Lubomirskiidae Research Articles

Heat shock protein 70 (Hsp70) response to elevated temperatures in the endemic Baikal sponge Lubomirskia baicalensis

A mass bleaching event affecting an endemic Baikal sponge points out the need to study stress responses in these organisms. To get the first data about Hsp70 content changes in endemic Baikal sponge Lubomirskia baicalensis during exposure to elevated temperatures which can occur in its natural habitat, we investigated the effect of two elevated temperatures on Hsp70 levels in the L. baicalensis and its cell aggregates (primmorphs). After increasing the water temperature from ambient 4 to 9 °C Hsp70 levels increased to protect against temperature stress. Incubation at 13 °C for one month resulted in bleaching and death of the sponges. The decreased level of HSP70 was recorded both in the case of bleaching after exposure to 13 °C, and in diseased individuals from the Lake Baikal. The L. baicalensis primmorph temperature stress response was not consistent with that of the sponge. In primmorphs, the Hsp70 level initially decreased at 4 °C and at 13 °C it increased compared with the levels observed in the sponge. Thus, our results show that primmorphs are not a good model system for the study of expression of stress proteins in sponges. Since the temperatures 9–13 °C were observed at L. baicalensis habitats, climate warming may increase vulnerability of this sponge to bleaching.

Mass disease and mortality of Baikal sponges

In recent years, signifcant changes in the ecological system of the coastal (littoral) zone, including mass death of the endemic representatives of the freshwater sponges of the Lubomirskiidae family, have been an urgent problem of Lake Baikal. Similar problems are known all over the world. Thus, mass disease and death of corals and sponges are indicated in the Mediterranean, Adriatic, Caribbean and other seas, which raises serious concerns about the future of these biocenoses (Olson et al., 2006; Webster, 2007; Wulff et al., 2007; Stabili et al., 2012). In Baikal, diseased sponges were frst found in 2011. The area of sponge disease is constantly expanding, and, to date, dying specimens have been found throughout the lake. The mass death of sponges occur in presence of the large-scale violation of the spatial distribution and the structure of phytocenoses in the littoral zone, but the causes of these phenomena are unknown. The relevance of the problem arises from the fact that changes in the littoral zone of Lake Baikal can signifcantly affect the productivity and composition of planktonic organisms and zoobenthos, which are the food base for fsh, as well as the quality of drinking water. The deterioration of the ecological state also affects the attractiveness of the lake for tourism. At the international level, serious intellectual and fnancial resources were mobilized to solve similar problems. Despite the obvious relevance, in Russia such studies are carried out irregularly by small groups of researchers.

Transformation of Baikal sponges (family Lubomirskiidae) after penetration into the Angara River

Genetic and morphological analysis of endemic sponges of the Lubomirskiidae family from Lake Baikal and the upper reaches of the Angara River was performed. Various sponge species acquired a number of similar morphological traits after the transition from the lake into the river. These traits enabled an increase of sponge skeleton strength under the conditions of elevated hydrodynamic activity. The changes significantly impeded morphology-based species identification of Angara sponges. Phylogenetic analysis of ITS regions and noncoding mitochondrial DNA fragments confirmed that the Angara sponges belonged to the Baikalian Lubomirskiidae family and demonstrated the polyphyletic origin of the sponges. The use of combined molecular and morphological data allowed for the clustering of some sponge samples into groups that corresponded to individual species. The absence of genetic isolation between the Baikalospongia intermedia and Lubomirskia baicalensis species was demonstrated, whereas the B. intermedia profundalis subspecies was well separated from B. intermedia. This finding pointed to the necessity of further studies for the clarification of the taxonomic status of this subspecies.

Molecular identification of filamentous cyanobacteria overgrowing the endemic sponge Lubomirskia baicalensis

In recent years, the surfaces of endemic Baikalian sponges have exhibited signs of damage and disease such as tissue bleaching, atypical microbial films, and necrotic areas. One of the most common sources of damage on branching sponges Lubomirskia baicalensis is a reddish-brown mucous film fully or partially covering the body of the sponge. Sequencing of the 16S rDNA gene revealed that the film on the surfaces of the sponge consists of Oscillatoriales cyanobacteria belonging to the following genera: Tychonema, Phormidium, and Leptolyngbya, with 44 of the 86 identified sequences belonging to Tychonema. Cyanobacterial fouling of the Baikalian sponge may be caused by changes in environmental conditions of the sponge habitat, in particular, temperature increases and eutrophication of the lakes.

Intraspecific and interspecific sequence variability in the ITS region of the rDNA of freshwater sponges of Lake Baikal and East Siberia

Sponge (Porifera) systematics and phylogeny are complicated due to the limited number of morphological features and lack of information employed for taxonomy. Baikal sponges occur in the families Lubomirskiidae and Spongillidae, with 13 and 4 species in each family, respectively. The aim of this research was to study the variability of internal transcribed spacers (ITS) of rDNA in the families Lubomirskiidae and Spongillidae to determine the effectiveness of using ITS sequences to differentiate species and study the evolution of sponges in Lake Baikal. ITS1 and ITS2 (~950 bp) were amplified and sequenced for 34 samples of Lubomirskiidae and 28 samples of Spongillidae. Phylogenetic reconstructions revealed monophyly of Ephydatia fluviatilis, Ephydatia muellery, and Spongilla lacustris. Intraspecific polymorphisms were much lower than interspecific variability among the species of Spongillidae, despite analyzed samples of each species originating from geographically distant locations. Our findings revealed that ITS1 and ITS2 sequences could be an appropriate tool for phylogenetic study and species identification of Spongillidae. ITS analyses did not support monophyly of the lubomirskiid genera and species, possibly because of incomplete species divergence, indicating that species delimitation is difficult in the case of rapid species radiation within Lubomirskiidae.

Phototrophic microorganisms in the symbiotic communities of Baikal sponges: Diversity of psbA gene (encoding D1 protein of photosystem II) sequences

Phototrophic microorganisms in the symbiotic communities of Baikal sponges: Diversity of psbA gene (encoding D1 protein of photosystem II) sequences

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.

Phototrophic microorganisms in the symbiotic communities of Baikal sponges: Diversity of psbA gene (encoding D1 protein of photosystem II) sequences

The psbA gene, which encodes a major photosystem II protein (protein II or D1), is a marker for the presence of phototrophic organisms in water communities. We have pioneered the use of this marker for studying the diversity of phototrophic microflora of freshwater invertebrates. The object of the study is the microbial associations accompanying the endemic Baikal sponge Baikalospongia intermedia and the surrounding aquatic microbial community. Analysis of the psbA gene sequences in the examined microbiomes demonstrates the presence of various phototrophic groups, such as Cyanobacteria, Chlorophyta, Heterokonta, Haptophyta, and Ochrophyta algae, as well as cyanophages. A total of 35 unique psbA gene sequences have been distinguished in the microbial communities of the endemic sponge B. intermedia and 32 unique sequences in the water community surrounding the sponge. These data demonstrate the involvement of sponge symbiotic communities in the accumulation of primary production and carbon cycle in the Lake Baikal ecosystem.

Identifying N-terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins.

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO2 ·nH2 O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N-terminal peptides were not identified. We found the sequences of these N-terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4-bromine-phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N-terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.

Comparison of bacterial diversity and species composition in three endemic Baikalian sponges

Baikalian sponges are unique organisms with many species harboring symbiotic microbes that produce novel bioactive compounds. To investigate bacterial diversity of three species of Baikalian sponges, specimens of Lubomirskia baicalensis , Baikalospongia intermedia and Swartschewskia papyracea collected from Lake Baikal were processed by pyrosequencing. We found differences in the species composition and diversity in bacteria among these sponges. Cyanobacteria accounted for the highest proportion and the second group was Proteobacteria in three sponges. The bacterial communities in B. intermedia and L. baicalensis were highly similar but less similar to the bacterial community associated with S. papyracea. Diversity of the bacterial community in S. papyracea was higher than in L. baicalensis and B. intermedia . In particular, the relative abundance of Actinobacteria was higher in S. papyracea . Bacterial species in phyla Acidobacteria and Gemmatimonadetes were only found in S. papyracea .

The complete mitochondrial genome of Baikalospongia intermedia (Lubomirskiidae): description and phylogenetic analysis

The complete mitochondrial genome of the Lake Baikal sponge Baikalospongia intermedia was sequenced. The circular mitochondrial genome is 28,327 bp in length and includes 14 protein-coding genes, 2 ribosomal RNA genes and 25 transfer RNA genes. Bayesian comparative analysis of molecular evolution rates was found no acceleration of the mtDNA evolution of B. intermedia. This species clustered with other species of the genus Baikalospongia on the Bayesian tree.

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, namely, Verrucomicrobia, Cyanobacteria, Actinobacteria, Bacteroidetes, Proteobacteria, and Planctomycetes. Unlike the microbial associations of other freshwater sponges, the community under study was dominated by the Verrucomicrobia (42.1%) and Cyanobacteria (17.5%) phyla, 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 homologs of which included the polyketide synthases of the microorganisms belonging to the bacterial phyla Cyanobacteria, Proteobacteria (Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria classes), and Acidobacteria and to the eukaryotic algae of the Heterokonta phylum (Eustigmatophyceae class). 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 homologs of the eukaryotic alga Nannochloropsis galiana. Notably, the identified polyketide synthase genes fragments showed only a 57-88% similarity to the sequences in 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 habitation conditions of S. papyracea affect the taxonomic composition of the microorganisms associated with the sponge, including the diversity of the producers of secondary metabolites.

Some aspects in the life cycle of freshwater sponges (Spongillidae, Porifera) from open Lake Baikal

A histological analysis of the tissue structure in different species of Baikal sponges was performed. The material was collected at a depth of 3–4 m at the Berezovyi test site (southern Lake Baikal) in various seasons of 2001–2003. During the entire year, representatives of the family Lubomirskiidae had a well-developed irrigation system with numerous choanocyte chambers. In Trochospongilla sp. (Spongillidae) from open Baikal, such a tissue pattern was observed only during the summer time. Annually, these sponges undergo tissue regression, i.e., a reduction of the irrigation system accompanied by changes in the cell composition in autumn with subsequent recovery in spring. The onset and termination of tissue regression are adapted to the environment of Lake Baikal.

Ultrastructural description of development and cell composition of primmorphs in the endemic Baikal sponge Lubomirskia baicalensis

The morphological description of morphogenesis accompanying primmorph formation from cell aggregates in the endemic Baikal sponge Lubomirskia baicalensis as well as the description of cell composition of these primmorphs was investigated. Development of small sponges from dissociated cells in L. baicalensis is a stepwise process that can be separated into three stages: 1—primary aggregates; 2—early-stage primmorphs; and 3—primmorphs or young sponges. Primary aggregates are irregular in shape and consist of loosely packed cells. Early-stage primmorph development is characterized by cell sorting, aggregate compactization, surface epithelization and cell differentiation. Primmorphs or young sponges have developed elements of the aquiferous system. The main morphogenesis occurring during primmorph development in L. baicalensis is the epithelization of the cell aggregate surface accompanied by the flattening of poorly connected spherical cells. The main sources of new exopinacoderm development during L. baicalensis primmorph formation are choanocytes and amoebocytes that transdifferentiate into exopinacocytes. The cellular composition of L. baicalensis primmorphs does not differ significantly when compared to intact sponges. The most abundant cells in both primmorphs and in intact sponges are amoebocytes and choanocytes. Our results provide a morphological basis for future investigations using primmorphs of this species as models in biotechnology, evo-devo, cytology and molecular biology approaches.

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, operation taxonomic unit), Verrucomicrobia (15.2%; n = 20; 4 OTU), Plactomycetes (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 of freshwater sponge microbiomes are present in the community. However, in contrast to previously studied L. baicalensis bacterial associations, a dominance of Cyanobacteria and a low number of representatives of the Bacteroidetes and Betaproteobacteria were observed in the bleached sponge community. 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.

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.

Genetic diversity of cyanophages of the myoviridae family as a constituent of the associated community of the Baikal sponge Lubomirskia baicalensis

Molecular-genetic analysis of cyanophages of the family Myoviridae from the associated community of the endemic Baikal sponge Lubomirskia baicalensis was carried out based on the g20 gene fragment. A large cyanophage diversity according to the g20 marker gene was found in the sponge. The Baikal sponge cyanophages were shown to be similar to those inhabiting plankton. Moreover, specific cyanophage groups that are significantly different from all of the known groups inhabiting the Lake Baikal were revealed.

Diversity of Bacterial Photosymbionts in Lubomirskiidae Sponges from Lake Baikal

Sponges are permanent benthos residents which establish complex associations with a variety of microorganisms that raise interest in the nature of sponge-symbionts interactions. A molecular approach, based on the identification of the 16S rRNA and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes, was applied to investigate diversity and phylogeny of bacterial phototrophs associated with four species of Lubomirskiidae in Lake Baikal. The phylogeny inferred from both genes showed three main clusters of Synechococcus associated with Baikalian sponges. One of the clusters belonged to the cosmopolitan Synechococcus rubescens group and the two other were not related to any of the assigned phylogenetic groups but placed as sister clusters to S. rubescens. These results expanded the understanding of freshwater sponge-associated photoautotroph diversity and suggested that the three phylogenetic groups of Synechococcus are common photosynthetic symbionts in Lubomirskiidae sponges.

Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia.

One of the fundamental methods for cultivating bacterial strains is conventional plating on solid media, but this method does not reveal the true diversity of the bacterial community. In this study, we develop a new technique and introduce a new device we term, I-tip. The I-tip was developed as an in situ cultivation device that allows microorganisms to enter and natural chemical compounds to diffuse, thereby permitting the microorganisms to grow utilizing chemical compounds in their natural environment. The new method was used to cultivate microorganisms from Baikalian sponges, and the results were compared with conventional plating as well as a pyrosequencing-based molecular survey. The I-tip method produced cultures of 34 species from five major phyla, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, 'missing' only two major phyla detected by pyrosequencing. Meanwhile, standard cultivation produced a smaller collection of 16 species from three major phyla, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, failing to detect over half of the major phyla registered by pyrosequencing. We conclude that the I-tip method can narrow the gap between cultivated and uncultivated species, at least for some of the more challenging microbial communities such as those associated with animal hosts.

Investigation of nuclear and mitochondrial DNA polymorphism in closely related species of endemic Baikal sponges

Investigation of nuclear and mitochondrial DNA polymorphism in closely related species of endemic Baikal sponges