Retinoic acid X receptor in the diploblast, Tripedalia cystophora

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (Kd = 4 × 10−10 M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His6)jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His6)jRXR showed that full-length jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

Fluorescent RNA cytochemistry: tracking gene transcripts in living cells

The advent of jellyfish green fluorescent protein and its spectral variants, together with promising new fluorescent proteins from other classes of the Cnidarian phylum (coral and anemones), has greatly enhanced and promises to further boost the detection and localization of proteins in cell biology. It has been less widely appreciated that highly sensitive methods have also recently been developed for detecting the movement and localization in living cells of the very molecules that precede proteins in the gene expression pathway, i.e. RNAs. These approaches include the microinjection of fluorescent RNAs into living cells, the in vivo hybridization of fluorescent oligonucleotides to endogenous RNAs and the expression in cells of fluorescent RNA-binding proteins. This new field of ‘fluorescent RNA cytochemistry’ is summarized in this article, with emphasis on the biological insights it has already provided. These new techniques are likely to soon collaborate with other emerging approaches to advance the investigation of RNA birth, RNA–protein assembly and ribonucleoprotein particle transport in systems such as oocytes, embryos, neurons and other somatic cells, and may even permit the observation of viral replication and transcription pathways as they proceed in living cells, ushering in a new era of nucleic acids research in vivo.

Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.

Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.

The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.

Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila.

We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time.

Evolução do veneno em cnidários baseada em dados de genomas e proteomas

A evolução do veneno, uma das misturas mais complexas da natureza, tem sustentado o sucesso da diversificação de inúmeras linhagens de animais. Serpentes deslizantes ou medusas flutuantes utilizam o veneno, um coquetel de peptídeos farmacologicamente ativos, sais e moléculas orgânicas. Esses animais surpreendentes têm provocado grande fascínio ao longo da história humana. Nesta dissertação propomos um estudo da evolução dos venenos no filo Cnidaria, englobando dados proteômicos e genômicos. Este projeto teve como objetivos: (1) caracterizar e elucidar a evolução da composição do veneno em Cnidaria por meio da comparação de listas de proteínas; (2) testar a hipótese de que a variação na família de toxinas específica de cnidários tem sido o resultado de um regime de seleção positiva; e (3) determinar a extensão em que a duplicação de genes pode ser considerada como a principal razão para a diversificação de toxinas em Cnidaria. O capítulo \"Comparative proteomics reveals common components of a powerful arsenal in the earliest animal venomous lineage, the cnidarians\" propõe o estudo comparado mais completo sobre a composição do veneno de cnidários e uma hipótese sobre a montagem evolutiva do complexo arsenal bioquímico de cnidários e do veneno ancestral desse grupo basal. Vinte e oito famílias de proteínas foram identificadas. Destas, 13 famílias foram registradas pela primeira vez no proteoma de Cnidaria. Pelo menos 15 famílias de toxinas foram recrutadas no proteoma de veneno de cnidários antes da diversificação dos grupos Anthozoa e Medusozoa. Nos capítulos \"Evidence of episodic positive selection in the evolution of jellyfish toxins of the cnidarian venom\" e \"Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)\", nossas análises demonstram que as famílias de toxinas nos cnidários se diversificam amplamente mediante a duplicação de genes. Além disso, em contraste com as famílias de toxinas do veneno na maioria das linhagens animais; nós identificamos um padrão diferente na família de toxinas específica de cnidários, em que há uma seleção purificadora por longos períodos seguindo longos tempos de diversificação ou vice-versa

Análise da influência de variáveis meteorológicas e da hidrodinâmica sobre os padrões espaciais e temporais da qualidade da água e de florescimentos de cianobactérias e macrófitas em um reservatório urbano

Os reservatórios urbanos estão suscetíveis a uma variedade de interferências antropogênicas que acarretam grande variabilidade espacial e temporal. Contudo, possuem uma dinâmica própria na qual o hidroclima e micro e macro-eventos meteorológicos atuam sobre os processos físicos, químicos e biológicos resultando em respostas particulares de cada corpo de água. No presente estudo a existência de padrões espaciais e temporais na formação de florescimentos de algas, cianobactérias e macrófitas no reservatório Guarapiranga, São Paulo, SP, foi avaliada por meio de experimento de curta escala de tempo durante o evento da entrada de uma frente fria. Foram amostrados 64 pontos em todo o reservatório, e o estudo intensivo de florescimento algal e de cianobactérias em dois ciclos nictemerais, em um ponto selecionado no reservatório. Um modelo tridimensional de hidrodinâmica foi aplicado ao estudo compartimentalizado dos tempos de residência e imagens de satélite foram analisadas para determinação de padrões temporais e espaciais durante períodos de tempo mais amplos. Os resultados revelaram que os períodos mais favoráveis ao surgimento de florescimentos de cianobactérias são geralmente os meses mais quentes, de dezembro e janeiro, ou aqueles em que ocorrem estratificações mais fortes como no fim do inverno, em julho, e após as primeiras chuvas nos meses de setembro e outubro. Existem padrões espaciais recorrentes na formação dos florescimentos, controlados em grande parte pela ação do vento, que no reservatório Guarapiranga é predominantemente nas direções leste e sudeste empurrando os florescimentos na direção da foz dos tributários Embu Mirim e Embu Guaçu e ocasionalmente na direção da foz do rio Parelheiros. As simulações hidrodinâmicas evidenciam as forçantes que determinam os padrões observados e reforçam a importância de se discretizarem os tempos de residência de diferentes compartimentos do reservatório. As séries temporais amplas permitiram a determinação da qualidade da água em cada região e fornecem subsídios para o futuro manejo do reservatório. Como esse comportamento não se restringe ao reservatório Guarapiranga, o tipo de modelagem aqui utilizada pode ser útil para obter informações importantes no processo de planejamento e seleção de medidas para o gerenciamento de reservatórios urbanos tropicais polimíticos, em geral.

Carybdea marsupialis (Cubozoa) in the Mediterranean Sea: The first case of a sting causing cutaneous and systemic manifestations

A woman stung by the box jellyfish Carybdea marsupialis (Cnidaria, Cubozoa) at a Spanish Mediterranean beach, showed systemic manifestations over several months (pain far from the inoculation point, arthralgia, paresthesia, hyperesthesia, increase of eosinophils and IgE) in addition to the skin condition.

Stable carbon and oxygen isotope record of live benthic foraminifera from Swedish fjords

In a previous 16-month seasonal study on living (stained) benthic foraminifera from two fjords on the Swedish west coast, it was reported that foraminifera proliferated in response to phytodetritus input; the strongest response came from the opportunistic species Stainforthia fusiformis. In this study, our objective was to find out if that phytodetritus input resulted in a change in the carbon isotopic composition of the foraminiferal tests. We also wanted to examine if variations in salinity and temperature (due to seasonality or deep-water exchanges) were reflected in the delta18O values. From S. fusiformis that were obtained from the Havstens Fjord (20 m) and the Gullmar Fjord (119 m) during the 16-month study, we developed a time series of delta18O and delta13C. After the spring blooms in the Havstens and the Gullmar Fjord, decreases of about 0.2 per mil to 0.3 per mil in the foraminiferal delta13C values were noted; in the Gullmar Fjord after the autumn blooms, decreases of the same order were also noted. Comparing the Havstens and the Gullmar Fjord, we found a 1 per mil difference in both delta13C and delta18O; we attribute this to hydrographic differences between the two fjords. Using calculated values of delta18O, together with the measured ones, we noticed that S. fusiformis in the Gullmar Fjord seems to calcify close to equilibrium with respect to the oxygen isotopes. During autumn, water temperatures were relatively high in the Havstens Fjord, and foraminiferal abundance in the fjord was also high after a phytodetritus input; but, the measured delta18O values do not reflect these higher temperatures. This apparently contradictory combination of results might be explained by a varying delta18O composition of the water during the year, which counterbalances the temperature effect.

Macerals in surface sediments of the Laptev Sea (Arctic Ocean)

Surface sediments from the Laptev Sea and adjacent continental slope were studied for their composition of particulate organic matter (OM) by means of maceral analysis. The composition of macerals in sediments gives information about the environment, terrigenous supply from the hinterland, and marine OM. With reference to their biological sources, we distinguish between terrigenous and marine macerals. We found that the particulate OM in the surface sediments of the Laptev Sea is predominantly of terrigenous origin (mean: 78%). However, distinct variations exist when looking in detail. In the shelf area, sediments may contain up to 99% terrigenous OM. Freshwater algae occur directly north of the river mouths, reflecting the strong fluvial influence. Relatively high amounts of marine OM (20-40%) are restricted to the upper continental slope, the Vilkitsky Strait and west of the New Siberian Islands, explained by increased surface-water productivity due to increased fluvial nutrient supply, open-water conditions, and phytoplankton blooms at the ice-edge.

Sinking rates of particles measured from deployments in the Atlantic Ocean based on seasonal data

The flux of materials to the deep sea is dominated by larger, organic-rich particles with sinking rates varying between a few meters and several hundred meters per day. Mineral ballast may regulate the transfer of organic matter and other components by determining the sinking rates, e.g. via particle density. We calculated particle sinking rates from mass flux patterns and alkenone measurements applying the results of sediment trap experiments from the Atlantic Ocean. We have indication for higher particle sinking rates in carbonate-dominated production systems when considering both regional and seasonal data. During a summer coccolithophorid bloom in the Cape Blanc coastal upwelling off Mauritania, particle sinking rates reached almost 570 m per day, most probably due the fast sedimentation of densely packed zooplankton fecal pellets, which transport high amounts of organic carbon associated with coccoliths to the deep ocean despite rather low production. During the recurring winter-spring blooms off NW Africa and in opal-rich production systems of the Southern Ocean, sinking rates of larger particles, most probably diatom aggregates, showed a tendency to lower values. However, there is no straightforward relationship between carbonate content and particle sinking rates. This could be due to the unknown composition of carbonate and/or the influence of particle size and shape on sinking rates. It also remains noticeable that the highest sinking rates occurred in dust-rich ocean regions off NW Africa, but this issue deserves further detailed field and laboratory investigations. We obtained increasing sinking rates with depth. By using a seven-compartment biogeochemical model, it was shown that the deep ocean organic carbon flux at a mesotrophic sediment trap site off Cape Blanc can be captured fairly well using seasonal variable particle sinking rates. Our model provides a total organic carbon flux of 0.29 Tg per year down to 3000 m off the NW African upwelling region between 5 and 35° N. Simple parameterisations of remineralisation and sinking rates in such models, however, limit their capability in reproducing the flux variation in the water column.

Raw data of SCADCP (self-contained Acoustic Doppler Current Profiler) from three moorings in the Eastern Weddell Sea, 2005-2008

The success of any efforts to determine the effects of climate change on marine ecosystems depends on understanding in the first instance the natural variations, which contemporarily occur on the interannual and shorter time scales. Here we present results on the environmental controls of zooplankton distribution patterns and behaviour in the eastern Weddell Sea, Southern Ocean. Zooplankton abundance and vertical migration are derived from the mean volume backscattering strength (MVBS) and the vertical velocity measured by moored acoustic Doppler current profilers (ADCPs), which were deployed simultaneously at 64°S, 66.5°S and 69°S along the Greenwich Meridian from February, 2005, until March, 2008. While these time series span a period of full three years they resolve hourly changes. A highly persistent behavioural pattern found at all three mooring locations is the synchronous diel vertical migration (DVM) of two distinct groups of zooplankton that migrate between a deep residence depth during daytime and a shallow depth during nighttime. The DVM was closely coupled to the astronomical daylight cycles. However, while the DVM was symmetric around local noon, the annual modulation of the DVM was clearly asymmetric around winter solstice or summer solstice, respectively, at all three mooring sites. DVM at our observation sites persisted throughout winter, even at the highest latitude exposed to the polar night. Since the magnitude as well as the relative rate of change of illumination is minimal at this time, we propose that the ultimate causes of DVM separated from the light-mediated proximal cue that coordinates it. In all three years, a marked change in the migration behaviour occurred in late spring (late October/early November), when DVM ceased. The complete suspension of DVM after early November is possibly caused by the combination of two factors: (1) increased availability of food in the surface mixed layer provided by the phytoplankton spring bloom, and (2) vanishing diurnal enhancement of the threat from visually oriented predators when the illumination is quasi-continuous during the polar and subpolar summer. Zooplankton abundance in the water column, estimated as the mean MVBS in the depth range 50-300 m, was highest end of summer and lowest mid to end winter on the average annual cycle. However, zooplankton abundance varied several-fold between years and between locations. Based on satellite and in situ data of chlorophyll and sea ice as well as on hydrographic measurements, the interannual and spatial variations of zooplankton mean abundance can be explained by differences in the magnitude of the phytoplankton spring bloom, which develops during the seasonal sea ice retreat. Whereas the vernal ice melt appears necessary to stimulate the blooming of phytoplankton, it is not the determinator of the blooms magnitude, its areal extent and duration. A possible explanation for the limitation of the phytoplankton bloom in some years is top-down control. We hypothesise that the phytoplankton spring development can be curbed by grazing when the zooplankton had attained high abundance by growth during the preceding summer.

Thorium-234, POC and PON fluxes during POLARSTERN cruise ANT-XXVIII/3, January-March 2012

Carbon fixation by phytoplankton plays a key role in the uptake of atmospheric CO2 in the Southern Ocean. Yet, it still remains unclear how efficiently the particulate organic carbon (POC) is exported and transferred from ocean surface waters to depth during phytoplankton blooms. In addition, little is known about the processes that control the flux attenuation within the upper twilight zone. Here, we present results of downward POC and particulate organic nitrogen fluxes during the decline of a vast diatom bloom in the Atlantic sector of the Southern Ocean in summer 2012. We used thorium-234 (234Th) as a particle tracer in combination with drifting sediment traps (ST). Their simultaneous use evidenced a sustained high export rate of 234Th at 100 m depth in the weeks prior to and during the sampling period. The entire study area, of approximately 8000 km**2, showed similar vertical export fluxes in spite of the heterogeneity in phytoplankton standing stocks and productivity, indicating a decoupling between production and export. The POC fluxes at 100 m were high, averaging 26 ± 15 mmol C/m**2/d, although the strength of the biological pump was generally low. Only <20% of the daily primary production reached 100 m, presumably due to an active recycling of carbon and nutrients. Pigment analyses indicated that direct sinking of diatoms likely caused the high POC transfer efficiencies (~60%) observed between 100 and 300 m, although faecal pellets and transport of POC linked to zooplankton vertical migration might have also contributed to downward fluxes.