WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2016 35 proteins into protein fragments and small peptides. The lack of a stomach does not hinder enzymatic digestion of easily digestible proteins in fish larvae because pancreatic and intestinal enzymes are highly active (Cahu and Zambonino-Infante 1997, 2001). Studies on larvae have demonstrated that the lack of a functional stomach affects the ability of larvae to use dietary proteins (Rust 1995, Tonheim et al. 2004, 2005). Digestibility of an intact model protein increases dramatically after metamorphosis in Atlantic halibut (Fig. 2B) (Tonheim et al. 2004, 2005, Rønnestad et al. 2007). The digestibility of this protein was dramatically reduced with increased dosage, indicating a low capacity to process proteins in larval stages. However, pre-hydrolysis of the model protein resulted in relatively high absorption, independent of the amount administered to larvae (Fig. 2A). Absorption rates of pepsin-hydrolysed protein and more extensively hydrolysed protein were more than three times faster than that of intact protein in Atlantic halibut larvae (Fig. 2C) (Tonheim et al. 2005). The capacity of Atlantic halibut larvae to digest and absorb dietary proteins is limited prior to metamorphosis and this may constitute a bottleneck for intensive larval production based on formulated diets that are rich in complex proteins. Fish Larvae May Be Voracious Feeders The early larva of Atlantic halibut is a visual predator and may ingest food continuously, with no apparent satiety signal from a full gut. Under these conditions ingestion rates appear to be more or less equal to evacuation rates and there are several reports that live prey may transit the whole gut alive! Such a rapid and uncontrolled gut passage may be at best problematic because only a short time is available for digestion and absorption of proteins from ingested feeds, and therefore a significant part of dietary nutrients may be lost in faeces. Intensive larval rearing conditions in aquaculture provide abundant prey availability, often in combination with continuous light. This may result in continuous ingestion of prey and consequently reduced time for digestion, less efficient nutrient absorption and potentially increased losses of nutrients in feces. Gut transit times of ingested food is an important aspect of digestive efficiency (Rønnestad et al. 2007) and feeding regimes that involve providing distinct meals may allow more time for digestion. In addition, recent research indicates that satiety signals may be low or non-existent in first-feeding Atlantic halibut larvae (Gomes et al. 2015), also supported by the lack of a fully developed functional stomach prior to metamorphosis (Gomes et al. 2014) and the late appearance of the important satiety signal cholecystokinin in the gut prior to initiation of metamorphosis (Kamisaka et al. 2001). In vivo Monitoring of Atlantic Halibut Stomach Development Stomach development is a crucial event in the life of fish larvae. In recent work on Atlantic halibut, we have started to explore development of various aspects of stomach function by analyzing gene expression in combination with experimental observations of live larvae (Gomes et al. 2014). The motility patterns and storage capacity of the digestive tract were studied by filming fish larva, using a camera connected to a dissecting microscope (Fig. 3). In vivo observations revealed strong, but slow muscular contractions and constricted waves that travel along the intestine and an area of the proximal intestine that is distended into a bulb. Observations of live fish suggest that the stomach’s short-term reservoir function is established before metamorphosis, although the midgut acts as the main storage compartment until this function shifts to the stomach as its volume increases at metamorphosis (Fig. 4C). Peristaltic wave activity is established well before metamorphosis, but the number of contractions in the midgut decreases synchronously with the stomach’s increasing peristaltic activity at metamorphic climax (Figs. 4D1 and D3). The production of gastric acid, essential for proteolytic activity in the stomach, was assessed by measuring pH in the lumen of the gastrointestinal tract (Fig. 3B). For this, pH indicator solutions (from alkaline to acidic ranges) were administered by tube-feeding through the mouth of the larvae. We photographed larvae under a stereo microscope and compared the color of the intestinal fluid with a set of standards (Figs. 4D2 and D4). In stage 9, the lumen pH of the stomach is clearly acidic (below 3.5) (Fig. 4D4) and we observed a rapid color change from acidic to alkaline when the pH indicator solution passed through the pyloric sphincter into the midgut. To verify this in vivo study, we applied molecular tools to quantify the expression of genes that act as specific markers for stomach proteolytic function (indicating ability to digest proteins) like pepsinogen and gastric proton pump. Proteolytic activity in the stomach starts during the climax of metamorphosis with synchronized expression of pepsinogen A2 and both gastric proton pump subunit transcripts (for secreting H+). Taken together, the morphological changes and key elements essential for the stomach’s proteolytic activity such as proton pump and pepsinogen expression and HCl production seems to be coordinated and occur in parallel. (CONTINUED ON PAGE 36) FIGURE 4. (A) Ontogeny of Atlantic halibut larvae reared at 11 C, the time in days post first feeding (dpff) and duration of metamorphosis is indicated. (B) A cartoon shows the external appearance of developing Atlantic halibut at stage 6 (premetamorphic) and stage 9 (metamorphic climax). (C) 3D models of the Atlantic halibut developing digestive tract during stage 6 (C1 and 2) and stage 9 (C3 and 4). The presumptive stomach (pSt) is in light blue and digestive tract lumen is in darker blue. (D) Motility patterns (D1 and 3) are indicated by a dashed line. Scale bar = 1 mm. pH changes in the digestive tract of Atlantic halibut larvae at stage 6 (D2) and stage 9 (D4) with special focus on the stomach (St) area. The color obtained inside the stomach is compared to the standards: pSt has a pH >7.5; midgut (Mg) and hindgut (Hg) has a pH > 8.0 for stage 6, while at stage 9 the halibut St has a pH <3.5 and the Mg and Hg has a pH > 6.5. Scale bar=0.5 mm. Modified from Gomes et al. (2014).
RkJQdWJsaXNoZXIy MjExNDY=