Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae)

The cyanobacterial symbionts of the marine sponge Chondrilla australiensis (Demospongiae) were examined using fluorescent microscopy and Transmission Electron Microscopy. Unicellular cyanobacteria with ultrastructure resembling Aphanocapsa feldmannii occur in the cortex and bacterial symbionts are located throughout the mesohyl. In C. australiensis, the developing eggs are distributed throughout the mesohyl and are surrounded by nurse cells attached to them by thin filaments. The nurse cells form cytoplasmic bridges with the eggs, apparently releasing their contents into the egg cytoplasm. The presence of cyanobacterial and bacterial symbionts inside developing eggs and nurse cells in 25% of female Chondrilla australiensiswas established using Transmission Electron Microscopy, suggesting that these symbionts are sometimes passed on to the next generation of sponges via the eggs.     

Differential Gene Expression in a Marine Sponge in Relation to Its Symbiotic State

The molecular mechanisms involved in the establishment and maintenance of sponge photosymbiosis, and in particular the association with cyanobacteria, are unknown. In the present study we analyzed gene expression in a common Mediterranean sponge (Petrosia ficiformis) in relation to its symbiotic (with cyanobacteria) or aposymbiotic status. A screening approach was applied to identify genes expressed differentially in symbiotic specimens growing in the light and aposymbiotic specimens growing in a dark cave at a short distance from the illuminated specimens. Out of the various differentially expressed sequences, we isolated two novel genes (here named PfSym1 and PfSym2) that were up-regulated when cyanobacterial symbionts were harbored inside the sponge cells. The sequence of one of these genes (PfSym2) was found to contain a conserved domain: the scavenger receptor cysteine rich (SRCR) domain. This is the first report on the expression of sponge genes in relation to symbiosis and, according to the presence of an SRCR domain, we suggest possible functions for one of the genes found in the sponge-cyanobacteria symbiosis.     

Continuous cell movements rearrange anatomical structures in intact sponges.

Time-lapse cinemicrography was used to record the active movements of cells in living intact sponges. Each of the three main cell types (pinacocytes, mesohyl cells, and choanocytes) continuously moved and rearranged themselves so that the internal anatomy of the sponge was continuously remodeled. The shape and appearance of the sponges anatomical structures often changed substantially within a few hours. The most motile were the mesohyl cells, with many moving as fast as one cell-length per minute (15 microns/min). Mesohyl cell locomotion was often accompanied by displacements of spicules, canals, and choanocyte chambers; the patterns of these displacements suggested that the mesohyl cells were providing the motive forces for these rearrangements. The locomotion of the pinacocytes varied according to position: those along the outer sponge margins were most active, whereas those in other parts of the surface moved relatively little. Choanocytes were never observed to undergo independent locomotion but were always found grouped together in choanocyte chambers. These choanocyte chambers interacted with pinacocytes and mesohyl cells to form excurrent canals, which continuously moved, fused with, and branched from one another. These observations suggest that the experimental phenomenon of sponge cell-reaggregation and reconstitution, discovered by H. V. Wilson, represents an extreme version of morphogenetic processes that normally go on continuously within intact sponges. The results from the present study also suggest that these cellular rearrangements are controlled by active cell movements and behavioral responses that include but are not limited to selective cell adhesion.     

Allogeneic cell interactions during graft rejection in Callyspongia diffusa (Porifera, Demospongia); a study with monoclonal antibodies

Many aspects of the cellular immune system in the marine sponge Callyspongia diffusa, have been defined by using artificially transplanted allogeneic tissues. Rejections show specificity of 'non-self' recognition, cytotoxic effector responses and short-term immunological memory. Histological investigations reveal a generalized mesohyl migration to the graft zone where archaeocytes line up at the allogeneic interface. Monoclonal antibodies (mAbs) raised to sponge cells have shown that little or no allogeneic cell mixing occurs at the graft interface and that certain mesohyl cell types do not appear to be directly involved in graft rejections. However, all mesohyl cell types are present in autograft fusion zones and in inflammatory responses to injury. The involvement of only some of the mesohyl cell types in graft rejections suggests specific interactions of an effector 'immunocyte'.     

The vacuolar cells of Oscarella lobularis (Porifera,Demospongiae): Ulatrastructural organization,origin, and function

This work is concerned with the ultrastructural organization and some histochemical features of the vacuolar cells of the demosponge Oscarella lobularis. Vacuolar cells are characterized by large clear vacuoles containing an aqueous fluid. They are commonly found in the mesohyl of this sponge and tend to constitute a sort of parenchyma in the choanosome. Mobile cells of the mesohyl appear to differetiate into vacuolar cells through the progressive formation of wide cytoplasmic lacunae. We have identified four types of cells showing progressive transformation toward the vacuolar cell type. Precursors (types 1–4) of the vacuolar cells probably derive from endopinacocytes, since they share several histochemical and ultrastructural characteristics with them. Our data support the notion that vacuolar cells are involved in the synthesis of collagen, act as a mechanical support of the sponge body, and are eventually extruded from the sponge through the canals of the aquiferous system.     

Allograft rejection, autograft fusion and inflammatory responses to injury in Callyspongia diffusa (Porifera; Demospongia)

Sponges exhibit a variety of swift, cellular defence responses to protect self integrity. The sponge Callyspongia diffusa has been used to characterize the cytological changes that occur during allograft rejection, autograft fusion, and inflammation. Allogeneic contact results in fusion of the two exopinacoderms followed by an infiltration of mesohyl cells into the graft zone. As mesohyl cells accumulate, they form tissue bridges that span the graft interface. After a few days, the tissue bridges and the nearby cellular infiltrate become necrotic and slough off, which separates the allogeneic tissues. Autograft fusion begins similarly but cellular infiltration does not follow exopinacoderm fusion. Contacted exopinacocytes are redistributed, the endopinacoderms and choanosomes come into contact, and the grafted sponge tissues merge. Tissue damage exposes internal regions of the sponge to the external environment. In many areas of injury, exposed choanosome is sealed by infiltrating mesohyl cells. In other areas, exposed endopinacoderm appears to serve as new exopinacoderm. Cellular debris is removed by phagocytic archaeocytes and new exopinacoderm is regenerated over the damaged choanosome. No scars remain once the inflammatory infiltrate has dispersed. In general, mesohyl cells are involved in defence responses without an observed enrichment of any specific cell type. However, archaeocytes from rejecting sponges appear to line both sides of the allogeneic interface.     

Unusual Symbiotic Cyanobacteria Association in the Genetically Diverse Intertidal Marine Sponge Hymeniacidon perlevis (Demospongiae,Halichondrida)

Cyanobacteria represent one of the most common members of the sponge-associated bacterial community and are abundant symbionts of coral reef ecosystems. In this study we used Transmission Electron Microscopy (TEM) and molecular techniques (16S rRNA gene marker) to characterize the spatial distribution of cyanobionts in the widely dispersed marine intertidal sponge Hymeniacidon perlevis along the coast of Portugal (Atlantic Ocean). We described new sponge associated cyanobacterial morphotypes (Xenococcus-like) and we further observed Acaryochloris sp. as a sponge symbiont, previously only reported in association with ascidians. Besides these two unique cyanobacteria, H. perlevis predominantly harbored Synechococcus sp. and uncultured marine cyanobacteria. Our study supports the hypothesis that the community of sponge cyanobionts varies irrespective of the geographical location and is likely influenced by seasonal fluctuations. The observed multiple cyanobacterial association among sponges of the same host species over a large distance may be attributed to horizontal transfer of symbionts. This may explain the absence of a co-evolutionary pattern between the sponge host and its symbionts. Finally, in spite of the short geographic sampling distance covered, we observed an unexpected high intra-specific genetic diversity in H. perlevis using the mitochondrial genes ATP6 (π = 0.00177), COI (π = 0.00241) and intergenic spacer SP1 (π = 0.00277) relative to the levels of genetic variation of marine sponges elsewhere. Our study suggests that genotypic variation among the sponge host H. perlevis and the associated symbiotic cyanobacteria diversity may be larger than previously recognized.     

Distribution of brominated compounds within the sponge<Emphasis Type="Italic"> Aplysina aerophoba</Emphasis>: coupling of X-ray microanalysis with cryofixation techniques

The major secondary metabolites of the sponge Aplysina aerophoba are brominated compounds. X-ray energy dispersive microanalysis was therefore used to locate secondary metabolites via the Br signal in energy emission spectra from sponge sections. To test the reliability of this method in the face of the loss or redistribution of metabolites during processing, we compared the results obtained by conventional aldehyde fixation with those obtained by cryofixation and cryosubstitution with and without cryoembedding. Bromine appeared to be concentrated in two sponge structures, viz. fibres and spherulous cells, when cryofixed material was examined. However, X-ray microanalysis failed to demonstrate the presence of bromine in spherulous cells in chemically fixed samples, showing the need for cryotechniques to avoid the loss of compounds. Cryofixation plus cryosubstitution methods performed best regarding structural preservation and the immobilization of metabolites. The presence of bromine in the spherulous cells suggests that this cell type is the producer of the secondary metabolites, as described for other sponge species. Nevertheless, the presence of bromine in sponge fibres indicates that they can accumulate metabolic substances, although we have been unable to assess whether the chemicals are in their original form or in a modified state within the fibres. A. aerophoba has both bacterial and cyanobacterial symbionts in its mesohyl; the absence of brominated compounds in them contrasts with previous findings in other sponges with prokaryote symbionts.     

Lineage-Specific Domain Fusion in the Evolution of Purine Nucleotide Cyclases in Cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.     

Differences Between Bacterial Communities Associated with the Surface or Tissue of Mediterranean Sponge Species

Bacterial communities associated with the surfaces of several Mediterranean sponge species (Agelas oroides, Chondrosia reniformis, Petrosia ficiformis, Geodia sp., Tethya sp., Axinella polypoides, Dysidea avara, and Oscarella lobularis) were compared to those associated with the mesohyl of sponges and other animate or inanimate reference surfaces as well as with those from bulk seawater. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified bacterial 16S ribosomal RNA genes obtained from the surfaces and tissues of these sponges demonstrated that the bacterial communities were generally different from each other. The bacterial communities from sponges were different from those on reference surfaces or from bulk seawater. Additionally, clear distinctions in 16S rDNA fingerprint patterns between the bacterial communities from mesohyl samples of "high-microbial abundance (HMA) sponges" and "low-microbial abundance sponges" were revealed by DGGE and cluster analysis. A dominant occurrence of particularly GC-rich 16S ribosomal DNA (rDNA) fragments was found only in the DGGE banding pattern obtained from the mesohyl of HMA sponges. Furthermore, sequencing analysis of 16S rDNA fragments obtained from mesohyl samples of HMA sponges revealed a dominant occurrence of sponge-associated bacteria. The bacterial communities within the mesohyl of HMA sponges showed a close relationship to each other and seem to be sponge-specific.     

Diversity and expression of nitrogen fixation genes in bacterial symbionts of marine sponges

Marine sponges contain complex assemblages of bacterial symbionts, the roles of which remain largely unknown. We identified diverse bacterial nifH genes within sponges and found that nifH genes are expressed in sponges. This is the first demonstration of the expression of any protein-coding bacterial gene within a sponge. Two sponges Ircinia strobilina and Mycale laxissima were collected from Key Largo, Florida and had delta(15)N values of c. 0-1 per thousand and 3-4 per thousand respectively. The potential for nitrogen fixation by symbionts was assessed by amplification of nifH genes. Diverse nifH genes affiliated with Proteobacteria and Cyanobacteria were detected, and expression of nifH genes affiliated with those from cyanobacteria was detected. The nifH genes from surrounding seawater were similar to those of Trichodesmium and clearly different from the cyanobacterial nifH genes detected in the two sponges. This study advances understanding of the role of bacterial symbionts in sponges and suggests that provision of fixed nitrogen is a means whereby symbionts benefit sponges in nutrient-limited reef environments. Nitrogen fixation by sponge symbionts is possibly an important source of new nitrogen to the reef environment that heretofore has been neglected and warrants further investigation.     

Phenotypic study of bacteria associated with the caribbean sclerosponge, Ceratoporella nicholsoni.

Heterotrophic bacteria associated with the Caribbean sclerosponge, Ceratoporella nicholsoni (Hickson), were found to occur extracellularly and were confined to the mesohyl regions of the sponge tissue. Physiological, metabolic, and morphological attributes of the culturable bacteria associated with the sponge were recorded by using numerical taxonomy methods for the analysis of 158 phenotypic attributes. Morphometric methods were used to determine the proportion of the total sponge-associated bacteria that were culturable by the methods employed, with the results ranging from 3 to 11% of the total bacteria inhabiting the sponge. Approximately 78% of the culturable bacteria clustered into four groups or phena, representing two previously undescribed Vibrio spp., an Aeromonas sp., and a coryneform- or actinomycete-like sp. Most of the bacteria were facultative anaerobes, fermenting sucrose and fucose but unusual in an inability to ferment glucose. This study was the first comprehensive study of heterotrophic bacteria associated with a sponge from the Caribbean basin, a region reputed to contain the most prolific sponge populations, with respect to biomass and diversity. The possible significance of these associations is discussed.     

Phenotypic study of bacteria associated with the caribbean sclerosponge, Ceratoporella nicholsoni

Heterotrophic bacteria associated with the Caribbean sclerosponge, Ceratoporella nicholsoni (Hickson), were found to occur extracellularly and were confined to the mesohyl regions of the sponge tissue. Physiological, metabolic, and morphological attributes of the culturable bacteria associated with the sponge were recorded by using numerical taxonomy methods for the analysis of 158 phenotypic attributes. Morphometric methods were used to determine the proportion of the total sponge-associated bacteria that were culturable by the methods employed, with the results ranging from 3 to 11% of the total bacteria inhabiting the sponge. Approximately 78% of the culturable bacteria clustered into four groups or phena, representing two previously undescribed Vibrio spp., an Aeromonas sp., and a coryneform- or actinomycete-like sp. Most of the bacteria were facultative anaerobes, fermenting sucrose and fucose but unusual in an inability to ferment glucose. This study was the first comprehensive study of heterotrophic bacteria associated with a sponge from the Caribbean basin, a region reputed to contain the most prolific sponge populations, with respect to biomass and diversity. The possible significance of these associations is discussed.     

Dynamic structure of the mesohyl in the sponge Chondrosia reniformis (Porifera, Demospongiae)

The common demosponge Chondrosia reniformis possesses the capacity to undergo an unusual creep process which results in the formation of long outgrowths from the parent body. These shape changes, which have been interpreted as adaptive strategies related to environmental factors, asexual reproduction or localised locomotor phenomena, are due mainly to the structural and mechanical adaptability of the collagenous mesohyl. This contribution describes the morphological correlates of mesohyl plasticisation in C. reniformis. The microscopic anatomy of the mesohyl was examined when it was in different physiological conditions: (1) standard ”resting” condition, (2) ”stiffened” condition and (3) dynamic ”creep” condition. In this last case four representative regions of the sponge body were analysed: the parent region, the elongation region, the transition region and the propagule region. The results show that the histological modification of the sponge mesohyl during plasticisation is limited and localised. The most significant structural changes involve mainly cytological features of specific cellular components characterised by granule inclusions (i.e. the spherulous cells) and the arrangement and density of the collagenous extracellular framework, though the integrity of the collagen fibrils themselves is not affected. Morphological and functional aspects of mesohyl plasticisation invite comparison with the mutable collagenous tissue of echinoderms. Possible functional analogies between these two tissues are hypothesised. Accepted: 29 June 2001     

Epilithic cyanobacterial communities and water quality: an alternative tool for monitoring eutrophication in the Alberche River (Spain)

Changes in epilithic cyanobacterial communities were determined in a river characterized by variations in nutrient content. The cyanobacterial community composition of the upstream sites was different from that of the downstream communities, where anthropogenic influences lead to an increase in nutrients (principally soluble reactive phosphate, SRP). There was a general trend in downstream sites towards a decrease in species richness, abundance, and diversity of cyanobacteria. The reduced cyanobacterial species richness in downstream locations was due largely to a marked decrease in the number of heterocystous species, although the number of non-heterocystous species also decreased. Epilithic phycobiliprotein content was positively correlated with the number of cyanobacterial cells, implying that this pigment provides information about the abundance of the cyanobacteria community in the epilithon. The lowest concentrations of phycobiliprotein in the epilithon were observed where concentrations of phosphate were highest. Similarly, the number of heterocystous and non-heterocystous species tended to decrease as the SRP increased, and as the DIN:SRP ratio decreased. However, no relation was found with dissolved inorganic nitrogen (DIN). The differences among cyanobacterial communities could be interpreted as being a consequence of variations in nutrient composition. Finally, the usefulness of cyanobacteria as an alternative tool for assessing changes in water quality is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Impact of carbon metabolism on 13C signatures of cyanobacteria and green non-sulfur-like bacteria inhabiting a microbial mat from an alkaline siliceous hot spring in Yellowstone National Park (USA)

Alkaline siliceous hot spring microbial mats in Yellowstone National Park are composed of two dominant phototropic groups, cyanobacteria and green non-sulfur-like bacteria (GNSLB). While cyanobacteria are thought to cross-feed low-molecular-weight organic compounds to support photoheterotrophic metabolism in GNSLB, it is unclear how this could lead to the heavier stable carbon isotopic signatures in GNSLB lipids compared with cyanobacterial lipids found in previous studies. The two groups of phototrophs were separated using percoll density gradient centrifugation and subsequent lipid and stable carbon isotopic analysis revealed that we obtained fractions with a approximately 60-fold enrichment in cyanobacterial and an approximately twofold enrichment in GNSLB biomass, respectively, compared with the mat itself. This technique was used to study the diel cycling and 13C content of the glucose pools in and the uptake of 13C-bicarbonate by the cyanobacteria and GNSLB, as well as the transfer of incorporated 13C from cyanobacteria to GNSLB. The results show that cyanobacteria have the highest bicarbonate uptake rates and accumulate glucose during the afternoon in full light conditions. In contrast, GNSLB have relatively higher bicarbonate uptake rates compared with cyanobacteria in the morning at low light levels. During the night GNSLB take up carbon that is likely derived through fermentation of cyanobacterial glucose enriched in 13C. The assimilation of 13C-enriched cyanobacterial carbon may thus lead to enriched 13C-contents of GNSLB cell components.     

四川江油渔洞子下三叠统飞仙关组巨鲕灰岩中疑似蓝细菌及早三叠世疑似蓝细菌的双幕式爆发

二叠纪末海洋生态环境的恶化导致海洋底栖生物的大灭绝及早三叠世蓝细菌的爆发,有关这一时期我国华南蓝细菌化石的报道主要来自早三叠世早期的微生物岩。四川江油渔洞子剖面下三叠统飞仙关组下部巨鲕灰岩中首次发现有丰富的疑似蓝细菌,在种类和结构上与以往所报道的蓝细菌有着明显区别。疑似蓝细菌在巨鲕内和围岩中的富集,表明巨鲕灰岩的成因与疑似蓝细菌的活动有关,与飞仙关组底部微生物岩在成因上有着密切联系,显示早三叠世早期在这一地区发生了疑似蓝细菌的双幕式爆发,同时,暗示着这一地区二叠纪-三叠纪之交至少发生了两次海洋环境恶化及动物灭绝事件。疑似蓝细菌的爆发性生长,对于海洋生态环境的修复和海洋含氧条件的改善,进而为早三叠世末、中三叠世的生物全面复苏、辐射有着重要意义。     

Molecular phylogeny and evogenomics of heterocystous cyanobacteria using rbcl gene sequence data

Taxonomic affiliations and molecular diversity of 41 heterocystous cyanobacteria representing 12 genera have been assessed on an evolutionary landscape using rbcl gene sequence data-based phylogenomics and evogenomics approaches. Phylogenetic affiliations have clearly demonstrated the polyphyly of the true branching cyanobacteria, along with a frequent intermixing amongst the heterocystous cyanobacteria. The monophyletic origin of the heterocystous cyanobacteria was also quite evident from maximum parsimony and neighbor joining analyses. Incongruency with the traditional scheme of cyanobacterial taxonomy was frequently observed, thus advocating towards some re-amendments in the cyanobacterial classificatory schemes. Evogenomics analyses of gene sequence data gave a clear indication about the greater evolutionary pace of the unbranched cyanobacteria as compared to the branched forms. It was evident that the order Nostocales would be controlling the future pace of evolution of heterocystous cyanobacteria. The cyanobacteria Nostoc was found to have the greatest genetic heterogeneity amongst the studied genera, along with some evidence towards events of lateral gene transfer amongst the heterocystous cyanobacteria in case of the rbcl gene. Thus, heterocystous cyanobacteria were found to be a fast evolving group, with estimates of gene conversion tracts pointing towards the unbranched heterocystous cyanobacteria being at the base of evolutionary diversifications of the complete heterocystous lineage.