Biochemical and proteomic response of the freshwater green alga Pseudochlorella pringsheimii to iron and salinity stressors.

BACKGROUND: Pseudochlorella pringsheimii (Ppr) is a green unicellular alga rich with chlorophyll, carotenoids, and antioxidants. As a widespread organism, Ppr must face, and adapt to, many environmental stresses and these are becoming more frequent and more extreme under the conditions of climate change. We therefore focused on salinity induced by NaCl and iron (Fe) variation stresses, which are commonly encountered by algae in their natural environment. RESULTS: The relatively low stress levels improved the biomass, growth rate, and biochemical components of Ppr. In addition, the radical-scavenging activity, reducing power, and chelating activity were stimulated by lower iron concentrations and all NaCl concentrations. We believe that the alga has adapted to the stressors by increasing certain biomolecules such as carotenoids, phenolics, proteins, and carbohydrates. These act as antioxidants and osmoregulators to protect cell membranes and other cellular components from the harmful effects of ions. We have used SDS-PAGE and 2D-PAGE in combination with tandem mass spectrometry to identify responsive proteins in the proteomes of stressed vs. non-stressed Ppr. The results of 2D-PAGE analysis showed a total of 67 differentially expressed proteins, and SDS-PAGE identified 559 peptides corresponding to 77 proteins. Of these, 15, 8, and 17 peptides were uniquely identified only under the control, iron, and salinity treatments, respectively. The peptides were classified into 12 functional categories: energy metabolism (the most notable proteins), carbohydrate metabolism, regulation, photosynthesis, protein synthesis, stress proteins, oxido-reductase proteins, transfer proteins, ribonucleic-associated proteins, hypothetical proteins, and unknown proteins. The number of identified peptides was higher under salinity stress compared to iron stress. CONCLUSIONS: A proposed mechanism for the adaptation of Ppr to stress is discussed based on the collected data. This data could serve as reference material for algal proteomics and the mechanisms involved in mediating stress tolerance.

Cooperative antioxidative defense of the blue-green alga Arthrospira (Spirulina) platensis under oxidative stress imposed by exogenous application of hydrogen peroxide.

Hydrogen peroxide (H2O2) is an environmentally-safe algaecide used to control harmful algal blooms and as a disinfectant in various domestic and industrial applications. It is produced naturally in sunny-water or as a by-product during growth, and metabolism of photosynthetic organisms. To assess the impact of H2O2 on Arthrospira platensis, several biochemical components, and antioxidant enzymes were analysed. The growth and biomass of A. platensis were decreased under the effect of H2O2. Whereas, the concentration up to 40 µM H2O2 non-significantly induced (at P < 0.05) the Chl a, C-phycocyanin (C-PC), total phycobiliprotein (PBP), and the radical scavenging activity of A. platensis. The half-maximal effective concentrations (EC50) for H2O2 were 57, 65, and 74 µM H2O2 with regards to the biomass yield, Chl a, and C-PC content, respectively. While, the total soluble protein, and soluble carbohydrates contents were significantly induced. However, the higher concentrations (60 and 80 µM) were lethal to these components, in parallel to the initiation of the lipid peroxidation process. Surprisingly, the carotenoids content was non-significantly increased by H2O2. Despite the relative consistency of catalase (CAT), the activities of superoxide dismutase (SOD) and peroxidase (POD) enzymes were boosted by all of the tested concentrations of H2O2. The relative transcript abundance of selected regulatory genes was also investigated. Except for the highest dose (80 µM), the tested concentrations had almost inhibitory effect on the relative transcripts of heat shock protein (HSP90), glutamate synthase (GOGAT), delta-9 desaturase (desC), iron-superoxide dismutase (FeSOD) and the Rubisco (the large subunit, rbcL) genes. The results demonstrated the importance of the non-enzymatic and enzymatic antioxidants for the cumulative tolerance of A. platensis.

Antioxidant enzymes of Pseudochlorella pringsheimii under two stressors: variation of SOD Isoforms activity.

Algae are always facing the challenge of exposure to different stress conditions, therefore raising challenges of adaptation for survival. In this context, the growth and the antioxidant enzymes of the green stress-tolerant alga Pseudochlorella pringsheimii were investigated under two environmental stresses viz. iron and salinity. The number of algal cells was moderately increased by iron treatment in the range of 0.025-0.09 mM of iron, yet, the number of cells decreased at high iron concentrations (0.18 to 0.7 mM Fe). Furthermore, the different NaCl concentrations (8.5-136.0 mM) had an inhibitory effect on the algal cell number, compared to the control.The superoxide dismutase (SOD) showed three isoforms namely; Mn, Fe, and Cu/Zn SOD. The in gel and in vitro (tube-test) activities of FeSOD were higher compared with the other SOD isoforms. The activity of total SOD and its isoforms increased significantly by the different concentrations of Fe and non-significantly by NaCl. The maximum SOD activity was recorded at 0.7 mM Fe (67.9% above control). The relative expression of FeSOD was high under iron and NaCl at 8.5 and 34 mM, respectively. However, FeSOD expression was reduced at the highest NaCl tested concentration (136 mM). In addition, the antioxidant enzyme activity of catalase (CAT) and peroxidase (POD) were accelerated by increasing iron and salinity stress which indicates the essential role of these enzymes under stress. The correlation between the investigated parameters was also investigated. A highly significant positive correlation between the activity of total SOD and its isoforms, and with the relative expression of FeSOD was observed.

Identification and analyses of the chemical composition of a naturally occurring albino mutant chanterelle.

White chanterelles (Basidiomycota), lacking the orange pigments and apricot-like odour of typical chanterelles, were found recently in the Canadian provinces of Québec (QC) and Newfoundland & Labrador (NL). Our phylogenetic analyses confirmed the identification of all white chanterelles from NL and QC as Cantharellus enelensis; we name these forma acolodorus. We characterized carotenoid pigments, lipids, phenolics, and volatile compounds in these and related chanterelles. White mutants of C. enelensis lacked detectable ß-carotene, confirmed to be the primary pigment of wild-type, golden-orange individuals, and could also be distinguished by their profiles of fatty acids and phenolic acids, and by the ketone and terpene composition of their volatiles. We detected single base substitutions in the phytoene desaturase (Al-1) and phytoene synthase (Al-2) genes of the white mutant, which are predicted to result in altered amino acids in their gene products and may be responsible for the loss of ß-carotene synthesis in that form.

How do genes flow? Identifying potential dispersal mode for the semi-aquatic lichen Dermatocarpon luridum using spatial modelling and photobiont markers.

BACKGROUND: Landscape genetics is an interdisciplinary field that combines tools and techniques from population genetics with the spatially explicit principles from landscape ecology. Spatial variation in genotypes is used to test hypotheses about how landscape pattern affects dispersal in a wide range of taxa. Lichens, symbiotic associations between mycobionts and photobionts, are an entity for which little is known about their dispersal mechanism. Our objective was to infer the dispersal mechanism in the semi-aquatic lichen Dermatocarpon luridum using spatial models and the spatial variation of the photobiont, Diplosphaera chodatii. We sequenced the ITS rDNA and the ß-actin gene regions of the photobiont and mapped the haplotype spatial distribution in Payuk Lake. We subdivided Payuk Lake into subpopulations and applied four spatial models based on the topography and hydrology to infer the dispersal mechanism. RESULTS: Genetic variation corresponded with the topography of the lake and the net flow of water through the waterbody. A lack of isolation-by-distance suggests high gene flow or dispersal within the lake. We infer the dispersal mechanism in D. luridum could either be by wind and/or water based on the haplotype spatial distribution of its photobiont using the ITS rDNA and ß-actin markers. CONCLUSIONS: We inferred that the dispersal mechanism could be either wind and/or water dispersed due to the conflicting interpretations of our landscape hypotheses. This is the first study to use spatial modelling to infer dispersal in semi-aquatic lichens. The results of this study may help to understand lichen dispersal within aquatic landscapes, which can have implications in the conservation of rare or threatened lichens.

Development and validation of a reliable LC-MS/MS method for quantitative analysis of usnic acid in Cladonia uncialis.

OBJECTIVE: The purpose of this study was to develop and validate a specific and sensitive liquid chromatography tandem mass-spectrometry method for quantification of usnic acid concentration in the lichen, Cladonia uncialis, suitable for detection of relatively small fluctuations of usnic acid concentration in response to environmental changes. RESULTS: The resulting method was fully validated according to international guidelines and demonstrated good selectivity and sensitivity with minor levels of a matrix effect and high accuracy.

Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta).

The FeSOD isoforms of Pseudochlorella pringsheimii were identified, a preliminary characterization of the enzyme was conducted, and the relationship among the FeSOD gene from P. pringsheimii and that of other organisms was examined. The FeSOD has an open reading frame of 612 bp that encodes 203 deduced amino acids with a molecular mass of 23 kDa. Expression of the recombinant FeSOD gene was done successfully in Escherichia coli. The purified FeSOD has a specific enzyme activity that reached 688 U mg-1 protein (in vitro assay). Alkaline conditions showed the highest activity for the recombinant FeSOD. Moreover, it showed a relative thermostability up to 50 °C, while at 50 and 70 °C, the activity was reduced by 32 and 68%, respectively, after 1 h as compared to the maximum. Phylogenetic analysis revealed three main clusters i.e., the prokaryotic Cyanophyta, bacteria, and the eukaryotic Chlorophyta intermingled with plant species and a dinoflagellate. P. pringsheimii was closely grouped with Chlorella pyrenoidosa, however, other species showed a relative disparity. Alignment of FeSOD gene sequences of the different species showed many conserved regions which could be used for FeSOD sequences among unexplored species and may be useful for the taxonomy of the revised coccoid Chlorella species.

Tissue-specific localization of polyketide synthase and other associated genes in the lichen, Cladonia rangiferina, using laser microdissection.

The biosynthesis of two polyketides, atranorin and fumarprotocetraric acid, produced from a lichen-forming fungus, Cladonia rangiferina (L.) F. H. Wigg. was correlated with the expression of eight fungal genes (CrPKS1, CrPKS3, CrPKS16, Catalase (CAT), Sugar Transporter (MFsug), Dioxygenase (YQE1), C2H2 Transcription factor (C2H2), Transcription Factor PacC (PacC), which are thought to be involved in polyketide biosynthesis, and one algal gene, NAD-dependent deacetylase sirtuin 2 (AsNAD)), using laser microdissection (LMD). The differential gene expression levels within the thallus tissue layers demonstrate that the most active region for potential polyketide biosynthesis within the lichen is the outer apical region proximal to the photobiont but some expression also occurs in reproductive tissue. This is the first study using laser microdissection to explore gene expression of these nine genes and their location of expression; it provides a proof-of-concept for future experiments exploring tissue-specific gene expression within lichens; and it highlights the utility of LMD for use in lichen systems.

Identification of 6-Hydroxymellein Synthase and Accessory Genes in the Lichen Cladonia uncialis.

A transcribed polyketide synthase (PKS) gene has been identified in the lichen Cladonia uncialis. The complete nucleotide sequence of this PKS was determined from the amplified cDNA, and an assignment of individual domains was accomplished by homology searching using AntiSMASH. A scan of the complete genome sequence of C. uncialis revealed the accessory genes associated with this PKS gene. A homology search has identified that several genes in this cluster are similar to genes responsible for the biosynthesis of terrein in Aspergillus terreus. This permitted assignment of putative function to each of the genes in this new C. uncialis cluster. It is proposed that this gene cluster is responsible for the biosynthesis of a halogenated iscoumarin. This is the first report linking a gene cluster to a halogenated metabolite in lichen.

Algal carbohydrates affect polyketide synthesis of the lichen-forming fungus Cladonia rangiferina.

Lichen secondary metabolites (polyketides) are produced by the fungal partner, but the role of algal carbohydrates in polyketide biosynthesis is not clear. This study examined whether the type and concentration of algal carbohydrate explained differences in polyketide production and gene transcription by a lichen fungus (Cladonia rangiferina). The carbohydrates identified from a free-living cyanobacterium (Spirulina platensis; glucose), a lichen-forming alga (Diplosphaera chodatii; sorbitol) and the lichen alga that associates with C. rangiferina (Asterochloris sp.; ribitol) were used in each of 1%, 5% and 10% concentrations to enrich malt yeast extract media for culturing the mycobiont. Polyketides were determined by high performance liquid chromatography (HPLC), and polyketide synthase (PKS) gene transcription was measured by quantitative PCR of the ketosynthase domain of four PKS genes. The lower concentrations of carbohydrates induced the PKS gene expression where ribitol up-regulated CrPKS1 and CrPKS16 gene transcription and sorbitol up-regulated CrPKS3 and CrPKS7 gene transcription. The HPLC results revealed that lower concentrations of carbon sources increased polyketide production for three carbohydrates. One polyketide from the natural lichen thallus (fumarprotocetraric acid) also was produced by the fungal culture in ribitol supplemented media only. This study provides a better understanding of the role of the type and concentration of the carbon source in fungal polyketide biosynthesis in the lichen Cladonia rangiferina.

Putative identification of the usnic acid biosynthetic gene cluster by de novo whole-genome sequencing of a lichen-forming fungus.

To identify the biosynthetic gene cluster responsible for the biosynthesis of the polyketide usnic acid we carried out the de novo genome sequencing of the fungal partner of Cladonia uncialis. This was followed by comprehensive in silico annotation of polyketide synthase (PKS) genes. The biosynthesis of usnic acid requires a non-reducing PKS possessing a carbon methylation (CMeT) domain, a terminal Claisen cyclase (CLC) domain, and an accompanying oxidative enzyme that dimerizes methylphloracetophenone to usnic acid. Of the 32 candidate PKS genes identified in the mycobiont genome, only one was identified as consistent with these biosynthetic requirements. This gene cluster contains two genes encoding a non-reducing PKS and a cytochrome p450, which have been respectively named methylphloracetophenone synthase (MPAS) and methylphloracetophenone oxidase (MPAO). Both mpas and mpao were demonstrated to be transcriptionally active by reverse transcriptase-PCR of the mRNA in a lichen sample that was observed by HPLC to produce usnic acid. Phylogenetic analysis of the bioinformatically identified ketosynthase (KS) and CLC domains of MPAS demonstrated that mpas grouped within a unique clade and that mpas could be used as a phylogenetic probe to identify other MPAS genes.

Substratum preference of two species of Xanthoparmelia.

The distribution of established saxicolous lichens has been previously studied but substratum preference and elemental composition has been relatively unexplored. The objectives of this study were to compare ascospore germination and growth for two species of Xanthoparmelia using media supplemented with pulverized rock and to explore photobiont selectivity relative to ecological guilds. Mature apothecia from X. cumberlandia and X. viriduloumbrina were subjected to five treatments, which include water agar supplemented with crushed granodiorite, basalt, mica schist, dolostone, and malt yeast agar as the control. The algal actin gene was sequenced and the closest algal matches were retrieved from GenBank and analysed to produce a haplotype network. X. cumberlandia exhibited substratum preference for the mica schist treatment, while X. viriduloumbrina grew better on granodiorite and malt yeast agar relative to dolostone. Ascospore germination for both species failed to progress beyond the initial swelling and protrusion stage on the dolostone treatment. The actin gene sequences for the algae were most similar to those of Trebouxia jamesii. The rock substrates did not correspond with the photobiont haplotypes, which does not support the ecological guild hypothesis. This study provided insights into substratum preference and the suitability of the substratum for algal selection.

Tellurite-, tellurate-, and selenite-based anaerobic respiration by strain CM-3 isolated from gold mine tailings.

The newly discovered strain CM-3, a Gram-negative, rod-shaped bacterium from gold mine tailings of the Central Mine in Nopiming Provincial Park, Canada, is capable of dissimilatory anaerobic reduction of tellurite, tellurate, and selenite. CM-3 possesses very high level resistance to these oxides, both aerobically and anaerobically. During aerobic growth, tellurite and tellurate resistance was up to 1500 and 1000 µg/ml, respectively. In the presence of selenite, growth occurred at the highest concentration tested, 7000 µg/ml. Under anaerobic conditions, resistance was decreased to 800 µg/ml for the Te oxides; however, much like under aerobic conditions, growth with selenite still took place at 7000 µg/ml. In the absence of oxygen, CM-3 couples oxide reduction to an increase in biomass. Following an initial drop in viable cells, due to switching from aerobic to anaerobic conditions, there was an increase in CFU/ml greater than one order of magnitude in the presence of tellurite (6.6 × 10(3)-8.6 × 10(4) CFU/ml), tellurate (4.6 × 10(3)-1.4 × 10(5) CFU/ml), and selenite (2.7 × 10(5)-5.6 × 10(6) CFU/ml). A control culture without metalloid oxides showed a steady decrease in CFU/ml with no recovery. ATP production was also increased in the presence of each oxide, further indicating anaerobic respiration. Partial 16S rRNA gene sequencing revealed a 99.0 % similarity of CM-3 to Pseudomonas reactans.

Recognition- and defense-related gene expression at 3 resynthesis stages in lichen symbionts.

Recognition and defense responses are early events in plant-pathogen interactions and between lichen symbionts. The effect of elicitors on responses between lichen symbionts is not well understood. The objective of this study was to compare the difference in recognition- and defense-related gene expression as a result of culture extracts (containing secreted water-soluble elicitors) from compatible and incompatible interactions at each of 3 resynthesis stages in the symbionts of Cladonia rangiferina. This study investigated gene expression by quantitative PCR in cultures of C. rangiferina and its algal partner, Asterochloris glomerata/irregularis, after incubation with liquid extracts from cultures of compatible and incompatible interactions at 3 early resynthesis stages. Recognition-related genes were significantly upregulated only after physical contact, demonstrating symbiont recognition in later resynthesis stages than expected. One of 3 defense-related genes, chit, showed significant downregulation in early resynthesis stages and upregulation in the third resynthesis stage, demonstrating a need for the absence of chitinase early in thallus formation and a need for its presence in later stages as an algal defense reaction. This study revealed that recognition- and defense-related genes are triggered by components in culture extracts at 3 stages of resynthesis, and some defense-related genes may be induced throughout thallus growth. The parasitic nature of the interaction shows parallels between lichen symbionts and plant pathogenic systems.

Evolution of ketosynthase domains of polyketide synthase genes in the Cladonia chlorophaea species complex (Cladoniaceae).

Lichen-forming fungi synthesize a diversity of polyketides, but only a few non-reducing polyketide synthase (PKS) genes from a lichen-forming fungus have been linked with a specific polyketide. While it is a challenge to link the large number of PKS paralogs in fungi with specific products, it might be expected that the PKS paralogs from closely related species would be similar because of recent evolutionary divergence. The objectives of this study were to reconstruct a PKS gene phylogeny of the Cladonia chlorophaea species complex based on the ketosynthase domain, a species phylogeny of the complex, and to explore the presence of PKS gene paralogs among members of the species complex. DNA was isolated from 51 individuals of C. chlorophaea and allies to screen for the presence of 13 PKS paralogs. A 128 sequence PKS gene phylogeny using deduced amino acid sequences estimated from the 13 PKS paralogs and sequences subjected to BLASTx comparisons showed losses of each of two PKS domains (reducing and methylation). This research provided insight into the evolution of PKS genes in the C. chlorophaea group, species evolution in the group, and it identified potential directions for further investigation of polyketide synthesis in the C. chlorophaea species complex.

Rehydration conditions for isolation of high quality RNA from the lichen Lobaria pulmonaria.

BACKGROUND: The poikilohydric nature of lichens enables them to survive repeated episodes of desiccation by utilizing water when it becomes available. During rehydration, RNA-degrading endonucleases may be released, reducing RNA quantity and quality. Re-generation of a steady-state condition where RNA quantity and quality no longer fluctuate establishes a framework for development of new hypotheses for future investigations. Using Lobaria pulmonaria as a model species, the objective of this study was to compare the effect of different rehydration conditions on the quantity and quality of RNA from the rehydrated thallus. FINDINGS: Spectrophotometric measurements of total RNA and cDNA were performed for samples prepared from dry lichen or lichen after rehydration (0.5 h, 1 h, 2 h, 4 h or 24 h), with limited light and dark conditions, and at three temperatures (15°C, 20°C or 32°C) for some of these conditions. The results showed that rehydration of the thallus for 4 h at 20°C in light yielded the highest concentration and quality of RNA. A higher RNA concentration was obtained in light than in dark conditions, but the RNA quality was unaffected. CONCLUSIONS: This study suggests that allowance of 4 h for thallus rehydration should be adequate to ensure complete recovery of transcription. After 4 h at 20°C further studies can be carried out on the RNA in this model species.

Identification and comparison of the 3 early stages of resynthesis for the lichen Cladonia rangiferina.

A lichen is an association between a biotrophic fungal partner and a green algal and (or) cyanobacterial partner, which may be considered a "controlled" parasitic interaction. While controlled parasitism implies benefit to both interacting partners, a parasitism that is not controlled implies that one partner benefits to the detriment of the other partner. The objective of this study was to compare morphological development of the interaction between Cladonia rangiferina with its compatible algal partner (Asterochloris glomerata/irregularis) and incompatible algae (Coccomyxa peltigerae and Chloroidium ellipsoideum) at 3 early resynthesis stages. The fungus was co-inoculated with each alga separately and the stages of development were compared using quantitative measures. The first 3 stages of development of the lichen thallus were identified in the compatible interaction as the "pre-contact" stage (1 day post co-inoculation (PCI)), "contact" stage (8 days PCI), and "growth together" stage (21 days PCI). Compatible interactions showed significantly shorter internode length, significantly more new lateral hyphal branches, significantly greater appressorial frequency, and no reduction in cell diameter of the algal cells, compared with incompatible interactions. At 21 days PCI, a parasitic interaction was observed between Cladonia rangiferina and Chloroidium ellipsoideum. These findings support the importance of recognition between compatible partners for successful lichenization. This study also revealed a strategy that may explain the success of this species in northern habitats. Identification of the resynthesis stages of Cladonia rangiferina is required before expression of the proteins involved in recognition and defense can be understood.

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata.

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.

Body fluid osmolytes and urea and ammonia flux in the colon of two chondrichthyan fishes, the ratfish, Hydrolagus colliei, and spiny dogfish, Squalus acanthias.

The present study has examined the role of the colon in regulating ammonia and urea nitrogen balance in two species of chondrichthyans, the ratfish, Hydrolagus colliei (a holocephalan) and the spiny dogfish, Squalus acanthias (an elasmobranch). Stripped colonic tissue from both the dogfish and ratfish was mounted in an Ussing chamber and in both species bi-directional urea flux was found to be negligible. Urea uptake by the mucosa and serosa of the isolated colonic epithelium through accumulation of (14)C-urea was determined to be 2.8 and 6.2 fold greater in the mucosa of the dogfish compared to the serosa of the dogfish and the mucosa of the ratfish respectively. Furthermore, there was no difference between serosal and mucosal accumulation of (14)C-urea in the ratfish. Through the addition of 2mM NH(4)Cl to the mucosal side of each preparation the potential for ammonia flux was also examined. This was again found to be negligible in both species suggesting that the colon is an extremely tight epithelium to the movement of both urea and ammonia. Plasma, chyme and bile fluid samples were also taken from the agastric ratfish and were compared with solute concentrations of equivalent body fluids in the dogfish. Finally molecular analysis revealed expression of 3 isoforms of the urea transport protein (UT) and an ammonia transport protein (Rhbg) in the gill, intestine, kidney and colon of the ratfish. Partial nucleotide sequences of the UT-1, 2 and 3 isoforms in the ratfish had 95, 95 and 92% identity to the equivalent UT isoforms recently identified in another holocephalan, the elephantfish, Callorhinchus milii. Finally, the nucleotide sequence of the Rhbg identified in the ratfish had 73% identity to the Rhbg protein recently identified in the little skate, Leucoraja erinacea.

Photobiont Relationships and Phylogenetic History of Dermatocarpon luridum var. luridum and Related Dermatocarpon Species.

Members of the genus Dermatocarpon are widespread throughout the Northern Hemisphere along the edge of lakes, rivers and streams, and are subject to abiotic conditions reflecting both aquatic and terrestrial environments. Little is known about the evolutionary relationships within the genus and between continents. Investigation of the photobiont(s) associated with sub-aquatic and terrestrial Dermatocarpon species may reveal habitat requirements of the photobiont and the ability for fungal species to share the same photobiont species under different habitat conditions. The focus of our study was to determine the relationship between Canadian and Austrian Dermatocarpon luridum var. luridum along with three additional sub-aquatic Dermatocarpon species, and to determine the species of photobionts that associate with D. luridum var. luridum. Culture experiments were performed to identify the photobionts. In addition, the question of the algal sharing potential regarding different species of Dermatocarpon was addressed. Specimens were collected from four lakes in northwestern Manitoba, Canada and three streams in Austria. Three Canadian and four Austrian thalli of D. luridum var. luridum were selected for algal culturing. The nuclear Internal Transcribed Spacer (ITS) rDNA gene of the fungal partner along with the algal ITS rDNA gene was sequenced to confirm the identity of the lichen/photobiont and afterwards the same data sets were used in phylogenetic analyses to assess algal sharing. The green algal photobiont was identified as Diplosphaera chodatii (Trebouxiophyceae). The phylogenetic analyses of Canadian and Austrian D. luridum var. luridum revealed that ITS sequences are identical despite the vast geographic distance. Phylogenetic placement of D. luridum var. decipiens and D. arnoldianum suggested that a re-examination of the species status might be necessary. This study concluded that additional photobiont culture experiments should be conducted to answer the question of whether multiple photobionts are present within the genus Dermatocarpon.