Calmodulin inhibition as a mode of action of antifungal imidazole pharmaceuticals in non-target organisms.

To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

Disparate effects of antibiotic-induced microbiome change and enhanced fitness in Daphnia magna.

It is a common view that an organism's microbiota has a profound influence on host fitness; however, supporting evidence is lacking in many organisms. We manipulated the gut microbiome of Daphnia magna by chronic exposure to different concentrations of the antibiotic Ciprofloxacin (0.01-1 mg L-1), and evaluated whether this affected the animals fitness and antioxidant capacity. In line with our expectations, antibiotic exposure altered the microbiome in a concentration-dependent manner. However, contrary to these expectations, the reduced diversity of gut bacteria was not associated with any fitness detriment. Moreover, the growth-related parameters correlated negatively with microbial diversity; and, in the daphnids exposed to the lowest Ciprofloxacin concentrations, the antioxidant capacity, growth, and fecundity were even higher than in control animals. These findings suggest that Ciprofloxacin exerts direct stimulatory effects on growth and reproduction in the host, while microbiome- mediated effects are of lesser importance. Thus, although microbiome profiling of Daphnia may be a sensitive tool to identify early effects of antibiotic exposure, disentangling direct and microbiome-mediated effects on the host fitness is not straightforward.

Multi-level toxicity assessment of engineered cellulose nanofibrils in Daphnia magna.

Cellulose nanofibril (CNF)-based materials are increasingly used in industrial and commercial applications. However, the impacts of CNF on aquatic life are poorly understood, and there are concerns regarding their potential toxicity. Using a combination of standard ecotoxicological tests and feeding experiments, we assessed the effects of CNF exposure (0.206-20.6 mg/L) on the feeding (food uptake and gut residence time) and life-history traits (growth and reproduction) in the cladoceran Daphnia magna. No mortality was observed in a 48 h acute exposure at 2060 mg/L. Moreover, a 21-day exposure at low food and moderate CNF levels induced a stimulatory effect on growth, likely driven by increased filtration efficiency, and, possibly, partial assimilation of the CNF by the animals. However, at low food levels and the highest CNF concentrations, growth and reproduction were negatively affected. These responses were linked to caloric restriction caused by dilution of the food source, but not an obstruction of the alimentary canal. Finally, no apparent translocation of CNF past the alimentary canal was detected. We conclude that CNF displays a low toxic potential to filter-feeding organisms and the expected environmental risks are low.

Bacteria-mediated effects of antibiotics on Daphnia nutrition.

In polluted environments, contaminant effects may be manifested via both direct toxicity to the host and changes in its microbiota, affecting bacteria-host interactions. In this context, particularly relevant is exposure to antibiotics released into environment. We examined effects of the antibiotic trimethoprim on microbiota of Daphnia magna and concomitant changes in the host feeding. In daphnids exposed to 0.25 mg L(-1) trimethoprim for 24 h, the microbiota was strongly affected, with (1) up to 21-fold decrease in 16S rRNA gene abundance and (2) a shift from balanced communities dominated by Curvibacter, Aquabacterium, and Limnohabitans in controls to significantly lower diversity under dominance of Pelomonas in the exposed animals. Moreover, decreased feeding and digestion was observed in the animals exposed to 0.25-2 mg L(-1) trimethoprim for 48 h and then fed 14C-labeled algae. Whereas the proportion of intact algal cells in the guts increased with increased trimethoprim concentration, ingestion and incorporation rates as well as digestion and incorporation efficiencies decreased significantly. Thus, antibiotics may impact nontarget species via changes in their microbiota leading to compromised nutrition and, ultimately, growth. These bacteria-mediated effects in nontarget organisms may not be unique for antibiotics, but also relevant for environmental pollutants of various nature.

Antibiotic-induced change of bacterial communities associated with the copepod Nitocra spinipes.

Environmental pressures, such as physical factors, diet and contaminants may affect interactions between microbial symbionts and their multicellular hosts. Despite obvious relevance, effects of antimicrobial contaminants on host-symbiont relations in non-target aquatic organisms are largely unknown. We show that exposure to antibiotics had negative effects on survival and juvenile development of the copepod Nitocra spinipes and caused significant alterations in copepod-associated bacterial communities. The significant positive correlations between indices of copepod development and bacterial diversity indicate that disruption of the microflora was likely to be an important factor behind retarded juvenile development in the experimental animals. Moreover, as evidenced by ribotype distribution in the bacterial clone libraries, the exposure to antibiotics caused a shift in dominance from Betaproteobacteria to Cardinium bacteria; the latter have been shown to cause reproductive manipulations in various terrestrial arthropods. Thus, in addition to providing evidence that the antibiotic-induced perturbation of the microbial community associates with reductions in fitness-related traits of the host, this study is the first record of a copepod serving as a host for endosymbiotic Cardinium. Taken together, our results suggest that (1) antimicrobial substances and possibly other stressors can affect micobiome and symbiont-mediated interactions in copepods and other hosts, and (2) Cardinium endosymbionts may occur in other copepods and affect reproduction of their hosts.