Spatial distribution of sponge-associated bacteria in the Mediterranean sponge Tethya aurantium

The local distribution of the bacterial community associated with the marine sponge Tethya aurantium Pallas 1766 was studied. Distinct bacterial communities were found to inhabit the endosome and cortex. Clear differences in the associated bacterial populations were demonstrated by denaturing gradient gel electrophoresis (DGGE) and analysis of 16S rRNA gene clone libraries. Specifically associated phylotypes were identified for both regions: a new phylotype of Flexibacteria was recovered only from the sponge cortex, while Synechococcus species were present mainly in the sponge endosome. Light conduction via radiate spicule bundles conceivably facilitates the unusual association of Cyanobacteria with the sponge endosome. Furthermore, a new monophyletic cluster of sponge-derived 16S rRNA gene sequences related to the Betaproteobacteria was identified using analysis of 16S rRNA gene clone libraries. Members of this cluster were specifically associated with both cortex and endosome of T. aurantium.     

Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)

The budding process has been studied in two congeneric Mediterranean species belonging to Tethya from different sampling sites: Marsala and Venice Lagoons (Tethya citrina); Marsala Lagoon and Porto Cesareo Basin (Tethya aurantium). Buds, connected to the adult by a spiculated stalk, differ between the two species in morphology and size, since those of T. citrina are small with elongated bodies, showing only a few spicules protruding from the apical region, whereas those of T. aurantium are round, larger, and show spicules radiating from the peripheral border. In T. citrina, cells with inclusions, varying in electron density and size, represent the main cell types of the buds. In T. aurantium, the cell component shows a major diversification, resulting from spherulous cells, grey cells, vacuolar cells and peculiar micro-vesicle cells. Neither canals nor choanocyte chambers were observed in the buds of the two species. In T. citrina, bud production is similar in both sampling sites. In T. aurantium, budding occurs more rarely in Porto Cesareo Basin, probably in relation with environmental factors, such as the covering of the cortex by sediment and micro-algae. Finally, in the buds of both species, the spicule size does not differ from that of the cortex of the adult sponges, further supporting the main involvement of the cortex in organizing the skeletal architecture of the buds.     

Multiple origins of the octoploid Scandinavian endemic Draba cacu minum: electrophoretic and morphological evidence

The octoploid Draba cacuminum (Brassicaceae) is one of the few endemic species in Scandinavia and has frequently been considered an example supporting the glacial survival theory. Two subspecies, ssp. cacuminum in southern Norway and ssp. angusticarpa in northern Scandinavia, have been described and suggested to be geographically diverged remnants of a more continuously distributed ancestor. To test an alternative hypothesis of independent origins of similar octoploids in different areas, we investigated populations of D. cacuminum and its possible progenitors using enzyme electrophoresis and morphological analysis. Electrophoretic analysis of progeny of D. cacuminum revealed high levels of fixed heterozygosity, suggesting that the species is a genetic alloploid. Fixed electrophoretic differences among the populations and additivity of electrophoretic phenotypes indicate that the octoploid D. cacuminum has originated at least three times by alloploidizations involving different populations of the hexaploid D. norvegica and a diploid species, possibly D. fladnizensis. Electrophoretic and morphometric data suggest that populations of D. cacuminum with broad siliculae have originated from populations of D. norvegica that had broad siliculae, and that populations of D. cacuminum with narrow siliculae have originated from populations of D. norvegica that had narrow siliculae. However, the electrophoretic and morphometric variation within D. cacuminum did not correspond to the geographic origin of the populations, and the previously described subspecies could not be recognized. Draba cacuminum gives no relevant information on the glacial survival theory; the polyploidizations may have occurred in postglacial time as well as in pre-Weichselian periods.     

Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncurium.

A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.     

Differences between two sympatric species of Tethya (Porifera, Demospongiae) concerning the growth and final form of their megasters

The megasters of T. aurantium and T. citrina consist, in their mature stage, of spherasters and spheroxyasters respectively. A study of the ratio between the ray length and the diameter of the centre (R/C), which determines the megaster shape, has been carried out on spicules of different size.
While the small megasters of these two species have a similar shape, successive stages show in T. aurantium an isometric growth and in T. citrina an allometric growth with rays growing faster than the centre. The two species differ also in the mean size, size frequency distribution and number of rays of the asters. Differences in the mean total diameter and shape were also detected among spicules coming from different regions of the sponge body. The taxonomic and evolutionary meaning of these phenomena are discussed.     

Interspecific variation in arrangement and morphology of micrasters of Tethya species (Porifera,Demospongiae)

Summary A new distinctive feature between the two Mediterranean species of Tethya, T. aurantium and T. citrina has been found in the body arrangement of different types of micrasters. Contrary to the previous assumptions, T. aurantium has two clearly distinct categories of micrasters: the chiaster-tylaster in the cortex and the larger, slender oxyaster in the choanosome. T. citrina has only slightly differentiated micraster sets in the cortex and choanosome; in the latter the shape of micrasters is close to that of oxyasters. SEM analysis shows that differences in micraster shape depend on the cylindrical or conical form of rays and on the distribution, density and strength of the microspines along their axis. The relationship between the degree of micraster differentiation and the development of the cortex in the two species is discussed.     

Metabolism of some carcinogenic aromatic amines in four species of marine sponges

Postmitochondrial fractions from marine sponges Geodia cydonium, Tethya aurantium, Verongia aerophoba and Pellina semitubulosa activate precarcinogenic aromatic amine 2-aminoanthracene, but not precarcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene, to Salmonella typhimurium TA 98 mutagens. All four sponge species lack a benzo(a)pyrene monooxygenase activity, but possesses the enzyme activity whose characteristics (selective activation of aromatic amines, NADPH-dependency, pH optimum at 8.4) are similar to FAD-containing monooxygenase. Tethya postmitochondrial fraction possesses an UDP-glucuronyl transferase activity which catalyzes the conjugation of a considerable part of metabolized 2-acetylamino [9-14C]fluorene to water soluble glucuronides. The possible ecological significance of exuded aromatic amine metabolites as well as the significance of the presence of the selective potential for the activation of aromatic amines to mutagens among sponges for our understanding of the fate and effects of carcinogens in the marine environment are discussed.     

Association of the sponge Tethya orphei (Porifera, Demospongiae) with filamentous cyanobacteria

Abstract. Specimens of the sponge Tethya orphei , collected in February 2005 on the underside of coral stones on Arì Athol (Maldives), have been processed for histological and ultrastructural investigations. The cortical layer of the sponge was found to be permeated by filamentous cyanobacteria, the trichomes of which measured 45–63 μm on average and were composed of 10–14 cells. The fine organization of the filaments was consistent with their taxonomic identification as Oscillatoria spongeliae. These filaments filled the cortical region of the sponge and penetrated inward into the upper choanosomal region, where they sometimes overlapped the siliceous spicule bundles. A budding specimen of T. orphei showed that the filaments were also present in the single bud protruding from the sponge surface, demonstrating that asexual reproduction can vertically transmit these symbionts from sponge to sponge. The occurrence of filaments in all the specimens studied is consistent with the assumption that filamentous cyanobacteria are not mere intruders but mutualistic symbionts with members of T. orphei.     

Chemical profile of adults and buds of the chemically defended marine sponge Tethya maza

Several adults and the corresponding larvae of marine sponges are known as rich sources of secondary metabolites. In this paper we will show the chemical profile of adults and buds of the sponge Tethya maza. Cholesterol, ergost-5,22-dienol-3, stigmast-5,24-dienol-3, cholest-5,22-dienol-3, cholest-5-enol-3-one-7, and stigmast-5,24(28)-dien-3-ol were the major compounds detected, two of them being detected only in crude extracts of the adults while one was present only in the buds. Due to this high similarity it is supposed that buds could be chemically protected with regards to predation as well as the adult individuals of T. maza. This is the first report of chemicals present in the structures (buds) of sponges produced by asexual reproduction.     

Observations on the moving colonies of the genus Tethya (Demospongia,Porifera)

Summary Observations on two species of sponges, Tethya seychellensis from the Red Sea, and T. aurantium from the Mediterranean Sea revealed that young colonies are able to detach from their sites of settlement and by means of filamentous podia, to move to other sites in the vicinity. These podia are 10–16 mm long extensions of the sponge body wall that bear an adhesive knob on their distal ends. After being attached, the contracting podia pull the spherical colonies of 2.0–3.0 cm in diameter, transporting them to a new site. EM observations showed that in the podia the matrix is rich in contractile myocytes, primary archaeocytes, nucleated archaeocytes and scleroblastic cells, each of which takes part in the moving ability of the podium. It was also shown that some of the archaeocytes go over a process of ripening within the podium and produce collagenic filaments deposited in the internal matrix.     

Functional morphology of Tethya species (Porifera): 1. Quantitative 3D-analysis of Tethya wilhelma by synchrotron radiation based X-ray microtomography

Rhythmic body contraction is a phenomenon in the Porifera, which is only partly understood. As a foundation for the understanding of the functional morphology of the highly contractile Tethya wilhelma, we performed a qualitative and quantitative volumetric 3D-analysis of the morphology of a complete non-contracted specimen at resolutions of 5.2 and 6.9 μm, using synchrotron radiation based X-ray computed microtomography (SR-μCT). For the first time, we were able to visualize all three major body structures of a complete poriferan without dissection of the shock-frozen, fixed and contrasted specimen in a near-to-life confirmation: poriferan tissue, mineral skeleton and aquiferous system. Applying a ‘virtual cast’ technique allowed us to analyze the structural details of the complete canal structure. Our results imply an extensive re-circulation of water inside the poriferan due to well-developed by-pass-canals, connecting excurrent and incurrent system. Nevertheless, the oscule region is strictly separated from the incurrent system. Based on our data, we developed a hypothetical flow regime for T. wilhelma, which explains the necessity of by-pass canals to minimize pressure boosts in the canal system during contraction. Additionally, re-circulation optimizes nutrient uptake, within small-sized poriferans, like T. wilhelma. Quantitative analysis allowed us to measure volumes and surfaces, displaying remarkable organizational differences between choanosome and cortex, by means of distribution of morphological elements. The surface-to-volume ratio proved to be very high, underlining the importance of the poriferan pinacoderm. We support a pinacoderm-contraction hypothesis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Dedicated to Prof. Dr. Michele Sarà (Genova, Italy), in honour of his 80th birthday in 2006.     

Lipids of North Atlantic krill

The seasonal variations in the total lipid content, lipid class composition, fatty acid composition, and fatty alcohol composition of Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Kr?yer), and T. raschii (M. Sars) have been examined. The total lipid content was highest in the autumn and early winter months and lowest in the spring. In M. norvegica, triacylglycerols served as the only depot lipids, whereas in T. inermis and T. raschii triacylglycerols, wax esters, and glycerophospholipids varied in proportion to the total lipid content. This suggests that glycerophospholipids, as well as wax esters and triacylglycerols, constitute depot lipids in these species. Wax esters and glycerophospholipids were the dominating depot lipids in T. inermis, whereas triacylglycerols and glycerophospholipids were most important in T. raschii. Results suggest that non-depot glycerophospholipids may constitute 3.5-4.5% of the dry weight of the three species of krill examined. T. inermis and T. raschii, from the same catches, had very similar fatty acid compositions for each of the major lipid classes, with the exception of a few minor fatty acids. The major lipid classes in all three species showed complex seasonal variations in the content of the fatty acids that typically reflect the diet, particularly in the case of the triacylglycerols. The results suggest that all the species examined are more herbivorous during the summer than during the autumn and winter. M. norvegica seemed to be significantly more carnivorous than the two Thysanoessa species. The degree of incorporation of individual fatty acids from the diet is probably specific for each lipid class in each krill species. The proportion of polyenoic fatty acids in the glycerophospholipids and the proportion of monoenoic fatty acids in the wax esters may be of importance for the temperature adaptation of T. inermis and T. raschii.     

The activator of human cerebroside sulphatase. Activating effect on the acidic forms of the sulphatases from invertebrates.

1) Acidic forms of the sulphatase were partially purified from the following invertebrate species: Tethya aurantium (Porifera), Patella vulgata (mollusca), Maja squinado (Arthropoda), Marthasterias glacialis (Echinodermata) and Microcosmus sulcatus (Tunicata). Enzyme preparations thus obtained cleaved cerebroside sulphates (sulphatides) only in the presence of either specific detergents (e.g. taurodeoxycholate) or an activator protein isolated from human liver. This corresponds to the findings on purified sulphatase A of human origin. 2) At low concentrations, the activating effect was proportional to the amount of activator protein applied; at higher concentrations, proportionality was obtained only in some cases. On a molar basis, less of the activator protein was required to achieve the same activation as taurodeoxycholate. At optimum concentrations of the detergent however, the activation was much higher. 3) The enzyme specificity of the activator and some evolutionary implications are discussed.     

中国美姿藓属植物的订正

根据文献记载,中国美姿藓属Ｔｉｍｍｉａ Ｈｅｄｗ。植物共有４种。通过实地采集和标本鉴定,确认中国产苔藓植物共有５种,其中的纤细美姿藓为中国新记录。本文对该属每种植物进行了描述,并给出了分种检索表,同时对它们的分类特征、地理分布也进行了简要讨论。     

COMPARATIVE MEASURES OF DESICCATION-TOLERANCE IN THE TORTULA RURALIS COMPLEX. I. VARIATION IN DAMAGE CONTROL AND REPAIR

A mechanism for desiccation-tolerance in bryophytes based on carbon balance, damage limitation, and cellular repair is proposed. These criteria are incorporated into an experimental framework to measure desiccation-tolerance comparatively. In this report we utilize measures of damage control and damage repair to determine relative differences in tolerance between populations of three tolerant moss species: Tortula caninervis, T. ruralis, and T. norvegica. The measurement of damage control by electrolyte leakage alone was demonstrated not to be useful in determining levels of tolerance by traditional criteria in these closely related and highly tolerant species. Measurement of protein synthetic differences between hydrated and desiccated-rehydrated treatments was used to distinguish between the capabilities of the three moss species to repair cellular damage and to formulate a measure of tolerance. The overall ranking of the three species in descending order of tolerance is calculated to be: Tortula caninervis, T. ruralis, and T. norvegica. However, individual populations of each of these species exhibit variation in tolerance levels that span this broader classification. These rankings correlate well with the perceived ranking of water stress in the species' natural habitat.     

Morphological, developmental and electrophoretic variation within and between obligately apomictic Taraxacum species

Interclonal variation in six morphometric characters and in the onset and rate of capitulum production, together with inter-and intra-clonal variation in esterase zymograms was examined in each of the two obligately apomictic species, Taraxacum pseudohamatum (section Hamata ) and T. unguilobum (section Naevosa ). Interclonal and interspecific variation in esterase zymograms was also examined within and between seven additional obligately apomictic species within the section Hamata. Within T. pseudohamatum , interclonal variation in the morphometric and electrophoretic characters was extensive; no variation was recorded in the onset and rate of capitulum production. Within T. unguilobum , interclonal variation in the morphometric characters and in the onset and rate of capitulum production was recorded; five of the six clones electrophoretically assayed shared the same esterase zymogram but one of these clones contained an individual which exhibited an aberrant zymogram. Within the section Hamata , considerable variation in esterase zymograms was recorded; this variation was both inter-and intra-specific although one zymogram predominated, being represented in each species and in 65% of the clones.     

Multiple species of the dinophagous dinoflagellate genus Amoebophrya infect the same host species

Populations of the dinoflagellate Dinophysis norvegica in the Baltic Sea and in the adjacent North Sea are infected by the endoparasite Amoebophrya sp. The high diversity recently unveiled within the genus Amoebophrya brings uncertainty about their identities. We applied molecular biology techniques--18S rDNA sequencing and fluorescent in situ hybridization (FISH)--to compare this host-parasite system from both environments. The North Sea Amoebophrya sp. 18S rDNA sequence was 89% identical to the previously described Baltic Sea Amoebophrya sp. sequence, suggesting they are different species. In spite of that, a phylogenetical analysis placed the North Sea parasite sequence in a well-supported cluster with other Amoebophrya sp. sequences. The D. norvegica 18S rDNA sequences from both environments were 100% identical, indicating that the hosts have not evolved independently. A DNA probe designed for the Baltic Sea Amoebophrya sp. 18S rRNA was used in FISH assays on infected D. norvegica populations from both environments. The probe stained all infected cells from the Baltic sample, whereas none from the North Sea were stained. The results indicate that D. norvegica is released from one parasite when entering the Baltic Sea, and become less infected by an alternative parasite species.     

Morphometric and allozyme variation in central and southern Greek populations ofMicrotus (Terricola) thomasi

The endemic Balkan vole taxonMicrotus (Terricola) thomasi (Barrett-Hamilton, 1903) exhibits great karyological variability in Greece. In this study, populations belonging to two different karyotypic forms (‘atticus’ and ‘thomasi’) are examined both morphometrically and electrophoretically. A total of 140 individuals ofM. (T.) thomasi were collected from 6 localities of south and central Greece. For the morphometric analysis, 27 variables (external body and cranial characters) were examined and evaluated according to multivariate analyses (PCA, MANOVA, CVA and CLUS). For the electrophoretic analysis, 18 putative genetic loci were examined and the allozymic data were treated by the biostatistical package BIOSYS-1. According to the results obtained, all the populations studied show little overall morphometric variability, whereas they are characterized by high electrophoretic variability. The populations studied are not grouped according to the karyotypic form. In almost all the cases, in the two UPGMA-dendrograms plotted according to morphometric and electrophoretic distances (Mahalanobis’ and Nei’s distances, respectively), the populations branching together belong to different karyotypic forms. Conclusively, the morphometric and electrophoretic results of this study revealed that the two karyotypic forms should not be considered separate species or subspecies, as it has been proposed by some authors in the past, and the populations studied can be considered only as different local populations of the rather variable vole speciesM. (T.) thomasi.     

A rearrangement of the family Tethyidae (Porifera Hadromerida) with establishment of new genera and description of two new species

The discovery of new species, a study of the type material of already described species and an analysis of the literature data call for an extensive rearrangement of the Family Tethyidae, whose last revision dates from Burton (1924). The rearrangement proposed here attributes to the Family eight genera: Tethya Lam. (type genus), Burtonitethya gen nov. , Columnitis Schmidt, Halicomeies Topsent, Stellitethya gen. nov., Tectnitethya gen. nov., Tethycometes gen. nov . and Xenospongia Gray. The genus Aaptos is excluded from the Tethyidae. Three of the new genera have been established for species previously attributed to the genus Tethya and two are new: Burtonitethya gemmiformis (Burton & Rao) and Tethycometes sibogae sp. nov . Five species, previously attributed to Tethya , are considered 'incertae sedis'. An analytical key and discussion of the evolution of the family are included.     

Lactate dehydrogenase from the northern krill Meganyctiphanes norvegica: comparison with LDH from the Antarctic krill Euphausia superba

Electrophoretic polymorphism of lactate dehydrogenase (LDH, EC 1.1.1.27) from abdominal muscle is reported in the northern krill Meganyctiphanes norvegica. In the population, from the Gullmarsfjord (west coast of Sweden), LDH was encoded for by two different Ldh-A* and -B* loci. The isoenzymes were named according to their electrophoretic mobilities. Ldh-A* locus was polymorphic. The allelic frequencies were a=0.99, a'=0.002, a"=0.004, a"'=0.004. The level of LDH polymorphism is low. Most individuals possess the same amount of two LDH homopolymers (LDH-A*(4) and LDH-B*(4)). The Meganyctiphanes norvegica LDH-A*(4) and LDH-B*(4) isoenzymes and the predominant LDH-A*(4) isoenzyme from Euphausia superba were purified to specific activities of 294, 306 and 464 micromol NADH min(-1) mg(-1), respectively. In both species the LDH isoenzymes were separated by chromatofocusing. All three isoenzymes are L-specific tetramers with molecular weight of approximately 160 kDa. Northern krill LDH-A*(4) has higher affinity for pyruvate and lactate and is more thermostable than LDH-B*(4). Both isoenzymes are inhibited significantly by high concentration of pyruvate but not lactate. Antarctic krill isoenzyme exhibits high substrate affinities, high NAD inhibition, high inhibition at 10 mM pyruvate, lack of lactate inhibition, and high heat stability and resembles northern krill LDH-A*(4) isoenzyme.