Structural evidence for extracellular silica formation by diatoms.

The silica cell wall of diatoms, a widespread group of unicellular microalgae, is an exquisite example for the ability of organisms to finely sculpt minerals under strict biological control. The prevailing paradigm for diatom silicification is that this is invariably an intracellular process, occurring inside specialized silica deposition vesicles that are responsible for silica precipitation and morphogenesis. Here, we study the formation of long silicified extensions that characterize many diatom species. We use cryo-electron tomography to image silica formation in situ, in 3D, and at a nanometer-scale resolution. Remarkably, our data suggest that, contradictory to the ruling paradigm, these intricate structures form outside the cytoplasm. In addition, the formation of these silica extensions is halted at low silicon concentrations that still support the formation of other cell wall elements, further alluding to a different silicification mechanism. The identification of this unconventional strategy expands the suite of mechanisms that diatoms use for silicification.

Sponge-associated bacteria mineralize arsenic and barium on intracellular vesicles.

Arsenic and barium are ubiquitous environmental toxins that accumulate in higher trophic-level organisms. Whereas metazoans have detoxifying organs to cope with toxic metals, sponges lack organs but harbour a symbiotic microbiome performing various functions. Here we examine the potential roles of microorganisms in arsenic and barium cycles in the sponge Theonella swinhoei, known to accumulate high levels of these metals. We show that a single sponge symbiotic bacterium, Entotheonella sp., constitutes the arsenic- and barium-accumulating entity within the host. These bacteria mineralize both arsenic and barium on intracellular vesicles. Our results indicate that Entotheonella sp. may act as a detoxifying organ for its host.

Culturable associated-bacteria of the sponge Theonella swinhoei show tolerance to high arsenic concentrations.

Sponges are potent filter feeders and as such are exposed to high fluxes of toxic trace elements, which can accumulate in their body over time. Such is the case of the Red Sea sponge Theonella swinhoei, which has been shown to accumulate up to 8500 mg/Kg of the highly toxicelement arsenic. T. swinhoei is known to harbor a multitude of sponge-associated bacteria, so it is hypothesized that the associated-bacteria will be tolerant to high arsenic concentration. This study also investigates the fate of the arsenic accumulated in the sponge to test if the associated-bacteria have an important role in the arsenic accumulation process of their host, since bacteria are key players in the natural arsenic cycle. Separation of the sponge to sponge cells and bacteria enriched fractions showed that arsenic is accumulated by the bacteria. Sponge-associated, arsenic-tolerant bacteria were cultured in the presence of 5 mM of either arsenate or arsenite (equivalent to 6150 mg/Kg arsenic, dry weight). The 54 isolated bacteria were grouped to 15 operational taxonomic units (OTUs) and isolates belonging to 12 OTUs were assessed for tolerance to arsenate at increased concentrations up to 100 mM. Eight of the 12 OTUs tolerated an order of magnitude increase in the concentration of arsenate, and some exhibited external biomineralization of arsenic-magnesium salts. The biomineralization of this unique mineral was directly observed in bacteria for the first time. These results may provide an explanation for the ability of the sponge to accumulate considerable amounts of arsenic. Furthermore arsenic-mineralizing bacteria can potentially be used for the study of bioremediation, as arsenic toxicity affects millions of people worldwide.

The elemental composition of demospongiae from the Red Sea, Gulf of Aqaba.

Trace elements are vital for the growth and development of all organisms. Little is known about the elemental content and trace metal biology of Red Sea demosponges. This study establishes an initial database of sponge elemental content. It provides the necessary foundation for further research of the mechanisms used by sponges to regulate the uptake, accumulation, and storage of metals. The metal content of 16 common sponge species was determined using ICP measurements. A combination of statistical methods was used to determine the correlations between the metals and detect species with significantly high or low concentrations of these metals. Bioaccumulation factors were calculated to compare sponge metal content to local sediment. Theonella swinhoei contained an extremely high concentration of arsenic and barium, much higher (at least 200 times) than all other species and local sediment. Hyrtios erecta had significantly higher concentration of Al, Cr, Fe, Mn, Ti and V than all other species. This is due to sediment accumulation and inclusion in the skeleton fibers of this sponge species. Suberites clavatus was found to contain significantly higher concentration of Cd, Co, Ni and Zn than all other species and local sediment, indicating active accumulation of these metals. It also has the second highest Fe concentration, but without the comparably high concentrations of Al, Mn and Ti that are evident in H. erecta and in local sediment. These differences indicate active uptake and accumulation of Fe in S. clavatus, this was also noted in Niphates rowi. A significantly higher B concentration was found in Crella cyatophora compared to all other species. These results indicate specific roles of trace elements in certain sponge species that deserve further analysis. They also serve as a baseline to monitor the effects of anthropogenic disturbances on Eilat's coral reefs.

Chemical defense against fouling in the solitary ascidian Phallusia nigra.

The solitary ascidian Phallusia nigra is rarely fouled by epibionts. Here, we tested the antifouling activity of its crude extracts in laboratory and field assays. P. nigra extracts inhibited the growth of all eight tested environmental bacteria and two of four laboratory bacteria. Extracts of the sympatric, but fouled solitary ascidian Herdmania momus inhibited only one test bacterium. Scanning electron microscopy confirmed that the tunic surface of P. nigra is largely bacteria-free. Both ascidian extracts significantly inhibited the larval metamorphosis of the bryozoan Bugula neritina at the tested concentration range of 0.05-2 mg ml(-1). Both crude extracts were toxic to larvae of the brine shrimp Artemia salina at natural volumetric whole-tissue concentrations, but only P. nigra showed activity at 2 mg ml(-1) and below (LC50 = 1.11 mg ml(-1)). P. nigra crude extracts also significantly reduced the settlement of barnacles, polychaetes, and algae in Mediterranean field assays and barnacle settlement in Red Sea trials. Comparisons between control experiments and pH values monitored in all experiments indicate that the observed effects were not due to acidity of the organic extracts. Our results show that P. nigra secondary metabolites have antifouling activities, which may act in synergy with previously proposed physiological antifouling mechanisms.

Unique crystallographic pattern in the macro to atomic structure of Herdmania momus vateritic spicules.

Biogenic vaterite is extremely rare. The only known example of a completely vateritic mineralized structure is the spicule of the solitary ascidian, Herdmania momus. In characterizing the structure of these spicules, using state-of-the-art techniques such as synchrotron X-ray diffraction and synchrotron micro- and nanotomography, we observed a continuous structural pattern from the macro down to the micro, nano, and atomic scales. We show that the spicules demonstrate a unique architecture composed of micron-sized, hexagonally faceted thorns organized in partial spirals along the cylinder-like polycrystalline body of the spicule, and tilted from it at an angle of about 26°. This morphological orientation coincides with the crystallographic orientation relationship between each thorn and the polycrystals within the spicule. Hence the entire spicule grows along the [011] direction of vaterite while the individual thorns grow along the [001] direction. This, together with the presence of both inter- and intra-crystalline organic phases, beautifully displays the organism's ability to achieve perfect control of mineralization biologically while employing an unstable polymorph of calcium carbonate: vaterite.

Vaterite crystals contain two interspersed crystal structures.

Calcite, aragonite, and vaterite are the three anhydrous polymorphs of calcium carbonate, in order of decreasing thermodynamic stability. Although vaterite is not commonly found in geological settings, it is an important precursor in several carbonate-forming systems and can be found in biological settings. Because of difficulties in obtaining large, pure, single crystals, the crystal structure of vaterite has been elusive for almost a century. Using aberration-corrected high-resolution transmission electron microscopy, we found that vaterite is actually composed of at least two different crystallographic structures that coexist within a pseudo-single crystal. The major structure exhibits hexagonal symmetry; the minor structure, existing as nanodomains within the major matrix, is still unknown.

Examination of marine-based cultivation of three demosponges for acquiring bioactive marine natural products.

Marine sponges are an extremely rich and important source of natural products. Mariculture is one solution to the so-called "supply problem" that often hampers further studies and development of novel compounds from sponges. We report the extended culture (767 days) at sea in depths of 10 and 20 m of three sponge species: Negombata magnifica, Amphimedon chloros and Theonella swinhoei that produce latrunculin-B, halitoxin and swinholide-A, respectively. Since sponge-associated microorganisms may be the true producers of many of the natural products found in sponges and also be linked to the health of the sponges, we examined the stability of the bacterial communities in cultured versus wild sponges. Growth rate of the sponges (ranging from 308 to 61 and -19 (%)(year(-1)) in N. magnifica, A. chloros and T. swinhoei, respectively) differed significantly between species but not between the two depths at which the species were cultivated. Survivorship varied from 96% to 57%. During culture all species maintained the content of the desired natural product. Denaturing gradient gel electrophoresis analysis of the sponge-associated bacterial consortia revealed that differences existed between cultured and wild sponges in T. swinhoei and A. chloros but the communities remained quite stable in N. magnifica. The cultivation technique for production of natural products was found to be most appropriate for N. magnifica, while for T. swinhoei and A. chloros it was less successful, because of poorer growth and survival rates and shifts in their bacterial consortia.

Marine-based cultivation of diacarnus sponges and the bacterial community composition of wild and maricultured sponges and their larvae.

Marine organisms including sponges (Porifera) contain many structurally diverse bioactive compounds, frequently in a low concentration that hampers their commercial production. Two solutions to this problem are: culturing sponge explants for harvesting the desired compound and cultivation of sponge-associated bacteria. These bacteria (often considered the source of the desired compounds) include the Actinobacteria, from which many novel drugs were developed. In a long-term experiment (lasting 767 days), we evaluated the culture amenability of the sponge Diacarnus erythraenus in a mariculture system, placed at 10- and 20-m depths. The growth and survival rates of sponge fragments were monitored. Wild and maricultured sponges from both depths and their larvae were sampled at different time intervals for denaturing gradient gel electrophoresis (DGGE) profiling of the bacterial community residing within them. 16S rRNA gene sequences of both cultured bacterial isolates and clone libraries of unculturable bacteria were composed and compared, focusing on Actinobacteria. Sponges from both depths did not differ significantly either in mean growth rates (percent weight change year⁻¹ ± S.E.) (64.5% ± 21% at 10 m and 79.3% ± 19.1% at 20 m) or in seasonal growth rates. Survival was also very similar (72% at 10 m and 70% at 20 m). There were 88 isolates identified from adults and 40 from their larvae. The isolates and clone libraries showed diverse bacterial communities. The DGGE profiles of wild and maricultured sponges differed only slightly, without a significant effect of depths or dates of sampling. This long-term experiment suggests that D. erythraenus probably remained healthy and indicates its mariculture suitability.