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Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23degrees22'N, 44degrees57'W) and the Guaymas Basin (27degrees01'N, 111degrees24'W) at a depth of 2,616 m. Positive enrichments were submitted to gamma-irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1(T) was isolated from chimney samples collected from the Mid-Atlantic Ridge after gamma-irradiation at 20 kGy, and strain EJ2(T) was isolated from the Guaymas Basin after gamma-irradiation at 30 kGy. Only strain EJ2(T) was motile, and both formed regular cocci. These new strains grew between 55 and 95 degreesC with the optimal temperature being 88 degreesC. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l(-1). These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1(T), such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2(T) was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2(T) growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1(T) and EJ2(T). The G+C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1(T) and EJ2(T), respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA-DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1(T) (=DSM 15227(T)=JCM 11825(T)) and the type strain of T. radiotolerans is strain EJ2(T) (=DSM 15228(T)=JCM 11826(T)).

A mesophilic, aerobic, non-flagellated, gliding bacterium, forming yellow colonies and designated Dsij(T), was isolated from a red alga on the sea-shore of Roscoff, Brittany, France. Dsij(T) was selected for its ability to actively degrade both agars and carrageenans. The Cram-negative cells occurred singly or in pairs as long rods. The temperature range for growth was 13-45 degreesC, with an optimum at 35 degreesC. The ph range for growth at 35 degreesC was from 6.0 to 8.5, with an optimum around ph 7.0. The NaCl concentrations required for growth at 35 degreesC and ph 7.0 ranged from 5 to 60 g l(-1), with an optimum around 25 g l(-1). The G+C content of the genomic DNA was 42-43 mol%. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Dsij(T) is closely related to [Cytophaga] uliginosa DSM 2061(T). Phenotypic features, however, allowed Dsij(T) and [Cytophaga] uliginosa strains to be distinguished on the basis of ten traits (spreading behaviour, assimilation of eight compounds and amylase production). Their total protein profiles were also different and DNA-DNA hybridization experiments confirmed that Dsij(T) constitutes a new species, distinct from [Cytophaga] uliginosa. Based on the phenotypic features and the phylogenetic relationships of the Flavobacteriaceae, a new genus designated Zobellia gen, nov, is proposed to include Zobellia galactanovorans gen. nov., sp. nov., while [Cytophaga] uliginosa becomes Zobellia uliginosa comb, nov. The type strain of Zobellia galactanovorans is Dsij(T) (= DSM 12802(T) = CIP 106680(T)).

An autotrophic, hyperthermophilic methanogen (M7(T)) was isolated from a deep-sea hydrothermal chimney sample collected on the East Pacific Rise at a depth of 2600 m. The coccoid-shaped cells are flagellated and exhibit a slight tumbling motility. The temperature range for growth at pH 6.5 was 49-89 degrees C, with optimum growth at 80 degrees C. The optimum ph for growth was 6.5, and the optimum NaCl concentration for growth was around 25 g l(-1). The new isolate used H-2 and CO2 as the only substrates for growth and methane production. Tungsten, selenium and yeast extract stimulated growth significantly. In the presence of CO2 and H-2, the organism reduced elemental sulphur to hydrogen sulphide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin and streptomycin. The G+C content of the genomic DNA was 31 mol%. As determined by 16S rDNA gene sequence analysis, this organism was closely related to Methanococcus jannaschii strain JAL-1(T). However, despite the high percentage of similarity between their 16S rDNA sequences (97.1%), the DNA-DNA hybridization levels between these strains were less than 5%. On the basis of these observations and physiological traits, it is proposed that this organism should be placed in a new species, Methanococcus vulcanius. The type strain is M7(T)(= DSM 12094(T)), During the course of this study, the 16S rDNA sequence analysis placed Methanococcus sp. strain AG86(T) (= DSM 4213(T)) as a close relative of M, jannaschii strain JAL-1T. However, the weak level of DNA-DNA hybridization with this strain (< 10%) allowed the proposal that strain AG86(T) also constitutes a new species, Methanococcus fervens.

International audience; High-throughput sequencing (HTS) technologies offer new promise to support surveillance programs targeting marine non-indigenous species (NIS). Metabarcoding might surpass traditional monitoring methods, for example through its ability to detect rare species, a key feature in early detection of NIS. Another interest of this approach is the identification of organisms difficult to identify based on morphology only (e.g., early developmental stages), making it relevant in the context of management programs. Because many marine benthic NIS have a biphasic bentho-pelagic life cycle, targeting their pelagic larval stages in zooplankton may allow early detection and assessment of their establishment and potential spread. We illustrate this approach with an analysis of bulk-DNA retrieved from a time-series of zooplankton samples collected over 22 months in one bay in Brittany (France). Using HTS of amplicons obtained with two markers (COI and 18S) and a metabarcoding approach, 12 NIS were identified and their temporal larval dynamics were monitored. Importantly, we chose to focus on a closed list of species, from four metazoan classes encompassing 52 NIS reported within the study area or nearby seas, with molecular references available or obtained locally for 42 of them. The use of a custom-designed database allowed the detection of three NIS otherwise not detected when using public databases. Interestingly, NIS known to have a short-lived larval stage were detected (e.g., the bryozoan Bugula neritina or the tunicate Corella eumyota). For two molluscs Ruditapes philippinarum and Crepidula fornicata, metabarcoding results were compared to those obtained using traditional methods (i.e., barcoding of individual larvae and morphology, respectively) to show the reliability of the approach in detecting and assessing the extent of their reproductive periods. Our results also revealed that the Pacific oyster Crassostrea gigas, a notorious invasive species, failed to reproduce in the study bay, showing that metabarcoding on larval stages also provides information regarding the establishment success (or failure) of NIS. While metabarcoding has its limitations and biases, this study demonstrates its effectiveness for surveillance of targeted NIS, notably to support management strategies like the European Marine Strategy Framework Directive (MSFD).

We have previously reviewed data standards and best practices in marine metagenomics and have published our review and three core recommendations (ten Hoopen et al., 2017). On our first recommendation - that scientists comply with data standards and recommended best practices, we assess in this Annex the status across sampling, sequencing, analysis and results archiving stages of metagenomics studies. We report for our second recommendation - that computational analysis process be described formally, our progress through collaborative work with the Interoperability platform on adopting the Common Workflow Language (CWL) We describe that in response to our third recommendation - that the results of computational metabarcoding and metagenomics analysis processes be archived.