Transcriptome Analysis Reveals the Immunosuppression in Tiger Pufferfish (Takifugu rubripes) under Cryptocaryon irritans Infection.

The tiger pufferfish (Takifugu rubripes), also known as fugu, has recently suffered from severe C. irritans infections under aquaculture environment, yet the underlying immune mechanisms against the parasite remain poorly understood. In this study, we conducted a comprehensive transcriptome analysis of the gill tissue from infected and uninfected fish using PacBio long-read (one pooled sample each for seriously infected and healthy individuals, respectively) and Illumina short-read (three pools for mildly infected, seriously infected, and healthy individuals, respectively) RNA sequencing technologies. After aligning sequence data to fugu's reference genome, 47,307 and 34,413 known full-length transcripts were identified and profiled in healthy and infected fish, respectively. Similarly, we identified and profiled 1126 and 803 novel genes that were obtained from healthy and infected fish, respectively. Interestingly, we found a decrease in the number of alternative splicing (AS) events and long non-coding RNAs (lncRNAs) after infection with C. irritans, suggesting that they may be involved in the regulation of the immune response in fugu. There were 687 and 1535 differentially expressed genes (DEGs) in moderately and heavily infected fish, respectively, compared to uninfected fish. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that immune-related DEGs in the two comparison groups were mainly enriched in cytokine-cytokine receptor interactions, ECM-receptor interactions, T-cell receptor signaling pathways, Th1 and Th2 cell differentiation, and Th17 cell differentiation pathways. Further analysis revealed that a large number of immune-related genes were downregulated in infected fish relative to uninfected ones, such as CCR7, IL7R, TNFRSF21, CD4, COL2A1, FOXP3B, and ITGA8. Our study suggests that C. irritans is potentially a highly efficient parasite that may disrupt the defense mechanisms of fugu against it. In addition, in combination of short-read RNA sequencing and previous genome-wide association analyses, we identified five key genes (NDUFB6, PRELID1, SMOX, SLC25A4, and DENND1B) that might be closely associated with C. irritans resistance. This study not only provides valuable resources of novel genic transcripts for further research, but also provides new insights into the immune mechanisms underlying C. irritans infection response in farmed fugu.

Comparison of specificity and sensitivity of diagnostic methods for Enteromyxum leei and Enteromyxum fugu detected from cultured tiger puffer, Takifugu rubripes in Korea.

Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real-time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real-time and two-step PCR for early diagnosis and follow-up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one-step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.

Parasites of pufferfish, Lagocephalus spp. and Torquigener flavimaculosus of the Israeli Mediterranean: A new case of Lessepsian endoparasites.

With the opening of the Suez Canal as a link between the Red Sea and the Mediterranean Sea in 1869, the biogeographical event of the Lessepsian migration has been starting. Aided by beneficial conditions in the new habitat, almost 500 marine species have immigrated and often established themselves in the Mediterranean Sea, including several pufferfish species, with all of them extending their range and becoming important components of the local fauna. The parasitic fauna of these pufferfish has scarcely been examined in the Mediterranean Sea or in their native range, which provides the opportunity to study host-parasite interaction in a new habitat. The present study describes the parasitic fauna in four alien invasive pufferfish species (Lagocephalus guentheri, L. sceleratus, L. suezensis, and Torquigener flavimaculosus) of various sizes and ages on the Israeli Mediterranean coast. The parasite fauna of these species was diverse (Maculifer dayawanensis Digenea; Calliterarhynchus gracilis, Nybelinia africana and Tetraphyllidea larvae Cestoda; Hysterothylacium reliquens, Hysterothylacium sp. and Raphidascaris sp. Nematoda; Trachellobdella lubrica Hirudinea and Caligus fugu and Taeniacanthus lagocephali Copepoda) and consisted of mostly generalist species, most likely acquired in the new habitat, and specialist copepod ectoparasites, having co-invaded with the pufferfish. Additionally, the oioxenic opecoelid digenean Maculifer dayawanensis was found in two pufferfish species. The genus was previously only known from the Indo-Pacific Ocean, representing the eighth reported case of a Lessepsian endoparasite so far. Our results suggest a change in parasite fauna to native Mediterranean species in the pufferfish like previously reported in other Lessepsian migrant predatory fish species and a wider spread of co-invasion of fish endoparasites to the Mediterranean Sea than previously assumed. The study also provides several new host records and the first report for parasites in T. flavimaculosus.

Accumulation of microplastics in fugu (Takifugu bimaculatus): A comparative study between fishing grounds and aquafarms.

Microplastics (MPs) in fish have attracted attention recently, for their ecological and food safety risks. However, knowledge gaps still exist regarding MPs in fugu, a special poisonous but precious seafood, especially that accumulated in its tissues. Accordingly, this study investigated the characteristics of MPs in cultured Takifugu bimaculatus which raised on three aquafarms and in wild individuals from three fishing grounds. More than 98.85 % of T. bimaculatus were contaminated by MPs and the average MPs abundance in wild fugu (4.25 ± 2.63 items/individual) was lower than that of cultured fugu (7.91 ± 2.16 items/individual). The abundance of MPs in fugu's tissues under different life patterns shows significant differences. There were marked differences in size of MPs presented in various tissues. This study adds to the knowledge on MPs accumulation in the tissues of wild and cultured fugu, providing warnings about its transmission and ecological risks in the food chain.

Tissue-targeted inorganic pyrophosphate hydrolysis in a fugu5 mutant reveals that excess inorganic pyrophosphate triggers developmental defects in a cell-autonomous manner.

Excess PPi triggers developmental defects in a cell-autonomous manner. The level of inorganic pyrophosphate (PPi) must be tightly regulated in all kingdoms for the proper execution of cellular functions. In plants, the vacuolar proton pyrophosphatase (H+-PPase) has a pivotal role in PPi homeostasis. We previously demonstrated that the excess cytosolic PPi in the H+-PPase loss-of-function fugu5 mutant inhibits gluconeogenesis from seed storage lipids, arrests cell division in cotyledonary palisade tissue, and triggers a compensated cell enlargement (CCE). Moreover, PPi alters pavement cell (PC) shape, stomatal patterning, and functioning, supporting specific yet broad inhibitory effects of PPi on leaf morphogenesis. Whereas these developmental defects were totally rescued by the expression of the yeast soluble pyrophosphatase IPP1, sucrose supply alone canceled CCE in the palisade tissue but not the epidermal developmental defects. Hence, we postulated that the latter are likely triggered by excess PPi rather than a sucrose deficit. To formally test this hypothesis, we adopted a spatiotemporal approach by constructing and analyzing fugu5-1 PDF1 pro ::IPP1, fugu5-1 CLV1 pro ::IPP1, and fugu5-1 ICL pro ::IPP1, whereby PPi was removed specifically from the epidermis, palisade tissue cells, or during the 4 days following seed imbibition, respectively. It is important to note that whereas PC defects in fugu5-1 PDF1 pro ::IPP1 were completely recovered, those in fugu5-1 CLV1 pro ::IPP1 were not. In addition, phenotypic analyses of fugu5-1 ICL pro ::IPP1 lines demonstrated that the immediate removal of PPi after seed imbibition markedly improved overall plant growth, abolished CCE, but only partially restored the epidermal developmental defects. Next, the impact of spatial and temporal removal of PPi was investigated by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). Our analysis revealed that the metabolic profiles are differentially affected among all the above transgenic lines, and consistent with an axial role of central metabolism of gluconeogenesis in CCE. Taken together, this study provides a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatio-temporal resolution. Finally, our findings suggest that excess PPi exerts its inhibitory effect in planta in the early stages of seedling establishment in a tissue- and cell-autonomous manner.

The functional verification and analysis of Fugu promoter of cardiac gene tnni1a in zebrafish.

Troponin I type 1b (Tnni1b) is thought to be a novel isoform that is expressed only in the zebrafish heart. Knocking down of tnni1b can lead to cardiac defects in zebrafish. Although both the zebrafish tnni1b and human troponin I1 (TNNI1) genes are thought to be closely associated with fatal cardiac development, the regulatory molecular mechanisms of these genes are poorly understood. Analyzing the functionally conserved sequence, especially in the noncoding regulatory region involved in gene expression, clarified these mechanisms. In this study, we isolated a 3 kb fragment upstream of Fugu tnni1a that can regulate green fluorescence protein (GFP) expression in a heart-specific manner, similar to the pattern of zebrafish homologue expression. Three evolutionarily conserved regions (ECRs) in the 5'-flanking sequence of Fugu tnni1a were identified by sequence alignment. Deletion analysis led to the identification of ECR2 as a core sequence that affects the heart-specific expression function of the Fugu tnni1a promoter. Interestingly, both the Fugu tnni1a promoter and ECR2 sequence were functionally conserved in zebrafish, although they shared no sequence similarity. Together, the findings of our study provided further evidence for the important role of tnni1a homologous in cardiac development and demonstrated that two functionally conserved sequences in the zebrafish and Fugu genomes may be ECRs, despite their lack of similarity.

Diel Variation in CC Chemokine Gene Expression in the Japanese Pufferfish Takifugu rubripes.

CC chemokines are key molecules in the regulation of leukocyte trafficking to the site of injury, infection, or inflammation. In recent years, some mammalian chemokines have been shown to exhibit rhythmic expression, regulated by clock genes. However, the rhythmic expression of chemokines in teleost fish remains unknown. In the present study, the diel variation of teleost CC chemokine genes was investigated using the model fish, Fugu (Takifugu rubripes). Diel variation analysis revealed that clock (bmal1, clock1, per2, rorα, and rev-erbß) and CC chemokine (ccl18l, ccl19, and ccl25l) genes show diel expression under 12:12 light-dark cycle (LD12:12) conditions. CC chemokine genes, which exhibit diel expression, contain RORE (ccl18l, ccl19, ccl25l) and/or E-box (ccl25l) motifs in their transcription regulatory region. Moreover, in vitro head kidney stimulation with lipopolysaccharide (LPS) at different zeitgeber times (ZT) under LD12:12 conditions affected the degree of ccl18l, ccl19, and ccl25l expression; high and low responsiveness to LPS stimulation at ZT12 and ZT0 (ccl25l), and ZT16 and ZT4 (ccl18l and ccl19), respectively, were observed. These results suggest that the expression of some fish CC chemokines is affected by the diel variation regulated by clock proteins, and that responsiveness against bacterial infection depends on the time zone.

Expression profile of kalliklectin, a soluble-type mannose receptor, during embryogenesis and early larval development in fugu (Takifugu rubripes).

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

Interleukin-17A/F1 from Japanese pufferfish (Takifugu rubripes) stimulates the immune response in head kidney and intestinal cells.

In mammals, interleukin (IL)-17A and IL-17F, mainly produced by Th17 cells, are hallmark inflammatory cytokines that play important roles in the intestinal mucosal immune response. In contrast, three mammalian IL-17A and IL-17F counterparts (IL-17A/F1-3) have been identified in teleosts, and most of their functions have been described in the lymphoid organs. However, their function in the intestinal mucosal immune response is poorly understood. In this study, a recombinant (r) tiger puffer fish fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using a mammalian expression system, and was used to stimulate cells isolated from fugu head kidney and intestines. The gene expression levels of TNF-α, IL-1ß, IL-6, and ß-defensin-like protein-1 (BD-1) genes were evaluated at 0, 3, 6 and 12 h post-stimulation (hps). Phagocytic activity and superoxide anion production were evaluated at the same time points using an NBT assay. The rIL-17A/F1 protein was shown to induce the expression of pro-inflammatory cytokines and antimicrobial peptides in both head kidney and intestinal cells. Expression levels for IL-1ß, TNF-α, and IL-6 were all up-regulated between 3 and 12 hps. In addition, stimulation with rIL-17A/F1 enhanced phagocytic activity at 24 hps. Superoxide anion production was increased at 48 hps in the head kidney cells and moderately increased at 48 hps in intestinal cells. This study suggests that fugu IL-17A/F1 plays an important role in promoting the innate immune response and may act as a bridge between innate and adaptive immunity in the head kidney and intestine.

Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thaliana.

In Arabidopsis thaliana, the vacuolar proton-pumping pyrophosphatase (H+-PPase) is highly expressed in young tissues, which consume large amounts of energy in the form of nucleoside triphosphates and produce pyrophosphate (PPi) as a byproduct. We reported that excess PPi in the H+-PPase loss-of-function fugu5 mutant severely compromised gluconeogenesis from seed storage lipids, arrested cell division in cotyledonary palisade tissue, and triggered compensated cell enlargement; this phenotype was recovered upon sucrose supply. Thus, we provided evidence that the hydrolysis of inhibitory PPi, rather than vacuolar acidification, is the major contribution of H+-PPase during seedling establishment. Here, examination of the epidermis revealed that fugu5 pavement cells exhibited defective puzzle-cell formation. Importantly, removal of PPi from fugu5 background by the yeast cytosolic PPase IPP1, in fugu5-1 AVP1pro::IPP1 transgenic lines, restored the phenotypic aberrations of fugu5 pavement cells. Surprisingly, pavement cells in mutants with defects in gluconeogenesis (pck1-2) or the glyoxylate cycle (icl-2; mls-2) showed no phenotypic alteration, indicating that reduced sucrose production from seed storage lipids is not the cause of fugu5 epidermal phenotype. fugu5 had oblong cotyledons similar to those of angustifolia-1 (an-1), whose leaf pavement cells display an abnormal arrangement of cortical microtubules (MTs). To gain insight into the genetic interaction between ANGUSTIFOLIA and H+-PPase in pavement cell differentiation, an-1 fugu5-1 was analyzed. Surprisingly, epidermis developmental defects were synergistically enhanced in the double mutant. In fact, an-1 fugu5-1 pavement cells showed a striking three-dimensional growth phenotype on both abaxial and adaxial sides of cotyledons, which was recovered by hydrolysis of PPi in an-1 fugu5-1 AVP1pro::IPP1. Live imaging revealed that cortical MTs exhibited a reduced velocity, were slightly fragmented and sparse in the above lines compared to the WT. Consistently, addition of PPi in vitro led to a dose-dependent delay of tubulin polymerization, thus supporting a link between PPi and MT dynamics. Moreover, mathematical simulation of three-dimensional growth based on cotyledon proximo-distal and medio-lateral phenotypic quantification implicated restricted cotyledon expansion along the medio-lateral axis in the crinkled surface of an-1 fugu5-1. Together, our data suggest that PPi homeostasis is a prerequisite for proper pavement cell morphogenesis, epidermal growth and development, and organ flattening.

Chromosomal-level assembly of Takifugu obscurus (Abe, 1949) genome using third-generation DNA sequencing and Hi-C analysis.

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome-level reference genome for T. obscurus was constructed using long-read Pacific Biosciences (PacBio) Sequel sequencing and a Hi-C-based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single-molecule real-time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome-scale scaffolds using a Hi-C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one-to-one orthologous relationship was observed between the two species, supporting the chromosome-level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu-specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.

Excess Pyrophosphate within Guard Cells Delays Stomatal Closure.

A variety of cellular metabolic reactions generate inorganic pyrophosphate (PPi) as an ATP hydrolysis byproduct. The vacuolar H+-translocating pyrophosphatase (H+-PPase) loss-of-function fugu5 mutant is susceptible to drought and displays pleotropic postgerminative growth defects due to excess PPi. It was recently reported that stomatal closure after abscisic acid (ABA) treatment is delayed in vhp1-1, a fugu5 allele. In contrast, we found that specific removal of PPi rescued all of the above fugu5 developmental and growth defects. Hence, we speculated that excess PPi itself, rather than vacuolar acidification, might delay stomatal closure. To test this hypothesis, we constructed transgenic plants expressing the yeast IPP1 gene (encoding a cytosolic pyrophosphatase) driven by a guard cell-specific promoter (pGC1::IPP1) in the fugu5 background. Our measurements confirmed stomatal closure defects in fugu5, further supporting a role for H+-PPase in stomatal functioning. Importantly, while pGC1::IPP1 transgenics morphologically mimicked fugu5, stomatal closure was restored in response to ABA and darkness. Quantification of water loss revealed that fugu5 stomata were almost completely insensitive to ABA. In addition, growth of pGC1::IPP1 plants was promoted compared to fugu5 throughout their life; however, it did not reach the wild type level. fugu5 also displayed an increased stomatal index, in violation of the one-cell-spacing rule, and phenotypes recovered upon removal of PPi by pAVP1::IPP1 (FUGU5, VHP1 and AVP1 are the same gene encoding H+-PPase), but not in the pGC1::IPP1 line. Taken together, these results clearly support our hypothesis that dysfunction in stomata is triggered by excess PPi within guard cells, probably via perturbed guard cell metabolism.

Transient Hemi-paresthesia after Eating Puffer Fish (Fugu): A Case Report.

The patient was a 64-year-old man who felt numbness of the tongue 30 minutes after eating puffer fish (fugu) prepared by an unqualified person. He then felt hotness on the left side of his face and head, followed by left hemi-paresthesia. The patient had obesity and dyslipidemia. On arrival at our hospital, 150 minutes after eating the fugu, his consciousness was clear, and his only abnormal vital sign was mild hypertension. At approximately four hours after eating the fugu, his hemi-paresthesia spontaneously subsided. He was admitted to our hospital and his post-admission course was uneventful. Brain magnetic resonance image revealed no specific findings. He was discharged on 2nd day of hospitalization without complaint. We presented the 1st case of transient hemi-paresthesia after eating fugu. The mechanism underlying the development of hemi-paresthesia may be pure sensory ischemic attack or fugu intoxication due to an asymmetric distribution of sodium channels.

Identification and immunoregulatory function of neuromedin U (Nmu) in the Japanese pufferfish Takifugu rubripes.

In this study, immunoregulatory function of neuromedin U (Nmu) in the teleost fish Fugu (Takifugu rubripes) was characterized. Three splicing variants of nmu mRNA encoding preproNMUs consisting of 164 (Nmu1), 139 (Nmu2), and 129 (Nmu3) amino acid residues were found in Fugu.The biologically active C-terminal region of Fugu Nmu showed high homology among fish and other vertebrate NMUs. The genomic organization of Fugu nmu differed from those of zebrafish and mammals. However, in phylogenetic analysis, Fugu Nmu formed a cluster with NMUs of other vertebrates, in addition to neuromedin S. The splicing variants of mRNA were identified in various tissues. Nmu-21 and Nmu-9 were purified as endogenous peptides from Fugu intestine. The synthetic Nmu-21 peptide activated phagocytic cells, and elevated the expression of cytokine mRNA in peripheral blood leukocytes.

Compensated Cell Enlargement in fugu5 is Specifically Triggered by Lowered Sucrose Production from Seed Storage Lipids.

To reveal the logic of size regulation in multicellular organisms, we have used Arabidopsis thaliana as a model organism and its leaves as a model organ. We discovered the existence of a compensatory system, whereby a decrease in leaf cell number often triggers unusual cell enlargement. However, despite the large number of compensation-exhibiting mutants analyzed to date, we have only a limited understanding of the detailed molecular mechanisms triggering the decrease in cell number and subsequent compensated cell enlargement (CCE). CCE in fugu5, the vacuolar type H+-pyrophosphatase loss-of-function mutant, is specific to cotyledons and completely suppressed when sucrose (Suc) is supplied or cytosolic pyrophosphate (PPi) is specifically removed. In addition, several lines of evidence suggest that excess cytosolic PPi in fugu5 impairs gluconeogenesis from triacylglycerol (TAG) to Suc. Here, detailed cellular phenotyping revealed that the loss-of-function mutants icl-2, mls-2 and pck1-2 triggered CCE in cotyledons. All double mutant combinations between fugu5-1 and the above three mutants exhibited compensation, but did not display a further increase in cell size. Importantly, similar phenotypes were observed in icl-2 mls-2, icl-2 pck1-2 and mls-2 pck1-2. Quantification of TAG breakdown and Suc contents further supported our findings. Taken together, we demonstrate that de novo Suc synthesis from TAG is fundamentally important for proper resumption of post-germinative cotyledon development. Moreover, provided that icl-2, mls-2 and pck1-2 are only compromised in Suc biosynthesis de novo from TAG, our findings clearly indicate that lowered Suc production in fugu5, rather than excess cytosolic PPi, is the direct trigger of CCE.

Embryonic development of the grass pufferfish (Takifugu niphobles): From egg to larvae.

Tetraodontidae (pufferfish) family members carry the smallest genomes among vertebrates, and these pocket-sized genomes have directly contributed to our understanding of the structure and evolution of higher animals. The grass pufferfish (Takifugu niphobles) could be considered a potential new model organism for comparative genomics and development due to the potential access to embryos, and availability of sequence data for two similar genomes: that of spotted green pufferfish (Tetraodon nigroviridis) and Fugu (Takifugu rubripes). In this study, we provide the first description of the normal embryonic development of T. niphobles, by drawing comparisons with the closely related species cited above. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored at a constant temperature consistent with natural conditions. T. niphobles development was divided into seven periods of embryogenesis: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods; and stages subdividing these periods are defined based on morphological characteristics. The developmental stage series described in this study aims to provide the utilization of T. niphobles as an experimental model organism for comparative developmental studies.

Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry.

A method based on liquid chromatography-electrospray mass spectrometric analysis of the enzymatically digested amplicons derived from the mitochondrial 16S rRNA gene was established for the discrimination of toxic pufferfish. A MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q Exactive mass spectrometer (Thermo Fisher) were employed for separation and detection, respectively. Monoisotopic masses of the oligonucleotides were calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm on average) and precision (relative standard deviation, 0.49 ppm on average) were achieved, and a mass accuracy of <2.8 ppm was maintained for at least 180 h without additional calibration. The present method was applied to 29 pufferfish samples, and results were consistent with Sanger sequencing.

Screening of candidate genes encoding proteins expressed in pectoral fins of fugu Takifugu rubripes, in relation to habitat site of parasitic copepod Caligus fugu, using suppression subtractive hybridization.

Caligus fugu is a parasitic copepod specific to the tetraodontid genus Takifugu including the commercially important Takifugu rubripes. Despite the rapid accumulation of knowledge on other aspects of its biology, the host and settlement-site recognition mechanisms of this parasite are not yet well understood. Since the infective copepodid stage shows preferential site selection in attaching to the fins, we considered it likely that the copepodid recognizes chemical cues released or leaking from the fins, and/or transmembrane protein present on the fins. To isolate molecules potentially related to attachment site specificity, we applied suppression subtractive hybridization (SSH) PCR by identifying genes expressed more highly in pectoral fins of T. rubripes than in the body surface skin. We sequenced plasmid DNA from 392 clones in a SSH library. The number of non-redundant sequences was 276, which included 135 sequences located on 117 annotated genes and 141 located in positions where no genes had been annotated. We characterized those annotated genes on the basis of gene ontology terms, and found that 46 of the identified genes encode secreted proteins, enzymes or membrane proteins. Among them nine showed higher expression in the pectoral fins than in the skin. These could be candidate genes for involvement in behavioral mechanisms related to the site specificity shown by the infective copepodids of C. fugu.

Fugu (Takifugu rubripes) serum GlcNAc-binding lectin is a kalliklectin but has different properties from those of a reported homologue.

A 40-kDa lectin with N-acetyl-d-glucosamine-binding ability was purified from the sera of fugu (Takifugu rubripes) by affinity chromatography and subsequent gel filtration. N-terminal amino acid sequencing, in silico cloning using the fugu genome database and cDNA cloning demonstrated that this lectin is a homologue of kalliklectin, a novel lectin that was previously found in the flathead teleost Platycephalus indicus and has structural similarity to mammalian plasma kallikreins and coagulation factor XI. This is the second report of a kalliklectin, but the fugu kalliklectin differs in its sugar-binding spectra, intersubunit association and tissue distribution from the previously identified flathead kalliklectin. These findings indicate that kalliklectins vary in properties among fish species.

Identification and analysis of a novel bmp4 enhancer in Fugu genome.

Spatiotemporal expression of bone morphogenetic protein 4 (Bmp4) in epithelial and mesenchymal cells is critical for the development of many organs including teeth. Since Bmp4 has a complex and widespread regulatory area in mammals, the tissue-specific enhancers that are responsible for mesenchymal expression of Bmp4 are difficult to identify in mammals. TakiFugu rubripes (Fugu, pufferfish) has a highly compact genome size and is widely used in comparative genomics studies of gene regulatory mechanisms. In this study, we used the Fugu genome to evaluate the 15kb promoter region upstream of the Fugu bmp4 gene. By DNA segmental cloning and luciferase assay with two dental odontoblast-like cell lines, a dental ameloblast-like cell line, and a kidney fibroblast cell line, we identified a 485bp cis-regulatory enhancer between -4213 and -3728bp of the Fugu bmp4 gene. This enhancer showed strong transcriptional activity in all three dental cell lines and, to a lesser extent, also in kidney fibroblast cells. Though not located in an evolutionary conserved region, the enhancer activity for the DNA segment is intense. This is the first time a bmp4 enhancer sequence with activity in both mesenchymal and epithelial cells has been identified, which will help to decode the mechanism of tooth development in vertebrates.