Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment - Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.

Accumulation of metal(loid)s in the digestive gland of the mussel Unio crassus Philipsson, 1788: A reliable detection of historical freshwater contamination.

The possible influence of historical contamination of water/sediments on the metal(loid) bioaccumulation in the digestive gland of mussel Unio crassus Philipsson, 1788, from two differently contaminated sites at the Mreznica River was studied in three seasons. The first data for this species on total/cytosolic concentrations of 27 (non)essential elements were obtained by HR ICP-MS. Higher bioaccumulation was observed at the historically contaminated site, with several nonessential elements (Bi, Cs, Pb, Sb, Tl, U) found in 5-6 times higher concentrations compared to the reference site. Although both total and cytosolic levels revealed the influence of water/sediment contamination, the latter showed association between bioaccumulation and exposure for larger number of studied elements. At the reference site, several elements (Ba, Ca, Cd, Cr, Mn, Sr) were also found in 2-10 times higher concentrations compared to contaminated one, but it was attributed to background levels characteristic for karst rivers (for Ca and Cd), and to coaccumulation due to chemical similarity (for Ba, Cr, Mn, Sr). The seasonal variability was also observed, with generally highest metal(loid) concentrations in mussel digestive glands found in autumn which was associated to mussels reproductive period. Our results confirmed that sediment-dwelling mussels, specifically U. crassus, represent a good bioindicators for detection of historical pollution due to their direct contact/exposure to contaminants stored in sediments, with concurrent consideration of physiological/chemical factors. Historical contamination potentially can have serious impact on freshwater environment even long time after its cessation, and, therefore, a careful continuous monitoring is recommended.

A PacBio Hi-Fi Genome Assembly of the Painter's Mussel Unio pictorum (Linnaeus, 1758).

The highly diverse group of freshwater mussels from order Unionida is found in the world's freshwater systems due to several fascinating evolutionary adaptations, including "parental care," and most notably, an obligatory parasitic phase in their early life cycle, called glochidia, which infests and uses fish for nutrition and dispersal. Freshwater mussels play essential ecological roles in freshwater habitats, including water filtration, sediment bioturbation, and nutrient cycling. However, these species are also highly threatened, being one of the faunal groups with the highest recorded extinction rate in the wild. Genomics methods have an incredible potential to promote biodiversity conservation, allowing the characterization of population health, identification of adaptive genetic elements, delineation of conservation units, and providing a framework for predictive assessments of the impact of anthropogenic threats and climate change. Unfortunately, only six freshwater mussel species have had their whole genomes sequenced to date, and only two of these are European species. Here, we present the first genome assembly of the Painter's Mussel, Unio pictorum (Linnaeus, 1758), the type species representative of the order and the most widespread species of the genus in Europe. We used long-read PacBio Hi-Fi sequencing reads to produce a highly contiguous assembly that will pave the way for the study of European freshwater mussels in the Genome Era.

A roadmap for the conservation of freshwater mussels in Europe.

Europe has a long history of human pressure on freshwater ecosystems. As pressure continues to grow and new threats emerge, there is an urgent need for conservation of freshwater biodiversity and its ecosystem services. However, whilst some taxonomic groups, mainly vertebrates, have received a disproportionate amount of attention and funds, other groups remain largely off the public and scientific radar. Freshwater mussels (Bivalvia, Unionida) are an alarming example of this conservation bias and here we point out six conceptual areas that need immediate and long-term attention: knowledge, threats, socioeconomics, conservation, governance and education. The proposed roadmap aims to advance research, policy and education by identifying the most pressing priorities for the short- and long-term conservation of freshwater mussels across Europe.

Benthos-drift relationships as proxies for the detection of the most suitable bioindicator taxa in flowing waters - a pilot-study within a Mediterranean karst river.

Mediterranean karst aquifers are sensitive systems vulnerable to contamination, exhibiting high rates of diversity and endemicity. In the present pilot-study, we aimed to detect the most suitable bioindicators of contaminant accumulation and mobilization within a Mediterranean karst river (Krka River, Croatia), whose lowermost sections belong to a designated protection area (national park). To meet our goal, we sampled water, drift and benthos (macroinvertebrates and periphytic microfauna) at the two Krka River sites, located upstream and downstream from town Knin and its urban influences. We compared: 1) environmental conditions (water physico-chemical parameters, trace- and macro-element concentrations); 2) abundance and diversity of periphyton and macroinvertebrate taxa constituting benthos; and 3) macroinvertebrate benthos-drift relationships between the two sites. Despite higher values of all measured physico-chemical parameters, and most trace- and macro-element concentrations at the urban-influenced site, the concentrations of contamination indicators (i.e., COD, nutrients, metals) at both sites were generally low. This is likely a result of specific "self-purification ability" of the Krka River, mediated by relatively high contaminant retention potential of the underlying tufa (i.e., calcareous) and/or macrophyte substrates. Between-site differences in water quality further affected the spatial variation of macrozoobenthos, drift, and periphytic microfauna. We suggest that increased COD and orthophosphate concentration, and macrophyte presence at the urban-influenced site, supported higher densities and diversity of benthic organisms dominated by eurivalent (i.e., contamination-tolerant) taxa. The most numerous macroinvertebrate taxa in benthos were amphipod Gammarus balcanicus and the representatives of the endemic Dinaric karst taxa - gastropods Emmericia patula and Radomaniola curta germari, and another amphipod Echinogammarus acarinatus. Although we expected to observe significantly increased drift at the urban-influenced site due to the degraded environmental conditions, it was not observed. The observed benthos-drift patterns suggest that freshwater amphipods (i.e., gammarids), which were found most numerous in drift, could be considered as the most suitable bioindicators of a contaminant (i.e., metal) accumulation and mobilization within karst aquifers comparable to Krka River.

Conservation status of freshwater mussels in Europe: state of the art and future challenges.

Freshwater mussels of the Order Unionida provide important ecosystem functions and services, yet many of their populations are in decline. We comprehensively review the status of the 16 currently recognized species in Europe, collating for the first time their life-history traits, distribution, conservation status, habitat preferences, and main threats in order to suggest future management actions. In northern, central, and eastern Europe, a relatively homogeneous species composition is found in most basins. In southern Europe, despite the lower species richness, spatially restricted species make these basins a high conservation priority. Information on freshwater mussels in Europe is unevenly distributed with considerable differences in data quality and quantity among countries and species. To make conservation more effective in the future, we suggest greater international cooperation using standardized protocols and methods to monitor and manage European freshwater mussel diversity. Such an approach will not only help conserve this vulnerable group but also, through the protection of these important organisms, will offer wider benefits to freshwater ecosystems.

Differences in tolerance to anthropogenic stress between invasive and native bivalves.

Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species can better endure unfavorable conditions compared with native species is limited and has yielded opposing results. In this study, we examined the tolerance to different stress conditions (thermal stress and trace metal zinc pollution stress) in two phylogenetically related and functionally similar freshwater bivalve species, the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed potential differences in response to stress conditions using several cellular response assays: efficiency of the multixenobiotic resistance mechanism, respiration estimate (INT reduction capacity), and enzymatic biomarkers. Our results demonstrated that the invasive species overall coped much better with unfavorable conditions. The higher tolerance of S. woodiana was evident from (i) significantly decreased Rhodamine B accumulation indicating more efficient multixenobiotic resistance mechanism; (ii) significantly higher INT reduction capacity and (iii) less pronounced alterations in the activity of stress-related enzymes (glutathione-S-transferase, catalase) and of a neurotoxicity biomarker (cholinesterase) in the majority of treatment conditions in both stress trials. Higher tolerance to thermal extremes may provide physiological benefit for further invasion success of S. woodiana in European freshwaters, especially in the context of climate change.