Effects of development of wind energy and associated changes in land use on bird densities in upland areas.

Wind energy development is the most recent of many pressures on upland bird communities and their habitats. Studies of birds in relation to wind energy development have focused on effects of direct mortality, but the importance of indirect effects (e.g., displacement, habitat loss) on avian community diversity and stability is increasingly being recognized. We used a control-impact study in combination with a gradient design to assess the effects of wind farms on upland bird densities and on bird species grouped by habitat association (forest and open-habitat species). We conducted 506 point count surveys at 12 wind-farm and 12 control sites in Ireland during 2 breeding seasons (2012 and 2013). Total bird densities were lower at wind farms than at control sites, and the greatest differences occurred close to turbines. Densities of forest species were significantly lower within 100 m of turbines than at greater distances, and this difference was mediated by habitat modifications associated with wind-farm development. In particular, reductions in forest cover adjacent to turbines was linked to the observed decrease in densities of forest species. Open-habitat species' densities were lower at wind farms but were not related to distance from turbines and were negatively related to size of the wind farm. This suggests that, for these species, wind-farm effects may occur at a landscape scale. Our findings indicate that the scale and intensity of the displacement effects of wind farms on upland birds depends on bird species' habitat associations and that the observed effects are mediated by changes in land use associated with wind-farm construction. This highlights the importance of construction effects and siting of turbines, tracks, and other infrastructure in understanding the impacts of wind farms on biodiversity.

Efectos del Desarrollo de la Energía Eólica y los Cambios Asociados al Uso de Suelo sobre las Densidades de Aves en Tierras Altas Resumen El desarrollo de la energía eólica es la más reciente de muchas presiones ejercidas sobre las comunidades de aves de tierras altas y sus hábitats. Los estudios sobre aves en relación con el desarrollo de la energía eólica se han enfocado en los efectos de la mortalidad directa, pero la importancia de los efectos indirectos (p. ej.: desplazamiento, pérdida de hábitat) sobre la diversidad y estabilidad de las comunidades aviares cada vez se reconoce más. Usamos un estudio de control-impacto combinado con un diseño de gradiente para evaluar los efectos de los campos eólicos sobre las densidades de aves de tierras altas y sobre las especies de aves agrupadas por asociación de hábitat (especies de bosque y de hábitat abierto). Realizamos 506 censos de conteo por puntos en 12 sitios de campos eólicos y 12 sitios control en Irlanda durante dos temporadas de reproducción (2012 y 2013). Las densidades de aves totales fueron más bajas en los campos eólicos que en los sitios control, con las diferencias más importantes ocurriendo cerca de las turbinas. Las densidades de las especies de bosque fueron significativamente más bajas a 100 m de las turbinas que a distancias mayores y esta diferencia estuvo mediada por modificaciones asociadas con el desarrollo de campos eólicos. De manera particular, las reducciones en la cobertura de bosque adyacente a las turbinas estuvieron vinculadas con la disminución observada en las densidades de las especies de bosque. Las densidades de las especies de hábitat abierto fueron más bajas en los campos eólicos pero no estuvieron relacionadas con la distancia a las turbinas y tuvieron una relación negativa con el tamaño del campo eólico. Lo anterior sugiere que, para estas especies, los efectos del campo eólico pueden ocurrir a la escala de paisaje. Nuestros hallazgos indican que la escala y la intensidad de los efectos de desplazamiento de los campos eólicos sobre las aves de tierras altas dependen de las asociaciones de hábitat de las especies de aves y que los efectos observados están mediados por cambios en el uso de suelo asociados con la construcción de campos eólicos. Esto remarca la importancia de los efectos de construcción y el sitiado de las turbinas, pistas y demás infraestructura en el entendimiento de los impactos que tienen los campos eólicos sobre la biodiversidad.

Breeding phenology, provisioning behaviour, and unusual patterns of life history variation across an anthropogenic heterogeneous landscape.

Understanding how resource use and life history variation influence a population's response to modified, fragmented landscapes is a major challenge for ecologists. We investigated the phenology, life history decisions and provisioning behaviour of a generalist passerine-the great tit-across a heavily managed woodland landscape. Contrary to most previous studies on this species, reproductive investment and success were lower in deciduous than in coniferous woodland fragments. This could not be explained by differences in provisioning behaviour; instead population density was considerably higher in deciduous woodlands, suggesting birds did not follow an ideal free distribution. Clutch size declined with lay date amongst populations breeding in coniferous woodland fragments, but these populations also displayed pronounced seasonal declines in the proportion of fledglings produced per egg and fledgling mass. In contrast, and against patterns observed in other similar study systems, clutch size did not change with lay date in mixed-species deciduous woodland fragments. Furthermore, the proportion of young fledged and fledgling condition remained stable throughout the season, even though the quality of food provisioned to nestlings increased over the season. Local recruitment was negligible, suggesting that plasticity rather than natural selection played a key role in driving the patterns observed. The unusual patterns we report are likely explained by the fragmented nature of the landscape, and unreliable phenological cues in a mixed-species tree community coupled with low food availability. They contrast with those reported from most other populations situated in continuous woodland, demonstrating that caution is needed when generalising across different contexts and ecosystems.

Pseudovibrio axinellae sp. nov., isolated from an Irish marine sponge.

A Gram-negative, motile, rod-shaped bacterial strain, designated Ad2(T), was isolated from a marine sponge, Axinella dissimilis, which was collected from a semi-enclosed marine lake in Ireland. Strain Ad2(T) grew optimally at 24 °C, at pH 7.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ad2(T) clustered with members of the genus Pseudovibrio, and showed 97.3-98.2 % sequence similarity to the type strains of recognized Pseudovibrio species. DNA-DNA relatedness values between strain Ad2(T) and the type strains of other Pseudovibrio species were <27 %. The DNA G+C content of strain Ad2(T) was 50.5 mol%. The major fatty acid was 18 : 1ω7c. Differences in phenotypic properties, together with phylogenetic and DNA-DNA hybridization analyses, indicated that strain Ad2(T) represented a novel species of the genus Pseudovibrio. The name Pseudovibrio axinellae sp. nov. is proposed, with Ad2(T) (= DSM 24994(T) = NCIMB 14761(T)) as the type strain.

Elucidation of the mass fragmentation pathways of the polyether marine toxins, dinophysistoxins, and identification of isomer discrimination processes.

RATIONALE: Most of the liquid chromatography/mass spectrometry (LC/MS) methods that have been developed for the analysis of Diarrhetic Shellfish Poisoning (DSP) toxins in shellfish and algae samples have been unable to differentiate the isomers okadaic acid (OA) and dinophysistoxin-2 (DTX2), unless separated by chromatography. Since there are many bioconversion products of these compounds it is imperative to determine characteristic product ions, which can provide unequivocal identification of OA and DTX2 and their analogs. METHODS: Using electrospray ionization, the fragmentation processes for two types of precursor ions, [M+Na](+) and [M-H](-), of the polyether marine toxins, dinophysistoxins (DTXs), were studied using a hybrid linear ion trap Orbitrap mass spectrometer which provided high mass accuracy data in combination with multiple tandem mass (MS(n)) spectra. Three structurally related toxins were compared; okadaic acid (OA), dinophysistoxin-2 (DTX2) and dinophysistoxin-1 (DTX1). A quick multiple reaction monitoring (MRM) LC/MS/MS method was developed utilizing the characteristic precursor/product ion mass transitions. RESULTS: Comparison of the high-resolution product ion, [M-H](-), spectra of these toxins featured dominant signals that resulted from two six-centered rearrangements and previously proposed fragmentation pathways for the ion of m/z 321 and 293 have been corrected and identified. By contrast, the [M+Na](+) product ion spectra only revealed distinctive ions for the isomers, OA (m/z 595, 443 and 151) and DTX2 (m/z 581, 429 and 165). To illustrate the benefits of this study, a mass selective LC/MS/MS method was developed in which the isomers OA and DTX2 co-eluted but were distinguished using the mass transitions, m/z 827/595, 827/443 (OA) and m/z 827/581, 827/429 (DTX2). CONCLUSIONS: Comparison of OA, DTX2 and DTX1 led to the correction of proposed negative ion mode fragmentation pathways. Through extensive study and comparison of the [M+Na](+) product ion spectra, distinctive product ions were identified which allowed for these compounds to be identified and distinguished without separation for the first time.

Mussels increase xenobiotic (azaspiracid) toxicity using a unique bioconversion mechanism.

Azaspiracid Poisoning (AZP) is a human toxic syndrome which is associated with the consumption of bivalve shellfish. Unlike other shellfish, mussels contain a large array of azaspiracid analogs, many of which are suspected bioconversion products. These studies were conducted to elucidate the metabolic pathways of azaspiracid (AZA1) in the blue mussel (Mytilus edulis) and revealed that the main biotransformation product was the more toxic demethyl analog, AZA3. To elucidate the mechanism of this C-demethylation, an unprecedented xenobiotic bioconversion step in shellfish, AZA1 was fed to mussels that contained no detectable azaspiracids. Triple quadrupole mass spectrometry (MS) and high resolution Orbitrap MS were used to determine the uptake of AZA1 and the toxin profiles in three tissue compartments of mussels. The second most abundant bioconversion product was identified as AZA17, a carboxyl analog of AZA3, which is a key intermediate in the formation of AZA3. Also, two pairs of isomeric hydroxyl analogs, AZA4/AZA5 and AZA7/AZA8, have been confirmed as bioconversion products for the first time. Ultra high resolution (100 k) MS studies showed that the most probable structural assignment for AZA17 is 22-carboxy-AZA3 and a mechanism for its facile decarboxylation to form AZA3 has been proposed.

Uptake and partitioning of zinc in Lemnaceae.

Macrophytes provide food and shelter for aquatic invertebrates and fish, while also acting as reservoirs for nutrients and trace elements. Zinc accumulation has been reported for various Lemnaceae species. However, comparative accumulation across species and the link between zinc accumulation and toxicity are poorly understood. Morphological distribution and cellular storage, in either bound or soluble form, are important for zinc tolerance. This study shows differences in the uptake and accumulation of zinc by three duckweed species. Landoltia punctata and Lemna minor generally accumulated more zinc than Lemna gibba. L. minor, but not L. gibba or L. punctata, accumulated greater concentrations of zinc in roots compared to fronds when exposed to high levels of zinc. The proportion of zinc stored in the bound form relative to the soluble-form was higher in L. minor. L. punctata accumulated greater concentrations of zinc in fronds compared to roots and increased the proportion of zinc it stored in the soluble form, when exposed to high zinc levels. L. gibba is the only species that significantly accumulated zinc at low concentrations, and was zinc-sensitive. Overall, internal zinc concentrations showed no consistent correlation with toxic effect. We conclude that relationships between zinc toxicity and uptake and accumulation are species specific reflecting, among others, zinc distribution and storage. Differences in zinc distribution and storage are also likely to have implications for zinc bioavailability and trophic mobility.

Beyond plastic microbeads - Short-term feeding of cellulose and polyester microfibers to the freshwater amphipod Gammarus duebeni.

Monitoring studies have revealed the presence of large numbers of natural as well as anthropogenic microfibers, plastic and non-plastic, in environmental samples. However, the interaction of organisms with microfibers is largely understudied. This is the first ecotoxicological study that compares short-term feeding of anthropogenic plastic and non-plastic microfibers on a consumer (leaf-shredding detritivores) species. The freshwater amphipod Gammarus duebeni was selected for this study as it is a model ecotoxicological species. After a 96-hour exposure, 58.3% and 41.7% of the amphipods contained cellulose or polyester fibers in their digestive tracts, respectively. Microfiber ingestion was analysed per polymers in presence or absence of food. The G. duebeni group exposed to 'polyester fibers in presence of food' accumulated highest numbers of microfibers in their digestive tracts (5.2 ±â€¯3.4 MFs/amphipod) followed by those exposed to 'cellulose in presence of food' (2.5 ±â€¯0.9 MFs/amphipod). A significantly (Three-way ANOVA, p-value <0.05) higher number of microfibers was found in the midgut-hindgut (posterior) sections, compared to the foregut (anterior) section. Microfiber uptake had no apparent short-term negative effect on amphipod survival at 96 h. Yet, as amphipods are both predators and prey, and therefore are key species in the aquatic food web, the rapid accumulation of anthropogenic microfibers in their digestive system has potentially further ecological implications. Future studies need to consider the possible transfer of ingested anthropogenic microfibers to higher trophic levels in freshwater communities.

Adsorption, uptake and toxicity of micro- and nanoplastics: Effects on terrestrial plants and aquatic macrophytes.

Plastic pollution is a new, pressing, environmental topic. Microplastics are considered contaminants of emerging concern and, consequently, microplastic research has grown exponentially in the last decade. Here, current knowledge regarding the impacts of micro- and nanoplastics on terrestrial plants and aquatic macrophytes is discussed, with a special focus on adsorption, uptake and toxicological effects. Our review reveals that a range of plants and macrophytes can adsorb or internalise plastic particles. Both processes depend on particle characteristics such as size and charge, as well as plant features including a sticky or hydrophobic surface layer. This finding is of concern given that plants and aquatic macrophytes are at the bottom of food webs and are a crucial component of the human diet. Therefore, there is a critical need for improved understanding of adsorption, uptake and impacts of micro- and nanoplastics, and the consequences thereof for trophic transfer, food safety and security. Also, a range of stress responses have been observed for many plant and macrophyte species after both short and long-term exposures to plastic particles. Given that some plastic particles can affect plant productivity, we surmise that plastic particles may potentially impact ecosystem productivity and function. Here we present a synthesis and a critical evaluation of the state of knowledge of micro- and nanoplastics and plants and macrophytes, identifying key questions for future research.

Food contaminant analysis at ultra-high mass resolution: application of hybrid linear ion trap - orbitrap mass spectrometry for the determination of the polyether toxins, azaspiracids, in shellfish.

The biotoxins, azaspiracids (AZAs), from marine phytoplankton accumulate in shellfish and affect human health by causing severe gastrointestinal disturbance, diarrhea, nausea and vomiting. Specific and sensitive methods have been developed and validated for the determination of the most commonly occurring azaspiracid analogs. An LTQ Orbitrap mass spectrometer is a hybrid instrument that combines linear ion trap (LIT) mass spectrometry (MS) with high-resolution Fourier transform (FT) MS and this was exploited to perform simultaneous ultra-high-resolution full-scan MS analysis and collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Using the highest mass resolution setting (100,000 FWHM) in full-scan mode, the methodology was validated for the determination of six AZAs in mussel (Mytilus galloprovincialis) tissue extracts. Ultra-high mass resolution, together with a narrow mass tolerance window of ±2 mDa, dramatically improved detection sensitivity. In addition to employing chromatographic resolution to distinguish between the isomeric azaspiracid analogs, AZA1/AZA6 and AZA4/AZA5, higher energy collisionally induced dissociation (HCD) fragmentation on selected precursor ions were performed in parallel with full-scan FTMS. Using HCD MS/MS, most precursor and product ion masses were determined within 1 ppm of the theoretical m/z values throughout the mass spectral range and this enhanced the reliability of analyte identity.For the analysis of mussels (M. galloprovincialis), the method limit of quantitation (LOQ) was 0.010 µg/g using full-scan FTMS and this was comparable with the LOQ (0.007 µg/g) using CID MS/MS. The repeatability data were; intra-day RSD% (1.8-4.4%; n = 6) and inter-day RSD% (4.7-8.6%; n = 3). Application of these methods to the analysis of mussels (M. edulis) that were naturally contaminated with azaspiracids, using high-resolution full-scan Orbitrap MS and low-resolution CID MS/MS, produced equivalent quantitative data.

Rapid fragmentation of microplastics by the freshwater amphipod Gammarus duebeni (Lillj.).

Microplastics have become ubiquitous in all environments. Yet, their environmental fate is still largely unknown. Plastic fragmentation is a key component of plastic degradation, which is mostly caused by abiotic processes over prolonged time scales. Here, it is shown that the freshwater amphipod Gammarus duebeni can rapidly fragment polyethylene microplastics, resulting in the formation of differently shaped and sized plastic fragments, including nanoplastics. Fragments comprised 65.7% of all observed microplastic particles accumulated in digestive tracts. Higher numbers of fragments were found in response to longer exposure times and/or higher microplastic concentrations. Furthermore, the proportion of smaller plastic fragments was highest when food was present during the depuration process. It is concluded that G. duebeni can rapidly fragment polyethylene microplastics and that this is closely associated with the feeding process. These results highlight the crucial role, currently understudied, that biota may play in determining the fate of microplastics in aquatic ecosystems.

Orthophosphate modulates the phytotoxicity of nano-ZnO to Lemna minor (L.).

Because of their applications in large numbers of products, Zinc Oxide nanoparticles (nano-ZnO) will inevitably enter into the environment. Nano-ZnO released into the environment will be present in a complex matrix which can cause various chemical and physical transformations and modulate the biological reactivity of these particles. Due to their rapid growth and small size, Lemna minor is recommended by OECD for toxicological testing. Here, we tested how nano-ZnO reactivity is modulated by the suite of macro- and micronutrients that are present in Lemna minor growth media. Specifically, we measured ex situ Reactive Oxygen Species (ROS) formation by nano-ZnO, and subsequent in planta toxicity. The data show how orthophosphate can modulate both ex situ ROS formation, and in planta toxicity. This has ramifications for phytotoxicity testing, which is commonly performed under controlled conditions and on media containing orthophosphate.

Functional shifts in bird communities from semi-natural oak forests to conifer plantations are not consistent across Europe.

While the area of plantation forest increased globally between 2010 and 2015, more than twice the area of natural forests was lost over the same period (6.5 million ha natural forest lost per year versus 3.2 million ha plantation gained per year). Consequently, there is an increasing need to understand how plantation land use affects biodiversity. The relative conservation value of plantation forests is context dependent, being influenced by previous land use, management regimes and landscape composition. What is less well understood, and of importance to conservation management, is the consistency of diversity patterns across regions, and the degree to which useful generalisations can be provided within and among bioregions. Here, we analyse forest birds in Ireland, France and Portugal, representing distinct regions across the Atlantic biogeographic area of Europe. We compared taxonomic, functional and phylogenetic diversity of bird communities among conifer plantations and semi-natural oak forests, and assessed correlations between species traits and forest type across these regions. Although bird composition (assessed with NMDS ordination) differed consistently between plantation and oak forests across all three regions, species richness and Shannon diversity did not show a consistent pattern. In Ireland and France, metrics of taxonomic diversity (richness and Shannon diversity), functional diversity, functional dispersion and phylogenetic diversity were greater in oak forests than plantations. However, in Portugal taxonomic and phylogenetic diversity did not differ significantly between forest types, while functional diversity and dispersion were statistically significantly greater in plantations. No single bird trait-forest type association correlated in a consistent direction across the three study regions. Trait associations for the French bird communities appeared intermediate between those in Ireland and Portugal, and when trait correlations were significant in both Ireland and Portugal, the direction of the correlation was always opposite. The variation in response of bird communities to conifer plantations indicates that care is needed when generalising patterns of community diversity and assembly mechanisms across regions.

Natural succession and clearcutting as drivers of environmental heterogeneity and beta diversity in North American boreal forests.

Clear-cutting alters natural ecosystem processes by reducing landscape heterogeneity. It is the dominant harvesting technique across the boreal zone, yet understanding of how environmental heterogeneity and beta diversity are structured in forest ecosystems and post-clear cut is lacking. We use ground-dwelling arthropods as models to determine how natural succession (progression from deciduous to mixed to coniferous cover types) and clear-cutting change boreal forests, exploring the role of environmental heterogeneity in shaping beta diversity across multiple spatial scales (between-cover types and between-stands of the same cover type (1600 to 8500 m), between-plots (100 to 400 m) and within-plots (20 to 40 m)). We characterise environmental heterogeneity as variability in combined structural, vegetational and soil parameters, and beta diversity, as variability in assemblage composition. Clear-cutting homogenised forest environments across all spatial scales, reducing total environmental heterogeneity by 35%. Arthropod beta diversity reflected these changes at larger scales suggesting that environmental heterogeneity is useful in explaining beta diversity both between-cover types and between-stands of the same cover type. However, at smaller scales, within- and between-plots spider beta diversity reflected the lower environmental heterogeneity in regenerating stands, whereas staphylinid and carabids assemblages were not homogenised 12 years post-harvest. Differences in environmental heterogeneity and staphylinid beta diversity between cover types were also important at small scales. In regenerating stands, we detected a subtle yet notable effect of pre-felling cover type on environmental heterogeneity and arthropods, where pre-felling cover type accounted for a significant amount of variance in beta diversity, indicating that biological legacies (e.g. soil pH reflecting pre-harvest conditions) may have a role in driving beta diversity even 12 years post-harvest. This study highlights the importance of understanding site history when predicting impacts of change in forest ecosystems. Further, to understand drivers of beta diversity we must identify biological legacies shaping community structure.

Wood ash residue causes a mixture of growth promotion and toxicity in Lemna minor.

The use of wood as a sustainable biofuel results in the generation of residual wood ash. The ash contains high amounts of plant macronutrients such as phosphorus, potassium, calcium as well as several micronutrients. To explore the potential use of wood ash as a fertiliser, the growth enhancing properties of Sitka spruce (Picea sitchensis Bong.) wood ash were contrasted with the potential toxic action, using common duckweed (Lemna minor L.) as a model test species. The growth of L. minor exposed to wood bottom and fly ash solids and corresponding leachates was assessed in ultra-oligotrophic and eutrophic media. Ash solids and leachates were also tested as neutralized preparations. Suspended ash solids promoted L. minor growth up to concentrations of 2.5-5g/L. Leachates promoted growth up to 10g ash equivalents per litre, but for bottom ash only. Beneficial effects of wood ash were most pronounced on ultra-oligotrophic medium. However, on such nutrient-deficient medium severe inhibition of L. minor biomass and frond growth was observed at relatively low concentrations of fly ash (EC50=14g/L). On standard, eutrophic medium, higher concentrations of fly ash (EC50=21g/L), or neutralized fly ash (EC50=37g/L) were required to impede growth. Bottom ash, or neutralized bottom ash retarded growth at concentrations of 51g/L and 74g/L (EC50), respectively, in eutrophic medium. It appears that phytotoxicity is due to the elemental composition of the ash, its alkaline character, and possible interactions between these two properties. Growth promotion was due to the substantial content of plant nutrients. This study underlines the importance of the receiving environment (nutrient status and pH) in determining the balance between toxicity and growth promotion, and shows that the margin between growth promoting and toxicity inducing concentrations can be enlarged through ash neutralization.

The orf4 gene of the enterobacterial ICE, R391, encodes a novel UV-inducible recombination directionality factor, Jef, involved in excision and transfer of the ICE.

The enterobacterial mobile genetic element R391, the prototype ICE (integrating-conjugative element) of the SXT/R391 family, shows increased conjugative transfer following UV irradiation. This is dependent on a functioning R391 orf4 gene, which is adjacent to the element encoded integrase gene, int. orf4 mutants fail to form a detectable circular transfer intermediate, do not show UV induced transfer and show a much reduced general transfer ability. The orf4 gene product, termed Jef (IncJ excision factor), shows little homology to anything currently in the nucleotide or protein databases. It is predicted to encode a 66 amino acid, 8.03 kDa, basic, DNA-binding protein with an iso-electric point of pH 8.1: these characteristics being similar to those of recombinational directionality factors involved in excision. Jef expression is up-regulated upon UV irradiation as demonstrated by real-time reverse transcriptase PCR and is controlled by two element encoded genes orf90 and orf91, which show similarity to the transcriptional activator complex FlhC and FlhD. orf4, orf90 and orf91 are conserved in all the SXT/R391-like elements sequenced to date including SXT, ICESpuPO1 and ICEVchMex1. orf4 is also conserved in other SXT/R391 family members such as R997, R392, R705 and pMERPH as shown by sequencing amplicons from these ICEs generated using orf4 specific primers.

In vivo exposure to microcystins induces DNA damage in the haemocytes of the zebra mussel, Dreissena polymorpha, as measured with the comet assay.

The Comet assay was used to investigate the potential of the biotoxin microcystin (MC) to induce DNA damage in the freshwater zebra mussel, Dreissena polymorpha. Mussels maintained in the laboratory were fed daily, over a 21-day period, with one of four strains of the cyanobacterium, Microcystis aeruginosa. Three of the strains produced different profiles of MC toxin, while the fourth strain did not produce MCs. The mussels were sampled at 0, 7, 14, and 21 days by withdrawing haemocytes from their adductor muscle. In addition, a positive control was performed by exposing a subsample of the mussels to water containing cadmium chloride (CdCl(2)). Cell viability, measured with the Fluorescein Diacetate/Ethidium Bromide test, indicated that the MC concentrations, to which the mussels were exposed, were not cytotoxic to the haemocytes. The Comet assay performed on the haemocytes indicated that exposure to CdCl(2) produced a dose-responsive increase in DNA damage, demonstrating that mussel haemocytes were sensitive to DNA-damaging agents. DNA damage, measured as percentage tail DNA (%tDNA), was observed in mussels exposed to the three toxic Microcystis strains, but not in mussels exposed to the nontoxic strain. Toxin analysis of the cyanobacterial cultures confirmed that the three MC-producing strains exhibit different toxin profiles, with the two MC variants detected being MC-LF and MC-LR. Furthermore, the DNA damage that was observed appeared to be strain-specific, with high doses of MC-LF being associated with a higher level of genotoxicity than low concentrations of MC-LR. High levels of MC-LF also seemed to induce relatively more persistent DNA damage than small quantities of MC-LR. This study is the first to demonstrate that in vivo exposure to MC-producing strains of cyanobacteria induces DNA damage in the haemocytes of zebra mussels and confirms the sublethal toxicity of these toxins.

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L.

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.

Analysis and comparative genomics of R997, the first SXT/R391 integrative and conjugative element (ICE) of the Indian Sub-Continent.

The aim of this study was to analyse R997, the first integrative and conjugative element (ICE) isolated from the Indian Sub-Continent, and to determine its relationship to the SXT/R391 family of ICEs. WGS of Escherichia coli isolate AB1157 (which contains R997) was performed using Illumina sequencing technology. R997 context was assessed by de novo assembly, gene prediction and annotation tools, and compared to other SXT/R391 ICEs. R997 has a size of 85 Kb and harbours 85 ORFs. Within one of the variable regions a HMS-1 ß-lactamase resistance gene is located. The Hotspot regions of the element contains restriction digestion systems and insertion sequences. R997 is very closely related to the SXT-like elements from widely dispersed geographic areas. The sequencing of R997 increases the knowledge of the earliest isolated SXT/R391 elements and may provide insight on the emergence of these elements on the Indian sub-continent.

Molecular tools to detect the IncJ elements: a family of integrating, antibiotic resistant mobile genetic elements.

The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and heavy metal resistance genes. They insert into a specific 17-bp site located in the prfC gene, encoding peptide release factor 3, in Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ attL and attR junction fragments. Alignment of over 30 enterobacterial prfC-like genes indicates that the primers designed to amplify attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise recA(+)E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.

Protein carbonylation and heat shock response in Ruditapes decussatus following p,p'-dichlorodiphenyldichloroethylene (DDE) exposure: a proteomic approach reveals that DDE causes oxidative stress.

Protein carbonylation and levels of heat shock proteins (hsp; 60, 70 and 90 kDa) were measured in gill, mantle and digestive gland of Ruditapes decussatus following exposure to p,p'-dichlorodiphenyldichloroethylene (DDE). Heat shock response was measured by immunoblotting using antibodies specific to heat shock proteins (hsps). Densitometry analysis of individual bands revealed no difference between control and treated samples except appearance of hsp90 in DDE-treated mantle. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and two-dimensional electrophoresis coupled with western blotting. Immunoblotting with dinitrophenol-specific antibody revealed extensive differences in both extent and number of carbonylated proteins in mantle and digestive gland in response to DDE while gill was unaffected. These results demonstrate for the first time that DDE causes tissue-specific formation of reactive oxygen species in clams.