Gemma Agell

Long-term culture of sponge explants: conditions enhancing survival and growth, and assessment of bioactivity

Sponges are an important source of secondary metabolites with pharmaceutical interest. This is the main reason for the increasing interest of sponge culture recent years. The optimal culture system depends on the species to be cultured: while some species easily produce sponge aggregates after dissociation (primmorphs), others show a great capacity to regenerate after fragmentation (explants). Corticium candelabrum is a Mediterranean bacteriosponge that can undergo asexual reproduction. We have taken advantage of this capability and cultured C. candelabrum explants under several experimental conditions. To find the best conditions for obtaining functional explants, we assayed a range of conditions, including seasons of collection, culture temperature, filtered versus filtered-sterile seawater, addition of antibiotics and proportion of ectosome. We monitored the changes in shape and ultrastructure during the formation of explants. After 24 h, TEM images showed the aquiferous system disarranged, in particular at the sponge periphery. From 2 to 4 weeks later, the aquiferous system regenerated, and fragments became functional sponges (explants). Explants were cultured under two regimes: in vitro and in a closed aquarium system. Antibiotics were only added to the in vitro culture to assess their effect on the symbiotic bacteria, which remained healthy despite the presence of antibiotics. Two food regimens (marine bacteria and green algae) were assayed for their ability to satisfy the metabolic requirements of explants. We monitored explant survival and growth. Explants showed a high long-term survival rate (close to 100%). Growth rates were higher in the closed aquarium system, without antibiotic addition, and fed with algae. Explants cultures were hardly contaminated because manipulation was reduced to a minimum and we used sterilized seawater. C. candelabrum produces bioactive molecules, which may play a defensive role in the sponge and may have pharmaceutical interest. The bioactivity of the explants was similar to that of wild sponges.

Endosymbiotic calcifying bacteria: A new cue to the origin of calcification in Metazoa?

Sponges show the highest diversity of associated bacteria among marine invertebrates. Immunological evidence traces the origin of the sponge bacterial symbioses to the Precambrian era. Hence, sponges appear to be ideally suited for studying the evolutionary origins of prokaryote–metazoan associations. Sponges produce either calcareous or siliceous skeletons, which only coexist in a relict group of demosponges, the sclerosponges. We report here, for the first time, intensive calcification in nonsclerosponge siliceous demosponges. Calcification is mediated by endosymbiotic bacteria (calcibacteria) located in archeocyte‐like sponge cells. These calcibacteria are devoid of bacterial walls and divide within sponge cells until they became surrounded by a calcitic sheet, being subsequently extruded to the sponge subectosomal (subepithelial) zone. Thousands of bacteria‐produced calcitic spherules cover the surface of the host sponges, forming a cortex‐like structure that mimics a rudimentary peripheral skeleton. Calcibacteria are vertically transferred to the sponge larvae during embryogenesis. Calcium detoxification may have generated this symbiotic association, with some additional benefits for the sponges, such as skeletal formation and deterrence from predation. This unique symbiosis holds implications for sponge biology and may advance discussions on the role of bacteria in early biocalcification processes in metazoans.

Ecosystem respiration increases with biofilm growth and bed forms: Flume measurements with resazurin

In a set of streamside mesocosms, stream ecosystem respiration (ER) increased with biofilm biomass and flow heterogeneity (turbulence) generated by impermeable bed forms, even though those bed forms had no hyporheic exchange. Two streamside flumes with gravel beds (single layer of gravel) were operated in parallel. The first flume had no bed forms, and the second flume had 10 cm high dune-shaped bed forms with a wavelength of 1.0 m. Ecosystem respiration was measured via resazurin reduction to resorufin in each flume at three different biomass stages during biofilm growth. Results support the hypothesis that ER increases with flow heterogeneity generated by bed forms across all biofilm biomass stages. For the same biofilm biomass, ER was up to 1.9 times larger for a flume with 10 cm high impermeable bed forms than for a flume without the bed forms. Further, the amount of increase in ER associated with impermeable bed forms was itself increased as biofilms grew. Regardless of bed forms, biofilms increased transient storage by a factor of approximately 4.

Isolation and characterization of microsatellite loci from the endangered Mediterranean sponge Spongia agaricina (Demospongiae: Dictyoceratida).

The abundance of the bath sponge Spongia agaricina has decreased drastically in recent years and it is now considered an endangered species under Annex 3 of Bern and Barcelona conventions. We describe eight microsatellite markers and present data on their allelic variation and utility as high resolution genetic markers. We analyzed 36 individuals from two populations and found that the number of alleles per locus ranged between 1 and 7. Observed heterozygosity ranged from 0 to 0.72. We found deviations from Hardy–Weinberg expectations for some loci. We exclusively detected null alleles for those loci that deviated from Hardy–Weinberg expectations. Also, distributions of allele frequencies differed significantly between the two populations, making them suitable for population genetic analyses.

Isolation and characterization of eight polymorphic microsatellite loci for the Mediterranean gorgonian Paramuricea clavata.

The gorgonian Paramuricea clavata is a benthic organism often included in conservation management plans since it creates complexity in the ecosystems and is extremely vulnerable to disturbances. Eight microsatellite markers isolated from an enriched genomic library were characterized in a total of 50 individuals from two north-western Mediterranean populations. All loci were polymorphic and the number of alleles per locus ranged between 2 and 14. Significant genetic differentiation was observed between populations. The polymorphic markers presently isolated will allow for the assessment of the spatial genetic structure between and within populations of this umbrella species of the Mediterranean Sea.

Phylogenetic Reassessment of Antarctic Tetillidae (Demospongiae, Tetractinellida) Reveals New Genera and Genetic Similarity among Morphologically Distinct Species

Species of Tetillidae are distributed worldwide. However, some genera are unresolved and only a few genera and species of this family have been described from the Antarctic. The incorporation of 25 new COI and 18S sequences of Antarctic Tetillidae to those used recently for assessing the genera phylogeny, has allowed us to improve the resolution of some poorly resolved nodes and to confirm the monophyly of previously identified clades. Classical genera such as Craniella recovered their traditional diagnosis by moving the Antarctic Tetilla from Craniella, where they were placed in the previous family phylogeny, to Antarctotetilla gen. nov. The morphological re-examination of specimens used in the previous phylogeny and their comparison to the type material revealed misidentifications. The proposed monotypic new genus Levantinella had uncertain phylogenetic relationships depending on the gene partition used. Two more clades would require the inclusion of additional species to be formally established as new genera. The parsimony tree based on morphological characters and the secondary structure of the 18S (V4 region) almost completely matched the COI M1-M6 and the COI+18S concatenated phylogenies. Morphological synapomorphies have been identified for the genera proposed. New 15 28S (D3-D5) and 11 COI I3-M11 partitions were exclusively sequenced for the Antarctic species subset. Remarkably, species within the Antarctic genera Cinachyra (C. barbata and C. antarctica) and Antarctotetilla (A. leptoderma, A. grandis, and A. sagitta), which are clearly distinguishable morphologically, were not genetically differentiated with any of the markers assayed. Thus, as it has been reported for other Antarctic sponges, both the mitochondrial and nuclear partitions used did not differentiate species that were well characterized morphologically. Antarctic Tetillidae offers a rare example of genetically cryptic (with the traditional markers used for sponges), morphologically distinct species.

Endosymbiotic calcifying bacteria across sponge species and oceans

From an evolutionary point of view, sponges are ideal targets to study marine symbioses as they are the most ancient living metazoans and harbour highly diverse microbial communities. A recently discovered association between the sponge Hemimycale columella and an intracellular bacterium that generates large amounts of calcite spherules has prompted speculation on the possible role of intracellular bacteria in the evolution of the skeleton in early animals. To gain insight into this purportedly ancestral symbiosis, we investigated the presence of symbiotic bacteria in Mediterranean and Caribbean sponges. We found four new calcibacteria OTUs belonging to the SAR116 in two orders (Poecilosclerida and Clionaida) and three families of Demospongiae, two additional OTUs in cnidarians and one more in seawater (at 98.5% similarity). Using a calcibacteria targeted probe and CARD-FISH, we also found calcibacteria in Spirophorida and Suberitida and proved that the calcifying bacteria accumulated at the sponge periphery, forming a skeletal cortex, analogous to that of siliceous microscleres in other demosponges. Bacteria-mediated skeletonization is spread in a range of phylogenetically distant species and thus the purported implication of bacteria in skeleton formation and evolution of early animals gains relevance.

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.

Characterization of nine polymorphic microsatellite loci for the calcareous sponge Paraleucilla magna Klautau et al. 2004 introduced to the Mediterranean Sea

Nine polymorphic microsatellite markers are described for the calcareous sponge Paraleucilla magna. Microsatellite were isolated from a genomic library enriched for AC15, AG15, CAA10 and GATA7 repeats. The microsatellite motifs were perfect, imperfect, and compound. The polymorphism of the microsatellite loci was screened in a total of 35 individuals from two populations: western Mediterranean (Blanes, NE Spain) and Atlantic Ocean (Rio de Janeiro, Brazil). All loci were polymorphic and the number of alleles per locus ranged from 2 to 9 with an average of ca. 5 alleles per locus. Significant genetic differentiation was observed between populations. These markers have been used successfully for analysing population structure at short spatial and temporal scales, and are promising for assessing the historic pathways of the introduction of this exotic sponge, which risks to become invasive in the Mediterranean. These are the first microsatellites developed for any calcareous sponge.

Redescription and establishment of a holotype and three paratypes for the species Hemimycale mediterranea sp. nov.

Background In a recent paper, we described a new sponge species named Hemimycale mediterranea Uriz, Garate & Agell, 2017. However, we failed to designate a holotype and a type locality, as required by the International Commission on Zoological Nomenclature (ICZN). Although the validity of the previous conclusions remains unchanged, the species name cannot be considered available according to ICZN regulations until a holotype is designated. Results The present work fulfills the requirements of the ICZN by designating a holotype, three paratypes and the type locality for the new species Hemimycale mediterranea and has been registered in ZooBank.