• Korean Journal of Environmental Biology
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• v.39 no.2
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• pp.160-168
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• 2021

Doublesex and mab-3 related transcription factor(dmrt) play crucial roles in sex determination and sex differentiation in vertebrates and invertebrates. Although dmrt genes have been identified in vertebrates, little is known about aquatic invertebrates. In this study, two dmrt genes, namely, Dc_dmrt93B and Dc_dmrt99B, were identified from brackish water flea, Diaphanosoma celebensis. Transcriptional changes were observed in the dmrt genes when the flea was exposed to bisphenol(BP), an endocrine disruptor. Sequence and phylogenetic analyses showed that both dmrt genes contained two conserved domains, namely, DM and DMA, closely clustered with those of Daphnia spp. Additionally, a significant increase in the Dc_dmrt99B mRNA expression level was observed upon exposure to intermediate concentrations of BP (bisphenol A>bisphenol S=bisphenol F, p<0.05), while the expression of Dc_dmrt93B mRNA was slightly modulated. These findings imply that the two dmrt genes may be involved in sex differentiation of D. celebensis. Furthermore, it was found that the ability of BP to modulate dmrt genes could affect development and reproduction. This study provides a basis for understanding the function of the dmrt genes and the molecular mode of action of BP in small crustaceans.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.43 no.2
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• pp.131-138
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• 2010

This study determined the optimum microalgae species and concentration for the brackish water flea, Diaphanosoma celebensis, in individual and community cultures by feeding it several different diets. Six single trials (Tetraselmis suecica, Isochrysis galbana, marine Chlorella ellipsoidea, freshwater Chlorella vulgaris, Scenedesmus sp., Selenastrum sp.) were conducted at 10 psu and $30^{\circ}C$. The community and individual cultures of the water flea were performed in 1-L beakers and 3-mL vessels (12-well culture plates), respectively. In the community cultures, the maximum density and specific growth rate were highest for water fleas fed T. suecica, reaching 60.0 individuals (ind.)/mL and 0.40, respectively. In the individual cultures, the most offspring and greatest life span of females were 56.9 ind. and 24.3 days, respectively, in the T. suecica trial. By contrast, diets of Scenedesmus sp. and Selenastrum sp. resulted in poor growth rates. In the T. suecica experiments examining a range of 10 to $200\times10^3$ cells/indl. the specific growth rate of the water flea tended to increase with the amount of supplement, while the life span decreased. The maximum density and number of offspring of females was highest at 53.5 ind./mL and 38.8 ind. respectively, at 40,000 cells/ind. These results suggest that the best microalgae species for the mass culture of D. celebensis is T. suecica and the optimum concentration is 40,000 cells per individual.

• Journal of Marine Life Science
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• v.6 no.2
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• pp.73-79
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• 2021

Bisphenol A is a representative endocrine disruptor and continuously detected in aquatic environment due to wide use, resulting in adverse effects on growth, development, and reproduction in diverse organisms as well as human. Structural analogs have been developed to substitute BPA are also suspected to have endocrine disrupting effects. In the present study, the time-dependent expression patterns of ecdysteroid synthesis (nvd, cyp314a1), receptors (EcRA, EcRB, USP, ERR), and downstream signaling pathway - related genes (HR3, E75, Vtg, VtgR) were investigated using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) in the brackish water flea Diaphanosoma celebensis exposed to Bisphenol analogs (BPs; BPA, BPF, and BPS) for 6, 12, and 24 h. As results, the expression of nvd, cyp314a1, EcRs, USP, ERR and E75 mRNA was upregulated at 6 h exposure to BPF, which is earlier than BPA and BPS (12 h). On the other hand, HR3, E75 and VtgR mRNA levels were elevated at 6 h earlier at BPS and BPF than at BPA (12 h), but Vtg mRNA level was slightly changed within 24 h. These findings suggest that like BPA, BPF and BPS can also modulate the transcription of ecdysteroid pathway - related genes with different mechanisms, and have a potential as endocrine disruptors. This study will provide a better understanding the molecular mode of action of bisphenols on ecdysteroid pathway in the brackish water flea.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.43 no.2
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• pp.139-145
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• 2010

This study investigated the optimum salinity and temperature conditions for mass culture of the brackish water flea, Diaphanosoma celebensis. Community and individual cultures of flea were maintained in 1 L beakers and 3 mL vessels (of a 12-well culture plate), respectively, and fed green algae, Tetraselmis suecica. In salinity experiments ranging from 5 to 34 psu, continuous growth of flea populations was found up to 34 psu. However, the specific growth rate and life span of females showed decreasing tendencies with the increase of salinity. The highest maximum density and offspring number were 33.6 individuals (ind.)/mL and 55.3 ind. at 10 psu, respectively. In the temperature experiments ranging from 20 to $40^{\circ}C$, population growth of D. celebensis increased continuously until $35^{\circ}C$ and then decreased over $40^{\circ}C$. The specific growth rate was significantly higher at 25 and $30^{\circ}C$ than at 20 and $40^{\circ}C$. Female life span tended to decrease with temperature increase. The highest maximum density and offspring number were 52.3 ind./mL and 46.0 ind. at $30^{\circ}C$, respectively. These results suggest that the optimum salinity and temperature for mass culture of D. celebensis may be 10 psu and $30^{\circ}C$, respectively.

• Korean Journal of Environmental Biology
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• v.37 no.1
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• pp.72-81
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• 2019

Bisphenol A (BPA), a representative endocrine disrupting chemicals, has adverse effects on growth, development and reproduction in aquatic organisms. The object of this study was to investigate the modulation of antioxidant enzyme-coding genes using quantitative real time RT-PCR (qRT-PCR), enzyme activity and total protein content, to understand oxidative stress responses after exposure to BPA for 48 h in brackish water flea Diaphanosoma celebensis. The BPA ($3mg\;L^{-1}$) significantly upregulated the expression of Cu/Zn-SOD, Mn-SOD, and catalase (CAT) mRNA. Three GST isoforms (GST-kappa, GST-mu, and GST-theta) mRNA levels significantly increased at the rate of $0.12mg\;L^{-1}$ of BPA. In particular, GST-mu showed the highest expression level, indicating its key role in antioxidant response to BPA. SOD activity was induced with a concentration-dependent manner, and total protein contents was reduced. These findings indicate that BPA can induce oxidative stress in this species, and these antioxidants may be involved in cellular protection against BPA exposure. This study will provide a better understanding of molecular mode of action of BPA toxicity in aquatic organisms.

• Korean Journal of Environmental Biology
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• v.40 no.3
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• pp.255-266
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• 2022

In crustaceans, molting is regulated by interactions between ecdysteroid and juvenile hormone (JH) signaling pathway-related genes. Unlike the ecdysteroid signaling pathway, little information on the role of JH signaling pathway-related genes in molting is available in zooplanktonic crustaceans. In this study, three genes (juvenile hormone acid O-methyltransferase (JHAMT), methoprene-tolerant (Met), and juvenile hormone epoxide hydrolase (JHEH)) which are involved in the synthesis, receptor-binding, and degradation of JH were identified using sequence and phylogenetic analysis in the brackish water flea, Diaphanosoma celebensis. Transcriptional changes in these genes during the molting cycle in D. celebensis were analyzed. Sequence and phylogenetic analysis revealed that these putative proteins may be functionally conserved along with those of insects and other crustaceans. In addition, the expression of the three genes was correlated with the molting cycle of D. celebensis, indicating that these genes may be involved in the synthesis and degradation of JH, resulting in normal molting. This study will provide information for a better understanding of the role of JH signaling pathway-related genes during the molting process in Cladocera.

• Journal of Marine Life Science
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• v.7 no.2
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• pp.145-153
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• 2022

Mercury (Hg) is a major concern in marine environment because of their bioaccumulation and biomagnification properties, and adverse effects to aquatic organisms at even a trace amount. However, little information on the effects of Hg, compared to other heavy metals, is available in marine small crustaceans. Here, we investigated the transcriptional modulation of metabolism-related genes in the brackish water flea, Diaphanosoma celebensis after exposure to sublethal concentration (0.2, 0.4, 0.8 ㎍/l) of HgCl₂ for 48 h. Relative mRNA expression levels of five detoxification enzyme-coding genes (cytochrome P450; cyp360A1, cyp361A1, cyp4AP3, cyp4C122, and cyp370C5) and six digestive enzyme-coding genes [alpha amylase (AMY), alpha amylase related protein (AMY-like), trypsin (TRYP), chymotrypsin-like protein (CHY), lipase (LIP), pancreatic lipase-related protein (PLRP)] were analyzed using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). As results, Hg increased the mRNA level of cyp370C5 (clan2) and cyp4AP3 (clan4) in a concentration dependent manner. A significant increase in TRYP mRNA was also concentration-dependently observed after exposure to Hg. These findings suggest that cyp370C5 and cyp4AP3 play a key role in Hg detoxification in D. celebensis, and Hg can affect energy metabolism by modulating the transcription of digestive enzyme. This study will provide better understanding the molecular effects of Hg in marine small crustacean.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.45 no.2
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• pp.146-153
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• 2012

This study examined the effect of formulated diets (composed of condensed freshwater $Chlorella$ or $Schizochytrium$ sp., $Crypthecodinium$ sp., and EPA oil sources derived from baker's yeast) on the population growth and fatty acid composition of the brackish water flea ($Diaphanosoma$ $celebensis$) to determine diet effectiveness for mass production. Results indicated that the highest maximum density, fecundity, and population specific growth rate (SGR) of the flea were observed using a diet of $Tetraselmis$ $suecica$ (control). However, results using condensed freshwater $Chlorella$ and the mixed treatment of $Schizochytrium$ sp.+$Crypthecodinium$ sp.+EPA oil did not significantly differ from the control treatment ($P$>0.05). Thus, $T.$ $suecica$ could be replaced with a combination diet of $Schizochytrium$ sp., $Crypthecodinium$ sp., and EPA oil in condensed freshwater $Chlorella$ without growth interruption of $D.$ $celebensis$. As the percentage of combination diet (mix of $Schizochytrium$ sp., $Crypthecodinium$ sp., and EPA oil) in condensed freshwater $Chlorella$ increased, the level of ARA, EPA, DHA, and n-3 HUFA in $D.$ $celebensis$ also increased. However, the population density of $D.$ $celebensis$ sharply decreased with the use of treated supplements C5M5 (CHL [50%] and $Crypthecodinium$ sp.+ $Schizochytrium$ sp. + EPA oil [MIX 50%]). The optimum proportions of CHL and MIX for $D.$ $celebensis$ within the mixture were 70% and 30%, respectively. Based on the results, the combination of $Schizochytrium$ sp., $Crypthecodinium$ sp., and EPA oil in condensed freshwater $Chlorella$ appears to be a stable and effective food source for mass production of $D.$ $celebensis$.

• Journal of Marine Life Science
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• v.5 no.2
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• pp.35-42
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• 2020

17β-estradiol (E₂) is a natural hormone secreted by ovary, and continuously discharged from household and livestock wastewater into aquatic environment. Due to its strong estrogenic activity, it has adverse effects on development and reproduction in crustacean as an endocrine disrupting chemical. Although ecdysteroid signaling pathway play a key role in development in crustacean, little information on transcriptional modulation of ecdysteroid-related genes in response to E₂ is available in small crustacean. Here, we investigated the acute toxicity of E₂ to obtain 24-h LC_x values in the brackish water flea Diaphanosoma celebensis. Time-dependent expression patterns of seven ecdysteroid pathway - related genes (CYP314a1, EcRA, EcRB, USP, ERR, Vtg, VtgR) were further examined using quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). As results, 24-h LC₅₀ and LC₁₀ values were 9.581 mg/l and 4.842 mg/l, respectively. The mRNA expression of CYP314a1, EcRA, USP, VtgR was significantly up-regulated at 12 or 24 h after exposure to E₂. These findings indicate that E₂ can affect their molting and reproduction by modulating the expression of ecdysteroid pathway - related in D. celebensis. This study will be useful for better understanding of molecular mode of action of endocrine disrupting chemicals on molting process in small crustacean.

• Journal of Marine Life Science
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• v.4 no.2
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• pp.45-52
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• 2019

Polycyclic aromatic hydrocarbons (PAHs) are persistent and commonly detected in marine ecosystem. They coexist with a various contaminants including heavy metals in real environment, but most studies have been still focused on single effects of these contaminants to aquatic organisms. In this study, the single and combined effects of benzy[a]pyrene (B[a]P) and HgCl₂ were investigated in the brackish water flea, Diaphanosoma celebensis. For combined effect study, three mixtures A (3:7), B (5:5), and C (7:3) of benzy[a]pyrene (B[a]P) and HgCl₂ were determined by 48 h - LC₅₀ values of single exposure. Combined effects of both chemicals were estimated by concentration addition (CA) model and independent action (IA) model. As results, 48 h - LC₅₀ values of benzy[a]pyrene (B[a]P) and HgCl₂ was 25.75 ㎍ l^-1 and 3.6 ㎍ l^-1, respectively. TU values of mixture A, B, and C were 1.06, 0.83, and 0.96, respectively, indicating additive effects of mixtures using CA model. This study will be helpful as basic data for understanding the combined effects of contaminants in marine environment.