Grass Puffer Research Articles

Gluconeogenesis during development of the grass puffer (Takifugu niphobles)

During the development of teleost fish, the sole nutrient source is the egg yolk. The yolk consists mostly of proteins and lipids, with only trace amounts of carbohydrates such as glycogen and glucose. However, past evidence in some fishes showed transient increase in glucose during development, which may have supported the development of the embryos. Recently, we found in zebrafish that the yolk syncytial layer (YSL), an extraembryonic tissue surrounding the yolk, undergoes gluconeogenesis. However, in other teleost species, the knowledge on such gluconeogenic functions during early development is lacking. In this study, we used a marine fish, the grass puffer (Takifugu niphobles) and assessed possible gluconeogenic functions of their YSL, to understand the difference or shared features of gluconeogenesis between these species. A liquid chromatography (LC) / mass spectrometry (MS) analysis revealed that glucose and glycogen content significantly increased in the grass puffer during development. Subsequent real-time PCR results showed that most of the genes involved in gluconeogenesis increased in segmentation stages and/or during hatching. Among these genes, many were expressed in the YSL and liver, as shown by in situ hybridization analysis. In addition, glycogen immunostaining revealed that this carbohydrate source was accumulated in many tissues at segmentation stage but exclusively in the liver in hatched individuals. Taken together, these results suggest that developing grass puffer undergoes gluconeogenesis and glycogen synthesis during development, and that gluconeogenic activity is shared in YSL of zebrafish and grass puffer.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography–mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Lunar Age-Dependent Oscillations in Expression of the Genes for Kisspeptin, GnIH, and Their Receptors in the Grass Puffer during the Spawning Season.

Grass puffer is a semilunar-synchronized spawner: spawning occurs on beaches only for several days of spring tide around new moon (lunar age 0) and full moon (lunar age 15) every 2 weeks from spring to early summer. To investigate the role of kisspeptin and gonadotropin-inhibitory hormone (GnIH) in the semilunar-synchronized spawning, lunar age-dependent expression of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kissr2), GnIH (gnih), GnIH receptor (gnihr), gonadotropin-releasing hormone 1 (GnRH1) (gnrh1), and three gonadotropin (GTH) subunits (gpa, fshb, lhb) was examined in the male grass puffer, which was kept in an aquarium under natural light condition in a lunar month during the spawning period. In the brain, both kiss2 and kissr2 showed lunar variations with a peak at lunar age 10, while both gnih and gnihr showed semilunar variations with two peaks at lunar age 0 and 20. On the other hand, gnrh1 showed semilunar variation with two peaks at lunar age 0 and 15. In the pituitary, kiss2, kissr2, gnih, and gnihr showed similar variations to those shown in the brain. The fshb and lhb mRNA levels showed semilunar variations with two peaks at lunar age 0 and 15. The present study shows lunar and semilunar oscillations of kiss2/kissr2 and gnih/gnihr expressions, respectively, with their peaks around spring tide in the brain and pituitary along with the semilunar expressions of gnrh1 and the pituitary GTH subunit genes. These results suggest that the lunar age-dependent expressions of the kisspeptin, GnIH, and their receptor genes may be primarily important in the control of the precisely timed semilunar spawning of the grass puffer.

An almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an odorant for the grass puffer

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding “islets” where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this TTX analog, which has been present in their body with TTX but whose function was unknown, as an odorant for chemical communication or effective TTX accumulation.

Kisspeptin Exhibits Stimulatory Effects on Expression of the Genes for Kisspeptin Receptor, GnRH1 and GTH Subunits in a Gonadal Stage-Dependent Manner in the Grass Puffer, a Semilunar-Synchronized Spawner.

Kisspeptin has an important role in the regulation of reproduction by directly stimulating the secretion of gonadotropin-releasing hormone (GnRH) in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and kisspeptin receptor types, and the two kisspeptins in teleosts have different effects depending on fish species and reproductive stages, serving reproductive and non-reproductive functions. In the grass puffer, Takifugu alboplumbeus, which has only a single pair of kiss2 and kissr2, both genes display seasonal, diurnal, and circadian oscillations in expression in association with the periodic changes in reproductive functions. To elucidate the role of kisspeptin in this species, homologous kisspeptin peptide (gpKiss2) was administered at different reproductive stages (immature, mature and regressed) and the expression levels of the genes that constitute hypothalamo-pituitary-gonadal axis were examined in male grass puffer. gpKiss2 significantly elevated the expression levels of kissr2 and gnrh1 in the brain and kissr2, fshb and lhb in the pituitary of the immature and mature fish. No noticeable effect was observed for kiss2, gnih, gnihr, gnrh2 and gnrh3 in the brain and gpa in the pituitary. In the regressed fish, gpKiss2 was ineffective in stimulating the expression of the gnrh1 and GTH subunit genes, while it stimulated and downregulated the kissr2 expression in the brain and pituitary, respectively. The present results indicate that Kiss2 has a stimulatory role in the expression of GnRH1/GTH subunit genes by upregulating the kissr2 expression in the brain and pituitary at both immature and mature stages, but this role is mostly ineffective at regressed stage in the grass puffer.

Anomalous Temperature Interdicts the Reproductive Activity in Fish: Neuroendocrine Mechanisms of Reproductive Function in Response to Water Temperature.

Fish are poikilotherm and small changes in water temperature can greatly affect physiological processes including reproduction, which is regulated by complex neuroendocrine mechanisms that respond to climatic events. This review provides evidence that anomalous high and low temperature may directly affect reproduction in fish by suppressing the expression of genes in the reproductive neuroendocrine system. The grass puffer, Takifugu alboplumbeus, is an excellent animal model for studying the thermal regulation of reproduction, for they exhibit periodic spawning activities, which are synchronized with seasonal, lunar and daily cycles. In the grass puffer, the expression of the genes encoding gonadotropin-releasing hormone (GnRH) 1, kisspeptin, gonadotropin-inhibitory hormone (GnIH) and their receptors were markedly suppressed in the diencephalon of fish exposed to high temperature (28°C) when compared to normal temperature (21°C), followed by the decrease in the pituitary mRNA levels for follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH). On the other hand, the exposure to low temperature (14°C) also inhibited the expression of gnrh1, kiss2, gnih and their receptor genes in the brain and fshb, lhb, gh and prl in the pituitary. Taken together, it is plausible that anomalous high and low temperature may be a proximate driver of termination of reproduction by suppressing the activity of the reproductive GnRH/kisspeptin/GnIH system, possibly through direct action of temperature signals at transcription level.

Enrichment of spermatogonia by density gradient centrifugation for use as a donor of surrogate production of tiger puffer

A method of producing functional gametes from cryopreserved testicular germ cells using surrogate broodstock technology is a powerful tool for preserving the aquacultural genetic resources of the tiger puffer (Takifugu rubripes). Among the various testicular cells, only type A spermatogonia (ASGs) can colonize recipient gonads and shift to gametogenesis; therefore, ASG enrichment is an important factor for successful surrogate production. This study examined the possibility of ASG enrichment by density gradient centrifugation for surrogate production, considering its practical use. Through density gradient centrifugation with Percoll media, the highest distribution of ASGs estimated on the cell morphology and vasa expression was counted at a fraction of 20% Percoll layer. These fractionated testicular cells were transplanted intraperitoneally and successfully incorporated into the gonads of the recipient, grass puffer (T. alboplumbeus); however, no colonization was observed in the control recipients transplanted with unfractionated testicular cells. Moreover, these recipients matured normally and produced functional donor-derived gametes from which tiger puffer offspring originated. Thus, the Percoll gradient centrifugation developed in this study enables the enrichment of ASGs from testicular cells, including many differentiated germ cells, and would be useful for efficiently managing broodstock with desirable genetic traits.

Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

Toxic puffers accumulate their defense substance (tetrodotoxin; TTX) through the food chain. Although the previous study suggests that 5,6,11-trideoxyTTX, a nontoxic TTX analog detected simultaneously with TTX in toxic puffers or their prey, acts as an olfactory chemoattractant for grass puffers, it is unclear whether toxic puffers are commonly attracted to 5,6,11-trideoxyTTX, and which types of olfactory sensory neurons (OSNs) detect 5,6,11-trideoxyTTX. Here, we demonstrated that green spotted puffer, a phylogenetically distant species from the grass puffer, is attracted to 5,6,11-trideoxyTTX. 5,6,11-TrideoxyTTX administration made green spotted puffers stay longer at the administered site, whereas a food odor (l-Arg) made them actively swim throughout the aquarium. Attractive responses were not observed when TTX or its vehicle was administered, nor when 5,6,11-trideoxyTTX was administered to anosmic fish. Furthermore, double immunohistochemistry with activity marker and crypt OSN marker antibodies labeled oval cells with apical invagination on the olfactory epithelium surface treated with 5,6,11-trideoxyTTX. These results suggest that 5,6,11-trideoxyTTX acts as an olfactory chemoattractant detected by crypt OSNs, and attraction to 5,6,11-trideoxyTTX odor appears to be a trait shared by toxic puffers for social communication or effective toxification.

Infections of <i>Kudoa septempunctata</i> (Myxozoa: Multivalvulida) in Wild Grass Puffer <i>Takifugu alboplumbeus</i> and Japanese Whiting <i>Sillago japonica</i>

Kudoa septempunctata is an important myxozoan parasite in culture and capture fisheries as it causes food poisoning in humans. ​This species is generally considered a parasite of olive flounder Paralichthys olivaceus because K. septempunctata-related food poisoning cases are associated with consumption of raw infected P. olivaceus. ​However, the host range of K. septempunctata remains unclear. ​Moreover, there is scarce information on the lifecycle of the parasite, and this hinders the development of a fundamental prophylactic measure in fish farms. ​During the survey of wild fishes at a K. septempunctata endemic area, kudoid myxospores were detected in the somatic muscle of grass puffer Takifugu alboplumbeus and Japanese whiting, Sillago japonica. ​Our morphological, molecular, and histological analyses identified them as K. septempunctata. ​While the infection levels in T. alboplumbeus were higher (up to 92.3% prevalence) than those in S. japonica (23.8%), the maximum spore density in the muscle tissue was about 7×104/g in both fishes, which is far less than the food poisoning risk value of 1 × 106/g. ​These findings indicate a wide host range of K. septempunctata and further attention is required for its possible infections in fishes other than P. olivaceus.

Light-induced and circadian expressions of melanopsin genes opn4xa and opn4xb in the eyes of juvenile grass puffer Takifugu alboplumbeus.

Animals regulate a variety of aspects of physiology according to environmental light conditions via nonvisual opsins such as melanopsin. In order to study photic regulation of fish physiology, expression changes of the genes for melanopsin (opn4xa and opn4xb) and effects of light on them were examined in juvenile grass puffer Takifugu alboplumbeus using quantitative real-time PCR. In the brain of juvenile fish, no significant diurnal nor circadian changes were observed in opn4x mRNA levels. On the other hand, in the eyes, the mRNA level of opn4xa showed a significant diurnal rhythm with a peak at Zeitgeber time (ZT) 4, while no apparent circadian changes were observed. The mRNA level of opn4xb in the eyes showed a diurnal change similar to that of opn4xa, while it showed a significant circadian change. Furthermore, continuous exposure to light during a subjective night significantly increased the mRNA levels of opn4xa in the eyes at ZT24, suggesting that light induces gene expression of opn4xa in the eyes and that the induction occurs only during the night-day transition period. These results suggest that Opn4xa and Opn4xb play differential roles in the eyes of juvenile grass puffer to mediate the physiological effects of environmental light information.

Induction of germ cell-deficiency in grass puffer by dead end 1 gene knockdown for use as a recipient in surrogate production of tiger puffer

The surrogate broodstock technique produces donor-derived gametes via transplantation of germ cells into surrogate fish. The technique is a promising approach for improving the establishment and management of aquaculture strains with desirable genetic traits. The tiger puffer (Takifugu rubripes) is one of the most popular aquaculture fishes in Japan. However, brood stocks have large body sizes and require a long time to reach sexual maturity. The current study established a sterilization technique using gene knockdown in the grass puffer (T. alboplumbeus), which has a small body size and matures in half the time taken by the tiger puffer, and assessed the possibility of using germ cell-deficient grass puffers as recipients to efficiently produce donor-derived tiger puffer gametes. Dead end 1, which has two transcribed variants, was identified in the grass puffer. Morphants resulting from the microinjection of antisense morpholino oligonucleotides against the transcribed variants, into fertilized eggs, showed germ cell deficiency. Germ cell-deficient morphants exhibited sexual dimorphism with respect to their morphological and gene expression patterns. Testicular germ cells prepared from the testes of tiger puffer were intraperitoneally transplanted into morphant hatchlings. These recipients matured normally and produced functional donor-derived gametes separately from endogenous gametes. Thus, the use of germ cell-deficient recipients that produce only donor-derived gametes improves the efficiency of surrogate production and may accelerate the breeding process in tiger puffer aquaculture.

D-mannose-specific Immunoglobulin M in Grass Puffer (Takifugu niphobles), a Nonhost Fish of a Monogenean Ectoparasite Heterobothrium okamotoi, Can Act as a Trigger for its Parasitism.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

Population genetic structure of the grass puffer (Tetraodontiformes: Tetraodontidae) in the northwestern Pacific revealed by mitochondrial DNA sequences and microsatellite loci

The genus Takifugu comprises approximately 25 species that are closely related due to fairly recent speciation in the northwestern Pacific. The variety of coloration patterns within Takifugu has led to taxonomic confusion. A recent study treated Takifugu niphobles (Jordan and Snyder, 1901) as a junior synonym of Takifugu alboplumbeus (Richardson, 1845) based on morphological comparison of the type specimens. In an attempt to review the taxonomic status of grass puffer at a molecular level, 209 individuals from nine localities in the northwestern Pacific (two localities in the East Sea, five in the Korea Strait, and two in the Yellow Sea) were analyzed using mitochondrial DNA (mtDNA) and microsatellites. The mtDNA sequence analyses showed two distinct lineages (Kimura-two-parameter distance = 1.5–2.6% and 2.2–3.6% in cytochrome c oxidase subunit I and cytochrome b, respectively). The distributions of the two lineages tended to be separated; lineage 1 was located in the East Sea and lineage 2 in the Yellow Sea, but both lineages in the Korea Strait. STRUCTURE analyses using 13 microsatellite markers showed two distinct groups, which was consistent with the two subdivisions in mtDNA, except for the western Korea Strait. Our findings suggest that T. niphobles is a distinct species differing from T. alboplumbeus, unlike previous study. This requires further careful taxonomic revision.

Annual patterns of ocular melatonin level in the female grass puffer, Takifugu alboplumbeus: possible involvement in seasonal reproductive response.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

Effects of high and low temperature on expression of GnIH, GnIH receptor, GH and PRL genes in the male grass puffer during breeding season

Gonadotropin-inhibitory hormone (GnIH) is a multifunctional hypophysiotropic neurohormone and has a stimulatory role in the control of reproduction in the grass puffer. To clarify the neuroendocrine mechanisms underlying the effect of changes in water temperature on reproduction in fish, we previously revealed that, in parallel to gonadal regression, both low and high temperature significantly decreased the expressions of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), gonadotropin-releasing hormone 1 (gnrh1) in the brain and gonadotropin (GTH) subunits (fshb and lhb) in the pituitary of sexually mature male grass puffer. In this study, we examined the changes in expression of gnih and GnIH receptor gene (gnihr) in the brain and pituitary along with the genes for growth hormone (gh) and prolactin (prl) in the pituitary of male grass puffer exposed to low temperature (14 °C), normal temperature (21 °C, as initial control) and high temperature (28 °C) conditions for 7 days. The levels of gnih and gnihr mRNAs were significantly decreased in both low and high temperature conditions compared to normal temperature in the brain and pituitary. Similarly, the gh mRNA levels were significantly decreased in both low and high temperature conditions. The prl mRNAs showed no significant changes at high temperature, whereas drastically decreased at low temperature possibly by dysfunctional cold stress. Taken together, the present results suggest that, in addition to the inhibitory effect of temperature changes on the Kiss2/GnRH1/GTH system, the suppression of GnIH/GH system may also be involved in the termination of reproduction by high temperature at the end of breeding season.

Effects of Different Light Spectra on the Oocyte Maturation in Grass Puffer Takifugu niphobles.

In order to examine the effects of four different light spectra (white, red, green, and blue) on the oocyte maturation, the change of reproductive parameters, via brain-pituitary-gonad (BPG) axis in grass puffer, were investigated. After exposure four different light spectra for 7 weeks, the abundance of gonadotropin-releasing hormone (GnRH) mRNA which is a type of seabream (sbGnRH) and two different subunit of gonadotropin hormones mRNAs, follicle-stimulating hormone (fshβ) mRNA and luteinizing hormone (lhβ) mRNA, were analyzed in the brain and pituitary. Histological analysis showed that the mature oocyte ratio in the green spectrum was higher than other light spectra-exposed groups. Gonadosomatic index (GSI) and oocyte developmental stage were also investigated in the gonad based on histological observations. GSI value with the presence of yolk stage oocytes was significantly increased in the green spectrum-exposed group when compared to that of the other light-exposed groups (white, red, and blue) (p˂0.05). The abundances of sbGnRH mRNA and fshβ mRNA in the green spectrum-exposed group were also significant higher than those of the other light spectra-exposed groups (p˂0.05). These results indicate that the maturation of oocyte in grass puffer can be accelerated by exposure to the spectrum of green. To better understand the molecular mechanism for the maturation of oocyte in grass puffer, further study examining the relationship between oocyte development and its related genes is required.

Production of tiger puffer Takifugu rubripes from cryopreserved testicular germ cells using surrogate broodstock technology

Tiger puffer Takifugu rubripes is one of most important fish in aquaculture. Recently, there has been progress in the production of tiger puffer aquaculture strains carrying economically desirable genetic traits. Cryopreservation techniques for the long-term preservation of their germ cells may reduce the risk of losing valuable genetic resources and the costs associated with maintenance of broodstock; however, cryopreservation methods for eggs and embryos, which contain both nuclear DNA and maternally inherited cytoplasmic compartments, have not yet been developed. In this study, we developed a methodology to produce functional gametes derived from cryopreserved testicular germ cells. Testes collected from tiger puffer were cryopreserved by a slow-freezing method, and the cryomedium for tiger puffer testis was optimized by testing several different cryoprotectants. Testicular germ cells prepared from frozen-thawed testes cryopreserved for at least two months were intraperitoneally transplanted into diploid or triploid hatchlings of grass puffer T. alboplumbeus. Transplanted germ cells migrated toward and were incorporated into recipient gonads. At 10 months post transplantation, 36.4% and 64.2% of diploid and triploid recipients, respectively, produced donor-derived sperm. In female recipients aged two years, production of donor-derived eggs was confirmed in one of 62 (1.6%) triploid recipients but not in diploid recipients. Insemination with the resultant sperm and eggs from triploid recipients generated viable offspring that originated from transplanted tiger puffer germ cells. Survival and growth potential of the offspring generated by the cross of both male and female triploid recipients were also comparable to those of control tiger puffer. This method is thus a breakthrough tool for the preservation of valuable genetic resources of tiger puffer and would help to efficiently manage broodstock with desirable genetic traits.

Temperature Dependent of Mitotic Interval for Grass Puffer, Takifugu niphobles.

ABSTRACTThe objective of this study was to determine the mitotic intervals (τ0) of two consecutive cell divisions and synchronous embryonic cleavage in grass puffer, Takifugu niphobles at different water temperatures (18, 20, 22, and 24℃). The color of the fertilized egg was light yellowish. The egg type was demersal and unadhesive. Egg weight was 0.09±0.002 mg. The sizes of unfertilized eggs were smaller than fertilized eggs in major axis and minor axis at 20℃ (p<0.05). The size of the fertilized egg of 18℃ water temperature group at the blastodisc stage was the smallest (p<0.05), but no significant differences were observed in the other water temperatures group except 18℃ water temperature group (p>0.05). The first cleavage stages at 18, 20, 22, and 24℃ were at 75, 90, 105, and 120 mins, respectively. As water temperature was increased, embryonic development and formation time of the first cleavage furrow were accelerated. There were negative correlation between τ0 and water temperature for grass puffer (Y=–1.225X+70.05, R2=0.988, n=10, where Y was τ0 and X was temperature). This study confirmed that successful hatching of grass puffer was related to water temperature. Chromosome manipulation will be helpful for this species using cleavage frequency and τ0.

Identification of the sex-determining locus in grass puffer (Takifugu niphobles) provides evidence for sex-chromosome turnover in a subset of Takifugu species.

There is increasing evidence for frequent turnover in sex chromosomes in vertebrates. Yet experimental systems suitable for tracing the detailed process of turnover are rare. In theory, homologous turnover is possible if the new sex-determining locus is established on the existing sex-chromosome. However, there is no empirical evidence for such an event. The genus Takifugu includes fugu (Takifugu rubripes) and its two closely-related species whose sex is most likely determined by a SNP at the Amhr2 locus. In these species, males are heterozygous, with G and C alleles at the SNP site, while females are homozygous for the C allele. To determine if a shift in the sex-determining locus occurred in another member of this genus, we used genetic mapping to characterize the sex-chromosome systems of Takifugu niphobles. We found that the G allele of Amhr2 is absent in T. niphobles. Nevertheless, our initial mapping suggests a linkage between the phenotypic sex and the chromosome 19, which harbors the Amhr2 locus. Subsequent high-resolution analysis using a sex-reversed fish demonstrated that the sex-determining locus maps to the proximal end of chromosome 19, far from the Amhr2 locus. Thus, it is likely that homologous turnover involving these species has occurred. The data also showed that there is a male-specific reduction of recombination around the sex-determining locus. Nevertheless, no evidence for sex-chromosome differentiation was detected: the reduced recombination depended on phenotypic sex rather than genotypic sex; no X- or Y-specific maker was obtained; the YY individual was viable. Furthermore, fine-scale mapping narrowed down the new sex-determining locus to the interval corresponding to approximately 300-kb of sequence in the fugu genome. Thus, T. niphobles is determined to have a young and small sex-determining region that is suitable for studying an early phase of sex-chromosome evolution and the mechanisms underlying turnover of sex chromosome.

Molecular and ecophysiological aspects of semilunar synchronized spawning migration in the grass puffer

Molecular and ecophysiological aspects of semilunar synchronized spawning migration in the grass puffer