Temperature modulates the interaction between fungicide pollution and disease: evidence from a Daphnia-microparasitic yeast model.

Temperature is expected to modulate the responses of organisms to stress. Here, we aimed to assess the influence of temperature on the interaction between parasitism and fungicide contamination. Specifically, using the cladoceran Daphnia as a model system, we explored the isolated and interactive effects of parasite challenge (yeast Metschnikowia bicuspidata) and exposure to fungicides (copper sulphate and tebuconazole) at two temperatures (17 and 20 °C), in a fully factorial design. Confirming a previous study, M. bicuspidata infection and copper exposure caused independent effects on Daphnia life history, whereas infection was permanently suppressed with tebuconazole exposure. Here, we show that higher temperature generally increased the virulence of the parasite, with the hosts developing signs of infection earlier, reproducing less and dying at an earlier age. These effects were consistent across copper concentrations, whereas the joint effects of temperature (which enhanced the difference between non-infected and infected hosts) and the anti-parasitic action of tebuconazole resulted in a more pronounced parasite × tebuconazole interaction at the higher temperature. Thus, besides independently influencing parasite and contaminant effects, the temperature can act as a modulator of interactions between pollution and disease.

Toxicity of two fungicides in Daphnia: is it always temperature-dependent?

The joint effect of increasing temperature and pollution on aquatic organisms is important to understand and predict, as a combination of stressors might be more noxious when compared to their individual effects. Our goal was to determine the sensitivity of a model organism (Daphnia spp.) to contaminants at increasing temperatures, allowing prior acclimation of the organisms to the different temperatures. Prior to exposure, two Daphnia genotypes (Daphnia longispina species complex) were acclimated to three temperatures (17, 20, and 23 °C). Afterwards, a crossed design was established using different exposure temperatures and a range of concentrations of two common fungicides (tebuconazole and copper). Daphnia life history parameters were analysed in each temperature × toxicant combination for 21 days. Temperature was the most influencing factor: Daphnia reproduced later and had lower fecundity at 17 °C than at 20 and 23 °C. Both copper and tebuconazole also significantly reduced the fecundity and survival of Daphnia at environmentally-relevant concentrations. Temperature-dependence was found for both toxicants, but the response pattern was endpoint- and genotype-specific. The combination of contaminant and high temperature often had severe effects on survival. However, unlike some literature on the subject, our results do not support the theory that increasing temperatures consistently foment increasing reproductive toxicity. The absence of a clear temperature-dependent toxicity pattern may result from the previous acclimation to the temperature regime. However, a proper framework is lacking to compare such studies and to avoid misleading conclusions for climate change scenarios.

Current trends and mismatches on fungicide use and assessment of the ecological effects in freshwater ecosystems.

Despite increasing evidence of off-site ecological impacts of pesticides and policy efforts worldwide, pesticide use is still far from being ecologically sustainable. Fungicides are among the most sold classes of pesticides and are crucial to ensure global food supply and security. This study aimed to identify potential gaps of knowledge and mismatches between research and usage data of fungicides by: (i) systematizing the current trends in global sales of fungicides, focusing on the European context in particular (where they are proportionally important); (ii) reviewing the scientific literature on the impacts of synthetic fungicides on non-target freshwater organisms. Sales data revealed important global and regional asymmetries in the relative importance of fungicides and the preferred active ingredients. The literature review on the ecological effects of fungicides disclosed a mismatch between the most studied and the most sold substances, as well as a bias towards the use of single species assays with standard test organisms. To ensure a proper evaluation, risk scenarios should focus on a regional scale, and research agendas must highlight sensitive aquatic ecorreceptors and improve the crosstalk between analytical and sales data.

Antiparasitic potential of agrochemical fungicides on a non-target aquatic model (Daphnia × Metschnikowia host-parasite system).

Pesticides are a major anthropogenic threat to the biodiversity of freshwater ecosystems, having the potential to affect non-target aquatic organisms and disrupt the processes in which they intervene. Important knowledge gaps have been recognised concerning the ecological effects of synthetic fungicides on non-target symbiotic aquatic fungi and the ecological processes where they intervene. The goal of this work was to assess the influence of three commonly used fungicides (myclobutanil, metalaxyl and cymoxanil), which differ in their mode of action, on a host (the crustacean Daphnia magna) × parasite (the yeast Metschnikowia bicuspidata) experimental model. Using a set of life history experiments, we evaluated the effect of each fungicide on the outcome of this relationship (disease) and on the fitness of both host and parasite. Contrasting results were observed: (i) cymoxanil and metalaxyl were overall innocuous to host and parasite at the tested concentrations, although host reproduction was occasionally reduced in the simultaneous presence of parasite and fungicide; (ii) on the contrary, myclobutanil displayed a clear antifungal effect, decreasing parasite prevalence and alleviating infection signs in the hosts. This antiparasitic effect of myclobutanil was further investigated with a follow-up experiment that manipulated the timing of application of the fungicide, to understand which stage of parasite development was most susceptible: while myclobutanil did not interfere in the early stages of infection, its antifungal activity was clearly observable at a later stage of the disease (by impairing the production of transmission stages of the parasite). More research is needed to understand the broader consequences of this parasite-clearance effect, especially in face of increasing evidence that parasites are ecologically more important than their cryptic nature might suggest.

Can parasites adapt to pollutants? A multigenerational experiment with a Daphnia × Metschnikowia model system exposed to the fungicide tebuconazole.

There is increasing evidence about negative effects of fungicides on non-target organisms, including parasitic species, which are key elements in food webs. Previous experiments showed that environmentally relevant concentrations of fungicide tebuconazole are toxic to the microparasite Metschnikowia bicuspidata, a yeast species that infects the planktonic crustacean Daphnia spp. However, due to their short-term nature, this and other experimental studies were not able to test if parasites could potentially adapt to these contaminants. Here, we tested if M. bicuspidata parasite can adapt to tebuconazole selective pressure. Infected D. magna lineages were reared under control conditions (no tebuconazole) and environmentally realistic tebuconazole concentrations, for four generations, and their performance was compared in a follow-up reciprocal assay. Additionally, we assessed whether the observed effects were transient (phenotypic) or permanent (genetic), by reassessing parasite fitness after the removal of selective pressure. Parasite fitness was negatively affected throughout the multigenerational exposure to the fungicide: prevalence of infection and spore load decreased, whereas host longevity increased, in comparison to control (naive) parasite lineages. In a follow-up reciprocal assay, tebuconazole-conditioned (TEB) lineages performed worse than naive parasite lineages, both in treatments without and with tebuconazole, confirming the cumulative negative effect of tebuconazole. The underperformance of TEB lineages was rapidly reversed after removing the influence of the selective pressure (tebuconazole), demonstrating that the costs of prolonged exposure to tebuconazole were phenotypic and transient. The microparasitic yeast M. bicuspidata did not reveal potential for rapid evolution to an anthropogenic selective pressure; instead, the long-term exposure to tebuconazole was hazardous to this non-target species. These findings highlight the potential environmental risks of azole fungicides on non-target parasitic fungi. The underperformance of these microbes and their inability to adapt to such stressors can interfere with the key processes where they intervene. Further research is needed to rank fungicides based on the hazard to non-target fungi (parasites, but also symbionts and decomposers), towards more effective management and protective legislation.

Interplay between fungicides and parasites: Tebuconazole, but not copper, suppresses infection in a Daphnia-Metschnikowia experimental model.

Natural populations are commonly exposed to complex stress scenarios, including anthropogenic contamination and their biological enemies (e.g., parasites). The study of the pollutant-parasite interplay is especially important, given the need for adequate regulations to promote improved ecosystem protection. In this study, a host-parasite model system (Daphnia spp. and the microparasitic yeast Metschnikowia bicuspidata) was used to explore the reciprocal effects of contamination by common agrochemical fungicides (copper sulphate and tebuconazole) and parasite challenge. We conducted 21-day life history experiments with two host clones exposed to copper (0.00, 25.0, 28.8 and 33.1 µg L-1) or tebuconazole (0.00, 154, 192 and 240 µg L-1), in the absence or presence of the parasite. For each contaminant, the experimental design consisted of 2 Daphnia clones × 4 contaminant concentrations × 2 parasite treatments × 20 replicates = 320 experimental units. Copper and tebuconazole decreased Daphnia survival or reproduction, respectively, whilst the parasite strongly reduced host survival. Most importantly, while copper and parasite effects were mostly independent, tebuconazole suppressed infection. In a follow-up experiment, we tested the effect of a lower range of tebuconazole concentrations (0.00, 6.25, 12.5, 25.0, 50.0 and 100 µg L-1) crossed with increasing parasite challenge (2 Daphnia clones × 6 contaminant concentrations × 2 parasite levels × 20 replicates = 480 experimental units). Suppression of infection was confirmed at environmentally relevant concentrations (> 6.25 µg L-1), irrespective of the numbers of parasite challenge. The ecological consequences of such a suppression of infection include interferences in host population dynamics and diversity, as well as community structure and energy flow across the food web, which could upscale to ecosystem level given the important role of parasites.

Concentration and timing of application reveal strong fungistatic effect of tebuconazole in a Daphnia-microparasitic yeast model.

Given the importance of pollutant effects on host-parasite relationships and disease spread, the main goal of this study was to assess the influence of different exposure scenarios for the fungicide tebuconazole (concentration×timing of application) on a Daphnia-microparasitic yeast experimental system. Previous results had demonstrated that tebuconazole is able to suppress Metschnikowia bicuspidata infection at ecologically-relevant concentrations; here, we aimed to obtain an understanding of the mechanism underlying the anti-parasitic (fungicidal or fungistatic) action of tebuconazole. We exposed the Daphnia-yeast system to four nominal tebuconazole concentrations at four timings of application (according to the predicted stage of parasite development), replicated on two Daphnia genotypes, in a fully crossed experiment. An "all-or-nothing" effect was observed, with tebuconazole completely suppressing infection from 13.5µgl-1 upwards, independent of the timing of tebuconazole application. A follow-up experiment confirmed that the suppression of infection occurred within a narrow range of tebuconazole concentrations (3.65-13.5µgl-1), although a later application of the fungicide had to be compensated for by a slight increase in concentration to elicit the same anti-parasitic effect. The mechanism behind this anti-parasitic effect seems to be the inhibition of M. bicuspidata sporulation, since tebuconazole was effective in preventing ascospore production even when applied at a later time. However, this fungicide also seemed to affect the vegetative growth of the yeast, as demonstrated by the enhanced negative effect of the parasite (increasing mortality in one of the host genotypes) at a later time of application of tebuconazole, when no signs of infection were observed. Fungicide contamination can thus affect the severity and spread of disease in natural populations, as well as the inherent co-evolutionary dynamics in host-parasite systems.

Progressive acclimation alters interaction between salinity and temperature in experimental Daphnia populations.

Environmental stressors rarely act in isolation, giving rise to interacting environmental change scenarios. However, the impacts of such interactions on natural populations must consider the ability of organisms to adapt to environmental changes. The phenotypic adaptability of a Daphnia galeata clone to temperature rise and salinisation was investigated in this study, by evaluating its halotolerance at two different temperatures, along a short multigenerational acclimation scenario. Daphniids were acclimated to different temperatures (20°C and 25°C) and salinities (0gL(-1) and 1gL(-1), using NaCl as a proxy) in a fully crossed design. The objective was to understand whether acclimation to environmental stress (combinations of temperature and salinity) influenced the response to the latter exposure to these stressors. We hypothesize that acclimation to different temperature×salinity regimes should elicit an acclimation response of daphniids to saline stress or its interaction with temperature. Acute (survival time) and chronic (juvenile growth) halotolerance measures were obtained at discrete timings along the acclimation period (generations F1, F3 and F9). Overall, exposure temperature was the main determinant of the acute and chronic toxicity of NaCl: daphniid sensitivity (measured as the decrease of survival time or juvenile growth) was consistently higher at the highest temperature, irrespective of background conditions. However, this temperature-dependent effect was nullified after nine generations, but only when animals had been acclimated to both stressors (high salinity and high temperature). Such complex interaction scenarios should be taken in consideration in risk assessment practices.

Assessment of river water quality using an integrated physicochemical, biological and ecotoxicological approach.

In order to maintain and improve the water quality in European rivers, the Water Framework Directive (WFD) requires an integrated approach for assessing water quality in a river basin. Although the WFD aims at a holistic understanding of ecosystem functioning, it does not explicitly establish cause-effect relationships between stressors and changes in aquatic communities. To overcome this limitation, the present study combines the typical WFD physicochemical and biological approaches with an ecotoxicological approach. The main goal was to assess river water quality through an integrated manner, while identifying potential risk situations for aquatic communities in the Cértima river basin (Portugal). To achieve this goal, surface water samples and macroinvertebrate specimens were collected under contrasting hydrological conditions (autumn and spring seasons) at three river sites exposed to distinct pollution levels defined according to the WFD (low, moderate and highly polluted). Physicochemical water quality status was defined according to the Portuguese classification for multipurpose surface waters, whereas biological water quality was assessed in accordance with the South Invertebrate Portuguese Index. Ecotoxicological assays included four standard species, a bacterial species (Vibrio fischeri), a unicellular algae (Pseudokirchneriella subcapitata), a macrophyte (Lemna minor) and a crustacean (Daphnia magna), which were exposed to different river water concentrations. The study sites represented a clear and pronounced gradient of pollution, from the unpolluted reference site to the sites under moderate to high anthropogenic pressure. In the latter sites, clear signs of organic pollution were found, such as low dissolved oxygen concentrations, high nutrient loads and prevalence of highly tolerant macroinvertebrate species. Despite the evident signs of pollution, no clear evidence of toxicity was observed in test species, suggesting that ecotoxicological assays using standard laboratory species and methodologies might not be suitable for assessing the effects of organic pollution. Nevertheless, the integrated methodology presented in this study provided important additional information on the Cértima's water quality status. Its wider use could contribute to a more comprehensive assessment of the effects of anthropogenic pollution on the status and functioning of aquatic ecosystems under the WFD and, thereby, improve the scientific foundations for the sustainable future management of surface water resources.