Freshwater Sponge Lubomirskia Baicalensis Research Articles

Patterns of distribution of polycyclic aromatic hydrocarbons in the system water–hydrobionts system of the lake Baikal

The results of model experiments designed to study the distribution patterns for polycyclic aromatic hydrocarbons (PAHs) of various structures and solubility (naphthalene, anthracene, pyrene, benz(a)pyrene) in the water system of the Lake Baikal (endemic species: freshwater sponge Lubomirskia baicalensis and haric algae Nitella sp.) are presented. The ability of hydrobionts to remove PAHs from aqueous solutions at the highest rate for the first 2-6 hours and with an efficiency of up to 70-95% of the initial content, depending on the mass and type of organism, exposure time, hydrophobicity (solubility) of the compound was revealed. The ability of hydrobionts to accumulate and convert PAHs was, which indicates the active participation of aquatic organisms in the processes of water self-purification from toxic and carcinogenic compounds. The ability to use hydrobionts as bioindicators for assessing the state of the aquatic environment of Lake Baikal was also demonstrated.

16S rRNA Gene Amplicon Sequencing Data of Bacterial Community of Freshwater Sponge Lubomirskia baicalensis.

ABSTRACTOur study was devoted to investigating the mass disease and mortality of freshwater sponges (Lubomirskiidae) in Lake Baikal. The first sights of the disease were discovered in 2011 and were associated with a shift in sponge microbial diversity. To study the microbiome, we performed sequencing of the 16S rRNA amplicon DNA extracted from the freshwater sponges.

Genome Analysis of the Janthinobacterium sp. Strain SLB01 from the Diseased Sponge of the Lubomirskia baicalensis.

The strain Janthinobacterium sp. SLB01 was isolated from the diseased freshwater sponge Lubomirskia baicalensis (Pallas, 1776) and the draft genome was published previously. The aim of this work is to analyze the genome of the Janthinobacterium sp. SLB01 to search for pathogenicity factors for Baikal sponges. We performed genomic analysis to determine virulence factors, comparing the genome of the strain SLB01 with genomes of other related J. lividum strains from the environment. The strain Janthinobacterium sp. SLB01 contained genes encoding violacein, alpha-amylases, phospholipases, chitinases, collagenases, hemolysin, and a type VI secretion system. In addition, the presence of conservative clusters of genes for the biosynthesis of secondary metabolites of tropodithietic acid and marinocine was found. We present genes for antibiotic resistance, including five genes encoding various lactamases and eight genes for penicillin-binding proteins, which are conserved in all analyzed strains. Major differences were found between the Janthinobacterium sp. SLB01 and J. lividum strains in the spectra of genes for glycosyltransferases and glycoside hydrolases, serine hydrolases, and trypsin-like peptidase, as well as some TonB-dependent siderophore receptors. Thus, the study of the analysis of the genome of the strain SLB01 allows us to conclude that the strain may be one of the pathogens of freshwater sponges.

Spiculogenesis in the siliceous sponge Lubomirskia baicalensis studied with fluorescent staining.

Siliceous sponges are the most primitive multicellular animals whose skeleton consists of spicules - needle-like constructions from silicon dioxide surrounding organic axial filaments. Mechanisms of spicule formation have been intensively studied due to the high ecological importance of sponges and their interest to materials science. Light and electron microscopy are not appropriate enough to display the process from silicon-enriched cells to mature spicules because of composite structure of the sponge tissues. In this article, spiculogenesis in the siliceous sponge has been studied for the first time with the use of fluorescent microscopy. Fluorescent vital dye NBD-N2 was applied to stain growing siliceous structures in the sponge and primmorph cell system. The main stages of spicule growth in the fresh-water sponge Lubomirskia baicalensis (Pallas, 1773) were visualized: silicon accumulation in sclerocytes; formation of an organic filament protruding from the cell; further elongation of the filament and growth of the spicule in a spindle-like form with enlargement in the center; merger with new sclerocytes and formation of the mature spicule. Fluorescent microscopy combined with SEM allows us to overcome the virtual differentiation between intra- and extracellular mechanisms of spicule growth. The growing spicule can capture silicic acid from the extracellular space and merge with new silicon-enriched cells. Visualization of the growing spicules with the fluorescent dye allows us to monitor sponge viability in ecological or toxicological experiments and to apply genomic, proteomic and biochemical techniques.

Diversity of 16S rRNA genes in metagenomic community of the freshwater sponge Lubomirskia baicalensis

Phylogenetic analysis of the nucleotide sequences of 16S rRNA genes in the metagenomic community of Lubomirskia baicalensis has revealed taxonomic diversity of bacteria associated with the endemic freshwater sponge. Fifty-four operational taxonomic units (OTUs) belonging to six bacterial phyla (Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Verrucomicrobia, Bacteroidetes, Cyanobacteria, and Nitrospiracea) have been identified. Actinobacteria, whose representatives are known as antibiotic producers, is the dominant phylum of the community (37%, 20 OTUs). All sequences detected shared the maximal homology with unculturable microorganisms from freshwater habitats. The wide diversity of bacteria closely coexisting with the Baikal sponge indicate the complex ecological relationships in the community formed under the unique conditions of Lake Baikal.

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.

Identification of silicateins in freshwater sponge Lubomirskia baicalensis

Siliceous spicules of Baikal freshwater sponge Lubomirskia baicalensis contain several proteins, including silicateins. Analysis of a L. baicalensis cDNA library revealed four different mRNAs coding for pro- teins related to marine sponge silicatein α ( α 1, α 2, α 3, and α 4). The intron-exon structure was determined for the genomic α 1 silicatein gene. The gene is 1988 bp from the initiation to the termination codon and consists of six intron (total size 1007 bp) and seven exons (total size 981 bp). Mass spectrometry of a tryptic digest of spicule proteins revealed peptides of two silicateins α . DOI: 10.1134/S002689330704005X

Silicateins, the major biosilica forming enzymes present in demosponges: Protein analysis and phylogenetic relationship

Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with p I's of 5.4, of 5.2, of 4.9 and of 4.7. Surprisingly silicatein not only mediates polymerization of silicate, but also displays proteolytic activity which is specific for cathepsin L enzymes, thus underscoring the high relationship of the silicateins to cathepsin L. The cDNAs from L. baicalensis for silicatein and cathepsin L, as well as the respective genes, were cloned. It was found that the five introns present in the sponge genes are highly conserved up to human cathepsin L. This analysis has been completed by sequencing of two silicatein genes (both for silicatein-α and -β) and of cathepsin L from another demosponge, Suberites domuncula. A comprehensive phylogenetic analysis with these new sequences shed new light upon the evolution of cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsin L.

Cold stress defense in the freshwater sponge Lubomirskia baicalensis

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.

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.

Isolation and Characteristic of Overlong-Chain Unsaturated Aldehydes from the Freshwater Sponge Lubomirskia baicalensis

Several unusual overlong-chain unsaturated aldehydes (22:1, 22:2, 23:1, 24:1, 24:2, and 25:2) were found in total lipids of the endemic sponge Lubomirskia baicalensis from Baikal Lake. Tetracos-17-enal was identified as the major aldehyde of the mixture using GC-MS and 1H and 13C NMR spectroscopy. A procedure for the isolation of total overlong-chain aldehydes was suggested. We think that the overlong-chain aldehydes defend the sponge from fouling and predators. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

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.

Comparative study of the endemic freshwater fauna of Lake Baikal—VI. Unusual fatty acid and lipid composition of the endemic sponge Lubomirskia baicalensis and its amphipod crustacean parasite Brandtia (Spinacanthus) parasitica

Lipids and phospholipids (both plasmalogen and alkyl forms) of the freshwater sponge Lubomirskia baicalensis and the sponge's gammarid parasite Brandtia (Spinacanthus) parasitica were examined. Composition of alkenyl-acyl (plasmalogen), alkylacyl and diacyl forms of major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine were determined. One hundred and eighty-three fatty acids were identified by GC-MS: 46 saturated, 55 monoenoic, 35 dienoic, 25 trienoic and 22 tetra-, penta- and hexaenoic. The freshwater sponges, belonging to the family Lubomirskiidae, were shown to contain unusual long-chain fatty acids: anteiso-5, 9–28:2, branched-5, 9–29:2, 5,9,23–29:3, 5,9,23–30:3, 15,18,21,24–30:4 and 15,18,21,24,27–30:5. Some from these fatty acids were found in lipids of the amphipod parasite.

Natural products from the Lake Baikal organisms—II. Sterols from the sponge Lubomirskia baicalensis

1. 1. The free sterol fraction of the Baikalian freshwater sponge Lubomirskia baicalensis was isolated, separated on AgNO 3 impregnated silica gel, and studied by GLC-MS and NMR analyses. 2. 2. It was shown that the sterol fraction contains at least 16 components belonging to the Δ 5- series ; 14 of them were identified. 25(26)-Unsaturated sterols, 24-norcholesta-5,22-dien-3β-ol and rare sterol baicalosterol (24-ethyl-26-norcholesta-5,22E,25-trien-3β-ol) were found as minor components of L. baicalensis.