The yellowtail (Seriola quinqueradiata) genome and transcriptome atlas of the digestive tract.

Seriola quinqueradiata (yellowtail) is the most widely farmed and economically important fish in aquaculture in Japan. In this study, we used the genome of haploid yellowtail fish larvae for de novo assembly of whole-genome sequences, and built a high-quality draft genome for the yellowtail. The total length of the assembled sequences was 627.3 Mb, consisting of 1,394 scaffold sequences (>2 kb) with an N50 length of 1.43 Mb. A total of 27,693 protein-coding genes were predicted for the draft genome, and among these, 25,832 predicted genes (93.3%) were functionally annotated. Given our lack of knowledge of the yellowtail digestive system, and using the annotated draft genome as a reference, we conducted an RNA-Seq analysis of its three digestive organs (stomach, intestine and rectum). The RNA-Seq results highlighted the importance of certain genes in encoding proteolytic enzymes necessary for digestion and absorption in the yellowtail gastrointestinal tract, and this finding will accelerate development of formulated feeds for this species. Since this study offers comprehensive annotation of predicted protein-coding genes, it has potential broad application to our understanding of yellowtail biology and aquaculture.

A novel C-type lectin gene is a strong candidate gene for Benedenia disease resistance in Japanese yellowtail, Seriola quinqueradiata.

Little is known about mechanisms of resistance to parasitic diseases in marine finfish. Benedenia disease is caused by infection by the monogenean parasite Benedenia seriolae. Previous quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2 of the Japanese yellowtail/amberjack Seriola quinqueradiata. To uncover the bioregulatory mechanism of Benedenia disease resistance, complete Illumina sequencing of BAC clones carrying genomic DNA for the QTL region in linkage group Squ2 was performed to reveal a novel C-type lectin in this region. Expression of the mRNA of this C-type lectin was detected in skin tissue parasitized by B. seriolae. Scanning for single nucleotide polymorphisms (SNPs) uncovered a SNP in the C-type lectin/C-type lectin-like domain that was significantly associated with B. seriolae infection levels. These results strongly suggest that the novel C-type lectin gene controls resistance to Benedenia disease in Japanese yellowtails.

Comparison of hatching mode in pelagic and demersal eggs of two closely related species in the order pleuronectiformes.

We compared several characteristics of the pelagic eggs of Verasper variegatus with those of demersal eggs of Pseudopleuronectes yokohamae, both in the order Pleuronectiformes (halibuts or flatfishes). V. variegatus eggs had about twice the diameter of P. yokohamae eggs. However, the total egg protein weight of P. yokohamae was similar to that of V. variegatus. The specific gravity of P. yokohamae eggs was calculated to be 7-fold that of V. variegatus. The difference in size is the main feature distinguishing the two types of egg. The thickness of the egg envelope of P. yokohamae- more than twice that of V. variegatus-must affect the manner of hatching. The amount of hatching enzyme synthesized in pre-hatching embryo was estimated to be larger in P. yokohamae than V. variegatus. The distribution of hatching gland cells differed between the species. In V. variegates embryos, these were located on the yolk sac as a narrow ring-shaped belt, resulting in cleavage of the egg envelope into two parts by digesting a limited region of the egg envelope, called "rim-hatching". The hatching gland cells of P. yokohamae embryos were distributed all over the surface of the yolk sac, forming a hole through which the embryo could escape. Thus, the location of the hatching gland cells in pre-hatching embryos varied during the evolution of the Pleuronectiformes, depending on the egg type and manner of hatching.

Population genetic structure of Scombrops boops (Percoid, Scombropidae) around the Japanese archipelago inferred from the cytochrome b gene sequence in mitochondrial DNA.

The gnomefish (Scombrops boops) is a member of the percoid family Scombropidae, which includes a single genus and three to four species worldwide. Since little is known about the ecology of this species, here, sequencing analysis of the cytochrome b gene (1141 bp) in mitochondrial DNA detected 101 haplotypes from 186 individuals of S. boops collected from waters at seven localities around the Japanese archipelago. A single haplotype (Sb2) was the most abundant in the combined populations of S. boops from various localities. Genetic population structure analyses revealed no significant differences among these populations (Fst = - 0.0313-0.0195; Φst = - 0.0505-0.0615) with high haplotype diversity and low nucleotide diversity. This suggests that S. boops around the Japanese archipelago constitutes a single population, and indicates that the genetic structure of this population may be influenced by larval and egg dispersal in association with warm currents.

Different hatching strategies in embryos of two species, pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, that belong to the same order Clupeiformes, and their environmental adaptation.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis. They were expressed specifically in hatching gland cells (HGCs), which differentiated from the pillow and migrated to the edge of the head in both species. HGCs of Japanese anchovy stopped migration at that place, whereas those of Pacific herring continued to migrate dorsally and distributed widely all over the head region. During evolution, the program for the HGC migration would be varied to adapt to different hatching timing. Analysis of the gene expression revealed that Pacific herring embryos synthesized a large amount of hatching enzyme when compared with Japanese anchovy. Chorion of Pacific herring embryo was about 7.5 times thicker than that of Japanese anchovy embryo. Thus, the difference in their gene expression levels between two species is correlated with the difference in the thickness of chorion. These results suggest that the hatching system of each fish adapted to its respective hatching environment. Finally, hatching enzyme genes were cloned from each genomic DNA. The exon-intron structure of group B genes basically conserved that of the ancestral gene, whereas group A genes lost one intron. Several gene-specific changes of the exon-intron structure owing to nucleotide insertion and/or duplication were found in Japanese anchovy genes.

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene.

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.

Immunomodulatory effects of mono-, di-, and trimethylphenols in mice.

The relationship of air pollutants with the increasing prevalence of allergic diseases is a matter of concern in developed countries. In this study, the immunomodulatory effects of mono-, di-, and trimethylphenols in mice were examined as regards two aspects. First, whether or not these chemicals act as sensitizers was evaluated by local lymph node assay. Of the 13 methylphenols tested, three dimethylphenol isomers (2,4-DMP, 2,5-DMP, and 3,4-DMP) were found to induce auricular lymphocyte proliferation after dermal application on both ears of mice. Cytokine production patterns in the supernatants of cultured auricular lymphocytes from mice showed these methylphenols to be contact sensitizers. Second, the effects of methylphenols on cytokine production profiles were examined using cultured splenocytes from immunologically naive mice. Under subtoxic conditions, eight methylphenols inhibited interferon-gamma (IFN-gamma) production significantly, while the effect on intreluekin-4 (IL-4) production was moderate, resulting in higher IL-4/IFN-gamma ratios in all of the tested chemicals, with the most prominent effect shown by 2,6-DMP. These results suggest that several methylphenols, especially dimethylphenol isomers, have potencies that affect the immune system, being immunogens themselves or modulators of the Th1/Th2 cytokine balance.

Allergenicity and cross-reactivity of naphthenic acid and its metallic salts in experimental animals.

The allergenicity and the cross-reactivity of naphthenic acid (NA) and its metallic salts were evaluated in experimental animals. In the guinea pig maximization test, sensitizing skin reactions were observed with cobalt naphthenate (CoN), zinc naphthenate (ZnN) and NA, but not with copper naphthenate, with CoN being the most potent sensitizer. Animals sensitized with 1 naphthenic compound cross-reacted to the other 3 as well. Furthermore, animals in the CoN-sensitized group reacted to the relevant metallic salt cobalt chloride (CoCl2). A dose-response study using the CoN-sensitized group showed that the concentration of CoCl2 required to elicit a skin reaction of similar extent in comparison with CoN was more than 10 times higher, when skin-reaction scores were compared on the basis of cobalt content. In the local lymph node assay, significant increases in stimulation index values without skin irritation were observed with CoN and ZnN, where the former was more potent than the latter. Although CoN is a reported skin sensitizer, this study showed that skin allergenicity of naphthenic compounds is not restricted solely to CoN. In addition, the results suggest the main antigenic determinant of naphthenic compounds to be the structure of NA, even though metal moieties modulate their allergenicity.

Quantitative comparison of the results obtained by the multiple-dose guinea pig maximization test and the non-radioactive murine local lymph-node assay for various biocides.

We compared the results of the multiple-dose guinea pig maximization test (GPMT) and the non-radioactive murine local lymph-node assay (LLNA) for various biocides. Thirteen out of 17 positive biocides in the GPMT gave positive results in the LLNA. In the GPMT, the minimum first induction doses ranged over four orders (0.00005-0.5%), while elicitation-threshold doses, which were evaluated using an optimally sensitized group of animals in the multiple-dose studies, ranged over five orders (0.00006-2.8%). In the LLNA, minimum induction doses ranged over more than three orders (0.01-30%). With respect to 13 biocides that were positive in both the GPMT and the LLNA, results were quantitatively compared. When compared after conversion to corresponding area doses (microg/cm), the minimum doses required to elicit skin reaction in guinea pigs were always lower than that for induction in mice with all biocides. Correlation between minimum induction doses from the GPMT and the LLNA seemed poor (r=0.57), while that between minimum induction doses in the LLNA and elicitation-threshold doses in the GPMT was relatively good (r=0.73). The results suggest the possibility to estimate human elicitation-threshold doses, which are definitely lacking in the process of risk assessment for skin-sensitizers, from the data of the LLNA.

Allergenicity evaluation of Bioban CS-1135 in experimental animals.

An industrial preservative, Bioban CS-1135, was evaluated for its contact allergenicity by means of multiple-dose guinea-pig maximization test and non-radioactive murine local lymph node assay. In the guinea-pig test, an induction dose of 0.5% Bioban CS-1135 sensitized all animals of the group. The dose-response study of the elicitation phase determined a minimum elicitation dose of 5% for positive skin reactions. In the murine assay, Bioban CS-1135 at doses of 10% and more exerted significant effects on lymphoid cell proliferation. Although the data clearly designated Bioban CS-1135 as a skin sensitizer, its relative potency was ranked lowest among skin-sensitizing biocides previously evaluated in this laboratory.

Allergenicity evaluation of p-chloro-m-cresol and p-chloro-m-xylenol by non-radioactive murine local lymph-node assay and multiple-dose guinea pig maximization test.

p-Chloro-m-cresol (PCMC) and p-chloro-m-xylenol (PCMX) are known to cause allergic contact dermatitis. For risk assessment of skin sensitizers, information on dose-response profiles in the induction and elicitation phases and cross-reactivity with analogous chemicals are important. In the non-radioactive local lymph-node assay (LLNA) using 5-bromo-2'-deoxyuridine instead of 3H-methyl thymidine, significant effect on lymph node cell proliferation was detected at 10% PCMC and 25% PCMX, while in the multiple-dose guinea pig maximization test (GPMT) at least one animal tested in the group was sensitized at a 5 ppm induction dose of either chemical. When mean skin reaction score in an animal group maximally sensitized with each allergen with the GPMT was plotted against log challenge concentration, linear regression lines with high correlations were obtained in both cases. The calculated elicitation threshold was lower for PCMC than PCMX. The area under the linear regression line between the threshold point and 1% of the elicitation concentration, another index of relative elicitation potency, was also greater for PCMC. Bidirectional cross-reactivity between PCMX and PCMC was detected in the GPMT. PCMC was thus identified in both LLNA and GPMT as a stronger sensitizer than PCMX in both the induction and elicitation phases. These results suggest that the non-radioactive LLNA is a simple and useful method for evaluating allergenicity in the induction phase, while the GPMT using a maximally sensitized animal group is more suitable for assessing the dose-response profile and cross-reactivity in the elicitation phase.

Simultaneous determination of metabolites of trimethylbenzenes, dimethylbenzylmercapturicacid and dimethylhippuric acid, in human urine by solid-phase extraction followed by liquid chromatography tandem mass spectrometry.

We describe a novel method for the determination of three kinds of dimethylbenzylmercapturic acids (DMM) and six kinds of dimethylhippuric acids (DMH), found in urine as metabolites of trimethylbenzenes, based on liquid chromatography/electrospray ionization tandem mass spectrometry. A solid-phase extraction procedure was used for the extractions of DMM and DMH from a urine sample, and the separation was performed on a reversed-phase C(30) column. The analytes were ionized by electrospray in the positive-ion mode. Operating in the multiple reaction monitoring mode, the linearity of the relative mass spectrometric responses to the internal standard versus analyte concentrations were established in the range 0.1-100 ng ml(-1). The extraction procedure was rapid and the relative standard deviations were below 5%. The detection limits of DMM and DMH in the urine by the proposed method were in the ranges 0.26-0.41 and 0.42-2.0 ng l(-1), respectively. Furthermore, DMM and DMH were detected in a urine sample from an individual who did not suffer from occupational exposure to trimethylbenzenes, by using this method.