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Two isoforms of Glutathione S-Transferase (GST) genes, belonging to mu (Dp-GSTm1 and Dp-GSTm2) and sigma (Dp-GSTs1 and Dp-GSTs2) classes, were cloned and characterised in the freshwater Daphnia pulex. No signal peptide was found in any of the four GST proteins, indicating that they were cytosolic GST. A highly conserved glutathione (GSH) binding site (G-site) occurred in the N-terminal sequence, and a substrate binding site (H-site), interacting non-specifically with the second hydrophobic substrate, was present in the C-terminal. A Tyr residue, for the stabilization of GSH, was found to be conserved in the analysed sequences. The secondary and tertiary structures indicated that these genes possess the typical cytosolic GST structure, including a conserved N-terminal domain with a βαβαββα motif. The μ loop (NVGPAPDYDR and NFIGAEWDR in Dp-GSTm1 and Dp-GSTm2, respectively) was identified between the βαβ (β1α1β2) and αββα motifs (α2β3β4α3) in the N-terminal domain. The expressions of Dp-GSTs1, Dp-GSTs2, and Dp-GSTm1 were higher in other age groups compared to the newly-born neonates (1 d); however, the expression of Dp-GSTm2 first increased and then decreased with age. Gene expression was significantly reduced by high concentration (1 and 2 mg/L) of 75 nm polystyrene nanoplastic. However, nanoplastic exposure at the predicted environmental concentration (1 μg/L) had a low effect. Exposure of mothers to nanoplastic (1 μg/L) elevated the Dp-GSTs2 level in their neonates. These results improve our understanding on the response of different types of Daphnid GST to environmental contaminants, especially nanoplastic., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recently, research on the biological effects of nanoplastics has grown exponentially. However, studies on the effects of nanoplastics on freshwater organisms and the mechanisms of the biological effects of nanoplastics are limited. In this study, the content of reactive oxygen species (ROS), gene and protein expression in the MAPK-HIF-1/NFkB pathway, and antioxidant gene expressions and enzyme activities were measured in Daphnia pulex exposed to polystyrene nanoplastic. In addition, the full-length extracellular signal-regulated kinases (ERK) gene, which plays an important role in the MAPK pathway, was cloned in D. pulex, and the amino acid sequence, function domain, and phylogenetic tree were analyzed. The results show that nanoplastic caused the overproduction of ROS along with other dose-dependent effects. Low nanoplastic concentrations (0.1 and/or 0.5 mg/L) significantly increased the expressions of genes of the MAPK pathway (ERK; p38 mitogen-activated protein kinases, p38; c-Jun amino-terminal kinases, JNK; and protein kinase B, AKT), HIF-1 pathway (prolyl hydroxylasedomain, PHD; vascular endothelial growth factor, VEGF; glucose transporter, GLUT; pyruvate kinase M, PKM; hypoxia-inducible factor 1, HIF1), and CuZn superoxide dismutase (SOD) along with the activity of glutathione-S-transferase. As the nanoplastic concentration increased, these indicators were significantly suppressed. The protein expression ratio of ERK, JNK, AKT, HIF1α, and NFkBp65 (nuclear transcription factor-kB p65) as well as the phosphorylation of ERK and NFkBp65 were increased in a dose-dependent manner. The activities of other antioxidant enzymes (catalase, total SOD, and CuZn SOD) were significantly decreased upon exposure to nanoplastic. Combined with our previous work, these results suggest that polystyrene nanoplastic causes the overproduction of ROS and activates the downstream pathway, resulting in inhibited growth, development, and reproduction. The present study fosters a better understanding of the biological effects of nanoplastics on zooplankton., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Polystyrene nanoplastic induces ROS production and affects the MAPK-HIF-1/NFkB-mediated antioxidant system in Daphnia pulex”., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The biological effects of nanoplastics are a growing concern. However, most studies have focused on exposure to high concentrations or short-term exposure. The potential effects of exposure to low environmental nanoplastic concentrations over the long-term and across multiple generations remain unclear. In the present study, Daphnia pulex was exposed over three 21-day generations to a typical environmental nanoplastic concentration (1 μg/L) and the effects were investigated at physiological (growth and reproduction), gene transcription and enzyme activity levels. Chronic exposure did not affect the survival or body length of D. pulex, whereas the growth rate and reproduction were influenced in the F2 generation. Molecular responses indicated that environmental nanoplastic concentrations can modulate the response of antioxidant defenses, vitellogenin synthesis, development, and energy production in the F0-F1 generations, and prolongation resulted in inhibitory effects on antioxidant responses in F2 individuals. Some recovery was observed in the recovery group, but reproduction and stress defenses were significantly induced. Taken together, these results suggest that D. pulex recovery from chronic exposure to nanoplastic may take several generations, and that nanoplastics have potent long-term toxic effects on D. pulex. The findings highlight the importance of multigenerational and chronic biological evaluations to assess risks of emerging pollution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cytochrome P450 (CYP) enzymes are one of the largest protein families, and they metabolise a wide range of lipophilic organic endogenous and exogenous compounds. Many cytochrome P450 genes have been cloned and characterised, and they are frequently used as biomarkers in environmental toxicology studies because of their sensitivity and inducibility. In the present study, the full-length cDNAs of DpCYP370B and DpCYP4 were cloned from Daphnia pulex for the first time. The sequence of DpCYP370B consisted of an ORF of 1515 bp that encoded a 504 amino acid polypeptide, while the sequence of DpCYP4 comprised an ORF of 1527 bp that encoded a 508 amino acid polypeptide. Homologous alignments revealed the presence of a conserved cysteine haeme-iron ligand signature, FxxGxxxCxG, located in the C-terminal portion. Both the proteins contained a sequence for a transmembrane region that was deduced to be located in the endoplasmic reticulum. Subsequently, the expression levels of DpCYP370B and DpCYP4, as well as those of CYP4AN1, CYP4C33, and CYP4C34, were investigated using quantitative real-time PCR after exposure to five polystyrene nanoplastic concentrations: 0 (control), 0.1, 0.5, 1, and 2 mg/L for 21 days. Except for DpCYP4, the highest mRNA expression was observed at 0.5 mg/L nanoplastics; next, the expression of three of the enzymes (DpCYP370B, CYP4AN1, CYP4C34,) decreased to that of the control level at 1 and 2 mg/L doses of nanoplastics. The expression of DpCYP4 did not significantly change compared with that of the control group. These results indicated that CYP genes might play an important role in protecting D. pulex against nanoplastic pollutants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The widespread occurrence and accumulation of plastic waste have been globally recognized as a critical issue. However, there is limited information on the adverse effects of nanoplastics on freshwater invertebrates. In this study, the effects of a polystyrene nanoplastic on physiological changes (e.g., survival, growth, and reproduction) and expression levels of stress defense genes (oxidative stress-mediated and heat shock proteins) in the freshwater flea Daphnia pulex were measured. The results showed that the digestive organs of D. pulex were strongly fluorescent after exposure to the nanoplastic particles, and the 48-h median lethal concentration (LC 50) of the nanoplastic was determined to be 76.69 mg/L. In the 21-day chronic toxicity test, dose- and time-dependent relationships were observed for body length, and the time to first eggs was significantly prolonged in the 0.5 and 1 mg/L groups. The time to clutch was delayed, and total offspring per female and number of clutches were decreased in all the treatment groups. In addition, the offspring per clutch were significantly decreased in the 0.1 mg/L group. As the nanoplastic concentration increased, expression of stress defense genes (SOD, GST, GPx, and CAT) was first induced and then inhibited. The gene expressions of heat shock proteins (HSP70 and HSP90) were induced in all the treatment groups. Our results suggest that nanoplastics can be ingested by the freshwater cladoceran D. pulex and affect its growth and reproduction as well as induce stress defense., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The widespread occurrence and accumulation of micro- and nanoplastics in aquatic environments has become a growing global concern. Generally, natural aquatic populations are characterized by a variety of multi-structured age groups, for which physiological and biochemical responses typically differ. The freshwater cladoceran, Daphnia pulex, is a model species used extensively in environmental monitoring studies and ecotoxicology testing. Here, the effects of a polystyrene nanoplastic on the physiological changes (i.e., survival) and expression levels of stress defense genes (i.e., those encoding antioxidant-mediated and heat shock proteins) in this freshwater flea were measured. Results from acute bioassays were used to determine the respective nanoplastic LC50 values for five age groups (1-, 4-, 7-, 14- and 21-day-old individuals): the obtained values for the 1- and 21-day-old D. pulex groups were similar (i.e., not significantly different). The expression levels of genes encoding key stress defense enzymes and proteins-SOD, CAT, GST, GPx, HSP70, and HSP90-were influenced by the nanoplastic in all the age groups, but not in the same way for each. Significant differences were observed among all age groups in their expression of the gene encoding the energy-sensing enzyme AMPK (adenosine monophosphate-activated protein kinase) α, β, and γ following exposure to the nanoplastic. Moreover, the expression of AMPK α was significantly increased in the 1-, 7-, and 21-day-old individuals exposed to nanoplastic relative to the control group. Together, these results indicate that age in D. pulex affects the sensitivity of its individuals to pollution from this nanoplastic, primarily via alterations to vital physiological and biochemical processes, such as cellular energy homeostasis and oxidation, which were demonstrated in vivo. We speculate that such age-related effects may extend to other nanoplastics and forms of pollution in D. pulex and perhaps similar marine organisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, one of Doublesex genes from the common freshwater cladoceran Daphnia carinata, designated DapcaDsx1, was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). qPCR was employed to quantify differences in DapcaDsx1 expression between the different sexual phases, with expression levels being higher in sexual females. The role of DapcaDsx1 in the reproductive transformation was further investigated in parthenogenetic-phase females and sexual-phase females using whole-mount in situ hybridization. This cellular localization study showed specific expression of DapcaDsx1 in the thoracic segments, second antenna and part of the ventral carapace. Higher expression levels were exhibited in sexual females compared to parthenogenetic females. This suggests that the DapcaDsx1 gene plays significant roles in switching modes of reproduction and during sexual differentiation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The cladoceran Daphnia carinata undergoes an unusual transition from asexual to sexual reproduction in response to environmental stimuli. Previously, a D. carinata cuticular protein (CP) was identified in an EST library. In this study, the full-length CP cDNA was cloned and sequenced (GenBank accession number: KF551931), and the expression levels in different reproductive states were assessed. Parthenogenetic and sexual female D. carinata were isolated, and CP expression was investigated using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). CP was expressed during both reproductive stages, but expression was higher in sexual females. Cellular localization was also investigated using digoxin-labeled RNA probes in RNA whole-mount in situ hybridization assays, and CP was mainly expressed in the first pair of thoracic appendages, the surface of the head, shell spines, and other parts of the epidermis in parthenogenetic organisms. In contrast, CP expression was restricted to the thoracic appendages in sexual females.

Daphnia (water fleas) are small crustaceans that undergo an unusual switch from asexual to sexual reproduction that is dependent on environmental conditions. In this study, a senescence-associated protein (SAP) from the common freshwater species Daphnia pulex was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). Real-time PCR was employed to quantify the expression of D. pulex SAP (DpSAP) in individual organisms. The role of DpSAP in the reproductive transformation was further investigated in both parthenogenetic and sexual females by using digoxin-labeled SAP RNA probes and RNA whole-mount in situ hybridization. DpSAP was more highly expressed in sexual females, indicating a role in growth and reproduction. Cellular localization studies using RNA whole-mount in situ hybridization showed specific expression in the second tentacle joints. These expression patterns suggest an important role for DpSAP in the reproductive transformation of D. pulex., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Daphnia carinata are unique freshwater crustaceans that undergo both sexual and asexual reproduction, depending on environmental factors. While the molecular mechanism behind the reproductive transformation has been unknown, chemosensory proteins (CSPs) may be involved. We have cloned the cDNA sequences of two CSP genes from D. carinata using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). The full-length cDNA of DacaCSP2 (GenBank accession no: KM624608 ) was 632 bp, with an ORF (open reading frame) of 330 bp encoding a 12.02 kDa protein; and the full-length cDNA of DacaCSP3 (GenBank accession no: KM624609 ) was 935 bp, with an ORF of 342 bp encoding a 12.78 kDa protein. Both CSPs encoded an N-terminal signal peptide, four conserved cysteines, an OS-D superfamily domain, a 2Fe–2S ferredoxin domain, an anaphylatoxin domain and an EGF-like domain. DaCaCSP2 and DaCaCSP3 proteins were most closely related to CSPs from Daphnia pulex and were more distantly related to CSPs from other insects. Using quantitative PCR, we found expression levels of DaCaCSP2 and DaCaCSP3 mRNA were highest in sexual females, followed by parthenogenetic females, and lowest in males. The expression levels of DaCaCSP2 and DaCaCSP3 mRNA also increased at lower temperatures, which suggested they could respond to environmental cues. Whole mount in situ hybridization (ISH) showed that DaCaCSP2 and DaCaCSP3 were expressed mainly in the ovaries, summer eggs, thoracic limbs, rectum and second antennae in sexual females; while they were expressed mainly in the ovaries, thoracic limbs, rectum and second antennae in parthenogenetic females. Together, these results suggest that DacaCSP2 and DacaCSP3 may respond to environmental cues and control the reproductive switch from sexual to asexual reproduction in D. carinata . [ABSTRACT FROM AUTHOR]