RESUMEN
This study aimed to investigate the effect of temperature on gonadal differentiation, growth, survival, and sex ratio of Leporinus friderici reared at 25 °C or 29 °C from 50 to 240 days after eclosion (DAE) in a water recirculation system. A total of 110 fish at 50 DAE (6.7 ± 0.1 cm and 6.1 ± 0.3 g) were equally and randomly distributed in 10 boxes (90 L) (11 fish/box, 5 boxes/temperature). One fish from each experimental unit was randomly sampled at 50, 70, 90, 110, 130, 150, 170, 190, 210 and 240 DAE. Female gonadal differentiation started at 150 DAE (11.4 ± 0.0 cm and 16.4 ± 0.0 g) at 25 °C and at 170 DAE (10.7 ± 0.7 cm and 27.7 ± 8.5 g) at 29 ºC, while testes differentiation only occurred at 29 °C from 190 DAE (12.1 ± 0.0 cm and 38.0 ± 0.0 g). Of 50 fishes sampled in each condition, 17 (12 females and five males) and three (three females) displayed gonadal differentiation at 29 °C and 25 °C, respectively. Final biometric values at 29 °C were twice those obtained at 25 °C, reaching 13.9 ± 0.65 cm and 57.3 ± 10.12 g versus 11.2 ± 0.39 cm and 28.5 ± 2.95 g, respectively. While temperature clearly influenced gonadal differentiation and growth, it had inconclusive effects on sex ratio. The higher temperature (29 °C) has direct implications for the production of this species, as it accelerates growth without causing mortality.
RESUMEN
Understanding microRNA (miRNA) functions has been hampered by major difficulties in identifying their biological target(s). Currently, the main limitation is the lack of a suitable strategy to identify biologically relevant targets among a high number of putative targets. Here we provide a proof of concept of successful de novo (i.e. without prior knowledge of its identity) miRNA phenotypic target (i.e. target whose de-repression contributes to the phenotypic outcomes) identification from RNA-seq data. Using the medaka mir-202 knock-out (KO) model in which inactivation leads to a major organism-level reproductive phenotype, including reduced egg production, we introduced novel criteria including limited fold-change in KO and low interindividual variability in gene expression to reduce the list of 2853 putative targets to a short list of 5. We selected tead3b, a member of the evolutionarily-conserved Hippo pathway, known to regulate ovarian functions, due to its remarkably strong and evolutionarily conserved binding affinity for miR-202-5p. Deleting the miR-202-5p binding site in the 3' UTR of tead3b, but not of other Hippo pathway members sav1 and vgll4b, triggered a reduced egg production phenotype. This is one of the few successful examples of de novo functional assignment of a miRNA phenotypic target in vivo in vertebrates.