Linking Pedigree Information to the Gene Expression Phenotype to Understand Differential Family Survival Mechanisms in Highly Fecund Fish: A Case Study in the Larviculture of Pacific Bluefin Tuna.

Mass spawning in fish culture often brings about a marked variance in family size, which can cause a reduction in effective population sizes in seed production for stock enhancement. This study reports an example of combined pedigree information and gene expression phenotypes to understand differential family survival mechanisms in early stages of Pacific bluefin tuna, Thunnus orientalis, in a mass culture tank. Initially, parentage was determined using the partial mitochondrial DNA control region sequence and 11 microsatellite loci at 1, 10, 15, and 40 days post-hatch (DPH). A dramatic proportional change in the families was observed at around 15 DPH; therefore, transcriptome analysis was conducted for the 15 DPH larvae using a previously developed oligonucleotide microarray. This analysis successfully addressed the family-specific gene expression phenotypes with 5739 differentially expressed genes and highlighted the importance of expression levels of gastric-function-related genes at the developmental stage for subsequent survival. This strategy demonstrated herein can be broadly applicable to species of interest in aquaculture to comprehend the molecular mechanism of parental effects on offspring survival, which will contribute to the optimization of breeding technologies.

Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes).

Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex determination gene in Thunnus fishes or at least functions at a crucial point in the sex-differentiation cascade.

Gonadal sex differentiation and early ovarian/testicular development in cultured Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel).

Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production.

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae.

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6-11, 8-11 and 6-9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40-50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception-increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10-12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.