Spatiotemporal dynamics of the bacterial microbiota on lacustrine Cladophora glomerata (Chlorophyta)

The branched periphytic green alga Cladophora glomerata, often abundant in nearshore waters of lakes and rivers worldwide, plays important ecosystem roles, some mediated by epibiotic microbiota that benefit from host‐provided surface, organic C, and O₂. Previous microscopy and high‐throughput sequencing studies have indicated surprising epibiont taxonomic and functional diversity, but have not included adequate consideration of sample replication or the potential for spatial and temporal variation. Here, we report the results of 16S rRNA amplicon‐based phylum‐to‐genus taxonomic analysis of Cladophora‐associated bacterial epibiota sampled in replicate from three microsites and at six times during the open‐water season of 2014, from the same lake locale (Picnic Point, Lake Mendota, Dane Co., WI, USA) explored by high‐throughput sequencing studies in two previous years. Statistical methods were used to test null hypotheses that the bacterial community: (i) is homogeneous across microsites tested, and (ii) does not change over the course of a growth season or among successive years. Results indicated a dynamic microbial community that is more strongly influenced by sampling day during the growth season than by microsite variation. A surprising diversity of bacterial genera known to be associated with the key function of methane‐oxidation (methanotrophy), including relatively high‐abundance of Crenothrix, Methylomonas, Methylovulum, and Methylocaldum–showed intraseasonal and interannual variability possibly related to temperature differences, and microsite preferences possibly related to variation in methane abundance. By contrast, a core assemblage of bacterial genera seems to persist over a growth season and from year to year, possibly transmitted by a persistent attached host resting stage.     

The epiphytic microbiota of the globally widespread macroalga Cladophora glomerata (Chlorophyta, Cladophorales)

? Premise of the study: The filamentous chlorophyte Cladophora produces abundant nearshore populations in marine and freshwaters worldwide, often dominating periphyton communities and producing nuisance growths under eutrophic conditions. High surface area and environmental persistence foster such high functional and taxonomic diversity of epiphytic microfauna and microalgae that Cladophora has been labeled an ecological engineer. We tested the hypotheses that (1) Cladophora supports a structurally and functionally diverse epiphytic prokaryotic microbiota that influences materials cycling and (2) mutualistic host-microbe interactions occur. Because previous molecular sequencing-based analyses of the microbiota of C. glomerata found as western Lake Michigan beach drift had identified pathogenic associates such as Escherichia coli, we also asked if actively growing lentic C. glomerata harbors known pathogens. ? Methods: We used 16S rRNA gene amplicon pyrosequencing to examine the microbiota of C. glomerata of Lake Mendota, Dane, Wisconsin, United States, during the growing season of 2011, at the genus- or species-level to infer functional phenotypes. We used correlative scanning electron and fluorescence microscopy to describe major prokaryotic morphotypes. ? Key results: We found microscopic evidence for diverse bacterial morphotypes, and molecular evidence for ca. 100 distinct sequence types classifiable to genus at the 80% confidence level or species at the 96-97% level within nine bacterial phyla, but not E. coli or related human pathogens. ? Conclusions: We inferred that bacterial epiphytes of lentic C. glomerata have diverse functions in materials cycling, with traits that indicate the occurrence of mutualistic interactions with the algal host.     

Zn uptake by Cladophora glomerata

The uptake of zinc by Cladophora glomerata at different pH-values, light conditions and exposure periods is reported. Zinc uptake was pH-dependent, with uptake increasing with rising pH. Uptake was greatest within the first two hours of exposure and was greater in light than in darkness due to increased pH. All results support the dominance of adsorptive uptake of zinc by Cladophora.     

Karyological studies of three species of Cladophora (Cladophorales,Chlorophyta) from Bermuda

Abstract

Chromosome numbers for three species of Cladophora from Bermuda are presented. C. laetevirens (Dillw.) Kuetz., C. prolifera (Roth) Kuetz., and C. conferta Crouan frat. ex Schramm & Maze were found to have similar karyotypes with 1 N = 2 X = 12 chromosomes belonging to size class III (0.8–2.2 μm). Correlations between each species' karyotype and its morphology and phytogeographic affinity are discussed. Estimates of the basic genome (1 X) for these and other Cladophora species indicate that nuclear DNA content, which shows a threefold variation in the genus, occurs in discontinuous increments. These findings are discussed in relation to reports of large scale, discontinuous DNA variation in vascular plant genera.     

Functional structure of algal mat (Cladophora glomerata) in a freshwater in western Poland

GAMs (Generalized Additive Models) present a growth pattern for the common filamentous algae (FA) taxa in freshwater ecosystems in Poland. Tribonema, Ulothrix taxa are an early spring species. In comparison with Rhzioclonium, Cladophora and Oedogonium taxa, the algae Zygnemataceae (e.g. Zygnema, Spirogyra) occurs in waters with lower levels of nutrients, total dissolved substances and electrical conductivity. Cladophora glomerata shows different responses to environmental gradients than C. rivularis, C. fracta. Furthermore, the algae Cladophora glomerata (Chlorophyta, Ulvophyceae) forms a thick monoalgae mat (from the surface to the bottom) in the shallow and eutrophic Lake Oporzynskie in Wielkopolska Region in Poland (Central Europe). Detailed algal mat characteristics, physical and chemical parameters of water, and pigments in thalli were measured vertically (surface, middle, bottom) and spatially (marginal and central part) in the stationary mat phase during maximal algal growth. Temperature, dissolved oxygen and PAR irradiation significantly declined as the depth of the mat increased. Water turbidity was greater in the surface layer than in deeper parts, in contrast to the concentrations of N-NO₃, N-NH₄ and TDS. Pigment content in the cells of C. glomerata, cell lengths and diameters of pyrenoids changed throughout the mat, from top to bottom. The obtained data were used for statistical analyses. In conclusion: dense mats of C. glomerata show a vertical stratification while the mats remain spatially homogenous. Our data suggest that mat structure be considered a biological indicator of freshwater eutrophication.     

Cladophora (Chlorophyta) spp. harbor human bacterial pathogens in nearshore water of Lake Michigan

Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 x 10(3) cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 x 10(2) Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.     

Cladophora (Chlorophyta) spp. Harbor Human Bacterial Pathogens in Nearshore Water of Lake Michigan

Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 × 10³ cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 × 10² Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.     

Evolution of nuclear rDNA its sequences in the Cladophora albida/sericea clade (Chlorophyta)

Ribosomal DNA ITS sequences were compared among 13 different species and biogeographic isolates from the monophyletic albida/sericea clade in the green algal genus Cladophora. Six distinct ITS sequence types were found, characterized by multiple insertions and deletions and high levels of nucleotide substitution. Conserved domains within the ITS regions indicate the presence of ITS secondary structure. Low transition/transversion ratios among the six types and nearly symmetrical tree-length frequency distributions indicate some saturation, and low phylogenetic signal. Although branching order among five of the six ITS sequence types could not be resolved, estimates of ITS sequence divergence as compared with 18S divergence in a subset of the taxa suggests that the origin of the different ITS types is probably in the mid-Miocene (12 Ma ago) but that biogeographic isolates within a single ITS type (including both Pacific and Atlantic representatives) have probably dispersed on a time scale of thousands rather than millions of years.Correspondence to: J.J. Olsen     

Studies on the growth of Cladophora glomerata in laboratory continuous-flow culture

Cladophora glomerata grown in continuous-flow culture was found to have optimal specific growth rate (μ) at, or near, 20°C. Specific growth rate increased linearly with increased duration of illumination per day up to 24h, and increased light intensity up to 6000 lx. Undissociated ammoniacal nitrogen (0·185 mg 1^-1) reduced μ to 50% of that at 0·010 mg 1^-1: 0·077–1·057 mg NO₂-N1^-1 and 7·2–15·2 mg NO₃-N1^-1 had no significant effect on μ. At 4·9 mg PO₄-P1^-1, μ was 48% of that at 1·9 mg1^-1. The critical medium PO₄-P concentration was less than 0·098 mg1^-1. Specific growth rate was reduced to 50% of that in the based medium by 0·036 mg Cu1^-1, 0·070 mg Zn1^-1 and 1·03 mg Pb1^-1. Results are discussed in the context of the natural distribution of the alga in the field situation.     

Cyclic tetrapyrrolic photosensitizers from Cladophora patentiramea (Cladophoraceae, Chlorophyta) and Turbinaria conoides (Sargassaceae, Phaeophyta) for photodynamic therapy

In screening for novel photosensitizers for photodynamic therapy, 14 seaweed samples from Port Dickson in Malaysia were collected. Methanolic extracts of these samples were prepared and evaluated for phototoxicity using a short-term cell viability assay, where promyelocytic leukemia cells, HL60 were incubated with the extracts prior to irradiation with a broad spectrum light at 9.6?J?cm^?2 (equivalent to 10.5?mW?cm^?2 for 10?min). Four of the methanolic extracts demonstrated moderate to strong phototoxicity and bioassay-guided isolation of photosensitizers was carried out on two selected seaweeds to yield a total of eight cyclic tetrapyrrolic compounds which are derivatives of chlorophyll-a and -b. Seven of these compounds showed >50% phototoxicity at 5?μg?mL^?1 while exhibiting minimal cytotoxicity in the dark, which is an important characteristic of an ideal photosensitizer.     

Purification and partial characterization of haloperoxidase from fresh water algae Cladophora glomerata

Various historical eras in the distant as well as the recent past of amphibian embryology are briefly reviewed. The concepts which emerged from the early years matured, then were laid to rest for several decades. A resurgence, driven by key discoveries with peptide growth factors, and fueled by modern molecular biology methods, is underway. The future for several amphibian research projects should be promising since interest in basic concepts remains strong, and application of frontier methodologies is yielding novel findings.     

Occurrence of Escherichia coli and Enterococci in Cladophora (Chlorophyta) in Nearshore Water and Beach Sand of Lake Michigan

Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (± standard errors) of 5.3 (± 4.8) and 4.8 (± 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R² = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4°C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.     

Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan

Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (+/- standard errors) of 5.3 (+/- 4.8) and 4.8 (+/- 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R(2) = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4 degrees C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.     

Ultrastructure of chloroplasts in'Marimo'(Cladophora aegagropila, Chlorophyta), and changes after exposure to light

This study compares the ultrastructure of the inner, dark-habituated cells of the green ‘Cladophora-ball’, or Marimo, to that of similar cells at the surface. Cells not exposed to light possess fewer, but larger and more irregular, chloroplasts than do the outer cells. Unexposed chloroplasts have a pyrenoid matrix lacking starch sheaths and containing unusually thick granal stacks. Despite prolonged exposure to darkness, the chloroplasts contain small starch grains. After exposure to light, such chloroplasts divide, become smaller and take on the appearance of those in the outer layer cells. Within 48 h, all of the chloroplasts develop substantial starch grains and the pyrenoids are surrounded by starch sheaths. This response is consistent with previous reports of the recovery of photosynthetic activity in inner cells exposed to light.     

Evolution of temperature responses in the Cladophora vagabunda complex and the C. albida/sericea complex (Chlorophyta)

Differentiation in temperature responses (survival and growth) was investigated among isolates of two tropical to temperate green algal lineages: the Cladophora vagabunda complex and the C. albida/sericea complex. The results were analysed in relation to published data on 18S rRNA and ITS sequence divergence, which have shown that the overall degree of genetic divergence is similar in the two lineages but that very different patterns of radiation have occurred. In the C. vagabunda complex, the two main clades in the well-resolved phylogenetic tree differed mainly in their tolerance to low temperatures. Within-clade variation was no stronger in the Atlantic/Pacific than in the all-Pacific clade. In the C. albida/sericea complex, six distinctive ITS types indicated early radiation. Although distinctive differences were found between some of these types, the thermal responses of others were very similar, indicating physiological stasis. In both lineages there was evidence for some adaptation to local temperature regimes but phylogenetic constraints were generally more important. Isolates with the same ITS sequences showed similar temperature responses even though collected from different climate zones. Evidence was found for a physiological trade-off between growth at high and at low temperatures in the C. albida/sericea complex, whereas, in the C. vagabunda complex, one clade showed more eurythermal growth responses than the other. In the C. vagabunda complex, which is the ancestral lineage of the C. albida/sericea complex, major differentiation was found in cold tolerance but not in heat tolerance, whereas the reverse pattern was found in the derived C. albida/sericea complex. These findings suggest that an acquisition of cold tolerance preceded the loss of heat tolerance during adaptation to colder climates.     

Antioxidant modulation in response to heavy metal induced oxidative stress in Cladophora glomerata

The present investigation was carried out to study the induction of oxidative stress subjected to heavy metal environment. Lipoperoxides showed positive correlation at heavy metal accumulation sites indicating the tissue damage resulting from the reactive oxygen species and resulted in unbalance to cellular redox status. The high activities of ascorbate peroxidase and superoxide dismutase probably counter balance this oxidative stress. Glutathione and soluble phenols decreased, whereas dehydroascorbate content increased in the algae from polluted sites. The results suggested that alga responded to heavy metals effectively by antioxidant compounds and scavenging enzymes.     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.