RNA-seq analysis reveals changes in mRNA expression during development in Daphnia mitsukuri.

Temporal transcriptional variation is a major contributor to functional evolution and the developmental process. Parthenogenetic water fleas of the genus Daphnia (Cladocera) provide an ideal model to characterize gene expression patterns across distinct developmental stages. Herein, we report RNA-seq data for female Daphnia mitsukuri at three developmental stages: the embryo, juvenile (three timepoints) and adult. Comparisons of gene expression patterns among these three developmental stages and weighted gene co-expression network analysis based on expression data across developmental stages identified sets of genes underpinning each of the developmental stages of D. mitsukuri. Specifically, highly expressed genes (HEGs) at the embryonic developmental stage were associated with cell proliferation, ensuring the necessary foundation for subsequent development; HEGs at the juvenile stages were associated with chemosensory perception, visual perception and neurotransmission, allowing individuals to enhance detection of potential environmental risks; HEGs at the adult stage were associated with antioxidative defensive systems, enabling adults to mount an efficient response to perceived environmental risks. Additionally, we found a significant overlap between expanded gene families of Daphnia species and HEGs at the juvenile stages, and these genes were associated with visual perception and neurotransmission. Our work provides a resource of developmental transcriptomes, and comparative analyses that characterize gene expression dynamics throughout development of Daphnia.

Polyester microplastic fibers induce mitochondrial damage, apoptosis and oxidative stress in Daphnia carinata, accompanied by changes in apoptotic and ferroptosis pathway.

With the widespread utilization of plastic products, microplastics (MPs) have merged as a newfound environmental contaminant in the United States, and the bulk of these MPs in the environment manifest as fibrous structures. Concerns have also been voiced regarding the potential hazards posed by microplastic fibers (MFs). However, research examining the toxicity of MFs, particularly in relation to planktonic organisms, remains severely limited. Meanwhile, polyester fiber materials find extensive applications across diverse industries. As a result, this investigation delved into the toxicology of polyester microplastic fibers (PET-MFs) with a focus on their impact on Daphnia carinata (D. carinata), a freshwater crustacean. Newly hatched D. carinata were subjected to varying concentrations of PET-MFs (0, 50, and 500 MFs/mL) to scrutinize the accumulation of PET-MFs within these organisms and their resultant toxicity. The outcomes revealed that D. carinata was capable of ingesting PET-MFs, leading to diminished rates of survival and reproduction. These effects were accompanied by mitochondrial impairment, heightened mitochondrial count, apoptosis, escalated generation of reactive oxygen species, augmented activity of antioxidant enzymes, and distinct patterns of gene expression. Interestingly, when comparing the group exposed to 50 MFs/mL with the one exposed to 500 MFs/mL, it was observed that the former triggered a more pronounced degree of mitochondrial damage, apoptosis, and oxidative stress. This phenomenon could be attributed to the fact that brief exposure to 500 MFs/mL resulted in greater mortality, eliminating individuals with lower adaptability. Those that survived managed to regulate elevated in vivo reactive oxygen species levels through an increase in glutathione S-transferase content, thereby establishing an adaptive mechanism. Low concentrations did not induce direct mortality, yet PET-MFs continued to inflict harm within the organism. RNA-seq analysis unveiled significant alterations in 279 and 55 genes in the 50 MFs/mL and 500 MFs/mL exposure groups, respectively. Functional enrichment analysis of the 50 MFs/mL group indicated involvement of the apoptosis pathway and ferroptosis pathway in the toxic effects exerted by PET-MFs on D. carinata. This study imparts valuable insights into the toxicological ramifications of PET-MFs on D. carinata, underscoring their potential risks within aquatic ecosystems.

Is a lower-toxicity strain of Microcystis aeruginosa really less toxic?

Some well-known hazards of blooming cyanobacteria are caused by toxic metabolites such as microcystins (MCs), though many other bioactive chemicals of unknown toxicity are present in their exudates. It is also unclear whether toxicity of cyanobacterial cells depends on growth phases in the life cycle. In this study, we compared toxicity to Daphnia magna of Microcystis aeruginosa - a common cyanobacterial species - exudates (MaE) from two MC-producing strains over both exponential growth and stationary phases in acute and chronic experiments. Specifically, we assessed mitochondrial dysfunction, oxidative stress and lipid peroxidation, and filtering activity and heartbeat rate of Daphnia exposed to MaE. All MaE treatments induced common characteristics of Microcystis toxicity including disorder in the mitochondrial membrane and aberrant heart rate. MaE from cells at stationary growth phase were more toxic than those at exponential phase. Surprisingly, the MC-lower strain had higher toxicity than MC-higher one. Microcystis at different stage of blooms may differentially affect waterfleas owing to variable MaE-induced physiological dysfunction, abundance and grazing rate. Our study suggested that Microcystis strains with lower microcystin-producing ability might release other detrimental chemicals and should not be ignored in harmful bloom monitoring.

Mechanistic Understanding toward the Maternal Transfer of Nanoplastics in Daphnia magna.

Nanoplastics induce transgenerational toxicity to aquatic organisms, but the specific pathways for the maternal transfer of nanoplastics remain unclear. Herein, laser scanning confocal microscopy (LSCM) observations identified the specific pathways on the maternal transfer of polystyrene (PS) nanoplastics (25 nm) in Daphnia magna. In vivo and in vitro experiments showed that PS nanoplastics could enter the brood chamber through its opening and then be internalized to eggs and embryos using LSCM imaging (pathway I). In addition, PS nanoplastics were observed in the oocytes of the ovary, demonstrating gut-ovary-oocyte transfer (pathway II). Furthermore, label-free hyperspectral imaging was used to detect the distribution of nanoplastics in the embryos and ovary of Daphnia, again confirming the maternal transfer of nanoplastics through the two pathways mentioned above. The contribution from pathway I (88%) was much higher than pathway II (12%) based on nanoflow cytometry quantification. In addition, maternal transfer in Daphnia depended on the particle size of PS nanoplastics, as demonstrated by using LSCM and hyperspectral imaging. Unlike 25 nm nanoplastics, 50 nm PS nanoplastics could enter the brood chamber and the eggs/embryos (pathway I), but were not detected in the ovary (pathway II); 100 nm PS nanoplastics were difficult to be internalized by eggs/embryos and could not enter the ovary either. These findings provide insight into the maternal transfer mechanisms of nanoplastics in Daphnia, and are critical for better understanding the transgenerational toxicity of aquatic organisms.

Exploring Proton-Only NMR Experiments and Filters for Daphnia In Vivo: Potential and Limitations.

Environmental metabolomics provides insight into how anthropogenic activities have an impact on the health of an organism at the molecular level. Within this field, in vivo NMR stands out as a powerful tool for monitoring real-time changes in an organism's metabolome. Typically, these studies use 2D 13C-1H experiments on 13C-enriched organisms. Daphnia are the most studied species, given their widespread use in toxicity testing. However, with COVID-19 and other geopolitical factors, the cost of isotope enrichment increased ~6-7 fold over the last two years, making 13C-enriched cultures difficult to maintain. Thus, it is essential to revisit proton-only in vivo NMR and ask, "Can any metabolic information be obtained from Daphnia using proton-only experiments?". Two samples are considered here: living and whole reswollen organisms. A range of filters are tested, including relaxation, lipid suppression, multiple-quantum, J-coupling suppression, 2D 1H-1H experiments, selective experiments, and those exploiting intermolecular single-quantum coherence. While most filters improve the ex vivo spectra, only the most complex filters succeed in vivo. If non-enriched organisms must be used, then, DREAMTIME is recommended for targeted monitoring, while IP-iSQC was the only experiment that allowed non-targeted metabolite identification in vivo. This paper is critically important as it documents not just the experiments that succeed in vivo but also those that fail and demonstrates first-hand the difficulties associated with proton-only in vivo NMR.

Detection of triclosan using 2-domain hemoglobin promoter of Daphnia magna.

Triclosan has been widely used as an antimicrobial agent. However, triclosan was found to cause toxicity, including muscle contraction disturbances, carcinogenesis, and endocrine disorders. In addition, it was found to affect central nervous system function adversely and even have ototoxic effects. Conventional methods for detecting such triclosan can be performed easily. However, the conventional detection methods are inadequate in precisely reflecting the impact of toxic substances on stressed organisms. Therefore, a test model for the toxic environment at the molecular level through the organism is needed. From that point of view, Daphnia magna is being used as a ubiquitous model. D. magna has the advantages of easy cultivation, a short lifespan and high reproductive capacity, and high sensitivity to chemicals. Therefore, the protein expression pattern of D. magna that appear in response to chemicals can be utilized as biomarkers for detecting specific chemicals. In this study, we characterized the proteomic response of D. magna following triclosan exposure via two-dimensional (2D) gel electrophoresis. As a result, we confirmed that triclosan exposure completely suppressed D. magna 2-domain hemoglobin protein and evaluated this protein as a biomarker for triclosan detection. We constructed the HeLa cells in which the GFP gene was controlled by D. magna 2-domain hemoglobin promoter, which under normal conditions, expressed GFP, but upon triclosan exposure, suppressed GFP expression. Consequently, we consider that the HeLa cells containing the pBABE-HBF3-GFP plasmid developed in this study can be used as novel biomarkers for triclosan detection.

Lipofuscin, amyloids, and lipid peroxidation as potential markers of aging in Daphnia.

Accumulation of autofluorescent waste products, amyloids, and products of lipid peroxidation (LPO) are important hallmarks of aging. Until now, these processes have not been documented in Daphnia, a convenient model organism for longevity and senescence studies. We conducted a longitudinal cohort study of autofluorescence and Congo Red (CR) fluorescent staining for amyloids in four clones of D. magna. Additionally, we used a single time point cross-sectional common garden experiment within a single clone in which autofluorescence and BODIPY C11 fluorescence were measured. We observed a robust increase in autofluorescent spots that show diagnostic co-staining by Sudan Black indicating lipofuscin aggregates, particularly in the upper body region. There was also a significant clone-by-age interaction indicating that some genotypes accumulated lipofuscins faster than others. Contrary to predictions, CR fluorescence and lipid peroxidation did not consistently increase with age. CR fluorescence demonstrated a slight non-monotonous relationship with age, achieving the highest values at intermediate ages, possibly due to elimination of physiological heterogeneity in our genetically uniform cohorts. LPO demonstrated a significant ovary status-by-age interaction, decreasing with age when measured in Daphnia with full ovaries (late phase ovarian cycle) and showing no significant trend or slight increase with age when measured during the early phase in the ovarian cycle.

Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors.

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

Daphnia magna sub-lethal exposure to phthalate pollutants elicits disruptions in amino acid and energy metabolism.

Phthalic acid esters (PAEs) are a class of chemicals that are usually incorporated as additives in the manufacturing of plastics. PAEs are not covalently bound to the material matrix and can, consequently, be leached into the environment. PAEs have been reported to act as endocrine disruptors, neurotoxins, metabolic stressors, and immunotoxins to aquatic organisms but there is a lack of information regarding the impact of sub-lethal concentrations to target organisms. The freshwater crustacean Daphnia magna, a commonly used model organism in aquatic toxicity, was exposed to four phthalate pollutants: dimethyl phthalate (DMP), diethyl phthalate (DEP), monomethyl phthalate (MMP), and monoethyl phthalate (MEP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed in a targeted metabolomic approach to quantify polar metabolites extracted from a single Daphnia body. Individual metabolite percent changes and hierarchical clustering heatmap analysis showed unique metabolic profiles for each phthalate pollutant. Metabolite percent changes were mostly downregulated or presented opposing responses for the low and high concentrations tested. Meanwhile, pathway analyses suggest the disruption of related and unique pathways, mostly connected with amino acid and energy metabolism. The pathways aminoacyl-tRNA biosynthesis, arginine biosynthesis, and glutathione metabolism were disrupted by most selected PAEs. Overall, this study indicates that although phthalate pollutants can elicit distinct metabolic perturbations to each PAE, they still impacted related biochemical pathways. These chemical-class based responses could be associated with a common toxic mechanism of action. The reported findings show how targeted metabolomic approaches can lead to a better understanding of sub-lethal exposure to pollutants, revealing metabolomic endpoints do not hold a close relationship with traditional acute toxicity endpoints.

A holistic NMR framework to understand environmental impact: Examining the impacts of superparamagnetic iron oxide nanoparticles (SPIONs) in Daphnia magna via imaging, spectroscopy, and metabolomics.

Superparamagnetic iron oxide nanoparticles (SPIONs) are a contaminant of emerging interest, often used in the medical field as an imaging contrast agent, with additional uses in wastewater treatment and as food additives. Although the use of SPIONs is increasing, little research has been conducted on the toxic impacts to living organisms beyond traditional lethal concentration endpoints. Daphnia magna are model organisms for aquatic toxicity testing with a well understood metabolome and high sensitivity to SPIONs. Thus, as environmental concentrations continue to increase, it is becoming critical to understand their sub-lethal toxicity. Due to the paramagnetic nature of SPIONs, a range of potential nuclear magnetic resonance spectroscopy (NMR) experiments are possible, offering the potential to probe the physical location (via imaging), binding (via relaxation weighted spectroscopy), and the biochemical pathways impacted (via in vivo metabolomics). Results indicate binding to carbohydrates, likely chitin in the exoskeleton, along with a decrease in energy metabolites and specific biomarkers of oxidative stress. The holistic NMR framework used here helps provide a more comprehensive understanding of SPIONs impacts on D. magna and showcases NMR's versatility in providing physical, chemical, and biochemical insights.

Differential susceptibility to arsenic in glutathione S-transferase omega 2 (GST-O2)-targeted freshwater water flea Daphnia magna mutants.

To examine the role of glutathione S-transferase omega class (GST-O2) genes in the biotransformation and detoxification in Daphnia magna, various responses such as in vivo endpoints, arsenic speciation, enzymatic activities, and gene expression pathways related to arsenic metabolism were investigated in wild-type (WT) and GST-O2-mutant-type (MT) fleas produced by CRISPR/Cas9. Sensitivity to arsenic in MT fleas was higher than in WT fleas. Also, the reduction rate of arsenate (AsV) to arsenite (AsIII) in the MT group was significantly lower and led to accumulation of higher arsenic concentrations, resulting in decreased protection against arsenic toxicity. Relative mRNA expression of other GST genes in the GST-O2-targeted MT group generally increased but the enzymatic activity of GST decreased compared with the WT group. Oxidative stress on arsenic exposure was more strongly induced in the MT group compared with the WT group, resulting in a decrease in the ability to defend against toxicity in GST-O2-targeted mutant D. magna. Our results suggest that GST-O2 plays an important role in arsenic biotransformation and detoxification functions in D. magna.

Sublethal Exposure of Per- and Polyfluoroalkyl Substances of Varying Chain Length and Polar Functionality Results in Distinct Metabolic Responses in Daphnia magna.

Per- and polyfluoroalkyl substances (PFAS) are a class of persistent organic pollutants used in industrial applications because of their physicochemical properties, which results in their ubiquitous presence across environmental matrices. To date, legacy PFAS have been well studied; however, the concentration of alternative PFAS may exceed the concentration of legacy pollutants, and more information is needed regarding the sublethal toxicity at the molecular level of aquatic model organisms, such as Daphnia magna. Perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorohexanesulfonic acid (PFHxS), and perfluorononanoic acid (PFNA) are four widely detected PFAS alternatives of varying chain length and polar functionality that are quantified in aquatic environments. The present study examines the metabolic perturbations of PFAS with varying chemistries to D. magna using targeted mass spectrometry-based metabolomics. Daphnia were acutely exposed to sublethal concentrations of PFBA, PFHxA, PFHxS, and PFNA before the polar metabolite profile was extracted from single organisms. Multivariate analysis demonstrated significant separation between the sublethal concentrations of PFHxA, PFHxS, and PFNA relative to the controls; in sum, longer chain lengths demonstrated greater overall perturbations to the extracted metabolic profiles. Univariate statistics revealed significant perturbations in the concentrations of several amino acids, nucleotides/nucleosides, and neurotransmitters with exposure to PFAS. These metabolic perturbations are consistent with disruptions in energy metabolism (pantothenate and coenzyme A metabolism, histidine metabolism) and protein synthesis (aminoacyl-transfer RNA biosynthesis and amino acid metabolism), which were identified through biochemical pathway analysis. These results provide evidence that although PFAS chemistry (chain length and polar functional group) invokes unique metabolic responses, there is also an underlying toxic mode of action that is common with select PFAS exposure. Overall, the present study highlights the capabilities of environmental metabolomics to elucidate the molecular-level perturbations of pollutants within the same chemical class to model aquatic organisms, which can be used to prioritize risk assessment of substituted PFAS alternatives. Environ Toxicol Chem 2023;42:242-256. © 2022 SETAC.

An Efficient Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Method for the Analysis of Methyl Farnesoate Released in Growth Medium by Daphnia pulex.

Methyl farnesoate (MF), a juvenile hormone, can influence phenotypic traits and stimulates male production in daphnids. MF is produced endogenously in response to stressful conditions, but it is not known whether this hormone can also be released into the environment to mediate stress signaling. In the present study, for the first time, a reliable solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed and validated for the ultra-trace analysis of MF released in growth medium by Daphnia pulex maintained in presence of crowding w/o MK801, a putative upstream inhibitor of MF endogenous production. Two different clonal lineages, I and S clones, which differ in the sensitivity to the stimuli leading to male production, were also compared. A detection limit of 1.3 ng/L was achieved, along with good precision and trueness, thus enabling the quantitation of MF at ultra-trace level. The achieved results demonstrated the release of MF by both clones at the 20 ng/L level in control conditions, whereas a significant decrease in the presence of crowding was assessed. As expected, a further reduction was obtained in the presence of MK801. These findings strengthen the link between environmental stimuli and the MF signaling pathway. Daphnia pulex, by releasing the juvenile hormone MF in the medium, could regulate population dynamics by means of an autoregulatory feedback loop that controls the intra- and extra-individual-level release of MF produced by endogenous biosynthesis.

Are Green Household Consumer Products Less Toxic than Conventional Products? An Assessment Involving Grass Shrimp (Palaemon pugio) and Daphnia magna.

Although it is generally assumed that green household consumer products (HCPs) contain individual compounds that are less toxic and/or more degradable than conventional HCPs, little research on this topic has been conducted. In our assessments, larval grass shrimp (Palaemon pugio) were used in a biodegradation study and juvenile freshwater cladocerans, Daphnia magna, were used in a photodegradation study. In each study, organisms were exposed to nondegraded and degraded treatments consisting of one green HCP and two conventional HCPs in six different categories (laundry detergent, dish detergent, mouthwash, insecticide, dishwasher gel, and all-purpose cleaner). Sensitivity to these products were assessed using 48-h static acute toxicity tests, and the median lethal concentrations (LC50s) then compared using an LC50 ratio test. For grass shrimp, only one green HCP (insecticide) was less toxic than both conventional HCPs. In one category (laundry detergent), the green HCP was the more toxic than either conventional HCP. Following a biodegradation treatment, none of the green product formulations became less toxic, whereas 44.4% of the conventional HCPs demonstrated decreased toxicity. For daphnids, green HCPs in three categories (dish detergent, insecticide, and all-purpose cleaner) were less toxic than both conventional products tested. Following a photodegradation treatment, two green product formulations (dish detergent and dishwasher gel) became less toxic (33.3%), whereas 87.5% of the conventional HCPs demonstrated decreased toxicity. The present study demonstrates that green HCPs are not necessarily less toxic and/or more degradable than their conventional counterparts. These results also suggest that the toxicity and degradability of end-product formulations need to be considered in the overall framework for green product evaluation. Environ Toxicol Chem 2022;41:2444-2453. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Multigenerational DNA methylation responses to copper exposure in Daphnia: Potential targets for epigenetic biomarkers?

Epigenetic mechanisms are moving to the forefront of environmental sciences, as environmentally induced epigenetic changes shape biological responses to chemical contamination. This work focused on Daphnia as a representative of potentially threatened freshwater biota, aiming to gain an insight into the involvement of epigenetic mechanisms in their response and eventual adaptation to metal contamination. Copper-induced DNA methylation changes, their potential transgenerational inheritance, and life-history traits were assessed. Organisms with different histories of past exposure to copper were exposed to toxic levels of the element for one generation (F0) and then monitored for three subsequent unexposed generations (F1, F2, and F3). Overall, methylation changes targeted important genes for counteracting the effects of metals and oxidative stress, including dynein light chain, ribosomal kinase and nuclear fragile X mental retardation-interacting protein. Also, contrasting overall and gene-specific methylation responses were observed in organisms differing in their history of exposure to copper, with different transgenerational methylation responses being also identified among the two groups, without apparent life-history costs. Taken together, these results demonstrate the capacity of copper to promote epigenetic transgenerational inheritance in a manner related explicitly to history of exposure, thereby supporting the development and incorporation of epigenetic biomarkers in risk assessment frameworks.

Gene expression study alerted to possible impairment in Daphnia magna individuals as a consequence of exposure to sublethal concentrations of prochloraz.

In the present study, Daphnia magna individuals were exposed for 21 days to 87, 130, 170, 230 and 380 µg/L of prochloraz. The expression of genes related to lipid metabolism (fabd), oxidative stress (cat and gst), heat shock proteins synthesis (hsp70 and hsp90), haemoglobin synthesis (hgb1 and hgb2), metallothioneins synthesis (mt-a, mt-b and mt-c), and vitellogenin synthesis (vgt1 y vgt2) were analyzed. Results showed that some gene expression in D. magna was altered as a consequence of the individual exposure to the fungicide. The genes fabd, vtg1 and vtg2, cat and gst resulted unaltered by the exposure of the daphnids to different fungicide concentrations. However, daphnid exposure to 380 µg/L of prochloraz resulted in an overexpression (p < 0.05) of hsp70 gene which indicated an alteration of the normal protein synthesis and its integrity maintenance. On the other hand, mt-b gene resulted significantly underexpressed (p < 0.05) in daphnids exposed to the lowest fungicide concentrations (87, 130 and 170 µg/L, respectively). In addition, hgb1 and hgb2 genes which are related with the haemoglobin synthesis weresignificantly overexpressed (p < 0.05). Results showed that hgb1 gene was overexpressed almost 100 times more in daphnids exposed for 21 days to 170, 230 and 380 µg/L than control values. However, many authors advocate for an association of these gene expression changes with the presence of contaminants in the medium, in fact they could be used as a good indicator of early contamination at low concentrations of toxicants.

Sulfoxaflor, Zn2+ and their combinations disrupt the antioxidant and osmoregulatory (Ca2+-ATPase) system in Daphnia magna.

BACKGROUND: The oxidative- and osmoregulatory stress-inducing potential of binary mixtures of sulfoxaflor (SUL), a recently developed sulfoximine insecticide, and Zn2+ was aimed to evaluate in Daphnia magna with different exposure regimes. METHODS: Animals were exposed to different SUL concentrations (1.25, 2.5, 10, and 25 mg/L) for 7 days. In vivo 48 h and in vitro effects of single and binary mixtures of SUL (25 and 50 mg/L) and Zn2+ (40 µg/L) were also determined. Furthermore, Ca2+-ATPase, oxidative stress biomarkers (catalase, CAT; superoxide dismutase, SOD; glutathione peroxidase, GPX; glutathione S-transferase, GST; reduced glutathione, GSH; thiobarbituric acid reactive substances, TBARS), and morphometric characteristics were measured. RESULTS: Variable response patterns were observed due to exposure duration and regime, toxicant type, and concentration. Marked effects of SUL were observed, especially in subacute exposure, and 25 mg/L SUL concentration can be considered as a threshold level. Stimulation of GST activity was the most typical response, followed by declined SOD activity and GSH levels. GPX activity and TBARS levels responded differently depending upon the exposure type. Subacute and in vitro effects of SUL and Zn2+ produced similar responses except for some cases. Ca2+-ATPase activity was altered differently upon subchronic duration, though inhibited by in vitro SUL+Zn effect. Subchronic SUL exposure increased body weight and length up to 25 mg/L, contrary to the observed decrease at higher concentrations. CONCLUSIONS: Single and binary mixtures of SUL and Zn2+ caused damage to the antioxidant and osmoregulatory system due to their oxidative potential on cellular targets (biomarkers). The current data emphasized that investigating the SUL toxicity with the Zn2+ combination based on the multi-biomarker approach is essential in the realistic evaluation of SUL toxicity in toxicological research.

The Daphnia carapace and other novel structures evolved via the cryptic persistence of serial homologs.

Understanding how novel structures arise is a central question in evolution. Novel structures are often defined as structures that are not derived from (homologous to) any structure in the ancestor.1 The carapace of the crustacean Daphnia magna is a bivalved "cape" of exoskeleton. Shiga et al.2 proposed that the carapace of crustaceans like Daphnia and many other plate-like outgrowths in arthropods are novel structures that arose through the repeated co-option of genes like vestigial that also pattern insect wings.2-4 To determine whether the Daphnia carapace is a novel structure, we compare previous functional work2 with the expression of genes known to pattern the proximal leg region (pannier, araucan, and vestigial)5,6 between Daphnia, Parhyale, and Tribolium. Our results suggest that the Daphnia carapace did not arise by co-option but instead derived from an exite (lateral leg lobe) that emerges from an ancestral proximal leg segment that was incorporated into the Daphnia body wall. The Daphnia carapace, therefore, appears to be homologous to the Parhyale tergal plate and the insect wing.5 Remarkably, the vestigial-positive tissue that gives rise to the Daphnia carapace appears to be present in Parhyale7 and Tribolium as a small, inconspicuous protrusion. Thus, rather than a novel structure resulting from gene co-option, the Daphnia carapace appears to have arisen from a shared, ancestral tissue (morphogenetic field) that persists in a cryptic state in other arthropod lineages. Cryptic persistence of unrecognized serial homologs may thus be a general solution for the origin of novel structures.

Metabolomic analysis predicted changes in growth rate in Daphnia magna exposed to acetaminophen.

As urbanization and the global population increases, pollutants associated with municipal wastewater such as pharmaceuticals are becoming more prevalent in aquatic environments. Acetaminophen (paracetamol) is a widely used drug worldwide and one of the most frequently detected pharmaceuticals in freshwater ecosystems. This study investigated the impact of acetaminophen on the metabolite profile of Daphnia magna at two life stages; and used these metabolomic findings to hypothesize a potential impact at a higher organismal level which was subsequently tested experimentally. Targeted polar metabolite analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure changes in the concentration of 51 metabolites in the neonate (> 24 h old) and adult (8 day-old) daphnids following a 48-h exposure to sub-lethal concentrations of acetaminophen. The impact of acetaminophen on the metabolic profile of neonates was widely different from adults. Also, acetaminophen exposure perturbed the abundance of nucleotides more extensively than other metabolites. The acute metabolomic experimental results led to the hypotheses that exposure to sub-lethal concentrations of acetaminophen upregulates protein synthesis in D. magna and subsequently increases growth during early life stages and has an opposite impact on adults. Accordingly, a 10 day growth rate experiment indicated that exposure to acetaminophen elevated biomass production in neonates but not in adults. These novel findings demonstrate that a targeted analysis and interpretation of the changes in the polar metabolic profile of organisms in response to environmental stressors could be used as a tool to predict changes at higher biological levels. As such, this study further emphasizes the incorporation of molecular-level platforms as critical and robust tools in environmental assessment frameworks and biomonitoring of aquatic ecosystems.

Daphnia magna egg piRNA cluster expression profiles change as mothers age.

BACKGROUND: PiRNAs prevent transposable elements wreaking havoc on the germline genome. Changes in piRNA expression over the lifetime of an individual may impact on ageing through continued suppression, or release, of transposable element expression. We identified piRNA producing clusters in the genome of Daphnia magna by a combination of bioinformatic methods, and then contrasted their expression between parthenogenetically produced eggs representing maternally-deposited germline piRNAs of young (having their 1st clutch) and old (having their 5th clutch) mothers. Results from eggs were compared to cluster expression in three generations of adults. RESULTS: As for other arthropods, D. magna encodes long uni-directionally transcribed non-coding RNAs consisting of fragmented transposable elements which account for most piRNAs expressed. Egg tissues showed extensive differences between clutches from young mothers and those from old mothers, with 578 and 686 piRNA clusters upregulated, respectively. Most log fold-change differences for significant clusters were modest, however. When considering only highly expressed clusters, there was a bias towards 1st clutch eggs at 41 upregulated versus eight clusters in the eggs from older mothers. F0 generation differences between young and old mothers were fewer than eggs, as 179 clusters were up-regulated in young versus 170 old mothers. This dropped to 31 versus 22 piRNA clusters when comparing adults in the F1 generation, and no differences were detected in the F3 generation. Inter-generational losses of differential piRNA cluster were similar to that observed for D. magna micro-RNA expression. CONCLUSIONS: Little overlap in differentially expressed clusters was found between adults containing mixed somatic and germline (ovary) tissues and germ-line representing eggs. A cluster encompassing a Tudor domain containing gene important in the piRNA pathway was upregulated in the eggs from old mothers. We hypothesise that regulation of this gene could form part of a feedback loop that reduces piRNA pathway activity explaining the reduced number of highly-expressed clusters in eggs from old mothers.