Identification and characterization of eleven glutathione S-transferase genes from the aquatic midge Chironomus tentans (Diptera: Chironomidae)

Eleven cDNAs encoding glutathione S-transferases (GSTs) were sequenced and characterized in Chironomus tentans, an ecologically important aquatic midge. Phylogenetic analysis revealed seven GSTs in three different cytosolic classes including 4 in sigma (CtGSTs1, CtGSTs2, CtGSTs3, CtGSTs4), 2 in delta (CtGSTd1, CtGSTd2), and 1 in omega (CtGSTo1). The remaining four GSTs (CtGSTu1, CtGSTu2, CtGSTu3, CtGSTu4) were unclassified due to their low relatedness to currently known classes of insect GSTs. Reverse-transcription (RT)-PCR analysis of the 11 GST genes showed that CtGSTd1, CtGSTu2, CtGSTu4, CtGSTs1, CtGSTs2, CtGSTs3, CtGSTs4 and CtGSTo1 were expressed in all tissues examined, including salivary glands, hemolymph, midgut, Malpighian tubules, fatbodies and carcass, whereas CtGSTd2 and CtGSTu1 were expressed in a limited number of tissues. CtGSTs1 and CtGSTs4 appeared to be the only two genes, of which expressions can be detected in eggs, whereas all the 11 GST genes showed various expression patterns in the four larval instars. However, expressions of CtGSTd2, CtGSTu1 and CtGSTu2 were not detectable in pupal and adult stages. Real-time quantitative PCR confirmed that the herbicide alachlor increased CtGSTd1, CtGSTs2 and CtGSTs3 gene expression by 2.1-, 2.8- and 4.3-fold, respectively, when fourth-instar midges were exposed to alachlor at 1000 μg/L for 72 h. Such increased gene expressions were associated with 2.2- and 1.8-fold decreases of total GST activities in vivo when CDNB and DCNB were used as substrates, respectively. Further studies showed that 65.5 and 73.5% of GST activities were inhibited in vitro by alachlor at 100 and 1000 μg/L, respectively. Because alachlor has been known as an electrophilic substrate that can be conjugated by glutathione (GSH), rapid in vitro inhibition of GST activities by alachlor suggested that decreased GST activities were likely caused by the depletion of GSH. However, alachlor may regulate different GST genes, as found in other organisms, leading to significantly increased transcriptional levels of CtGSTd1, CtGSTs2 and CtGSTs3 in out of 11 GST genes examined in this study.