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.     

A blue carotenoprotein from the marine sponge Suberites domuncula: Purification and properties

A blue carotenoprotein, extracted from the marine spongia Suberites domuncula has been purified. The apoprotein has a molecular weight of about 31,000; the chromophore is not astaxanthin but it is similar to a monohydroxy, mono-expoxycarotene. The amino acid composition of the protein is briefly discussed.     

The systematic position of some boring sponges (Demospongiae,Hadromerida) studied by molecular analysis

A phylogenetic analysis of some bioeroding sponges of the family Clionaidae (Order Hadromerida) was performed to resolve some taxonomic problems both at the specific and the supraspecific level using the D2 and D3 regions of 28S rDNA. Species belonging to the genera Cliona, Cliothosa, Spheciospongia (fam. Clionaidae) and Diplastrella (fam. Spirastrellidae) from the Mediterranean Sea and Celebes Sea (Indonesia) were analysed. In the phylogenetic tree, the species clustered on two main branches, one comprising Cliona celata, C. rhodensis, C. utricularis, and Cliothosa hancocki, and the other made up of C. viridis, C. nigricans, C. schmidti, C. jullieni, Spheciospongia solida and S. vagabunda. Above the species level, data do not support the separation of the genus Cliothosa from Cliona, while they do support the inclusion of some massive boring species, previously assigned to the genus Spirastrella, in the family Clionaidae. At the species level, data demonstrated the genetic identity of taxa C. viridis and C. nigricans, in spite of their considerable morphological differences. In contrast, the yellow species commonly attributed to C. celata are probably to be considered as a complex of sibling species with a number of distinct taxa present in the Mediterranean. Data also showed the identity of the Mediterranean and Pacific populations of C. schmidti, suggesting the status of a Tethyan relict for this species.     

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.     

Genetic, morphological, and chemical divergence in the sponge genus Latrunculia (Porifera: Demospongiae) from New Zealand

We undertook a comprehensive study of Latrunculia in New Zealand to determine the relationship between taxonomic, environmental, and chemical variation within the genus. Sponges were collected from five locations around New Zealand: Three Kings Islands, Tutukaka, Wellington, Kaikoura, and Doubtful Sound. Allozyme electrophoresis at nine polymorphic loci indicated that sponges from each geographic location were genetically distinct, and that they displayed genetic differences of the magnitude usually associated with reproductively isolated species (Nei's D between locations =0.375-2.476). Additionally, the comparisons revealed that the green and brown colour morphs of Latrunculia that are sympatric at Three Kings Islands and Kaikoura are distinct from each other, and that there are two genetic groups within the green sponges in Doubtful Sound. On the basis of genetic data we conclude that there are at least eight species of Latrunculia in New Zealand waters, not one to four as had been previously thought. Morphological comparisons of the eight genetic species based on skeletal characters (i.e. skeletal organisation of the choanosome, spicule composition, size, and geometry) indicated that the eight Latrunculia species fell into only two morphological groups that could be easily diagnosed on the basis of discorhabd type. Within these two primary morphological groups, skeletal characteristics among the eight species largely overlap and are not diagnostic. These findings emphasise the limitations of traditional taxonomic methods based solely on skeletal characters for distinguishing species of Latrunculia. However, multivariate analysis (MANOVA and CDA) based on six measured skeletal variables did reveal significant morphological variation among the species (Pillai's Trace=3.28, F=6.90, P<0.0001), supporting the division of the genus into eight species. Comparisons of chemical extracts from Latrunculia also showed that the amounts of five different bioactive compounds (discorhabdins A, B, C, D, and J) varied predictably among the eight species. This finding suggests that discorhabdin variation within Latrunculia, previously thought to be associated with intra-specific environmental variability, is more likely to reflect differences among species rather than phenotypic plasticity. Our results also highlight the importance of thorough taxonomic studies associated with marine natural products research to understand fully the variation in bioactive properties among individuals. The potential processes underlying the unusually high speciation rates in New Zealand Latrunculia that are indicated in our study are discussed.     

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.     

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.     

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.     

Molecular response of the sponge Suberites domuncula to bacterial infection

The aim of this study was the documentation of the molecular immune response of Suberites domuncula upon bacterial infection. Additionally, the bacteria that are naturally present in the sponge after prolonged aquarium maintenance were characterized. After 6 months of maintenance of S. domuncula in seawater aquaria, only one bacterial 16S rDNA sequence could be recovered, which belongs to the genus Pseudomonas. Concomitantly, morphologically uniform bacteria were found encapsulated in bacteriocytes. These findings indicate that certain bacteria, possibly of the genus Pseudomonas, are able to persist for long periods in host bacteriocytes. Subsequent to performing a previously established infection assay with S. domuncula, a potentially pathogenic Vibrio sp. was isolated from the tissues. Furthermore, the host tissue disintegrated and asexual propagation bodies (gemmules) were formed. In order to gain insights into the molecular events occurring after bacterial infection, the stress-response kinases, p38 protein kinase and JNK protein kinase, were analyzed. It is demonstrated that these two kinases are activated (phosphorylated) upon incubation of the tissue with the bacterial endotoxin lipopolysaccharide (LPS). Moreover, LPS strongly inhibits protein synthesis. It is concluded that there are many functionally different interactions between S. domuncula and bacteria and that the animal possesses mechanisms to differentiate between bacteria and to respond accordingly.     

Endofauna differences between two temperate marine sponges (Demospongiae; Haplosclerida; Chalinidae) from southwest Australia

The endofaunal assemblages associated with two species of sponge from the family Chalinidae (Haliclona sp. 1 and Haliclona sp. 2) were studied at four locations along the south west coast of Australia. The species have distinct morphologies and inhabit similar microhabitats; there is also considerable scientific interest in Haliclona sp. 1 (green Haliclona) due to the unique bioactive compound it produces. A total of 948 and 287 endofaunal individuals were found associated with 16 specimens of both the green Haliclona and Haliclona sp. 2 (brown Haliclona), respectively. Twenty-four endofaunal taxa were found (from mysid shrimps to teleost fish), with the brown Haliclona having a greater density of endofaunal species and individuals than the green Haliclona. The endofaunal assemblages of both species of sponge were significantly different, but only the endofaunal assemblage within the green Haliclona differed significantly among locations. Differences in the abundance and biomass of associated endofauna of each species of sponge can be related to differences in their morphologies, size and internal structure. In the green Haliclona, differences in endofaunal assemblages among locations are unlikely to be due to environmental influences as taxa discriminating each locations assemblage were common to both species of sponge. Numerous endofaunal individuals were found to be reproductively active, and it is clear that the species of sponge provide important habitats for their associated endofauna. This provision of habitat needs to be taken into account when harvesting green Haliclona biomass for supply of its target bioactive compound for further pharmaceutical development.     

Genetic analysis of sympatric morphotypes of common dolphins (genus Delphinus)

Sympatric populations of two forms of the common dolphin, currently recognized collectively as Delphinus delphis Linnaeus, occur in several areas of the world's oceans. A molecular genetic study was initiated to determine whether these forms are genetically distinct in the Northeast Pacific. We compared mitochondrial DNA sequences from the control region and cytochrome b gene between specimens of the long-beaked and the short-beaked morphotypes collected between 1986 and 1989 off the coast of southern California. Additional short-beaked specimens collected from the eastern tropical Pacific (in 1978 and 1982) and the Black Sea (in 1989) were also compared. There were no shared mitochondrial DNA haplotypes between the two morphotypes, and both gene regions exhibited frequency and fixed nucleotide substitutions between the two morphotypes. This genetic differentiation, coupled with unique morphological characters of the short-beaked and long-beaked morphotypes determined in a parallel study, indicate that although sympatric, these populations of common dolphin are reproductively isolated from one another and may represent separate species.     

The multixenobiotic resistance mechanism in the marine sponge Suberites domuncula: its potential applicability for the evaluation of environmental pollution by toxic compounds

Experiments were carried out with the marine sponge Suberites domuncula to determine whether sponges may express-like mammalian tumor cells-a multidrug-like transporter system. The results demonstrate that sponge cells possess such a protective system termed multixenobiotic resistance (MXR) pump or P-glycoprotein-like pump. The protein was identified by antisera for the mammalian P170 multidrug resistance protein as a 130 kDa molecule. Binding studies were performed with ³H-vincristine (³H-VCR) and membrane vesicles; this process is ATP-dependent and inhibited by verapamil, which is known to reverse the multidrug-resistance phenotype in mammalian systems. Accumulation experiments were performed to demonstrate that the uptake of ³H-VCR is time-dependent, and increases at elevated extracellular levels of ³H-VCR. Application of the dyeing technique with calcein-AM, a suitable functional assay for multidrug transporter systems in mammal cells, also revealed the existence of the MXR pump in S. domuncula plasma membranes. These data demonstrate that S. domuncula is provided with a multidrug-like transporter, the MXR pump, which might function as a protection system for sponges in polluted environments.     

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     

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 differentiation in Littorina saxatilis (Gastropoda)

Littorina saxatilis, an ovoviviparous gastropod, displays considerable morphological variability, perhaps due to limited dispersal abilities. Gene and genotype frequencies of 2 polymorphic enzymatic loci were determined electrophoretically for 7 populations of L. saxatilis, to estimate degree of population differentiation. Results show that significant population differentiation may occur over distances of as little as 2 km, while widely separated populations may be nearly identical. An index of probability of genotypic identity calculated for populations of L. saxatilis shows variable and heterogeneous values. Comparison of this index for L. saxatilis with an index for 12 populations of Nassarius obsoletus —which possesses strong dispersal abilities due to long-term veliger larva and high, homogeneous indices of probability of genotypic identity between populations — indicates a strong correlation between population differentiation and dispersal ability of the organism. Possible factors influencing population differentiation in L. saxatilis are discussed.     

Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by fluorescence in situ hybridization (FISH)

We have employed electronmicroscopical methods (SEM, TEM) to document the microbial community associated with the marine sponge Aplysina cavernicola (formerly Verongia cavernicola, class Demospongiae). Five dominant bacterial types were identified, three of which resemble the morphotypes originally described by Vacelet (1975). One bacterial type possesses morphological properties that are characteristic of the genus Planctomyces. In addition, morphologically uniform bacteria which reside inside the nuclei of host cells were observed. Using in situ hybridization with fluorescently labelled rRNA probes directed against known bacterial groups, the phylogenetic affiliation of the mesohyl bacteria was assessed. It could be shown that the vast majority of mesohyl bacteria belongs to the domain Bacteria with a low GC content. Among the Bacteria, the delta-Proteobacteria were most abundant, followed by the gamma-Proteobacteria and representatives of the Bacteroides cluster. Clusters of Gram-positive bacteria with a high GC content were also found consistently in low amounts. No hybridization signal was obtained with probes specific to the domain Archaea, to the alpha- and beta-Proteobacteria and to the Cytophaga/Flavobacterium cluster. This study describes for the first time the application of the “top-to-bottom approach” using 16S rRNA probes and in situ hybridization to assess the microbial diversity in Aplysina sponges. Received: 18 December 1998 / Accepted: 12 March 1999     

Genetic differentiation between populations of Talitrus saltator and Talorchestia deshayesii (Crustacea: Amphipoda) from coastal areas of the north-western European continent

Electrophoretic studies of gene-enzyme variation in the littoral talitrids Talitrus saltator (Montagu) and Talorchestia deshayesii (Audouin) were undertaken to estimate the amount of divergence among geographically separated populations. Samples of both species were taken from sandy beaches over a transect of approximately 3 500 km along the coast of the European continent including Baltic, North Sea and Atlantic locations. A total of 22 T. saltator and 15 T. deshayesii populations were analysed for genetic variation at various enzyme loci. Both amphipods revealed relatively low levels of polymorphism and heterozygosity. Among the loci studied, phosphoglucose isomerase (Pgi) and phosphoglucomutase (Pgm) were highly polymorphic. Patterns of micro- and macrogeographic variation in terms of distributions of allele frequencies at these particular loci are compared. Interpopulation allozymic variation was shown to be lower in T. deshayesii than in T. saltator. As demonstrated by T. saltator populations sampled in coastal sites ranging from Denmark to western France, clinal variation in frequencies of two alleles became evident at the PGI locus, exhibiting a steady increase in the level of polymorphism with decreasing latitudes. It is argued that limited gene flow and, to some extent, random genetic drift may account for the gene pool structure of the talitrid species investigated.     

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     

Genetic differentiation in the intertidal zone in populations of the alga Enteromorpha linza (Ulvales: Chlorophyta)

Genetic differentiation within the intertidal zone was examined in six populations of the asexually reproducing alga Enteromorpha linza growing in the Long Island Sound, USA. Four of the five populations sampled in 1981 showed significant differentiation between high and low intertidal positions with respect to the GOT-2 locus. The pattern of differentiation was consistent for samples collected at several times during the year with some seasonal modifications. Four additional polymorphic loci, resolved in 1982, identified a total of 13 five-locus genotypes or clones. Four of the six populations sampled in 1982 showed significant differences in clone frequency between high and low intertidal positions. Laboratory experiments revealed differences in response to temperature among the clones. At 24°C a high intertidal associated clone showed an increase in growth, while low intertidal associated clones showed decreased growth compared to growth at 15°C. These results suggest that the microgeographic differentiation observed for E. linza in the intertidal zone may in part be due to the differential adaptation of clones to different intertidal environments. Additional demographic information is needed for individual clones in order to determine the role longevity, reproductive output, recruitment and interclonal competition play in maintaining the observed differentiation.