ry: C ist urr m en e t h R C Chernogor et al. Organic Chem Current Res 2011, S:2 e c s i e n a DOI: 10.4172/2161-0401.S2-001 a r c g r h O Organic Chemistry ISSN: 2161-0401 Current Research ResearchResearch Article Article OpenOpen Access Access Formation of Spicules During the Long-term Cultivation of Primmorphs from the Freshwater Baikal Sponge Lubomirskia baikalensis L.I. Chernogor1*, N.N. Denikina1, S.I. Belikov1 and A.V. Ereskovsky2,3 1Limnological Institute of the Siberian Branch of the Russian Academy of Sciences, Ulan-Batorskaya 3, Irkutsk 664033, Russia 2Department of Embryology, Faculty of Biology and Soils, Saint-Petersburg State University, Universitetskaja nab. 7/9, St. Petersburg 199034, Russia 3Centre d’Océanologie de Marseille, Station marine d’Endoume - CNRS UMR 6540-DIMAR, rue de la Batterie des Lions, 13007 Marseille, France Abstract Sponges (phylum Porifera) are phylogenetically ancient Metazoa that use silicon to form their skeletons. The process of biomineralization in sponges is one of the important problems being examined in the field of research focused on sponge biology. Primmorph cell culture is a convenient model for studying spiculogenesis. The aim of the present work was to produce a long-term primmorph culture from the freshwater Baikal sponge Lubomirskia baikalensis (class Demospongiae, order Haplosclerida and family Lubomirskiidae) in both natural Baikal water and artificial Baikal water to study the influence of silicate concentration on formation and growth of spicules in primmorphs. Silicate concentration plays an important role in formation and growth of spicules, as well as overabundance of silica leads to destruction of cell culture primmorphs. We also found that the composition of chemical elements (Si, O, C, and Na) varied along the length of growing spicules at cultivation in different media. The long-term culture of Baikal sponge primmorphs will be necessary for further investigations, and this system may serve as a powerful in vitro model to study spiculogenesis in Baikal siliceous sponges during the early stages of intracellular spicule formation to identify genes that affect biomineralization. Keywords: Primmorphs, Spiculogenesis, Silica spicules, situated in South-Eastern Siberia (51–56° N, 104–110° E), the world’s Microanalysis, Baikal sponges largest (23,000 square kilometres), deepest (1,643 m), and oldest (> 24 million years [MiY]) freshwater body [23]. Sponges dominate the Introduction littoral zone of Lake Baikal, covering 47% of the available surfaces [24]. Sponges (phylum Porifera) are the phylogenetically oldest Metazoa. A percentage cover of 47% for sponges is unusual for a freshwater Demosponges recorded before the end the Marinoan glaciation ecosystem and is difficult to compare with reported sponge biomass (635Myr ago) exist at present [1]. Processes of biomineralization and and occurrence for freshwater ecosystems [25,26]. Habitats of Baikal biosilification in sponges are the subject of active research and debate. sponges differ considerably from those of other freshwater sponges Three sponge clades (class Demospongiae, order Hexactinellida) because of hydrological and hydrochemical peculiarities of Lake Baikal: produce silica skeletons [2]. Their skeletons consist of spicules that great depths, long ice period, low water temperature (10–12°С) in the form species-specific shapes. Their mineral components consist of upper layers in summer, high oxygen content, and low concentration silicon dioxide, silicon in amorphous opal-A and the bonded protein of organic matter [27,28]. Moreover, Lake Baikal contains relatively spongin (an analogue of collagen) [3,4]. The secretion of silica spicules low levels of nutrients, but the nutrients are stably balanced relative in sponges is an intracellular process [3,5]. Many silica spicule-forming to the chemical composition ratios in phytoplankton [29]. Another sponges have been characterized as containing and expressing silicatein peculiar characteristic of Lake Baikal is relatively high level of dissolved genes [6-9]. The exact mechanism(s) of the action of silicatein remain silicic acid. It is known, that the silicon concentration in Lake Baikal undetermined, but the investigation of this compound is considered is 100 μМ [27], whereas in marine coastal areas the mean silicon to be particularly important for the use of silica-based materials in concentration at the surface is less than 3 μМ [30] gradually increasing vitro [10]. Formation of spicules is a genetically controlled process and with depth [31]. The high content of silicic acid in Lake Baikal is caused highly relevant to nanobiotechnology [6,11-13]. by heavy influx of silicon from the rivers [32]. It is very difficult to investigate the mechanism of spicule formation The main aim of this work was to study how the concentration of (spiculogenesis) at the cellular and molecular levels in adult sponges silicate influenced the formation and growth of spicules in primmorphs because spicules contain a wide variety of proteins that are involved in metabolism of silica and formation of spicules (biogenic silica). To avoid this complexity, the use of sponge cell cultures (primmorphs) *Corresponding author: Lubov I. Chernogor, Limnological Institute of the Siberian has been suggested [14-16]. Primmorphs are 3D cell aggregates from Branch of Russian Academy of Sciences, 3 Ulan-Batorskaya Street, Irkutsk 664033, Russia, Tel: +7-395-2511874; Fax: +7-395-2425405; E-mail: lchernogor@ sponge cells being a suitable model for studying spiculogenesis [8,17- mail.ru 21]. They are formed by dissociation of sponge cells and subsequent growth of aggregates. Previously, a primmorph system from Suberites Received September 22, 2011; Accepted October 18, 2011; Published October 20, 2011 domuncula was used to demonstrate that silicatein (a biosilica- synthesising enzyme) and silicase (a catabolic enzyme) are colocalized Citation: Chernogor LI, Denikina NN, Belikov SI, Ereskovsky AV (2011) Formation of Spicules During the Long-term Cultivation of Primmorphs from the Freshwater at the surfaces of growing spicules and in axial filaments 22[ ]. Baikal Sponge Lubomirskia baikalensis. Organic Chem Current Res S2:001. doi:10.4172/2161-0401.S2-001 The endemic freshwater Baikal sponge Lubomirskia baikalensis (Pallas 1776) was the subject of our investigation. These sponges are Copyright: © 2011 Chernogor LI, et al. This is an open-access article distributed a good model for applied studies because L. baikalensis is the only under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the Baikal sponge species that can easily be identified. Lake Baikal is original author and source are credited. Organic Chem Current Res Bioorganic Chemistry ISSN:2161-0401 OCCR an open access journal Citation: Chernogor LI, Denikina NN, Belikov SI, Ereskovsky AV (2011) Formation of Spicules During the Long-term Cultivation of Primmorphs from the Freshwater Baikal Sponge Lubomirskia baikalensis. Organic Chem Current Res S2:001. doi:10.4172/2161-0401.S2-001 Page 2 of 9 from the freshwater sponge L. baikalensis (as model system) in both day during the first week, then once a week, replacing only 50% of natural Baikal water (NBW) and artificial Baikal water (ABW). The the medium. We tested the influence of silicate (Na2SiO4×7H2O) on L. baikalensis primmorphs were cultivated in a previous study in primmorph development and spicule growth by varying the silicate both NBW and ABW for >10 months [33]. The prior investigations concentration between 70 and 120 μМ, pH was adjusted to 7.9. The have indicated that neither 1.0% or 1.5% foetal bovine serum (FBS) primmorphs were cultured in ABW without silicate. As a control, we (HyClone Laboratories, Inc.) nor classical Dulbecco’s modified Eagle’s cultivated primmorphs in vitro in NBW. Silicate concentrations in the medium (DMEM; Sigma) supplemented with antibiotics is necessary wells permanently were controlled by spectrophotometric analysis for the cultivation or growth of Baikal sponge primmorphs, as during the experiments. The protocol was adapted from Strickland & symbiotic green algae in cells of L. baikalensis and specific symbiotic Parsons [40]. Subsequently the extinction of standards and samples microbial flora provide the freshwater sponge with necessary nutrition was measured with a spectrophotometer (Spectronic-20 Genesys, [34,35]. Moreover, a distinctive feature of Baikal sponges compared Spectronic Instruments, Rochester, USA) at a wavelength of 810 nm. A to other freshwater sponges is their ability to live symbiotically with calibration curve was made from 0 to 100 μM. various zoochlorellae and dinoflagellates [36,37]. Primmorphs are likely to grow due to photosynthetic products of their symbionts. Spicule preparation and imaging Materials and Methods In our study, spicules totalling more than 1050 were measured in cell culture primmorphs L. baikalensis. Spicules were prepared on a Sponge sampling single glass slide for each sample cultured either in ABW in the absence L. baikalensis (class Demospongiae, order Haplosclerida and family of silicate, ABW in the presence of 70 μМ silicate and in NBW. A scalpel Lubomirskiidae) (Figure 1) has a branched shape and an encrusting was used to cut 1-mm-thick sections that ran perpendicular to the base with erect (30–60 cm, up to 1 m high) dichotomous branches surfaces of the specimens, which had been stored in 96% ethanol. Several and rounded