3-D Culture of Marine Sponge Cells for Production of Bioactive Compounds

Production of sponge-derived bioactive compounds in vitro has been proposed as an alternative to wild harvest, aquaculture, and chemical synthesis to meet the demands of clinical drug development and manufacture. Until recently, this was not possible because there were no marine invertebrate cell lines. Recent breakthroughs in the development of sponge cell lines and rapid cell division in improved nutrient media now make this approach a viable option. We hypothesized that three-dimensional (3-D) cell cultures would better represent how sponges function in nature, including the production of bioactive compounds. We successfully cultured sponge cells in 3-D matrices using FibraCel® disks, thin hydrogel layers, and gel microdroplets (GMDs). For in vitro production of bioactive compounds, the use of GMDs is recommended. Nutrients and sponge products rapidly diffuse into and out of the 3-D matrix, the GMDs may be scaled up in spinner flasks, and cells and/or secreted products can be easily recovered. Research on scale-up and production is in progress in our laboratory.

CRISPR/Cas12a-Mediated Gene Editing in Geodia barretti Sponge Cell Culture

Sponges and their associated microorganisms are the most prolific source of marine natural products, and many attempts have been made at creating a marine sponge cell line to produce these products efficiently. However, limited knowledge on the nutrients sponge cells require to grow and poor genetic accessibility have hampered progress toward this goal. Recently, a new sponge-specific nutrient medium M1 has been shown to stimulate sponge cells in vitro to divide rapidly. In this study, we demonstrate for the first time that sponge cells growing in M1 can be genetically modified using a CRISPR/Cas12a gene editing system. A short sequence of scrambled DNA was inserted using a single-stranded oligodeoxynucleotide donor template to disrupt the 2′,5′-oligoadenylate synthetase gene of cells from the boreal deep-sea sponge Geodia barretti. A blue fluorescent marker gene appeared to be inserted in an intron of the same gene and expressed by a small number of G. barretti cells. Our results represent an important step toward developing an optimized continuous sponge cell line to produce bioactive compounds.

The buds of Oscarella lobularis (Porifera): a new convenient model for sponge cell and developmental biology

The comparative study of the four non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, Porifera) should provide insights into the origin of bilaterian traits. Except for Cnidaria, present knowledge on the cell biology and development of these animals is so far limited. Non-bilaterian models are needed to get further into cell architecture and molecular mechanisms.Given the developmental, histological, ecological and genomic differences between the four sponge classes, we develop a new sponge model: the buds of the Oscarella lobularis (class Homoscleromorpha). This experimental model supplements the two other most famous sponge models Amphimedon queenslandica and Ephydatia muelleri, both belonging to the class Demospongiae.Budding is a natural and spontaneous asexual reproduction mean, but budding can be triggered in vitro ensuring availability of biological material all year long. We provide a full description of buds, from their formation to their development into juveniles. Their transparency enables fluorescent and live imaging, and their abundance allows for experimental replicates. Moreover, regeneration and cell reaggregation capabilities provide interesting experimental morphogenetic contexts. The numerous techniques now mastered on these buds make it a new suitable sponge model.Summary statementStudying sponge biology is needed to understand the evolution of metazoans. We developed a new model suitable for experimental biology that allows to study morphogenetic processes with modern tools.

Breakthrough in Marine Invertebrate Cell Culture: Sponge Cells Divide Rapidly in Improved Nutrient Medium

Sponges (Phylum Porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases.

The Contribution of Calcium to Changes in Leaf Anatomical Character of Oil Palm Seedlings (Elaeis guineensis Jacq.) under Drought Stress

The research was purposed (1) to know the effects of drought stress on changes in leaf anatomical character of oil palm seedlings (2) to know the contribution of calcium in cell compactness and increase the structural strength of leaf tissue so that oil palm seedlings were more tolerant to drought stress. This experiment was laid out following a split plot design with three blocks as replication. Main plot consists of drought stress levels, that are field capacity (FTSW 1.00), moderate drought stress (FTSW 0.35) and severe drought stress (FTSW 0.15). The fraction of transpirable soil water (FTSW) is a method for evaluating gradually increasing drought stress based on the amount of water loss due to transpiration. Meanwhile, subplot consists of four doses of calcium (Ca), that are of 0.0 g/seedlings, 0.04 g/seedlings, 0.08 g/seedlings, and 0.12 g/seedlings. Calcium fertilizer used is calcium sulfate (CaSO4) pure analysis. Leaf anatomical character was observed including the epidermal length and epidermal width; hypodermal length and hypodermal width; palisade cell length and palisade cell width; sponge cell length and sponge cell width; mesophyll tissue thickness; xylem and phloem diameter. The results showed that moderate and severe drought stress reduced epidermal cell length, upper hypodermal cell width, mesophyll thickness, palisade width and phloem diameter of leaf vessels. The applications of calcium to the leaf of oil palm seedlings under drought stresses were able to increased in the sponge cell length at a Ca dosage of 0.04 g/seedlings; increased lower hypodermal width and diameter phloem at a Ca dosage of 0.04 g/seedlings; and increased diameter xylem of leaves vessel at a Ca dosage of 0.12 g/seedlings.

Pluripotency and the origin of animal multicellularity

The most widely held, but rarely tested, hypothesis for the origin of animals is that they evolved from a unicellular ancestor with an apical cilium surrounded by a microvillar collar that structurally resembled present-day sponge choanocytes and choanoflagellates1–4. Here we test this traditional view of the origin of the animal kingdom by comparing the transcriptomes, fates and behaviours of the three primary sponge cell types – choanocytes, pluripotent mesenchymal archeocytes and epithelial pinacocytes – with choanoflagellates and other unicellular holozoans. Unexpectedly, we find the transcriptome of sponge choanocytes is the least similar to the transcriptomes of choanoflagellates and is significantly enriched in genes unique to either animals or to sponges alone. In contrast, pluripotent archeocytes upregulate genes controlling cell proliferation and gene expression, as in other metazoan stem cells and in the proliferating stages of two closely-related unicellular holozoans, including a colonial choanoflagellate. In the context of the body plan of the sponge, Amphimedon queenslandica, we show that choanocytes appear late in development and are the result of a transdifferentiation event. They exist in a metastable state and readily transdifferentiate into archeocytes, which can differentiate into a range of other cell types. These sponge cell type conversions are similar to the temporal cell state changes that occur in many unicellular holozoans5. Together, these analyses offer no support for the homology of sponge choanocytes and choanoflagellates, nor for the view that the first multicellular animals were simple balls of cells with limited capacity to differentiate. Instead, our results are consistent with the first animal cell being able to transition between multiple states in a manner similar to modern transdifferentiating and stem cells.

MORPHOLOGICAL AND PHENOTYPE ANALYSIS OF MICROSYMBIONT AND BIOMASS MARINE SPONGE FROM MELAWAI BEACH, BALIKPAPAN, EAST KALIMANTAN

Determination biomass and phenotypic analysis of microsymbionts sponge is a comprehensive effort to discover the specificity of the sponge, not only on the identification and characterization studies that have been growing. Research directed at diversification of knowledge of the functions and benefits of a sponge for the life and welfare of mankind. The purpose of this research is the analysis of biomass morphology and phenotype test microsymbionts sponge. Histomorfologi analysis method to determine the type, components and composition biomass. Isolates obtained by sponge microsymbiont isolation- purification followed by phenotypic analysis through Gram staining and biochemical tests. Histomorfologi analysis results obtained sponge species is Callyspongia sp. Components and composition consists of sponge biomass fraction skeleton (spicules and cell debris) reached 69.8 %, 18.8 % sponge cell fraction and 11.3 % bacterial pellet fraction. The results of the isolation-purification microsymbiont obtained two isolates. Staining test results both isolates are Gram-positive bacteria and biochemical tests is Bacillus subtilis isolates the spherical shape large size, beige and white, while the isolates two clustered colonies are bacillus flexus jagged shape elongated, white-purple color and a separate colony