SUMMARY Copepods play an important role in the aquatic food web in marine and fresh water environments. Among the various species in copepods, Apocyclops royi and Acartia bilobata are very good live feed for fish larvae because of their small size and rich in long chain unsaturated fatty acid. In this study, we used the NGS approach to study the transcriptome of A. royi and A. bilobata, the codon usage bias was also analyzed, and another 3 copepods were selected (Calanus rogercresseyi, Lepeophtheirus salmonis and Tigriopus japonicus) to reconstruct the phylogeny of these species with outgroup of Daphnia pulex. Base on the results, we suggested that 1) A. royi may be able to synthesize DHA and EPA by using linoleic acid (C18:3 n-3) through fatty acid synthesizing pathway; 2) A. bilobata showed a higher expression of Na+/K+-ATPase, H+-ATPase and ATP synthase in diluted seawater; 3) In the codon usage bias pattern, genes of A. bilobata were biased, whereas A. royi was less biased, indicating that A. bilobata might be under compositional translational selection; 4) The estimated coalescence time for the five copepod species was about 454.3-457.4 myr (million years). Our data provided much important information for future copepod study. INTRODUCTION Copepods (Arthropoda: Crustacea) are among the most abundant metazoans in the oceans. They are a very ancient group and are considered to have diverged from other arthropod taxa about 388-522 million years ago. Ten orders comprise the subclass Copepoda, including Calanoida, Cyclopoida, and Harpacticoida, and a total of about 13,000 species are recognized. Copepods play an important role in the aquatic food web in marine and fresh water environments. They are also the best live feed for early stage of fish larvae because of their small size and rich in long chain unsaturated fatty acid, such as DHA and EPA. However, genome resources for copepods are only available for very few species. The information on genomic DNA sequences is increasingly important, especially for non-model organisms such as copepods. Recently, next generation sequencing (NGS) technology enables us to obtain extensive DNA or cDNA sequence information in a short period of time. In this study, we used an NGS approach to study the transcriptome for Apocyclops royi and Acartia bilobata, the codon usage bias was also analyzed, and another 3 copepods were selected (Calanus rogercresseyi, Lepeophtheirus salmonis and Tigriopus japonicus) to reconstruct the phylogeny of these species with outgroup of Daphnia pulex. MATERIALS AND METHODS A. royi and A. bilobata were collected from Tungkang Biotechnology Research Center of Fisheries Research Institute, Taiwan. The experiment was started with salinity acclimation of 20 psu and 30 psu for A. royi and A. bilobata, respectively, and transferred to 15 psu for 5 days. mRNAs were isolated from total RNA using oligo-dT and then converted to double-stranded cDNA. The samples were sequenced using Illumina HiSeqTM 2000. De-novo assembly of transcriptome was carried out using software Trinity. The GO, COG and KEGG annotations for the two copepod species were performed using the automatic annotation tool Blast2GO. The expression level of a transcript was quantified in read per kilobase of the transcript per million mapped reads (RPKM). We analyzed the codon usage bias in A. royi and A. bilobata, the frequency of 59 codons code for 18 amino acids (exclude Met, Trp and termination codons) were determined for all the selected genes. Five codon usage indices, relative synonymous codon usage (RSCU), G+C content at the third position of synonymous codons (GC3s), effective number of codons (ENc), correspondence analysis (COA), and optimal codons, were used to analyze the codon usage in this study. The sequences of A. royi and A. bilobata from annotated unigenes, as well as the sequences of C. rogercresseyi, L. salmonis, T. japonicus and D. pulex (outgroup) obtained from NCBI, were used in reconstructing phylogenetic trees with neighbor-joining method and maximum likelihood method in software MEGA 5.2. Maximum likelihood bootstrap analyses were conducted with 1000 replicates, while the values greater than 70% were shown on trees. The time to the most recent common ancestor (TMRCA) under an MCMC Bayesian approach was estimated with the BEAST 1.7.5. All simulations were performed using the HKY+G model and strict molecular clock with Fixed Rate Model. RESULTS AND DISCUSSION In this study, we obtained 53,363,934 and 55,433,040 NGS reads for A. royi and A. bilobata, respectively, generating 35,363 unigenes (mean size: 941 bp, N50 size: 1,547 bp) and 35,442 unigenes (mean size: 878 bp, N50 size: 1,600 bp) by de novo assembly. By mapping to databases, 20,062 and 17,214 unigenes could be annotated to NCBI Nr, 15,017 and 13,771 unigenes hadKEGG annotations, 8,317 and 8,219 had COG annotations, 4,064 and 3,327 had GO annotations. Some related enzymes involving in synthesizing long chain polyunsaturated fatty acids were identified, revealing that A. royi may be able to synthesize DHA and EPA through fatty acid synthesizing pathway. Since salinity is an important environmental factor for crustacean survival, we further analyzed the genes (Na+/K+-ATPase, H+-ATPase and ATP synthase) related to osmoregulation and found significant differences in gene expression between the two species. A. bilobata showed a higher expression of Na+/K+-ATPase, H+-ATPase and ATP synthase in diluted seawater. Analysis of codon usage bias indicated some biases occurred at the genes of A. bilobata, whereas genes of A. royi was less biased, suggesting a complex pattern in codon usage in copepods. The result implied that A. bilobata was under compositional translational selection. The genealogical analysis of 31 orthologous genes displayed 20 tree topologies, 14 trees of which showed that C. rogercressyi and L. salmonis were most closely related. Furthermore, the estimated coalescence time for the five species was about 454.3-457.4 myr (million years) ago. CONCLUSION The A. royi and A. bilobata transcriptome analyzed using the next generation sequencing technique enhance our knowledge on the copepods genes, which will facilitate our understanding of the crustacean genome, and advances the studies on copepod.
|Contributors||Tzen-Yuh Chiang, 蔣鎮宇|
|Source Sets||National Digital Library of Theses and Dissertations in Taiwan|
|Type||學位論文 ; thesis|
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