The microbiome of the sponge Aplysina caissara in two sites with different levels of anthropogenic impact.

Despite the important roles that marine sponges play in ecosystem functioning and structuring, little is known about how the sponge holobiont responds to local anthropogenic impacts. Here we assess the influence of an impacted environment (Praia Preta) on the microbial community associated with the endemic sponge Aplysina caissara in comparison to a less-impacted area (Praia do Guaecá) from the coast of São Paulo state (Brazil, southwestern Atlantic coast). We hypothesized that the local anthropogenic impacts will change the microbiome of A. caissara and that the community assembly will be driven by a different process (i.e. deterministic versus stochastic) under distinct levels of impact. The microbiome at the amplicon sequence variants level was found to be statistically distinct between sponges from the different sites, and this was also seen for the microbial communities of the surrounding seawater and sediments. Microbial communities of A. caissara from both sites were found to be assembled by deterministic processes, even though the sites presented distinct anthropogenic impacts, showing a pivotal role of the sponge host in selecting its own microbiome. Overall, this study revealed that local anthropogenic impacts altered the microbiome of A. caissara; however, assembly processes are largely determined by the sponge host.

Different Species of Marine Sponges Diverge in Osteogenic Potential When Therapeutically Applied as Natural Scaffolds for Bone Regeneration in Rats.

A highly porous structure, and an inorganic (biosilica) and collagen-like organic content (spongin) makes marine sponges potential candidates to be used as natural scaffolds in bone tissue engineering. The aim of this study was to characterize (through SEM, FTIR, EDS, XRD, pH, mass degradation and porosity tests) scaffolds produced from two species of marine sponges, Dragmacidon reticulatum (DR) and Amphimedon viridis (AV), and to evaluate the osteogenic potential of these scaffolds by using a bone defect model in rats. First, it was shown that the same chemical composition and porosity (84 ± 5% for DR and 90 ± 2% for AV) occurs among scaffolds from the two species. Higher material degradation was observed in the scaffolds of the DR group, with a greater loss of organic matter after incubation. Later, scaffolds from both species were surgically introduced in rat tibial defects, and histopathological analysis after 15 days showed the presence of neo-formed bone and osteoid tissue within the bone defect in DR, always around the silica spicules. In turn, AV exhibited a fibrous capsule around the lesion (19.9 ± 17.1%), no formation of bone tissue and only a small amount of osteoid tissue. The results showed that scaffolds manufactured from Dragmacidon reticulatum presented a more suitable structure for stimulation of osteoid tissue formation when compared to Amphimedon viridis marine sponge species.

Comparison of Different Methods for Spongin-like Collagen Extraction from Marine Sponges (Chondrilla caribensis and Aplysina fulva): Physicochemical Properties and In Vitro Biological Analysis.

This study aimed to compare different protocols (Protocol 1: P1; Protocol 2: P2; Protocol 3: P3; Protocol 4: P4) for the extraction of spongin-like collagen (SC) from marine sponges. The SEM micrographs demonstrated a fibrillar structure for the extracts from Chondrilla caribensis and the nodular/particulate aggregates for Aplysina fulva. FTIR showed for all samples peaks similar to collagen for both species. For C. caribensis, the extracts obtained using P2, P3, and P4 protocols presented higher values of extraction yield, TPQ, and GAGs. P2 and P4 showed higher values of SC concentration and for antioxidant analysis. For A. fulva, P2, P3, and P4 provided a higher extraction yield besides an increase in the antioxidant assay. For both species, no difference was observed for Col quantification and TPQ analysis; also, higher values of GAGs were found using P2 and P4. Fibroblast proliferation observed for C. caribensis was lower for P1 on day 1 and for P2 and P3 on day 3 (for 50%) compared to the control group. There was a significant reduction in fibroblast cell proliferation for all A. fulva extracts evaluated. It can be concluded that protocols P2 and P4 were more efficient for extracting SC from C. caribensis.

Prokaryotic, Fungal, and Unicellular Eukaryotic Core Communities Across Three Sympatric Marine Sponges From the Southwestern Atlantic Coast Are Dominated Largely by Deterministic Assemblage Processes.

Marine sponges are known to harbor a diverse and complex microbiota; however, a vast majority of surveys have been investigating the prokaryotic communities in the north hemisphere and Australia. In addition, the mechanisms of microbial community assembly are poorly understood in this pivotal player of the ecosystem. Thus, this survey addressed the holobiome of the sponge species in the São Paulo region (Brazil) for the first time and investigated the contribution of neutral and niche processes of prokaryotic, fungal, and unicellular eukaryotic assemblage in three sympatric species Aplysina caissara, Aplysina fulva, and Tedania ignis along with environmental samples. The compositions of the holobiome associated with the sponges and detected in environmental samples were strikingly different. Remarkably, between 47 and 88% of the assigned operational taxonomic units (OTUs) were specifically associated with sponge species. Moreover, around 77, 69, and 53% of the unclassified OTUs from prokaryotic, fungal, and unicellular eukaryotic communities, respectively, showed less than 97% similarity with well-known databases, suggesting that sponges from the southwestern Atlantic coast are an important source of microbial novelty. These values are even higher, around 80 and 61% of the unclassified OTUs, when excluding low abundance samples from fungal and unicellular eukaryotic datasets, respectively. Host species were the major driver shaping the sponge-associated microbial community. Deterministic processes were primarily responsible for the assembly of microbial communities in all sponge species, while neutral processes of prokaryotic and fungal community assembly were also detected in the sympatric A. caissara and T. ignis replicates, respectively. Most of the species-rich sponge-associated lineages from this region are also found in the Northern seas and many of them might play essential roles in the symbioses, such as biosynthesis of secondary metabolites that exhibit antimicrobial and antiviral activities, as well as provide protection against host predation. Overall, in this study the microbiota was assembled by interactions with the host sponge in a deterministic-based manner; closely related sponge species shared a strong phylogenetic signal in their associated prokaryotic and fungal community traits and Brazilian sponges were a reservoir of novel microbial species.

Cytocentrifugation as an additional method to study echinoderm coelomocytes: a comparative approach combining living cells, stained preparations, and energy-dispersive x-ray spectroscopy

Introduction: Echinoderm coelomocytes have traditionally been investigated through a morphological approach using light microscopy, which relies on the idea of constant cell shape as a stable character. However, this can be affected by biotic or abiotic conditions. Objective: To analyze if the consistency in cell morphology offered by the cytocentrifugation method, might be used as a convenient tool to study echinoderm coelomocytes. Methods: Cells of Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus, and Echinometra lucunter (Echinoidea) were spread on microscope slides by cytocentrifugation, stained, and analyzed through light microscopy. Additionally, fluorescence microscopy, scanning electron microscopy, and energy-dispersive x-ray spectroscopy were applied to cytospin preparations, to complement the analysis of granular and colorless spherulocytes of Eucidaris tribuloides. Results: Altogether, 11 cell types, including phagocytes, spherulocytes, vibratile cells, and progenitor cells were identified in the samples analyzed. The granular spherulocyte, a newly-described cell type, was observed in all Echinoidea and was very similar to the acidophilic spherulocytes of Holothuria (Holothuria) tubulosa. Conclusions: Cytocentrifugation proved to be versatile, either as the main method of investigation in stained preparations, or as a framework on which other procedures may be performed. Its ability to maintain a constant morphology allowed accurate correspondence between live and fixed/stained cells, differentiation among similar spherulocytes as well as comparisons between similar cells of Holothuroidea and Echinoidea.

Introducción: Los celomocitos de equinodermos se han investigado tradicionalmente a través de un enfoque morfológico utilizando microscopía óptica, que se basa en la idea de la forma celular constante como un carácter estable. Sin embargo, esto puede verse afectado por condiciones bióticas o abióticas. Objetivo: Analizar si la consistencia en la morfología celular que ofrece el método de citocentrifugación podría utilizarse como una herramienta conveniente para estudiar los celomocitos de equinodermos. Métodos: Células de Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus y Echinometra lucunter (Echinoidea) se esparcieron en portaobjetos de microscopio por citocentrifugación, se tiñeron y analizaron mediante microscopía óptica. Adicionalmente, se aplicó microscopía de fluorescencia, microscopía electrónica de barrido y espectroscopía de rayos X con dispersión de energía a las preparaciones de citoespina, para complementar el análisis de los esferulocitos granulares e incoloros de Eucidaris tribuloides. Resultados: En total, se identificaron en las muestras analizadas 11 tipos de células, incluidos fagocitos, esferulocitos, células vibrátiles y células progenitoras. El esferulocito granular, un tipo de célula recién descrito, se observó en todos los Echinoidea y fue muy similar a los esferulocitos acidófilos de Holothuria (Holothuria) tubulosa. Conclusiones: La citocentrifugación demostró ser un método bastante versátil, ya sea como el método principal de investigación en preparaciones teñidas o como un marco en el que se pueden realizar otros procedimientos. Su capacidad para mantener una morfología constante permitió una correspondencia precisa entre las células vivas y las células fijas/teñidas, la diferenciación entre esferulocitos similares, así como comparaciones entre células similares de Holothuroidea y Echinoidea.

Comparative study of coelomocytes from Arbacia lixula and Lythechinus variegatus: Cell characterization and in vivo evidence of the physiological function of vibratile cells.

The knowledge on echinoderm coelomocytes has increased in recent years, but researchers still face a complex problem: how to obtain purified cells. Even flow cytometry being useful to address coelomocytes in suspension, the need for a method able to provide isolated cells is still noteworthy. Here, we use Imaging Flow Cytometry (IFC) to characterize the coelomocytes of two sea urchin species - Arbacia lixula and Lytechinus variegatus - and obtain gates to isolate cell populations. Then, we used these gates to study the physiological response of A. lixula coelomocytes during an induced immune challenge with Escherichia coli. An analysis of area and aspect ratio parameters of the flow cytometer allowed the identification of two main cell populations in the coelomic fluid: circular and elongated cells. A combination of this method with nucleus labeling using propidium iodide allowed the determination of gates containing isolated subpopulations of vibratile cells, red spherulocytes, and two phagocytes subpopulations in both species. We observed that during an induced bacterial immune challenge, A. lixula was able to modulate coelomocyte frequencies, increasing the phagocytes and decreasing red spherulocytes and vibratile cells. These results indicate that vibratile cells and red spherulocytes act by immobilizing and stoping bacterial growth, respectively, cooperating with phagocytes in the immune response. The use of IFC was fundamental not only to identify specific gates for the main coelomic subpopulations but also allowed the investigation on how echinoids modulate their physiological responses during immune challenges. Furthermore, we provide the first experimental evidence about the role of vibratile cells, corroborating its involvement with the immune system.

Characterization of cadmium transport in hepatopancreatic cells of a mangrove crab Ucides cordatus: The role of calcium.

Cadmium is a toxic metal, present in batteries and discarded in estuaries and mangrove habitats. Apart from that, it is a non-essential metal that causes toxic effects in many organisms. Cadmium accumulates in gills and hepatopancreas of crustaceans and its route into the cell is unknown. It is possible that occurs by calcium channels or calcium transporters. The objective of this study was to characterize the transport of cadmium and the role of calcium in different cell types from hepatopancreas of the mangrove crab Ucides cordatus. For this, the hepatopancreas was dissociated by magnetic stirring and after that separated by a sucrose gradient. Then, the cells were labeled with FluoZin-3 AM and different CdCl2 concentrations were added together with a variety of inhibitors. The results showed that Cd2+ transport occurs differently in each cell type from hepatopancreas and is partially explained by the function the cells perform in this organ. Embryonic (E) and Resorptive (R) cells transported more Cd2+ compared to Fibrillar (F) and Blister (B) cells. R cells responded to Ca2+ channel inhibitors and intracellular Ca2+ manipulations positively, as the other cell types and in a stronger way. B cells were the least responsive to Ca2+ channel inhibitors and, unlike the other cells, showed a competition of Cd2+ with intracellular Ca2+ manipulations. The results indicate that Ca2+ affects the transport of Cd2+ in hepatopancreatic cells of Ucides cordatus and uses Ca2+ channels to enter these cells. In addition, information about Ca concentration could be used as a mitigating factor for Cd accumulation in crabs' hepatopancreas.

Bioleaching of electronic waste using bacteria isolated from the marine sponge Hymeniacidon heliophila (Porifera).

The bacteria isolated from Hymeniacidon heliophila sponge cells showed bioleaching activity. The most active strain, Hyhel-1, identified as Bacillus sp., was selected for bioleaching tests under two different temperatures, 30°C and 40°C, showing rod-shaped cells and filamentous growth, respectively. At 30°C, the bacteria secreted substances which linked to the leached copper, and at 40°C metallic nanoparticles were produced inside the cells. In addition, infrared analysis detected COOH groups and linear peptides in the tested bacteria at both temperatures. The Hyhel-1 strain in presence of electronic waste (e-waste) induced the formation of crust, which could be observed due to bacteria growing on the e-waste fragment. SEM-EDS measurements showed that the bacterial net surface was composed mostly of iron (16.1% w/w), while a higher concentration of copper was observed in the supernatant (1.7% w/w) and in the precipitated (49.8% w/w). The substances linked to copper in the supernatant were sequenced by MALDI-TOF-ms/ms and identified as macrocyclic surfactin-like peptides, similar to the basic sequence of Iturin, a lipopeptide from Bacillus subtilis. Finally, the results showed that Hyhel-1 is a bioleaching bacteria and cooper nanoparticles producer and that this bacteria could be used as a copper recovery tool from electronic waste.

Physiological differences in the crab Ucides cordatus from two populations inhabiting mangroves with different levels of cadmium contamination.

Crustaceans found in metal-contaminated regions are able to survive, and the authors investigated the physiological mechanisms involved by comparing populations from contaminated and noncontaminated areas. The objective of the present study was to measure the cellular transport of a nonessential metal (cadmium [Cd]) in gills and hepatopancreas of Ucides cordatus, together with cell membrane fluidity, metallothionein levels, and lipid peroxidation. The 2 populations compared were from a polluted and a nonpolluted mangrove area of São Paulo State, Brazil. The authors found, for the first time, larger Cd transport in gills and hepatopancreatic cells from crabs living in polluted mangrove areas. The cells also had lower plasma membrane fluidity, increased lipid peroxidation and less metallothionein compared to those from nonpolluted regions. The authors also found larger amounts of Cd in intracellular organelles of gills, but not in the hepatopancreas, from crabs in polluted regions. Therefore, in polluted areas, these animals showed higher Cd transport and lower plasma membrane fluidity and storage of Cd intracellularly in gill cells, whereas hepatopancreatic cells used metallothionein as their main line of defense. The findings suggest that crabs from polluted areas can accumulate Cd more easily than crabs from nonpolluted areas, probably because of an impairment of the regulatory mechanisms of cell membrane transport. Environ Toxicol Chem 2017;36:361-371. © 2016 SETAC.

Isolation and cultivation of fungal strains from in vitro cell cultures of two marine sponges (Porifera: Halichondrida and Haplosclerida)

Despite the large number of reports describing sponge-microbe associations, limited knowledge is available about associated fungi and their relationships with the hosts. In this work, specific fungal strains were obtained directly from in vitro sponge cell cultures (primmorphs) and single sponge cells (cytospins) and compared with those obtained from whole tissue preparations. A total of 27 fungal strains were isolated from the marine sponges Hymeniacidon heliophila and Haliclona melana. Fifteen strains, nine from H. heliophila and six from H. melana, were obtained from whole tissue and were considered as possible mesohyl associated or transient fungi. Twelve strains were isolated from in vitro sponge cell cultures (primmorphs) and were, therefore, considered as cell associated. From these, five different strains were obtained from H. heliophila isolated cells, while five were identified from cytospins and two from primmorphs of H. melana. The fungal strains obtained from cell cultures from both sponge species were different, and none of them were detected in the whole tissue preparations of the same species. Nine H. heliophila and seven H. melana strains shows low similarity with the sequences available in public databases and belong to potentially new species. This is the first report of fungi isolated directly from sponge cells, which allowed the observation and selection of specific strains that probably would not be obtained by usual culture dependent techniques.

Separation and viability of gill and hepatopancreatic cells of a mangrove crab Ucides cordatus.

The gills contain essential cells for respiration and osmoregulation, whereas the hepatopancreas is the site of digestion, absorption, and nutrients storage. The aim of this work was to separate and characterize gill and hepatopancreatic cells of the mangrove crab, Ucides cordatus. For gills, the methodology consisted of an enzymatic cellular dissociation using Trypsin at 0.5%, observation of cellular viability with Tripan Blue, and separation of cells using discontinuous sucrose gradient at concentrations of 10%, 20%, 30%, and 40%. The hepatopancreatic cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at the same concentrations above. For gills, a high cellular viability was observed (92.5±2.1%), with hemocyte cells in 10% sucrose layer (57.99 ± 0.17%, *P < 0.05), principal cells in the 20% sucrose layer (57.33 ± 0.18, *P < 0.05), and thick cells and pillar cells in the 30% and 40% sucrose layers, respectively (39.54 ± 0.05%, *P < 0.05; and 41.81 ± 0.04%, *P < 0.05). The hepatopancreatic cells also showed good viability (79.22 ± 0.02%), with the observation of embryonic (E) cells in the 10% sucrose layer (67.87 ± 0.06%, **P < 0.001), resorptive (R) and fibrillar (F) cells in the 20% and 30% sucrose layers (44.71 ± 0.06%, **P < 0.001, and 43.25 ± 0.01%, *P < 0.05; respectively), and blister (B) cells in the 40% sucrose layer (63.09 ± 0.03%, **P < 0.001). The results are a starting point for in vitro studies of heavy metal transport in isolated cells of the mangrove crab U. cordatus, subjected to contamination by metals in the mangrove habitat where they are found.

Isolation and cultivation of fungal strains from in vitro cell cultures of two marine sponges (Porifera: Halichondrida and Haplosclerida).

Despite the large number of reports describing sponge-microbe associations, limited knowledge is available about associated fungi and their relationships with the hosts. In this work, specific fungal strains were obtained directly from in vitro sponge cell cultures (primmorphs) and single sponge cells (cytospins) and compared with those obtained from whole tissue preparations. A total of 27 fungal strains were isolated from the marine sponges Hymeniacidon heliophila and Haliclona melana. Fifteen strains, nine from H. heliophila and six from H. melana, were obtained from whole tissue and were considered as possible mesohyl associated or transient fungi. Twelve strains were isolated from in vitro sponge cell cultures (primmorphs) and were, therefore, considered as cell associated. From these, five different strains were obtained from H. heliophila isolated cells, while five were identified from cytospins and two from primmorphs of H. melana. The fungal strains obtained from cell cultures from both sponge species were different, and none of them were detected in the whole tissue preparations of the same species. Nine H. heliophila and seven H. melana strains shows low similarity with the sequences available in public databases and belong to potentially new species. This is the first report of fungi isolated directly from sponge cells, which allowed the observation and selection of specific strains that probably would not be obtained by usual culture dependent techniques.