Fluoxetine may interfere with learning in fish.

Our study aimed to test whether fluoxetine impairs learning in fish and whether this potential impairment is reversible. Learning efficiency, with no aversive stimuli, of the Carassius carassius was analysed under different pharmaceutical conditions: (i) fish cultured without antidepressant (control), (ii) fish exposed to fluoxetine for 21 days (fluoxetine), and (iii) fish exposed to fluoxetine for 21 days and then cultured without fluoxetine for another 21 days (recovery). We exposed animals to environmental concentrations (360 ng L-1) of antidepressant. The learning rate was measured by timing how long it took the individual fish to find food and start feeding, six days in a row. The control and recovery fish took significantly less time to start eating over the six days. Control fish start eating 14 times faster than the fluoxetine fish. Fluoxetine can significantly affect learning and 21-day recovery period is not enough to fully restore the original learning abilities.

Impact of Fluoxetine on Herbivorous Zooplankton and Planktivorous Fish.

The contamination of freshwater environments by pharmaceuticals is a growing problem. Modern healthcare uses nearly 3000 substances, many of which are designed to work at low dosages and act on physiological systems that have been evolutionarily conserved across taxa. Because drugs affect the organisms from different trophic levels, pharmaceutical pollution is likely to disturb species interactions. However, such effects are still only poorly understood. We investigated the impacts of environmentally relevant concentrations of the common drug fluoxetine (Prozac), an increasingly common contaminant of European waters, on predation behavior of crucian carp (Carassius carassius), a common planktivorous European fish, and the somatic growth of its prey, the water flea (Daphnia magna), a widespread planktonic crustacean. We exposed these two organisms to environmentally relevant levels of fluoxetine (360 ng L-1 ): the fish for 4 weeks and the water fleas for two generations. We tested the growth of the daphnids and the hunting behavior (reaction distance at which fish attacked Daphnia and feeding rate) of the fish under drug contamination. We found that Daphnia exposed to fluoxetine grew larger than a nonexposed cohort. The hunting behavior of C. carassius was altered when they were exposed to the drug; the reaction distance was shorter, and the feeding rate was slower. These effects occurred regardless of Daphnia size and the treatment regime they were subjected to. Our results suggest that contamination of freshwater environments with fluoxetine can disrupt the top-down ecological control of herbivores by reducing the hunting efficiency of fish and, as a consequence, may lead to increases in cladoceran population numbers. Environ Toxicol Chem 2023;42:385-392. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Contamination of aquatic environment with anticancer reagents influences Daphnia magna - Ecotoxicogenomics approach.

Pharmaceuticals used in human medicine contaminate freshwater ecosystems. Chemotherapeutics applied in cancer treatment are found in freshwaters at low concentrations (in the range of ng L-1) which, however, can be toxic or mutagenic to aquatic organisms. The aim of this study was to determine the impact of the alkylating/crosslinking anticancer agents, cyclophosphamide (CP) and cisplatin (CDDP), at the concentration detected in water, on Daphnia magna life history, transcriptome, and proteome. This filter feeding cladoceran is an important member of the aquatic food webs controlling algal biomass and forming basic food for planktivorous fish. Here, observations of the D. magna growth rate, age at first reproduction, and the number of eggs produced were performed in the presence of CP or CDDP. The D. magna proteins and RNA were isolated and analysed by mass spectrometry and the mRNA-seq method, respectively. Five generations of contact with the pharmaceuticals in question significantly influenced the D. magna life history parameters with the growth rate and number of laid eggs decreased, whereas age at first reproduction was increased. A decrease in survivorship was observed when daphnids were exposed to CP. These changes are the result of modifications in the gene/transcript expression followed by differences in the proteome profile in comparison to the untreated control. The proteome changes were generally in accordance with the modified transcriptome. The ecotoxicogenomics approach makes it possible to get closer to a complete picture of the influence of CP and CDDP on Daphnia. We have gathered evidence that animals in the presence of anticancer pharmaceuticals attempt to cope with permanent stress by changing their proteome and transcriptome profile. Additionally, our analyses indicate that CDDP showed a stronger effect on tested organisms than CP.

Photosynthetic activity in both algae and cyanobacteria changes in response to cues of predation.

A plethora of adaptive responses to predation has been described in microscopic aquatic producers. Although the energetic costs of these responses are expected, with their consequences going far beyond an individual, their underlying molecular and metabolic mechanisms are not fully known. One, so far hardly considered, is if and how the photosynthetic efficiency of phytoplankton might change in response to the predation cues. Our main aim was to identify such responses in phytoplankton and to detect if they are taxon-specific. We exposed seven algae and seven cyanobacteria species to the chemical cues of an efficient consumer, Daphnia magna, which was fed either a green alga, Acutodesmus obliquus, or a cyanobacterium, Synechococcus elongatus (kairomone and alarm cues), or was not fed (kairomone alone). In most algal and cyanobacterial species studied, the quantum yield of photosystem II increased in response to predator fed cyanobacterium, whereas in most of these species the yield did not change in response to predator fed alga. Also, cyanobacteria tended not to respond to a non-feeding predator. The modal qualitative responses of the electron transport rate were similar to those of the quantum yield. To our best knowledge, the results presented here are the broadest scan of photosystem II responses in the predation context so far.

Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance (mcr) genes in various environmental settings.

Antibiotic resistance is considered one of the biggest threats to public health and has become a major concern for governments and international organizations. Combating this problem starts with improving global surveillance of antibiotic resistance genes (ARGs) and applying standardized protocols, both in a clinical and environmental context, in agreement with the One Health approach. Exceptional efforts should be directed to controlling ARGs conferring resistance to Critically Important Antimicrobials (CIA). In this study, a systematic literature review to synthesize data on the identification of mcr genes using a PCR technique was performed. Additionally, a novel set of PCR primers for mcr-1 - mcr-9 genes detection was proposed. The developed primers were in silico and experimentally validated by comparison with mcr-specific PCR primers reported in the literature. This validation, besides being a proof-of-concept for primers' usefulness, provided insight into the distribution of mcr genes in municipal wastewater, clay and river sediments, glacier moraine, manure, seagulls and auks feces and daphnids from four countries. This analysis proved that commonly used primers may deliver false results, and some mcr genes may be overlooked in tested samples. Newly-developed PCR primers turned out to be relevant for the screening of mcr genes in various environments.

Fluoxetine results in misleading conclusions on fish behavior.

Fluoxetine is an antidepressant medicine causing relaxation and mood improvement in people, with silencing certain personality traits in some cases. The question arise if such phenomena can be observed in nontarget organisms such as fish. Fluoxetine affects fishes behavior; however, it is not known if the medicine affects its "personality." This study aimed to evaluate the reaction of the invasive Neogobius fluviatilis and native Gobio gobio individuals to fluoxetine at environmental concentration of 360 ng/L. We prepared three variants of the experiments: (a) behavioral trials with unexposed fishes, (b) behavioral trials with the same fishes after 21 days of fluoxetine exposure, and (c) behavioral trials with the same fishes after 21-day depuration period, that is, without fluoxetine. The fishes reaction time (RT), that is, difference in time spent on reaching food with and without the necessity of overcoming the obstacle, was analyzed. Additionally, the personality, bold or shy, traits of each fish individual, was assigned. The results indicated that environmental concentrations of the antidepressant influenced RT. The average RT of the fishes cultured with fluoxetine was by 7-min shorter in comparison with the nonexposed control. Share of individuals exposed to fluoxetine assigned as bold raised to 71.4% in comparison with 46.4% in nonexposed control. This sheds new light on wild fishes behavior caught from freshwater. Environmental concentrations of the antidepressant influenced the time of fishes reaction and share individuals assigned as bold. Moreover, 21-day recovery lasting might be not enough to get fluoxetine effect on fishes.

Replacing centrifugation with mixing in urine analysis enriches protein pool in the urine samples.

Urine is the basic diagnostic material, easy to collect, not requiring invasive approach. During standard procedure the urine samples are centrifuged and the supernatant analysed physically, biochemically, and microscopically. The centrifugation step removes proteins including those forming aggregates especially in the state of illness and after transplantation. Here, we analysed the effect of urine centrifuging on specific protein content in urine samples obtained from cardiovascular patients (CVD) and after kidney or liver transplantation. We tested homogeneous whole urine samples, standardly centrifuge one, and the pellet after centrifuging. Protein content was examined using Western blot analysis and mass spectrometry (MS) of samples from CVD patients or the one after transplantation. The average of 21% proteins from non-centrifuged samples were found in the pellet removed after standard centrifugation. MS analysis confirmed that diagnostically important proteins were located there in. In 90% of cases whole urine samples contained more proteins than standard supernatant, among them e.g. proteins involved in immunological response like immunoglobulins and complement compounds secreted by leucocytes. Replacing centrifuging with intensive mixing of urine samples provides a method of enriching the samples with proteins removed during standard procedure, thus increasing possibility of finding new biomarkers for diseases undiagnosable with classic urine analysis.

Diversity and phylogenetic affinities of Bradyrhizobium isolates from Pampa and Atlantic Forest Biomes.

In this work, we investigated Bradyrhizobium strains isolated from soils collected from the rhizosphere of native and exotic legumes species inhabiting two ecoclimatic zones - asubtropical-lowland pasture (Pampa Biome) and a volcanic plateau covered by Araucaria Moist Forests (Atlantic Forest Biome). The rhizobial strains were isolated from the nodules of seven native and one exotic legume species used as rhizobium traps. Single-gene (recA, glnII, dnaK) and combined-gene MLSA analyses (dnaK-glnII-gyrB-recA-rpoB) revealed that nearly 85% of the isolates clustered in B. elkanii supergroup, while the remaining (except for two isolates) in B. japonicum supergroup, albeit, in most cases, separately from the type strains of Bradyrhizobium species. As a symbiotic gene marker, a portion of nifD gene was sequenced for 194 strains. In the nifD-tree, an American branch III.3D (104 isolates), was the most numerous among the isolates. A significant portion of the isolates clustered in American groups; subclade III.4 (40 strains), Clade VII (3 strains), and a new Clade XX (4 strains). Most of the remaining strains belonged to a pantropical III.3C branch (39 isolates). On the other hand, identification of isolates belonging, respectively, to Clade I and Clade II may result of spreading of the Australian (Clade I) and European (Clade II) bradyrhizobia following the introduction of their legume hosts. Our study indicated that the American groups predominated in the symbiotic Bradyrhizobium communities in southern Brazil. However, there is a significant component of exotic lineages, resulting from the dispersal of pantropical Fabaceae taxa and the introduction of exotic legumes.

Morphological deformation of Daphnia magna embryos caused by prolonged exposure to ibuprofen.

The effect of chronic exposure of freshwater cladoceran Daphnia magna to low, environmentally relevant concentrations i.e 4 µgL-1of ibuprofen (a nonsteroidal anti-inflammatory drug) in a laboratory experiment was studied. We observed the key life history traits of first and fifth generation individuals: age and size at first reproduction, number of first clutch eggs and individual growth rate. Moreover, chosen molecular/subcellular markers of experimental animals stress response such as triglyceride content, heat shock proteins (HSP) expression and DNA:RNA ratio were collected. Overall, chronic exposure to ibuprofen had no significant effect on the molecular markers nor on the life history parameters of the Daphnia. It did, however, caused lethal morphological deformities in embryos and juvenile daphnids. Depending on the clonal affiliation, exposure to a low dosage of ibuprofen over five generations resulted in the deformation of ∼3%-∼10% of the first clutch of offspring. Also, up to 90% of females carried at least one deformed embryo. This is the first time that research has revealed such an effect of ibuprofen on D. magna.

Anticancer agents found in environment affect Daphnia at population, individual and molecular levels.

Pharmaceuticals are used in medical treatment on a large scale and as a waste contaminate freshwater ecosystems. Growing amount of so-called civilization diseases, such as different type of cancer, significantly contribute to this form of pollution. The aim of the present study was to determine how the exposure to chemotherapeutics: cyclophosphamide (CP) and cisplatin (CDDP), at detected in environment concentrations, influence proteome profile, life history and population parameters of naturally setting surface waters Daphnia pulex and Daphnia pulicaria. The parameters important for crustaceans, survivorship and population growth rate, were importantly decreased by CDDP treatment but not influenced by CP. On the contrary, the individual growth rate was affected only by CP and exclusively in the case of D. pulicaria. In both clones treated with CP or CDDP, decreased number of eggs was observed. Interestingly, Daphnia males were less sensitive to tested chemotherapeutic than females. Proteome profile revealed that tested anticancer pharmaceuticals modified expression of some proteins involved in Daphnia metabolism. Moreover, males exposed to CDDP showed increased level of enzymes participating in DNA repair. Summing up, the contaminating environment chemotherapeutics reduced fitness of naturally occurring Daphnia species. In consequence this may affect functioning of the aquatic food webs.

When males outlive females: Sex-specific effects of temperature on lifespan in a cyclic parthenogen.

Lifespans of males and females frequently differ as a consequence of different life history strategies adopted to maximize fitness. It is well visible in cyclic parthenogens, such as water fleas of the genus Daphnia, where males appear in the population usually only for periods when receptive females are available. Moreover, even within one sex, different life history strategies and mechanisms regulating lifespan may exist. Previous studies suggested that Daphnia males may regulate their lifespan by staying in colder waters than females. We hypothesize that such behavioral mechanism should be associated with stronger reaction to low temperature-that is greater lifespan extension in males than in females. In this study, we monitored survivorship of Daphnia magna females and males of three clonal lines cultured at 16 or 20°C. The results did not provide a species-level corroboration of our hypothesis; instead, they revealed very strong intraspecific differences in the responses of male and female lifespan to temperature change. They further suggest the existence of parallel life history strategies, hypothesis whose tests would bring new insights into the ecology of males in cyclic parthenogens.

Correction: How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome.

[This corrects the article DOI: 10.1371/journal.pone.0195366.].

How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome.

The waste of commonly used medicines is known to contaminate freshwater ecosystems. Pharmaceuticals can be toxic, mutagenic, or modifying to freshwater organisms even at low concentrations if consider their permanent presence in the environment. Chemotherapeutics used to treat cancer, and in particular alkylating agents, contribute significantly to this form of pollution, the latter introducing cytotoxic and/or mutagenic lesions to the DNA and RNA of organisms which can be disruptive to their cells. The aim of the present study was to investigate the influence of the alkylating anticancer agent cyclophosphamide (CP) on Daphnia magna clones. We evaluated the life history parameters and protein profiles of this crustacean following exposure to environmentally relevant CP concentration of 10 ng L-1. Even at this low concentration, the alkylating agent caused modification of the life history parameters and proteome profile of the Daphnia. These changes were clone-specific and involved growth rate, age at first reproduction, neonate number, and proteins related to cell cycle and redox state regulation. The disturbance caused by pharmaceuticals contaminating freshwater ecosystem is probably weaker and unlikely to be cytotoxic in character due to the high dilution of these substances in the water. However, our results indicate that prolonged exposure of organisms to these toxins may lead to modifications on the organismal and molecular levels with unpredictable significance for the entire ecosystem.

Environmental concentrations of pharmaceuticals directly affect phytoplankton and effects propagate through trophic interactions.

Pharmaceuticals are found in freshwater ecosystems where even low concentrations in the range of ng L-1 may affect aquatic organisms. In the current study, we investigated the effects of chronic exposure to three pharmaceuticals on two microalgae, a potential modulation of the effects by additional inorganic phosphorus (Pi) limitation, and a potential propagation of the pharmaceuticals' effect across a trophic interaction. The latter considers that pharmaceuticals are bioaccumulated by algae, potentially metabolized into more (or less) toxic derivates and consequently consumed by zooplankton. We cultured Acutodesmus obliquus and Nannochloropsis limnetica in Pi-replete and Pi-limited medium contaminated with one of three commonly human used pharmaceuticals: fluoxetine, ibuprofen, and propranolol. Secondly, we tested to what extent first level consumers (Daphnia magna) were affected when fed with pharmaceutical-grown algae. Chronic exposure, covering 30 generations, led to (i) decreased cell numbers of A. obliquus in the presence of fluoxetine (under Pi-replete conditions) (ii) increased carotenoid to chlorophyll ratios in N. limnetica (under Pi-limited conditions), and (iii) increased photosynthetic yields in A. obliquus (in both Pi-conditions). In addition, ibuprofen affected both algae and their consumer: Feeding ibuprofen-contaminated algae to Pi-stressed D. magna improved their survival. We demonstrate, that even very low concentrations of pharmaceuticals present in freshwater ecosystems can significantly affect aquatic organisms when chronically exposed. Our study indicates that pharmaceutical effects can cross trophic levels and travel up the food chain.

Photosynthetic sensitivity of phytoplankton to commonly used pharmaceuticals and its dependence on cellular phosphorus status.

Recently pharmaceuticals have become significant environmental pollutants in aquatic ecosystems, that could affect primary producers such as microalgae. Here we analyzed the effect of pharmaceuticals on the photosynthesis of microalgae commonly found in freshwater-two species of Chlorophyceae and a member of the Eustigmatophyceae, via PAM fluorometry. As pharmaceuticals, three medicines often consumed in households were chosen: (i) fluoxetine, an antidepressant, (ii) propranolol, a ß-blocker and (iii) ibuprofen, an anti-inflammatory and analgesic medicine. The EC50 for the quantum yield of photosystem II in phytoplankton acclimated to inorganic phosphorus (Pi)-replete and Pi-limited conditions was estimated. Acute toxicity experiments over a 5 h exposure revealed that Nannochloropsis limnetica was the least sensitive to pharmaceuticals in its photosynthetic yield out of all species tested. Although the estimation of sub-lethal effects can be vital in contrast to that of LC50s, the EC50 values in all species and for all medicines were orders of magnitude higher than concentrations found in polluted surface water. Chlamydomonas reinhardtii was the most sensitive to fluoxetine (EC50 of 1.6 mg L(-1)), and propranolol (EC50 of 3 mg L(-1)). Acutodesmus obliquus was most sensitive to ibuprofen (EC50 of 288 mg L(-1)). Additionally, the sensitivity to the pharmaceuticals changed under a Pi-limitation; the green algae became less sensitive to fluoxetine and propranolol. In contrast, Pi-limited algal species were more sensitive to ibuprofen. Our results suggest that the sensitivity of algae to pharmaceuticals is (i) highly compound- and species-specific and (ii) dependent on the cellular P status.

Differential levels of stress proteins (HSPs) in male and female Daphnia magna in response to thermal stress: a consequence of sex-related behavioral differences?

In two independent experiments, we compared: (1) water depth selection (and accompanying temperature selection) by male and female Daphnia magna under different kinds of environmental stress, including the presence of filamentous cyanobacteria, the risk of predation from fish, and the presence of toxic compounds; and (2) sex-dependent production of heat shock proteins (HSP60, 70, and 90) in response to a sudden change in temperature. Male D. magna selected deep water strata, which offer a relatively stable environment, and thereby avoided the threat of predation and the presence of toxic compounds in surface waters. Correlated with this behavior, males reduce their molecular defenses against stress, such as the production of heat shock proteins (HSPs), and do not maintain the physiological machinery that triggers an increase in HSP levels in response to stress. In contrast, female D. magna actively select habitats that offer optimal conditions for growth and production of offspring. Consequently, females are exposed to variable environmental conditions that may be associated with increased stress. To permit survival in these different habitats, D. magna females require molecular mechanisms to protect their cells from rapid changes in stress levels. Thus, they maintain high constitutive levels of the heat shock proteins from HSP 60, 70, and 90 families, and they have the potential to further enhance the production of the majority of these proteins under stress conditions. The results of this study indicate that the separate habitats selected by male and female D. magna result in different patterns of HSP production, leading us to hypothesize that that male and female Daphnia magna adopt different strategies to maximize the fitness of the species.