Three species of Lagenidiales isolated from the eggs and zoeae of the marine crabPortunus pelagicus

Three species of Lagenidiales were isolated from the eggs and zoeae of the marine crabPortunus pelagicus. One of them,Lagenidium callinectes, is reported for the first time in Japan, with fungal infection in the eggs.Haliphthoros milfordensis was discovered from a zoea of the crab.Atkinsiella okinawaensis was also isolated from a zoea of the crab and described as a new species. Growth temperature range and optimum temperature of the fungi were examined. All of the isolates grew at various concentrations of NaCl or KCl, and optimum growth was observed on PYGS agar containing seawater. Pathogenicity to zoeae of the swimming crabPortunus trituberculatus was demonstrated by using a zoospore suspension of each fungus at 25°C.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

Phylogenetic Analysis of the Complete Genome Sequence of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> Supports the Fungal Origin of Microsporidia and Reveals a High Frequency of Fast-Evolving Genes

Microsporidia are unicellular eukaryotes living as obligate intracellular parasites. Lacking mitochondria, they were initially considered as having diverged before the endosymbiosis at the origin of mitochondria. That microsporidia were primitively amitochondriate was first questioned by the discovery of microsporidial sequences homologous to genes encoding mitochondrial proteins and then refuted by the identification of remnants of mitochondria in their cytoplasm. Various molecular phylogenies also cast doubt on the early divergence of microsporidia, these organisms forming a monophyletic group with or within the fungi. The 2001 proteins putatively encoded by the complete genome of Encephalitozoon cuniculi provided powerful data to test this hypothesis. Phylogenetic analysis of 99 proteins selected as adequate phylogenetic markers indicated that the E. cuniculi sequences having the lowest evolutionary rates preferentially clustered with fungal sequences or, more rarely, with both animal and fungal sequences. Because sequences with low evolutionary rates are less sensitive to the long-branch attraction artifact, we concluded that microsporidia are evolutionarily related to fungi. This analysis also allowed comparing the accuracy of several phylogenetic algorithms for a fast-evolving lineage with real rather than simulated sequences.This article contains online supplementary material.Reviewing Editor: Dr. Wen-Hsiung LiSupplementary material is available at     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Pathogenicity of fungi isolated from the larvae of the mangrove crab,Scylla serrata, in Indonesia

Heavy mortality reaching almost 100% occurred in the larvae of the mangrove crab,Scylla serrata, from July to December 1997 at the hatchery of Gondol Research Station for Coastal Fisheries in Bali, Indonesia. Mortality was observed in larvae after hatching from the eggs. The affected larvae were whitish and filled with numerous aseptate hyphae. Three fungi belonging to the order Lagenidiales,Lagenidium callinectes, Haliphthoros milfordensis, andHalocrusticida sp., were isolated from the infected larvae. Pathogenicity tests of the infected fungi against the larvae of mangrove crab demonstrated that all isolates were pathogenic.     

Molecular and phylogenetic analysis of PCR-amplified cyclin-dependent kinase (CDK) family sequences from representatives of the earliest available lineages of eukaryotes

Cyclin-dependent kinase (CDK) and cell division control (CDC2) sequences are strongly conserved among eukaryotes and may complement the use of other sequence families in eukaryotic phylogenetic inference. We synthesized degenerate PCR primers to amplify the catalytic region of CDK homologs in representatives of the earliest available lineages of eukaryotes. CDK family sequence-based, maximum-likelihood distance measurements with neighbor joining, and Fitch-Margoliash least-squares analyses produced unrooted dendrograms that included protists, yeasts, and higher eukaryotes. Bootstrap confidence estimates supported CDK-based phylogenetic groupings among the protists, fungi, and vertebrates although resolution within these groups was often insignificant. However, Trichomonas vaginalis and Giardia lamblia exhibited two of the most divergent CDK-like sequences consistent with rRNA-phylogenetic inference of early divergence of these eukaryotic lineages. In the evolution from unicellular to multicellular organisms, a constellation of amino acid residues aligning with the human, CDK N-terminal sheet may have undergone abrupt replacement.Abbreviations CDC cell division control - CDK cyclin-dependent kinase family - PCR polymerase chain reaction Correspondence to: D.E. Riley     

Identification of fungi for sweet whey permeate utilization and eicosapentaenoic acid production

Summary Eighteen selected organisms of theEumycota division of the fungi kingdom were examined for eicosapentaenoic acid production and utilization of sweet whey permeate. The organisms belong to the subdivisionsMastigomycotina, Zygomycotina, Ascomycotina andDeuteromycotina. Seven organisms were initially identified as lactose utilizers (the predominant sugar in sweet whey permeate_ and eicosapentaenoic acid (EPA) producers. Utilization of lactose was demonstrated and EPA production was confirmed for four organisms, all of the subdivisionMastigomycotina. Growth studies showed thatP. ultimum had the best potential for future work.     

<Emphasis Type="Italic">Penicillium frequentans</Emphasis> isolated from<Emphasis Type="Italic"> Picea glehnii</Emphasis> seedling roots as a possible biological control agent against damping-off

Picea glehnii seedlings are affected by damping-off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.     

Evolutionary changes reflected by the cellular amino acid composition

Summary Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli,Klebsiella,Proteus, andVibrio alginolyticus) was identical. However, the amino acid composition ofStaphylococcus aureus andBacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium,Chroococidiopsis) was quite similar to that ofE. coli. A marked difference in the amino acid composition was observed betweenE. coli and green alga (Chlorella), and significant differences were observed betweenE. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.     

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protistan diseases of commercially important crabs: a review

Protists are a diverse group of eukaryotes that possess a unicellular level of organization. As unicellular organisms, the differentiation of cells into tissues does not occur, although when cell differentiation does occur, it is limited to sexual reproduction, alternate vegetative morphologies or quiescent life history stages. Protistan parasites may possess simple or complex life histories that are important factors to consider when investigating protistan diseases of decapods. Unfortunately, the life histories of many protistan parasites of decapods are insufficiently described, resulting in the fact that modes of infection and transmission are often unidentified. This is surprising considering the economic importance of many marine decapods and the ability of protistan parasites to produce significant, but generally transient and area limited mortalities. However, the marine disease landscape is changing and will continue to change as climate change and ocean acidification will play important roles in disease occurrence and distribution. As a result, the following discussion attempts to summarize current knowledge on several crab diseases, their protistan etiological agents, the impact of disease on economically important crab populations and draw attention to areas of needed research. The discussion is not complete as only selected diseases are addressed, or perfect as the Microsporidia are included in the discussion (a traditional error continued in this summary) despite the recent, but controversial placement of the taxon with the fungi.     

Dekkera and Brettanomyces growth and utilisation of hydroxycinnamic acids in synthetic media

Dekkera and Brettanomyces yeast are important spoilage organisms in a number of food and beverage products. Isolates of both genera were cultured in a defined medium and supplemented with hydroxycinnamic acids and vinylphenols to investigate their influence on growth and the formation of ethyl phenol derivatives. The growth rate of Brettanomyces species in the presence of acids was reduced, and no significant conversion to vinyl or ethyl derivatives was observed. The growth rate and substrate utilisation rates of Dekkera anomala and Dekkera bruxellensis yeast differed depending on strain and the acid precursor present. Growth of D. bruxellensis was slowed by the presence of ferulic acid with the addition of 1 mM ferulic acid completely inhibiting growth. This study provides an insight into the spoilage potential of these organisms and possible control strategies involving hydroxycinnamic acids.     

Incorporation of ω6 polyunsaturated fatty acids into phospholipids of the crab Carcinus maenas

The incorporation in vivo of ¹⁴C-18:2 ω6 and ³H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle.     

Biochemical identification of thymosin alpha-1: its phylogenetic distribution and evolutionary implications

1. Thymosin alpha-1, like reactivity, was found in several different species (insects, crab, protozoan, fungus and bacteria) by radioimmunoassay and immune fluorescence and as an extracellular product from the bacterial genus Mycobacterium. 2. Biochemically, thymosin alpha-1 has been isolated from combined crab visceral and nervous tissue by reverse phase HPLC. 3. The identification of thymosin alpha-1 in lower life forms suggests a more generalized exocrine origin in unicellular organisms prior to the development of the immune system or exocrine differentiation.     

Impact Scenario for an Introduced Decapod on Arctic Epibenthic Communities

The intentional introduction of a species for the enhancement of stock or establishment of new fisheries, often has unforeseen effects. The red king crabs, Paralithodes camtschaticus, which was introduced into the Barents Sea by Russian scientists, has established a self-sustaining population that has expanded into Norwegian waters. As top benthic predators, the introduced red king crabs may have possible effects upon native epifaunal scallop (Chlamys islandica) communities. These benthic communities may be a source of prey species in late spring, when the red king crabs feed most intensively. Foraging rates (consumption, killing or severely damaging) of red king crab on native prey organisms were measured by factorial manipulation of crab density (0.5, 1.5 and 3 per m ²), size classes (immature, small mature, and large mature crabs), and by evaluating prey consumption after 48 h, in order to extrapolate a scenario of the likely impacts. Foraging rates of the red king crab on scallops ranged between 150 and 335 g per m² within 48 h. These rates did not change when crab density was altered, though an increased amount of crushed scallops left uneaten at the tank floor, were correlated with high density of small mature crabs. Foraging rate changed significantly with crab size. Consequently, the susceptibility of native, shallow water epibenthic communities to red king crab predation in the early life history stages, and during the post-mating/molting spring period, must be considered significant when foraging rates are contrasted with natural scallop biomass between 400 and 1200 g scallops per m².     

Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae

The non-polar lipid content and fatty acid (FA) composition of 11 mushroom species of the family Boletaceae were determined. The non-polar lipid content ranged from 2.0 (Leccinum aurantiacum and Boletus erythropus) to 5.4 % (w/w) d.w. (Suillus grevillei) with an average value of 2.9 %. More than 25 different FAs were found in the mushroom lipids. Unsaturated FAs, mainly linoleic and oleic acids, accounted for about 83 % of the total FAs, while palmitic acid was the main saturated FA. Some FAs are identified for the first time in Boletaceae and in higher Basidiomycetes (cis-11,12-methyleneoctadecanoic acid, 7-cis,10-cis hexadecadienoic) or in fungi (cis-11,12-methyleneoctadecanoic acid). There were significant differences (P < 0.05) in the contents of specific FAs between mushroom species.     

The effect of tree-root exudates on the growth rate of ectomycorrhizal and saprotrophic fungi

Summary The object of this investigation was the promotion by root exudates of the growth rate of ectomycorrhizal fungi, discovered by Elias Melin in 1954. Eight ectomycorrhizal and ten non-mycorrhizal species were used as test fungi in the experiments. Different species often reacted differently: none of the eight isolated strains of Suillus luteus were promoted by pineroot exudate, whereas the growth rates of all seven strains of S. granulatus were increased. Among the other ectomycorrhizal species, S. variegatus, Laccaria bicolor, Pisolithus tinctorius and Thelephora terrestris, each represented by only one, two or three strains, usually reacted to the pine root exudate with an increased growth rate; S. bovinus and Paxillus involutus did not respond at all. Hitherto, studies of root-exudate effects on fungi have been based exclusively on the responses of ectomycorrhizal species; in the present study saprotrophic fungi were also used as test organisms. Seven out of ten saprotrophic species reacted with markedly accelerated growth when exposed to a pineroot exudate. Melin's assumption that a constituent of the root exudate, the M-factor, could replace the exudate growth-promoting activity was verified. By means of TLC fraction it was found that the fatty acid palmitic acid alone caused an increase in growth rate equal to that of the pine-root exudate. In line with previously published data by Gogala (1970), we also showed that certain cytokinins, especially isopentenylaminopurine, could act as substitutes for the total root exudate. Thus both palmitic acid and isopentenylaminopurine are able to function as M-factors equivalent to a root exudate.     

AN HERBICIDE (SULFOMETURON METHYL) RESISTANT MUTANT IN PORPHYRIDIUM (RHODOPHYTA)1

The unicellular red alga Porphyridium sp. is, like many bacteria, fungi and higher plants, sensitive to the sulfonylurea herbicide sulfometuron methyl (SMM). However, the minimal inhibitory concentration for SMM in Porphyridium (55 μM) is higher than in green plants. We isolated a spontaneous SMM-resistant mutant SMR1 of Porphyridium sp. by use of a double-layered agar technique. The mutation frequency and the similarity of the mutant's morphology and growth patterns to the parent strain strongly suggest that SMR1 is a single gene mutation. The activity of the enzyme acetohydroxy acid synthase (AHAS) in crude extracts of the mutant SMR1 is at least two orders of magnitude less sensitive to SMM than that of the parent strain, which indicates that AHAS is the target of SMM (as has been shown in a variety of organisms). We propose that such a mutation, the first isolated in a unicellular rhodophyte, would be a useful marker for genetic studies of Porphyridium. It may also be useful for maintaining unialgal cultures in large scale open ponds.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

Effect of soil liming and vesicular-arbuscular-mycorrhizal inoculation on the growth and micronutrient content of the teff plant

In a greenhouse experiment involving an acid soil teff [Eragrostis tef (Zucc.) Trotter] plants failed to grow unless the soil was limed or inoculated with either of two vesicular-arbuscular-mycorrhizal (VAM) fungi,Glomus mosseae orGlomus macrocarpum. Plant growth increased by liming and to a lesser extent by VAM fungal inoculation. Liming also enhanced root colonization by VAM fungi. Shoot micronutrient content generally increased as a result of inoculation, and decreased by increased lime applications.