Duck-Hyun Kim

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- (0.05u2009μm) and micro-sized (0.5 and 6u2009μm) 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.

Rotifers in Ecotoxicology

Rotifers are widespread and abundant in aquatic ecosystems and are valuable live feeds in aquaculture and fisheries. They are particularly useful in evaluating full life cycle and population-level effects in response to pollutant exposures. Rotifers have, therefore, been considered as a promising species in aquatic ecotoxicology. The importance of using rotifers in aquatic ecotoxicology was constantly reviewed (Snell and Janssen Hydrobiologia 313/314:231–247, 1995; Dahms et al. Aquat Toxicol 101:1–12, 2011). This chapter will focus on discussing, with diverse examples, why rotifers became promising model species in ecotoxicology, taking into account their biological, physiological, and genomic information. Ecotoxicogenomic approaches using RNA-seq and genomic approaches will be demonstrated. In particular, the monogonont rotifer Brachionus sp. will be introduced as a target taxon for ecotoxicogenomic studies with substantial genetic information.

Multi-walled carbon nanotubes (MWCNTs) lead to growth retardation, antioxidant depletion, and activation of the ERK signaling pathway but decrease copper bioavailability in the monogonont rotifer (Brachionus koreanus).

To examine the toxic effects of multi-walled carbon nanotubes (MWCNTs) in the marine environment, we first exposed the monogonont rotifer (Brachionus koreanus) to MWCNTs in the presence of copper. The acute toxicity of copper decreased significantly with a decrease in copper bioavailability resulting from MWCNT exposure. Furthermore, we examined the effects of MWCNT exposure on reproductive capacity, population growth rate, growth patterns, antioxidant systems, and mitogen-activated protein kinase (MAPK) activation. Reproductive capacity, population growth rate, and body growth rate were significantly suppressed in B. koreanus in response to 1.3-4mg/L MWCNT exposure. Furthermore, MWCNTs induced the generation of reactive oxygen species (ROS) and decreased the antioxidant enzymatic activities of catalase (CAT) and glutathione reductase (GR). However, the enzymatic activity of glutathione S-transferase (GST) was up-regulated after a 24 h-exposure to 100mg/L MWCNTs. Exposure to 100mg/L MCWNTs induced extracellular signal-regulated kinase (ERK) activation in B. koreanus, suggesting that p-ERK may mediate the adverse effects of MWCNTs in B. koreanus via the MAPK signaling pathway. Our results provide insight into the mechanistic basis of the ecotoxicological effects of MWCNTs in the marine environment.

BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus

2,2,4,4-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5-6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure.

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.

Effects of trimethoprim on life history parameters, oxidative stress, and the expression of cytochrome P450 genes in the copepod Tigriopus japonicus.

Trimethoprim (TMP) is an antibiotic that has been detected in various environments including marine habitats; however, the toxic effects of TMP are poorly understood in non-target marine organisms. In this study, the effects of TMP on mortality, development, reproduction, intracellular reactive oxygen species (ROS) levels, and transcription levels of antioxidant and xenobiotic detoxification-related enzyme genes were investigated in the copepod Tigriopus japonicus. The TMP half lethal dose at 48xa0h (LC50-48xa0h) in nauplius and TMP LC50-96xa0h in adult T.xa0japonicus copepods was determined as 156xa0mg/L and 200xa0mg/L, respectively. In TMP-exposed T.xa0japonicus, delayed developmental time and impaired reproduction were observed as harmful effects on the life history parameters. Increased ROS levels were also shown in response to TMP exposure at the highest concentration (100xa0mg/L TMP) and the expression of antioxidant- (e.g. GST-kappa, GST-sigma) and xenobiotic detoxification (e.g. CYPs)-related genes were upregulated in a time and/or dose-dependent manner in response to TMP. Particularly, significant upregulation of three CYP genes (Tj-CYP3024A2, Tj-CYP3024A3 and Tj-CYP3027C2) were examined, suggesting that these CYP genes are likely playing an important role in the TMP detoxification metabolism in T.xa0japonicus. In summary, we found that TMP induced oxidative stress via the transcriptional regulation of antioxidant- and xenobiotic detoxification-related genes, leading to changes in life history parameters such as developmental delay and reproduction impairment. Three Tj-CYP genes (Tj-CYP3024A2, Tj-CYP3024A3 and Tj-CYP3027C2) could be useful as potential T.xa0japonicus biomarkers in response to antibiotics.

Immune gene discovery in the crucian carp Carassius auratus.

The crucian carp Carassius auratus (Cyprinidae) is one of the important fish species in aquaculture. Although the crucian carp has several economic benefits, their immune system and gene information have not been investigated in depth as yet. Here, we performed the transcriptome analysis of C.xa0auratus using the pyrosequencing method and selected several immune-related genes. Of unigenes obtained in this species, we identified a number of immune system-related genes (e.g. adhesive protein, antimicrobial protein, apoptosis- and cell cycle-related protein, cellular defense effector, immune regulator, pattern recognition protein, protease, protease inhibitor, reduction/oxidation-related protein, signal transduction-related protein and stress protein) that are potentially useful for studies on fish immunity. To be of public and practical use, we designed primer pairs of each gene from the crucian carp for real-time RT-PCR application and tested the amplicon identity of entire gene sets with the total RNA sample. For comparative analysis, we measured tissue-preferential transcript profiles of selected genes. This study will be helpful to extend our knowledge on the immune system of the crucian carp in comparative aspects and to develop the crucian carp as a potential model organism for aquatic quality monitoring in fish farming.

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.

Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the copepod Tigriopus japonicus

Methylmercury (MeHg) is a concerning environmental pollutant that bioaccumulates and biomagnifies in the aquatic food web. However, the effects of MeHg on marine zooplankton are poorly understood even though zooplankton are considered key mediators of the bioaccumulation and biomagnification of MeHg in high-trophic marine organisms. Here, the toxicity of MeHg in the benthic copepod Tigriopus japonicus was assessed, and its adverse effects on growth rate and reproduction were demonstrated. Antioxidant enzymatic activities were increased in the presence of MeHg, indicating that these enzymes play an important role in the defense response to MeHg, which is regulated by a complex mechanism. Subsequent activation of different patterns of mitogen-activated protein kinase (MAPK) pathways was demonstrated, providing a mechanistic approach to understand the signaling pathways involved in the effects of MeHg. Our results provide valuable information for understanding the toxicity of MeHg and the underlying defense mechanism in response to MeHg exposure in marine zooplankton.

Genome-wide identification of 31 cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and analysis of their benzo[α]pyrene-induced expression patterns

While marine invertebrate cytochrome P450 (CYP) genes and their roles in detoxification mechanisms have been studied, little information is available regarding freshwater rotifer CYPs and their functions. Here, we used genomic sequences and RNA-seq databases to identify 31 CYP genes in the freshwater rotifer Brachionus calyciflorus. The 31 Bc-CYP genes with a few tandem duplications were clustered into CYP 2, 3, 4, mitochondrial, and 46 clans with two marine rotifers Brachionus plicatilis and Brachionus koreanus. To understand the molecular responses of these 31 Bc-CYP genes, we also examined their expression patterns in response to benzo[α]pyrene (B[α]P). Three Bc-CYP genes (Bc-CYP3044B3, Bc-CYP3049B4, Bc-CYP3049B6) were significantly upregulated (P<0.05) in response to B[α]P, suggesting that these CYP genes can be involved in detoxification in response to B[α]P exposure. These genes might be useful as biomarkers of B[α]P exposure in B. calyciflorus. Overall, our findings expand the repertoire of known CYPs and shed light on their potential roles in xenobiotic detoxification in rotifers.