Endemic Lake Baikal sponges from deep water. 2: Taxonomy and Bathymetric Distribution.

Unique samples of deep-water sponges of Lake Baikal were collected between 120 and 1450 m depth and their taxonomy and bathymetric distribution were studied. Based on morphological studies with scanning electron microscopy (SEM) and molecular analyses (CO1, ITS) we describe a new species, Baikalospongia abyssalis sp. nov. Spicule morphology of this new species is similar to Palaeoephydatia sp., a species previously known only from fossils in Late Pliocene (3.2-2.8 mya) sediments. Other sponge samples collected were identified as Baikalospongia intermedia intermedia, B. intermedia profundalis, B. bacillifera, B. fungiformis, B. martinsoni and Swartschewskia papyracea, all from the family Lubomirskiidae. Sponge specimens with giant spicules, identified as B. fungiformis, were found at great depths. B.i. intermedia and B. i. profundalis are dominating species at great depth. Light is a limiting factor for distribution of Lubomirskia baicalensis, possibly due to its symbiosis with photosynthetic protists. The current study extends our knowledge on the distribution boundaries of Lubomirskiidae at great depths.

Endemic Lake Baikal sponges from deep water. 1: Potential cryptic speciation and discovery of living species known only from fossils.

We revealed new deep-water species and cryptic speciation within freshwater sponges of the endemic family Lubomirskiidae (Porifera; Demospongiae; Spongillina) based on molecular and spicule morphology analyses of ITS and CO1 mtDNA. Lubomirskiidae contains a group of closely related species which are a dominant component of the benthos in Lake Baikal, the world's deepest and most ancient lake. Spicule morphology was similar between two Recent samples and species only known previously from fossils in Late Pliocene (3.2-2.8 mya) sediments. Despite the morphological similarity with the cosmopolitan family Spongillidae, molecular analysis of ITS sequences has reliably assigned these species to Lubomirskiidae. This not only indicates that species identification of freshwater fossil sponge spicules should be made with caution, but also suggests that the structure of megascleres may not be a reliable character for interpretations of paleoclimatic reconstructions for the Baikal region. Our results do not support the current classification of Lubomirskiidae into its morphologically defined genera and species, suggesting a strong discrepancy between molecular and morphological variation in Baikalian sponges. This present contribution is the first part of a study on the phylogenetic relationships of the Lake Baikal deep water sponge fauna.

Towards a molecular systematics of the Lake Baikal/Lake Tuva sponges.

Lake Baikal is famous for its extensive biodiversity that is equaled only by few other lakes. Fascinatingly, about 80% of all the animals the lake hosts are endemic. Sponges (Porifera) that live in symbiosis with photosynthetic algae are the most abundant animal taxon found in the littoral zone of Lake Baikal and have been grouped to the family Lubomirskiidae. In recent years, several attempts to determine the phylogenetic relationship between Lubomirskiidae and cosmopolitan freshwater sponges have been undertaken. Yet the results obtained remain inconclusive. Here, we strive to determine the phylogeny of freshwater sponges with the focus on endemic Lake Baikal species, also taking into account two poriferan species that were collected during an expedition in 2006 in two other isolated Siberian lakes, Lake Chagytai and Lake Tore-Khol. Since its discovery at the beginning of the twentieth century, the Lake Chagytai species was grouped to the Lubomirskiidae and called Baikalospongia dzhegatajensis. However, analyses of molecular sequence data [internal transcribed spacer 2 (ITS2), ribosomal DNA (rDNA)] and morphological markers (spicules, habitus) inferred a close relationship to the cosmopolitan genus Ephydatia and also to the Lake Tore-Khol species that had not so far been described. Thus, both species were tentatively termed Ephydatia tuva (Lake Chagytai) and E. altaiensis (Lake Tore-Khol). We hypothesize that these new species might have evolved from Ephydatia-like ancestors through adaptation to the unique environmental conditions of both lakes. To test the ITS data, an unlinked genetic locus was chosen for further phylogenetic analyses, the protein-coding gene silicatein. These analyses provided not only a more robust resolution between the Lubomirskiidae, but also corroborated the grouping of the Lake Chagytai and Lake Tore-Khol species to the genus Ephydatia. In addition, the phylogenetic analyses suggest a Spongilla-like founder generation of poriferan species in Lake Chagytai and Lake Tore-Khol. In conclusion, we propose that the process of speciation in Lake Baikal and Lake Chagytai/Lake Tore-Khol, from a cosmopolitan Spongilla-like ancestor to more than ten endemic species follows allopatric speciation patterns and is of the peripatric type.

Symbiotic interaction between dinoflagellates and the demosponge Lubomirskia baicalensis: aquaporin-mediated glycerol transport.

Lake Baikal is rich in endemic sponge species, among them the arborescently growing species Lubomirskia baicalensis. During winter when the lake is covered by ice, this species reproduces sexually, reflecting a high metabolic activity. Throughout the year, L. baicalensis lives in association with dinoflagellates, which - according to the data presented herein - are symbiotic. The dinoflagellates have been determined on the basis of their rDNA/ITS characteristics and were found to display high sequence similarity to Gymnodinium sanguineum. The dinoflagellates give the sponge its characteristic green color, reflecting the high chlorophyll content (chlorophyll-a content in March and September of 3.2 +/- 0.6 microg/g and 1.9 +/- 0.5 microg/g of protein, respectively). With the in vitro cell culture system for sponges, the primmorphs, it could be demonstrated that [(14)C] glycerol is readily taken up by sponge cells; this process can be inhibited by phloretin, an aquaporin channel blocker. In order to prove the effect of cholesterol on the intermediate metabolism of the sponge cells, molecule probes, cDNAs for key enzymes in gluconeogenesis, glycolysis, and citric acid, have been applied in Northern blot studies. The data revealed that the genes coding for the enzymes citrate synthase and fructose-1,6-bisphosphatase are strongly upregulated after exposure of primmorphs to glycerol. This effect is abolished by phloretin. The genes encoding the phosphoglucose isomerase and pyruvate dehydrogenase do not respond to glycerol supply, suggesting that their expression is not under genetic control in L. baicalensis. To prove the assumption that the aquaporin channel is involved in the influx of glycerol in sponge cells, this cDNA was cloned and applied for in situ hybridization studies. The results obtained show that cells surrounding the dinoflagellates become brightly stained after hybridization with the aquaporin this probe. This demonstrates that L. baicalensis cells respond to glycerol, a metabolite which might be supplied by the dinoflagellates and imported via the aquaporin channel into the sponge cells.

Regional and modular expression of morphogenetic factors in the demosponge Lubomirskia baicalensis.

Some sponges [phylum Porifera], e.g. the demosponges Lubomirskia baicalensis or Axinella polypoides, show an arborescent growth form. In the freshwater sponge L. baicalensis this morphotype is seen mostly in depths below 4m while in more shallow regions it grows as a crust. The different growth forms are determined in nature very likely by water current and/or light. The branches of this species are composed of modules, arranged along the apical-basal axis. The modules are delimited by a precise architecture of the spicule bundles; longitudinal bundles originate from the apex of the earlier module, while at the basis of each module these bundles are cross-linked by traverse bundles under formation of annuli. Genes encoding putative morphogenetic factors, myotrophin and epidermal growth factor (EGF)-like molecules, and one gene of an antagonist for the Wnt signaling pathway, the soluble frizzled molecule, have been identified and characterized. Their expression levels as well as those of silicatein, one major spicule-forming molecule, have been studied in the crusts and the modules. The data revealed that at the apices of each module higher level of expression of myotrophin and EGF can be detected, while the base of each module is characterized by a high steady-state expression level of soluble frizzled molecule. These results suggest that module formation in L. baicalensis is controlled by a tuned interaction of agonistic (e.g., myotrophin and EGF) as well as antagonistic morphogenetic factors (e.g., soluble frizzled molecule).

Cold stress defense in the freshwater sponge Lubomirskia baicalensis. Role of okadaic acid produced by symbiotic dinoflagellates.

The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0 degrees C, and in summer in open water at 17 degrees C (September). In March, specimens show high metabolic activity as reflected by the production of gametes. L. baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum. Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state. In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1). Only cDNA corresponding to PP1 could be identified in L. baicalensis and subsequently isolated from a L. baicalensis cDNA library. The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases. As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer). PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition. A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 degrees C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 degrees C. In the enriched extract, PP1 activity at 4 degrees C is close to that measured at 17 degrees C. Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at 4 degrees C. From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge.

Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal demosponge Lubomirskia baicalensis.

Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical-basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of the branches and comparably low at the top, the pro-survival gene follows an opposite gradient. Parallel with the tuned expression of these genes, the activities of the apoptosis-executing enzymes caspase-8 (IETDase activity) and caspase-3 (DEVDase activity) are lowest at the top of the branch and highest at their base. This characteristic expression/activity pattern of the genes/enzymes, which had been determined in a few specimens, collected from an unpolluted, natural site, appears reversed in specimens collected from an anthropogenically polluted site. These findings indicate the involvement of apoptotic proteins in the axis formation (branches) in L. baicalensis.

Magnetic resonance imaging of the siliceous skeleton of the demosponge Lubomirskia baicalensis.

The skeletal elements (spicules) of the demosponge Lubomirskia baicalensis were analyzed; they are composed of amorphous, non-crystalline silica, and contain in a central axial canal the axial filament which consists of the enzyme silicatein. The axial filament, that orients the spicule in its longitudinal axis exists also in the center of the spines which decorate the spicule. During growth of the sponge, new serially arranged modules which are formed from longitudinally arranged spicule bundles are added at the tip of the branches. X-ray analysis revealed that these serial modules are separated from each other by septate zones (annuli). We describe that the longitudinal bundles of spicules of a new module originate from the apex of the earlier module from where they protrude. A cross section through the oscular/apical-basal axis shows that the bundle rays are organized in a concentric and radiate pattern. High resolution magnetic resonance microimaging studies showed that the silica spheres of the spicules in the cone region contain high amounts of 'mobile' water. We conclude that the radiate accretive growth pattern of sponges is initiated in the apical region (cones) by newly growing spicules which are characterized by high amounts of 'mobile' water; subsequently spicule bundles are formed laterally around the cones.

Molecular control of serial module formation along the apical-basal axis in the sponge Lubomirskia baicalensis: silicateins, mannose-binding lectin and mago nashi.

The freshwater sponge Lubomirskia baicalensis (from Lake Baikal) is characterized by a body plan composed of serial modules which are arranged along an apical-basal axis. In shallow water, the sponge occurs only encrusting, while in deeper environment (>3 m), this species forms branches and grows in an arborescent manner. Each module is stabilized by bundles of spined oxeas (amphioxeae spicules). The spicules are surrounded by an organic matrix. cDNAs for structural proteins (silicatein and mannose-binding lectin (MBL)) as well as for one regulatory protein (mago nashi) were isolated from L. baicalensis. Surprisingly the silicatein alpha molecule exists in several, at least four, isoforms (a1 to a4). Expression studies revealed that the steady-state levels of transcripts for the silicateins, the mannose-binding lectin, and mago nashi are highest at the top of the branches, while only very low levels are found in cells at the base. Based on in situ hybridization studies, evidence is presented that the spicule formation (1) starts and is completed inside of the bundles, and (2) occurs together with the mannose-binding lectin from the surfaces of the bundles. The data suggest that the modules are sequentially formed. It is speculated that the expression of the silicateins and the mannose-binding lectin might be (partially) controlled by mago nashi.

Dynamics of skeleton formation in the Lake Baikal sponge Lubomirskia baicalensis. Part II. Molecular biological studies.

In a preceding study it has been reported that the freshwater sponge Lubomirskia baicalensis, living in Lake Baikal (East Siberia), is composed of spicules forming a characteristic pattern which follows radiate accretive growth. Here we report that the spicules are synthesized by the enzyme silicatein, a protein which is related to cathepsin L. The cDNAs for silicatein and the related cathepsin L were isolated and used as probes to show that the mRNA levels of silicatein in the bases of the spicule skeleton of the animals are low, while the mRNA level of cathepsin L in this region exceeds that of the growing zone. This is the first comprehensive study on the importance of the axial filament/silicatein as an essential structural and functional component determining the growth and stability of demosponge spicules.

Dynamics of skeleton formation in the Lake Baikal sponge Lubomirskia baicalensis. Part I. Biological and biochemical studies.

In ancient Lake Baikal (East Siberia), freshwater sponges have diversified to an extraordinary degree. The skeleton of Lubomirskia baicalensis, which attains a size of up to 1 m, is constructed from spicules, which are cemented into longitudinal bundles. Our X-ray analysis revealed that the architecture of the specimens follows a highly ordered radiate accretive growth pattern. The spicules have a central axial canal with an axial filament inside. This organic filament is composed of silicatein, the major enzyme involved in silica formation of the spicules. We found that the specific activity of silicatein in samples from the non-growing (basal) zone is much lower than in those from the growth zone (tips) and that even the composition of this molecule differs in these regions. The present study shows for the first time that the turnover of silicatein, the major element of the axial canal of sponge spicules, changes within a sponge specimen depending on the region in which it is found.