Effects of water accommodated fractions (WAFs) of crude oil in two congeneric copepods Tigriopus sp.

Oil pollution has deleterious effects on marine ecosystems. However, the toxicity of crude oil towards Antarctic marine organisms has not been well studied. We compared the deleterious effects of water accommodated fractions (WAFs) of crude oil on reproduction, intracellular reactive oxygen species (ROS) levels, and antioxidant enzymatic activity in Antarctic (Tigriopus kingsejongensis) and temperate (Tigriopus japonicus) copepods. Reproductive rates of T. kingsejongensis and T. japonicus were significantly reduced (P < 0.05) in response to WAFs. Furthermore, T. kingsejongensis showed elevated levels of ROS and higher antioxidant enzyme (glutathione peroxidase [GPx]) activity than T. japonicus in response to WAFs. CYP genes from congeneric copepods were identified and annotated to better understand molecular detoxification mechanisms. We observed significant up-regulation (P < 0.05) of Tk-CYP3024A3 and Tj-CYP3024A2 in response to WAFs, suggesting that CYP genes may contribute to the detoxification mechanism in response to WAF exposure. These finding also suggest that WAFs may induce oxidative stress, leading to reproductive impairment in copepods. Furthermore, Tk-CYP3024A3 and Tj-CYP3024A2 genes can be considered as potential biomarkers of WAF toxicity in the congeneric copepods T. kingsejongensis and T. japonicus. This study will be helpful for enhancing our knowledge on the harmful effects of WAFs in Antarctic and temperate copepods and provides insight into the underlying molecular mechanisms.

Assessing the identity and expression level of the cytochrome P450 20A1 (CYP20A1) gene in the BPA-, BDE-47, and WAF-exposed copepods Tigriopus japonicus and Paracyclopina nana.

CYP20A1 is a member of the cytochrome P450 (CYP) superfamily, identified as an orphan P450 without any assigned biological function; hence, its continued status as an "orphan" gene. In order to address this shortcoming in our understanding of this superfamily, we sought to characterize the CYP20A1 gene in the copepods Tigriopus japonicus (Tj-CYP20A1) and Paracyclopina nana (Pn-CYP20A1) at their mRNA transcriptional level. We assessed the response of this gene's expression in various developmental stages and in response to treatment with bisphenol A (BPA), 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47), and water accommodated fractions (WAFs) of crude oil. As shown in the vertebrate CYP20A1, both Tj-CYP20A1 and Pn-CYP20A1 contained characteristic conserved motifs and domain regions (I helix, K helix and heme-binding motifs) with unusual amino acid sequences apparent in their gene structure. Also molecular characterization of the putative responsive elements in the promoter regions was performed. We observed transcriptional up-regulation of these genes during post-embryonic developmental stages including sex-specific up-regulation in adults. In addition, concentration- and time-dependent mRNA transcripts in response to xenobiotics (BPA, BDE-47, and WAFs) were seen. This study focuses on the molecular elucidation of CYP20A1 genes and their interactions with xenobiotics in the copepods T. japonicus and P. nana that provides important insight into the biological importance of CYP20A1 in invertebrates.

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.

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.

Identification of the Full 46 Cytochrome P450 (CYP) Complement and Modulation of CYP Expression in Response to Water-Accommodated Fractions of Crude Oil in the Cyclopoid Copepod Paracyclopina nana.

The 46 cytochrome P450 (CYP) gene superfamily was identified in the marine copepod Paracyclopina nana after searching an RNA-seq database and comparing it with other copepod CYP gene families. To annotate the 46 Pn-CYP genes, a phylogenetic analysis of CYP genes was performed using a Bayesian method. Pn-CYP genes were separated into five different clans: CYP2, CYP3, CYP20, CYP26, and mitochondrial. Among these, the principal Pn-CYP genes involved in detoxification were identified by comparing them with those of the copepod Tigriopus japonicus and were examined with respect to their responses to exposure to a water-accommodated fraction (WAF) of crude oil and to the alkylated forms of two polycyclic aromatic hydrocarbons (PAHs; phenanthrene and fluorene). The expression of two Pn-CYP3027 genes (CYP3027F1 and CYP3027F2) was increased in response to WAF exposure and also was upregulated in response to the two alkylated PAHs. In particular, Pn-CYP3027F2 showed the most notable increase in response to 80% WAF exposure. These two responsive CYP genes (Pn-CYP3027F1 and CYP3027F2) were also phylogenetically clustered into the same clade of the WAF- and alkylated PAH-specific CYP genes of the copepod T. japonicus, suggesting that these CYP genes would be those chiefly involved in detoxification in response to WAF exposure in copepods. In this paper, we provide information on the copepod P. nana CYP gene superfamily and also speculate on its potential role in the detoxification of PAHs in marine copepods. Despite the nonlethality of WAF, Pn-CYP3027F2 was rapidly and significantly upregulated in response to WAF that may serve as a useful biomarker of 40% or higher concentration of WAF exposure. This paper will be helpful to better understand the molecular mechanistic events underlying the metabolism of environmental toxicants in copepods.

Crude oil exposure results in oxidative stress-mediated dysfunctional development and reproduction in the copepod Tigriopus japonicus and modulates expression of cytochrome P450 (CYP) genes.

In this study, we investigated the effects of the water-accommodated fraction (WAF) of crude oil on the development and reproduction of the intertidal copepod Tigriopus japonicus through life-cycle experiments. Furthermore, we investigated the mechanisms underlying the toxic effects of WAF on this benthic organism by studying expression patterns of cytochrome P450 (CYP) genes. Development of T. japonicus was delayed and molting was interrupted in response to WAF exposure. Hatching rate was also significantly reduced in response to WAF exposure. Activities of antioxidant enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), and catalase (CAT) were increased by WAF exposure in a concentration-dependent manner. These results indicated that WAF exposure resulted in oxidative stress, which in turn was associated with dysfunctional development and reproduction. To evaluate the involvement of cytochrome P450 (CYP) genes, we cloned the entire repertoire of CYP genes in T. japonicus (n=52) and found that the CYP genes belonged to five different clans (i.e., Clans 2, 3, 4, mitochondrial, and 20). We then examined expression patterns of these 52 CYP genes in response to WAF exposure. Three TJ-CYP genes (CYP3024A2, CYP3024A3, and CYP3027C2) belonging to CYP clan 3 were significantly induced by WAF exposure in a time- and concentration-dependent manner. We identified aryl hydrocarbon responsive elements (AhRE), xenobiotic responsive elements (XREs), and metal response elements (MRE) in the promoter regions of these three CYP genes, suggesting that these genes are involved in detoxification of toxicants. Overall, our results indicate that WAF can trigger oxidative stress and thus induce dysfunctional development and reproduction in the copepod T. japonicus. Furthermore, we identified three TJ-CYP genes that represent potential biomarkers of oil pollution.