N-acyl homoserine lactone production by bacteria within the sponge Suberites domuncula (Olivi, 1792) (Porifera, Demospongiae)

Many bacteria live in close association with sponges. Within these consortia, molecules of communication such as quorum-sensing and hormone-like molecules may occur in order to regulate the partnership. Of particular interest, bacterial N-acyl-l-homoserine lactones (AHLs) were screened in supernatants from Suberites domuncula-associated bacteria using an E. coli bioluminescent reporter system. These sponge-associated bacteria were beforehand isolated on several media supplemented or not with a sponge extract to attempt to isolate sponge-specific bacteria. Out of 81 AHL-producing bacteria, three strains requiring sponge extract to grow were selected for AHL characterization. The in vitro produced AHLs, that is, in bacterial culture supernatants, were identified as N-(3-butanoyl)-l-homoserine lactone and N-(3-oxododecanoyl)-l-homoserine lactone and quantified using LC–ESI–MS/MS. The in vivo production of AHLs by sponge-associated bacteria has also been demonstrated in a healthy host for the first time: N-(3-oxododecanoyl)-l-homoserine lactone, N-(3-hexanoyl)-l-homoserine lactone, and N-(3-heptanoyl)-l-homoserine lactone. This AHL production in sponges may suggest a potential role of these molecules between sponge-associated bacteria and/or between sponge-associated bacteria and the sponge.     

Predictable annual mass release of gametes by the coral reef sponge Neofibularia nolitangere (Porifera: Demospongiae)

Mass release of gametes of the sponge Neofibularia nolitangere (Duch. & Mich., 1864) occurs simultaneosly along the leeward coast of Curaçao over a period of three subsequent days, without any apparent spatial pattern in the sperad of activity. A population of 99 individual sponges was monitored from August through November 1984 for development and subsequent release of gametes. Release started every day of 3 3-d period (12 to 14 October) at about 1400 hrs and lasted until just after sunset (1830 hrs). Ninety percent of the population showed reproductive activity. Exactly one lunar month later (11 to 12 November), a second release of gametes occurred. In the following year the same sequence of events was observed for the original population (2 to 4 October and 1 to 2 November, 1985). In all instances the first gamete release began on the third day after the full moon. These and earlier observations on this phenomenon show a strong correlation between moon phase and the time of gamete release. Histological and field observations show separate development and release of gametes (sex-ratio males: females 1.5:1) with external fertilization. No sex-reversal occurred in the population during release activity the following year, demonstrating a clear gonochoristic and oviparous type of reproduction for N. nolitangere. First development of oocytes and spermatocytes has been observed at 36 and 7 d, respectively, before the date of first release. In both males and females, a major part of the mesohyl of the total sponge was involved in gamete development. Spermatozoa are released through the osculum as a dense white smoke; eggs consisting of oocyte-nurse cell globules, reinforced with spicules, are separately released en masse with the outgoing water stream of the female sponge. Shortly after release the eggs become sticky and show a negative buoyancy. The reproductive strategy of N. nolitangere appears to be directed at maximizing the number of surviving recruits by maintaining a high reproductive output at a short specific time interval.     

Reproductive strategies of Mycalecontarenii (Porifera: Demospongiae)

The sexual and asexual phases of the reproductive cycle of the sponge Mycalecontarenii (Martens, 1824) were studied from samples collected over a period of 2 years (June 1994 to May 1996) in a Mediterranean coastal basin (Porto Cesareo, southwestern Apulia). The species is viviparous; year round it shows a discontinuous oocyte production. No males were found, possibly owing to the very short period of spermatogenesis. M.␣contarenii produces asexual buds during autumn and winter, and this process seems to be related to changes in water temperature. Even though, at present, we cannot evaluate the incidence of larval recruitment, we postulate that asexual reproduction in M.contarenii plays a crucial role in maintaining the sponge population. Received: 8 July 1997 / Accepted: 5 November 1997     

Genetic differentiation between morphotypes of the marine sponge Suberites ficus (Demospongiae: Hadromerida)

Sponges of three morphotypes of Suberites ficus (Johnston, 1842) were collected during February and March 1985 off the south-west of the Isle of Man, and were compared by using spicule size distributions and genetic allele frequencies of isozyme loci. The populations did not show any significant differences of spicule size or type, but could be easily differentiated into three separate species based on isozyme patterns. Samples of pale orange S. ficus growing on gastropod shells inhabited by hermit crabs (Pagurus spp.) were reproductively isolated from the redorange and the pale yellow colour morphs encrusting the bivalve Chlamys opercularis. These latter two colour morphs were genetically similar, but significant differences were observed at two of the 19 gene loci assayed. All the sponges studied were sympatric, and therefore the genetic differences, indicating reproductive isolation, are strong evidence for separate gene pools and, hence, that they are different species. The genetic identity between the two colour morphs of S. ficus on C. opercularis shells was 0.977, whilst between each of these and S. ficus on hermit crabs it was about 0.65. In all three species genetic variability was high, with mean expected and observed heterozygosity values per locus ranging from 0.17 to 0.36.     

Silica content and spicule size variations in Pellina semitubulosa (Porifera: Demospongiae)

 The variations in both silica content and spicular size were studied in two populations of the demosponge Pellina semitubulosa (Lieberkühn). Samples were collected over a period of 1 year (June 1994 to May 1995) in two Mediterranean coastal basins: Porto Cesareo (southwestern Apulia) and Marsala (northwestern Sicily). The values of spicule size (length and width) and sponge silica content were significantly higher in the population of Porto Cesareo, where the highest water silica concentration was recorded. In both Porto Cesareo and Marsala the sponge silica content showed a seasonal trend, positively correlated with water temperature values. In both populations, the smallest spicules were found in specimens collected from summer to late autumn, after sexual reproduction. Secretion of new spicules may be connected with the process of remodelling occurring in sponges after gamete and larval release. Received: 10 October 1999 / Accepted: 13 April 2000     

Modelling growth forms of the spongeHaliclona oculata (Porifera,Demospongiae) using fractal techniques

The radiate-accretive growth process of the spongeHaliclona oculata, under different environmental conditions, is simulated in a two-dimensional model with fractal modelling techniques. In this model material is added in layers to the object, and growth velocities attain highest values at its protrusions. With this model some aspects of the growth process can be explained. It is possible to simulate thin-branching growth forms, which are normally found under sheltered conditions, and plate-like forms, which are typical for sites more exposed to water movement. These simulated forms are compared with actual growth forms in order to test the validity of the model.     

Sponge-specific bacterial symbionts in the Caribbean sponge, Chondrilla nucula (Demospongiae, Chondrosida)

Marine sponges harbor dense and highly diverse bacterial communities, and some percentage of the microflora appears to be specialized for the sponge habitat. Bacterial diversity was examined in Chondrilla nucula Schmidt to test the hypothesis that some subset of sponge symbiont communities is highly similar regardless of the species of host or habitat requirements of the host. C. nucula was collected from a mangrove channel on Lower Matcumbe Key in the Florida Keys (25°53′N; 80°42′W) in August 1999. Domain-specific universal bacterial primers were used to amplify the 16S rDNA gene from genomic DNA that had been extracted from sponges and the surrounding water. An RFLP technique was used to assess diversity of sponge-associated and environmental bacterial communities. The clone library from C. nucula contained 21 operational taxonomic units (OTUs). None of the 53 OTUs from adjacent water samples were found in the C. nucula library indicating that a distinct community was present in the sponge. Sequence analysis indicated that C. nucula harbors a microbial community as diverse as the microbes from other sponges in different habitats around the world. Phylogenetic analysis placed several C. nucula clones in clades dominated by bacteria that appear to be sponge specialists (e.g., Acidobacteria, Bacteroidetes, and Cyanobacteria). Proportional representation of major bacterial taxonomic groups represented in symbiont communities was compared as a function of geographic location of sponge hosts. This study supports the hypothesis that sponges from different oceans existing in dissimilar habitats harbor closely related bacteria that are distinct from other bacterial lineages and appear specialized for residing within sponges.     

Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon

The sexual and asexual phases of reproductive cycles of two sponges, Tethya citrina and T. aurantium, living sympatrically in a Mediterranean coastal lagoon (Stagnone di Marsala, NW Sicily) were studied from samples collected over an 18-mo period. Both species are oviparous and gonochoric. They have a summer, partially overlapping, period of oocyte production, although T. citrina appear to mature earlier. No males were found, possibly due to the very short period of spermatogenesis. Both species produce asexual buds during the autumn/winter months. However, they seem to follow different reproductive strategies, with T. citrina showing a significantly lower production of buds than T. aurantium; by contrast, egg production is significantly lower in the latter species. The difference in the reproductive resource allocation is consistent with data reported in the literature on the anatomy features, genetic population structure and ecological distribution.     

Sessile and non-sessile morphs of Geodia cydonium (Jameson) (Porifera, Demospongiae) in two semi-enclosed Mediterranean bays

Morphological plasticity and ecological aspects of the demosponge Geodia cydonium (Jameson) were studied from seasonal samples collected over 1 year in two semi-enclosed Mediterranean bays of the Southern Italian coast (Marsala lagoon and Porto Cesareo basin). Sponge specimens present two morphs: sessile and non-sessile, both of which showed constant size distribution and density over the studied year. Sessile specimens were larger in size than non-sessile ones. This feature is particularly evident at Porto Cesareo, where these sponges have a more compact skeletal network than at Marsala (evident both in the cortical spicule size and sponge silica content). Sessile specimens adhere to hard rocky substrates (Porto Cesareo) or phanerogam rhizomes (Marsala); non-sessile ones occur on soft bottom areas. Several morphological and structural features of the non-sessile forms differ in the two environments, but the difference in body shape seems to play the most relevant role in enhancing the colonization of incoherent substrates. Indeed, at Marsala, where the large amount of silt and clay determines the occurrence of a markedly reduced anoxic layer just below the surface of the sediment, non-sessile specimens of G. cydonium are fairly spherical and thus able to roll, dragged by slow circular currents. In addition, the usual association with the red alga Rytyphlöea tinctoria, which almost constantly forms a thick and continuous layer around the sponge, allows them to avoid contact with the substrate. The non-sessile specimens from Porto Cesareo inhabit sandy soft bottoms and are flattened. In such an environment, affected by moderate wave turbulence, the flattened shape widens the contact surface between the body and the substrate, thereby reducing the risk of stranding. The evident signs of abrasion, provided by scanning electron microscopy investigations, on both cortical spicules and outermost sponge surface suggest that sponges rub on the bottom. Sediment, epibiontic organisms, and the phanerogam leaves protect this sciaphilous sponge from high solar radiation, allowing the specimens to live in these shallow environments.     

The intrasponge fauna of Spheciospongia vesparia (Porifera,Demospongiae) at Curaçao and bonaire

The infauna of 35 individuals of Spheciospongia vesparia (Lamarck, 1814) of different volumes and from different sites and depths have been inventoried and compared. The number of sponge-inhabiting taxa is logarithmically related to sponge volume. Biomass and total number of the animals contained in the sponge are directly proportional to sponge volume. Numerical and taxonomic composition of infaunas from different sampling sites is fairly constant. Biomass and total number of sponge-inhabiting animals is not significantly different for any of the 4 sampling sites. Several taxa, however, are more abundant in sponges from one or more localities. The ratio of total biomass to total number of intrasponge fauna is found to be significantly smaller for sponges collected in deep water than in shallow water. Differences from and similarities with Pearse's results (1932, 1950) on the infauna of the same sponge species at Dry Tortugas and Bimini are discussed. The relation of the number of contained taxa and the volume of a sponge is compared with the relation of island size and number of taxa present according to MacArthur and Wilson's island-theory (MacArthur, 1972). Finally the erratic occurrence of some taxa as opposed to the highly regular occurrence of some other taxa is discussed. It is concluded that the composition of the sponge-infauna in specimens larger than 11 is highly constant and that the sponge-inhabiting fauna constitutes an ecological community.     

Unravelling host and symbiont phylogenies of halichondrid sponges (Demospongiae, Porifera) using a mitochondrial marker

We present the first comparative phylogenetic analysis of a selected set of marine sponges and their bacterial associates. The Halichondrida form an important order in demosponge systematics and are of a particular interest due to the production of secondary metabolites. We sequenced a fragment of the cytochrome oxidase subunit 1 (CO1) gene of the sponges and their bacterial associates, compared the reconstructed phylogenies and found evidence for radiation in coevolution. The tree of six host-species associations showed four supported cospeciation events between the sponges and the bacteria. In addition, we present the first gene tree of sponges based on a mitochondrial marker. The tree shows major congruences with previous morphological studies and suggests the applicability of a mitochondrial marker in sponge molecular systematics.     

Location of toxicity within the Mediterranean sponge Crambe crambe (Demospongiae: Poecilosclerida)

Within-specimen location of toxicity in Crambe crambe (Schmidt) has been addressed by complementary procedures on specimens collected in north-east Spain (Western Mediterranean) in winter of 1993. The toxicity of the distal (ectosome) and basal (choanosome) sponge parts have been analysed and the main cellular types present in these two layers have been studied by light and electron microscopy. The toxicity of the three main cell types, separated by the gradient-density method, has also been analysed. Three main fractions, each of them enriched in a different cellular type, were obtained: Fraction 1 (interface between 2 and 5% Ficoll) contained 90±0.9% (mean±SE) of spherulous cells and 10% of different cell types consisting of choanocytes (5±0.54%), and unidentified cells or cell debris (5±0.84%); Fraction 2 (interface between 5 and 8% Ficoll) was enriched in choanocytes (70±0.95%), and also contained spherulous cells (11.8±0.73%), archeocytes (6.2±0.74%) and unidentified sponge cells (12±0.74%); Fraction 3 (interface between 8 and 11% Ficoll) mainly consisted of archeocytes and archeocyte-like cells (75±0.66%), together with spherulous cells (7±0.74%) and other unidentified sponge cells and cell aggregates mainly formed by choanocytes (18±0.41%). Toxicity [measured in toxicity units, TU, using the Microtox^® procedure] was significantly higher in the sponge ectosome (12.45±1.4 TU) than in the choanosome (2.58±0.92 UT). Only the abundance of spherulous cells in the sponge tissues correlated well with the pattern of toxicity observed, and this was corroborated by the toxic behaviour of the three cellular fractions obtained: the one enriched in spherulous cells was highly toxic (9.08 UT), whereas those enriched in choanocytes and in archeocytes were almost inactive (0.48 UT) or totally innocuous, respectively. All these results point to the spherulous cells being responsible for the storage (and possibly production) of the toxic compounds in C. crambe. Toxicity is concentrated in the sponge periphery. Spherulous cells are also concentrated in this area and can also be observed outside the sponge exopinacoderm. These results correlate well with the assumption of a defensive role of toxicity, since encounters with potential epibionts, predators and competitive neighbours take place through this peripheral zone. However, we found two types of spherulous cells (orange and colourless, respectively) coexisting in the same sponge zones as well as in Cell Fraction 1. Thus, we cannot at present determine whether one or both types are responsible for the toxicity encountered, although it is likely that the two correspond to different states of the same cell type.     

Isolation of a new toxin from the sponge Latrunculia magnifica in the Gulf of Aquaba (Red Sea)

The sponge Latrunculia magnifica was found to be toxic for fish. A toxin has been isolated from this sponge by organic extraction-column chromatography and thin-layer chromatography. Several chemical properties of the toxin were determined by spectroscopic analysis. Toxicity tests were performed on the fish Gambusia affinis. In vitro experiments showed the toxin to be a cholinesterase inhibitor.     

Growth dynamics and mortality of the intertidal encrusting sponge Halichondria okadai (Demospongiae, Halichondrida)

A periodical survey of the growth of an intertidal encrusting sponge Halichondria okadai was conducted on a rocky intertidal shore on the Izu Peninsula, southern Japan. The area covered by each of 16 marked sponges was monitored, and the fusion and fission of specimens were recorded from June 1995 to June 1998. During the investigation, 15 of the original specimens managed to survive accompanying fusions or fissions and only one colony completely disappeared. Ten fusions and 23 fissions were observed in total. The maximum death rate during a semilunar period was 16.7% and cumulative mortality throughout the study period was 46.1%, indicating that the mortality of H. okadai is lower than that of other species previously reported. There was no particular seasonal pattern in the occurrences of fusion, fission and death of sponges. However, fissions were frequently observed in the latter half of the study period and were sometimes followed by fusions or death of specimens. Positive growth rates were often observed during warmer months, and specimens showed little growth or regression in winter. Seasonal patterns in surface area were thought to be related to seasonality in reproductive activity.     

Carotenoid patterns in twenty-nine species of sponges in the order Poecilosclerida (Porifera: Demospongiae): a possible tool for chemosystematics

We assessed the utility of separated carotenoid mixtures for sponge systematics. Carotenoids were extracted from 29 species of 22 genera of 6 families for the demosponge order Poecilosclerida. Mixtures were separated (by thinlayer chromatography on silica gel) without chemical modification and after alteration by saponification, reduction or acetylation or a combination thereof. This approach allowed analysis of individual colonies of less than 1 g wet weight. Relationships among taxa were determined through use of Lawson's similarity index and discriminative analysis. Analysis of multiple runs of fractions from one individual of Ophlitaspongia pennata showed the method to be consistent and reliable. Comparison of specimens of species collected at different localities and times showed high correspondence of carotenoid patterns except in those species suspected of representing species complexes. Analysis of members of the family Clathriidae and of relationships within the order Poecilosclerida suggest that fatty acids esterified to carotenoids may be family-specific and of more taxonomic value than carotenoids. The importance of testing for seasonal, geographic and habitat variability in biochemical patterns is stressed. We discuss the significance of these methods to sponge systematics and their use in comparative studies.     

Biochemical genetic divergence and systematics in sponges of the genera Corticium and Oscarella (Demospongiae: Homoscleromorpha) in the Mediterranean Sea

The sponge sub-class Homoscleromorpha is generally considered to include just two families, the Oscarellidae (without spicules) and the Plakinidae (with simple spicules). In May 1990, an unusual sponge was found deep inside a submarine cave in the western Mediterranean Sea. On the basis of externally visible characters this sponge appeared indistinguishable from the common plakinid species Corticium candelabrum Schmidt, 1862. However, on closer examination in the laboratory the new sponge proved to be devoid of spicules. Therefore, despite great morphological similarities to C. candelabrum, the new sponge should, by taxonomic convention, have been placed in the Oscarellidae. On the basis of other criteria, the similarities to C. candelabrum were great and the new sponge was at first considered to be conspecific. Thus, the taxonomic position of the new sponge and its relationship to C. candelabrum are highly confusing. It could be an aspiculate morph of C. candelabrum, or a new and undescribed related species or, lacking spicules, it could justifiably be placed in a different family (Oscarellidae). The relationship of the new sponge to C. candelabrum and also to two species of Oscarella (Oscarellidae) was assessed by the use of enzyme electrophoresis to estimate genetic divergence between species. It was found that the new sponge was reproductively isolated from sympatric C. candelabrum, with 6 of 16 loci proving diagnostic. Thus it is clear that the new sponge belongs to a different biological species. Surprisingly it was also found that, although this new species was fairly closely related to C. candelabrum (level of genetic identity, I0.47), the two Oscarella species were similarly closely related to C. candelabrum (I0.31 to 0.41) and rather less closely to the new species (I0.17 to 0.28). Indeed from genetic identity estimates, O. tuberculata is more closely related to C. candelabrum than it is to O. lobularis. It is concluded that all homoscleromorph sponges should be placed in the single family Plakinidae.     

Verification of fractal growth models of the sponge Haliclona oculata (Porifera) with transplantation experiments

The growth form of the sponge Haliclona oculata is to a significant extent determined by the environmental conditions in which the form emerges. One of the main environmental parameters affecting the growth form is exposure to water movement. In this study, a morphological growth model is used to simulate the effect on the growth process of a change in exposure to water movement. Predictions based on the model are verified by experiments in which sponges from a sheltered growth site are transplanted to an exposed site, and vice versa. The effect of the transplantation on growth forms is determined by morphological comparisons. By combining the morphological simulation model with interpretation of growth forms, it becomes possible to use the growth form of H. oculata for bio-monitoring purposes. This form reflects the environmental conditions governing the growth process.     

Ultrastructural aspects of the symbiosis between two species of the genus Aphanocapsa (Cyanophyceae) and Ircinia variabilis (Demospongiae)

The association between two species of the genus Aphanocapsa (Cyanophyceae) and the sponge Ircinia variabilis has been studied by electron microscopy. A. feldmanni is localized in the mesohyl or inside the cells of the sponge, while the larger A. raspaigellae is located only in an extracellular position inside cavities of the mesohyl. Both algae differ from other symbiotic Cyanophyceae in having a normal cell wall. They are able to reproduce in symbiotic condition, but also undergo, in their various extracellular and intracellular positions, a massive process of disintegration. A large amount of algal material is dispersed in the sponge tissues, which is a confirmation, at the ultrastructural level, of trophic relationships in the symbiosis Aphanocapsa-Ircinia.     

Genetic heterogeneity among New Zealand species of hydrothermal vent mussels (Mytilidae: Bathymodiolus)

Molecular systematic studies provide evidence for three new species of Bathymodiolus-like hydrothermal vent mussels (Bivalvia: Mytilidae) from relatively shallow waters (depth less than 750 m) associated with the Kermadec Arc off northern New Zealand. Mitochondrial COI sequences from the three putative new species differed substantially from those of other known bathymodiolin species from the Pacific and Indian Oceans. Population genetic analysis of one of these species (Bathymodiolus new species NZ-1) revealed heterogeneity in allozyme gene frequencies between samples collected from two seamounts about 50 km apart. Factors that might contribute to genetic differentiation between neighbouring seamounts are discussed.Communicated by M.S. Johnson, Crawley     

Genetic evidence for two species of sprat (Sprattus) in New Zealand waters

The New Zealand sprat Sprattus antipodum (Hector) is phenotypically variable, with two body forms (deep and slender) being recognised in the Canterbury Bight of New Zealand. Electrophoretic analysis of tissue samples from the two body types showed them to be fixed for different alleles at two loci and to have very different allele frequencies at a further three loci. Heterozygosities of 0.088 and 0.104 were found for the deep type and 0.056 for the slender type. Genetic distances of 0.421 and 0.403 were found between samples of deep and slender types and 0.014 between samples of deep types. It is concluded that the two body types are separate species.