Thraustochytrids as novel parasitic protists of marine free-living flatworms: <Emphasis Type="Italic">Thraustochytrium caudivorum</Emphasis> sp. nov. parasitizes <Emphasis Type="Italic">Macrostomum lignano</Emphasis>

The Labyrinthulomycota are a relatively poorly studied group of heterotrophic unicellular eukaryotes. They comprise three lineages, labyrinthulids, thraustochytrids, and aplanochytrids, which are all primarily marine organisms and considered to be important components of marine microbial communities. Recently a number of Labyrinthulomycota have been implicated as parasites of marine (but also terrestrial) plants and marine molluscs. Here we describe a new species of thraustochytrid, Thraustochytrium caudivorum sp. nov. that we have isolated from laboratory cultures of Macrostomum lignano (Rhabditophora, Macrostomorpha), a marine free-living flatworm. In these worms T. caudivorum can cause lesions, which start at the tip of the tail plate and which can lead to the dissolution of the posterior part of the animal. Although the worms can frequently cure these lesions and regenerate the lost parts, the lesions can also result in the complete dissolution of the animal. We describe this thraustochytrid based on pure agar cultures and infestations in the worm cultures. Moreover, we describe its pathological effects on the worms and its morphology using both light and electron microscopy. In addition, we report a phylogenetic analysis using a partial 18S rDNA sequence that allows us to place this new species within the thraustochytrids. Finally, we outline a protocol that allows to permanently remove the parasites from infested worm cultures. We conclude that thraustochytrids represent a novel group of parasites of free-living flatworms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of a PCR strategy for thraustochytrid identification based on 18S rDNA sequence

The identification and characterization of the thraustochytrids, an emerging economically and biotechnologically important group of marine heterotrophic protists, is usually based on morphological characters. In this research we used molecular markers to identify thraustochytrids. We designed three sets of primers based on the 18S rDNA sequence alignment of known strains and employed a PCR (polymerase chain reaction) strategy to identify unknown thraustochytrid isolates. DNA from 26 thraustochytrids (three isolated from primary cell cultures of the tunicate Botryllus schlosseri and 23 from a coral holding aquarium) were amplified by these primers, revealing 21 isolates with three bands each, which were assigned to two groups according to PCR fragment sizes. Taxonomic characterizations were deduced by comparing with GenBank data. Four isolates were further studied by sequencing their 18S rDNA. Sequence alignments and phylogenetic analysis revealed that isolates from the coral aquarium (7-5 and 8-7) were highly similar to each other and 95.0-97.0% similar to Thraustochytrium multirudimentale and Schizochytrium minutum. Isolates from the tunicate primary cell cultures (BS1 and BS2) were also closely related to each other and 84.3-86.0% similar to labyrinthulid quahog parasite and Thraustochytrium pachydermum. AFLP (amplified fragment-length polymorphism) analysis revealed 2.5-3.6% differences within the genomic DNA of each group, showing that each isolate is different, although isolates within each group may belong to the same species, in spite of differences found in the general morphology.     

Detection of the thraustochytrid protist Ulkania visurgensis in a hydroid,using immunofluorescence

An immunofluorescence technique was employed to detect the thraustochytrid protist Ulkenia visurgensis (Ulken) Gaertner, originally isolated from an algal bed, in natural samples in Goa, India. Collections were made monthly from September 1985 to November 1986. By treating the samples with antiserum prepared against this organism and conjugated with FITC stain, the protist was regularly found to occur in association with a hydroid. Several cells of the organism were observed in the coelenteron and the hydranth of the hydroid. The thraustochytrid could be repeatedly isolated in cultures from the hydroid. The cells of the protist differed from those in culture in that they lacked ectoplasmic net elements.     

Bacterivory: a novel dual role for thraustochytrids in the sea

Two thraustochytrids, Thraustochytrium striatum Schneider and Schizochytrium mangrovei Raghukumar, isolated in 1989 in axenic cultures using pine pollen in seawater from mangroves in Goa, India, were observed in continuous-flow chambers. When grown in the presence of bacteria, both species produced amoebae, which moved using pseudopodia and phagocytosed bacterial cells. Several amoebae were produced in sporangia, or isolated vegetative cells transformed directly into amoebae. These findings were confirmed with several single-cell isolations of the protists. The demonstration of phagotrophic amoebae in these organisms suggests a dual role for thraustochytrids in nature, namely a fungus-like breakdown of complex organic molecules as well as bacterivory. This phenomenon also places the thraustochytrics in a unique relationship with many other protistan groups.     

Inhibition of attachment of larval barnacles,Balanus amphitrite,by bacterial surface films

Films of bacteria on solid substrata can positively or negatively influence the attachment of marine invertebrate larvae. Effects of marine bacteria on the attachment of cypris larvae of the barnacle Balanus amphitrite Darwin were examined in the laboratory. Bacteria, grown to mid-exponential phase and allowed to adsorb irreversibly to polystyrene petri dishes, attached in densities of 10⁷ cells cm^-2. Assays (22h) were used to compare the effects of adsorbed cells of 18 different bacterial species on larval barnacle attachment. Most of the adsorbed bacteria either inhibited or had no effect on larval attachment compared to clean surfaces. Experiments testing the effect of larval age on barnacle attachment were conducted with six species of bacteria and showed that older larvae attached in higher percentages to clean surfaces and that bacterial films generally inhibited larval attaschment. Both the species of bacteria and the in situ age of the adsorbed bacteria affected barnacle attachment response: older films of Deleya (Pseudomonas) marina were more inhibitory. Bacterial extracellular materials may be involved in the inhibitory process.     

Docosahexaenoic acid accumulation in thraustochytrids: search for the rationale

Thraustochytrid protists characteristically accumulate high amounts of lipids, much of which is comprised of the polyunsaturated fatty acid, docosahexaenoic acid (DHA). Since DHA is important in human health, thraustochytrids have attracted much attention from the point of view of biotechnology. However, the biological rationale of DHA production in the storage lipids of these organisms is not clear. We carried out three experiments to study this. (1) The fate of lipids in thraustochytrids during development was studied by staining lipids of vegetative cells with the fluorescent vital stain for lipids, namely Nile blue, following the growth of the cells. The fluorescent lipid bodies decreased in abundance in freshly formed motile limaciform amoeboid cells and subsequently disappeared when they moved away. When vegetative cells produced vast extensions of plasma membrane, the ectoplasmic net elements (EN), the fluorescent lipids appeared to be transported to them. (2) Changes in lipids and DHA during starvation were examined in cells with enhanced lipid and DHA contents and those in which they were not enhanced. Cells in which lipids and DHA were enhanced by refrigerating them for 48 h survived starvation for a longer period. Compared to cells that had not been refrigerated, total fatty acids, as well as DHA were marginally higher in pre-refrigerated cells, while palmitic acid levels were lower. Starvation of cells resulted in a gradual decrease of absolute concentrations and percentage levels of DHA after 8 days, while percentage of palmitic acid levels increased. (3) The relationship between DHA and specific gravity of cells was studied by comparing cells pre-refrigerated as above with those which were not pre-refrigerated. Refrigerated cells with increased lipids and DHA showed higher specific gravities than non-refrigerated cells. We suggest the following roles for storage lipids and DHA in thraustochytrids: (1) lipids serve as energy sources during movement of cells and for production of EN; (2) DHA in storage lipids become distributed in the plasma membrane when EN are formed; (3) DHA is preferably utilized as a fatty acid energy reserve during starvation and (4) an unknown mechanism results in negative buoyancy of cells when total lipids and DHA are marginally enhanced. DHA in storage lipids might be crucial to the survival of thraustochytrid cells.     

Noncovalent bonds are key mechanisms for the cohesion of barnacle (Balanus crenatus) adhesive proteins

Cement of the barnacle species, Balanus crenatus, was isolated and separated by SDS-PAGE. Reductive and nonreductive conditions yielded the same result of six major proteins (in the range up to 100 kDa) occurring as two triplets. A third triplet of lower molecular mass was faintly visible. The presence of a reductive agent had only little impact on the solubility of B. crenatus cement. The strong effect of the denaturing conditions (the presence of SDS and the incubation at elevated temperature) on the solubility of the barnacle cement and the absence of disulfide signals in Raman spectra suggest a complex structure of the barnacle cement noncovalently bound. It can be speculated that this protein complex in its quaternary structure is formed by small subunits and is equivalent to the globular cement structures previously detected.     

Sugar compound products of the periphytic diatom <Emphasis Type="Italic">Navicula ramosissima</Emphasis> induce larval settlement in the barnacle, <Emphasis Type="Italic">Amphibalanus amphitrite</Emphasis>

This study investigated the response of cyprids of the barnacle Amphibalanus amphitrite to 23 strains of laboratory cultured periphytic diatoms isolated from microbial biofilms that formed on glass slides immersed in Tachibana bay, Nagasaki, and those from mass-production tanks in the Fisheries Center of Nagasaki City, Japan. In addition, periphytic diatoms were subjected to various treatments, in order to investigate the nature of the chemical cue in periphytic diatoms. Cyprids of A. amphitrite responded differently to the 23 different periphytic diatom strains and settled in high percentages on Cocconeis sp. and Navicula ramosissima strain A. On the other hand, nine strains of diatoms significantly inhibited settlement. The settlement inducing activity of N. ramosissima strain A increased linearly with diatom density, and its activity was enhanced by culturing the diatom under a bacteria-free condition, suggesting that specific diatom species, i.e., N. ramosissima strain A, may play an important role on larval settlement of the barnacle. Subjecting N. ramosissima strain A biofilm to hydrochloric acid (HCl) and ethanol (EtOH) treatments or heating it at 100°C did not inactivate the film, indicating that the settlement cue was a stable surface bound compound that did not decompose from the above treatments. Moreover, of the various lectins, enzymes, and drugs [H₅IO₆ and sodium dodecyl sulfate or (SDS)] used, only Lentil Agglutinin (LCA) treatment of N. ramosissima strain A biofilm resulted in the reduction its settlement inducing activity. A positive correlation was observed between the settlement inducing activity and the amount of LCA conjugated fluorescein isothiocyanate (FITC-LCA) of N. ramosissima strain A. On the other hand, subjecting biofilms of N. ramosissima strain B, an inactive strain, to various types of treatments resulted in the induction of A. amphitrite larval settlement but LCA treatment also reduced the activity of these treated N. ramosissima strain B biofilms. These findings suggest that a cue containing an LCA-binding sugar chain is present in both A and B strains of N. ramosissima but the large amount of mucous substance covering N. ramosissima strain B biofilm probably makes the sugar chain containing active subunit in strain B unavailable to A. amphitrite cyprids. In conclusion, periphytic diatoms such as N. ramosissima play an important role in larval settlement of the barnacle A. amphitrite. The cue in the diatom was an LCA-binding sugar chain(s) compound that may have similarities to the settlement inducing protein complex (SIPC) from adult shell of the barnacle.     

Larval settlement and metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> in response to biofilms

Biofilms were allowed to develop on glass slips immersed 1.0–1.5 m below the sea surface in Tachibana Bay, Nagasaki, Japan, for different periods of time from November 2003 to January 2005. The effects of age, immersion month, dry weight, bacterial and diatom densities of these biofilms on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Furthermore, biofilms were subjected to various treatments to investigate the nature of the settlement and metamorphosis cue in the biofilm. Pediveliger larvae of the mussel settled and metamorphosed in response to biofilms. Settlement and metamorphosis to the post-larval stage significantly increased with the biofilm age. In addition, the biofilm activity varied depending on the immersion month (season), e.g., for biofilms with the same age, those immersed between June and August had higher activities than those immersed between November and March. The activity of the biofilm also positively correlated with the dry weight, bacterial and diatom densities. These three quantitative parameters of the biofilm were significantly affected by the film age but were not affected by the immersion month, suggesting that other parameters (e.g., community structures, extracellular products) also affected the inductive activity of the biofilm. The fixative agents (formalin and glutaraldehyde), heat, ethanol, ultraviolet irradiation and antibiotics treatments of the biofilm resulted in significant reduction or loss of its inductive activity. The survival of bacterial cells in the treated films where activities were either reduced or lost also decreased significantly. No settlement and metamorphosis were obtained when larvae were exposed to the conditioned water of the biofilm. Thus, larvae of M. galloprovincialis settled and metamorphosed in response to a cue produced by living bacteria in the biofilm. The cue may be a bacterial extracellular product which was susceptible to the above treatments.     

Influence of substratum heterogeneity and settled barnacle density on the settlement of cypris larvae

On the Atlantic coast of Canada, Semibalanus balanoides (L.) is widely distributed in the mid-intertidal zone, whereas in the Gulf of St. Lawrence, this species is mostly limited to crevices. We tested the hypothesis of regional differences in microhabitat selection by barnacle larvae at settlement in 1984 and 1985 at St. Andrews, New Brunswick, Canada. Since larvae settle in microhabitats already colonized by adults, the relative influence of settled barnacle density and of different scales of substratum heterogeneity on settlement were evaluated experimentally at Capucins, Québec, (Gulf of St. Lawrence) and at St. Andrews, New Brunswick (Atlantic coast). On a large scale (>10 cm deep crevices) of heterogeneity, results show that, in the Gulf, cypris larvae settled nearly exclusively (93%) in natural crevices rather than on adjacent horizontal surfaces. On the Atlantic coast, settlement was more important outside than inside of crevices, when the substrata were either natural or artificial. This result is unique and contrasts sharply with all known reports on barnacle settlement in relation to surface contour. The influence of barnacle density on settlement was greater than that of large scale heterogeneity. On a small scale (<1.5 cm deep cracks), the presence of conspecifics had a stronger effect on settlement than heterogeneity in both regions. Field observations showed a relationship between larval settlement density and percentage of adult cover. Settlement increased up to 22 or 30% (Gulf and Atlantic coast) of adult cover and decreased afterwards. The results confirm the hypothesis of larval selection for cryptic habitats in the Gulf and the opposite behaviour (preferences for horizontal surfaces) on the Atlantic coast. This microhabitat selection is apparent at large scales of heterogeneity, whereas at small scales, the presence of conspecifics is the predominant factor.Contribution to the programme of GIROQ (Groupe interuniversitaire de recherches océanographiques du Québec)     

Larval metamorphosis of the sea urchins,<Emphasis Type="Italic"> Pseudocentrotus depressus</Emphasis> and<Emphasis Type="Italic"> Anthocidaris crassispina</Emphasis> in response to microbial films

In Japan, mass-production of sea urchin juveniles involves the culture of periphytic diatom films on plastic plates in 5- to 15-tonne tanks for the induction of larval metamorphosis. This study focused on the larval response of sea urchins, Pseudocentrotus depressus and Anthocidaris crassispina, to natural microbial films in the sea and diatom-based films formed in the tanks. The effect of diatoms and bacteria on larval metamorphosis was also examined using laboratory-cultured diatom-based films in the presence of germanium dioxide and antibiotics during film culture. Furthermore, the nature of the cue of the cultured diatom-based film was also investigated. Results showed that P. depressus and A. crassispina metamorphosed both on natural microbial films and diatom-based films in a tank. In the sea, the metamorphosis (%) of P. depressus increased gradually in accordance with the immersion period of film formed on glass slides, whereas the larval metamorphosis of A. crassispina had a bell-shaped response curve. In the tank, although the diatom-based films showed a low inducing activity for larval metamorphosis of A. crassispina, the metamorphosis of P. depressus larvae increased linearly in accordance with the diatom density. These results suggest that diatom-based films could promote the larval metamorphosis of P. depressus, but are less important in A. crassispina. In a simultaneous larval assay (May), P. depressus showed a higher percentage of metamorphosis than A. crassispina. We concluded that the former is more sensitive to diatom-based film than the latter and that this is due to differences in their natural habitats. For laboratory-cultured diatom-based film, both species of sea urchins showed a similar response, in which reduction in diatom and bacteria density resulted in a decrease in the original inducing activity. There seems to be a synergistic effect between diatom and bacteria in inducing larval metamorphosis. Films subjected to treatment with 0.1 N HCl were no longer inductive for either sea urchin, while those films treated with 40^°C heat or EtOH (5% and 10% EtOH) showed a significant reduction in the inducing activity. Thus the surface-associated cues may be highly susceptible to the above treatments.Communicated by T. Ikeda, Hakodate     

Impact of conditions of cultivation and adsorption on antimicrobial activity of marine bacteria

The potency of free-living and animal-associated marine bacteria to produce antimicrobial substances has been studied in 491 strains isolated from northern and southern parts of the Pacific Ocean. A total of 26% (126 out of 491) of the strains examined produced antimicrobial compounds against 11 test bacterial strains (TBS) including the fish pathogens Aeromonas hydrophila and Vibrio anquillarum. Antimicrobial substances (AS) produced by marine bacteria were especially active against Staphylococcus epidermidis, Proteus vulgaris, Enterococcus faecalis, and Candida albicans. Twelve strains, isolated from different sources, were chosen as promising candidates, producing a number of AS. Production of AS varied within 24 to 72 h, increasing in a culture medium based on natural sea water with Br-ions, and after attachment to polymeric surfaces. In order to study the influence of adsorption, selected strains with a high potential for antimicrobial production were cultivated on polymeric surfaces with different hydrophobicities and chemical functionalities. These parameters of the surface hydrophobicity (measured by means of water contact angles) and chemical functionality of the surfaces were manipulated using the photo- and thermochemistry of a polymeric system (diazo-naphto-quinone/novolak) commonly used as a photoresistant material in semiconducto-manufacturing. The highest antimicrobial activities occurred on hydrophilic surfaces (standard exposed photoresistant films), whereas the number of attached cells was greater on hydrophobic surfaces, characterized as unexposed resistant films. These results suggest that the chemical nature of induced hydrophilicity may also be a major factor in controlling antimicrobial activity of adsorbed bacteria. Received: 5 March 1997 / Accepted: 24 August 1997     

Pre-immersion salinity-choice behaviour in Porcellana platycheles

The ability of the littoral porcelain crab Porcellana platycheles (Pennant) to recognise and avoid low-salinity levels when immersed in media has already been demonstrated in previous experiments. It was thought that the ability to identify and avoid diluted sea water before complete immersion would also be of survival value, since this would prevent exposure of the permeable body surfaces (especially the gills) to the swelling effects of the osmotic uptake of water. The experiments reported here demonstrate that such pre-immersion salinity choice behaviour may be elicited from P. platycheles in the laboratory, and that salinity reception is apparently sited upon the walking limbs. Choice between salinity levels is shown to be based on the osmotic concentration of media rather than on the concentration of a particular ion or group of ions.     

Chemical mediation of invertebrate defensive behaviors and the ability to distinguish between foraging and inactive predators

When the predatory sea star Pycnopodia helianthoides was placed upstream, the sea urchin Strongylocentrotus purpuratus responded defensively by extending and opening its globiferous pedicellariae. No pedicellaria response was given in control seawater or when the sea star was downstream. The snail Tegula funebralis responded by moving up vertical surfaces when Pycnopodia helianthoides or when Pisaster ochraceus were placed upstream. When these sea stars were introduced downstream, the snail's response was not significantly different from that in control seawater. Water collected from an aquarium containing a single sea star was sufficient to trigger the response of S. purpuratus and T. funebralis; the physical presence of the sea star was not essential. This indicated that a chemical stimulus was involved, and the lack of responses when sea stars were downstream argued strongly against the possible additional involvement of visual or vibrational stimuli. S. purpuratus gave stronger pedicellaria responses to water flowing over an active Pycnopodia helianthoides than to water flowing over the same sea star when it was inactive. The significance of the ability to distinguish between actively foraging and inactive predators is discussed, and a mechanism is proposed to explain differences in the amount of stimulatory chemicals released by active and inactive sea stars.     

Strobilation,budding and initiation of scyphistoma morphogenesis in the rhizostome Cassiopea andromeda (Cnidaria: Scyphozoa)

Scyphistomae of Cassiopea andromeda Forskål, 1775 containing symbiontic zooxanthellae did not develop medusae at a constant temperature of 20°C, but monodisc strobilation was initiated after transfer of the polyps to 24°C. After release of the ephyrae and regeneration of the hypostome and tentacular region, the recovered polyps either produced vegetative buds or entered a new strobilation phase. Formation of motile, planula-like buds was not found to be indicative of unfavourable environmental conditions. Intensity of budding was positively correlated with available food and with increase of temperature. Budding was negatively correlated with the number of polyps maintained per dish and with the conditioning of the sea water. Under optimal feeding and temperature conditions, polyps could simultaneously produce chains of buds at 2 to 4 budding regions. Settlement and development of buds into scyphistomae was suppressed in pasteurized sea water and in pasteurized sea water containing antibiotics, but polyps developed from buds in the presence of algal material taken from the aquarium, debris or egg shells of Artemia salina, or on glass slides which had been incubated in used A. salina culture medium. Several species of marine bacteria were detected after staining these slides. One, a Gramnegative coccoid rod, which was identified as a nonpathogenic Vibrio species, was isolated, cultivated as a pure strain, and was proved to induce the development of C. andromeda buds into polyps. Millipore filter-plates coated with Vibrio sp. cells grown in suspension culture were ineffectual, but diluted filtrate initiated polyp morphogenesis. The inducing factor is obviously not a constituent of the bacterial cell surface, but is a product of growing Vibrio sp. cells released into the medium. This product was found to be relatively heat-stable and dialyzable. As to the basic mechanism involved in the induction of polyp formation, it is suggested that the inducing factor (s) acts bimodally by inducing pedal disc development and by eliminating a head inhibitor originating from the basal end of the bud. The life history, and various aspects of medusa-formation and of vegetative reproduction in scyphozoans are reviewed and discussed with particular reference to rhizostome species. Special attention has been paid to some reports of larval metamorphosis controlled by marine bacteria.     

Physiological tolerance predicts species composition at different scales in a barnacle guild

This study examined how the species composition of an intertidal barnacle guild varied according to physical gradients in the environment at small scales governed by microclimates, medium scales of wave exposure and large scales of latitude. Barnacle distributions at small and medium scales were sampled in Ireland between 51°29′ and 52°44′N and 6°50′ and 10°08′W. Sampling on European shores spanned ~18° latitude from 37°05′ to 55°16′N. Barnacle surveys mainly took place in 2003–2004. An index of wave fetch was calculated along the wave exposure gradient using a digital coastline-based model that was supported by a biological exposure scale. A ‘dryness’ index was defined according to mean monthly wind speed, fetch along the average wind direction and mean monthly air or sea surface temperatures for 2 years (January 2001–December 2002) which is the period when the most recent adults in the barnacle community would have settled and grown to adulthood. The proportion of the dry-loving barnacle Chthamalus montagui Southward increased within the barnacle guild at all scales as the habitat became warmer and drier. Barnacle densities were high in all habitats, mean densities ranged from a minimum of 4.16 cm⁻² on moderately exposed shores to a maximum of 6.27 cm⁻² in sunlit or south-facing microclimates. Percentage cover of barnacles across the gradient of latitudes was usually >70%. The results suggest that the distribution and abundance of interacting barnacle species on European coasts is strongly controlled by abiotic factors, most likely temperature and desiccation.     

Tolerance of some cirripede nauplii to fluctuating salinities

The effects of fluctuating salinity on the mortality of newly released nauplii of three species of barnacle, Elminius modestus (Darwin), Balanus balanoides (L) and B. hameri (Ascanius) have been measured. The larvae were exposed to three different types of salinity regime; in all cases, a regime which fluctuated gently between full-strength sea water and a reduced concentration was found to be least damaging. The contributions of the abruptness of salinity shock, and the degree of exposure to reduce salinity, are considered as factors influencing mortality. Survival depends upon the interaction of both these factors, but the abruptness of the shock which the barnacles receive is of major importance in determining their survival range.     

Colonization and early succession on artificial hard substrata by meiofauna

An experiment was undertaken at Farol Island, Brazil, to examine colonization of bare aluminium surfaces by microbes and meiofauna. It was hypothesized that a primary source of meiofaunal colonists was sediment resuspended during upwelling events, two of which occurred during the experiment. Microbial biofilms formed on the experimental substrata within 1 day, and continued to develop throughout the experimental period. Among meiofaunal groups copepods also appeared on the first day, and nematodes on the second. Meiofaunal community structure developed in three main phases: an initial phase of 2 days, characterized by low abundances of copepods; a second phase during the first upwelling period characterized by higher abundances of copepods and also by turbellarians; and a third phase from day 13 onwards characterized by relatively stable abundances of a range of taxa including copepods, cirripedes, nematodes and ostracods. Nematode assemblages also developed in three phases, but with different timings coinciding with upwelling events: an initial phase, from the beginning of the experiment to day 9, characterized by few species and low (or no) abundances; a second phase following the first upwelling characterized by moderate abundances of Chromadorina, Chromadorella, Daptonema and Euchromadora sp. 3; a third phase following the second upwelling period (from day 26 onwards) in which Daptonema disappeared and the assemblage was characterized by moderate to high abundances of Euchromadora (species 1 and 2) and Chromadorella. Although shifts in nematode assemblage structure coincided with upwelling events no evidence was found for sediments being the primary source of colonizers on the aluminium substrata, in contrast to our hypothesis.     

Hypoxic life of intertidal acorn barnacles

Oxygen levels were monitored within the mantle cavities of three barnacle species ( Chthamalus stellatus, Semibalanus balanoides, Elminius modestus), using optode microsensors. Conditions were always hypoxic, even when barnacles were actively using the prosoma and cirri to pump aerated seawater into the mantle cavity. Mantle fluid oxygen concentrations were extremely variable; behaviour and oxygen concentrations were not closely coupled. Ventilation of the mantle cavity depended partially on external water flow, with higher and more stable mantle fluid oxygen concentrations being sustained when the water around barnacles was agitated. During emersion, barnacles initially pumped seawater between the mantle cavity and the cone above the opercular plates to achieve ventilation. As water was lost it was replaced by air bubbles, eventually resulting in an air-filled mantle cavity. In S. balanoides and E. modestus, once the mantle cavity was filled with air, the barnacle usually used up the oxygen within the bubble within 2–3 h and did not regain oxic conditions until the barnacle was reimmersed in seawater. In C. stellatus, the air bubble was repeatedly refreshed for many hours by pneumostome formation. In response to low environmental salinity, all three species closed the opercular plates firmly and rapidly used up oxygen within the mantle fluid.     

Ultrastructure of olfactory mucosa in the homing baltic sea trout Salmo trutta trutta

Scanning and transmission electron microscopy techniques have been used to study the olfactory organs of adult fresh-water adapted Baltic sea trout Salmo trutta trutta L. homing back to the River Umeälven in Västerbotten, Sweden. Parr and smolt show only minor differences in comparison to the adult mucosa. All fish have polymorphism of receptors, supporting cells and basal cells, and a dynamic cell population with primary cells basally. These mature and die, and are either extruded or phagocytized. The mucosa has a remarkably large population of loose connective tissue cells (plasma cells, macrophages and lymphoid wandering cells) as well as all types of leucocytes. The olfactory organs of sea trout have labyrinth cells, a cell type unique for vertebrates. These cells probably help to maintain an optimal ion balance, which is of great ecological importance for this migrating species. The mucosa cilia have small tubercles arranged in a spiral pattern and correlated with transitional filaments from the 9 peripheral fibrils. A hypothesis on the relationship of primary supporting cells, primary nonsensory cells, immature fibroblasts and Schwann cells is presented.