iTRAQ-based proteomic profiling, pathway analyses, and apoptotic mechanism in the Antarctic copepod Tigriopus kingsejongensis in response to ultraviolet B radiation.

iTRAQ proteomic profiling was conducted to examine the proteomic responses of the Antarctic copepod Tigriopus kingsejongensis under ultraviolet B (UVB) exposure. Of the 5507 proteins identified, 3479 proteins were annotated and classified into 25 groups using clusters of orthologous genes analysis. After exposing the T. kingsejongensis to 12 kJ/m2 UVB radiation, 77 biological processes were modulated over different time periods (0, 6, 12, 24, and 48 h) compared with the control. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that UVB exposure in T. kingsejongensis downregulated ribosome and glyoxylate and dicarboxylate metabolism at all time points. Furthermore, antioxidant and chaperone proteins were highly downregulated in response to UVB exposure, causing protein damage and activating apoptotic processes in the 48 h UVB exposure group. These proteomic changes show the mechanisms that underlie the detrimental effects of UVB on the cellular defense systems of the Antarctic copepod T. kingsejongensis.

An improved genome assembly and annotation of the Antarctic copepod Tigriopus kingsejongensis and comparison of fatty acid metabolism between T. kingsejongensis and the temperate copepod T. japonicus.

Copepods in the genus Tigriopus are widely distributed in the intertidal zone worldwide. To assess differences in fatty acid (FA) metabolism among congeneric species in this genus inhabiting polar and temperate environments, we analyzed and compared FA profiles of the Antarctic copepod Tigriopus kingsejongensis and the temperate copepod T. japonicus. Higher amounts of total FAs were found in the Antarctic copepod T. kingsejongensis than the temperate copepod T. japonicus under administration of the identical amount of Tetraselmis suecica. To determine the genomic basis for this, we identified fatty acid metabolism-related genes in an improved genome of T. kingsejongensis. The total length of the assembled genome was approximately 338 Mb with N50 = 1.473 Mb, 938 scaffolds, and a complete Benchmarking Universal Single-Copy Orthologs value of 95.8%. A total of 25,470 genes were annotated using newly established pipeline. We identified eight elongation of very long-chain fatty acid protein (Elovl) genes and nine fatty acid desaturase (Fad) genes in the genome of T. kingsejongensis. In addition, fatty acid profiling suggested that the duplicated Δ5/6 desaturase gene in T. kingsejongensis is likely to play an essential role in synthesis of different FAs in T. kingsejongensis to those in T. japonicus. However, further experimental research is required to validate our in silico findings. This study provides a better understanding of fatty acid metabolism in the Antarctic copepod T. kingsejongensis.

Effects of water accommodated fractions (WAFs) of crude oil in two congeneric copepods Tigriopus sp.

Oil pollution has deleterious effects on marine ecosystems. However, the toxicity of crude oil towards Antarctic marine organisms has not been well studied. We compared the deleterious effects of water accommodated fractions (WAFs) of crude oil on reproduction, intracellular reactive oxygen species (ROS) levels, and antioxidant enzymatic activity in Antarctic (Tigriopus kingsejongensis) and temperate (Tigriopus japonicus) copepods. Reproductive rates of T. kingsejongensis and T. japonicus were significantly reduced (P < 0.05) in response to WAFs. Furthermore, T. kingsejongensis showed elevated levels of ROS and higher antioxidant enzyme (glutathione peroxidase [GPx]) activity than T. japonicus in response to WAFs. CYP genes from congeneric copepods were identified and annotated to better understand molecular detoxification mechanisms. We observed significant up-regulation (P < 0.05) of Tk-CYP3024A3 and Tj-CYP3024A2 in response to WAFs, suggesting that CYP genes may contribute to the detoxification mechanism in response to WAF exposure. These finding also suggest that WAFs may induce oxidative stress, leading to reproductive impairment in copepods. Furthermore, Tk-CYP3024A3 and Tj-CYP3024A2 genes can be considered as potential biomarkers of WAF toxicity in the congeneric copepods T. kingsejongensis and T. japonicus. This study will be helpful for enhancing our knowledge on the harmful effects of WAFs in Antarctic and temperate copepods and provides insight into the underlying molecular mechanisms.

Characterization of Stearoyl-CoA Desaturases from a Psychrophilic Antarctic Copepod, Tigriopus kingsejongensis.

Stearoyl-CoA desaturase is a key regulator in fatty acid metabolism that catalyzes the desaturation of stearic acid to oleic acid and controls the intracellular levels of monounsaturated fatty acids (MUFAs). Two stearoyl-CoA desaturases (SCD, Δ9 desaturases) genes were identified in an Antarctic copepod, Tigriopus kingsejongensis, that was collected in a tidal pool near the King Sejong Station, King George Island, Antarctica. Full-length complementary DNA (cDNA) sequences of two T. kingsejongensis SCDs (TkSCDs) were obtained from next-generation sequencing and isolated by reverse transcription PCR. DNA sequence lengths of the open reading frames of TkSCD-1 and TkSCD-2 were determined to be 1110 and 681 bp, respectively. The molecular weights deduced from the corresponding genes were estimated to be 43.1 kDa (TkSCD-1) and 26.1 kDa (TkSCD-2). The amino acid sequences were compared with those of fatty acid desaturases and sterol desaturases from various organisms and used to analyze the relationships among TkSCDs. As assessed by heterologous expression of recombinant proteins in Escherichia coli, the enzymatic functions of both stearoyl-CoA desaturases revealed that the amount of C16:1 and C18:1 fatty acids increased by greater than 3-fold after induction with isopropyl ß-D-thiogalactopyranoside. In particular, C18:1 fatty acid production increased greater than 10-fold in E. coli expressing TkSCD-1 and TkSCD-2. The results of this study suggest that both SCD genes from an Antarctic marine copepod encode a functional desaturase that is capable of increasing the amounts of palmitoleic acid and oleic acid in a prokaryotic expression system.

De novo assembly and annotation of the Antarctic copepod (Tigriopus kingsejongensis) transcriptome.

The whole transcriptome of the Antarctic copepod (Tigriopus kingsejongensis) was sequenced using Illumina RNA-seq. De novo assembly was performed with 64,785,098 raw reads using Trinity, which assembled into 81,653 contigs. TransDecoder found 38,250 candidate coding contigs which showed homology to other species by BLAST analysis. Functional gene annotation was performed by Gene Ontology (GO), InterProScan, and KEGG pathway analyses. Finally, we identified a number of expressed gene catalog for T. kingsejongensis that is a useful model animal for gene information-based polar research to uncover molecular mechanisms of environmental adaptation on harsh environments. In particular, we observed highly developing lipid metabolism in T. kingsejongensis directly compared to those of the Far East Pacific coast copepod Tigriopus japonicus at the transcriptome level.