The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.

Menorca Channel (Balearic Islands, western Mediterranean) comprises 98,700 Ha of continental shelf. It has been proposed to include this area in the Natura 2000 network due to the wide range of species and habitats of high conservation value found here, such as Posidonia oceanica meadows and maërl and coralligenous beds. This study aimed to establish a scientific basis for managing and protecting the continental shelf bottoms in Menorca Channel. Sampling was carried out with side-scan sonar, beam trawls, box corers, a remote-operated vehicle and an underwater drop camera. The information collected was used to map the habitat distribution between 50 and 100 m depth, as well as make an inventory and describe the spatial patterns of both the specific and functional diversity. A total of 636 species was recorded in a mosaic of habitats in which Corallinacea calcareous algae and other soft red algae (Osmundaria volubilis and Peyssonnelia spp.) were the most abundant groups. Hotspots of specific and functional diversity were located in areas with high habitat heterogeneity and complexity. Protection of Menorca Channel should not only include the habitats and species in the European directives, but also the habitats that are not currently protected, such as O. volubilis and Peyssonnelia beds, due to Electronic supplementary material The online version of this article (

Marine Crenarchaeota represent an abundant component of the oceanic microbiota that play an important role in the global nitrogen cycle. Here we report the association of the colonial ascidian Cystodytes dellechiajei with putative ammonia-oxidizing Crenarchaeota that could actively be involved in nitrification inside the animal tissue. As shown by 16S rRNA gene analysis, the ascidian-associated Crenarchaeota were phylogenetically related to Nitrosopumilus maritimus, the first marine archaeon isolated in pure culture that grows chemolithoautotrophically oxidizing ammonia to nitrite aerobically. Catalysed reporter deposition (CARD)-FISH revealed that the Crenarchaeota were specifically located inside the tunic tissue of the colony, where moreover the expression of amoA gene was detected. The amoA gene encodes the alpha-subunit of ammonia monooxygenase, which is involved in the first step of nitrification, the oxidation of ammonia to nitrite. Sequencing of amoA gene showed that they were phylogenetically related to amoA genes of N. maritimus and other putative ammonia-oxidizing marine Crenarchaeota. In order to track the suspected nitrification activity inside the ascidian colony under in vivo conditions, microsensor profiles were measured through the tunic tissue. Net NO(x) production was detected in the tunic layer 1200-1800 microm with rates of 58-90 nmol cm(-3) h(-1). Oxygen and pH microsensor profiles showed that the layer of net NO(x) production coincided with O(2) concentrations of 103-116 microM and pH value of 5.2. Together, molecular and microsensor data indicate that Crenarchaeota could oxidize ammonia to nitrite aerobically, and thus be involved in nitrification inside the ascidian tissue.

A B S T R A C TDesalination of seawater has been considered as a potential solution for the water shortage problem in coastal areas and the number of projected and constructed desalination plants has significantly increased in recent years. The challenge of the desalination industry is to produce new water resources without increasing the pressure on the marine environment. Environmental impact of SWRO desalination plants is mainly associated with the discharge into the sea of the brine produced. To estimate the area of influence of the brine several models have been proposed, but validation with real data is needed. The objective of this paper is to present the results of the monitoring of the brine effluent emanating from several SWRO desalination plants in the western Mediterranean Sea in order to estimate the area of influence of the hypersaline plume. We also illustrate how the behavior of these brine discharges can differ significantly according to discharge characteristics. This information may be useful to predict effluent distribution in order to minimize the harmful effects of brine discharges into the sea.

Symbiotic crustaceans are expected to live solitarily with their hosts when members of their host species are small (relative to symbiont body size) and structurally simple. We tested the hypothesis of a solitary lifestyle in Ascidonia flavomaculata, a symbiotic shrimp that inhabits the branchial chamber of the relatively small and structurally simple tunicate Ascidia mentula in the subtidal zone of the Islas Baleares, Spain. We found that members of A. flavomaculata dwell as solitary individuals in the branchial chamber of ascidians at a higher frequency than expected by chance alone. Given this host use pattern of A. flavomaculata, we hypothesized that males actively move among host individuals in search of receptive females. We provide several lines of evidence consistent with that hypothesis. First, a positive correlation between shrimp and host body size was detected for females, but not for males, during one of the sampling seasons. If males as well as females spend long periods of time within their host individuals, a positive correlation between shrimp and host body size should have been found for both males and females. Second, the body sizes of individuals in the few male-female pairs observed during this study were poorly correlated. If males of A. flavomaculata shared their host individuals with females for long periods of time, a positive correlation between the sizes of males and females in a pair should have been found. Lastly, the body sizes of paired males were larger than those of solitary males during summer, when reproduction was more intense. This difference in body size between paired and solitary males additionally suggests competition among putatively roaming males for receptive females. Manipulative experiments and behavioral observations are necessary to reveal the details of the mating system of A. flavomaculata and other symbiotic crustaceans with a solitary lifestyle.