A discovery of two new Tetrahymena species parasitizing slugs and mussels: morphology and multi-gene phylogeny of T. foissneri sp. n. and T. unionis sp. n.

The presence of parasitic ciliates of the hymenostome genus Tetrahymena was examined in 150 mollusks belonging to six bivalve and 13 gastropod species in Slovakia, Central Europe. Tetrahymenids were detected only in two species, viz., in the invasive Lusitanian slug (Arion vulgaris) and in the native swollen river mussel (Unio tumidus). Although only 10.52% of the examined mollusk taxa were positive, their Tetrahymena infections were very intensive accounting for several hundreds of ciliates per host. Phylogenetic analyses of the 16S and 18S rRNA genes as well as of the barcoding region of the gene encoding for cytochrome c oxidase subunit I revealed that both isolates represent new taxa, T. foissneri sp. n. and T. unionis sp. n. The former species belongs to the 'borealis' clade and its nearest relative is T. limacis, a well-known parasite of slugs and snails. Besides molecular data, T. foissneri can be distinguished from T. limacis also morphologically by the body shape of the parasitic-phase form, dimensions of micronuclei, and the silverline system. On the other hand, T. unionis was classified within the 'paravorax' clade along with T. pennsylvaniensis, T. glochidiophila, and T. nigricans. Although these four species are genetically distinct, T. unionis could be morphologically separated only from T. nigricans by body shape and size. The present study suggests that both aquatic and terrestrial mollusks represent interesting hosts for the discovery of novel Tetrahymena lineages.

Biochemical responses of freshwater mussel Unio tumidus to titanium oxide nanoparticles, Bisphenol A, and their combination.

Multiple interactions between different pollutants in the surface waters can cause unpredictable consequences. The aim of the study was to evaluate the combined effect of two widespread xenobiotics, titanium oxide nanoparticles (TiO2) and bisphenol A (BPA), on freshwater bivalve Unio tumidus. The specimens were exposed for 14 days to TiCl4 (Ti, 1.25 µM), TiO2 (1.25 µM), BPA (0.88 nM), or their combination (TiO2 + BPA). Every type of exposure resulted in a particular oxidative stress response: TiO2 had antioxidant effect, decreasing the generation of reactive oxygen species (ROS) and phenoloxidase (PhO) activity, and doubling reduced glutathione (GSH) concentration in the digestive gland; Ti caused oxidative changes by increasing levels of ROS, PhO and superoxide dismutase; BPA decreased the GSH level by a factor of two. In the co-exposure treatment, these indices as well as lysosomal membrane stability were not affected. All Ti-containing exposures caused elevated levels of metalated metallothionein (Zn,Cu-MT), its ratio to total metallothionein protein, and lactate/pyruvate ratio. Both BPA-containing exposures decreased caspase-3 activity. All exposures, and particularly co-exposure, up-regulated CYP450-dependent oxidation, lipid peroxidation and lipofuscin accumulation, lysosomal cathepsin D and its efflux, as well as alkali-labile phosphates in gonads and caused DNA instability (except for TiO2). To summarize, co-exposure to TiO2 + BPA produced an overlap of certain individual responses but strengthened the damage. Development of water purification technologies using TiO2 requires further studies of the biological effects of its mixtures. U. tumidus can serve as a sentinel organism in such studies.

Differential effects of antidepressant sertraline in glochidia-fish interactions involving drug transfer from parasite to host.

This study examined the impact of sertraline, an antidepressant common in treated wastewater, on the host-parasite dynamics between parasitic freshwater mussel (Unio tumidus, Unionidae) larvae (glochidia) and their host fish (Squalius cephalus, Cyprinidae). Employing a full-factorial design, both fish and glochidia were subjected to sertraline at the combinations of 0 µg L-1 (control), 0.2 µg L-1 (environmentally relevant concentration), and 4 µg L-1 (elevated concentration, short-term exposure of the parasite). The results showed that long-term host exposure (involving intensive sertraline accumulation in the fish brain) marginally increased subsequent glochidia attachment success by 2 %, while parasite exposure at the same environmentally relevant concentrations had no detectable effect. There was also no effect of exposure of glochidia to 0.2 µg L-1 of sertraline on their viability and encapsulation success during the initial parasitic stage. However, a significant alteration in attachment behavior, marked by a 3.3 % increase in attachment success and changes in the glochidia spatial distribution on the host body, was noted after 24 h of glochidia exposure to 4 µg L-1 of sertraline. Importantly, this study provides the first evidence of sertraline transfer from exposed glochidia to nonexposed host fish, as indicated by elevated levels of sertraline (12.8 ng g-1) in the brain tissue of nonexposed hosts. These findings highlight the subtle yet significant effects of pharmaceutical pollutants on freshwater ecosystems but also underscore the importance of understanding the unexpected dynamics of such contamination to predict and address future ecological changes.

A comparison of the concentrations of heavy metals in modern and medieval shells of swollen river mussels (Unio tumidus) from the Szczecin Lagoon, SW Baltic basin.

The shells of mussels, live-collected bivalves or during archaeological excavations, can be used as bioindicators of current and historical levels of heavy metal contamination. In this study, we examined the shells of Unio tumidus, commonly found in the Baltic Sea region, and determined the concentrations of Zn, Cu, Fe, Pb, Ni, and Cd in samples from the 10th, 11th, and 21st century from the area of the Szczecin Lagoon. The average levels of heavy metals (in µg g-1 dry weight) in the shells from the Middle Ages were: 137.5 (Fe), 3.87 (Zn), 0.789 (Cu), 0.012 (Pb), 0.047 (Ni), and 0.0009 (Cd). Shells from the 21st century were significantly (P<0.05) more abundant in Fe, Cu, Ni, Pb, and Cd (rates of increase: 1.96×, 3.54×, 2.71×, 2.08×, and 3.55×, respectively) than shells from the Middle Ages. These results reflect contemporary anthropogenic pollution of the environment with heavy metals and confirm the possibility of using U. tumidus shells in the assessment of heavy metal pollution levels.

Flooding modifies the genotoxic effects of pollution on a worm, a mussel and two fish species from the Sava River.

Extreme hydrological events, such as water scarcity and flooding, can modify the effect of other stressors present in aquatic environment, which could result in the significant changes in the ecosystem functioning. Presence and interaction of various stressors (genotoxic pollutants) in the environment can influence the integrity of DNA molecules in aquatic organisms which can be negatively reflected on the individual, population and community levels. Therefore, in this study we have investigated the impact of flooding, in terms of genotoxicity, on organisms belonging to different trophic levels. The study was carried out on the site situated in the lower stretch of the Sava River which faced devastating effects of severe flooding in May 2014. The flooding occurred during our field experiment and this event provided a unique opportunity to assess its influence to the environment. The in situ effects of this specific situation were monitored by measuring physical, chemical and microbiological parameters of water, and by comparing the level of DNA damage in coelomocytes and haemocytes of freshwater worms Branchiura sowerbyi, haemocytes of freshwater mussels Unio tumidus and blood cells of freshwater fish Abramis bjoerkna/Abramis sapa, by means of the comet assay. Our study indicated that the flooding had a significant impact on water quality by decreasing the amount and discharge rate of urban wastewaters but simultaneously introducing contaminants from the nearby fly ash disposal field into river by runoff, which had diverse effects on the level of DNA damage in the studied organisms. This indicates that the assessment of genotoxic pollution in situ is strongly affected by the choice of the bioindicator organism.

Assessment of the genotoxic potential along the Danube River by application of the comet assay on haemocytes of freshwater mussels: The Joint Danube Survey 3.

In this study we assessed the level of genotoxic pollution along the Danube River by measuring the level of DNA damage in the haemocytes of freshwater mussels of Unio sp. (Unio pictorum/Unio tumidus) and Sinanodonta woodiana. The comet assay was used for the assessment of DNA damage. The research was performed on 34 out of 68 sites analysed within the Joint Danube Survey 3 - the world's biggest river research expedition of its kind in 2013. During research, 2285 river kilometres were covered with an average distance of 68 km between the sites. The complex data set on concentrations of various substances present in water, suspended particulate matter and sediment on investigated sites gave the opportunity to identify the groups of xenobiotics which mostly affect the studied biomarker - DNA damage. The highest levels of DNA damage were recorded in the section VI (Panonnian Plain), which is under the impact of untreated wastewater discharges. Both positive and negative influences of the large tributaries on the level of genotoxicity in the Danube River were evident. Significant correlation in response was detected between the studied species of freshwater mussels. The level of DNA damage in mussels correlated with concentrations of compounds from the group of hazardous priority substances (polycyclic aromatic hydrocarbons), persistent organic pollutants (dioxins) and emerging pollutants (Oxazepam, Chloridazon-desphenyl).

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 µM) with 0.4 µM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 µM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.