ALBERT

Description: The GeoChip 4.2 gene array was employed to interrogate the microbial functional gene repertoire of sponges and seawater collected from the Red Sea and the Mediterranean. Complementary amplicon sequencing confirmed the microbial community composition characteristic of high microbial abundance (HMA) and low microbial abundance (LMA) sponges. By use of GeoChip, altogether 20 273 probes encoding for 627 functional genes and representing 16 gene categories were identified. Minimum curvilinear embedding analyses revealed a clear separation between the samples. The HMA/LMA dichotomy was stronger than any possible geographic pattern, which is shown here for the first time on the level of functional genes. However, upon inspection of individual genes, very few specific differences were discernible. Differences were related to microbial ammonia oxidation, ammonification, and archaeal autotrophic carbon fixation (higher gene abundance in sponges over seawater) as well as denitrification and radiation-stress-related genes (lower gene abundance in sponges over seawater). Except for few documented specific differences the functional gene repertoire between the different sources appeared largely similar. This study expands previous reports in that functional gene convergence is not only reported between HMA and LMA sponges but also between sponges and seawater.

Description: Ferredoxins are electron shuttles harbouring iron–sulfur clusters that connect multiple oxido-reductive pathways in organisms displaying different lifestyles. Some prokaryotes and algae express an isofunctional electron carrier, flavodoxin, which contains flavin mononucleotide as cofactor. Both proteins evolved in the anaerobic environment preceding the appearance of oxygenic photosynthesis. The advent of an oxygen-rich atmosphere proved detrimental to ferredoxin owing to iron limitation and oxidative damage to the iron–sulfur cluster, and many microorganisms induced flavodoxin expression to replace ferredoxin under stress conditions. Paradoxically, ferredoxin was maintained throughout the tree of life, whereas flavodoxin is absent from plants and animals. Of note is that flavodoxin expression in transgenic plants results in increased tolerance to multiple stresses and iron deficit, through mechanisms similar to those operating in microorganisms. Then, the question remains open as to why a trait that still confers plants such obvious adaptive benefits was not retained. We compare herein the properties of ferredoxin and flavodoxin, and their contrasting modes of expression in response to different environmental stimuli. Phylogenetic analyses suggest that the flavodoxin gene was already absent in the algal lineages immediately preceding land plants. Geographical distribution of phototrophs shows a bias against flavodoxin-containing organisms in iron-rich coastal/freshwater habitats. Based on these observations, we propose that plants evolved from freshwater macroalgae that already lacked flavodoxin because they thrived in an iron-rich habitat with no need to back up ferredoxin functions and therefore no selective pressure to keep the flavodoxin gene. Conversely, ferredoxin retention in the plant lineage is probably related to its higher efficiency as an electron carrier, compared with flavodoxin. Several lines of evidence supporting these contentions are presented and discussed.

Description: Epibiotic biofilms have the potential to control major aspects of the biology and ecology of their hosts. Their composition and function may thus be essential for the health of the host. We tested the influence of salinity on the composition of epibacterial communities associated with the brown macroalga Fucus vesiculosus. Algal individuals were incubated at three salinities (5, 19, and 25) for 14days and nonliving reference substrata (stones) were included in the experiment. Subsequently, the composition of their surface-associated bacterial communities was analyzed by 454 pyrosequencing of 16S rRNA gene sequences. Redundancy analysis revealed that the composition of epiphytic and epilithic communities significantly differed and were both affected by salinity. We found that 5% of 2494 epiphytic operational taxonomic units at 97% sequence similarity were responsible for the observed shifts. Epibacterial -diversity was significantly lower at salinity 5 but did not differ between substrata. Our results indicate that salinity is an important factor in structuring alga-associated epibacterial communities with respect to composition and/or diversity. Whether direct or indirect mechanisms (via altered biotic interactions) may have been responsible for the observed shifts is discussed.

Description: In spite of considerable insights into the microbial diversity of marine sponges, quantitative information on microbial abundances and community composition remains scarce. Here, we established qPCR assays for the specific quantification of four bacterial phyla of representative sponge symbionts as well as the kingdoms Eubacteria and Archaea. We could show that the 16S rRNA gene numbers of Archaea, Chloroflexi, and the candidate phylum Poribacteria were 4–6 orders of magnitude higher in high microbial abundance (HMA) than in low microbial abundance (LMA) sponges and that actinobacterial 16S rRNA gene numbers were 1–2 orders higher in HMA over LMA sponges, while those for Cyanobacteria were stable between HMA and LMA sponges. Fluorescence in situ hybridization of Aplysina aerophoba tissue sections confirmed the numerical dominance of Chloroflexi, which was followed by Poribacteria. Archaeal and actinobacterial cells were detected in much lower numbers. By use of fluorescence-activated cell sorting as a primer- and probe-independent approach, the dominance of Chloroflexi, Proteobacteria, and Poribacteria in A. aerophoba was confirmed. Our study provides new quantitative insights into the microbiology of sponges and contributes to a better understanding of the HMA/LMA dichotomy.

Description: Dinoflagellates harboring diatom endosymbionts (termed “dinotoms”) have undergone a process often referred to as “tertiary endosymbiosis”—the uptake of algae containing secondary plastids and integration of those plastids into the new host. In contrast to other tertiary plastids, and most secondary plastids, the endosymbiont of dinotoms is distinctly less reduced, retaining a number of cellular features, such as their nucleus and mitochondria and others, in addition to their plastid. This has resulted in redundancy between host and endosymbiont, at least between some mitochondrial and cytosolic metabolism, where this has been investigated. The question of plastidial redundancy is particularly interesting as the fate of the host dinoflagellate plastid is unclear. The host cytosol possesses an eyespot that has been postulated to be a remnant of the ancestral peridinin plastid, but this has not been tested, nor has its possible retention of plastid functions. To investigate this possibility, we searched for plastid-associated pathways and functions in transcriptomic data sets from three dinotom species. We show that the dinoflagellate host has indeed retained genes for plastid-associated pathways and that these genes encode targeting peptides similar to those of other dinoflagellate plastid-targeted proteins. Moreover, we also identified one gene encoding an essential component of the dinoflagellate plastid protein import machinery, altogether suggesting the presence of a functioning plastid import system in the host, and by extension a relict plastid. The presence of the same plastid-associated pathways in the endosymbiont also extends the known functional redundancy in dinotoms, further confirming the unusual state of plastid integration in this group of dinoflagellates.

Description: The macroalga Fucus vesiculosus carries a specific community of surface bacteria. To identify chemical compounds that possibly mediate abundance and community composition of algae-associated bacteria, we tested the effect of surface-available algal compounds on bacterial settlement and community composition under field conditions. Compounds on algal thalli were separated from the surface by extraction with organic solvents and investigated on growth inhibition and settlement of bacterial isolates. Based on in vitro data, partially purified extract fractions were then exposed to bacterial colonizers in vivo followed by bacterial enumeration and community analysis. The polar fraction of the algal surface extract revealed a significant profouling effect for Vibrionales, whereas the nonpolar fraction containing the xanthophyll pigment fucoxanthin and other unidentified nonpolar surface compounds revealed a significant 80% reduction of surface colonizing bacteria. The analysis of bacterial surface communities by 454 pyrosequencing demonstrated that the antifouling activity of nonpolar algal surface compounds was targeting the abundance of natural bacterial colonizers rather than the relative composition of bacterial members within the community. Moreover, the bacterial community composition on F.vesiculosus was markedly different from artificial control substrates and chemically manipulated experimental treatments, suggesting that other, nonextractable surface features and/or physical properties render algal-specific epiphytic bacterial communities.

Description: Following their transmission from the human to the mosquito with the bloodmeal, malaria parasites have to persevere in the mosquito midgut for approximately 1 d. During this period the parasites are highly vulnerable to factors of the mosquito midgut, including bacteria. We here aimed at determining the microbial diversity of gut bacteria of the Asian malaria vector Anophebs stephensi (Liston) during development and under different feeding regimes, including feeds on malaria parasite-infected blood. 16S rRNA and denaturing gradient gel electrophoresis analyses demonstrated an increasing reduction in the microbial diversity during mosquito development from egg to adult and identified the gram-negative bacterium Elizabethkingia meningoseptica King as the dominant species in the midgut of lab-reared male and female mosquitoes. E. meningoseptica is transmitted between generations and its predominance in the mosquito midgut was not altered by diet, when the gut microbiota was compared between sugar-fed and blood-fed female mosquitoes. Furthermore, feeds on blood infected with malaria parasites did not impact the presence of E. meningoseptica in the gut. Extracts from cultured E. meningoseptica were active against gram-positive and negative bacteria and yeast and against the blood and gametocyte transmission stages of the malaria parasite Plasmodium falciparum Welch. The antimicrobial and antiplasmodial activities of E. meningoseptica may account for its dominance in the midgut of the malaria vector.

Description: The concentration of CO2 in global surface ocean waters is increasing due to rising atmospheric CO2 emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO2 concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO2 concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO2 concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO2 and was highest in algae grown at 665 µatm CO2. Nitrate and phosphate uptake rates were inversely related to CO2, while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO2. The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO2 due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO2 are discussed.

Description: The sheer scale of the metagenomic and metatranscriptomic datasets that are now available warrants the development of automated protocols for organizing, annotating and comparing the samples in terms of their metabolic profiles. We describe a user-friendly java program FROMP (Fragment Recruitment on Metabolic Pathways) for mapping and visualizing enzyme annotations onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways or custom-made pathways and comparing the samples in terms of their Pathway Completeness Scores,their relative Activity Scores or enzyme enrichment odds ratios. This program along with our fully-configurable PERL based annotation organization pipeline Meta2Pro (METAbolic PROfiling of META-omic data) offers a quick and accurate standalone solution for metabolic profiling of environmental samples or cultures from different treatments. Apart from pictorial comparisons, FROMP can also generate score matrices for multiple meta-omics samples which can be used directly by other statistical programs

Description: We investigated the gelatinous carnivore zooplankton community in the coastal waters of northeast Taiwan during the period 20072010. The community assemblage was composed of 45 species, of which only 14 appeared recurrent in Taiwanese waters. Although there was no clear seasonality, higher richness and abundances occurred in spring and autumn. Examination of potential physical drivers of coastal biomass accumulation did not show any link with water mass transport; instead, peak events were associated with typhoon disturbances, suggesting a potential resource pulse effect.

Description: This study aimed to examine interactive effects between ocean acidification and temperature on the photosynthetic and growth performance of Neosiphonia harveyi. N. harveyi was cultivated at 10 and 17.5 °C at present (~380 µatm), expected future (~800 µatm), and high (~1500 µatm) pCO2. Chlorophyll a fluorescence, net photosynthesis, and growth were measured. The state of the carbon-concentrating mechanism (CCM) was examined by pH-drift experiments (with algae cultivated at 10 °C only) using ethoxyzolamide, an inhibitor of external and internal carbonic anhydrases (exCA and intCA, respectively). Furthermore, the inhibitory effect of acetazolamide (an inhibitor of exCA) and Tris (an inhibitor of the acidification of the diffusive boundary layer) on net photosynthesis was measured at both temperatures. Temperature affected photosynthesis (in terms of photosynthetic efficiency, light saturation point, and net photosynthesis) and growth at present pCO2, but these effects decreased with increasing pCO2. The relevance of the CCM decreased at 10 °C. A pCO2 effect on the CCM could only be shown if intCA and exCA were inhibited. The experiments demonstrate for the first time interactions between ocean acidification and temperature on the performance of a non-calcifying macroalga and show that the effects of low temperature on photosynthesis can be alleviated by increasing pCO2. The findings indicate that the carbon acquisition mediated by exCA and acidification of the diffusive boundary layer decrease at low temperatures but are not affected by the cultivation level of pCO2, whereas the activity of intCA is affected by pCO2. Ecologically, the findings suggest that ocean acidification might affect the biogeographical distribution of N. harveyi.

Description: The vast majority of microbes inhabiting the subseafloor remain uncultivated and their energy sources unknown. Thus, a focus of ocean drilling expeditions over the past decade has been to characterize the distribution of microbes associated with specific metabolic reactions. An important question has been whether microbes involved in key microbial processes, such as sulfate reduction and methanogenesis, differ fundamentally from their counterparts in surface environments. To this end, functional genes of anaerobic methane cycling (mcrA), sulfate reduction (dsrAB), acetogenesis (fhs), and dehalorespiration (rdhA) have been examined. A compilation of existing functional gene data suggests that subseafloor microbes involved in anaerobic methane cycling, sulfate reduction, acetogenesis, and dehalorespiration are not fundamentally different from their counterparts in the surface world. Moreover, quantifications of mcrA and dsrAB suggest that, unless the majority of subseafloor microbes involved in methane cycling and sulfate reduction are too genetically divergent to be detected with conventional methods, these processes only support a small fraction (〈 1%) of total microbial biomass in the deep biosphere. Ecological explanations for the observed trends, target processes and methods for future investigations, and strategies for tackling the unresolved issue of microbial contamination in samples obtained by ocean drilling are discussed.

Description: Trophodynamics of meso-zooplankton in the North Sea (NS) were assessed at a site in the southern NS, and at a shallow and a deep site in the central NS. Offshore and neritic species from different ecological niches, including Calanus spp., Temora spp. and Sagitta spp., were collected during seven cruises over 14 months from 2007 to 2008. Bulk stable isotope (SI) analysis, phospholipid-derived fatty acid (PLFA) compositions, and δ 13CPLFA data of meso-zooplankton and particulate organic matter (POM) were used to describe changes in zooplankton relative trophic positions (RTPs) and trophodynamics. The aim of the study was to test the hypothesis that the RTPs of zooplankton in the North Sea vary spatially and seasonally, in response to hydrographic variability, with the microbial food web playing an important role at times. Zooplankton RTPs tended to be higher during winter and lower during the phytoplankton bloom in spring. RTPs were highest for predators such as Sagitta sp. and Calanus helgolandicus and lowest for small copepods such as Pseudocalanus elongatus and zoea larvae (Brachyura). δ 15NPOM-based RTPs were only moderate surrogates for animals’ ecological niches, because of the plasticity in source materials from the herbivorous and the microbial loop food web. Common (16:0) and essential (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) structural lipids showed relatively constant abundances. This could be explained by incorporation of PLFAs with δ 13C signatures which followed seasonal changes in bulk δ 13CPOM and PLFA δ 13CPOM signatures. This study highlighted the complementarity of three biogeochemical approaches for trophodynamic studies and substantiated conceptual views of size-based food web analysis, in which small individuals of large species may be functionally equivalent to large individuals of small species. Seasonal and spatial variability was also important in altering the relative importance of the herbivorous and microbial food webs.

Description: Effects of epibiotic bacteria associated with macroalgae on barnacle larval attachment were investigated. Eight bacterial isolates obtained from samples of three macroalga species were cultured as monospecies bacterial films and tested for their activity against barnacle (Amphibalanus improvisus) attachment in field experiments (Western Baltic Sea). Furthermore, natural biofilm communities associated with the surface of the local brown alga, Fucus vesiculosus, which were exposed to different temperatures (5, 15 and 20 °C), were harvested and subsequently tested. Generally, monospecies bacterial biofilms, as well as natural microbial assemblages, inhibited barnacle attachment by 20-67%. denaturing gradient gel electrophoresis fingerprints showed that temperature treatment shifted the bacterial community composition and weakened the repellent effects at 20 °C. Repellent effects were absent when settlement pressure of cyprids was high. Nonviable bacteria tended to repel cyprids when compared to the unfilmed surfaces. We conclude that biofilms can have a repellent effect benefiting the host by preventing heavy fouling on its surface. However, severe settlement pressure, as well as stressful temperature, may reduce the protective effects of the alga's biofilm. Our results add to the notion that the performance of F. vesiculosus may be reduced by multiple stressors in the course of global warming.

Description: The aim of this study was to examine sponge orange band (SOB) disease affecting the prominent Caribbean sponge Xestospongia muta. Scanning and transmission electron microscopy revealed that SOB is accompanied by the massive destruction of the pinacoderm. Chlorophyll a content and the main secondary metabolites, tetrahydrofurans, characteristic of X. muta, were significantly lower in bleached than in healthy tissues. Denaturing gradient gel electrophoresis using cyanobacteria-specific 16S rRNA gene primers revealed a distinct shift from the Synechococcus/Prochlorococcus clade of sponge symbionts towards several clades of unspecific cyanobacteria, including lineages associated with coral disease (i.e. Leptolyngbya sp.). Underwater infection experiments were conducted by transplanting bleached cores into healthy individuals, but revealed no signs of SOB development. This study provided no evidence for the involvement of a specific microbial pathogen as an etiologic agent of disease; hence, the cause of SOB disease in X. muta remains unidentified.

Description: Shallow coastal waters, where phototrophic purple sulfur bacteria (PSB) regularly form massive blooms, are subjected to massive diurnal and event-driven changes of physicochemical conditions including temperature and salinity. To analyze the ability of PSB to cope with these environmental factors and to compete in complex communities we have studied changes of the environmental community of PSB of a Baltic Sea lagoon under experimental enrichment conditions with controlled variation of temperature and NaCl concentration. For the first time, changes within a community of PSB were specifically analyzed using the photosynthetic reaction center genes pufL and M by RFLP and cloning experiments. The most abundant PSB phylotypes in the habitat were found along the NaCl gradient from freshwater conditions up to 7.5% NaCl. They were accompanied by smaller numbers of purple nonsulfur bacteria and aerobic anoxygenic phototrophic bacteria. Major components of the PSB community of the brackish lagoon were affiliated to PSB genera and species known as marine, halophilic or salt-tolerant, including species of Marichromatium, Halochromatium, Thiorhodococcus, Allochromatium, Thiocapsa, Thiorhodovibrio, and Thiohalocapsa. A dramatic shift occurred at elevated temperatures of 41 and 44°C when Marichromatium gracile became most prominent which was not detected at lower temperatures.

Description: The activity of the human armpit microbiota triggers the formation of body odor. We used differential 16S rRNA gene (rDNA)- and rRNA-based terminal-restriction fragment length polymorphism fingerprinting in combination with cloning and sequencing to identify active members of the human armpit microbiota. DNA and RNA were isolated from skin scrub samples taken from both armpits of 10 preconditioned, healthy males. The fingerprint profiles indicated pronounced similarities between the armpit microbiota in the right and the left axillae of an individual test person, but larger differences between the axilla microbiota of different individuals. Using 16S rDNA and rRNA sequence data, the majority of peaks in the armpit profiles were assigned to bacteria affiliated with well-known genera of skin bacteria. The relative abundances of all groups were similar among the rDNA and rRNA samples, suggesting that all groups of armpit bacteria were active. Surprisingly, the relative abundance of sequences affiliated with Peptoniphilus sp. was by far and with statistical significance the highest in the rRNA samples of the right armpits. Thus, bacteria affiliated with Peptoniphilus sp. might have been particularly active in the right axillae of the test persons, possibly owing to the handedness of the test persons, which might cause different environmental conditions in the right axillae.

Description: Mixing processes of reduced hydrothermal fluids with oxygenated seawater and fluid-rock reactions contribute to the chemical signatures of diffuse venting and likely determine the geochemical constraints on microbial life. We examined the influence of fluid chemistry on microbial diversity and activity by sampling diffuse fluids emanating through mussel beds at two contrasting hydrothermal vents. The H(2) concentration was very low at the basalt-hosted Clueless site, and mixing models suggest O(2) availability throughout much of the habitat. In contrast, effluents from the ultramafic-hosted Quest site were considerably enriched in H(2) , while O(2) is likely limited to the mussel layer. Only two different hydrogenase genes were identified in clone libraries from the H(2) -poor Clueless fluids, but these fluids exhibited the highest H(2) uptake rates in H(2) -spiked incubations (oxic conditions, at 18 °C). In contrast, a phylogenetically diverse H(2) -oxidizing potential was associated with distinct thermal conditions in the H(2) -rich Quest fluids, but under oxic conditions, H(2) uptake rates were extremely low. Significant stimulation of CO(2) fixation rates by H(2) addition was solely illustrated in Quest incubations (P-value 〈0.02), but only in conjunction with anoxic conditions (at 18 °C). We conclude that the factors contributing toward differences in the diversity and activity of H(2) oxidizers at these sites include H(2) and O(2) availability.

Description: Phototrophic bacteria are important primary producers of salt lakes in the Salar de Atacama and at times form visible mass developments within and on top of the lake sediments. The communities of phototrophic bacteria from two of these lakes were characterized by molecular genetic approaches using key genes for the biosynthesis of the photosynthetic apparatus in phototrophic purple bacteria (pufLM) and in green sulfur bacteria (fmoA). Terminal restriction fragment length polymorphism of the pufLM genes indicated high variability of the community composition between the two lakes and subsamples thereof. The communities were characterized by the dominance of a novel, so far undescribed lineage of pufLM containing bacteria and the presence of representatives related to known halophilic Chromatiaceae and Ectothiorhodospiraceae. In addition, the presence of BChl b-containing anoxygenic phototrophic bacteria and of aerobic anoxygenic bacteria was indicated. Green sulfur bacteria were not detected in the environmental samples, although a bacterium related to Prosthecochloris indicum was identified in an enrichment culture. This is the first comprehensive description of phototrophic bacterial communities in a salt lake of South America made possible only due to the application of the functional pufLM genes.

Description: Analyses of clone libraries from water and sediments of different sites from Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano, revealed the presence of five unique clusters of uncultured Archaea that have not been previously reported or specifically assigned. These sequences were distantly related (83–96% sequence identity) to a limited number of other clone sequences and revealed no identity to cultured Archaea. The abundance of Archaea and Bacteria was estimated using qPCR and community composition was examined through the construction of clone libraries of archaeal 16S rRNA gene. Archaea were found to be dominant over Bacteria in sediments from two saline sites (sites H4: 6.31 × 104 and site H6: 1.37 × 104 μS cm−1) and in one of the water samples (freshwater from site H0: 607 μS cm−1). Euryarchaeotal sequences were more abundant than crenarchaeotal sequences. Many of the clone sequences (52%) were similar to uncultured archaeal groups found in marine ecosystems having identity values between 99% and 97%. A major fraction of the sequences (40%) were members of Methanobacteria, while others were included in the Marine Benthic Groups B and D, the Miscellaneous Crenarchaeotic Group, the Terrestrial Miscellaneous Euryarchaeotal Group, Marine Group I and Halobacteria. The presence of uncultured archaeal groups in Salar de Huasco extends their known distribution in inland waters, providing new clues about their possible function in the environment.

Description: Life at deep-sea hydrothermal vents depends on chemolithoautotrophic microorganisms as primary producers mediating the transfer of energy from hydrothermal fluids to higher trophic levels. A comprehensive molecular survey was performed with microbial communities in a mussel patch at the Irina II site of the Logatchev hydrothermal field by combining the analysis of 16S rRNA gene sequences with studies of functional key genes involved in biochemical pathways of sulfur oxidation–reduction (soxB, aprA) and autotrophic carbon fixation (aclB, cbbM, cbbL). Most significantly, major groups of chemoautotrophic sulfur oxidizers in the diffuse fluids differed in their biosynthetic pathways of both carbon fixation and sulfur oxidation. One important component of the community, the Epsilonproteobacteria, has the potential to grow chemoautotrophically by means of the reductive tricarboxylic acid cycle and to gain energy through the oxidation of reduced sulfur compounds using the Sox pathway. The majority of soxB and all retrieved aclB gene sequences were assigned to this group. Another important group in this habitat, the Gammaproteobacteria, may use the adenosine 5'-phosphosulfate pathway and the Calvin–Benson–Bassham cycle, deduced from the presence of aprA and cbbM genes. Hence, two important groups of primary producers at the investigated site might use different pathways for sulfur oxidation and carbon fixation.

Description: The prasinophyte order Mamiellales contains several widespread marine picophytoplankton (≤2 μm diameter) taxa, including Micromonas and Ostreococcus. Complete genome sequences are available for two Micromonas isolates, CCMP1545 and RCC299. We performed in silico analyses of nitrogen transporters and related assimilation genes in CCMP1545 and RCC299 and compared these with other green lineage organisms as well as Chromalveolata, fungi, bacteria, and archaea. Phylogenetic reconstructions of ammonium transporter (AMT) genes revealed divergent types contained within each Mamiellales genome. Some were affiliated with plant and green algal AMT1 genes and others with bacterial AMT2 genes. Land plant AMT2 genes were phylogenetically closer to archaeal transporters than to Mamiellales AMT2 genes. The Mamiellales represent the first green algal genomes to harbor AMT2 genes, which are not found in Chlorella and Chlamydomonas or the chromalveolate algae analyzed but are present in oomycetes. Fewer nitrate transporter (NRT) than AMT genes were identified in the Mamiellales. NRT1 was found in all but CCMP1545 and showed highest similarity to Mamiellales and proteobacterial NRTs. NRT2 genes formed a bootstrap-supported clade basal to other green lineage organisms. Several nitrogen-related genes were colocated, forming a nitrogen gene cluster. Overall, RCC299 showed the most divergent suite of nitrogen transporters within the various Mamiellales genomes, and we developed TaqMan quantitative polymerase chain reaction primer-probes targeting a subset of these, as well as housekeeping genes, in RCC299. All those investigated showed expression either under standard growth conditions or under nitrogen depletion. Like other recent publications, our findings show a higher degree of "mixed lineage gene affiliations" among eukaryotes than anticipated, and even the most phylogenetically anomalous versions appear to be functional. Nitrogen is often considered a regulating factor for phytoplankton populations. This study provides a springboard for exploring the use and functional diversification of inorganic nitrogen transporters and related genes in eukaryotic phytoplankton. © 2010 The Author.

Description: The metabolic demands of a rare paper nautilus, Argonauta nouryi, in the eastern tropical Pacific (ETP) are evaluated. After adjusting for temperature and size, the rates of oxygen consumption and of aerobic and anaerobic metabolic potential (as evidenced by citrate synthase and octopine dehydrogenase activities, respectively) of A. nouryi were much higher than those in holopelagic octopods that exhibit float-and-wait predation strategies. In fact, the rates were similar to those found in small epipelagic squids and benthic octopods. The critical oxygen partial pressure was 4.9 kPa at 20°C, suggesting that the strong oxygen minimum layer found at intermediate depths in the ETP may constrain the vertical distribution of A. nouryi to the upper few metres of the water column. We also report the occurrence of a chain of shelled females at the surface, in which each animal was attached, as if on the benthos, to the next individual in the chain. Although it may constitute an effective strategy to increase the rates of mate encounter in the vast open ocean, there may be an important ecological trade-off for such behaviour, namely the increase in visibility at the surface with concomitant attraction of predators.

Description: The distribution of paralarvae and small juvenile cephalopods sampled by a rectangular midwater trawl (opening area 8 m2) over the continental shelf off Cape Santa Marta Grande, southern Brazil (28°09′S–29°56′S) during spring 1989 is discussed. An intrusion of Brazil Current Tropical Water (22°C; 36.5) separates warm, less-saline water (22°C; 35.2) from cooler, more-saline water (15°C; 36.4). Prevailing northeasterly winds led to upwelling of South Atlantic Central Water over the shelf, promoting high Chl a concentrations. Three species constituted 99% of the 628 cephalopods collected: Illex argentinus (n = 540; 4–40 mm mantle length, ML), Argonauta nodosa (n = 46; 2–19 mm ML), and Loligo sanpaulensis (n = 42, 2–21 mm ML). Segregation of I. argentinus juveniles of similar size suggests school formation as small as 10 mm ML. The presence of mature males along with fertilized female A. nodosa indicates mating early in life. There was a consistent and direct link between high plankton production and high densities of juvenile cephalopods through a short and ecologically efficient food chain. The relationship between production, pycnocline intensity, and the density of paralarvae and juveniles revealed suitable conditions for survival and growth during the upwelling season.

Description: As ecosystem engineers, seagrasses are angiosperms of paramount ecological importance in shallow shoreline habitats around the globe. Furthermore, the ancestors of independent seagrass lineages have secondarily returned into the sea in separate, independent evolutionary events. Thus, understanding the molecular adaptation of this clade not only makes significant contributions to the field of ecology, but also to principles of parallel evolution as well. With the use of Dr. Zompo, the first interactive seagrass sequence database presented here, new insights into the molecular adaptation of marine environments can be inferred. The database is based on a total of 14 597 ESTs obtained from two seagrass species, Zostera marina and Posidonia oceanica, which have been processed, assembled and comprehensively annotated. Dr. Zompo provides experimentalists with a broad foundation to build experiments and consider challenges associated with the investigation of this class of non-domesticated monocotyledon systems. Our database, based on the Ruby on Rails framework, is rich in features including the retrieval of experimentally determined heat-responsive transcripts, mining for molecular markers (SSRs and SNPs), and weighted key word searches that allow access to annotation gathered on several levels including Pfam domains, GeneOntology and KEGG pathways. Well established plant genome sites such as The Arabidopsis Information Resource (TAIR) and the Rice Genome Annotation Project are interfaced by Dr. Zompo. With this project, we have initialized a valuable resource for plant biologists in general and the seagrass community in particular. The database is expected to grow together with more data to come in the near future, particularly with the recent initiation of the Zostera genome sequencing project. The Dr. Zompo database is available at http://drzompo.uni-muenster.de/

Description: The taxonomy of two sympatric surf clams Donax marincovichi Coan, 1983 and Donax obesulus Reeve, 1854, inhabiting the coastal Humboldt Current Upwelling System is revisited. Because both species are exploited by artisanal fisheries, it is essential to verify that they are indeed distinct species that have to be managed separately. In this study, both taxa were sampled across their shared distributional area and specimens were indentified according to their respective morphological characteristics. Although width/height and height/length ratios revealed significant differences within sampling areas, the two morphotypes were frequently incongruent for taxonomically important morphometric parameters. In addition, they showed no significant mitochondrial genetic differentiation within or among populations and exhibited indistinguishable sperm ultrastructure. We conclude that the two morphotypes do not represent distinct species and should be included together under D. obesulus.

Description: The diversity of Cyanobacteria in water and sediment samples from four representative sites of the Salar de Huasco was examined using denaturing gradient gel electrophoresis and analysis of clone libraries of 16S rRNA gene PCR products. Salar de Huasco is a high altitude (3800 m altitude) saline wetland located in the Chilean Altiplano. We analyzed samples from a tributary stream (H0) and three shallow lagoons (H1, H4, H6) that contrasted in their physicochemical conditions and associated biota. Seventy-eight phylotypes were identified in a total of 268 clonal sequences deriving from seven clone libraries of water and sediment samples. Oscillatoriales were frequently found in water samples from sites H0, H1 and H4 and in sediment samples from sites H1 and H4. Pleurocapsales were found only at site H0, while Chroococcales were recovered from sediment samples of sites H0 and H1, and from water samples of site H1. Nostocales were found in sediment samples from sites H1 and H4, and water samples from site H1 and were largely represented by sequences highly similar to Nodularia spumigena. We suggest that cyanobacterial communities from Salar de Huasco are unique - they include sequences related to others previously described from the Antarctic, along with others from diverse, but less extreme environments.

Description: The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Schöhsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Schöhsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Schöhsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environments

Description: Bacterial communities associated with the brown alga Laminaria saccharina from the Baltic Sea and from the North Sea were investigated using denaturing gradient gel electrophoresis and 16S rRNA gene clone libraries. The rhizoid, cauloid, meristem and phyloid revealed different 16S rRNA gene denaturing gradient gel electrophoresis banding patterns indicating a specific association of bacterial communities with different parts of the alga. Associations with cauloid and meristem were more specific, while less specific associations were obtained from the old phyloid. In addition, seasonal and geographical differences in the associated communities were observed. Results from 16S rRNA gene libraries supported these findings. Bacterial phylotypes associated with the alga were affiliated with the Alphaproteobacteria (nine phylotypes), Gammaproteobacteria (nine phylotypes) and the Bacteroidetes group (four phylotypes). A number of bacteria associated with other algae and other marine macroorganisms were among the closest relatives of phylotypes associated with L. saccharina.

Description: Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study.

Description: We studied the effect of the quorum-sensing (QS) blockers 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-2(5H)-one (FUR1), (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl]furan-2(5H)-one (FUR2) and triclosan (TRI) on the formation of bacterial biofilms, and the effect of these biofilms on the larval attachment of the polychaete Hydroides elegans and the bryozoan Bugula neritina. 14-day-old subtidal biofilms were harvested from artificial substrata and were allowed to develop in the laboratory with and without QS blockers. QS blockers inhibited the production of violacein by the QS reporter strain Chromobacterium violaceum CV026 and did not affect the metabolic activity of bacteria in multispecies biofilms. At a concentration of 10(-3) M all three tested compounds inhibited the establishment of microbial communities, but at one of 10(-4) M only FUR2 inhibited establishment. The tested QS blockers caused changes in bacterial density and bacterial community structure, as revealed by terminal restriction fragment length polymorphism and FISH. The groups most affected by QS blockers were Alphaproteobacteria, Gammaproteobacteria and the Cytophagales. Larvae of H. elegans and B. neritina avoided settling on biofilms that had developed in the presence of QS blockers. Our results suggest that QS blockers directly control the formation of multi-species biofilms, and indirectly - by means of biofilm properties - affect larval attachment on these modified biofilms.

Description: Microbial isolate Z143-1 found to be associated with an unidentified tunicate was characterized due to its significant antimicrobial activity. Z143-1 is similar to Pseudovibrio ascidiaceicola and Pseudovibrio denitrificans in morphological, physiological and biochemical characteristics, except for its ability to ferment glucose and produce a characteristic red pigment. Fatty acid methyl ester analysis revealed a predominance of the fatty acid 18:1 ω7c at 80.55%, at levels slightly lower than the Pseudovibrio denitrificans type strain DN34T (87.7%). The mol% G+C of Z143-1 is 54.02, relatively higher than the Pseudovibrio denitrificans type strain DN34T and Pseudovibrio ascidiaceicola with mol% G+C of 51.7 and 51.4, respectively. However, phylogenetic analysis of the 16S rRNA gene sequence of Z143-1 showed 100% similarity with the Pseudovibrio denitrificans type strain DN34T. In this study, the bacterium Z143-1 is reported as a new strain of Pseudovibrio denitrificans. While there is no report of a secondary metabolite for Pseudovibrio denitrificans, Z143-1 produces the red pigment heptylprodigiosin, also known as 16-methyl-15-heptyl-prodiginine, which shows anti-Staphylococcus aureus activity.

Description: Mechanisms that affect thermal tolerance of ectothermic organisms have recently received much interest, mainly due to global warming and climate-change debates in both the public and in the scientific community. In physiological terms, thermal tolerance of several marine ectothermic taxa can be linked to oxygen availability, with capacity limitations in ventilatory and circulatory systems contributing to oxygen limitation at extreme temperatures. The present review briefly summarizes the processes that define thermal tolerance in a model cephalopod organism, the cuttlefish Sepia officinalis, with a focus on the contribution of the cephalopod oxygen-carrying blood pigment, hemocyanin. When acutely exposed to either extremely high or low temperatures, cuttlefish display a gradual transition to an anaerobic mode of energy production in key muscle tissues once critical temperatures (Tcrit) are reached. At high temperatures, stagnating metabolic rates and a developing hypoxemia can be correlated with a progressive failure of the circulatory system, well before Tcrit is reached. However, at low temperatures, declining metabolic rates cannot be related to ventilatory or circulatory failure. Rather, we propose a role for hemocyanin functional characteristics as a major limiting factor preventing proper tissue oxygenation. Using information on the oxygen binding characteristics of cephalopod hemocyanins, we argue that high oxygen affinities (= low P50 values), as found at low temperatures, allow efficient oxygen shuttling only at very low venous oxygen partial pressures. Low venous PO2s limit rates of oxygen diffusion into cells, thus eventually causing the observed transition to anaerobic metabolism. On the basis of existing blood physiological, molecular, and crystallographical data, the potential to resolve the role of hemocyanin isoforms in thermal adaptation by an integrated molecular physiological approach is discussed.

Description: Recent molecular studies investigating higher-level phylogenetics of coleoid cephalopods (octopuses, squids and cuttlefishes) have produced conflicting results. A wide range of sequence alignment and analysis methods are used in cephalopod phylogenetic studies. The present study investigated the effect of commonly used alignment and analysis methods on higher-level cephalopod phylogenetics. Two sequence homology methods: (1) eye alignment, (2) implied alignment, and three analysis methods: (1) parsimony, (2) maximum likelihood, (3) Bayesian methodologies, were employed on the longest sequence dataset available for the coleoid cephalopods, comprising three mitochondrial and six nuclear loci. The data were also tested for base composition heterogeneity, which was detected in three genes and resolved using RY coding. The Octopoda, Argonautoidea, Oegopsida and Ommastrephidae are monophyletic in the phylogenies resulting from each of the alignment and analysis combinations. Furthermore, the Bathyteuthidae are the sister taxon of the Oegopsida in each case. However many relationships within the Coleoidea differed depending upon the alignment and analysis method used. This study demonstrates how differences in alignment and analysis methods commonly used in cephalopod phylogenetics can lead to different, but often highly supported, relationships.

Description: The basal position of the Mamiellales (Prasinophyceae) within the green lineage makes these unicellular organisms key to elucidating early stages in the evolution of chlorophyll a/b-binding light-harvesting complexes (LHCs). Here, we unveil the complete and unexpected diversity of Lhc proteins in Ostreococcus tauri, a member of the Mamiellales order, based on results from complete genome sequencing. Like Mantoniella squamata, O. tauri possesses a number of genes encoding an unusual prasinophyte-specific Lhc protein type herein designated "Lhcp". Biochemical characterization of the complexes revealed that these polypeptides, which bind chlorophylls a, b, and a chlorophyll c-like pigment (Mg-2,4-divinyl-phaeoporphyrin a 5 monomethyl ester) as well as a number of unusual carotenoids, are likely predominant. They are retrieved to some extent in both reaction center I (RCI)- and RCII-enriched fractions, suggesting a possible association to both photosystems. However, in sharp contrast to previous reports on LHCs of M. squamata, O. tauri also possesses other LHC subpopulations, including LHCI proteins (encoded by five distinct Lhca genes) and the minor LHCII polypeptides, CP26 and CP29. Using an antibody against plant Lhca2, we unambiguously show that LHCI proteins are present not only in O. tauri, in which they are likely associated to RCI, but also in other Mamiellales, including M. squamata. With the exception of Lhcp genes, all the identified Lhc genes are present in single copy only. Overall, the discovery of LHCI proteins in these prasinophytes, combined with the lack of the major LHCII polypeptides found in higher plants or other green algae, supports the hypothesis that the latter proteins appeared subsequent to LHCI proteins. The major LHC of prasinophytes might have arisen prior to the LHCII of other chlorophyll a/b-containing organisms, possibly by divergence of a LHCI gene precursor. However, the discovery in O. tauri of CP26-like proteins, phylogenetically placed at the base of the major LHCII protein clades, yields new insight to the origin of these antenna proteins, which have evolved separately in higher plants and green algae. Its diverse but numerically limited suite of Lhc genes renders O. tauri an exceptional model system for future research on the evolution and function of LHC components. © The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

Description: Comparable values have been obtained for endogenous fecal calcium in cattle by an “isotope dilution” method and a “comparative balance” method. Both procedures involve the use of labeled calcium, but they are different in principle. The “isotope dilution” method offers advantages in being independent of the availability of the dietary calcium and of the uncertainties of balance determinations.

Description: Endogenous fecal calcium was estimated directly in normal cattle by a method which consisted of 10 daily intravenous injections of radiocalcium, comparison of the specific activities of blood and feces taken on the 4th to 8th day post-injection, and routine determination of calcium balance. The daily endogenous fecal values for three 8-year-old dairy cows averaged 7 gm and for two yearling Hereford steers about 4 gm. The endogenous fecal calcium was not significantly changed when the dietary calcium was varied from normal to a high level. In two animals on a low calcium diet a decrease in endogenous fecal calcium was indicated.