Comparison of the Estimation Methods from Acute to Chronic Biotic Ligand Model-Based Predicted No-Effect Concentrations for Nickel in Freshwater Species.

Biotic ligand models (BLMs) and the sensitivities of indigenous species are used to assess the environmental risk considering the bioavailability of metals, such as nickel. However, the BLM-based acute-to-chronic ratio (ACR) is required if the predicted no-effect concentration (PNEC) cannot be derived from the chronic species sensitivity distribution (SSD). The applicability of the ACR approach for estimating BLM-based PNEC for nickel from acute toxicity data was evaluated in the present study. The BLM-based acute SSD for nickel was built using the sensitivities of 21 indigenous species and different taxon-specific BLMs for each taxonomic group. To predict the acute sensitivity of invertebrates, the chronic crustacean nickel BLM with pH effect term, which can account for nickel toxicity at high pH levels, was used. This was used instead of the existing acute BLM for crustacean, which has too narrow a pH range to cover the pH dependency of toxicity. The final BLM-based ACR of nickel, determined within a factor of 1.53 from the species-specific acute and chronic sensitivities of the six species, was more reliable than the typical ACR estimated within a factor of 1.84. A linear relationship (r2 = 0.95) was observed between the PNECs using BLM-based ACR and the PNECs derived from the BLM-based chronic SSD of the European Union Risk Assessment Reports. In conclusion, the BLM-based PNEC for nickel could be derived using the ACR approach, unlike when copper BLM was applied. The BLM-based ACR for nickel is the first result calculated by directly comparing acute and chronic species sensitivities, and will contribute to the application of BLM-based risk assessment in broader ecoregions. Environ Toxicol Chem 2023;42:914-927. © 2023 SETAC.

Derivation of acute copper biotic ligand model-based predicted no-effect concentrations and acute-chronic ratio.

The copper biotic ligand model (BLM) can quantitatively describe the bioavailability depending on various environmental factors and has been used to derive the predicted no-effect concentrations (PNECs). The commonly employed acute BLM tool, HydroQual, which applies the biotic ligand constants of fathead minnow in the same model structure for all taxonomic groups, estimates lower acute copper toxicity values compared to the chronic copper PNECs of the European Union Risk Assessment Reports (EU-RAR), which are based on taxon-specific model structures and biotic ligand constants for vertebrates, invertebrates, and algae. In this study, the full-BLM approach was applied using an appropriate acute BLM for each taxonomic group to derive acute HC5s (fifth percentile value in the species sensitivity distribution [SSD]) and an acute-chronic ratio for copper. Two acute BLMs for vertebrates and invertebrates were used and validated against site waters using the new method to estimate the intrinsic sensitivity for each species across different environmental conditions. To derive acute copper full BLM-based HC5s in Korean freshwater, acute toxicity tests were performed with 10 indigenous species, which were used to build the acute BLM-based SSD at each site. The final estimated acute full-BLM HC5s were higher than the EU-RAR chronic PNECs within the BLM calibration range. Furthermore, a linear relationship was observed between the acute full-BLM HC5s and the EU-RAR chronic PNECs. This linear regression function was suggested as an acute to chronic transformation function that can be applied to calculate chronic PNEC values. In conclusion, if the chronic ecotoxicity database of indigenous aquatic organisms for copper is lacking, it may be more efficient to derive chronic PNECs using an acute-chronic ratio after deriving BLM-based acute copper SSDs for indigenous species within representative taxonomic groups. This study provides a scientific foundation for the derivation of water quality criteria for copper in freshwater.

Complete mitochondrial genome of the calanoid copepod Eurytemora affinis (Calanoida, Temoridae).

The complete mitochondrial genome was sequenced from the calanoid copepod Eurytemora affinis. The sequenced total genome size was 18,553 bp. The mitochondrial genome of E. affinis has 13 protein-coding genes (PCGs), two rRNAs, and 22 tRNAs. Of 13 PCGs, ND1, ND5, and ATP6 genes had incomplete stop codons TA-, T-, and TA-, respectively. Furthermore, the stop codons of the remaining eleven PCGs were TAG or TAA while the start codon of 13 PCGs was ATG (Cytb, ATP8, ATP6, and CO3 genes), ATT (CO1, ND2, ND3, ND4L, ND5, and ND6 genes), and ATA (ND1, ND4, and CO2 genes), respectively. The ratio of A + T and G + C nucleotides of 13 PCGs of E. affinis mitogenome showed 63.9% and 36.1%, respectively while those ratio of the entire sequences were 65.5% and 34.5%, respectively.

Complete mitochondrial genome of the marine mysid Neomysis awatschensis (Mysida, Mysidae).

The complete mitochondrial genome was sequenced from the marine mysid Neomysis awatschensis. The sequenced total genome size was 19,135 bp. The mitochondrial genome of N. awatschensis contained 13 protein-coding genes (PCGs), two rRNAs, and 22 tRNAs. Of 13 PCGs, all the genes had complete stop codons TAA and TAG, respectively, while the start codon of 13 PCGs was ATG (CO1, Cytb, ND4L, ATP8, ATP6, and ND4 genes), ATT (CO3, ND2, and ND5 genes), and ATA (CO2, ND3, ND6, and ND1 genes), respectively. The ratio of A + T and G + C nucleotides of 13 PCGs of N. awatschensis mitogenome showed 68.8% and 31.2%, respectively, while those ratio of all the sequences were 70.8% and 29.2%, respectively.

The genome of the marine medaka Oryzias melastigma.

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field-based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole-genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.

The genome of the freshwater monogonont rotifer Brachionus calyciflorus.

Monogononta is the most speciose class of rotifers, with more than 2,000 species. The monogonont genus Brachionus is widely distributed at a global scale, and a few of its species are commonly used as ecological and evolutionary models to address questions related to aquatic ecology, cryptic speciation, evolutionary ecology, the evolution of sex and ecotoxicology. With the importance of Brachionus species in many areas of research, it is remarkable that the genome has not been characterized. This study aims to address this lacuna by presenting, for the first time, the whole-genome assembly of the freshwater species Brachionus calyciflorus. The total length of the assembled genome was 129.6 Mb, with 1,041 scaffolds. The N50 value was 786.6 kb, and the GC content was 24%. A total of 16,114 genes were annotated with repeat sequences, accounting for 21% of the assembled genome. This assembled genome may form a basis for future studies addressing key questions on the evolution of monogonont rotifers. It will also provide the necessary molecular resources to mechanistically investigate ecophysiological and ecotoxicological responses.

Identification and characterization of homeobox (Hox) genes and conservation of the single Hox cluster (324.6 kb) in the water flea Daphnia magna.

We report the complete sequence analysis of the entire complement of eight typical homeobox (Hox) genes (Lab, Pb, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) and two other genes (Hox3 and Ftz) in a 324.6-kb region in the water flea Daphnia magna. In the cluster of D. magna Hox genes, we found one long interspersed nuclear element (LINE)/R2-NeSL between Ubx and Abd-A that was not present in Daphnia pulex Hox genes. In basal expression of Hox genes at different developmental stages, biothorax complex genes (Ubx, Abd-A, and Abd-B) and some antennapedia complex genes (Lab, Scr, Antp) were moderately expressed, but the Hox3 gene was barely expressed. Three homeobox genes (Antp, Ubx, Abd-A) were highly expressed at 6-7 days after release from the brood chamber and/or in the adult stage. The structural array and transcribed orientation of Dm-Hox genes were identical to those of the sister species D. pulex (∼340 kb), indicating that the Hox gene structure in daphnids is highly conserved. However, Dm- and Dp-Hox3, -deformed (Dfd), and -fushi tarazu (Ftz) genes varied from orthologous genes in pancrustacean species.

Genome-wide identification of nuclear receptor (NR) genes and the evolutionary significance of the NR1O subfamily in the monogonont rotifer Brachionus spp.

Nuclear receptors (NRs) are a large family of transcription factors that are involved in many fundamental biological processes. NRs are considered to have originated from a common ancestor, and are highly conserved throughout the whole animal taxa. Therefore, the genome-wide identification of NR genes in an animal taxon can provide insight into the evolutionary tendencies of NRs. Here, we identified all the NR genes in the monogonont rotifer Brachionus spp., which are considered an ecologically key species due to their abundance and world-wide distribution. The NR family was composed of 40, 32, 29, and 32 genes in the genomes of the rotifers B. calyciflorus, B. koreanus, B. plicatilis, and B. rotundiformis, respectively, which were classified into seven distinct subfamilies. The composition of each subfamily was highly conserved between species, except for NR1O genes, suggesting that they have undergone sporadic evolutionary processes for adaptation to their different environmental pressures. In addition, despite the dynamics of NR evolution, the significance of the conserved endocrine system, particularly for estrogen receptor (ER)-signaling, in rotifers was discussed on the basis of phylogenetic analyses. The results of this study may help provide a better understanding the evolution of NRs, and expand our knowledge of rotifer endocrine systems.

Adverse effects of BDE-47 on life cycle parameters, antioxidant system, and activation of MAPK signaling pathway in the rotifer Brachionus koreanus.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is widely dispersed endocrine disrupting chemicals (EDCs) in the aquatic ecosystem. Due to its devastating effect on marine organisms and insufficient database on toxicology, we investigated the adverse effects of BDE-47 on life parameters and antioxidant defense system following the reactive oxygen species (ROS) production in the monogonont rotifer Brachionus koreanus. In B. koreanus, the reduction in life cycle, fecundity, and population growth were observed in response to BDE-47. 50µg/L BDE-47 significantly reduced (P<0.05) life expectancy and net reproductive rate. In response to 10-50µg/L BDE-47 exposure, the oxidative stress was elicited via the generation of ROS, while the antioxidant related enzymes (e.g. glutathione S-transferase [GST] and glutathione reductase [GR]) have demonstrated significant activity levels (P<0.05) to further alleviate the oxidative stress in a concentration dependent manner. Furthermore, transcript profiles of antioxidant function (GST-A, -O, and -S1-S8)-related genes have shown the significant increase over 24h in response to BDE-47 (0, 10, 25, and 50µg/L). As for MAPK signaling pathway analysis, up-regulation of their activities was observed at 25µg/L BDE-47 but their activities have reduced at adult NOEC concentration of 50µg/L. This study provides a better understanding of the effects of BDE-47 on life parameters, molecular defense system, and activation of MAPK signaling pathway against generated oxidants in the rotifer.

Ultraviolet B radiation induces impaired lifecycle traits and modulates expression of cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus.

To evaluate the effects of ultraviolet B (UV-B) radiation at the developmental, reproductive, and molecular levels in aquatic invertebrates, we measured UV-B-induced acute toxicity, impairments in developmental and reproductive traits, and UV-B interaction with the entire family of cytochrome P450 (CYP) genes in the intertidal benthic copepod Tigriopus japonicus. We found a significant, dose-dependent reduction (P<0.05) in the survival of T. japonicus that began as a developmental delay and decreased fecundity. The 48h LD10 and LD50 were 1.35 and 1.84kJ/m2, and the CYP inhibitor (PBO) elevated mortality, confirming the involvement of CYP genes in UV-B induced toxicity. Low-dose UV-B (1.5kJ/m2) induced developmental delays, and higher doses (6-18kJ/m2) caused reproductive impairments in ovigerous females. The significant up-regulation of CYP genes belonging to clans 2/3/MT/4/20 in T. japonicus exposed to UV-B (12kJ/m2) confirmed molecular interaction between UV-B and CYP genes. Moreover, orphan CYPs, such as CYP20A1, provide good insight on the deorphanization of invertebrate CYPs. Overall, these results demonstrate the involvement of UV-B radiation in the expression of all the CYP genes in T. japonicus and their susceptibility to UV-B radiation. This will provide a better understanding of the mechanistic effects of UV-B in copepods through the predicted AhR-mediated up-regulation of CYP genes.

Adverse effects of microplastics and oxidative stress-induced MAPK/Nrf2 pathway-mediated defense mechanisms in the marine copepod Paracyclopina nana.

Microplastic pollution causes a major concern in the marine environment due to their worldwide distribution, persistence, and adverse effects of these pollutants in the marine ecosystem. Despite its global presence, there is still a lack of information on the effect of microplastics on marine organisms at the molecular level. Herein we demonstrated ingestion and egestion of nano- (0.05 µm) and micro-sized (0.5 and 6 µm) polystyrene microbeads in the marine copepod Paracyclopina nana, and examined molecular responses to exposure to microbeads with in vivo endpoints such as growth rate and fecundity. Also, we proposed an adverse outcome pathway for microplastic exposure that covers molecular and individual levels. This study provides the first insight into the mode of action in terms of microplastic-induced oxidative stress and related signaling pathways in P. nana.

Marine copepod cytochrome P450 genes and their applications for molecular ecotoxicological studies in response to oil pollution.

Recently, accidental spills of heavy oil have caused adverse effects in marine organisms. Oil pollution can induce damages on development and reproduction, linking with detrimental effects on diverse molecular levels of genes and proteins in plankton and fish. However, most information was mainly focused on marine vertebrates and consequently, limited information was available in marine invertebrates. Furthermore, there is still a lack of knowledge bridging in vivo endpoints with the functional regulation of cytochrome P450 (CYP) genes in response to oil spill pollution in marine invertebrates. In this paper, adverse effects of oil spill pollution in marine invertebrates are summarized with the importance of CYP genes as a potential biomarker, applying for environmental monitoring to detect oil spill using marine copepods.

Ecdysone receptor (EcR) and ultraspiracle (USP) genes from the cyclopoid copepod Paracyclopina nana: Identification and expression in response to water accommodated fractions (WAFs).

Ecdysteroid hormones are pivotal in the development, growth, and molting of arthropods, and the hormone pathway is triggered by binding ecdysteroid to a heterodimer of the two nuclear receptors; ecdysone receptors (EcR) and ultraspiracle (USP). We have characterized EcR and USP genes, and their 5'-untranslated region (5'-UTR) from the copepod Paracyclopina nana, and studied mRNA transcription levels in post-embryonic stages and in response to water accommodated fractions (WAFs) of crude oil. The open reading frames (ORF) of EcR and USP were 1470 and 1287bp that encoded 490 and 429 amino acids with molecular weight of 121.18 and 105.03kDa, respectively. Also, a well conserved DNA-binding domain (DBD) and ligand-binding domain (LBD) were identified which confirmed by phylogenetic analysis. Messenger RNA transcriptional levels of EcR and USP were developmental stage-specific in early post-embryonic stages (N3-4). However, an evoked expression of USP was observed throughout copepodid stage and in adult females. WAFs (40 and 80%) were acted as an ecdysone agonist in P. nana, and elicited the mRNA transcription levels in adults. Developmental stage-specific transcriptional activation of EcR and USP in response to WAFs was observed. USP gene was down-regulated in the nauplius in response to WAF, whereas up-regulation of USP was observed in the adults. This study represents the first data of molecular elucidation of EcR and USP genes and their regulatory elements from P. nana and the developmental stage specific expression in response to WAFs, which can be used as potential biomarkers for environmental stressors with ecotoxicological evaluations in copepods.

Complete mitochondrial genome of the monogonont rotifer Brachionus rotundiformis (Rotifera, Brachionidae).

The two complete mitochondrial genomes were sequenced from the monogonont rotifer Brachionus rotundiformis. The genome sequences were 10,268 bp and 11,703 bp in size, and the gene order and contents were identical with those of B. koreanus but were different in tRNA-Cys with B. plicatilis mitochondrial genomes. Of the 12 protein-coding genes (PCGs), five genes (ND1, ATP6, ND5, CO3, ND3) had incomplete stop codons. Furthermore, the start codon of ND4 and CO3 gene was ATA, while the start codon of other PCGs was ATG. The base composition of B. rotundiformis mitogenome shows an anti-G bias (12.05% and 10.24%) on the second and third position of the PCGs, respectively.

Cross-reactivities of mammalian MAPKs antibodies in rotifer and copepod: Application in mechanistic studies in aquatic ecotoxicology.

The mitogen-activated protein kinases (MAPKs) family is known to mediate various biological processes in response to diverse environmental pollutants. Although MAPKs are well characterized and studied in vertebrates, in invertebrates the cross-reactivities of MAPKs antibodies were not clearly known in response to environmental pollutants due to limited information of antibody epitopes with material resources for invertebrates. In this paper, we performed phylogenetic analysis of MAPKs genes in the marine rotifer Brachionus koreanus and the copepods Paracyclopina nana and Tigriopus japonicus. Also in rotifer and copepods, several studies of Western blot of MAPK signaling pathways were shown in response to environmental pollutants, including multi-walled carbon nanotubes (MWCNTs), water-accommodated fractions (WAFs) of crude oil, and microplastics. This paper will provide a better understanding of the underlying mechanistic scenario in terms of cross-reactivities of mammalian antibodies in rotifer and copepod.

WAFs lead molting retardation of naupliar stages with down-regulated expression profiles of chitin metabolic pathway and related genes in the copepod Tigriopus japonicus.

Oil pollution is considered being disastrous to marine organisms and ecosystems. As molting is critical in the developmental process of arthropods in general and copepods, in particular, the impact will be adverse if the target of spilled oil is on molting. Thus, we investigated the harmful effects of water accommodated fractions (WAFs) of crude oil with an emphasis on inhibition of chitin metabolic pathways related genes and developmental retardation in the copepod Tigriopus japonicus. Also, we analysed the ontology and domain of chitin metabolic pathway genes and mRNA expression patterns of developmental stage-specific genes. Further, the developmental retardation followed by transcriptional modulations in nuclear receptor genes (NR) and chitin metabolic pathway-related genes were observed in the WAFs-exposed T. japonicus. As a result, the developmental time was found significantly (P<0.05) delayed in response to 40% WAFs in comparison with that of control. Moreover, the NR gene, HR3 and chitinases (CHT9 and CHT10) were up-regulated in N4-5 stages, while chitin synthase genes (CHS-1, CHS-2-1, and CHS-2-2) down-regulated in response to WAFs. In brief, a high concentration of WAFs repressed nuclear receptor genes but elicited activation of some of the transcription factors at low concentration of WAFs, resulting in suppression of chitin synthesis. Thus, we suggest that WAF can lead molting retardation of naupliar stages in T. japonicus through down-regulations of chitin metabolism. These findings will provide a better understanding of the mode of action of chitin biosynthesis associated with molting mechanism in WAF-exposed T. japonicus.

Identification and conservation of gene loss events of Hox gene clusters in the marine medaka (Oryzias melastigma).

In this study, the identification of the whole Hox gene clusters (46 Hox genes) in the marine medaka Oryzias melastigma was investigated using genome assembly and RNA-seq information. Moreover, the gene loss events of Hox gene clusters, which may occur during fish evolution, were examined for a better understanding of the evolutionary status of the gene lost events of the Hox gene cluster across fish species, particularly in the genus Oryzias.

BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana.

Brominated flame retardant, 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana. Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress (e.g. reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR, SREBP, ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene (e.g. ELO3, Δ5-DES, Δ9-DES) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain.

Adverse effects of MWCNTs on life parameters, antioxidant systems, and activation of MAPK signaling pathways in the copepod Paracyclopina nana.

Engineered multi-walled carbon nanotubes (MWCNTs) have received widespread applications in a broad variety of commercial products due to low production cost. Despite their significant commercial applications, CNTs are being discharged to aquatic ecosystem, leading a threat to aquatic life. Thus, we investigated the adverse effect of CNTs on the marine copepod Paracyclopina nana. Additional to the study on the uptake of CNTs and acute toxicity, adverse effects on life parameters (e.g. growth, fecundity, and size) were analyzed in response to various concentrations of CNTs. Also, as a measurement of cellular damage, oxidative stress-related markers were examined in a time-dependent manner. Moreover, activation of redox-sensitive mitogen-activated protein kinase (MAPK) signaling pathways along with the phosphorylation pattern of extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinases (JNK) were analyzed to obtain a better understanding of molecular mechanism of oxidative stress-induced toxicity in the copepod P. nana. As a result, significant inhibition on life parameters and evoked antioxidant systems were observed without ROS induction. In addition, CNTs activated MAPK signaling pathway via ERK, suggesting that phosphorylated ERK (p-ERK)-mediated adverse effects are the primary cause of in vitro and in vivo endpoints in response to CNTs exposure. Moreover, ROS-independent activation of MAPK signaling pathway was observed. These findings will provide a better understanding of the mode of action of CNTs on the copepod P. nana at cellular and molecular level and insight on possible ecotoxicological implications in the marine environment.

Microplastic Size-Dependent Toxicity, Oxidative Stress Induction, and p-JNK and p-p38 Activation in the Monogonont Rotifer (Brachionus koreanus).

In this study, we evaluated accumulation and adverse effects of ingestion of microplastics in the monogonont rotifer (Brachionus koreanus). The dependence of microplastic toxicity on particle size was investigated by measuring several in vivo end points and studying the ingestion and egestion using 0.05-, 0.5-, and 6-µm nonfunctionalized polystyrene microbeads. To identify the defense mechanisms activated in response to microplastic exposure, the activities of several antioxidant-related enzymes and the phosphorylation status of mitogen-activated protein kinases (MAPKs) were determined. Exposure to polystyrene microbeads of all sizes led to significant size-dependent effects, including reduced growth rate, reduced fecundity, decreased lifespan and longer reproduction time. Rotifers exposed to 6-µm fluorescently labeled microbeads exhibited almost no fluorescence after 24 h, while rotifers exposed to 0.05- and 0.5-µm fluorescently labeled microbeads displayed fluorescence until 48 h, suggesting that 6-µm microbeads are more effectively egested from B. koreanus than 0.05- or 0.5-µm microbeads. This observation provides a potential explanation for our findings that microbead toxicity was size-dependent and smaller microbeads were more toxic. In vitro tests revealed that antioxidant-related enzymes and MAPK signaling pathways were significantly activated in response to microplastic exposure in a size-dependent manner.