Changes in Macrozoobenthos Community after Aquatic Plant Restoration in the Northern Venice Lagoon (IT).

Responses of the macrozoobenthic community to an ecological restoration activity in the northern Venice lagoon were studied, within the scope of the project LIFE SEagrass RESTOration aimed at recreating aquatic phanerogam meadows largely reduced in recent decades. Transplants were successful in almost all project areas. Macrozoobenthos was sampled in eight stations before (2014) and after (2015, 2016, 2017) transplanting activities. An increase in abundance and fluctuations in richness and univariate ecological indices (Shannon's, Margalef's, Pielou's indices) resulted during the years. Comparing non-vegetated and vegetated samples in 2017, every index except Pielou's increased in the latter. Multivariate analysis (hierarchical cluster analysis, MDS, PERMDISP, SIMPER) grouped samples by localization rather than years, with differences between stations due to the abundance of common species. In 2017, results were also grouped by the presence or absence of aquatic plants, with differences in the abundance of grazer and filter-feeding species. Results of ecological index M-AMBI depicted conditions from moderate to good ecological status (sensu Dir.2000/60/EC) with similar fluctuations, as presented by univariate indices from 2014 to 2017. Responses of the macrozoobenthic community were more evident when comparing vegetated and non-vegetated samples, with the vegetated areas sustaining communities with greater abundance and diversity than non-vegetated samples, thus demonstrating the supporting function of aquatic plants to benthic communities.

Environmental restoration by aquatic angiosperm transplants in transitional water systems: The Venice Lagoon as a case study.

The paper reports the results obtained after 4 years of aquatic angiosperm transplants in areas of the Venice Lagoon (North Adriatic Sea, Mediterranean) where meadows almost disappeared due to eutrophication, pollution and overexploitation of clam resources. The project LIFE12 NAT/IT/000331-SeResto, funded by the European Union, allowed to recolonize the Habitat 1150* (coastal lagoons) in the northernmost part of the lagoon, by extensive manual transplants of small sods or single rhizomes of Zostera marina, Zostera noltei, Ruppia cirrhosa and, in some stations also of Cymodocea nodosa. Over the 4 years of the project more than 75,000 rhizomes were transplanted in 35 stations with the support of local stakeholders (fishermen, hunters and sport clubs). Plants took root in 32 stations forming extensive meadows on a surface of approx. 10 km2 even if some failures were recorded in areas affected by outflows of freshwater rich in nutrients and suspended particulate matter. The rapid recovery of the ecological status of the involved areas was the result of this meadow restoration, which was in compliance with Water Framework Directive (WFD 2000/60/EC) objectives. Moreover, the monitoring of environmental parameters in the water column and in surface sediments allowed to identify the best conditions for successful transplants. Small, widespread interventions and the participation of local stakeholders in the environmental recovery, make this action economically cheap and easily transposable in other similar environments.

Imposex levels and butyltin compounds (BTs) in Hexaplex trunculus (Linnaeus, 1758) from the northern Adriatic Sea (Italy): Ecological risk assessment before and after the ban.

The aim of this study was to compare imposex and butyltin compounds (BTs) data, collected before and after the organotin ban in 2008, in order to assess temporal and spatial variation of the phenomenon, the decline of BT contamination, and the effects on Hexaplex trunculus population in the coastal area of the northern Adriatic Sea, close to the Venice Lagoon. Both in marine and in lagoon sites, the results obtained in 2013-2015 showed a significant decline in the incidence of imposex in respect to those from the 2002 survey. In 2002, lagoon samples exhibited Relative Penis Size Index (RPSI) higher than marine samples, whereas no differences were detected in the recent survey, when all RPSI values were below 0.6%. Vas Deference Sequence Index (VDSI) mean values were over 4 before the ban introduction and below this value after that, indicating more critical conditions for gastropod population in 2002 rather than in 2013-15. Percentage of sterile females was up to 69% in 2002, whilst in the more recent survey no sterile female was found. Range of BT concentrations in gastropods decreased from 252 to 579 to 16-31ng∑BT/g d.w. BT body burdens varied according to a gender dependant pattern, with higher concentrations observed in females than in males. A first attempt to propose a classification based on BT impact on H. trunculus, according to the Water Framework Directive, revealed that most sites were in Bad ecological status before the ban and attained a Poor/Moderate status after that.

Imposex in Nassarius nitidus (Jeffreys, 1867) as a possible investigative tool to monitor butyltin contamination according to the Water Framework Directive: A case study in the Venice Lagoon (Italy).

Butyltin (TBT, DBT, and MBT) effects on molluscs, especially endocrine disruption in bivalves and gastropods, have been widely investigated. Imposex, the superimposition of male characters onto female gonochoristic Caenogastropods, is the most studied biological effect of TBT. TBT compounds are among the priority hazardous substances within Directives 2000/60/EC (WFD) and 2008/105/EC. The Environmental Quality Standards (EQSs) set by the WFD for TBT are quite difficult to quantify by means of chemical analysis, without the use of expensive and high performance methods. Assuming that EQSs set for TBT were derived from evidence of imposex development at very low concentrations, this specific biomarker could be used as an indirect measure of assessing levels of bioavailable BTs. Therefore, this study aims to validate the use of imposex development as an investigative tool to monitor the bioavailable fraction of BTs within the WFD, by comparing imposex levels and BT concentrations in Nassarius nitidus from the Venice Lagoon. BT concentrations and imposex levels in N. nitidus, collected in 2013, had decreased when compared to previous studies in the same area. Both VDSI and RPLI correlated positively with BT body burden in females, confirming that imposex is a valid tool to monitor bioavailable BTs. However, TBT is still a matter of concern in the Venice Lagoon, as TBT concentrations were still higher than its degradation products suggesting recent fresh TBT inputs in the studied area. To propose imposex levels as an indicator of the impact of BTs within the WFD, classification class boundaries and Ecological Quality Ratios were introduced. As a preliminary attempt, imposex levels were also compared to the OSPAR Commission EcoQOs which linked imposex levels in Nassarius reticulatus with TBT concentrations in water. Based on this comparison the degree of imposex development in the Venice Lagoon suggested that TBT concentrations in water should be over the EQS-AA concentration and, at one site, also over the EQS-MAC. From all the results obtained in this work, it appears that imposex evaluation in N. reticulatus could give information about the ecological status regarding BT compounds under the WFD and also be used to monitor their effects and support chemical analyses until more sensitive methods become available.

PAH (Polycyclic Aromatic Hydrocarbon) bioaccumulation and PAHs/shell weight index in Ruditapes philippinarum (Adams & Reeve, 1850) from the Vallona lagoon (northern Adriatic Sea, NE Italy).

The Vallona lagoon is a transitional area located in the Po River delta (NE, ITALY) traditionally exploited for Manila clam (Ruditapes philippinarum) farming. During 2007-2008, a pipeline was buried in the middle of the lagoon to connect an off-shore structure to facilities on land. PAH levels were monitored in Manila clams and sediments before, during and after the pipeline construction to assess the impact of the activities through the pattern of distribution of the PAH compounds. PAH bioaccumulation in clams displayed seasonal fluctuations with higher levels in autumnal and wintry surveys than in spring-summer. Principal component analysis applied to PAHs in clams highlighted a petrogenic input during ante operam period and a pyrolytic origin during the burying activities. On the contrary, sediment PAH concentrations resulted quite similar both among sites and periods. Biota-Sediment-Accumulation-Factor values also confirmed that sediments were not the major source of PAH pollution for clams in this study. The welfare of clams was examined through two physiological indices (condition index and survival in air) to check the effects of the activities on a commercial resource. Both physiological indices exhibited seasonal variations connected to natural endogenous and exogenous factors; however survival in air was the most sensitive index in highlighting the effects of the pipeline burying activities. Finally, to ensure that PAH bioavailability assessment was not affected by seasonal variation of soft tissues of molluscs, PAHs/shell weight index was applied. Higher levels of this index were observed before and during the burying activities, whilst, after that, values significantly lowered. Moreover, the normalization enabled us to highlight the PAH uptake from clams in some particular periods and to compare different populations in a long-term biomonitoring program with data obtained from different periods of the year.

A fast and effective routine method based on HS-SPME-GC-MS/MS for the analysis of organotin compounds in biota samples.

This work validated an automated, fast, and low solvent- consuming methodology suited for routine analysis of tributyltin (TBT) and degradation products (dibutyltin, DBT; monobutyltin, MBT) in biota samples. The method was based on the headspace solid-phase microextraction methodology (HS-SPME), coupled with gas chromatographic separation and tandem mass-spectrometry (GC-MS/MS). The effectiveness of the matrix-matched signal ratio external calibration was tested for quantification purposes. The exclusion of matrix influences in the calibration curves proved the suitability of this versatile quantification method. The method detection limits obtained were of 3 ng Sn g(-1) dw for all the analytes. The analysis of references materials showed satisfying accuracy under optimum calibration conditions (% recovery between 87-111%; |Z-scores|<2). The repeatability RSD% and intra-laboratory reproducibility RSD% were lower than 9.6% and 12.6%, respectively. The work proved the remarkable analytical performances of the method and its high potential for routine application in monitoring organotin compounds (OTC).

Structure-activity relationships of 2,4-diphenyl-1H-imidazole analogs as CB2 receptor agonists for the treatment of chronic pain.

A series of 2,4-diphenyl-1H-imidazole analogs have been synthesized and displayed potent human CB2 agonist activity. Many of these analogs showed high functional selectivity over human CB1 receptors. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described.

Detecting constitutive activity and protean agonism at cannabinoid-2 receptor.

Since the cannabinoid system is involved in regulating several physiological functions such as locomotor activity, cognition, nociception, food intake, and inflammatory reaction, it has been the subject of intense study. Research on the pharmacology of this system has enormously progressed in the last 20years. One intriguing aspect that emerged from this research is that cannabinoid receptors (CBs) express a high level of constitutive activity. Investigation on this particular aspect of receptor pharmacology has largely focused on CB1, the CB subtype highly expressed in several brain regions. More recently, research on constitutive activity on the other CB subtype, CB2, was stimulated by the increasing interest on its potential as target for the treatment of various pathologies (e.g., pain and inflammation). There are several possible implications of constitutive activity on the therapeutic action of both agonists and antagonists, and consequently, it is important to have valuable methods to study this aspect of CB2 pharmacology. In the present chapter, we describe three methods to study constitutive activity at CB2: two classical methods relying on the detection of changes in cAMP level and GTPγS binding and a new one based on cell impedance measurement. In addition, we also included a section on detection of protean agonism, which is an interesting pharmacological phenomenon strictly linked to constitutive activity.

Evaluation of cannabinoid receptor 2 and metabotropic glutamate receptor 1 functional responses using a cell impedance-based technology.

Recently, new technologies based on biosensors and called label free have been developed. These technologies eliminate the need for using markers and dyes. The authors applied one of these technologies, based on measurement of cell impedance variation, to study the pharmacological profiles of ligands for the cannabinoid receptor 2 (CB2), a Gi-coupled receptor, and for the metabopotropic glutamate receptor 1 (mGluR1), a Gq-coupled receptor. Reference agonists and antagonists/inverse agonists for the 2 receptors were applied to recombinant cell lines and impedance monitored over time. Agonists (JWH133 and CP55940 for CB2; quisqualate, glutamate, 1S-3R-ACPD, and S-3,5-DHPG for mGluR1) triggered a variation of impedance consistent in both potency and efficacy with data obtained using classical assays measuring cAMP or Ca(2+) levels. This effect was not present in the parental nontransfected cell line, confirming specific receptor-mediated response. Application of antagonists (AM630 for CB2; YM298198, SCH1014222, J&J16259685, and CPCCOEt for mGluR1) reduced agonist-induced impedance changes. The only exception was the mGluR1 antagonist BAY367620 that, while active in the Ca(2+) assay, was inactive in the impedance assay. Overall, these results confirm the possibility of using cell impedance-based technology to study the pharmacological profile of ligands acting at G-protein-coupled receptors coupled to different downstream signaling pathways.

Ecdysone-based system for controlled inducible expression of metabotropic glutamate receptor subtypes 2, 5, and 8.

Stable and inducible expression of human metabotropic glutamate receptor types 2, 5, and 8 was achieved in HEK293 cells using the ecdysone inducible system. Treatment of the respective cell lines with ponasterone A resulted in time and concentration-dependent induction of receptor expression. In all cases, the functional activation of receptors was determined by measuring increases in intracellular calcium. The physiologically GalphaI-coupled receptors mGluR2 and mGluR8 were successfully coupled to phospholipase C activation using the chimeric G protein Galphaq/o. The pharmacological properties of recombinant receptors were characterized and proved to be similar to native receptors. Our data suggest that the ecdysone system has a number of characteristics that make it well suited for expressing mGluRs and that the combined use of this system and chimeric G proteins allows receptors to be characterized using a rapid and straightforward Ca2+ assay.

Age-progressing cognitive impairments and neuropathology in transgenic CRND8 mice.

Patients with Alzheimer's disease suffer from progressive cognitive impairments and show distinct post-mortem neuropathology, including beta-amyloid plaques. Transgenic (Tg) CRND8 mice carry a mutated human amyloid precursor protein gene and show age-related increases in beta-amyloid production and plaque deposition. It was previously reported that during the early stages of plaque deposition, Tg CRND8 mice demonstrated Morris maze impairments. However, it is unknown if Tg mice would be impaired at an earlier age prior to plaque deposition or more impaired at a later age with more extensive plaque deposition. In the current study, we describe Tg CRND8 age-progressing beta-amyloid neuropathology and cognitive abilities in greater detail. At all ages, Tg mice showed normal short-term memory in the Y-maze. Pre-plaque Tg and age-matched Non-Tg mice did not differ in learning the spatial Morris water maze. However, both early and late plaque Tg mice showed impairments during acquisition. In addition, although early plaque Tg mice performed well in the probe trial, late plaque Tg mice demonstrated impaired probe trial performance. Therefore compared to their Non-Tg littermates, Tg CRND8 mice demonstrate cognitive impairments that progressed with age and seemed to coincide with the onset of beta-amyloid plaque deposition.