Wastewater and marine bioindicators surveillance to anticipate COVID-19 prevalence and to explore SARS-CoV-2 diversity by next generation sequencing: One-year study.

This study presents the results of SARS-CoV-2 surveillance in sewage water of 11 municipalities and marine bioindicators in Galicia (NW of Spain) from May 2020 to May 2021. An integrated pipeline was developed including sampling, pre-treatment and biomarker quantification, RNA detection, SARS-CoV-2 sequencing, mechanistic mathematical modeling and forecasting. The viral load in the inlet stream to the wastewater treatment plants (WWTP) was used to detect new outbreaks of COVID-19, and the data of viral load in the wastewater in combination with data provided by the health system was used to predict the evolution of the pandemic in the municipalities under study within a time horizon of 7 days. Moreover, the study shows that the viral load was eliminated from the treated sewage water in the WWTP, mainly in the biological reactors and the disinfection system. As a result, we detected a minor impact of the virus in the marine environment through the analysis of seawater, marine sediments and, wild and aquacultured mussels in the final discharge point of the WWTP.

High-Throughput Sequencing of Environmental DNA as a Tool for Monitoring Eukaryotic Communities and Potential Pathogens in a Coastal Upwelling Ecosystem.

The marine environment includes diverse microeukaryotic organisms that play important functional roles in the ecosystem. With molecular approaches, eukaryotic taxonomy has been improved, complementing classical analysis. In this study, DNA metabarcoding was performed to describe putative pathogenic eukaryotic microorganisms in sediment and marine water fractions collected in Galicia (NW Spain) from 2016 to 2018. The composition of eukaryotic communities was distinct between sediment and water fractions. Protists were the most diverse group, with the clade TSAR (Stramenopiles, Alveolata, Rhizaria, and Telonemida) as the primary representative organisms in the environment. Harmful algae and invasive species were frequently detected. Potential pathogens, invasive pathogenic organisms as well as the causative agents of harmful phytoplanktonic blooms were identified in this marine ecosystem. Most of the identified pathogens have a crucial impact on the aquacultural sector or affect to relevant species in the marine ecosystem, such as diatoms. Moreover, pathogens with medical and veterinary importance worldwide were also found, as well as pathogens that affect diatoms. The evaluation of the health of a marine ecosystem that directly affects the aquacultural sector with a zoonotic concern was performed with the metabarcoding assay.

Integrated transcriptomic and functional immunological approach for assessing the invasiveness of bivalve alien species.

Biological invasions started when humans moved species beyond their normal geographic limits. Bivalves are the most notoriously invasive species in subtidal aquatic environments. Next-generation sequencing technologies are applied to understand the molecular mechanisms involved in the invasion. The ecological immunology focuses on the role of immunity in invasion, and its magnitude could help to predict the invasiveness of alien species. A remarkable case of invasion has been reported in the Ría de Vigo (Spain) by the black pygmy mussel Xenostrobus securis. In Galicia, the Mediterranean mussel Mytilus galloprovincialis is the predominant cultured bivalve species. Can we predict the invasiveness of alien bivalve species by analyzing their immune response? Can X. securis represent a risk for the autochthonous mussel? We evaluated the suitability of the immune-related hypotheses in our model by using an integrated transcriptomic and functional immunological approach. Our analysis suggests lower immune capabilities in X. securis compared to M. galloprovincialis, probably due to the relocation of energetic resources from the immune response to vital physiological processes to cope with salinity stress. This multidisciplinary approach will help us understand how the immune response can be influenced by the adaptive process and how this immune response can influence the invasion process.

Parasites in two coexisting bivalves of the Patagonia coast, southwestern Atlantic Ocean: The Puelche oyster (Ostrea puelchana) and false oyster (Pododesmus rudis).

The purpose of this study was to compare the parasites of two coexisting bivalves, the edible Puelche oyster (Ostrea puelchana) and the false oyster (Pododesmus rudis) that lives attached to O. puelchana shells, and to investigate their host specificity. Samples from wild populations, 465 O. puelchana and 131 P. rudis, were collected seasonally during two years in the San José Gulf (northern Patagonia, Argentina) and were processed using standard histological techniques. To increase the natural low prevalences of Bonamia spp. and Perkinsus spp. that are present in wild populations, an in situ experiment was performed by maintaining captive sentinel bivalves at high densities inside a plastic mesh bag to enhance parasite transmission. Polymerase chain reaction (PCR) assays were used to test for apparent Bonamia sp. infections among captive sentinel O. puelchana specimens (n = 80), and Ray's fluid thioglycollate medium (RFTM) assays and histological immunoassays tested for apparent Perkinsus sp. infections among captive sentinel P. rudis specimens (n = 100). Despite histological observations that revealed the presence of microcells resembling Bonamia sp. infecting hemocytes of some Puelche oysters, PCR assays did not confirm that parasite identification. Among captive sentinel P. rudis that showed histological evidence of Perkinsus sp. infections, neither RFTM nor immunoassays confirmed such parasites. Ostrea puelchana from wild populations were occasional hosts for both Rickettsia-like organism (RLOs) and Urastoma-like turbellarians. In contrast, six parasite taxa infected P. rudis from coexisting populations, including RLOs, Urastoma-like turbellarians, an intracellular gregarine species, Nematopsis-like oocysts, an unidentified coccidian and a Perkinsus qugwadi-like protozoan. These results demonstrated specific infection patterns of the identified parasites in relation to their hosts.

Moving from Histopathology to Molecular Tools in the Diagnosis of Molluscs Diseases of Concern under EU Legislation.

One of the main factors limiting molluscs production is the presence of pathogens and diseases. Disease agent transfer via transfers of live molluscs has been a major cause of disease outbreaks and epizootics. Because of that, the European Union has adopted several decisions and directives, the last in 2006 (2006/88/EC) to control movements of marine organisms over the European countries. Once the disease is established in a determined area its eradication is a complicated task because life cycle of pathogens are not completely known and only a good and early diagnosis of the disease could be the most appropriate way to deal with it. Besides, molluscs do not have an adaptive immune response and vaccination strategies are not possible. Molluscs listed diseases under EU legislation are mainly protozoan parasites, that's why histological techniques are recognized for their diagnosis. However, molecular techniques are being increasingly used primarily as confirmatory techniques of the presence of the pathogens but also in disease monitoring programs. Research perspectives are mainly focussed in the optimization, of the already described techniques to gain in sensitivity and sensibility and in the development of new molecular biology techniques (quantitative real time PCRs), that are faster and easier to apply and that allow a positive diagnosis even in early stages of infection. However, molecular tools detect DNA sequences of the pathogen which does not imply that pathogen is viable in the cell host and the infection is established. Consequently, it needs to be validated against other techniques, such as histology or in situ hybridization, so that its reliability can be determined.

Scrapie infectivity is quickly cleared in tissues of orally-infected farmed fish.

BACKGROUND: Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible spongiform encephalopathy (TSE). BSE epidemic in the UK and elsewhere in Europe has been linked to the use of bovine meat and bone meals (MBM) in the feeding of cattle. There is concern that pigs, poultry and fish bred for human consumption and fed with infected MBM would eventually develop BSE or carry residual infectivity without disease. Although there has been no evidence of infection in these species, experimental data on the susceptibility to the BSE agent of farm animals other than sheep and cow are limited only to pigs and domestic chicken. In the framework of a EU-granted project we have challenged two species of fish largely used in human food consumption, rainbow trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus), with a mouse-adapted TSE strain (scrapie 139A), to assess the risk related to oral consumption of TSE contaminated food. In trout, we also checked the "in vitro" ability of the pathological isoform of the mouse prion protein (PrPSc) to cross the intestinal epithelium when added to the mucosal side of everted intestine. RESULTS: Fish challenged with a large amount of scrapie mouse brain homogenate by either oral or parenteral routes, showed the ability to clear the majority of infectivity load. None of the fish tissues taken at different time points after oral or parenteral inoculation was able to provoke scrapie disease after intracerebral inoculation in recipient mice. However, a few recipient mice were positive for PrPSc and spongiform lesions in the brain. We also showed a specific binding of PrPSc to the mucosal side of fish intestine in the absence of an active uptake of the prion protein through the intestinal wall. CONCLUSION: These results indicate that scrapie 139A, and possibly BSE, is quickly removed from fish tissues despite evidence of a prion like protein in fish and of a specific binding of PrPSc to the mucosal side of fish intestine.

Ultrastructural and molecular characterization of Haplosporidium montforti n. sp., parasite of the European abalone Haliotis tuberculata.

A new member of the parasitic phylum Haplosporidia, which was found infecting the connective tissue, gill, digestive gland, and foot muscle of Haliotis tuberculata imported from Ireland and experimentally grown in Galicia (NW Spain), is described. Scanning electron microscopy, transmission electron microscopy, and molecular characterization of the small subunit ribosomal RNA (SSU rRNA) gene were carried out to confirm the description of this species. The ultrastructural morphology of the spores and their surrounding ornaments attached to the spore wall was described from light, scanning, and transmission electron microscopy observations. Systemic infection with uninucleated and multinucleated plasmodia containing spherical nuclei was observed among several sporocysts containing the different spore maturation stages. The spores were spherical to slightly ellipsoidal (2.42 +/- 0.5 x 2.31 +/- 0.6 microm). The apical zone of the spore wall was modified into a complex opercular system covering a circular orifice that measured about 0.5 microm in diameter. The operculum was connected to the spore wall by a hinge. The spore wall was about 110 nm thick, with 4 filaments (20-28 microm long). The filaments were composed of the same material that formed the wall. The cross-sections through the base of these filaments showed T-like and X-like sections. Internally, the uninucleated endosporoplasm contained typical haplosporidian structures, such as, haplosporosomes, a spherulosome, and mitochondria with vesicular cristae. The SSU rRNA gene sequence was different from previously reported haplosporidian SSU rRNA gene sequences, corroborating morphological data that this was an undescribed species. Based on differences from previously described haplosporidians in ultrastructural characteristics of the spore and SSU rRNA gene sequence, we describe the abalone haplosporidian as Haplosporidium montforti n. sp.