An evolutionarily ancient mechanism for regulation of hemoglobin expression in vertebrate red cells.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.

A Novel Virus Causes Scale Drop Disease in Lates calcarifer.

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Differential transcriptomic response in the spleen and head kidney following vaccination and infection of Asian seabass with Streptococcus iniae.

Vaccination is an important strategy in the protection of aquaculture species from major diseases. However, we still do not have a good understanding of the mechanisms underlying vaccine-induced disease resistance. This is further complicated by the presence of several lymphoid organs that play different roles when mounting an immune response. In this study, we attempt to elucidate some of these mechanisms using a microarray-based approach. Asian seabass (Lates calcarifer) were vaccinated against Streptococcus iniae and the transcriptomic changes within the spleen and head kidney at one and seven days post-vaccination were profiled. We subsequently challenged the seabass at three weeks post-vaccination with live S. iniae and similarly profiled the transcriptomes of the two organs after the challenge. We found that vaccination induced an early, but transient transcriptomic change in the spleens and a delayed response in the head kidneys, which became more similar to one another compared to un-vaccinated ones. When challenged with the pathogen, the spleen, but not the head kidneys, responded transcriptomically at 25-29 hours post-challenge. A unique set of genes, in particular those involved in the activation of NF-κB signaling, was up-regulated in the vaccinated spleens upon pathogen challenge but not in the un-vaccinated spleens. A semi-quantitative PCR detection of S. iniae using metagenomic DNA extracted from the water containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. This result indicated that vaccination not only led to a successful immune defense against the infection, but also reduced the chances for horizontal transmission of the pathogen. In conclusion, we have provided a transcriptomic analysis of how the teleost spleen and head kidneys responded to vaccination and subsequent infection. The different responses from the two organs are suggestive of their unique roles in establishing a vaccine-induced disease resistance.

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, Paralichthys olivaceus.

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.

Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami.

Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.