Fish Physiology and Biochemistry22: 225–235, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Ontogeny of digestive tract functionality in Japanese flounder,Paralichthys olivaceusstudied by in vivo microinjection: pH andassimilation of free amino acids

I. Rønnestad1, R. Perez Dominguez2 and M. Tanaka21Department of Zoology, University of Bergen, All´egt 41, N-5007 Bergen Norway (Phone: +47 55 58 35 86; Fax:+47 55 58 96 73; E-mail: ivar. ronnestad@zoo.uib.no);2Division of Applied Biosciences, Graduate School ofAgriculture, Kyoto University, Kyoto 606-01, Japan

Accepted: November 25, 1999

Key words:development, digestion, fish larvae, functionality, gut, nutrition

Abstract

To specifically study the functional ontogeny of the digestive tract, larvae of Japanese flounder at various devel-opmental stages were injected with liquid solutions using tube feedingin vivo. The survival in the stages testedwas on average 93%. The injected solutions were almost completely transferred from the presumptive stomach,or stomach to the midgut within 10 min of injection. The passage to the hindgut in some cases started 10 minafter injection and over 90% of the solution had passed from the midgut to the hindgut after 1 h. In most cases thehindgut seemed to be completely empty after 3 h.

Two different mixtures of pH indicators with sensitivities in the alkaline (7.5 to 9) and acid (4 to 6) rangesrespectively were used for assessment of pH in the various gut segments. The pH of the stomach remained alkalineduring the larval period, but had fallen close to 4 during late metamorphosis, an indication of active HCl secretionand progressive stomach differentiation. In mid and late metamorphosing fish a rapid colour change in the pHindicator was observed once it had passed the pyloric sphincter. This demonstrates that there was also activesecretion of alkaline fluid, most likely HCO−3 , from the pancreas into the pylorus lumen.

A single injection of liquid solution of14C-FAA showed that assimilation of FAA was high in all stages tested(79.5± 7.1%; SD;n = 91). The presently reported data for Japanese flounder support earlier studies that FAA areabsorbed with a high efficiency in the early stages of marine fish.

Introduction

The early larval stages represent the major bottleneckfor a large-scale intensive production of marine fishspecies (e.g., Watanabe and Kiron 1994). It is gener-ally believed that future cost effective cultivation ofmarine fish requires feeding of larvae with formulatedfeeds from first feeding. A present, no artificial dietsare available that can support survival and growth ratescomparable to that of live feed. Several of the prob-lems associated with formulated diets are related toproduction technology and to the acceptance of arti-ficial feed by the larvae. In some cases first feedingmarine fish larvae will ingest artificial feed but fail to

grow (Person-Le Ruyet et al. 1993) although recentadvances show improving results (Yúfera et al. 1995;1996). There is a lack of knowledge in diet compo-sition with respect to the quantitative requirement ofnutrients and the form that the nutrients should bepresented. Understanding the development of diges-tive function especially in quantitative terms regardingthe capacity to digest and absorb various nutrients willserve as an important basis for developing appropriateartificial diets for these life stages.

There are several direct and indirect literature datasuggesting that intestinal function, and especially pro-teolytic ability is not fully developed at the time ofstart-feeding in fish larvae. Rust (1995) examined the

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development of nutrient assimilation in six freshwa-ter fish species, using a method of controlled tubefeeding radio-labelled nutrients. His results indicatedthat different assimilation patterns exist according tothe life history of the fish. Altrical larvae that laterdevelop gastric digestion (most marine fish of com-mercial interest belong to this group) initially assim-ilated simple forms of amino acids more efficientlythan more complex forms (assimilation order; FAA>peptides> protein). Interestingly, the differences inassimilation rate decreased as the larvae approachedmetamorphosis.

It has been suggested that bioavailable amino acidsare a lacking commodity in the larval diet (Fyhn 1989;Walford et al. 1991; Rønnestad and Fyhn 1993; Rust1995) and it has been proposed that freely dissolvedamino acids may assuage nutritional deficiency duringthe developmental window from first feeding and untilthe intestine is fully differentiated and able to digestingested proteins (Fyhn 1989, 1993). Fish larvae havebeen shown to pinocytotically absorb large moleculesand proteins in the mucosa with intracellular diges-tion of the hind gut (Iwai and Tanaka 1968; Watanabe1984; Deplano et al. 1991; Kurokawa et al. 1996).Watanabe (1983) showed that Horseradish peroxidasewas intactly absorbed and then digested intracellu-larly. In smelt (Hypomesus transpacificus) it took 10to 24 h before the protein was hydrolysed intracellu-larly. Based on such findings some claim that the lackof gastric proteolysis are compensated for by hindgutprotein pinocytotis (e.g., Govoni et al. 1986). How-ever, the pinocytotic protein uptake rate has not beenquantified and questions can be raised whether theamino acids absorbed through this mechanisms aresufficient to satisfy the amino acid requirement of thefast growing fish larvae.

Growth is primarily an increase in body musclemass by protein accretion. This accretion is the netresult of protein synthesis and catabolism (Houlihanet al. 1995; Conceição 1997). Due to their fast growthrate fish larvae have a high requirement for aminoacids for protein synthesis. In addition, in marine fishlarvae amino acids represent one of the main fuels intheir energy dissipation (Fyhn 1989; Rønnestad et al.1992, 1994, 1998, 1999; Rønnestad and Fyhn 1993;Finn et al. 1995, 1996). Together, this results in anexceptional demand for amino acids for fish larvae.

Taken together, this implies that the delivery ofamino acids from intestinal protein digestion in marinefish larvae may be insufficient to cover the metabolicdemands for energetic and synthetic purposes. A sup-

ply of free amino acids (FAA) in the diet should there-fore the beneficial for survival and further growth. It isnoteworthy that the fish larvae in the ocean consumesuch a supply by their intake of plankton organismsafter start feeding (Fyhn 1989, 1993; Fyhn et al. 1993,1995).

To specifically test intestinal assimilation rates ofFAA, developing larvae of Japanese flounder,Par-alichthys olivaceuswere tube fed controlled amountsof 14C-amino acids. In addition, the pH in differentsections of the digestive tract was assessed in orderto determine the functional ontogeny of the stomachwith respect to protein denaturation and hence proteindigestibility.

Materials and methods

Larvae

Eggs from captive brood-stock of Japanese floun-der (Paralichthys olivaceus) were transferred fromYamagataya-Suisan (Awaji Island, Hyogo Prefecture)to Fisheries Research Station of Kyoto University atMaizuru. The larvae were reared in a polycarbonatetank (500 l) with running seawater. Water temperaturewas maintained at 18.0± 1.5◦C and salinity rangedfrom 30.6 to 32.0 g l−1. The larvae were initially fedrotifers (Brachionus plicatilis) cultivated with Nan-nochloropsissp. andω-Yeast (Kyowa Hakko Kogyo,Japan), and laterArtemia sp. naupii enriched withEster-85 (Nippon Chemical Feed, Japan) as describedby Seikai et al. (1986).

Once a week about 400 larvae were transferredby car from the main rearing tank at Maizuru to themain campus of Kyoto University for experimentation.The larvae were kept in polycarbonate tanks (5l) at18± 1◦C, 32 g l−1 salinity, and offered newly hatchedArtemia nauplii every morning. Survival was almost100% in these transferred groups.

The larvae used for thein vivo studies were trans-ferred form the holding tank and kept overnight inclean seawater, thus ensuring only larvae with emptyguts. At the day of experimentation, groups of 50larvae were transferred to the Radioisotope ResearchCentre at Kyoto University were the tube feeding ofradioactive compounds were conducted.

Developmental stages of experimental fish wereclassified according to Minami (1982): D and E areonset stages of metamorphosis, F, G and H are corre-sponding to early, mid and late phases of metamorpho-

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sis, respectively (see Figure 2). Stage I correspond tosettled fish.

Experimental set-up

To specifically test the functionality of the digestivesystem, we used anin vivo-method for controlled tube-feeding of fish larvae modified after Rust et al. (1993).The in vivo set-up comprised a stereo dissecting mi-croscope, with camera and two micromanipulators. Ananoliter injector (World Precision Instruments) wasfastened to one of the micromanipulators while a hold-ing pipette was fastened to the other. A glass capillarymade according to the larval mouth and oesophagusdiameter was fastened to the nanoliter injector. Toavoid damage to the gut epithelium the capillary tipwas polished to a conical shape using a fine gradecarborundum paper. Prior to injection the larvae wereanaesthetised (MS-222; 10 mg l−1 final concentration)and thereafter gently placed on a microscopic slide ina droplet of clean seawater. At the early larval stagesthe fish were kept in position for injection with thetail sucked into the holding pipette using a catheterof appropriate size. At later stages when the fish ap-proached metamorphosis the anaesthetised fish couldbe kept in proper position for injection by the watersurface tension without any holding pipette. With thefish in position the capillary was gently passed throughthe mouth and oesophagus into the presumptive stom-ach area of early larvae and into the stomach of olderlarvae. In all stages tested the presumptive stomach orthe stomach could be identified due to the presenceof a strong pyloric sphincter. Graded samples of thetest diet was then deposited into the pre-pyloric lu-men with the nanoliter-injector. After sample deliveryand capillary withdrawal the larvae were gently rinsedfor any spillage by six successive transfers to wells(20 ml) containing clean seawater for 30 s immersionin each well. The larvae were transferred between thewells with a pipette with a minimum of seawater. Fi-nally, single larva was incubated in wells with 2 mlof clean seawater. Visual observations showed that theventilation movements of the operculi effectively re-moved any contamination in the mouth and branchialchambers during the first steps of the rinsing series.All experiments was performed in a termostated roomat 18± 1 ◦C. All experimental fish were killed aftertreatment.

FAA assimilation

To determine FAA assimilation rates, an aqueous so-lution of 14C-protein hydrolysate (1.85 MBq ml−1;>1.85 GBq milliatom C−1; Amersham Int., England)was injected. To allow visual observation of the dietduring injection as well as assessment of gut retentiontime (see below) a colour indicator 0.1% M-Creosol(Sigma) was added to the hydrolysate. The exactvolume of the labelled diet remaining in the larvalintestine was back-calculated from the experimentalresults (dpm) by summing up all labels in larvae andincubation water and using the data provided by themanufacturer.

Groups were tube fed and incubated individually asdescribed above. One series consisted of 5–7 groupsof 6 larvae that were incubated for l5 min, 30 min,1 h, 2 h, 4 h and 6 h, except for differences as com-mented. After the set time the larvae were lifted bythe tail using a forceps, rinsed once in distilled waterand transferred to a scintillation vial containing 0.5 mltissue solubilizer (Soluene-350, Packard). When thesolution was clear (24–30 h) 4.5 ml of scintillationcocktail (Ultima Gold, Packard) was added. The wellincubation water (1.8 ml) was transferred to a scintil-lation vial and added 3.2 ml of scintillation cocktail(Ultima Gold XR, Packard). All samples were storedfor at least 10 min at room temperature before beingcounted for 10 min using an Aloka LSC-3050 liquidscintillation counter.

A refinement to this protocol was used during thelast four of totally eight experimental days. At sam-pling time the larvae were lifted by the tail usinga forceps, rinsed once in distilled water and trans-ferred to a scintillation vial containing 0.25 ml 6%tri chlor acetic acid (TCA). After 48 h the sampleswere centrifuged and the supernatant with the ex-tracted solubles including FAA, was transferred intonew scintillation vials. The vials were rinsed once with0.25 ml 6% TCA, and the rinsed solutions were addedto the new scintillation vials. The precipitated larvalbody containing the protein was added 0.5 ml tissuesolubilizer (Soluene-350, Packard) and left for 24 huntil the solution was clear. Thereafter 4.5 ml of scin-tillation cocktail (Ultima Gold, Packard) was added.The well water (1.8 ml) and the TCA solution (0.5 ml)were added scintillation cocktail (Ultima Gold XR,Packard) to a total volume of 5 ml. All samples werestored for at least 10 min before being counted for10 min using an Aloka LSC-3050 liquid scintillationcounter.

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The data were calculated from cpm (counts min−1)to dpm (disintegrations min−1) after subtraction ofblanks and correcting for counting efficiency whichwas 95% and 90% for Ultima Gold and Ultima GoldXR, respectively.

During the first four experimental days,14C re-tention in the fish larvae (RL) was calculated usingformula (1). During the last four experimental days,retention in the fish larvae was divided into that of thefree pool (RLFAA and the protein pool (RLProt ) usingformula (2) and (3):

RL = [L/(L+W)] × 100, (1)

whereL = dpm of the whole larvae,W= dpm of theincubation water;

RLFAA=[LFAA/(LFAA+LProt+W)]×100, (2)

RLProt=[LProt/(LFAA+LProt+W)]×100, (3)

where LFAA = dpm of the TCA solute andLProt = dpm of the TCA precipitate

The 14C-protein hydrolysate contain amino acidsin the following %-activity: alanine: 9.5, arginine:6.9, aspartic acid: 10, glutamic acid: 9, glycine:5.8, histidine: 1.6, leucine: 12.7, isoleucine: 5.8,lysine: 4.8, methionine: 0.6, phenylalanine: 7.4, pro-line: 5.3, serine: 3.2, threonine: 5.7, tyrosine: 5.8,valine: 5.5 (Amersham, Technical data sheet). As-suming a uniform labelling of the C, an averageFAA in the hydrolysate will have the following for-mula C4.5H9.0O2.5N1.2S0.01 with a molecular weightof 121.5 g. Calculations using the specific activityof the mix and the dpm data show that on average84± 36 nL (SD;n = 260; Range: 24–200 nL) wereeffectively injected. Using a calculated total FAA con-centration of 0.26 mM in the injected fluid this meansthat 0.8–6.3 ng of FAA was injected pr. larva (average2.6± 1.2 ng).

Injection of pH indicator solutions

To assess the pH of the various gut-compartments oneof two pH indicator solutions were injected into a totalof 12 larvae at each experimental date. The first solu-tion (mix 1) consisted of 0.1% M-Creosol (Sigma) indistilled water (pH range 7.5–9). The second solution(mix 2) contained 0.1% of Chlorphenol Red and Bro-mophenol Blue (Sigma) in distilled water (pH range4–6). The colour of the intestinal fluid was comparedto that of a set of mix 1 and 2 standards prepared in pHbuffers from pH 2.0 to pH 9.5 in steps of 0.5 and con-tained in sealed glass capillaries. The standards were

immersed in water and under the same light conditionas the larvae under the dissecting microscope.

Gut transit time

Gut passage was examined in larvae (stage G-H) thatwere visually observed for presence of pH indicatorat regular intervals from 0 to 3 h after injection. Toallow a semi-quantitative assessment of the gut transittime, the gut was divided into three compartments:stomach/presumptive stomach, midgut, and hindgut.The filling in each compartment was based on visualassessment of volume and presence of colour, andgraded from 0 (empty and without colour) to 10 (filledand with strong colour).

Results

Treatment recovery

Larvae of Japanese flounder proved to be tolerantto the moderate stress imposed by handling and themicro-tube feeding technique used in the presentstudy. The overall survival in the stages tested wason average 93%. On the earliest stages injected (D-E to F-G stage; 17 to 22 DPH), four of 30 injectedlarvae died in the incubation well on each injection dayrepresenting a survival rate of 89%. After the larvaereached stage (F)-G (24 DPH) there was no mortalityduring the incubation. As a separate test for treatmentrecovery, six larvae (stage E) were injected with pHsolution and all of them remained alive for three dayswhen they were sacrificed.

FAA assimiliation

The FAA solution injected into the digestive tractseemed to be absorbed from the lumen into the lar-val body compartment with a high efficiently. Twohours post injection about 70 to 90% of the injectedlabelled diet was retained in the larval body, while theremaining 10 to 30% had passed unabsorbed throughthe digestive tract and into the incubation water. Inmost cases there was no significant increase in thefraction lost to the incubation seawater from 2 to 6 hpost-injection (Figure 1). This suggests that all thebolus of liquid diet injected into the lumen was com-pletely processed by the digestive system during thefirst 2 h after injection. Analysis of the pooled data2 and 6 h post-diet injection showed that a liquid so-lution of 14C-FAA showed was assimilated with anaverage efficiency of 79.5± 7.1% (SD;n = 91).

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Figure 1. Assimilation of14C-FAA (free amino acids) injected as a bolus of aqueous solution in developing Japanese flounder. Note that fromDay 26 the pool retained in the larvae was separated into two compartments (1) the TCA supernatant with the extracted solubles including FAAand (2) the TCA precipitated larval body containing the protein.

Regression analysis of the pooled data 2 and 6 hpost diet injection showed that the absorption effi-ciency was somewhat lower in the younger larvalstages (y = 0.7431x + 60.619; R2 = 0.3121)increasing from 72% at 17 DPH to 89% at 33 DPH(p<0.005).

From 26 DPH the larval body was separated intotwo pools; a TCA precipitate fraction containing thelarval body protein and a TCA soluble fraction con-taining FAA. The data from this compartmentalisationshow that there was a trend towards slower rate ofincorporation of the injected FAA into body proteinas the larvae grew older. In the G stage (26 DPH)about 70% of the injected FAA had been incorporated

into the TCA precipitate fraction 30 min after injec-tion (Figure 1). Of the remaining, 20% was found inthe TCA soluble fraction and 10% had passed throughthe gut unabsorbed. After 1 h 75% of the FAA wasincorporated into the TCA precipitate fraction andthereafter no significant increase occurred in this pool.In older fish (I stage; 33 DPH) only about 45% of theinjected FAA had been incorporated into TCA precip-itate fraction 30 min after injection while more than40% was still present in the TCA soluble fraction. Atthis stage the TCA precipitate fraction continued toincrease even 2 h after the diet was injected. Six hafter injection 65% of the fed FAA was found in the

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Figure 2. Japanese flounder larvae injected with pH indicator solutions at various developmental stages. pH mix 1 consisted of 0.1% M-Creosol(Sigma) while pH mix 2 contained 0.1% Chlorphenol Red and Bromophenol Blue. The standards were immersed in water and photographedunder the same light condition as the larvae under the dissecting microscope. Note that due to variation in growth and development, stages G toH-I were all present at 29 DPH. S: stomach/presumptive stomach, M: midgut, H: hindgut

TCA precipitate fraction. At this time the TCA solublefraction contained 20% of the injected amount.

pH

The pH of the stomach in D and D-E stage (18 to 20DPH) was evidently alkaline (8< pH< 8.5; Table 1;Figure 2) followed by a gradual acidification with de-

velopment until pH was< 4 in larvae at H stage (31DPH). The present method cannot quantify the low-ering exactly but the observed colour of the injectedpH indicators suggested a pH in the range from 6.5–8.0 at F-G stage and 6.0–7.0 at G-H stage (i.e., slightacidification). A difference in the pH between stomachand midgut was clearly seen in G-stage (Figure 2). The

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Table 1. pH in the gut of developingJapanese flounder. pH is presented as arange based on visual observations oncolour changes in two pH mixtures in-jected. See text for further explanations

DPH Stage Fed larvae

Stomach Midgut

18 D 8.0–8.5 8.0–8.5

24 (F)-G 6.5–8.0 8.0–8.5

26 G 6.5–7.0 8.0–8.5

29 G-H 6.0–7.0 8.0–8.5

31 H <4.0 8.0–8.5

33 I <4.0 8.0–8.5

pH indicator mix 1 was bright yellow (pH ca. 7.5) inthe stomach and as soon as the solution passed intothe midgut it became purple (pH>8). The pH in thestomach did not go below 4 until the H-stage and atthis developmental stage the larvae injected with pHsolution B showed a bright yellow colour (pH< 4.0)at the very moment it was injected. This indicates thatthe HCl secreting cells become functional at the Hstage and that the secretion rates of HCl are sufficientto reduce pH of ingested diets rapidly.

Gut transit time

The presumptive stomach was a separate compartmentand served as a reservoir from the earliest recordeddevelopmental stage (Stage D, Figure 2). At the timeof injection the pyloric sphincter was in most casesfirmly closed and remained so for several min unlessthe volume and/or pressure in the stomach increasedabove what seemed to be an upper filling capacity.In some cases the injection capillary initiated trainsof small reverse peristaltic waves in the oesophagusarea which resulted in partial expulsion of the stomachcontent back into the branchial cavity. More forcefulactivity resembling vomiting where the stomach wasalmost completely emptied was only observed in afew cases. Neither the reverse peristalsis nor vomitingwas observed after the capillary was removed from thedigestive tract.

The injected coloured solution was not retained inthe stomach for long, and on average 90% of the con-tent was passed down to the midgut within 10 min(Figure 3). Most of the solution (about 90%) waspassed through the midgut in about 1 h and the passageto the hindgut started less than 10 min after the injec-

Figure 3. Gut passage of a single injected bolus of a coloured aque-ous solution in Japanese flounder. The content in each compartmentis based on visual assessment of the relative volume and presenceof colour in each compartment (see text for further explanations). S:stomach, M: midgut, H: hindgut, ES: oesophageal sphincter, PS :pyloric sphincter.

tion (Figure 3). Thirty min after injection the solutionwas equally distributed between mid- and hindgut.The transport of the solution along the digestive tractinvolved both diffusion and, more common, peristalticmovements of variable strength. In one observed casethe peristaltic waves were initiated every 8.5 s. Itwas observed that the contraction started in the areawith high filling and then proceeded rectally to be-come extinct after some sec. The peristalsis sometimesceased for a long time, and could also be replaced bynon-propulsive, mixing activity (segmentation). Themixing activity consisted of local contractions of a fewseconds in duration.

Three hours after injection the coloured solutiononly remained in the hindgut and in many cases thehindgut seemed to be completely empty at this time.Of the larvae collected 6 h after injection 85% of thelarvae was completely empty, while the other had onlya small hint of colour in the hind gut.

Discussion

Larvae of Japanese flounder proved to be tolerant tothe moderate stress imposed by handling, anaestheticsand the micro-injection technique used in the presentstudy as shown by the high overall survival rate of93%. In the studies reported by Rust (1995) the au-thors claimed that the main cause of post-operativemortality was due to an inappropriate concentrationof the anaesthetics. In the present study the final con-centration of 10 mg l−1 MS 222 was chosen as acompromise between (1) the anaesthetic effect of the

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larvae to allow handling and injection, (2) the impor-tance to maintain continuous opercular movements toensure survival (Rust et al. 1993), and (3) a rapid re-covery to ensure proper physiological function of thedigestive system.

The presumptive stomach/stomach of the Japaneseflounder larvae served as a functional storage chamberwith sphincters in the cardiac (oesophagus) and caudal(pyloric sphincter) end for all stages tested (Figures 2,3). The short retention time of less than 10 min forthe bolus in the stomach may be due to the liquid dietwith its low viscosity. Observations showed that assoon as the pyloric sphincter opened a large fraction ofthe stomach content was emptied into the midgut. Theemptying behaviour of the presumptive stomach whenfilled with particulate food remains to be studied.

The alkaline pH of the presumptive stomach in theD and D-E stages shows that there is no HCl pro-duction at these early developmental stages. This sup-ports histological observations that altrical fish larvae(Tanaka 1973) including Japanese flounder (Tanakaet al. 1996), do not posses the HCl producing cells(gastric glandular acini) at this stage. A gradualdecline in the pH from F-G stage and onward suggestsa low, but increasing, capacity for HCl production andsecretion into the stomach lumen. When the larvaereached the H-stage the pH in the stomach was lowerthan 4. This supports the study of Tanaka et al. (1996)who suggested that the gastric gland was not func-tional until G stage. Walford and Lam (1993) workingon Asian seabass (Lates calcarifer) showed that priorto day 14 post hatch there was no acid secretion inthe presumptive stomach. These authors showed agradual decline in the pH in the presumptive stomachdecreasing from about 8 at day 14 to about 3.7 at day22.

Several authors have previously shown a slightlyacidic anterior intestine of larval fishes shortly af-ter onset of first feeding. The methods used in suchstudies include micro- electrode measurement in dis-sected sections of the gut of freshly killed larvae (Mahret al. 1983; Buddington 1985; Walford and Lam 1993)and immersion in, or injection of pH indicatorsinvivo (Verreth et al. 1992; Bengtson et al. 1993; Rustet al.1993; Rust 1995).

A difference in the pH between the stomach andthe midgut was evident from stage F-G (Table 1; Fig-ure 2) since the pH solution A was bright yellow(pH < 7.5) in the stomach and as soon as the so-lution passed the pyloric valve and into the midgutit instantly became purple (pH≥ 8.5). Histological

studies have shown that the pancreas differentiationis complete at onset of first feeding with an openingof the pancreatic duct into the pyloric area (Tanaka1972). Immunohistochemical analysis for trypsin-likeenzymes has shown active secretion into the gut lu-men from 3 DPH (Kurokawa and Suzuki 1996). Thepresently reported rapid change in colour when the pHindicator passed the pyloric sphincter shows that thereis also active secretion of alkaline fluid most likelyHCO−3 the pancreas into the pyloric area lumen. Thealkaline pH observed in the midgut, which also havebeen described earlier (Walford and Lam 1993), istherefore not due to drinking of seawater (pH about8.2) but results from active secretion of alkaline fluid.We cannot draw any conclusions at what stage the se-cretion starts since the pH in the stomach, midgut andseawater all are in the alkaline area before the F stage.

Data for proteolytic enzymes in Japanese flounder(Alvarez et al. 1999) showed that, at hatching, boththe total and specific gastric pepsin-like activities arevery low. The total pepsin-like activity in developinglarvae increase slowly from 10 units (assessed asµgtyrosine produced 30 min−1 ind−1) in the D stage toca 30 units in the H stage (climax of metamorpho-sis), more than 150 units in I stage (just settled) andmore than 300 units in I4 stage (completely metamor-phosed) (Alvarez et al. 1999). Thus, there seems tobe synchronised activation of the mechanisms respon-sible for production and secretion of pepsin and HCl.This would assure pepsin activation from its zymogenform, pepsinogen, by HCl mediated hydrolysis as wellas producing the optimum pH range for pepsin activ-ity. A pattern of declining pH and increasing pepsinactivity was also found in Asian seabass (Walford andLam 1993).

Since natural folded proteins present a smaller sur-face area than denatured proteins, they may be lessreadily attacked by alkaline proteases (Jany 1976).Thus, due to the absence of HCl and pepsin secre-tion, there is no preparatory acid denaturation of in-gested proteins in the early feeding larvae of Japaneseflounder before stage G-H. This may imply lowerproteolytic efficiency in the gut with resulting lowerassimilation of proteins from the diets. Alvarez et al.(1999) suggested that the digestive mechanism on pro-tein change during the H stage, which is recognisedas a critical stage for settlement of Japanese flounder(Tanaka et al. 1996).

FAA seemed to be effectively absorbed from thegut lumen and incorporated into the body tissues ofJapanese flounder even in the earliest tested stages. It

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is important to emphasise that the presently reporteddata show the relative amount retained in the body andmay in fact underestimate the total amount absorbedfrom the gut. The reason is that the method used doesnot discriminate between two possible sources for14Cin the water (W). The first being unabsorbed14C-FAA emptied from the gut while the other is14C-CO2originating from catabolism of absorbed14C-FAA. Weare currently carrying out studies to separate thesecomponents.

Two hours post injection the bolus of liquid dietwas almost completely processed by the digestive sys-tem. At the earliest stages (D-E) examined 72% ofthe injected FAA was assimilated into the body at thistime. Although not directly comparable, this is higherthan the assimilation of FAA described for the ear-liest recorded stages of other altrical fish larvae, thestriped bass and zebrafish, which was 42% and 59%,respectively (Rust 1995). The striped bass and zebrafish larvae where examined at 17 and 5 days post firstfeeding respectively. This is well before metamorpho-sis for these fishes which occurs 45–55 and 14 dayspost first feeding, respectively (Rust 1995).

The assimilation efficiency of FAA increasedslightly with development and at stage I about 85% ofthe FAA wa.s retained in the Japanese flounder larvae.An increase in the assimilation of FAA with develop-ment was also found in the larvae of striped bass andzebrafish (Rust 1995) although the reported values forassimilation efficiency in Japanese flounder are higher.In striped bass the assimilation efficiency increasedto 70% at 47 days post first feeding while in larvaeof zebrafish assimilation efficiency increased to 82%at 7 days post first feeding. The increase in assimila-tion efficiency with time suggests increasing efficiencyfor absorbing FAA as the larvae develop. Gwak et al.(1999) demonstrated a substantial reduction of the gutepithelial cell height when the Japanese flounder lar-vae transferred into I stage juveniles and suggestedit may be a replacement of gut epithelium from lar-val to adult types associated with metamorphosis. Itseems reasonable to speculate that the gut replacementwould relate to the increasing efficiency. The physio-logical mechanisms underlying these observations arethe focus for our further studies.

It is unknown whether the absorption of FAA fromthe gut in larvae is a saturable phenomenon obey-ing classic Michaelis–Menten kinetics or whether theabsorption is a passive event following concentrationgradients. Methionine is often selected as a modelfor FAA absorption since it is one of the first lim-

iting essential amino acid for protein synthesis infish (Ash 1985) including their larval stages feedingon Artemiaand zooplankton (Conceição 1997). Rust(1995) showed that free methionine was assimilatedwith an efficiency of about 90% over the course of thestudy on striped bass. This was higher than the absorp-tion of a mix of FAA and he suggested that one or morespecific carriers for methionine became functional be-fore the active transport systems for other amino acids.In adult fishKt (the apparentKm) for L-methioninein the gut have been reported to be 1.9 mM (rainbowtrout, Oncorhyncus mykiss; Ingham and Arme 1977),and 1.8 (Atlantic salmon,Salmo salar; Olsen 1998)with a Vmax in the latter species of 0.13 nmol min−1

mg gut−1 DW (Olsen 1998). The presently used mixof amino acids has a calculated concentration of me-thionine equal to 1.5µM and was therefore most likelywell below saturation levels for the active transportsystems into the gut epithelia. Further studies are nec-essary to determine the maximal absorption rates ofFAA from the digestive tract into the larval tissues.

In their study, Rust and co-workers (Rust et al.1993; Rust 1995), concluded that in early larvalstages amino acids in free form were more efficientlyassimilated than amino acids in polymerised forms(assimilation order: FAA> peptides> protein). Pro-tein assimilation was around 30% in the first stagesrecorded for striped bass. Interestingly, the differencesin assimilation rate decreased as the larvae approachedmetamorphosis reaching about 60, 80 and 90% forprotein, peptides and FAA, respectively (Rust 1995).

It has been suggested that FAA (Fyhn 1989) or lowmolecular peptides (Walford and Lam 1993) are vitalcomponents of the diet of first-feeding marine fish lar-vae. Indeed, Rønnestad and Naas (1993) concludedthat fed Atlantic halibut larvae derived about 60%of its energy need from amino acids during the firstmonth of exogenous feeding. Data collected for fourspecies of marine fish larvae (Atlantic halibut,Hip-poglossus hippoglossus; Atlantic cod,Gadus morhua;turbot,Scophthalmus maximusand gilthead seabream,Sparus aurata), supports that 60% or greater of theenergy metabolism is based on catabolism of aminoacids (R.N. Finn, University of Bergen, pers. comm).The presently reported data for larvae of Japaneseflounder support those of Rust (1995) that FAA areabsorbed with a high efficiency. Current studies are inprogress to quantify the maximal absorption rates ofFAA and protein and compare these with the demandfor amino acids in fast growing larvae for growth andenergy dissipation at various developmental stages.

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Acknowledgements

We thank Mr. N. Hriai for rearing the larvae, As-sociate Professors M. Tagawa for help during theexperiment and O. Garatun-Tjeldstø for initial dis-cussions on the scintillation technique. IR gratefullyacknowledge a visiting scientist grant from the KyotoUniversity Foundation, and additional support fromthe Norwegian Research Council grant 115876/122.

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