3-Benzylmenadiones and their Heteroaromatic Analogues Target the Apicoplast of Apicomplexa Parasites: Synthesis and Bioimaging Studies.

The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii, which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodiumsuch as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum, the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.

Trophic effects of Bti-based mosquito control on two top predators in floodplain pond mesocosms.

Chironomid (Diptera: Chironomidae) larvae play a key role in aquatic food webs as prey for predators like amphibian and dragonfly larvae. This trophic link may be disrupted by anthropogenic stressors such as Bacillus thuringiensis var. israelensis (Bti), a biocide widely used in mosquito control. In a companion study, we recorded a 41% reduction of non-target larval chironomids abundance in outdoor floodplain pond mesocosms (FPMs) treated with Bti. Therefore, we examined the diet of two top predators in the FPMs, larvae of the palmate newt (Salamandridae: Lissotriton helveticus) and dragonfly (Aeshnidae: predominantly Anax imperator), using bulk stable isotope analyses of carbon and nitrogen. Additionally, we determined neutral lipid fatty acids in newt larvae to assess diet-related effects on their physiological condition. We did not find any effects of Bti on the diet proportions of newt larvae and no significant effects on the fatty acid content. We observed a trend in Aeshnidae larvae from Bti-FPMs consuming a higher proportion of large prey (Aeshnidae, newt, damselfly larvae; ~42%), and similar parts of smaller prey (chironomid, mayfly, Libellulidae, and zooplankton), compared to controls. Our findings may suggest bottom-up effects of Bti on aquatic predators but should be further evaluated, for instance, by using compound-specific stable isotope analyses of fatty acids or metabarcoding approaches.

Elevated Fungicide and Nutrient Concentrations Change Structure but not Function of Aquatic Microbial Communities.

Leaf decomposition is a key process in stream ecosystems within forested catchments; it is driven by microbial communities, particularly fungi and bacteria. These microorganisms make nutrients and energy bound in leaves available for wider parts of the food web. Leaf-associated microorganisms are subjected to anthropogenic pressures, such as the increased exposure to nutrients and fungicides associated with land-use change. We assessed the sensitivity of leaf-associated microbial communities with differing exposure histories, namely, from pristine (P) streams, and streams impacted by wastewater (W) and agricultural run-off (vineyards; V). In the laboratory, microbial communities were exposed to elevated nutrient (NO3-N: 0.2-18.0 mg/L, PO4-P: 0.02-1.8 mg/L) and fungicide concentrations (sum concentration 0-300 µg/L) in a fully crossed 3 × 4 × 4-factorial design over 21 days. Leaf decomposition and exoenzyme activity were measured as functional endpoints, and fungal community composition and microbial abundance served as structural variables. Overall, leaf decomposition did not differ between fungicide treatments or exposure histories. Nonetheless, substantial changes in the fungal community composition were observed after exposure to environmentally relevant fungicide concentrations. Elevated nutrient concentrations assisted leaf decomposition, and the effect size depended on the exposure history. The observed changes in the fungal community composition support the principle of functional redundancy, with highly efficient decomposers maintaining leaf decomposition. Environ Toxicol Chem 2024;43:1300-1311. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Aging of nanosized titanium dioxide modulates the effects of dietary copper exposure on Daphnia magna - an assessment over two generations.

Nanosized titanium dioxide (nTiO2) is widely used in products, warranting its discharge from various sources into surface water bodies. However, nTiO2 co-occurs in surface waters with other contaminants, such as metals. Studies with nTiO2 and metals have indicated that the presence of natural organic matter (NOM) can mitigate their toxicity to aquatic organisms. In addition, "aging" of nTiO2 can affect toxicity. However, it is a research challenge, particularly when addressing sublethal responses from dietary exposure over multiple generations. We, therefore exposed the alga Desmodesmus subspicatus to nTiO2 (at concentrations of 0.0, 0.6 and 3.0 mg nTiO2/L) in nutrient medium aged for 0 or 3 days with copper (Cu) at concentrations of 0 and 116 µg Cu/L and with NOM at concentrations equivalent to 0 and 8 mg total organic carbon (TOC) per litre. Subsequently, the exposed alga was fed to Daphnia magna for 23 days over two generations and survival, reproduction and body length were assessed as endpoints of toxicity. In parallel, Cu accumulation and depuration from D. magna were measured. The results indicate that the reproduction of D. magna was the most sensitive parameter in this study, being reduced by 30% (at both parental (F0) and filial (F1) generations) and 50% (at F0 but not F1) due to the dietary Cu exposure in combination with nTiO2 for 0 and 3 days aging, respectively. There was no relationship between the effects observed on reproduction and Cu body burden in D. magna. Moreover, D. magna from the F1 generation showed an adaptive response to Cu in the treatment with 3.0 mg nTiO2/L aged for 3 days, potentially due to epigenetic inheritance. Unexpectedly, the presence of NOM hardly changed the observed effects, pointing towards the function of algal exopolymeric substances or intracellular organic matter, rendering the NOM irrelevant. Ultimately, the results indicate that the transferability of the impacts observed during the F0 to the responses in the F1 generation is challenging due to opposite effect directions. Additional mechanistic studies are needed to unravel this inconsistency in the responses between generations and to support the development of reliable effect models.

Microbial community history and leaf species shape bottom-up effects in a freshwater shredding amphipod.

Arable land use and the associated application of agrochemicals can affect local freshwater communities with consequences for the entire ecosystem. For instance, the structure and function of leaf-associated microbial communities can be affected by pesticides, such as fungicides. Additionally, the leaf species on which these microbial communities grow reflects another environmental filter for community structure. These factors and their interaction may jointly modify leaves' nutritional quality for higher trophic levels. To test this assumption, we studied the structure of leaf-associated microbial communities with distinct exposure histories (pristine [P] vs vineyard run off [V]) colonising two leaf species (black alder, European beech, and a mixture thereof). By offering these differently colonised leaves as food to males and females of the leaf-shredding amphipod Gammarus fossarum (Crustacea; Amphipoda) we assessed for potential bottom-up effects. The growth rate, feeding rate, faeces production and neutral lipid fatty acid profile of the amphipod served as response variable in a 2 × 3 × 2-factorial test design over 21d. A clear separation of community history (P vs V), leaf species and an interaction between the two factors was observed for the leaf-associated aquatic hyphomycete (i.e., fungal) community. Sensitive fungal species were reduced by up to 70 % in the V- compared to P-community. Gammarus' growth rate, feeding rate and faeces production were affected by the factor leaf species. Growth was negatively affected when Gammarus were fed with beech leaves only, whereas the impact of alder and the mixture of both leaf species was sex-specific. Overall, this study highlights that leaf species identity had a more substantial impact on gammarids relative to the microbial community itself. Furthermore, the sex-specificity of the observed effects (excluding fatty acid profile, which was only measured for male) questions the procedure of earlier studies, that is using either only one sex or not being able to differentiate between males and females. However, these results need additional verification to support a reliable extrapolation.

Effects of flow reduction and artificial light at night (ALAN) on litter decomposition and invertebrate communities in streams: A flume experiment.

River ecosystems are heavily impacted by multiple stressors, where effects can cascade downstream of point sources. However, a spatial approach to assess the effects of multiple stressors is missing. We assessed the local and downstream effects on litter decomposition, and associated invertebrate communities of two stressors: flow reduction and artificial light at night (ALAN). We used an 18-flow-through mesocosm system consisting of two tributaries, where we applied the stressors, merging in a downstream section. We assessed the changes in decomposition rate and invertebrate community structure in leaf bags. We found no effect of ALAN or its interaction with flow reduction on the litter decomposition or the invertebrate community in the tributaries. Flow reduction alone led to a 14.8 % reduction in decomposition rate in the tributaries. We recorded no effect of flow reduction on the macroinvertebrates community composition in the litter bags. We also observed no effects of the spatial arrangement of the stressors on the litter decomposition and macroinvertebrate community structure downstream. Overall, our results suggest the impact of stressors on litter decomposition and macroinvertebrate communities remained local in our experiment. Our work thus calls for further studies to identify the mechanisms and the conditions under which spatial effects dominate over local processes.

Nitrogen retention in stream biofilms - A potential contribution to the self-cleaning capacity.

Surface waters are under increasing pressure due to human activities, such as nutrient emissions from wastewater treatment plants (WWTPs). Using the retention of nitrogen (N) released from WWTPs as a proxy, we assessed the contribution of biofilms grown on inorganic and organic substrates to the self-cleaning capacity of second-order streams within the biosphere reserve Vosges du Nord/Palatinate forest (France/Germany). The uptake of N from anthropogenic sources, which is enriched with the heavy isotope 15N, into biofilms was assessed up- and downstream of WWTPs after five weeks of substrate deployment. Biofilms at downstream sites showed a significant positive linear relationship between δ15N and the relative contribution of wastewater to the streams' discharge. Furthermore, δ15N substantially increased in areas affected by WWTP effluent (∼8.5‰ and ∼7‰ for inorganic and organic substrate-associated biofilms, respectively) and afterwards declined with increasing distance to the WWTP effluent, approaching levels of upstream sections. The present study highlights that biofilms contribute to nutrient retention and likely the self-cleaning capacity of streams. This function seems, however, to be limited by the fact that biofilms are restricted in their capacity to process excessive N loads with large differences between individual reaches (e.g., δ15N: -3.25 to 12.81‰), influenced by surrounding conditions (e.g., land use) and modulated through climatic factors and thus impacted by climate change. Consequently, the impact of WWTPs located close to the source of a stream are dampened by the biofilms' capacity to retain N only to a minor share and suggest substantial N loads being transported downstream.

Diatoms Reduce Decomposition of and Fungal Abundance on Less Recalcitrant Leaf Litter via Negative Priming.

Heterotrophic microbial decomposers colonize submerged leaf litter in close spatial proximity to periphytic algae that exude labile organic carbon during photosynthesis. These exudates are conjectured to affect microbial decomposers' abundance, resulting in a stimulated (positive priming) or reduced (negative priming) leaf litter decomposition. Yet, the occurrence, direction, and intensity of priming associated with leaf material of differing recalcitrance remains poorly tested. To assess priming, we submerged leaf litter of differing recalcitrance (Alnus glutinosa [alder; less recalcitrant] and Fagus sylvatica [beech; more recalcitrant]) in microcosms and quantified bacterial, fungal, and diatom abundance as well as leaf litter decomposition over 30 days in absence and presence of light. Diatoms did not affect beech decomposition but reduced alder decomposition by 20% and alder-associated fungal abundance by 40% in the treatments including all microbial groups and light, thus showing negative priming. These results suggest that alder-associated heterotrophs acquired energy from diatom exudates rather than from leaf litter. Moreover, it is suggested that these heterotrophs have channeled energy to alternative (reproductive) pathways that may modify energy and nutrient availability for the remaining food web and result in carbon pools protected from decomposition in light-exposed stream sections.

Leaf Species-Dependent Fungicide Effects on the Function and Abundance of Associated Microbial Communities.

Microbially-mediated leaf litter decomposition is a critical ecosystem function in running waters within forested areas, which can be affected by fungicides. However, fungicide effects on leaf litter decomposition have been investigated almost exclusively with black alder leaves, a leaf species with traits favourable to consumers (i.e., low recalcitrance and high nutrient content). At the same time, little is known about fungicide effects on microbial colonisation and decomposition of other leaf species with less favourable traits. In this 21 day lasting study, we explore the effects of increasing fungicide sum concentrations (0-3000 µg/L) on microbial colonisation and decomposition of three leaf species (black alder, Norway maple and European beech) differing in terms of recalcitrance and nutrient content. Leaf litter decomposition rate, leaf-associated fungal biomass and bacterial density were quantified to observe potential effects at the functional level. Beech, as the species with the least favourable leaf traits, showed a substantially lower decomposition rate (50%) in absence of fungicides than alder and maple. In the presence of high fungicide concentrations (300-3000 µg/L), beech showed a concentration-related decrease not only in microbial leaf litter decomposition but also fungal biomass. This suggests that favourable traits of leaf litter (as for alder and maple) enable leaf-associated microorganisms to acquire leaf-bound energy more easily to withstand potential effects induced by fungicide exposure. Our results indicate the need to deepen our understanding on how leaf species' traits interact with the impact of chemical stressors on the leaf decomposition activity of microbial communities.

Interactions between titanium dioxide nanoparticles and polyethylene microplastics: Adsorption kinetics, photocatalytic properties, and ecotoxicity.

The present study investigated the adsorption mechanism of titanium dioxide nanoparticles (nTiO2) on polyethylene microplastics (MPs) and the resulting photocatalytic properties. This effort was supported by ecotoxicological assessments of MPs with adsorbed nTiO2 on the immobility and behaviour of Daphnia magna in presence and absence of UV irradiation. The results showed that nTiO2 were rapidly adsorbed on the surface of MPs (72% of nTiO2 in 9 h). The experimental data fit well with the pseudo-second order kinetic model. Both suspended nTiO2 and nTiO2 immobilized on MPs exhibited comparable photocatalytic properties, with the latter showing a lower effect on Daphnia mobility. A likely explanation is that the suspended nTiO2 acted as a homogeneous catalyst under UV irradiation and generated hydroxyl radicals throughout the test vessel, whereas the nTiO2 adsorbed on MPs acted as a heterogeneous catalyst and generated hydroxyl radicals only locally and thus near the air-water interface. Consequently, Daphnia, which were hiding at the bottom of the test vessel, actively avoided exposure to hydroxyl radicals. These results suggest that the presence of MPs can modulate the phototoxicity of nTiO2 - at least the location at which it is active - under the studied conditions.