Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in Cyprinidae fish: Towards hints of their arrangements using advanced classification methods.

To tackle the ever-present global concern regarding human exposure to persistent organic pollutants (POPs) via food products, this study strived to indicate associations between organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in lake-fish tissue depending on the species and sampling season. Apart from the monitoring initiatives recommended in the Global Monitoring Plan for POPs, the study discussed 7 OCPs and 18 PCB congeners determined in three Cyprinidae species (rudd, carp, and Prussian carp) from Vransko Lake (Croatia), which are widely domesticated and reared as food fish across Europe and Asia. We exploit advanced classification algorithms, the Kohonen self-organizing maps (SOM) and Decision Trees (DT), to search for POP patterns typical for the investigated species. As indicated by SOM, some of the dioxin-like and non-dioxin-like PCBs (PCB-28, PCB-74, PCB-52, PCB-101, PCB-105, PCB-114, PCB-118, PCB-156 and PCB-157), α-HCH and ß-HCH caused dissimilarities among fish species, but regardless of their weight and length. To support these suggestions, DT analysis sequenced the fish species and seasons based on the concentration of heavier congeners. The presented assumptions indicated that the supplemental application of SOM and DT offers advantageous features over the usually rough interpretation of POPs pattern and over the single use of the methods.

Trace Metals in Noah's Ark Shells (Arca noae Linnaeus, 1758): Impact of Tourist Season and Human Health Risk.

Commercially important bivalve Noah's Ark shell (Arca noae Linnaeus, 1758) represents a high-quality seafood product, but the data on levels of metal contaminants that could pose a human health risk and also on some essential elements that are important for health protection are lacking. This study examined the concentrations of Cd, Pb, Cr, Ni, Cu, Co, and Zn in the soft tissue of A. noae from harvesting area in the central Adriatic Sea, to survey whether heavy metals are within the acceptable limits for public health and whether tourism could have an impact on them. The concentrations of analysed metals varied for Cd: 0.15-0.74, Pb: 0.06-0.26, Cr: 0.11-0.34, Ni: 0.09-0.22, Cu: 0.65-1.95, Co: 0.04-0.09, and Zn: 18.3-74.7 mg/kg wet weight. These levels were lower than the permissible limits for safe consummation of seafood, and only for Cd, some precautions should be taken into account if older shellfish were consumed. Increase of Cd, Cr, and Cu in shell tissue was observed during the tourist season at the site closest to the marine traffic routes, indicating that metal levels in shellfish tissue should be monitored especially carefully during the peak tourist season to prevent eventual toxic effects due to increased intake of metals, specifically of Cd.

Supplementation with imuno-2865® in gilthead sea bream (Sparus aurata Linnaeus, 1758): Effects on hematological and antioxidant parameters.

The aim of this study was to evaluate the effects of IMUNO-2865(®) on hematological and antioxidative parameters in sea bream. Total of 640 sea bream were fed with diets containing 0 (Group 1), 1 (Group 2), 10 (Group 3) and 25 (Group 4) g of IMUNO-2865(®) kg(-1) feed during 90 days. Samples were taken each month and three months after the supplementation. A significant heterophils increase was observed in group 4 compared to group 1 after two months, and an increase in monocytes number was observed in group 4 compared to the other groups after one month. Glutathione peroxidase (GSH-Px) and paraoxonase-1 (PON1) were significantly increased in groups 3 and 4 compared to the control group three months into the experiment. Superoxide dismutase (SOD) was increased in group 4 compared to the control group from day 60 until the end of the experiment, and in groups 2 and 3 compared to the control after three months. Based on the differences in the cellular immunity and oxidative stress parameters, with an overall absence of mortality, the results of this study suggest that the use of IMUNO-2865(®) in aquaculture is safe and possess a cumulative immunostimulatory effect on sea bream.

Anatomically and morphologically unique dark septate endophytic association in the roots of the Mediterranean endemic seagrass Posidonia oceanica.

Roots of terrestrial plants host a wide spectrum of soil fungi that form various parasitic, neutral and mutualistic associations. A similar trend is evident in freshwater aquatic plants and plants inhabiting salt marshes or mangroves. Marine vascular plants (seagrasses), by contrast, seem to lack specific root-fungus symbioses. We examined roots of two Mediterranean seagrasses, Posidonia oceanica and Cymodocea nodosa, in the northwestern Mediterranean Sea for fungal colonization using light and scanning and transmission electron microscopy. We found that P. oceanica, but not C. nodosa, is regularly associated with melanized septate hyphae in a manner resembling colonization by the ubiquitous dark septate endophytes (DSE) in roots of most terrestrial plants. P. oceanica roots were found to be colonized by sparse dematiaceous running hyphae as well as dense parenchymatous nets/hyphal sheaths on the root surface, intracellular melanized microsclerotia and occasionally also intra- and intercellular hyphae. The colonization was most prominent in the thick-walled hypodermis of the thinnest healthy looking roots, and the mycobiont seemed to colonize both living and dead host cells. Dark septate hyphae infrequently occurred also inside rhizodermal cells, but never colonized vascular tissues. The biological significance of this overlooked marine symbiosis remains unknown, but its morphology, extent, distribution across the NW Mediterranean Sea and absence in C. nodosa indicate an intriguing relationship between the dominant Mediterranean seagrass and its dark septate root mycobionts.

Epidemiology of Noble Pen Shell (Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression.

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such "lower risk" areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

Glycymeris pilosa (Bivalvia) - A high-potential geochemical archive of the environmental variability in the Adriatic Sea.

Due to its outstanding longevity (decades), the shallow-water bivalve Glycmeris pilosa represents a prime target for sclerochronological research in the Mediterranean Sea. In the present study, we analyzed the microgrowth patterns and the stable carbon (δ13Cshell) and oxygen (δ18Oshell) isotopes of the outer shell layer of live-collected G. pilosa specimens from four different sites along the Croatian coast, middle Adriatic Sea. Combined analysis of shell growth patterns and temporally aligned δ18Oshell data indicated that the main growing season lasts from April to December, with fastest growth rates occurring during July and August when seawater temperatures exceeded 22 °C. Slow growth in the cold season (<12 °C) coincided with the formation of winter growth lines on the outer shell surface. The growth cessation occurred in winter, but on the outer shell surface the brown summer bands are more pronounced than the winter lines. Mutvei-staining of cross-sections facilitated the recognition of the growth lines. δ13Cshell values reflect ontogenetic changes in physiology as well as seasonal changes in primary production and salinity.

Tracking a mass mortality outbreak of pen shell Pinna nobilis populations: A collaborative effort of scientists and citizens.

A mass mortality event is devastating the populations of the endemic bivalve Pinna nobilis in the Mediterranean Sea from early autumn 2016. A newly described Haplosporidian endoparasite (Haplosporidium pinnae) is the most probable cause of this ecological catastrophe placing one of the largest bivalves of the world on the brink of extinction. As a pivotal step towards Pinna nobilis conservation, this contribution combines scientists and citizens' data to address the fast- and vast-dispersion and prevalence outbreaks of the pathogen. Therefore, the potential role of currents on parasite expansion was addressed by means of drift simulations of virtual particles in a high-resolution regional currents model. A generalized additive model was implemented to test if environmental factors could modulate the infection of Pinna nobilis populations. The results strongly suggest that the parasite has probably dispersed regionally by surface currents, and that the disease expression seems to be closely related to temperatures above 13.5 °C and to a salinity range between 36.5-39.7 psu. The most likely spread of the disease along the Mediterranean basin associated with scattered survival spots and very few survivors (potentially resistant individuals), point to a challenging scenario for conservation of the emblematic Pinna nobilis, which will require fast and strategic management measures and should make use of the essential role citizen science projects can play.

Bivalve trophic ecology in the Mediterranean: Spatio-temporal variations and feeding behavior.

The trophic ecology of two bivalves, the clam Callista chione and the cockle Glycymeris bimaculata was studied using environmental and biochemical variables of the suspended particulate matter and the sediment. Samples were collected from two shallow sites, Pag and Cetina, in the coastal oligotrophic Mediterranean Sea, during a 17 month period. The temporal variation of the particulate matter reflected a mixture between marine and terrestrial sources throughout the year, with a clear marine influence during summer and fall, and predominance of terrestrial inputs during spring and winter. The digestive gland was a useful rapid turnover tissue, where the carbon isotope signal was species-specific and the nitrogen isotope one was site-specific. FA markers in the digestive gland revealed a mixed diet where Callista chione fed more upon fresh material than G. bimaculata which relied largely on bacteria-derived detritus. Overall, little feeding niche overlap was observed between the two species during the year, indicating resource partitioning, expected for a food-limited system. The present trophic ecology study in co-occurring species allowed identifying species-specific feeding adaptations to environmental variability.

Changes in the tissue concentrations of trace elements during the reproductive cycle of Noah's Ark shells (Arca noae Linnaeus, 1758).

Concentrations of 23 trace elements (TEs; essential: Co, Cu, Fe, Mn, Mo, Se, V, Zn; non-essential: Ag, Al, As, Ba, Cd, Cr, Cs, Li, Ni, Pb, Rb, Sr, Ti, Tl, U) in whole soft tissues of Noah's Ark shell (Arca noae) were determined monthly during one year (March 2013-February 2014) at two sampling sites in the central part of the Eastern Adriatic Sea. Our aim was to detect the influence of reproductive cycle and changes in the environmental factors on the variabilities of TEs' contents. Higher concentrations of Pb, Ba, V, Mo, Mn and Fe were found at potentially contaminated site in Pasman channel, whereas higher concentrations of Tl, Ni, Li, Cr, Cd, Ti and Se were found at reference site in Nature Park Telascica. Since several bioaccumulated TEs were associated to mean gonadal index, in TEs monitoring in A. noae, animal gonadal status has to be considered.

Elemental profiling of Noah's Ark shell (Arca noae, Linnaeus, 1758) by plasma optical spectrometry and chemometric tools.

Determination of metal content in biominerals provides essential information with respect to relations between biomineralization processes and environmental status. Mussels are species that have a great potential as bio-marker species and therefore, they are in the focus of numerous biomineralization and ecological studies. In this study, elemental profile of mussel shell of Noah's Ark (Arca noe, Linnaeus, 1758), which inhabit eastern Adriatic Sea was obtained by determination of seventeen elements content using inductively coupled plasma optical emission spectrometry (ICP-OES). Shell samples were collected from marine protected area and from marine shipping route in eastern Adriatic Sea. The accuracy of applied analytical procedure based on microwave decomposition of shell samples was tested by use of reference materials of limestone and by matrix-matched standards. By aid of chemometric methods, the elemental profile along with variability of elements content of shell was obtained. The impact of different environment on elements content was established by use of multivariate statistical PCA method. Discernment between two groups of samples was manifested. Among results of main, minor and trace elements content, the last one which denoted to Cd, Co, Cu, Pb, and Mn was expressed as principal distinctive feature of shell samples collected from different sampling sites. Elemental profiling of mussel shell Noah's Ark provides novel insight in species status as well as in environmental status on the observed locations.