title: Spliced leader-based analyses reveal the effects of polycyclic aromatic hydrocarbons on gene expression in the copepod Pseudodiaptomus poplesia.
abstract: Polycyclic aromatic hydrocarbons (PAHs) are a group of toxic and carcinogenic pollutants that can adversely affect the development, growth and reproduction of marine organisms including copepods. However, knowledge on the molecular mechanisms regulating the response to PAH exposure in marine planktonic copepods is limited. In this study, we investigated the survival and gene expression of the calanoid copepod Pseudodiaptomus poplesia upon exposure to two PAHs, 1, 2-dimethylnaphthalene (1, 2-NAPH) and pyrene. Acute toxicity responses resulted in 96-h LC50of 788.98μgL-1and 54.68μgL-1for 1, 2-NAPH and pyrene, respectively. Using the recently discovered copepod spliced leader as a primer, we constructed full-length cDNA libraries from copepods exposed to sublethal concentrations and revealed 289 unique genes of diverse functions, including stress response genes and novel genes previously undocumented for this species. Eighty-three gene families were specifically expressed in PAH exposure libraries. We further analyzed the expression of seven target genes by reverse transcription-quantitative PCR in a time-course test with three sublethal concentrations. These target genes have primary roles in detoxification, oxidative defense, and signal transduction, and include different forms of glutathione S-transferase (GST), glutathione peroxidases (GPX), peroxiredoxin (PRDX), methylmalonate-semialdehyde dehydrogenase (MSDH) and ras-related C3 botulinum toxin substrate (RAC1). Expression stability of seven candidate reference genes were evaluated and the two most stable ones (RPL15 and RPS20 for 1, 2-NAPH exposure, RPL15 and EF1D for pyrene exposure) were used to normalize the expression levels of the target genes. Significant upregulation was detected in GST-T, GST-DE, GPX4, PRDX6 and RAC1 upon 1, 2-NAPH exposure, and GST-DE and MSDH upon pyrene exposure. These results indicated that the oxidative stress was induced and that signal transduction might be affected by PAH exposure in P. poplesia. However, gene upregulation was followed by a reduction in expression level towards 96h, indicating a threshold value of exposure time that leads to depressed gene expression. Prolonged exposure may cause dysfunction of detoxification and antioxidant machinery in P. poplesia. The transcriptional responses of GST-T, GPX2 and GPX4 upon pyrene exposure were minimal. Our results reveal the different sensitivity of P. poplesia to two PAHs at both the individual and transcriptional levels. As the first attempt, this study proved that copepod spliced leader is useful for obtaining full-length cDNA in P. poplesia exposed to PAHs and provided a valuable gene resource for this non-model species. This approach can be applied to other calanoid copepods exposed to various stressors, particularly under field conditions.
authors.affiliation: Key Laboratory of Marine Environment and Ecology, Ministry of Education,Ocean University of China, Qingdao 266100, China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Zhuang
authors.affiliation: Key Laboratory of Marine Environment and Ecology, Ministry of Education,Ocean University of China, Qingdao 266100, China; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Yang
authors.affiliation: Key Laboratory of Marine Environment and Ecology, Ministry of Education,Ocean University of China, Qingdao 266100, China.
authors.forename: Donghui
authors.initials: D
authors.lastname: Xu
authors.affiliation: Key Laboratory of Marine Environment and Ecology, Ministry of Education,Ocean University of China, Qingdao 266100, China.
authors.forename: Hongju
authors.initials: H
authors.lastname: Chen
authors.affiliation: Key Laboratory of Marine Environment and Ecology, Ministry of Education,Ocean University of China, Qingdao 266100, China; Department of Marine Sciences, University of Connecticut, Groton, CT 06340, USA. Electronic address: huan.zhang@uconn.edu.
authors.forename: Huan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Marine Environment and Ecology, Ministry of Education,Ocean University of China, Qingdao 266100, China. Electronic address: gxliu@ouc.edu.cn.
authors.forename: Guangxing
authors.initials: G
authors.lastname: Liu
mesh_terms: D000818:Animals; D033342:Copepoda / Q000187:drug effects* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D015723:Gene Library; D007928:Lethal Dose 50; D009281:Naphthalenes / Q000633:toxicity*; D011721:Pyrenes / Q000633:toxicity*; D020038:RNA, Spliced Leader / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D018076:DNA, Complementary; D009281:Naphthalenes; D011721:Pyrenes; D020038:RNA, Spliced Leader; D014874:Water Pollutants, Chemical; C117619:1,2-dimethylnaphthalene; C030984:pyrene
keywords: Copepod; Full-length cDNA; Gene expression; Polycyclic aromatic hydrocarbons (PAHs); Pseudodiaptomus poplesia; Spliced leader