From shells to sequences: A proof-of-concept study for on-site analysis of hemolymphatic circulating cell-free DNA from sentinel mussels using Nanopore technology.

Blue mussels are often abundant and widely distributed in polar marine coastal ecosystems. Because of their wide distribution, ecological importance, and relatively stationary lifestyle, bivalves have long been considered suitable indicators of ecosystem health and changes. Monitoring the population dynamics of blue mussels can provide information on the overall biodiversity, species interactions, and ecosystem functioning. In the present work, we combined the concept of liquid biopsy (LB), an emerging concept in medicine based on the sequencing of free circulating DNA, with the Oxford Nanopore Technologies (ONT) platform using a portable laboratory in a remote area. Our results demonstrate that this platform is ideally suited for sequencing hemolymphatic circulating cell-free DNA (ccfDNA) fragments found in blue mussels. The percentage of non-self ccfDNA accounted for >50 % of ccfDNA at certain sampling Sites, allowing the quick, on-site acquisition of a global view of the biodiversity of a coastal marine ecosystem. These ccfDNA fragments originated from viruses, bacteria, plants, arthropods, algae, and multiple Chordata. Aside from non-self ccfDNA, we found DNA fragments from all 14 blue mussel chromosomes, as well as those originating from the mitochondrial genomes. However, the distribution of nuclear and mitochondrial DNA was significantly different between Sites. Similarly, analyses between various sampling Sites showed that the biodiversity varied significantly within microhabitats. Our work shows that the ONT platform is well-suited for LB in sentinel blue mussels in remote and challenging conditions, enabling faster fieldwork for conservation strategies and resource management in diverse settings.

Species- and site-specific circulating bacterial DNA in Subantarctic sentinel mussels Aulacomya atra and Mytilus platensis.

Impacts of climate changes are particularly severe in polar regions where warmer temperatures and reductions in sea-ice covers threaten the ecological integrity of marine coastal ecosystems. Because of their wide distribution and their ecological importance, mussels are currently used as sentinel organisms in monitoring programs of coastal ecosystems around the world. In the present study, we exploited the concept of liquid biopsy combined to a logistically friendly sampling method to study the hemolymphatic bacterial microbiome in two mussel species (Aulacomya atra and Mytilus platensis) in Kerguelen Islands, a remote Subantarctic volcanic archipelago. We found that the circulating microbiome signatures of both species differ significantly even though their share the same mussel beds. We also found that the microbiome differs significantly between sampling sites, often correlating with the particularity of the ecosystem. Predictive models also revealed that both species have distinct functional microbiota, and that the circulating microbiome of Aulacomya atra was more sensitive to changes induced by acute thermal stress when compared to Mytilus platensis. Taken together, our study suggests that defining circulating microbiome is a useful tool to assess the health status of marine ecosystems and to better understand the interactions between the sentinel species and their habitat.

GAS1: A New ß-Glucan Immunostimulant Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Bacterial Infections With Aeromonas salmonicida achromogenes.

ß-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two ß-glucan types extracted from wild-type baker's yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard®) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard®, Gas1, and Wild type-ß-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that ß-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each ß-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard ß-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT ß-glucan, at D36, WT-ß-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-ß-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of ß-glucans (p<0.05). In conclusion, our results suggest that Gas1-ß-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other ß-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.

Liquid biopsies for omics-based analysis in sentinel mussels.

Liquid biopsy of plasma is a simple and non-invasive technology that holds great promise in biomedical research. It is based on the analysis of nucleic acid-based biomarkers with predictive potential. In the present work, we have combined this concept with the FTA technology for sentinel mussels. We found that hemocytes collected from liquid biopsies can be readily fixed on FTA cards and used for long-term transcriptome analysis. We also showed that liquid biopsy is easily adaptable for metagenomic analysis of bacterial profiles of mussels. We finally provide evidence that liquid biopsies contained circulating cell-free DNA (ccfDNA) which can be used as an easily accessible genomic reservoir. Sampling of FTA-fixed circulating nucleic acids is stable at room temperature and does not necessitate a cold-chain protection. It showed comparable performance to frozen samples and is ideally adapted for sampling in remote areas, most notably in polar regions threatened by anthropogenic activities. From an ethical point of view, this minimally-invasive and non-lethal approach further reduces incidental mortality associated with conventional tissue sampling. This liquid biopsy-based approach should thus facilitate biobanking activities and development of omics-based biomarkers in mussels to assess the quality of aquatic ecosystems.

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha.

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10-6 M to 10-3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC50 of 3.71±0.53×10-4M for phagocytic activity and 2.79±0.19×10-4M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies.

Acclimation capacity of the three-spined stickleback (Gasterosteus aculeatus, L.) to a sudden biological stress following a polymetallic exposure.

To get closer to the environmental reality, ecotoxicological studies should no longer consider the evaluation of a single pollutant, but rather combination of stress and their interaction. The aim of this study was to determine if responses of a fish to a sudden biological stress could be modified by a prior exposure to a chemical stress (a polymetallic contamination). For this purpose, in situ experiment was conducted in three ponds in the Haute-Vienne department (France). One pond was chosen for its high uranium concentration due to uranium mine tailings, and the two other ponds, which were not submitted to these tailings. Three-spined sticklebacks (Gasterosteus aculeatus) were caged in these ponds for 14 days. After this period, fish were submitted to a biological stress, exerted by lipopolysaccharides injection after anesthesia, and were sacrificed 4 days after these injections for multi-biomarkers analyses (leucocyte viability, phagocytic capacity and reactive oxygen species production, antioxidant peptide and enzymes, lipid peroxidation and DNA damage). The pond which received uranium mine tailings had higher metallic concentrations. Without biological stress, sticklebacks caged in this pond presented an oxidative stress, with increasing of reactive oxygen species levels, modification of some parts of the antioxidant system, and lipid peroxidation. Caging in the two most metal-contaminated ponds resulted in an increase of susceptibility of sticklebacks to the biological stress, preventing their phagocytic responses to lipopolysaccharides and modifying their glutathione contents and glutathione-S-transferase activity.