Enhancing seafood traceability: tracking the origin of seabass and seabream from the tuscan coast area by the analysis of the gill bacterial communities.

BACKGROUND: The seafood consumption and trade have increased over the years, and along its expected expansion pose major challenges to the seafood industry and government institutions. In particular, the global trade in fish products and the consequent consumption are linked to reliable authentication, necessary to guarantee lawful trade and healthy consumption. Alterations or errors in this process can lead to commercial fraud and/or health threats. Consequently, the development of new investigative tools became crucial in ensuring unwanted scenarios. Here we used NGS techniques through targeted metagenomics approach on the V3-V4 region of the 16S rRNA genes to characterize the gill bacterial communities in wild-caught seabream (Sparus aurata) and seabass (Dicentrarchus labrax) within different fisheries areas of the "Costa degli Etruschi'' area in the Tuscan coast. Our challenge involved the possibility of discriminating between the microbiota of both fish species collected from three different fishing sites very close to each other (all within 100 km) in important areas from a commercial and tourist point of view. RESULTS: Our results showed a significant difference in the assembly of gill bacterial communities in terms of diversity (alpha and beta diversity) of both seabass and seabream in accordance with the three fishing areas. These differences were represented by a unique site -related bacterial signature, more evident in seabream compared to the seabass. Accordingly, the core membership of seabream specimens within the three different sites was minimal compared to the seabass which showed a greater number of sequence variants shared among the different fishing sites. Therefore, the LRT analysis highlighted the possibility of obtaining specific fish bacterial signatures associated with each site; it is noteworthy that specific taxa showed a unique association with the fishing site regardless of the fish species. This study demonstrates the effectiveness of target-metagenomic sequencing of gills in discriminating bacterial signatures of specimens collected from fishing areas located at a limited distance to each other. CONCLUSIONS: This study provides new information relating the structure of the gill microbiota of seabass and seabream in a fishing area with a crucial commercial and tourist interest, namely "Costa degli Etruschi". This study demonstrated that microbiome-based approaches can represent an important tool for validating the seafood origins with a central applicative perspective in the seafood traceability system.

The use of polarized light in the zonal orientation of the sandhopper Talitrus saltator (Montagu).

It is well known that the celestial polarization is used as a compass cue by many species of insects and crustaceans. Although it has been shown that the sandhopper Talitrus saltator perceives polarized light and possesses an arrangement of the rhabdomeres that could allow e-vector interpretation and utilization, T. saltator does not use the e-vector of the skylight polarization as a compass cue when making excursions along the sea-land axis of sandy shores. We performed tests in confined conditions to clarify if skylight polarization is somehow involved in the zonal recovery of T. saltator. We observed the directional responses of sandhoppers in a transparent bowl under an artificial sky (an opaline Plexiglas dome). The bowl was covered by a blue gelatin filter with a grey filter (control condition) and a linear polarizing filter (experimental conditions) positioned under the blue one in such a way as to occupy half of the upper surface of the Plexiglas bowl so as to create a linear polarization gradient. Our experiments confirm that T. saltator perceives polarized light and highlight that this visual capability determines the perception, or perhaps the increase, of the radiance and/or spectral gradient and their use as compass cues in the zonal orientation. Moreover, our findings confirm that the radiance gradient is used as a chronometric compass orienting reference in the absence of other celestial orienting cues.

Bioplastics on marine sandy shores: Effects on the key species Talitrus saltator (Montagu, 1808).

Talitrid amphipods are an important component of detritus web, playing a key role in the fragmentation of organic matters of marine and terrestrial origin, and it is well known that sandhoppers ingest microplastics. To assess the effective consumption of bioplastics and their effects on survival rate and on pollutants transfer (i.e. phthalates) on supralittoral arthropods, laboratory experiments were conducted by feeding adult T. saltator with two different types of bioplastic commonly used in the production of shopping bags. Groups of about 20 individuals were fed with 10 × 10 cm sample sheets of the two types of bioplastic for four weeks. The results show that amphipods ingest bioplastics even in the absence of microbial film and that ingestion of bioplastic can have effects on talitrid amphipods. Microtomographic analyses of faecal pellets seem consistent with this finding. The high phthalate concentrations in freshly collected individuals suggest the presence in the environment of these compounds, and the ability of amphipods to assimilate them, while the decrease in phthalate concentrations in bioplastic-fed individuals could be attributed to the scavenging effect of virgin plastic, as already observed in a previous study. In summary, the results indicate that different bioplastics may have effects on T. saltator (i.e. survival rate and faecal pellets structure) and confirm a potential role of amphipods in the degradation of bioplastics in supralittoral zone of marine sandy beaches, even when bioplastics are not colonized by bacterial biofilm that seems to improve palatability.

Action Observation and Motor Imagery administered the day before surgery enhance functional recovery in patients after total hip arthroplasty: A randomized controlled trial.

OBJECTIVE: To investigate the effects of Action Observation and Motor Imagery administered the day before surgery on functional recovery in patients after total hip arthroplasty. DESIGN: Randomised controlled trial. SETTING: Humanitas Clinical and Research Center, Milan, Italy. PARTICIPANTS: Eighty inpatients with end-stage hip osteoarthritis undergoing total hip arthroplasty. INTERVENTIONS: All patients followed a standardized postoperative rehabilitation program. Experimental group (AO + MI) performed two 12-minute Action Observation and Motor Imagery sessions on the preoperative day, whereas control group underwent usual care consisting of education without any additional preoperative activity. OUTCOME MEASURES: A blinded physiotherapist assessed participants for functional mobility (Timed Up and Go - TUG) (primary outcome), maximum walking speed (10-Meter Walk Test - 10MWT), pain (Numeric Pain Rating Scale - NPRS) and fear of movement (Tampa Scale of Kinesiophobia - TSK) the day before and at four days after surgery. RESULTS: No between-group differences were found at baseline. Although TUG and 10MWT worsened in both groups (p < 0.001), better TUG was found for AO + MI group at four days (mean difference -5.8 s, 95% confidence interval from -11.3 to -0.3 s, p = 0.039). NPRS (p < 0.001) and TSK (p = 0.036 for AO + MI group, p = 0.003 for control group) improved after surgery without between-group differences. CONCLUSIONS: Patients undergoing Action Observation and Motor Imagery on the day before surgery showed less functional decline than control group in the first days after total hip arthroplasty. This intervention may contribute to a safer discharge with higher functional abilities in patients hospitalized for total hip arthroplasty.

Occurrence and characterization of microplastic and mesoplastic pollution in the Migliarino San Rossore, Massaciuccoli Nature Park (Italy).

Microplastics pollution is progressively threatening natural parks across the world. In the framework of monitoring this concerning trend, the present study focuses on the occurrence and identification of mesoplastics (MEPs) and microplastics (MPs) in sand samples collected before and after the summer season from the beach of the Nature Park of Migliarino San Rossore Massaciuccoli (Pisa, Italy). Meso- and microplastics were identified using Fourier transform infrared spectroscopy 2D Imaging, and detected in all samples with average concentrations of 207 ± 30 MPs/kg d.w., and 100 ± 44 MEPs/kg d.w., respectively. Seasonal changes of flow of the Arno River, industrial activities, and urban footprint were considered as the major sources of plastic pollution. Our results showed the occurrence of both natural and synthetic polymers including cellulose, polyethylene, polypropylene, polyamides, polyethylene terephthalate, and acrylonitrile.

Sky radiance and spectral gradient are orienting cues for the sandhopper Talitrus saltator (Crustacea, Amphipoda).

The sandhopper Talitrus saltator relies on both the sun and the moon compasses to return to the belt of damp sand on the beach in which it lives buried during the day. In addition to the sun, the gradient of radiance and the spectral distribution across the sky could provide directional information that T. saltator can potentially use to orient itself during the day even when the sun is not visible (e.g. cloudy sky). The scope of this work was (1) to determine the intensity levels of sky radiance that the sandhoppers use in their zonal recovery and (2) to investigate whether this species relies on the celestial spectral gradient in its zonal recovery. Sandhoppers were tested in the laboratory under artificial radiance or spectral gradients. Our results show that under an artificial sky simulating the natural radiance gradient on a cloudless day, sandhoppers orientated toward the correct seaward direction of their home beach; however, individuals lost their ability to use the intensity gradient as an orientation cue when the radiance was attenuated by at least 40%. Sandhoppers were also able to head in the correct seaward direction of their home beach at any time of the day by using the spectral gradient as their only source of visual orientation reference.

Specializations in the compound eye of Talitrus saltator (Crustacea, Amphipoda).

We investigated the eye regionalization in Talitrus saltator by morphological, electrophysiological and behavioural experiments. Each ommatidium possesses five radially arranged retinular cells producing a square fused rhabdom by R1-R4 cells; the smaller R5 exists between R1 and R4. The size of R5 rhabdomere is larger in the dorsal part and becomes smaller in the median and ventral parts of the eye. Spectral-sensitivity by electroretinograms were recorded from dorsal or ventral parts of the eye. The dorsal part possesses maxima at green and UV-blue region. The main response region in the ventral part is only from UV (390 nm) to blue (430 nm) decreasing at longer wavelengths. To evaluate the sandhoppers' celestial orientation, their eyes were painted black either in the dorsal or ventral part, under the natural sky or a blue filter with or without the vision of the sun. Sandhoppers with the dorsal region of the eyes painted and tested under the screened sun were more dispersed and their directions varied more than in other groups of individuals. Sandhoppers with this area of the eye obscured display considerable difficulties to head in a specific direction. This work suggests the existence of regional specializations in the eye of T. saltator.

Ingested microplastic as a two-way transporter for PBDEs in Talitrus saltator.

The presence and accumulation of plastic waste into the marine environment are well known environmental issues. Microplastics (MPs) end up in sea waters and, due to their hydrophobicity and high surface/volume ratio, POPs tend to sorb and accumulate to their surface. The supralittoral amphipod Talitrus saltator (T. saltator) was selected to study the role of MPs in the transfer of organic pollutants and to investigate if ingested MPs could either transfer contaminants to biota or clean it adsorbing pollutants taken from the diet. T. saltator is an established POPs (Persistent Organic Pollutants) biomonitor in coastal environments and it is able to swallow microplastics in natural condition. Two laboratory experiments were performed and T. saltator was exposed to a labelled polybrominated diphenyl ether (13C-labelled BDE-47) to investigate the opposite gradient role of MPs. X Ray Micro-CT (Micro-Computed Tomography) analyses were also performed on sandhopper samples to evaluate the uptake of MPs via digestive tract. The results showed that MPs ingestion could whether transfer and remove contaminants from T. saltator, indicating a partial balance among positive and negative effects. This study has underlined MP potential double role demonstrating that MP can act both as a carrier and scavenger for the bioaccumulation of organic pollutants (i.e. PBDEs), suggesting that chemicals leaching from MPs could have a limited impact to biota.

Exploring the bacterial gut microbiota of supralittoral talitrid amphipods.

Talitrid amphipods (sandhoppers and beach fleas) are typical of the supralittoral zone. They are known to thrive on stranded materials, including detrital marine angiosperms and macroalgae, as well as occasional dead animals. In this work, the gut microbiota of five species of talitrid amphipods (Talitrus saltator, Talorchestia ugolinii, Sardorchestia pelecaniformis, Orchestia montagui and Orchestia stephenseni) collected in Sardinia (Italy) has been investigated through: i) metabarcoding analysis of the amplified 16S rRNA V4 region; and ii) quantification of family 48 glycosyl hydrolase genes (GHF48), involved in cellulose degradation. Results indicate that, though talitrid gut biodiversity is not directly related to taxon or sampling locality, the animals' digestive tracts may host species-specific bacterial communities. In particular, gut microbiota of O. montagui, an inhabitant of Posidonia banquettes and macro-algae mat, showed the greatest differences in taxonomic composition and the highest proportion of GHF48 genes with respect to 16S rRNA genes. These results suggest that the different talitrid species may host species-specific bacterial communities whose function could partially reflect the different microhabitats and food preferences of their host.

Evidence for discrete solar and lunar orientation mechanisms in the beach amphipod, Talitrus saltator Montagu (Crustacea, Amphipoda).

Animals that use astronomical cues to orientate must make continuous adjustment to account for temporal changes in azimuth caused by Earth's rotation. For example, the Monarch butterfly possesses a time-compensated sun compass dependent upon a circadian clock in the antennae. The amphipod Talitrus saltator possesses both a sun compass and a moon compass. We reasoned that the time-compensated compass mechanism that enables solar orientation of T. saltator is located in the antennae, as is the case for Monarch butterflies. We examined activity rhythms and orientation of sandhoppers with antennae surgically removed, or unilaterally occluded with black paint. Removing or painting the antennae did not affect daily activity rhythms or competence to orientate using the sun. However, when tested at night these animals were unable to orientate correctly to the moon. We subsequently measured circadian gene expression in the antennae and brain of T. saltator and show the clock genes period and cryptochrome 2 are rhythmically expressed in both tissues, reminiscent of other arthropods known to possess antennal clocks. Together, our behavioural and molecular data suggest that, T. saltator has anatomically discrete lunar and solar orientation apparatus; a sun compass, likely located in the brain and a moon compass in the antennae.

Landscape vision and zonal orientation in the Equatorial sandhopper Talorchestia martensii.

We investigate the role of the landscape in the zonal recovery of the Equatorial sandhopper Talorchestia martensii Weber. It is known that this species uses the sun and the magnetic compasses to return to the belt of damp sand of the beach following the shortest route (the sea-land axis). However, the sun is not always easy to use at Equatorial latitudes because of astronomical reasons (its zenithal culmination and its hourly azimuthal speed) at least during the central time of the day (around noon) and close to the equinox. Our experiments were performed in Kenya, during the equinoctial period. We tested adult individuals, belonging to Malindi (Kenya) population, in a confined environment with and without the vision of the landscape of their home beach and with the vision of the prominent landscape of a different-orientated shore (Temple Point). Releases were carried out with either natural or zeroed magnetic field. Results clearly show the importance of the landscape as an orienting factor mainly during the central hours of the day when it seems to assume a greater importance than magnetic cues.

Salt concentration and solar orientation in two supralittoral sandhoppers: Talitrus saltator (Montagu) and Talorchestia ugolinii Bellan Santini and Ruffo.

The influence of salt concentration in the seawater on solar orientation in Talitrus saltator and Talorchestia ugolinii was studied in a confined environment (transparent plexiglass bowls). Sodium and calcium concentrations strongly affect both sea-land orientation and the sun compass mechanism in T.saltator, whereas the behaviour of T. ugolinii is less influenced. The absence of Na(+) does not influence the sun compass mechanism, but causes an inversion in the mean direction of orientation in T. saltator. In T. ugolinii, there was no influence on the compass mechanism for solar orientation and no inversion in the directional choice. In the absence of Ca(2+), a photonegative tendency was observed for T saltator together with marked reduction in the capacity to go in any direction. However, the effect of Ca(2+) absence on the orientation capacity of T. saltator is reversible and the orientation capacity can be reduced in a few minutes. The different behaviour of the two species of sandhoppers is discussed.

Optic flow and sea-land orientation in the sandhopper Talitrus saltator.

The problem sandhoppers face when they find themselves on the dry sand is to reach as quickly as possible the belt of moist sand near the water. In the present study, I ask whether, alongside many other orienting factors, sandhoppers use the optic flow they experience to maintain their bearing relative to the sea-land axis. Adult individuals of Talitrus saltator were released in a transparent Plexiglas bowl, horizontally placed between four walls with a pattern of vertical black and white stripes. The orientation of one pair of opposite walls was south-north, orthogonal to the sea-land axis of the home beach, whilst the second pair of walls was oriented east-west. The black and white striped pattern of opposite walls could be moved in pairs and in the same direction (speed=4.8 cm s(-1)). The results demonstrate that the optic flow sandhoppers experience when moving on the sea-land axis of their home beach influences their direction of travel and could help sandhoppers in maintaining a straight path to reach favourable ground by the shortest route.

Sandhopper Talitrus saltator (Montagu) as a bioindicator of contamination by polycyclic aromatic hydrocarbons.

This study evaluates the use of the sandhopper Talitrus saltator as a bioindicator of PAHs contamination of the supralittoral zone of sandy beaches. Adult amphipods were collected at eight localities on the Tuscany shore in summer. Concentrations of 16 PAHs indicated as priority pollutants by the Environmental Protection Agency were determined. T. saltator is able to accumulate PAHs (total PAHs ranging from 0.75 to 62.1 ng g(-1)), with concentrations generally greater than in the sand (total PAHs ranging from 0.04 to 38 ng g(-1)). In particular, it accumulates Phe, Cry, and BbF. Preliminary laboratory tests indicate food as a possible route of PAHs intake by sandhoppers. Despite the need of further studies to clarify the variability of the PAH concentrations found in the substrata and in sandhoppers, T. saltator could be used as a bioindicator of PAHs contamination of the supralittoral zone of sandy shores.

The skylight gradient of luminance helps sandhoppers in sun and moon identification.

To return to the ecologically optimal zone of the beach, the sandhopper Talitrus saltator (Montagu) maintains a constant sea-land direction based on the sun and moon compasses. In this study, we investigated the role of the skylight gradient of luminance in sun and moon identification under natural and artificial conditions of illumination. Clock-shifted (inverted) sandhoppers tested under the sun (during their subjective night) and under the full moon (during their subjective day) exhibit orientation in accordance with correct identification of the sun and the moon at night. Tested in artificial conditions of illumination at night without the artificial gradient of luminance, the artificial astronomical cue is identified as the moon even when the conditions of illumination allow sun compass orientation during the day. When the artificial gradient of luminance is added, the artificial astronomical cue is identified as the sun. The role of the sky gradient of luminance in sun and moon identification is discussed on the basis of present and past findings.

Behavioural responses of the supralittoral amphipod Talitrus saltator (Montagu) to trace metals contamination.

We investigated behavioural responses (locomotor activity and substrate selection) of the amphipod Talitrus saltator after laboratory exposure to different concentrations of Hg, Cu and Cd. Locomotor activity, measured with a microwave radar device, was assessed in animals exposed to contaminated sand and in sandhoppers previously kept for 48 h in contaminated seawater and tested in clean sand. The contents of Hg, Cu and Cd in T. saltator tissues were measured at the end of pre-exposure to contaminated seawater and after 7 days of exposure to contaminated sand. Substrate selection tests were carried out in a binary choice arena. Tests in contaminated sand showed that sandhopper locomotor activity varied in a dose-dependent manner in the presence of Cu and Hg (at lower Hg concentration they were more active during daytime) but did not show significant changes in Cd-exposed animals except for disappearance of the typical circadian activity pattern. Pre-exposure to trace metals in seawater induced a significant decrease of movements for all metals, although the effects varied according to the toxicity of the metal. Trace metals analyses showed that tissue concentrations of Hg, Cu and Cd were related to their concentrations in the test medium. Substrate selection tests showed avoidance of contaminated sand only when sandhoppers were tested in the presence of both concentrations of Hg and at the higher concentration of Cu. In conclusion, locomotor activity and avoidance behaviour of T. saltator could be used as behavioural biomarkers of trace metals exposure.

Trace metal contamination of Tuscan and eastern Corsican coastal supralittoral zones: The sandhopper Talitrus saltator (Montagu) as a biomonitor.

The aim of this study was to determine trace metal concentrations (Hg, Cd, Cu, Zn, Al, Fe, Mn, Cr and Pb) in samples of Talitrus saltator collected at 14 localities along the Tuscan and eastern Corsican coasts. This talitrid amphipod is a well-known biomonitor of trace metal contaminations of the supralittoral zone of European sandy beaches. The results show the ability of T. saltator to accumulate Cu, Cd, Zn, Hg, Al, Fe and Mn, but not Cr or Pb. Moreover, the sites can be distinguished on the basis of the accumulated metals. Finally, comparison with literature data regarding the same species and the same sampling sites shows a general decrease in Cd, Zn and Hg contamination. In conclusion, this study confirms that T. saltator can be used as an indicator of the spatial distribution of trace metals contamination.

Seawater Ca2+ concentration influences solar orientation in Talitrus saltator (Crustacea, Amphipoda).

The role of salinity in the ecophysiology of many intertidal invertebrates has been extensively investigated. Calcium (Ca(2+)), magnesium (Mg(2+)), potassium (K(+)) and sodium (Na(+)) are the major constituents of seawater and it has been demonstrated that sandhoppers tested under the sun in diluted seawater (3.5 per thousand) head seaward, instead of going landward as expected. Therefore, the variation in seawater salinity (from 35 per thousand to 3.5 per thousand) influences their directional choice. This paper investigates the contribution of different cations to the sea-land directional choice of Talitrus saltator (Crustacea, Amphipoda) by the sun compass orientation mechanism. Results of releases carried out in basic seawater selectively deprived of Ca(2+), Mg(2+) or K(+) and containing the same concentration of Na(+) indicate that only the reduction in Ca(2+) concentration affects the capacity of solar orientation. The pH does not influence the directional choice of sandhoppers and nor do small variations in salinity in the range 32-39 per thousand. Moreover, the clear photopositive tendency registered in experiments of phototaxis in Ca(2+)-deprived seawater indicates that the absence of Ca(2+) does not affect the normal functioning of the visual cells. Therefore, our results show that Ca(2+) seawater concentration is important for the correct functioning of one of the principal mechanisms of orientation in supralittoral amphipods and it could affect their survival in the field.

Sandhopper solar orientation as a behavioural biomarker of trace metals contamination.

Although many studies have focused on trace metals accumulation, investigations of talitrid amphipods as biomarkers are rare. This study explores the possibility of using the solar orientation capacity of Talitrus saltator as a behavioural marker of exposure to two essential (Cu and Zn) and two non-essential (Cd and Hg) metals. LC(50) analyses performed before the solar orientation tests showed that the 72 h LC(50) for Hg was 0.02 ppm while the 96 h LC(50) values for Cu, Cd and Zn were 13.28 ppm, 27.66 ppm, and 62.74 ppm, respectively. The presence of metals in seawater affects the solar orientation capacity of T. saltator in a concentration-dependent manner and according to the toxicity ranking of the metals (Hg > Cu > Cd > Zn). Therefore, the solar orientation capacity of T. saltator seems to be a promising behavioural marker for exposure to trace metals.