Mucor rouxii ultrastructure: cyclic AMP and actin cytoskeleton

Summary. A comparative analysis of the effect of two compounds, dibutyryl–cyclic-AMP (dbcAMP) and latrunculin B, on the morphology and ultrastructure of the dimorphic fungus Mucor rouxii under aerobic growth conditions is presented. dbcAMP acts through the sustained activation of protein kinase A, and latrunculin B through the disruption of the actin cytoskeleton. Upon addition of these compounds to the growth medium at any stage of the germination process, cells lost polarised growth and switched to isodiametric growth. The effect was reversible. The morphologies, visualised by light microscopy or scanning electron microscopy (SEM), were alike. A switch from a rough to a smooth surface was observed by SEM when cells were repolarised by removal of the added compound. Ultrastructural changes under both conditions, as observed by transmission electron microscopy, were similar, the main feature being the enlargement of the cell wall, with irregular depositions, and detachment from the cell membrane. dbcAMP-treated cells showed a decrease in the number of glycogen granules compared with control and latrunculin B-treated cells. F-actin staining with fluorescein isothiocyanate–phalloidin showed that both dbcAMP- and latrunculin B-treated cells displayed a much lower fluorescence than control cells, with only a few pale plaques. The results suggest that the sustained activation of protein kinase A, which impairs polarised growth, might exert its effect through a modification of actin cytoskeleton organisation, very probably also involving an integrinlike pathway, as judged by the cell wall detachment and loss of cell adhesiveness of the dbcAMP-treated isodiametric cells. Correspondence and reprints: Departamento de Química Biológica, Pabellón 2, Piso 4, Ciudad Universitaria, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.     

Apical vesicles and microtubules in rhizoids ofBlastocladiella emersonii: Effects of actinomycin D and cycloheximide on development during germination

Summary The ultrastructure of untreated germinating cells ofBlastocladiella emersonii was compared with that of cells inhibited by Actinomycin D and cycloheximide. The rhizoids and germ tubes of the fungus contained longitudinally oriented microtubules and apical clusters of cytoplasmic vesicles. The vesicles, apparently derived from Golgi apparatus equivalents also described in this paper, were involved in the tip growth of the germ tubes and rhizoids. Zoospores germinated in Actinomycin D encysted and developed short germ tubes containing microtubules and apical vesicles before further development was arrested. Zoospores treated with cycloheximide encysted but did not develop germ tubes.     

<Emphasis Type="Italic">Mucor rouxii</Emphasis> Rho1 protein; characterization and possible role in polarized growth

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70–85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the β-1,3-glucan synthase complex in fungi in which β-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of β-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of β-1,3-glucans on the regulation of morphology of zygomycetes.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

Plant communities and phytogeographical position of a large depression in the Great Chaco,Argentina

A survey is presented of the vegetation of the central region of the Santafesinian Chaco (Argentina), a scarcely populated flat area of 20 000 km², with seasonal flooding. Soils have a strong halo-hydromorphic character and vegetation is basically halophilous. Trees are scarce and most communities are savannas, grasslands or swampy vegetation. Twenty-three communities are described, some of them with several variants. The most widespread communities areSpartina argentinensis grasslands,Elyonurus muticus savannas and a complex of hygrophilous communities. The most important communities are distributed in relation to a topographical gradient, and their structure is shaped by recurrent flooding and fire disturbance. Most of the area is virgin land with very little human interference. The phytogeographical position of the area is discussed. Nomenclature: Burkart, A. 1969, 1974, 1979. Flora ilustrada de Entre Rios, INTA, Buenos Aires, and Cabrera, 1963, 1965a, b, 1967, 1968, 1970. Flora de la Provincia de Buenos Aires, INTA, Buenos Aires.     

COEVOLUTION AND AVIAN BROOD PARASITISM: COWBIRD EGGS SHOW EVOLUTIONARY RESPONSE TO HOST DISCRIMINATION

The Rufous Hornero (Furnarius rufus) is an important host of the brood parasitic Shiny Cowbird (Molothrus bonariensis) in Uruguay, but not in nearby Buenos Aires Province, Argentina. Eggs of the Shiny Cowbird are extremely variable in size, and horneros eject cowbird eggs with widths less than about 88% of the widths of their own eggs. Uruguayan cowbird eggs are, on average, 12% larger than those from Buenos Aires, a geographic pattern in egg size that corresponds to the pattern of successful host use. Uruguayan cowbird eggs are also wider per unit volume than eggs from Buenos Aires. Allometric analyses of egg width and volume indicate that this shape change is apparently an evolutionary response to selection exerted by the hornero. Such a response is absent in Buenos Aires because interactions between the cowbird and the hornero are probably of recent origin there.     

Orientation of cells during slug formation inDictyostelium

Summary Scanning electron microscopic observations ofDictyostelium discoideum cell masses during slug formation revealed two populations around the anterior tip; one group of cells resembled elongated aggregation stream cells and their orientation suggested that they move to the tip, whereas the other group of cells were isodiametric and showed no obvious orientation. In seeking further evidence for a role of differential cAMP chemotaxis in the orientation and movement of slug cells the anterior prestalk cells were compared to the posterior prespore cells in two chemotaxis tests. When a cell mass is placed on cAMP agar the prestalk cells exhibited better movement to cAMP sources but when the gradient was generated in a diffusion chamber the prestalk cells did not. This evidence suggested that the cells which are better able to generate a cAMP gradient might form part of the anterior zone of the slug.     

Contribution à l'etude des thecamoebiens de la Province de Buenos Aires

Résumé La faune des Thécamoebiens fut étudiée dans deux étangs (La Brava et De los Padres) situe dans la Province de Buenos Aires (Argentina). Le nombre d'espèces trouvé en tout a été de 14, quelques-unes desquelles représentées par de différentes formae. Une nouvelle forma Centropyxis compressa (Cushman), forma brava n.f. a été trouvée et décrite, et une espèce de Pontigulasia a été proposée en nomenclatura aperta.

---

Summary The Thecamoebiens of two lakes (La Brava and de los Padres) located in the Province of Buenos Aires (Argentina) were studied. The total number of species found is 14, several of them are represented by various formae. A new forma was found and described, namely Centropyxis compressa (Cushman), forma brava n.f. and one species of Pontigulasia was put in nomenclatura aperta.

---

---

Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires     

Coupled saccharification and fermentation of pre-treated Eucalyptus wood: a simple kinetic model

Coupled saccharification and fermentation of Eucalyptus globulus wood, pre-treated by acid hydrolysis and sodium hypochlorite, was carried out in two column reactors: one for enzymatic hydrolysis of the substrate at 50°C and the other for fermentation of sugars with calcium alginate-immobilized Saccharomyces cerevisiae at 30°C. A buffered solution containing cellulases at pH 4.8 was recycled through both reactors. The maximum yields were about 0.26 g ethanol per g of substrate. The results were reproduced reasonably well using a simple kinetic model consisting of two successive pseudo-first-order reactions.C. Albornoz and D. M. Ferrari are with the Centro de Investigaciones Tecnológicas (CIT), Administración Nacional de Combustibles, Alcohol y Portland (ANCAP), Pando, Canelones, C.P. 91000, Uruguay. S. Blanco and G. Ellenrieder are with Instituto de Investigaciones para la Industria Química (INIQUI), Universidad Nacional de Salta (UNSa), Buenos Aires 177, 4400 Salta, Argentina.     

Effect of methyl-cyclodextrin on adenylate cyclase activity of Bordetella pertussis

The activity of Bordetella pertussis extracytoplasmic adenylate cyclase (AC) decreased during decelerating growth phase in a Stainer-Scholte medium. Neither proteolytic activity nor virulence variation (phase variation; antigenic modulation) appears to be responsible for the observed activity fall. The addition of methyl--cyclo-dextrin enhances AC activity and prevents the inhibition of AC activity by fatty acids. Cyclodextrin could entrap inhibitors increasing in this way the AC activity. These results show that the inclusion of cyclodextrin in the culture medium increases the AC activity.D.F. Hozbor and O.M. Yantorno are with the Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, (1900) La Plata, Argentina. A. Samo is with the Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires.     

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).     

Resistance to freezing temperatures in Aedes (Ochlerotatus) albifasciatus (Macquart) eggs (Diptera: Culicidae) from two different climatic regions of Argentina

Aedes (Ochlerotatus) albifasciatus (Macquart) has the capacity to proliferate in different kinds of climates within its distribution range in South America. With the aim of studying local thermal adaptations of eggs, we exposed egg stocks from two climatically different localities: temperate humid pampa (Buenos Aires) and cold arid Patagonian (Sarmiento), to freezing conditions and then evaluated the effect on some features at this level. First, we thermally described the substrate where this species lays its eggs in the arid region. A typical thermal condition during winter was 10 h at ?12° C. Second, we evaluated the effect of freezing on primary hatching (vs total hatching) and embryo survival. We also compared the proportion of embryonated eggs from both populations. The proportions of embryonated eggs were not different between localities, with averages of 78% and 83% in Sarmiento and Buenos Aires, respectively. Survival was equally successful after freezing in the two localities with an average range between 94–99%. Whether or not the eggs from Buenos Aires and Sarmiento were under freezing conditions, hatching was more than 98% after the first flooding. The results suggest that eggs of Ae. albifasciatus from Sarmiento and Buenos Aires have the same ability to survive at extreme temperatures (<0° C), showing a regional thermal adaptation rather than a local one.     

Detection of dehydrin-like proteins in embryos and endosperm of mature Euterpe edulis seeds

Summary. Euterpe edulis Martius, a tropical palm species characterized as highly recalcitrant, accumulated dehydrin proteins in both the endosperm and the embryo of the mature seed, as detected by Western blot analysis and immunogold electron microscopy. Three major bands at molecular masses of approximately 16, 18, and 24 kDa were identified in both samples analysed. Immunogold electron microscopy studies detected the presence of dehydrins in the embryo and endosperm. In both cases, dehydrins were immunolocalized in cytoplasm and chromatin. No labelling associated with either membranes or organelles was detected. It is known that dehydrins are produced as part of the developmental program of orthodox seeds and are also present in some recalcitrant seeds of temperate regions. The constitutive presence of dehydrins in embryos of extremely recalcitrant species of tropical origin has not been previously reported. Correspondence and reprints: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA, Ciudad Autónoma de Buenos Aires, Argentina.     

Predation upon the subterranean rodent Ctenomys talarum (tuco-tucos) by Buteo polyosoma (red-backed hawks) in coastal grasslands of Argentina

Differential predation upon tuco-tucos (Ctenomys talarum) by red-backed hawks (Buteo polyosoma) is reported, considering the characteristics that could lead to prey vulnerability and to predator selectivity. Predation was evaluated through analysis of 100 pellets and 20 prey remains collected in coastal grasslands at Mar de Cobo (Buenos Aires, Argentina) during winter (May to August) 2003–2004. The tuco-tucos represented the main component in the diet of red-backed hawks, registering a high consumption of sub-adult individuals (49.3%). Juvenile individuals (42.0%) were overrepresented and adults (8.7%) were underrepresented in relation to their field frequencies (14.4 and 43.1%, respectively), whereas sub-adults did not show significant differences. This high consumption of smaller tuco-tucos would be based on the higher vulnerability of young individuals to predation rather than raptor's handling constraints on bigger individuals. Our results indicate that a strong predator–prey relationship between tuco-tucos and red-backed hawks is established at the study area, and that tuco-tucos represent a key component for the survival of wintering hawks.

Reportamos la depredación diferencial sobre el tuco-tuco (Ctenomys talarum) por el aguilucho común (Buteo polyosoma), considerando las características que podrían determinar la vulnerabilidad de la presa y la selectividad del predador. La depredación fue evaluada mediante el análisis de 100 egagrópilas y 20 restos presa recolectados en pastizales costeros de la localidad de Mar de Cobo (Buenos Aires, Argentina) durante el periodo invernal Mayo–Agosto de 2003–2004. El tuco-tuco representó el principal componente en la dieta del aguilucho común, registrándose un alto consumo de individuos subadultos (49.3%). Los individuos juveniles (42.0%) fueron sobrerrepresentados y los adultos (8.7%) fueron subrepresentados en la dieta en relación a sus frecuencias en el campo (14.4 y 43.1%, respectivamente), mientras que los subadultos no mostraron diferencias significativas. Este elevado consumo de individuos más pequeños radicaría en una mayor vulnerabilidad a la depredación de los juveniles más que en restricciones de manipuleo del predador sobre las presas más grandes. Nuestros resultados indican que existe una estrecha relación predador–presa entre el tuco-tuco y el aguilucho común, y que los tuco-tucos representan un componente clave para la supervivencia invernal de los aguiluchos.     

Distributional and biometrical study of appendicularians from the west south Atlantic ocean

This study deals with appendicularians from 21 superficial plankton samples collected in the coast of Argentina, Uruguay and Brazil in October–November, 1972. Seventeen species were identified and their distribution established and compared with the results of previous authors. Megalocercus abyssorum received particular attention because it is a very infrequent species in the Atlantic Ocean. Specimens of the family Oikopleuridae were measured, taking data from trunk and tail. From the state of development of the gonads three different stages were established. The data from each station, grouped by species and state of maturity, were subjected to a conventional statistical treatment, obtaining the mean and standard error for each parameter. In addition, the use of statistical testing techniques validated the three developmental stages established, because from the analysis of variance for all the measurements highly significant results were obtained. An exception must be made for Oikopleura dioica since the only difference is in the height of the trunk. The same pertains to the difference between males and females. Oikopleura longicauda showed significant differences between specimens from coastal and pelagic samples in all measurements of the tail.Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires.Departamento de Ciencias Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.     

Enhanced chitosan production and modeling hyphal growth of Mucor rouxii interpreting the dependence of chitosan yields on processing and cultivation time

Chitosan is a major structural component of fungal cell walls and has diverse medical and other applications. However, cost‐effective culture and extraction methods for fungi need to be developed. Therefore, Mucor rouxii was grown on YPG‐media in both submerged batch and semi‐continuous cultures. Chitosan was extracted from the mycelia to explore strategies to enhance yields and production rates. As observed in earlier studies, M. rouxii is able to adapt to shear stress when cultured semi‐continuously. Modeling the hyphal growth of batch experiments shows that the mycelia were ruptured by shear forces within a short cultivation time shown by a decreased hyphal length. However, an increasing chitosan content was observed with an increasing cultivation period in semi‐continuous cultures, which is an indication for the adaption to shear stress. Semi‐continuous culture resulted in the highest contents of extractable chitosan. The results and models of hyphal growth, including tip extension and branching, suggest that repeated batch cultures may be optimal for chitosan production.     

Effects of fire frequency on survival,growth and fecundity of Paspalum quadrifarium (Lam.) in a grassland of the Flooding Pampa (Argentina)

Abstract Fire and grazing are important factors in the regulation of the grassland structure of the Flooding Pampa (Buenos Aires Province, Argentina dominated by Paspalum quadrifarium plants (henceforth ‘pajonal’)). Winter burning of pajonal is a practice that increases P. quadrifarium growth and its nutritious value for cattle. The objective of this work was to determine the responses of different demographic variables of P. quadrifarium growing in a grassland managed with different fire frequencies. The work was carried out in a pajonal situated in San Ignacio (Ayacucho county, Buenos Aires Province, Argentina). The treatments were: high fire frequency (0.8 fires per year) and low fire frequency (0.4 fires per year). The population of P. quadrifarium was affected by fire frequency. Biomass, number of tillers, and the relative production of tillers per plant did not change with fire frequency. Plant height was negatively affected by fire frequency. Whereas no plant mortality was observed in high fire frequency, 30% of the mature plants died at the end of the experiment in low fire frequency. In both treatments, plant mortality was size‐dependent and juvenile plants were more affected than mature ones. Burning had an immediate negative effect on mature and juvenile plants in terms of fecundity, survival of tillers and basal cover. Tiller survival and fecundity were significantly lower in burned plants than in unburned ones. In the high fire frequency treatment the number of ramets and/or genets and basal cover were greater than in the low fire frequency treatment.     

The Late Endosomal HOPS Complex Anchors Active G-Protein Signaling Essential for Pathogenesis in Magnaporthe oryzae

In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate) highly dynamic tubulo-vesicular network, scaffold active MagA/Gα_S, Rgs1 (a GAP for MagA), Adenylate cyclase and Pth11 (a non-canonical GPCR) in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation) of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.     

Exocellular hydrolases from Penicillium ulaiense

Penicillium ulaiense exhibited carboxymethylcellulase, pectinase, protease, amylase and phenolase activities, while no xylanase, cellulase, lipase or ligninase activities were found. Pectinolytic action was studied in liquid medium, showing low levels of pectinesterase and pectinase production. No mycotoxins were detected by thin-layer-chromatography.The authors are with INIQUI, Universidad Nacional de Salta, Buenos Aires 177, 4400 Salta, Argentina     

Influence of applied electrical fields on yeast and hyphal growth of Candida albicans

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.