Genetic Dissection of a Precocious Phenotype in Male Tiger Pufferfish (Takifugu rubripes) using Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di).

The novel non-targeted PCR-based genotyping system, namely Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di), is characterized by the simplicity in library construction and robustness against DNA degradation and is expected to facilitate advancements in genetics, in both basic and applied sciences. In this study, we tested the utility of GRAS-Di for genetic analysis in a cultured population of the tiger pufferfish Takifugu rubripes. The genetic analyses included family structure analysis, genetic map construction, and quantitative trait locus (QTL) analysis for the male precocious phenotype using a population consisting of four full-sib families derived from a genetically precocious line. An average of 4.7 million raw reads were obtained from 198 fish. Trimmed reads were mapped onto a Fugu reference genome for genotyping, and 21,938 putative single-nucleotide polymorphisms (SNPs) were obtained. These 22 K SNPs accurately resolved the sibship and parent-offspring pairs. A fine-scale linkage map (total size: 1,949 cM; average interval: 1.75 cM) was constructed from 1,423 effective SNPs, for which the allele inheritance patterns were known. QTL analysis detected a significant locus for testes weight on Chr_14 and three suggestive loci on Chr_1, Chr_8, and Chr_19. The significant QTL was shared by body length and body weight. The effect of each QTL was small (phenotypic variation explained, PVE: 3.1-5.9%), suggesting that the precociousness seen in the cultured pufferfish is polygenic. Taken together, these results indicate that GRAS-Di is a practical genotyping tool for aquaculture species and applicable for molecular breeding programs, such as marker-assisted selection and genomic selection.

Production of Tiger Puffer Takifugu rubripes Offspring from Triploid Grass Puffer Takifugu niphobles Parents.

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2-5 kg) much larger than average market size (0.6-1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.

Transitional reactive oxygen species (ROS) production in fertilized egg embryos of devil stinger (Inimicus japonicus), a marine fish species.

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.

Estradiol-17beta treatment induces intersexual gonadal development in the pufferfish, Takifugu rubripes.

Estrogens are responsible for most characteristics of the female sex of a species, such as metabolic, behavioral, and morphological changes during reproduction. Artificial estradiol-17beta (E2) treatment Induces sex reversal in some fish. The Japanese pufferfish (Takifugu rubripes) has the most compact genome among vertebrates and great pottial for comparative genome analysis. In this paper, we describe the Influence of E2 treatment during gonadal development in the pufferfish. After hatching, fry were treated with no (control) or a 0.1, 1, 10, or 100 microg/g diet from 21 to 80 days after hatching (dah). Doublesex-mab3-related transcription factor (DMRT1) is Involved in testicular development. VASA is responsible for germ cell development, and CYP19A plays a role in E2 biosynthesis during ovarian development across animal phyla as well as in gonadal morphology after E2 treatment. DMRT1, VASA, and CYP19A were Investigated in the gonads of E2-treated pufferfish. Fish fed with the highest dose (E2 100 microg/g diet) developed Intersexual gonads in the testis; the majority of germ cells were oocytes, but some spermatocytes were detected. RT-PCR results showed the expression of VASA and CYP19A in all intersexual gonads and DMRT1 in some. Furthermore, abnormalities in the epithelium-tunica layer were detected, and gonadal somatic cells (e.g., granulosa cells, theca cells, or germinal epithelium) proliferated extensively in the intersexual gonad. These results suggest that E2 treatment Induces ovarian development in the bipotential gonads of genetic males by modification of gonadal somatic cells and E2 production, mediated by CYP19A.

Production of reactive oxygen species (ROS) by various marine fish species during the larval stage.

Previous studies have indicated that the devil stinger produces reactive oxygen species (ROS) during early development from fertilized egg to larva. To determine whether ROS generation is a common feature in marine fish species, we conducted chemiluminescence analysis using ROS specific probe (L012) on larvae of six marine fish species. Marbled rockfish, black rockfish, and devil stinger showed higher levels of chemiluminescence response (CR), whereas the levels of CR of sevenband grouper, tiger puffer, and red seabream were fairly lower. These CRs were inhibited by the addition of superoxide dismutase. Hypersensitive photon-counting microscopic observation of black rockfish suggested that ROS production was concentrated in the head area. Our results suggest that the larvae of these six marine fishes produce ROS to considerably different extents depending on species, and that rockfish species, belonging to ovoviviparous fish, tend to produce much higher levels of ROS especially at the later larval stage.

Production of reactive oxygen species (ROS) by devil stinger (Inimicus japonicus) during embryogenesis.

In aqua-cultural industry, the seed production of devil stinger, a valuable fish in Japan, has not succeeded yet due to the cryptogenic mass mortality. We found that survival rate of the larvae of devil stinger increased by the addition of green tea extract rich in catechin into rearing tank. Generation of reactive oxygen species (ROS) was detected in the embryo of devil stinger by chemiluminescence analysis under the normal growth conditions without addition of specific stimulants. Even in the unfertilized egg, certain level of ROS was detected. ROS were continuously detected during the development from fertilized egg to larva and tended to increase gradually. Observation of embryos and post-hatching larvae with hypersensitive photon-counting microscopy indicated that ROS were produced on the surface of embryo and the head region of larva especially peripheries of eyes. When the embryo proteins were analyzed by immunoblotting using antibody against the human neutrophil cytochrome b558 large subunit (gp91 phox), a main band of approximately 91 kDa was detected, suggesting the presence of NADPH oxidase-like ROS generating system in the embryo of devil stinger. After treatment with streptomycin and penicillin G for 1 day, the level of ROS production in larvae decreased with increase in the survival rate of larvae. Our results suggest that devil stinger has ROS generation system that is already activated at fairly early stage of development before the maturation of usual immune system.

Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes.

DMRT1, a gene containing the Doublesex/Mab-3 DNA-binding motif (DM domain), encodes a transcription factor that regulates early differentiation of Sertoli cells in the testes of vertebrates. Here, we describe the pattern of expression of six DMRT genes (DMRT1, 2a, 2b, 3, 4, and 5) during gonadal development in the Japanese pufferfish, Takifugu rubripes, for the first time. Conventional histological analysis showed that a primordial gonad is present 2 weeks after hatching and that sexual differentiation had occurred by 6 weeks after hatching, as determined by the formation of cavities in the ovaries. RT-PCR analysis showed that the DMRT1 transcript was abundant in the testes, but not in the ovaries, of 3-month-old fish. DMRT3 transcript was also detected in the testes, but not in the ovaries, indicating that the patterns of gonadal expression of DMRT3 are very similar to DMRT1. The other four DMRT genes examined did not exhibit sex-specific expression patterns. In situ hybridization analysis revealed that, after gonadal differentiation was complete, DMRT1 was expressed in the Sertoli cells that were in proximity to proliferating spermatogonia. These results indicate that DMRT1 involved in gonadal development rather than sex differentiation and that its expression correlates with the proliferation of spermatogonia in T. rubripes. The expression profile of DMRT1 in T. rubripes was similar to that in the teleost species Medaka.