Polymer aging affects the bioavailability of microplastics-associated contaminants in sea urchin embryos.

Microplastics (MPs) in the marine environment undergo complex weathering factors that can affect their ability to interact with different coexisting environmental contaminants (termed here co-contaminants). In this study, the influence of artificially aging using UV on the sorption of a complex mixture of co-contaminants onto MPs was investigated in order to provide meaningful hypotheses on their individual and combined toxicities on sea urchin embryos. A mixture of artificially aged MPs (PS particles and PA microfibers) combined with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), or Cd or Cu, both alone and in a mix, were used to expose embryos of Paracentrotus lividus. The effects of polymer aging on co-contaminants bioavailability were assessed by measuring changes in the transcriptional profile of genes involved in oxidative-stress response and skeletogenic and endo-mesodermal specification. Changes in the sorption ability of MPs to co-contaminants in the aqueous phase highlighted that aging did not affect the sorption of BDE-47 and Cd on MPs, although a certain influence on Cu sorption was found. Despite no morphological effects in embryos at the gastrula stage after MPs/contaminants combinatorial exposure emerged, the greatest influence of the aging process was mainly found for combined exposures which included BDE-47. Finally, the exposure to multiple contaminants generated transcriptional profiles poorly related to those activated by single contaminant, at times suggesting a mixture-dependent different aging influence. These results open new scenarios on the controversial role of vector of co-contaminants for MPs, especially when complex and different types of mixtures were considered.

Signals from the deep-sea: Genetic structure, morphometric analysis, and ecological implications of Cyclothone braueri (Pisces, Gonostomatidae) early life stages in the Central Mediterranean Sea.

Cyclothone braueri (Stomiiformes, Gonostomatidae) is a widely distributed fish inhabiting the mesopelagic zone of marine tropical and temperate waters. Constituting one of the largest biomasses of the ocean, C. braueri is a key element in most of the ecological processes occurring in the twilight layer. We focused on the ecological processes linked to early life stages in relation to marine pelagic environmental drivers (temperature, salinity, food availability and geostrophic currents) considering different regions of the Central Mediterranean Sea. A multivariate morphometric analysis was carried out using six parameters with the aim of discerning different larval morphotypes, while a fragment of 367 bp representing the 12S ribosomal RNA gene was used to perform molecular analyses aimed at determining the intraspecific genetic variability. Analysis highlighted two geographically distinct morphotypes not genetically discernible and related to the different nutritional conditions due to spatial heterogeneities in terms of temperature and food availability. The body depth (BD) emerged as an appropriate morphometric parameter to detect the larval condition in this species. Molecular analysis highlighted a moderate genetic divergence in the fish population, showing the recurrence of two phylogroups not geographically separated.

Gene Expression Changes after Parental Exposure to Metals in the Sea Urchin Affect Timing of Genetic Programme of Embryo Development.

It is widely accepted that phenotypic traits can be modulated at the epigenetic level so that some conditions can affect the progeny of exposed individuals. To assess if the exposure of adult animals could result in effects on the offspring, the Mediterranean sea urchin and its well-characterized gene regulatory networks (GRNs) was chosen as a model. Adult animals were exposed to known concentrations of zinc and cadmium (both individually and in combination) for 10 days, and the resulting embryos were followed during the development. The oxidative stress occurring in parental gonads, embryo phenotypes and mortality, and the expression level of a set of selected genes, including members of the skeletogenic and endodermal GRNs, were evaluated. Increased oxidative stress at F0, high rates of developmental aberration with impaired gastrulation, in association to deregulation of genes involved in skeletogenesis (dri, hex, sm50, p16, p19, msp130), endodermal specification (foxa, hox11/13b, wnt8) and epigenetic regulation (kat2A, hdac1, ehmt2, phf8 and UBE2a) occurred either at 24 or 48 hpf. Results strongly indicate that exposure to environmental pollutants can affect not only directly challenged animals but also their progeny (at least F1), influencing optimal timing of genetic programme of embryo development, resulting in an overall impairment of developmental success.

Aberrant gene expression profiles in Mediterranean sea urchin reproductive tissues after metal exposures.

Marine organisms are simultaneously exposed to numerous pollutants, among which metals probably represent the most abundant in marine environments. In order to evaluate the effects of metal exposure at molecular level in reproductive tissues, we profiled the sea urchin transcriptional response after non-lethal exposures using pathway-focused mRNA expression analyses. Herein, we show that exposures to relatively high concentrations of both essential and toxic metals hugely affected the gonadic expression of several genes involved in stress-response, detoxification, transcriptional and post-transcriptional regulation, without significant changes in gonadosomatic indices. Even though treatments did not result in reproductive tissues visible alterations, metal exposures negatively affected the main mechanisms of stress-response, detoxification and survival of adult P. lividus. Additionally, transcriptional changes observed in P. lividus gonads may cause altered gametogenesis and maintenance of heritable aberrant epigenetic effects. This study leads to the conclusion that exposures to metals, as usually occurs in polluted coastal areas, may affect sea urchin gametogenesis, thus supporting the hypothesis that parental exposure to environmental stressors affects the phenotype of the offspring.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues.

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.

Partially Purified Extracts of Sea Anemone Anemonia viridis Affect the Growth and Viability of Selected Tumour Cell Lines.

In the last few years, marine species have been investigated for the presence of natural products with anticancer activity. Using reversed phase chromatography, low molecular weight proteins were fractionated from the sea anemone Anemonia viridis. Four different fractions were evaluated for their cytotoxic activity by means of erythrocyte haemolysis test, MTS, and LDH assays. Finally, the antiproliferative activities of three of these fractions were studied on PC3, PLC/PRF/5, and A375 human cancer cell lines. Our analysis revealed that the four fractions showed different protein contents and diverse patterns of activity towards human PBMC and cancer cell lines. Interestingly, fractions III and IV exerted cytotoxic effects on human cells. Conversely, fractions I and II displayed very low toxic effects associated with antiproliferative activities on cancer cell lines.

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii.

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5'-untranslated region (UTR) of 63 bp and a 3'-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection.

Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.

Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP.

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu(2+), Cd(2+), Pb(2+) and Ni(2+) concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 µM). Moreover, starting from these data, using the combination of molecular biology techniques and optical setup, we developed a device for the detection of Cu(2+) in water solutions. The quenching effect detected with the device showed that the relative attenuation of the signal (0.46 ± 0.02 AU) was slightly larger than the data measured by fluorescence spectra (0.65 ± 0.03 AU). The good sensitivity in the span of two orders of the magnitude of Cu(2+) concentration, the fact that the instrument is made up of low-cost and sturdy parts and the selective quenching of rAsGFP to copper ions make this setup suited as a low cost, on-the-field, copper ion-specific biosensor.