Characterization and localization of primordial germ cells in Totoaba macdonaldi.

The totoaba, Totoaba macdonaldi, is an endangered fish of the Gulf of California with high economic and ecological potential. Therefore, our purpose was to characterize the Primordial Germ Cells (PGCs) of this Sciaenid with two objectives: (1) to provide the basis for PGCs cryopreservation to preserve the genetic resources and (2) to take the first step to know the gonadal genesis and sex differentiation of totoaba. Immunofluorescence analysis performed from 2-cell stage to 8-day after hatch (DAH) shows that Vasa protein is specific for PGCs. These cells were first observed in the peripheral and dorsal regions of the blastodisc at approximately the 50%-epiboly stage and migrated to both sides of embryo body during the development. Finally, at 7 DAH the PGCs of the hatching embryo reached the place where the gonad will be developed. Histology analysis of larvae showed a genital ridge with enclosed PGCs on the dorsal side of the peritoneum at 9 DAH, gonadal primordium growth was observed at 11 DAH as a result of the interaction between PGCs and somatic cells derived from the peritoneum. Results of qPCR showed that vasa expression was restricted to the embryonic and early larval development, highest values were observed in 2-cell and mid-blastula stage suggesting the maternal inheritance of vasa mRNA. These findings support the hypothesis of preformation in T. macdonaldi PGCs. The migration pattern of PGCs allow us to recommend the isolation and subsequent cryopreservation of these cells before 7 DAH when the embryonic and larval development is given at 21 °C.

Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny.

In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24h cycle in larvae reared under a 12h light/12h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60days post-hatch. In each sampling day, larvae were sampled every 3h during a complete 24h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes.

Cloning and molecular ontogeny of digestive enzymes in fed and food-deprived developing gilthead seabream (Sparus aurata) larvae.

We have determined the expression pattern of key pancreatic enzymes precursors (trypsinogen, try; chymotrypsinogen, ctrb; phospholipase A2, pla2; bile salt-activated lipase, cel; and α-amylase, amy2a) during the larval stage of gilthead seabream (Sparus aurata) up to 60days after hatching (dph). Previously, complete sequences of try, cel, and amy2a were cloned and phylogenetically analyzed. One new isoform was found for cel transcript (cel1b). Expression of all enzyme precursors was detected before the mouth opening. Expression of try and ctrb increased during the first days of development and then maintained high values with some fluctuations during the whole larval stage. The prolipases pla2 and cel1b increased from first-feeding with irregular fluctuation until the end of the experiment. Contrarily, cel1a maintained low expression values during most of the larval stage increasing at the end of the period. Nevertheless, cel1a expression was negligible as compared with cel1b. The expression of amy2a sharply increased during the first week followed by a gradual decrease. In addition, a food-deprivation experiment was performed to find the differences in relation to presence/absence of gut content after the opening of the mouth. The food-deprived larvae died at 10dph. The expression levels of all digestive enzymes increased up to 7dph, declining sharply afterwards. This expression pattern up to 7dph was the same observed in fed larvae, confirming the genetic programming during the early development. Main digestive enzymes in gilthead seabream larvae exhibited the same expression profiles than other marine fish with carnivorous preferences in their juvenile stages.

Daily rhythms of clock gene expression and feeding behavior during the larval development in gilthead seabream, Sparus aurata.

Light is the main environmental time cue which synchronizes daily rhythms and the molecular clock of vertebrates. Indeed, alterations in photoperiod have profound physiological effects in fish (e.g. reproduction and early development). In order to identify the changes in clock genes expression in gilthead seabream larvae during ontogeny, three different photoperiods were tested: a regular 12L:12D cycle (LD), a continuous light 24L:0D (LL) and a two-phases photoperiod (LL + LD) in which the photoperiod changed from LL to LD on day 15 after hatching (dph). Larvae were sampled on 10, 18, 30 and 60 days post-hatch (dph) during a 24 h cycle. In addition to the expression of clock genes (clock, bmal1, cry1 and per3), food intake was measured. Under LD photoperiod, larvae feed intake and clock genes expression showed a rhythmic pattern with a strong light synchronization, with the acrophases occurring at the same hour in all tested ages. Under LL photoperiod, the larvae also showed a rhythmic pattern but the acrophases occurred at different times depending on the age, although at the end of the experiment (60 dph) clock genes expression and feed intake rhythms were similar to those larvae exposed to LD photoperiod. Moreover, the expression levels of bmal1 and cry1 were much lower than in LD photoperiod. Under the LL + LD photoperiod, the 10 dph larvae showed the same patterns as LL treatment while 18 and 30 dph larvae showed the same patterns as LD treatment. These results revealed the presence of internal factors driving rhythmic physiological responses during larvae development under constant environmental conditions. The LL + LD treatment demonstrates the plasticity of the clock genes expression and the strong effect of light as synchronizer in developing fish larvae.