Biomarker responsiveness in Norwegian Sea mussels, Mytilus edulis, exposed at low temperatures to aqueous fractions of crude oil alone and combined with dispersant.

Biological effects of aqueous fractions of a crude oil, alone or in combination with dispersant, were investigated in mussels, Mytilus edulis, exposed at three temperatures (5, 10 and 15 °C). Polycyclic aromatic hydrocarbons (PAHs) tissue concentrations were determined, together with genotoxicity, oxidative stress and general stress biomarkers and the Integrated Biological Response (IBR) index. The bioaccumulation of individual PAHs varied depending on the exposure temperature, with relevant bioaccumulation of phenantrene and fluoranthene at 5 °C and heavier (e.g. 5-rings) PAHs at 15 °C. The values and response profiles of each particular biomarker varied with exposure time, concentration of the oil aqueous fraction and dispersant addition, as well as with exposure temperature. Indeed, PAH bioaccumulation and biomarker responsiveness exhibited specific recognizable patterns in mussels exposed at low temperatures. Thus, genotoxicity was recorded early and transient at 5 °C and delayed but unremitting at 10-15 °C. Catalase activity presented a temperature-dependent response profile similar to the genotoxicity biomarker; however, glutathione-S-transferase responsiveness was more intricate. Lysosomal membrane stability in digestive cells decreased more markedly at 5 °C than at higher temperatures and the histological appearance of the digestive gland tissue was temperature-specific, which was interpreted as the combined effects of PAH toxicity and cold stress. It can be concluded that the profile and level of the biological effects are definitely different at low temperatures naturally occurring in the Arctic/Subarctic region (e.g. 5 °C) than at higher temperatures closer to the thermal optimum of this species (10-15 °C).

Integrated assessment of biological responses to pollution in wild mussels (Mytilus edulis) from subarctic and arctic areas in the Norwegian sea.

North Atlantic and Arctic Oceans contain large amount of undiscovered oil and gas reserves. Therefore threat of oil spills and its hazardous ecological consequences are of great importance to the marine environment. Although mussels (Mytilus sp.) respond clearly to contaminants, biomarkers have shown variability linked to biological and environmental changes. In order to help avoiding misinterpretation of biological responses the aim of this study was to reveal the effect of natural variability in the responsiveness to pollution of a battery of cell and tissue-level biomarkers in mussels. Mussels were collected in relatively non-impacted and potentially impacted sites at ports and the vicinity of a waste water treatment plant in Trondheim and Tromsø in autumn of 2016. Although the battery of biomarkers used herein proved to be useful to discriminate impacted and non-impacted mussel populations, some confounding factors altering the biological responses were identified. Geographical/latitudinal factors seemed to be critical regarding the reproductive cycle, reserve material storage and the prevalence of parasites such as Gymnophallus cf. Bursicola trematodes. Mussels from the reference site in Tromsø displayed general stress responses at different levels, which could be influenced by the pathogenic effect of the Gymnophallus cf. Bursicola trematode and by a more advanced gametogenic developmental stage compared to the mussels from Trondheim, which could lead to misinterpretation of the reasons behind the measured stress levels in those mussels. Despite these confounding effects, the use of integrative tools such as IBR index helped to discriminate mussel populations from chemically impacted and non-impacted sites. Overall, this work serves as an anchor point both as a reference of the baseline level values of the analyzed endpoints in the studied geographical area and time of the year, and as an indication of the potential extent of the environmental confounding factors in monitoring programs causing stress on the analyzed mussel populations.

The influence of temperature in sea urchin embryo toxicity of crude and bunker oils alone and mixed with dispersant.

This investigation deals with how temperature influences oil toxicity, alone or combined with dispersant (D). Larval lengthening, abnormalities, developmental disruption, and genotoxicity were determined in sea urchin embryos for assessing toxicity of low-energy water accommodated fractions (LEWAF) of three oils (NNA crude oil, marine gas oil -MGO-, and IFO 180 fuel oil) produced at 5-25 °C. PAH levels were similar amongst LEWAFs but PAH profiles varied with oil and production temperature. The sum of PAHs was higher in oil-dispersant LEWAFs than in oil LEWAFs, most remarkably at low production temperatures in the cases of NNA and MGO. Genotoxicity, enhanced after dispersant application, varied depending on the LEWAF production temperature in a different way for each oil. Impaired lengthening, abnormalities and developmental disruption were recorded, the severity of the effects varying with oil, dispersant application and LEWAF production temperature. Toxicity, only partially attributed to individual PAHs, was higher at lower LEWAF production temperatures.

Variability and distribution of parasites, pathologies and their effect on wild mussels (Mytilus sp) in different environments along a wide latitudinal span in the Northern Atlantic and Arctic Oceans.

Histopathological examination in mussels can provide useful information for the diagnosis of ecosystem health status. The distribution of parasites in mussels can be conditioned by several environmental factors, including mussels collecting sites or the presence/absence of other species necessary to complete the complex life cycle of certain parasites. Thus, these variables could not only govern the parasitic burden of mussels but also the presence of pathologies associated to parasitism. The aim of this study was to identify the histopathological alterations which could be indicative of a health status distress along a wide latitudinal span in the Northern Atlantic and Arctic Oceans in mussels of two size-classes sampled in clean and impacted sites. A latitudinal gradient is clearly observed in gamete developmental stages as northern and southern mussels presented different conditions at the same period. Furthermore, mussels of the same size in different latitudes presented differences in the reproductive cycle and the appearance of related pathologies, which probably meant the age of individuals was different. In addition, specific parasitic profiles ruled by latitudinal conditions and the settlement of mussels in the shore (horizontal/vertical) have been demonstrated to be significantly influential in the health condition of mussels. Furthermore, the present work provides the first histological description of Gymnophallus cf. bursicola parasite causing a considerable host response in Tromsø and Iceland plus the report of grave histopathological status that included high prevalence of granulocytomas in Scotland and Germany.

Toxicity to sea urchin embryos of crude and bunker oils weathered under ice alone and mixed with dispersant.

A multi-index approach (larval lenghthening and malformations, developmental disruption, and genotoxicity) was applied using sea-urchin embryos as test-organisms. PAH levels measured in the under-ice weathered aqueous fraction (UIWAF) were lower than in the low-energy water accommodated fraction (LEWAF) and similar amongst UIWAFs of different oils. UIWAFs and LEWAFs caused toxic effects, more markedly in UIWAFs, that could not be attributed to measured individual PAHs or to their mixture. Conversely, UIWAF was less genotoxic than LEWAF, most likely because naphthalene concentrations were also lower. In agreement, NAN LEWAF, the most genotoxic, exhibited the highest naphthalene levels. Dispersant addition produced less consistent changes in PAH levels and embryo toxicity in UIWAFs than in LEWAFs, and did not modify LEWAF genotoxicity. Overall, under ice weathering resulted in lowered waterborne PAHs and genotoxicity but augmented embryo toxicity, not modified by dispersant application.

Txikispora philomaios n. sp., n. g., a micro-eukaryotic pathogen of amphipods, reveals parasitism and hidden diversity in Class Filasterea.

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting amphipod genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly hemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host hemocytes, resulting in hemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Influence of dispersant application on the toxicity to sea urchin embryos of crude and bunker oils representative of prospective oil spill threats in Arctic and Sub-Arctic seas.

This study deals with the toxicity assessment of crude and bunker oils representative of prospective oil spill threats in Arctic and Sub-Arctic seas (NNA: Naphthenic North-Atlantic crude oil; MGO: Marine Gas Oil; IFO: Intermediate Fuel Oil 180), alone or in combination with a third-generation dispersant (Finasol OSR52®). Early life stages of sea urchin, Paracentrotus lividus, were selected for toxicity testing of oil low-energy water accommodated fractions. A multi-index approach, including larval size increase and malformation, and developmental disruption as endpoints, was sensitive to discriminate from slight to severe toxicity caused by the tested aqueous fractions. IFO (heavy bunker oil) was more toxic than NNA (light crude oil), with MGO (light bunker oil) in between. The dispersant was toxic and further on it enhanced oil toxicity. Toxic units revealed that identified PAHs were not the main cause for toxicity, most likely exerted by individual or combined toxic action of non-measured compounds.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles by waterborne exposure to benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are priority contaminants in coastal and estuarine ecosystems under anthropogenic pressure. Although PAHs tend to accumulate in the sediment, toxicity for benthic flat fish such as soles may be caused by PAHs released from the sediment to the water column. Within this context, the present investigation aims at recognizing toxicopathic effects elicited after waterborne exposure to benzo[a]pyrene B[a]P, a model individual PAH compound, in juvenile Solea senegalensis. Sole juveniles were exposed to various concentrations of waterborne B[a]P for 3 and 7 days. Brain, liver, gills and gonad were the target tissues selected to determine biochemical and lysosomal biomarkers, and histopathology. Biological responses and toxicopathic effects were consistent with B[a]P concentration and exposure time. From day 3, hepatic catalase inhibition indicated potential oxidative effects of B[a]P. At day 7, contaminant exposure produced hepatic glutathione-S-transferase induction at low concentrations and inhibition at higher levels, evidencing a bell-shaped response. A clear gradient in lysosomal membrane destabilisation was observed in relation with B[a]P concentrations. Histopathological lesions were more frequent at day 7 and at higher contaminant levels. It seems that environmentally relevant waterborne concentrations of B[a]P (1000 ng/l) would suffice to cause toxicopathic effects on sole juveniles in relatively short exposure times. In agreement, the Integrative Biological Response index (IBR/n) indicated a dose-dependent decline in health condition upon exposure to B[a]P (IBR/nHighB[a]P > IBR/nMidB[a]P > IBR/nLowB[a]P > IBR/nDMSO > IBR/nControl). Overall, changes in antioxidant enzymes activity, lysosomal biomarkers and gill and liver histopathology are responsive early-warning signs of health disturbance in sole juveniles exposed to waterborne PAHs.

Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles on exposure to contaminated sediments under laboratory conditions.

Whole-sediment toxicity assays contribute to elucidating the intricate association between the presence of contaminants in sediments and their toxicopathic effects in benthic fish. In the present study, Solea senegalensis juveniles were exposed under laboratory conditions to contaminated whole-sediments for 7 and 28 days. Sediments were obtained from a low to moderately polluted estuary, a highly polluted harbour and from the mixture of both field-collected sediments. Biometry data were recorded. Liver, brain, gills, and gonads were dissected out and processed to determine markers of oxidative stress, neurotoxicity and lysosomal biomarkers, and histopathology. Analyses of sediment granulometry and chemical profiles indicated different degrees of toxicity and suggested a distinct release of pollutants from each sediment in relation with their physicochemical properties. Interestingly, biological responses were in agreement with contaminant levels reported in source sediments. The most distinct toxicopathic effects were detected upon exposure to the harbour's sediment and particularly on day 28. Overall, enhanced hepatic glutathione-S-transferase activity and lysosomal enlargement were detected in all experimental groups, demonstrating a toxic effect from all sediments whilst catalase inhibition, lysosomal membrane destabilisation, changes in lysosomal content and liver histopathology were most pronounced in soles exposed to the harbour's sediment. The Integrative Biomarker Response index (IBR/n) evidenced that exposure to the three sediments caused an impact of diverse magnitude in sole health (IBR/nHarbour > IBR/nMixture > IBR/nEstuary). The magnitude of biological responses essentially depended on the presence of contaminants in source sediments, which seemed to be altered by the conditions imposed by whole-sediment toxicity assays.