Developing sound breeding programs for aquaculture species may be challenging when matings cannot be controlled due to communal spawning. We developed a genotyping-by-sequencing marker panel of 300 SNPs for parentage testing and sex determination by using data from an in-house reference genome as well as a 90 K SNP genotyping array based on different populations of yellowtail kingfish (Seriola lalandi). The minimum and maximum distance between adjacent marker pairs were 0.7 Mb and 13 Mb, respectively, with an average marker spacing of 2 Mb. Weak evidence of the linkage disequilibrium between adjacent marker pairs was found. The results showed high panel performance for parental assignment, with probability exclusion values equaling 1. The rate of false positives when using cross-population data was null. A skewed distribution of genetic contributions by dominant females was observed, thus increasing the risk of higher rates of inbreeding in subsequent captive generations when no parentage data are used. All these results are discussed in the context of breeding program design, using this marker panel to increase the sustainability of this aquaculture resource.

The longfin yellowtail Seriola rivoliana is an emerging species for aquaculture diversification worldwide and production relies on fertilized eggs from captive broodstock. Temperature is the main factor that influences the developmental process and success during fish ontogeny. However, the effects of temperature on the utilization of the main biochemical reserves and bioenergetics are scarcely investigated in fish, whereas protein, lipid and carbohydrate metabolism have critical roles in maintaining cellular energy homeostasis. In this context, we aimed to evaluate metabolic fuels (protein, lipids, triacylglicerides, carbohydrates), adenylic nucleotides and derivates (ATP, ADP, AMP, IMP), and the adenylate energy charge (AEC) during embryogenesis and in hatched larvae in S. rivoliana at different temperatures. For this purpose, fertilized eggs were incubated at six constant (20, 22, 24, 26, 28 and 30 °C) and two oscillating (21⇄29 °C) temperatures. Biochemical analyses were made at blastula, optic vesicles, neurula, prehatch and hatch periods. Results indicated that the developmental period had a major influence on the biochemical composition at any temperature regime tested during the incubation. Protein content decreased only at hatching mainly due to the loss of the chorion, total lipids tended to increase at the neurula period and variations in carbohydrates depended on the particular spawn analyzed. Triacylglicerides were a critical egg fuel during hatching. The high AEC during embryogenesis and even in hatched larvae suggested an optimal energy balance regulation. The lack of critical biochemical changes from different temperature regimes during embryo development confirmed that this species exhibits a high adaptive capacity in response to constant and fluctuating temperatures. However, the timing of hatching was the most critical period of development, where biochemical components and energy utilization significantly changed. The oscillating temperatures tested may have physiological advantages without detrimental energetic effects that will require further research on larval quality after hatching.

The buoyancy of eggs and embryos is associated with successful development in pelagic fish. Buoyancy is the result of oocyte hydration, which depends on the osmotic force exerted by free amino acids (FAA) generated by yolk proteolysis, and cathepsins are the main enzymes involved in this process. Seriola lalandi is a pelagic fish whose farming has been hampered by development failure that have been partially attributed to decreased buoyancy of embryos. Therefore, the aim of this study was to compare the mRNA expression and activity of cathepsins B, D, and L, as well as the FAA content in floating and low-floating embryos at different developmental stages. The chosen stages were eggs, morula, blastula, gastrula and 24 h embryos. Complementary assessments showed that there were no differences attributed to buoyancy status in embryo and oil droplet diameters, as well as the transcriptional status at any developmental stage. Cathepsin B did not show differences in mRNA expression or activity related to buoyancy at any stage. Cathepsin D displayed higher transcript and activity levels only in low-floating eggs compared with those floating. Cathepsin L showed higher expression in floating eggs and 24 h embryos compared with that of low-floating, but the activity of this enzyme was higher in floating eggs and morula. Total FAA content constantly decreased throughout development in floating embryos, but it was always higher than low-floating embryos until gastrula stage. In 24 h embryos floating and low-floating embryos share similar quantities of FAA. In summary, differences in the expression and activity of cathepsins between floating and low-floating embryos could be revealed at specific embryonic stages, suggesting different functions of these enzymes throughout development. Besides 24 h embryos, FAA content seems to be a decisive factor for buoyancy of embryos during early development of S. lalandi. Overall, considering the main role of cathepsins and FAA in buoyancy acquisition process and therefore in both embryo quality and viability, our study identifies good marker candidates to evaluate embryo quality in the farming of this species.

We investigated a time-course larval transcriptional analysis (RNA-seq) in the longfin yellowtail Seriola rivoliana, from hatching to day four at 22 °C, without providing zooplankton as food. Larval starvation is a critical physiological stage that must be prevented to ensure survival. However, the transcriptional mechanisms to endure starvation have not been investigated in marine fish. Differential gene expression showed newly day-specific transcriptome events during larval development. On day 1 (yolk sac absorption), the predominant upregulated developmental processes were larval growth, muscle and vision development, cytoskeletal structure, protein synthesis, protein and fat digestion-absorption, and hormone biosynthesis, whereas the cell cycle was suppressed. On day 2 (yolk sac exhaustion), a new stage of energy regeneration (ATP) was supplied by the oil drop reserve, whereas protein digestion-absorption and growth were suppressed. On day 3 (mouth opening and starvation), stress signals and nutrition deprivation upregulated the p53 signal and triggered autophagy and the AMP-activated protein kinase (AMPK) pathways as an alternative catabolic pathway to enduring starvation, and the circadian rhythm was established. On day 4 (starving and weakened larvae condition), autophagy supported subsequent protein synthesis, activated the immune system, and promoted estrogen signaling and skeleton renovation. However, larvae suppressed muscle development, vision and carbohydrate, and fat digestion-absorption and became lethargic, evidencing limited physiological support by autophagy to maintain survival without exogenous nutrition in this species.

In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.

Interferon (IFN)γ is a pivotal cytokine that promotes and orchestrates innate cellular and adaptive cell-mediated immunity against intracellular pathogens. The capacity of T cells in mammals to produce IFNγ has been measured using specific antibodies in order to analyze cell-mediated immune responses against infection or immuno-stimulants. In fish, however, measurement of IFNγ protein levels has not been possible due to a lack of research tools. In the present study, therefore, we established antibodies that react with endogenous amberjack IFNγ. An enzyme-linked immunosorbent assay (ELISA) for IFNγ in amberjack species was developed using these antibodies. The ELISA could detect endogenous IFNγ at concentrations less than 100 pg/mL in PMA/ionomycin-stimulated leukocytes culture supernatant. IFNγ production was enhanced and lasted a long time following intracellular bacterial infection with Nocardia seriolae, which is thought to be targeted by cell-mediated immunity. These results demonstrate that quantification of IFNγ using the reported ELISA can be used to estimate the status of cell-mediated immunity in amberjack species.

There is accumulating evidence that insulin-like growth factors (IGFs), primary mediators of somatic growth, play an important role in fish reproduction. Previously, we showed that IGF-1 and IGF-2 are expressed in the ovarian follicle cells of the yellowtail (Seriola quinqueradiata) during the vitellogenic phase, suggesting that IGFs may be involved in ovarian steroidogenesis. In this study, we examined the effects of IGF-1 and IGF-2 on gene expression and activity of steroidogenic enzymes in yellowtail ovary in vitro. IGF-1 and IGF-2 had no effect on mRNA levels of several steroidogenesis-related genes (star, cyp11a1, hsd3b, cyp17a2, and cyp19a1). However, both IGFs enhanced the transcription of cyp17a1 in vitellogenic ovaries, although such up-regulation was not found in the ovary at the pre-vitellogenic stage. The stage-dependent effects of IGFs were correlated with changes in ovarian cyp17a1 mRNA levels during the reproductive cycle: transcript abundances increased in conjunction with ovarian development. In addition, IGF-induced cyp17a1 gene expression was significantly inhibited by wortmannin, suggesting that PI3 kinase plays an essential role in IGF-mediated ovarian steroidogenesis. Furthermore, IGF-1 and IGF-2 promoted the conversion of both progesterone and 17α-hydroxyprogesterone to androstenedione in vitellogenic ovaries, suggesting that both IGFs stimulated 17α-hydroxylase and C17-20 lyase activities. Taken together, these findings suggest that IGF-1 and IGF-2 act directly on follicle cells to stimulate steroid production through an increase in gene expression and enzymatic activity of cyp17a1 via induction of PI3 kinase.

Vertebrate sex development consists largely of two processes: \"sex determination,\" the initial bifurcation of sexual identity, and \"sex differentiation,\" which subsequently facilitates maleness or femaleness according to the sex determination signal. Steroid hormones promote multiple types of sexual dimorphism in eutherian mammals and avians [1-3], in which they are indispensable for proper sex differentiation. By contrast, in many poikilothermic vertebrates, steroid hormones have been proposed to be key players in sex determination as well as sex differentiation [4-8]. This hypothesis was introduced more than 50 years ago but has never been rigorously tested due to difficulties in discriminating the roles of steroids in sex determination and differentiation. We found that a missense SNP in the gene encoding the steroidogenic enzyme 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1) was perfectly associated with ZZ/ZW sex determination in Seriola fishes. Biochemical analyses revealed that a glutamate residue present specifically in Z-type HSD17B1 attenuated interconversion between 17-keto and 17β-hydroxy steroids relative to the allelic product from the W chromosome, which harbors glycine at that position, by disrupting the hydrogen bond network between the steroid and the enzyme\'s catalytic residues. Hsd17b1 mRNA is constitutively expressed in undifferentiated and differentiating gonads of both genotypic sexes, whereas W-type mRNA is expressed only in genotypic females. Meanwhile, Cyp19a1 is predominantly expressed in differentiating ovary. We conclude that the combination of Hsd17b1 alleles determines sex by modulating endogenous estrogen levels in Seriola species. These findings strongly support the long-standing hypothesis on steroids in sex determination.

Seriola lalandi is an economically important species that is globally distributed in temperate and subtropical marine waters. Aquaculture production of this species has had problems associated with intensive fish farming, such as disease outbreaks or nutritional deficiencies causing high mortalities. Intestinal microbiota has been involved in many processes that benefit the host, such as disease control, stimulation of the immune response, and the promotion of nutrient metabolism, among others. However, little is known about the potential functionality of the microbiota and the differences in the composition between wild and aquacultured fish. Here, we assayed the V4-region of the 16S rRNA gene using high-throughput sequencing. Our results showed that there are significant differences between S. lalandi of wild and aquaculture origin (ANOSIM and PERMANOVA, P < 0.05). At the genus level, a total of 13 genera were differentially represented between the two groups, all of which have been described as beneficial microorganisms that have an antagonistic effect against pathogenic bacteria, improve immunological parameters and growth performance, and contribute to nutrition. Additionally, the changes in the presumptive functions of the intestinal microbiota of yellowtail were examined by predicting the metagenomes using PICRUSt. The most abundant functional categories were those corresponding to the metabolism of cofactors and vitamins, amino acid metabolism and carbohydrate metabolism, revealing differences in the contribution of the microbiota depending on the origin of the animals. To our knowledge, this is the first study to characterize and compare the intestinal microbiota of S. lalandi of wild and aquaculture origin using high-throughput sequencing.

This work contributes basic knowledge on larval development of Seriola rivoliana. A histological study describes the development of the digestive tract and accessory glands in S. rivoliana larvae reared under laboratory conditions at 24 °C from hatching to 30 days post-hatching (DPH). At hatching (2.6 ± 0.12 mm), larvae had an undifferentiated digestive tract with a closed straight tube and a large yolk sac with an oil globule. The liver and pancreas were observed at 1 and 2 days, and the mouth and anus opened at day 2. Enriched rotifers were visible in their digestive tract. At the beginning of the pre-flexion stage, a mixed nutritional period was observed. At day 3, exogenous feeding began; the digestive tract became differentiated into the buccopharynx, esophagus, an undifferentiated stomach, and the intestines. Zymogen granules were visible in the exocrine pancreas. At day 4, supranuclear vacuoles were present in the posterior intestine, indicating the beginning of intracellular digestion. At day 5, goblet cells were present in the esophagus and became functional at day 7 in the esophagus and intestine. The buccopharynx goblet cells developed at day 15. The presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions during the post-flexion stage occurred at day 20. This was the onset of the juvenile period and the beginning of weaning; however, a long co-feeding phase is recommended. Pyloric caeca were observed at day 30 (13.6 ± 1.6 mm). These results provide valuable information on S. rivoliana larvae biology and digestive physiology, which should be useful to improve cultivation techniques and identify ecological features involved in ontogeny.