Evidence for a symbiosis between bacteria of the genus Rhodobacter and the marine sponge Halichondria panicea : harbor also for putatively toxic bacteria?

Halichondria panicea (Pallas) is a marine sponge, abundantly occurring in the Adriatic Sea, North Sea, and Baltic Sea. It was the aim of the present study to investigate if this sponge species harbors bacteria. Cross sections through H. panicea were taken and inspected by electron microscopy. The micrographs showed that this sponge species is colonized by bacteria in its mesohyl compartment. To identify the bacteria, polymerase chain reaction (PCR) analysis of the 16S rRNA gene segment, typical for bacteria, was performed. DNA was isolated from sponge material that had been collected near Rovinj (Adriatic Sea), Helgoland (North Sea), and Kiel (Baltic Sea) and was amplified with bacterial primers by PCR. The data gathered indicate that in all samples bacteria belonging to the genus Rhodobacter (Proteobacteria, subdivision α) are dominant, suggesting that these bacteria live in symbiotic relationship with the sponge. In addition, the results show that the different samples taken contain further bacterial species, some of them belonging to the same genus even though found in sponges from different locations. The possibility of the presence of toxic bacteria was supported by the finding that organic extracts prepared from sponge samples displayed toxicity, when analyzed in vitro using leukemia cells. Received: 7 March 1997 / Accepted: 2 October 1997     

Sponge-specific bacterial symbionts in the Caribbean sponge, Chondrilla nucula (Demospongiae, Chondrosida)

Marine sponges harbor dense and highly diverse bacterial communities, and some percentage of the microflora appears to be specialized for the sponge habitat. Bacterial diversity was examined in Chondrilla nucula Schmidt to test the hypothesis that some subset of sponge symbiont communities is highly similar regardless of the species of host or habitat requirements of the host. C. nucula was collected from a mangrove channel on Lower Matcumbe Key in the Florida Keys (25°53′N; 80°42′W) in August 1999. Domain-specific universal bacterial primers were used to amplify the 16S rDNA gene from genomic DNA that had been extracted from sponges and the surrounding water. An RFLP technique was used to assess diversity of sponge-associated and environmental bacterial communities. The clone library from C. nucula contained 21 operational taxonomic units (OTUs). None of the 53 OTUs from adjacent water samples were found in the C. nucula library indicating that a distinct community was present in the sponge. Sequence analysis indicated that C. nucula harbors a microbial community as diverse as the microbes from other sponges in different habitats around the world. Phylogenetic analysis placed several C. nucula clones in clades dominated by bacteria that appear to be sponge specialists (e.g., Acidobacteria, Bacteroidetes, and Cyanobacteria). Proportional representation of major bacterial taxonomic groups represented in symbiont communities was compared as a function of geographic location of sponge hosts. This study supports the hypothesis that sponges from different oceans existing in dissimilar habitats harbor closely related bacteria that are distinct from other bacterial lineages and appear specialized for residing within sponges.     

Rapid tissue reduction and recovery in the sponge <Emphasis Type="Italic">Aplysinella</Emphasis> sp.

We observed a pronounced, yet reversible tissue reduction in the tropical sponge Aplysinella sp. under non-experimental conditions in its natural habitat, after transfer into seawater tanks, as well as after transplantation from deep to shallow water in the field. Tissue reduction resulted in the formation of small “reduction bodies” tightly attached to the sponge skeleton. Although volume loss and gain were substantial, both tissue reduction and regeneration were often remarkably rapid, occurring within few hours. Microscopic analysis of the reduction bodies revealed morphological similarities to previously described sponge primmorphs, with densely packed archaeocytes and spherulous cells enclosed by a thin layer of epithelial-like cells. Denaturing gradient gel electrophoresis (DGGE) revealed pronounced changes in the sponge-associated microbial community upon tissue reduction during laboratory and field experiments and following changes in ambient conditions after transplantation in the field. Generally, the microbial community associated with this sponge proved less stable, less abundant, and less diverse than those of other, previously investigated Verongid sponges. However, one single phylotype was consistently present in DGGE profiles of Aplysinella sp. This phylotype clustered with γ-proteobacterial sequences found previously in other sponge species of different taxonomic affiliations and geographic provenances, as well as in sponge larvae. No apparent changes in the total secondary metabolite content (per dry weight) occurred in Aplysinella sp. upon tissue reduction; however, comparative analysis of intact and reduced tissue suggested changes in the concentrations of two minor compounds. Besides being ecologically interesting, the tissue reduction phenomenon in Aplysinella sp. provides an experimentally manipulable system for studies on sponge/microbe symbioses. Moreover, it may prove useful as a model system to investigate molecular mechanisms of basic Metazoan traits in vivo, complementing the in vitro sponge primmorph system currently used in this context.     

Spatial distribution of bacteria associated with the marine sponge Tethya californiana

Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge Tethya californiana was analyzed based on 16S rRNA gene sequences. One candidate division and nine bacterial phyla were detected, including members of all five subdivisions of Proteobacteria. Moreover, chloroplast-derived Stramenopiles- and Rhodophyta-affiliated 16S rRNA gene sequences were found and Stramenopiles represented the most abundant clones (30%) in the clone library. On the phylum-level, the microbial fingerprint of T. californiana showed a similar pattern as its Mediterranean relative T. aurantium. An interesting difference was that Cyanobacteria that were abundantly present in T. aurantium were not found in T. californiana, but that the latter sponges harbored phototrophic Stramenopiles instead. Surprisingly, the phototrophic microorganisms were evenly distributed over the inner and outer parts of the sponge tissue, which implies that they also reside in regions without direct light exposure. The other phyla were also present in both the outer cortex and the mesohyl of the sponges. These results were confirmed by analysis on the operational taxonomic unit level. This leads to the conclusion that from a qualitative point of view, spatial distribution of microorganisms in T. californiana tissue is quite homogeneous. Thirty-two percent of the operational taxonomic units shared less than 95% similarity with any other known sequence. This indicates that marine sponges are a rich source of previously undetected microbial life.     

The culturable microbial community of the Great Barrier Reef sponge Rhopaloeides odorabile is dominated by an α-Proteobacterium

The microbial community cultured from the marine sponge Rhopaloeides odorabile Thompson et al. is dominated by a single bacterium, designated strain NW001. Sequence analysis of 1212 bp of the16S rRNA gene of strain NW001 indicates that it is a member of the α-subgroup of the class Proteobacteria. The association between this bacterium and its host sponge was observed in healthy R. odorabile collected from six different reefs in the Great Barrier Reef representing a geographic distance of 460 km, and in four collections in different seasons in 1997–1998 at Davies Reef (18°49.6′S; 147°34.49′E). The proportion of colonies of strain NW001 in samples from R. odorabile, expressed as a percentage of the total heterotrophic bacterial colony count, showed no significant spatial (range: 81–98%) or temporal differences (range: 81–99%), although colony counts of strain NW001 varied by up to two orders of magnitude between reef sites and sampling periods. The location of strain NW001 within the sponge mesohyl was visualized by in situ hybridization, using fluorescently labeled probes based on the 16S rRNA gene sequence of this strain. Cells of strain NW001 surround the choanocyte chambers, suggesting that these bacteria may play a role in nutrient uptake by the sponge. The absence of strain NW001 from corresponding seawater samples indicates that it has a specific, intimate relationship with R. odorabile and is not being utilized as a food source. A unique cyanobacterium related to the genera Leptolyngbya and Plectonema was also isolated from R. odorabile and characterized by 16S rRNA gene sequencing. Received: 19 May 2000 / Accepted: 18 November 2000     

Limited population structure in Northern and Spotted Wolffishes (<Emphasis Type="Italic">Anarhichas denticulatus</Emphasis> and <Emphasis Type="Italic">A. minor</Emphasis>) despite low apparent dispersal potential

Northern and Spotted Wolffishes (Anarhichas denticulatus and A. minor) are demersal marine fishes listed as “threatened” in Canadian waters. Both species have unusually large benthic eggs and large size at hatch, which should reduce passive dispersal. We examined population differentiation with microsatellite and AFLP loci across the ranges of both species in the North Atlantic Ocean. Although significant population structure was documented, differentiation was less than expected based on knowledge of life history characteristics. Significant differentiation was found in Northern Wolffish between the Barents Sea and other samples based on both microsatellite and AFLP data. In contrast, population structure in the Spotted Wolffish was notably weaker, particularly with microsatellites. Both species were characterized by low genetic diversity for marine fishes and had significantly lower genetic diversity than the congeneric Atlantic Wolffish. This finding was consistent with the conservation status of these three species and suggests potential vulnerability to over-exploitation in Northern and Spotted Wolffishes.     

Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by fluorescence in situ hybridization (FISH)

We have employed electronmicroscopical methods (SEM, TEM) to document the microbial community associated with the marine sponge Aplysina cavernicola (formerly Verongia cavernicola, class Demospongiae). Five dominant bacterial types were identified, three of which resemble the morphotypes originally described by Vacelet (1975). One bacterial type possesses morphological properties that are characteristic of the genus Planctomyces. In addition, morphologically uniform bacteria which reside inside the nuclei of host cells were observed. Using in situ hybridization with fluorescently labelled rRNA probes directed against known bacterial groups, the phylogenetic affiliation of the mesohyl bacteria was assessed. It could be shown that the vast majority of mesohyl bacteria belongs to the domain Bacteria with a low GC content. Among the Bacteria, the delta-Proteobacteria were most abundant, followed by the gamma-Proteobacteria and representatives of the Bacteroides cluster. Clusters of Gram-positive bacteria with a high GC content were also found consistently in low amounts. No hybridization signal was obtained with probes specific to the domain Archaea, to the alpha- and beta-Proteobacteria and to the Cytophaga/Flavobacterium cluster. This study describes for the first time the application of the “top-to-bottom approach” using 16S rRNA probes and in situ hybridization to assess the microbial diversity in Aplysina sponges. Received: 18 December 1998 / Accepted: 12 March 1999     

Genetic evidence for cryptic speciation in allopatric populations of two cosmopolitan species of the calcareous sponge genusClathrina

Many sponge species are considered to be cosmopolitan. However, the systematics of marine sponges are very difficult because of the paucity of taxonomically useful characters, and hence the apparently cosmopolitan nature of many species may be simply a consequence of this. In this paper, geographically distant populations of two pairs of cosmopolitan calcareous sponges of the genusClathrina were compared genetically.C. clathrus andC. cerebrum were collected by SCUBA diving between January and March 1989 from two localities: the Mediterranean Sea at La Vesse, near Marseille, Frances, at 9 to 12 m depth, and from the South West Atlantic at Arraial do Cabo, about 200 km east of Rio de Janeiro, at 2 to 10 m depth. Very high levels of gene divergence were found between the allegedly conspecific populations. The levels of genetic identity,I, observed are so low (I=0.128 and 0.287) that the populations clearly cannot be considered conspecific. New species names ofC. aurea sp. nov. andC. brasiliensis sp. nov. are therefore assigned to the southwest Atlantic counterparts ofC. clathrus andC. cerebrum, respectively. It is concluded that, at least for the species studied, and probably for many other species in taxonomically difficult groups, the actual distributions of single species may be far more geographically restricted than is generally assumed.     

Microalgal communities epibiontic on the marine hydroid <Emphasis Type="Italic">Eudendrium racemosum</Emphasis> in the Ligurian Sea during an annual cycle

The microalgal community associated with Eudendrium racemosum, a marine hydroid widely distributed in the Mediterranean Sea, was studied during an annual cycle, at monthly frequency, in a coastal station of the Ligurian Sea. Microalgae were represented mainly by diatoms, which exhibited higher abundance and biomass values between autumn and spring (max 46,752 cells mm⁻² and 1.94 μg C mm⁻², respectively), while during summer a significant decrease was observed (min 917 cells mm⁻² and 0.013 μg C mm⁻²). High levels of abundance of filamentous cyanobacteria were observed in summer. Spatial distribution of epibiontic microalgae showed a markedly increasing gradient from the basal to the apical part of hydroid colonies. Considering the growth forms of diatom communities, motile diatoms (mainly small naviculoid taxa) were the most abundant in all the periods. Adnate (Amphora and Cocconeis spp.) were distributed mainly in the basal and central part of hydroid colonies and showed two peaks (autumn and summer). Erect forms (mainly Tabularia tabulata, Licmophora spp., Cyclophora tenuis) were mainly distributed in the apical part of the colonies and showed their maximum densities in spring–summer. Tube-dwelling (Berkeleya rutilans, Parlibellus sp.) were observed at low densities throughout the study period, without any significant temporal or spatial variability. Comparing the microalgal communities on marine hydroid to those grown on mimic substrata placed in the sampling station during summer, significantly higher values of abundance were observed in the hydroid, suggesting that microalgae may benefit from the polyp catabolites. This fact was particularly evident for the adnate diatoms, whose temporal trend paralleled the cycle of hydroid host.     

Time and space: genetic structure of the cohorts of the common sea urchin <Emphasis Type="Italic">Paracentrotus</Emphasis><Emphasis Type="Italic">lividus</Emphasis> in Western Mediterranean

Spatio-temporal variability in settlement and recruitment, high mortality during the first life-history stages, and selection may determine the genetic structure of cohorts of long-lived marine invertebrates at small scales. We conducted a spatial and temporal analysis of the common Mediterranean Sea urchin Paracentrotus lividus to determine the genetic structure of cohorts at different scales. In Tossa de Mar (NW Mediterranean), recruitment was followed over 5 consecutive springs (2006–2010). In spring 2008, recruits and two-year-old individuals were collected at 6 locations along East and South Iberian coasts separated from 200 to over 1,100 km. All cohorts presented a high genetic diversity based on a fragment of mtCOI. Our results showed a marked genetic homogeneity in the temporal monitoring and a low degree of spatial structure in 2006. In 2008, coupled with an abnormality in the usual circulation patterns in the area, the genetic structure of the southern populations studied changed markedly, with arrival of many private haplotypes. This fact highlights the importance of point events in renewing the genetic makeup of populations, which can only be detected through analysis of the cohort structure coupling temporal and spatial perspectives.     

Cellulose degradation by Lulworthia floridana and other lignicolous marine fungi

Gravimetric analyses of cellulose (Solka Floc) utilization by representative marine Ascomycetes, including Lulworthia floridana, Lindra sp., Torpedospora sp. and Halosphaeria mediosetigera, have shown noteworthy activity based on loss of weight of cellulose by the various fungi. Particularly striking activity is evidenced by L. floridana and Torpedospora sp., with >50% cellulose degradation after 3 weeks of fungal growth. Comparable studies with the deuteromycete, Dendryphiella salina, showed >50% loss within 6 days. Dissimilar responses by various L. floridana isolates are noted. Intensive degradative activity at pH's of 6 to 8 is common along with negligible amounts of cellulase (Cx units). Adsorption of the enzyme to the mycelia or to the cellulose particles in the medium is suggested. Earlier laboratory analyses of fungal degradation of Manila cordage compare favorably with present gravimetric studies and support field observations on the significance of fungal infestation of wood, particularly that incited by the Lulworthia floridana group.This work was supported by Grant GM 12482 from the National Institute of Health and Office of Naval Research Contract No. 137-792 to the Institute of Marine Sciences, University of Miami. Contribution No. 950 from the Institute of Marine Sciences, University of Miami.     

Analysis of global and local population stratification of finless porpoises <Emphasis Type="Italic">Neophocaena phocaenoides</Emphasis> in Chinese waters

The existence of three distinct populations is widely accepted for the finless porpoise (Neophocaena phocaenoides) in Chinese waters: the Yellow Sea, Yangtze River, and South China Sea populations. Here, we use nine species-specific microsatellite loci, the complete mitochondrial DNA control region (912 bp), and the complete mitochondrial cytochrome b gene (1,140 bp) to further investigate potential population stratification in the Yellow Sea using 147 finless porpoise samples from the Bohai Sea and adjacent northern Yellow Sea, two regions that were largely underrepresented in previous genetic studies. Our F-statistics analyses confirm the previously described three populations, but further demonstrate significant genetic differentiation between the [Bohai + northern Yellow] Sea and the southern Yellow Sea. On the other hand, median-joining network analyses do not exhibit well-differentiated haplotype groups among different geographic populations, suggesting the existence of shared ancestral haplotypes. Levels of microsatellite diversity are moderate to high (mean H _E = 0.794) among the 147 [Bohai + northern Yellow] Sea finless porpoises and no recent bottleneck was detected, whereas mtDNA control region and cytochrome b gene diversity is low to moderate. The microsatellite genotypic and mtDNA haplotypic data also confirm the presence of mother-calf pairs in single-net bycatch cases. The results presented here highlight the necessity to treat the [Bohai + northern Yellow] Sea population (highly impacted by anthropogenic threats) as a separate Management Unit.     

Arsenic detoxification potential of <Emphasis Type="Italic">aox</Emphasis> genes in arsenite-oxidizing bacteria isolated from natural and constructed wetlands in the Republic of Korea

Arsenic is subject to microbial interactions, which support a wide range of biogeochemical transformations of elements in natural environments such as wetlands. The arsenic detoxification potential of the bacterial strains was investigated with the arsenite oxidation gene, aox genotype, which were isolated from the natural and constructed wetlands. The isolates were able to grow in the presence of 10 mM of sodium arsenite (As(III) as NaAsO₂) and 1 mM of d+glucose. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that these isolated strains resembled members of the genus that have arsenic-resistant systems (Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Comamonas sp., Enterobacter sp., Pantoea sp., and Pseudomonas sp.) with sequence similarities of 81–98%. One bacterial isolate identified as Pseudomonas stutzeri strain GIST-BDan2 (EF429003) showed the activity of arsenite oxidation and existence of aoxB and aoxR gene, which could play an important role in arsenite oxidation to arsenate. This reaction may be considered as arsenic detoxification process. The results of a batch test showed that P. stutzeri GIST-BDan2 (EF429003) completely oxidized in 1 mM of As(III) to As(V) within 25–30 h. In this study, microbial activity was evaluated to provide a better understanding of arsenic biogeochemical cycle in both natural and constructed wetlands, where ecological niches for microorganisms could be different, with a specific focus on arsenic oxidation/reduction and detoxification.     

Distribution of secondary metabolites in the sponge Oceanapia sp. and its ecological implications

The Micronesian sponge Oceanapia sp. has an unusual growth form that consists of an irregular turnip-shaped base, which is buried in the substrate. One to several fistules, which protrude through the sand, are attached to the base of the sponge. On top of each fistule is a small fragile capitum. We examined whether this conspicuous red-colored sponge was chemically defended and if intraspecimen variation existed in the distribution of secondary metabolites between different parts of the sponge. Furthermore we assessed the deterrent properties of the secondary metabolites to generalist and more specialized fish predators. We also wanted to see if the optimal defense theory holds in the case of a marine invertebrate. According to the theory, organisms evolve and allocate defenses in a way that maximizes individual fitness, assuming that defenses are costly to the fitness of the organisms. We were able to evaluate this hypothesis, since the different sponge parts in Oceanapia sp. were at different risk to damage by predators and had a different value in terms of fitness loss to the sponge (the capitum probably plays a role in asexual propagation). Concentrations of crude organic extract increased from the base to the capitum of the sponge. The major secondary metabolites kuanoniamine C and D also showed a sharp increase from the basal root to the capitum. There was no difference in structural material or ash content between the base and the fistule of the sponge, but fiber and protein content were significantly higher in the fistule. The methanol fraction was highly deterrent in field feeding assays towards generalist reef fish at base concentration. It also deterred feeding by the spongivorous angelfish Pomacanthus imperator in laboratory feeding experiments at the same concentration. The field feeding assays with pure compounds showed that kuanoniamine C and D deterred feeding by natural assemblages of reef fishes at fistule concentrations, confirming their role as defensive agents. The intraspecimen variation of secondary metabolites in Oceanapia sp. supports the optimal defense theory by showing the highest concentrations in those parts of the sponge that are most visible to predators and are likely to be most important for inclusive fitness. Received: 5 May 1999 / Accepted: 16 September 1999     

Phylogeographic structure of <Emphasis Type="Italic">Octopus vulgaris</Emphasis> in South Africa revisited: identification of a second lineage near Durban harbour

In a previous study that investigated genetic structure of Octopus vulgaris along the South African coast by sequencing the mitochondrial cytochrome oxidase III gene (COIII), all sequences generated were identical. Such a finding is unusual, because mitochondrial DNA mutates quickly, and several marine invertebrates present in southern Africa show considerable genetic variation and structure. We reanalysed the samples using two different mitochondrial markers, namely cytochrome oxidase I (COI) and the large ribosomal subunit (16S rRNA). Sequences of both these markers showed variation. The conclusion of the previous study, that South Africa’s O. vulgaris population is characterised by a lack of genetic structure along the coast, is rejected. Some specimens from Durban (southeast Africa) were genetically more different from those found in the remainder of the country than were specimens from other regions (Tristan da Cunha and Senegal). We suggest that the lineage in Durban may have been recently introduced.     

Phylogenetic analyses of potentially free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> spp. isolated from coral reef sand in Okinawa,Japan

The existence of “free-living” Symbiodinium that can form symbioses with hosts is implied by the presence of hosts that produce Symbiodinium-free gametes and expulsion and/or expelled symbiotic algae from host. However, it is still unclear if potentially symbiotic Symbiodinium are found “free-living” in the coral reef environment. Sixteen Symbiodinium strains were established from samples taken from three sampling locations of coral reef sand in Okinawa, Japan. Phylogenetic analyses of the partial large subunit ribosomal DNA (28S-rDNA) and the internal transcribed spacer of ribosomal DNA (ITS-rDNA) conclusively showed that all 16 isolates belonged to Symbiodinium clade A sensu Rowan and Powers (1991). The lack of other Symbiodinium clades besides clade A in this study may be due to other clades not being readily culturable under culture conditions used here. The new isolates could be phylogenetically divided into four groups, though no sequences were identical to previously reported Symbiodinium. Two of the four groups were closely related to symbiotic Symbiodinium clade A isolated from a variety of host species. One isolate group formed a highly supported monophyly with Symbiodinium types that have previously been characterized as “free-living”. The remaining isolate group, although within clade A, was quite divergent from other clade A Symbiodinium. These results indicate that novel diversity of free-living Symbiodinium exists in coral sand.     

Population structure of the common sole (<Emphasis Type="Italic">Solea solea</Emphasis>) in the Northeastern Atlantic and the Mediterranean Sea: revisiting the divide with EPIC markers

Spatial and temporal population genetic structures of the common sole, Solea solea, were studied in Northeastern Atlantic and Mediterranean Sea populations, using three polymorphic exon-primed intron-crossing (EPIC) markers. Results demonstrated significant multilocus differentiation among Eastern Mediterranean and a group composed by Western Mediterranean and Atlantic populations (θ = 0.150, P < 0.001), but also suggested unrecorded genetic differentiation of the Adriatic Sea population. No pattern of isolation-by-distance was recorded across the range covered by sampling, from the Kattegat to the Aegean Sea. Conversely to genetically structured Mediterranean populations, Atlantic populations ranging from Denmark to Portugal could be considered as representative of the same panmictic unit (θ = 0.009, not significant). Results further demonstrated stability of multilocus genetic structure among temporarily replicated cohort samples [0+, 1+, subadults] from several coastal and estuarine locations from Bay of Biscay, excepted for the amylase locus Am2B3-2 at one location (Pertuis d’Antioche). Despite coherence of such observed patterns of multilocus differentiation with previous allozymic surveys in sole, and with patterns generally obtained for other marine fish species, single-locus results from EPICs indicated divergent coalescence schemes supporting a complex response to ecology and history of sole’s populations. Results stress the use of nuclear genes such as EPIC markers to investigate population structure, but also historical, demographic, and possibly selective processes in marine fishes.     

Evaluation of combined morphological and molecular techniques for marine nematode (<Emphasis Type="Italic">Terschellingia</Emphasis> spp.) identification

Marine nematodes, which play an important role in many ecosystems, include a number of apparently cosmopolitan taxa that exhibit broad biogeographic ranges even though there is no obvious dispersal phase in their lifecycle. In this study, standard taxonomic approaches to marine nematode identification in conjunction with multivariate statistical analysis of morphometric data were compared with molecular techniques. Specimens of the marine nematode Terschellingia longicaudata that had been identified by their morphological features were investigated from a range of localities (East and West Atlantic, Bahrain, Malaysia) and habitats (estuarine, intertidal, subtidal) using molecular approaches based on the amplification and sequencing of the small subunit ribosomal RNA (18S rRNA). The study revealed that the majority of the morphologically defined T. longicaudata specimens share a single 18S rRNA sequence and apparently belong to a single taxon distributed from the British Isles to Malaysia. In addition, 18S rRNA analysis also revealed two additional sequences. One of these sequences was found in both the British Isles and Mexico, the other was recorded only from British waters. Individuals collected in Bahrain and identified from their morphology as T. longicaudata had two highly divergent 18S rRNA sequences. Separate morphological and morphometric approaches to identification of specimens from the same sites that had been formalin-preserved did not support evidence of multiple genotypes revealed previously by molecular analysis. Current taxonomy based on morphological characters detected using light-microscopy may be unable to discriminate possible species complexes. Biodiversity of marine nematodes may often be underestimated due to the presence of morphologically cryptic species complexes. High-throughput techniques such as DNA barcoding would aid in species identification but may require thorough analysis of multiple nuclear and mitochondrial molecular markers.     

<Emphasis Type="Italic">Alexandrium catenella</Emphasis> (Dinophyceae), a toxic ribotype expanding in the NW Mediterranean Sea

The presence of the paralytic shellfish poisoning (PSP) dinoflagellate Alexandrium catenella in the north western (NW) Mediterranean Sea has been known since 1983. From this date on, the species has spread along the Spanish and Italian coastlines. Information concerning A. catenella isolates in the NW Mediterranean Sea was gained through phylogenetic studies. Twenty established toxic cultures of A. catenella taken from various NW Mediterranean Sea locations were analysed by nucleotide sequencing of the 5.8S rDNA and internal transcribed spacer regions. These rDNA ribosomal markers resulted useful in delineating the phylogenetic position of this species in the genus Alexandrium as well as in determining relationships between A. catenella isolates from different geographic areas. The phylogenetic position of the Mediterranean A. catenella ribotype, when compared to the “Alexandrium tamarense/catenella/fundyense species complex”, fits this species complex well. All the Mediterranean A. catenella isolates were constituted by only one genetic ribotype. By comparing the isolate sequences with those of other geographic areas, it revealed that the Mediterranean A. catenella ribotype was closely related to the A. catenella from Japan, Western Pacific Ocean. It was also evident that in temperate Japanese waters, a genetic variability was detected within A. catenella isolates; in fact, all strains resulted divergent showing as many as 15 mutational steps. The possibility that A. catenella has been recently introduced into the Mediterranean basin from temperate Asian areas is discussed.     

Rapid tissue reduction and recovery in the sponge Aplysinella sp.

We observed a pronounced, yet reversible tissue reduction in the tropical sponge Aplysinella sp. under non-experimental conditions in its natural habitat, after transfer into seawater tanks, as well as after transplantation from deep to shallow water in the field. Tissue reduction resulted in the formation of small “reduction bodies” tightly attached to the sponge skeleton. Although volume loss and gain were substantial, both tissue reduction and regeneration were often remarkably rapid, occurring within few hours. Microscopic analysis of the reduction bodies revealed morphological similarities to previously described sponge primmorphs, with densely packed archaeocytes and spherulous cells enclosed by a thin layer of epithelial-like cells. Denaturing gradient gel electrophoresis (DGGE) revealed pronounced changes in the sponge-associated microbial community upon tissue reduction during laboratory and field experiments and following changes in ambient conditions after transplantation in the field. Generally, the microbial community associated with this sponge proved less stable, less abundant, and less diverse than those of other, previously investigated Verongid sponges. However, one single phylotype was consistently present in DGGE profiles of Aplysinella sp. This phylotype clustered with γ-proteobacterial sequences found previously in other sponge species of different taxonomic affiliations and geographic provenances, as well as in sponge larvae. No apparent changes in the total secondary metabolite content (per dry weight) occurred in Aplysinella sp. upon tissue reduction; however, comparative analysis of intact and reduced tissue suggested changes in the concentrations of two minor compounds. Besides being ecologically interesting, the tissue reduction phenomenon in Aplysinella sp. provides an experimentally manipulable system for studies on sponge/microbe symbioses. Moreover, it may prove useful as a model system to investigate molecular mechanisms of basic Metazoan traits in vivo, complementing the in vitro sponge primmorph system currently used in this context.