10:45 AM - 11:00 AM
[BCG05-01] Morphological plasticity and morphogenic process of siliceous skeletons in silicoflagellates (Dictyochophyceae)
★Invited Papers
Keywords:biosilica, cell coverings, morphogenesis, silicate, skeleton
Introduction
Silicoflagellates are unicellular algae characterised by a basket-like external siliceous skeleton. Their skeletons are well preserved in marine sediments, providing a useful fossil record for paleoceanographic reconstructions. Their taxonomic classification relies on skeletal morphology, and many varieties have been described. Our understanding of morphological plasticity of silicoflagellate skeleton is however limited and experimental validation is required. Silicate, a material of siliceous skeletons, is a candidate environmental factor that affects their morphological plasticity. In this study, we examined the effects of silicate concentration on the growth and morphology of cultured strains of the silicoflagellates. In addition, we established an experimental system in which skeleton presence/absence was readily changed by altering silicate concentration in the culture medium. Using this system, we elucidated the skeletogenic process, which has been a mystery for more than 180 years since the first description of silicoflagellates.
Methods
Silicoflagellates Octactis octonaria and Dictyocha fibula cultures were established from in Kagoshima Bay and Ago Bay, Japan, respectively. The experiments were conducted in a batch culture system, and artificial seawater media containing varying concentrations of sodium metasilicate nonahydrate (<0.6–200 µM) was used. Skeletogenesis was detected using the fluorescent dye PDMPO, which labels newly formed silica structures. The detailed morphology of the skeleton was observed via scanning electron microscopy (SEM).
Results & Discussion
We first observed that the specific growth rate μ (d−1) was 0.317±0.036 in silicate-replete medium (10–190 µM) and 0.302±0.006 in silicate-deplete medium (<0.6 µM) during the exponential growth phase (n=3, p>0.05). This suggests that silicate depletion does not affect the growth of silicoflagellates. However, skeletal morphology of silicoflagellates changed with the silicate concentrations. For example, the thickness of the basal spine was 1.6±0.2 µm in silicate-replete medium (186–190 µM) and 1.2±0.2 µm in silicate-deplete medium (0.7–2.3 µM); the thickness in silicate-deplete medium was 25% thinner (n=30, p<0.05). These results indicate that some varieties, which were classified on the basis of skeletal thickness, reflect the silicate concentration in the environment. Most notably, upon silicate depletion, both strains grew with no skeletal structure. This loss of skeleton was reversible: when skeleton-less cells were transferred to a silicate-replete medium, skeleton regeneration occurred.
Morphological analyses during skeletal regeneration were further performed using D. fibula. The use of skeleton-less cells allowed the identification of initial skeletal structures. In the early incubation times, spherical structures and their development were observed. Elemental analysis by SEM coupled with energy dispersive X-ray spectrometry revealed that these structures were composed of silicon and oxygen indicating that they are siliceous skeletal precursors. The proportion of each structure was analysed over time. After 1 h incubation with silicate-replete medium, almost half of the cells developed the PDMPO-stained structure, in which almost all were the single spherical structure (diameter: 5–10 µm). After 18 h, the number of spherical structures decreased to less than half, while the developing and completed structures increased to one-fifth and a quarter of the total, respectively. Given these results, skeletogenesis begins with the formation of spherical silica structures that further undergo morphogenesis and develop into a final skeleton. This morphogenetic development is unprecedented in that polymerized silica structures show a drastic alteration in morphology during morphogenesis. Hence the experimental system of silicoflagellates is useful for exploring novel mechanisms and biomolecules involved in the biosilica morphogenesis.
Silicoflagellates are unicellular algae characterised by a basket-like external siliceous skeleton. Their skeletons are well preserved in marine sediments, providing a useful fossil record for paleoceanographic reconstructions. Their taxonomic classification relies on skeletal morphology, and many varieties have been described. Our understanding of morphological plasticity of silicoflagellate skeleton is however limited and experimental validation is required. Silicate, a material of siliceous skeletons, is a candidate environmental factor that affects their morphological plasticity. In this study, we examined the effects of silicate concentration on the growth and morphology of cultured strains of the silicoflagellates. In addition, we established an experimental system in which skeleton presence/absence was readily changed by altering silicate concentration in the culture medium. Using this system, we elucidated the skeletogenic process, which has been a mystery for more than 180 years since the first description of silicoflagellates.
Methods
Silicoflagellates Octactis octonaria and Dictyocha fibula cultures were established from in Kagoshima Bay and Ago Bay, Japan, respectively. The experiments were conducted in a batch culture system, and artificial seawater media containing varying concentrations of sodium metasilicate nonahydrate (<0.6–200 µM) was used. Skeletogenesis was detected using the fluorescent dye PDMPO, which labels newly formed silica structures. The detailed morphology of the skeleton was observed via scanning electron microscopy (SEM).
Results & Discussion
We first observed that the specific growth rate μ (d−1) was 0.317±0.036 in silicate-replete medium (10–190 µM) and 0.302±0.006 in silicate-deplete medium (<0.6 µM) during the exponential growth phase (n=3, p>0.05). This suggests that silicate depletion does not affect the growth of silicoflagellates. However, skeletal morphology of silicoflagellates changed with the silicate concentrations. For example, the thickness of the basal spine was 1.6±0.2 µm in silicate-replete medium (186–190 µM) and 1.2±0.2 µm in silicate-deplete medium (0.7–2.3 µM); the thickness in silicate-deplete medium was 25% thinner (n=30, p<0.05). These results indicate that some varieties, which were classified on the basis of skeletal thickness, reflect the silicate concentration in the environment. Most notably, upon silicate depletion, both strains grew with no skeletal structure. This loss of skeleton was reversible: when skeleton-less cells were transferred to a silicate-replete medium, skeleton regeneration occurred.
Morphological analyses during skeletal regeneration were further performed using D. fibula. The use of skeleton-less cells allowed the identification of initial skeletal structures. In the early incubation times, spherical structures and their development were observed. Elemental analysis by SEM coupled with energy dispersive X-ray spectrometry revealed that these structures were composed of silicon and oxygen indicating that they are siliceous skeletal precursors. The proportion of each structure was analysed over time. After 1 h incubation with silicate-replete medium, almost half of the cells developed the PDMPO-stained structure, in which almost all were the single spherical structure (diameter: 5–10 µm). After 18 h, the number of spherical structures decreased to less than half, while the developing and completed structures increased to one-fifth and a quarter of the total, respectively. Given these results, skeletogenesis begins with the formation of spherical silica structures that further undergo morphogenesis and develop into a final skeleton. This morphogenetic development is unprecedented in that polymerized silica structures show a drastic alteration in morphology during morphogenesis. Hence the experimental system of silicoflagellates is useful for exploring novel mechanisms and biomolecules involved in the biosilica morphogenesis.