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.

Cytotoxic Potential of Bioactive Compounds from Aspergillus flavus, an Endophytic Fungus Isolated from Cynodon dactylon, against Breast Cancer: Experimental and Computational Approach.

Endophytic fungi are a diverse group of microorganisms that colonize the inter- or intracellular spaces of plants and exhibit mutual benefits. Their interactions with the host plant and other microbiomes are multidimensional and play a crucial role in the production of secondary metabolites. We screened bioactive compounds present in the extracts of Aspergillus flavus, an endophytic fungus isolated from the roots of the medicinal grass Cynodon dactylon, for its anticancer potential. An in vitro analysis of the Ethyl acetate extract from A. flavus showed significant cytostatic effects (IC50: 16.25 µg/mL) against breast cancer cells (MCF-7). A morphological analysis of the cells and a flow cytometry of the cells with annexin V/Propidium Iodide suggested that the extract induced apoptosis in the MCF-7 cells. The extract of A. flavus increased reactive oxygen species (ROS) generation and caused a loss of mitochondrial membrane potential in MCF-7 cells. To identify the metabolites that might be responsible for the anticancer effect, the extract was subjected to a gas chromatography-mass spectrometry (GC-MS) analysis. Interestingly, nine phytochemicals that induced cytotoxicity in the breast cancer cell line were found in the extract. The in silico molecular docking and molecular dynamics simulation studies revealed that two compounds, 2,4,7-trinitrofluorenone and 3α, 5 α-cyclo-ergosta-7,9(11), 22t-triene-6beta-ol exhibited significant binding affinities (-9.20, and -9.50 Kcal/mol, respectively) against Bcl-2, along with binding stability and intermolecular interactions of its ligand-Bcl-2 complexes. Overall, the study found that the endophytic A. flavus from C. dactylon contains plant-like bioactive compounds that have a promising effect in breast cancer.

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.

Acute aquatic toxicity of arsenic-based chemical warfare agents to Daphnia magna.

Sea dumping of chemical warfare (CW) took place worldwide during the 20th century. Submerged CW included metal bombs and casings that have been exposed for 50-100 years of corrosion and are now known to be leaking. Therefore, the arsenic-based chemical warfare agents (CWAs), pose a potential threat to the marine ecosystems. The aim of this research was to support a need for real-data measurements for accurate risk assessments and categorization of threats originating from submerged CWAs. This has been achieved by providing a broad insight into arsenic-based CWAs acute toxicity in aquatic ecosystems. Standard tests were performed to provide a solid foundation for acute aquatic toxicity threshold estimations of CWA: Lewisite, Adamsite, Clark I, phenyldichloroarsine (PDCA), CWA-related compounds: TPA, arsenic trichloride and four arsenic-based CWA degradation products. Despite their low solubility, during the 48 h exposure, all CWA caused highly negative effects on Daphnia magna. PDCA was very toxic with 48 h D. magna LC50 at 0.36 µg × L-1 and Lewisite with EC50 at 3.2 µg × L-1. Concentrations at which no immobilization effects were observed were slightly above the analytical Limits of Detection (LOD) and Quantification (LOQ). More water-soluble CWA degradation products showed no effects at concentrations up to 100 mg × L-1.

Kairomone-like activity of bile and bile components: A step towards revealing the chemical nature of fish kairomone.

Despite the efforts of a number of research groups worldwide, we still have a poor understanding of the chemical nature of the fish kairomones which induce defensive morphology, life history and behavior in their planktonic prey. Bile excreted by foraging fish play a crucial role in their signaling systems. Using high-performance liquid chromatography (HPLC), we revealed the presence of primary and secondary bile acids and bile salts in fish-conditioned water, similar as in carp bile. Upon exposure to either fish bile or commercially acquired bile salts, Daphnia demonstrated similar changes in life history and behavior as when exposed to fish kairomones. The synergic effect of the injured Daphnia alarm substance with fish bile on Daphnia life history is similar to the adaptive effect of the same alarm substance combined with fish kairomones. This strongly supports the view that fish bile or selected bile acids/salts may be responsible for the biological activity of kairomones.

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.

The role of the refractory period in diapause length determination in a freshwater crustacean.

We investigate here the mechanism of allochronic resumption of development by the dormant forms in organisms inhabiting temporary habitats. The cohorts of resting eggs of a short living freshwater crustacean Daphnia magna collected in two temporary waters at two occasions (spring and autumn) were exposed after different storage periods (0-16 weeks) spent either in wet or dry conditions to a given set of hatching stimuli announcing appearance of favourable conditions. Freshly formed resting eggs did not hatch or hatched occasionally. The resting eggs formed in autumn hatched more eagerly than the spring ones when exposed to favourable conditions after wet storage. The hatching proportion increased linearly up to 68-82% in autumn resting eggs while to 33-44% in the spring ones over 16 weeks of storage that might have covered several generations of the active forms. Dry storage of the resting eggs reduced their hatching proportion considerably. We suspect that the length variation of a refractory period (initial phase of developmental arrest when resting forms remain insensitive to hatching stimuli) followed by a reactivation period may constitute the simplest two-step physiological mechanism allowing staggering revival of the dormant forms in subsequent generations that maximise chances for survival in unpredictably changing habitats.

Population density shapes patterns of survival and reproduction in Eleutheria dichotoma (Hydrozoa: Anthoathecata).

Budding hydromedusae have high reproductive rates due to asexual reproduction and can occur in high population densities along the coasts, specifically in tidal pools. In laboratory experiments, we investigated the effects of population density on the survival and reproductive strategies of a single clone of Eleutheria dichotoma. We found that sexual reproduction occurs with the highest rate at medium population densities. Increased sexual reproduction was associated with lower budding (asexual reproduction) and survival probability. Sexual reproduction results in the production of motile larvae that can, in contrast to medusae, seek to escape unfavorable conditions by actively looking for better environments. The successful settlement of a larva results in starting the polyp stage, which is probably more resistant to environmental conditions. This is the first study that has examined the life-history strategies of the budding hydromedusa E. dichotoma by conducting a long-term experiment with a relatively large sample size that allowed for the examination of age-specific mortality and reproductive rates. We found that most sexual and asexual reproduction occurred at the beginning of life following a very rapid process of maturation. The parametric models fitted to the mortality data showed that population density was associated with an increase in the rate of aging, an increase in the level of late-life mortality plateau, and a decrease in the hidden heterogeneity in individual mortality rates. The effects of population density on life-history traits are discussed in the context of resource allocation and the r/K-strategies' continuum concept.

Temporal Expression of the Clock Genes in the Water Flea Daphnia pulex (Crustacea: Cladocera).

The timekeeping mechanisms that operate at the core of circadian clocks (oscillators) are based on interacting molecular feedback loops consisting of clock and clock-associated genes. However, there is a lack of comprehensive studies on the expression of clock genes (particularly those forming its core) in single crustacean species at the mRNA and protein levels, and these studies could serve as a basis for constructing a model of the crustacean molecular oscillator. Studies on Daphnia pulex are well suited to fill this gap because this species is the only representative crustacean whose genome has been sequenced. We analyzed the abundance of 20 gene transcripts throughout the day in the whole bodies of D. pulex (single clone); we found that 15 of these genes were transcriptionally active, and most had daily expression level changes. According to the functional classification of their homologues in insects, these genes may represent elements of the Daphnia molecular oscillator core and its input and output pathways. Studies of PERIOD (PER) protein, one of the main clock components, revealed its rhythmic expression pattern in the epidermis, gut, and ovaries. Finally, the cycling levels of many of these clock components observed in animals reared in continuous light led to the conclusion that the Daphnia oscillator, even if it is structurally similar to the oscillators of other arthropods, can be considered a particularly important adaptive mechanism for living in environments with extreme photoperiods.

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.

Daphnia response to biotic stress is modified by PCBs.

The aim of this study was to examine the influence of xenobiotics (PCBs) on the responses of Daphnia to biotic factors such as the presence of a predator (fish kairomone) or filamentous cyanobacteria. Both behaviour (depth selection) and life history (body size at first reproduction and fecundity) were affected by these stressors. Though there was no direct effect of PCBs, their influence resulted in disruption of the "natural" reaction to the presence of fish or cyanobacteria, leading to inadequate responses of Daphnia to these biotic threats. Examined clones of Daphnia showed significant diversity in their reaction to these stress factors, which was greater than that between Daphnia clones exposed to different environmental conditions. PCB pollution may change the frequency of Daphnia clones in favour of those whose responses to biotic stress are similar in both the absence and presence of these toxic chemicals.

Daphnia response to predation threat involves heat-shock proteins and the actin and tubulin cytoskeleton.

Of all the environmental pressures that all organisms across all kingdoms must face, one of the greatest is the risk of predation. The unpredictability of predation events from the perspective of a single individual is one of the major components of a changing, unstable environment (Gliwicz and Pijanowska, 1989; Lampert, 1987). The panoply of antipredator defenses among terrestrial and aquatic organisms involves a variety of morphological, behavioral, and life-history adaptations that even if they are not life-saving, may enable organisms to complete reproduction before predation occurs. Most of these phenotypic changes are directly induced by cues associated with the biotic agent, in the case of aquatic organisms, the chemical compounds (kairomones) released by a predator into the water. Herein we show that exposure of Daphnia to invertebrate and vertebrate kairomones results in changes in motion, behavior, and life history and at the molecular level involves changes in heat-shock proteins (HSPs) level and the actin and tubulin cytoskeleton. In addition, some of these effects are transgenerational, i.e., they are passed on from the mother to her offspring.

Alarm signals in Daphnia?

Daphnia magna can respond to chemical cues from freshly crushed conspecifics with various behavioural reactions. A shift in vertical distribution towards the bottom, the formation of aggregations and direct escape responses can all be induced by water-borne signals released from crushed Daphnia. The pattern and strength of the first two behavioural responses (i.e. the persistent tendency to occupy deeper strata in the experimental columns and to stay within patches) indicate that Daphnia perceive the signal from crushed conspecifics as nonspecific information, not necessarily associated with any particular kind of danger from either vertebrate or invertebrate predators. The adaptive value and possible costs associated with performing these two behavioural reactions are discussed. The adaptive value of the induced escape response was directly tested: Daphnia which had experienced the presence of a cue from crushed conspecifics avoided attacks by common bream more efficiently than naive Daphnia. The recognition of the signal originating from crushed conspecifics can be especially adaptive in encounters with unfamiliar predators and with predators that undergo ontogenetic shifts in their diet. Under natural conditions, the combination of such a signal with a predator cue can, very reliably, advertise the local scale of the predatory impact.

Predator-mediated genotypic shifts in a prey population: experimental evidence.

We demonstrate the effect of fish predation on genotype frequencies in a laboratory population composed of two Daphnia magna clones, with historically contrasting exposures to fish predation. The two clones differed in their responsiveness to predation via differential avoidance/escape behavior. The clone which coexists with fish in nature is more responsive to the presence of a fish predator, while the clone not exposed to fish predation does not exhibit the defensive reaction. Fish caused a rapid (within 18 h) and significant shift in Daphnia clonal composition, from 1:1 to 8:1, in favor of the responsive clone. Genotype-specific defensive abilities (modus defendi) can contribute greatly to the phenomenon of genotype replacement under selective predation.