Two Delta9-stearic acid desaturases are required for Aspergillus nidulans growth and development

Unsaturated fatty acids are important constituents of all cell membranes and are required for normal growth. In the filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives also influence asexual (conidial) and sexual (ascospore) sporulation processes. To investigate the relationship between fatty acid metabolism and fungal development, we disrupted the A. nidulans sdeA and sdeB genes, both encoding Delta9-stearic acid desaturases responsible for the conversion of palmitic acid (16:0) and stearic acid (18:0) to palmitoleic acid (16:1) and oleic acid (18:1). The effects of sdeA deletion on development were profound, such that growth, conidial and ascospore production were all reduced at 22 and 37 degrees C. Total fatty acid content was increased over 3-fold in the DeltasdeA strain, reflected in up-regulation of the expression of the fasA gene encoding the alpha chain of the fatty acid synthase, compared to wild type. Stearic acid accumulated approximately 3-fold compared to wild type in the DeltasdeA strain, while unsaturated fatty acid production was decreased. In contrast, disruption of sdeB reduced fungal growth and conidiation at 22 degrees C, but did not affect these processes at 37 degrees C compared to wild type. Interestingly, ascospore production was increased at 37 degrees C for DeltasdeB compared to wild type. Total fatty acid content was not increased in this strain, although stearic acid accumulated 2-fold compared to wild type, and unsaturated fatty acid production was decreased. Combining the DeltasdeA and DeltasdeB alleles created a synthetic lethal strain requiring the addition of oleic acid to the medium for a modicum of growth. Taken together, our results suggest a role for sdeA in growth and development at all temperatures, while sdeB is involved in growth and development at lower temperatures.     

Serologic evidence of influenza A infection in marine mammals of arctic Canada.

A serologic survey of influenza A antibodies was undertaken on 1,611 blood samples from five species of marine mammals collected from Arctic Canada from 1984-98. Sampling was done in 24 locations throughout the Canadian Arctic encompassing Sachs Harbor (72 degrees N, 125 degrees W), Northwest Territories in the west to Loks Land (63 degrees N, 64 degrees W), Nunavut in the east, to Eureka (80 degrees N, 86 degrees W), Nunavut in the north to Sanikiluaq (56 degrees N, 79 degrees W), Nunavut in the south. A competitive ELISA using a monoclonal antibody (Mab) against influenza A nucleoprotein (NP) was used. Five of 418 (1.2%) belugas (Delphinapterus leucas) and 23 of 903 (2.5%) ringed seals (Phoca hispida) were serologically positive. None of the 210 walruses (Odobenus rosmarus rosmarus), 76 narwhals (Monodon monoceros) and four bowhead whales (Balaena mysticetus) had detectable antibodies to influenza A. Positive belugas were identified from communities on southeast Baffin Island while positive ringed seals came from communities in the eastern, western and high Arctic. Virus isolation attempts on lung tissue from a seropositive beluga were unsuccessful. We believe that influenza A infection in marine mammals is sporadic, the infection is probably self-limiting, and it may not be able to be maintained in these animals. Although the predominant hemagglutinin (H) type was not determined and therefore the pathogenicity of the strains to humans is unknown, the hunting and consumption of marine mammals by the Inuit, may put them at risk for influenza A infection.     

Meiosis and ascospore development in Cochliobolus heterostrophus.

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

CHROMOSOME NUMBERS IN THE HYPOCREALES. II. ASCUS DEVELOPMENT IN NECTRIA CINNABARINA

The cytology of ascus development in Nectria cinnabarina was investigated with the orceinsmear technique, from crozier formation to ascospore maturation. At prophase I synapsis occurs while the chromosomes are still contracted, and the nucleus passes through dictyotene, a diffuse stage rarely seen in plants. A haploid complement of five chromosomes has been precisely determined. The first two divisions in the ascus constitute meiosis, and the third (mitotic) is followed by ascospore delimitation. A fourth division takes place in the ascospore, which is subsequently divided by a septum into two uninucleate cells. Of all species of Nectria thus far investigated N. cinnabarina is the only species in which additional nuclear divisions in the ascus do occur, accounting for the multinucleate condition in the ascospore cells. The bearing that this distinctive nuclear condition has on phylogeny and evolution in the Hypocreales is discussed.     

A phylogenetic study of the genus Haligena (Halosphaeriales, Ascomycota)

The genus Haligena (Halosphaeriales, Ascomycota), with two accepted species, is encountered frequently in marine habitats, especially on wood in temperate regions. Phylogenetic analyses of Haligena elaterophora (type species) and H. salina were undertaken, with partial large subunit ribosomal DNA sequences, to determine their relationships with other closely related genera in the order. The genus was shown to be polyphyletic within the Halosphaeriales with the type species forming a basal clade to the order. Haligena salina constituted a sister clade with weak support of Neptunella longirostris in all analyses. Haligena elaterophora and H. salina differ significantly in the nature of their ascospore appendages: wider, more sticky and strap-like in H. elaterophora and spoon-shaped at the point of attachment; in H. salina they are longer and narrower, finely drawn out filaments. A new genus, Morakotiella, is introduced to accommodate H. salina.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.     

Ascus development in two temperature-sensitive four-spore mutants of Neurospora crassa

Two nonallelic Four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. Expression depends on temperature. The Four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees C) for Fsp-1 or to low temperatures (15-20 degrees C) for Fsp-2. Heterozygous Fsp-1 X Fsp-1+ crosses make eight-spored asci at 15-20 degrees C but produce many four-spored asci at 25 degrees C and mostly four-spored asci at 30 degrees C. Homozygous Fsp-1 X Fsp-1 crosses respond similarly to increasing temperature but make 40-50% four-spored asci even at 20 degrees C. Heterozygous Fsp-2 X Fsp-2+ crosses produce almost exclusively four-spored asci at 15 degrees C but a mixture of four- and eight-spored asci at 20, 25, and 30 degrees C. Homozygous Fsp-2 X Fsp-2 crosses make all four-spored asci at 15 and 20 degrees C and a mixture of four- and eight-spored asci at 25 and 30 degrees C. When both Fsp-1 and Fsp-2 are present in a cross, either homozygous or heterozygous, no asci contain more than four ascospores at any temperature. Limited temperature-shift experiments with Fsp-1 and Fsp-2 show that the sensitive period for Four-spore expression is sometime after meiotic prophase, possibly at interphase II.     

Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan

In a preceding paper (Briza, P., Winkler, G., Kalchhauser, H., and Breitenbach, M. (1986) J. Biol. Chem. 261, 4288-4294), we reported the presence of dityrosine in the outer layers of yeast ascospore walls. Both outer layers seen in electron micrographs of yeast ascospore walls are sporulation-specific. Here we show that the second of these two outer layers consists of chitosan. In intact spores, it is shielded from staining with primulin by the outermost layer. However, in purified spore walls, the second layer is brightly stained by primulin, and hydrolysates of such preparations contain about 10% glucosamine relative to spore wall dry weight. The spore wall material staining with primulin is resistant to chitinase, but readily degraded by treatment with HNO2. Acetylation prior to HNO2 treatment completely prevents its degradation. A partial acid hydrolysate of spore walls contains predominantly soluble poly-beta-(1,4)-glucosamine as determined by 13C NMR spectroscopy. By these criteria, the glucosamine polymer of yeast ascospore walls is chitosan. As spore walls treated with alkali lack the inner layers but contain chitosan and as chitosan is not exposed at the surface of the spore, we conclude that it is localized in the second outer layer of the spore wall.     

Purification and characterization of a natural agglutinin in the hemolymph of the prawn Penaeus indicus H. Milne Edwards.

A natural agglutinin in the hemolymph of the marine prawn Penaeus indicus was isolated by gel filtration chromatography, purified using polyacrylamide gel electrophoresis, and characterized. Prawn agglutinin has a native molecular mass of 181 kDa and consists of two monomeric units (97 and 84 kDa), maintains some agglutinating activity over a wide pH range (7-9), and is inactivated at 85 degrees C. The agglutinin was denatured upon mixing with trichloroacetic acid, phenol, chloroform, and 45% ammonium sulfate. It was also sensitive to trypsin digestion. The results indicate that prawn agglutinin is proteinaceous in nature, with agglutinating, hemolytic, and antibacterial properties against marine bacteria and erythrocytes with carbohydrate binding sites.     

Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans

In the Ascomycete fungus Aspergillus nidulans, the ratio of conidia (asexual spores) to ascospores (sexual spores) is affected by linoleic acid moieties including endogenous sporogenic factors called psi factors. Deletion of odeA (Delta odeA), encoding a Delta-12 desaturase that converts oleic acid to linoleic acid, resulted in a strain depleted of polyunsaturated fatty acids (18:2 and 18:3) but increased in oleic acid (18:1) and total percent fatty acid content. Linoleic acid-derived psi factors were absent in this strain but oleic acid-derived psi factors were increased relative to wild type. The Delta odeA strain was reduced in conidial production and mycelial growth; these effects were most noticeable when cultures were grown at 26 degrees C in the dark. Under these environmental conditions, the Delta odeA strain was delayed in ascospore production but produced more ascospores than wild type over time. This suggests a role for oleic acid-derived psi factors in affecting the asexual to sexual spore ratio in A. nidulans. Fatty acid composition and spore development were also affected by veA, a gene previously shown to control light driven conidial and ascospore development. Taken together our results indicate an interaction between veA and odeA alleles for fatty acid metabolism and spore development in A. nidulans.     

Ascospore discharge in Pleospora herbarutn (Pers.) Rabenh. (Dothideales) stimulated by near-ultraviolet radiation

An isolate of P. herbarum from beet seed failed to discharge ascospores in darkness but did so when exposed to light either continuously or cyclically (12 h light/12 h dark). When colonies with mature asci were subjected to a regime of alternating light and darkness for 54 days at a constant temperature of 20^°C, ascospores were discharged over the entire period. Maximum discharge occurred on the 23rd day; few spores were liberated towards the end of the period. Light-induced spore discharge occurred over a wide temperature range (10–30^°C) with the optimum being approximately 14–23^°C. When light of different wavelengths (300 nm-infrared) was tested, only near-ultraviolet (310–330 nm) radiation stimulated ascospore discharge. Vertical height of ascospore discharge was also determined. When ascospores were trapped above colonies over a range of heights (2–80 mm), most spores were caught at 2 mm; none was caught at heights above 30 mm. The number of spores trapped at 30 mm was only 1.3% of the capture at 2 mm.     

Meiosis and ascospore development in Cochliobolus heterostrophus

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

Metal reduction and iron biomineralization by a psychrotolerant Fe(III)-reducing bacterium, Shewanella sp. strain PV-4

A marine psychrotolerant, dissimilatory Fe(III)-reducing bacterium, Shewanella sp. strain PV-4, from the microbial mat at a hydrothermal vent of Loihi Seamount in the Pacific Ocean has been further characterized, with emphases on metal reduction and iron biomineralization. The strain is able to reduce metals such as Fe(III), Co(III), Cr(VI), Mn(IV), and U(VI) as electron acceptors while using lactate, formate, pyruvate, or hydrogen as an electron donor. Growth during iron reduction occurred over the pH range of 7.0 to 8.9, a sodium chloride range of 0.05 to 5%, and a temperature range of 0 to 37 degrees C, with an optimum growth temperature of 18 degrees C. Unlike mesophilic dissimilatory Fe(III)-reducing bacteria, which produce mostly superparamagnetic magnetite (<35 nm), this psychrotolerant bacterium produces well-formed single-domain magnetite (>35 nm) at temperatures from 18 to 37 degrees C. The genome size of this strain is about 4.5 Mb. Strain PV-4 is sensitive to a variety of commonly used antibiotics except ampicillin and can acquire exogenous DNA (plasmid pCM157) through conjugation.     

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.     

Analyses of air samples for ascospores ofLeptosphaeria maculans andL.biglobosa by light microscopy and molecular techniques

Spores of many fungal pathogens are dispersed by wind. Detection of these airborne inocula is important in forecasting both the onset and the risk of epiphytotics. Species-specific primers targeted at the internal transcribed spacer (ITS) region ofLeptosphaeria maculans andL. biglobosa — the causal organisms of phoma stem canker and stem lesions ofBrassica spp., including oilseed rape — were used to detect DNA extracted from particles deposited on tapes obtained from a spore trap operated in Rarwino (northwest Poland) from September to November in 2004 and 2006. The quantities of DNA assessed by traditional end-point PCR and quantitative real-time PCR were compared to microscopic counts of airborne ascospores. Results of this study showed that fluctuations in timing of ascospore release corresponded to the dynamics of combined concentrations of DNA fromL. maculans andL. biglobosa, with significant positive correlations between ascospore number and DNA yield. Thus the utilization of PCR-based molecular diagnostic techniques enabled the detection, identification, and accurate quantification of airborne inoculum at the species level. Moreover, real-time PCR was more sensitive than traditional PCR, especially in years with low ascospore numbers.     

Ultrastructure of germination and mucilage production inHalosphaeria appendiculata (Halosphaeriaceae)

The germination of ascospores of the marine fungusHalosphaeria appendiculata was investigated with transmission electron microscopy. Prior to germination, settled ascospores became surrounded by a fibro-granular layer. Small, membrane-bounded vesicles and larger electron-dense membrane-bounded vesicles aggregated at the site of germ tube formation where the plasmalemma adjacent to the aggregation was convoluted. The vesicles appeared to fuse with the plasmalemma, releasing their contents. Enzymatic digestion of the spore wall probably occurred at the time of germ tube emergence. After the nucleus had migrated into the newly formed germ tube, a septum was formed to delimit the germ tube from the ascospore. The growing germ tube can be divided into 3 morphological regions, namely the apical, sub-apical and vacuolated regions, and is typical of other fungi. A mucilaginous sheath was associated with the older mycelium. The germ tube displaced the polar appendage, and the ascospore, germ tube and appendage were enclosed in a mucilaginous sheath. In ascospores which subtended old germ tubes, the nucleus and lipid body became irregular in shape and the cytoplasm was more vacuolated. Microbody-like structures remained associated with the lipid throughout development, and were present in old ascospores.     

Ultrastructure of Weddellomyces epicallopisma (Dothideales, Dacampiaceae, Ascomycota), especially of its ascospores]

An ultrastructural study of Weddellomyces epicallopisma (ascomata wall, asci, ascospores and vegetative hyphae), the first done on the family Dacampiaceae, confirms most of the observations made in light microscopy. Moreover it shows that ascospores are provided with an endospore (not visible in light microscope) and that the structure of the ascospore septum is more complex. The similarity of the wall structure between the ascospore and the hyphoid appendages, developed on the upper part of the ascoma, is emphasized.     

Historical ecology of the Raja Ampat Archipelago, Papua Province, Indonesia

This work presents a review of the status of marine resources of the Raja Ampat Archipelago, Papua Province, Indonesia, based on narratives of early European expeditions in various museums and libraries in Europe, Canada, and local archives in Papua. More than 500 pertinent documents on the study area were identified and located in various European museums and at the University of British Columbia library. About half of these were scanned (25,000 pages), which yielded the equivalent of 900 pages of text (or 4% of the total number of pages scanned) with observations on abundance and impact of the human population on the marine ecosystem within 2 degrees North and 2 degrees South between 127 degrees and 132 degrees East. In general, these observations, which spanned the period from 1810 to the present, suggest a decrease in the perceived occurrences of turtles, fish, and invertebrates; perceived abundance of turtles, fish, and algae; percieved subsistence exploitation of marine resources; and an increase in perceived commercial exploitation of marine resources. We conclude with a discussion of the problems and potential of contents analysis, and its use in the historical reconstruction of broad biodiversity trends.     

Dityrosine is a prominent component of the yeast ascospore wall. A proof of its structure

The yeast ascospore wall consists of four morphologically distinct layers. The hydrophobic surface layers are biogenically derived from the prospore wall and appear dark after OsO4 staining. They seem to be responsible for the stability of the spores against attack by lytic enzymes. By amino acid analysis of acid hydrolysates of ascospore walls, two new peaks were detected, which were shown to be the racemic and meso form, respectively, of dityrosine. The identity of this hitherto unknown component of the yeast ascospore wall with standard dityrosine was proven by 1H NMR and by mass spectrometry. A 13C NMR spectroscopic investigation of the structure of dityrosine confirmed that, in natural dityrosine, the biphenyl linkage is located ortho, ortho to the hydroxyl groups. Following digestion of the inner layers of isolated ascospore walls it was shown that dityrosine is very probably located only in the surface layers. The same conclusion was reached independently by an investigation of spores of a strain homozygous for the mutation gcn1, which lack the outermost layers of the spore wall and were practically devoid of dityrosine. In sporulating yeast, L-tyrosine was readily incorporated into the dityrosine of the ascospore wall. Control experiments involving vegetative a/alpha cells and nonsporulating alpha/alpha cells under sporulation conditions showed that dityrosine is indeed sporulation-specific.     

Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores.

Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate. It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain. In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose. Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate. None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate. The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5. Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.