Mitochondrial DNA of the demosponge Acanthella acuta: Linear Architecture and Other Unique Features.

While Acanthella acuta (Schmidt 1862), a common demosponge found in the Mediterranean Sea and Atlantic Ocean, is-morphologically-little distinguishable from other sponges, its mitochondrial DNA (mtDNA) is unique within the class. In contrast to all other studied demosponges, mtDNA of A. acuta is inferred to be linear and displays several unusual features: inverted terminal repeats, group II introns in three mt-genes, and two unique ORFs. One of the ORFs (ORF1535) combines a DNA-polymerase domain with a DNA-directed RNA-polymerase domain, while the second bears no discernible similarity to any reported sequences. The group II intron within the cox2 gene is the first such intron reported in an animal. Our phylogenetic analyses indicate that the cox1 intron is related to similar introns found in other demosponges, while the cox2 intron is likely not of animal origin. The two domains found within ORF1535 do not share a common origin and, along with the cox2 intron, were likely acquired by horizontal transfer. The findings of this paper open new avenues of exploration in the understanding of mtDNA linearization within Metazoa.

The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae).

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

Marine heatwave-driven mass mortality and microbial community reorganisation in an ecologically important temperate sponge.

Marine heatwaves (MHWs) are increasing in frequency, duration and intensity, disrupting global marine ecosystems. While most reported impacts have been in tropical areas, New Zealand experienced its strongest and longest MHW in 2022, profoundly affecting marine sponges. Sponges are vital to rocky benthic marine communities, with their abundance influencing ecosystem functioning. This study examines the impact of this MHW on the photosynthetic sponge Cymbastella lamellata in Fiordland, New Zealand. We describe the extent, physiological responses, mortality, microbial community changes and ecological impact of this MHW on C. lamellata. The Fiordland MHW reached a maximum temperature of 4.4°C above average, lasting for 259 days. Bleaching occurred in >90% of the C. lamellata Fiordland population. The population size exceeded 66 million from 5 to 25 m, making this the largest bleaching event of its kind ever recorded. We identified the photosynthetic symbiont as a diatom, and bleached sponges had reduced photosynthetic efficiency. Post-MHW surveys in 2023 found that over 50% of sponges at sampling sites had died but that the remaining sponges had mostly recovered from earlier bleaching. Using a simulated MHW experiment, we found that temperature stress was a driver of necrosis rather than bleaching, despite necrosis only rarely being observed in the field (<2% of sponges). This suggests that bleaching may not be the cause of the mortality directly. We also identified a microbial community shift in surviving sponges, which we propose represents a microbial-mediated adaptive response to MHWs. We also found that C. lamellata are key contributors of dissolved organic carbon to the water column, with their loss likely impacting ecosystem function. We demonstrate the potential for MHWs to disrupt key marine phyla in temperate regions, highlighting how susceptible temperate sponges globally might be to MHWs.

Unlocking Antarctic molecular time-capsules - Recovering historical environmental DNA from museum-preserved sponges.

Marine sponges have recently emerged as efficient natural environmental DNA (eDNA) samplers. The ability of sponges to accumulate eDNA provides an exciting opportunity to reconstruct contemporary communities and ecosystems with high temporal and spatial precision. However, the use of historical eDNA, trapped within the vast number of specimens stored in scientific collections, opens up the opportunity to begin to reconstruct the communities and ecosystems of the past. Here, we define the term 'heDNA' to denote the historical environmental DNA that can be obtained from the recent past with high spatial and temporal accuracy. Using a variety of Antarctic sponge specimens stored in an extensive marine invertebrate collection, we were able to recover information on Antarctic fish biodiversity from specimens up to 20 years old. We successfully recovered 64 fish heDNA signals from 27 sponge specimens. Alpha diversity measures did not differ among preservation methods, but sponges stored frozen had a significantly different fish community composition compared to those stored dry or in ethanol. Our results show that we were consistently and reliably able to extract the heDNA trapped within marine sponge specimens, thereby enabling the reconstruction and investigation of communities and ecosystems of the recent past with a spatial and temporal resolution previously unattainable. Future research into heDNA extraction from other preservation methods, as well as the impact of specimen age and collection method, will strengthen and expand the opportunities for this novel resource to access new knowledge on ecological change during the last century.

Exploring depth-related patterns of sponge diversity and abundance in marginal reefs.

Marine sponges play a vital role in the reef's benthic community; however, understanding how their diversity and abundance vary with depth is a major challenge, especially on marginal reefs in areas deeper than 30 m. To help bridge this gap, we used underwater videos at 24 locations between 2- and 62-meter depths on a marginal reef system in the Southwestern Atlantic to investigate the effect of depth on the sponge metacommunity. Specifically, we quantified the abundance, density, and taxonomic composition of sponge communities, and decomposed their gamma (γ) diversity into alpha (α) and beta (ß) components. We also assessed whether beta diversity was driven by species replacement (turnover) or by nesting of local communities (nestedness). We identified 2020 marine sponge individuals, which belong to 36 species and 24 genera. As expected, deep areas (i.e., those greater than 30 m) presented greater sponge abundance and more than eightfold the number of sponges per square meter compared to shallow areas. About 50% of the species that occurred in shallow areas (<30 m) also occurred in deep areas. Contrarily to expectations, alpha diversity of rare (0 D α), typical (1 D α), or dominant (2 D α) species did not vary with depth, but the shallow areas had greater beta diversity than the deep ones, especially for typical (1 D ß) and dominant (2 D ß) species. Between 92.7% and 95.7% of the beta diversity was given by species turnover both inside and between shallow and deep areas. Our results support previous studies that found greater sponge abundance and density in deep areas and reveal that species sorting is stronger at smaller depths, generating more beta diversity across local communities in shallow than deep areas. Because turnover is the major driver at any depth, the entire depth gradient should be considered in management and conservation strategies.

Potential of Polar Lipids Isolated from the Marine Sponge Haliclona (Halichoclona) vansoesti against Melanoma.

Marine sponges represent a good source of natural metabolites for biotechnological applications in the pharmacological, cosmeceutical, and nutraceutical fields. In the present work, we analyzed the biotechnological potential of the alien species Haliclona (Halichoclona) vansoesti de Weerdt, de Kluijver & Gomez, 1999, previously collected in the Mediterranean Sea (Faro Lake, Sicily). The bioactivity and chemical content of this species has never been investigated, and information in the literature on its Caribbean counterpart is scarce. We show that an enriched extract of H. vansoesti induced cell death in human melanoma cells with an IC50 value of 36.36 µg mL-1, by (i) triggering a pro-inflammatory response, (ii) activating extrinsic apoptosis mediated by tumor necrosis factor receptors triggering the mitochondrial apoptosis via the involvement of Bcl-2 proteins and caspase 9, and (iii) inducing a significant reduction in several proteins promoting human angiogenesis. Through orthogonal SPE fractionations, we identified two active sphingoid-based lipid classes, also characterized by nuclear magnetic resonance and mass spectrometry, as the main components of two active fractions. Overall, our findings provide the first evaluation of the anti-cancer potential of polar lipids isolated from the marine sponge H. (Halichoclona) vansoesti, which may lead to new lead compounds with biotechnological applications in the pharmaceutical field.

Diversity and structure of the deep-sea sponge microbiome in the equatorial Atlantic Ocean.

Sponges (phylum Porifera) harbour specific microbial communities that drive the ecology and evolution of the host. Understanding the structure and dynamics of these communities is emerging as a primary focus in marine microbial ecology research. Much of the work to date has focused on sponges from warm and shallow coastal waters, while sponges from the deep ocean remain less well studied. Here, we present a metataxonomic analysis of the microbial consortia associated with 23 individual deep-sea sponges. We identify a high abundance of archaea relative to bacteria across these communities, with certain sponge microbiomes comprising more than 90 % archaea. Specifically, the archaeal family Nitrosopumilaceae is prolific, comprising over 99 % of all archaeal reads. Our analysis revealed that sponge microbial communities reflect the host sponge phylogeny, indicating a key role for host taxonomy in defining microbiome composition. Our work confirms the contribution of both evolutionary and environmental processes to the composition of microbial communities in deep-sea sponges.

Analysis and Visualization of Single-Cell Sequencing Data with Scanpy and MetaCell: A Tutorial.

The emergence and development of single-cell RNA sequencing (scRNA-seq) techniques enable researchers to perform large-scale analysis of the transcriptomic profiling at cell-specific resolution. Unsupervised clustering of scRNA-seq data is central for most studies, which is essential to identify novel cell types and their gene expression logics. Although an increasing number of algorithms and tools are available for scRNA-seq analysis, a practical guide for users to navigate the landscape remains underrepresented. This chapter presents an overview of the scRNA-seq data analysis pipeline, quality control, batch effect correction, data standardization, cell clustering and visualization, cluster correlation analysis, and marker gene identification. Taking the two broadly used analysis packages, i.e., Scanpy and MetaCell, as examples, we provide a hands-on guideline and comparison regarding the best practices for the above essential analysis steps and data visualization. Additionally, we compare both packages and algorithms using a scRNA-seq dataset of the ctenophore Mnemiopsis leidyi, which is representative of one of the earliest animal lineages, critical to understanding the origin and evolution of animal novelties. This pipeline can also be helpful for analyses of other taxa, especially prebilaterian animals, where these tools are under development (e.g., placozoan and Porifera).

Methylmercury exposure of the sponge O. lobularis induces strong tissue and cell defects.

Mediterranean marine biota suffers from various anthropogenic threats. Among them, pollutants such as mercury (Hg) represent important environmental issues that are exacerbated by bioaccumulation and bioamplification along food webs via its organic form, monomethylmercury (MMHg). To date, very little is known regarding the impact of mercury on Porifera and the few available studies have been exclusively focused on Demospongiae. This work studies the effect of MMHgCl at different biological levels of Oscarella lobularis (Porifera, Homoscleromorpha). Bioaccumulation assays show that MMHgCl significantly accumulated in sponge tissues after a 96-h exposure to 0.1 µg L-1. Toxicity assays (LC5096h) show a sensibility that depends on life-stage (adult vs bud). Additionally, we show that the exposure to 1 µg L-1 MMHgCl negatively impacts the epithelial integrity and the regeneration process in buds, as shown by the loss of cell-cell contacts and the alteration of osculum morphogenesis. For the first time in a sponge, a whole set of genes classically involved in metal detoxification and in antioxidant response were identified. Significant changes in catalase, superoxide dismutase and nuclear factor (erythroid-derived 2)-like 2 expressions in exposed juveniles were measured. Such an integrative approach from the physiological to the molecular scales on a non-model organism expands our knowledge concerning sensitivity and toxicity mechanisms induced by MMHg in Porifera, raising new questions regarding the possible defences used by marine sponges.

Marine-Derived Peptides with Anti-Hypertensive Properties: Prospects for Pharmaceuticals, Supplements, and Functional Food.

Hypertension, a major health concern linked to heart disease and premature mortality, has prompted a search for alternative treatments due to side effects of existing medications. Sustainable harvesting of low-trophic marine organisms not only enhances food security but also provides a variety of bioactive molecules, including peptides. Despite comprising only a fraction of active natural compounds, peptides are ideal for drug development due to their size, stability, and resistance to degradation. Our review evaluates the anti-hypertensive properties of peptides and proteins derived from selected marine invertebrate phyla, examining the various methodologies used and their application in pharmaceuticals, supplements, and functional food. A considerable body of research exists on the anti-hypertensive effects of certain marine invertebrates, yet many species remain unexamined. The array of assessments methods, particularly for ACE inhibition, complicates the comparison of results. The dominance of in vitro and animal in vivo studies indicates a need for more clinical research in order to transition peptides into pharmaceuticals. Our findings lay the groundwork for further exploration of these promising marine invertebrates, emphasizing the need to balance scientific discovery and marine conservation for sustainable resource use.

Brief History of Ctenophora.

Ctenophores are the descendants of the earliest surviving lineage of ancestral metazoans, predating the branch leading to sponges (Ctenophore-first phylogeny). Emerging genomic, ultrastructural, cellular, and systemic data indicate that virtually every aspect of ctenophore biology as well as ctenophore development are remarkably different from what is described in representatives of other 32 animal phyla. The outcome of this reconstruction is that most system-level components associated with the ctenophore organization result from convergent evolution. In other words, the ctenophore lineage independently evolved as high animal complexities with the astonishing diversity of cell types and structures as bilaterians and cnidarians. Specifically, neurons, synapses, muscles, mesoderm, through gut, sensory, and integrative systems evolved independently in Ctenophora. Rapid parallel evolution of complex traits is associated with a broad spectrum of unique ctenophore-specific molecular innovations, including alternative toolkits for making an animal. However, the systematic studies of ctenophores are in their infancy, and deciphering their remarkable morphological and functional diversity is one of the hot topics in biological research, with many anticipated surprises.

The marine sponge, Hymeniacidon sinapium, displays allorecognition of siblings during post-larval settling and metamorphosis to juveniles.

Marine sponges, including the crumb of bread sponge, Hymeniacidon sinapium, display allorejection responses to contact with conspecifics in both experimental and natural settings. These responses have been used to infer immunocompetence in a variety of marine invertebrates. However, larvae and juveniles from several marine sponge species fuse and form chimeras. Some of these chimeras persist, whereas others eventually break down, revealing a period of allogeneic non-responsiveness that varies depending on the species. Alternatively, for H. sinapium, most pairs of sibling post-larvae and juveniles that settle in contact initiate immediate allorecognition and show the same morphological response progression as the adults. This indicates that allorecognition and response occurs during early metamorphosis. Results from H. sinapium and other sponge species, in addition to annotations of sponge genomes, suggest that allorecognition and immunocompetence in sponges are mediated by distinct systems and may become functional at different times during or after metamorphosis for different species. Consequently, allorecognition may not be a good proxy for the onset of immunocompetence.

Parallel Evolution of Transcription Factors in Basal Metazoans.

Transcription factors (TFs) play a pivotal role as regulators of gene expression, orchestrating the formation and maintenance of diverse animal body plans and innovations. However, the precise contributions of TFs and the underlying mechanisms driving the origin of basal metazoan body plans, particularly in ctenophores, remain elusive. Here, we present a comprehensive catalog of TFs in 2 ctenophore species, Pleurobrachia bachei and Mnemiopsis leidyi, revealing 428 and 418 TFs in their respective genomes. In contrast, morphologically simpler metazoans have a reduced TF representation compared to ctenophores, cnidarians, and bilaterians: the sponge Amphimedon encodes 277 TFs, and the placozoan Trichoplax adhaerens encodes 274 TFs. The emergence of complex ctenophore tissues and organs coincides with significant lineage-specific diversification of the zinc finger C2H2 (ZF-C2H2) and homeobox superfamilies of TFs. Notable, the lineages leading to Amphimedon and Trichoplax exhibit independent expansions of leucine zipper (BZIP) TFs. Some lineage-specific TFs may have evolved through the domestication of mobile elements, thereby supporting alternative mechanisms of parallel TF evolution and body plan diversification across the Metazoa.

Purification of an Acidic Polysaccharide with Anticoagulant Activity from the Marine Sponge Sarcotragus spinosulus.

Thromboembolic conditions are the most common cause of death in developed countries. Anticoagulant therapy is the treatment of choice, and heparinoids and warfarin are the most adopted drugs. Sulphated polysaccharides extracted from marine organisms have been demonstrated to be effective alternatives, blocking thrombus formation by inhibiting some factors involved in the coagulation cascade. In this study, four acidic glycan fractions from the marine sponge Sarcotragus spinosulus were purified by anion-exchange chromatography, and their anticoagulant properties were investigated through APTT and PT assays and compared with both standard glycosaminoglycans and holothurian sulphated polysaccharides. Moreover, their topographic localization was assessed through histological analysis, and their cytocompatibility was tested on a human fibroblast cell line. A positive correlation between the amount of acid glycans and the inhibitory effect towards both the intrinsic and extrinsic coagulation pathways was observed. The most effective anticoagulant activity was shown by a highly charged fraction, which accounted for almost half (about 40%) of the total hexuronate-containing polysaccharides. Its preliminary structural characterization, performed through infrared spectroscopy and nuclear magnetic resonance, suggested that it may consist of a fucosylated chondroitin sulphate, whose unique structure may be responsible for the anticoagulant activity reported herein for the first time.

Microbiome changes through the ontogeny of the marine sponge Crambe crambe.

BACKGROUND: Poriferans (sponges) are highly adaptable organisms that can thrive in diverse marine and freshwater environments due, in part, to their close associations with internal microbial communities. This sponge microbiome can be acquired from the surrounding environment (horizontal acquisition) or obtained from the parents during the reproductive process through a variety of mechanisms (vertical transfer), typically resulting in the presence of symbiotic microbes throughout all stages of sponge development. How and to what extent the different components of the microbiome are transferred to the developmental stages remain poorly understood. Here, we investigated the microbiome composition of a common, low-microbial-abundance, Atlantic-Mediterranean sponge, Crambe crambe, throughout its ontogeny, including adult individuals, brooded larvae, lecithotrophic free-swimming larvae, newly settled juveniles still lacking osculum, and juveniles with a functional osculum for filter feeding. RESULTS: Using 16S rRNA gene analysis, we detected distinct microbiome compositions in each ontogenetic stage, with variations in composition, relative abundance, and diversity of microbial species. However, a particular dominant symbiont, Candidatus Beroebacter blanensis, previously described as the main symbiont of C. crambe, consistently occurred throughout all stages, an omnipresence that suggests vertical transmission from parents to offspring. This symbiont fluctuated in relative abundance across developmental stages, with pronounced prevalence in lecithotrophic stages. A major shift in microbial composition occurred as new settlers completed osculum formation and acquired filter-feeding capacity. Candidatus Beroebacter blanensis decreased significatively at this point. Microbial diversity peaked in filter-feeding stages, contrasting with the lower diversity of lecithotrophic stages. Furthermore, individual specific transmission patterns were detected, with greater microbial similarity between larvae and their respective parents compared to non-parental conspecifics. CONCLUSIONS: These findings suggest a putative vertical transmission of the dominant symbiont, which could provide some metabolic advantage to non-filtering developmental stages of C. crambe. The increase in microbiome diversity with the onset of filter-feeding stages likely reflects enhanced interaction with environmental microbes, facilitating horizontal transmission. Conversely, lower microbiome diversity in lecithotrophic stages, prior to filter feeding, suggests incomplete symbiont transfer or potential symbiont digestion. This research provides novel information on the dynamics of the microbiome through sponge ontogeny, on the strategies for symbiont acquisition at each ontogenetic stage, and on the potential importance of symbionts during larval development.

Chemical and microbiological insights into two littoral Antarctic demosponge species: Haliclona (Rhizoniera) dancoi (Topsent 1901) and Haliclona (Rhizoniera) scotti (Kirkpatrick 1907).

Introduction: Antarctic Porifera have gained increasing interest as hosts of diversified associated microbial communities that could provide interesting insights on the holobiome system and its relation with environmental parameters. Methods: The Antarctic demosponge species Haliclona dancoi and Haliclona scotti were targeted for the determination of persistent organic pollutant (i. e., polychlorobiphenyls, PCBs, and polycyclic aromatic hydrocarbons, PAHs) and trace metal concentrations, along with the characterization of the associated prokaryotic communities by the 16S rRNA next generation sequencing, to evaluate possible relationships between pollutant accumulation (e.g., as a stress factor) and prokaryotic community composition in Antarctic sponges. To the best of our knowledge, this approach has been never applied before. Results: Notably, both chemical and microbiological data on H. scotti (a quite rare species in the Ross Sea) are here reported for the first time, as well as the determination of PAHs in Antarctic Porifera. Both sponge species generally contained higher amounts of pollutants than the surrounding sediment and seawater, thus demonstrating their accumulation capability. The structure of the associated prokaryotic communities, even if differing at order and genus levels between the two sponge species, was dominated by Proteobacteria and Bacteroidota (with Archaea abundances that were negligible) and appeared in sharp contrast to communities inhabiting the bulk environment. Discussions: Results suggested that some bacterial groups associated with H. dancoi and H. scotti were significantly (positively or negatively) correlated to the occurrence of certain contaminants.

Looking for the sponge loop: analyses of detritus on a Caribbean forereef using stable isotope and eDNA metabarcoding techniques.

Coral reefs are biodiverse ecosystems that rely on trophodynamic transfers from primary producers to consumers through the detrital pathway. The sponge loop hypothesis proposes that sponges consume dissolved organic carbon (DOC) and produce large quantities of detritus on coral reefs, with this turn-over approaching the daily gross primary production of the reef ecosystem. In this study, we collected samples of detritus in the epilithic algal matrix (EAM) and samples from potential sources of detritus over two seasons from the forereef at Carrie Bow Cay, Belize. We chose this location to maximize the likelihood of finding support for the sponge loop hypothesis because Caribbean reefs have higher sponge abundances than other tropical reefs worldwide and the Mesoamerican barrier reef is an archetypal coral reef ecosystem. We used stable isotope analyses and eDNA metabarcoding to determine the composition of the detritus. We determined that the EAM detritus was derived from a variety of benthic and pelagic sources, with primary producers (micro- and macroalgae) as major contributors and metazoans (Arthropoda, Porifera, Cnidaria, Mollusca) as minor contributors. None of the sponge species that reportedly produce detritus were present in EAM detritus. The cnidarian signature in EAM detritus was dominated by octocorals, with a scarcity of hard corals. The composition of detritus also varied seasonally. The negligible contribution of sponges to reef detritus contrasts with the detrital pathway originally proposed in the sponge loop hypothesis. The findings indicate a mix of pelagic and benthic sources in the calmer summer and primarily benthic sources in the more turbulent spring.

The ancestral type of the R-RAS protein has oncogenic potential.

BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Cell cycle dynamics of food-entrapping cells of sponges: an experimental approach.

Sponges (phylum Porifera) exhibit surprisingly complex tissue dynamics, maintaining constant cell turnover and migration, rearranging internal structures, and regenerating after severe injuries. Such tissue plasticity relies on the activity of proliferating cells represented primarily by the food-entrapping cells, choanocytes. Although there are plenty of studies regarding the dynamics of regeneration and tissue rearrangement in sponges, cell cycle kinetics of choanocytes in intact tissues remains a controversial issue. This study is devoted to the comparative description of choanocyte cell cycle dynamics in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia corallorrhiza (class Calcarea). We have identified populations of proliferating cells and synchronized them in the S-phase to estimate the growth fraction of cycling cells. Using continuous exposure to labeled thymidine analog ethynyl deoxyuridine (EdU), we calculated choanocyte cell cycle duration and the length of the S phase. We also applied double labeling with EdU and antibodies against phosphorylated histone 3 to estimate the lengths of choanocyte M and G2 phases. Finally, flow-cytometry-based quantitative analysis of DNA content provided us with the lengths of G2 and G1 phases. We found that tissue growth and renewal in the studied sponges are generally maintained by a relatively large population of slowly cycling choanocytes with a total cell cycle duration of 40 h in H. dujardinii and 60 h in L. corallorrhiza. In both species, choanocytes are characterized by an extremely short M-phase and heterogeneity in the duration of the G2 phase.

Novel microsatellite markers suggest significant genetic isolation in the Eastern Pacific sponge Aplysina gerardogreeni.

BACKGROUND: The Eastern Tropical Pacific (ETP) harbors a great diversity of Porifera. In particular, the Aplysina genus has acquired biotechnological and pharmacological importance. Nevertheless, the ecological aspects of their species and populations have been poorly studied. Aplysina gerardogreeni is the most conspicuous verongid sponge from the ETP, where it is usually found on rocky-coralline ecosystems. We evaluated the polymorphism levels of 18 microsatellites obtained from next-generation sequencing technologies. Furthermore, we tested the null hypothesis of panmixia in A. gerardogreeni population from two Mexican-Pacific localities. METHODS AND RESULTS: A total of 6,128,000 paired reads were processed of which primer sets of 18 microsatellites were designed. The loci were tested in 64 specimens from Mazatlan, Sinaloa (N = 32) and Isabel Island, Nayarit (N = 32). The microsatellites developed were moderately polymorphic with a range of alleles between 2 and 11, and Ho between 0.069 and 0.785. Fifteen loci displayed significant deviation from the Hardy-Weinberg equilibrium. No linkage disequilibrium was detected. A strong genetic structure was confirmed between localities using hierarchical Bayesian analyses, principal coordinates analyses, and fixation indices (FST = 0.108*). All the samples were assigned to their locality; however, there was a small sign of mixing between localities. CONCLUSIONS: Despite the moderate values of diversity in microsatellites, they showed a strong signal of genetic structure between populations. We suggest that these molecular markers can be a relevant tool to evaluate all populations across the ETP. In addition, 17 of these microsatellites were successfully amplified in the species A. fistularis and A. lacunosa, meaning they could also be applied in congeneric sponges from the Caribbean Sea. The use of these molecular markers in population genetic studies will allow assessment of the connectivity patterns in species of the Aplysina genus.