The impact of silver nanoparticles on marine plankton dynamics: Dependence on coating, size and concentration.

During this study, three microcosm experiments were carried out with natural coastal seawater, collected in the Eastern Mediterranean Sea, in order to assess the effect of silver nanoparticle (AgNP) exposure to natural plankton communities. The impact of coating (branched-polyethyleneimine: BPEI vs. poly-vinylpyrrolidone: PVP), size (40 vs. 60nm), concentration (200, 500, 2000, 5000 and 10,000ng Ag L-1) and silver form (dissolved Ag+ vs. AgNPs) were tested. The results of chlorophyll a concentration revealed that PVP AgNPs caused a higher toxicity than BPEI AgNPs, and this was possibly related to the measured higher dissolution rate. Additionally, toxicity of BPEI AgNPs was size-dependent, with 40 being more toxic than 60 nm AgNPs, which was nevertheless not seen clearly for PVP AgNPs. Interestingly, community composition altered in response to AgNP exposure: cyanobacterial abundance was negatively affected at concentrations ≥200ng Ag L-1, and dinoflagellate abundance and composition were altered at a 2000ng Ag L-1 concentration. Specifically, dinoflagellate (Gymnodinium, Prorocentrum and Gyrodinium) and diatom (Nitzschia, Navicula and Climacosphenia) genera either increased or decreased, highlighting taxa-specific effects, with some of them being able to tolerate, compensate or even benefit from AgNPs. Silver in either form (dissolved Ag+ or in NPs) caused almost identical results in the plankton community, further indicating that Ag+ release is the primary cause of AgNP toxicity. This study employed for the first time environmentally relevant AgNP concentrations (minimum 200ng Ag L-1) in natural seawater without pre-filtration steps and showed that community changes were driven by the exposure but were largely dependent on ambient physico-chemical characteristics and should be further investigated.

An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus.

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen-associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome. We obtained more than 2 x 10(6) reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immune response against Vibrio vulnificus, a septicemic pathogen that uses the gills as the portal of entry into the blood, as well as the role of the main toxin of this species (RtxA13) on this early interaction. The gill transcriptomic profiles obtained after bath infecting eels with the wild type strain or with a mutant deficient in rtxA13 were analyzed and compared. Results demonstrate that eels react rapidly and locally against the pathogen and that this immune-response is rtxA13-dependent as transcripts related with cell destruction were highly up-regulated only in the gills from eels infected with the wild-type strain. Furthermore, significant differences in the immune response against the wild type and the mutant strain also suggest that host survival after V. vulnificus infection could depend on an efficient local phagocytic activity. Finally, we also found evidence of the presence of an interbranchial lymphoid tissue in European eel gills although further experiments will be necessary to identify such tissue.

Early steps in the European eel (Anguilla anguilla)-Vibrio vulnificus interaction in the gills: role of the RtxA13 toxin.

Vibrio vulnificus is an aquatic gram-negative bacterium that causes a systemic disease in eels called warm-water vibriosis. Natural disease occurs via water born infection; bacteria attach to the gills (the main portal of entry) and spread to the internal organs through the bloodstream, provoking host death by haemorrhagic septicaemia. V. vulnificus produces a toxin called RtxA13 that hypothetically interferes with the eel immune system facilitating bacterial invasion and subsequent death by septic shock. The aim of this work was to study the early steps of warm-water vibriosis by analysing the expression of three marker mRNA transcripts related to pathogen recognition (tlr2 and tlr5) and inflammation (il-8) in the gills of eels infected by immersion with either the pathogen or a mutant deficient in rtxA13. Results indicate a differential response that is linked to the rtx toxin in the expression levels of the three measured mRNA transcripts. The results suggest that eels are able to distinguish innocuous from harmful microorganisms by the local action of their toxins rather than by surface antigens. Finally, the cells that express these transcripts in the gills are migratory cells primarily located in the second lamellae that re-locate during infection suggesting the activation of a specific immune response to pathogen invasion in the gill.

Prostaglandin (F and E, 2- and 3-series) production and cyclooxygenase (COX-2) gene expression of wild and cultured broodstock of Senegalese sole (Solea senegalensis).

Prostaglandin levels in different tissues and cyclooxygenase (COX-2) gene expression were compared between wild and cultured Senegalese sole (Solea senegalensis) broodstock in which a significantly different fatty acid profile, particularly lower tissue levels of arachidonic acid (ARA, 20:4n-6) and higher levels of eicosapentaenoic acid (EPA, 20:5n-3) in the cultured fish compared to wild had already been described. This is the first report of the COX-2 mRNA expression in Senegalese sole. Cyclooxygenase (COX-2) mRNA expression and prostaglandin (2- and 3-series) levels were determined in tissues from 32 broodstock fish, 16 (8 males and 8 females) from each origin wild and cultured (G1). Transcripts of COX-2 were highly expressed in gills, sperm-duct (s-duct), testis, oviduct and spleen compared to liver, kidney and muscle. Differences in COX-2 transcripts expression were found in response to the origin of the fish and expression was significantly higher in s-duct and gills from wild fish compared to cultured. Wild fish showed significantly higher levels of total 2-series PGs and lower levels of 3-series compared to cultured fish. The significance of the lower COX-2 expression and lower PG 2-series production in some of the tissues of cultured fish was discussed in relation to the previously described differences in fatty acid profile (lower tissue levels of ARA and higher levels of EPA and EPA/ARA ratio in cultured fish) and the reproductive failure to spawn viable eggs from G1 cultured Senegalese sole compared to successful spawning from captive wild broodstock.

RNA-Seq reveals an integrated immune response in nucleated erythrocytes.

BACKGROUND: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. METHODOLOGY/PRINCIPAL FINDINGS: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. CONCLUSIONS/SIGNIFICANCE: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.

Endotoxin recognition in fish results in inflammatory cytokine secretion not gene expression.

Macrophages are phagocytes that have a central role in the organization of the immune system after an infection. These cells can recognize specific molecular components of micro-organisms (pathogen-associated molecular patterns, PAMPs) via specific receptors (PRRs) and elicit specific cellular responses. In the past, the expression of immune genes in response to different PAMPs has been characterized in different fish species. However, little is known about actual cytokine release. We characterized the secretion of tumour necrosis factor (TNF)-α in primary macrophage cultures of rainbow trout (Oncorhynchus mykiss) in response to several PAMPs by Western blot and compared this to the induction of TNF-α gene expression as well as other pro-inflammatory cytokines such as interleukin (IL)-1ß and IL-6 and anti-viral molecules such as INF-α and Mx protein (Mx). We show that lipopolysaccharide (LPS) and zymosan are major inducers of TNF-α secretion, which is not initially linked to the induction of TNF-α mRNA expression. The secretion of TNF-α, but intriguingly not the expression, is also stimulated by ultrapure LPS meaning that, in fish, contaminants of commercial LPS preparations are better inducers of the inflammatory response. Moreover, we have characterized the signaling pathways that are activated by different PAMPs and the link between those pathways and the final step of TNF-α secretion: TNF-α shedding by TNF-α converting enzyme (TACE/ ADAM17). For the first time, we show that, in fish macrophages, TNF-α is processed by TACE-like activity and this cleavage is dependent upon the activation of ERK, p38MAPK and JNK signaling pathways by LPS.

The effects of immunostimulation through dietary manipulation in the rainbow trout; evaluation of mucosal immunity.

Immunostimulant-containing diets are commonly used in aquaculture to enhance the resistance of cultured fish to disease and stress. Although widespread in use, there have been conflicting results published, and surprisingly little is known about the regulation of immune response-related genes in tissues key to mucosal immunity induced by immunostimulant dietary feeding. Using a salmonid-specific microarray platform enriched with immune-related genes and in situ hybridization, we investigated dietary acclimation in two organs relevant to mucosal immunity, the gills and the intestine, in the rainbow trout (Oncorhynchus mykiss). Immunostimulant diets significantly changed gene expression profiles and gene distribution in a tissue-specific manner: genes and functional Gene Ontology categories involved in immunity were differently expressed at portals of entry where significant changes in genes and functional groups related to remodeling processes and antigen presentation were observed. Furthermore, genes involved in chemotaxis, cell differentiation, antigen-presenting capacity and tissue remodeling were localized in both organs.