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 LC₅₀ of 788.98μgL^-1 and 54.68μgL^-1 for 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.

Keywords: Copepod; Full-length cDNA; Gene expression; Polycyclic aromatic hydrocarbons (PAHs); Pseudodiaptomus poplesia; Spliced leader.