Cliona Varians Research Articles

Ocean acidification influences the gene expression and physiology of two Caribbean bioeroding sponges

IntroductionCoral reef ecosystems are experiencing increased rates of carbonate dissolution due to losses in live coral cover coupled with the impacts of ocean acidification (OA) on coral reef calcifiers and bioeroders. While the stimulating effect of OA on bioerosion has been demonstrated experimentally, predominantly in the Pacific, the underlying physiological and molecular mechanisms behind the response are still poorly understood.MethodsTo address this, we subjected common zooxanthellate (Cliona varians) and azooxanthellate (Pione lampa) Caribbean sponges to pre-industrial (8.15 pH), present-day (8.05 pH), and two future OA scenarios (moderate OA, 7.85 pH; extreme OA, 7.75 pH) and evaluated their physiological and transcriptomic responses.ResultsThe influence of OA on sponge bioerosion was nonlinear for both species, with the greatest total bioerosion and chemical dissolution rates found in the 7.85 pH treatment, then not increasing further under the more extreme 7.75 pH conditions. A trend towards reduced bioerosion rates in the 7.75 pH treatment occurred regardless of the presence of algal symbionts and suggests that the sponges may become physiologically impaired under prolonged OA exposure, resulting in diminished bioerosion potential. These findings were supported by the RNA-seq analysis, which revealed differentially expressed genes involved in a stress response to OA, in particular, suppressed metabolism.DiscussionThis may indicate that the sponges had reallocated energy resources towards more critical physiological needs in response to OA as a survival mechanism under stressful conditions. These data reveal that while the bioerosion rates of excavating sponges in Caribbean reef ecosystems may increase under moderate OA scenarios, this OA-stimulation may plateau or be lost at extreme end-of-century pH conditions, with implications for the dissolution and long-term persistence of reef habitat structures.

Checklist, diversity descriptors and selected descriptions of a highly diverse intertidal sponge (Porifera) assemblage at Costa do Descobrimento (Bahia, Brazil).

Costa do Descobrimento is located in southern Bahia, Brazil, and only 22 species of sponges (Porifera) were known from the area until now, despite its important reef formations. In the present study, we report a checklist of the sponges of Costa do Descobrimento and their distribution in the studied reefs, with several species illustrated in life. We also describe nine new sponge records for this region, some of which are new records for Brazil, or new records of species previously only poorly known. We present alpha and beta diversity indices and compare the sponge assemblage of the sampled locations. Four reefs were considered: two fringing reefs (Arraial D'Ajuda-AA and Coroa Vermelha / Mutá-CVM) and two offshore Municipal Marine Park "Parque Municipal Marinho- (P.M.M. Coroa Alta-CA and P.M.M. Recife de Fora-RF). A total of 229 specimens were collected (224 Demospongiae, 2 Homoscleromorpha and 3 Calcarea). These were classified in 101 morphotypes. Studied materials included representatives of 15 orders, 34 families and 48 genera. The richest orders are Haplosclerida (29 spp.), Poecilosclerida (15 spp.) and Tetractinellida (11 spp.). The richest families were Chalinidae (24 spp.), Clionaidae (7 spp.) and Mycalidae and Suberitidae (6 spp. each). The richest genus is, by far, Haliclona (20 spp.). Only 13 species were shared among all four reefs surveyed, namely Amphimedon viridis, Cinachyrella alloclada, C. apion, Cladocroce caelum, Cliona varians, Dysidea robusta, Mycale (Naviculina) diversisigmata, Niphates erecta, Spirastrella hartmani, Tedania (Tedania) ignis, Terpios fugax, Tethya bitylastra and T. maza. The reefs with the highest richness were CA and CVM, and the lowest richness was observed in RF. The most similar reefs in terms of species composition were CA and CVM, while AA and RF were more dissimilar to the previous reefs, but also from each other. While the difference among CA, CVM and AA was mainly explained by species turnover, RF differed from the previous based on its lower richness (nestedness component). Even though CA and CVM were the richest reefs, AA presented the highest number of exclusive species, highlighting the uniqueness of this reef, and urging the inclusion of local beachrock fringing reefs in a more holistic conservation strategy at Costa do Descobrimento.

The influences of diurnal variability and ocean acidification on the bioerosion rates of two reef-dwelling Caribbean sponges.

Ocean acidification (OA) is expected to modify the structure and function of coral reef ecosystems by reducing calcification, increasing bioerosion, and altering the physiology of many marine organisms. Much of our understanding of these relationships is based on experiments with static OA treatments, although evidence suggests that the magnitude of diurnal fluctuations in carbonate chemistry may modulate the calcification response to OA. These light-mediated swings in seawater pH are projected to become more extreme with OA, yet their impact on bioerosion remains unknown. We evaluated the influence of diurnal carbonate chemistry variability on the bioerosion rates of two Caribbean sponges: the zooxanthellate Cliona varians and azooxanthellate Cliothosa delitrix. Replicate fragments from multiple colonies of each species were exposed to four precisely controlled pH treatments: contemporary static (8.05 ± 0.00; mean pH ± diurnal pH oscillation), contemporary variable (8.05 ± 0.10), future OA static (7.80 ± 0.00), and future OA variable (7.80 ± 0.10). Significantly enhanced bioerosion rates, determined using buoyant weight measurements, were observed under more variable conditions in both the contemporary and future OA scenarios for C. varians, whereas the same effect was only apparent under contemporary pH conditions for C. delitrix. These results indicate that variable carbonate chemistry has a stimulating influence on sponge bioerosion, and we hypothesize that bioerosion rates evolve non-linearly as a function of pCO2 resulting in different magnitudes and directions of rate enhancement/reduction between day and night, even with an equal fluctuation around the mean. This response appeared to be intensified by photosymbionts, evident by the consistently higher percent increase in bioerosion rates for photosynthetic C. varians across all treatments. These findings further suggest that more variable natural ecosystems may presently experience elevated sponge bioerosion rates and that the heightened impact of OA enhanced bioerosion on reef habitat could occur sooner than prior predictions.

Microbiome composition of the marine sponge Cliona varians at the neotropical southern Caribbean Sea displays a predominant core of Rhizobiales and Nitrosopumilaceae.

This work aims to characterize the microbial diversity of the encrusting sponge Cliona varians, a pore-forming and coral reef bioeroding marine sponge of emerging spread related to ocean acidification. We analysed the microbiome composition by 16S V4 amplicon next-generation sequencing in a community of the bioeroding coral reef encrusting/excavating marine sponge Cliona varians thriving at the Southern Caribbean Sea. About 87.21% and 6.76% of the sequences retrieved were assigned to the domain Bacteria and Archaea. The most predominant operational taxonomic units were classified as members of the order Rhizobiales and family Nitrosopumilaceae, representing members of not yet characterized genera. Features found strictly conserved in the strain/genomic representatives reported in those microbial taxa are nitrogen fixation and transformation. Our results suggest, in accordance with recent results, that these microbiome members and associated functions could be contributing to the biological fitness of the sponge to be able to colonize and bioerode in environments with low access and scarce availability of nitrogen sources. Coral reefs bioresources such as sponge holobionts are intriguing and complex ecosystem units. This study contributes to the knowledge of how C. varians microbiota is composed or shaped, which is crucial to understand its ecological functions.

Comparative transcriptomics reveals altered species interaction between the bioeroding sponge Cliona varians and the coral Porites furcata under ocean acidification.

Bioeroding sponges interact and compete with corals on tropical reefs. Experimental studies have shown global change alters this biotic interaction, often in favour of the sponge. Ocean acidification in particular increases sponge bioerosion and reduces coral calcification, yet little is known about the molecular basis of these changes. We used RNA-Seq data to understand how acidification impacts the interaction between the bioeroding sponge, Cliona varians, and the coral, Porites furcata, at the transcriptomic level. Replicate sponge and coral genets were exposed to ambient (8.1 pH) and acidified (7.6 pH) conditions in isolation and in treatments where they were joined for 48h. The coral had a small gene expression response (tens of transcripts) to the sponge, suggesting it does little at the transcriptomic level to deter sponge overgrowth. By contrast, the sponge differentially expressed 7320 transcripts in response to the coral under ambient conditions and 3707 transcripts in response to acidification. Overlap in the responses to acidification and the coral, 2500 transcripts expressed under both treatments, suggests a similar physiological response to both cues. The sponge expressed 50× fewer transcripts in response to the coral under acidification, suggesting energetic costs of bioerosion, and other cellular processes, are lower for sponges under acidification. Our results suggest how acidification drives ecosystem-level changes in the accretion/bioerosion balance on coral reefs. This shift is not only the result of changes to the thermodynamic balance of these chemical reactions but also the result of active physiological responses of organisms to each other and their abiotic environment.

Microbiome structure of ecologically important bioeroding sponges (family Clionaidae): the role of host phylogeny and environmental plasticity

The potential of increased bioerosion by excavating sponges in future environmental scenarios represents a potential threat to coral reef structure and function. Little is known about prokaryotic associations in excavating sponges despite the fact that evidence indicates they contribute to the sponge growth through their heterotrophic metabolism and may even act as microborers. Here, we provide the first detailed description of the microbial community of multiple bioeroding sponges from the Clionaidae family (Cliona varians, C. tumula, C. delitrix, Spheciospongia vesparium, Cervicornia cuspidifera) collected in inshore and offshore coral reefs in the Florida Keys. A total of 6811 prokaryote OTUs identified using 16S rRNA gene sequencing was detected in the samples studied, including ambient water, belonging to 39 bacterial phyla and 3 archaeal phyla. The microbiomes of species harboring Symbiodinium (C. varians, C. tumula, C. cuspidifera) and the azooxanthellate S. vesparium were dominated by Alphaproteobacteria that represented from 83 to 96% of total sequences. These clionaid sponges presented species-specific core microbiomes, with 4 OTUs being shared by all sponge samples, albeit with species-specific enrichments. The microbiomes of C. varians and S. vesparium were stable but showed certain plasticity between offshore and inshore reefs. The distantly related C. delitrix does not harbor Symbiodinium, and had a microbiome dominated by Gammaproteobacteria, which represented 82% of all sequences. Most of the sponge-enriched OTUs are found in low abundance and belong to the ‘rare biosphere’ category, highlighting the potential importance of these microbes in the ecology of the holobiont. Sponge microbiomes may enhance functional redundancy for the sponge holobiont and allow it to respond to shifting environments over much short time scales than evolutionary change would permit. This work establishes the basis for future research to explore how microbial shifts in bioeroding sponges contribute to bioerosion in the face of a changing environment.

Biotechnological Properties of Sponges from Northeast Brazil: Cliona varians as a Biocatalyst for Enantioselective Reduction of Carbonyl Compounds

To research the potential ability of whole marine sponges to act as biocatalysts, this paper describes for the first time the employment of whole Cliona varians sponge in the stereoselective reduction of prochiral a-keto esters and isatin to the corresponding chiral alcohols. The addition of D-fructose, D-glucose or sucrose remarkably increased the conversion ratios and stereoselectivities by this marine sponge. Furthermore, in the presence of D-glucose and D-maltose, the reduction of isatin by C. varians afforded the corresponding 3-hydroxyindolin-2-one with high conversions (85-90%) and good enantioselectivities (60-74%). These results showed that the marine sponge presents great potential to be used as biocatalyst for stereoselective reduction of carbonyl compounds.

Sibling species of mutualistic Symbiodinium clade G from bioeroding sponges in the western Pacific and western Atlantic oceans.

Dinoflagellates in the genus Symbiodinium associate with a broad array of metazoan and protistian hosts. Symbiodinium-based symbioses involving bioeroding sponge hosts have received less attention than those involving popular scleractinian hosts. Certain species of common Cliona harbor high densities of an ecologically restricted group of Symbiodinium, referred to as Clade G. Clade G Symbiodinium are also known to form stable and functionally important associations with Foraminifera and black corals (Antipatharia) Analyses of genetic evidence indicate that Clade G likely comprises several distinct species. Here, we use nucleotide sequence data in combination with ecological and geographic attributes to formally describe Symbiodinium endoclionum sp. nov. obtained from the Pacific boring sponge Cliona orientalis and Symbiodinium spongiolum sp. nov. from the congeneric western Atlantic sponge Cliona varians. These species appear to be part of an adaptive radiation comprising lineages of Clade G specialized to the metazoan phyla Porifera and Cnidaria, which began prior to the separation of the Pacific and Atlantic Oceans.

Cliona acephala (Porifera: Demospongiae: Clionaida), a new encrusting excavating reef sponge from the Colombian Caribbean belonging to the Cliona viridis species complex.

Several groups of sponges are able to excavate galleries and tunnels in calcareous substrata such as limestone rock, shells, calcareous algae and coral skeletons. Within the genus Cliona, some species share the common traits of being brown to olive-green in color, and harboring photosynthetic, unicellular dinoflagellates (zooxanthellae). These Cliona spp. have been grouped as the Cliona viridis species complex. Several species of this complex completely encrust the excavated substratum with a thin veneer of tissue and, when colonizing dead exposed parts of live coral colonies, they are able to undermine or overgrow and thus kill live coral tissue as they advance predominantly laterally. In the course of our taxonomic and ecological studies of Caribbean brown to brown-black encrusting Cliona, we found an as yet undescribed species that stands out by having tylostyle megasclere spicules with narrow heads and lacking the usual microsclere spicule complement of spirasters. This species, named and described here Cliona acephala n. sp., has so far been found exclusively in the Santa Marta area, Caribbean coast of Colombia. Previous studies with ITS2 ribosomal DNA showed it to be genetically distinct from other Caribbean encrusting species belonging to the Cliona viridis species complex, vis. Cliona aprica, Cliona caribbaea, Cliona tenuis and Cliona varians, but making it genetically closer to Indo-Pacific Cliona orientalis. An intriguing possibility, to be addressed with further studies, is that C. acephala n. sp. may have been introduced to the Caribbean. However, until proved otherwise, we regard the material presently described as distinct.

The potential of azooxanthellate poriferan hosts to assess the fundamental and realized Symbiodinium niche: evaluating a novel method to initiate Symbiodinium associations

On coral reefs, Symbiodinium spp. are found in most cnidarian species, but reside in only a small number of sponge species. Of the sponges that do harbor Symbiodinium, most are found in the family Clionaidae, which represents a minor fraction of the poriferan diversity on a reef. Our goal was to determine whether Symbiodinium can be taken up by sponge hosts that do not typically harbor these algal symbionts, and then to follow the fate of any Symbiodinium that enter the intracellular space. We used the filter-feeding capacity of sponges to initiate intracellular interactions between sponge-specialist clade G Symbiodinium and six sponge species that do not associate with Symbiodinium. Using a pulse-chase experimental design, we determined that all of the species we examined captured Symbiodinium, and undamaged intracellular algae were found up to 1 h after inoculation. In a longer-term experiment, Symbiodinium populations in Amphimedon erina persisted in sponge cells for at least 5 d post-inoculation. While no evidence of digestion was detected, the population decreased exponentially after inoculation. We contrast these data with the characteristics of symbiont acquisition and establishment in Cliona varians, which normally harbors Symbiodinium. Explants from experimentally derived aposymbiotic sponges were placed in the field where they acquired Symbiodinium from ambient sources (i.e., we did not inoculate them as in the pulse-chase experiments). We began to detect Symbiodinium cells in C. varians after 12 d, and the algal population increased exponentially until densities approached those typically found in this host (after ~128 d). We discuss the implications of this work in light of growing interest in the evolution of specificity between hosts and symbionts, and the fundamental and realized niche of Symbiodinium.

Secondary metabolites, letalithy and antimicrobial activity of six marine sponges from Mochima Bay, Sucre state, Venezuela.

he specimens of the sponges Aplysina lacunosa, Aplysina fulva, Cliona varians, Cinachyrella kuekenthali, Amphimedon viridis and Aaptos pernucleata, were collected in Mochima Bay, Venezuela, in October 2009 with the purpose of identifying families of secondary metabolites and evaluating the antibacterial properties, antifungal and brine shrimp lethal study of the ethyl acetate soluble fractions of these sponges. The chemical evaluation showed the presence of alkaloids, sterols, triterpenes, methylene ketone and tannins. The ethyl acetate soluble fractions of A. pernucleata, A. fulva and A. lacunosa sponges were the only ones that showed antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli; the soluble fraction of A. lacunosa showed the highest inhibition zones. The antifungal activity was not significant, except for A. pernucleata sponge wich showed mild activity against Candida albicans. The most prominent lethal activity in Artemia salina, were observed for C. kuekenthali, A. viridis and A. pernucleata sponges, with LC50 values of 4,10, 1,13 and < 0,01µg/mL respectively. These results allow profiling the sponges A. lacunosa, A. pernucleata and A. fulva as promising sources of antimicrobial compounds and the sponges C. kuekenthali, A. viridis and A. pernucleata as sources of antitumor compounds.

Sponge erosion under acidification and warming scenarios: differential impacts on living and dead coral.

Ocean acidification will disproportionately impact the growth of calcifying organisms in coral reef ecosystems. Simultaneously, sponge bioerosion rates have been shown to increase as seawater pH decreases. We conducted a 20-week experiment that included a 4-week acclimation period with a high number of replicate tanks and a fully orthogonal design with two levels of temperature (ambient and +1°C), three levels of pH (8.1, 7.8, and 7.6), and two levels of boring sponge (Cliona varians, present and absent) to account for differences in sponge attachment and carbonate change for both living and dead coral substrate (Porites furcata). Net coral calcification, net dissolution/bioerosion, coral and sponge survival, sponge attachment, and sponge symbiont health were evaluated. Additionally, we used the empirical data from the experiment to develop a stochastic simulation of carbonate change for small coral clusters (i.e., simulated reefs). Our findings suggest differential impacts of temperature, pH and sponge presence for living and dead corals. Net coral calcification (mgCaCO3 cm(-2) day(-1) ) was significantly reduced in treatments with increased temperature (+1°C) and when sponges were present; acidification had no significant effect on coral calcification. Net dissolution of dead coral was primarily driven by pH, regardless of sponge presence or seawater temperature. A reevaluation of the current paradigm of coral carbonate change under future acidification and warming scenarios should include ecologically relevant timescales, species interactions, and community organization to more accurately predict ecosystem-level response to future conditions.

Hemagglutinating/Hemolytic activities in extracts of marine invertebrates from the Brazilian coast and isolation of two lectins from the marine sponge Cliona varians and the sea cucumber Holothuria grisea.

Twenty species of marine invertebrates from the Brazilian coast were screened for hemagglutinating/hemolytic activity. In at least twelve tested species, hemagglutinating activity was different for different blood types, suggesting the presence of lectins. Extracts from four species showed hemolytic activity. Two new lectins were purified from the marine sponge Cliona varians (CvL-2) and sea cucumber Holothuria grisea (HGL). CvL-2 was able to agglutinate rabbit erythrocytes and was inhibited by galactosides. The hemagglutinating activity was optimal in pH neutral and temperatures below 70 °C. CvL-2 is a trimeric protein with subunits of 175 kDa. On the other hand, HGL showed both hemagglutinating and hemolytic activity in human and rabbit erythrocytes, but hemolysis could be inhibited by osmotic protection, and agglutination was inhibited by mucin. HGL was stable in pH values ranging from 4 to 10 and temperatures up to 90 °C. In electrophoresis and gel filtration, HGL was a monomeric protein with 15 kDa. CvL-2 and HGL showed different levels of toxicity to Artemia naplii. CvL-2 showed LC50 of 850.1 μg/mL, whereas HGL showed LC50 of 9.5 µg/mL.

Effects of pCO2 on the interaction between an excavating sponge, Cliona varians, and a hermatypic coral, Porites furcata

Rising dissolved pCO2 is a mounting threat to coral reef ecosystems. While the biological and physiological impacts of increased pCO2 are well documented for many hermatypic corals, the potential effects on bioerosion processes remain largely unknown. Increases in pCO2 are likely to modify the direct interactions between corals and bioeroders, such as excavating sponges, with broad implications for the balance between biologically mediated deposition and erosion of carbonate in reef communities. This study investigated the effects of three levels of CO2 (present-day, mid-century and end-of-century projections) on the direct interaction between a bioeroding sponge, Cliona varians, and a Caribbean coral, Porites furcata. Increased pCO2 concentrations had no effect on the attachment rates of C. varians to the corals, and we observed no significant impact of pCO2 on the survival of either the coral or sponges. However, exposure to end-of-century levels of CO2-dosing (~750 μatm) reduced calcification in P. furcata and led to a significant increase in sponge-mediated erosion of P. furcata. These findings demonstrate that pCO2 can enhance erosional efficiency without impacting survival or competitive vigor in these two species. While few studies have considered the influence of pCO2 on the competitive outcomes of interactions between corals and other reef organisms, our study suggests that assessing the impacts of changing pCO2 on species interactions is crucial to adequately predict ecosystem-level responses in the future.

Transcriptomic analysis of differential host gene expression upon uptake of symbionts: a case study with Symbiodinium and the major bioeroding sponge Cliona varians.

BackgroundWe have a limited understanding of genomic interactions that occur among partners for many symbioses. One of the most important symbioses in tropical reef habitats involves Symbiodinium. Most work examining Symbiodinium-host interactions involves cnidarian partners. To fully and broadly understand the conditions that permit Symbiodinium to procure intracellular residency, we must explore hosts from different taxa to help uncover universal cellular and genetic strategies for invading and persisting in host cells. Here, we present data from gene expression analyses involving the bioeroding sponge Cliona varians that harbors Clade G Symbiodinium.ResultsPatterns of differential gene expression from distinct symbiont states (“normal”, “reinfected”, and “aposymbiotic”) of the sponge host are presented based on two comparative approaches (transcriptome sequencing and suppressive subtractive hybridization (SSH)). Transcriptomic profiles were different when reinfected tissue was compared to normal and aposymbiotic tissue. We characterized a set of 40 genes drawn from a pool of differentially expressed genes in “reinfected” tissue compared to “aposymbiotic” tissue via SSH. As proof of concept, we determined whether some of the differentially expressed genes identified above could be monitored in sponges grown under ecologically realistic field conditions. We allowed aposymbiotic sponge tissue to become re-populated by natural pools of Symbiodinium in shallow water flats in the Florida Keys, and we analyzed gene expression profiles for two genes found to be increased in expression in “reinfected” tissue in both the transcriptome and via SSH. These experiments highlighted the experimental tractability of C. varians to explore with precision the genetic events that occur upon establishment of the symbiosis. We briefly discuss lab- and field-based experimental approaches that promise to offer insights into the co-opted genetic networks that may modulate uptake and regulation of Symbiondinium populations in hospite.ConclusionsThis work provides a sponge transcriptome, and a database of putative genes and genetic pathways that may be involved in Symbiodinium interactions. The relative patterns of gene expression observed in these experiments will need to be evaluated on a gene-by-gene basis in controlled and natural re-infection experiments. We argue that sponges offer particularly useful characteristics for discerning essential dimensions of the Symbiodinium niche.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-376) contains supplementary material, which is available to authorized users.

A lactose specific lectin from the sponge Cinachyrella apion: Purification, characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 degrees C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.

Growth inhibitory activity of a novel lectin from Cliona varians against K562 human erythroleukemia cells

In this study, the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians was studied in different cancer cell lines. CvL cytotoxicity was evaluated in mammalian tumor cells and in normal human peripheral blood lymphocytes by the MTT assay using the same range of concentrations (1-150 microg ml(-1)). The mechanisms involved in K562 cell death were investigated by confocal fluorescence microscopy, flow cytometry and immunoblot. CvL inhibited the growth of human leukemia cells, with IC(50) values of 70 and 100 microg ml(-1) for K562 and JURKAT cells, respectively, but it was ineffective on blood lymphocytes and solid tumor cell lines. K562 cell death occurred 72 h after exposure to the lectin and with signs of apoptosis, as analyzed by DAPI and annexin V/PI staining. Investigation of the possible mediators of this process showed that cell death occurred via a caspase-independent pathway. Confocal fluorescence microscopy indicated a pivotal role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished CvL cytotoxic effect. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFkappaB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and reduced expression of pRb, suggesting that CvL can induce cell cycle arrest. Collectively, these findings indicate an antileukemic effect for CvL and suggest that cathepsin B acts as a death mediator in CvL-induced cytotoxicity possibly in an uncharacterized connection with the membrane death receptor pathway.

Pro-inflammatory effect in mice of CvL, a lectin from the marine sponge Cliona varians

CvL, a lectin from the marine sponge Cliona varians agglutinated type A papainized erythrocytes and was strongly inhibited by d-galactose and sucrose. Models of leukocyte migration in vivo were used to study the inflammatory activity of CvL through of mouse paw oedema and peritonitis. Effect of CvL on peritoneal macrophage activation was analysed. Effects of corticoids and NSAIDS drugs were also evaluated on peritonitis stimulated by CvL. Results showed that mouse hind-paw oedema induced by subplantar injections of CvL was dose dependent until 50 μg/cavity. This CvL dose when administered into mouse peritoneal cavities induced maxima cell migration (9283 cells/μL) at 24 h after injection. This effect was preferentially inhibited by incubation of CvL with the carbohydrates d-galactose followed by sucrose. Pre-treatment of mice with 3% thioglycolate increases the peritoneal macrophage population 2.3 times, and enhanced the neutrophil migration after 24 h CvL injection (75.8%, p < 0.001) and no significant effect was observed in the presence of fMLP. Finally, pre-treatment of mice with dexamethasone (cytokine antagonist) decreased (65.6%, p < 0.001), diclofenac (non-selective NSAID) decreased (34.5%, p < 0.001) and Celecoxib (selective NSAID) had no effect on leukocyte migration after submission at peritonitis stimulated by CvL, respectively. Summarizing, data suggest that CvL shows pro-inflammatory activity, inducing neutrophil migration probably by pathway on resident macrophage activation and on chemotaxis mediated by cytokines.

CvL, a lectin from the marine sponge Cliona varians: Isolation, characterization and its effects on pathogenic bacteria and Leishmania promastigotes

CvL, a lectin from the marine sponge Cliona varians was purified by acetone fractionation followed by Sepharose CL 4B affinity chromatography. CvL agglutinated papainized treated human erythrocytes with preference for type A erythrocytes. The lectin was strongly inhibited by monosaccharide d-galactose and disaccharide sucrose. CvL is a tetrameric glycoprotein of 28 kDa subunits linked by disulphide bridges with a molecular mass of 106 kDa by SDS-PAGE and 114 kDa by Sephacryl S300 gel filtration. The lectin was Ca2+ dependent, stable up to 60 degrees C for 60 min, with optimum pH of 7.5. CvL displays a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus. However, CvL did not affect gram negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Leishmania chagasi promastigotes were agglutinated by CvL up to 2(8) titer. These findings are indicative of the physiological defense roles of CvL and its possible use in the antibiosis of bacteria and protozoa pathogenic.