Ihn-Sil Kwak

Expression of stress response HSP70 gene in Asian paddle crabs, Charybdis japonica, exposure to endocrine disrupting chemicals, bisphenol A (BPA) and 4-nonylphenol (NP)

The Asian paddle crab, Charybdis japonica, is a potential bio-indicator reflecting marine sediment toxicity as well as a commercially important species living along coastal areas in Korea. This study investigated its stress response by looking at the heat shock protein (HSP70) gene of C. japonica when the organism is exposed to bisphenol A (BPA) and 4-nonylphenol (NP). We characterized partial sequence of HSP70 as the stressresponse gene of C. japonica. The nucleotide sequence of C. japonica HSP70 is over 90% homologous with the corresponding gene of other crabs. Phylogenetic tree analysis revealed a close relationship between C. japonica HSP70 and HSP70 in other species of lobster and shrimps. HSP70 mRNA transcripts were detected in all the examined tissues of C. japonica, with the highest level in gills, the organ that most frequently came into contact with the external BPA or NP-laden water. As no reference data were available for C. japonica crab exposure, the BPA and NP 24-h LC50 values have not been previously determined. The expression of the C. japonica HSP70 gene to various BPA or NP concentrations during short and longer times was assessed. Gene expression was significantly induced in concentration- and time-dependent manners after BPA or NP exposures. These results support the postulation that crab C. japonica HSP70 could be a potential stress response molecular marker to monitor marine ecosystems.

Vitellogenin gene characterization and expression of Asian paddle crabs (Charybdis japonica) following endocrine disrupting chemicals

Vitellogenin (VTG), the yolk-precursor lipoprotein, has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. The Asian paddle crab, Charybdis japonica, is a potential bio-indicator for monitoring marine environments. The aim of this study was to identify the possibility of using C. japonica VTG as biomarkers of stress caused by endocrine disrupting chemicals (EDCs). We characterized a partial sequence of the VTG cDNA in the C. japonica crab and evaluated the crab’s mRNA expression profiles following exposure to different concentrations of bisphenol A (BPA) and 4-nonylphenol (NP) for 24 or 96 h. The sequence homology of C. japonica VTG is over 93% in nucleotide and over 98% in amino acid with the corresponding gene of other crabs. Phylogenetic analysis revealed that the C. japonica VTG is an ortholog of other species of lobster and shrimp. Tissue distribution analysis of the C. japonica VTG mRNA revealed that the expression of VTG mRNA was highest in the ovary of females and hepatopancreas. The expression of the C. japonica VTG gene in various BPA or NP concentrations during shorter and longer times was assessed. The expression of VTG transcripts was significantly increased in the C. japonica crab exposed to BPA and NP at different concentrations for 24 h. The mRNA expression of the VTG gene was significantly induced in concentration- and time-dependent manners after BPA or NP exposures for 96 h. These results indicate that crab C. japonica VTG could be used as a potential biomarker of EDCs in marine environment monitoring.

Cascading effects from survival to physiological activities, and gene expression of heat shock protein 90 on the abalone Haliotis discus hannai responding to continuous thermal stress

Increasing temperatures can be a significant stressor for aquatic organisms. Abalones, a type of large marine gastropods, are the most commercially important species in aquaculture for Asia. To evaluate the potential ecological risk posed by temperature stress, we measured biological responses such as survival rate, adhesion ability (falling rate), and foot abnormalities in the abalone Haliotis discus hannai. Additionally, biochemical and molecular responses were evaluated in H. discus hannai exposed to various temperature gradients. The survival rate was reduced in abalones exposed to relative high temperatures (more than 26 °C). Increased temperature stress induced a higher falling rate and abnormal foot structure. Furthermore, increased antioxidant enzyme activities were observed in abalones exposed to relative high temperatures (26 and 28 °C). The activities of superoxide dismutase were induced in a time-dependent manner after high temperature stress. Generally, heat shock protein 90 also increased significantly in H. discus hannai exposed to temperature gradients (more than 24 °C) for 12 h. These results provide valuable information regarding stress responses to increased temperatures, in H. discus hannai: adverse biological and molecular outcomes could be utilized as risk assessments and stress monitoring of marine ecosystems under increased water temperatures.

The effect of temperature gradients on endocrine signaling and antioxidant gene expression during Chironomus riparius development

Temperature is one of the most important environmental factors affecting the biological processes of aquatic species. To investigate the potential effects of temperature on the developmental processes of aquatic invertebrates, we analyzed biological and molecular transcriptional responses during Chironomus riparius development, including five stages spanning from embryo to adult stages. We assessed the temperature change-induced reduction of survival rate, changes in biological development including the male:female ratio in emerged adults, the success rates of pupation and emergence, and the developmental timing of pupation and emergence. The increased temperature induced expression of endocrine signaling genes, such as the ecdysone receptor, ultraspiracle (ortholog of the RXR), and the estrogen-related receptor in the fourth-instar larval and pupal stages of C. riparius development. Altered temperature also affected the activity of antioxidant genes, including catalase, peroxidase, glutathione peroxidase, and superoxide dismutase during the fourth-instar larval to adult stages of C. riparius development, as a result of altered development. Increased temperature during the fourth-instar larval stage increased oxidative stress in pupae and adults. Responses of antioxidant genes to increased temperature occurred in a developmental stage-dependent manner. However, reduced temperature did not induce the expression of antioxidant genes in a developmental stage-dependent manner, although it did induce oxidative stress during C. riparius development. Increased temperature also caused greater toxicity of di-ethylhexyl phthalate (DEHP) in fourth-instar larvae. Our findings suggest that altered temperatures may disturb the invertebrate hormone system and developmental processes by inducing oxidative stress in aquatic environments.

Identification of potential markers and sensitive tissues for low or high salinity stress in an intertidal mud crab (Macrophthalmus japonicus)

Macrophthalmus japonicus is an intertidal mud crab is an ecologically important species in Korea, can tolerate a wide range of natural and anthropogenic stressors. Environmental changes especially salinity cause physiological stress to the marine habitats. Differential gene transcription of M. japonicus tissues provided information about tissue specific responses against salinity. Five potential genes were identified and their transcription levels were determined quantitatively comparison to seawater (SW: 31 ± 1 psu) in M. japonicus gills and hepatopancreas after exposed them to different salinities. Ecdysteroid receptor (Mj-EcR), trypsin (Mj-Tryp), arginine kinase (Mj-AK), lipopolysaccharide and β-1,3-glucan binding protein (Mj-LGBP) and peroxinectin (Mj-Prx) in hepatopancreas up-regulated against different salinities. In contrast, the gills, Mj-EcR, Mj-Tryp and Mj-AK showed late up-regulated responses to 40 psu compared to SW. All genes except Mj-LGBP showed up regulation in the gills as time dependent manner. These genes can be considered as potential markers to assess responses in salinity changes. This study suggests hepatopancreas is a suitable tissue for transcriptional, biochemical and physiological responses analysis on M. japonicus in low and high salinity stress.

Characterization and gene expression of heat shock protein 90 in marine crab Charybdis japonica following bisphenol A and 4-nonylphenol exposures

Objectives Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. Methods This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. Results The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Conclusions Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments.

Antioxidative-related genes expression following perfluorooctane sulfonate (PFOS) exposure in the intertidal mud crab, Macrophthalmus japonicus

Perfluorooctane sulfonate (PFOS) is a persistent environmental contaminant that is used as a surfactant in various industries and consumer products. The intertidal mud crab, Macrophthalmus japonicus, is one of the most abundant macrobenthic creatures. In this study, we have investigated the effect of PFOS on the molecular transcription of antioxidant and detoxification signaling in M. japonicus crab. The selected stress response genes were superoxide dismutases (CuZnSOD and MnSOD), catalase (CAT), glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx), peroxiredoxin (Prx), and thioredoxin reductase (TrxR). Significant up-regulation of SODs and CAT was observed after 24 and 96 h exposure to PFOS at different concentrations. The gene expression levels of GPx, PHGPx, and TrXR were significantly up-regulated after exposure to PFOS for 96 h. The transcript levels of CAT and PHGPx were induced in dose- and time-dependent manners after PFOS treatments. However, Prx gene expression was significantly up-regulated in M. japonicus crabs exposed to 10 and 30 μg L-1 PFOS for 96 h. Additionally, PFOS toxicity in M. japonicus induced reduced survival rates at relatively high concentrations of PFOS exposure. Our findings support the contention that exposures to PFOS induced the response of genes related to oxidative stress and detoxification in M. japonicus crabs.

Changes of exoskeleton surface roughness and expression of crucial participation genes for chitin formation and digestion in the mud crab (Macrophthalmus japonicus) following the antifouling biocide irgarol

Irgarol is a common antifoulant present in coastal sediment. The mud crab Macrophthalmus japonicus is one of the most abundant of the macrobenthos in the costal environment, and its exoskeleton has a protective function against various environmental threats. We evaluated the effects of irgarol toxicity on the exoskeleton of M. japonicus, which is the outer layer facing the environment. We analyzed transcriptional expression of exoskeleton, molting, and proteolysis-related genes in the gill and hepatopancreas of these exposed M. japonicus. In addition, changes in survival and exoskeleton surface characteristics were investigated. In the hepatopancreas, mRNA expression of chitinase 1 (Mj-chi1), chitinase 4 (Mj-chi4), and chitinase 5 (Mj-chi5) increased in M. japonicus exposed to all concentrations of irgarol. Mj-chi1 and Mj-chi4 expressions from 1 to 10μgL(-1) were dose- and time-dependent. Ecdysteroid receptor (Mj-EcR), trypsin (Mj-Tryp), and serine proteinase (Mj-SP) in the hepatopancreas were upregulated in response to different exposure levels of irgarol at day 1, 4, or 7. In contrast, gill Mj-chi5, Mj-Tryp, and Mj-SP exhibited late upregulated responses to 10μgL(-1) irgarol compared to the control at day 7. Mj-chi1 showed early upregulation upon exposure to 10μgL(-1) irgarol and Mj-chi4 showed no changes in transcription in the gill. Gill Mj-EcR presented generally downregulated expression patterns. In addition, decreased survival and change of exoskeleton surface roughness were observed in M. japonicus exposed to the three concentrations of irgarol. These results suggest that exposure to irgarol induces changes in the exoskeleton, molting, and proteolysis metabolism of M. japonicus.

Development of ELID Monitoring System and its Application to ELID Grinding of Structural Ceramics

This study has focused on development of ELID monitoring system and its application to ELID grinding of structural ceramics. ELID monitoring system was consisted of grinding equipment, ELID power supply, grinding wheel, electrode and monitoring program. It can give a real time data to check spindle grinding resistance, wheel revolution, dressing current and voltage in ELID grinding process. The performance of developed system was evaluated by applying to grinding of structural ceramics, silicon carbide and alumina. As the results of experiments, monitored data for spindle resistance and ELID dressing current was useful to check steady-state ELID grinding process. From the comparison of spindle resistance between ELID grinding and conventional grinding process according to change of depth of cut, it could be confirmed that the spindle resistance in ELID grinding was lower than conventional grinding process.

Effect of Reference Current and Depth of Cut on Electro-Discharge Energy in Electro- Discharge Truing Process for Metal-Bonded Grinding Wheel

This study focused on developing an electro-discharge truing method for the ELID grinding process using a metal-bonded grinding wheel. The truing process in grinding plays important roles in enhancing the quality of the ground surface. In this study, a reference current in the electro-discharge truing process was confirmed to be a viable solution for efficient truing through performance experiments. Current and voltage variation experiments were carried out and the variation output data were collected with a monitoring program to understand the electrodischarge phenomenon that occurs between metal-bonded grinding wheels and copper electrodes. The experimental results showed that as the reference current decreased, the average electro-discharge energy decreased. Therefore, the reference current can be used as an indicator to estimate the size of the gap between the truer and grinding wheel.