BACKGROUND
the divergent gastrointestinal  tract morphology and feeding strategies between larval and adult phases are adaptations to fundamentally different habitats and food resources  <cit> .
the post-embryonic maturation of the digestive system is a key event in the life history of vertebrates and essential for survival.
thyroid hormone  driven metamorphosis plays a crucial role in the functional maturation of the gi-tract and in shaping its morphology to the adult form  <cit> .
remodelling of the gi-tract from larva to adult has been extensively studied in xenopus <cit> .
in this organism, the intestine is transformed under the influence of ths from a long coiled tube into a complex organ with a differentiated stomach and small intestine  <cit> .
this involves epithelial and mesenchymal proliferation, smooth muscle thickening and the formation of intestinal folds.
several studies have described the cellular mechanisms responsible for this remodelling in amphibians  <cit> , yet little is known about their impact on tissue function in vertebrates, particularly the multiple functions integrated in the stomach.
a striking feature of vertebrate metamorphosis is the organogenesis of the stomach.
in early developmental stages of fishes and anurans the stomach is often absent and part of its function may be carried out by the intestine.
the main roles of the vertebrate stomach are storage of ingested food, secretion of hydrochloric acid  and pepsinogen, and mechanical breakdown and mixing of food with gastric secretions  <cit> .
thus, in larvae of altricial-gastric species, such as the atlantic halibut, the absence of a stomach limits the ability to digest dietary protein when exogenous feeding is initiated  <cit> .
this is one of the reasons why most studies of gi-tract development during metamorphosis have focused on stomach development and consider the appearance of gastric glands as an adequate indicator of a fully developed stomach  <cit> .
however, it has become clear that the histological identification of gastric glands does not indicate that the stomach is fully functional.
hence, the stomach’s proteolytic function is best indicated by pepsin activity  <cit>  and pepsinogen content  <cit> .
to better understand the efficiency of digestive processing during fish ontogeny, several studies compared expression profiles of pepsinogen and the gastric proton pump , localized in the hcl secreting oxynticopeptic cells  <cit> .
murray et al.
<cit>  have used histology and pepsinogen transcript analysis to study the ontogeny of the stomach in atlantic halibut and showed that the appearance at 66 days post-hatching  of gastric glands preceded expression of pepsinogens a <dig> and a <dig> transcripts at  <dig> dph.
however, the impact of metamorphosis on other important functions of the stomach or gi-tract development in general has largely been overlooked in flatfish.
in addition to acid production and proteolysis the vertebrate stomach also has reservoir functions.
after ingestion, the stomach stores and predigests food, then delivers the chyme to the midgut for further digestion and subsequent nutrient absorption  <cit> .
the storage function of the stomach relieves juvenile and adult fish from the need to constantly feed like the stomachless larval stages.
establishment of the stomach as a reservoir requires functional sphincters  and well developed neural and smooth muscle layers.
the mechanical mixing and transport of food through the gi-tract is achieved by specific motility patterns and by matching peristalsis with the release of digestive enzymes.
this process plays a central role in effective food processing , though very few studies have targeted gi-tract movements in fish larvae.
the advantage of using fish larvae, such as atlantic halibut, is their optic transparency that is maintained until metamorphosis.
this permits direct visual observations of the gi-tract and its motility patterns in live animals.
pittman et al.
<cit>  reported peristaltic contractions in atlantic halibut larvae, in the anterior intestine at  <dig> dph.
in juvenile atlantic halibut gi-tract both anterograde  and retrograde  contraction waves were described  <cit> , and identical patterns were also observed in embryos and larvae of the stomachless zebrafish   <cit> .
the stomach produces hormones involved both in the regulation of appetite and digestion.
ghrelin is an example of a hormone that is mainly produced in the stomach and acts as a stimulator of food intake  <cit> .
in mammals, ghrelin has also been suggested to stimulate gastric acid secretion and motility  <cit> .
the function of ghrelin in fish larvae is still poorly described, but it has been proposed as an indicator of the stomach’s involvement in appetite regulation in developing fish  <cit> .
in atlantic halibut, ghrelin gene expression increases during the climax of metamorphosis, coinciding with stomach development  <cit> .
ghrelin is abundant in the developing gastric glands and several osmoregulatory tissues.
additionally, its co-expression with na+/k+-atpase suggests a putative role in hydromineral balance  <cit> .
yet, the role of ghrelin in appetite regulation, motility and osmoregulation is unknown, as well as its link to the proteolytic and reservoir function of the stomach in atlantic halibut during metamorphosis.
this study aims to establish the impact of the agastric-gastric transition on the functional role of the post-embryonic gi-tract remodelling that occurs during metamorphosis in atlantic halibut, a flatfish species of high commercial interest for the northern european and north american aquaculture industry.
to map the changes in gi-tract development and establish events linked to th-driven metamorphosis we constructed a series of 3d models of the morphological and spatial organization of the digestive organs in representative developmental stages.
we tested the hypothesis that the development of the multiple stomach functions is synchronous and linked to its physical appearance at metamorphosis.
the proteolytic function of the stomach was studied using in vivo ph analyses combined with expression profiles of the specific gene markers h+/k+-atpase α and β subunit and pepsinogen a <dig> using quantitative pcr .
stomach filling and reservoir function were assessed by in vivo visual studies of the transparent larvae at prometamorphosis and climax of metamorphosis.
the putative role of a fully functional stomach in appetite regulation was assessed by measuring ghrelin transcript abundance.
the establishment of gi-tract motility patterns during development was determined by in vivo observations and the involvement of the gi-tract in osmoregulation was assessed by measuring the abundance of na+/k+-atpase α subunit transcripts.
RESULTS
3d reconstruction of digestive organs
3d models of the morphology of the digestive system during development were reconstructed from a series of histological sections.
location and size of the gi-tract and its associated organs, such as liver, endocrine and exocrine pancreas, and gallbladder, were observed from stage  <dig>  until the post-metamorphic stage  <dig> .
the gi-tract includes a narrow foregut , midgut, and a short hindgut  .
the anterior region of the midgut, just after the pyloric sphincter , was larger in diameter, i.e.
more voluminous, compared to the rest of the midgut.
this feature was maintained during gi-tract ontogeny .
both ps  and ileorectal sphincter  were identified from stage  <dig> onwards .
pyloric caeca became evident as projections from the most anterior part of the midgut at stage  <dig> .
the stomach was well-differentiated at stage  <dig> and the gastric glands were visible on histological sections .
the luminal volume of the gi-tract increased during development, particularly in the two last stages analysed  .
the stomach volume from 9a to  <dig> increased from  <dig> to  <dig> nl, respectively and corresponded to an  <dig> fold increment .
the values were calculated from the 3d models using imaris measurementspro.
agi-tract tissue volume = gi-tract outer layer - gi-tract inner layer.
the liver was positioned under the foregut and anterior to the ascending loop of the midgut  and its volume steadily increased during development .
the exocrine pancreas was observed between the presumptive stomach and the anterior part of the midgut at stage  <dig> and it surrounded this midgut area throughout ontogeny .
in the endocrine pancreas, a clearly distinguishable islet of langerhans was observed close to the gallbladder at stage  <dig> .
in contrast to the other digestive organs, the increment in the normalized volume of endocrine and exocrine pancreas was low and negative, respectively, between stages 9a and  <dig> .
the yolk-sac, positioned under the gi-tract at stage  <dig>  decreased in size after the initiation of exogenous feeding and a small vestige remained besides the liver at stage  <dig> .
the gallbladder was observed on the right-hand side between the exocrine pancreas and the liver, and maintained this position in all the developmental stages analysed .
the pancreatic duct and the bile duct opened next to each other into the lumen at the median plane of the anterior midgut, just after the ps .
cloning and phylogenetic characterization of pepsinogen a <dig>  ghrelin, gastric proton pump subunits and na+/k+-atpase subunit α
the complete coding sequence  of atlantic halibut pepsinogen a <dig> was  <dig> bp and was submitted to genbank under accession no.
kf <dig> .
the amino acid  sequence of pepsinogen is relatively well-conserved among teleost fish and, as expected, more variable when compared to other vertebrate pepsinogens.
for instance, halibut pepsinogen a <dig> shared respectively 88% and 64% aa sequence identity with winter flounder  pepsinogen a form iib and iia, but only 52% and 48% identity with homologues from xenopus laevis and human, respectively .
the cdna fragments cloned for atlantic halibut h+/k+-atpase α subunit  and na+/k+-atpase α subunit  were deposited in genbank with the accession numbers kf <dig> and kf <dig>  respectively .
the cds for h+/k+-atpase β subunit of  <dig> bp was cloned and submitted to genbank with the accession no.
kf <dig> .
phylogenetic analysis of the α subunit of the gastric proton pump and na+/k+-atpase, and vertebrate homologues  generated two major clades, one corresponding to h+/k+-atpase and the other to na+/k+-atpase.
phylogenetic analysis of the β subunit  generated a tree with two major clades that shared the same general topology as the phylotree for the α subunit with the h+/k+-atpase and na+/k+-atpase clustered independently.
atlantic halibut h+/k+-atpase α subunit clustered most closely with teleost homologues, with which it shared 94% aa sequence identity, and increased to 98% identity with winter flounder and atlantic cod .
lower aa sequence identity  was found when atlantic halibut h+/k+-atpase α subunit was compared to atlantic halibut na+/k+-atpase α subunit  and to other vertebrate counterparts .
the atlantic halibut na+/k+-atpase α subunit clustered with an antarctic eelpout  homologue  and shared approximately 88% aa identity with other teleost gene homologues.
h+/k+-atpase β subunit clustered as expected within the teleost clade  and shared rather low identity with its human homologue .
atlantic halibut h+/k+-atpase β subunit did not share more than 39% aa sequence identity with the atlantic halibut na+/k+-atpase β subunit.
ontogenetic expression pattern and correlation analysis
the developmental expression profiles of pepsinogen a <dig>  h+/k+-atpase α and β subunits, na+/k+-atpase α subunit and ghrelin were analysed by qpcr in the gi-tract of individual atlantic halibut larvae .
the gene expression of both gastric proton pump subunits were significantly  correlated  and had parallel expression patterns, with a sharp and significant  increase at climax and in post-metamorphic stages .
pepsinogen a <dig> was significantly  correlated with the expression profile of the gastric proton pump α  and β  subunits .
a significant  increase during stage  <dig> was observed for pepsinogen a <dig> and its expression peaked in the post-metamorphic stage.
ghrelin mrna transcript abundance increased gradually and significantly  during the proclimax/climax of metamorphosis, and attained a maximum in the post-metamorphic stage .
moreover, ghrelin transcript abundance and proteolytic activity during gi-tract ontogeny were significantly correlated .
expression of na+/k+-atpase α subunit mrna was detected in all developmental stages, with significantly  more transcripts at stage  <dig>
estimation of ph in the lumen of stomach and detection of acid production
the ph assessment in the lumen of the stomach and midgut/hindgut during post-embryonic development was based on the colour observed after the injection of ph indicator solutions .
the ph in the midgut/hindgut remained alkaline  in all the developmental stages analysed .
the presumptive stomach also had an alkaline ph with values above  <dig>  until stage  <dig>  gradual acidification was observed in the stages corresponding to the climax of metamorphosis.
transition from an alkaline to an acidic ph in the stomach lumen was evident at stage 9a, when the injected sol cpr remained purple but the sol mcp gave a yellow coloration .
the lumen of the stomach was clearly in the acidic range  at stage 9b, as revealed by the yellow colour in the stomach following administration of both cpr and bpb solutions.
the presented ph values are based on visual observations of colour changes after the administration of three ph indicator solutions.
analysis of gi-tract motility
spontaneous propagating contractions were observed in the gi-tract at prometamorphosis  and climax of metamorphosis  .
due to considerable individual variation, number and frequency of contractions could not be grouped and are presented for each individual analysed .
two types of contractions were observed in the midgut region  <dig>  and  <dig> : phasic and propagating waves .
the propagating contractions observed in mg <dig> were retrograde waves that originated in area “a” and moved towards the mouth.
however, in mg <dig> most of the propagating contractions originated just under the ps and were anterograde waves that moved in an anal direction.
motility activity in both midgut regions was detected at stage  <dig> with a frequency ranging from  <dig>  to  <dig>  min- <dig>  depending on the individual and type of contraction.
at stage  <dig>  relatively few spontaneous contractions of short duration were observed in the midgut.
during the climax of metamorphosis, contractions in the stomach were registered in all individuals, in contrast to stage  <dig> when motility in the presumptive stomach was only observed in one larva.
the rectal contraction  was a mixture of retro- and anterograde contractions and were observed in both stages  <dig> and  <dig> with similar frequencies in most of the individuals analysed.
propagating
phasic
propagating
phasic
n
n
n
n
n
n
n
propagating and phasic contractions are stated for midgut  <dig> and midgut  <dig> regions.
frequency is the number of contractions registered  per min.
*not possible to quantify phasic and propagating wave contractions.
the affected gi-tract segments were constantly  contracted during the whole observation period .
discussion
in all altricial-gastric species, the gi-tract undergoes dramatic remodelling during th-driven metamorphosis.
the gi-tract changes from a simple tubular form into a more complex folded structure.
at the same time the stomach becomes a distinct compartment and continues to acquire its multiple functions through metamorphosis.
there are surprisingly few studies examining and integrating the anatomical and functional changes in the gi-tract associated with this th-driven event.
in amphibians such as xenopus, it is well established that ths induce gi-tract remodelling leading to intestinal shortening and the development of crypts and villi  <cit> .
the remodelling of the xenopus digestive tract is a consequence of changes in th regulated genes, including sonic hedgehog/bone morphogenetic protein- <dig>  and tolloid/bmp- <dig> <cit> .
however, in contrast to amphibians, few studies have linked gi-tract remodelling with th-driven metamorphosis in teleost fish, in particular the pleuronectiformes.
the present study used a series of 3d models to reconstruct the ontogeny of atlantic halibut gi-tract morphology and its volume changes during larval development.
using molecular and functional analysis, the modification of gi-tract function from the premetamorphic stage  <dig> until the end of metamorphosis  was established.
insight was obtained into the way in which post-embryonic differentiation of the gi-tract and the growth of a new organ, the stomach, affects the function of the whole digestive system.
moreover, the results can be extrapolated to other altricial-gastric teleosts to further understand how functional remodelling of the digestive system affects feeding behaviour.
our 3d models of the digestive organs showed a general trend of volume increase during atlantic halibut larvae development.
during metamorphic climax , when ths  levels were high  <cit> , the atlantic halibut gi-tract tissue volume increased almost four fold, and the stomach volume increased more than 11-fold.
the peak in ths in stage  <dig> atlantic halibut coincided with maximal gi-tract remodelling and the changes were reminiscent of what occurs in xenopus .
in contrast to the gi-tract and liver, the exocrine and endocrine pancreas appeared to have no growth or only a two fold increase in size, respectively, between the two oldest stages analysed .
a similar tendency was observed for the pancreas in atlantic cod during stomach differentiation  <cit> .
the authors hypothesized that this trend may be related to the importance of the pancreatic digestive enzymes in early agastric stages, particularly trypsin  <cit> .
the similarity of the changes in the pancreas and stomach of atlantic halibut and cod gives credence to the hypothesis, but experimental proof is still required.
the correlation between th level increments and stomach development during metamorphosis has previously been documented for the pleuronectiformes, japanese flounder  and summer flounder   <cit> .
in these flatfish, the development of the gastric glands started during pre-metamorphosis, but pepsinogen was only detected after metamorphosis.
in atlantic halibut, the gastric glands appeared prior to the th peak and pepsinogen transcripts were detected before metamorphic climax  <cit> .
however, the results were dependent on the method used: with rt-pcr pepsinogen a <dig> expression was observed at  <dig> dph  and with in situ hybridization  at  <dig> dph .
murray et al.
<cit>  suggested that pepsinogen expression in atlantic halibut can only occur when the gastric glands are completely developed.
in the present study, pepsinogen transcripts were detected in atlantic halibut gi-tract before the metamorphic climax, suggesting that the proteolytic capacity of the presumptive stomach was triggered by the rise in ths at the start of metamorphosis.
nonetheless, it remains to be demonstrated that pepsinogen transcript abundance correlates with proteolytic activity.
future studies discriminating between pepsinogen and pepsin activity will be needed to clarify this issue.
acid secretion in the stomach requires the gastric h+/k+-atpase, an α,β-heterodimeric enzyme, which exchanges a proton with potassium using energy from atp to generate hcl  <cit> .
the α subunit of the enzyme contains the catalytic site and the β subunit is required for proper maturation and targeting of the enzyme to the apical membrane  <cit> .
in the present study the expression of α and β subunit transcripts of gastric h+/k+-atpase were synchronous and correlated, as previously reported in mammalian stomach development .
in atlantic halibut the expression of pepsinogen a <dig> and both gastric proton pump subunits were also correlated, and similar observations have been made during larval development of the winter flounder, red porgy  and yellow catfish ,  <cit> .
the synchronous expression of these genes was proposed to be a physiological strategy to promote quick conversion of pepsinogen into pepsin  <cit> .
however, it remains to be established if the mechanism for the release of both enzymes from the oxynticopeptic cells is the same or occurs independently  <cit> .
the identification of pepsinogen and gastric h+/k+-atpase transcripts or protein indicate the stomachs proteolytic potential but not its actual activity.
for this reason in the present study, in vivo ph analysis was carried out and revealed the increase in h+/k+-atpase α and β subunit transcripts occurred simultaneously with increasing acidic capacity in atlantic halibut larvae during the climax of metamorphosis , when th levels rise.
from the first slight acidification observed in the lumen of the stomach during stage 9a, the lumen ph decreased below  <dig>  in stage 9b.
an increase of hcl production capacity during larval development has previously been reported in several other teleost species  <cit> .
however, the present study revealed that there was co-ordination between morphological changes and the key elements essential for the stomachs proteolytic activity such as h+/k+-atpase and hcl production and the ths most likely orchestrate this change.
during the climax of metamorphosis, a rapid colour change from acidic to alkaline was observed in the midgut when the ph indicator solution passed through the pyloric sphincter.
similar findings were reported for seabass  and japanese flounder  <cit> .
this observation in atlantic halibut at metamorphic climax suggests active secretion of hco3- into the midgut, most likely via cl-/hco3- exchange in the apical membrane of enterocytes  <cit> , and also alkaline bile and liver secretions.
future studies will be required to determine whether ths trigger the events that lead to the development of a functional stomach in other altricial-gastric species as well.
previous studies have shown that atlantic halibut ghrelin was predominantly expressed in the stomach area but was also detected in pyloric caeca, immature gonads and intestine  <cit> .
in newly hatched yolk-sac larvae, ghrelin protein was widely distributed in the gi-tract and was present in the anterior gi-tract before the gastric glands and pepsinogen production appeared  <cit> .
in the present study ghrelin mrna expression levels greatly increased during stomach differentiation in metamorphosis proclimax and climax.
the significant correlation between ghrelin mrna expression and pepsinogen a <dig> and gastric proton pump subunit transcript expression appears to confirm the link between ghrelin and the acquisition of stomach proteolytic function.
intriguingly, in weanling pigs the physiological role of ghrelin in appetite stimulation has been correlated with the initiation of the stomachs proteolytic activity  <cit>  and a similar association may also occur in halibut.
the presence of the pyloric sphincter from stage  <dig> onwards and its functional activity - to hold ingested food in the stomach - were observed in the 3d models and in the in vivo studies.
this indicated a small storage function that was already established in the atlantic halibut presumptive stomach during early developmental stages.
although before metamorphosis, this function was mainly assumed by the anterior midgut , which has a bulb-like shape with a much larger volume.
the presence of a “physiological” sphincter  in the mg <dig> of the gi-tract allowed it to assume a reservoir function or at least to delay the chyme transit so that sufficient mixing with bile and digestive enzymes from the pancreas can occur.
the lack of a fully developed stomach at stage  <dig> to mix the ingested food may be functionally compensated by the strong peristaltic activity  observed in the mg <dig>  which contributes to the mechanical degradation of the ingested food.
this supports earlier notions in zebrafish , proposing that the retrograde contractions observed in the anterior part of the midgut generate a similar mechanical mixing as the stomach  <cit> .
considered in the context of a chemical reactor  <cit>  the atlantic halibut gi-tract changed from a plug-flow reactor  operating system, in which ingested food flowed continuously through the intestine to a continuous-flow stirred-tank reactor , with food entering and exiting continuously through the reaction vessel .
it will be insightful in the future to model halibut gi-tract function during development in order to identify when critical changes occur and the regulatory processes that control them.
CONCLUSIONS
in conclusion, this study contributes to our understanding of how th-driven metamorphosis affects the morphology and the function of the gi-tract.
the remodelling of atlantic halibut gi-tract, specifically the stomach development and volume growth, is linked to the surge of th levels during the climax of metamorphosis, and the morphological modifications are connected with a set of functional changes.
we show that the proteolytic activity in the stomach starts during the climax of metamorphosis with the synchronized expression of pepsinogen a <dig> and both gastric proton pump subunit transcripts.
this ensures pepsinogen activation and creates the optimum ph range for pepsin activity.
furthermore, we demonstrate that stomach ghrelin, a key element for the gastric involvement in appetite regulation, is correlated with the emergence of proteolytic activity.
the presumptive stomach has a storage capacity in early development, however the main storage function is assumed by the anterior part of the midgut before metamorphic climax.
during the metamorphic climax the main short term storage capacity shifts to the stomach, when its volume increases, and the gi-tract motility patterns change with a decrease in contractions of the midgut due to the functional development of the stomach.
considering the generally conserved nature of the post-embryonic modifications of the gi-tract in altricial-gastric species, our results are likely a general characteristic of teleost fish and potentially other vertebrates.
however, further research is required to substantiate this general hypothesis and elucidate the molecular mechanisms regulating the functional development of the gi-tract.

 the divergent gastrointestinal tract morphology and feeding strategies between larval and adult phases are adaptations to fundamentally different habitats and food resources. the post-embryonic maturation of the digestive system is a key event in the life history of vertebrates and essential for survival. thyroid hormone driven metamorphosis plays a crucial role in the functional maturation of the gi-tract and in shaping its morphology to the adult form. remodelling of the gi-tract from larva to adult has been extensively studied in xenopus. in this organism, the intestine is transformed under the influence of ths from a long coiled tube into a complex organ with a differentiated stomach and small intestine. this involves epithelial and mesenchymal proliferation, smooth muscle thickening and the formation of intestinal folds. several studies have described the cellular mechanisms responsible for this remodelling in amphibians, yet little is known about their impact on tissue function in vertebrates, particularly the multiple functions integrated in the stomach. a striking feature of vertebrate metamorphosis is the organogenesis of the stomach. in early developmental stages of fishes and anurans the stomach is often absent and part of its function may be carried out by the intestine. the main roles of the vertebrate stomach are storage of ingested food, secretion of hydrochloric acid and pepsinogen, and mechanical breakdown and mixing of food with gastric secretions. thus, in larvae of altricial-gastric species, such as the atlantic halibut, the absence of a stomach limits the ability to digest dietary protein when exogenous feeding is initiated. this is one of the reasons why most studies of gi-tract development during metamorphosis have focused on stomach development and consider the appearance of gastric glands as an adequate indicator of a fully developed stomach. however, it has become clear that the histological identification of gastric glands does not indicate that the stomach is fully functional. hence, the stomach’s proteolytic function is best indicated by pepsin activity and pepsinogen content. to better understand the efficiency of digestive processing during fish ontogeny, several studies compared expression profiles of pepsinogen and the gastric proton pump, localized in the hcl secreting oxynticopeptic cells. murray et al. have used histology and pepsinogen transcript analysis to study the ontogeny of the stomach in atlantic halibut and showed that the appearance at 66 days post-hatching of gastric glands preceded expression of pepsinogens a and a transcripts at dph. however, the impact of metamorphosis on other important functions of the stomach or gi-tract development in general has largely been overlooked in flatfish. in addition to acid production and proteolysis the vertebrate stomach also has reservoir functions. after ingestion, the stomach stores and predigests food, then delivers the chyme to the midgut for further digestion and subsequent nutrient absorption. the storage function of the stomach relieves juvenile and adult fish from the need to constantly feed like the stomachless larval stages. establishment of the stomach as a reservoir requires functional sphincters and well developed neural and smooth muscle layers. the mechanical mixing and transport of food through the gi-tract is achieved by specific motility patterns and by matching peristalsis with the release of digestive enzymes. this process plays a central role in effective food processing, though very few studies have targeted gi-tract movements in fish larvae. the advantage of using fish larvae, such as atlantic halibut, is their optic transparency that is maintained until metamorphosis. this permits direct visual observations of the gi-tract and its motility patterns in live animals. pittman et al. reported peristaltic contractions in atlantic halibut larvae, in the anterior intestine at dph. in juvenile atlantic halibut gi-tract both anterograde and retrograde contraction waves were described, and identical patterns were also observed in embryos and larvae of the stomachless zebrafish. the stomach produces hormones involved both in the regulation of appetite and digestion. ghrelin is an example of a hormone that is mainly produced in the stomach and acts as a stimulator of food intake. in mammals, ghrelin has also been suggested to stimulate gastric acid secretion and motility. the function of ghrelin in fish larvae is still poorly described, but it has been proposed as an indicator of the stomach’s involvement in appetite regulation in developing fish. in atlantic halibut, ghrelin gene expression increases during the climax of metamorphosis, coinciding with stomach development. ghrelin is abundant in the developing gastric glands and several osmoregulatory tissues. additionally, its co-expression with na+/k+-atpase suggests a putative role in hydromineral balance. yet, the role of ghrelin in appetite regulation, motility and osmoregulation is unknown, as well as its link to the proteolytic and reservoir function of the stomach in atlantic halibut during metamorphosis. this study aims to establish the impact of the agastric-gastric transition on the functional role of the post-embryonic gi-tract remodelling that occurs during metamorphosis in atlantic halibut, a flatfish species of high commercial interest for the northern european and north american aquaculture industry. to map the changes in gi-tract development and establish events linked to th-driven metamorphosis we constructed a series of 3d models of the morphological and spatial organization of the digestive organs in representative developmental stages. we tested the hypothesis that the development of the multiple stomach functions is synchronous and linked to its physical appearance at metamorphosis. the proteolytic function of the stomach was studied using in vivo ph analyses combined with expression profiles of the specific gene markers h+/k+-atpase α and β subunit and pepsinogen a using quantitative pcr. stomach filling and reservoir function were assessed by in vivo visual studies of the transparent larvae at prometamorphosis and climax of metamorphosis. the putative role of a fully functional stomach in appetite regulation was assessed by measuring ghrelin transcript abundance. the establishment of gi-tract motility patterns during development was determined by in vivo observations and the involvement of the gi-tract in osmoregulation was assessed by measuring the abundance of na+/k+-atpase α subunit transcripts.  3d reconstruction of digestive organs 3d models of the morphology of the digestive system during development were reconstructed from a series of histological sections. location and size of the gi-tract and its associated organs, such as liver, endocrine and exocrine pancreas, and gallbladder, were observed from stage until the post-metamorphic stage. the gi-tract includes a narrow foregut, midgut, and a short hindgut. the anterior region of the midgut, just after the pyloric sphincter, was larger in diameter, i.e. more voluminous, compared to the rest of the midgut. this feature was maintained during gi-tract ontogeny. both ps and ileorectal sphincter were identified from stage onwards. pyloric caeca became evident as projections from the most anterior part of the midgut at stage. the stomach was well-differentiated at stage and the gastric glands were visible on histological sections. the luminal volume of the gi-tract increased during development, particularly in the two last stages analysed. the stomach volume from 9a to increased from to nl, respectively and corresponded to an fold increment. the values were calculated from the 3d models using imaris measurementspro. agi-tract tissue volume = gi-tract outer layer - gi-tract inner layer. the liver was positioned under the foregut and anterior to the ascending loop of the midgut and its volume steadily increased during development. the exocrine pancreas was observed between the presumptive stomach and the anterior part of the midgut at stage and it surrounded this midgut area throughout ontogeny. in the endocrine pancreas, a clearly distinguishable islet of langerhans was observed close to the gallbladder at stage. in contrast to the other digestive organs, the increment in the normalized volume of endocrine and exocrine pancreas was low and negative, respectively, between stages 9a and. the yolk-sac, positioned under the gi-tract at stage decreased in size after the initiation of exogenous feeding and a small vestige remained besides the liver at stage. the gallbladder was observed on the right-hand side between the exocrine pancreas and the liver, and maintained this position in all the developmental stages analysed. the pancreatic duct and the bile duct opened next to each other into the lumen at the median plane of the anterior midgut, just after the ps. cloning and phylogenetic characterization of pepsinogen a ghrelin, gastric proton pump subunits and na+/k+-atpase subunit α the complete coding sequence of atlantic halibut pepsinogen a was bp and was submitted to genbank under accession no. kf. the amino acid sequence of pepsinogen is relatively well-conserved among teleost fish and, as expected, more variable when compared to other vertebrate pepsinogens. for instance, halibut pepsinogen a shared respectively 88% and 64% aa sequence identity with winter flounder pepsinogen a form iib and iia, but only 52% and 48% identity with homologues from xenopus laevis and human, respectively. the cdna fragments cloned for atlantic halibut h+/k+-atpase α subunit and na+/k+-atpase α subunit were deposited in genbank with the accession numbers kf and kf respectively. the cds for h+/k+-atpase β subunit of bp was cloned and submitted to genbank with the accession no. kf. phylogenetic analysis of the α subunit of the gastric proton pump and na+/k+-atpase, and vertebrate homologues generated two major clades, one corresponding to h+/k+-atpase and the other to na+/k+-atpase. phylogenetic analysis of the β subunit generated a tree with two major clades that shared the same general topology as the phylotree for the α subunit with the h+/k+-atpase and na+/k+-atpase clustered independently. atlantic halibut h+/k+-atpase α subunit clustered most closely with teleost homologues, with which it shared 94% aa sequence identity, and increased to 98% identity with winter flounder and atlantic cod. lower aa sequence identity was found when atlantic halibut h+/k+-atpase α subunit was compared to atlantic halibut na+/k+-atpase α subunit and to other vertebrate counterparts. the atlantic halibut na+/k+-atpase α subunit clustered with an antarctic eelpout homologue and shared approximately 88% aa identity with other teleost gene homologues. h+/k+-atpase β subunit clustered as expected within the teleost clade and shared rather low identity with its human homologue. atlantic halibut h+/k+-atpase β subunit did not share more than 39% aa sequence identity with the atlantic halibut na+/k+-atpase β subunit. ontogenetic expression pattern and correlation analysis the developmental expression profiles of pepsinogen a h+/k+-atpase α and β subunits, na+/k+-atpase α subunit and ghrelin were analysed by qpcr in the gi-tract of individual atlantic halibut larvae. the gene expression of both gastric proton pump subunits were significantly correlated and had parallel expression patterns, with a sharp and significant increase at climax and in post-metamorphic stages. pepsinogen a was significantly correlated with the expression profile of the gastric proton pump α and β subunits. a significant increase during stage was observed for pepsinogen a and its expression peaked in the post-metamorphic stage. ghrelin mrna transcript abundance increased gradually and significantly during the proclimax/climax of metamorphosis, and attained a maximum in the post-metamorphic stage. moreover, ghrelin transcript abundance and proteolytic activity during gi-tract ontogeny were significantly correlated. expression of na+/k+-atpase α subunit mrna was detected in all developmental stages, with significantly more transcripts at stage estimation of ph in the lumen of stomach and detection of acid production the ph assessment in the lumen of the stomach and midgut/hindgut during post-embryonic development was based on the colour observed after the injection of ph indicator solutions. the ph in the midgut/hindgut remained alkaline in all the developmental stages analysed. the presumptive stomach also had an alkaline ph with values above until stage gradual acidification was observed in the stages corresponding to the climax of metamorphosis. transition from an alkaline to an acidic ph in the stomach lumen was evident at stage 9a, when the injected sol cpr remained purple but the sol mcp gave a yellow coloration. the lumen of the stomach was clearly in the acidic range at stage 9b, as revealed by the yellow colour in the stomach following administration of both cpr and bpb solutions. the presented ph values are based on visual observations of colour changes after the administration of three ph indicator solutions. analysis of gi-tract motility spontaneous propagating contractions were observed in the gi-tract at prometamorphosis and climax of metamorphosis. due to considerable individual variation, number and frequency of contractions could not be grouped and are presented for each individual analysed. two types of contractions were observed in the midgut region and: phasic and propagating waves. the propagating contractions observed in mg were retrograde waves that originated in area “a” and moved towards the mouth. however, in mg most of the propagating contractions originated just under the ps and were anterograde waves that moved in an anal direction. motility activity in both midgut regions was detected at stage with a frequency ranging from to  min- depending on the individual and type of contraction. at stage relatively few spontaneous contractions of short duration were observed in the midgut. during the climax of metamorphosis, contractions in the stomach were registered in all individuals, in contrast to stage when motility in the presumptive stomach was only observed in one larva. the rectal contraction was a mixture of retro- and anterograde contractions and were observed in both stages and with similar frequencies in most of the individuals analysed. propagating phasic propagating phasic n n n n n n n propagating and phasic contractions are stated for midgut and midgut regions. frequency is the number of contractions registered per min. *not possible to quantify phasic and propagating wave contractions. the affected gi-tract segments were constantly contracted during the whole observation period. discussion in all altricial-gastric species, the gi-tract undergoes dramatic remodelling during th-driven metamorphosis. the gi-tract changes from a simple tubular form into a more complex folded structure. at the same time the stomach becomes a distinct compartment and continues to acquire its multiple functions through metamorphosis. there are surprisingly few studies examining and integrating the anatomical and functional changes in the gi-tract associated with this th-driven event. in amphibians such as xenopus, it is well established that ths induce gi-tract remodelling leading to intestinal shortening and the development of crypts and villi. the remodelling of the xenopus digestive tract is a consequence of changes in th regulated genes, including sonic hedgehog/bone morphogenetic protein- and tolloid/bmp-. however, in contrast to amphibians, few studies have linked gi-tract remodelling with th-driven metamorphosis in teleost fish, in particular the pleuronectiformes. the present study used a series of 3d models to reconstruct the ontogeny of atlantic halibut gi-tract morphology and its volume changes during larval development. using molecular and functional analysis, the modification of gi-tract function from the premetamorphic stage until the end of metamorphosis was established. insight was obtained into the way in which post-embryonic differentiation of the gi-tract and the growth of a new organ, the stomach, affects the function of the whole digestive system. moreover, the results can be extrapolated to other altricial-gastric teleosts to further understand how functional remodelling of the digestive system affects feeding behaviour. our 3d models of the digestive organs showed a general trend of volume increase during atlantic halibut larvae development. during metamorphic climax, when ths levels were high, the atlantic halibut gi-tract tissue volume increased almost four fold, and the stomach volume increased more than 11-fold. the peak in ths in stage atlantic halibut coincided with maximal gi-tract remodelling and the changes were reminiscent of what occurs in xenopus. in contrast to the gi-tract and liver, the exocrine and endocrine pancreas appeared to have no growth or only a two fold increase in size, respectively, between the two oldest stages analysed. a similar tendency was observed for the pancreas in atlantic cod during stomach differentiation. the authors hypothesized that this trend may be related to the importance of the pancreatic digestive enzymes in early agastric stages, particularly trypsin. the similarity of the changes in the pancreas and stomach of atlantic halibut and cod gives credence to the hypothesis, but experimental proof is still required. the correlation between th level increments and stomach development during metamorphosis has previously been documented for the pleuronectiformes, japanese flounder and summer flounder. in these flatfish, the development of the gastric glands started during pre-metamorphosis, but pepsinogen was only detected after metamorphosis. in atlantic halibut, the gastric glands appeared prior to the th peak and pepsinogen transcripts were detected before metamorphic climax. however, the results were dependent on the method used: with rt-pcr pepsinogen a expression was observed at dph and with in situ hybridization at dph. murray et al. suggested that pepsinogen expression in atlantic halibut can only occur when the gastric glands are completely developed. in the present study, pepsinogen transcripts were detected in atlantic halibut gi-tract before the metamorphic climax, suggesting that the proteolytic capacity of the presumptive stomach was triggered by the rise in ths at the start of metamorphosis. nonetheless, it remains to be demonstrated that pepsinogen transcript abundance correlates with proteolytic activity. future studies discriminating between pepsinogen and pepsin activity will be needed to clarify this issue. acid secretion in the stomach requires the gastric h+/k+-atpase, an α,β-heterodimeric enzyme, which exchanges a proton with potassium using energy from atp to generate hcl. the α subunit of the enzyme contains the catalytic site and the β subunit is required for proper maturation and targeting of the enzyme to the apical membrane. in the present study the expression of α and β subunit transcripts of gastric h+/k+-atpase were synchronous and correlated, as previously reported in mammalian stomach development. in atlantic halibut the expression of pepsinogen a and both gastric proton pump subunits were also correlated, and similar observations have been made during larval development of the winter flounder, red porgy and yellow catfish,. the synchronous expression of these genes was proposed to be a physiological strategy to promote quick conversion of pepsinogen into pepsin. however, it remains to be established if the mechanism for the release of both enzymes from the oxynticopeptic cells is the same or occurs independently. the identification of pepsinogen and gastric h+/k+-atpase transcripts or protein indicate the stomachs proteolytic potential but not its actual activity. for this reason in the present study, in vivo ph analysis was carried out and revealed the increase in h+/k+-atpase α and β subunit transcripts occurred simultaneously with increasing acidic capacity in atlantic halibut larvae during the climax of metamorphosis, when th levels rise. from the first slight acidification observed in the lumen of the stomach during stage 9a, the lumen ph decreased below in stage 9b. an increase of hcl production capacity during larval development has previously been reported in several other teleost species. however, the present study revealed that there was co-ordination between morphological changes and the key elements essential for the stomachs proteolytic activity such as h+/k+-atpase and hcl production and the ths most likely orchestrate this change. during the climax of metamorphosis, a rapid colour change from acidic to alkaline was observed in the midgut when the ph indicator solution passed through the pyloric sphincter. similar findings were reported for seabass and japanese flounder. this observation in atlantic halibut at metamorphic climax suggests active secretion of hco3- into the midgut, most likely via cl-/hco3- exchange in the apical membrane of enterocytes, and also alkaline bile and liver secretions. future studies will be required to determine whether ths trigger the events that lead to the development of a functional stomach in other altricial-gastric species as well. previous studies have shown that atlantic halibut ghrelin was predominantly expressed in the stomach area but was also detected in pyloric caeca, immature gonads and intestine. in newly hatched yolk-sac larvae, ghrelin protein was widely distributed in the gi-tract and was present in the anterior gi-tract before the gastric glands and pepsinogen production appeared. in the present study ghrelin mrna expression levels greatly increased during stomach differentiation in metamorphosis proclimax and climax. the significant correlation between ghrelin mrna expression and pepsinogen a and gastric proton pump subunit transcript expression appears to confirm the link between ghrelin and the acquisition of stomach proteolytic function. intriguingly, in weanling pigs the physiological role of ghrelin in appetite stimulation has been correlated with the initiation of the stomachs proteolytic activity and a similar association may also occur in halibut. the presence of the pyloric sphincter from stage onwards and its functional activity - to hold ingested food in the stomach - were observed in the 3d models and in the in vivo studies. this indicated a small storage function that was already established in the atlantic halibut presumptive stomach during early developmental stages. although before metamorphosis, this function was mainly assumed by the anterior midgut, which has a bulb-like shape with a much larger volume. the presence of a “physiological” sphincter in the mg of the gi-tract allowed it to assume a reservoir function or at least to delay the chyme transit so that sufficient mixing with bile and digestive enzymes from the pancreas can occur. the lack of a fully developed stomach at stage to mix the ingested food may be functionally compensated by the strong peristaltic activity observed in the mg which contributes to the mechanical degradation of the ingested food. this supports earlier notions in zebrafish, proposing that the retrograde contractions observed in the anterior part of the midgut generate a similar mechanical mixing as the stomach. considered in the context of a chemical reactor the atlantic halibut gi-tract changed from a plug-flow reactor operating system, in which ingested food flowed continuously through the intestine to a continuous-flow stirred-tank reactor, with food entering and exiting continuously through the reaction vessel. it will be insightful in the future to model halibut gi-tract function during development in order to identify when critical changes occur and the regulatory processes that control them.  in conclusion, this study contributes to our understanding of how th-driven metamorphosis affects the morphology and the function of the gi-tract. the remodelling of atlantic halibut gi-tract, specifically the stomach development and volume growth, is linked to the surge of th levels during the climax of metamorphosis, and the morphological modifications are connected with a set of functional changes. we show that the proteolytic activity in the stomach starts during the climax of metamorphosis with the synchronized expression of pepsinogen a and both gastric proton pump subunit transcripts. this ensures pepsinogen activation and creates the optimum ph range for pepsin activity. furthermore, we demonstrate that stomach ghrelin, a key element for the gastric involvement in appetite regulation, is correlated with the emergence of proteolytic activity. the presumptive stomach has a storage capacity in early development, however the main storage function is assumed by the anterior part of the midgut before metamorphic climax. during the metamorphic climax the main short term storage capacity shifts to the stomach, when its volume increases, and the gi-tract motility patterns change with a decrease in contractions of the midgut due to the functional development of the stomach. considering the generally conserved nature of the post-embryonic modifications of the gi-tract in altricial-gastric species, our results are likely a general characteristic of teleost fish and potentially other vertebrates. however, further research is required to substantiate this general hypothesis and elucidate the molecular mechanisms regulating the functional development of the gi-tract. 
in vivo ph analysis in the gi-tract lumen combined with quantitative pcr of α and β subunits of the gastric proton pump and pepsinogen a indicated that gastric proteolytic capacity is established during the climax of metamorphosis. a 3d models series of the gi-tract development indicated a functional pyloric sphincter prior to first feeding. 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. 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. 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. 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. observations of fed larvae in vivo confirmed that stomach reservoir function was established before metamorphosis, and was thus independent of this event. the functional specialization of the gi-tract was not exclusive to metamorphosis, and its osmoregulatory capacity and reservoir function were established before first feeding. transcript abundance of ghrelin, a putative orexigenic signalling molecule produced in the developing stomach, correlated with the emergence of gastric proteolytic activity, suggesting that the stomach’s role in appetite regulation occurs simultaneously with the establishment of proteolytic function. 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 tract occur.
 flatfish 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 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.  onset of gastric function was determined with several approaches. in vivo ph analysis in the gi-tract lumen combined with quantitative pcr of α and β subunits of the gastric proton pump and pepsinogen a 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 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.  the 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. atlantic halibutgastric proton pumpgastrointestinal tractghrelinmotilityna+/k+-atpasepepsinogenontogenyphstomach 