WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2016 29 (CONTINUED ON PAGE 30) of a few species reveal that gut contents are not the same over a daily cycle. Larvae feed preferably during crepuscular periods and during the daytime, each species having a particular feeding pattern. Although larvae of most marine fish species are selective visual feeders that depend on light for feeding success, not all species require good illumination to feed. Nocturnal habits or feeding in turbid waters have also been described in some species, while others transition from diurnal to nocturnal feeding as development progresses. The belief that longer illumination leads directly to greater ingestion and faster growth leads many hatcheries to set up a 24-h illumination-and-feeding practice during the first weeks of larval rearing. Nevertheless, it is necessary to look more in depth at ingestion and digestion processes to know whether photoperiod and feeding protocol affect larval growth and fitness. The first step to advance this understanding is to elucidate whether food is being ingested and digested during a 24-h cycle. In fish larvae, the best approximation of the digestive reaction to a given feed is to determine the level of activity of digestive enzymes. Our research on larvae and early juvenile biology focuses primarily on two species of commercial interest in Mediterranean aquaculture, the gilthead seabream Sparus aurata and the Senegal sole Solea senegalensis. The larvae of gilthead seabream are pelagic throughout larval development and swim continuously during the juvenile stage. Senegal sole is a benthic flatfish, but during the early larval stage it has pelagic life for approximately two weeks. After eye migration and change of anatomical symmetry, post-larvae settle on the bottom, adopting a sedentary benthic habit with just occasional swimming. Our results with gilthead seabream demonstrate that, under continuous food availability and alternating light-dark photoperiod, fish larvae feed according to a circadian rhythm. We found a progressive increase in gut contents during the light period, reaching a maximum when the light is turned off. Then the gut is evacuated during the dark period, becoming empty in a few hours. This pattern is maintained during the whole larval stage, lasting about two months in this species. The expression of the positive elements of the molecular clock also oscillates with a phase similar to that of the food level in the gut (Fig. 2). It is very clear that it is the light-dark cycle that is synchronizing this 24-h feeding cadence. Interestingly, when the alteration of day and night is absent under continuous illumination, the clock genes and the feeding pattern maintains a circadian rhythm, although asynchronous with the theoretical natural illumination cycle (Mata-Sotres et al. 2015). In some other fish species, periodic oscillation of clock genes tends to disappear with time when the environmental pacemaker is absent. In our study, it was maintained during the whole larval stage. In accord with the rhythmicity of feed intake, it would be expected that some kind of rhythm also occurs during digestion. A battery of pancreatic and intestinal digestive enzymes participates in hydrolysis of different macronutrients during their passage through the digestive tract. The question is whether or not different enzymes are acting also in a cyclic mode. The alkaline proteases, specifically trypsin, follow an almost parallel pattern to that exhibited by feed intake under a light-dark photoperiod (Fig. 3, Mata-Sotres et al. 2016). Trypsin is considered the key digestive enzyme in fish larvae, taking into account the large amount of amino acids that are necessary to build new tissues during fast growth. In contrast, lipases and amylases, the enzymes responsible for hydrolysis of fats and carbohydrates, do not follow a clear daily rhythm associated with feeding pattern. They exhibit irregular changes during the cycle, although always tending to decrease in the last hours of the dark period. However, at the end of the larval period, when larvae are nearly juveniles, the activity of all digestive enzymes follows a rhythm associated with the feeding pattern. Like feeding behavior, we found that the rhythmicity of digestive enzyme activities associated with the feeding pattern also occurs under continuous illumination. At the molecular level, the temporal expression pattern of mRNA transcript of proenzymes is different from corresponding activities. Such discordance is not surprising because molecular expression and biochemical activity are the net result of the produced and used molecules and do not necessarily exhibit similar patterns. Nevertheless, the greatest gene expression occurs at the transition from night to day, in concordance with negative elements of the molecular clock. This may indicate regulation of genes for digestive enzymes by clock genes to prepare the enzymatic machinery for the coming feeding cycle. This anticipatory response to feeding could allow better utilization of the nutrients. In Senegal sole, the same daily rhythm mentioned for seabream in ingestion and trypsin activity is observed during the pelagic stage of larvae. However, after eye migration and acquisition of benthic life, post-larvae start to eat throughout the daily period, changing first to a higher ingestion during the night and finally to clear nocturnal feeding in the juvenile stage (Navarro-Guillén et al. 2015). During this benthic post-larval stage, trypsin activity still maintains part of the rhythmicity. The activity is practically constant throughout the whole day, except for a short break during the transition from night to day. The activity of lipases and carbohydrases also exhibit this pattern. FIGURE 3. Daily patterns of gut content and trypsin activity in gilthead seabream larvae under continuous feeding and light/dark (LD) or continuous illumination (LL) photoperiod. Modified from Mata-Sotres et al. (2015) and Yúfera et al. (2015). Dph: days after hatching.
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