V. B. Itskovich

Heat shock protein Hsp70 expression and DNA damage in Baikalian sponges exposed to model pollutants and wastewater from Baikalsk Pulp and Paper Plant

Lake Baikal, a unique habitat for a great number of endemic species, is the largest freshwater reservoir in the world which is still largely unaffected by anthropogenic pollution, except for some shore regions with industrial activity. The expressions of a biomarker of exposure (heat shock protein Hsp70) and a biomarker of effect (DNA single-strand breaks) were measured for the first time in endemic Baikalian sponge species (Baikalospongia intermedia, Lubomirskia fusifera, and Lubomirskia abietina). Tissue cubes of B. intermedia and dissociated cells of L. fusifera and L. abietina reacted to temperature stress (10-16 degrees C above ambient temperature) with a time-dependent increase in expression of Hsp70. In B. intermedia, the effects of model pollutants (lead, copper, and zinc, and the organochlorines tetrachloroguaiacol, TCG, and pentachlorophenol, PCP) and of the wastewater from the final refinement and aeration reservoirs of the Baikalsk Pulp and Paper Plant (BPPP), located at the shore of the southern basin of Lake Baikal, on the expression of Hsp70 and the extent of DNA damage were investigated. It was found that lead and zinc but not copper cause a strong induction of Hsp70 in this sponge, while the frequency of DNA single-strand breaks increased after exposure to all these heavy metals tested. Induction of DNA single-strand breaks was also observed after exposure to TCG and PCP, but these compounds did not (consistently) enhance Hsp70 expression. Wastewater taken from the final water aeration pond of BPPP caused a concentration-dependent increase in Hsp70 expression in B. intermedia. However, there was no difference in the basal levels of Hsp70 between sponges collected in the shallow water at an unpolluted site near Baikalsk City and at a polluted site where the wastewaters of BPPP are discharged into the lake. There was also no clear difference in the wastewater concentration-dependent induction of Hsp70 expression between sponges collected at these sites, indicating no adaptation to continuous stress exposure.

Ribosomal ITS Sequences Allow Resolution of Freshwater Sponge Phylogeny with Alignments Guided by Secondary Structure Prediction

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatiamuelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.

Monophyletic origin of freshwater sponges in ancient lakes based on partial structures of COXI gene

The phylogenetic relationships between freshwater sponges (families Lubomirskiidae, Spongillidae and Potamolepidae) are still poorly understood. Partial sequences of the cytochrome oxidase subunit I (COXI) gene were obtained from seven freshwater sponge species and one marine sponge. Phylogenetic analyses showed that all the freshwater sponge species formed one common clade and they appeared to be closely related. These results indicate that freshwater sponges are of monophyletic origin. Potamolepidae and Spongillidae are situated at the base of the tree and are assumed to be phylogenetically older than Lubomirskiidae.

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, Proteobacteria (class α-Proteobacteria and β-Proteobacteria) Verrucomicrobia, Bacteroidetes, Cyanobacteria, and Nitrospira) 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.

Conservation and phylogeny of a novel family of non-Hox genes of the Antp class in demospongiae (Porifera)

A survey across the most basal animal phylum, the Porifera, for the presence of homeobox-containing genes led to the isolation of 24 partial or complete homeobox sequences from 21 sponge species distributed in 15 families and 6 orders of Demospongiae. All the new sequences shared a high identity/similarity with EmH-3 (Ephydatia muelleri), a non-Hox gene from the Antp class. The Demox sequences, EmH-3, and related homeodomains formed a well-supported clade with no true affinity with any known bilaterian family, including the Tlx/Hox11 family, suggesting that the EmH-3 family of genes, comprising 31 members, represents a novel family of non-Hox genes, called the Demox family, widespread among Demospongiae. The presence of the Tlx/Hox11 specific signature in the Demox family and common regulatory elements suggested that the Demox and Tlx/Hox11 families are closely related. In the phylogenetic analyses, freshwater Haplosclerida appeared as monophyletic, and Haplosclerida and Halichondrida as polyphyletic, with a clade comprising Agelas species and Axinella corrugata. As for their expression, high levels of Demox transcripts were found in adult tissues. Our data add to the number of published poriferan homeobox sequences and provide independent confirmation of the current Demospongiae phylogenies.

Phylogenetic diversity of microorganisms associated with the deep-water sponge Baikalospongia intermedia

The diversity of bacteria associated with deep-water sponge Baikalospongia intermedia was evaluated by sequence analysis of 16S rRNA genes from two sponge samples collected in Lake Baikal from depths of 550 and 1204 m. A total of 64 operational taxonomic units, belonging to nine bacterial phyla, Proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria), Actinobacteria, Planctomycetes, Cloroflexi, Verrucomicrobia, Acidobacteria, Chlorobi, and Nitrospirae, including candidate phylum WS5, were identified. Phylogenetic analysis showed that the examined communities contained phylotypes exhibiting homology to uncultured bacteria from different lake ecosystems, freshwater sediments, soil and geological formations. Moreover, a number of phylotypes were relative to psychrophilic, methane-oxidizing, sulfate-reducing bacteria, and to microorganisms resistant to the influence of heavy metals. It is noted that the unusual habitation conditions of deep-water sponges contribute to the taxonomic diversity of associated bacteria and have an influence on the presence of functionally important microorganisms in bacterial communities.

Bleaching of Baikalian Sponge Affects The Taxonomic Composition of Symbiotic Microorganisms

The diversity of 16S rRNA genes in the microbial community of endemic sponge Lubomirskia baicalensis with bleached patches of tissue was studied. Eight bacterial phyla were identified in the sponge microbiome: Cyanobacteria (27.3%; n = 36; 2 OTU, operational taxonomic unit), Proteobacteria (22.7%; n = 30; 5 OTU), Actinobacteria (16.7%; n = 22; 7 OTU), Verrucomicrobia (15.2%; n = 20; 4 OTU), Planctomycetes (9%; n = 12; 3 OTU), Bacteroidetes (4.5%; n = 6; 3 OTU), WS5 (3%; n = 4; 1 OTU), and TM7 (1.5%; n = 2; 1 OTU). The basic phyla typical for freshwater sponge microbiomes are present in the community. However, in contrast to previously studied L. baicalensis bacterial associations in the bleached sponge community, a dominance of Cyanobacteria and a low number of representatives of the Bacteroidetes and Betaproteobacteria were observed. Phylotypes exhibiting a high percentage of similarity with the microorganisms inhabiting substrates rich in organic matter were also found. Clearly, the bleaching processes of Baikal sponges affect the composition and the ratio of the major taxonomic groups of sponge-associated bacteria.

Distinctive features of the microbial diversity and the polyketide synthase genes spectrum in the community of the endemic Baikal sponge Swartschewskia papyracea

The diversity of the symbiotic community of the endemic Baikal sponge Swartschewskia papyracea was studied, and an analysis of the polyketide synthases genes spectrum in sponge-associated microorganisms was carried out. Six bacterial phyla were detected in the S. papyracea microbiome: Verrucomicrobia, Cyanobacteria, Actinobacteria, Bacteroidetes, Proteobacteria, and Planctomycetes. Unlike the microbial associations of other freshwater sponges, the community under study was dominated by the phylaVerrucomicrobia (42.1%) and Cyanobacteria (17.5%), while the proportion of the Proteobacteria was unusually low (9.7%). In the S. papyracea community metagenome, there were identified 18 polyketide synthases genes fragments, the closest homologues of which included the polyketide synthases of the microorganisms belonging to the bacterial phyla Cyanobacteria, Proteobacteria (classes Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria), and Acidobacteria as well as the eukaryotic algae of the phylum Heterokonta (class Eustigmatophyceae). Polyketide synthase sequences from S. papyracea formed three groups on the phylogenetic tree: a group of hybrid NRPS/PKS complexes, a group of cyanobacterial polyketide synthases, and a group of homologues of the eukaryotic alga Nannochloropsis gaditana. Notably, the identified polyketide synthase genes fragments showed only a 57–88% similarity to the sequences from the databases, which implies the presence of genes controlling the synthesis of the novel, still unstudied, polyketide compounds in the S. papyracea community. It was proposed that the habitat conditions of S. papyracea affect the taxonomic composition of the microorganisms associated with the sponge, including the diversity of the producers of secondary metabolites.

Diversity of Polyketide Synthase (PKS) genes in metagenomic community of freshwater sponge Lubomirskia baicalensis

The screening of metagenomic DNA of the microbial community associated with the Baikalian sponge Lubomirskia baicalensis was performed in order to investigate the presence of polyketide synthase (PKS) genes. PKS enzyme systems take part in the synthesis of a great number of biologically active substances. The cloning and sequencing of amplified products of the ketosynthase domain section of the PKS gene cluster revealed 15 fragments of PKS genes with amino acid sequences differing from each other by 35–65%. A BLASTX analysis showed that all of these sequences belong to KS domains identified in various groups of microorganisms, i.e., Alpha-, Beta-, and Deltaproteobacteria; Verrucomicrobia; Cyanobacteria; and Chlorophyta. Some sequences were related to genes that participate in the biosynthesis of curacin A (CurI, CurJ), stigmatellin (StiC, StiG), nostophycin (NpnB), and cryptophycin (CrpB). The homology of the found sequences with those of the EMBL database lies in the range of 50–82%, which indicates that the freshwater sponge community contains genes that encode new, not yet studied polyketide substances of potential biotechnological significance.

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 worlds 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.