Identification and response of cytochrome P450 genes in the brackish water flea Diaphanosoma celebensis after exposure to benzo[α]pyrene and heavy metals.

The cytochrome P450 (CYP) enzyme family is extensive; these enzymes participate in phase I enzyme metabolism and are involved in xenobiotic detoxification in all living organisms. Despite their significance in xenobiotic detoxification, little is known about the species-specific comparison of CYPs and their molecular responses in aquatic invertebrates. We identified 31 CYPs in the brackish water flea Diaphanosoma celebensis via thorough exploration of transcriptomic databases and measured the transcript profiles of 9 CYPs (within full sequences) in response to benzo[α]pyrene (B[α]P) and two heavy metals (cadmium [Cd] and copper [Cu]). Through phylogenetic analysis, the CYPs were separated and clustered into four clans: mitochondrial, CYP2, CYP3, and CYP4. The expression of 9 CYPs were differentially modulated (up- and/or downregulated) in response to B[α]P, Cd, and Cu. In particular, CYP370A15 was significantly upregulated in response to B[α]P, Cd, and Cu, suggesting that the identified CYPs are involved in xenobiotic detoxification and are useful as biomarkers in response to B[α]P, Cd, and Cu. This study aimed to comprehensively annotate cladoceran CYPs; our results will add to the existing knowledge on the potential roles of CYPs in xenobiotic detoxification in cladocerans.

De novo transcriptome assembly of brackish water flea Diaphanosoma celebensis based on short-term cadmium and benzo[a]pyrene exposure experiments.

To develop a brackish water flea as a promising model for marine monitoring, Diaphanosoma celebensis were exposed to two pollutants, cadmium (Cd) and benzo[a]pyrene (BaP), which have different chemical characteristics and distinct modes of metabolic action on aquatic animals. Twenty-four hours after exposure to Cd (2 mg/L) or BaP (25 µg/L), whole body transcriptomes were analyzed. In total, 99.6 Mbp were assembled from nine libraries, resulting in 98,458 transcripts with an N50 of 1883 bp and an average contig length of 968 bp. Functional gene annotations were performed using Gene Ontology, Eukaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Cd significantly modulated endocrine and digestive enzyme system. Following BaP treatment, DNA repair and circadian rhythm related metabolisms were significantly modulated. Both the chemicals induced stress response and detoxification metabolism. This brackish water flea genomic information will be useful to monitor estuaries and coastal regions, as water fleas have been confirmed as promising sentinel models in freshwater ecosystems.

The comparative toxicity of biobased, modified biobased, biodegradable, and petrochemical-based microplastics on the brackish water flea Diaphanosoma celebensis.

The escalating production and improper disposal of petrochemical-based plastics have led to a global pollution issue with microplastics (MPs), which pose a significant ecological threat. Biobased and biodegradable plastics are believed to mitigate plastic pollution. However, their environmental fate and toxicity remain poorly understood. This study compares the in vivo effects of different types of MPs, poly(butylene adipate-co-terephthalate) as a biodegradable plastic, polylactic acid (PLA) as a biobased plastic, ß-cyclodextrin-grafted PLA as a modified biobased plastic, and low density polyethylene as the reference petrochemical-based plastic, on the key aquatic primary consumer Diaphanosoma celebensis. Exposure to MPs resulted in significant reproductive decline, with comparable effects observed irrespective of MP type or concentration. Exposure to MPs induced distinct responses in redox stress, with transcriptional profiling revealing differential gene expression patterns that indicate varied cellular responses to different types of MPs. ATP-binding cassette transporter activity assays demonstrated altered efflux activity, mainly in response to modified biobased and biodegradable MPs. Overall, this study highlights the comparable in vivo and in vitro effects of biobased, biodegradable, and petrochemical-based MPs on aquatic primary consumers, highlighting their potential ecological implications.

Oxidative stress-mediated deleterious effects of hypoxia in the brackish water flea Diaphanosoma celebensis.

In this study, we investigated the acute toxicity, in vivo effects, oxidative stress, and gene expression changes caused by hypoxia on the brackish water flea Diaphanosoma celebensis. The no-observed-effect concentration (NOEC) of 48 h of hypoxia exposure was found to be 2 mg/L O2. Chronic exposure to NOEC caused a significant decline in lifespan but had no effect on total fecundity. The induction of reactive oxygen species increased in a time-dependent manner over 48 h, whereas the content of antioxidant enzymes (superoxide dismutase and catalase) decreased. The transcription and translation levels were modulated by hypoxia exposure. In particular, a significant increase in hemoglobin level was followed by up-regulation of hypoxia-inducible factor 1α gene expression and activation of the mitogen-activated protein kinase pathway. In conclusion, our findings provide a better understanding of the molecular mechanism of the adverse effects of hypoxia in brackish water zooplankton.

Pathway-dependent toxic interaction between polystyrene microbeads and methylmercury on the brackish water flea Diaphanosoma celebensis: Based on mercury bioaccumulation, cytotoxicity, and transcriptomic analysis.

Given their worldwide distribution and toxicity to aquatic organisms, methylmercury (MeHg) and microplastics (MP) are major pollutants in marine ecosystems. Although they commonly co-exist in the ocean, information on their toxicological interactions is limited. Therefore, to understand the toxicological interactions between MeHg and MP (6-µm polystyrene), we investigated the bioaccumulation of MeHg, its cytotoxicity, and transcriptomic modulation in the brackish water flea Diaphanosoma celebensis following single and combined exposure to MeHg and MP. After single exposure to MeHg for 48-h, D. celebensis showed high Hg accumulation (34.83 ± 0.40 µg/g dw biota) and cytotoxicity, which was reduced upon co-exposure to MP. After transcriptomic analysis, 2, 253, and 159 differentially expressed genes were detected in the groups exposed to MP, MeHg, and MeHg+MP, respectively. Genes related to metabolic pathways and the immune system were significantly affected after MeHg exposure, but the effect of MeHg on these pathways was alleviated by MP co-exposure. However, MeHg and MP exhibited synergistic effects on the expression of gene related to DNA replication. These findings suggest that MP can reduce the toxicity of MeHg but that their toxicological interactions differ depending on the molecular pathway.

Microplastics disrupt energy metabolism in the brackish water flea Diaphanosoma celebensis.

Energy metabolism is crucial for normal biological processes, such as growth, development, and reproduction. Microplastics disrupt energy homeostasis by modulating the digestive capacity and contents of energy reserves to overcome stress. This study investigated the modulation of digestive enzyme activity and energy reserves in the brackish water flea Diaphanosoma celebensis exposed to polystyrene (PS) beads (0.05-, 0.5-, 6-µm) for 48 h, and examined transcriptional changes in digestive enzyme-coding genes and AMP-activated protein kinase (AMPK) signaling pathway genes. PS particle size differentially modulated digestive enzyme activity, energy molecule content (glycogen, protein, and lipids), and metabolism-related gene expression. In particular, the 0.5-µm PS had the most significant effect on digestive enzyme activity. In contrast, the 0.05-µm PS caused significant metabolic disorder following a decrease in total energy budget (Ea). These findings suggest that PS beads can modulate energy metabolism through different modes depending on the bead size.

Modulation of ecdysteroid and juvenile hormone signaling pathways by bisphenol analogues and polystyrene beads in the brackish water flea Diaphanosoma celebensis.

Owing to its high production and world-wide usage, plastic pollution is an increasing concern in marine environments. Plastic is decomposed into nano- and micro-sized debris, which negative affect reproduction and development in aquatic organisms. Bisphenol A (BPA), an additive of plastic, is released into the water column upon plastic degradation, and is known as a representative endocrine-disrupting chemical. However, the reproductive effects of plastics and bisphenols at the molecular level have not yet been explored in small marine crustaceans. In this study, we investigated the effects of polystyrene (PS) beads (0.05, 0.5, and 6 - µm) and bisphenol analogues (BPs; BPA, BPS, and BPF) on reproduction and development of small marine crustaceans. Effects on transcriptional changes in ecdysteroid and juvenile hormone (JH) signaling pathway-related genes were examined in the brackish water flea Diaphanosoma celebensis exposed to PS beads and BPs for 48 h. As results, BPs and PS beads delayed emergence time of first offspring, and increased fecundity in a concentration-dependent manner. BPs differentially modulated the expression of ecdysteroid and JH signaling pathway-related genes, indicating that BP analogs can disrupt endocrine systems via mechanisms different from those of BPA. PS beads was also changed the gene expression of both pathway, depending on their size and concentration. Our findings suggest that BP analogues and PS beads disrupt the endocrine system by modulating the hormonal pathways, affecting reproduction negatively. This study provides a better understanding of the molecular mode of action of BPs and PS beads in the reproduction of small crustaceans.

Genome-wide identification of 216 G protein-coupled receptor (GPCR) genes from the marine water flea Diaphanosoma celebensis.

G protein-coupled receptors (GPCRs) are considered to have originated from early evolution of eukaryotic species, therefore, the genome-wide identification of GPCR genes can provide insight into the adaptive strategy and evolutionary tendency in an animal taxon. Here, we identified a total 216 full-length GPCR genes in the marine water flea Diaphanosoma celebensis genome, which were classified into five distinct classes (A, B, C, F, and other). Phylogenetic comparison of GPCRs in D. celebensis to those in humans (Homo sapiens), fruitfly (Drosophila melanogaster), and freshwater water flea (Daphnia magna) reveals a high level of orthological relationship of amine, neuropeptide, and opsin receptor repertoire, while purinergic and chemokine receptors were highly differentiated in humans. Our findings suggest sporadic evolutionary processes within the GPCR gene families identified in D. celebensis. In this study, these results may provide a better understanding on the evolution of GPCRs, and expand our knowledge of the cladoceran GPCR gene repertories which in part, mediate cell physiological mechanisms in response to various environmental stimuli.

Combined effects of heavy metals (Cd, As, and Pb): Comparative study using conceptual models and the antioxidant responses in the brackish water flea.

The combined effect of toxic inducers has emerged as a challenging topic, particularly due to their inconsistent impacts on the environment. Using toxic unit (TU) based on LC50 value, we investigated the 48 h acute toxicities of the following combinations: Cd + As, Cd + Pb, As + Pb, and Cd + As + Pb, and binary and ternary combined effects were interpreted using concentration addition (CA) and independent action (IA) model. The molecular effects of these combinations were further examined on the basis of gene expression (four GST and two SOD isoforms) and antioxidant enzymes activity (SOD and GST). The CA-predicted LC50 was similar to the observed results, indicating that the CA model is more applicable for evaluating the combined effects of the metal mixtures. Synergistic effects (ΣTULC50 < 0.8) were observed for the mixtures As + Pb and Cd + Pb, while additive effects (0.8 < ΣTULC50 < 1.2) were observed for the mixtures Cd + As + Pb and Cd + As. No antagonistic effects were observed in this study. Molecular biomarkers for oxidative stress caused by metals, as well as traditional endpoints such as lethality, have shown a clear response in assessing the toxicity of binary and ternary mixtures. This study opens up a new avenue for the use of biomarkers to assess the combined effects of metals in aquatic environments.

Oxidative stress responses in brackish water flea exposed to microcystin-LR and algal bloom waters from Nakdong River, Republic of Korea.

Microcystis blooms and the impact of their toxins, particularly microcystin (MC), in coastal ecosystems is an emerging threat, but the species-specific effects of MC and the potential for bioconcentration are not fully understood. We exposed the brackish water flea, Diaphanosoma celebensis, to MC-LR, which showed antioxidant responses measured at the molecular to enzyme levels but no acute toxicity. We extended our experimental investigation to measure the released MC and its uptake by D. celebensis exposed to river water. In a short-term exposure (48 h) experiment, D. celebensis exposed to water from an algal bloom (approximately 2 µg L-1 MC) assimilated more than 50 pg MC per individual. The significant increase of MCs suggests the potential for the species to accumulate MCs. The dose-dependent increase in the antioxidant response observed in the mRNA levels also showed that D. celebensis exposed to diluted algal bloom waters were affected by toxins from cyanobacteria.

Complete mitochondrial DNA of the marine water flea Diaphanosoma celebensis (Cladocera, Sididae).

The complete mitochondrial genome was sequenced from the marine water flea Diaphanosoma celebensis. The sequenced mitochondrial genome size was 17,060 bp, possessing identical gene order of 13 protein-coding genes (PGCs) to those of the congeneric freshwater species Diaphanosoma dubium in the genus Diaphanosoma. The mitochondrial genome of D. celebensis had 13 PGCs, two rRNAs, and 22 tRNAs. Of 13 PGCs, three genes (CO3, ND3, and ND4) had incomplete stop codons. Furthermore, the stop codons of the remaining ten PGCs were TAA (for CO1, ATP8, ATP6, ND5, ND6, and ND1) and TAG (for NL4L, Cytb, and ND2). The second and third base composition of codon on 9 PCGs on the L strand in D. celebensis mitogenome showed an anti-G bias (11.0% and 15.0%), respectively.

Cloning and molecular characterization of estrogen-related receptor (ERR) and vitellogenin genes in the brackish water flea Diaphanosoma celebensis exposed to bisphenol A and its structural analogues.

Although it has previously been shown that bisphenol (BP) analogues may interfere with the normal hormonal regulation by acting as endocrine disrupting chemicals (EDCs), little information is available on effects of BP analogues in invertebrates, particularly on cladocerans. In the present study, we identified estrogen-related receptors (EER), vitellogenin (VTG), and VTG receptor (VtgR) from the brackish water flea Diaphanosoma celebensis, and examined the effects of BPA and the substitutes, BPF and BPS, in different sublethal concentrations. Gene expression varied with time well matched with brooding, suggesting that DcEER, DcVTG, and DcVtgR play a role in reproduction in D. celebensis. qRT-PCR analysis showed that BPA and its substitutes differently modulated mRNA expressions of DcEER, DcVTG, and DcVtgR, indicating that these compounds adversely affect the normal reproduction-related pathway. This study facilitates better understanding of the molecular mode of action of BP analogues on the reproductive system of D. celebensis.