaluated by applying the 90 degrees Dynamic Adhesive Strength Peel Test (90 degrees DASPT) and 1800 Release Liner Peel Test (180 degrees RLPT). Coating thickness, concentration of enhancer, and type of adhesive did affect the 90 degrees DASPT. For the 180 degrees RLPT, the most significant factors were coating thickness and concentration of enhancer, with a strong interaction observed between the two. Coating thickness and concentration of enhancer were also used to create mathematical models that correlated these factors with the mechanical properties of the PSAs. For this purpose, the optimization technique 3(2) was applied. It was found that the correlation of the above factors can be adequately described with polynomial equations, which can be used for predicting the mechanical properties of the laminates containing the above PSAs and methyl laurate (0%-10%).     

Wood penetration ability of hydrogen cyanide and its efficacy for fumigation of Anoplophora glabripennis,Hylotrupes bajulus (Coleoptera), and Bursaphelenchus xylophilus (Nematoda)

Pinewood nematodes (Bursaphelenchus xylophilus) and Asian longhorned beetles (Anoplophora glabripennis) are the primary regulated pests for packaging wood and timber in the EU, while the house longhorned beetle (Hylotrupes bajulus) is the most important cosmopolitan pest of construction wood. Gaseous hydrogen cyanide (HCN) is one of the few fumigation alternatives to the banned ozone-depleting chemical methyl bromide (MBr). This study reports the results of HCN fumigation experiments in a hermetically sealed steel chamber regarding (1) the penetration and absorption rates of HCN in wooden blocks, and (2) the biological efficacy of HCN against the wood-infesting pests B. xylophilus (in sawdust), A. glabripennis, and H. bajulus (in wooden blocks). A concentration equilibrium for HCN (at 20 g m⁻³) between the fumigation chamber headspace and the center of the treated spruce blocks (100 × 100 × 120 mm) was reached after 48 h in the saturated atmosphere. A dose of 10 g m⁻³ in the center of the spruce blocks was reached for both saturated and non-saturated atmospheres after 24 h of fumigation. The wood tested absorbed approximately 40–45% of the HCN, until equilibrium was reached. The highest tested HCN dose (20 g m⁻³) led to 100% mortality of the A. glabripennis and H. bajulus larvae after less than 1 h of exposure. For 20 g m⁻³ and 1 h exposure the Ct product was <18.66 g*h/m³ for H. bajulus and <17.67 g*h/m³ for A. glabripennis. Hydrogen cyanide doses of 10 g m⁻³ and 20 g m⁻³ led to 100% B. xylophilus mortality in 40 and 18 h, respectively. For B. xylophilus the Ct product was <424.00 g*h/m³ for 20 °C and 10 g m⁻³ and <349.51 g*h/m³ for 25 °C and 20 g m⁻³. The initial results are promising in terms of establishing an alternative technology and protocol to MBr for timber fumigation.     

Schistosoma mansoni: cercaricidal effect of 2-tetradecenoic acid, a penetration stimulant

Schistosoma mansoni cercariae are stimulated by 2-tetradecenoic acid (TDA) to penetrate agar substrates. TDA simultaneously causes tegumental transformation similar to that seen when cercariae transform to schistosomula, reduces the Cercarienhüllen reaction in immune human serum, and reduces larval tolerance to water. TDA damages cercariae that fail to penetrate or have no opportunity to do so. This damage apparently stems from increased tegumental permeability to water. Preincubation in TDA for 60 min reduces the percutaneous infectivity of cercariae to mice by from 95% at 0.2 ppm to 100% at 0.7 ppm TDA, but does not reduce the infectivity of subcutaneously injected cercariae. The interference with percutaneous infection seems to be entirely due to osmotic damage. TDA does not induce premature secretion of the acetabular glands or block host-recognition chemoreceptors. TDA may be a promising cercaricide for schistosomiasis control. It is highly specific for schistosome cercariae and is effective at low concentrations (0.2 to 0.7 ppm). Both cercariae and TDA tend to collect in the upper few millimeters of standing water. It is unlikely that cercariae can evolve resistance to a chemical that triggers the host penetration mechanisms.     

Ultrastructure of infection of Cokeromyces recurvatus by Piptocephalis unispora (Mucorales)

Infection of the mucoraceous host Cokeromyces recurvatus by Piptocephalis unispora was studied ultrastructurally, using a new technique involving yeast-phase cells of the host to obtain large numbers of infection sites for thin-sectioning. Morphologically, the haustorial apparatus was similar to that of fungi parasitic on higher plants, and comprised an appressorium, a neck region with a collar and a neck ring, and a lobed region surrounded by a sheath matrix enclosed in an extra-haustorial membrane. Penetration of the host by the infection peg probably involved both enzymatic degradation and physical pressure. Reaction of the host to infection is described and the results related to the theory of host infection by haustorial fungal parasites.