Molecular biology and functional genomics of immune-endocrine interactions in the Japanese eel, Anguilla japonica.

Immune-endocrine interactions are an important pathogen resistance mechanism in fish. We review the immune-endocrine interactions in the Japanese eel, Anguilla japonica, with special reference to high throughput gene sequencing. These data may be relevant to the significant decrease in the eel harvest in recent years and will aid in the selection of appropriate disease-resistant strains for aquaculture. More than 1000 sequences that whose expression in elvers responded to air exposure were identified through comprehensive gene expression analysis using next-generation sequencing. These included transcription factors within the MAPK pathway. Significant changes in expression after air exposure were detected by quantitative polymerase chain reaction analysis in many genes related to disease resistance. These factors include innate immune system factors and cytokines that interact with the endocrine system during the stress response. Other applications of immune-endocrine interactions in eel culture are discussed.

Transcriptomic features associated with energy production in the muscles of Pacific bluefin tuna and Pacific cod.

Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication.

BACKGROUND: Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. RESULTS: We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. CONCLUSIONS: The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.

Rainbow trout hspb1 (hsp27): identification of two mRNA splice variants that show predominant expression in muscle tissues.

Here, I investigate features of the heat-shock protein beta1 (HspB1) that may be unique to cold-water fish. cDNAs encoding HspB1 were cloned from the rainbow trout Oncorhynchus mykiss, and two splice variants, Hspb1_tv1 and Hspb1_tv2, were identified. The C-terminus of the deduced proteins had a polyglutamic acid (polyE) stretch that is not found in other vertebrate HspB1s. In fish exposed to a continuous heat shock, the mRNA level of Hspb1_tv1 increased whereas that of b1_tv2 decreased. Northern blot and RT-PCR analyses showed that under normal physiological conditions Hspb1_tv1 mRNA is predominantly expressed in muscle tissues, although it is present in all organs. In contrast, Hspb1_tv2 mRNA is selectively expressed in muscle tissues, particularly in the heart. Distinctive features of rainbow trout Hspb1, such as having two splice variants and a polyE stretch, may contribute to the function of the protein under the typical low-temperature habitat of cold-water fish.

Rhodopsin gene copies in Japanese eel originated in a teleost-specific genome duplication.

BACKGROUND: Gene duplication is considered important to increasing the genetic diversity in animals. In fish, visual pigment genes are often independently duplicated, and the evolutionary significance of such duplications has long been of interest. Eels have two rhodopsin genes (rho), one of which (freshwater type, fw-rho) functions in freshwater and the other (deep-sea type, ds-rho) in marine environments. Hence, switching of rho expression in retinal cells is tightly linked with eels' unique life cycle, in which they migrate from rivers or lakes to the sea. These rho genes are apparently paralogous, but the timing of their duplication is unclear due to the deep-branching phylogeny. The aim of the present study is to elucidate the evolutionary origin of the two rho copies in eels using comparative genomics methods. RESULTS: In the present study, we sequenced the genome of Japanese eel Anguilla japonica and reconstructed two regions containing rho by de novo assembly. We found a single corresponding region in a non-teleostean primitive ray-finned fish (spotted gar) and two regions in a primitive teleost (Asian arowana). The order of ds-rho and the neighboring genes was highly conserved among the three species. With respect to fw-rho, which was lost in Asian arowana, the neighboring genes were also syntenic between Japanese eel and Asian arowana. In particular, the pattern of gene losses in ds-rho and fw-rho regions was the same as that in Asian arowana, and no discrepancy was found in any of the teleost genomes examined. Phylogenetic analysis supports mutual monophyly of these two teleostean synteny groups, which correspond to the ds-rho and fw-rho regions. CONCLUSIONS: Syntenic and phylogenetic analyses suggest that the duplication of rhodopsin gene in Japanese eel predated the divergence of eel (Elopomorpha) and arowana (Osteoglossomorpha). Thus, based on the principle of parsimony, it is most likely that the rhodopsin paralogs were generated through a whole genome duplication in the ancestor of teleosts, and have remained till the present in eels with distinct functional roles. Our result indicates, for the first time, that teleost-specific genome duplication may have contributed to a gene innovation involved in eel-specific migratory life cycle.

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress.

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.

Cold-inducible expression of the cell division cycle gene CDC48 and its promotion of cell proliferation during cold acclimation in zebrafish cells.

A member of the ATPases associated with diverse cellular activities (AAA) family, the cell division cycle gene CDC48/VCP (valosin-containing protein)/p97, was cloned from zebrafish and found to be a major cold-inducible protein in fish cells. CDC48 mRNA levels increased significantly after reducing the temperature from 30 to 15 degrees C for 25 days. CDC48 protein levels also increased 2.5-fold after 30 days at cold temperatures. When fish cells overexpressing CDC48 were exposed to a temperature of 15 degrees C, cell proliferation was markedly enhanced in comparison with control cells. By contrast, expression of a mutant molecule with a tyrosine-805 to alanine substitution at the C-terminal phosphorylation site inhibited cell proliferation and induced apoptosis at low temperatures. Therefore, CDC48 may promote cell cycling and cell proliferation via C-terminal tyrosine phosphorylation during cold acclimation in fish cells.

The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis).

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.

Isolation and characterization of a genomic fosmid clone containing hspb1 (hsp27) from the Pacific bluefin tuna Thunnus orientalis.

We constructed a fosmid library of genomic DNA from the Pacific bluefin tuna Thunnus orientalis and isolated a clone containing the complete gene hspb1 (hsp27) from the library. To screen the library, we first obtained a full-length hspb1 cDNA by RACE-PCR. This cDNA encodes a putative protein of 202 amino acids. Phylogenetic analysis indicated that the protein belongs to the Hsp27 family. Northern blot analysis demonstrated that the gene transcript was expressed in tuna muscle tissues. Using a combination of PCR screening and colony hybridization, we isolated a 39,422-bp fosmid clone containing hspb1 from an arrayed library. Sequence alignment showed that hspb1 contains three exons and two introns. Comparative genomic analysis revealed that hspb1 lies in a region of conserved synteny in the genomes of fish and human. Our finding of conserved regions within and around hspb1 in fish species will help to identify functional elements such as promoter regions.

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells.

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.

Cloning and characterization of two distinct isoforms of rainbow trout heat shock factor 1. Evidence for heterotrimer formation.

To elucidate the molecular mechanism underlying the heat shock response in cold-water fish species, genes encoding heat shock transcription factors (HSFs) were cloned from RTG-2 cells of the rainbow trout Oncorhynchus mykiss. Consequently, two distinct HSF1 genes, named HSF1a and HSF1b, were identified. The predicted amino acid sequence of HSF1a shows 86.4% identity to that of HSF1b. The two proteins contained the general structural motifs of HSF1, i.e. a DNA-binding domain, hydrophobic heptad repeats and nuclear localization signals. Southern blot analysis showed that each HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were coexpressed in unstressed rainbow trout RTG-2 cells and in various tissues. In an electrophoretic mobility shift assay, each in vitro translated HSF1 bound to the heat shock element. Chemical cross-linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers as well as homotrimers. Taken together, these results demonstrate that in rainbow trout cells there are two distinct HSF1 isoforms that can form heterotrimers, suggesting that a unique molecular mechanism underlies the stress response in tetraploid and/or cold-water fish species.