Los polizones ignorados: dispersión por el mundo de las especies de microalgas exóticas mediante el «biofouling» que recubre el fondo de los barcos / The ignored stowaways: worldwide dispersion of exotic microalgae species through the biofouling recovering the ships underwater body

Invasion by introduced species cause huge environmental damageand economic (estimated in $138 billion in USA). Marine ecosystemsare specially affected by introduced species of toxin-producingmicroalgae. Ships ballast water has been considered the major vectorin dispersion of phytoplankton. However, most ships do not useballast water. Alternatively, we propose that the biofouling recoveringthe underwater body of ships is the main cause of microalgaldispersion. Antifouling paints (containing tributyltin, TBT or othertoxics) are used to coat the bottoms of ships to prevent biofouling.After sampling biofouling recovering the underwater body of shipswe demonstrate that numerous coastal, oceanic and toxin-producingmicroalgae species proliferates attached on bottoms of ships directlyon TBT antifouling paint. These microalgae species should beresistant variants because antifouling paints rapidly destroy sensitivewild type microalgae. Consequently, the key to explain microalgaespecies transport via ships biofouling is know the mechanisms thatallow to these species to survive long time attached to antifoulingpaint. A fluctuation analysis demonstrate that genetic adaptationby rare spontaneous mutation, which occurs by chance prior toantifouling exposure is the mechanism allowing adaptation ofmicroalgae to antifoulig paints and their dispersion in the shipsbiofouling. Around 3 TBT-resistant mutants per each 10–4 wild typesensitive cells occurs in microalgal population. This assures a rapidcolonization of ships bottoms to travel long-distances(AU)

La introducción de especies invasoras puede causar grandes problemasmedioambientales y económicos (estimados en 138 billonesde $ en USA). Los ecosistemas marinos se ven especialmente afectadospor la introducción de microalgas tóxicas. El agua de lastre delos barcos está considerada como el mayor vector de dispersión defitoplancton. Sin embargo, la mayoría de los barcos no tienen lastrede agua. Como alternativa, proponemos que el biofouling que recubrelos barcos es la principal causa de dispersión de microalgas. Seutilizan pinturas antifouling (conteniendo tributil-estaño, TBT uotros tóxicos) para recubrir la obra viva de los barcos previniendo elbiofouling. Despues de diversos muestreos de la obra viva en barcosdemostramos que numerosas especies de microalgas costeras, oceánicasy productoras de toxinas son capaces de proliferar adheridasa la obra viva de los barcos, directamente sobre la pintura TBT antifouling.Estas microalgas deben ser variantes resistentes porque elTBT rápidamente destruye las microalgas sensibles. Consecuentemente,la clave para explicar el transporte de las especies de microalgasen el biofouling de los barcos es conocer los mecanismos quepermiten a las especies sobrevivir mucho tiempo sobre la pinturaantifouling. Un análisis de fluctuación ha demostrado que la adaptacióngenética debida a raras mutaciones espóntaneas, que ocurrenanteriormente a la exposición al TBT, es el mecanismo que le permitea las microalgas adaptarse a la pintura antifouling y su posteriordispersión en el biofouling. Hay alrededor de tres mutantes resistentesal TBT por cada 10–4 células sensibles en la población. Estoasegura la rápida colonización de la obra viva de los barcos paraviajar largas distancias(AU)

Novel bisabolane sesquiterpenes from the marine-derived fungus Verticillium tenerum.

Chemical investigations of the marine-derived fungus Verticillium tenerum yielded two new hydroxylated bisabolane-type sesquiterpenes verticinol A (1) and B (2). The planar structures of the new compounds were elucidated by employing spectroscopic (NMR, UV, and IR) and mass spectrometric techniques. The absolute configuration of the cyclohexenyl moiety was deduced by a combination of CD spectroscopy and NOESY measurements.

Microbial ecology of corals, sponges, and algae in mesophotic coral environments.

Mesophotic coral ecosystems that occur at depths from 30 to 200 m have historically been understudied and yet appear to support a diverse biological community. The microbiology of these systems is particularly poorly understood, especially with regard to the communities associated with corals, sponges, and algae. This lack of information is partly due to the problems associated with gaining access to these environments and poor reproducibility across sampling methods. To summarize what is known about the microbiology of these ecosystems and to highlight areas where research is urgently needed, an overview of the current state of knowledge is presented. Emphasis is placed on the characterization of microbial populations, both prokaryotic and eukaryotic, associated with corals, sponges, and algae and the factors that influence microbial community structure. In topic areas where virtually nothing is known from mesophotic environments, the knowledge pertaining to shallow-water ecosystems is summarized to provide a starting point for a discussion on what might be expected in the mesophotic zone.

Methods for isolation of marine-derived endophytic fungi and their bioactive secondary products.

Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some of them featuring new carbon frameworks hitherto unprecedented in nature. These compounds are of interest as new lead structures for medicine as well as for plant protection. The aim of this protocol is to give a detailed description of methods useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on rapid 'in house' screening systems. Some results exemplifying the positive outcomes of the protocol are given at the end. From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at least 3 months has to be scheduled.

Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade.

The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC-MS, and several MeSH-derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]-3-(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH- and MeSeH-derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH-derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

Flavobacterium algicola sp. nov., isolated from marine algae.

A rod-shaped Gram-staining-negative, non-motile, aerobic and fucoidan-digesting strain, designated TC2(T), was isolated from marine algae collected from the coast of the Sea of Okhotsk at Abashiri, Hokkaido, Japan. The bacterium formed yellow, translucent, circular and convex colonies. Comparative 16S rRNA gene sequence analysis indicated that the strain belonged to the genus Flavobacterium, with the highest sequence similarities of 97.1 to 97.3 % to the type strains of Flavobacterium frigidarium, Flavobacterium frigoris, Flavobacterium limicola and Flavobacterium psychrolimnae. DNA-DNA relatedness values between strain TC2(T) and the above-mentioned species were lower than 28 %. The genomic DNA G+C content was 33.9 mol%. The major respiratory quinone was menaquinone-6 and the predominant fatty acids were iso-C(15 : 1) G, iso-C(15 : 0), iso-C(15 : 0) 3-OH and summed feature 3 (which comprises iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c). Strain TC2(T) could be differentiated from related species by several phenotypic characteristics. Thus, on the basis of these results, strain TC2(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium algicola sp. nov. is proposed. The type strain is TC2(T) (=NBRC 102673(T) =CIP 109574(T)).

The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker's guide to life in the sea.

Dinoroseobacter shibae DFL12(T), a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12(T) is able to synthesize the vitamins B(1) and B(12) for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B(12) are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B(12) was confirmed to be functional, and D. shibae DFL12(T) was shown to provide the growth-limiting vitamins B(1) and B(12) to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12(T) is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12(T) has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12(T) shows the most complex viral defense system of all Rhodobacterales sequenced to date.

A cluster of amyotrophic lateral sclerosis in New Hampshire: a possible role for toxic cyanobacteria blooms.

Cyanobacteria produce many neurotoxins including beta-methylamino-L-alanine (BMAA) that has been liked to amyotrophic lateral sclerosis (ALS) and neurodegenerative disease. A number of ALS cases have been diagnosed among residents of Enfield, NH, a town encompassing a lake with a history of cyanobacteria algal blooms. To investigate an association between toxic cyanobacterial blooms in New Hampshire and development of ALS, we reviewed records from our institution and other community databases to obtain demographic information on patients diagnosed with ALS within New England. We identified nine ALS patients who lived near Lake Mascoma in Enfield, NH, an incidence of sporadic ALS that is 10 to 25 times the expected incidence of 2/100,000/year. We suggest that the high incidence of ALS in this potential cluster could be directly related to chronic exposure to cyanobacterial neurotoxins such as BMAA. Possible routes of toxin exposure include inhalation of aerosolized toxins, consuming fish, or ingestion of lake water. Further investigation, including analysis of brain tissue for cyanobacterial toxins, will be helpful to test for an association between BMAA and ALS.

Complex coevolutionary history of symbiotic Bacteroidales bacteria of various protists in the gut of termites.

BACKGROUND: The microbial community in the gut of termites is responsible for the efficient decomposition of recalcitrant lignocellulose. Prominent features of this community are its complexity and the associations of prokaryotes with the cells of cellulolytic flagellated protists. Bacteria in the order Bacteroidales are involved in associations with a wide variety of gut protist species as either intracellular endosymbionts or surface-attached ectosymbionts. In particular, ectosymbionts exhibit distinct morphological patterns of the associations. Therefore, these Bacteroidales symbionts provide an opportunity to investigate not only the coevolutionary relationships with the host protists and their morphological evolution but also how symbiotic associations between prokaryotes and eukaryotes occur and evolve within a complex symbiotic community. RESULTS: Molecular phylogeny of 31 taxa of Bacteroidales symbionts from 17 protist genera in 10 families was examined based on 16S rRNA gene sequences. Their localization, morphology, and specificity were also examined by fluorescent in situ hybridizations. Although a monophyletic grouping of the ectosymbionts occurred in three related protist families, the symbionts of different protist genera were usually dispersed among several phylogenetic clusters unique to termite-gut bacteria. Similar morphologies of the associations occurred in multiple lineages of the symbionts. Nevertheless, the symbionts of congeneric protist species were closely related to one another, and in most cases, each host species harbored a unique Bacteroidales species. The endosymbionts were distantly related to the ectosymbionts examined so far. CONCLUSION: The coevolutionary history of gut protists and their associated Bacteroidales symbionts is complex. We suggest multiple independent acquisitions of the Bacteroidales symbionts by different protist genera from a pool of diverse bacteria in the gut community. In this sense, the gut could serve as a reservoir of diverse bacteria for associations with the protist cells. The similar morphologies are considered a result of evolutionary convergence. Despite the complicated evolutionary history, the host-symbiont relationships are mutually specific, suggesting their cospeciations at the protist genus level with only occasional replacements.

The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors.

Only a limited number of bacterial pathogens evade destruction by phagocytic cells such as macrophages. Legionella pneumophila is a Gram-negative gamma-proteobacterial species that can infect and replicate in alveolar macrophages, causing Legionnaires' disease, a severe pneumonia. L. pneumophila uses a complex secretion system to inject host cells with effector proteins capable of disrupting or altering the host cell processes. The L. pneumophila effectors target multiple processes but are essentially aimed at modifying the properties of the L. pneumophila phagosome by altering vesicular trafficking, gradually creating a specialized vacuole in which the bacteria replicate robustly. In nature, L. pneumophila is thought to parasitize free-living protists, which may have selected for traits that promote virulence of L. pneumophila in humans. Indeed, many effector genes encode proteins with eukaryotic domains and are likely to be of protozoan origin. Sustained horizontal gene transfer events within the protozoan niche may have allowed L. pneumophila to become a professional parasite of phagocytes, simultaneously giving rise to its ability to infect macrophages, cells that constitute the first line of cellular defence against bacterial infections.

Broad-spectrum antimicrobial epiphytic and endophytic fungi from marine organisms: isolation, bioassay and taxonomy.

In the search for new marine derived antibiotics, 43 epi- and endophytic fungal strains were isolated from the surface or the inner tissue of different marine plants and invertebrates. Through preliminary and secondary screening, 10 of them were found to be able to produce broad-spectrum antimicrobial metabolites. By morphological and molecular biological methods, three active strains were characterized to be Penicillium glabrum, Fusarium oxysporum, and Alternaria alternata.

Morphology, phylogeny, and diversity of Trichonympha (Parabasalia: Hypermastigida) of the wood-feeding cockroach Cryptocercus punctulatus.

Trichonympha is one of the most complex and visually striking of the hypermastigote parabasalids-a group of anaerobic flagellates found exclusively in hindguts of lower termites and the wood-feeding cockroach Cryptocercus-but it is one of only two genera common to both groups of insects. We investigated Trichonympha of Cryptocercus using light and electron microscopy (scanning and transmission), as well as molecular phylogeny, to gain a better understanding of its morphology, diversity, and evolution. Microscopy reveals numerous new features, such as previously undetected bacterial surface symbionts, adhesion of post-rostral flagella, and a distinctive frilled operculum. We also sequenced small subunit rRNA gene from manually isolated species, and carried out an environmental polymerase chain reaction (PCR) survey of Trichonympha diversity, all of which strongly supports monophyly of Trichonympha from Cryptocercus to the exclusion of those sampled from termites. Bayesian and distance methods support a relationship between Trichonympha species from termites and Cryptocercus, although likelihood analysis allies the latter with Eucomonymphidae. A monophyletic Trichonympha is of great interest because recent evidence supports a sister relationship between Cryptocercus and termites, suggesting Trichonympha predates the Cryptocercus-termite divergence. The monophyly of symbiotic bacteria of Trichonympha raises the intriguing possibility of three-way co-speciation among bacteria, Trichonympha, and insect hosts.

Microbulbifer variabilis sp. nov. and Microbulbifer epialgicus sp. nov., isolated from Pacific marine algae, possess a rod-coccus cell cycle in association with the growth phase.

Phylogenetic and taxonomic characterization was performed for 14 strains of bacteria that produce anticancer antibiotics (pelagiomicins) (represented by strain Ni-2088(T)) and one strain that produces UV-absorbing substances (strain F-104(T)), isolated from marine algae and seagrass collected from coastal areas of tropical Pacific islands and a subtropical island of Japan. All 15 isolates were Gram-negative, strictly aerobic, non-motile and non-spore-forming. Sequence analysis of the 16S rRNA gene showed that the isolates occupied positions in the phylogenetic radiation of the genus Microbulbifer, with similarities of 93.6-97.6 %. The cells possessed a clearly discernible rod-coccus cell cycle in association with the growth phase; cells were rods during the growth phase and all converted to coccoid-ovoid cells when proliferation ceased. The coccoid-ovoid cells were optically denser than the rod cells and were viable for extended periods. They were considered to constitute a resting form. The type strains of described species of Microbulbifer were also found to possess identical rod-coccus cell cycles. The G+C content of the DNA was 48.1-49.7 mol%. The major respiratory quinone system was ubiquinone-8. The major fatty acids were C(18 : 1)omega7c and C(16 : 0), and the hydroxy acids comprised C(10 : 0) 3-OH, C(12 : 0) 3-OH and iso-C(11 : 0) 3-OH. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The group of 14 pelagiomicin-producing strains and strain F-104(T) each constituted a single genomic species. Based on phylogenetic affiliation, phenotypic characteristics and genomic distinctness, the isolates represent two novel species in the genus Microbulbifer, for which the names Microbulbifer variabilis sp. nov. (type strain Ni-2088(T) =MBIC01082(T) =ATCC 700307(T)) and Microbulbifer epialgicus sp. nov. (type strain F-104(T) =MBIC03330(T) =DSM 18651(T)) are proposed.

Alkalibacterium thalassium sp. nov., Alkalibacterium pelagium sp. nov., Alkalibacterium putridalgicola sp. nov. and Alkalibacterium kapii sp. nov., slightly halophilic and alkaliphilic marine lactic acid bacteria isolated from marine organisms and salted foods collected in Japan and Thailand.

We describe 10 new strains of marine lactic acid bacteria isolated from decaying marine algae, decaying seagrass, raw fish, salted fish and salted and fermented shrimp paste ('ka-pi') collected from a temperate area of Japan and Thailand. The isolates are Gram-positive and non-sporulating. They have motility with peritrichous flagella depending on the strains. They lack catalase and quinones. Under anaerobic conditions, lactate yields were 64-93 % of the glucose consumed; residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. The pH of the fermentation medium markedly affected the product composition; at higher pH, the yield of lactate decreased (15-48 % at pH 9.0) and yields of other products increased, retaining the molar ratio. Under aerobic conditions, acetate and lactate were produced from carbohydrates and related compounds. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 0.5 and 4.0 % (w/v), depending on the strain, with a growth range of between 0 and 17-21 % (11 % for one isolate). The optimum pH for growth ranged between 8.0 and 9.5, with a growth range of 6.0-11.0, depending on the strains. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied three phylogenetic positions within the genus Alkalibacterium, showing 97.1-99.8 % similarity to Alkalibacterium indicireducens. DNA-DNA hybridization values (<46 %) among the 10 isolates and phylogenetically related taxa resulted in the identification of four genomic species (designated groups GS1-GS4). The G+C contents of the DNA were 41.7 mol% (group GS1), 42.2 mol% (group GS2), 41.0-43.0 mol% (group GS3) and 38.4-39.4 mol% (group GS4). The cell-wall peptidoglycan was type A4beta, Orn-d-Asp, for three genomic species (groups GS1, GS2 and GS3), and type A4beta, Orn-d-Glu, for the remaining species (group GS4). The major components of cellular fatty acids were C(16 : 0), C(16 : 1)omega9c and C(18 : 1)omega9c (oleic acid). On the bases of phenotypic characteristics, genetic distinctiveness and phylogenetic affiliations, the four genomic species, groups GS1, GS2, GS3 and GS4, were classified as four novel species within the genus Alkalibacterium, for which the names Alkalibacterium thalassium sp. nov., Alkalibacterium pelagium sp. nov., Alkalibacterium putridalgicola sp. nov. and Alkalibacterium kapii sp. nov., respectively, are proposed. The respective type strains are T117-1-2(T) (=DSM 19181(T)=NBRC 103241(T)=NRIC 0718(T)), T143-1-1(T) (=DSM 19183(T)=NBRC 103242(T)=NRIC 0719(T)), T129-2-1(T) (=DSM 19182(T)=NBRC 103243(T)=NRIC 0720(T)) and T22-1-2(T) (=DSM 19180(T)=NBRC 103247(T)=NRIC 0724(T)).

A Legionella effector acquired from protozoa is involved in sphingolipids metabolism and is targeted to the host cell mitochondria.

Legionella pneumophila infects alveolar macrophages and protozoa through establishment of an intracellular replication niche. This process is mediated by bacterial effectors translocated into the host cell via the Icm/Dot type IV secretion system. Most of the effectors identified so far are unique to L. pneumophila; however, some of the effectors are homologous to eukaryotic proteins. We performed a distribution analysis of many known L. pneumophila effectors and found that several of them, mostly eukaryotic homologous proteins, are present in different Legionella species. In-depth analysis of LegS2, a L. pneumophila homologue of the highly conserved eukaryotic enzyme sphingosine-1-phosphate lyase (SPL), revealed that it was most likely acquired from a protozoan organism early during Legionella evolution. The LegS2 protein was found to translocate into host cells using a C-terminal translocation domain absent in its eukaryotic homologues. LegS2 was found to complement the sphingosine-sensitive phenotype of a Saccharomyces serevisia SPL-null mutant and this complementation depended on evolutionary conserved residues in the LegS2 catalytic domain. Interestingly, unlike the eukaryotic SPL that localizes to the endoplasmic reticulum, LegS2 was found to be targeted mainly to host cell mitochondria. Collectively, our results demonstrate the remarkable adaptations of a eukaryotic protein to the L. pneumophila pathogenesis system.

Functional GacS in Pseudomonas DSS73 prevents digestion by Caenorhabditis elegans and protects the nematode from killer flagellates.

The success of biocontrol bacteria in soil depends in part on their ability to escape predation. We explored the interactions between Pseudomonas strain DSS73 and two predators, the nematode Caenorhabditis elegans and the flagellate Cercomonas sp. Growth of the nematode in liquid culture was arrested when it was feeding on DSS73 or a DSS73 mutant (DSS73-15C2) unable to produce the biosurfactant amphisin, whereas a regulatory gacS mutant (DSS73-12H8) that produces no exoproducts supported fast growth of the nematode. The flagellate Cercomonas sp. was able to grow on all three strains. The biosurfactant-deficient DSS73 mutant caused severe dilation of the nematode gut. In three-species systems (DSS73, Cercomonas and C. elegans), the nematodes fed on the flagellates, which in turn grazed the bacteria and the number of C. elegans increased. The flagellates Cercomonas sp. usually kill C. elegans. However, DSS73 protected the nematodes from flagellate killing. Soil microcosms inoculated with six rhizobacteria and grazed by nematodes were colonized more efficiently by DSS73 than similar systems grazed by flagellates or without grazers. In conclusion, our results suggest that C. elegans and DSS73 mutually increase the survival of one another in complex multispecies systems and that this interaction depends on the GacS regulator.

Legionella, protozoa, and biofilms: interactions within complex microbial systems.

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.

Real-time PCR detection and quantification of fish probiotic Phaeobacter strain 27-4 and fish pathogenic Vibrio in microalgae, rotifer, Artemia and first feeding turbot (Psetta maxima) larvae.

AIMS: To develop a SYBR Green quantitative real-time PCR protocol enabling detection and quantification of a fish probiotic and two turbot pathogenic Vibrio spp. in microcosms. METHODS AND RESULTS: Phaeobacter 27-4, Vibrio anguillarum 90-11-287 and Vibrio splendidus DMC-1 were quantified as pure and mixed cultures and in presence of microalgae (Isochrysis galbana), rotifers (Brachionus plicatilis), Artemia nauplii or turbot (Psetta maxima) larvae by real-time PCR based on primers directed at genetic loci coding for antagonistic and virulence-related functions respectively. The optimized protocol was used to study bioencapsulation and maintenance of the probiont and pathogens in rotifers and for the detection and quantification of Phaeobacter and V. anguillarum in turbot larvae fed rotifers loaded with the different bacteria in a challenge trial. CONCLUSIONS: Our real-time PCR protocol is reproducible and specific. The method requires separate standard curve for each host organism and can be used to detect and quantify probiotic Phaeobacter and pathogenic Vibrio bioencapsulated in rotifers and in turbot larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our method allows monitoring and quantification of a turbot larvae probiotic bacteria and turbot pathogenic vibrios in in vivo trials and will be useful tools for detecting the bacteria in industrial rearing units.

Metabolites from symbiotic bacteria.

This review describes secondary metabolites that have been shown to be synthesized by symbiotic bacteria, or for which this possibility has been discussed. It includes 365 references.

Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters.

Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.