Yuko Kamisaka

The supply of amino acids during early feeding stages of marine fish larvae: a review of recent findings

In marine fish larvae, the sum of protein deposition, turnover and catabolism necessary for their rapid growth dictates a high amino acid (AA) requirement. Once the yolk is exhausted, the digestive tract becomes the vital organ that ensures a steady supply of dietary AA to the growing larval tissues. In this paper, we discuss the demand and availability of AA (free and polymerised pools) in relation to larval digestive capacity. The sources of AA from compound and live diets are described, and the early regulatory roles of cholecystokinin (CCK) and a retrograde peristaltic activity are highlighted.

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects.

SUMMARY A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2838 bp long, with an open reading frame of 2190 bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylation-rich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8–12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.

Cholecystokinin mRNA in Atlantic herring, Clupea harengus--molecular cloning, characterization, and distribution in the digestive tract during the early life stages.

The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.

Distribution of cholecystokinin-immunoreactive cells in the digestive tract of the larval teleost, ayu, Plecoglossus altivelis.

The ontogenetic development of cholecystokinin-immunoreactive (CCK-IR) cells was studied in larval ayu, Plecoglossus altivelis. This species has a straight digestive tract during the larval phase. CCK-IR cells were present in all the larvae from the day of hatching (0 days after hatching, DAH). An immunoreaction to anti-trypsinogen antibody was also detected in the pancreas at this stage. The distribution pattern of the CCK-IR cells was quantified by recording the location of CCK-IR cells at 1, 16, and 76 DAH. Although the number of CCK-IR cells increased during development, the distribution pattern of CCK-IR cells did not change until 76 DAH. The CCK-IR cells were scattered throughout the midgut, with the exception of the regions adjacent to the pyloric and rectal sphincters. No CCK-IR cells were detected in the foregut or the hindgut. This distribution pattern differs from species with rotated digestive tracts, whose CCK-IR cells are only found in the anterior part of the midgut. CCK-IR cells seem to be located in regions where the ingested food is retained and thus can easily receive chemical signals from the food and the digestive process in order to control the release of the hormone.

Expression of the oligopeptide transporter, PepT1, in larval Atlantic cod (Gadus morhua)

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract.

Ontogeny of cholecystokinin-immunoreactive cells in the digestive tract of bluefin tuna, Thunnus thynnus , larvae

The appearance and distribution of cholecystokinin (CCK)-immunoreactive (IR) cells in the digestive tract of cultured developing larvae of bluefin tuna, Thunnus thynnus , were investigated by immunohistochemistry. The CCK-IR cells had an elongated triangular shape and a thin apex pointing towards the lumen of the gut. Very few weakly stained CCK-IR cells were found at hatching (0 days after hatching, DAH) and first feeding (3 DAH), while strongly stained CCK-IR cells were detected in all the larvae examined after 12 DAH. CCK-IR cells were scattered both in the pyloric caeca and the anterior intestine at 12 DAH, while the distribution was restricted to the pyloric caeca at 22 DAH. The relationships between the ontogeny of CCK and pancreatic enzymes are discussed.

Distribution of cholecystokinin-immunoreactive cells in the gut of developing Atlantic cod Gadus morhua L. larvae fed zooplankton or rotifers

One of the main gastrointestinal hormones, cholecystokinin (CCK), was studied in order to advance understanding of the control of the digestive process in Atlantic cod Gadus morhua larvae after onset of first feeding. Larvae were fed either natural zooplankton or enriched rotifers in similar rearing systems and sampled from hatching to 22 days post-hatch (dph). CCK was visualized by immunohistochemistry and the first CCK-immunoreactive (IR) cells were detected at 8 dph corresponding to 6 days after first feeding. The CCK-IR cells were mostly found in the anterior midgut, and the number of CCK-IR cells was lower in the posterior midgut. They were also present in the hindgut of some of the larvae, but not in the foregut. No clear differences were found in the ontogenetic appearance and the distribution pattern of CCK-IR cells between the two dietary treatments. This indicates that the onset of CCK production in the gut as well as the spatial distribution of the CCK-IR cells is not differentially affected by these diets. To what extent the hormone production itself is influenced by dietary factors needs to be studied by more sensitive methods.

Functional modifications associated with gastrointestinal tract organogenesis during metamorphosis in Atlantic halibut (Hippoglossus hippoglossus)

BackgroundFlatfish metamorphosis is a hormone regulated post-embryonic developmental event that transforms a symmetric larva into an asymmetric juvenile. In altricial-gastric teleost fish, differentiation of the stomach takes place after the onset of first feeding, and during metamorphosis dramatic molecular and morphological modifications of the gastrointestinal (GI-) tract occur. Here we present the functional ontogeny of the developing GI-tract from an integrative perspective in the pleuronectiforme Atlantic halibut, and test the hypothesis that the multiple functions of the teleost stomach develop synchronously during metamorphosis.ResultsOnset of gastric function was determined with several approaches (anatomical, biochemical, molecular and in vivo observations). In vivo pH analysis in the GI-tract lumen combined with quantitative PCR (qPCR) of α and β subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicated that gastric proteolytic capacity is established during the climax of metamorphosis. Transcript abundance of ghrelin, a putative orexigenic signalling molecule produced in the developing stomach, correlated (p < 0.05) with the emergence of gastric proteolytic activity, suggesting that the stomach’s role in appetite regulation occurs simultaneously with the establishment of proteolytic function. A 3D models series of the GI-tract development indicated a functional pyloric sphincter prior to first feeding. Observations of fed larvae in vivo confirmed that stomach reservoir function was established before metamorphosis, and was thus independent of this event. Mechanical breakdown of food and transportation of chyme through the GI-tract was observed in vivo and resulted from phasic and propagating contractions established well before metamorphosis. The number of contractions in the midgut decreased at metamorphic climax synchronously with establishment of the stomach’s proteolytic capacity and its increased peristaltic activity. Putative osmoregulatory competence of the GI-tract, inferred by abundance of Na+/K+-ATPase α transcripts, was already established at the onset of exogenous feeding and was unmodified by metamorphosis.ConclusionsThe functional specialization of the GI-tract was not exclusive to metamorphosis, and its osmoregulatory capacity and reservoir function were established before first feeding. Nonetheless, acid production and the proteolytic capacity of the stomach coincided with metamorphic climax, and also marked the onset of the stomach’s involvement in appetite regulation via ghrelin.