Effect of development
It is suggested that the onset of exogenous feeding in fish larvae can be considered when a great part of the population has assimilated the prey, and larval growth is detected (Yúfera and Darias 2007). According to this definition, S. rivoliana larvae did not reach the onset of exogenous feeding, since total length even shrank during oil droplet exhaustion (Fig. 5a) which is a common sign of starvation in fish larvae. In Lutjanus peru larvae, changes in body form due to starvation were a consequence of body mass loss resulting from the summed changes in organs and tissues (Peña et al. 2021). However, in that species, organs such as head height do not undergo significant changes in body size, suggesting that this segment is preferentially spared from catabolic processes or catabolized more slowly than other tissues. It may be explained by the fact that efficiency in prey capture relies upon the head and associated organs that are likely to be critical for survival at the early stages of fish larval development (Von et al. 1996). This coincides with the present study since the eye diameter of S. rivoliana larvae never shrank during development, while head dimensions decreased until oil droplet exhaustion.
In this study, we suggest some potential causes of food deprivation e.g. poor illumination, white color of containers and small size of larvae, which possibly prevented the S. rivoliana larva from being unable to learn to feed. Additionally, abnormality in morphological development, alimentary tract deterioration and truck musculature may cause food deprivation after yolk reabsorption even when live food is available (Heming et al. 1982).
Effect of temperature regime
Daily temperature fluctuation (DTF) was applied in the very early embryo of S. rivoliana. The development rate was not affected by DTF compared to the control at 24°C constant (CTE). This phenomenon has already been reported for this species (Pacheco-Carlón et al. 2021) and is widely observed in other fishes (Johns 1981; Coulter et al. 2015; Lim et al. 2017; Pisano et al. 2019).
To our knowledge, there are no reports of hatching success from embryos of marine fish species incubated in daily temperature fluctuation. However, the lack of differences in the hatching success herein observed agrees with studies performed in freshwater species such as Zebrafish (Villamizar et al. 2012; de Alba et al. 2022), Fathead minnows (Coulter et al. 2015) and Nile tilapia (Santo et al. 2020). On the other hand, yolk sac conversion efficiency into larval tissue was also unaffected by temperature regime since there were no differences in body length and yolk sac volume in hatched larvae, which agrees with the observations on Fathead minnows (Coulter et al. 2015).
Pericardial/yolk-sac edema incidence at mouth-opening larvae was not reduced by DTF as expected. In a previous study, a relatively lower incidence of edema was found in embryos incubated under fluctuating temperatures and a high constant of 28°C, compared to a higher edema incidence at a constant of 20–26°C (Pacheco Carlón et al. 2022). Therefore, it will be interesting to test a daily temperature fluctuation with an average > 24°C in further experiments.
Daily temperature fluctuation has been seen to improve larval growth in advanced stages. For example, Senegalese sole larvae present higher growth at 25 DAH when reared in thermocycles (19.2 ⇄ 22.1°C) compared to constant temperatures (Blanco-Vives et al. 2010). On the other hand, Zebrafish larvae from thermocycles grew and survived better than in constant temperature at 15 days after fertilization (de Alba et al. 2022). In the case of the present study, daily temperature fluctuation caused a lower degree of body shrinkage, coupled with improved survivability at oil droplet exhaustion (5.5 DAH) (Fig. 6a, 7c). This adds to the existing evidence indicating that fish larvae tend to perform better under cycling temperatures. However, little is reported about the molecular mechanisms activated in early development by thermocycles. According to Santo et al. (2020), thermocycles lead to higher growth performance in Nile Tilapia larvae at 13 DPF and they observed that most of analyzed genes (chymotrypsinogen, lipase, maltase) displayed daily rhythms in the thermocycle group but not at constant temperature. They hypothesized that the higher growth rate observed in the thermocycle larvae could have happened due to improved synchronization of feed digestion and utilization.
On the other hand, it is worth noting that larval survival was beneficially affected only by daily temperature fluctuation (no effect by rotifer enrichment), which supports previous findings that pointed out that during the transition to exogenous feeding, the impact of abiotic factors meanly temperature and photoperiod are more important than feeding frequency and food quality on fish survival (McGurk 1984; Gardeur et al. 2007).
Effect of rotifer enrichment
Larvae fed rotifer enriched with Domestic and S.presso emulsions suffered to a lesser degree the ravages of starvation compared to larvae from Ori-green in terms of larval size (Fig. 6). In this regard, larvae of Florida pompano (Trachionus carolinus) fed with rotifer enriched with Ori-green were significantly smaller than the other rotifer enrichment treatments at 6 DPH, without significant differences in survival between larvae-fed rotifers enriched with Protein Selco Plus (S.presso) (34.4%) and Ori-green (30.6%) at 9 DPH (Cavalin and Weirich 2009). In pigfish, the survival of larvae-fed rotifers enriched with DHA Protein Selco was significantly higher than Ori-green, however, no significant differences in larval lengths at 5 DPH were observed between treatments (Broach et al. 2017).
Good levels and ratios of essential fatty acids in S.presso emulsion and poor levels of DHA (22:6n-3) and ARA (20:4 m-6) in Ori-green have been previously documented by Partridge et al. (2014). Watanabe (1993) suggested that deficiency in DHA will result in reduced growth due to poor dietary value. On the other hand, ARA is vital in the formation of eicosanoids in early stages of marine fish larvae, improving stress tolerance, pigmentation, growth, and survival (Park et al. 2006). A deficiency of these essential nutrients may have led to the worst growth performance in Ori-green in the present study.
Digestive enzyme study is the first approach to describing and refining the nutritional status during fish ontogeny (Lavajoo et al. 2023). In general, little information is available on the regulatory mechanisms at the molecular level related to diet quality (Yúfera et al. 2018). In the present study, the expression of trypsin gen was greater than lipase in larvae aged 3.7 DAH. This pattern is supported by previous results of a major trypsin-specific activity of 0.97 U mg protein− 1 compared to a lower lipase-specific activity of 0.60 U mg protein− 1 at 5 DAH in S. rivoliana (Teles et al. 2019). In turn, a higher expression of try gen was observed in Domestic emulsion treatment, where a larger larva was obtained at the end of the experiment. In this respect, trypsin is the most important enzyme linked to early digestive capacity in fish (Ronnestad et al. 2013), making larvae capable of digesting proteins from mouth opening until the stomach fully develops (Ribeiro et al. 2002). Regarding lipase, the expression was higher in the Ori-green treatment, however, this rotifer enricher is characterized by a low lipid content (Partigrade et al. 2014). Rather, we suggest that the greater lp expression was given due to increased metabolism of the oil droplet from this treatment (Teles et al. 2019).
The study of potential consequences of early nutritional and environmental challenges on later digestive capacity is still in its early steps (Yúfera et al. 2018). It will be necessary to evaluate rotifer enrichment and temperature oscillations under improved experimental conditions which could improve the survival of larvae in advanced stages where they acquire a greater digestion capacity. In the case of Seriola rivoliana, the presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions occurred on day 20 after hatching where they reach an efficient digestive process (Teles et al. 2017).