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The interaction of nanomaterials with pollutants in the marine environment might alter bioavailability, as well as toxicity, of both nanomaterials and pollutants, representing a risk, not only for marine organisms, but also for consumers through the marine food chain.The aim of this study was to evaluate the effect of titanium dioxide nanoparticles (TiO2NPs) in terms of bioaccumulation and toxicity on Mediterranean mussels (Mytilus galloprovincialis) exposed to six-indicator non-dioxin-like polychlorinated biphenyls (ndl-PCBs). Mussels were exposed to ndl-PCBs (20 µg/mL) (groups 3–4) or to a combination of ndl-PCBs (20 µg/mL) and TiO2NPs (100 µg/mL) (groups 5–6) for four consecutive days. TiO2NPs was detected in groups 5–6 (3247 ± 567 and 1620 ± 223 µg/kg respectively), but their presence did not affect ndl-PCBs bioaccumulation in mussels. In fact, in groups 3–4, the concentration of ndl-PCBs (ranging from 3818.4 ± 166.0–10,176 ± 664.3 µg/kg and 2712.7 ± 36.1–9498.0 ± 794.1 µg/kg respectively) was not statistically different from that of groups 5–6 (3048.6 ± 24.0–14,635.9 ± 1029.3 and 5726.0 ± 571.0–9931.2 ± 700.3 µg/kg respectively). Histological analyses showed alterations to the structure of the gill tissue with respect to the control groups, with more severe and diffuse dilatation of the central hemolymphatic vessels of the gill lamellae in groups 5–6 (treated with TiO2NPs and ndl-PCBs concurrently) compared to groups 3–4 (ndl-PCBs only). Finally, in mussels submitted to a seven-day depuration process, most TiO2NPs were eliminated, and NPs had a synergistic effect on ndl-PCBs elimination; as a matter of fact, in groups 5–6, the percentage of concentration was statically inferior to the one observed in groups 3–4. In any case, consumers might be exposed to TiO2NPs and ndl-PCBs (both concurrently and separately) if edible mussels, harvested in a contaminated environment, are consumed without a proper depuration process.

In endangered crayfish conservation projects, it is paramount to map the distribution of the causative agent of crayfish plague, Aphanomyces astaci, in native populations. Considering the inapplicability of the destructive cuticular sampling protocol for monitoring endangered populations, we explored the use of non-invasive sampling techniques to detect this pathogen with molecular assays. In the present study, we exploited environmental DNA (testing increasing water volumes combined with different filter porosities) and cuticular swabs to collect A. astaci DNA. In addition, we evaluated the impact of the storage method on DNA preservation during field activities. After the first evaluations performed on both highly infected Austropotamobius pallipes and carrier Procambarus clarkii specimens in laboratory conditions, these sampling techniques were applied to wild populations of white-clawed crayfish. Our findings highlight better results with the filtration of 5 L of water with filters of 2.7 µm porosity for eDNA analysis and demonstrate that cuticular swabbing is equally effective as the World Organisation of Animal Health’s protocol. Storage in absolute ethanol proved to be the best solution to preserve swabs and filter samples for up to a week at room temperature. In conclusion, we suggest an integration of both sampling methods when monitoring A. astaci for conservation purposes.

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus “Red-Spotted Grouper Nervous Necrosis Virus” (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

This investigation focused on an episode of chronic mortality observed in juvenile Huso huso sturgeons. The examined subjects underwent pathological, microbiological, molecular, and chemical investigations. Grossly severe body shape deformities, epaxial muscle softening, and multifocal ulcerative dermatitis were the main observed findings. The more constant histopathologic findings were moderate to severe rarefaction and disorganization of the lymphohematopoietic lymphoid tissues, myofiber degeneration, atrophy and interstitial edema of skeletal epaxial muscles, and degeneration and atrophy of the gangliar neurons close to the myofibers. Chemical investigations showed a lower selenium concentration in affected animals, suggesting nutritional myopathy. Other manifestations were nephrocalcinosis and splenic vessel wall hyalinosis. Septicemia due to bacteria such as Aeromonas veronii, Shewanella putrefaciens, Citrobacter freundii, Chryseobacterium sp., and pigmented hyphae were found. No major sturgeon viral pathogens were detected by classical methods. Next-generation sequencing (NGS) analysis confirmed the absence of viral pathogens, with the exception of herpesvirus, at the order level; also, the presence of Aeromonas veronii and Shewanella putrefaciens was confirmed at the family level by the metagenomic classification of NGS data. In the absence of a primary yet undetected biological cause, it is supposed that environmental stressors, including nutritional imbalances, may have led to immune system impairment, facilitating the entry of opportunistic bacteria and mycotic hyphae.

Cherax destructor is a crayfish endemic to south-eastern Australia and one of the last alien crayfish to be introduced in Italy. In the Laghi di Ninfa Natural Reserve (Latium region, Central Italy), the species was probably introduced in 1999, but only reported for the first time in 2008. Nearby this area, the most widespread alien crayfish is the red swamp crayfish Procambarus clarkii. In the Natural Reserve, between 2008 and 2013 and during spring and summer, crayfish sampling was carried out with baited traps to assess the distribution of C. destructor and its possible relationship with P. clarkii. Cherax destructor was first recorded in 2008; few P. clarkii were detected in the cultivation ponds where C. destructor was present in 2012 and 2013. Moreover, crayfish plague analyses evidenced a positive result in two out of the 12 sampled P. clarkii. Cherax destructor is now completely absent from the Natural Reserve, while P. clarkii has spread in the area and was probably responsible for this eradication since C. destructor is vulnerable to crayfish plague which was also detected in the area. An ecosystem restoration project in the area favoured the spread of. P. clarkii; the implications of this intervention are discussed.