Inn-Sil Kwak

Molecular effects of endocrine-disrupting chemicals on the Chironomus riparius estrogen-related receptor gene.

Endocrine-disrupting chemicals (EDCs) mimic the action of endogenous estrogen hormones; consequently, they can interfere with the endocrine systems of a variety of organisms. There is global concern regarding the potential impacts of EDCs on the aquatic environment. To evaluate the effects of EDCs on the estrogen-related receptors (ERR) of Chironomus, we characterized full-length cDNA sequences of the ERR gene from Chironomus riparius. The complete cDNA sequence of the ERR gene was found to be 1332bp in length. The results of our phylogenetic analysis demonstrated that C. riparius ERR was most closely related to that of mosquitoes. The basal level of ERR mRNA was expressed abundantly during different life-history stages, with the exception of adult males. Additionally, ERR gene expression was upregulated significantly in C. riparius exposed to bisphenol A (BPA) and 4-nonylphenol (NP) at all concentrations assayed after 24h of exposure. The ERR gene was significantly upregulated following short periods of exposure to di(2-ethylhexyl)phthalate (DEHP) concentrations of only 50mgL(-1). However, under long-term exposure conditions, ERR expression was induced to a significant degree after BPA, NP, and DEHP exposure at all concentrations assayed. Furthermore, the luciferase reporter gene assay revealed increased ERR expression following exposure to these compounds. Collectively, these findings indicate that EDCs influence the expression of ERR in Chironomus species.

Characterization of heat shock protein 40 and 90 in Chironomus riparius larvae: effects of di(2-ethylhexyl) phthalate exposure on gene expressions and mouthpart deformities.

Di(2-ethylhexyl) phthalate (DEHP) is a widely distributed phthalate that organisms are frequently exposed to due to its wide range of commercial and manufacturing uses as a plasticizer. Indeed, DEHP is often found in freshwater systems that receive domestic waste water discharges. Therefore, we evaluated the effects of DEHP on the mRNA levels of heat shock protein (HSP) 40 and 90 in chironomids. In addition, we evaluated the effects of exposure to DEHP on the induction of morphological deformities in chironomids. To accomplish this, partial sequences of HSP 40 and 90 from Chironomus riparius larvae were amplified and sequenced. The inferred amino acid sequences were then aligned with those of other insect HSP 40 and 90 genes. The results of this alignment revealed that there was a high degree of similarity among the homologues. In addition, the HSP 40 and 90 mRNA expressions were significantly upregulated in response to short and long-term exposure to DEHP at concentrations of 1, 10, and 30 microgL(-1). Furthermore, the occurrence of mouthpart deformities was significantly higher in chironomids that were treated with DEHP (12-20%) than in controls (3-5%). Taken together, these results indicate that HSP 40 and 90 play important roles in the physiological changes related to metabolism and cell protection that occur in C. riparius larvae that have been exposed to DEHP.

Ecotoxicological multilevel-evaluation of the effects of fenbendazole exposure to Chironomus riparius larvae

Veterinary antibiotics may find their way into the aquatic environment through direct or indirect pathways due to their widespread use. Fenbendazole is a benzimidazole anthelmintic that is widely used in veterinary medicine. To evaluate the potential ecological risk of fenbendazole, we examined the molecular and biochemical responses of biomarker genes such as heat shock proteins (HSPs), cytochrome P450 (CYP450), glutathione S-transferases (GSTs) and hemoglobins (Hbs) in Chironomus riparius for long periods. The expression of HSP70, HSP40, HSP90 and CYP450 in C. riparius increased significantly after exposure to all concentrations of fenbendazole evaluated, while the levels of GST and HbA only increased in C. riparius exposed to relatively high concentrations of fenbendazole (30 microg L(-1)). HbB expression did not differ significantly between the control and treatment groups. Exposure to 30 microg L(-1) fenbendazole had significant effects on the survival, growth, sex balance of emergent adults and development of mouthpart deformity in C. riparius. These results should constitute an important contribution to the understanding of the toxicology of fenbendazole in C. riparius. Moreover, the responses of the biomarker genes also provide valuable information that will aid in understanding the effects of fenbendazole in aquatic ecosystems.

Biological and molecular responses of Chironomus riparius (Diptera, Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10microgL(-1) of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10microgL(-1) of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1microgL(-1) of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10microgL(-1) concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.

Patterning and short-term predictions of benthic macroinvertebrate community dynamics by using a recurrent artificial neural network

Dynamic features of community data were extracted by training with a recurrent artificial neural network. Field data collected monthly from an urbanized stream consisted of densities of selected taxa in benthic macroinvertebrate communities. Sets of time-sequence data for communities were provided as the input for the network. The connectivity of computation nodes was arranged in such a way that the previous community data have recurrent feedback. In concurrence with the input of biological data, corresponding sets of environmental data such as water velocity and depth, sedimented organic matter, and volume of small substrates were also provided for the network. Through the connectivity of the network, environmental data were used as input to produce continuous, independent effects on determining community abundance. A trained pattern effectively represented the effects of habitat types and environmental impact on determining community dynamics. Short-term predictions of changes in the densities of selected taxa were made possible by a trained network after new sets of data were provided to the network.

Characterization of benthic macroinvertebrate communities in a restored stream by using self-organizing map

Abstract The Self-Organizing Map (SOM) was used for revealing the ecological states of streams in recovery through patterning of benthic macroinvertebrate communities. SOM was capable of showing different clusters of the sample sites in a small scale according to changes in environmental variables such as water velocity, depth, substrate roughness and the amount of silt. Community abundance correspondingly varied in different clusters of the sample sites. Within each cluster, data for community abundance were further grouped according to temporal changes in water quality. The patterns of benthic macroinvertebrate communities in the trained SOM were efficient in assessing recovery processes in the polluted sample sites, revealing the effects of river restoration projects in stream ecosystems. The study showed that spatial heterogeneity at the local level plays an important role in characterizing community patterns and consequently biological water quality assessment.

Gene expression of ribosomal protein mRNA in Chironomus riparius: Effects of endocrine disruptor chemicals and antibiotics

Ribosomal protein genes are essential for cellular development. To examine the effects of ribosomal protein genes under various cellular stress conditions in chironomids, ribosomal protein S3 (RpS3) and S6 (RpS6) cDNA from Chironomus riparius were characterized and their expression was analyzed during development. A comparative and phylogenetic study among different orders of insects was carried out by analysis of sequence databases. C. riparius RpS3 was highly conserved at the protein level and shared over 85% amino acid identity with homologous sequences from other insects. RpS6 also showed approximately 80% amino acid identity. The RpS3 and S6 transcripts were present during different developmental stages but were most abundant during the embryonic stage. Furthermore, expression of the previously reported ribosomal proteins RpL11, L13, and L15, as well as RpS3 and S6 was analyzed following exposure to various concentrations of three endocrine disruptor chemicals (EDCs), di(2-ethylhexyl) phthalate, bisphenol A, and 4-nonylphenol (4NP), and the veterinary antibiotics (VAs) fenbendazole, sulfathiazole, and lincomycin. Only RpS3 gene expression was up-regulated significantly in response to EDCs and fenbendazole. However, the C. riparius ribosomal proteins showed a limited response to cellular stress, following exposure to EDCs and VAs.

Expression of Chironomus riparius serine-type endopeptidase gene under di-(2-ethylhexyl)-phthalate (DEHP) exposure

Environmental stressors can induce changes in gene expression that can be useful as biomarkers. To identify potential biomarkers of water quality, we characterized full-length cDNA sequences of the serine-type endopeptidase (SP) gene from Chironomus riparius. Their expression was analyzed during different life-history stages and in response to treatment with various concentrations of di(2-ethylhexyl) phthalate (DEHP) for short and long periods of time. A comparative molecular and phylogenetic investigation was then conducted among different orders of insects using sequence database analysis. The sequence of the C. riparius SP gene was found to be most closely related to the sequence of SPs isolated from Aedes aegypti. In addition, the basal level of C. riparius SP mRNA was more highly expressed in larvae than in other life-history stages. However, the expression of C. riparius SP was primarily limited to the gut in larvae. When the effects of short-term exposure to DEHP were evaluated, C. riparius SP gene expression decreased within 1 h of treatment, regardless of dose. We also investigated expression of the C. riparius SP gene following long-term DEHP exposure (10 days) and found that it decreased significantly across all DEHP dosages. Finally, the response of the SP gene was more sensitive in C. riparius that were exposed to low concentrations of DEHP than in those that were exposed to high concentrations. These results show that suppression of the C. riparius SP gene by DEHP is as a potential biomarker that could be useful for monitoring aquatic quality.

Alcohol dehydrogenase gene expression in Chironomus riparius exposed to di(2-ethylhexyl) phthalate.

Di(2-ethylhexyl) phthalate (DEHP) is an industrial additive that is widely used as a plasticizer. Due to its widespread use, DEHP is often found in freshwater ecosystems and many freshwater species have been exposed to various levels of DEHP in natural aquatic systems. Alcohol dehydrogenase (ADH) is a metabolizing enzyme produced in response to exposure to DEHP. To evaluate the effects of DEHP exposure on the ADH metabolizing process of Chironomus, the full-length cDNA of ADH from Chironomus riparius was determined through molecular cloning and rapid amplification of cDNA ends (RACE). The expression of ADH was then analyzed during different life-cycle developmental stages and under various DEHP concentrations. In addition, a comparative and phylogenetic study among different orders of insects and vertebrates was conducted through analysis of sequence databases. The complete cDNA sequence of the ADH gene was 1134 bp in length. The amino acid sequence of C. riparius ADH was found to have a low degree of homology (around 70%) with other insects available in the databases. ADH mRNA was highly expressed during various developmental stages. ADH gene expression by C. riparius increased significantly after short-term exposure (24 h) to DEHP, regardless of the exposure concentration. ADH gene expression also increased in C. riparius following exposure to DEHP for 7 days. These results suggest that DEHP affects the metabolism associated with ADH in Chironomus species.

Detecting points as developmental delay based on the life-history development and urosome deformity of the harpacticoid copepod, Tigriopus japonicus sensu lato, following exposure to benzo(a)pyrene.

To identify ecotoxicological responses to an endocrine disrupter, benzo(a)pyrene, we examined the life-history of the harpacticoid copepod, Tigriopus japonicus sensu lato. Based on the life-history of copepods, survival rate of nauplii (NSR) and copepodites (CSR), copepodite emergence day (CED) and adult male emergence day (AMED), sex ratio (MER), brooding success rate (BSR), and first brooding day of adult females (FBD) were measured. Significant differences were observed in the survival and development of nauplii (NSR and CED) and sex ratio (MER) of exposed and non-exposed copepods. Moreover, high concentration of BaP can be lethal to copepodite and exhibited a delay of growth. In this study, the CED and AMED among ecotoxicological response based on life-history developments were delayed and the body characteristics decreased in response to exposure to benzo(a)pyrene. The dwarfism and urosome deformity of the T. japonicus s.l. was exhibited in response to chemical exposure. Specifically, the body characteristics and biomass of dwarf copepods that had been exposed to benzo(a)pyrene were 30% and 50% lower than the control group, respectively. The incidence of abnormal urosomes was divided into two types. The first deformity type was signs of shrinkage in the middle of the urosome or the entire urosome was narrower than those of the control organisms. In the second type, the anal somite and the distal side of the urosome had abnormally swelled. Taken together, the nauplii and copepodid development of T. japonicus s.l. can be used as a useful biomaker for detecting developmental delay based on their entire life-history. In addition, the urosome deformity was used a good potential monitoring tool invading various chemicals and environmental contamination into water system.