Producción de pólipos clonales del organismo modelo Exaiptasia diaphana (Rapp, 1829)

Background. The sea anemone Exaiptasia diaphana (Order Actiniaria) is an ideal model organism to study diverse biological, physiological, and immune processes in corals (Order Scleractinia) due to its close phyletic relationship and shared traits. E. diaphana is widely distributed along the world’s tropical coastal areas. This species is easy to grow in aquariums under diverse experimental conditions since reproduces asexually and can be rendered aposymbiotic. However, there are a variety of methods to propagate them, making difficult comparisons of results. A standardized propagation protocol for E. diaphana can also contribute to improving the understanding of its biology. Goal. Determine the most rapid method of clonal production in controlled conditions. Results. In the micro-laceration treatment, 50% of the remnant tissue gave rise to a new clonal polyp, while all the amputated anemones resulted in two polyps with tentacles and a pedal disc. Amputated clonal polyps developed their tentacles from the third day, being this treatment the most rapid compared with the control group and the micro-laceration treatment. In both cases, the tentacles started to develop from the sixth day of the experiment. The control group naturally released five clonal polyps with tentacles in the ten-day experiment Conclusion. Transversal amputation was the most rapid method to obtain developed clonal polyps. We, therefore, propose transversal amputation as a standard method for the efficient artificial propagation of the clonal polyps of the model organism E. diaphana.Antecedentes. La anémona marina Exaiptasia diaphana (Orden Actiniaria) es un organismo modelo ideal para estudiar procesos biológicos, fisiológicos e inmunitarios en corales (Orden Scleractinia), debido a su estrecha relación filética y sus rasgos compartidos. E. diaphana tiene una distribución amplia en las zonas tropicales costeras. Esta especie es fácil de mantener en acuarios bajo diversas condiciones experimentales, ya que se reproduce asexualmente y puede transformarse en aposimbiótica. Sin embargo, los métodos de propagación son diversos, lo que dificulta la comparación directa de resultados. Un protocolo estandarizado de propagación de E. diaphana puede contribuir a mejorar el entendimiento de su biología. Objetivo. Determinar el método más rápido de producción de pólipos clonales en condiciones controladas. Resultados. En el tratamiento de micro laceración, el 50% del tejido remanente dio lugar a un nuevo pólipo clonal, mientras que cada anémona amputada formó dos pólipos con tentáculos y pie desarrollados. Los pólipos clonales amputados desarrollaron tentáculos a partir del tercer día, mientras que los pies del grupo control y el tejido remanente de la micro laceración desarrollaron tentáculos a partir del sexto día. El grupo control liberó naturalmente cinco pólipos con tentáculos bien desarrollados en los diez días del experimento. Conclusión. La amputación transversal fue el método más rápido para obtener pólipos clonales desarrollados, por lo que se propone como método estándar para la propagación artificial eficiente de pólipos clonales del organismo modelo E. diaphana

Contrasting Antibacterial Capabilities of the Surface Mucus Layer From Three Symbiotic Cnidarians

Coral reefs are affected by the deterioration of the oceans due to global warming and other anthropogenic perturbations, increasing the frequency, and severity of bleaching and disease. To overcome some of these conditions, reef corals and other cnidarians rely on a mucus layer housing a diverse community of beneficial microorganisms and mechanisms of innate immune response. The antimicrobial defense has been associated with the bacterial community in these organisms, but the potential antimicrobial activity of the mucus layer itself has not been explored fully. We hypothesized that the bacteria-free mucus layer of different cnidarians would show differential and specific antimicrobial and immunological responses when challenged with two potentially pathogenic bacteria. We evaluated this capability through antimicrobial properties, immune response and biochemical composition of the mucus. Results clearly showed that the mucus of healthy cnidarians has the capability of inhibiting the growth of Serratia marcescens and Aurantimonas sp. in a species-specific way that includes differences in the potency of the response. The anemone Exaiptasia pallida was particularly potent against Aurantimonas sp. while the coral Pseudodiploria strigosa and the medusa Cassiopea xamachana had similar capabilities against both bacterial strains. In coral affected by black band disease, this antibacterial capability diminished in the mucus layer, but the associated bacteria remained potent. Results showed that hydroxyproline and phenoloxidase increased in the transition zone of diseased corals, although melanin was not detected in any of the animals tested. Bleaching of anemones and medusas also diminished the antibacterial capability of the surface mucus layer, but in anemones, the associated bacteria did not show a significant reduction in their ability to inhibit the growth of the bacterial strains. The mucus of bleached medusas showed an increased inhibitory activity against Aurantimonas sp. that may be associated with a specific bacterial strain we isolated. Mucus collected from bleached anemones and medusas did not show a significant immune response. In this work, we show that the surface mucus layer itself has antibacterial properties not associated with the bacteria this layer houses; such properties diminished due to disease or bleaching, while immunological responses increased in the mucus of diseased animals

Indomethacin reproducibly induces metamorphosis in Cassiopea xamachana scyphistomae

Cassiopea xamachana jellyfish are an attractive model system to study metamorphosis and/or cnidarian–dinoflagellate symbiosis due to the ease of cultivation of their planula larvae and scyphistomae through their asexual cycle, in which the latter can bud new larvae and continue the cycle without differentiation into ephyrae. Then, a subsequent induction of metamorphosis and full differentiation into ephyrae is believed to occur when the symbionts are acquired by the scyphistomae. Although strobilation induction and differentiation into ephyrae can be accomplished in various ways, a controlled, reproducible metamorphosis induction has not been reported. Such controlled metamorphosis induction is necessary for an ensured synchronicity and reproducibility of biological, biochemical, and molecular analyses. For this purpose, we tested if differentiation could be pharmacologically stimulated as in Aurelia aurita, by the metamorphic inducers thyroxine, KI, NaI, Lugol’s iodine, H2O2, indomethacin, or retinol. We found reproducibly induced strobilation by 50 μM indomethacin after six days of exposure, and 10–25 μM after 7 days. Strobilation under optimal conditions reached 80–100% with subsequent ephyrae release after exposure. Thyroxine yielded inconsistent results as it caused strobilation occasionally, while all other chemicals had no effect. Thus, indomethacin can be used as a convenient tool for assessment of biological phenomena through a controlled metamorphic process in C. xamachana scyphistomae

Induction of glycerol synthesis and release in cultured Symbiodinium.

Symbiotic dinoflagellates transfer a substantial amount of their photosynthetic products to their animal hosts. This amount has been estimated to represent up to 90% of the photosynthetically fixed carbon and can satisfy in some instances the full respiratory requirements of the host. Although in several cnidarian-dinoflagellate symbioses glycerol is the primary photosynthetic product translocated to the host, the mechanism for its production and release has not been demonstrated conclusively.Using Symbiodinium cells in culture we were able to reproduce the synthesis and release of glycerol in vitro by employing an inductor for glycerol synthesis, osmotic up-shocks. Photosynthetic parameters and fluorescence analysis of photosystem II showed that the inductive conditions did not have a negative effect on photosynthetic performance, suggesting that the capacity for carbon fixation by the cells was not compromised. The demand for glycerol production required to attain osmotic balance increased the expression of ribulose 1,5-bisphosphate and of glycerol 3-phosphate dehydrogenase, possibly competing with the flux of fixed carbon necessary for protein synthesis. In longer exposures of cultured Symbiodinium cells to high osmolarity, the response was analogous to photoacclimation, reducing the excitation pressure over photosystem II, suggesting that Symbiodinium cells perceived the stress as an increase in light. The induced synthesis of glycerol resulted in a reduction of growth rates.Our results favor a hypothetical mechanism of a signaling event involving a pressure sensor that may induce the flux of carbon (glycerol) from the symbiotic algae to the animal host, and strongly suggest that carbon limitation may be a key factor modulating the population of symbionts within the host

Expression of genes associated to carbon fixation (Rubisco, <i>rbcA</i>) and synthesis of glycerol (glycerol 3-phosphate dehydrogenase, <i>GPD</i>).

<p>RT-PCR from 1 µg RNA extracted from <i>Symbiodinium</i> type B17 (upper panel) or type A1 cultures (lower panel) exposed to control (left lanes) or osmotic stress conditions (right lanes) for 1 hr. The amplification products were separated in agarose gels photographed, scanned and quantified with the ImageJ program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>, 1997–2011). Color was inverted in the scans for clearer visualization. Numbers represent relative intensity of bands normalized to transcript levels for <i>GAPDH</i> (glyceraldehyde 3-phosphate dehydrogenase, see methods).</p

Photosynthetic parameters for <i>Symbiodinium</i> cultures exposed to high osmolarity conditions for 7 days.

<p>P_max and respiration (R) values expressed as µmol O₂ min⁻¹ cell⁻⁹ or µmol O₂ mg⁻¹ Chl <i>a</i> min⁻¹ respectively. Chl <i>a</i> values expressed in mg mL⁻¹. Values are the means of 3 independent experiments ± SD. Significant probabilities (P) in bold.</p>‡<p><b> = </b>values×10⁻⁸;</p>‡‡<p><b> = </b>values×10⁻⁶.</p

Total and extracellular glycerol levels in <i>Symbiodinium</i> cells exposed to an osmotic up-shock.

<p>Control cultures with no additions, stressed cultures with 9% PEG added. Cells were collected after 1 hr treatment. Glycerol concentration given in mg mL⁻¹ in 10⁵ cells. Values are the means ± SD from three independent experiments. Significant probabilities (P) between control and stressed treatments for each cell type in bold (<b><0.05</b>).</p

Photosynthesis <i>vs</i> irradiance (PE) curves normalized to cell density.

<p>Cells cultured for two weeks were incubated for 1 hr in control medium (solid line) or with the addition of 9% PEG (broken line). O₂ evolution was assessed under increasing light intensity, in <i>Symbiodinium</i> type B17 (<b>A</b>) and type A1 (<b>B</b>) cells. Each value represents the mean ± SD (bars) from 3 independent experiments. Data were fitted to a hyperbolic tangent function.</p