Specificity of LSU rRNA-targeted oligonucleotide probes for Pseudo-nitzschia species tested through dot-blot hybridisation

In the scope of the development of a microarray PhyloChip for the detection of toxic phytoplankton species, we designed a large series of probes specific against targets in the nuclear large subunit (LSU) rRNA of a range of Pseudo-nitzschia species and spotted these onto the microarray. Hybridisation with rRNA extracted from monoclonal cultures and from plankton samples revealed many cross-reactions. In the present work, we tested the functionality and specificity of 23 of these probes designed against ten of the species, using a dot-blot procedure. In this case, probe specificity is tested against the target region in PCR products of the LSU rRNA gene marker region blotted on nitrocellulose filters. Each filter was incubated with a species-specific oligoprobe. Eleven of the tested probes showed specific responses, identifying seven Pseudo-nitzschia species. The other probes showed non-specific responses or did not respond at all. Results of dot-blot hybridisations are more specific than those obtained with the microarray approach and the possible reasons for this are discussed.     

EFFECTS OF A DAIRY LOAFING LOT-BUFFER STRIP ON STREAM WATER QUALITY1

ABSTRACT: A loafing or sacrifice lot is an area located outside of the free stall barn, where a dairy herd spends several hours per day. Sacrifice lots are usually denuded of vegetation and have high concentrations of manure and urine that can contribute significant amounts of sediment, nutrients, and pathogens to nearby surface waters. In this study, stream water quality impacted by direct runoff from a sacrifice lot was monitored for a period of 20 months. Ambient stream water quality was monitored by grab sampling upstream and downstream of the sacrifice lot. During runoff events, stream water quality downstream of the sacrifice lot was monitored with an automatic sampler. Laboratory analyses were conducted for total suspended solids and nutrients (nitrogen and phosphorus compounds). A grass filter strip (GFS) was installed as a buffer downslope of the sacrifice lot 10 months into the study period. The impact of the buffer strip on the standardized pollutant concentrations and loads was evaluated using the non-parametric Wilcoxon test. The Wilcoxon test indicated that there was no significant difference (α= 0.05) in the standardized yield of sediment and dissolved pollutants before and after the GFS installation, except for phosphate-phosphorus and filtered total phosphorus concentrations, and sediment-bound total phosphorus and total kjeldahl nitrogen loads that decreased significantly. However, load decrease could have been partially caused by the smaller rainfall volumes after the GFS installation as compared to the existing condition.     

Phylogenetic diversity of epibiotic bacteria in the accessory nidamental glands of squids (Cephalopoda: Loliginidae and Idiosepiidae)

Bacterial communities were identified from the accessory nidamental glands (ANGs) of European and Western Pacific squids of the families Loliginidae and Idiosepiidae, as also in the egg capsules, embryo and yolk of two loliginid squid species, and in the entire egg of one idiosepiid squid species. The results of phylogenetic analyses of 16S RNA gene (rDNA) confirmed that several phylotypes of α-proteobacteria, γ-proteobacteria and Cytophaga–Flavobacteria–Bacteroides phylum were present as potential symbiotic associations within the ANGs. Several identified clones were related to reference strains, while others had no known close relatives. Gram positive strains were rare in loliginid squids. Several bacterial groups may play important roles in the function of the ANGs, such as production of the toxic compounds involved in egg protection and carotenoid pigments. Within the eggs, no bacteria were associated with embryo or yolk of Loligo vulgaris and Sepioteuthis lessoniana, but α- and γ-proteobacteria were present in the egg capsules. Most bacterial strains detected in the egg capsules were the same as those found in the ANGs. The cephalopods of temperate regions (European cuttlefishes and the squid L. vulgaris) appear to be associated with one Agrobacterium strain (Agro2) while tropical-subtropical strains (Asian and Australian loliginids) are associated with Silicibacter-related strains, suggesting a biogeographic clustering for the Agrobacterium-like strains.     

Genotoxicity of gold nanoparticles functionalized with indolicidin towards Saccharomyces cerevisiae

The toxic effects of gold nanoparticles surface-functionalized with the antimicrobial peptide indolicidin(Au NPs-indolicidin) towards the yeast Saccharomyces cerevisiae, one of the major eukaryotic model organisms, have been evaluated. Growth and survival,genotoxicity, as measured by comet assay, and expression of the YCA1, an apoptosis indicating gene, following 72 hr exposure of yeast to Au NPs-indolicidin, and to Au NPs and indolicidin alone have been examined. The gold nanoparticles exerted toxicity with DNA damage, accompanied by reactive oxygen species production(ROS), but they do not inhibit yeast growth and viability. Genotoxicity was less pronounced for surface-functionalized nanoparticles, showing that S. cerevisiae is quite resistant to the complex Au NPs-indolicidin.A progressive reduction of the genotoxic effect was observed along 72 hr exposure,presumably due to the activation of DNA repair mechanisms. These findings suggest the occurrence of a physiological protective response of S. cerevisiae towards nanoparticles,thereby providing useful information to the assessment of the environmental impact of metal nanoparticles.     

Instrumental and bio-monitoring of heavy metal and nanoparticle emissions from diesel engine exhaust in controlled environment

In the present article we characterized the emissions at the exhaust of a Common Rail (CR) diesel engine, representative of lightduty class, equipped with a catalyzed diesel particulate filter (CDPF) in controlled environment. The downstream exhausts were directly analyzed (for PM, CO, CO2, O2, HCs, NOx) by infrared and electrochemical sensors, and SEM-EDS microscope; heavy metals were chemically analyzed using mosses and lichens in bags, and glass-fibre filters all exposed at the engine exhausts. The highest particle emission value was in the 7–54 nm size range; the peak concentration rose until one order of magnitude for the highest load and speed. Particle composition was mainly carbonaceous, associated to noticeable amounts of Fe and silica fibres. Moreover, the content of Cu, Fe, Na, Ni and Zn in both moss and lichen, and of Al and Cr in moss, was significantly increased. Glass-fibre filters were significantly enriched in Al, B, Ba, Cu, Fe, Na, and Zn. The role of diesel engines as source of carbonaceous nanoparticles has been confirmed, while further investigations in controlled environment are needed to test the catalytic muffler as a possible source of silica fibres considered very hazardous for human health.     

Long-term changes in phytoplankton phenology and community structure in the Bahía Blanca Estuary, Argentina

The phytoplankton of the Bahía Blanca Estuary, Argentina, has been surveyed since 1978. Chlorophyll a, phytoplankton abundance, species composition and physico-chemical variables have been fortnightly recorded. From 1978 to 2002, a single winter–early spring diatom bloom has dominated the main pattern of phytoplankton interannual variability. Such pattern showed noticeable changes since 2006: the absence of the typical winter bloom and changes in phenology, together with the replacement of the dominant blooming species, i.e. Thalassiosira curviseriata, and the appearance of different blooming species, i.e. Cyclotella sp. and Thalassiosira minima. The new pattern showed relatively short-lived diatom blooms that spread throughout the year. In addition, shifts in the phytoplankton size structure toward small-sized diatoms, including the replacement of relatively large Thalassiosira spp. by small Cyclotella species and Chaetoceros species have been noticed. The changes in the phenology and composition of the phytoplankton are mainly attributed to warmer winters and the extremely dry weather conditions evidenced in recent years in the Bahía Blanca area. Changing climate has modified the hydrological features in the inner part of the estuary (i.e. higher temperatures and salinities) and potentially triggered the reorganization of the phytoplankton community. This long-term study provides evidence on species-specific and structural changes at the bottom of the pelagic food web likely related to the recent hydroclimatic conditions in a temperature estuary of the southwestern Atlantic.     

Mapping and quantifying habitat fragmentation in small coastal areas: a case study of three protected wetlands in Apulia (Italy)

In the Mediterranean Region, habitat loss and fragmentation severely affect coastal wetlands, due to the rapid expansion of anthropogenic activities that has occurred in the last decades. Landscape metrics are commonly used to define landscape patterns and to evaluate fragmentation processes. This investigation focuses on the performance of a set of landscape pattern indices within landscapes characterized by coastal environments and extent below 1,000 ha. The aim is to assess the degree of habitat fragmentation for the monitoring of protected areas and to learn whether values of landscape metrics can characterize fine-resolution landscape patterns. The study areas are three coastal wetlands belonging to the Natura 2000 network and sited on the Adriatic side of Apulia (Southern Italy). The Habitat Maps were derived from the Vegetation Maps generated integrating phytosociological relevés and Earth Observation data. In the three sites, a total of 16 habitat types were detected. A selected set of landscape metrics was applied in order to investigate their performance in assessing fragmentation and spatial patterns of habitats. The final results showed that the most significant landscape patterns are related to highly specialized habitat types closely linked to coastal environments. In interpreting the landscape patterns of these highly specialized habitats, some specific ecological factors were taken into account. The shape indices were the most useful in assessing the degree of fragmentation of habitat types that usually have elongated morphology along the shoreline or the coastal lagoons. In all the cases, to be meaningful, data obtained from the application of the selected indices were jointly assessed, especially at the class level.