Similar sponge-associated bacteria can be acquired via both vertical and horizontal transmission.

Marine sponges host diverse communities of microorganisms that are often vertically transmitted from mother to oocyte or embryo. Horizontal transmission has often been proposed to co-occur in marine sponges, but the mechanism is poorly understood. To assess the impact of the mode of transmission on the microbial assemblages of sponges, we analysed the microbiota in sympatric sponges that have previously been reported to acquire bacteria via either vertical (Corticium candelabrum and Crambe crambe) or horizontal transmission (Petrosia ficiformis). The comparative study was performed by polymerase chain reaction-denaturing gradient gel electrophoresis and pyrosequencing of barcoded PCR-amplified 16S rRNA gene fragments. We found that P. ficiformis and C. candelabrum each harbour their own species-specific bacteria, but they are similar to other high-microbial-abundance sponges, while the low-microbial-abundance sponge C. crambe hosts microbiota of a very different phylogenetic signature. In addition, nearly 50% of the reads obtained from P. ficiformis were most closely related to bacteria that were previously reported to be vertically transmitted in other sponges and comprised vertical-horizontal transmission phylogenetic clusters (VHT clusters). Therefore, our results provide evidence for the hypothesis that similar sponge-associated bacteria can be acquired via both vertical and horizontal transmission.

Removing environmental sources of variation to gain insight on symbionts vs. transient microbes in high and low microbial abundance sponges.

In this study, we pursue unravelling the bacterial communities of 26 sponges, belonging to several taxonomical orders, and comprising low microbial abundance (LMA) and high microbial abundance (HMA) representatives. Particularly, we searched for species-specific bacteria, which could be considered as symbionts. To reduce temporal and spatial environmentally caused differences between host species, we sampled all the sponge species present in an isolated small rocky area in a single dive. The bacterial communities identified by pyrosequencing the 16S rRNA gene showed that all HMA species clustered separated from LMA sponges and seawater. HMA sponges often had highest diversity, but some LMA sponges had also very diverse bacterial communities. Network analyses indicated that no core bacterial community seemed to exist for the studied sponges, not even for such a space and time-restricted sampling. Most sequences, particularly the most abundant ones in each species, were species-specific for both HMA and LMA sponges. The bacterial sequences retrieved from LMA sponges, despite being phylogenetically more similar to seawater, did not represent transient seawater bacteria. We conclude that sponge bacterial communities depend more on the host affiliation to the HMA or LMA groups than on host phylogeny.

"Living together apart": the hidden genetic diversity of sponge populations.

Intraorganism genetic stability is assumed in most organisms. However, here we show for the first time intraorganism genetic heterogeneity in natural populations of marine sponges. A total of 36 different multilocus genotypes (MLGs) were detected in 13 individuals of Scopalina lophyropoda sampled at 4 distant points within each sponge. All genotypes (showing a mosaic distribution), were transmitted to the progeny, thus contributing to the high genetic diversity and low clonality reported for this species, although its populations are small and structured and show high fission rates. There did not seem to be intraindividual genotype conflicts; on the contrary, chimeric individuals are expected to show low mortality thanks to the differential mortality of their different MLGs. This novel mechanism may also counterbalance genetic constraints in other benthic invertebrate species. The presence of sponge chimerism also suggests that results from previous population genetics studies could have been misinterpreted.

Sponge ecology in the molecular era.

Knowledge of the functioning, health state, and capacity for recovery of marine benthic organisms and assemblages has become essential to adequately manage and preserve marine biodiversity. Molecular tools have allowed an entirely new way to tackle old and new questions in conservation biology and ecology, and sponge science is following this lead. In this review, we discuss the biological and ecological studies of sponges that have used molecular markers during the past 20 years and present an outlook for expected trends in the molecular ecology of sponges in the near future. We go from (1) the interface between inter- and intraspecies studies, to (2) phylogeography and population level analyses, (3) intra-population features such as clonality and chimerism, and (4) environmentally modulated gene expression. A range of molecular markers has been assayed with contrasting success to reveal cryptic species and to assess the genetic diversity and connectivity of sponge populations, as well as their capacity to respond to environmental changes. We discuss the pros and cons of the molecular gene partitions used to date and the prospects of a plentiful supply of new markers for sponge ecological studies in the near future, in light of recently available molecular technologies. We predict that molecular ecology studies of sponges will move from genetics (the use of one or some genes) to genomics (extensive genome or transcriptome sequencing) in the forthcoming years and that sponge ecologists will take advantage of this research trend to answer ecological and biological questions that would have been impossible to address a few years ago.

Population genetics at three spatial scales of a rare sponge living in fragmented habitats.

BACKGROUND: Rare species have seldom been studied in marine habitats, mainly because it is difficult to formally assess the status of rare species, especially in patchy benthic organisms, for which samplings are often assumed to be incomplete and, thus, inappropriate for establishing the real abundance of the species. However, many marine benthic invertebrates can be considered rare, due to the fragmentation and rarity of suitable habitats. Consequently, studies on the genetic connectivity of rare species in fragmented habitats are basic for assessing their risk of extinction, especially in the context of increased habitat fragmentation by human activities. Sponges are suitable models for studying the intra- and inter-population genetic variation of rare invertebrates, as they produce lecitotrophic larvae and are often found in fragmented habitats. RESULTS: We investigated the genetic structure of a Mediterranean sponge, Scopalina lophyropoda (Schmidt), using the allelic size variation of seven specific microsatellite loci. The species can be classified as "rare" because of its strict habitat requirements, the low number of individuals per population, and the relatively small size of its distribution range. It also presents a strong patchy distribution, philopatric larval dispersal, and both sexual and asexual reproduction. Classical genetic-variance-based methods (AMOVA) and differentiation statistics revealed that the genetic diversity of S. lophyropoda was structured at the three spatial scales studied: within populations, between populations of a geographic region, and between isolated geographic regions, although some stochastic gene flow might occur among populations within a region. The genetic structure followed an isolation-by-distance pattern according to the Mantel test. However, despite philopatric larval dispersal and fission events in the species, no single population showed inbreeding, and the contribution of clonality to the population makeup was minor (only ca. 4%). CONCLUSIONS: The structure of the S. lophyropoda populations at all spatial scales examined confirms the philopatric larval dispersal that has been reported. Asexual reproduction does not seem to play a relevant role in the populations. The heterozygote excess and the lack of inbreeding could be interpreted as a hitherto unknown outcrossing strategy of the species. The envisaged causes for this strategy are sperm dispersal, a strong selection against the mating of genetically related individuals to avoid inbreeding depression or high longevity of genets combined with stochastic recruitment events by larvae from other populations. It should be investigated whether this strategy could also explain the genetic diversity of many other patchy marine invertebrates whose populations remain healthy over time, despite their apparent rarity.

In situ aquaculture methods for Dysidea avara (Demospongiae, Porifera) in the northwestern Mediterranean.

Marine sponges produce secondary metabolites that can be used as a natural source for the design of new drugs and cosmetics. There is, however, a supply problem with these natural substances for research and eventual commercialisation of the products. In situ sponge aquaculture is nowadays one of the most reliable methods to supply pharmaceutical companies with sufficient quantities of the target compound. In this study, we focus on the aquaculture of the sponge Dysidea avara (Schmidt, 1862), which produces avarol, a sterol with interesting pharmaceutical attributes. The soft consistency of this species makes the traditional culture method based on holding explants on ropes unsuitable. We have tested alternative culture methods for D. avara and optimized the underwater structures to hold the sponges to be used in aquaculture. Explants of this sponge were mounted on horizontal ropes, inside small cages or glued to substrates. Culture efficiency was evaluated by determination of sponge survival, growth rates, and bioactivity (as an indication of production of the target metabolite). While the cage method was the best method for explant survival, the glue method was the best one for explant growth and the rope method for bioactivity.

Multipartner Symbiosis across Biological Domains: Looking at the Eukaryotic Associations from a Microbial Perspective.

Sponges establish tight associations with both micro- and macroorganisms. However, while studies on sponge microbiomes are numerous, nothing is currently known about the microbiomes of sponge-associated polychaetes and their relationships with those of their host sponges. We analyzed the bacterial communities of symbiotic polychaetes (Haplosyllis spp.) and their host sponges (Clathria reinwardti, Amphimedon paraviridis, Neofibularia hartmani, and Aaptos suberitoides) to assess the influence of the sponges on the polychaete microbiomes. We identified both eukaryote partners by molecular (16S and COI genes) and morphological features, and we identified their microbial communities by high-throughput sequencing of the 16S rRNA gene (V4 region). We unravel the existence of six Haplosyllis species (five likely undescribed) associated at very high densities with the study sponge species in Nha Trang Bay (central Vietnam). A single polychaete species inhabited A. paraviridis and was different from the single species that inhabited A. suberitoides Conversely, two different polychaete species were found in C. reinwardti and N. hartmani, depending on the two host locations. Regardless of the host sponge, polychaete microbiomes were species specific, which is a widespread feature in marine invertebrates. More than half of the polychaete bacteria were also found in the host sponge microbiome but at contrasting abundances. Thus, the associated polychaetes seemed to be able to select, incorporate, and enrich part of the sponge microbiome, a selection that appears to be polychaete species specific. Moreover, the bacterial diversity is similar in both eukaryotic partners, which additionally confirms the influence of food (host sponge) on the structure of the polychaete microbiome.IMPORTANCE The symbiotic lifestyle represents a fundamental cryptic contribution to the diversity of marine ecosystems. Sponges are ideal targets to improve understanding the symbiotic relationships from evolutionary and ecological points of view, because they are the most ancient metazoans on earth, are ubiquitous in the marine benthos, and establish complex symbiosis with both prokaryotes and animals, which in turn also harbor their own bacterial communities. Here, we study the microbiomes of sponge-polychaete associations and confirm that polychaetes feed on their host sponges. The study worms select and enrich part of the sponge microbiome to shape their own species-specific bacterial communities. Moreover, worm microbiome diversity runs parallel to that of its food host sponge. Considering our results on symbiotic polychaetes and previous studies on fishes and mammals, diet appears to be an important source of bacteria for animals to shape their species-specific microbiomes.

Sponges and Their Microbiomes Show Similar Community Metrics Across Impacted and Well-Preserved Reefs.

Sponge diversity has been reported to decrease from well-preserved to polluted environments, but whether diversity and intra-species variation of their associated microbiomes also change as function of environmental quality remains unknown. Our study aimed to assess whether microbiome composition and structure are related to the proliferation of some sponges and not others under degraded conditions. We characterized the most frequent sponges and their associated bacteria in two close areas (impacted and well-preserved) of Nha Trang Bay (Indo-Pacific). Sponge assemblages were richer and more diverse in the well-preserved reefs, but more abundant (individuals/m. transect) in the impacted environments, where two species (Clathria reinwardti and Amphimedon paraviridis) dominated. Sponge microbiomes from the polluted zones had, in general, lower bacterial diversity and core size and consequently, higher intra-species dispersion than microbiomes of sponges from the well-preserved environments. Microbial communities reflect the reduction of diversity and richness shown by their host sponges. In this sense, sponges with less complex and more variable microbiomes proliferate under degraded environmental conditions, following the ecological paradigm that negatively correlates community diversity and environmental degradation. Thereby, the diversity and structure of sponge microbiomes might indirectly determine the presence and proliferation of sponge species in certain habitats.

Biodiversity loss in a Mediterranean ecosystem due to an extreme warming event unveils the role of an engineering gorgonian species.

Stochastic perturbations can trigger major ecosystem shifts. Marine systems have been severely affected in recent years by mass mortality events related to positive thermal anomalies. Although the immediate effects in the species demography affected by mortality events are well known, information on the mid- to long-term effects at the community level is much less documented. Here, we show how an extreme warming event replaces a structurally complex habitat, dominated by long-lived species, by a simplified habitat (lower species diversity and richness) dominated by turf-forming species. On the basis of a study involving the experimental manipulation of the presence of the gorgonian Paramuricea clavata, we observed that its presence mitigated the effects of warming by maintaining the original assemblage dominated by macroinvertebrates and delaying the proliferation and spread of the invasive alga Caulerpa cylindracea. However, due to the increase of sediment and turf-forming species after the mortality event we hypothesize a further degradation of the whole assemblage as both factors decrease the recruitment of P.clavata, decrease the survival of encrusting coralligenous-dwelling macroinvertebrates and facilitate the spreading of C. cylindracea.

Isomeric furanosesquiterpenes from the Portuguese marine sponge Fasciospongia sp.

This paper reports the chemical study of a sample of Fasciospongia sp. collected along the Atlantic Portuguese coast. Three new isomeric furanosesquiterpenes, isomicrocionin-3 ( 1), (-)-microcionin-1 ( 2), and (-)-isomicrocionin-1 ( 3), were isolated along with the known (-)- ent-pallescensin A ( 4) and (-)-pallescensin-1 ( 5) from the ethyl acetate-soluble portion of the methanolic extract. The structures of the new compounds were elucidated by extensive spectroscopic studies. (-)-Microcionin-1 ( 2) tested positive against several Gram-negative bacteria.

Environmental flow regimes for Dysidea avara sponges.

The aim of our research is to design tank systems to culture Dysidea avara for the production of avarol. Flow information was needed to design culture tanks suitable for effective production. Water flow regimes were characterized over a 1-year period for a shallow rocky sublittoral environment in the Northwestern Mediterranean where D. avara sponges are particularly abundant. Three-dimensional Doppler current velocities at 8-10-m depths ranged from 5 to 15 cm/s over most seasons, occasionally spiking to 30-66 cm/s. A thermistor flow sensor was used to map flow fields in close proximity ( approximately 2 cm) to individual sponges at 4.5-, 8.8-, and 14.3-m depths. These "proximal flows" averaged 1.6 cm/s in calm seas and 5.9 cm/s during a storm, when the highest proximal flow (32.9 cm/s) was recorded next to a sponge at the shallowest station. Proximal flows diminished exponentially with depth, averaging 2.6 cm/s +/- 0.15 SE over the entire study. Flow visualization studies showed that oscillatory flow (0.20-0.33 Hz) was the most common regime around individual sponges. Sponges at the 4.5-m site maintained a compact morphology with large oscula year-around despite only seasonally high flows. Sponges at 8.8 m were more erect with large oscula on tall protuberances. At the lowest-flow 14.3-m site, sponges were more branched and heavily conulated, with small oscula. The relationship between sponge morphology and ambient flow regime is discussed.

Description of two new genera (Antarctotetilla, Levantiniella) and a new species of Tetillidae.

Tetillidae is a sponge family distributed all over the world but with some genera apparently endemic from the Antarctic and Subantarctic (the "Antarctic clade"). Species identification results tricky due to the similarities of their morphological characters. However, molecular phylogenies have helped to resolve the family taxonomy. The last phylogenetic study on Tetillidae suggested the creation of two new genera: Levantiniella and Antarctotetilla. Lenvantiniella, from Middle East Mediterranean Sea, was previously classified within Cinachyrella, from which it differs in the small rounded surface cavities, distinctive from true porocalices. Antarctotetilla has up to now an Antarctic distribution, and harbors species wrongly classified within Tethya, Craniella, or Tetilla. The main differences of Antarctotetilla to other Tetillidae genera are the presence of pores grouped in small areas, and a poorly-defined cortex (pseudocortex). This study aims to re-describe in detail the species of Tetillidae that belong in the two above mentioned new genera, and to highlight that molecular phylogenies should be combined with morphological analyses to improve taxonomical decisions. We also describe a new Tetillidae species with a hair-like hispidation, which we name Antarctotetilla pilosa nov. sp. Furthermore, the types of Tethya coactifera and T. crassispicula (Lendenfeld, 1907) were reexamined because of some morphological similarities with Antarctotetilla. The minibarcode sequences (a small COI fragment) placed them within the Antarctic clade harboring Antarctotetilla and Cinachyra, but did not resolve their genus position. A morphological revision, however, suggests placing T. coactifera in Antarctotetilla, while T. crassispicula, which owns porocalices and a spicule-reinforced cortex, appeared to belong in Cinachyra.

-Reproductive strategies of two common sympatric Mediterranean sponges: Dysidea avara (Dictyoceratida) and Phorbas tenacior (Poecilosclerida).

Despite their abundance in benthic ecosystems, life cycles and reproductive features of most sponge species remain unknown. We have studied the main reproductive features of two demosponges, Dysidea avara and Phorbas tenacior, belonging to phylogenetically distant groups: Orders Dictyoceratida and Poecilosclerida, respectively. Both sponges are abundant and share habitat in the Mediterranean rocky sublittoral. They brood parenchymella larvae with different morphology and behaviour. Sampling was conducted monthly over a two-year period in a locality where both species coexist. The two species reproduced in spring-summer, and presented species-specific reproductive features despite being subject to the same environmental conditions. D. avara has a shorter reproductive period than P. tenacior, ending before the peak of temperature in summer, while the reproductive period of P. tenacior lasts until beginning of autumn. Brooding larvae were present in June-July in D. avara, and in August-October in P. tenacior. Larval size, reproductive effort and number of larvae produced (measured the month with the maximum production) were significantly higher in D. avara than in P. tenacior. A higher reproductive effort and larval traits point to a more opportunistic life strategy in D. avara than in P. tenacior. A lack of overlap in the timing of larval release, as well as different reproductive traits, may reduce competition and facilitate the coexistence of these two sympatric and abundant sponges.

Showcasing the role of seawater in bacteria recruitment and microbiome stability in sponges.

We studied the core bacterial communities of 19 sponge species from Nha Trang Bay (Central Vietnam), with particular emphasis on the contribution of planktonic seawater bacteria to the sponge core microbiomes. To ensure consistent sponge-microbe associations and accurate identification of planktonic bacteria transmitted from seawater, we were very restrictive with the definition of the sponge core microbiomes (present in all the replicates), and with the identification of valid biological 16S rRNA gene sequences (100% sequence identity) that belonged to potentially different bacterial taxa. We found a high overlap (>50% relative abundance) between the sponge species core microbiome and the seawater bacterial core in ca. a half of the studied species, including representatives of both, HMA and LMA sponges. From our restrictive analysis, we point to horizontal transmission as a relevant way of symbiont acquisition in sponges. Some species-specific recognition mechanisms may act in sponges to enrich specific seawater bacteria in their tissues. These mechanisms would allow the maintenance of bacterial communities in a species across geographical ranges. Moreover, besides contrasting preferences in bacteria selection from seawater, divergent physiological traits may also account for the different microbiomes in species of HMA and LMA sponges.

Cell culture from sponges: pluripotency and immortality.

Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a new source of sponge material for cell culture. Stem cells are present in high amounts in embryos and are more versatile and resistant to infections than adult cells. Additionally, genetic engineering and cellular research on apoptotic mechanisms are promising new fields that might help to improve cell survival in sponge-cell lines. We propose that one topic for future research should be how to reduce apoptosis, which appears to be very high in sponge cell cultures.

Cultivation of sponge larvae: settlement, survival, and growth of juveniles.

The aim of this study was to culture sponge juveniles from larvae. Starting from larvae we expected to enhance the survival and growth, and to decrease the variation in these parameters during the sponge cultures. First, settlement success, morphological changes during metamorphosis, and survival of Dysidea avara, Ircinia oros, Hippospongia communis, under the same culture conditions, were compared. In a second step, we tested the effects of flow and food on survival and growth of juveniles from Dysidea avara and Crambe crambe. Finally, in a third experiment, we monitored survival and growth of juveniles of D. avara and C. crambe transplanted to the sea to compare laboratory and field results. The results altogether indicated that sponge culture from larvae is a promising method for sponge supply and that laboratory culture under controlled conditions is preferred over sea cultures in order to prevent biomass losses during these early life stages.

Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern mediterranean: multispecies comparison.

Contamination by heavy metals has increased drastically in the coastal Mediterranean during the last 20 years. A comparative study on metal bioaccumulation by four widespread sponge species (Crambe crambe, Chondrosia reniformis, Phorbas tenacior, and Dysidea avara) has been performed to select the most suitable species for metal monitoring. Copper bioaccumulation fits an accumulation strategy while Pb concentration seems to be regulated in most sponges. Crambe crambe was the only studied species that bioaccumulated Pb and Cu as a function of the available metal, proving its suitability for monitoring purposes. Then, we examined its effectiveness as a bioindicator at large spatial and temporal scales, comparing metal accumulation in this species and in sediments. Crambe crambe provided accurate information on the background levels of metals in the area at both spatial and temporal scales, and furthermore it reflected seasonal fluctuations of the bioavailable metals, which would be impossible to assess by means of a sediment survey.

Molecular phylogenies confirm the presence of two cryptic Hemimycale species in the Mediterranean and reveal the polyphyly of the genera Crella and Hemimycale (Demospongiae: Poecilosclerida).

BACKGROUND: Sponges are particularly prone to hiding cryptic species as their paradigmatic plasticity often favors species phenotypic convergence as a result of adaptation to similar habitat conditions. Hemimycale is a sponge genus (Family Hymedesmiidae, Order Poecilosclerida) with four formally described species, from which only Hemimycale columella has been recorded in the Atlanto-Mediterranean basin, on shallow to 80 m deep bottoms. Contrasting biological features between shallow and deep individuals of Hemimycale columella suggested larger genetic differences than those expected between sponge populations. To assess whether shallow and deep populations indeed belong to different species, we performed a phylogenetic study of Hemimycale columella across the Mediterranean. We also included other Hemimycale and Crella species from the Red Sea, with the additional aim of clarifying the relationships of the genus Hemimycale. METHODS: Hemimycale columella was sampled across the Mediterranean, and Adriatic Seas. Hemimycale arabica and Crella cyathophora were collected from the Red Sea and Pacific. From two to three specimens per species and locality were extracted, amplified for Cytochrome C Oxidase I (COI) (M1-M6 partition), 18S rRNA, and 28S (D3-D5 partition) and sequenced. Sequences were aligned using Clustal W v.1.81. Phylogenetic trees were constructed under neighbor joining (NJ), Bayesian inference (BI), and maximum likelihood (ML) criteria as implemented in Geneious software 9.01. Moreover, spicules of the target species were observed through a Scanning Electron microscope. RESULTS: The several phylogenetic reconstructions retrieved both Crella and Hemimycale polyphyletic. Strong differences in COI sequences indicated that C. cyathophora from the Red Sea might belong in a different genus, closer to Hemimycale arabica than to the Atlanto-Mediterranean Crella spp. Molecular and external morphological differences between Hemimycale arabica and the Atlanto-Mediterranean Hemimycale also suggest that Hemimycale arabica fit in a separate genus. On the other hand, the Atlanto-Mediterranean Crellidae appeared in 18S and 28S phylogenies as a sister group of the Atlanto-Mediterranean Hemimycale. Moreover, what was known up to now as Hemimycale columella, is formed by two cryptic species with contrasting bathymetric distributions. Some small but consistent morphological differences allow species distinction. CONCLUSIONS: A new family (Hemimycalidae) including the genus Hemimycale and the two purported new genera receiving C. cyathophora and Hemimycale arabica might be proposed according to our phylogenetic results. However, the inclusion of additional Operational Taxonomic Unit (OTUs) appears convenient before taking definite taxonomical decisions. A new cryptic species (Hemimycale mediterranea sp. nov.) is described. Morphologically undifferentiated species with contrasting biological traits, as those here reported, confirm that unidentified cryptic species may confound ecological studies.