Parasitic Chytrids Upgrade and Convey Primary Produced Carbon During Inedible Algae Proliferation.

Microbial parasites have only recently been included in planktonic food web studies, but their functional role in conveying dietary energy still remains to be elucidated. Parasitic fungi (chytrids) infecting phytoplankton may constitute an alternative trophic link and promote organic matter transfer through the production of dissemination zoospores. Particularly, during proliferation of inedible or toxic algal species, such as large Cyanobacteria fostered by global warming, parasites can constitute an alternative trophic link providing essential dietary nutrients that support somatic growth and reproduction of consumers. Using phytoplankton-parasites associations grown under laboratory controlled conditions we assessed the fatty acids and biochemical composition of species with different nutritional quality and followed the metabolic pathway from the algal host and their parasites zoospores using compound-specific stable isotope analysis. This study demonstrated that chytrids are trophic upgraders able to retain essential nutrients that can be transferred to upper trophic levels both in terms of organic matter quantity and nutritional quality. Through the production of zoospores, nutritionally important long-chain polyunsaturated fatty acids that can be consequently assimilated by consumers. We conclude that parasitism at the base of aquatic food webs may represent a crucial trophic link for dietary nutrients and essential biomolecules alternative to herbivory or bacterivory, which can be particularly crucial during the proliferation of inedible or nutritionally inadequate algal species fostered by climate change.

Isolation and Identification of Alkaloids from Poisons of Fire Salamanders ( Salamandra salamandra).

Fire salamanders ( Salamandra salamandra) are conspicuously colored amphibians secreting a skin poison that contains unique steroid alkaloids such as samandarine (1) and samadarone (2), exhibiting toxic as well as antimicrobial activities. Because of their antipredatory and anti-infectious functions, alkaloids from Salamandra poison are of interest with regard to the threat that the lethal fungus Batrachochytrium salamandrivorans ( Bsal) poses to salamanders. Nevertheless, reliable data on the biological activity of Salamandra alkaloids are scarce, in part due to the difficulty to obtain and study those substances. Thus, isolation of pure salamander alkaloids is an important task that might directly contribute to the understanding of Bsal infections. Here we present a noninvasive isolation procedure for samandarine (1) and O-acetylsamandarine (3), as well as for two new alkaloids, O-3-hydroxybutanoylsamandarine (4) and samanone (6), using HPLC. For the first time, high-field NMR data are presented for these alkaloids. Analysis using GC/MS and ESI+-MS, provided important information on the structural variability of these salamander alkaloids.

Unique odd-chain polyenoic phospholipid fatty acids present in chytrid fungi.

Chytrid fungi are ubiquitous components of aquatic and terrestrial ecosystems yet they remain understudied. To investigate the use of phospholipid fatty acids as phenotypic characteristics in taxonomic studies and biomarkers for ecological studies, 18 chytrid fungi isolated from soil to freshwater samples were grown in defined media and their phospholipid fatty acid profile determined. Gas chromatographic/mass spectral analysis indicated the presence of fatty acids typically associated with fungi, such as 16:1(n-7), 16:0, 18:2(n-6), 18:3(n-3) 18:1(n-9), and 18:0, as well as, a number of odd-chain length fatty acids, including two polyunsaturated C-17 fatty acids. Conversion to their 3-pyridylcarbinol ester facilitated GC-MS determination of double-bond positions and these fatty acid were identified as 6,9-17:2 [17:2(n-8)] and 6,9,12-17:3 [17:3(n-5)]. To the best of our knowledge, this is the first report of polyunsaturated C-17 fatty acids isolated from the phospholipids of chytrid fungi. Cluster analysis of PLFA profiles showed sufficient correlation with chytrid phylogeny to warrant inclusion of lipid analysis in species descriptions and the presence of several phospholipid fatty acids of restricted phylogenetic distributions suggests their usefulness as biomarkers for ecological studies.

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells.

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10(®) Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.

Chytrid fungus Batrachochytrium dendrobatidis has nonamphibian hosts and releases chemicals that cause pathology in the absence of infection.

Batrachochytrium dendrobatidis, a pathogenic chytrid fungus implicated in worldwide amphibian declines, is considered an amphibian specialist. Identification of nonamphibian hosts could help explain the virulence, heterogeneous distribution, variable rates of spread, and persistence of B. dendrobatidis in freshwater ecosystems even after amphibian extirpations. Here, we test whether mosquitofish (Gambusia holbrooki) and crayfish (Procambarus spp. and Orconectes virilis), which are syntopic with many amphibian species, are possible hosts for B. dendrobatidis. Field surveys in Louisiana and Colorado revealed that zoosporangia occur within crayfish gastrointestinal tracts, that B. dendrobatidis prevalence in crayfish was up to 29%, and that crayfish presence in Colorado wetlands was a positive predictor of B. dendrobatidis infections in cooccurring amphibians. In experiments, crayfish, but not mosquitofish, became infected with B. dendrobatidis, maintained the infection for at least 12 wk, and transmitted B. dendrobatidis to amphibians. Exposure to water that previously held B. dendrobatidis also caused significant crayfish mortality and gill recession. These results indicate that there are nonamphibian hosts for B. dendrobatidis and suggest that B. dendrobatidis releases a chemical that can cause host pathology, even in the absence of infection. Managing these biological reservoirs for B. dendrobatidis and identifying this chemical might provide new hope for imperiled amphibians.

Evidence for acquisition of virulence effectors in pathogenic chytrids.

BACKGROUND: The decline in amphibian populations across the world is frequently linked to the infection of the chytrid fungus Batrachochytrium dendrobatidis (Bd). This is particularly perplexing because Bd was only recently discovered in 1999 and no chytrid fungus had previously been identified as a vertebrate pathogen. RESULTS: In this study, we show that two large families of known virulence effector genes, crinkler (CRN) proteins and serine peptidases, were acquired by Bd from oomycete pathogens and bacteria, respectively. These two families have been duplicated after their acquisition by Bd. Additional selection analyses indicate that both families evolved under strong positive selection, suggesting that they are involved in the adaptation of Bd to its hosts. CONCLUSIONS: We propose that the acquisition of virulence effectors, in combination with habitat disruption and climate change, may have driven the Bd epidemics and the decline in amphibian populations. This finding provides a starting point for biochemical investigations of chytridiomycosis.

Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs.

The effect of different extraction techniques on the recovery of fatty acids from freeze-dried biomass of two lipid-producing microheterotrophs was examined. Two procedures were used: the extraction of lipids from biomass followed by transesterification of the fatty acids (extraction-transesterification); and the direct transesterification of biomass to produce fatty acid methyl esters (i.e. without the initial extraction step). Variable factors in the extraction-transesterification experiment were the sequence in which solvents were added to the samples, the relative amount of methanol in the solvent mix, and sonication of biomass while in the solvent mix. Variable factors in the direct transesterification experiment were sample size, and reaction duration. Statistical analysis of data (level of significance P<0.05) showed that: (1) extraction of total fatty acids prior to transesterification was significantly more efficient when solvents were added in the order of increasing polarity; (2) neither sonication nor increasing the proportion of methanol in the extraction solvent significantly affected extraction of fatty acids prior to transesterification; (3) efficiency of direct transesterification of fatty acids increased significantly with reaction time; (4) efficiency of direct transesterification of fatty acids was not significantly affected by sample size; (5) the most efficient method for extraction of fatty acids prior to transesterification yielded significantly less fatty acids than the most effective direct transesterification method. While the study examined only two strains, our results suggest that fatty acid analysis methodology for microheterotrophs under consideration for biotechnological exploitation requires optimisation and validation.

Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin.

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.