Endosymbiosis in the lucinid clams Lucinoma aequizonata,Lucinoma annulata and Lucina floridana: a reexamination of the functional morphology of the gills as bacteria-bearing organs

A three-dimensional representation of the structure of the endosymbiont-containing gills of Lucinoma aequizonata, L. annulata and Lucina floridana was constructed using light and electron microscopy of fresh and plastic-embedded thin-sectioned samples. The gills of these lucinids are identical in overall structure, each being composed of three structurally and functionally distinct regions here called the ctenidial filament zone (CFZ), the transition zone (TZ), and the bacteriocyte zone (BZ). Rather than a simple medial extension of the filament tissue, the bacteriocyte tissue is organized as an array of cylindrical tubes, the walls of which are composed primarily of bacteriocyte cells covered by a thin microvillar epithelium. The physical relationship between the symbionts, the host tissues and the external environment are examined, and structural constraints on the potential functions of bacteria in this host-symbiont system are discussed.     

Phenotypic variations in the gills of the symbiont-containing bivalve Lucinoma aequizonata

The marine bivalve Lucinoma aequizonata (Lucinidae) maintains a population of sulfide-oxidizing chemoautotrophic bacteria in its gill tissue. These are housed in large numbers intracellularly in specialized host cells, termed bacteriocytes. In a natural population of L. aequizonata, striking variations of the gill colors occur, ranging from yellow to grey, brown and black. The aim of the present study was to investigate how this phenomenon relates to the physiology and numbers of the symbiont population. Our results show that in aquarium-maintained animals, black gills contained fewer numbers of bacteria as well as lower concentrations of sulfur and total protein. Nitrate respiration was stimulated by sulfide (but not by thiosulfate) 33-fold in homogenates of black gills and threefold in yellow gill homogenates. The total rates of sulfide-stimulated nitrate respiration were the same. Oxygen respiration could be measured in animals with yellow gills but not in animals with black gills. The cumulative data suggest that black-gilled clams maintained in the aquarium represent a starvation state. When collected from their natural habitat black gills contain the same number of bacteria as yellow gills. Also, no significant difference in glycogen concentrations of the host tissues was observed. Therefore, starvation is unlikely the cause of black gill color in a natural population. Alternative sources of nutrition to sulfur-based metabolism are discussed. Denaturing gradient gel electrophoresis (DGGE) performed on the different gill tissues, as well as on isolated symbionts, resulted in a single gill symbiont amplification product, the sequence of which is identical to published data. These findings provide molecular evidence that one dominant phylotype is present in the morphologically different gill tissues. Nevertheless, the presence of other phylotypes cannot formally be excluded. The implications of this study are that the gill of L. aequizonata is a highly dynamic organ which lends itself to more detailed studies regarding the molecular and cellular processes underlying nutrient transfer, regulation of bacterial numbers and host–symbiont communication. Received: 1 September 1999 / Accepted: 1 February 2000     

The bacterial symbiont from the hydrothermal vent tubewormRiftia pachyptila is a sulfide specialist

We have investigated the metabolic adaptations of the chemolithotrophic bacterial symbionts ofRiftia pachyptila. Specimens of the tubeworm were collected by submersible from depths of 2600 m at 13°N on the East Pacific Rise in 1987, and 2450 m at the Galápagos Rift in 1988. Isolated bacteria utilize sulfide, but not thiosulfate or sulfite, as their sole reduced-sulfur energy source. The bacteria rapidly oxidize a wide range of sulfide concentrations (5µM to 2 mM), with maximal respiration rates at concentrations >1 mM, and unlike many sulfur-oxidizing bacteria, show no inhibition in oxygen consumption at sulfide concentrations up to 2 mM. Incubations of freshly homogenized trophosome tissue or isolated bacteria with sodium [³⁵S] sulfide and subsequent analysis of sulfur products by high-performance liquid chromatography and flow-through scintillation counting showed that sulfide disappeared almost completely within 1 min. Both soluble and insoluble products of sulfide oxidation were produced. The soluble fraction contained sulfate and polysulfides, with no thiosulfate produced. However, the majority of the radioactivity was in the water-insoluble fraction, mostly as elemental sulfur. Whole-worm experiments under pressure showed a rapid removal of³⁵S-sulfide from the incubation water, with sulfide, sulfate, and polysulfides appearing in the blood within 4 h. There was no utilization of thiosulfate by the whole worms, freshly homogenized trophosome tissue, or isolated bacteria.     

Sulfide-oxidizing bacteria in the burrowing echinoid,Echinocardium cordatum (Echinodermata)

Symbiotic filamentous bacteria thrive in the intestinal caecum of the deposit-feeding echinoid Echinocardium cordatum. Specimens of E. cordatum were collected at Wimereux (Nord Pas-de-Calais, France) in 1991. Their symbiotic bacteria build nodules by forming multilayered mats around detrital particles that enter the caecum. The morphological features of the bacteria are those of Thiothrix, a sulfide-oxidizing genus. The filaments, which may form rosettes, are sheathed and made by a succession of hundreds of rod-shaped bacteria which store elemental sulfur in the presence of external sulfide. Live bacteria are restricted to the outer layers of the nodules. Their sulfide-oxidizing activity was investigated, using a Biological Oxygen Monitor, by measuring the O₂-consumption when reduced sulfur compounds are provided. They oxidize thiosulfate and sulfide. Optimal sulfide oxidation occurs at intermediary pO₂ (100 to 160 M O₂l^-1). Spectrophotometry has confirmed that the sulfur content of the filamentous symbiotic sulfideoxidizing bacteria depends on the presence of external sulfide. This is the first report of symbiotic intradigestive Thiothrixspp.-like bacteria; it lengthens the list of symbioses between sulfide-oxidizing bacteria and invertebrates from sulfide-rich habitats.     

Contrasting effects of sulfide and thiosulfate on symbiotic CO2-assimilation of Phallodrilus leukodermatus (Annelida)

Experiments were conducted in order to specify the reductants responsible for the carbon dioxide fixation of the symbiotic sulfur bacteria in the gutless marine oligochaete Phallodrilus leukodermatus (Annelida) from shallow calcareous sediments in Bermuda. Carbon dioxide-uptake rates were suppressed by S⁼ and stimulated by S₂O₃ ⁼. Individuals which hosted bacteria containing reserve energy substances maintained a high short-term CO₂-uptake activity, while bacteria in worm homogenates and in worms treated with an antibiotic (Baypen) did not show any significant metabolic activity. Absolute uptake rates in P. leukodermatus were usually considerably higher than those reported for other animals harbouring prokaryotic sulfuroxidizing symbionts. Utilization of thiosulfate rather than sulfide is compatible with the preferred occurrence of the worms around the redox discontinuity layer and has been confirmed in other thiobiotic animals. Sulfur stored in the symbiotic bacteria appears to be oxidized to sulfate and be excreted when the worms are held under energy-limited conditions. The data emphasize the complexity of the possible metabolic pathways involved in the oxidation of reduced-sulfur compounds by bacterial symbionts in marine invertebrates.     

Localization of RubisCO and sulfur in endosymbiotic bacteria of the gutless marine oligochaete Inanidrilus leukodermatus (Annelida)

 The chemoautotrophic potential of the two co-occurring larger and smaller bacterial endosymbionts of the gutless marine oligochaete Inanidrilus leukodermatus was determined using immunocytochemistry. An antibody directed against the Form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key CO₂-fixing enzyme of the Calvin–Benson cycle, consistently labeled the larger symbionts. Electron microscopic spectroscopy showed that the larger symbionts contained sulfur in intracellular globules and to a lesser degree in the cytoplasm. The presence of RubisCO and sulfur indicates that the larger endosymbionts of I. leukodermatus are chemoautotrophic sulfur-oxidizing bacteria. In contrast, no RubisCO or sulfur was detected in the smaller endosymbionts of this host. Received: 28 September 1999 / Accepted: 29 May 2000     

Response of hydrothermal vent vestimentiferan <Emphasis Type="Italic">Riftia pachyptila</Emphasis> to differences in habitat chemistry

Vestimentiferan tubeworms, which rely on intracellular sulfide-oxidizing autotrophic bacteria for organic carbon, flourish at deep-sea hydrothermal vents despite the erratic nature of their habitat. To assess the degree to which differences in habitat chemistry (sulfide, pH/CO₂) might impact host and symbiont metabolic activity, Riftia pachyptila tubeworms were collected from habitats with low (H₂S < 0.0001 mM) and high (up to 0.7 mM) sulfide concentrations. The elemental sulfur content of the symbiont-containing trophosome organ was lower in specimens collected from the low-sulfide site. Symbiont abundance, RubisCO activity, and trophosome carbon fixation rates were not significantly different for individuals collected from low- versus high-sulfide habitats. Carbonic anhydrase activities were higher in the anterior gas exchange organs of R. pachyptila from the low-sulfide habitat. Despite large differences in habitat chemistry, symbiont abundance and autotrophic potential were consistent, while the host appears to tailor carbonic anhydrase activity to environmental CO₂ availability.     

Biochemical correlates of dissolved mercury uptake by the oyster Ostrea edulis

From previous work, the equilibrium concentration factor for dissolved mercury in the digestive gland of Ostrea edulis Linnaeus was found to be three to four times higher than that in the gills. In the present study, an analysis of soluble protein revealed values of 49.3±14.2 mg g wet tissue^-1 for the digestive gland and 0.7 ±0.1 mg g wet tissue^-1 for the gills. Starvation significantly reduces the soluble protein level of the digestive gland to 31.1±6.4 mg g^-1 and that of the gills to below the limit of detection. These results suggest that the difference in concentration factors between the gills and digestive gland may be based on a quantitative difference in macromolecular binding sites. However, the uptake of dissolved mercury over a period of 48 h was considerably greater in the gills, so that although the soluble protein content of the tissue may influence the final concentration factor, it does not appear to affect the rate at which this equilibrium is achieved. A more detailed investigation of the mechanism of dissolved mercury uptake by oyster gills has been carried out using isolated tissues. The process is inhibited by 5mM 2–4 dinitrophenol, by the absence of a readily metabolizable substrate (dextrose) in the uptake medium, and by 30mM K⁺. The effect of K⁺ necessitated further investigation with a specific inhibitor of K⁺ transport. Strophanthin G (ouabain), at a concentration of 0.01 mM, caused a significant increase in mercury uptake.     

The importance of methane and thiosulfate in the metabolism of the bacterial symbionts of two deep-sea mussels

Undescribed hydrocarbon-seep mussels were collected from the Louisiana Slope, Gulf of Mexico, during March 1986, and the ultrastructure of their gills was examined and compared to Bathymodiolus thermophilus, a mussel collected from the deep-sea hydrothermal vents on the Galápagos Rift in March 1985. These closely related mytilids both contain abundant symbiotic bacteria in their gills. However, the bacteria from the two species are distinctly different in both morphology and biochemistry, and are housed differently within the gills of the two mussels. The symbionts from the seep mussel are larger than the symbionts from B. thermophilus and, unlike the latter, contain stacked intracytoplasmic membranes. In the seep mussel three or fewer symbionts appear to be contained in each host-cell vacuole, while in B. thermophilus there are often more than twenty bacteria visible in a single section through a vacuole. The methanotrophic nature of the seep-mussel symbionts was confirmed in ¹⁴C-methane uptake experiments by the appearance of label in both CO₂ and acid-stable, non-volatile, organic compounds after a 3 h incubation of isolated gill tissue. Furthermore, methane consumption was correlated with methanol dehydrogenase activity in isolated gill tissue. Activity of ribulose-1,5-biphosphate (RuBP) carboxylase and ¹⁴CO₂ assimilation studies indicate the presence of either a second type of symbiont or contaminating bacteria on the gills of freshly captured seep mussels. A reevaluation of the nutrition of the symbionts in B. thermophilus indicates that while the major symbiont is not a methanotroph, its status as a sulfur-oxidizing chemoautotroph, as has been suggested previously, is far from proven.     

Multiple trophic resources for a chemoautotrophic community at a cold water brine seep at the base of the Florida Escarpment

The biological community that surrounds the hypersaline cold water brine seeps at the base of the Florida Escarpment is dominated by two macrofaunal species: an undescribed bivalve of the family Mytilidac and a vestimentiferan worm, Escarpia laminata. These animals are apparently supported by the chemoautotrophic fixation of carbon via bacterial endosymbionts. Water column and sediment data indicate that high levels of both sulfide and methane are present in surface sediments around the animals but absent from overlying waters. Stable isotopic analyses of pore water indicate that there are two sources of sulfide: the first is geothermal sulfide carried in groundwater leaching from the base of the escarpment, and the second is microbial sulfide produced in situ. The vestimentiferan E. laminata, and the mytilid bivalve (seep mussel) live contiguously but rely on different substrates for chemoautotrophy. Enzyme assays, patterns of elemental sulfur storage and stable isotopic analyses indicate that E. laminata relies on sulfide oxidation and the seep mussel on methane oxidation for growth.     

Anatomical and experimental evidence for particulate feeding in<Emphasis Type="Italic"> Lucinoma aequizonata</Emphasis> and<Emphasis Type="Italic"> Parvilucina tenuisculpta</Emphasis> (Bivalvia: Lucinidae) from the Santa Barbara Basin

Previous nutritional models for adults of the lucinid bivalve Lucinoma aequizonata contend that symbiotic chemoautotrophic bacteria provide most of the organic carbon for the host. The existence of this symbiosis, coupled with the hosts distinctive anatomical features, shaped the impression that particulate feeding was not a significant part of L. aequizonata nutrition. Here, we use several techniques to show that particulate feeding is a consistent and important part of the L. aequizonata nutritional strategy. Histological and scanning electron microscopy observations reveal that the gills of L. aequizonata, like those of the lucinid Parvilucina tenuisculpta, have functional mucociliary epithelia, able to transport captured particles to the mouth. Observations of gut content and radiolabeled feeding experiments indicate that L. aequizonata does ingest and assimilate carbon from particulate organic matter. Furthermore, molecular identification of a broad spectrum of organisms in the guts of native adult specimens demonstrates that L. aequizonata is non-selective when ingesting organic material, and has a mixotrophic diet.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P.W. Sammarco, Chauvin     

Gill structure in Lucina pectinata (Bivalvia: Lucinidae) with reference to hemoglobin in bivalves with symbiotic sulphur-oxidizing bacteria

Lucina pectinata is a large tropical Lucinidae which is characterized by abundant tissue hemoglobin in its deep-red gills. In the present paper, hemoglobin is described as being located in cytoplasmic dark patches of the bacteriocytes together with a cystine-rich protein. Large microbodies contain a non-hemoglobin heme-compound which is identified with a previously described non-protein-bound hematin; however, it has not been established whether this heme is involved in a sulphur-oxidizing system or represents a catabolic by-product of hemoglobin. Electron-lucent vesicles are associated with the basal microbodies but their function is, so far, unknown. In addition, the bacteriocytes have been observed to have direct contact with sea water, modulated by large intercalary cells which overlap the bacteriocytes on their margin. Such relationships between bacteriocytes and intercalary cells, as well as their cytological features, are different from those observed in lucinid species inhabiting sea-grass beds, but very similar to those observed in Calyptogena magnifica. From the congruence between the shallow-water Lucinidae L. pectinata, inhabiting mangrove swamps, and the deep-sea Vesicomyidae C. magnifica, found at hydrothermal vents, we conclude that such features are likely to be adaptative to high-sulphide environments, notwithstanding the phylogenetic distance.     

The color of the trophosome: elemental sulfur distribution in the endosymbionts of<Emphasis Type="Italic"> Riftia pachyptila</Emphasis> (Vestimentifera; Siboglinidae)

Riftia pachyptila Jones, 1981, lives in association with a chemoautotrophic, sulfide-oxidizing -Proteobacterium that occurs in a specialized organ, the trophosome. Ultrastructurally different bacterial subpopulations occur in different regions of the trophosome lobules (central rods, median small cocci, peripheral large cocci) and contain vesicles, which have been proposed to be sites of elemental sulfur storage. Differently colored trophosomes have been suggested to reflect different amounts of elemental sulfur in the tissue. In this study, the presence of elemental sulfur (S⁰) was confirmed in the vesicles of the symbionts of R. pachyptila by electron energy loss spectrography (EELS). The proportion of (two-dimensional) area occupied by sulfur vesicles in the cells was found to be strongly correlated with trophosome color, both in individuals with uniformly colored trophosomes and individuals that exhibited a gradual color change along the length of their trophosomes. Elemental sulfur content was highly variable between individuals from a single collection, suggesting a high degree of microhabitat heterogeneity within vestimentiferan aggregations.Communicated by O. Kinne, Oldendorf/Luhe     

Sulfide tolerance and detoxification in shallow-water marine fishes

Hydrogen sulfide is a potent inhibitor of aerobic respiration. Sulfide is produced in sediments, and many species of fish live in association with the bottom. Tolerance tests, enzyme assays, and chromatography of sulfur compounds in thirteen species of shallow-water marine fishes (collected in San Diego, California, USA in 1987–1988) indicate adaptations to sulfide that vary with habitat and lifestyle. Tidal-marsh inhabitants, like Gillichthys mirabilis and Fundulus parvipinnis, have higher tolerance to sulfide (96 h LC₅₀ at 525 to 700 M) relative to outer-bay and open-coast inhabitants (surviving <12 h at much lower concentrations). The cytochrome c oxidase of all species shows high activity and susceptibility to sulfide poisoning, with 50% inhibition at 30 to 500 nM in various tissues. The two marsh species are able to survive at sulfide concentrations already inhibitory to their cytochrome c oxidase and fatal to other species. All species detoxify sulfide by oxidizing it to thiosulfate. All have sulfide-oxidizing activity in the blood, spleen, kidney, liver and gills, which correlates significantly with heme content. Thiosulfate appears in the tissues of sulfide-exposed fish and builds up to high concentrations (up to 2 mM) with stronger and longer exposure. Unexposed fish contain little or no thiosulfate. Sulfide is barely detectable in the tissues, even in high-sulfide exposure tests. We suggest that fish blood, in having high sulfide-oxidizing activity and no cytochrome c oxidase, can act as a short-term first line of defense against sulfide, and thus minimize the amount that reaches the vital organs. The results of this study indicate that sulfide is a significant environmental factor influencing the ecological distribution of marine fishes.     

Oxygen dependent sulfide detoxification in the lugwormArenicola marina

The lugwormArenicola marina L. oxidizes entering sulfide to thiosulfate. After 8 h of normoxic incubations with sulfide concentrations of 0.2 to 1.0 mmoll^-1 thiosulfate in the coelomic fluid amounted up to about 4 mmoll^-1 whereas sulfite concentrations were 100-fold lower and no accumulation of sulfate in the coelomic fluid was found. The sulfide oxidation was highly oxygen dependent. An increase of oxygen partial pressure ( ) in the medium was followed by enhanced thiosulfate production and by a decrease of sulfide concentration in the coelomic fluid. Under normoxia, the sulfide oxidation rate was sufficient to compensate the influx of sulfide into the coelomic fluid when the sulfide concentration in the medium was below 0.33 mmoll^-1. When external sulfide was raised beyond this level, sulfide up to 5 moll^-1 in the coelomic fluid appeared. Succinate in the body wall tissue was low as long as no sulfide appeared in the coelomic fluid, indicating the maintenance of an aerobic metabolism. The oxidation of sulfide to thiosulfate was localized in the mitochondria of the body wall tissue. The oxygen consumption of mitochondria was stimulated by the addition of sulfide. The mitochondrial sulfide oxidation rate depended on the amount of mitochondrial protein and followed a Michaelis-Menten kinetic. An apparentK _m of 0.68±0.29 moll^-1 and aV _max of 41.9±22.3 nmol min^-1 mg^-1 protein was calculated. Sulfide was stoichiometrically oxidized to thiosulfate with 1 mol sulfide consuming 1 mol oxygen. Sulfide oxidation was not inhibited by sulfide concentrations as high as 100 moll^-1. At low concentrations of cyanide or azide, when respiration without sulfide was already inhibited, sulfide oxidation could still be stimulated, tentatively indicating the existence of an alternative terminal oxidase. Specimens examined in the present study were collected near St. Pol de Leon, France, from 1989 to 1992.     

Resource acquisition in two intertidal fucoid seaweeds, Fucus serratus and Himanthalia elongata : seasonal variation and effects of reproductive development

Seasonal variations and the effect of reproductive development on resource acquisition by two intertidal fucoid species, the iteroparous Fucus serratus L. and the semelparous Himanthalia elongata (L.) S. F. Gray were examined. The oxygen-exchange characteristics of vegetative apical tissue of both non-fertile and fertile plants and receptacle tissue were compared at monthly intervals throughout reproductive development. Respiratory rates in non-fertile F. serratus varied seasonally between 1.5 and 8.0 μmol g⁻¹ fresh wt h⁻¹; in fertile plants the receptacle had a significantly lower respiratory rate than the vegetative tissue. The respiratory rate of the vegetative button of fertile H. elongata displayed less seasonal variation and was lower than that of the receptacle, which varied from a maximum of 9.5 μmol g⁻¹ fresh wt h⁻¹ at receptacle initiation in October to a minimum of 2.0 μmol g⁻¹ fresh wt h⁻¹ in February. The maximum photosynthetic rate (P _max) of non-fertile plants of both species did not vary in a distinct seasonal manner (∼60 μmol g⁻¹ fresh wt h⁻¹ for F. serratus and ∼12 μmol g⁻¹ fresh wt h⁻¹ for H. elongata). In fertile plants, the P _max of the receptacle tissue was (∼50% lower in F. serratus, and at its peak three times higher in H. elongata, than that of vegetative tissue. The stable carbon-isotope ratio (δ¹³C) did not differ between different tissue types in F. serratus, but values did vary seasonally, being less negative in the summer than in the winter (−13.5‰ compared to −18‰). The receptacle tissue of H. elongata also displayed a distinct seasonal variation in δ¹³C values (−12‰ in summer, −16‰ in winter), whilst the δ¹³C of the vegetative button did not vary seasonally. The rate of uptake of inorganic nitrogen by the vegetative thallus was lower in H. elongata than in F. serratus. The receptacle tissue of F. serratus had lower uptake rates than the vegetative tissue, whilst the uptake rate by H. elongata receptacle tissue was higher than that of the vegetative button. Received: 14 March 1997 / Accepted: 22 April 1997     

Anatomy of structures associated with air-breathing in Orchestia gammarellus (Crustacea: Amphipoda: Talitridae): coxal plates and gills

The structure of the coxal gills and coxal plates of the semi-terrestrial beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda: Talitridae) is described in relation to their possible use for aerial gas exchange and ion exchange. Anatomical evidence is presented to support the hypothesis that the medial surface of the coxal plates functions as an extrabranchial aerial gas-exchange site in O. gammarellus. Thus, the effective diffusion distance across the medial (or inside-facing) surface of O. gammarellus coxal plates (mean±SD=5.4±0.3 m; n=9, cuticle thickness 4.4±0.5 m, n=21) is only a third of the equivalent distance across both the coxal gills (18.4±6.0 m, n=10; cuticle thickness 1.7±0.6 m, n=7) and the lateral (or external) surface of the coxal plates (19.4±0.7 m, n=5; cuticle thickness 8.7±0.8 m, n=7). Chloride-ion-permeable areas were located using a silver-staining technique. All ten coxal gills appeared to be equally permeable to chloride ions after examination with a light microscope. However, the coxal plates and the rest of the integument do not appear to be chloride-permeable.     

The tunicate Salpa thompsoni ecology in the Southern Ocean. II. Proximate and elemental composition

Detailed determination of Salpa thompsoni elemental composition has been carried out on specimens collected in the Eastern Bellingshausen Sea and at the northern edge of the Weddell Gyre during austral autumn (April and May) of 1996 and 2001. More than 170 Antarctic tunicates S. thompsoni were analysed to determine wet weight (WW), dry weight (DW), ash-free dry weight (AFDW) and elemental composition (C, N content, proteins, carbohydrates and lipids) of different sizes and stages. Dry weight comprised 6.4% (aggregate form) to 7.7% (solitary form) of the WW. AFDW amounted to ~44% of the DW. Carbon and nitrogen contents (Carbon: 17–22%, Nitrogen: 3–5% of the DW) of both aggregate and solitary forms were found to be high relative to data reported in the literature. Although some unidentified organic compounds are not included in our carbon budget, the findings of this study show higher than previously reported nutritional values of S. thompsoni. In spite of this, a shift from a krill-dominated towards a salp-dominated ecosystem would have dramatic consequences for organisms at higher trophic levels.     

Elemental composition (C,N, H) and energy in the development of Pagurus bernhardus (Decapoda: Pagurida) megalopa

No differences in development time and mortality were detected between starved and fed laboratory raised megalopa of Pagurus bernhardus. The average time of development in 138 megalopa was determined as 7.3±0.1 (95% CI) days. During megalopa development P. bernhardus loses about 7% in dry weight (DW), 17% in carbon (C), 6% in nitrogen (N) and 17% in hydrogen (H). During development C/N ratio and individual energy content descend about 14 and 22% respectively. Weight specific energy content decreases by 17% in the first 3 d and remains constant at 12.3±0.3 (95% CI) J·(mg DW)^-1 thereafter. About another 25% in individual energy content was lost by molting to crabs. The measured compounds do not follow a steady decrease. The possibility is discussed that a period of low energy cost (about the first half of development) alternates with times of higher energy expenditure mainly based on lipids. A fixed physiological program different from starvation capability is indicated for P. bernhardus megalopae. By comparing megalopae hatched in two different seasons and years reference is given to the variability in growth pattern.     

Presence of hydrocarbon-degrading bacteria in the gills of mussel Mytilus galloprovincialis in a contaminated environment: a mesoscale simulation study

A number of previous studies have shown that the relationships of symbiosis existing between mussels and microorganisms are directly dependent on the environmental conditions. However, little is known about existing relationships between mussels and bacteria in hydrocarbon-impacted marine environments. The aim of this preliminary study is to investigate the presence of oil-degrading bacteria in the mussel Mytilus galloprovincialis during growth in polluted ecosystems. All the experiments were carried out in a mesocosm system designed to simulate chronic pollution and to enable direct exposure of mussels to chemicals. Quantitative (4′,6-diamidino-2-phenylindole, colony-forming units, Most Probable Number) analyses and screening (presence/absence) of metabolic functional genes were performed to analyse bacterial populations inside the gills of mussels exposed and not exposed to hydrocarbons. The data obtained show that the presence of hydrocarbons affected the abundance of bacteria inside the gills of specimens and determines selection for specific (hydrocarbon-degrading) bacteria (i.e. Alcanivorax sp. and Marinobacter sp.). However, is not yet clear whether the presence of such genera of bacteria inside the mussel is due to symbiosis or as a result of filtration.